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ultrasonic technology for cutting and sealing soft tissue was introduced > 20 years ago as a safer alternative to monopolar electrosurgery.1 during the ensuing period , the advantages of ultrasonic sealing and cutting devices over conventional electrosurgery , such as excellent hemostasis with minimal thermal damage,2 reduced risk of nerve damage via the strictly mechanical mechanism of action of ultrasound,3 and low visual obstruction from mist or smoke,4 have become well documented . furthermore , its adoption as a mainstay in the armamentarium of surgeons was solidified by improvements in ergonomics , delivery mechanisms , and dissection capabilities , which resulted in a large portfolio of tools used by surgeons in all areas of surgery for their multifunctionality , precision , and control.5 early versions of the 5 mm ultrasonic shears were limited to sealing vessels up to 3 mm in outer diameter.6 later versions , notably harmonic ace ( harmonic ace+7 shears ; ethicon inc . , cincinnati , oh , usa ) ( harmonic 7 ) extended the range of reliable sealing capability to 5 mm.7 advanced bipolar devices introduced in the mid to late 1990s , such as ligasure ( ligasure 5 mm blunt tip lf1537 laparoscopic instrument ; covidien , mansfield , ma , usa ) , gyrus ( olympus corporation , tokyo , japan ) , and enseal ( ethicon inc . ) , were developed and cleared by the us food and drug administration ( fda ) to seal vessels up to 7 mm in diameter . until now , no tissue sealer using ultrasonic technology alone has been approved to seal vessels of this size , giving users the impression that there is a 5 mm glass ceiling for ultrasonic vessel - sealing capability . even the recent combination of advanced bipolar energy with ultrasonic technology relies on bipolar energy to produce vessel seals.8 sealing vessels with ultrasonic technology , like all thermal sealing , is under the control of three critical and interdependent variables : compression , heat , and time.911 furthermore , because cutting occurs simultaneously with coagulation , unlike with bipolar technologies , the frequency , geometry , and compression of the device are critical determinants of seal strength as well as cutting ability.1,12 recent advances in the control of ultrasonic energy delivery through adaptive tissue technology furnished the possibility of sealing vessels up to 7 mm in diameter by precise modulation of energy delivery and time without needing to alter device geometry . this was supported by evidence that vessel seals in the 35 mm diameter range demonstrated increased burst pressures and decreased thermal damage when adaptive tissue technology was included in the device.13 adaptive tissue technology is a platform capability within the harmonic system that actively monitors the instrument during use and enables the system to sense and respond appropriately to changes in tissue conditions using a set of proprietary algorithms . these algorithms have been designed to deliver improved hemostasis , thermal management , and precision , depending on the settings . harmonic 7 ( figure 1 ) is a recently fda - cleared device that leverages adaptive tissue technology with the addition of predictive analytics to modulate energy delivery during the sealing cycle . herein we report on the successful development of an ultrasonic device , harmonic 7 , which is able to seal vessels up to 7 mm in diameter with burst pressures significantly greater than those observed with advanced bipolar technologies . using benchtop testing , the ultrasonic device was compared with advanced bipolar devices for both rate of hemostatic sealing and burst pressure , and the impact of the harmonic 7 activation time on seal strength was evaluated . in addition , the ultrasonic and advanced bipolar devices were tested in preclinical porcine and caprine studies to examine initial hemostasis and seal durability during simulated hypertensive crisis ( blood pressure challenge ) , both acutely and after a 30-day survival period . histological evaluation was also performed to ensure that the historically low thermal damage of ultrasonic devices was not significantly altered in a clinically meaningful way by the changes in energy delivery . ultrasonic and bipolar devices were used to seal and transect porcine carotid arteries ( animal biotech industries , inc . , danboro , pa , usa ) distributed into two groups by outer diameter : small ( 35 mm ) and large ( > 57 mm ) . porcine carotid arteries are believed to better represent human arteries than arteries of other locations in pigs.14 harmonic 7 was operated at power level 3 only for vessels 5 mm in diameter , and in the advanced hemostasis ( ah ) mode for vessel sizes 7 mm . ligasure was operated in the default mode indicated by the instructions for use ( two green bars ) . sealing of all vessels was performed under an axial tension of 50 g on the vessel , which was filled with saline at a pressure of 60120 mmhg . the test devices were applied and activated and the transection sites were observed visually for any signs of leakage at transection . sealed vessels were tested by filling with saline solution at a rate of 47 ml / min while monitoring pressure until a fall in pressure occurred or the maximum pressure ( ~2,000 mmhg ) was reached . if a vessel leaked at transection , it was assigned a burst pressure of 0 mmhg . time to transection was recorded as the amount of time from initial activation to complete transection of the vessel . in the case of harmonic 7 , vessel transection occurred as a direct result of the activation . in the case of ligasure , vessel transection included the time to throw the knife blade after the completion tone was registered . the effect of time on harmonic 7 seal strength and the influence of time on vessel seal were evaluated in the in vitro porcine carotid arteries by activating each device 14 times : twice at each of 2 , 4 , 6 , 8 , 10 , and 12 second durations , as well as two full transections . each time duration was evaluated in 40 vessels for a total of 280 experimental data points . vessels were defined as recannulated if no pressure was required to observe saline flow through the vessel in other words , the lumen of the vessel opened back up spontaneously with no pressure required to do so . recannulated at pressure was defined as saline flowing through the seal only when pressure was applied . a partial transection was defined as a vessel that was not completely divided along the seal area and separated like a full transection but some of the vessel had begun to separate into a full transection after the device was activated for a period of time and then pulled away . no transection is an event defined as the vessel not being separated after device removal when the device was stopped at a prespecified time . despite no transection , it is conceivable that a vessel was sealed well enough to have a burst pressure . burst is an event defined as bursting upon pressurization after complete transection of the vessel with an intact seal . all in vivo procedures were reviewed and the animals approved for use in the studies by the ethicon endo - surgery institutional animal care and use committee . the harmonic 7 ultrasonic devices were evaluated in multiple in vivo porcine studies and compared with advanced bipolar devices in several in vivo caprine subjects . for the acute porcine studies , after standard induction of anesthesia , the animal was placed in dorsal recumbency , and a ventral midline neck incision and laparotomy were performed . the devices were then applied to transect the vessels , which included the gastroepiploic veins and artery ( isolated or as a pedicle ) , carotid artery ( unilateral ) , short gastric pedicles , and splenic vein and artery , completing a splenectomy . for the acute study , hemostasis was evaluated intraoperatively after initial transection and again after a simulated hypertensive crisis ( overstress test ) post - transection in which a vasopressor agent ( phenylephrine ) was titrated intravenously to increase the systolic blood pressure to at least 200 mmhg for a period of at least 10 minutes . after euthanasia via intravenous injection of pharmaceutical grade potassium choride while under a surgical plane of anesthesia , transection seals with 12 cm of adjacent untreated artery were nonthermally excised and fixed in 10% neutral buffered formalin . the tissue was processed and lateral thermal damage was assessed via histological staining with hematoxylin and eosin ( h&e ) by a blinded pathologist . the survival porcine study followed the same surgical preparation and procedures for splenectomy and unilateral carotid artery transection , and hemostasis was evaluated intraoperatively after initial transection and observed for 10 minutes before the incisions were closed using a standard surgical technique ( sutures / tissue glue ) . at 30 days postoperatively , vessel seal integrity was challenged with the pig under general anesthesia by simulating an acute hypertensive crisis as described . following completion of the blood pressure challenge period , necropsy all vessel transection sites were observed for hemorrhage . for the caprine studies , similar surgical preparation and a ventral midline neck incision for the acute study , following a bilateral carotid artery transection with the device , hemostasis was evaluated intraoperatively at initial transection and again after a simulated hypertensive crisis ( overstress test ) . a vasopressor agent ( epinephrine or vasopressin ) after each challenge period , the vasopressor agent was discontinued to allow the blood pressure to return to baseline . each goat underwent four rounds of bilateral carotid artery transection and subsequent blood pressure challenges . at the completion of the study , hemostasis was evaluated intraoperatively after initial transection and observed for 10 minutes before the incision was closed . at 30 days postoperatively , vessel seal integrity was challenged with the goat under general anesthesia by simulating an acute hypertensive crisis as described . following completion of the blood pressure challenge period , continuous variables such as burst pressure or thermal damage were compared using student s t - test , analysis of covariance , or the mann whitney test , as appropriate , and binomial variables such as hemostasis and adhesions were compared using fisher s exact test . ultrasonic and bipolar devices were used to seal and transect porcine carotid arteries ( animal biotech industries , inc . , danboro , pa , usa ) distributed into two groups by outer diameter : small ( 35 mm ) and large ( > 57 mm ) . porcine carotid arteries are believed to better represent human arteries than arteries of other locations in pigs.14 harmonic 7 was operated at power level 3 only for vessels 5 mm in diameter , and in the advanced hemostasis ( ah ) mode for vessel sizes 7 mm . ligasure was operated in the default mode indicated by the instructions for use ( two green bars ) . sealing of all vessels was performed under an axial tension of 50 g on the vessel , which was filled with saline at a pressure of 60120 mmhg . the test devices were applied and activated and the transection sites were observed visually for any signs of leakage at transection . sealed vessels were tested by filling with saline solution at a rate of 47 ml / min while monitoring pressure until a fall in pressure occurred or the maximum pressure ( ~2,000 mmhg ) was reached . if a vessel leaked at transection , it was assigned a burst pressure of 0 mmhg . time to transection was recorded as the amount of time from initial activation to complete transection of the vessel . in the case of harmonic 7 , vessel transection occurred as a direct result of the activation . in the case of ligasure , vessel transection included the time to throw the knife blade after the completion tone was registered . the effect of time on harmonic 7 seal strength and the influence of time on vessel seal were evaluated in the in vitro porcine carotid arteries by activating each device 14 times : twice at each of 2 , 4 , 6 , 8 , 10 , and 12 second durations , as well as two full transections . each time duration was evaluated in 40 vessels for a total of 280 experimental data points . vessels were defined as recannulated if no pressure was required to observe saline flow through the vessel in other words , the lumen of the vessel opened back up spontaneously with no pressure required to do so . recannulated at pressure was defined as saline flowing through the seal only when pressure was applied . a partial transection was defined as a vessel that was not completely divided along the seal area and separated like a full transection but some of the vessel had begun to separate into a full transection after the device was activated for a period of time and then pulled away . no transection is an event defined as the vessel not being separated after device removal when the device was stopped at a prespecified time . despite no transection , it is conceivable that a vessel was sealed well enough to have a burst pressure . burst is an event defined as bursting upon pressurization after complete transection of the vessel with an intact seal . all in vivo procedures were reviewed and the animals approved for use in the studies by the ethicon endo - surgery institutional animal care and use committee . the harmonic 7 ultrasonic devices were evaluated in multiple in vivo porcine studies and compared with advanced bipolar devices in several in vivo caprine subjects . for the acute porcine studies , after standard induction of anesthesia , the animal was placed in dorsal recumbency , and a ventral midline neck incision and laparotomy were performed . the devices were then applied to transect the vessels , which included the gastroepiploic veins and artery ( isolated or as a pedicle ) , carotid artery ( unilateral ) , short gastric pedicles , and splenic vein and artery , completing a splenectomy . for the acute study , hemostasis was evaluated intraoperatively after initial transection and again after a simulated hypertensive crisis ( overstress test ) post - transection in which a vasopressor agent ( phenylephrine ) was titrated intravenously to increase the systolic blood pressure to at least 200 mmhg for a period of at least 10 minutes . after euthanasia via intravenous injection of pharmaceutical grade potassium choride while under a surgical plane of anesthesia , transection seals with 12 cm of adjacent untreated artery were nonthermally excised and fixed in 10% neutral buffered formalin . the tissue was processed and lateral thermal damage was assessed via histological staining with hematoxylin and eosin ( h&e ) by a blinded pathologist . the survival porcine study followed the same surgical preparation and procedures for splenectomy and unilateral carotid artery transection , and hemostasis was evaluated intraoperatively after initial transection and observed for 10 minutes before the incisions were closed using a standard surgical technique ( sutures / tissue glue ) . at 30 days postoperatively , vessel seal integrity was challenged with the pig under general anesthesia by simulating an acute hypertensive crisis as described . following completion of the blood pressure challenge period , necropsy all vessel transection sites were observed for hemorrhage . for the caprine studies , similar surgical preparation and a ventral midline neck incision for the acute study , following a bilateral carotid artery transection with the device , hemostasis was evaluated intraoperatively at initial transection and again after a simulated hypertensive crisis ( overstress test ) . a vasopressor agent ( epinephrine or vasopressin ) after each challenge period , the vasopressor agent was discontinued to allow the blood pressure to return to baseline . each goat underwent four rounds of bilateral carotid artery transection and subsequent blood pressure challenges . at the completion of the study , hemostasis was evaluated intraoperatively after initial transection and observed for 10 minutes before the incision was closed . at 30 days postoperatively , vessel seal integrity was challenged with the goat under general anesthesia by simulating an acute hypertensive crisis as described . following completion of the blood pressure challenge period , the animal was euthanized as described . at continuous variables such as burst pressure or thermal damage were compared using student s t - test , analysis of covariance , or the mann whitney test , as appropriate , and binomial variables such as hemostasis and adhesions were compared using fisher s exact test . during development of harmonic 7 , the strength of the vessel seal was evaluated over a range of power levels in 57 mm porcine carotid arteries , with the ah mode producing the highest burst pressures ( figure 2 ) . in this series of experiments , the high level was similar to power level 5 , and the low level was similar to power level 3 , which is indicated for sealing vessels up to 5 mm in diameter . the highest power level used is not available on generators and is evaluated only for the purpose of these experiments . the results demonstrated that modulated power delivery with the ah mode created the strongest seals in vessels of diameters up to 7 mm . transection time at the highest power level averaged 2.6 seconds , whereas in ah mode transection time was variable due to energy modulation but up to 17.1 seconds in duration . based on these initial studies , the ah mode was chosen as the best means to achieve optimal blood vessel seals without changing instrument geometry and was therefore compared with existing technologies of ultrasonics and advanced bipolar for efficacy . results for median burst pressures for harmonic and ligasure in both 35 mm and 57 mm porcine carotid arteries are given in table 1 and for mean burst pressures in figure 3 . harmonic 7 in ah mode had significantly higher median burst pressures than ligasure for both 35 mm ( p=0.001 ) and 57 mm ( p<0.001 ) vessels . harmonic 7 at power level 3 also had significantly higher median burst pressure than ligasure for 35 mm vessels ( p=0.046 ) but less than that observed with the ah mode ( p=0.031 ) . conversely , harmonic 7 in ah mode delivered longer transection times than ligasure for vessels 57 mm in diameter . mean transection time for harmonic 7 was 12.7 seconds ( range 7.818.5 seconds ) versus 5.0 seconds ( range 3.46.5 seconds ) for ligasure , depending upon tissue type and thickness . in general , longer sealing times corresponded to higher burst pressures and more reliable sealing . both ultrasonic and advanced bipolar sealing modalities achieved a high rate of complete seals that failed only by bursting under pressure ( figure 4 ) . harmonic 7 in ah mode had a lower rate of leaks at transection for 57 mm vessels . unlike ligasure , harmonic 7 in either power level 3 or ah mode did not experience any adventitial leaks . as some surgeons may attempt to seal a vessel prior to transection using the so - called ultrasonic clip method that seals not to complete vessel transection but to a specific amount of time or visual cue , the security of such seals was evaluated by applying energy for defined periods of time to 57 mm porcine carotid arteries using the harmonic 7 ah mode ( figure 5 ) . the rate of vessel recannulation decreased as the time interval of sealing increased and was highly correlated with time . in contrast , 97.4% were completely sealed when the device was allowed to reach full transection . that is , although there was a visual seal seen with shorter durations of activation , these seals , when placed under pressure , allowed fluid to pass : ie , the compressed lumen recannulated . no vessel was fully transected with a seal that would withstand a burst pressure similar to that of full activation unless at least 8 seconds of activation was applied . furthermore , recannulation with pressure was observed only after a minimum of 4 seconds of activation and demonstrated a burst pressure that although in many cases was greater than physiologic pressures was always less than half that observed when vessels were sealed enough to burst . burst pressure was also dependent upon transection time , with the highest burst pressures achieved when energy was activated through the complete vessel transection . evaluation of harmonic 7 in an in vivo porcine study ( table 2 ) showed high rates of hemostasis at initial transection that remained secure during a simulated hypertensive crisis for both acute and survival studies . in the ah mode , all seals were hemostatic both initially and upon challenge . in an acute in vivo caprine study , harmonic 7 and ligasure had similar rates of hemostasis ( table 3 ) , but harmonic 7 displayed significantly less thermal damage than ligasure ( p=0.003 ) , as evaluated by h&e histological staining ( figures 6 and 7 ) . in a caprine 30-day survival study of harmonic 7 using ah mode , carotid artery vessel seals in all five animals were hemostatic at initial sealing ( 100% ) and after a simulated hypertensive crisis at the completion of the survival period ( 100% ) . during development of harmonic 7 , the strength of the vessel seal was evaluated over a range of power levels in 57 mm porcine carotid arteries , with the ah mode producing the highest burst pressures ( figure 2 ) . in this series of experiments , the high level was similar to power level 5 , and the low level was similar to power level 3 , which is indicated for sealing vessels up to 5 mm in diameter . the highest power level used is not available on generators and is evaluated only for the purpose of these experiments . the results demonstrated that modulated power delivery with the ah mode created the strongest seals in vessels of diameters up to 7 mm . transection time at the highest power level averaged 2.6 seconds , whereas in ah mode transection time was variable due to energy modulation but up to 17.1 seconds in duration . based on these initial studies , the ah mode was chosen as the best means to achieve optimal blood vessel seals without changing instrument geometry and was therefore compared with existing technologies of ultrasonics and advanced bipolar for efficacy . results for median burst pressures for harmonic and ligasure in both 35 mm and 57 mm porcine carotid arteries are given in table 1 and for mean burst pressures in figure 3 . harmonic 7 in ah mode had significantly higher median burst pressures than ligasure for both 35 mm ( p=0.001 ) and 57 mm ( p<0.001 ) vessels . harmonic 7 at power level 3 also had significantly higher median burst pressure than ligasure for 35 mm vessels ( p=0.046 ) but less than that observed with the ah mode ( p=0.031 ) . conversely , harmonic 7 in ah mode delivered longer transection times than ligasure for vessels 57 mm in diameter . mean transection time for harmonic 7 was 12.7 seconds ( range 7.818.5 seconds ) versus 5.0 seconds ( range 3.46.5 seconds ) for ligasure , depending upon tissue type and thickness . in general , longer sealing times corresponded to higher burst pressures and more reliable sealing . both ultrasonic and advanced bipolar sealing modalities achieved a high rate of complete seals that failed only by bursting under pressure ( figure 4 ) . harmonic 7 in ah mode had a lower rate of leaks at transection for 57 mm vessels . unlike ligasure , harmonic 7 in either power level 3 or ah mode did not experience any adventitial leaks . as some surgeons may attempt to seal a vessel prior to transection using the so - called ultrasonic clip method that seals not to complete vessel transection but to a specific amount of time or visual cue , the security of such seals was evaluated by applying energy for defined periods of time to 57 mm porcine carotid arteries using the harmonic 7 ah mode ( figure 5 ) . the rate of vessel recannulation decreased as the time interval of sealing increased and was highly correlated with time . in contrast , 97.4% were completely sealed when the device was allowed to reach full transection . that is , although there was a visual seal seen with shorter durations of activation , these seals , when placed under pressure , allowed fluid to pass : ie , the compressed lumen recannulated . no vessel was fully transected with a seal that would withstand a burst pressure similar to that of full activation unless at least 8 seconds of activation was applied . furthermore , recannulation with pressure was observed only after a minimum of 4 seconds of activation and demonstrated a burst pressure that although in many cases was greater than physiologic pressures was always less than half that observed when vessels were sealed enough to burst . burst pressure was also dependent upon transection time , with the highest burst pressures achieved when energy was activated through the complete vessel transection . evaluation of harmonic 7 in an in vivo porcine study ( table 2 ) showed high rates of hemostasis at initial transection that remained secure during a simulated hypertensive crisis for both acute and survival studies . in the ah mode , all seals were hemostatic both initially and upon challenge . in an acute in vivo caprine study , harmonic 7 and ligasure had similar rates of hemostasis ( table 3 ) , but harmonic 7 displayed significantly less thermal damage than ligasure ( p=0.003 ) , as evaluated by h&e histological staining ( figures 6 and 7 ) . in a caprine 30-day survival study of harmonic 7 using ah mode , carotid artery vessel seals in all five animals were hemostatic at initial sealing ( 100% ) and after a simulated hypertensive crisis at the completion of the survival period ( 100% ) . the benefits of ultrasonic cutting and coagulation in comparison with conventional electrosurgery are well documented in numerous studies . based on lower heat production in the tissue , incisions made with ultrasonic devices result in less inflammation than those made with electrosurgery,15 along with enhanced revascularization and faster fibrosis remodeling.16 the purely mechanical action of ultrasonic devices , without the flow of electrical current that occurs in electrosurgery , allows usage of the devices in the vicinity of nerves , with greatly reduced risk of injury.3 recently , the differences between ultrasonic and electrosurgery have been examined at a molecular level via transcriptomics and proteomics.17 these studies demonstrated that in the acute phase of wound healing following incisions in subcutaneous tissue , ultrasonic energy produced lower levels of immunological and inflammatory mediators , indicating a reduced amount of iatrogenic trauma . in addition , there was an early shift to wound - healing proteins with ultrasonic energy observed in these studies . adaptive tissue technology is a transformative capability of the harmonic ultrasonic platform that actively monitors the instrument during use and enables the system to sense and respond appropriately to changes in tissue conditions using a set of proprietary algorithms.18 these algorithms can be designed and modified to deliver improved hemostasis , thermal management , and precision , depending on how they are set . the introduction of adaptive tissue technology to ultrasonic devices has notably improved the performance of these devices . for example , the addition of adaptive tissue technology to existing devices is associated with lower peak device temperatures , increased seal strength , faster cutting , and improved visualization from reduction in mist.13 the results observed with adaptive tissue technology are consistent with those observed in piezoelectric surgery in which modulation of energy delivery results in reduced heat and improves the effectiveness of cutting in bone surgery.19 harmonic 7 introduces the use of predictive analytics within the adaptive tissue technology algorithms to modulate the delivery of energy to tissue . based on data obtained from over 18,000 seals performed on vessels up to 7.5 mm in diameter , the system was trained to optimize energy delivery using numerous inputs received by the generator in the ah mode . as shown in figure 8 , the device is monitoring during the three phases of preheating , vessel sealing ( compression ) , and transection . based on monitoring of multiple parameters , the amount and length of time energy is delivered is altered to result in the best - predicted seal . the ah mode of harmonic 7 , used under the conditions specified in benchtop testing , produced a higher success rate and statistically higher burst pressures than an advanced bipolar device for vessels up to 7 mm in diameter . the benchtop data also demonstrate the importance of using the ah mode for large vessels ( > 57 mm ) , because seals were not as reliable and did not reach the same burst pressures when other power settings were used . in in vivo preclinical testing , harmonic 7 at power level 3 had a complete seal success rate that was equivalent to advanced bipolar devices in sealing vessels up to 5 mm in diameter , and in the ah mode for vessels and pedicles up to 7 mm in diameter . most importantly , there was no evidence of hemorrhage at any vessel seal for harmonic 7 at the completion of a 30-day survival period and after simulated hypertensive crisis . temperature and lateral thermal damage are important considerations for surgeons when using energy - based technologies . first , and most importantly , surgeons are concerned about injury to nearby structures either by direct contact or by the visually unrecognizable transmission of energy . second , surgeons are concerned about the potential impact of tissue damage on the inflammatory response and overall recovery of the patient . both harmonic 7 modes ( power level 3 and ah mode ) have demonstrated histologic thermal damage that was statistically less compared with advanced bipolar devices , with no differences in tissue sticking or adhesions between the tissue - sealing methods . however , these differences were small and the potential clinical impact unknown . the generation of heat by an advanced energy device is a critical component to vessel sealing and tissue transection . the temperature the instrument reaches is dependent on multiple variables , including tissue type , tissue thickness , energy used , and power setting . most , if not all , currently marketed advanced energy devices reach instrument temperatures of at least 100c during activation on tissue . there are , however , situations when the temperature of an ultrasonic device can reach well beyond the 100c range due to tissue conditions , sticking of tissue to the blade , and excessive activation times beyond those needed to actually seal and transect the tissue . in this regard , it is important to recognize that the temperature of the instrument ( ultrasonic or advanced bipolar ) is not the same as the temperature of the tissue . in the case of ultrasonic devices , the tissue temperature is usually much lower than the device because heat flows from the device to the tissue following a thermal gradient.20 in the case of bipolar devices , the temperature is often higher in the tissue because of the ohmic heating effect immediately adjacent to the jaws of the device.21 in large part , the increase in temperature observed with ultrasonic devices occurs after transection but before the tissue is completely separated from the device . the rapid increase in temperature using an ultrasonic device is largely a function of the active blade coming into contact and running on the nonactive tissue pad , especially when there is a tissue tag left or partial transection.20 adaptive tissue technology monitors blade conditions through proprietary algorithms and provides surgeons with a secondary auditory tone when transection is near completion . although blade temperature is not directly measured , changes in the blade temperature result in very small changes in the resonant frequency of the device that can be monitored . specific changes in frequency result in generation of a tone to notify the surgeon that additional surgical action , such as increasing tension of the tissue on the active blade , may be required to complete the transection . the secondary tone also indicates that adaptive tissue technology has reduced the power level to deliver enhanced thermal management of the blade . the addition of adaptive tissue technology has been shown to reduce the peak temperatures observed with harmonic devices in previous studies.13 studies also demonstrate that despite increases in blade temperature , minimal conductive heat is transmitted to surrounding tissue , as evidenced by the low amount of lateral thermal damage measured for all harmonic devices and the ability to operate close to nerves ( 2 mm ) without impacting the electrophysiological function of the nerve.3 nevertheless , surgeons must always be mindful that the instrument is hot following activation , and heat mitigation techniques should be employed prior to touching or grasping tissue . the harmonic 7 creates strong and durable vessel seals by modulating the delivery of energy and extending the compression and thermal sealing time . this is consistent with the current understanding of how thermal seals are formed , namely via compression , heat , and time , but also expands our understanding of the process by simplifying the relationship to two components : heat over time and compression over time . harmonic 7 handles these differently by modulating the energy delivery so that the amount of heat is not the same throughout the sealing cycle ( figure 8) . these studies further document the importance of time in achieving the optimal seal by varying the activation duration without achieving transection . reliable and durable sealing occurred only when energy delivery continued through the complete cycle to vessel transection , which raises serious doubt as to the validity of ultrasonic clips . the ability of the device to modulate energy delivery was furthermore substantiated by the variability of time required from vessel to vessel . the concept of extending the length of time to produce better seals is in direct juxtaposition to the desire of surgeons to experience the fastest seal formation possible . although surgeons first and foremost want hemostatic security and safety , they also want this to occur as fast as possible . in fact , all available technologies to date have placed a premium on the fastest sealing time . despite this desire , previous investigators , as well as our own testing , have shown the positive impact of time on obtaining optimal seals both with ultrasonics and electrosurgery . for example , using bipolar energy , longer periods of energy activation resulted in better seals , with periods of 1520 seconds being ideal for traditional bipolar technologies.2124 similar findings have been shown with advanced bipolar technologies.10,11 we , as well as others , have shown the importance of seal time on vessel - sealing security . for example , a significant difference has been shown in burst pressures with harmonic ace at power level 3 versus power level 5 for vessels in the 5 mm range , which correlates with length of the transection time.25 furthermore , recent advances in the understanding of device tissue interactions with surgical staplers have documented the importance of extending compression time in order to deliver better hemostasis and more consistent staple formation.26 the ability to seal 7 mm diameter vessels is not the only important aspect of this device , as surgeons rarely , if ever , seal 67 mm vessels with any energy device . even though the inferior mesenteric artery is often cited as the reason for 7 mm vessel - sealing capacity , the inferior mesenteric artery is , on average , only 34 mm in size.27 in fact , no human vessel in the abdomen is > 5 mm in diameter other than the renal , splenic , and iliac arteries , and arteries > 5 mm are usually divided with a belt and suspenders approach using multiple ties , clips , and/or staples . the key aspect of the ability to seal 7 mm vessels may therefore rest in the confidence , safety margin , and control it provides the surgeon . indeed , burst pressures in the ah mode were greater than those observed with bipolar devices for vessels from 3 mm to 7 mm in diameter . in that regard , it is noteworthy that burst pressures in the ah mode were greater than those observed at power level 3 . thus , the ah mode can also be used under specific situations where the surgeon wants greater confidence in the security of the seal . for example , surgeons faced with sealing a uterine artery or an inflamed vessel can choose to use the ah mode to further optimize the sealing cycle , even if the vessels are 5 mm in diameter . burst pressure studies show that a 5 mm vessel divided at a 45 angle has an equivalent burst pressure to that of a 7 mm vessel divided perpendicularly.28 in other words , even though the vessel is 5 mm in diameter , the area of seal is equivalent to a 7 mm vessel . as a result , the need for 7 mm vessel sealing is as much about size as the angle of sealing when it comes to vessel security . surrounding tissue also plays a role in the visibility of the vessel being fully placed in the jaws of the instrument . an important surgical technique is to ensure that the tips of the device can be seen and that the vessel being transected is fully captured within the jaws . regardless of the modality , advanced bipolar or ultrasonic , a partially transected vessel will not deliver the same vessel security as a fully sealed and transected vessel . therefore , there may be times when skeletonizing the vessel is appropriate to ensure adequate visibility of the vessel and tips of the instrument prior to energy activation and subsequent transection . these studies have also begun to explore an important area in respect of ultrasonic energy , namely the creation of so - called ultrasonic clips . in this process , surgeons apply energy to the vessel proximal and distal to the intended transection site , but do not activate the device long enough to produce transection . the device is then activated between these two clips to a complete transection . the belief is that this will strengthen the overall seal by providing two sites of closure , as one might do with two metallic clips . the recannulation data presented in this study show that for vessels 57 mm , an ultrasonic clip provides no additional security , as almost all vessels were recannulated or recannulated at pressure if the complete seal cycle to transection did not occur . in fact , one could argue that it provides the surgeon with a false sense of security and could potentially reduce the reliability of the seal by weakening the vessel by thermal damage in the area proximal to the transection . these studies document the ability of ultrasonic energy alone to reliably seal large vessels 57 mm in diameter with significantly higher initial burst pressure than with an advanced bipolar technology in benchtop studies . seals created in ah mode are demonstrated to be long - lasting and resistant to fluctuations in blood pressure . these studies also add to the existing body of literature regarding seal formation and support the importance of time in the seal formation process and the resulting security of the seal . using a longer period of compression and heating leads to superior results in respect of burst pressures that were consistently observed with the ultrasonic device . these results , however , should not be interpreted to prove that longer sealing times are always necessary under all conditions . clearly , other factors , such as the type of energy used , the time pattern of tissue heating , end effector geometry , compression force applied , and tissue characteristics , must also be considered . | introductionultrasonic energy is a mainstay in the armamentarium of surgeons , providing multifunctionality , precision , and control when dissecting and sealing vessels up to 5 mm in diameter .
historically , the inability to seal vessels in the 57 mm range has been perceived as an inherent limitation of ultrasonic technology .
the purpose of this study was to evaluate sealing of vessels up to 7 mm in diameter with an ultrasonic device that modulates energy delivery during the sealing period.methodsin ex vivo benchtop and in vivo acute and survival preclinical models , a new ultrasonic device , harmonic ace+7 shears ( harmonic 7 ) , was compared with advanced bipolar devices in sealing vessels 17 mm in diameter with respect of burst pressure , seal reliability , and seal durability .
lateral thermal damage and transection time were also evaluated.resultsex vivo tests of harmonic 7 demonstrated significantly greater median burst pressures than an advanced bipolar device both for vessels < 5 mm in diameter ( 1,078 mmhg and 836 mmhg , respectively , p=0.046 ) and for those in the range of 57 mm ( 1,419 mmhg and 591 mmhg , p<0.001 ) . in vivo tests in porcine and caprine models demonstrated similar rates of hemostasis between harmonic 7 and advanced bipolar devices , with high success rates at initial transection and seal durability of 100% after a 30-day survival period.conclusionsealing 57 mm vessels is not a limitation of the type of energy used but of how energy is delivered to tissue .
these studies document the ability of ultrasonic energy alone to reliably seal large vessels 57 mm in diameter , with significantly greater burst pressure observed in in vitro studies than those observed with an advanced bipolar technology when energy delivery is modulated during the sealing cycle .
furthermore , the seals created in 57 mm vessels are shown to be reliable and durable in in vivo preclinical studies . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the mediterranean diet ( mediet ) is a unique plant - based dietary pattern , an expression of different food cultures of the mediterranean region , and a group of practices , representations , knowledge , and skills that mediterranean population have historically built and recreated in a sustainable interaction with nature [ 13 ] . scientific research has established a beneficial role for the major components of the mediet , such as fatty acids , vitamins , minerals , fiber , and bioactive compounds in cardiovascular diseases ( cvd ) and other chronic degenerative conditions [ 47 ] . however , the possible beneficial role of carbohydrates has been little studied in comparison with existing literature regarding different types of fat . actually , there is epidemiological evidence linking consumption of whole grains and characteristics of the mediet , with decreased risk of cvd and type 2 diabetes mellitus ( t2 dm ) . it has been known for some time that healthy and diabetic subjects show different postprandial glycemic responses to carbohydrates of different quality . dietary glycemic index ( gi ) is an indicator of the quality of carbohydrates consumed in terms of glycemic response , while glycemic load ( gl ) , the mathematical product of the gi of a food and its carbohydrate content , integrates the quantity and quality of carbohydrates consumed [ 1113 ] . these two indices have been used in epidemiological studies to evaluate associations with chronic disease risk , but results have not been fully consistent . in a meta - analysis of 37 prospective studies , the effect of gl and gi on chronic diseases in general was modest , but more evident for t2 dm , coronary heart disease , and gallbladder disease . a more recent meta - analysis of 8 prospective studies focusing on coronary heart disease showed that higher dietary gl and gi significantly increased risk among women and the unfavourable effects were more pronounced in overweight and obese subjects . many foods that are at the core of the mediet have a low gi , such as fruits , vegetables , legumes , nuts , and seeds , and this could play a role in the salutary effects of this dietary model , but only a few studies have reported the blood glucose response to carbohydrate - rich foods typically consumed in the mediterranean area . this prompted us to explore the effect of two dietary interventions with mediet on gl and gi in the predimed study ( prevencin con dieta mediterrnea ) and evaluate their relationship with adherence to the mediet at one year of follow - up . this report is a longitudinal analysis within the frame of the predimed study , a dietary intervention , parallel group , multicenter , single - blinded , randomized trial , designed to ascertain whether a mediet supplemented with olive oil or mediet supplemented with nuts prevents major cardiovascular events ( cardiovascular death , myocardial infarction , and/or stroke ) compared with a low - fat diet in a high risk population . participants were randomly assigned to one of the three diet groups by using a computer - generated random - number sequence . subjects allocated in the mediet groups received individual and group dietary training at baseline and every three months during intervention . additionally , participants received supplemental foods at no cost ; extra virgin olive oil ( 1 l / week ) was provided to the first group ( mediet + evoo ) and 30 g / day of mixed nuts ( 15 g walnuts , 7.5 g hazelnuts , and 7.5 g almonds ) to the second group ( mediet + nuts ) . the group sessions with the mediet groups were run by predimed registered dietitians with up to 20 participants per session and separate sessions for each group . each session consisted of informative talks and provision of written material with elaborate descriptions of typical mediet foods and seasonal shopping lists , meal plans , and cooking recipes . in the control group , dietary advice with the same periodicity and methods and a leaflet recommending the national cholesterol education program adult treatment panel iii the research and ethic committee of the hospital clinic in barcelona , spain , accredited by the department of health and human services and regulated by the federalwide assurance for the protection of human subjects of international ( non - us ) institutions no . the original study sample included 7447 participants , aged 55 to 80 y , at high risk of cvd , with at least one of the following criteria : presence of t2 dm , 3 cardiovascular risk factors ( current smoking , hypertension , and high ldl cholesterol ( 160 mg / dl ) , low hdl cholesterol ( 40 mg / dl in men and 50 mg / dl in women ) , overweight or obesity ( bmi 25 kg / m ) , or family history of premature cvd . incident cases of diabetes at 1 year of follow - up were excluded ( n = 61 ) . subjects with incomplete dietary data at baseline ( n = 37 ) and at one year of follow - up ( n = 773 ) were also excluded . finally , we excluded subjects with values of total energy intake outside predefined limits at baseline ( n = 63 ) and at one year of follow - up ( n = 33 ) . overall , 2866 participants ( 1085 men and 1781 women ) were analyzed in this study . we excluded participants with t2 dm since this population usually has nutritional recommendations to follow a low gl / gi diet and could lead to a bias in our analysis . information on dietary habits was collected by trained dietitians in face - to - face interviews using a 137-item food frequency questionnaire ( ffq ) that was previously validated for a similar population . in spite of the fact that ffq was not designed to evaluate dietary gl and gi , intraclass correlation coefficients between two repeated ffq were 0.85 for gl and 0.32 for gi . from the information collected in the ffq , the daily intake of 131 food and beverage items was obtained in grams per day . spanish food composition tables were used to estimate energy ( kcal per day ) and nutrient ( grams per day ) intake . a 14-item dietary screener was used to assess adherence to the mediet at baseline and every three months [ 21 , 22 ] . this tool was useful in evaluating the compliance with mediet , allowing personalized dietary advice to be provided to subjects allocated in mediet groups by adapting it to the participant 's clinical condition , preferences , and beliefs . twelve questions set a cut - off point expressed in serving units per day or per week and indicate the typical serving size for each food . one point was given for the use of olive oil as the principal source of fat for cooking , for preferring white meat over red meat , and for each of the following 12 food consumption patterns : ( a ) 4 or more tablespoons of olive oil per day ( including that used in frying , salads , meals eaten away from home , etc . ) , ( b ) 2 or more servings of vegetables per day , ( c ) 3 or more pieces of fruit per day , ( d ) less than 1 serving of red meat or sausages per day , ( e ) less than 1 serving of butter , cream , or margarine per day , ( f ) less than 1 cup of sugar - sweetened beverages per day , ( g ) 7 or more servings of red wine per week , ( h ) 3 or more servings of pulses per week , ( i ) 3 or more servings of fish per week , ( j ) fewer than 2 commercial pastries per week , ( k ) 3 or more servings of nuts per week , and ( l ) 2 or more servings per week of sofrito which is a mediterranean sauce made with onion , garlic , tomato , and spices sauted in olive oil . if the condition was not met , 0 points were recorded for any particular item . we determined the amount of digestible carbohydrates in each meal using spanish food composition tables . then , gi of each food present in the ffq was assigned through a protocol described by louie et al . , using data available in the international tables of gi and gl values 2002 and the sydney university gi research service . published values were extracted from studies in normal subjects , using 50 g of glucose as reference food and 2 hour testing periods . step 1 : we determined whether there was a direct link to a food in a gi database with gi values obtained from studies conducted on healthy subjects . of the 131 food items of the ffq , the gi values of 60 foods were assigned . step 2 : a gi value of 0 was given to 59 food items due to their low carbohydrate content ( < 5 g of carbohydrates per 100 g ) . step 3 : we searched for a closely related food item in the databases used . the gi of 10 foods was specified according to values of related items from the database . step 4 : we determined whether the median gi value of the food subgroup was available . the median group in the food group of pastries and cakes was determined and assigned for 1 item . step 5 : finally , for the rest of the items that were not assigned with a gi value in the previous steps , we evaluated whether or not the item was a top carbohydrate contributor . if so , a gi value of 0 , 50 or a gi value of an appropriate closely matched item as decided by the research nutritionists was assigned . only one food item reached this stage and it was assigned with a gi value of 0 because it was not one of the main contributors of carbohydrates to the diet . after the gi assignment , we estimated dietary gl and gi by the following equations :
( 1)dietary gl=i=1n[giichoi]100dietary gi=i=1n[giichoi]i=1nchoi ,
where gii is the value of the food i obtained from the gi database ; choi is the amount of available carbohydrates from food i ( g / g ) multiplied by food intake ( grams per day ) . and bmi was estimated as weight ( kg ) divided by the square of height ( m ) . leisure time physical activity was appraised using the validated spanish version of the minnesota questionnaire [ 28 , 29 ] . qualitative and quantitative variables among groups were compared by using chi - square tests and analysis of variance ( anova ) , respectively . the intakes of carbohydrate , protein , fat , alcohol , and fiber and dietary values of gl and gi were adjusted by the residuals method proposed by willett et al . . in order to describe dietary gl and gi of our population at 1 year of follow - up , sample was distributed in quintiles of adherence to the mediet . means were compared using anova and p - trend was estimated using anova - trend . generalized linear models were fitted to assess the relationship between the intervention groups and changes in dietary gl and gi at one year of follow - up , using the control group as reference . multiple linear regression models were constructed to assess the relationship between adherence to mediet and gl and gi according to tertiles of adherence to mediet considering the lowest adherence as reference . in two further models , the associations were adjusted for potential confounders including sex , age , physical activity ( continuous ) , smoking ( nonsmokers , smokers ) , total energy intake ( continuous ) , and bmi ( continuous ) . analyses were performed using spss software ( version 18 , 2009 , spss inc . ) and a p value < 0.05 was considered statistically significant . we explored the effect of this intervention at one year of follow - up because in this clinical trial , trained dietitians achieved a high adherence to the mediet in such period of time . participants allocated to both mediet groups increased their intake of low gi foods : virgin olive oil , nuts , vegetables , legumes , and fruits ( p < 0.05 for all within- and between - group differences ) . participants in all three groups decreased their intake of meat and pastries and cakes and sweets ( p < 0.05 for all ) . in table 1 are presented the baseline characteristics of the population according to the study group . subjects in the mediet groups were more likely to be younger and more physically active than those in the control group . in this study , subjects allocated in both mediet groups had slightly higher adherence to mediet and higher values of total energy intake than those in the control group . mediet groups consumed more polyunsaturated fat than the control group and mediet + nuts group had higher alcohol intake than control diet . dietary gl was lower in the mediet + nuts group than in the control group . dietary gi was similar in the three groups . in this study , the mean gl was 118.0 ( 24.2 ) and average gi at baseline was 57.5 ( 4.7 ) . average gl in women and men was 118.5 ( 22.3 ) and 117.3 ( 27.0 ) , respectively . mean gi in women and men was 56.7 ( 4.6 ) and 58.8 ( 4.6 ) , respectively . table 2 exhibits the regression coefficients and 95% ci for gl and gi changes according to the intervention groups at one year of follow - up . the multivariate - adjusted models showed a significant inverse association between gl and gi with mediet + evoo ( for gl = 8.52 ; 95% ci : 10.83 to 6.20 and for gi = 0.93 ; 95% ci : 1.38 to 0.49 ) and a more pronounced effect in the mediet + nuts group ( for gl = 10.34 ; 95% ci : 12.69 to 8.00 and for gi = 1.06 ; 95% ci : 1.51 to 0.62 ) , when compared to control diet . figure 1 illustrates the relative contribution of food groups to dietary gl and gi at one year of follow - up . the group of cereals and cereal products was the main contributor , providing 45.0% to the total dietary gl and 44.3% to total dietary gi . within this cereals group , the contribution of gl for white and whole grain bread was 28.0% and 6.9% , respectively . regarding gi , white bread supplied 26.9% of total dietary gi , followed by whole grain bread with 7.2% . tubers , particularly potatoes , contributed 8.6% and 9.0% to total gl and gi , respectively . in this population , 8.3% and 7.9% of total dietary gl and gi the contribution of legumes to total dietary gl and gi was minor , as they supplied only 1.2% of gl and 1.3% of gi . in order to describe in detail dietary gl and gi of the population the mean ( sd ) gl of the highest adherence group was significantly lower than those of the rest of categories ( 106.1 ( 20.5 ) ; p for trend < 0.001 ) ( figure 2(a ) ) . similarly , the mean ( sd ) gi for the highest category was significantly lower than that of the first category ( 56.0 ( 4.4 ) and 58.1 ( 5.0 ) , resp . ) we found a significant linear trend for dietary gi across categories of adherence to mediet ( p for trend < 0.001 ) . table 3 shows the regression coefficients with 95% confidence intervals ( ci ) of gl and gi values by categories of adherence to the mediet . the unadjusted linear regression model showed significant negative associations between dietary gl and the second and third categories of adherence to the mediet compared to the lowest ( reference ) category . in a large sample of older nondiabetic subjects at high risk of cvd participating in the predimed study , we found that an intervention with mediet supplemented either with evoo or nuts lowers the gl and gi of the diet . we observed that changes in dietary gl after one year of intervention were more pronounced than dietary gi changes , because gl contemplates both the quality and amount of carbohydrates consumed . although there have been several studies describing gl and gi in different countries and population groups [ 12 , 13 , 31 ] , to our knowledge , this relationship had not been assessed before . in this study , an inverse association was found between gl / gi and mediet + evoo when compared with the control group . this could be explained due to intervention in both mediet groups , which included dietary advice to increase consumption of vegetables , fruits , legumes , fish , olive oil , and nuts ( in the mediet + nuts groups ) , and these foods frequently consumed in the mediet have a low gi . on the other hand , patients were advised to reduce their sweetened and/or carbonated beverages intake and commercial sweets or pastries intake , therefore , reducing the servings of foods with high gi values . furthermore , studies have demonstrated that common foods such as spaghetti or potato dumplings have a low gi in spite of their low fiber content . however , in our study population , white bread and whole grain bread supplied a considerable proportion of the total dietary gl and gi ( around 35% ) . previous studies have shown that bread consumption in a mediet pattern , especially whole grain bread , has a beneficial effect on adiposity , while increased consumption of white bread has the opposite effect . in our study , the estimated mean dietary gl at baseline was 118 and the mean dietary gi was 58 . thus , the dietary gl and gi in a spanish population sample were lower compared with our study , probably because of a wider age range . in the epic study , the mean gl was also higher , which could be explained by a higher intake of carbohydrate ( 222 ( 31 ) g / day ) in the population studied . plant foods are the main source of nutrients such as slow - release carbohydrate and fiber , vegetable protein , beneficial minerals , antioxidant vitamins , and polyphenols that contribute to an optimal nutrition , satiety , and maintenance of a balanced diet . the group of cereals is found in the main meals , preferably consumed as whole grains , emphasizing the importance of a high fiber content in the diet [ 1 , 3 , 36 ] . in our study , greater fiber intake in mediet groups may be related to lower dietary gl and gi . a recent meta - analysis reported that many interventions directed at lowering the gi of a diet also resulted in increased intakes of fiber , usually causing a decreased gl and that gl acts independently from fiber on fasting blood glucose . the relevance of assessing the effect on gl and gi relies on their relationship with chronic disease , assessed in several studies . for instance , high dietary gl and gi were associated with an increased risk of coronary heart disease events in women [ 14 , 38 ] . also , a recent meta - analysis showed a significant association between gl and gi and the risk of colorectal and endometrial cancer , while the relationship with other types of cancer was inconsistent . moreover , it has been demonstrated that diets with lower values of gl and gi reduce t2 dm risk with a protective effect similar to that of whole grain and high fiber intakes . recently , the combined effect of dietary gl and mediet on the risk of incident t2 dm has been evaluated in greek population . the authors found a positive but weak correlation between mds and gl ( r spearman = 0.28 ) . however , subjects with a high mds and a low gl tended to have an 18% lower risk of diabetes when compared with participants with a diet characterized by a high gl and a low mds . furthermore , various similar clinical trials have evaluated the effect of a modified mediterranean - style low gl diet on prevalence of metabolic syndrome ( mets ) components in women . the second study demonstrated beneficial changes in waist circumference , plasma triglycerides , ldl cholesterol , and systolic blood pressure in female participants . significant decreases were also found in plasma insulin , tnf- levels , and hmg - coa reductase expression . the latter finding indicates decreased cholesterol biosynthesis , although the trigger is unclear . in the third study , in addition to ldl cholesterol reduction , decreases were observed in some atherogenic subfractions with different particle diameters : large very low - density lipoprotein ( vldl , > 60 nm ) , small ldl ( 1819.8 nm ) , and medium high - density lipoprotein ( 8.28.8 nm ) . . in addition , another investigation showed that apolipoprotein b was reduced in women with a low gl and mediterranean - type diet . also , the mediet with a low gl seems to have beneficial effects on weight , waist circumference , and systolic blood pressure . moreover , a mediet supplemented with nuts during 1 year was associated with a 14% reduction in prevalence of mets . a cross - sectional study in the canary islands showed that fruit intake had a protective effect on the triglyceride criteria of mets and that cereal intake protected from insulin resistance . recently , it was found that mediet supplemented with extra virgin olive oil or nuts can reduce by 30% the incidence of major cardiovascular events when comparing to a low - fat diet . the mechanisms of action by which low gl / gi diets possibly decrease chronic disease risk could be related to postprandial glucose and its related endocrine responses . after consumption of a high gi food , a dramatic increase in blood glucose occurs ; this is followed by a large insulin response and inhibition of glucagon release . on the contrary , intake of low gi foods results in an attenuated glucose response due to delayed intestinal glucose absorption . therefore , the resulting hormone responses and their effects are more stable , reducing postprandial hyperglycemia and hyperinsulinemia and attenuating late postprandial rebounds in circulating nonesterified fatty acids . these effects of a low gi diet could attenuate oxidative stress , which is associated with inflammation , and other risk factors . although low gl and gi have been associated with a reduced risk of chronic diseases , there is still insufficient evidence to include them in dietary recommendations to the general population . the main strengths of this study are its large sample size , use of a comprehensive and validated ffq with assignment of gi values through an established protocol , use of a validated mediet screener , and adjustment for all possible confounders in multivariate analyses . as it was conducted in older individuals at high cvd risk , the results can not be easily extrapolated to other populations . another limitation is that values of gi for spanish food are scarce ; we assigned them using general gi tables obtained mainly from australian and us studies ; estimations could be misrepresented because the properties of foods with the same name may fluctuate between countries . in summary , our findings suggest that mediet supplemented with evoo or nuts lowers the gl and gi of the diet . at any rate , gl and gi of the mediet could explain other mechanisms for protection against cvd apart from the mufa / sfa ratio and the antioxidant capacity involved in this beneficial dietary pattern . more researches are needed to clarify the role of these indexes in future dietary recommendations . | objective . to compare the one year effect of two dietary interventions with mediet on gl and gi in the predimed trial
. methods .
participants were older subjects at high risk for cardiovascular disease .
this analysis included 2866 nondiabetic subjects .
diet was assessed with a validated 137-item food frequency questionnaire ( ffq ) .
the gi of each ffq item was assigned by a 5-step methodology using the international tables of gi and gl values .
generalized linear models were fitted to assess the relationship between the intervention group and dietary gl and gi at one year of follow - up , using control group as reference . results .
multivariate - adjusted models showed an inverse association between gl and mediet + extra virgin olive oil ( evoo ) group : = 8.52 ( 95% ci : 10.83 to 6.20 ) and mediet + nuts group : = 10.34 ( 95% ci : 12.69 to 8.00 ) , when comparing with control group .
regarding gi , = 0.93 ( 95% ci : 1.38 to 0.49 ) for mediet + evoo , = 1.06 ( 95% ci : 1.51 to 0.62 ) for mediet + nuts when comparing with control group . conclusion .
dietary intervention with mediet supplemented with evoo or nuts lowers dietary gl and gi . |
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paenibacillus species were originally classified within the bacillus genus . members of this genus are often gram variable , facultatively anaerobic , and endospore forming . these bacteria are frequently isolated from environments such as soil , water , vegetable matter , forage , larvae and insects but could be detected from clinical samples , , . bacteria belonging to this genus are able to produce polysaccharide - degrading enzymes and proteases , are beneficial to agriculture and horticulture and have industrial and medical applications . some species of this genus may be involved in human infections , , , . recently high - throughput genome sequencing and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) analyses of bacteria have given unprecedented access to an abundance of genetic and proteomic information , . thus , a polyphasic approach is currently proposed to describe new bacterial taxa that includes their genome sequence , maldi - tof spectrum and major phenotypic characteristics such as gram staining , culture , metabolic characteristics , habitat and , if applicable , pathogenicity . the strain ff9 (= csur p1429 = dsm 29777 ) was isolated from a blood culture of a 16-month - old child presenting at the hpital principal de dakar , senegal . strain ff9 is a gram - variable bacterium , facultatively anaerobic , motile and rod shaped . here we present a summary classification and a set of features for paenibacillus dakarensis sp . in march 2014 a blood culture was performed on a 16-month - old child presenting at the hpital principal de dakar , senegal . strain ff9 ( table 1 ) was isolated from this blood culture by culture on 5% sheep 's blood enriched columbia agar ( biomrieux , marcy letoile , france ) . identification was not obtained using maldi - tof because the scores obtained by this strain were low . moreover , strain ff9 exhibited 98.18% 16s rrna sequence similarity with paenibacillus uliginis
( genbank accession no . fn556467 ) , the phylogenetically closest bacterial species with standing in nomenclature ( fig . 1 ) . these values were lower than the 98.7% 16s rrna sequence threshold recommended by meier - kolthoff et al .
in 2013 to delineate a new species within the firmicutes phylum without carrying out dna - dna hybridization . different growth temperatures ( 25 , 28 , 37 , 45 and 56c ) were tested . growth of the strain was also tested under anaerobic and microaerophilic conditions using genbag anaer and genbag microaer systems ( biomrieux ) , respectively , and under aerobic conditions , with or without 5% co2 . optimal growth was observed under aerobic conditions , but weak growth was observed under anaerobic and microaerophilic conditions . cells are gram - variable , endospore - forming rods with rounded ends ( fig . 2 ) and have a mean diameter of 0.6 m ( range , 0.50.7 m ) and a mean length of 2.8 m ( range , 2.13.5 m ) ( fig . 3 ) . paenibacillus dakarensis is catalase positive and oxidase negative . using an api 50ch strip ( biomrieux ) , positive reactions were observed for d - ribose , d - glucose , d - mannose , n - acetyl - d - glucosamine , amygdaline , esculin , d - cellobiose , d - lactose , d - saccharose , d - trehalose , d- melezitose , gentiobiose and d - lyxose . using a api 20ne strip ( biomrieux ) , positive reactions were observed for esculin , -galactosidase , glucose and mannose . using a api zym strip ( biomrieux ) , negative reactions were observed for alkaline phosphatase , esterase , esterase - lipase , leucine arylamidase , acid phosphatase , naphthol - as - bi - phosphohydrolase , cystine arylamidase , valine arylamidase , trypsin , -glucosidase , -glucosidase , -galactosidase , -galactosidase , -glucuronidase , -mannosidase , -fucosidase and n - acetyl--glucosaminidase . strain ff9 is susceptible to amoxicillin / clavulanic acid , ticarcillin , ceftriaxone , cefalotin , imipenem , gentamicin and doxycycline but is resistant to penicillin , metronidazole and trimethoprim / sulfamethoxazole . the minimum inhibitory concentrations ( mics ) for some antibiotics tested by paenibacillus dakarensis strain ff9 sp . nov . a comparison of phenotypic characteristics with paenibacillus polymyxa
, paenibacillus massiliensis and paenibacillus sanguinis
is summarized in table 3 . maldi - tof protein analysis was performed using a microflex lt ( bruker daltonics , leipzig , germany ) , as previously reported , . the scores previously established by bruker to identify or validate species compared to the instrument 's database were applied . in short , a score 2.000 with a species with a validly published name allows for identification at the species level ; a score 1.700 and < 2.000 allows for identification at the genus level ; and a score < 1.700 does not allow for any identification to be made . two microlitres of matrix solution ( saturated solution of -cyano-4-hydroxycinnamic acid ) in 50% acetonitrile and 2.5% trifluoroacetic acid were distributed on each smear and subjected to air drying for 5 minutes . the spectra from the 12 different colonies were then imported into the maldi biotyper 2.0 software ( bruker ) and analysed by standard pattern matching ( with default parameter settings ) against the main spectra of 6252 bacteria . scores ranging from 1.225 to 1.456 were obtained for the ff9 , suggesting that this strain was not a member of any known species . the reference mass spectrum from strain ff9 was incremented in our database ( fig . 4 ) . the organism was selected for sequencing on the basis of its phylogenetic position , 16s rrna similarity and phenotypic differences with other members of the paenibacillaceae family . there are more than 15 genomes for the paenibacillus genus available in public genomic collections . here table 4 shows the project information and its association with minimum information about a genome sequence ( migs ) 2.0 compliance . paenibacillus dakarensis strain ff9 (= csur p1429 = dsm 29777 ) was grown on 5% sheep 's blood bacteria grown on four petri dishes were resuspended in 5 100 l of tris - edta ( te ) buffer ; 150 l of this suspension was diluted in 350 l te buffer 10 , 25 l proteinase k and 50 l sodium dodecyl sulfate for lysis treatment . after centrifugation , dna was suspended in 65 l elution buffer ( eb ) buffer . the genomic dna concentration was measured at 452.7 ng/l using the qubit assay with the high sensitivity kit ( life technologies , carlsbad , ca , usa ) . the mate pair library was prepared with 1.5 g of genomic dna using the nextera mate pair illumina guide ( illumina , san diego , ca , usa ) . the genomic dna sample was simultaneously fragmented and tagged with a mate pair junction adapter . the pattern of fragmentation was validated on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) using a dna 7500 lab chip . the dna fragments ranged in size from 1.5 to 11 kb , with an optimal size of 5.773 kb . no size selection was performed , and 600 ng of tagmented fragments were circularized . the circularized dna was mechanically sheared into small fragments with an optimal size of 932 bp on the covaris device s2 in t6 tubes ( covaris , woburn , ma , usa ) . the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent ) , and the final concentration library was measured at 19.07 nmol / l . after a denaturation step and dilution at 15 pm , the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell . automated cluster generation and sequencing runs were performed in a single 39-hour run in a 2 251 bp read length . total information of 4.9 gb was obtained from a 506k / mm cluster density with a cluster passing quality control filters of 97% ( 9 954 000 clusters ) . within this run , open reading frames ( orfs ) were predicted using prodigal with default parameters , but the predicted orfs were excluded if they spanned a sequencing gap region . the predicted bacterial protein sequences were searched against the genbank database and the clusters of orthologous groups ( cogs ) database using blastp . the trnascanse tool was used to find trna genes , while ribosomal rnas were found using rnammer and blastn against the genbank database . lipoprotein signal peptides and the number of transmembrane helices were predicted using signalp and tmhmm respectively . orfans were identified if their blastp e value was lower than 1e-03 for alignment length greater than 80 amino acids . if alignment lengths were smaller than 80 amino acids , we used an e value of 1e-05 . artemis was used for data management and dna plotter for visualization of genomic features . the mauve alignment tool ( version 2.3.1 ) was used for multiple genomic sequence alignment . to estimate the mean level of nucleotide sequence similarity at the genome level , we used the magi homemade software to calculate the average genomic identity of gene sequences ( agios ) among compared genomes . briefly , this software combines the proteinortho software for detecting orthologous proteins in pairwise genomic comparisons , then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous orfs using the needleman - wunsch global alignment algorithm . genomes from the paenibacillus genus and closely related genera were used for the calculation of agios values . the script created to calculate agios values was named magi ( marseille average genomic identity ) and is written in perl and bioperl modules . genome - to - genome distance calculator ( ggdc ) analysis was also performed using the ggdc web server ( http://ggdc.dsmz.de ) as previously reported , . here , we compared the genome sequences of p. dakarensis strain ff9 ( genbank accession no . cdse01000001 ) with those of paenibacillus lactis strain 154 ( agip00000000 ) , paenibacillus polymyxa strain atcc 842 ( afox00000000 ) , paenibacillus massiliensis strain 2301065 ( aril00000000 ) , paenibacillus sabinae strain t27 ( cp004078 ) , paenibacillus borealis strain dsm 13188 ( cp009285 ) and paenibacillus forsythiae strain t98 ( assc00000000 ) . the genome of the p. dakarensis strain ff9 is 4 569 428 bp long with a 45.7% g+c content ( fig . of the 4427 predicted genes , 4352 were protein - coding genes and 75 were rna genes . six rrna genes ( two 16s rrna , two 23s rrna and two 5s rrna ) and 69 predicted trna genes were identified in the genome . the draft genome of p. dakarensis is smaller than that of p. lactis , p. polymyxa , p. massiliensis , p. sabinae , p. borealis and p. forsythiae ( 4.56 , 6.81 , 5.9 , 6.39 , 5.27 , 8.16 and 5.08 mb , respectively ) . the g+c content of p. dakarensis is higher than those of p. polymyxa ( 45.7 and 44.9% , respectively ) but lower than those of p. lactis , p. massiliensis , p. sabinae , p. borealis and p. forsythiae ( 49.1 , 48.5 , 52.6 , 51.4 and 52.9 respectively ) . the gene content of p. dakarensis is lower than those of p. lactis , p. massiliensis , p. sabinae , p. borealis and p. forsythiae ( 4427 , 6234 , 5206 , 5193 , 4896 , 6382 and 5103 respectively ) . however , the distribution of genes into cogs categories was similar in all compared genomes ( fig . 7 ) . in addition , p. dakarensis shared 4352 , 6149 , 5068 , 5055 , 4788 , 6213 and 5011 orthologous genes with p. lactis , p. polymyxa , p. massiliensis , p. sabinae , p. borealis and p. forsythiae respectively ( table 7 ) . among species with standing in nomenclature , agios values ranged from 69.19% between p. polymyxa and p. forsythiae to 84.00% between p. forsythiae and p. sabinae . on the basis of phenotypic , phylogenetic and genomic analyses the strain was isolated from a blood sample taken from a 16-month - old senegalese child presenting at the hpital principal de dakar . n. dakarensis , or originating from dakar , the capital of senegal , where the type strain was isolated ) . the strain ff9 is a facultative anaerobic , gram variable bacterium , with small , white colonies on 5% sheep 's blood cells have a mean diameter of 0.6 m ( range , 0.50.7 m ) and a mean length of 2.8 m ( range , 2.13.5 m ) . positive reactions were observed for d - ribose , d - glucose , d - mannose , n - acetyl - d - glucosamine , amygdaline , esculin , d - cellobiose , d - lactose , d - saccharose , d - trehalose , d - melezitose , gentiobiose , d - lyxose and -galactosidase . paenibacillus dakarensis strain ff9 is susceptible to amoxicillin / clavulanic acid , ticarcillin , ceftriaxone , cefalotin , imipenem , gentamicin and doxycycline but resistant to metronidazole , penicillin and trimethoprim / sulfamethoxazole . the 16s rrna and genome sequences are deposited in genbank under accession numbers lm652718 and cdse01000001 , respectively . the type strain ff9 (= csur p1429 = dsm 29777 ) was isolated from a blood sample taken from a 16-month - old child presenting at the hpital principal de dakar , senegal . | strain ff9 t was isolated in dakar ( senegal ) from a blood - culture taken from a 16-month - old child .
maldi - tof analysis did not allow for identification .
after sequencing , strain ff9 t exhibited 98.18% similarity with the 16srrna sequence of paenibacillus uliginis .
a polyphasic study of phenotypic and genomic analyses showed that strain ff9 t is gram variable , catalase - positive , and presents a genome of 4,569,428 bp ( one chromosome but no plasmid ) with 4,427genes ( 4,352 protein - coding and 75 rna genes ( including 3 rrna operons ) .
the g+c content is 45.7% . on the basis of these genomic and phenotypic data analyses
, we propose the creation of paenibacillus dakarensis strain ff9 t . |
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non - alcoholic fatty liver disease ( nafld ) and its more severe form , non - alcoholic steatohepatitis ( nash ) , have become the metabolic epidemics of the modern world . these have been attributed to the growing prevalence of obesity and insulin resistance in the population . roughly a third of obese people are prone to and develop nafld / nash in their lifetime . on the other hand , this has been described extensively and we know that some lean people are prone to nafld / nash as well . therefore , it is logical to think of other , yet unknown , factors triggering the process of fatty liver in the liver of obese and lean people .
the epidemic of obesity and nafld / nash has occurred alongside economic development and adopting a more western type of diet in most parts of the world . meanwhile the metabolic syndrome has risen as a serious threat to the health of communities and a new burden on the health economy . gastro - esophageal reflux disease ( gerd ) has also been considered as the gastro - intestinal manifestation of the metabolic syndrome . interestingly gerd is also on the rise in parallel with nafld / nash and obesity is a risk factor for gerd too . again not all obese people develop gerd and many patients with gerd are not obese . having common epidemiologic backgrounds , it looks plausible that these conditions may have a common trigger or mechanism for their development . the exact pathophysiologic mechanisms causing gerd are not known , but recent , animal , ecologic , and human data have linked excess dietary nitrate to gerd . nitrate is an inert compound and can only take part in biochemical reactions after being reduced to nitrite by the nitrate reductase of oral bacteria residing on the dorsum of the tongue . the ingested nitrite then enters an enterosalivary circulation and is re - secreted into the mouth by salivary glands . it has been recently shown that oral nitrate reductase activity ( nra ) is increased in patients with erosive gerd . therefore , oral flora may play a role in development of erosive gastro - esophageal reflux disease . as gerd is considered a gastrointestinal ( gi ) manifestation of the metabolic syndrome which is in turn related to nash / nafld , common pathways may exist . having these in mind , we hypothesized that oral nra may have an effect on development of nafld / nash . nash was defined as having at least grade - i fatty liver on abdominal ultrasound examination and either aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) more than 1.5 times the upper normal limit on two occasions during the six months before enrollment . all patients were checked for hepatitis b and c , autoimmune hepatitis , wilson s disease , alpha-1 anti - trypsin deficiency , and hemochromatosis and required to be negative for all these . in addition , those who took herbal medicine or supplements for bodybuilding regularly were excluded from the study . controls were selected from patients referring to one of the authors ( sm ) and were matched for age . the following data were obtained from each participant : height ( in centimeters ) , weight ( in kilograms ) , waist circumference ( in centimeters ) , hip circumference ( in centimeters ) , aspartate aminotransferase ( ast , iu / l ) , alanine aminotransferase ( alt , iu / l ) , fasting blood sugar ( fbs , mg / dl ) , total cholesterol ( mg / dl ) , serum triglyceride ( tg , mg / dl ) , high density lipoprotein ( hdl , mg / dl ) ) , low density lipoprotein ( ldl , mg / dl ) , and fasting serum insulin level ( u / ml ) . body mass index ( bmi , kg / m ) and homeostasis model assessment - insulin resistance ( homa - ir ) were calculated , the latter using fbs and fasting serum insulin level . participants underwent abdominal ultrasound examination for assessment of fatty liver as well and their fat estimation reported as grades of fatty change . all participants were asked to report to the clinic after an overnight fast and avoiding food containing high nitrate ( including lettuce , cabbage , celery , turnip , beetroot , radish , and rhubarb ) for at least eight hours . after filling in a questionnaire for demographic , clinical and paraclinical information , oral nra briefly , particiapants rinsed their mouth with 22 ml of deionized , sterile water initially . then he / she was asked to hold 22 ml of a 10 mg nitrate - n / l solution in the mouth for 3 min . the subject was instructed to mix the solution in the mouth at the beginning and every 1 min over the 3 min , and 1.5 ml of the solution incubated in the mouth was sampled with a sterile syringe after each mixing ( i.e. , at the beginning and at 1-min intervals ) . the samples were gathered in special tubes and the rest of the nitrate reductase in the samples was inactivated to prevent in vitro activity . the nitrite was then measured immediately according to the previously described method and the slope of nitrite concentration was used to determine the nra accordingly and reported as g nitrite - n formed per person per minute . twenty - three people ( 11 cases and 12 controls , all male ) were enrolled . the patients heights were comparable but cases weighed
significantly heavier than controls ( table-1 ) . total cholesterol , hdl and ldl were comparable in the two groups , but tg was significantly higher among cases than controls ( 187.5 vs. 118.1 mg / dl respectively , p=0.014 ) . there was also a trend towards higher fbs among cases ( 91.0 vs. 83.7 mg / dl respectively , p=0.081 ) . two cases had grade - i , one had grade - iii , and the rest had grade - ii fatty liver on abdominal ultrasound examination . one control was reported to have a grade - i fatty liver with normal liver enzyme levels . mean oral nra was not different between the two groups ( 2.82 vs. 3.51 g nitrite - n formed per person per minute for cases and controls respectively , p=0.46 ) . there were two outliers among controls ( nra level>8 g nitrite - n formed per person per minute ) . nafld and nash are among the common problems for which people seek medical advice and their prevalence is increasing worldwide . although frequency of nafld / nash increases with increasing bmi , but most obese people never develop these conditions . besides , lean people may develop nafld / nash . is it a genetic predisposition ( which probably is ) or are there environmental factors affecting this ? it is not possible to answer this question yet , but hypotheses can be generated . considering the vast metabolic activities of these microbes and their extensive involvement in human physiology , it is plausible to hypothesize that alterations in this milieu may have effects on disease behavior in different patients . in other words , , the oral and gi flora may be more influential in metabolic diseases which involve diet and its constituents in some way . an important point in such studies is that it may not be a given microbe which affects the outcome , but the metabolic effect of the given microbe in a given milieu of other microbes and their topographical distribution in an environment determines the final effect . therefore , despite the fact that looking for individual microbes is informative , but checking metabolic pathways may be more enlightening . we tested the oral nitrate reductase activity as a marker of oral flora composition in individuals with and without nash . nra was selected as it has been recently shown that it may contribute to development of erosive reflux disease which itself is considered a gi manifestation of the metabolic syndrome just like nash . according to our data , oral nitrite production is not different between people with nafld / nash and age - matched controls . in other words , strengths of our study include that we measured nra with an accurate and reproducible method in vivo . another point which may have affected our data is that nra measurements for controls have been done in the colder months of the year . another point which may have affected our results is that nash patients in our study had significantly higher bmi levels . the question to be answered is that if bmi is associated with development of nash , why some obese / overweight people develop nash / nafld , while other people with similar bmi do not . for this to be answered , we need cases and controls to be comparable in their bmis . in addition , controlling or matching for other confounders may in further studies may give us better insight to this concept . therefore , it is still possible that oral nra and excessive dietary nitrate can have a role in development of nafld / nash . hence , studies with higher numbers of cases and controls with a more meticulous methodology are warranted . | background
nafld / nash is a manifestation of metabolic syndrome and is associated with obesity / overweight .
not all
obese / overweight individuals develop nash .
gastro - esophageal reflux disease ( gerd ) is considered a
gastrointestinal manifestation of the metabolic syndrome and is associated with obesity / overweight . again
not all
obese / overweight individuals develop gerd .
recent data show association of dietary nitrate content and oral nitrate
reductase activity ( nra ) with gerd .
nitrates need to be converted to nitrite ( done in human beings by nitrate reductase
of oral bacteria exclusively ) to be active in metabolic pathways .
objective
to assess the relation between nash / nafld and
oral nra .
methods
oral nra was measured in individuals with nash ( compatible abdominal ultrasound and two elevated alt / ast
levels over six months ) and was compared with that of those without nash .
oral nra was measured according to a previously
reported protocol .
results
eleven nash patients and twelve controls were enrolled .
mean oral nra activity were 2.82 vs.
3.51 g nitrite - n formed per person per minute for cases and controls respectively ( p=0.46 ) .
conclusion
according to our data , oral nitrite production is not different between individual swith
and without nash . |
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purified herba epimedii extract ( chuan - ke - zhi , ckz ) , a chinese medicine preparation , has already attracted more and more attention in recent years for its curative effects on respiratory system diseases , such as bronchial asthma , chronic bronchitis , and chronic obstructive pulmonary emphysema [ 14 ] . and this purified extract was also reported to be able to regulate human humoral and cellular immunity [ 59 ] ; therefore , it has been gradually extended to the treatment of various cancers and immunological diseases [ 1013 ] . although ckz has been clinically applied for many years , there are still some difficulties and challenges on its in vivo process and mechanism of action . nowadays the pharmacokinetic research of traditional chinese medicine ( tcm ) are usually carried out in accordance with the research method of western medicine ; however , not like western medicine tcm always contains numerous compounds , which generally display different pharmacokinetic behaviour and may interact with each other in organism . therefore in order to evaluate the rationality and compatibility of chinese herbal compound formula , it is obviously important and necessary to investigate the pharmacokinetics of marker component as well as the possible interaction of other ingredients contained in tcm . according to the existing phytochemical and pharmacological studies [ 1420 ] , herba epimedii contains a large number of flavonoid glycosides . most of them are characterized by an isopentenyl group substitution at the c8 position of flavonoid skeleton . these 8-isopentenyl flavonoid glycosides , of which epimedins a , b , and c and icariin are the principal and representative , are thought to be main bioactive components of herba epimedii and its extract preparation . as a purified extract , ckz contains more abundant 8-isopentenyl flavonoid glycosides than this crude drug , and epimedin c is the most dominant compound , which is visually exhibited in figure 1 . in the present study , therefore , the pharmacokinetic research of ckz firstly focused on the pharmacokinetic behaviour of epimedin c in rat , and then on the base of established and validated quantitative method for epimedin c in rat plasma , we compared the pharmacokinetic differences of epimedin c after intramuscular administration of individual epimedin c , combination solution of epimedins a , b , and c and icariin , and ckz to rats . finally , considering the poor oral absorption of epimedin c [ 22 , 23 ] , the absolute bioavailability of epimedin c administrated intramuscularly was investigated for the first time in this study . overall , our study could facilitate the understanding of absorption mechanism of flavonoid glycosides in herbal formulations and provide reference for the clinical application of this purified herba epimedii extract . epimedin a ( purity 98% ) and epimedin b ( purity 98% ) were purchased from shanghai ronghe pharmaceutical technology development co. , ltd . epimedin c ( purity 98% ) , icariin ( purity 98% ) , and naringin ( purity 98% ) used as internal standard ( is ) were provided by the national institute for the control of pharmaceutical and biological products ( beijing , china ) . ( lot number 11072502 , guangzhou , china ) , and the concentration of epimedins a , b , and c and icariin was determined to be 81.38 , 68.49 , 667.5 , and 132.5 g / ml , respectively . hplc - grade methanol and acetonitrile were obtained from fisher company ( fisher scientific , fairlawn , nj ) . the deionized water was prepared by a millipore purification system ( millipore , milford , ma , usa ) and filtered with 0.2 m membranes . hplc was performed on an agilent eclipse xdb - c18 ( 150 2.1 mm i.d . 5 m ) column using an agilent 1100 series hplc system ( agilent technologies , inc . , usa ) equipped with a g1311a binary pump , a g1379a vacuum degasser unit and a g1313a autosampler , and a g1316a sl thermostated column compartment . the temperature of column chamber was set at 40c . the mobile phase consisted of acetonitrile and 0.1% formic acid ( 35 : 65 , v / v ) . an isocratic elution mode was adopted with a flow rate of 220 l / min . the hplc system was coupled to a triple - quadruple tandem mass spectrometer ( api-3000 pulsar , applied biosystems , foster city , usa ) equipped with an electrospray ionization ( esi ) source . analyst 1.4 workstation ( applied biosystems , foster city , usa ) was used for the control of equipment , data acquisition , and analysis . for the optimization of ms / ms parameters , the standard solutions of epimedin c and is prepared with 50% acetonitrile were infused at a flow rate of 20 l / min using a syringe pump ( harvard apparatus , holliston , ma , usa ) . finally , the instrument was operated with the ionspray voltage , deflector voltage ( df ) and multiplier voltage ( cem ) at 5000 , 200 , and 2200 v , respectively . nitrogen was used as nebulizer gas ( 8 l / min ) , as auxiliary gas ( 7.5 l / min at 350c ) , and as curtain gas ( 8 l / min ) . the optimized mrm fragmentation transitions were m / z 867.3 659.3 for epimedin c and m / z 579.2 271.1 for is . the experimentation on rats obtained the approval from an independent ethics committee at guangdong provincial hospital of chinese medicine ( approval number 2012038 ) . the experiment was performed at an spf level laboratory , authorized by guangdong provincial government . sprague - dawley rats ( 300 20 g ) were purchased from guangdong medicinal laboratory animal center . the animals fasted for 12 h with free access to water prior to administration . the four groups were administrated as follows : two groups dosed with epimedin c aqueous solution by intramuscular and intravenous administration , respectively , one group dosed with ckz by intramuscular administration , and one group intramuscularly administrated with the combination solution of epimedins a , b , and c and icariin whose contents are the same as those in ckz . the four aqueous solutions were administered to rat at the same epimedin c dosage ( approximately 0.33 mg / kg ) . about 350 l of blood sample was collected from the suborbital vein into heparinized tubes before drug administration ( 0 h ) and at 3 , 6 , 10 , 15 , 25 , 35 , and 45 min and 1 , 2 , 3 , 4 , 6 , and 8 h after intramuscular and intravenous administration . and then each blood sample was centrifuged at 3000 rcf for 15 min at 4c , and the plasma was transferred into 1.5 ml centrifuge tube and stored at 80c until analysis . during routine analysis , each analytical run included blank rat plasma , a set of calibration samples , and a set of qc samples . a simple solid - phase extraction ( spe ) method was used for extracting epimedin c from rat plasma . the agilent ods - c18 spe cartridge was preconditioned with 1 ml methanol followed with 1 ml water . a 100 l aliquot of plasma sample was spiked with 10 l of is working solution ( 1 g / ml ) . the combination was vortexed for 60 s and then added to a pretreated cartridge . after the sample was drawn through the spe bed , the cartridge was washed with 1.0 ml water , followed by vacuumizing for 1 min to remove the aqueous effluent . the methanol eluate was transferred to a clean test tube and evaporated to dryness under a gentle stream of nitrogen at 40c . the residue was redissolved in 150 l of mobile phase ( 35% acetonitrile containing 0.1% formic acid ) and centrifuged by a microspin filter tube ( 0.22 m nylon , alltech ) at 10,800 g for 1 min . a 10 l aliquot of the solution was injected into lc - ms / ms for analysis . blank rat plasma samples were spiked with epimedin c standard solutions to determine the final concentration ranges ( 1 , 3 , 10 , 30 , 100 , 300 , and 1000 ng / ml ) . the curves were fitted by a weighted ( 1/x ) least - squares linear regression method through the measurement of the peak - area ratio of epimedin c to is . the acceptance criterion for a calibration curve was a correlation coefficient ( r ) of 0.99 or better , and each back - calculated standard concentration must be within 15% deviation from the nominal value except at the lower limit of quantitation ( lloq ) , for which the maximum acceptable deviation was set at 20% . the lloq was defined as the lowest concentration on the standard curve at which the standard deviation was within 20% and accuracy was within 100 20% , and it was established using five samples independent of standards . the analyte response at this concentration level should be > 5 times the baseline noise . to evaluate the accuracy and precision of established method , qc samples at four concentration levels ( 3 , 30 , 300 , and 800 ng / ml ) were analyzed in five replicates on three validation days . the assay accuracy was expressed as ( observed concentration / nominal concentration ) 100% . the accuracy was required to be within 85115% , and the intra- and interday precision are not to exceed 15% . to investigate the effect of the sample preparation , the extraction recovery and matrix effect were determined by comparing the peak areas between three types of samples : ( 1 ) plasma spiked with known amount of epimedin c before sample preparation ; ( 2 ) plasma spiked with known amount of epimedin c after sample preparation ; ( 3 ) the standard solution of epimedin c. the difference between peak areas of sample 2 and sample 3 reflects the extent of ion suppression . the accuracy ( trueness ) of the method was calculated by comparing theoretical and experimentally measured analyte levels . the stability of epimedin c in rat plasma was evaluated using 20 qc samples ( five samples at each concentration ) . the stability was tested under the following conditions : ( 1 ) freeze - thaw stability of epimedin c in rat plasma through three freeze - thaw cycles ; ( 2 ) short - term stability of epimedin c in rat plasma at room temperature for 6 h ; ( 3 ) long - term stability of epimedin c in rat plasma stored at 80c for 30 days ; ( 4 ) postpreparative stability of epimedin c in rat plasma stored in the autosampler at 25c for 24 h. the validated method was applied to the pharmacokinetic and bioavailability studies of epimedin c in rats . the maximum plasma concentration ( cmax ) and time to cmax ( tmax ) were obtained directly from the concentration - time curves . the area under the concentration - time curve ( auc0 ) was calculated using linear trapezoidal rule . the mean residence time ( mrt ) was calculated as aumc0/auc0 , where aumc0 represents the area under the first moment plasma concentration - time curve . the mass spectrometric behaviour of epimedin c and is was studied using both positive and negative ion esi . it was found that epimedin c and is had good responses in negative ion detection mode with a low background noise level . the ms / ms spectra of epimedin c and is were shown in figure 3 . the former was discarded because of its high noise level and the interference of endogenous substances . the latter offered a very clean sample which made the quantitative method robust and scalable . consequently , the spe method was used for sample preparation and further optimized by testing and comparing the influence of different solid - phase extraction cartridges ( c8 , c18 , and hlb ) on the recovery of analyte and is . agilent c18 spe cartridge was the best owing to its low background noise , ease of sample preparation , and relatively high extraction recovery . the optimal conditions were presented above . in order to improve the peak shape and enhance the signal response of analytes , and to reduce the run time , different analytical columns and mobile phase compositions were tried to achieve good resolution and symmetric peak shapes for epimedin c and is . by comparison with shiseido capcell pak c18 ( 150 2.0 mm i.d . 5 m ) column , agilent xdb c18 ( 150 2.1 mm i.d . 5 m ) column could obtain better chromatographic behaviour and higher signal response for the analytes . at last , the agilent xdb c18 ( 150 2.1 mm i.d . 5 m ) column was selected for analysis . the modifier of mobile phase of acetonitrile - water binary solvent system was screened from ammonium acetate , acetate acid , and formic acid . as a result , the mobile phase consisting of acetonitrile - water containing 0.1% formic acid with an isocratic elution could improve the symmetry of peak shape and enhance the signal response . the flow rate was also optimized and finally set at 0.22 ml / min . under the above - mentioned hplc conditions , the retention time of epimedin c and is was within 3 min , and no interfering substance was detected at the retention time of analytes in blank rat plasma samples ( shown in figure 4 ) . the specificity of the analytical method was evaluated by analyzing individual blank plasma samples from six different sources . all samples were found to have no interference with endogenous substances and is at the respective retention position of epimedin c. typical mrm chromatograms of blank plasma spiked with is , plasma spiked with epimedin c , and the rat plasma sample after intramuscular administration of epimedin c , flavone combination , and ckz were shown in figure 4 . the regression equation , linear range , and correlation coefficient of epimedin c were y = 0.00461x + 0.002 ( 1.001000 ng / ml ) and r = 0.9981 . the llod with a signal - to - noise ( s / n ) ratio of > 5 of epimedin c was 0.04 ng / ml , while the lloq with an s / n ratio of > 10 was 0.05 ng / ml , which was sensitive enough for pharmacokinetic study of epimedin c in rat plasma . intra- and interday precision and accuracy were calculated by analysis of variances , based on replicate analyses ( three days , four concentrations , each n = 5 ) of qc samples . the mean extraction recovery rates of epimedin c from rat plasma were 86.6 3.1 , 80.7 4.3 , 80.1 2.6 , and 78.4 1.1 at low , medium , and high concentrations , and the matrix effects were 90.71 5.3 , 95.95 2.49 , 94.57 0.82 , and 100.9 2.21 , respectively . the result of stability experiment showed that epimedin c was stable in autosampler ( 24 h ) at 25c , bench - top ( 6 h ) at room temperature , and under repeated three freeze / thaw cycles and frozen condition at 80c within 30 days ( table 2 ) , as rsds were within 15% for both the low and high concentrations . the method showed good precision , repeatability , and stability and was suitable for determination of epimedin c in biological samples . after administration of epimedin c , ckz , and combination of epimedins a , b , and c and icariin , the plasma epimedin c concentrations were successfully determined by lc - ms / ms method described above . the mean plasma concentration - time profiles of epimedin c were illustrated in figure 5 . the result indicated that epimedin c exhibited rapid absorption with the plasma peak concentrations occurring at around 10 min and were quickly eliminated thereafter . in addition , the plasma concentration - time curves of epimedin c after intramuscular administration showed a second peak or shoulder in most rats . as shown in table 3 , the data indicated that many pharmacokinetic parameters were significantly different among three intramuscular administration groups . values of the area under the plasma concentration versus the time curve ( auc0 ) were 271.03 44.39 , 311.41 128.11 , and 448.45 137.74 for intramuscular administration of epimedin c , combination of four flavones , and ckz , respectively . compared with administration of individual epimedin c , auc of epimedin c in ckz group was statistically different ( p < 0.05 ) , while after administration of combination of four flavones , the auc value was also higher than that of individual epimedin c but the difference was not significant . the mrt0 and t1/2 values of epimedin c after administration of ckz and combination of four flavones were longer than corresponding values after administration of epimedin c , and the mrt0 value was significantly different ( p < 0.01 ) among three groups . drug clearance ( clz ) is defined as the volume of plasma that would contain the amount of drug excreted per minute . in the present study , drug clearance ( clz ) of epimedin c was 1.26 0.21 , 1.20 0.41 , and 0.79 0.19 for epimedin c , combination , and ckz group , respectively . the data demonstrated that the systemic clearance of epimedin c is significantly different ( p < 0.01 ) between epimedin c group and ckz group . in summary , longer mrt0 and t1/2 , higher auc , and lower clz values of epimedin c in ckz group than epimedin c group implied that the purified herbal preparation may be superior to single compound in prolonging the duration of action of epimedin c. the bioavailability of flavonoids has received extensive attention for many years due to its poor oral absorption characteristic . information on the comparative kinetic and bioavailability studies of flavonoid glycosides from herba epimedii is important for understanding the biological effects of the herbal drug and its preparation . it has been reported that the oral bioavailability of individual epimedin c was higher than that of herba epimedii extracts . our results , on the contrary , turned out that , after intramuscular administration of the purified extract to rats , the absorption and bioavailability of epimedin c enhanced significantly in comparison with those of individual epimedin c administered intramuscularly , which suggests other components in cki could improve the bioavailability of epimedin c. because of the synergistic effects of other herbs / components , chinese herbal formula is usually more potent than individual herb / component , which has been confirmed by more and more researches on chinese medicine formula such as huangqin decoction [ 2427 ] . the purified herbal preparation might be more effective than individual epimedin c via intramuscular administration in clinical application . moreover , the above - mentioned report also demonstrated that epimedin c is poorly absorbed following oral administration of both individual compound and herbal extract and the absolute bioavailability of epimedin c is not more than 0.58% in rat . in the present study , we observed , compared to the oral administration , that epimedin c was quickly absorbed with an extremely high bioavailability after intramuscular administration ; the absolute bioavailability of this flavonoid glycoside was about 100% . clearly , an injectable form of epimedin c may be more effective and reasonable for the clinical application of the purified herba epimedii extract preparation . our study was designed to evaluate the pharmacokinetic parameters of epimedin c after a single dose of epimedin c , combination of four flavonoid glycosides , and ckz administered , respectively , to rats via intramuscular and intravenous injection and to compare the relative and absolute bioavailability of epimedin c. although there have been investigations on the pharmacokinetics of epimedin c administrated orally [ 22 , 23 ] , to the best of our knowledge , this is the first study to reveal the comparative pharmacokinetic and absolute bioavailability of epimedin c administrated intramuscularly . the results showed that among three intramuscular administration groups the differences of pharmacokinetic parameters such as clz , mrt0 , and auc0 were statistically significant ( p < 0.05 ) . with the obtained pharmacokinetic results , it can be concluded that ( 1 ) after intramuscular administration of the purified herba epimedii extract to rats , the absorption and bioavailability of epimedin c enhanced significantly in comparison with those of epimedin c administered individually . in brief , other ingredients in the purified extract can affect the pharmacokinetic behaviour of epimedin c in rats ; ( 2 ) as for epimedin c , intramuscular administration is superior to oral administration due to higher absolute bioavailability ; ( 3 ) the results presented in this study implicated that the purified herbal preparation might be more effective and safer than individual compound . in conclusion , results from this work could provide some guidance for the clinical applications of this herbal drug extract . and in terms of the bioavailability of epimedin c | chuan - ke - zhi ( ckz ) , a purified herba epimedii extract , is a potent chinese medicine preparation whose main bioactive components are a class of flavonoid glycosides such as epimedins a , b , and c and icariin . and epimedin c is far more abundant than other flavones in this extract .
this study aims to investigate the pharmacokinetic and bioavailability of epimedin c and what effects , if any , other ingredients in ckz have on its pharmacokinetics .
epimedin c , ckz , and a combination of epimedins a , b , and c and icariin were , respectively , administrated to rats , and then the pharmacokinetic parameters of epimedin c in the three groups were calculated and compared .
the result indicated that clz , mrt0 , and auc0 of epimedin c were significantly different among the three groups ( p < 0.05 ) , and compared with the epimedin c group , the absorption of epimedin c significantly increased in the ckz group . furthermore , in this study the absolute bioavailability of epimedin c was also investigated by comparing intramuscular and intravenous administration of epimedin c. as a result , epimedin c could be quickly absorbed with extremely high absolute bioavailability after intramuscular administration . |
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a 38-year - old woman with a history of tobacco abuse and oral contraceptive use presented to the emergency department with transient loss of vision in the right eye , which lasted for 30 min , with a second episode that respected her horizontal meridian . the patient initially developed a non - productive cough for 2 weeks , followed by right - sided neck pain prior to the episode of vision loss . vital signs were notable for a blood pressure of 124/85 , heart rate 63 bpm , and respiratory rate 16 . eye examination revealed vision 20/20 , 3 mm pupils , equal and brisk to direct and consensual light , reactive to accommodation . cardiopulmonary examination revealed regular rate and rhythm , with a 2/6 systolic murmur loudest at the left lower sternal border , without rubs or gallops , and lungs were clear to auscultation . neurologic examination revealed intact cranial nerves , no loss of hemifacial sweating , and preserved strength in all extremities . electrocardiogram revealed normal sinus rhythm , no acute st changes . computed tomography of the head was negative . the patient was discharged home with a follow - up appointment with an ophthalmologist , who noted a normal eye exam with an unremarkable external and dilated retinal exam . carotid ultrasound revealed decreased velocity in the right internal carotid artery with evidence of an intimal flap . ct angiogram of the neck confirmed diffuse narrowing and string sign of the right intracranial internal carotid artery due to carotid dissection ( figs . 1 and 2 ) . dissection of the right internal carotid artery , which was the etiology for amaurosis fugax . this case describes the initial signs and symptoms of carotid dissection , with resultant amaurosis fugax . notably , this patient did not have a history of diabetes , hyperlipidemia , hypertension , or migraines which further warranted workup for an atypical source of symptoms . the majority will have symptoms of neck pain and headache ( > 90% ) , followed by focal hemispheric ischemic symptoms ( 5090% ) , horner 's syndrome ( < 50% ) , monocular blindness ( 630% ) , and dysgeusia ( 1019% ) ( 3 ) . the incidence of carotid dissection is estimated to be two to three cases per 100,000 for all age groups , with a mean age of onset noted to be early 40s ( 4 ) . there are four types of amaurosis fugax : embolic , hypoperfusion , angiospasm , and idiopathic ( 5 ) . given the most common symptoms being non - specific , early diagnosis can be difficult , as symptoms are overlooked . common non - traumatic causes of carotid dissection include fibromuscular dysplasia , marfan 's syndrome , atheroma , cystic medial necrosis , and systemic hypertension . in cases with carotid dissection , hemorrhage into the carotid wall could lead to intimal injury . hematomas may form as the dissection progresses into the media , which can occlude the lumen . early diagnosis and treatment can decrease the progression of the dissection . diagnosis is typically initiated with carotid ultrasound as symptoms raise suspicion for source of possible emboli . internal carotid artery dissection can produce cerebral infarction in 3668% of patients , as occlusion of the lumen near the dissection or embolization of thrombus occurs ( 3 ) . an echogenic intimal flap or thrombus , along with decreased velocity of the carotid artery , may be noted on doppler ultrasound . ct angiogram is the gold standard for diagnosing cervico - cephalic carotid dissection , noting tapered narrowing of the lumen . in reference to the american heart association , initial treatment of aspirin 75325 mg daily clopidogrel and aspirin with extended release dipyridamole may be considered in patients with aspirin allergy . anticoagulation may be considered in patients with recurrence of symptoms , especially in the setting of a mechanical valve or atrial fibrillation . treatment regimen also includes medication for lowering of blood pressure to less than 140/90 and ldl to less than 100 , medication for diabetes mellitus , along with smoking cessation ( 6 ) . in cases of recurrence despite appropriate anticoagulation with coumadin , surgical intervention may be considered . surgical options include endovascular repair such as angioplasty , stent placement , embolization , surgical revascularization , and bypass . the authors have not received any funding or benefits from industry or elsewhere to conduct this study . | this case report describes a patient found to have amaurosis fugax as a result of non - traumatic internal carotid dissection .
monocular blindness can be due to multiple causes including keratitis , acute glaucoma , vitreous hemorrhage , uveitis , retinal vascular occlusion , retinal detachment , optic neuropathy , trauma , or vascular malformations . in the setting of headache , neck pain , and an otherwise normal ophthalmic examination ,
this case report highlights the importance of recognizing transient ischemic attack and carotid artery dissection in the differential diagnosis . to further clarify the diagnosis ,
carotid ultrasound may aid diagnosis as was seen in this case , where decreased internal carotid artery velocities were found and subsequent ct angiography of the neck confirmed a diagnosis of carotid dissection . if a dissection is present , progression of symptoms may indicate impending cerebral infarction and warrant immediate attention .
antiplatelet therapy is the first - line treatment with anticoagulation , thrombolysis , and surgery reserved for cases of recurrent , progressive symptomatic episodes .
surgical options include endovascular repair such as angioplasty , stent placement , embolization , surgical revascularization , and bypass . |
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isocratic elution exhibits some advantages over the gradient one , such as greater simplicity , lower cost , simpler instrumentation , and no need of column reequilibration between consecutive injections . however , gradient elution is becoming almost unavoidable in conventional liquid chromatography , including ion chromatography ( ic ) . in gradient mode the elution strength usually increases during analysis , thus providing much narrower chromatographic peaks and significantly shorter analysis time . such characteristics are favored in cases with multicomponent samples which span a wide retention range . the implementation of gradient elution implies finding the suitable gradient program . the major criterion for optimal elution is a good resolution between analytes accompanied with an acceptable analysis time . existence of a good model to predict the column output is a crucial item in optimization of chromatographic elution . ic development in the last several decades was accompanied with the increasing number of models , either theoretical or empirical , which can be used to predict or to explain experimental chromatograms [ 318 ] . among numerous approaches , the retention isocratic - to - gradient ( iso - to - grad ) model [ 12 , 16 ] appears to be particularly interesting . this model is primarily developed for systems with a single - competing eluent ; it is based on the transfer of retention information from isocratic to gradient elution mode . the linear solvent strength model is the first and practically the most important model that describes the relation between component retention and concentration of competing ion for isocratic elution . this theoretical model , developed by snyder et al . in 1979 , in its origin considers only electrostatic effects leading to ion exchange retention . therefore , the presence of other mechanisms or occurrence of factors influencing the retention will result in deviations from the linear model . to overcome this problem some authors include an additional factor as a correction for nonlinearity [ 12 , 19 , 20 ] in ic and other lc techniques . such empirically obtained polynomial models were shown to fit isocratic experimental data significantly better than the linear one . the selection of polynomial instead of linear model does not have significant influence on the calculation time with contemporary computers . therefore the authors decided to describe the isocratic behavior with the better fitting model , that is , the polynomial dependence between the logarithm of retention coefficient , k , and concentration of competing ion in eluent , c :
( 1)logk = a0+a1logc+a2log2c .
in principle only three isocratic experiments are sufficient for the determination of regression coefficients , a02 , of the model described by ( 1 ) . the retention coefficient is calculated as
( 2)k = trtmtm ,
where tr is a component retention time and tm is the column 's holdup time . for the ion chromatography case the column 's hold - up time is equal to the retention time of unretained compounds , that is , column void time t0 . on the other hand , gradient elution can be described generally by the integral elution equation
( 3)t0=0trt0dtk[c(t ) ] . the integral from ( 3 ) can be approximated with a sum of integrals over small time intervals . inside those intervals the coefficient k can be assumed constant and can be calculated as an average of k values at the interval boundaries , obtained according to ( 1 ) . in this way the iso - to - grad approach allows for the prediction of retention time for practically any gradient using only several isocratic experiments . nevertheless , the optimization of gradient elution remains a severe and complex problem , especially knowing that the number of different gradient programs in any gradient domain is practically unlimited . the problem can be simplified by setting different constraints on the domain ( i.e. , defining the sets of finite intervals in which the gradient curve may change , finite sets of gradient curve shapes , or finite sets of allowed gradient slopes [ 2225 ] ) . unfortunately , the usage of any constraint involves the possibility of missing the true optimum . an increase of the number of allowable options inside each finite set will produce a finer description of the real experimental domain . however , it will at the same time increase significantly the number of possible gradient profiles . this leads to another problem , a domain of profiles that is too large substantially increases both the experimental effort and required modeling time , which may even exceed the performance of modern computers . a mathematical model that is able to describe the optimization surface for gradient ic is generally a nonlinear and very complex function . the fact that there are often multiple solutions ( local extremes ) complicates the situation even more . there are several classes of approaches available for solving this kind of problems among which are sequential searching procedures and evolutionary algorithms . the user defines a very restricted number of simplex points , that is , vertexes , usually one more than the number of factors to be varied , nf . the function to be optimized is evaluated at the simplex vertexes , either experimentally or by calculation , using a previously constructed model . then a decision is made about the worst vertex , which is replaced by a vertex that is reflected through the nf - dimensional surface ( the reflection being defined by the rest nf vertexes ) . according to the situation evolutionary algorithms mimic different biological processes in an attempt to optimize highly complex functions . genetic algorithms [ 29 , 30 ] ( figure 1(b ) ) , as a representative of the evolutionary algorithms , are based on genetic inheritance and darwinian strive to survival . in other words they allow a population composed of many individuals to evolve under specified selection rules to a state that optimizes the predefined objective function value . this work is focused on the application of simplex methodology and genetic algorithm in the optimization of gradient elution in ic . the conventional application of these two methodologies in gradient ic implies the search for the optimum in the real experiment domain . a limited set of experiments is performed initially and , subsequently , the experiments with the worst output are replaced by new , better ones . this might take a long time and a lot of experimental effort . in the particular approach described by this paper , the number of experiments was practically minimized ; the novel experiments to replace the old ones are not performed in the real domain , but by applying the initially constructed iso - to - grad model , that is , on a computer . the experiments were performed on a dionex ics-5000 ( thermo fisher scientific ) ion chromatographic system , equipped with a dual pump ( dp-5 ) , eluent generator module ( eg-5 ) with egc iii koh cartridge , degas unit on eluent generator , continuously regenerated anion trap column ( cr - atc ) , thermostatically controlled detection module ( dc-5 ) , and an autosampler ( as - ap ) . for performing the separation , the system was equipped with a dionex strong anion exchange column carbopac pa20 ( 3 150 mm ) and a respective guard column ( 3 30 mm ) . the detection mode was pulse amperometry , with reference electrode ag / agcl and working gold electrode . the separation was performed at constant temperature of 30c , while the detection temperature was 20c . the eluent flow rate was 0.5 ml / min , the sample loop volume was 10 l , and the data collection rate was 1 hz . standard solutions of 8 sugars : arabitol ( 150 ppm ) , cellobiose ( 600 ppm ) , fructose ( 1000 ppm ) , fucose ( 100 ppm ) , n - acetyl - d - glucosamine ( 500 ppm ) , lactulose ( 3000 ppm ) , melibiose ( 600 ppm ) , and raffinose ( 1000 ppm ) were prepared by diluting appropriate amounts of solid compounds in deionized water ( the first six sugars were from sigma - aldrich , usa , and the last two from dr . working mix solutions were prepared from standard solutions in concentrations 100 times lower than the standard ones . working eluent solutions were hydroxide solutions prepared online in the eluent generator module by pumping water through the eluent generator chamber . in all cases , in order to determine parameters of isocratic retention model ( 1 ) for each sugar , a set of 5 isocratic experiments was performed ; the experiments were equidistantly distributed within the range from 2 to 98 mm koh . peak maximum retention was modeled as common ; however , isocratic models were also defined for the 50% peak height point at the fronting side as well as for the 50% peak height point at the tailing side . these three retention times associated with each peak were used to predict the resolution between the analyzed sugars according to [ 32 , 33 ]
( 4)rs=1.18tr(2)tr(1)w0.5(1)+w0.5(2 ) ,
where tr(1 ) and tr(2 ) are retention times of two adjacent sugars and w0.5(1 ) and w0.5(2 ) are corresponding peak widths at half peak heights . the void time needed for prediction of component retention under gradient simplex vertexes or units from the genetic population were valued by the criterion function , cf :
( 5)cf=(1rs)ta.
two optimization goals were implemented into the criterion : the maximal sum of resolutions between eluted components rs [ brackets in ( 5 ) ] and the shortest analysis time ta . the desired balance between the goals was adjusted by the proper choice of criterion function weights and . a possible misbalance would produce an elution that is too long at one extreme or a peak overlapping at another . therefore it was necessary to select the appropriate weight values . for the selection purpose the same gradient domain was searched for the optimal separation conditions with both the simplex and genetic algorithms . the eluent concentration was ranging from 5 to 95 mm koh ; only the gradients producing elution times shorter than 30 min were taken as the acceptable ones . in the case of simplex approach , the acceptable time range of 30 min was split into nt equidistant intervals ( with varying nt ) . this provided nt + 1 simplex factors ( time points ) in which the change of eluent concentration was possible . in the genetic algorithm case the equivalents to the simplex factors were termed genes . in the applied genetic algorithm , the 30-minute time range was split into 3 min intervals which provided 11 genes in which the concentration change was possible . the gradients were defined by eluent concentrations ( the values of factors or genes ) at start and end of each interval . for both applied approaches , the elution continued isocratically after the first 30 minutes , taking the last factor or gene value . the optimization procedure implied searching for the optimal eluent concentration for each factor or gene . therefore , an appropriate design in creation of initial matrix of points or units was needed . in the simplex optimization approach , the initial matrix of points was created using the doehlert design [ 34 , 35 ] , which provides the mesh of lattice points uniformly and equidistantly distributed in the space around some central point . if there are multiple optima in the searched domain , local optima ( instead of the global optimum ) will probably be reached using different initial matrices . to test this , we varied the parameters of the doehlert design . these were ( 1 ) the number of simplex factors , ( 2 ) characteristic distance ( concentration interval ) , and ( 3 ) central point position ( central concentration ) . the number of simplex factors was varied from 1 to 5 , using step 1 . the concentration intervals were selected equidistantly within the predefined gradient domain range of 90 mm koh to take values of 5 , 10 , 15 , 30 , 45 , and 90 mm koh . central point was selected as an isocratic elution at a midpoint concentration within the selected concentration interval . for the three characteristic nelder - mead parameters , that is , coefficient of reflection , expansion , and contraction , standard values of 1 , 2 , and 0.5 were taken , respectively [ 3739 ] . the optimization was set to terminate when the sum of absolute differences between the factor values of two consecutive iterations became lower than the predefined objective value of 10 . in the genetic algorithm approach , the first 30 minutes of elution were divided into 10 equally sized time intervals providing the set of 11 genes . the set of all genes is commonly termed the chromosome ; it completely describes a unit with its peculiar elution characteristics . the initial population of 100 units was created by randomly assigning the concentration values from the predefined gradient domain range to all genes in the population this means that every unit from the population was valued according to ( 5 ) ; afterwards the worst 70% were removed . we applied the uniform crossover procedure ; the genes forwarded from the first parent to the offspring were chosen randomly , and the rest of the genes came from the other parent . the new - born population was joined with the parents ; afterwards such a created population was valued again . the worst 50% of units was removed and the rest became the new parent population . crossover without mutations restricts the characteristics of the new - born population from the characteristics of their parents ; mutations bring new characteristics to the population . the percentage of mutations in new - born population may be considered an adjustable parameter . the number of mutations in new - born population was varied from 5 to 110 ( using step 5 ) of overall 330 genes . the cf value ( 5 ) of the best unit was recorded after each crossover + mutation cycle . the optimization stopped after a predefined number of 200 cycles . since the random setting of initial population and application of mutations generally produces diverse results ( different optima ) , a set of 10 consecutive runs was performed in all cases of genetic algorithm calculations . the generalized logistic distribution function was used for the description of peak shape of individual components in calculated chromatograms :
( 6)f(t)=cbe(at)/b(1+e(at)/b)c+1 . the median of distribution is associated with parameter a , parameter b characterizes the distribution width , and parameter c bears information about the distribution skewness [ 40 , 41 ] . peak description according to generalized logistic distribution function requires only four experimental ( or calculated ) data . these are the retention time of peak maximum , retention times of peak half - heights at fronting and tailing peak sides and the stretching factor , sf :
( 7)h(t)=sff(t ) . simplex and genetic algorithms were applied for the optimization of ic separation of the solution of 8 sugars . the conventional approach of simplex and genetic algorithms was improved by incorporating the iso - to - grad model . thus the simplex and genetic algorithms were allowed to search for the optimum in the virtual experiment domain , instead of the real experimental space , which produced the significant reduction of time and costs . in principle , only three isocratic runs are needed to obtain coefficients of the quadratic dependence described by ( 1 ) . nevertheless , the higher number of isocratic experiments generally provides a more reliable isocratic model . in addition , it can not be known a priori how the competing anion in eluent would affect the peak area and this is the feature essential for the peak shape prediction . where overlapping of the components was observed , working solutions of pure analytes were eluted as well . thus a set of isocratic retention data was obtained to allow for the calculation of model parameters of ( 1 ) . apparently , the chosen polynomial model fits the retention behavior well for 7 of the 8 investigated sugars ( r 0.9989 ) . in the case of arabitol the observed error may be explained by comparing the retention times of all the 8 sugars at the lowest eluent concentration ( 2 mm koh ) . the elution order observed was arabitol 1.53 min , fucose 3.85 , n - acetyl - d - glucosamine 15.83 , fructose 18.87 , melibiose 23.98 , raffinose 27.92 , lactulose 40.12 , and cellobiose 61.28 and void time was 0.93 . in comparison to other sugars , the difference between arabitol elution time and void time is particularly small , only 0.60 min . it is important to recur that the data collection rate of the detector was only 1 hz . such a small rate could probably incorporate some error in the retention prediction , which would be especially noticeable for extremely fast eluting components like arabitol . some constrains were set on the investigated gradient domain in both applied optimization approaches . in the case of simplex optimization we had to limit the number of factors as well as the concentration interval investigated . constrains of the genetic algorithm were posed by the number of genes ( 11 ) in population units as well as by the allowed concentration values for each gene ( integer encoding of the population ) . although the acceptable time of ic analysis was set to 30 min for both approaches , it should be pointed out that the procedure was allowed to predict the optimal separation with elution longer than 30 min . however , such events were then simply excluded from further considerations . as for the eluent concentration range , concentrations higher than 95 mm produced poor separation of the several least retained sugars ; concentrations below 5 mm led to very long and economically unjustified elutions . although the negative gradients are quite uncommon in ic , since they deteriorate the baseline behavior , the authors did not exclude them from the simulation . in some cases , after reaching the satisfactory separation of a few first eluting components the negative gradient may , in principle , slow down the elution of the remaining components that would otherwise lead to overlapping of their peaks . as mentioned before , we had to assign the appropriate weights to the two contributions incorporated in the criterion function cf ( 5 ) that were selected for the valuation of simplex vertexes or genetic units . an appropriate balance between the weights should diminish the possibility of obtaining elution times that are too long or elutions with overlapped peaks . in addition , the simplex methodology always produces the same output for the same starting conditions , which is not the case in genetic approach due to mutations . therefore , the selection of cf weights was performed according to the results obtained by simplex calculations ; 25 simplex runs with different weight values were performed . the number of simplex factors was held constant at the value of 5 and the concentration interval was 15 mm koh . the calculated optimal elutions were compared according to the existence of peak overlapping or analysis time that is too long . the peak overlapping threshold was set to rs < 3 , which is somewhat higher than the common values 1.5 or 2 . we decided to set a more rigorous overlapping criterion since we were dealing with the retention models associated with possible errors . the obtained results , as shown in figure 2 , indicate that contribution of analysis time must be favored over the contribution of overlapping for the acceptable separation ; that is , must be higher than . the unacceptable weight combinations are marked with hollow circles in figure 2 . we decided to select the combination of weight factors = 1 and = 2 for further calculations . the size of simplex was varied in the simplex optimization ; 6 different concentration intervals and 5 different factor numbers were used . for the 2- and 3-factor cases the optimizations eventually reached the isocratic chromatograms , regardless of the size of the concentration interval applied ( an example is presented in figure 3(a ) ) . in the case of 4-factor optimization , four different chromatograms were obtained depending on the starting point ( figures 3(a)3(d ) ) . the acceptable separation was obtained for the 5 mm concentration interval case , although the last sugar eluted at the edge of the tolerated period of 30 minutes ( figure 3(b ) ) . with further increase of the concentration interval the analysis time decreased ; at the same time the peak overlapping was observed ( figures 3(a ) , 3(c ) , and 3(d ) ) . the results for the 5-factor optimization eventually produced five different chromatograms ( figures 3(a ) and 3(e)3(h ) ) . the smallest concentration interval provided the peak overlapping and analysis that is too long at the same time ( figure 3(e ) ) . good separations were predicted for the 10 and 15 mm concentration intervals ( figures 3(f ) and 3(g ) ) with a distinction of significantly shorter analysis time for the 15 mm case . further increase of the concentration interval produced peak overlapping ( figures 3(a ) and 3(h ) ) . therefore , the best optimization result occurred when gradient domain was scanned by the algorithm characterized with 5 simplex factors and the concentration interval of 15 mm koh as the starting point . this indicates that the applied simplex algorithm did not reach the global optimum in every case ; that is , it had serious problems when dealing with local minima . it can be noticed that gradients shown in figure 3 continue even after the last component eluted from the column . the concentration and time domains were defined prior to the search for optimal separation conditions . therefore , even for those cases where components eluted very fast , the remaining part of eluent concentration profile must exist ( regardless of the fact that it has no influence on separation ) , but only as an artifact of the applied calculation procedure . the specific eluent concentration profiles shown in figure 3 after the last eluted component are simply the first ones that the algorithm found . the selection of any other concentration profile continuing the last eluted component will have no effect on separation since it is already achieved . in the case of genetic algorithm , we were dealing with the percentage of mutations in new - born population and the number of crossover cycles as the important issues . although the final number of crossover cycles was set to 200 , the improvement of the best unit 's cf value vanished normally after a much lower number of cycles . we termed it as the threshold number of cycles and we recorded it when the cf value dropped below 10 . the gray circles in figure 4 represent the median of the threshold number of cycles for 10 consecutive runs and the bars represent the corresponding span of values . according to the results plotted , however , considerably more scattering in the threshold number of cycles was observed when more than 90 genes were allowed to mutate . in conclusion , the crossover with 60 mutated genes ( i.e. , 18.2% of mutations ) produced the smallest median of the threshold number of cycles ( the shortest calculation time ) . at the same time it produced the minimum scattering of results . therefore , this percentage of mutations was taken as the best one . among the results calculated with this percentage of mutation , the gradient elution profile with the shortest elution time ( 18.89 min ) was selected as the optimal one . it is important to point out that all 220 calculations using the genetic algorithm , regardless of the number of mutations applied or diverse initial populations randomly selected , produced extremely similar resolutions of adjacent components ( rs values , table 2 ) . the elution order of the analyzed sugars was always the same and none of the peaks overlapped . the standard deviation of predicted resolutions for all sugars and all 220 calculations was not higher than 1.28 . also , the standard deviation of maximal elution time was only 0.39 . in order to test the results obtained by the simplex and genetic algorithm optimization , the real ic analyses were performed under the calculated optimal koh gradients ( figure 5 ) . the experimental chromatograms are compared with the predicted ones . however , for the full description of predicted chromatogram one has to choose an appropriate distribution function to model the peak shape . the generalized logistic function proved a good choice for the peak shape prediction of inorganic anions in ic [ 16 , 25 ] due to its simplicity and fair representation of experimental chromatograms . therefore , in order to create a better presentation of the separated sugars , it was applied in this case as well . all the data needed for calculation of a , b , and c parameters of the generalized logistic distribution function are presented in table 1 , and the calculation procedure is described elsewhere [ 16 , 25 ] . therefore , only the stretching factor of the generalized logistic distribution function ( 7 ) will be discussed here . as mentioned before the stretching factor in this case equals the peak area of a real chromatogram . as said before , in this research this detector is a concentration selective detector , an influence of competing anion concentration on the peak area is observed ( figure 6 ) . for all the analyzed sugars a decrease of peak area with the increase of eluent concentration is observed . isocratic experimental data were used to estimate the stretching factor , that is , peak area . since the stretching factor depends on the competing anion concentration , the appropriate values for every peak were calculated in the following manner . second , competing anion concentration at that retention time is detected from the gradient profile calculated . third , peak area is estimated at that competing anion concentration using the nearby experimental values from figure 6 and linear interpolation . the predicted chromatograms have much broader bandwidths in comparison to the experimental ones , offering slight apparent overlapping of the two last eluted components ( figures 5(a ) and 5(b ) ) . such overlapping was not a consequence of invalid optimization ( all the calculated rs values were higher than 3 ) but simply the product of the chosen peak - shape function , which seems to be less adequate for sugars than it was for inorganic anions . despite that , the chromatograms generally match each other , confirming the applicability of both methodologies in the optimization of ic separation . although the genetic algorithm may be considered somewhat better with respect to the analysis time ( the predicted retention of the last eluting sugar of 18.98 min and 19.19 min for genetic and simplex optimization , resp . ) , it is obvious that both approaches provide similar analysis time . in both cases the last two eluted sugars ( raffinose and cellobiose ) have very close retention times . their predicted resolutions are significantly smaller than for the other adjacent sugar pairs and almost equal to the predefined threshold value of rs = 3 . the required separation of these two sugars is , therefore , the most probable reason of such a long calculated analysis time . this work describes the application of the two methodologies , that is , simplex and genetic algorithms , combined with the iso - to - grad retention model in the optimization of gradient ic separation . this combination allowed the finding of the optimum gradient profile in the virtual domain , thus minimizing the experimental effort and costs . the cf function weight factors were selected to favor the analysis time contribution ( = 1 for the resolution and = 2 for the analysis time ) . the optimal gradient profile for the simplex approach was obtained using the 5 simplex factors and the concentration interval of 15 mm koh in initiating the search . in the case of genetic algorithm , 18.2% of mutations in each offspring population were found to be the optimal percentage in fast and reliable finding of the optimum profile . the simplex methodology exhibited problems when dealing with local minima ; that is , different starting conditions resulted in significantly different component elution times . at the same time , the genetic algorithm did not suffer from the local minima problem : 220 calculations provided 220 almost identical separations . the real sample experimental chromatograms obtained by applying the calculated optimal gradient profiles were compared with the predicted ones and good agreement was found . both simplex and genetic algorithms in combination with iso - to - grad model proved a potential to be applied in gradient ic optimization . the developed approach offers a significant reduction of the experimental effort ( only 5 isocratic experiments needed + few more for the overlapping peaks ) . in addition , there is no practical limitation on the gradient profile to be tested . based on the results of this study , the genetic algorithm in combination with the iso - to - grad retention modeling may be recommended as the method of choice for optimization in gradient ion chromatography . | gradient ion chromatography was used for the separation of eight sugars : arabitol , cellobiose , fructose , fucose , lactulose , melibiose , n - acetyl - d - glucosamine , and raffinose .
the separation method was optimized using a combination of simplex or genetic algorithm with the isocratic - to - gradient retention modeling .
both the simplex and genetic algorithms provided well separated chromatograms in a similar analysis time . however , the simplex methodology showed severe drawbacks when dealing with local minima .
thus the genetic algorithm methodology proved as a method of choice for gradient optimization in this case .
all the calculated / predicted chromatograms were compared with the real sample data , showing more than a satisfactory agreement . |
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strachan in an article that claimed an inverse relationship between the occurrence of hay fever and numbers of siblings . according to the hypothesis , atopic disorders are due to reduced exposure to microorganisms in childhood . nowadays , the concept is becoming more accepted with accumulating evidence not only in atopic diseases but also in autoimmune inflammatory diseases . for instance , the incidence of multiple sclerosis ( ms ) is higher in high latitude countries (= westernized developed countries ) than in equatorial areas . not only residents of western countries but also immigrants from developing countries are at high risk of developing inflammatory bowel diseases ( ibds ) and asthma . in the case of type 1 diabetes ( t1d ) , a similar geographical distribution to the diseases above and an inverse correlation to hygiene conditions are observed . a population - based ecologic study in canada showed that ibd , including ulcerative colitis ( uc ) and crohn 's disease ( cd ) , correlated with a high socioeconomic status , low rate of enteric infection , and high rate of ms . for instance , an inverse correlation between autoimmune liver diseases and strongyloides stercoralis infection was demonstrated in okinawa , japan . cross - sectional studies on the relationship between skin prick tests and helminth infections suggested a general protective effect on the atopic reaction . the authors summarized effects of geohelminths on the risk of asthma according to previous studies ; that is , hookworm lowered but ascaris increased the risk of asthma and trichuris had no effect . collectively , it is concluded that at least some helminths seem to have anti - allergic or anti - inflammatory effects in humans . experimental studies have also shown protective effects of helminth infections in animal models of autoimmunity ( e.g. , colitis , arthritis , and diabetes ) and allergy ( e.g. , airway hypersensitivity ) [ 3 , 810 ] . in this review , we discuss possible mechanisms of anti - allergic / anti - inflammatory effects of helminths in animal models including autoimmune arthritis . based on t cell skewing patterns and their relative importance in the pathogenesis , disorders with excessive immune responses had been briefly classified as th1 diseases and th2 diseases according to the th1/th2 paradigm . however , the recent discovery of a novel pathogenic t cell subset ( th17 ) led investigators to the concept of th17 disease . while most atopic immune disorders ( e.g. , hay fever and bronchial asthma ) can be classified as th2 diseases , the classification of autoimmune diseases is relatively difficult . experimental autoimmune encephalomyelitis ( eae ) as a model of ms was long thought to be a th1 disease ; however , the recent studies using il-12/23 subunit ( p35 , p19 , or p40 ) deficient mice revealed the progression of the disease to be dependent on the il-23/il-17 axis (= th17 response ) rather than il-12/ifn axis (= th1 response ) . the pathogenic role of il-17 was shown directly by the finding that eae development was significantly suppressed in il-17-deficient mice . regarding t1d , the diabetes observed in nod mice ( a model of t1d ) has been classified as a th1 disease despite the presence of some controversial study results [ 1416 ] . recent reports demonstrated that th17 cells could also cause diabetes , but only after their conversion to th1-type cells [ 17 , 18 ] . regarding ibd , the pathogenic roles of both th1 and th17 responses in tnbs - induced colitis ( a model of ibd ) are still controversial [ 1923 ] . collectively , some experimental autoimmune disorders can not yet be distinctly classified as either th1 or th17 disease . the effects of schistosomes and other helminths on experimental autoimmunity / allergy are summarized in table 1 . surprisingly , helminths have been shown to suppress all types ( th1 , th2 , and th17 ) of disease in the models described above [ 2445 ] . considering classical th1/th2 paradigm , it is reasonable to speculate that helminth - induced th2-skewing with downregulation of th1 immune responses results in an amelioration of th1 diseases . as il-4 is known to suppress th17 development , th17 response could also be suppressed as well as th1 response in helminth - infected or helminth antigen - treated animals . in fact , stat6-dependent il-4/il-13 signaling was shown to be essential in the suppression of tnbs - induced colitis and eae by schistosome eggs , although the authors did not measure changes of il-17 . given that the involvement of th1 and th17 in some forms of autoimmunity is still controversial , downregulation of both t helper responses may be beneficial for the amelioration of various kinds of autoimmunity . along with other investigators , we recently found that schistosome - infected mice became resistant to experimental arthritis accompanying down - regulation of both th1 and th17 responses of splenocytes . reported suppression of tnbs - induced colitis by schistosome antigens , accompanying down - regulation of il-17 gene expression in the colon and mesenteric lymph node ( mln ) . an intestinal nematode ( heligmosomoides polygyrus ) infection was also reported to suppress il-17 production in mln cells and lamina propria mononuclear cells . the authors showed that the blocking of both il-4 and il-10 restored il-17 production in vitro . another study revealed that fasciola hepatica - induced down - regulation of autoantigen - specific th1 and th17 responses ( and protection from eae ) was dependent on tgf- , not il-10 . although the mechanisms of th17 's down - regulation by helminths are not yet established , some of the mechanisms might be common to those of th1 's down - regulation ( e.g. , through induction of il-4 and il-10 , down - regulation of il-12p40 ) and others might be distinct . in our study on experimental arthritis in mice , we found no increase of treg - related gene expression ( foxp3 , tgf- and il-10 ) in the paws of s. mansoni - infected mice compared to the paws of uninfected control mice . as treg cell population was known to expand in schistosome - infected or egg - treated mice [ 31 , 32 , 35 , 50 ] , the cells might participate in the regulation of the disease systemically rather than locally . to confirm the essential involvement of treg cells in the antiarthritic effects of schistosome , further studies ( e.g. , persistent treg depletion experiments ) are necessary . in contrast to our result , in the case of diabetes in nod mice , schistosome egg antigens induce infiltration of treg cells at a local inflamed site ( pancreas ) . likewise , filarial nematode ( litomosoides sigmodontis ) infection induced treg cells and protected mice from the diabetes . h. polygyrus also protected mice from the diabetes ; however , the protection was not dependent on treg cells . in addition to schistosome and nematodes , tapeworm ( taenia crassiceps ) infection also has anti - diabetic effects in multiple low - dose streptozotocin - induced diabetes ( mlds ) in mice [ 45 , in this issue ] . in the study , taken together , these studies suggest that there may be various mechanisms in anti - diabetic effects of helminths . we also found that schistosome - induced down - regulation of th1 and th17 occurred in the same period after the infection , corresponding to the beginning of egg - laying ( unpublished observation ) . this result suggests that egg deposition is the major stimulus to lower th17 responses ( as well as th1 response ) in murine experimental schistosomiasis . some epidemiological studies support that helminth infections are protective against atopic reactions and/or symptoms [ 5153 ] . the helminth - induced suppression of th2 diseases ( atopic disorders etc . ) is difficult to explain in terms of the th1/th2 paradigm . in the paradigm , theoretically , helminth infections are expected to cause ige overproduction and hypereosinophilia , followed by exacerbation of allergic reactions . indeed , persistent bronchoalveolar eosinophilia , airway hyperresponsiveness , and exacerbation of allergic airway inflammation were observed in toxocara - infected mice . one interesting explanation of anti - allergic effects of helminths is introduced in a review by fallon and mangan , in which th2 responses are subdivided to allergic and modified , with helminth - induced th2 responses corresponding to the latter type characterized by predominant treg and il-10 responses and a relatively weak il-5 response . ( the authors , however , describe that such modifications are localized in the lungs and different from systemic responses . ) along with this hypothesis , administration of ascaris extract was shown to inhibit not only il-5 production but also eosinophilic inflammation in a murine asthma model . the suppressive effect of the filarial worms on hyperreactivity was dependent on treg cells and tgf-. as helminths seem to have both allergenic and immunomodulatory components in their bodies , the balance of them may determine the outcomes ( i.e. , exacerbation or amelioration ) of allergic disorders . helminthic infections often result in expansion and/or activation of treg cells [ 31 , 32 , 35 , 36 , 50 ] . however , the importance of il-10 in helminth - induced suppression of atopic diseases is not fully established ; that is , mesenteric lymph node cells from heligmosomoides - infected il-10-deficient mice could confer protection against allergic airway inflammation . likewise , in humans , anti - allergic effect of ascaris lumbricoides is associated with treg , but not with il-10 . in the case of mice , il-35 , a recently identified effecter molecule of treg cells , might be involved in the suppression . other than the regulation of lymphocytes , helminth - derived products have potential anti - allergic effects on various kinds of cells . for instance , the filarial product es-62 was shown to suppress the release of mediators from bone marrow - derived mast cells . such macrophages not only suppress the pathogenesis by the parasites themselves but also may suppress pathological immune responses to autoantigens or allergens . the involvement of aam in anti - diabetic effects was suggested in experimental tapeworm infection . on the other hand , schistosome - modulated macrophages ( but not aam ) immune cells and mediators possibly involved in the helminth - induced immunomodulation are illustrated in figure 1 . collagen - induced arthritis ( cia ) is an autoimmune arthritis in mice and rats widely used as a model of rheumatoid arthritis ( ra ) . although the role of the il-12/ifn axis in cia has been controversial [ 66 , 67 ] , recent findings suggest that ifn has ameliorating rather than exacerbating effects [ 6871 ] . instead , the il-23/il-17 axis was recently shown to be important in the pathogenesis of cia [ 7176 ] as well as ra in humans . regarding anti - arthritic effects of helminths , a filarial es product es-62 and porcine roundworm ascaris suum extract were shown to suppress cia in mice . as described , we examined effects of s. mansoni infection on cia in mice . in humans , schistosomiasis has been reported as arthritogenic rather than anti - arthritic [ 79 , 80 ] . in our experiments , however , s. mansoni infection lowered arthritis scores and numbers of arthritic paws . histopathological examination revealed that cell infiltration and bone / cartilage destruction were diminished in the infected mice . in cia and ra , we observed that the marked augmentation of il-1 and il-6 gene expression in the paws was almost completely abrogated by s. mansoni infection . it was especially noteworthy that receptor activator of nfb ligand ( rankl ) gene expression in the inflamed paws was also abrogated by s. mansoni infection . as rankl expression is induced by proinflammatory cytokines including il-17 , tnf- , and il-1 and essential to osteoclast development followed by bone destruction [ 8688 ] , this result suggests that schistosome infection has anti - arthritic effects preventing bone destruction . interestingly , we also found that intraperitoneally administered schistosome worm antigens ( swap ) or egg antigens ( sea ) did not affect the progress of cia ( unpublished observation ) . in the antigen - administered mice , levels of il-10 , tnf , and il-17 produced by splenocytes were comparable to those in antigen nonadministered control mice . thus , regarding schistosome , there is a considerable difference in immunomodulating effects between infection and antigen administration . as described above , our experiments with schistosome showed that anti - arthritic effects were observed only in the viable worm infected mice . likewise , hunter et al . showed that anticolitic effects of hymenolepis diminuta were dependent on a viable infection , and surprisingly , melon et al . showed that therapeutic efficacy of the viable tapeworm was superior to dexamethazone treatment [ 44 , in this issue ] . because of these observations in experimental models , attenuated or non / lowly - pathogenic helminths are worth testing directly for therapeutic effects in fact , porcine whipworm ( trichuris suis ) eggs and necator americanus infective larvae have been clinically tested for the treatment of chronic inflammatory or allergic diseases . reddy and fried summarized the recent progress in clinical trials using these two intestinal nematodes . summers et al . reported that the administration of t. suis eggs effectively ameliorated both uc and cd [ 90 , 91 ] without adverse effects . this worm is considered superior to porcine whipworm in that repeated administration is not needed . although it is not permissible to directly apply highly pathogenic helminths ( e.g. , schistosome ) to clinical use , purified or synthetic immunomodulatory products from such worms can be considered for clinical purposes . various immunomodulatory molecules ( carbohydrates , proteins , and lipids ) have been identified ; for example , lacto - n - fucopentaose iii ( lnfp iii ) contained in schsitosome eggs is an oligosaccalide as the molecule affecting b cells ( especially b-1 cells ) to induce il-10 production . a chemokine - binding protein ( cbp ) from schsitosome eggs inhibited the recruitment of neutrophils to inflammatory foci . peroxiredoxin ( prx ) is an antioxidant protein found in various species including helminths [ 96 , 97 ] . the molecules from s. mansoni and f. hepatica alternatively activate macrophages and are involved in the induction of a th2-type immune response . the il-4-inducing principle of s. mansoni eggs ( ipse ) is the molecule that induces primary il-4 production from basophils . regarding anti - arthritic effects , a glycoprotein from spirometra erinaceieuropaei was shown to suppress rankl - induced osteoclastogenesis , suggesting that there may be more anti - arthritic substances in various helminths . it should be pointed out that helminths do not always suppress autoimmune / allergic disorders . apart from th2-biasing abilities of helminths , allergens contained in the worms may partially explain the mechanisms of exacerbation . mice infected with toxocara canis showed exacerbation of allergic airway inflammation whereas hookworm ( nippostrongylus brasiliensis ) infection persistently reduced airway responsiveness in mice , suggesting that there are considerable differences of outcomes even among lung - migratory nematodes . thus the roles of th1/th17/treg - related regulatory cytokines ( il-25 , il-27 , il-35 , etc . ) in the helminth - induced suppression of allergy / autoimmunity have not been sufficiently studied . it is well known that the roles of cytokines in human th17 differentiation are very different from those in mice [ 12 , 102 ] . for clinical applications in the future , we should ascertain changes in th17-related cytokine patterns not only in animal models but also in patients with helminthiasis . it was reported that concurrent filarial infection suppressed both th1 and th17 responses to mycobacterium tuberculosis . this implies that helminths have down - regulating activity of both th1 and th17 in humans as well as in mice . in human ra , the relative importance of th1 and th17 is still unclear , and therefore , suppression of both t helper responses is recommended . thus , at present we can conclude that regulatory effects on both th1 and th17 are promising characteristics of helminths as anti - inflammatory agents . however , the interpretation of experimental results of helminth - induced immunomodulation may change depending on changes in basic immunological knowledge . indeed , it was recently reported that t - bet expression was more important in the pathogenesis of eae than the th1/th17 balance . therefore , further studies of helminth - induced amelioration of immune disorders should strictly follow the studies of the diseases ' pathogenesis . as noted , in experimental animal studies , viable helminth infections seem to be superior to the administration of worm antigens or killed worms in the therapeutic effects . this might be due to that viable helminths can regulate secretion of immunomodulatory molecules in the most appropriate conditions for their survival . taken together with successful treatment of uc and cd with viable porcine whipworms [ 90 , 91 ] , the optimal attenuation of human parasites by gene manipulation may be useful for the clinical application of parasitic helminths in the future . | the prevalence of allergic and autoimmune diseases is increasing in developed countries , possibly due to reduced exposure to microorganisms in childhood ( hygiene hypothesis ) . epidemiological and experimental evidence in support of this hypothesis
is accumulating . in this
context , parasitic helminths are now important candidates for antiallergic / anti - inflammatory agents . here
we summarize antiallergic / anti - inflammatory effects of helminths together along with our own study of the effects of schistosoma mansoni on th17-dependent experimental arthritis .
we also discuss possible mechanisms of helminth - induced suppression according to the recent advances of immunology . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
membranous nephropathy ( mn ) is one of the most common causes of nephrotic syndrome ( ns ) in adults [ 1 , 2 ] . most cases are considered idiopathic ( imn ) , where auto - antibodies react with one or more podocyte antigens ( e.g. , the m - type phospholipase a2 receptor ) to form the subepithelial deposits characteristic of all types of mn . however , in about 25% of the cases a secondary cause can be found , including lupus , viral infections ( most notably hepatitis b ) , cancer , and medications . sometimes , a specific relevant antigen can be detected in the subepithelial deposits . these include the hepatitis e antigen in cases related to hepatitis b , carcinoembryonic antigen in colon carcinoma , and cationic bovine serum albumin in certain pediatric cases . the pathologic features of both imn and secondary mn are otherwise similar , but subtle differences do exist . for example , in lupus mn there may be mesangial proliferation by light microscopy , full house positivity by immunofluorescence microscopy , and mesangial electron dense deposits by electron microscopy ; features not usually found in imn . in imn , igg4 is the most prominent subclass detected , whereas in secondary cases another subclass is usually found . guillain - barr syndrome ( gbs ) is a heterogeneous group of disorders with similar clinical presentations . typically , it is an acute , self - limited , paralyzing illness , which peaks in 2 to 4 weeks and then subsides . most cases in the usa ( about 85% ) result from a reversible , immunologically mediated , peripheral nerve demyelination . ( about 15% ) , the immunologic attack is against axons , with sparing of myelin . if just motor neurons are involved , it is called acute motor axonal neuropathy ( aman ) ; if sensory fibers are affected as well , the term is acute motor and sensory axonal neuropathy ( asman ) . glomerulonephritis has been found in association with gbs [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ] . some patients had pathologic confirmation but only mild clinical manifestations . more commonly , however , reported cases had ns , and the most common lesion was mn [ 15 , 17 , 18 , 19 ] . it is unclear whether this results from autoantibodies against podocyte antigens as in imn or rather against an extrinsic ( to the podocyte ) antigen as in secondary cases . we present a case of severe ns occurring simultaneously with severe gbs of the axonal variety . this suggests that the mn was indeed secondary , perhaps to an antigen released by the primary nerve damage . we discuss this in detail in light of the current knowledge of the imn pathophysiology . a 69-year - old man with a history of hypertension , hypothyroidism , dyslipidemia , obstructive sleep apnea , benign prostatic hypertrophy , and stroke was in his usual state of health until bilateral lower extremity edema developed rapidly over a 2-week period . he developed shortness of breath and was admitted to an outside hospital . on examination , blood pressure was 142/112 , pulse 69 , respirations 18 , temperature 37c , and oxygen saturation 96% on 2 liters oxygen by nasal cannula . there were 2 + lower extremity edema and mild right hand weakness , but no other focal neurologic findings . a chest radiograph revealed cardiomegaly ; however , no infiltrates or evidence of interstitial edema . creatinine was 1.4 mg / dl and albumin 2.4 g / dl , but liver function tests were otherwise normal . several days after admission , the patient began to complain about arm and leg numbness . lumbar puncture showed a glucose level of 169 mg / dl , a protein level of 35 mg / dl , wbc of 0/l , and rbc of 1/l . the patient stated his right hand weakness began about 3 weeks prior to the lower extremity edema . he described slow worsening of this weakness and progressive right leg weakness . on neurological exam , he had no cranial nerve abnormalities , and his sensory exam was normal except for decreased pin - prick sensation on the left leg up to the knee . motor exam showed 2/5 strength in his right deltoid and 4/5 in the left , biceps 4/5 bilaterally , triceps 4/5 bilaterally , wrist flexors 4/5 bilaterally , interossei 3/5 on the right and 4/5 on the left , hip flexors 3/5 on the right and 4/5 on the left , knee extensors 3/5 on the right and 4/5 on the left , and dorsiflexion 4/5 bilaterally . reflex exam was 1 + to 2 + with the exception of 0 bilateral ankle reflex ; there was no babinski reflex . the patient was treated with a 5-day course of intravenous immunoglobulin , which showed no significant improvement . laboratory results were negative for neuromyelitis optica antibodies , rheumatoid factor , complements , antinuclear antibodies , and lyme titers . the renal biopsy showed mn , with diffuse thickening of glomerular basement membranes with spikes on the epithelial side and interstitial fibrosis in about 20% of the cortical surface area ( fig . igg1 , igg2 , and igg3 subclass stains are not routinely done at our institution . electron microscopy revealed subepithelial electron dense deposits uniformly distributed throughout the glomerular capillary walls ( fig . the patient received a second round of intravenous immunoglobulin , which had to be discontinued due to acute kidney injury , however . the patient developed facial diplegia , ophthalmoplegia as well as decreased corneal reflexes , and was found to be quadriplegic . the next day , he became hypotensive and bradycardic and proceeded to polymorphic ventricular tachycardia , which responded to cardioversion . our case presented with concurrent ns and an axonal variety of gbs , which proved fatal . the complete absence of igg4 staining suggests that this was not the chance occurrence of imn , but rather a true secondary case . renal disease in the setting of gbs is not common clinically , but it has been known for many years , first described in a clinicopathologic report from 1918 . in the ensuing years , renal involvement was only occasionally noted . in 1973 , described a consecutive series of 9 patients of whom 8 had biopsy evidence of glomerulonephritis . however , the clinical manifestations were minor , with either transient hypertension , hematuria or both . that same year , 2 case reports described ns in association with gbs [ 15 , 16 ] . both showed underlying mn , and 1 characterized the subclass as igg4 . various case reports of ns have appeared since then , and the association has extended to the subacute and chronic inflammatory polyneuropathies [ 21 , 22 , 23 ] as well as gbs . most commonly , the glomerular lesion is mn , although minimal change disease has also been reported [ 24 , 25 ] . mn is the most common cause of ns in adults , although it may present with subnephrotic proteinuria . these include viruses ( hepatitis b or rarely c ) , cancers , medications ( mercury , gold , penicillamine , nsaids , etc . ) , autoimmune diseases ( lupus , sarcoid , etc . ) , and others . included in the latter is gbs . the issue is whether this is merely a chance association without pathogenic relationship or not . imn is usually characterized by a strong igg4 reaction detectable by immunofluorescence [ 9 , 26 ] . it has recently been determined that these igg4 antibodies may react with antigens normally expressed on the podocyte cell membrane ( m - type phospholipase a2 receptor ) , or only abnormally so ( aldose reductase , superoxide dismutase , -enolase ) . the antigen - antibody complexes are then shed to form the characteristic subepithelial deposits seen on electron microscopy . in general , an igg4 response occurs after many months of chronic antigenic exposure , such as may occur with bee keepers or parasitic infestations . these include hepatitis b viral antigens , tumor - related antigens , dna , and cationic bovine serum albumin . the antigen may first plant in the glomerulus , with in situ formation of the immune complex , or , less likely , circulating immune complexes may deposit . in any case , the immunoglobulins are usually not of the igg4 subclass . there are several potential mechanisms by which gbs and secondary mn may be pathogenically related . antibodies against the ganglioside gq1b are found in most patients with the miller fisher variant of acute inflammatory demyelinating polyradiculoneuropathy , and antibodies against gm1 , gd1a , and gd1b may be found with axonal variants . these antibodies are thought to arise from molecular mimicry from a preceding infectious agent such as campylobacter jejuni . it is possible that in occasional patients , these antibodies react , also through molecular mimicry , with one or more antigens expressed on podocyte cell membranes . alternatively , damage to peripheral nerves may result in the release of an antigen(s ) , which deposits in the subepithelial space . the respective antibodies then bind , resulting in in situ immune complex formation . finally , preformed circulating immune complexes involving one or more neural antigens may deposit in the subepithelial space . the rather acute nature of the apparent exposure would be consistent with a non - igg4 response . in the case of a true infectious cause of gbs , the infectious agent itself could similarly deposit as the inciting antigen , form in situ immune complexes , and cause mn as well . in mn associated with gbs , the renal lesion usually appears to be self - limiting , resolving temporally along with improvement of the neuropathy . unfortunately , in cases associated with the chronic and/or relapsing neuropathies , the renal disease appears to persist as well . | membranous nephropathy ( mn ) is one of the most common causes of nephrotic syndrome ( ns ) in adults .
it may be primary , usually mediated by igg4 anti - phospholipase a2 autoantibodies or secondary to various other conditions .
guillain- barr syndrome ( gbs ) has been associated with mn , but a cause and effect relation has not been proven .
we present a case of concurrent development of gbs and severe ns , with renal biopsy demonstrating mn .
igg4 stain was negative , indicating that most likely , the mn was secondary and probably caused by the underlying gbs . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
despite continued advancements in laparoscopic surgery allowing increasingly complicated procedures to be performed in a minimally invasive fashion , laparoscopic ureteral surgery has not received adequate attention in the literature . the majority of the literature reports regarding laparoscopic ureteroureterostomy comprise case reports and small series wherein these procedures were performed for the management of various conditions including retrocaval ureters , midureteral strictures , and iatrogenic ureteral injuries . beyond these limited reports , studies specifically evaluating technical considerations are lacking . because a suboptimal repair carries the risks of urine leakage , stricture , or even complete occlusion of the ureter , refinement and reviews of various techniques are necessary to allow better outcomes in the hands of experienced surgeons and guidance for those without experience in laparoscopic ureteral surgery . surgical ureteroureterostomy has traditionally been performed through an open incision appropriate for the level of the ureteral lesion in the repair of traumatic or iatrogenic injuries or after partial ureterectomy for benign disease , such as strictures . ureteral stents are often left in place to assist with healing and urinary drainage , but when placed for this reason , the stent can be placed prior to open exposure of the ureter or after reconstruction . we sought to determine whether laparoscopic placement of a ureteral stent impacts the quality or ease of performing a ureteroureterostomy by a novice or an experienced laparoscopic surgeon . the research design was reviewed and approved by the animal review committee at our institution prior to beginning the protocol . eight ureteroureteral anastomoses were performed laparoscopically on each ureter of a 50 kg female pig for a total of 16 anastomoses ( figure 1 ) . eight anastomoses were performed with a 6 french stent in place during division and spatulation of the ureter , and 8 anastomoses were performed without a stent . half of the anastomoses with and without a stent were performed by a novice with training in intracorporeal suturing in a dry laboratory environment only ( jp ) and the other half by a surgeon experienced in laparoscopic ureteral and other urologic reconstruction ( ra ) . a single 6-inch long , 4 0 vicryl suture with an rb-1 needle ( ethicon , somerville , new jersey ) was used to complete the anastomosis in a running fashion . stented and nonstented anastomoses were performed in an alternating manner extending between the proximal and distal ureter . the times required for ureteral division and spatulation , initial stitch placement , and completion of the anastomosis were recorded . at the conclusion of the surgical procedure , the quality of the anastomosis was determined based on the presence of gaps between any of the sutures in the anastomosis as well as the size of the defects and the patency of the lumen . the ureters were also perfused with saline at a pressure of 30 cm h2o to evaluate the size of defects and patency . the absence of defects was noted as none , while small and large defects were quantified as 1 mm to 2 mm and > 2 mm in length , respectively . a 2-sided , paired student t test was used in analysis of the data with significance defined as a p - value of less than or equal to 0.05 . the mean time in minutes required for completion of the ureteral division and spatulation , initial stitch placement , completion of the anastomosis , and total time for the stented and nonstented procedures was 4.3 vs. 2.2 , 4.2 vs. 4.4 , 10.4 vs. 13.5 , and 18.3 vs. 20.1 minutes , respectively . in reviewing the performance of the novice and experienced surgeons independently , the time in minutes required for completion of the ureteral division and spatulation , initial stitch placement , completion of the anastomosis , and total procedure time for the stented versus the nonstented procedures was 4.4 vs. 2.3 , 3.2 vs. 4.5 , 13.1 vs. 16.4 , and 20.7 vs. 23.2 minutes , respectively , for the novice laparoscopist and 4.2 vs. 2.2 , 5.3 vs. 4.2 , 7.6 vs. 10.6 , and 16.0 vs. 17.0 minutes for the more experienced laparoscopist ( table 1 ) . mean operative times for various individual steps of laparoscopic ureteroureterostomy procedure and total time in the stented and nonstented ureters , 3 vs. 5 anastomoses were found to have no or small gaps , 5 vs. 1 anastomosis were found to have large gaps , and 0 vs. 2 anastomoses were found to have occluded lumens , respectively ( table 2 ) . the feasibility of laparoscopic ureteroureterostomy has been demonstrated by multiple authors in the management of obstruction secondary to retrocaval ureters or ureteral strictures with varying techniques of stent usage . retrocaval ureteral reconstruction has been described by 2 groups both using an open - ended ureteral catheter placed preoperatively followed by division of the ureter , repositioning of the proximal and distal ureter anterior to the vena cava , and advancement of the ureteral catheter into the renal pelvis until replacement by a double - j ureteral stent after completion of the anastomosis . bhandarkar et al repaired a congenital midureteral stricture laparoscopically using a double - j stent advanced to the level of the stricture until resection and reconstruction with the stent then advanced to the renal pelvis . there is still a paucity of data in the literature regarding outcomes after laparoscopic ureteroureterostomy . nezhat et al reported follow - up of 2 months to 6 years on 9 patients who had undergone either a laparoscopic ureteroureterostomy ( 8 procedures ) or laparoscopic ureteroneocystostomy ( 2 procedures ) after iatrogenic injuries . seven patients had successful outcomes , while one patient required transvesical ureteral dilatation and another developed a recurrent ureteral stricture distal to the anastomotic site requiring laparoscopic ureteroneocystostomy . simmons et al described their series of 46 ureteral procedures for benign ureteral stricture disease including open and laparoscopic ureteroureterostomy as well as other procedures . thirty - one patients underwent 34 open procedures ( including 9 ureteroureterostomies ) , and 12 patients underwent 12 laparoscopic procedures ( including 5 ureteroureterostomies ) with 6-french , double - j stents placed in all cases . with a median follow - up of 34 months for the open group ( range , 11 to 79 ) and 23 months ( range , 4 to 70 ) for the laparoscopic group , respectively , success rates and complications were not different between the 2 groups . a review of the thus - far limited literature supports not only the feasibility of laparoscopic ureteroureterostomy but also suggests successful outcomes similar to those with standard open repair . widespread adoption of laparoscopic reconstruction by the urologic community will require that surgeons learn the complex skills required and that more experienced surgeons learn how to teach these techniques to those in training . as with all procedures , technique varies among surgeons and most notably among surgeons of different levels of experience . our goal was to determine whether an indwelling stent or ureteral catheter facilitates or hinders the performance of laparoscopic ureteroureterostomy and whether this depends on the experience of the laparoscopist . in our experimental model , the mean time required for ureteral division and spatulation was significantly less for the unstented ureter , as might be expected given that the stent seemed to interfere with cutting of the ureter while trying not to cut the stent . in regards to times for initial stitch placement , completion of the anastomosis , and total time for the stented and nonstented procedures , though , there was no statistical difference . in reviewing the performance of the novice and experienced surgeons independently , the mean time required for anastomoses was not significantly different for either one . the data did suggest a trend towards faster anastomoses with a stent , but statistical significance may have been hindered by sample size . this finding , regardless of the surgeon 's laparoscopic experience , may be due to an increased ability to identify the ureteral lumen during completion of the anastomosis ( figure 2 ) . laparoscopic ureteral division without stent ( a ) versus with stent ( b ) where navigation around stent is necessary , first ureteral anastomotic stitch without stent ( c ) versus with stent ( d ) where needle must be passed alongside stent but where back - walling is not possible , and final anastomotic suture without stent ( e ) versus with stent ( f ) where stent may aid with identification of lumen . anastomotic quality was impacted by the presence of a ureteral stent , whereby in the stented group , gaps between any of the sutures in the stented anastomoses were slightly more common overall at 8 versus 6 , but no stented anastomoses were found to have occluded lumens compared with lumens in 2 of the nonstented group . there was a trend toward tighter anastomoses when a stent was not present in the ureter , which is advantageous , but with 2 occluded anastomoses the expected outcome in a human patient would likely be reoperation or loss of the renal unit . we attribute the absence of occlusion with a stent present to an increased ability to identify the ureteral lumen and thereby minimize the risk of back - walling any of the stitches . a larger cohort of laparoscopists and more total anastomoses may have better represented the differences between performing an anastomosis with and without a stent in place . additionally , although a 50 kg porcine model is a decent representation of the human genitourinary tract , the kidneys and ureters are smaller than typically found in the adult human , and we only tested the most commonly used caliber of ureteral stent in our study . additionally , the port placement in this experiment was centered at the level of the mid to proximal ureter , which increased the level of difficulty when performing ureteroureterostomy on the distal ureter . nevertheless , we believe that the findings of at least as good of an operative time for anastomoses and lower likelihood of occlusion with a stent would hold true for a procedure on a human ureter . in this study , a ureteral stent was either placed prior to ureteral division and suturing of the anastomosis or was not placed at any point during the ureteral division and suturing of the anastomosis . alternatives to this in a true operation might include ureteral division and spatulation without a stent followed by stent placement and suturing with it in position or ureteral division with spatulation and suturing of a portion of the anastomosis without a stent followed by stent placement and completion . this last approach , which has become our method of choice ( figure 3 ) , may indeed take advantage of the ease of ureteral division with spatulation without a ureteral stent and retain the advantage of improved visualization of the ureteral lumen and minimization of the risk of back - walling . based on our findings and personal , subjective conclusions from this experiment , we would recommend having a stent in place particularly during the final stitches of the anastomosis regardless of when the stent is placed so as to prevent occlusion at this point when the lumen is difficult to identify . with the advent of robotic ureteral surgery , further study will be necessary to identify whether similar or unique benefits or limitations of stenting exist in the setting of laparoscopy with robotic instrumentation . laparoscopic ureteroureterostomy after iatrogenic injury during laparoscopic partial colectomy with stent placed after anastomosis partially completed . although more time was needed for division and spatulation of the ureter , the presence of a ureteral stent did not affect the overall time for completing laparoscopic ureteroureteral anastomoses , despite a trend towards shorter procedure times with the stent in place . additionally , there were no instances of ureteral occlusion with a ureteral stent in place during suturing . this may represent a reduced risk of back - walling the ureter during suture placement . preoperative ureteral stent placement when performing laparoscopic ureteroureterostomy may be advantageous both for the experienced laparoscopist and the novice , but further refinement and investigation in technique are necessary to optimize results in human patients . | objective : laparoscopic ureteral surgery is becoming increasingly common ; however , advanced laparoscopic skills are required due to the precise suturing involved . because of the size of the ureter and need for careful mucosal apposition to prevent stricturing , there is less room for error than with larger lumens , as in pyeloplasty .
we sought to identify whether the presence of a stent is beneficial or a hindrance in performing ureteroureterostomy both for the novice and more experienced laparoscopist.materials and methods : eight ureteroureteral anastomoses were performed on each ureter of a 50 kg female pig for a total of 16 anastomoses .
eight were performed with a stent in place , and 8 were performed without a stent . an equal number with and without a stent
were performed by a novice and an experienced laparoscopist .
anastomoses were graded by time to complete and quality of the anastomosis .
quality was graded by the presence and size of defects and patency of the lumen.results:the overall times required for ureteral division and spatulation , initial stitch placement , completion of the anastomosis , and total time for the stented vs. nonstented procedures were 4.3 vs. 2.2 minutes ( p=0.05 ) , 4.2 vs. 4.4 minutes ( p=0.16 ) , 10.4 vs. 13.5 ( p=0.22 ) minutes , and 18.3 vs. 20.1 minutes ( p=0.49 ) , respectively . for stented and nonstented ureters , 3 vs. 5 anastomoses were found to have no or very small gaps , 5 vs. 1 anastomosis were found to have large gaps , and 0 vs. 2 anastomoses were found to have occluded lumens , respectively.conclusions:for both the novice and experienced surgeon , presence of a stent did not affect the overall time to complete a ureteroureteral anastomosis despite the significantly longer time needed to divide and spatulate the ureter .
there were no occlusions when the ureteral stent was placed prior to suturing , which may indicate a reduced risk of back - walling the ureter . |
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cadherins comprise a family of calcium - dependent transmembrane cell - cell adhesion molecules , generally thought to be homophilic cell adhesion proteins . the homophilic binding of cadherins is regulated by extracellular and intracellular signals , which modulate cadherin activity without a concomitant changes in cadherin expression . protein - linked carbohydrates determine protein stability , activity and specificity of interaction , and they are also involved in cell - cell and cell - matrix recognition . disturbances in cadherin - based adhesion contribute to tumor progression in a range of epithelium tumors . yoshimura et al . have shown that the suppression of metastasis in murine melanoma b16-hm cells expressing n - acetylglucosaminyltransferase iii was at least partly due to increased level of glycosylated e - cadherin . the authors imply that glycosylation is one of the important events in the process of metastasis . despite the increasing number of studies on structure and biological function of cadherins , little it is indispensable for us to know the changes in glycosylation pattern of adhesion molecules , for example cadherins , in both normal and malignant tissues in order to promote a better understanding of the roles of these carbohydrate structures in physiological and pathological processes . thus , the aim of our study was to perform a preliminary characterization of carbohydrate structure of cadherins from human non - malignant epithelial cells of ureter ( hcv29 ) , v - raf transfected hcv29 cell line ( bc 3726 ) and human transitional cell cancers of urinary bladder ( hu456 and t24 ) using highly specific digoxigenin - labeled lectins . the results revealed some differences in glycosylation patterns of cadherins from normal and cancer cell lines . the presence of high mannose type oligosaccharides was ascertained in cadherins from bc3726 , hu456 and t24 cell lines as indicated by the positive reaction with gna . the specific reaction with pha - l indicated the existence of glcnac1,6-branched triantennary and/or tetraantennary complex type glycans on cadherins from hu456 and t24 cell lines . the dsa binding to cadherin from hcv29 cell line indicated the presence of terminal disaccharide(s ) gal1,4glcnac ( n - acetyllactosamine unit ) in biantennary complex type and/or in 2,4-branched triantennary species . moreover , positive reaction with both dsa and pha - l , observed for cadherins from bladder cancer hu456 and t24 cell lines , suggested the presence of poly - n - acetyllactosamine units on 2,6-branched triantennary and/or tetraantennary structures . the sialic acid residue(s ) occupying the terminal position in n - linked oligosaccharides of cadherins were found to be in gal2,3-linkage in hu456 and t24 cell lines ( positive reaction with maa ) , or in gal2,6-linkage in hcv29 and t24 cell lines ( positive reaction with sna ) . additionally , positive reaction with aaa confirmed the existence of fucose residue(s ) on cadherin from t24 cell line . aaa binds fuc1,6-linked to the proximal glcnac residue as well as a fuc1,2gal1,4glcnac- sequence ( blood group h(o ) determinant ) and a gal1,4(fuc1,3)glcnac- sequence ( le determinant ) . no evidence for the presence of bisected species and/or branching poly - n - acetyllactosamine species on examined cadherins from all cell lines was found ( negative reaction with wga , data not shown ) . extracts from cell lines : hcv29 , bc3726 , hu456 and t24 ( 100 g of total protein ) were run on 8% page / sds and blotted onto immobilon p membrane and probed with lectins : gna , maa , sna , pha - l , dsa and aaa . ( + ) positive reaction with lectin , ( - ) negative reaction with lectin . sugar binding specificities of lectin used for lectin blotting studies ( lectin assays ) . immunodevelopment of the western blots of protein cell extracts with anti - pan cadherin antibodies revealed a position of cadherin molecules on the blots and it allowed to estimate the molecular weight of examined glycoproteins . cadherin from non - malignant hcv29 cell line showed lower apparent molecular weight ( 130 kda ) than its cancer counterparts ( 131 kda in hu456 and 135 kda in bc3726 and t24 ) . previously , we had established that the adhesion molecules , expressed in all mentioned above cell lines , which reacted with anti - pan cadherin monoclonal antibodies were n - cadherins except the hcv29 non - malignant ureter cell line . in this cell line only trace amounts of n - cadherin were detected . moreover , neither this nor any other examined cancer cell lines expressed e - cadherin . found that differentiated human cancer cell lines , including bladder cell lines , generally expressed e - cadherin and were noninvasive in vitro , whereas dedifferentiated cell lines did not express this cell - cell adhesion molecule and were invasive . it is well - documented that , in a wide range of cancers , e - cadherin expression is loss or downregulated , resulting in a reduced level of intracellular adhesion , and perturbation in e - cadherin - mediated cell adhesion is involved in tumor progression and metastasis [ 7,11 - 15 ] . giroldi et al . showed that n - cadherin become predominantly expressed in bladder - cancer cell lines that have lost e - cadherin expression . these findings suggested that n - cadherin could play a role in bladder carcinogenesis , especially in e - cadherin - negative , poorly differentiated cells . in various cancers [ 18 - 20 ] ) , including bladder cancer reduced e - cadherin expression has been shown to correlate with progression of disease . . hypothesized that , n - cadherin expressed in t24 bladder cell , a highly invasive tumor , could play a major role in acquisition of invasive phenotype . thus , it is not surprising , that bladder cancer cell line which we examined did not express e - cadherin molecules . however , the lack of e - cadherin expression in the non - malignant hcv29 cell line was not expected since normal epithelium strongly expresses e - cadherin . it is conceivable that a lack of e - cadherin may be a phenomenon characteristic for in vitro culture of hcv29 cell line . it is well known that tumorigenesis and metastasis are frequently associated with altered structure and expression of oligosaccharides on cell surface glycoproteins and glycolipids [ 8,22 - 25 ] . therefore , we suspected that the cadherins from human cancers cell lines originated from ureter and bladder tissues , might represent , in comparison with cadherin from non - malignant hcv29 cell line different glycosylation patterns . our study confirmed this expectation and demonstrated that cadherin from non - malignant hcv29 cell line possessed bi- and/or 2,4-branched triantennary complex type glycans , some of which were 2,6-sialylated . the glycosylation pattern observed for n - cadherin from bc3726 cell line was , in comparison with cadherin from parental hcv29 cell line , dramatically different . this finding indicated that v - raf transfection of parental cells suppressed the synthesis of complex type glycans on n - cadherin molecules . the contrary , bladder cancer cells of hu456 and t24 cell lines showed ability to generate more complex glycans on cadherins than hcv29 cell line . the basic n - oligosaccharide structures recognised on cadherins from bladder cancer hu456 and t24 cell lines were high mannose type as well as 2,6-branched tri- and/or tetrantennary poly - n - acetyllactosamine complex type oligosaccharides . some of complex species were 2,3- or 2,3- and 2,6-sialylated in n - cadherins from hu456 or t24 cell lines , respectively . additionally , n - cadherin from highly invasive tumor t24 , possessed also core fucosylated and/or fuc1,2 and fuc1,3 substituted carbohydrates . it has been demonstrated that the presence of poly - n - acetyllactosamine structures appears to be essential for metastatic potential of lymphoid tumor cell line and sublines of human colon cancers . also , increased branching of n - linked glycans is a common feature of most malignant cells . in several model systems , malignant transformation , tumor cell invasiveness and metastatic potential were shown to be associated with increased levels of glcnac6man6man4-r branches of complex n - glycans . it was suggested that poly - n - acetyllactosamine chains contributed to the metastatic potential by diminishing cell - substratum adhesion and thereby facilitating tumor cell invasion . examining the glycosylation pattern of hu456 and t24 we could observe that the higher - grade classification had a cell line in which the glycosylation pattern was more changed . the donor of hu456 cells was a male patient , aged 72 , with urinary bladder cancer grade i , whereas the line t24 was obtained from a 82-year - old female patient with urinary bladder cancer grade iii . both cancer lines were enriched in high mannose type glycans , but only cadherins from t24 were core fucosylated and also possessed 2 - 6 linked glycans . thus , fucosylation and 2,6-sialylation in bladder cancer correlate with poor prognosis and patient survival . we found quite similar results for n - cadherins from human metastatic melanoma cell lines . at present we can only speculate on the physiological role that the cadherins glycans play . although it is well established that covalentely linked oligosaccharide chains can be involved in such fundamental biological process as cellular adhesion , their role in the case of individual glycoprotein is usually enigmatic . moreover , more invasive cell lines are characterized by fewer cell - to - cell junctions . it was suggested that n - cadherin mediates a less stable or more dynamic intercellular adhesion than that of e - cadherin and may make possible detachment and heterotypic interactions with surrounding cells . we postulate that altered glycosylation can repel intercellular interaction and sterically prevent cell adhesion molecules such as cadherins from achieving intermolecular distances necessary for effective interactions . human bladder cancer cell lines ( hu456 , t24 , bc3726 ) and human non - malignant ureter epithelium cell line ( hcv29 ) were kindly donated by prof . danuta du , institute of immunology and experimental therapy , pan , wrocaw , poland . equal amounts of total protein ( 100 g ) from all cell extracts were electrophoresed on 8% sds - polyacrylamide gels under reducing conditions according to laemmli . western - blotting on pvdf membranes ( millipore ) was performed according to at 250 ma for 18 h at 4c . the immunodetection of cadherins was performed with a 1/500 dilution of mouse anti - pan cadherin monoclonal antibodies ( sigma ) in 0.1% tween / tbs , 1% bsa for 18 hs at room temperature and with alkaline phosphate coupled goat anti - mouse immunoglobulin ( roche ) ( a 1/500 dilution of igg in 0.1% tween / tbs , 1% bsa ) for 1 h at room temperature . glycan chains analysis of cadherins was performed according to the procedure described by the manufacturer of the glycan differentiation kit ( roche ) . human bladder cancer cell lines ( hu456 , t24 , bc3726 ) and human non - malignant ureter epithelium cell line ( hcv29 ) were kindly donated by prof . danuta du , institute of immunology and experimental therapy , pan , wrocaw , poland . equal amounts of total protein ( 100 g ) from all cell extracts were electrophoresed on 8% sds - polyacrylamide gels under reducing conditions according to laemmli . western - blotting on pvdf membranes ( millipore ) was performed according to at 250 ma for 18 h at 4c . the immunodetection of cadherins was performed with a 1/500 dilution of mouse anti - pan cadherin monoclonal antibodies ( sigma ) in 0.1% tween / tbs , 1% bsa for 18 hs at room temperature and with alkaline phosphate coupled goat anti - mouse immunoglobulin ( roche ) ( a 1/500 dilution of igg in 0.1% tween / tbs , 1% bsa ) for 1 h at room temperature . glycan chains analysis of cadherins was performed according to the procedure described by the manufacturer of the glycan differentiation kit ( roche ) . this work was supported by the state committee for scientific research ( kbn , poland ) grant no . 6 p04a 021 20 , and the institute of zoology , jagiellonian university ( bw / iz/2001 , ds/22/iz/2002 ) . | backgroundthe aim of the present study was to determine whether stage of invasiveness of bladder cancer cell lines contributes to alterations in glycan pattern of their cadherins.resultshuman non - malignant epithelial cell of ureter hcv29 , v - raf transfected hcv29 line ( bc3726 ) and transitional cell cancers of urine bladder hu456 and t24 were grown in cell culture .
equal amounts of protein from each cell extracts were separated by sds - page electrophoresis and were blotted on an immobilon p membrane .
cadherins were immunodetected using anti - pan cadherin mab and lectin blotting assays were performed , in parallel .
n - oligosaccharides were analysed by specific reaction with galanthus nivalis agglutinin ( gna ) , sambucus nigra agglutinin ( sna ) , maackia amurensis agglutinin ( maa ) , datura stramonium agglutinin ( dsa ) , aleuria aurantia agglutinin ( aaa ) , phaseolus vulgaris agglutinin ( pha - l ) and wheat germ agglutinin ( wga ) . the cadherin from hcv29 cell line possessed bi- and/or 2,4-branched triantennary complex type glycans , some of which were 2,6-sialylated .
the cadherin from bc3726 cell line exhibited exclusively high mannose type glycans .
cadherins from hu456 and t24 cell lines expressed high mannose type glycans as well as 1,6-branched oligosaccharides with poly - n - acetyllactosamine structures and 2,3-linked sialic acid residues .
additionally , the presence of fucose and 2,6-sialic acid residues on the cadherin from t24 cell line was detected.conclusionsthese results indicate that n - glycosylation pattern of cadherin from bladder cancer cell line undergoes modification during carcinogenesis . |
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bacteria collection : campylobacter isolates were
recovered from laboratories and centers participating in the korean veterinary antimicrobial
resistance monitoring system ( kvarms ) . isolated from chicken and swine animal feces and carcasses in 2010 : 73 c.
jejuni from chicken feces ( n=43 ) and chicken carcasses ( n=30 ) , and 121 c.
coli from pig feces ( n=46 ) , pig carcasses ( n=12 ) , chicken feces ( n=38 ) and
chicken carcasses ( n=25 ) . campylobacter were isolated using bolton
broth ( thermo scientific , basingstoke , u.k . ) and campylobacter blood - free
selective agar ( thermo scientific ) and confirmed by polymerase chain reaction ( pcr ) . antimicrobial resistance : minimum inhibitory concentrations ( mics ) for
campylobacter were determined by the broth dilution method using
commercially available sensititre panel campy ( trek diagnostic systems , west
sussex , u.k . ) according to the manufacturer s instructions . briefly , the antimicrobials ,
azithromycin , ciprofloxacin , clindamycin , erythromycin , florfenicol , gentamicin , nalidixic
acid , telithromycin and tetracycline , were tested . the interpretation of mics was carried
out according to the national antimicrobial resistance monitoring system ( narms , 2011 )
. analysis of the molecular mechanisms of macrolide resistance : domain v of
the 23s rrna , l4 protein and l22 protein were
amplified by the pcr . amplified pcr products were purified , and the products were then
directly sequenced at macrogen ( seoul , korea ) . dna sequences of resistant and susceptible
strains were compared with the sequence of the c. coli jv20 genome
( genebank accession number nz _ detection of virulence genes : the presence of 12
campylobacter virulence genes , flaa , flha , cadf , doca , cdta ,
cdtb , cdtc , ciab , iam , wlan , virb11 and
ceue , in 194
campylobacter spp . was detected by pcr as in previously described work
[ 1 , 4 ] . antimicrobial resistance : the mics at which 50% and 90% of 194
campylobacter isolates were inhibited ( mic50 and
mic90 , respectively ) , and the proportions of resistant isolates for the
different antimicrobial agents are summarized in table
1table 1.antimicrobial resistance among campylobacter jejuni and
campylobacter coli isolates from chicken and swine feces and
carcassesantimicrobialsc . jejuni ( n=73)c . coli ( n=121)chicken feces ( n=43)chicken carcasses ( n=30)pig feces ( n=46)pig carcasses ( n=12)chicken feces ( n=38)chicken carcasses ( n = 25)mic50mic90r ( % ) mic50mic90r ( % ) mic50mic90r ( % ) mic50mic90r ( % ) mic50mic90r ( % ) mic50mic90r ( % ) azithromycin0.030.060 ( 0)0.0320 ( 0)0.036411 ( 23.9)0.0644 ( 33.3)0.03648 ( 21.1)0.0611 ( 4.0)ciprofloxacin83235 ( 81.4)166429 ( 96.7)163239 ( 84.8)8168 ( 66.7)83238 ( 100)163225 ( 100)clindamycin0.060.254 ( 9.3)0.13163 ( 10)0.5168 ( 17.4)0.5643 ( 25.0)0.1340 ( 0)0.2541 ( 4.0)erythromycin0.250.53 ( 7.0)0.2582 ( 6.7)0.256411 ( 23.9)0.5643 ( 25.0)0.25647 ( 18.4)1642 ( 8.0)florfenicol0.514 ( 9.3)1644 ( 13.3)122 ( 4.3)0.510 ( 0)0.510 ( 0)110 ( 0)gentamicin0.250.54 ( 9.3)0.25324 ( 13.3)0.512 ( 4.3)0.5323 ( 25.0)0.50.50 ( 0)0.5327 ( 28.0)nalidix acid646435 ( 81.4)646429 ( 96.7)646439 ( 84.8)64648 ( 66.7)646438 ( 100)646425 ( 100)telithromycin0.2510 ( 0)0.540 ( 0)0.540 ( 0)180 ( 0)0.540 ( 0)10.50 ( 0)tetracycline646434 ( 79.1)646426 ( 86.7)26433 ( 71.7)32649 ( 75.0)26416 ( 42.1)326415 ( 60.0)mic50 and mic90 , the lowest concentration to inhibit 50% and
90% of the isolates tested , respectively ; r , resistant isolates ( % ) .. no differences were identified in the frequency of the same antimicrobial
profiles between c. jejuni and c. coli or between strains
of the same species that originated either from feces or carcasses . resistance to ( fluoro )
quinolone antimicrobials was highest ( ranging from 67% to 100% ) in both c.
jejuni and c. coli from all samples except pig carcasses . resistance rates to tetracycline were the second highest , ranging from 42% to 87% among the
campylobacter isolates . erythromycin
resistance varied among bacterial species and source animals . c. coli
isolates from pig feces ( 23.9% ) and pig carcasses ( 25.0% ) showed higher macrolide resistance
than did isolates from chicken feces ( 18.4% ) and chicken carcasses ( 8.0% ) . mic50 and mic90 , the lowest concentration to inhibit 50% and
90% of the isolates tested , respectively ; r , resistant isolates ( % ) . sequence analysis
of the internal 316-bp amplicon of the 23s rrna gene revealed an a2075 g transition in all
high - level erythromycin - resistant isolates ( table
2table 2.ribosomal mutations and virulence factors in erythromycin - resistant
campylobacter jejuni and campylobacter coli
isolated from chicken and swine feces and carcassesisolatesamplemic
( g / ml)nucleotide / amino acid substitutionvirulence factoremazitel23s rrnal4l22c . jejuniv01 - 10-a03 - 008 - 016chicken feces3212-v196a - flaa , cdtb , cadf , racr , cdta , cdtcv01 - 10-a03 - 008 - 017chicken feces3212-v196a - flaa , cdtb , cadf , racr , cdta , cdtcv01 - 10-a03 - 008 - 014chicken feces3224-t91k , v176i , t177s , v196aa73 t , s109aflaa , cdtb , cadf , racr , cdta , cdtcv09-cj-02chicken carcass3224-v196a - flaa , cdtb , cadf , cdta , cdtcv06-cj-04chicken carcass 3228-v196a - flaa , cdtb , cdta , cdtcc . coli v01 - 10-a03 - 027 - 027chicken feces64328a2075gv196ai65v , a74 g , s109 t , e111a , t114aflaa , cadf , ceuev01 - 10-a03 - 027 - 029chicken feces64644a2075gv196ai65v , a74 g , s109 t , e111a , t114aflaa , cadf , ceuev01 - 10-a03 - 027 - 031chicken feces32641a2075gv196ai65v , a74 g , s109 t , e111a , t114aflaa , cadf , ceuea03 - 008 - 007chicken feces64644a2075gv196aq24r , s109aflaa v01 - 10-a03 - 027 - 026chicken feces64644a2075gv196ai65v , a74 g , s109 t , e111a , t114aflaa , cadf , ceuev01 - 10-a03 - 027 - 025chicken feces64648a2075gv196ai65v , a74 g , s109 t , e111a , t114aflaa , cadf v01 - 10-a03 - 027 - 028chicken feces64648a2075gv196ai65v , a74 g , s109 t , e111a , t114aflaa , cadf v06 - 10-s03 - 027 - 009chicken carcass3244-v196ai65v , a74 g , s109 t , e111a , t114acadf v06 - 10-s03 - 027 - 015chicken carcass64644a2075gv196a--24pig feces64328a2075gv196ai65v , a74 g , s109 t , e111a , t114aflaa , cdaf , ceuea02 - 008 - 017pig feces32640.12a2075gv196ai65v , a74 g , s109 t , e111a , t114aflaaa02 - 008 - 024pig feces64644a2075gm192i , v196ai65v , a74 g , s109 t , e111a , t114aflaa , virb1141pig feces64648a2075gm192i , v196ai65v , a74 g , s109 t , e111a , t114aflaaa02 - 008 - 006pig feces64648a2075gv121a , a140 t , m192i , v196ai65v , a74 g , s109 t , e111a , t114aa02 - 008 - 009pig feces64648a2075gv121a , a140 t , m192i , v196ai65v , a74 g , s109 t , e111a , t114aflaaa02 - 008 - 010pig feces64648a2075gv176i , t177s , v184i , m192i , v196ai65v , a74 g , a103v , s109 t , e111a , t114aflaa , cadfa02 - 008 - 018pig feces64648a2075gv196aq24r , s109av14 - 10-s02 - 028 - 001pig feces64648a2075gv121a , v176i , t177s , v184i , m192i , v196ai65v , a74 g , a103v , s109 t , e111a , t114aflaa , cadfv01 - 10-a02 - 027 - 018pig feces64648a2075gv196ai65v , a74 g , s109 t , e111a , t114aceuev11-cc-01pig carcass64328a2075gv121a , m192i , v196a - flaav02-cc-02pig carcass64648a2075gp28s , v196ai65v , a74 g , s109 t , e111a , t114aflaa , cdaf v11-cc-03pig carcass64648a2075gv121a , m192i , v196a - flaaa02 - 008 - 013pig feces32648a2075gp28s , v196ai65v , a74 g , s109 t , e111a , t114aflaaa ) abbreviations : em , erythromycin ; azi , azithromycin , tel , telithromycin . dna sequences of rpld and rplv
genes coding l4 and l22 ribosomal proteins , respectively , were compared with the
sequence in the c. coli jv20 genome . ) . no mutation was identified in this region in any of the intermediate - level
resistant isolates ( mic 32 g / ml ) . a comparison of the
amino acid sequences of the ribosomal proteins l4 and l22 in the c. jejuni
and c. coli strains with type strains revealed several different amino acid
substitutions and a combination of such substitutions . four amino acid substitutions in the
l4 ribosomal protein and two in the l22 ribosomal protein were observed in c.
jejuni with intermediate - level resistance to erythromycin ( mic 32
g / ml ) : t91k ( n=1 ) , v176i ( n=1 ) , t177s ( n=1 ) and v196a
( n=4 ) in l4 , and a73 t ( n=1 ) and s109a ( n=1 ) in l22 . in erythromycin - resistant c.
coli ( mic 32 g / ml ) , eight amino acid
substitutions in the l4 ribosomal protein and seven in the l22 ribosomal protein were
observed : v196a ( n=23 ) , m192i ( n=8 ) , v121a ( n=5 ) , p28s ( n=2 ) , v176 ( n=2 ) , t177 ( n=2 ) , v184
( n=2 ) and a140 t ( n=2 ) in l4 , and i65v ( n=18 ) , a74 g ( n=18 ) , s109 t ( n=18 ) , e111a ( n=18 ) , t114a
( n=18 ) , q24r ( n=2 ) and s109a ( n=2 ) in l22 . a ) abbreviations : em , erythromycin ; azi , azithromycin , tel , telithromycin . dna sequences of rpld and rplv
genes coding l4 and l22 ribosomal proteins , respectively , were compared with the
sequence in the c. coli jv20 genome . prevalence of virulence factors : the erythromycin - resistant
campylobacter isolates were analyzed for the presence of 12 virulence
markers that are associated with human invasion and infection . c.
jejuni isolates possessed more virulence genes than did c. coli ;
all c. jejuni isolates carried four to six virulence genes , whereas
c. coli isolates had zero to three such genes . almost all c.
jejuni and c. coli isolates possessed the flaa
gene ; however , three gene subunits , cdta , cdtb and cdtc ,
were found in 100% of c. jejuni isolates , but in none of the c.
coli isolates . furthermore , the cadf gene was more prevalent in
samples from chickens [ c. jejuni 80% ( 4/5 ) and c. coli
77.8% ( 7/9 ) ] than in samples from pigs [ c. coli 28.6% ( 4/14 ) ] , irrespective
of the bacterial species . in the present study , campylobacter isolates from animals and carcasses
that were tested against nine antimicrobials were most commonly resistant to ciprofloxacin
and nalidixic acid ( 81.496.7% for c. jejuni and 66.7100% for c.
coli ) . we noted higher resistance to ( fluoro ) quinolones in c.
coli from pigs and c. jejuni from chickens than has been
reported by european union countries ( spain 90.9% and 94.5% , respectively ; hungary 52.6% and
86.1% ; switzerland 41.1% and 40.7% ; france 46.3% and 56.9% ; and the netherlands 10.9% and
67.3% ) . although most of the c.
jejuni isolates were susceptible to erythromycin , c. coli
isolates from pigs ( 23.9% ) and pig carcasses ( 25.0% ) showed a relatively high rate of
resistance . this finding agreed with the results of other studies [ 7 , 22 ] . generally , resistance to
macrolides is more prevalent in c. coli isolates of pig origin than in
c. coli from chickens or c. jejuni from pigs or chickens
[ 7 , 22 ] . in
korea , fluoroquinolones ( enrofloxacin ) and macrolides ( tylosin ) are routinely given to
chickens and pigs to prevent and treat enteric and respiratory diseases , respectively . this practice , which is also followed by other
countries , may favor the selection of resistant bacteria [ 6 , 7 ] . in the present study , mutations in highly resistant strains were identified at position
2,075 in the 23s rrna gene in campylobacter spp . the primary mechanism of
macrolide resistance was due to a single point mutation in the 23s rrna gene , as previously
reported by researches in poland [ 2 , 3 , 18 ] and korea
. mutations in five c. jejuni
and one c. coli showing intermediate - level resistance ( mic 32
g / ml ) were not identified at position 2,075 in the 23s
rrna gene . thus , a further study on low - level resistance mechanisms , such as the cmeabc
efflux pump and mutation of other ribosomal proteins , is required . the 50s ribosomal subunit proteins l4 and l22 , encoded for by the rpld and
rplv genes , respectively , were characterized in erythromycin - resistant
isolates [ 2 , 18 ] . amino acids spanning positions 6374 are reported to be the most important
target regions of the l4 protein . mutations within
this region confer high - level macrolide resistance in various bacterial species . in the present study , four and eight amino acid
substitutions in the l4 ribosomal protein were identified in c. jejuni and
c. coli , respectively . the mutations at positions , 196 and 121 , were
reported by previous studies [ 3 , 18 ] ; however , the present study is the first to report mutations at
positions , at 28 , 91 , 140 , 192 , 176 , 177 , 184 and 841 . in the l22 ribosomal protein , we
noted two amino acid substitutions in c. jejuni and seven in c.
coli , respectively . although in the present study , erythromycin - susceptible
strains were not included for mutations , such amino acid substitutions were reported in both
susceptible and resistant isolates in other studies [ 3 , 11 , 18 ] . thus , these substitutions may have little direct involvement in erythromycin
resistance in campylobacter spp . so far , the significance of the amino acid
substitutions in the ribosomal proteins l4 and l22 remains unknown and warrants further
evaluation . the presence of virulence factor genes in erythromycin - resistant
campylobacter isolates varied by bacteria species and source . the
c. jejuni isolates carried more virulence genes than did the c.
coli isolates that we tested . although further studies on the relationship
between virulence genes in bacteria and pathogenicity in the host are needed , our results
may explain why c. jejuni is a more common cause of human infections
( 9095% ) than c. coli ( 510% ) . the most common virulence gene in both c. jejuni and c.
coli was a flagellin - coding flaa gene . motility expression via
the flagella is essential for cell adhesion and invasion to achieve infection [ 10 , 14 , 20 ] . the second most common virulence gene in this study was cadf , which is
responsible for adhensin , and the fibronectin - binding protein involved in invasion ,
influencing microfilament organization in host cells [ 1 , 20 ] . furthermore , this gene had a high
prevalence in campylobacter isolates in human cases and chicken carcasses
. in the present study , this gene in c.
coli was more prevalent in isolates from chickens than those from pigs . although
the presence or absence of key genes in campylobacter spp . can not be used
to predict the virulence of strains , further
studies on virulence genes in c. coli from different origins are needed in
order to develop effective intervention strategies to prevent transmission of resistant
strains via the food chain . we discovered a high rate of antimicrobial resistance in both c. jejuni
and c. coli , with a mutation in the 23s rrna gene mainly responsible for
erythromycin resistance in campylobacter isolates and more virulence genes
in c. jejuni than in c. coli . the effect of the amino acid
substitutions in the l4 and l22 proteins on macrolide resistance and the relationship
between the presence of virulence genes and pathogenicity require further evaluations . to
prevent the transmission to humans of resistant and virulent campylobacter
spp . via the food chain , we urge more prudent use of critically important antimicrobials ,
such as fluoroquinolones and macrolides , in swine and poultry production , as well as
constant monitoring of resistance among campylobacter isolates in animals
and animal products . | resistance to antimicrobials was measured in 73 isolates of campylobacter
jejuni ( c. jejuni ) and 121 isolates of campylobacter
coli ( c. coli ) from chicken and swine feces and carcasses in
korea .
both bacterial species showed the highest resistance to ( fluoro ) quinolones
( ciprofloxacin and nalidixic acid ) out of the nine antimicrobials tested .
erythromycin
resistance was much higher in c. coli ( 19.0% , 23/121 ) than in c.
jejuni ( 6.8% , 5/73 ) .
the mutation in the 23s rrna gene was primarily
responsible for macrolide resistance in campylobacter isolates .
several
amino acid substitutions in the l4 and l22 ribosomal proteins may play a role in the
mechanism of resistance , but the role requires further evaluation .
a total of eight
virulence genes were detected in 28 erythromycin - resistant campylobacter
isolates .
all c. jejuni isolates carried more than four such genes , while
c. coli isolates carried fewer than three such genes .
the high rate of
resistance highlights the need to employ more prudent use of critically important
antimicrobials , such as fluoroquinolones and macrolides , in swine and poultry production ,
and to more carefully monitor antimicrobial resistance in campylobacter
isolates in food animals . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
pcr performed at a miniaturized scale in l - nl volumes continue to attract the attention of the research community ; a diverse range of chip , card and array devices have been created . this article focuses on developments that have occurred in this field during the last five years . a number of recent reviews have surveyed different aspects of miniaturized pcr and pcr microchips ( 1 - 4 ) . more extensive coverage of the earlier literature on these types of analytical devices is presented elsewhere ( 5 - 7 ) . chip fabrication materials and design - the first pcr microchips were fabricated from silicon and glass ( 8) , but subsequently other types of material , especially polymers have been evaluated . this has been driven in part by issues concerning both ease of fabrication and production cost . in this context polyester has been utilized for pcr microchip fabrication by laser - printing of toner onto polyester films ( 9 ) . this technique produces microfluidic devices with channel depths on the order of tens of micrometers . deeper channels ( e.g. , 270 m ) can be produced using a co2 laser to cut microchannels in a uniform layer of printed toner coated onto polyester sheets . the final chip is constructed by sandwiching the channel layer between uncoated cover sheets of polyester in which access holes have been precut . this type of chip has been used to perform solid phase extraction of dna from blood ( > 65% recovery ) and amplify a 520 bp fragment of lambda - phage dna . the low thermal conductivity of glass or polymers used to fabricate microchips limits both heat transfer rates and the speed of thermal cycling . reducing the volume of a pcr reaction chamber can improve the speed of thermal cycling , but this strategy is not suited to analyzing samples with low analyte concentrations . faster thermal transitionscan be achieved by providing a heat sink . in the context of glass chips , a heat sink component has been shown to substantially reduce thermal resistance opposing heat dissipation into the ambient environment , and eliminates the parasitic thermal capacitance of other microchip regions that do not need to be heated ( 10 ) . on - chip components and integration - the scope of on - chip integration continues to expand and includes chips that combine solid phase extraction and pcr ( 500 nl reaction chamber ) ( 11 ) ; template purification , polymerase chain reaction ( 250 nl ) , post - pcr cleanup and inline injection , and capillary electrophoresis ( ce ) ( 12 ) ; cell lysis , dna binding , washing , elution and pcr in the same reaction chamber ( 13 ) ; dna extraction and pcr ( 14 - 16 ) ; and cell pre - concentration ( via immunomagnetic beads ) , purification , pcr ( 100 nl ) , and capillary electrophoretic analysis ( 17,18 ) . cell isolation is often required as part of an integrated pcr microchip assay and different strategies have been implemented ( 19 ) such as bead capture ( 20 ) and electrokinetic capture ( 21 ) . this has been accomplished on a chip by fabricating two interconnected chambers each containing electrodes . the first set of electrodes inside a larger chamber ( 0.6 l ) diverts bacterial cells from a flowing stream into a smaller chamber ( 0.4 nl ) that contains interdigitated electrodes that actively trap and concentrate the bacterial cells using dielectrophoresis . one strategy for microchip pcr has been to have separate zones for each of the steps in a pcr reaction ( melting , annealing , extension ) and move the reaction mixture between zones . a closed - loop ferrofluid - driven pcr chip provides an elegant solution to fluid movement in this type of multi - zone chip ( 23 ) . in this prototype , the pcr reaction mixture was contained in a circular closed microchannel in a polymethyl methacrylate chip . flow of the reaction mixture was driven by the magnetic force generated by an external magnet through a small oil - based ferrofluid plug and amplification of genetically modified soya or maize was achieved in < 13 minutes . this has been achieved in a pcr chip by means of just - in - time releasable , paraffin - passivated , dry reagents ( 24 ) . the paraffin protects the stored reagents during storage in the chip and during the sample preparation phase of the assay . in the final analytic phase heating the pcr chamber to the denaturation temperature melts the paraffin and releases the reagents that then rehydrate in the target dna solution and subsequently initiate pcr . a benefit of this strategy is that it reduces the number of analytical operations and simplifies the flow control on the chip . nanotechnology and the applications of nanofabricated objects have assumed considerable importance in many branches of science ( 25 ) . poly(quaternary ammonium)-modified gold nanoparticles have been used for efficient on - chip cell lysis prior to microchip pcr for the rapid detection of bacteria ( 3 ) . the nanoparticles remain in the pcr solution thus facilitating integration of cell lysis and pcr as both processes are accomplished in the same reaction chamber ; however , one problem was pcr inhibition caused by the gold nanoparticles which was eventually overcome by treating the pcr chamber with 1 - 10% bsa and increasing the annealing temperature . gold nanorods can also be used to lyse cells for one step dna extraction and real - time pcr of pathogens in a single chamber ( 32 ) . the longitudinal resonance of gold nanorods transforms near infrared energy into thermal energy within a chip , and the heat generated causes cell lysis . another application of nanotechnology in the context of pcr microchips has been the development of a magnetic nanoparticle - based microheater embedded in polydimethylsiloxane ( pdms ) microchips ( 29 ) . heat generated by the microheater could be controlled by the number of embedded particles and by the intensity of the applied ac magnetic field . this new type of chip with an embedded magnetic nanoparticle - based heater was shown to amplify a 732 bp target dna with good efficiency ( > 90% ) compared to a conventional pcr thermocycler . real time pcr - one target of microchip research has been the development of small portable or handheld instruments for point - of - care dna analysis that could be used for medical diagnosis or environmental monitoring . to this end an electrochemical real- time pcr has been implemented on a silicon - glass microchip for simultaneous dna amplification and detection ( 33 ) . the onset thermal cycle at which the analytical signal became distinguishable from the background , was found to be much lower for the electrochemical real - time pcr compared to a fluorescence - based counterpart ( template dna 3 x 106 copies/l ) . a two - step ultra - rapid real - time ( urrt ) pcr has been described using a commercial silicon microchip system , the genspector tmc-1000(34 ) . rapid detection of enterohemorrhagic e. coli was accomplished in a chip with a 6 l total reaction volume using 1 sec denaturation and 3 sec combined annealing / extension steps . the stx2 gene target of the enterohemorrhagic e. coli was detected in 7 minutes including melting point analysis ( detection limit 3 cfu / pcr ) ( 35 ) . reverse transcription pcr ( rt - pcr ) - rt - pcr in an integrated microdevice has also been developed for single - cell gene expression analysis . the microchip comprises integrated nanoliter metering pumps , a 200-nl rt - pcr reactor with a pad for single - cell capture , and an affinity capture matrix for the purification and concentration of products . the latter is coupled to a microfabricated capillary electrophoresis separation channel for analysis of the product . this microchip was successfully used to measure sirna knockdown of the gapdh gene in single jurkat cells ( 26 ) . point - of - care testing ( poct ) detection of human immunodeficiency virus ( hiv ) has also motivated development of microchips for rt - pcr and an associated analyzer ( 36 ) . this was achieved using a polymer lab - on - a - chip device coupled with a portable analyzer that provides non - contact infrared - based temperature control and chemiluminescence detection . analysis of hiv ( primer sets for p24 and go120 ) with this system was completed in < 1 hour . similarly , development of a low cost ( $ 1000 in component costs ) , portable and integrated microfluidic instrument has been the motivation for other microchip - based rt- pcr devices ( 37 ) . reactions were performed in a tri - layered glass - pdms microchip that consists of integrated pneumatically - actuated valves and pumps , a thin - film resistive combined heater and temperature sensor , and channels for capillary electrophoresis . the chip fits onto a platform that houses a laser diode and a charged coupled device ( ccd ) camera , circuitry for thermal control , and mini - pumps to operate the on - chip pumps and valves . one route to real - time pcr miniaturization has been to design cards that contain arrays of microwells linked to sample application reservoirs ( e.g. , taqman array card , 384 wells , applied biosystems , foster city , ca ) ( 38 ) . the fluidigm digital array ( fluidigm corp , south san franscisco , ca ) integrated fluidic circuit is another example of a device that partitions a pcr reaction mix into hundreds of individual pcr reactions in order to perform digital pcr these devices have an on - chip network of microfluidic channels , chambers , and valves that automatically assemble individual pcr reactions ranging from 2304 to 39,960 reactions per device in volumes ranging from 0.85 - 10 nl . partitioning of pcr reaction mixtures localizes individual nucleic acid molecules in separate regions so that each location will contain either zero molecules or one molecule , i.e. , a negative or positive reaction . quantification of target nucleic acid is then simply achieved by counting the positive regions ( 42 ) . droplets of emulsified pcr reaction mixtures have become a popular choice for miniaturizing pcr to perform digital pcr . for example , the biorad qx100 droplet digital pcr system ( bio - rad laboratories inc , hercules , ca ) produces 20,000 monodisperse droplets from a 20 l sample of pcr reaction mixture ( 43 ) . , lexington , ma ) produces up to 80 million partitions per run with a volume of only 5 pl per partition ( 44 ) . another approach is the beaming ( beads , emulsions , amplification , and magnetics ) technology ( inostics inc . , baltimore , md ) ( http://www.inostics.com ) ( 45,46 ) . this performs single - molecule pcrs on magnetic beads in an emulsion droplet ( average diameter of emulsion compartment 5 m ) . miniaturization of pcr reactions , especially in emulsion droplets , has progressed rapidly in order to reap the benefits of digital pcr and several commercial systems are available . however , commercial development of pcr lab - on - a - chip devices has not kept pace with research and development activities in this area and the full potential of lab - on- a - chip devices for pcr - based analysis remains in the future . | microminiaturization of assays and lab - on - a - chip devices hold considerable promise for the future of analysis , especially in point - of - care testing .
this article focuses on developments that have occurred during the last five years in the specific area of microchip pcr and miniaturized pcr in arrays of reaction vessels and droplets .
although , this area continues to be an active focus of research and development and the variety and ingenuity of microchip pcr and integrated microchip pcr devices continue to increase , commercialization lags behind the progress being made in digital pcr and arrays for real - time pcr . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
a spinal extradural arachnoid cyst ( seac ) , which is mostly found at the thoracic spine , is a rare disease , accounting for approximately 1% to 3% of spinal tumors.123456789 ) seac patients are generally men in their 20s,3 ) 30 s , or 50s.6 ) seacs are most commonly found in the middle or lower thoracic spine , and less frequently found in the lumbar region , although they can be detected at any lesion of the spine.1568 ) a seac results from a small defect in the dura matter that induces cerebrospinal fluid ( csf ) accumulation and arachnoid membrane herniation.12569 ) the cause of this herniation , which is still under debate , may be either congenital or acquired.13456789 ) the neurological symptoms of a seac , including myelopathy , can be triggered by the mass effect of cord or root compression.1234567 ) a seac can be enlarged during exercise or other actions because there is small defect between the cysts and subarachnoid space that allows increased cranial pressure to stimulate the flow of csf from the intradural space to the cyst.13 ) a seac is a meningeal cyst that can be classified into three major categories . type i cysts are composed of extradural arachnoid cysts ( eac ) without nerve root fibers , type ii cysts are composed of eac with nerve root fibers , and type iii cysts are composed of intradural meningeal cyst . type 1 meningeal cysts can be subcategorized as eac ( type ia ) and sacral meningoceles ( type ib).256 ) a seac can be caused by herniation of the dura matter , which can be the result of a congenital anomaly , trauma , infection , or inflammation . however , the causes of such herniation remain unclear.158 ) to treat a seac , diverse surgical techniques have been introduced , although there is no consensus regarding the most reasonable treatment.125 ) most investigators agree that the dural defect must be repaired , but total removal of a seac remains controversial.25 ) here we report our experience with 2 seac cases and review the relevant current literature . a 72-year - old woman presented with low back pain for 10 years and bilateral leg radiating pain for 4 months . she was first diagnosed with a seac 10 years ago at the local medical center and treated with medication . we performed a radiological examination of the patient , including myelography , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) . preoperative myelography showed multi - level spinal canal stenosis and distortion from t12 to l5 on the spinal level . no connection site was found between the cyst and the thecal sac ( figure 1a ) . a spinal mri revealed an elongated well - defined mass - like lesion with a high t2 signal and a low t1 signal at t12 to l4 on the spinal level causing spinal stenosis ( figure 1b and c ) . we suspected that the dural defect was at l1 on the vertebral level because the presence of a csf flow artifact . we extended the incision line to the t11 level , and a t12 l1 hemi - laminectomy was performed . eventually , we found a dural defect on the right side at the l1 vertebral level . we removed both the l1 spinous process and the l1 lamina to expose the defect site ( figure 2a ) . we performed a cyst penetration and a primary dural repair with the cyst wall ( figure 2b ) . the postoperative histologic findings were compatible with the presence of an arachnoid cyst with dystrophic calcification . the patient s symptoms gradually subsided , and follow - up mri scans taken 4 months after the surgery showed a complete disappearance of the cyst with no evidence of cord compression due to a residual cyst ( figure 3 ) . a 33-year - old woman presented with low back pain and right leg radiating pain for 7 months . she has no motor weakness and no pathological reflexes , but there was generalized paresthesia of the right leg . the local hospital transferred the patient to our care for surgical treatment of the seac . to prepare the operation , we performed an additional radiological examination , including a thoracolumbar spine simple x - ray and ct thoracic myelography scan . myelography revealed an arachnoid cyst at the posterior epidural space of t12 to l2 , and indicated that communication of the cyst and the thecal sac occurred at the l1 level ( figure 4a ) . an mri revealed an elongated well - defined mass - like lesion with a high t2 signal and a low t1 signal at t12 to l2 on the spinal level indicating thecal sac compression ( figure 4b and c ) . first , we opened the t12 lamina site and performed a left partial hemi - laminectomy . the cyst wall was identified , and we identified a dural defect at the left l1 nerve root sleeve . we performed a cyst penetration and a primary dural repair with the cyst wall fragment ( figure 5 ) . postoperative histologic findings were compatible the presence of an arachnoid cyst with fibrous cyst wall - like tissue . radiating leg pain and back pain in the patient were gradually improved , and a follow - up mri taken 2 months after the surgery showed a complete disappearance of the cyst with no evidence of cord compression due to a residual cyst ( figure 6 ) . a 72-year - old woman presented with low back pain for 10 years and bilateral leg radiating pain for 4 months . she was first diagnosed with a seac 10 years ago at the local medical center and treated with medication . we performed a radiological examination of the patient , including myelography , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) . preoperative myelography showed multi - level spinal canal stenosis and distortion from t12 to l5 on the spinal level . no connection site was found between the cyst and the thecal sac ( figure 1a ) . a spinal mri revealed an elongated well - defined mass - like lesion with a high t2 signal and a low t1 signal at t12 to l4 on the spinal level causing spinal stenosis ( figure 1b and c ) . we suspected that the dural defect was at l1 on the vertebral level because the presence of a csf flow artifact . we extended the incision line to the t11 level , and a t12 l1 hemi - laminectomy was performed . eventually , we found a dural defect on the right side at the l1 vertebral level . we removed both the l1 spinous process and the l1 lamina to expose the defect site ( figure 2a ) . we performed a cyst penetration and a primary dural repair with the cyst wall ( figure 2b ) . the postoperative histologic findings were compatible with the presence of an arachnoid cyst with dystrophic calcification . the patient s symptoms gradually subsided , and follow - up mri scans taken 4 months after the surgery showed a complete disappearance of the cyst with no evidence of cord compression due to a residual cyst ( figure 3 ) . a 33-year - old woman presented with low back pain and right leg radiating pain for 7 months . she has no motor weakness and no pathological reflexes , but there was generalized paresthesia of the right leg . the local hospital transferred the patient to our care for surgical treatment of the seac . to prepare the operation , we performed an additional radiological examination , including a thoracolumbar spine simple x - ray and ct thoracic myelography scan . myelography revealed an arachnoid cyst at the posterior epidural space of t12 to l2 , and indicated that communication of the cyst and the thecal sac occurred at the l1 level ( figure 4a ) . an mri revealed an elongated well - defined mass - like lesion with a high t2 signal and a low t1 signal at t12 to l2 on the spinal level indicating thecal sac compression ( figure 4b and c ) . first , we opened the t12 lamina site and performed a left partial hemi - laminectomy . the cyst wall was identified , and we identified a dural defect at the left l1 nerve root sleeve . we performed a cyst penetration and a primary dural repair with the cyst wall fragment ( figure 5 ) . postoperative histologic findings were compatible the presence of an arachnoid cyst with fibrous cyst wall - like tissue . radiating leg pain and back pain in the patient were gradually improved , and a follow - up mri taken 2 months after the surgery showed a complete disappearance of the cyst with no evidence of cord compression due to a residual cyst ( figure 6 ) . a seac is a rare spinal tumor that does not expand within the spinal canal.123456789 ) a seac may develop as a cystic formation that is caused by a dural defect , and may be enlarged by the flow of csf that originates from the intradural arachnoid space.16 ) a seac may grow extraspinally or intraspinally , and is also typically located on the posterolateral portion of the spinal canal , compressing the spinal cord toward the anterior side.1689 ) as enlargement continues , a seac can aggravate spinal cord compression or nerve root compression , which leads to patient symptoms such as pain or weakness.1689 ) although many have studied the etiology of theses cysts , such mechanisms remain uncertain.123456789 ) for nearly all seac cases , communication between the subarachnoid spaces and cysts have been reported.12358 ) this communication can be congenital or acquired . a small dural tear that is induced by a congenital anomaly , trauma , arachnoiditis , or iatrogenic events allows for csf flow to initiate herniation of the arachnoid membrane . the dural defect is usually found near the nerve root sleeve.126 ) the most reliable theory of etiology of such tears suggests that stretching force between the movable thecal sac and the relatively fixed roots are a major factor.1 ) the possibility of a dural tear increases if the patients has underlying structural problems ( e.g. , marfan syndrome or dural ectasia ) or thecal sac movements ( trauma or csf leakage).167 ) our current understanding is that this small dural tear appears to act as a ball - valve , and active csf secretion from the residual arachnoid membrane plays a role in cyst expansion.1 ) this cyst expansion can cause compressive myelopathy in our cases , neither patient had a history of trauma or arachnoiditis , or a previous spine surgery . the cysts observed in both cases showed a single small dural tear at the l1 right root level , and a trapped rootlet was found near the dural defect . we suggest that the csf flow via the dural defect and the rootlet trapped in the defect likely acts as a one - way - valve mechanism , which has been described in several studies . the cysts in our cases are similar to a type 2 meningeal cyst that was observed in another study . histological investigation showed that the cyst wall in our cases only contains calcified tissue and layered collagenous fibers , but not the glands or secreting tissues since the clinical symptoms of seacs are similar to those for other degenerative spine diseases , seacs are easy to misdiagnose . an mri is the most useful tool to diagnose a seac.6 ) radiological studies report that a seac appears with a low signal intensity on a t1-weighted image ( wi ) and with a high signal intensity on a t2-wi , similar to cerebrospinal fluid.1268 ) a ct myelography is also a useful diagnostic tool because it can more reliably detect the anatomical location of the cyst , and measures the severity of compression of the spinal cord and nerve roots.1 ) in addition , some reports indicate that a myelogram and ct myelography can help locate the dural defective site between the spinal subarachnoid space and the cyst cavity.19 ) mri methods that detect csf flow can help to localize a defective site . a study reported that csf flow mri can identify the pulsating turbulent flow void of a defective site.1 ) in our cases , preoperative ct myelography could not help to detect the dural defect site . however , we could predict where the location of the dural defect before surgery using a csf flow mri , and we could identify the exact location during surgery . the standard treatment for seacs remains uncertain.1235689 ) some recommend non - surgical care for the treatment of seacs and have reported good outcomes with this approach.16 ) however , if the patient has a neurologic symptom that is caused by nerve compression due to a growing seac , then a surgical procedure would be needed.25 ) there is a consensus among investigators to repair the dural defect in the treatment of a seac.12369 ) however , there is still disagreement regarding the treatment of the cyst . some insist that the complete removal of the cyst is necessary to prevent cyst recurrence.5 ) other insist that a seac can be removed with only fenestration of the cystic wall.1 ) yet another investigator implanted a shunt or aspirated the cyst.5 ) traditionally , a complete microsurgical resection of a seac with multilevel laminectomy and repair of dural defect has been promoted as the standard treatment of choice for a seac.1 ) however , if the cyst is large , an extensive laminectomy would be necessary to remove the entire cyst wall for neural decompression . however , such a procedure can cause postoperative complications such as bleeding and postoperative instability.1235689 ) selective laminectomy with closure of the dural defect site alone has been reported as effective for treating an seac.16 ) in our cases , we performed selective laminectomy with interlaminar fenestration at the transdural communication site that was observed as a pulsating flow , voiding on a preoperative mri . with the repair of the dural defect site in our cases , the regression of the cyst was confirmed in the postoperative radiological finding , and the clinical symptoms were significantly improved . our procedure of selective laminectomy with interlaminar fenestration has the advantage of being a limited laminectomy , which can prevent complications related to wide surgical operation . because of the technical precision of current radiological diagnostic devices , we could accurately localize the communication site , and minimize the extent of laminectomy and risk of complication . our approach of focal laminectomy for cyst fenestration and repair of the dural defect produced good clinical and radiological outcomes . a seac , which is mostly found at the thoracic spine , is a rare disease . a seac can cause spinal cord or nerve root compression and may present with myelopathy . a seac is caused by the herniation of the arachnoid membrane , which is due to a dural defect that can be generated by congenital anomaly , trauma , infection , or inflammation . diverse surgical techniques have been introduced . however , there is no consensus regarding the most reasonable treatment . most investigators agree that the dural defect must be repaired , but total removal of the seac is controversial . we penetrated the cyst and performed a primary repair of the dural defect for two seac cases . compared to the classical complete resection of the cyst wall with multilevel laminectomy , our approach of fenestration to treat a seac poses fewer complications . | a spinal extradural arachnoid cyst ( seac ) results from a rare small defect of the dura matter that leads to cerebrospinal fluid accumulation and communication defects between the cyst and the subarachnoid space .
there is consensus for the treatment of the dural defect , but not for the treatment of the cyst .
some advocate a total resection of the cysts and repair of the communication site to prevent the recurrence of a seac , while others recommended more conservative therapy . here
we report the outcomes of selective laminectomy and closure of the dural defect for a 72-year - old and a 33-year - old woman .
magnetic resonance imaging of these patients showed an extradural cyst from t12 to l4 and an arachnoid cyst at the posterior epidural space of t12 to l2 .
for both patients , we surgically fenestrated the cyst and repaired the dural defect using a partial hemi - laminectomy .
the patient s symptoms dramatically subsided , and follow - up radiological images show a complete disappearance of the cyst in both patients .
our results suggest that fenestration of the cyst can be a safe and effective approach in treating seacs compared to a classical complete resection of the cyst wall with multilevel laminectomy . |
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use of this method is associated with fewer complications such as aneurysms , acute ischemia in the spinal cord , and recoarctation compared to surgery and/or balloon dilatation . however , recoarctation has been reported as its major complication.1 in previous studies , attempts were made to define clinical indications for using this type of treatment and to evaluate the efficacy and outcome of stent implantation in different groups of patients . yet , first , currently used indications for stenting have been mostly made based on a group of patients with similar demographic conditions such as age , weight , and comorbidities rather than each patient`s status individually.2 , 3 second , most of the tools drawn upon in the follow - up of these patients are mainly based on evaluating anatomical changes after stent implantation rather than focusing on the hemodynamic status of the stent - inserted aorta . hypertension and decreased blood pressure in the lower extremities are assumed as hemodynamic changes which are common findings in patients with coarctation.4 both factors are associated with a decreased pulsatility of the abdominal aorta . pulsatility of the blood flow in the aorta causes continuous blood flow to the end organs and is a significant factor which is believed to decrease in coarctation . the pressure gradient across the aortic coarctation site results in an altered flow pattern in the abdominal aorta5 , 6 with loss of pulsatility . as a result of decreased pulsatility of the aorta , the flow becomes continuous throughout diastole.5 this phenomenon can be assessed via doppler echocardiography using the pulsatility index ( pi ) [ ( vmax - vmin)/vmean ] ( where vmax , vmin and vmean represent maximum , minimum , and mean velocities , respectively ) . correction of this factor could be an important surrogate of coarctation repairing . there is a dearth of data on the trend of changes in the pi after stent implantation . also , the association between the pi and other echocardiographic indices in patients undergoing stent implantation is unclear . recognition of these issues can be useful to predict early and late hemodynamic outcomes in the aorta before stent placement . this study was designed to evaluate changes in the pi after stent implantation and its correlation with other echocardiographic parameters . also , the feasibility of some baseline characteristics of patients in predicting the trend of changes in the pi after stent implantation was investigated . twenty - three patients with a diagnosis of congenital aortic coarctation were consecutively enrolled in this prospective study . the patients were referred to rajaei cardiovascular , medical and research center ( affiliated to tehran university of medical sciences ) , tehran , iran between april 2008 and august 2009 . all the patients had type iii ( postductal ) congenital coarctation in the abdominal aorta . the exclusion criteria consisted of any other concomitant lesions , including aortic stenosis or regurgitation , patent ductus arteriosus , anomalies of the head and neck vessels , and long segment aortic coarctation or hypoplastic arch . stenting of the abdominal aortic coarctation was performed for all the patients enrolled in the study . in addition to baseline and demographic variables ( e.g. age , gender , blood pressure , and length of stenosis ) , the characteristics of stenting , including length and width of the stent , length and width of the balloon , and first and second gradients of the cash , were recorded for all the patients . informed written consent was obtained from all the patients , and the research project was approved by the ethics committee of tehran university of medical sciences . echocardiographic evaluation ( both abdominal and descending aortas ) was done twice for all the patients , before stenting and 24 hours after it via two - dimensional and doppler echocardiographic imaging modalities using a vivid 3 imaging system ( ge , usa ) , in accordance with the institutional guidelines ( figure 1 ) . for this purpose , standard suprasternal position was considered to measure the maximum velocity across the coarctation site and then continuous wave doppler recordings were obtained . pulsed - wave doppler from the standard subcostal view was also performed to document the flow pattern of the abdominal aorta . all the echocardiographic studies were performed by a single echo - cardiologist before and after stenting . moreover , a simultaneous electrocardiographic monitoring was also utilized . the onset of diastole was assumed at the end of the electrocardiographic t - wave , and the three measurements were taken from three consecutive cycles each time . thereafter , the mean of the three measurements was calculated and reported as the main records . the continuous values are expressed as mean sd , and frequency percentages are used to describe the categorical variables . the kolmogorov - smirnov test was employed to assess the normality of the distribution of the numerical variables , and the mann - whitney u - test was performed to compare the significance of the difference in the baseline doppler echocardiographic profile between the two groups of the patients regarding increase - percentage of the pi of < 50% or 50% ( 50 was the mean percentage of pi increase in these patients after stenting ) . the receiver operating characteristic curve ( roc ) analysis was performed to assess the predictability of 50% increase in the pi after stenting with quantitative indices of the study , and thereafter to compare the area under curve ( auc ) of these variables . the best predictive cut - off value was that which gave the highest product of sensitivity and specificity . diagnostic values of each cut - off point , including sensitivity and specificity , were calculated and reported . all the p values were two - tailed and a p value < 0.05 was considered statistically significant . spss v.17 software ( chicago , il , usa ) was used for the analytic procedures . twenty - three patients , comprised of 16 ( 69.6% ) males and 7 ( 30.4% ) females , who had coarctation stenting were recruited in this study . the mean age of the patients was 26.14 ( sd = 10.17 ) years and the mean duration of the disease was 36.50 ( sd = 69.02 ) months . all these 23 patients underwent coarctation stenting with the mean stent size of 37.16 ( sd = 3.67 ) mm 8.47 ( sd = 1.94 ) mm . the pi was increased from 0.89 ( sd = 0.30 ) to 1.75 ( sd = 0.51 ) , which showed improvement of 119.57% ( p value < 0.001 ) . based on the percentage of increase in the pi ( < 50% or 50% ) , the patients were divided into two groups . as is shown in table 2 , the baseline diastolic / systolic velocity ( d / s velocity ) ratio of the abdominal aorta was significantly higher in the patients with 50% increase in the pi after stenting [ 0.77 ( sd = 0.25 ) vs. 0.56 ( sd = 0.09 ) , p value = 0.013 ] . additionally , the mean velocity of the descending aorta before stenting was significantly higher in the patients with aortic coarctation who had 50% increase in the post - stenting pi [ 1.97 ( sd = 0.36 ) vs. 1.57 ( sd = 0.56 ) , p value = 0.033 ] . the other baseline echocardiographic index which was significantly different between these two groups was the mean peak gradient of the descending aorta . as is shown in table 2 , the mean peak gradient ( pg ) of the descending aorta at baseline was significantly higher in the patients with 50% increase in the pi after stenting [ 21.77 ( sd = 6.43 ) vs. 14.57 ( sd = 9.85 ) , p value = 0.033 ] . more analysis was conducted to evaluate the diagnostic values of the different baseline characteristics of the patients to predict 50% increase in the pi after stenting . the significant baseline indices to differentiate the percentage of increase in the pi as < 50% or 50% are listed in table 3 . the baseline d / s ratio velocity of the abdominal aorta , mean velocity and peak gradient of the descending aorta , and baseline systolic blood pressure were the statistically significant indices to predict 50% increase in the pi in the patients with aortic coarctation ( all p values < 0.05 ) . as is illustrated in figure 2 , the baseline d / s ratio velocity of the abdominal aorta had the greatest area under curve ( auc = 0.865 , p value = 0.010 ) to predict 50% increase in the post - stenting pi . the cut - off point of 0.67 for this index had 75% sensitivity and 100% specificity . the pi was increased from 0.89 ( sd = 0.30 ) to 1.75 ( sd = 0.51 ) , which showed improvement of 119.57% ( p value < 0.001 ) . based on the percentage of increase in the pi ( < 50% or 50% ) , the patients were divided into two groups . as is shown in table 2 , the baseline diastolic / systolic velocity ( d / s velocity ) ratio of the abdominal aorta was significantly higher in the patients with 50% increase in the pi after stenting [ 0.77 ( sd = 0.25 ) vs. 0.56 ( sd = 0.09 ) , p value = 0.013 ] . additionally , the mean velocity of the descending aorta before stenting was significantly higher in the patients with aortic coarctation who had 50% increase in the post - stenting pi [ 1.97 ( sd = 0.36 ) vs. 1.57 ( sd = 0.56 ) , p value = 0.033 ] . the other baseline echocardiographic index which was significantly different between these two groups was the mean peak gradient of the descending aorta . as is shown in table 2 , the mean peak gradient ( pg ) of the descending aorta at baseline was significantly higher in the patients with 50% increase in the pi after stenting [ 21.77 ( sd = 6.43 ) vs. 14.57 ( sd = 9.85 ) , p value = 0.033 ] . more analysis was conducted to evaluate the diagnostic values of the different baseline characteristics of the patients to predict 50% increase in the pi after stenting . the significant baseline indices to differentiate the percentage of increase in the pi as < 50% or 50% are listed in table 3 . the baseline d / s ratio velocity of the abdominal aorta , mean velocity and peak gradient of the descending aorta , and baseline systolic blood pressure were the statistically significant indices to predict 50% increase in the pi in the patients with aortic coarctation ( all p values < 0.05 ) . as is illustrated in figure 2 , the baseline d / s ratio velocity of the abdominal aorta had the greatest area under curve ( auc = 0.865 , p value = 0.010 ) to predict 50% increase in the post - stenting pi . the cut - off point of 0.67 for this index had 75% sensitivity and 100% specificity . previous studies have emphasized the need for creating an available , accurate , and reliable profile which can assist in the follow - up of patients who undergo stent implantation for the treatment of aortic coarctation . to date , several non - invasive and invasive methods have been used for this purpose . for example , although magnetic resonance imaging was first introduced as a reliable modality for assessing patients after stent insertion , later it was shown that it has limitations in demonstrating the stented portion of the aorta due to artificial noising produced by the metallic stents.7 , 8 the other mostly used modality in the follow - up of these patients is angiography , which has its known intra- and postoperative complications . by advancing echocardiography and its newly emerged branches , including doppler echocardiography , some studies have suggested this non - invasive and available modality for assessing the feasibility of stent implantation in patients with aortic coarctation.911 this non - invasive modality evaluates the hemodynamic changes of the aorta rather than the anatomical changes evaluated by angiography . there are a large number of echocardiography indices which have been proven to change after the treatment of coarctation , but few of them have been found to be closely related to the clinically important hemodynamic status of the aorta . studies on the hepatic , renal , human placental , carotid , mesenteric , and other vascular circulations have suggested that assessment of these arterial circulations can be made by evaluating the pi of the corresponding arteries.1218 thus , in the present study , the pi of the abdominal aorta was considered as a marker of the aortic hemodynamic status . evaluation of this index after stenting and comparing it with baseline values can determine the efficacy of stent insertion . also as a simple , available , and non - invasive method , it could potentially be used as a clinically useful tool in the long - term follow - up of patients . based on our results , impaired pulsatile systolic abdominal aortic flow is associated with impaired continuous diastolic flow in patients with significant aortic coarctation . moreover , we found that increase in the pi following stent implantation is statistically significant . as a result , the pi of the abdominal aorta was investigated as an echocardiographic index in the early follow - up of these patients . most of these studies have defined these indications based on the complications seen in the same age groups of their study , such as femoral access problems and re - stenosis due to intimal growth , seen mostly in younger patients . coarctation of the aorta is a congenital disease that has complex hemodynamic effects on each patient . thus , it seems unreasonable to consider the same indications of stenting for all patients . in other words , none of the previous studies could introduce an assessment tool which can reliably predict the efficacy and outcome of stent implantation in each patient based on his / her own baseline hemodynamic status . in the present study , we succeeded in defining some doppler echocardiographic indices which are able to predict the effects of stenting on the hemodynamic status of the aorta . four baseline ( before stenting ) doppler echocardiographic indices were found to predict the increase in the pi . after sorting the patients based on the percentage of increase in their pi after stent implantation , i.e. more or less than 50% , these findings were reported : d / s ratio velocity of the abdominal aorta before treatment could be considered as a helpful index to predict the pi elevation after stenting . on the other hand , the d / s ratio before stenting was significantly different between the patients with post - pi of lower and higher than 50% . the mean velocity of the descending aorta is the other predicting index in the evaluation of stent implantation outcome based on the degree of increase in the pi . its difference between the two groups of the pi was also statistically significant at baseline . mean pg of the descending aorta was an index found to be a significant predictor of the pi and it varied significantly between the patients with various indices of pulsatility . systolic blood pressure was also a helpful predictor of the pi ; however , its change based on the pi was not assessed early after treatment because of the effects of postoperative stress on the blood pressure . as is shown by the present study , when patients are sorted into two groups of more or less than 50% increase in the pi after stent implantation , some important echocardiographic indices differ significantly between these two groups . as a result , the pi of 50% could be a reasonable cut - off point to compare the efficacy of this intervention . in this study , the results of stent implantation were found to be predictable and the predictors were suggested to be considered as indications for this type of intervention in the treatment of coarctation . further studies are required to evaluate the effect of the pi in the late follow - up of hemodynamic indices . furthermore , studies comparing the degree of pi elevation after different methods of treatment ( surgery , balloon dilatation , and stenting ) can be helpful in decision - making for this population of patients . | background : the pulsatility index ( pi ) shows continuous blood flow to the end organs and is a significant factor believed to decrease in aortic coarctation . correction of this factor is of great importance in the treatment of stenotic lesions of the aorta . however , there are minimal data regarding the trend of changes in the pi after stent implantation .
furthermore , the association between the pi and other echocardiographic indices in patients undergoing stent implantation is unclear .
this study was designed to evaluate changes in the pi following stenting and its correlation with other echocardiographic indices.methods:twenty-three patients with a diagnosis of aortic coarctation consecutively underwent two - dimensional and doppler echocardiographic imaging modalities twice ( before and after stenting ) .
the patients were divided into two groups based on the percentage of increase in the pi after stenting ( < 50% or 50% ) .
the relation between the post - stenting pi and the baseline echocardiographic indices was assessed.results:the pi was increased from 0.89 ( sd = 0.30 ) to 1.75 ( sd = 0.51 ) after stenting ( p value < 0.001 ) .
baseline diastolic / systolic velocity ( d / s velocity ) ratio of the abdominal aorta ( p value = 0.013 ) , mean velocity ( p value = 0.033 ) , and peak gradient of the descending aorta ( p value = 0.033 ) were significantly higher in the patients with 50% increase in the pi after stenting.conclusion:our findings showed that elevation in the pi after stenting was a predictable criterion in patients with aortic coarctation : it was predicted by some baseline clinical and echocardiographic indices . baseline d / s ratio velocity of the abdominal aorta , mean velocity and peak gradient of the descending aorta , and baseline systolic blood pressure were the statistically significant indices to predict 50% increase in the pi in our patients . |
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the patient was a 13-day - old male infant 1,350 g at birth , who was born by cesarean delivery . pulmonary surfactant was administered via an endotracheal tube twice because of respiratory distress syndrome and the newborn was provided with mechanical ventilation for 35 hours . the patient received oxygen by hood until the sixth day of life , and received 2.5 mg injections of aminophylline every 12 hours because of apnea diagnosed on the eighth day of life . gavage feeding was discontinued and bile excretion was noted . before the surgery , the vital signs were stable . there were no abnormalities on the ecg or chest x - ray . on the day of surgery , standard monitoring ( electrocardiogram , noninvasive blood pressure and peripheral pulse oxygen saturation ) was performed . injection of 500 ml of 10% dextrose water + nacl 15 meq + heparin 500 iu 8 ml / hr was carried out via the internal jugular vein using a 24 gauge catheter and was maintained at 10 - 0 ml / hr during the surgery . an 8 vol% of sevoflurane was administered and then maintained with a 3 vol% of sevoflurane . fresh gas flow was maintained with o2 1 l / min and air 4 l / min . injection of 1 mg of rocuronium was administered because there was no difficulty with mask ventilation . two minutes later , intubation with a 2.0 mm i d ( internal diameter ) endotracheal tube was attempted . the tube was checked and intubation attempted again with the same endotracheal tube , two minutes later . the tube was fixed at 7 cm the tube was cut from 12 cm to reduce the dead space . after cutting the tube , there was no change in the depth ; ventilation could not be provided and there were no breath sounds . the endotracheal tube was removed and mask ventilation was provided again . the tube was fixed at 7 cm and cut from 12 cm to reduce the dead space . after cutting the tube , one - lung ventilation was suspected , so manual ventilation was attempted , changing the depth of the endotracheal tube . after the intubation , ventilation was successful and breath sounds were detected bilaterally , in addition , to regular capnography of the etco2 . the tube was fixed at 7 cm and cut from 12 cm to reduce the dead space . however , this time the ventilation was successful . injections with 0.05 mg of pyridostigmine and 0.01 mg of glycopyrrolate were provided and spontaneous ventilation occurred . after surgery , the duration for anesthesia was 2 hr 30 min and the operation was 2 hr . there was no specific change in the bp or hr . two hours after surgery , the endotracheal tube was removed because the respirations were stable . however , rigid bronchoscopy was not performed for confirmation , because there were no specific symptoms at that time . cough , inspiratory stridor , and recurrent pneumonia are symptoms associated with tracheomalacia ; the most severe symptom is apnea . in most cases , symptoms improve spontaneously and patients require conservative care . however , if severe apnea develops , recurrent pneumonia , or extubation failure , surgery is needed . tracheomalacia is frequently associated with other congenital anomalies such as an esophageal stricture or tracheoesophageal fistula . it is also associated with tumors that can press on the trachea , connective tissue disorders and long - term high pressure ventilator care . common symptoms associated with a tracheoesophageal fistula include dyspnea during feeding , cyanosis , and aspiration pneumonia caused by regurgitation of food or saliva ; this can be diagnosed by failure of attempts to insert a nasogastric tube . the diagnosis is usually made immediately after birth or within 15 days of life . the h - type of tracheoesophageal fistula , which is not associated with an esophageal stricture , may have no specific symptoms until adulthood . however , in this case , a tracheoesophageal fistula was ruled out because the patient had gavage tube feeding and there were no specific signs on the gastrograffin test . ventilation was not successful with a 2.0 mm i d tube or a cut 2.5 mm i d tube in this case . the relationship between the flow , the tube and gas in a straight tube is explained by the hagen - poiseuille equation . the hagen - poiseuille equation is as follows : v = pr4/8ul ( v = flow and = 3.1416 , p = pressures , r = radius of tube , l = length of tube , u = viscosity of gas ) . a tube with a smaller radius has greater flow ; the flow was greatest for the 2.0 mm i d tube and slowest with the 3.0 mm i d tube . according to the bernoulli effects , pressure decreases as the flow increases , at narrow points . turbulent flow occurs at the end of the tube where the radius of the tube changes . the relationship between the flow and the pressure is as follows : v p . it is assumed that the obstruction of the airway was caused by the pressure outside of the tube . flow increases with the 2.0 mm i d tube more than with 3.0 mm i d tube . the faster flow passes to the end of the tube and the pressure inside of the tube decreases when the faster flow passes the narrow point caused by the tracheomalacia . at that moment , obstruction of the airway occurs due to pressure outside of the tube . in this case , ventilation was not successful immediately after the tube was cut to reduce the dead space when a 2.5 mm i d tube was used . ventilation failure after cutting the tube was caused by obstruction of the airway due to a decrease in the pressure . the decrease in pressure was caused by shortening of the length of the tube which increases the flow rate ; then , faster flow passes through the narrow segment . reported that with a 4.0 mm i d tube , there is a 22% decrease in the resistance when the length of tube was changed to 11.3 cm from 20.7 cm . the decrease of the pressure by 22% was associated with shortening of the length of the endotracheal tube . for endotracheal tubes smaller than the 4.0 mm i d tube , the decrease in the radius is associated with a rapid increase in the pressure . an increase in the gas flow from 5 l / min to 10 l / min causes an increase of pressure from 81.2 h2o / l / sec to 139.4 h2o / l / sec in a 2.5 mm i d tube while the pressure changes from 3.1 h2o / l / sec to 4.6 h2o / l / sec in a 6.0 mm i d tube . in this case , the 2.5 mm i d tube length was changed from 18 cm to 12 cm and the 3.0 mm i d tube length was changed from 19 cm to 12 cm . the flow in the tube was not laminar flow but turbulent flow , which is not directly proportional to the pressure or length . the decrease in pressure was 18% and 19% , each , according to the relationship between the length of laminar flow and the pressure . disappearance of the parabola form , with turbulent flow , can lead to an increase in the pressure . it is assumed that the decrease of pressure is lower with turbulent flow than with laminar flow . during manual ventilation , 5 l / min was used ; however , the fresh gas flow rate was briefly more than 10 l / min . this patient received mechanical ventilator care and oxygen hood treatment before surgery ; there were no abnormal symptoms associated with respiration . reported that 95% of patients with tracheomalacia have no symptoms until two to three months after birth . thus , when obstruction of the airway occurs during induction of general anesthesia , tracheomalacia should be suspected . it is important to select the radius and length of a tube carefully when endotracheal intubation is performed in a patient with tracheomalacia . | tracheomalacia is a malformation of the tracheal membranosa .
it is maintained during spontaneous breathing but can be altered by bronchoscopy or positive airway pressure .
tracheomalacia is associated with a high mortality and may cause prolonged intubation and ventilation . here , the case of a 13-day - old infant with jejunoileal stenosis that had surgery is reported . during induction of general anesthesia , endotracheal intubation
was attempted several times with different sized endotracheal tubes .
airway obstruction occurred after the endotracheal intubation .
after the airway was maintained , the operation was completed .
tracheomalacia was diagnosed after otolaryngology evaluation postoperatively . |
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patients within this group are at risk of energy imbalance and an increased prevalence of obesity . studies of childhood cancer survivors who were treated in the 1970s and 1980s demonstrate a clear association between cranial radiotherapy ( crt ) and an increased prevalence of obesity , especially with higher radiation doses [ 24 ] . contemporary treatment protocols have removed crt for the majority of children with all and , where it is still utilized , reduced the doses delivered . despite these improvements in treatment protocols , obesity is still a concern during treatment and follow - up [ 58 ] . various risk factors have been identified including younger age at diagnosis [ 5 , 7 ] , elevated body mass index ( bmi ) at diagnosis , dose of corticosteroid , treatment risk [ 9 , 10 ] , and female sex [ 8 , 11 ] but these observations are not consistent among all reports [ 9 , 10 ] . in a recent meta - analysis of obesity in survivors of pediatric all , zhang et al . concluded that such modifiers of bmi remain speculative . for those studies of patients on modern treatment protocols for all with multiple observations during treatment , few have adjusted for or included all these predictors in the same analysis . our aim was to identify those patients at greatest risk of becoming obese during treatment for all . we therefore performed a retrospective chart review of all children and adolescents diagnosed with all in western australia between 2003 and 2007 to evaluate longitudinal changes in bmi during treatment for all and to identify risk factors that would inform future intervention strategies . between 1 january 2003 and 31 december 2007 , a total of 89 children ( 017 y ) were newly diagnosed with all in western australia . all patients were treated at princess margaret hospital ( pmh ) for children , the sole tertiary pediatric hospital in the state of western australia . patients were treated on a variety of children 's cancer group ( ccg ) or children 's oncology group ( cog ) protocols . exclusion criteria included patients with infant all ( n = 3 ) , mature b cell all ( n = 2 ) , and trisomy 21 ( n = 2 ) and patients who did not proceed to maintenance therapy due to relapse ( n = 1 ) or death ( n = 1 ) . thus , 80 patients were eligible for inclusion in this study ( see figure 1 ) . medical records were retrospectively reviewed and information pertaining to auxology and cancer treatment was abstracted . data collected included height ( meters ) and weight ( kilograms ) ; risk stratification , which correlates with therapeutic intensity , according to national cancer institute ( nci ) criteria ( standard / high ) ; cumulative corticosteroid exposure ; and receipt of crt . heights ( measured using a stadiometer ) and weights were obtained at the start of each treatment phase by accredited oncology nurses for the purpose of calculating chemotherapy doses , where available heights and weights from other oncology visits were included . during treatment , height and weight were collected at least every three months . cumulative steroid doses during treatment were calculated in mg / m for both dexamethasone ( dex ) and prednisone ( pdn ) . the total cumulative steroid dose was expressed as pdn equivalents after multiplying the dex dose by 6.67 to account for the differences in steroid potency . for the analysis , total steroid dose was cumulated separately for the duration of maintenance treatment and for the early treatment period prior to maintenance . heights and weights were therefore collected for 3.35 years after the start of treatment for all patients unless data were censored at the date of relapse , transferring out of western australia , or transition to adult services ; see figure 1 for summary . this audit was approved by the child and adolescent health service , quality and safety committee with delegated authority from the pmh institutional review board . it conforms to the provisions of the declaration of helsinki in 1995 ( as revised in tokyo 2004 ) and the national statement on ethical conduct in human research , australian national health and medical research council . heights and weights of children from the raine study were used as a comparison population to the all cohort . the raine study ( http://www.rainestudy.org.au/ ) briefly , 2900 pregnancies were recruited at king edward memorial hospital ( perth , western australia ) at 18 weeks ' gestation from 1989 to 1991 . auxological review of this pregnancy cohort was carried out at ages 1 , 2 , 3 , 6 , 8 , 10 , 14 , 17 , and 21 years . the current raine cohort has been shown to be representative of the ethnicity and demographics of the general population of perth , western australia . this study was approved by the raine executive committee and the ethics committees of king edward memorial hospital and pmh . written consent was provided by adolescent participants and their accompanying parent or guardian . in comparison with the raine cohort , 33% of the patient cohort were born at the same time of within 5 years of the raine cohort , 35% of the patient cohort were born within 510 years of the raine cohort , and 32% were born within 1015 years of the raine cohort . the outcome of interest was bmi , which was calculated using the standard formula : weight ( kg)/height ( m ) . bmi values for patients aged between 2 and 20 years were converted to age and sex - adjusted z - scores using the formula ( ( ( x / m ) ) 1)/ls , where x is the bmi measurement and l , m , and s are the age and sex specific values for the power in the box - cox transformation and the median and the coefficient of variation , respectively , by the sas macros for the center for disease control ( cdc ) 2000 growth charts [ 15 , 16 ] . bmi z - score categories are defined as underweight ( < 5th percentile ) , healthy weight ( 5th percentile to < 85th percentile ) , overweight ( 85th to < 95th percentile ) , and obese ( 95th percentile ) . who 2006 growth charts were used to calculate the bmi z - score at diagnosis for the six children who were less than two years of age . these values were not included in the longitudinal response variable but were included in a covariate variable ( bmi z - score at start of treatment ) to ensure that all individuals were included in the statistical analysis . proportions were compared using fisher 's exact test ( independent samples ) or mcnemar 's test ( paired data ) . longitudinal bmi z - scores for the all cohort were analyzed using linear mixed - effects , including fixed effects for time since start of treatment ( quartic polynomial ) and time - dependent interactions with sex , age , and bmi z - score at the start of treatment , nci risk , and total maintenance steroid dose received . random effects for intercept and time and a compound symmetry correlation structure for the errors using the lme function from the statistical package nlme in r were included in the models [ 17 , 18 ] . p values were calculated for each covariate , including associated interaction terms , using the likelihood ratio test . sample - mean predicted bmi z - scores were estimated using the pred function ( package nlme ) and associated 95% confidence intervals were calculated , which were graphed using the ggplot2 package . longitudinal bmi z - scores for the raine cohort were analyzed similarly , including fixed effects for age ( quartic polynomial ) and age - dependent sex interaction and random effects for intercept and age . a significance level of 5% selected characteristics of the 51 male and 29 female patients included in the study are presented in table 1 . the median age at diagnosis was 5.49 years ( range : 1.0216.66 ) , with six children diagnosed before 2 years of age . the majority of the children were diagnosed with pre - b all ( 88.8% ) , of which 73% ( n = 52/71 ) were standard risk . patients were treated on ccg and cog protocols : ccg1961 ( n = 10 ) , ccg1991 ( n = 24 ) , all0031 ( n = 4 ) , all0232 ( n = 12 ) , all0331 ( n = 29 ) , and all0434 ( n = 1 ) . the duration of treatment was greater for males ( median 3.18 years ) than females ( median 2.18 years ) , as males receive an additional year of maintenance therapy on these protocols . twelve patients ( 15% ) ( 10 male , 2 female ) received crt as part of their therapy . the majority of patients received dex ( n = 62 , 78% ) as the only corticosteroid , one patient received pdn only , and the remaining 17 patients received both dex and pdn during treatment . no patient received growth hormone during treatment or in the two years immediately following the end of therapy . at the time of diagnosis the bmi z - scores for individuals with all predominantly fall within the 95% prediction interval for the healthy ( raine ) cohort ( figure 2(a ) ) ; 77.5% ( 62/80 ) of the patients were in the healthy weight category , 3.8% ( 3/80 ) were in the underweight category , and 8.8% ( 7/80 ) were classified as overweight and 10% ( 8/80 ) as obese according to cdc reference standards . there was no sex difference in the proportion of males and females in each of the bmi categories . at the end of treatment ( figure 2(b ) ) , 46.3% ( 37/80 ) of the patients were in the healthy weight category , 2.5% ( 2/80 ) were in the underweight category , 26.3% ( 21/80 ) were classified as overweight , and 25% ( 20/80 ) were classified as obese . at the end of treatment , there was an increase in bmi z - scores for individuals with all diagnosed before 10 years of age , whereas individuals diagnosed after 10 years of age predominantly were within or below the 95% prediction interval for the healthy ( raine ) cohort . by the end of treatment there were significant differences in the proportions of males and females in each bmi category ( p = 0.006 ; fisher 's exact test ) . during treatment , increases in bmi z - scores were greater for females than males ; the prevalence of obesity increased from 10.3% to 44.8% ( p < 0.004 ) for females but remained relatively unchanged for males ( 9.8% to 13.7% , p = 0.7 ) . the mean number of bmi observations per individual was 34.8 ( sd 19.7 ) for females and 27.9 ( sd 17.0 ) for males . the peak increase in bmi z - scores occurs around two years posttreatment , for both sexes , and thereafter gradually declines ( figure 3 ) . the acceleration in bmi z - scores in females , compared to males , predominantly occurs between 6 and 12 months posttreatment , whereas from two years posttreatment an approximately constant mean difference in bmi z - scores is maintained between the sexes . bmi z - scores were associated with sex , nci risk , age and bmi z - score at diagnosis , and total maintenance therapy steroid dose ( see table s1 in supplementary material available online at http://dx.doi.org/10.1155/2015/386413 ) . no associations were detected between bmi z - scores and either total steroid dose in premaintenance therapy or cranial radiotherapy . significant associations with bmi z - scores were identified for time - dependent interactions with sex ( lrt p = 0.0005 ) , bmi z - score at diagnosis ( p = 0.0005 ) , and total dose of steroid during the maintenance phase ( p = 0.004 ) , in addition to sex and time interactions for nci risk ( lrt p < 0.0001 ) and age at diagnosis ( lrt p = 0.0001 ) . these interactions are illustrated in figure 4 and contrasted with mean bmi z - score for healthy ( raine ) males and females at the same age . standard risk and earlier age at diagnosis were associated with the greatest increases in mean bmi z - score , which is more pronounced in females . bmi z - scores for high - risk individuals were not significantly different from healthy individuals for either sex . table 2 illustrates that , on average , standard risk females have bmi z - scores 1 sd higher than healthy females of the same age . a similar effect size was seen in standard risk males for the youngest age group ( 2 years ) but this effect rapidly diminished across the older age groups . to further investigate the relationship between bmi z - score and steroid intake we predicted bmi trajectories based on identical steroid doses ( total maintenance therapy ) in males and females using the fully adjusted model described above ( figure s1 in supplementary material ) . although slightly higher mean bmi z - score values are predicted with higher steroid doses , the greatest increases are based on female sex and earlier ages at diagnosis . obesity is a recognized consequence of treatment for all ; however , risk factors for this increase remain speculative . obesity may exacerbate late - effects of cancer therapy such as cardiovascular and metabolic health ; it is therefore important to identify survivors of all who are at greatest risk of becoming obese . our longitudinal analysis which includes a median of 30 auxological measurements per patient over a period of 3.35 years has confirmed that bmi increases during treatment for all in childhood . the prevalence of obesity in the western australian all cohort increased from 10% at the start of treatment to 25% by the end of treatment for the whole cohort , similar to other reports of 11% to 21% and 14% to 23% , where increases in mean bmi were expressed as z - scores based on the cdc reference population and increases of 0.61.0 sd units [ 6 , 7 , 10 ] are similar to our observed increase of 0.6 sd units from the start to the end of treatment . while there was a significant increase in mean bmi z - score during maintenance treatment in the study by esbenshade and colleagues , the prevalence of obesity at the start and end of treatment ( 19% versus 21% ) was not significantly different , which may be explained by a higher prevalence of obesity at diagnosis in this study compared with other studies . in our cohort , female sex was a risk factor for increased bmi during treatment for all . in the longitudinal mixed - effects model adjusted for sex only , there were differences in predicted mean bmi z - scores between males and females . although mean bmi z - scores increased during the first two years of treatment for both sexes , this increase was greater for females than males . consistent with our observation , greater increases in bmi during treatment for females compared to males have been identified during treatment in several studies [ 5 , 6 , 8 , 20 ] , whereas other studies have found no differences between the sexes [ 9 , 10 , 21 ] . analysis of adult bmi in one of the largest cohorts of childhood cancer survivors , the childhood cancer survivor study , has confirmed that there is a greater prevalence of obesity in adult female survivors of all compared to males . in addition , a uk cohort treated from 1997 to 2003 , which excluded patients who received crt , also found increased bmi z - scores in females , which persisted 6 years after end of treatment . taken together , these data raise the possibility that increases in bmi during treatment for all in childhood persist into adulthood . longitudinal changes in bmi z - scores in children treated for all were associated not only with sex but also with age at diagnosis and nci risk / therapeutic intensity . for patients with high - risk all , mean bmi z - scores did not increase with treatment irrespective of age at diagnosis or sex . for patients with standard risk all , mean bmi z - scores increased for standard risk females irrespective of age but only younger standard risk males had an increase in bmi z - score . thus , for standard risk patients predictors for increased bmi during treatment are younger age at diagnosis and being female . studies typically investigate the association of bmi with either age at diagnosis or risk profile due to the potential confounding effects of age at diagnosis , which is one of the determinants of disease risk . younger age at diagnosis has been associated with increased bmi in several studies [ 7 , 8 , 23 ] . in addition , an association between treatment for higher risk all and lower bmi during treatment has also been reported [ 9 , 10 ] . although they are administered in high doses during treatment for all , few studies provide data on corticosteroid exposure during treatment for all , of which some [ 23 , 25 ] support a role for corticosteroids in the development of obesity whereas others do not [ 3 , 9 , 26 ] . a recent meta - analysis did not include corticosteroid exposure in its analysis due to the small number of studies with the availability of this data . in our cohort we identified a small , albeit significant , effect of cumulative steroid dose during maintenance and bmi . there was no interaction between corticosteroid dose and sex and therefore this association does not explain why females who are treated with one year less maintenance therapy compared with males have a higher prevalence of obesity by the end of treatment . corticosteroids are widely used for other pediatric illnesses and while their use is associated with side effects such as adrenal suppression and altered bone metabolism [ 2729 ] there are no reports of increased prevalence of obesity in females compared to males due to corticosteroid exposure . it is known that cancer survivors who were treated in the 1970s and 1980s are at increased risk of becoming obese with risk persisting well into adulthood . children treated for all or brain tumors with high doses of crt are most at risk , especially females or those who were younger at diagnosis [ 30 , 31 ] . increased risk of obesity has been associated with crt doses 20 gy or 18 gy . various studies have suggested that crt is associated with hypothalamic damage which leads to hyperleptinemia and/or gh deficiency in young adulthood [ 3235 ] . however , analysis of cohorts treated since 1990 that include patients exposed to crt does not show an association with crt and increased bmi [ 5 , 8 , 9 ] . cranial radiation was not identified as a risk factor for increased bmi in our cohort . however , the 15% of the patients that received crt were administered a relatively low ( 1218 gy ) dose compared to earlier studies where over half of the study cohorts received much higher doses of crt . further studies are required to elucidate the etiology of obesity in children treated for all on contemporary protocols , in particular why females are more susceptible to the late - effects of treatment compared with males , an effect that is consistent among a variety of cancer diagnoses and treatment protocols . the ccg / cog treatment protocols for all typically include one year less maintenance therapy for females compared with males , yet paradoxically they are at an increased risk of obesity , suggesting that treatment regimes per se may not explain the sex difference and that further studies should focus on life style factors in addition to treatment modalities . younger age at diagnosis is consistently a risk factor for development of endocrine late - effects of treatment in cancer survivors including obesity . one study has reported that children diagnosed with all who were younger than 2.5 years of age were more likely to experience an earlier adiposity rebound which is an important risk factor in adult obesity . long - term survivors of all are at risk of elevated abdominal obesity , an atherogenic ldl phenotype , and diabetes . additional follow - up of recent cohorts of all survivors beyond the end of treatment and into adulthood may determine incidence of metabolic syndrome and other morbidities . recent studies that focus on energy balance through eating behavior and physical activity in all survivors suggest obesity in survivors is a result of both disordered / increased energy intake and reduced physical activity associated with fatigue , with the decrease in physical activity greater for females compared to males [ 42 , 43 ] . the strengths of our study include the frequent measurement of heights and weights and analysis by longitudinal modelling . thus , female patients in our cohort who received standard risk protocols for all as well as younger males who received standard risk protocols for all were at the greatest risk of becoming obese towards the end of treatment . our patient cohort was compared to the raine cohort which is from the same geographical location . all children and adolescents diagnosed with all in the state of western australia are treated at pmh and therefore our cohort has 100% ascertainment of the selection criteria for the state . we have compared the changes in bmi of our all cohort to both the cdc reference population and the raine cohort which is representative of the western australian population . although the mean bmi z - scores were slightly greater in the raine population compared to the cdc reference , the increases in the bmi z - scores of the cohort with all were greater than those observed for children in the raine cohort over the same time frame . a third of our patient cohort was born 1015 years after the raine . however , a meta - analysis of trends in the prevalence of childhood overweight and obesity in australia between 1985 and 2008 identified an increase in mean bmi z - score of no more than 0.1 sd units in any 3-year period with no significant differences in the rates of change between boys and girls . it seems unlikely therefore that the increases in bmi observed in our patient cohort reflect secular changes in our local population . in conclusion , this study has confirmed that there is an increased prevalence of obesity at the end of treatment for childhood all . in the statewide cohort of western australian children treated on ccg / cog protocols between 2003 and 2007 , the biological risk factors for increased bmi / obesity during standard risk therapy are younger age at diagnosis and female sex . the philosophy of nutritional / dietician support in oncology units concentrates on detecting underweight children and adding high calorie feeds . while further data is required to determine the causes of obesity in survivors and the female propensity , our findings support further consideration of appropriate nutrition and energy balance during therapy and empowering of parents to exert control over excess calorie intake in their children with leukemia in the same manner as for their healthy children . | objective & design .
we undertook a retrospective review of children diagnosed with acute lymphoblastic leukemia ( all ) and treated with modern cog protocols ( n = 80 ) to determine longitudinal changes in body mass index ( bmi ) and the prevalence of obesity compared with a healthy reference population .
results . at diagnosis ,
the majority of patients ( 77.5% ) were in the healthy weight category . during treatment ,
increases in bmi z - scores were greater for females than males ; the prevalence of obesity increased from 10.3% to 44.8% ( p < 0.004 ) for females but remained relatively unchanged for males ( 9.8% to 13.7% , p = 0.7 ) .
longitudinal analysis using linear mixed - effects identified associations between bmi z - scores and time - dependent interactions with sex ( p = 0.0005 ) , disease risk ( p < 0.0001 ) , age ( p = 0.0001 ) , and bmi z - score ( p
< 0.0001 ) at diagnosis and total dose of steroid during maintenance ( p = 0.01 ) . predicted mean bmi z - scores at the end of therapy were greater for females with standard risk all irrespective of age at diagnosis and for males younger than 4 years of age at diagnosis with standard risk all .
conclusion .
females treated on standard risk protocols and younger males may be at greatest risk of becoming obese during treatment for all .
these subgroups may benefit from intervention strategies to manage bmi during treatment for all . |
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alzheimer s disease ( ad ) is characterized by the presence in the brain of hallmark lesions such as a plaques , abnormal these lesions follow a specific pattern of regional and cellular vulnerability , with characteristic distribution patterns of a deposits and subtype - specific neuronal susceptibility to nft pathology and degeneration . cytoskeletal changes in dystrophic neurites near plaques resemble the filamentous changes seen in cell body nfts , suggesting that a plaques may induce cytoskeletal alterations . however , there is also significant heterogeneity among dystrophic neurites at different stages of ad , which may provide further insight into the relationship between a plaque and neuronal cytoskeletal pathology . focal lesions in the brain , how this may result in wider patterns of neuronal degeneration requires elucidation . it is also clear that neurons may have some capacity to react or adapt to such lesions , making the relationship between overt pathology and functional disruption dynamic and complex . this review focuses on the effects of a plaque formation on neurons at different stages of ad , and explores the capacity of commonly used transgenic mouse models expressing familial ad ( fad ) mutated human genes to recapitulate such pathology , as well as to provide further insight into mechanisms and therapy . to date , there are a number of strong indications that a abnormalities and plaque accumulation are an early and potentially necessary event in the sequence of brain changes that lead to ad . these include familial forms of ad involving mutations in the amyloid precursor protein ( app ) , down syndrome in which the presence of three copies of the app gene leads to an ad - like syndrome ; studies of the staging of brain pathology ; and in vivo human brain imaging for a. however , there is no clear consensus on whether the critical damage is caused by abnormal a species as intracellular accumulations , or as extracellular monomers , aggregates or plaques . the neocortex is an early site of a accumulation , where it tends to localize in particular layers , indicating that misprocessing of a leading to extracellular deposits may be specific to synaptic pathways terminating in these layers . however , the origin of abnormal a and the formation of oligomers or plaques are still controversial , having been variably attributed to blood vessels , glial secretion , neuronal secretion from terminals and cell bodies , lysis of neurons or dystrophic neurites , and fibrillation by microglia ( reviewed in ) . while it is commonly assumed that extracellular a deposits mature from diffuse forms to more dense , fibrillar plaques , staging studies in human cases indicate that these plaque subtypes develop separately , with a higher proportion of fibrillar a deposits in later stages . in vivo imaging studies of fad transgenic mice show that a plaques can form rapidly and are then relatively stable in morphology and that smaller plaque deposits can occur in clusters and then merge into larger plaques . it is not well understood why some amyloid protein deposits remain diffuse , whereas others densely aggregate into highly fibrillar and dense forms . analysis by confocal microscopy also indicates that the more fibrillar a plaques are spheres with a complex internal geometry , often around a dense amyloid core ; the factors that influence the morphology and size of these deposits are also unknown . furthermore , human fad involving ps1 mutations produces a larger and morphologically distinct plaque form ( cotton wool plaques ) , indicating that the means of a production can influence plaque formation and morphology . human pathology staging and recent human in vivo imaging using radio ligands for a deposits , strongly indicate that a deposition in the brain occurs early in ad , perhaps even decades before overt symptoms . however , it is also clear that there is substantial individual heterogeneity in the damaging effects of such deposits , since a load by itself does not correlate well with cognitive deficits in established ad . low a load can accompany overt dementia , whereas some individuals show few cognitive and behavioural alterations despite relatively high a deposition . while it is possible that a oligomers have a more distributed role in neurotoxicity or compromising neuronal function , it is also clear that the more dense and fibrillar a plaques act as discrete lesions , causing local damage to axons , dendrites and synapses and focal stimulation of astrocytes and microglia . in this regard , an accumulating burden of more dense , damaging , plaques concentrated in association areas of the cerebral cortex would likely have substantial effects on higher level processing capacity . interestingly , plaque formation does not appear to be directly related to cell death of adjacent neurons , although neurons can be deflected to the margins of such deposits . dendrites of pyramidal neurons within and proximal to a plaques demonstrate deflection around the plaque , as well as withering and dendritic spine loss [ 12 , 13 ] . those dystrophic neurites appear to derive principally from axons undergoing reactive , aberrant regenerative and/or frank degenerative changes near plaques [ 14 - 16 ] . in human cases , dystrophic neurites can be classified by morphology , neurochemistry and association with different stages of ad ( fig . 1 ) . in end - stage ad , the major subtypes are distinguished by their complement of specific cytoskeletal proteins and synaptic markers . angular dystrophic neurites commonly labelled with antibodies directed to tau ( including abnormally phosphorylated tau isoforms , closely resembling the cytoskeletal pathology of neurofibrillary tangles ) are usually seen within and very near plaques , and are probably a degenerative form of dystrophic neurite . these tau - immunolabelled dystrophic neurites likely correspond to the plaque - associated abnormal neurites seen in thioflavine s staining , further reinforcing their identity as end - stage pathology involving a substantially transformed cytoskeleton . another dystrophic neurite subtype is immunolabelled for neurofilament ( nf ) proteins , the nf triplet and alpha - internexin . these neurites are typically larger than the tau - labelled structures , and appear as swellings or torturous tubular structures localized within the pores of plaques or around the corona [ 15 , 16 , 18 ] . interestingly , a subset of these larger nf labeled dystrophic neurites has a core of abnormal tau which stains with thioflavine s , which we hypothesized may represent a transition from abnormally reactive and regenerative axons responding to a plaques , into tau - abundant degenerative forms . the nf abundant dystrophic neurites also colocalise with growth - related proteins ( eg gap43 ) and often have a long neurite tail that can be traced out to the neuropil [ 15 , 16 ] . in this regard , they closely resemble the sprouting axons near plaques that have been described with neurofibrillar silver staining ( eg cajal , 1928 , in ) and by golgi staining . another type of dystrophic neurite observed in human cases with a plaques has a swollen globular morphology , and predominantly contains synaptic markers such as synaptophysin , chromogranin a , and , potentially , app [ 16 , 23 - 25 ] . these appear to form largely independently of those containing neurofilaments or altered tau , although some nf labeled dystrophic neurites show co - labelling for synaptic markers . numerous other proteins have been implicated in dystrophic neurite formation , including gap-43 , ubiquitin , ubiquilin , prion protein , cytochrome c , c9 or f72 , reticulon-3 and bace-1 [ 26 - 32 ] . in this regard , many of these markers correspond to proteins implicated in amyloidogenic and neuropathological pathways , potentially supporting the view that dystrophic neurite formation may lead to , for example , abnormal a processing , release of abnormal a oligomers and subsequent a fibrillization into plaques . notably , bace1 , an enzyme critical for generating a fragments , may have a role in axon and synapse development , and also accumulates in axons and terminals in association with a plaques , and there is interest in potentially reducing this axonal pathology and subsequent amyloid pathology by bace1 inhibition ( see for a review ) . ultrastructural studies have also indicated that dystrophic neurites can contain filamentous structures including nfs and paired helical filaments ( the latter only in human cases ) , abundant organelles such as mitochondria and lysosomes , abnormal swollen vesicles , and multilamellar and dense bodies [ 14 , 34 - 36 ] . accumulation of proteins and organelles within dystrophic neuritis suggests an interruption of normal axonal transport in damaged axons . in addition , the abnormal structures may accumulate because of disruption in normal autophagic - lysosomal pathways within dystrophic neurites . the major form arises from long axons reacting and/or aberrantly regenerating around a plaques , causing progressive changes to the neuronal cytoskeleton . the second subtype resembles swollen axon terminals , which do not show regenerative features or substantial cytoskeletal pathology . it may be that the a plaques have a differential effect on axonal or terminal compartments ; or , given the localization of app and potential autophagic - lysosomal dysfunction , these swollen terminals may be abnormally processing a , leading to plaque formation . with respect to neuronal susceptibility to dystrophic neurite formation , there may also be differences between subsets of neurons responding to a plaque formation . as noted above , many dystrophic neurites may correspond to long axons , including corticocortical axons ( see also ) , as well as from neighbouring pyramidal neurons [ 22 , 38 ] and from specific subcortical brain regions . nf abundant axons may also correspond to neurons that preferentially express , for example , nf triplet proteins in the cerebral cortex . a subset of pyramidal neurons show high levels of nf triplet proteins across mammalian species ( reviewed in ) and are likely to contribute to long corticocortical projections , as seen in studies of non - human primate species . these nf triplet containing neurons are particularly susceptible to nft formation [ 42 , 43 ] and degeneration in ad . conversely , non - pyramidal neurons , generally lacking nf triplet proteins , show very little propensity to nft formation and overt cell loss in ad , and also very little reactive changes or dystrophic neurite formation around a plaques . this indicates that nf content may be a predisposing factor for axons to undergo a substantial reactive and regenerative response to plaque - related injury . a deposits probably accumulate in the brain for many years before cognitive deficits and behavioural changes are discernible . in this regard , pathological staging studies indicate that the clinical features of ad are closely associated with the presence of a plaques throughout the neocortex and the spread of neurofibrillary pathology from medial temporal regions into other neocortical association areas . increasing a imaging studies also indicate that a accumulation in the brain may represent a key early brain change , significantly increasing the risk of developing ad . however , it is not yet clear whether all ad cases follow a lengthy period of a accumulation , or whether all people who accumulate a will necessarily transform into fulminant disease . we have also demonstrated that overall a deposition load may be less critical than a change in the proportion of plaque types from predominantly diffuse forms to more compact , dense structures able to induce neuronal pathology . in this regard , we have hypothesized that fibrillar plaques precipitate aberrant regenerative changes preceding classic neurofibrillary pathology , in neurons whose axons and terminal fields are impinged by plaque formation . the dystrophic neurite profile of a plaques also differs in prodromal disease compared to end - stage ad . for example , abnormal tau in dystrophic neurites is rarely seen near fibrillar a plaques of preclinical , which are instead surrounded by abundant nf immunoreactive dystrophic neurites , including large spherical structures with axons that can be traced out into the neuropil , and smaller ring - like neurofilament structures [ 16 , 47 ] . both of these resemble the reactive and regenerative changes in axons subjected to structural injury in vivo and in vitro [ 48 , 49 ] . the accumulation of nfs , but not abnormal tau , in these dystrophic neurites supports the proposal that they are a relatively early form of abnormality , and that the comprehensive cytoskeletal changes of tau pathology take a relatively long time to develop . a plaques and associated dystrophic neurites have been described in a small number of non - human species , such as aged primates and dogs ( e.g. , [ 50 , 51 ] ) , whereas transgenic mice expressing human fad mutated app ( often in combination with mutated human ps1 ) typically develop a deposits and plaques without substantial neuronal degenerationor neurofibrillary pathology such as paired helical filaments and highly modified tau . some models combine human app and ps1 mutations with a tau mutation ( p301l ) implicated in a subset of frontotemporal dementia cases . although the latter shows an augmentation of the tau pathology , it does not develop paired helical filament pathology , or significant cell loss and atrophy resembling human ad . notwithstanding these shortcomings , we have demonstrated that commonly used transgenic ad models such as the appswe / ps1de9 , tg2576 and crnd8 lines , develop a pattern of pathology that is most reminiscent of early or preclinical ad . as they age , these lines develop dense , fibrillar plaques surrounded by nf labeled dystrophic neurites that appear identical to those in preclinical human cases [ 45 , 53 ] . hence , such transgenic mice can model the earliest pathogenic events of ad , and will be useful for suggesting and exploring potential disease - modifying strategies . the mammalian cerebral cortex has a highly conserved , repetitive organization including columnar arrangements of neurons which dynamically group into units to enable sensory processing , integration of information and intentional behavior . in the human neocortex , a plaques cluster in layers involved in corticocortical connectivity , and are more abundant in association areas relative to primary motor and sensory regions . cortical a plaques are comparatively sparse in preclinical ad , but during disease progression , their spread throughout cortical layers may damage and disrupt most of the neuron groups in association areas , reducing the capacity for compensation in disrupted information processing . ad is also associated with the degeneration and death of specifically susceptible neurons , as well as a generalized loss of brain . there has also been substantial interest in how damage to particular cortical circuits may represent a critical degenerative change that triggers progressive deterioration in cognitive function and alterations in behavior . earlier work by terry and associates emphasized the critical role played by synaptic loss in ad , correlating more closely than either plaques or neurofibrillary tangles with indices of cognitive decline . the extent of synapse loss in higher association neocortex has been reported to vary between 30 - 45% with a predilection for layers involved in corticocortical connectivity . the particular vulnerability of connection - related neurons and synapses likely gives rise to a pattern of disconnection between higher cortical areas . with respect to cortical microcircuitry , as noted above , densely fibrillar plaques cause dendritic withering , spine loss , loss of normal axons , demyelination , reactive axonal changes and swelling of synaptic terminals ( fig . , these plaques also represent a focus of synapse loss [ 54 , 55].the greatest degree of synapse degeneration occurs in the central region of the plaque , with a substantial loss of both excitatory and inhibitory synapses . however , in the periphery of plaques and throughout the neuropil , there is a selective loss of excitatory synapses , as demonstrated with the presynaptic marker , vglut-1 , whereas , inhibitory synapses appear unaffected , in the cortical neuropil between plaques in both established human ad and in transgenic models . however , inhibitory synapses are reduced on cortical neuron cell body surfaces and initial axon segments near plaques [ 56 , 57 ] . in preclinical ad cases , synapse loss is restricted to the plaques , with no discernible decreased density in the wider neuropil . collectively , this indicates that plaques can cause substantial localized damage primarily to excitatory cortical connections in preclinical stages , but that further disease progression , perhaps associated with neuronal degeneration and increased a plaque load , is necessary for more widespread synapse loss . in end - stage ad cases , remaining vglut-1 labelled puncta in and around plaquesare relatively reduced in size , whereas vgat labeled inhibitory boutons are larger . interestingly , in preclinical ad cases , both vglut-1 and vgat labeled boutons were larger in the neuropil and at the periphery of plaques . these specific and stage - specific alterations in bouton size may reflect type - specific adaptive changes in response to the disruption in cortical circuitry . aside from synaptic changes , recent studies have suggested that substantial reactive changes may also occur in glial gabaergic systems . increased glutamate decarboxylase ( gad ) activity was detected in glial membrane fraction preparations from 12 month old app / ps1 mice , but not from neural synaptosomes . more recent studies show increased gaba labeling in reactive astrocytes in human ad brains , as well as release of gaba by astrocytes in transgenic ad models [ 58 , 59 ] . this may be linked with increased , and synchronized , activity of glial cells as shown by in vivo calcium imaging of transgenic models . in human ad , the combination of specific synapse vulnerability , synaptic remodeling and altered inhibitory glio transmission , may contribute to local processing abnormalities in the vicinity of a plaques . in vivo calcium imaging of ad transgenic models has demonstrated abnormal hypo- and hyperactivity in subsets of neurons , the latter specifically in neurons and neurites near a plaques [ 61 , 62 ] . while there is limited evidence of nerve cell body degeneration around plaques , increased calcium in neurites adjacent to plaques could contribute to degeneration , since calcineurin inhibition in experimental models reduces peri - plaque neurite beading . in this regard , the relative preservation of inhibitory synaptic structures , including increased bouton size and gaba production and release by reactive astrocytes may partly compensate for abnormal excitation and hyperactivity around plaques . the large spatial extent of astrocytes compared to neurons could explain the wider spread of abnormally silent or hypoactive neurons between plaques in transgenic models . in vivo calcium imaging of transgenic models has shown hypoactive neurons in the visual cortex which were unresponsive to visual stimuli , whereas visual processing alterations were associated with abnormally hyperactive subsets of neurons . increased epileptiform activity in ad and transgenic models has been well described , and likely represents an altered balance between excitatory and inhibitory transmission , for which the system may compensate by means such as sprouting of inhibitory processes and increased miniature inhibitory postsynaptic currents in the hippocampus . furthermore , gabaa antagonism is capable of restoring a degree of activity in previously abnormally hypoactive neurons , further supporting the proposition that this pathological milieu involves excessive inhibition . plaques also locally reduce experience - induced expression of the immediate early gene arc following visual stimulation in transgenic models , indicating that excitatory plasticity is also disrupted . taken together , studies of early human ad and corresponding transgenic models indicate a disruption in normal cortical processing in the vicinity of a plaques , consisting of altered and degenerating synapses , cytoskeletal and aberrant regenerative changes in axons , but little overt neuronal degeneration . ad progression is linked to an increased density of fibrillar a plaques , a wider loss of synapses throughout affected cortical regions and the substantial transformation of the normal neuronal cytoskeleton in subsets of vulnerable neurons . the evolution of these initial neuronal changes and network disruption into frank degeneration of associative cortical pathways suggests multiple points of potential intervention , from pharmacological manipulation of synaptic activity through to inhibition of cytoskeletal pathology that results in disconnection . common animal models involving familial ad mutant transgenes broadly reflect initial stages of ad , and may provide insights into how a pathology results in structural and physiological changes in neurons and network dynamics , as well as informing potential new therapeutic approaches for early intervention and/or prevention of fulminant disease . | the prospects for effectively treating well - established dementia , such as alzheimer s disease ( ad ) , are slim , due to the destruction of key brain pathways that underlie higher cognitive function .
there has been a substantial shift in the field towards detecting conditions such as ad in their earliest stages , which would allow preventative or therapeutic approaches to substantially reduce risk and/or slow the progression of disease .
ad is characterized by hallmark pathological changes such as extracellular a plaques and intracellular neurofibrillary pathology , which selectively affect specific subclasses of neurons and brain circuits .
current evidence indicates that a plaques begin to form many years before overt dementia , a gradual and progressive pathology which offers a potential target for early intervention .
early a changes in the brain result in localized damage to dendrites , axonal processes and synapses , to which excitatory synapses and the processes of projection neurons are highly vulnerable . a pathology is replicated in a range of transgenic models overexpressing mutant human familial ad genes ( eg app and presenilin 1 ) .
studying the development of aberrant regenerative and degenerative changes in neuritic processes associated with a plaques may represent the best opportunity to understand the relationship between the pathological hallmarks of ad and neuronal damage , and to develop early interventions to prevent , slow down or mitigate against a pathology and/or the neuronal alterations that leads to cognitive impairment . |
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the loss of muscle mass with aging ( i.e. , sarcopenia ) has a negative effect on strength and the ability to perform tasks of daily living . females typically experience an accelerated decline in muscle mass ( 0.6 % /year ) after menopause , possibly because of an imbalance between muscle protein synthesis and protein catabolism . resistance exercise increases the rates of muscle protein synthesis , which may lead to significant muscle accretion and strength over time in older adults . in addition to resistance exercise , nonsteroidal anti - inflammatory drugs ( nsaids ) such as ibuprofen may also benefit aging muscle . ibuprofen inhibits cyclooxygenase ( cox-1 , cox-2 ) activity , which decreases the synthesis of prostaglandin e2 ( pge2 ) , a biological regulator of protein catabolism . in a recent study by trappe et al . , healthy older adults ( nine males , four females ; 64 years ) who ingested the maximum daily recommended dosage of ibuprofen ( 1,200 mg ) during structured resistance exercise for 12 weeks experienced a significant increase in quadriceps muscle volume and muscle strength over a placebo group who performed the same training program . the authors speculate that ibuprofen inhibited cox and pge2 activity and decreased muscle protein catabolism more so than protein synthesis following exercise , which resulted in net muscle protein retention and muscle accretion over time . previous work by the same researchers showed that 1,200 mg of ibuprofen following an acute bout of resistance exercise inhibited the fractional synthetic rate of muscle proteins and prostaglandin f2 , a regulator of muscle protein synthesis . based on these findings , higher - dose ibuprofen ( i.e. , 1,200 mg ) following resistance exercise may suppress the rates of muscle protein synthesis and protein catabolism , which may increase , decrease , or have no change on muscle accretion over time . the maximum recommended single dosage of ibuprofen ( 400 mg ) immediately following resistance exercise sessions for 9 months had no effect on lean tissue mass in premenopausal women . furthermore , ingesting 400 mg of ibuprofen or placebo during 6 weeks of unilateral resistance training resulted in similar gains in elbow flexor muscle thickness and biceps maximal strength in young adults . results across studies indicate that 400 mg of ibuprofen is not effective in younger populations for improving muscle mass or strength ; however , postmenopausal women may be more responsive to the physiological effects of 400 mg of ibuprofen due to elevated inflammation and oxidative stress . therefore , the purpose of this study was to examine the effects of low - dose ibuprofen ( i.e. , 400 mg ) immediately following resistance exercise sessions in postmenopausal women . it was hypothesized that ibuprofen would lead to an increase in muscle mass and strength compared to placebo and training over time . thirty - four healthy postmenopausal women , who were not performing supervised resistance exercise , were recruited for the study . participants were excluded if they had taken bisphosphonates , hormone replacement therapy , selective estrogen receptor modulators , parathyroid hormone , or calcitonin 12 months prior to the start of the study , if they suffered from severe osteoarthritis , if they were taking nsaids , and if they were smokers . prior to group allocation , participants were given a physical activity readiness questionnaire , which assessed their readiness to participate in resistance exercise . this questionnaire included questions related to heart conditions , angina at rest or during physical exercise , balance , and bone or joint problems that may affect exercise performance . if the participant indicated a contraindication to exercise , they were required to get medical approval before participating in the study . participants were also required to fill out a leisure time exercise questionnaire where the number of times on average per week strenuous ( i.e. , heart beats rapidly ) , moderate ( i.e. , not exhausting ) , and mild ( i.e. , minimal effort ) exercise was performed . participants were instructed not to change their diet , engage in additional physical activity that was not part of their normal daily routine , or ingest other nsaids that were not part of the study intervention . the study was approved by the university ethics review board at the university of regina . participants were informed of any risks and of the purpose of the study before their written consents were obtained . the study used a double - blind , placebo - controlled , repeated - measures design where participants were randomized to ingest either ibuprofen ( 400 mg ) or placebo ( micronized cellulose crystals ) immediately following resistance exercise ( 3 days / week ) for 9 weeks . we have previously shown that 8 weeks of whole - body resistance exercise is sufficient to increase muscle mass and strength [ 1720 ] . ibuprofen and placebo tablets were administered to each participant by a research supervisor and were similar in size , taste , and texture . peak plasma concentrations of ibuprofen occur between 1 and 4 h after ibuprofen ingestion . inflammatory cytokines are elevated within 15 min postexercise in postmenopausal women and return to baseline within 2 h . we , therefore , administered ibuprofen immediately after resistance exercise sessions so that elevated plasma concentrations of ibuprofen would coincide with increased inflammation . participants were instructed to refrain from food or drink , excluding water , for 60 min following each training session so that a valid estimate of the effects of ibuprofen on muscle could be made . prior to the first visit to the laboratory for initial testing and data collection , participants were instructed to refrain from physical activity and alcohol for 24 h , caffeine for 6 h , and food and drink for 2 h. the primary dependent variables measured before and after the 9-week intervention were ( 1 ) whole - body lean tissue mass , ( 2 ) muscle thickness of the elbow and knee flexors and extensors and ankle dorsiflexors and plantar flexors , and ( 3 ) strength ( leg press and chest press one - repetition maximum [ 1-rm ] ) . after a familiarization training session , participants followed the same supervised , whole - body resistance exercise program combined with ibuprofen or placebo for 9 weeks . prior to the start of each resistance exercise session , participants performed a 5-min aerobic warm - up ( i.e. , stationary cycle , elliptical trainer , treadmill ) at a self - selected intensity . participants then completed three sets of 10 repetitions to muscle fatigue with a 2-min rest between sets for each exercise at an intensity corresponding to their 10-repetition maximum for each exercise . we have previously used this training protocol successfully to increase muscle mass and strength in older adults [ 23 , 24 ] . resistance exercises included leg press , chest press , lat pull - down , shoulder press , leg ( knee ) extension , leg curl ( knee flexion ) , triceps extension , biceps curl , calf press , low back extension , and abdominal curl . participants maintained daily training logs where the load and the number of sets and repetitions performed was recorded . resistance was increased by 25 kg once a participant could complete three sets of 10 repetitions to muscle fatigue for an exercise . once the resistance was increased , the participant maintained this load until a subsequent three sets of 10 repetitions to fatigue was completed . whole - body lean tissue mass was assessed using dual - energy x - ray absorptiometry ( hologic wi system , christie group , winnipeg , mb , canada ) in array mode . before scanning , participants were required to remove all objects containing metal ( i.e. , jewelry ) . scans were performed with participants lying in a supine position along the scanning table s centerline longitudinal axis . feet were taped together at the toes ( i.e. , phalanges ) to immobilize the legs , while the hands maintained a pronated position within the scanning region . muscle size of the elbow and knee flexors and extensors and ankle dorsiflexors and plantar flexors was measured using b - mode ultrasound ( aloka ssd-500 , tokyo , japan ) . briefly , a 5-mhz scanning transducer head was coated with water - soluble transmission gel to provide acoustic contact with the muscle surface . when the image produced on the screen was visible , a cursor was enabled to quantify muscle thickness ( in centimeters ) at three sites : the proximal , the mid , and the distal , as determined by divisions ( 1 cm ) on the monitor . muscle thickness measurements were extrapolated from the monitor screen by measuring the distance from the bottom of the subcutaneous adipose layer to the surface of the humerus for elbow flexor and extensor muscle thickness , to the surface of the femur for knee flexor and extensor muscle thickness , and to the surface of the tibia for ankle dorsiflexor and plantar flexor muscle thickness . the coefficients of variation were 2.5 % ( elbow flexors ) , 2.2 % ( elbow extensors ) , 3.6 % ( knee flexors ) , 2.1 % ( knee extensors ) , 3.2 % ( ankle plantar flexors ) , and 4.0 % ( ankle dorsiflexors ) [ 24 , 25 ] . leg press and chest press strength was assessed using a 1-rm testing protocol . following 5 min of cycling on a stationary cycle ergometer , participants performed two warm - up sets in order : one set of 10 repetitions using a light weight determined by each participant to be comfortable and another set of five repetitions using slightly increased weight . following the warm - up sets , weight was progressively increased by approximately 10 kg for leg press and 5 kg for chest press for each subsequent 1-rm attempt with a 2-min rest interval between attempts . the 1-rm was reached in four to six trials , independent of the two warm - up sets . the leg press and chest press strength measures have coefficients of variation of 3.8 and 3.1 % , respectively . sample size was calculated using the coefficient of variation for whole - body lean tissue mass and a change in lean tissue mass that would be clinically significant . a 3 % change in lean tissue mass was considered clinically significant because a 3 % reduction in muscle mass in postmenopausal women is associated with 11 % lower muscle strength compared to young premenopausal women . based on these calculations , six postmenopausal women were required per group to demonstrate a 3 % increase in lean tissue mass with a power of 0.9 and alpha of 0.05 . a 2 ( groups : ibuprofen vs. placebo ) 2 ( time : pretest and posttest periods ) anova with repeated measures on the second factor was used to determine differences between groups over time for each of the dependent variables of lean tissue mass , muscle thickness , and strength . an independent sample t test was used to determine differences in training volume between groups . statistical analyses were carried out using spss version 18.0 for windows xp ( spss , chicago , il , usa ) . significance was set at p < 0.05 . all results are expressed as the mean standard deviation . thirty - four healthy postmenopausal women , who were not performing supervised resistance exercise , were recruited for the study . participants were excluded if they had taken bisphosphonates , hormone replacement therapy , selective estrogen receptor modulators , parathyroid hormone , or calcitonin 12 months prior to the start of the study , if they suffered from severe osteoarthritis , if they were taking nsaids , and if they were smokers . prior to group allocation , participants were given a physical activity readiness questionnaire , which assessed their readiness to participate in resistance exercise . this questionnaire included questions related to heart conditions , angina at rest or during physical exercise , balance , and bone or joint problems that may affect exercise performance . if the participant indicated a contraindication to exercise , they were required to get medical approval before participating in the study . participants were also required to fill out a leisure time exercise questionnaire where the number of times on average per week strenuous ( i.e. , heart beats rapidly ) , moderate ( i.e. , not exhausting ) , and mild ( i.e. , minimal effort ) exercise was performed . participants were instructed not to change their diet , engage in additional physical activity that was not part of their normal daily routine , or ingest other nsaids that were not part of the study intervention . the study was approved by the university ethics review board at the university of regina . participants were informed of any risks and of the purpose of the study before their written consents were obtained . the study used a double - blind , placebo - controlled , repeated - measures design where participants were randomized to ingest either ibuprofen ( 400 mg ) or placebo ( micronized cellulose crystals ) immediately following resistance exercise ( 3 days / week ) for 9 weeks . we have previously shown that 8 weeks of whole - body resistance exercise is sufficient to increase muscle mass and strength [ 1720 ] . ibuprofen and placebo tablets were administered to each participant by a research supervisor and were similar in size , taste , and texture . peak plasma concentrations of ibuprofen occur between 1 and 4 h after ibuprofen ingestion . inflammatory cytokines are elevated within 15 min postexercise in postmenopausal women and return to baseline within 2 h . we , therefore , administered ibuprofen immediately after resistance exercise sessions so that elevated plasma concentrations of ibuprofen would coincide with increased inflammation . participants were instructed to refrain from food or drink , excluding water , for 60 min following each training session so that a valid estimate of the effects of ibuprofen on muscle could be made . prior to the first visit to the laboratory for initial testing and data collection , participants were instructed to refrain from physical activity and alcohol for 24 h , caffeine for 6 h , and food and drink for 2 h. the primary dependent variables measured before and after the 9-week intervention were ( 1 ) whole - body lean tissue mass , ( 2 ) muscle thickness of the elbow and knee flexors and extensors and ankle dorsiflexors and plantar flexors , and ( 3 ) strength ( leg press and chest press one - repetition maximum [ 1-rm ] ) . after a familiarization training session , participants followed the same supervised , whole - body resistance exercise program combined with ibuprofen or placebo for 9 weeks . prior to the start of each resistance exercise session , participants performed a 5-min aerobic warm - up ( i.e. , stationary cycle , elliptical trainer , treadmill ) at a self - selected intensity . participants then completed three sets of 10 repetitions to muscle fatigue with a 2-min rest between sets for each exercise at an intensity corresponding to their 10-repetition maximum for each exercise . we have previously used this training protocol successfully to increase muscle mass and strength in older adults [ 23 , 24 ] . resistance exercises included leg press , chest press , lat pull - down , shoulder press , leg ( knee ) extension , leg curl ( knee flexion ) , triceps extension , biceps curl , calf press , low back extension , and abdominal curl . participants maintained daily training logs where the load and the number of sets and repetitions performed was recorded . resistance was increased by 25 kg once a participant could complete three sets of 10 repetitions to muscle fatigue for an exercise . once the resistance was increased , the participant maintained this load until a subsequent three sets of 10 repetitions to fatigue was completed . whole - body lean tissue mass was assessed using dual - energy x - ray absorptiometry ( hologic wi system , christie group , winnipeg , mb , canada ) in array mode . before scanning , participants were required to remove all objects containing metal ( i.e. , jewelry ) . scans were performed with participants lying in a supine position along the scanning table s centerline longitudinal axis . feet were taped together at the toes ( i.e. , phalanges ) to immobilize the legs , while the hands maintained a pronated position within the scanning region . muscle size of the elbow and knee flexors and extensors and ankle dorsiflexors and plantar flexors was measured using b - mode ultrasound ( aloka ssd-500 , tokyo , japan ) . briefly , a 5-mhz scanning transducer head was coated with water - soluble transmission gel to provide acoustic contact with the muscle surface . when the image produced on the screen was visible , a cursor was enabled to quantify muscle thickness ( in centimeters ) at three sites : the proximal , the mid , and the distal , as determined by divisions ( 1 cm ) on the monitor . muscle thickness measurements were extrapolated from the monitor screen by measuring the distance from the bottom of the subcutaneous adipose layer to the surface of the humerus for elbow flexor and extensor muscle thickness , to the surface of the femur for knee flexor and extensor muscle thickness , and to the surface of the tibia for ankle dorsiflexor and plantar flexor muscle thickness . the coefficients of variation were 2.5 % ( elbow flexors ) , 2.2 % ( elbow extensors ) , 3.6 % ( knee flexors ) , 2.1 % ( knee extensors ) , 3.2 % ( ankle plantar flexors ) , and 4.0 % ( ankle dorsiflexors ) [ 24 , 25 ] . leg press and chest press strength was assessed using a 1-rm testing protocol . following 5 min of cycling on a stationary cycle ergometer , participants performed two warm - up sets in order : one set of 10 repetitions using a light weight determined by each participant to be comfortable and another set of five repetitions using slightly increased weight . following the warm - up sets , weight was progressively increased by approximately 10 kg for leg press and 5 kg for chest press for each subsequent 1-rm attempt with a 2-min rest interval between attempts . the 1-rm was reached in four to six trials , independent of the two warm - up sets . the leg press and chest press strength measures have coefficients of variation of 3.8 and 3.1 % , respectively . sample size was calculated using the coefficient of variation for whole - body lean tissue mass and a change in lean tissue mass that would be clinically significant . a 3 % change in lean tissue mass was considered clinically significant because a 3 % reduction in muscle mass in postmenopausal women is associated with 11 % lower muscle strength compared to young premenopausal women . based on these calculations , six postmenopausal women were required per group to demonstrate a 3 % increase in lean tissue mass with a power of 0.9 and alpha of 0.05 . a 2 ( groups : ibuprofen vs. placebo ) 2 ( time : pretest and posttest periods ) anova with repeated measures on the second factor was used to determine differences between groups over time for each of the dependent variables of lean tissue mass , muscle thickness , and strength . an independent sample t test was used to determine differences in training volume between groups . statistical analyses were carried out using spss version 18.0 for windows xp ( spss , chicago , il , usa ) . of the original 34 participants who volunteered for the study , 28 participants ( 15 ibuprofen , 13 placebo ; 5068 years of age ) completed the study . average resistance training compliance was 83.1 % ( 22 of 27 sessions performed ) for the ibuprofen group and 75 % ( 20 of 27 sessions performed ) for the placebo group.table 1subject characteristics and physical activity performed at baseline for the ibuprofen and placebo groupsgroupage ( years)body mass ( kg)height ( cm)strenuous activity ( times per week)moderate activity ( times per week)mild activity ( times per week)total activity ( times per week)ibuprofen ( n = 15)57.8 ( 5.1)75.9 ( 9.0)165.9 ( 6.2)1.9 ( 2.5)2.4 ( 1.5)4.6 ( 2.8)9.0 ( 3.1)placebo ( n = 13)56.5 ( 4.4)73.0 ( 10.4)163.1 ( 5.9)1.2 ( 1.7)3.7 ( 3.3)4.4 ( 3.6)9.3 ( 6.4)values are expressed as the mean ( standard deviation ) subject characteristics and physical activity performed at baseline for the ibuprofen and placebo groups values are expressed as the mean ( standard deviation ) there was a significant decrease in whole - body lean tissue mass over time ( ibu , 1.1 1.0 kg ; pla , 0.7 1.4 kg ; p < 0.05 ; fig . 1 ) , with no differences between groups . there was a significant increase ( p < 0.05 ) in muscle size of the knee extensors ( ibu , 0.3 0.6 cm ; pla , 0.2 0.7 cm ) , ankle dorsiflexors ( ibu , 0.5 0.8 cm ; pla , 0.1 0.5 cm ) , and ankle plantar flexors ( ibu , 0.3 0.9 cm ; pla , 0.5 0.9 cm ; table 2 ) , and leg press ( ibu , 20.6 18.0 kg ; pla , 20.0 20.0 kg ; fig . 2 ) and chest press strength ( ibu , 5.1 9.5 kg ; pla , 8.1 7.6 kg ; fig . 3 ) over 9 weeks of training , with no differences between groups.fig . 1change in lean tissue mass after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 13 ) groups . p < 0.05 , significant decrease over timetable 2muscle thickness measurements ( in centimeters ) for the elbow and knee flexors and extensors and ankle dorsiflexors and plantar flexors before and after 9 weeks of resistance exercisemuscle groupibuprofen ( n = 15)placebo ( n = 13)prepostprepostelbow flexors3.1 ( 0.8)3.0 ( 0.7)2.5 ( 0.6)3.1 ( 0.6)elbow extensors3.6 ( 0.7)3.8 ( 0.8)3.7 ( 0.8)3.9 ( 0.6)knee flexors5.0 ( 0.6)5.1 ( 0.5)5.3 ( 0.5)5.2 ( 0.9)knee extensors3.3 ( 0.7)3.6 ( 0.6)*3.5 ( 0.6)3.7 ( 0.5)*ankle plantar flexors4.6 ( 0.8)5.0 ( 1.0)*4.2 ( 0.6)4.7 ( 0.8)*ankle dorsiflexors3.3 ( 0.5)3.7 ( 0.9)*3.3 ( 0.4)3.4 ( 0.6)*values are expressed as the mean ( standard deviation)*p < 0.05 , significant increase with trainingfig . 2change in leg press strength after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 12 ) groups . 3change in chest press strength after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 12 ) groups . * p < 0.05 , significant increase over time change in lean tissue mass after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 13 ) groups . * p < 0.05 , significant decrease over time muscle thickness measurements ( in centimeters ) for the elbow and knee flexors and extensors and ankle dorsiflexors and plantar flexors before and after 9 weeks of resistance exercise values are expressed as the mean ( standard deviation ) * p < 0.05 , significant increase with training change in leg press strength after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 12 ) groups . * p < 0.05 , significant increase over time change in chest press strength after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 12 ) groups . * p < 0.05 , significant increase over time muscle size of the elbow flexors , elbow extensors , and knee flexors ( table 2 ) and body mass ( ibu : pre , 75.9 9.0 kg ; post , 74.2 8.1 kg ; pla : pre , 73.0 10.4 kg ; post , 72.4 10.8 kg ) did not change over time in either group . the total amount of exercise performed over the 9 weeks of training was similar between groups ( ibu , 9,344 1,700 kg ; pla , 10,286 1,321 kg ) . there were no adverse effects reported from the resistance exercise program , ibuprofen , or placebo . of the original 34 participants who volunteered for the study , 28 participants ( 15 ibuprofen , 13 placebo ; 5068 years of age ) completed the study . average resistance training compliance was 83.1 % ( 22 of 27 sessions performed ) for the ibuprofen group and 75 % ( 20 of 27 sessions performed ) for the placebo group.table 1subject characteristics and physical activity performed at baseline for the ibuprofen and placebo groupsgroupage ( years)body mass ( kg)height ( cm)strenuous activity ( times per week)moderate activity ( times per week)mild activity ( times per week)total activity ( times per week)ibuprofen ( n = 15)57.8 ( 5.1)75.9 ( 9.0)165.9 ( 6.2)1.9 ( 2.5)2.4 ( 1.5)4.6 ( 2.8)9.0 ( 3.1)placebo ( n = 13)56.5 ( 4.4)73.0 ( 10.4)163.1 ( 5.9)1.2 ( 1.7)3.7 ( 3.3)4.4 ( 3.6)9.3 ( 6.4)values are expressed as the mean ( standard deviation ) subject characteristics and physical activity performed at baseline for the ibuprofen and placebo groups values are expressed as the mean ( standard deviation ) there was a significant decrease in whole - body lean tissue mass over time ( ibu , 1.1 1.0 kg ; pla , 0.7 1.4 kg ; p < 0.05 ; fig . 1 ) , with no differences between groups . there was a significant increase ( p < 0.05 ) in muscle size of the knee extensors ( ibu , 0.3 0.6 cm ; pla , 0.2 0.7 cm ) , ankle dorsiflexors ( ibu , 0.5 0.8 cm ; pla , 0.1 0.5 cm ) , and ankle plantar flexors ( ibu , 0.3 0.9 cm ; pla , 0.5 0.9 cm ; table 2 ) , and leg press ( ibu , 20.6 18.0 kg ; pla , 20.0 20.0 kg ; fig . 2 ) and chest press strength ( ibu , 5.1 9.5 kg ; pla , 8.1 7.6 kg ; fig . 3 ) over 9 weeks of training , with no differences between groups.fig . 1change in lean tissue mass after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 13 ) groups . values are expressed as the mean standard deviation . * p < 0.05 , significant decrease over timetable 2muscle thickness measurements ( in centimeters ) for the elbow and knee flexors and extensors and ankle dorsiflexors and plantar flexors before and after 9 weeks of resistance exercisemuscle groupibuprofen ( n = 15)placebo ( n = 13)prepostprepostelbow flexors3.1 ( 0.8)3.0 ( 0.7)2.5 ( 0.6)3.1 ( 0.6)elbow extensors3.6 ( 0.7)3.8 ( 0.8)3.7 ( 0.8)3.9 ( 0.6)knee flexors5.0 ( 0.6)5.1 ( 0.5)5.3 ( 0.5)5.2 ( 0.9)knee extensors3.3 ( 0.7)3.6 ( 0.6)*3.5 ( 0.6)3.7 ( 0.5)*ankle plantar flexors4.6 ( 0.8)5.0 ( 1.0)*4.2 ( 0.6)4.7 ( 0.8)*ankle dorsiflexors3.3 ( 0.5)3.7 ( 0.9)*3.3 ( 0.4)3.4 ( 0.6)*values are expressed as the mean ( standard deviation)*p < 0.05 , significant increase with trainingfig . 2change in leg press strength after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 12 ) groups . 3change in chest press strength after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 12 ) groups . * p < 0.05 , significant increase over time change in lean tissue mass after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 13 ) groups . values are expressed as the mean standard deviation . * p < 0.05 , significant decrease over time muscle thickness measurements ( in centimeters ) for the elbow and knee flexors and extensors and ankle dorsiflexors and plantar flexors before and after 9 weeks of resistance exercise values are expressed as the mean ( standard deviation ) * p < 0.05 , significant increase with training change in leg press strength after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 12 ) groups . * p < 0.05 , significant increase over time change in chest press strength after 9 weeks of resistance exercise for ibuprofen ( ibu , n = 15 ) and placebo ( pla , n = 12 ) groups . p < 0.05 , significant increase over time muscle size of the elbow flexors , elbow extensors , and knee flexors ( table 2 ) and body mass ( ibu : pre , 75.9 9.0 kg ; post , 74.2 8.1 kg ; pla : pre , 73.0 10.4 kg ; post , 72.4 10.8 kg ) did not change over time in either group . the total amount of exercise performed over the 9 weeks of training was similar between groups ( ibu , 9,344 1,700 kg ; pla , 10,286 1,321 there were no adverse effects reported from the resistance exercise program , ibuprofen , or placebo . to our knowledge , this is the first study to determine the effects of low - dose ibuprofen ( i.e. , 400 mg ) after resistance exercise sessions in postmenopausal women . contrary to our hypotheses , ibuprofen had no greater effect on muscle mass or strength compared to placebo . old healthy rats ( 20 months of age ) who were given an ibuprofen - enriched chow ( 700 mg / kg body mass ) for 5 months experienced a significant reduction in inflammation , which corresponded with an increase in hind limb muscle mass ( + 1.32 g ) . in the postprandial state , ibuprofen - treated rats experienced a significant increase in phosphorylation of forkhead 03a ( foxo3a ) , a transcription factor that activates genes that express proteins such as ubiquitin - activated ligases which are involved in protein catabolism in the ubiquitin proteasome pathway [ 29 , 30 ] . phosphorylation of foxo3a prevents its entry into the nucleus , which inhibits muscle protein catabolism . using a double - blind , placebo - controlled , repeated - measures design , trappe et al . found that healthy older adults ( n = 13 , nine males , four females ; 64 years ) who ingested the maximal daily recommended dosage of ibuprofen ( 1,200 mg ) during structured resistance exercise ( bilateral knee extension , three sets of 10 repetitions to fatigue ; 3 days / week ; 12 weeks ) experienced a significant increase in quadriceps muscle volume ( ibu , + 11 % ; pla , + 8.4 % ) and muscle strength ( ibu , + 17.5 % ; pla , + 15 % ) . the greater increase in muscle mass from ibuprofen may be the result of a decrease in cox and pge2 activity and muscle protein catabolism , leading to greater net protein retention and muscle accretion over time . previous findings in rats have also shown that cox inhibition reduced the production of pge2 and suppressed muscle protein catabolism , leading to greater net protein balance . in the present study , ibuprofen had no effect on muscle size of the elbow flexors and extensors or knee flexors . perhaps a longer training period ( > 9 weeks ) with more frequent ( daily ) ingestion of ibuprofen ( > 400 mg ) is required to produce significant increases in muscle mass and strength compared to placebo in postmenopausal women . there was a small , yet significant , decrease in whole - body lean tissue mass over the 9-week training period . while it is somewhat puzzling as to why lean tissue mass did not increase with repeated training sessions , it is possible that acute caloric deficit ( i.e. , dietary protein ) for 1 h postexercise influenced our results . however , in the postabsorptive state following exercise , muscle protein balance remains negative ( i.e. , protein catabolism > protein synthesis ) until amino acids are consumed . protein ingestion following resistance exercise increases the rates of muscle protein synthesis [ 3234 ] , which may lead to muscle hypertrophy over time . in healthy older adults , protein supplementation immediately following resistance exercise for 12 weeks increased muscle cross - sectional area , mean fiber area , and muscular strength . however , delaying dietary protein intake for 2 h resulted in no beneficial effects . these results imply that protein intake after exercise is crucial for muscle protein synthesis to proceed and delaying protein intake jeopardizes muscle protein accretion over time in older adults . postmenopausal women in our study were instructed to refrain from food intake ( i.e. , calorie deficit ) for 1 h postexercise so that a valid estimate of the effects of ibuprofen on muscle could be made . however , this postabsorptive protocol may have limited the muscle protein synthetic response from resistance exercise , which may have impaired muscle accretion over time . unfortunately , no direct measure of muscle protein synthesis or protein catabolism was made and habitual dietary intake was not assessed , which limits our ability to determine whether ibuprofen or diet influenced muscle protein balance over time . ingestion of low - dose ibuprofen ( i.e. , 400 mg ) in the postabsorptive state following resistance exercise does not lead to greater gains in muscle mass or strength compared to placebo in postmenopausal women . these results are in contrast to previous research showing that 1,200 mg / day of ibuprofen was effective for increasing muscle mass and strength in older men and women . it is , therefore , likely that the effective ibuprofen dose for increasing muscle mass is higher than 400 mg in postmenopausal women . future research should investigate the effects of different doses of ibuprofen ( 4001,200 mg ) during longer - term ( > 9 weeks ) resistance exercise on the rates of muscle protein synthesis and protein catabolism in postmenopausal women in the postprandial state . | backgroundpostmenopausal women typically experience accelerated muscle loss which has a negative effect on strength .
the maximum daily recommended dosage of ibuprofen ( 1,200 mg ) following resistance exercise has been shown to increase muscle hypertrophy and strength in older adults .
this study aimed to determine the effects of low - dose ibuprofen ( 400 mg ) immediately following resistance exercise sessions on muscle mass and strength in postmenopausal women.methodsparticipants were randomized to ingest ibuprofen ( ibu : n = 15 , 57.8 5.1 years , 75.9 9.0 kg , 165.9 6.2 cm , bmi = 28 4 kg / m2 ) or placebo ( pla : n = 13 , 56.5 4.4 years ,
73.0 10.4 kg , 163.1 5.9 cm , bmi = 26 9 kg / m2 ) immediately following resistance exercise ( 11 whole - body exercises ) , which was performed 3 days / week , on nonconsecutive days , for 9 weeks . prior to and following training , measures were taken for lean tissue mass ( dual - energy x - ray absorptiometry ) , muscle size of the elbow and knee flexors and extensors and ankle dorsiflexors and plantar flexors ( ultrasound ) , and strength ( one - repetition maximum leg press and chest press).resultsover the 9 weeks of training , there were significant changes ( p < 0.05 ) in lean tissue mass ( ibu , 1.1 1.0 kg ; pla , 0.7 1.4 kg ) , muscle size of the knee extensors ( ibu , 0.3 0.6 cm ; pla , 0.2 0.7 cm ) , ankle dorsiflexors ( ibu , 0.5 0.8 cm ; pla , 0.1 0.5 cm ) , and ankle plantar flexors ( ibu , 0.3 0.9 cm ; pla , 0.5 0.9 cm ) , leg press strength ( ibu , 20.6 18.0 kg ; pla , 20.0 20.0 kg ) , and chest press strength ( ibu , 5.1 9.5 kg ; pla , 8.1 7.6 kg ) , with no differences between groups.conclusionlow-dose ibuprofen following resistance exercise has no greater effect on muscle mass or strength over exercise alone in postmenopausal women . |
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evidence from numerous epidemiological studies indicates that type 2 diabetes ( t2d , a noninsulin - dependent form of diabetes mellitus ) is associated with a two- to three - fold increase in the relative risk for alzheimer 's disease ( ad ) , independent of the risk for vascular dementia [ 19 ] . experimental evidence suggests that abnormalities in insulin metabolism under diabetic conditions could mechanistically influence the onset of ad via modulation of the synthesis and degradation of amyloidogenic beta - amyloid ( a ) peptides . for example , insulin itself may significantly promote a accumulation by accelerating amyloid precursor protein / a trafficking from the trans - golgi network , a major cellular site for a generation , to the plasma membrane . moreover , elevated circulating insulin contents under diabetic conditions may also promote amyloid accumulation by direct competition with a for the insulin - degrading enzyme ( ide ) , and therefore may limit a degradation by ide [ 11 , 12 ] . in addition to the direct roles of insulin and ide , accumulating evidence shows that under diabetic conditions , impairments in certain insulin receptor- ( ir- ) responsive cellular signaling pathways might also mechanistically promote ad - related neuropathology and cognitive deterioration [ 1318 ] . building on this observation , a recent hypothesis implicates impaired insulin signaling in the brain as a common underlying cause of sporadic ad , regardless of diabetic or nondiabetic status . cellular insulin signaling is initiated by the coupling of extracellular insulin with the insulin receptor in the plasma membrane , which leads to ir activation and subsequent promotion of cellular ir - signaling processes . despite the central role of ir activation in cellular ir - signaling processes , there is limited and conflicting information available on the regulation and activity of ir in the brains of sporadic ad cases . in particular , frlich et al . reported significantly increased ir - binding activity in the brains of sporadic ad cases . moloney et al . recently reported no change in the levels of total ir and ir subunits , but found an aberrant subcellular distribution of ir and ir in temporal cortex specimens from cases characterized by severe ad neuropathology , suggesting the presence of compromised ir signaling in surviving ad neurons . none of the studies indicate the diabetic status of the study subjects . a recent study by liu et al . reported no change in the total ir subunit level in postmortem frontal cortex specimens from ad cases without diabetes , but there is little information given on the criteria by which the absence of diabetes was determined , and there is no information regarding the activation status of the insulin receptor . accumulating epidemiological and experimental evidence suggests that in the ad brain , impairments in select cellular signaling pathways associated with ( but not necessarily limited to ) ir signaling might mechanistically promote ad phenotypes [ 2 , 3 , 6 , 7 , 1417 ] . among these , impaired glycogen synthase kinase 3 ( gsk3 ) function in the ad brain has been considered pivotal for disease development [ 2427 ] . gsk3 is a ubiquitously expressed , highly conserved serine / threonine kinase involved in numerous cellular processes . there are two mammalian gsk3 isoforms , gsk3 and gsk3 , with gsk3 being particularly abundant in the central nervous system . gsk3 and are constitutively active , but are inactivated by ir - responsive akt - mediated phosphorylation at [ ser21]-gsk3 and [ ser9]-gsk , respectively . some studies argue that overactivity of gsk3 plays a critical role in the pathogenesis of both sporadic and familial ad ( for review , see ) . accordingly , gsk3 hyperactivity may contribute to ad by increasing tau hyperphosphorylation , promoting a production , and/or stimulating brain inflammatory responses . however , contrary to this hypothesis there are studies that show evidence of reduced total gsk3 contents and activity in the ad brain [ 24 , 25 ] . in particular , a study by baum et al . revealed significantly reduced contents of total ( nonphosphorylated ( active ) and phosphorylated ( inactive ) ) gsk3 and gsk3 in the ad brain . a second study by griffin et al . observed significantly reduced contents of gsk3 , coupled with a significantly elevated ratio of ser9-phospho - gsk3/total gsk3 , implicating inactivation of gsk3 in ad compared to control brain specimens . none of the studies on the regulation of gsk3 in the ad brain indicate the diabetic status of the study subjects . in a more recent paper , liu et al . reported no significant change in total gsk3 or phosphorylated gsk3 protein levels in the brains of nondiabetic , sporadic ad cases . while t2d is a risk factor for ad , there is little information available on the regulation and activity of ir in the ad brain , either in the absence or in the presence of comorbid diabetic conditions . insulin binding to ir leads to rapid autophosphorylation of specific tyrosine residues in the ir subunit , which converts ir into a catalytic active conformation that is necessary for ir signal transduction . for example , ir autophosphorylation at tyr1162/1163 is critical for stabilizing ir in a catalytically active conformation . the present study was designed to explore the regulation of ir contents and ir activation in the brains of nondiabetic ad cases . we assessed the contents of total ( nonphosphorylated and phosphorylated ) ir and [ tyr1162/1163]-phosphorylated ir as surrogate indices of , respectively , total ir content and ir activation in the brains of nondiabetic ad cases as a function of clinical ad dementia and ad neuropathology . results from our studies demonstrated that there is no detectable change in ir content and activation in the brain . nonetheless , we found significantly lower levels of total gsk3 protein in the brains of nondiabetic ad cases , suggesting that impaired ir signaling mechanisms might contribute to the onset and/or progression of ad dementia in the absence of diabetes . mice were sacrificed by cervical dislocation and freshly isolated brain specimens were either immediately frozen or stored postmortem for up to 6 hours at room temperature before homogenization for analysis of total and phosphorylated ir contents . tissue specimens were homogenized in tris / triton solution : 250 mm sucrose , 50 mm tris - hcl ( ph 7.4 ) , 1 mm edta , 2 mm egta , 1% triton x100 containing 1 mm pmsf and cocktails of proteinase / phosphatase inhibitors ( pierce biotech inc , rockford , il , usa ) . total protein concentration in the tissue homogenates was determined with a cbqca quantitation kit ( molecular probes inc , eugene , or , usa ) . aliquot samples of total protein contents ( 15 g ) were loaded in triplicates onto pre - cast 8% precise protein gels ( pierce biotech inc , rockford , il , usa ) under reducing conditions . total ( nonphosphorylated and phosphorylated ) ir and phosphorylated ir were detected , respectively , using mouse monoclonal l55b10 antibodies for total ir and rabbit monoclonal 19h7 antibodies for [ tyr 1150/1151]-phosphorylated ir ; both antibody preparations were obtained from cell signaling technology inc . image detection was conducted using infrared fluorescence detection ( irdye 680 or 800 goat antiappropriate species igg , li - cor biosciences , lincoln , ne , usa ) and scanned using the odyssey infrared imaging system ( li - cor biosciences , lincoln , ne , usa ) . images were analyzed and quantified using odyssey software ver.3 ( li - cor biosciences , lincoln , ne , usa ) . human postmortem temporal muscle and hippocampal formation specimens from ad and age - matched non - ad cases were obtained from the alzheimer 's disease brain bank of the mount sinai school of medicine . the cases selected had no significant neuropathological features or had only neuropathological features associated with ad [ 32 , 33 ] . cognitive status of the cases was assessed based on the cognitive dementia rating ( cdr ) , which is generated using a multistep evaluation of cognitive and functional status during the last 6 months of life , as previously reported . moreover , only nondiabetic cases were selected for this study ; cases with a premorbid history of diabetes were excluded . diabetic ( t2d ) or nondiabetic cases were identified using criteria previously described [ 35 , 36 ] . our analysis included only cases with no record of diabetes ( absence of reported history and failure to meet blood chemistry - based criteria ) ; cases with a premorbid history of diabetes were excluded ( i.e. , plasma glucose concentration > 200 mg / dl , fasting glucose > 126 mg / dl , 2-hour plasma glucose > 200 mg / dl during oral glucose test , and impaired fasting glucose was defined as 110125 mg / dl ( 6.17.0 mmol / l ) ) . cdr 0 : cognitive normal ( n = 10 ) ; cdr 0.5 , at high risk of developing ad dementia ( n = 9 ) ; cdr 1 , mild ad dementia ( n = 11 ) ; cdr 2 , moderate ad dementia ( n = 13 ) ; cdr 5 , severe ad dementia ( n = 19 ) . the extent of neuritic plaque ( np ) and neurofibrillary tangles ( nfts ) staining in the brain ( entorhinal cortex ) was assessed in accord with the consortium to establish a registry for alzheimer 's disease ( cerad ) neuropathologic battery . the density of nps and nfts were rated on a 4-point scale : 0 , absent ; 1 , sparse ; 3 , moderate , and 5 , severe . multiple ( ~5 ) high power ( 200 , 0.5-mm ) fields were examined in each histological slide from multiple regions according to the cerad regional sampling scheme . all investigators were masked to the clinical diagnosis of each case until all histological and biochemical analyses were completed and values were assigned to each specimen . the contents of a
140 and a
142 in the hippocampal formation were assessed as previously described . briefly , frozen tissue samples were homogenized in a buffer containing 70% formic acid and 100 mmol / l betaine , and soluble a
140 and a
142 were quantified by enzyme - linked immunosorbent assays ( elisas ) using , respectively , synthetic a
140 and a
142 ( us peptides , fullerton , ca , usa ) as standards . microtiter plates were coated with 2 mg / ml monoclonal antibody 4g8 ( senetek , maryland heights , mo , usa ) , which recognizes an epitope between residues 17 and 20 of a. unoccupied binding sites on the plates were blocked by incubation with casein . samples and standards were applied in quadruplicate and incubated for 48 hours at 4c . after the a
140 and a
142 capture phase , the plates were probed with , respectively , an a
140 or an a
142 c - terminal - specific antibody , followed by incubation with a reporter antibody ( alkaline phosphatase - conjugated anti - rabbit igg , -chain - specific ) ( jbl scientific , san luis obispo , ca , usa ) . the assay was developed using an alkaline phosphatase substrate ( attophos ; jbl scientific ) , yielding a fluorescent product , and analyzed with a 96-well fluorescence reader ( cytofluor ; millipore , bedford , ma , usa ) . frozen banked tissue ( hippocampal formation or temporal muscle ) specimens were powderized under liquid nitrogen and were then homogenized in ice - cold cell lysis buffer ( 20 m tris / hcl , ph7.5 , 150 mm nacl , 1 mm ecta , 1 mm egta , 1% triton x-100 , 2.5 mm sodium pyrophosphate , 1 mm beta - glycerophosphate , 1 mm na3vo4 , 1 ug / ml leupeptin and 1 mm phenyl sulphonyl fluoride ) using a hand held biovortexer or pellet pestle motor ( kontes , northbrook , il , usa ) as previously described [ 14 , 39 ] . the homogenates were sonicated three times for 10 seconds each ( sonic dismembrator model 500 , fisher scientific ) and were then centrifuged at 13,000 xg for 15 min . supernatants were collected and protein concentrations were determined using bradford protein assays ( bio - rad laboratories , hercules , ca , usa ) . protein extracts ( 25 g ) were separated on 10% sds - page under reducing conditions and transferred to pvdf membranes using 10 mm caps ph11 , 10% methanol at 4c . the membranes were blocked with 5% blocking grade nonfat dry milk in 10 mm tris / hcl ph7.6 , 140 mm nacl , 0.1% tween-20 , before being incubated with a primary anti - ir antibody ( rabbit polyclonal igg , c-19 , 1 : 500 dilution ; santa cruz biotechnology inc . , santa cruz , ca , usa ) . membranes were washed and incubated with an hrp - conjugated secondary antibody , washed , and bands were detected using chemiluminescence methodology ( amersham ecl plus western blotting detection system , ge healthcare , uk ) followed by exposure to kodak x - ray films . films were scanned and appropriate protein band densities were quantified with bio - rad quantity - one software ( bio - rad laboratories , hercules , ca , usa ) . louis , mo , usa ) on the same blots served as a loading control . assessments of [ tyr1162/1163]-phosphorylated ir protein contents were conducted using a commercial sandwich [ tyr1162/1163]-phosphorylated ir elisa assay ( biosource international , inc . , camarillo , ca , usa ) that is specific for ir and does not cross - react with igf-1r. in this study , the [ tyr1162/1163]-phosphorylated ir elisa was conducted according to the manufacturer 's recommendations . a lyophilized lysate from insulin - stimulated human ir transfected chinese hamster ovary cells provided by the manufacturer served as a quantitative standard ; 1 unit of standard is equivalent to the amount of ir [ tyr1162/1163 ] derived from 0.6 g of ir ( -subunit ) in transfected chinese hamster ovary cells stimulated with 100 nm insulin . contents of total gsk3 / [ including both phosphorylated ( inactive ) and nonphosphorylated ( active ) forms ] in hippocampal formation specimens were assessed by western blot . in this study , 25 g of lysate proteins was assayed using a commercial anti - gsk3 / antibody ( mouse monoclonal 1h8 antibody , dilution 1 : 3,500 ; calbiochem , san diego , ca , usa ) that simultaneously detects total gsk3 and total gsk3 ( inactive phosphorylated and active nonphosphorylated gsk3 / ) ; identification of gsk3 and gsk3 is based on their unique molecular sizes : 51 kda for gsk3 and 47 kda for gsk3. statistical analysis was performed using the prism software package ( graphpad software , inc , san diego , ca , usa ) . analysis of variance ( anova ) was used to evaluate differences in mean values among three or more groups , and the dunnett t - test was used to test the significance of the differences in means . correlation analysis between two variables was done using the pearson parametric method followed by 2-way analysis of the p value . patient information including age , postmortem interval , gender , and neuropathological findings for cases assessed in this study is summarized in table 1 . only nondiabetic cases were selected for this study ; cases with a premorbid history of diabetes were excluded . analysis of variance indicated that there were no significant differences among the cdr groups with respect to age ( p = .40 ) and postmortem interval ( p = .82 ) at the time of death . postmortem interval ( pmi ) is known to affect the phosphorylation status of a number of signaling proteins . for example , li et al . examined a number of signaling proteins , such as erk , jnk , rsk , creb , and atf-2 proteins , in mouse brain specimens at 0 , 8 , 24 , and 48 hrs postmortem , and demonstrated dramatically reduced contents of phosphorylated species for each of these proteins by 8 hrs postmortem . the cohort of 62 nondiabetic cases selected for our present study were characterized by a relatively shorter than average postmortem interval , ranging from a minimal average postmortem interval of 4.3 0.4 h for the cdr 0.5 cases to a maximal average postmortem interval of 5.44 0.91 h for the cdr 0 cases ( table 1 ) . in a series of control studies using mouse brain specimens , we explored the potential impact of similarly short postmortem intervals on the detection of tyrosine phosphorylated ir in brain specimens . we dissected mouse brain tissue and assessed [ tyr1150/1151]-phosphorylated ir contents from tissue specimens kept at room temperature and found no significant changes in the detection of tyrosine phosphorylated ir levels ( normalized to total ir ) from mouse brain specimens that were kept at room temperature for up to 6 hrs postmortem ( figure 1 ) . this suggests that the relatively short postmortem intervals that are associated with the human brain specimens used in our present study likely have no appreciable impact on the detection of tyrosine - phosphorylated ir contents from these specimens . we assessed temporal muscle and hippocampal formation specimens from the same cases to explore the regulation of ir in the periphery and in the brain among nondiabetic cases across cdrs . in these studies , total ir content was assessed by western blot analysis of total ir peptide contents using a specific antibody that does not cross - react with igf-1r. the content of [ tyr1162/1163]-phosphorylated ir , assessed using a specific elisa that does not cross - react with igf-1r , was used as a surrogate index of ir activation . consistent with the selection of nondiabetic cases for this study , we found no difference in the contents of total ir ( figure 2(a ) ; anova , p = .976 ) and [ tyr1162/1163]-phosphorylated ir ( figure 2(b ) ; anova , p = .478 ) in peripheral temporal muscle across the cdr groups . we found no significant difference in the contents of total ir ( figure 2(c ) ; anova , p = .220 ) and [ tyr1162/1163]-phosphorylated ir ( figure 2(d ) ; anova , p = .425 ) in the hippocampal formation across the cdr groups among the nondiabetic cases assessed in this study . we continued to explore potential interrelationships between total ir and [ tyr1162/1163]-phosphorylated ir contents in the brain and ad neuropathology among the nondiabetic cases . we found no correlation between total ir content and the contents of a
142 ( figure 3(a ) ; p = .205 ) or a
140 ( figure 3(b ) ; p = .271 ) peptides in the hippocampal formation . more importantly , we found that the content of [ tyr1162/1163]-phosphorylated ir in the hippocampal formation is not correlated with the contents of a
142 ( figure 3(c ) ; p = .684 ) or a
140 ( figure 3(d ) ; p = .681 ) peptides . consistent with our observation that ad - dementia in nondiabetic cases is not associated with significant changes in the contents of total ir or [ tyr1162/1163]-phosphorylated ir in the brain ( figures 2(c)-2(d ) ) , we found that total or [ tyr1162/1163]-phosphorylated ir contents are not correlated with ad - type amyloid neuritic plaque ( np ) or neurofibrillary tangle ( nft ) neuropathology in the brain ( figures 4(a)4(d ) ) . in particular , based on histological assessments of neuritic plaques and neurofibrillary tangles using the 4-point cerad rating , we found no correlation between the content of total ir in the hippocampal formation and either nps ( figure 4(a ) ; p = .749 ) or nfts ( figure 4(c ) ; p = .516 ) . similarly , we found no correlation between the contents of [ tyr1162/1163]-phosphorylated ir in the hippocampal formation and either nps ( figure 4(b ) ; p = .283 ) or nfts ( figure 4(d ) ; p = .912 ) . numerous studies have documented changes in ir - responsive cellular signaling pathways in the brain . for example , data has shown reduced gsk3 and contents and activities [ 24 , 25 ] in the ad brain . consistent with these observations , we observed significantly lower contents of total gsk3 ( figure 5(a ) ; p < .05 ) and gsk3 ( figure 5(b ) ; p < .005 ) in the hippocampal formation of cdr 1 , 2 and 5 cases in comparison to neurological control ( cdr 0 ) cases . interestingly , we found no correlation between the contents of [ tyr1162/1163]-phosphorylated ir and either total gsk3 ( figure 4(c ) ; pearson correlational analysis , p = .318 ) or total gsk3 ( figure 4(d ) ; pearson correlation analysis , p = .308 ) in the hippocampal formation . thus , our evidence suggests that downregulation of total gsk3 / contents in the brains of the nondiabetic ad cases analyzed in this study might be mediated by mechanisms independent of ir activation . recent hypotheses raised the possibility that impaired ir signaling in the brain might be a common underlying cause of sporadic ad [ 19 , 23 , 41 ] . although cellular ir activation is the first , and a necessary , step in cellular ir - signaling processes , there is no consensus on the regulation of ir content and ir activation in the brains of sporadic ad cases [ 19 , 21 , 23 ] . with the exception of a recent publication by liu et al . , , it is not known whether any of the ad and control cases used in previously reported studies are characterized by t2d . it is possible that the outcomes in these reports might be complicated by inclusion of t2d cases . the recent publication by liu et al . reported no significant change in ir levels in the brains of nondiabetic ad cases , but did not report the status of ir activation . this study was designed to investigate the contents of ir and [ tyr1162/1163]-phosphorylated ir as surrogate indices of , respectively , total ir contents and ir activation in the brains of nondiabetic ad cases as a function of ad dementia and ad - type neuropathology . among the nondiabetic cases examined in this study , we found that total ir contents in postmortem hippocampal specimens from cases characterized by mild cognitive impairment ( cdr 0.5 ) , mild ad dementia ( cdr 1 ) , moderate ad dementia ( cdr 2 ) and severe ad dementia ( cdr 5 ) were comparable to levels that were found in cognitive normal ( cdr 0 ) control cases . our findings are consistent with observations by moloney et al . and liu et al . , who reported comparable levels of total ir and ir proteins in postmortem temporal cortex specimens from severe ad and control cases in addition to total ir protein contents , evidence from our nondiabetic cohort also revealed similar levels of [ tyr1162/1163]-phosphorylated ir in hippocampal specimens from cdr 0.5 , 1 , 2 , and 5 cases compared to control cdr 0 cases . moreover , we found that the severity of amyloid and tau ad - neuropathology among nondiabetic ad cases was not correlated with the contents of either total ir or [ tyr1162/1163]-phosphorylated ir in the hippocampal formation . collectively , our observations tentatively suggest that nondiabetic sporadic ad is characterized by normal ir content and ir activation in the brain . interestingly , moloney et al . observed aberrant subcellular distributions of ir and ir proteins among surviving neurons in brain specimens from severe ad cases , without the consideration of the diabetic / nondiabetic status of these cases . future studies will be necessary to examine whether nondiabetic cdr 0.5 , 1 , 2 , and 5 cases might also be characterized by similar aberrant subcellular distribution of ir/ and [ tyr1162/1163]-phosphorylated ir in the brain . activation of the ir leads to the modulation of a large number of cellular signaling processes [ 4244 ] . however , many of these cellular signaling molecules such as akt and gsk3 / are also regulated by other signaling processes [ 4549 ] . for example , activation of ir or insulin - like growth factor 1 receptor ( igf-1r ) both lead to receptor - mediated tyrosine phosphorylation of adaptor proteins such as insulin receptor substrate proteins that , in turn , modulate the activation of akt , gsk3 [ 50 , 51 ] , extracellular signal - regulated kinase ( erk ) , and other signaling pathways . accumulating epidemiological and experimental evidence suggests that impairments in select ir - associated cellular signaling pathways in the ad brain might mechanistically promote the ad phenotype [ 2 , 3 , 6 , 7 , 1417 , 25 ] . among cellular processes that are typically associated with ir - signaling , impaired gsk3 / function in the ad brain consistent with previous reports [ 24 , 25 ] , we observed significantly lower levels of total gsk3 / in brain specimens from nondiabetic sporadic ad cases examined in this study . our observation is consistent with evidence from griffin et al . , which demonstrated increased akt activation coinciding with elevated levels of inactive ser9-phosphorylated gsk-3 in the temporal cortex of ad cases . ir ( as well as the igf-1r ) signaling pathways are known to regulate akt , gsk3 / and other signal transduction mediators , primarily by modulating the phosphorylation status and thereby the activities of these signal transduction components [ 5052 , 54 ] . based on this consideration and on our observation suggesting normal ir contents and ir activation in brain specimens from our study cohort , downregulation of total gsk3 / contents in the brains of nondiabetic sporadic ad cases is likely mediated by mechanisms independent of ir activation . additional studies will be necessary to clarify whether there might be changes in the regulation of other ir - associated cellular signaling mechanisms in the brains of nondiabetic cases , and the mechanisms by which cellular contents and activities of akt , gsk3 / , and other ir mediators might be modulated in the ad brain . nonetheless , consistent with a recent report by moloney et al . , our observation suggests that , in spite of our evidence suggesting normal ir contents and ir activation , impaired ir signaling mechanisms in the brains of nondiabetic sporadic ad cases might contribute to the onset and/or progression of ad dementia . numerous epidemiological studies have linked t2d with an increased risk for ad [ 2 , 3 , 6 , 7 ] . we and others demonstrated that diet - induced t2d in the tg2576 ad mouse model leads to the promotion of ad - type amyloid neuropathology and cognitive deterioration , which are both associated with impaired ir activity and ir signaling the brain . while our present studies suggest the existence of impaired ir signaling in the brains of nondiabetic sporadic ad cases , a recent study by liu et al . suggests that ad and t2d may induce impaired ir signaling in the brain via different mechanisms than those implicated in our studies , and that the presence of t2d may exacerbate ir signaling impairments in the ad brain . there is an increasing effort to develop novel ad therapeutics based on the promotion of ir - signaling processes by either directly inducing ir activation ( e.g. , nasal insulin inhalation [ 5557 ] ) or by applying insulin - sensitization measures ( e.g. , ppar activators [ 41 , 58 , 59 ] ) that stimulate downstream ir - signaling . prior studies have not yet explored the potential impact of comorbid diabetic versus nondiabetic conditions on the regulation of ir activation in the ad brain . results from our study demonstrating reduced contents of total gsk3 / in the brains of nondiabetic sporadic ad cases suggest that , even in the absence of comorbid diabetic conditions , impaired downstream ir signaling processes in the ad brain may contribute to the onset and/or progression of ad phenotypes . this would support the application of insulin - sensitization therapeutic strategies in nondiabetic , sporadic ad . while accumulating experimental evidence suggests that diabetic conditions could lead to reduced ir activity in the brain [ 14 , 15 ] , our present study found no detectable changes in ir activity in the brains of nondiabetic sporadic ad cases . based on this , we suggest that , in comparison to nondiabetic sporadic ad cases , sporadic ad cases with concomitant diabetic conditions may respond better to therapeutic strategies such as intranasal insulin administration that are designed to directly target ir in the brain . | we investigated the contents of the insulin receptor - beta subunit ( ir ) and [ tyr1162/1163]-phosphorylated ir as surrogate indices of total ir content and ir activation in postmortem hippocampal formation brain specimens from nondiabetic sporadic alzheimer 's disease ( ad ) cases .
we found no significant changes in the brain contents of total ir or [ tyr1162/1163]-phosphorylated ir , suggesting normal ir content and activation in the brains of nondiabetic sporadic ad cases .
moreover , total ir and [ tyr1162/1163]-phosphorylated ir levels in the hippocampal formation are not correlated with the severity of amyloid or tau - neuropathology .
exploring the regulation of glycogen synthase kinase 3 ( gsk3 ) / , key ir - signaling components , we observed significantly lower levels of total gsk3 / in brain specimens from nondiabetic ad cases , suggesting that impaired ir signaling mechanisms might contribute to the onset and/or progression of ad dementia .
outcomes from our study support the development of insulin - sensitizing therapeutic strategies to stimulate downstream ir signaling in nondiabetic ad cases . |
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aortic root dilatation is a hazardous complication in patients with marfan syndrome ( mfs ) , an heritable connective tissue disorder equally prevalent all over the world [ 1 , 2 ] . these mutations induce abnormal or deficient fibrillin-1 protein affecting the structural integrity of the vascular extracellular matrix , and have been described to enhance the release of transforming growth factor- ( tgf- ) . we have shown that plasma tgf- was indeed elevated in mfs patients , which correlated to increased aortic root diameter and aortic root dilatation rate ( aodr ) . current treatment comprises prophylactic aortic root replacement and -blocker therapy , which has significantly improved the life expectancy of patients with mfs . however , cardiovascular complications remain a problem [ 6 , 7 ] . in a mfs mouse model , this losartan effect was attributed to reduced tgf- expression , which was mimicked by treatment with neutralising tgf- antibodies . these findings in mice have resulted in the initiation of multiple studies assessing the effect of losartan on aodr in mfs patients . recently , we demonstrated that losartan reduced aodr in a randomised and prospective cohort of adult patients with mfs ( compare trial ) [ 9 , 10 ] . in addition , a small study in 20 children revealed beneficial effects of losartan on aodr . [ 11 , 12 ] however , the responsiveness to losartan treatment was diverse , which may depend on variability in expression and release of tgf-. in order to determine the role of tgf- as a therapeutic biomarker for the effectiveness of losartan therapy on aodr , we performed a sub - study of the compare trial and revealed that tgf- is an indirect effector of aortic dilatation . for this sub - study we measured baseline tgf- levels of 99 mfs patients , all monitored by the academic medical centre amsterdam , of whom 55 were randomised to 100 mg losartan and 44 to no losartan . in 42 patients on losartan therapy , plasma tgf- levels in order to determine aodr , mfs patients underwent magnetic resonance imaging of the aorta at baseline and after 3 years of follow-up.fig . 1
a flowchart shows an overview of marfan syndrome patients and controls . b in 15 of the 42 mfs patients losartan normalised plasma tgf- levels to that of controls . in 27 of mfs patients c decrease in tgf- level is associated with an increase in aodr in patients using losartan therapy ( r = 0.47 , p = 0.02 ) . aodr = aortic root dilatation rate ; tgf- = transforming growth factor
a flowchart shows an overview of marfan syndrome patients and controls . b in 15 of the 42 mfs patients losartan normalised plasma tgf- levels to that of controls . in 27 of mfs patients c decrease in tgf- level is associated with an increase in aodr in patients using losartan therapy ( r = 0.47 , p = 0.02 ) . aodr = aortic root dilatation rate ; tgf- = transforming growth factor despite inter - laboratory variations in tgf- measurements throughout the medical world , our tgf- measurements show that mfs patients had significantly higher tgf- levels compared with healthy controls ( 121 pg / ml versus 54 pg / ml , p = 0.006 ) , which is in line with our previous results . surprisingly , 1 month of losartan therapy did not reduce circulating tgf- levels ( 101 pg / ml ; 95% ci : 27:229 pg / ml ; p = 0.12 ) ( fig tgf- levels were only normalised to the control level in 15 of the 42 patients ( p = 0.26 ) . in the remaining 27 patients tgf- did not decrease after one - month losartan therapy , and showed a significantly higher tgf- compared with the healthy controls ( 292 pg / ml versus 54 pg / ml , p = 0.028 ) . unexpectedly , after 3 years of losartan therapy , patients with a decrease in tgf- had a significantly higher aodr compared with patients without reduced tgf- levels ( 1.5 0.8 mm/3 years versus 0.5 1.2 mm/3 years , respectively ; p = 0.04 ) . in addition , 91% of the patients with a decrease in tgf- showed a significant increase in aodr despite losartan therapy , compared with 33% of the patients with increased / stable tgf- levels after losartan ( p = 0.013 ) ( fig . 1a , b ) . in order to explain these unexpected results , we compared baseline characteristics between these groups . patient groups were comparable , with the exception of baseline tgf- levels ( table 1 ) . patients showing a decrease in tgf- after losartan therapy had significantly elevated baseline tgf- levels compared with patients who did not show this decrease ( 189 166 pg / ml versus 94 113 pg / ml , p = 0.05 ) ( fig . interestingly , we found a linear association between the change in tgf- level and the increase in aodr in patients using losartan therapy ( r = 0.47 , p = 0.02 ) ( fig.1c ) . this effect could not be ascribed to -blocker therapy , because -blocker therapy + / losartan therapy was not different at any level ( data not shown).table 1patient characteristics according to the change in tgf- after losartan therapydecreased tgf-increased / stable tgf-mfs patients with:(n = 15)(n = 27 )
p - value
clinical features
mean age ( sd)38 ( 10)35 ( 11 ) sex ( male)7 ( 47)18 ( 67 ) baseline tgf- level*189 ( 166)94 ( 113)0.05cardiovascular aor dilatation15 ( 100)22 ( 81 ) mean aor diameter ( sd)45 ( 4)43 ( 6 ) aor operation4 ( 27)9 ( 33 ) mean age aor operation ( sd)31 ( 11)31 ( 15 ) mv prolapse12 ( 80)17 ( 63 ) mv repair1 ( 7)3 ( 11 ) fh of dissection6 ( 40)12 ( 44)dilatation of distal aorta2 ( 13)3 ( 11)-blocker10 ( 67)21 ( 78)dosage > 100 mg5 ( 33)14 ( 52)dosage < 100 mg5 ( 33)7 ( 26)values are given in absolute numbers ( percentage ) if not otherwise indicatedsd : standard deviation ; aor : aortic root ; mv : mitral valve ; fh : family history patient characteristics according to the change in tgf- after losartan therapy values are given in absolute numbers ( percentage ) if not otherwise indicated sd : standard deviation ; aor : aortic root ; mv : mitral valve ; fh : family history this sub - study highlights a paradoxical topic of tgf- in the pathogenesis of mfs . although 3 years of losartan therapy reduces the overall aodr in mfs patients , only one - third of mfs patients responded by a reduction of plasma tgf- after 1 month of losartan therapy . these responders had elevated baseline tgf- levels and an increase in aodr after 3 years , despite losartan therapy . considering treatment with 100 mg losartan is a sufficient dose to reduce aodr in mfs , and assuming that one - month treatment is sufficient to initiate a tgf- response , we have three possible explanations for our findings . the first explanation for the fact that losartan did not reduce overall tgf- levels is that tgf- is a readout of the diseased state of the aorta . losartan did reduce tgf- levels in a subgroup of mfs patients , yet these patients revealed a higher aodr after 3 years of therapy . this increase in aodr may be explained by the elevated baseline tgf- levels and the slightly , but non - significant larger aortic root dimension at baseline ( 45 4 mm versus 43 6 mm , p = 0.215 ) . these results suggest that elevated plasma tgf- is a marker for aortic damage , such as fibrosis , rather than the initial cause of aortic dilatation . a second explanation for the variable response of tgf- to losartan therapy comprises the multitude of fbn1 mutations . at present , more than 2900 different mutations have been described in the universal mutation database . most fbn1 mutations result in expression of mutated fibrillin-1 proteins , which are improperly folded . the abnormal fibrillin-1 protein may have a dominant - negative effect on the structure of the extracellular fibrillin network when it interacts with normal fibrillin-1 protein of the non - mutated allele and other extracellular matrix proteins . both the strength of the fibrillin-1 matrix may be changed as well as the release of tgf- that is captured in the fibrillin-1 network . mutations in one of the seven tgf- binding protein - like ( tb ) domains of the fbn1 gene may especially alter tgf- levels . other fbn1 mutations will lead to reduced fibrillin-1 protein levels as a result of deletion of the entire fbn1 gene on one allele or for example upon deletion of the first exon of fbn1 ( such that the mrna is not translated ) , causing haploinsufficiency. the reduced level of normal fibrillin-1 protein presumably results in a thinner fibrillin-1 matrix in the vasculature and thus in reduced aortic wall strength . in such patients , angiotensin ii ( angii ) activation the angii - mediated signalling cascade is a common inducer of tgf- production in the vessel wall and thus involved in the increased plasma tgf- levels in these patients . blocking the angii receptor-1 ( at1 ) with losartan will diminish tgf- production as well as other angii - mediated detrimental processes in the vessel wall such as blood pressure increase , enhanced pro - inflammatory responses , myofibroblast differentiation and reactive oxygen species ( ros ) generation . therefore , we hypothesise that in patients with an fbn1 mutation leading to haploinsufficiency , the effect of losartan on aortic dilatation is better compared with patients with a dominant - negative mutation . we anticipate that genetics plays a critical role in the tgf- response to losartan . a third explanation for the variable tgf- response to losartan therapy comprises variation in the abundance of at1 expression or activity , or by polymorphisms in the renin - angiotensin - aldosterone system ( raas ) . increased angii - mediated signalling as a result of this type of polymorphisms will coincide with increased tgf- production . for example , the ace deletion / insertion polymorphism is significantly associated with an increased risk for thoracic aortic aneurysm formation . however , in most studies tgf- signalling has been extrapolated from the abundance of smad2 activation ( psmad2 ) in the dilated aortic tissue . smad signalling is best known from its role in the tgf--induced signalling cascade , where it transfers the extracellular tgf- signal to the nucleus to act as a transcription factor and to regulate gene expression . before birth , tgf- is essential for the development of the cardiovascular system . in later life , this accentuates the possibility that tgf- is a result and not the cause of aortic damage . interestingly , angii can induce smad2 activation directly through its receptor at1 [ 20 , 21 ] , as well as indirectly by enhancing tgf- expression ( fig . 2 ) . thus , increased levels of tgf- and psmad2 levels in mfs may both result from increased angii - mediated signalling . when angii is considered a primary cause of aortic disease in mfs , other detrimental angii - mediated pathways may be responsible for initiation of arterial damage ( fig . 2 ) . 2schematic overview of proposed mechanism involving angii- and tgf--mediated signalling in aortic dilatation in mfs . angii directly activates smad2 ( psmad2 ) and increases tgf- production , which can be secreted and subsequently binds to its cell surface receptor and thereby increases smad2 activation further . losartan blocks at1 and thus inhibits angii - mediated signalling including smad2 activation , tgf- production , blood pressure increase , pro - inflammatory responses , myofibroblast differentiation and ros generation . angii = angiotensin ii ; at1 = angiotensin ii receptor type 1 ; psmad2 = phosphorylated smad2 ; ros = reactive oxygen species ; tgf- = transforming growth factor schematic overview of proposed mechanism involving angii- and tgf--mediated signalling in aortic dilatation in mfs . angii directly activates smad2 ( psmad2 ) and increases tgf- production , which can be secreted and subsequently binds to its cell surface receptor and thereby increases smad2 activation further . losartan blocks at1 and thus inhibits angii - mediated signalling including smad2 activation , tgf- production , blood pressure increase , pro - inflammatory responses , myofibroblast differentiation and ros generation . angii = angiotensin ii ; at1 = angiotensin ii receptor type 1 ; psmad2 = phosphorylated smad2 ; ros = reactive oxygen species ; tgf- = transforming growth factor in mice , chronic infusion of angii is known to affect the integrity of the vasculature resulting in aneurysms in the ascending and descending aorta [ 22 , 23 ] . we propose that the beneficial effect of losartan on aodr in mfs mice and patients [ 5 , 6 , 8 ] is obtained through inhibition of the unfavourable angii - mediated signalling cascades , involving tgf- synthesis and psmad2 signalling , blood pressure increase , myofibroblast differentiation , ros generation and pro - inflammatory responses [ 22 , 24 ] . we wish to emphasise that the power of our study was limited , therefore a prospective trial with a larger patient cohort is needed to confirm our results . furthermore , tgf- levels are known to vary between different laboratories . in order to prevent these variations , we collected plasma samples and performed all tgf- measurements in the same laboratory at the same time . finally , it would have been interesting to have the follow - up tgf- measurements after 3 years of losartan therapy . despite these study limitations , in conclusion , the variable effect of losartan on plasma tgf- levels probably reflects , at least in part , the heterogeneity in fbn1 mutations or raas modifiers . we showed that mfs patients who responded with a decrease in plasma tgf- level during losartan therapy had higher baseline tgf- levels . most likely , tgf- levels may be considered to be a readout of the disease state of the aorta . significantly , the effectiveness of losartan on the aodr in mfs patients [ 6 , 8 ] proves that ang ii - mediated signalling is crucial in the vascular pathology of mfs . we propose that increased angii signalling is the initiator of aorta dilatation and is responsible for the increased tgf- levels in mfs . the concept of tgf- as the initiator of aortic dilatation in mfs patients should be nuanced now it is clear that angii - mediated signalling is instructive and affects more than just tgf- levels . this work was supported by the netherlands heart foundation ( grant 2008b115 ) and fighting aneurismal disease ( fad ) european project . | backgroundrecently , we demonstrated that losartan reduced the aortic root dilatation rate ( aodr ) in adults with marfan syndrome ( mfs ) ; however , responsiveness was diverse .
the aim was to determine the role of transforming growth factor- ( tgf- ) as therapeutic biomarker for effectiveness of losartan on aodr.methodsbaseline plasma tgf- levels of 22 healthy controls and 99 mfs patients , and tgf- levels after 1 month of losartan treatment in 42 mfs patients were measured .
aodr was assessed by magnetic resonance imaging at baseline and after 3 years of follow-up.resultspatients with mfs had higher tgf- levels compared with healthy controls ( 121 pg / ml versus 54 pg / ml , p = 0.006 ) .
after 1 month of therapy , losartan normalised the tgf- level in 15 patients ( 36% ) ; the other 27 patients ( 64% ) showed a significant increase of tgf-. after 3 years of losartan therapy , patients with a decrease in tgf- had significantly higher aodr compared with patients with increased tgf- ( 1.5 mm/3 years versus 0.5 mm/3 years , p = 0.04 ) .
patients showing a decrease in tgf- after losartan therapy had significantly elevated baseline tgf- levels compared with patients with increased tgf- ( 189 pg / ml versus 94 pg / ml , p = 0.05).conclusionpatients responding to losartan therapy with a reduction of the plasma tgf- level had higher baseline tgf- levels and a higher aodr .
most likely , tgf- levels may be considered to be a readout of the disease state of the aorta .
we propose that increased angiotensin ii is the initiator of aorta dilatation and is responsible for increased tgf- levels in mfs .
the concept of tgf- as initiator of aortic dilatation in mfs patients should be nuanced . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the nih - nigms - sponsored protein structural initiative ( 1 ) initially sponsored seven pilot projects in structural genomics , including the southeast collaboratory for structural genomics ( secsg ) ( 2 ) . the structural genomics of caenorhabditis elegans ( sgce ) project established a pipeline for high - throughput protein expression , microarray crystallization screening , x - ray data collection and user - friendly bioinformatics . the initial sgce focus was the nematode worm c.elegans , one of the best - studied multicellular model organisms ( 3 ) whose complete genomic sequence is known ( 4 ) . the sgce project is facilitated by the c.elegans orfeome project ( 5 ) , which aims at cloning all predicted protein - encoding open reading frames ( orfs ) as gateway entry clones ( 6 ) . the 96-well plates of cdna supplied enabled a high - throughput approach to recombinant protein expression and analysis . however , a pipeline to express , purify and crystallize over 10 000 c.elegans orfs ( 7 ) required more comprehensive tracking for meaningful analysis . additional requirements for the project included sample tracking and monitoring group progress for a smooth transition between the expression , purification and crystallization groups . plates of cdna orfs for robotic screening needed to be prioritized based on the expectation of results . in order to prioritize , established external databases and analysis algorithms must be applied on a genome - wide basis . finally , the methods used to accomplish each step were updated on a regular basis ; therefore , the database and interface system needed to be flexible enough to rearrange and modify experiments before the data stored became obsolete with respect to the experiments performed . the resulting sgcedb is a database and interface framework that has been applied to a variety of genomes and heterogeneous experiment methodologies . the target i d used for selecting a single protein is generally the wormbase ( 8) aceid , familiar to c.elegans researchers . unlike wormbase , which concentrates on genomic information , the sgcedb concentrates on information of the putative proteins . the sgce was among the first structural genomics groups to make its protein production data publicly available . the most detailed way of browsing the sgcedb is by viewing one of the 16 566 c.elegans individual proteins . in this view links are provided to wormbase website ( 10 ) , the orfeome project ( 5 ) and the protein information resource ( 11 ) if available . blast ( 12 ) results against the protein data bank ( pdb ) structures ( 13 ) are updated weekly . blast results against the non - redundant database ( 14 ) and pfam ( 15 ) results are periodically updated . theoretical values , such as isoelectric point and hydrophobicity , are calculated from the expasy site ( 16 ) . transmembrane protein ( 17 ) , signal peptide ( 18 ) and prosite ( 19 ) results are available . for each of the 11 727 proteins with experiments an example web page of a protein view with a completed structure ( 20 ) is shown in figure 1 . the xml reports are updated weekly and are intended to provide improved interoperability with external databases . database queries are used to format all results according to the targetdb and the protein expression , purification and crystallization ( pepcdb ) xml standards for structural genomics maintained at the pdb ( 13 ) . individuals interested in detailed results of a specific step can go directly to that page , e.g. structures. the modeling page contains secondary structures and other predictions available from proteinpredict ( 21 ) . the modeling page also provides distributions per plate and histograms over the entire database for calculated protein parameters . the parameter distribution per plate is used internally to prioritize efforts . a variety of per plate experimental reports are also available . the complete system was developed in close collaboration with the sgce project scientists and technicians . the infrastructure was constructed entirely with open - source tools including the apache web server , the python language and the postgresql database system . a more detailed summary of the design and data entry including figures is provided in supplementary data . the database of sgcedb is separated into two distinct parts : protein source and experiment tracking . each schema implements advanced database methods to efficiently and comprehensively track proteins , results and analyses throughout the experimental process . when a protein source is initially received or considered for study , the sequences are organized into plates and wells . the protein source schema has configurable plate geometry allowing it to track protein production , 96-well solubility screens and 384-well crystallization trials . by keeping plates , wells and sequences separate in the protein source schema data attributes can be assigned at an appropriate level . for instance , the pfam ( 15 ) algorithm is initially executed and stored once per sequence . experiment parameters common to an entire screening plate are stored once per plate whereas individual experiment results are stored at the plate level . this design provides efficient queries of experimental data by eliminating duplicate information and allowing the design to scale to experiments over the entire c.elegans genome . experiment history is stored in a condensed form , called parenthetical tree notation ( 22 ) . this notation is exceptionally useful for querying lineage information . populating the history tree is handled by the database during data entry , with the scientists only having to indicate a major benefit of storing an experiment history tree is the ability to efficiently retrieve every experiment that has influenced an experiment of interest . generic experiment table when tracking the sequence of experiments . by tracking experiment history using a generic experiment i d any combination of available experiments can be combined and tracked without a major database re - design . in general the sequence of experiments is fixed by protocol . however , in the event of an unexpected result , quality control experiments such as mass spectrometry and additional chromatography column experiments may be inserted anywhere in the sequence of experiments . all subsequent experiments will include the unplanned mass spectrometry in a query of its history . a benefit of zope is the ability to reuse common data entry components to quickly assemble entry forms for the bench scientist . the target i d used for selecting a single protein is generally the wormbase ( 8) aceid , familiar to c.elegans researchers . unlike wormbase , which concentrates on genomic information , the sgcedb concentrates on information of the putative proteins . the sgce was among the first structural genomics groups to make its protein production data publicly available . the most detailed way of browsing the sgcedb is by viewing one of the 16 566 c.elegans individual proteins . in this view links are provided to wormbase website ( 10 ) , the orfeome project ( 5 ) and the protein information resource ( 11 ) if available . blast ( 12 ) results against the protein data bank ( pdb ) structures ( 13 ) are updated weekly . blast results against the non - redundant database ( 14 ) and pfam ( 15 ) results are periodically updated . theoretical values , such as isoelectric point and hydrophobicity , are calculated from the expasy site ( 16 ) . transmembrane protein ( 17 ) , signal peptide ( 18 ) and prosite ( 19 ) results are available . for each of the 11 727 proteins with experiments an example web page of a protein view with a completed structure ( 20 ) is shown in figure 1 . the xml reports are updated weekly and are intended to provide improved interoperability with external databases . database queries are used to format all results according to the targetdb and the protein expression , purification and crystallization ( pepcdb ) xml standards for structural genomics maintained at the pdb ( 13 ) . individuals interested in detailed results of a specific step can go directly to that page , e.g. structures. the modeling page contains secondary structures and other predictions available from proteinpredict ( 21 ) . the modeling page also provides distributions per plate and histograms over the entire database for calculated protein parameters . the parameter distribution per plate is used internally to prioritize efforts . a variety of per plate experimental reports are also available . the complete system was developed in close collaboration with the sgce project scientists and technicians . the infrastructure was constructed entirely with open - source tools including the apache web server , the python language and the postgresql database system . a more detailed summary of the design and data entry including figures is provided in supplementary data . the database of sgcedb is separated into two distinct parts : protein source and experiment tracking . each schema implements advanced database methods to efficiently and comprehensively track proteins , results and analyses throughout the experimental process . when a protein source is initially received or considered for study , the sequences are organized into plates and wells . the protein source schema has configurable plate geometry allowing it to track protein production , 96-well solubility screens and 384-well crystallization trials . by keeping plates , wells and sequences separate in the protein source schema data attributes can be assigned at an appropriate level . for instance , the pfam ( 15 ) algorithm is initially executed and stored once per sequence . experiment parameters common to an entire screening plate are stored once per plate whereas individual experiment results are stored at the plate level . this design provides efficient queries of experimental data by eliminating duplicate information and allowing the design to scale to experiments over the entire c.elegans genome . experiment history is stored in a condensed form , called parenthetical tree notation ( 22 ) . this notation is exceptionally useful for querying lineage information . populating the history tree is handled by the database during data entry , with the scientists only having to indicate a major benefit of storing an experiment history tree is the ability to efficiently retrieve every experiment that has influenced an experiment of interest . generic experiment table when tracking the sequence of experiments . by tracking experiment history using a generic experiment i d any combination of available experiments can be combined and tracked without a major database re - design . in general the sequence of experiments is fixed by protocol . however , in the event of an unexpected result , quality control experiments such as mass spectrometry and additional chromatography column experiments may be inserted anywhere in the sequence of experiments . all subsequent experiments will include the unplanned mass spectrometry in a query of its history . a benefit of zope is the ability to reuse common data entry components to quickly assemble entry forms for the bench scientist . currently the sgcedb framework has the capability to handle individual and plate - based experiments with external database reference and algorithm analysis . an experiment creation tool would generate table definitions , entry forms and standard html views based on data types specified in a graphical interface . the modular visualization methods would utilize java display layers to allow the user to superimpose additional information where it is most useful for instance , secondary structure under the sequence and information content over a 3d structure ( 24 ) or gel markers by a gel image . the left - hand side shows the information available for all targets . the right - hand side shows the experimental data generated . | the sgcedb ( ) database / interface serves the primary purpose of reporting progress of the structural genomics of caenorhabditis elegans project at the university of alabama at birmingham .
it stores and analyzes results of experiments ranging from solubility screening arrays to individual protein purification and structure solution . external databases and algorithms
are referenced and evaluated for target selection in the human , c.elegans and pneumocystis carinii genomes . the flexible and reusable design permits
tracking of standard and custom experiment types in a scientist - defined sequence .
the database coordinates efforts between collaborators and is adaptable to a wide range of biological applications . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
obstructive sleep apnea ( osa ) is a chronic condition that affects 3 - 7% of the population . despite increasing public awareness over the last 20 years , osa is still underdiagnosed and untreated . the negative health consequences of untreated sleep apnea continue to be elucidated , but currently include significant morbidity - related entities such as cardiovascular disease , hypertension , and abnormal glucose metabolism . more readily available and cost - effective methods of diagnosing osa could have significant impact on public health and reduce health - care expenditures . factors known to increase risk of osa include neck size , age , male sex , obesity , family history , and craniofacial anatomy . upper airway anatomy is involved in the pathophysiology of osa . decreased cross - sectional area and increased compliance of the upper airway contribute to osa . neck circumference measurement reflects neck tissue mass and serves as a surrogate measure of neck mass . hypopnea index ( ahi ) than did the measure of body mass index ( bmi ) . neck circumference corrected for by height is more useful . in 1990 , katz et al . published an article entitled do patients with obstructive sleep apnea have thick necks ? our study restates the question to read , do patients with thick necks have obstructive sleep apnea ? we hypothesized that a simple and objective test using proportionate neck size ( subject spanning their own neck with their own hands ) rather than measured circumference would simplify the screening and increase the recognition and diagnosis of osa . this simple objective maneuver may eliminate the need to rely on first- and third - person subjective reporting of snoring quality , possible apnea , sleep quality , and sleepiness that is currently heavily relied upon for suspicion of osa . we evaluated a simple neck grasp maneuver as a predictor of osa in a general sleep clinic . the institutional review board at bassett medical center approved this retrospective chart review and waived the need for informed consent from patients . a retrospective review of charts was performed to obtain the following data : esap status , age , gender , bmi , and polysomnogram / home sleep test ( psg / hst ) result . fifty consecutive adult patients ( aged > 18 years ) requiring psg for clinical evaluation were assessed with the easy sleep apnea predictor ( esap ) test [ figure 1 ] at the time of the consultation . the patients were instructed to place their thumbs together at the anterior neck and to wrap their fingers around their neck until they met in the posterior . a positive esap test was defined as the inability to easily ( without excess squeezing and choking ) encircle the neck completely [ figure 2 ] . a negative esap test was defined as the ability to easily encompass their hands around the neck [ figure 3 ] . the result of the esap test was recorded in the medical record as positive or negative . clinical testing with psg or hst level 3 was dictated by the consulting clinician 's clinical determination and insurance coverage . psg was performed and scored according to current the american academy of sleep medicine ( aasm ) guidelines . in medicare patients ( n = 7 ) , the hypopnea definition of 30% reduction in flow with 4% desaturation was applied ; all others were scored using the 30% reduction with either a 3% desaturation or an arousal . hst ( n = 1 ) was performed and scored by manufacturer 's setting and reviewed and interpreted by a board certified sleep physician . for the purpose of the main study analysis , osa was defined as an ahi of 5 . subanalyses were conducted using ahi cutoffs of ahi of > 10 and ahi of > 15 . the easy sleep apnea predictor is performed by touching the thumbs in the anterior of the neck and encircling the neck with the digits in the posterior easy sleep apnea predictor positive is the inability to easily encircle the neck so that the patient 's digits meet on the posterior neck easy sleep apnea predictor negative is the ability to encircle the neck without excessive choking with digits touching at the posterior neck demographic variables were compared between esap positive and esap negative patients using chi - square ( for gender ) and the t - test ( for bmi , age , and ahi ) . using ahi as the gold standard for osa diagnosis , the sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of esap to predict osa were calculated . sensitivity , specificity , ppv and npv of obesity classes ( bmi 30 and bmi 35 ) to predict osa were also calculated . demographic variables were compared between esap positive and esap negative patients using chi - square ( for gender ) and the t - test ( for bmi , age , and ahi ) . using ahi as the gold standard for osa diagnosis , the sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of esap to predict osa were calculated . sensitivity , specificity , ppv and npv of obesity classes ( bmi 30 and bmi 35 ) to predict osa were also calculated . two of the 47 subjects had hst rather than psg by insurance requirement [ table 1 ] . the mean age was 51.6 years ( sd 14.4 , range 29 - 81 ) . the mean bmi was 38.8 ( sd 9.9 , range 20.4 - 69.5 ) . in our population , characteristics and demographics of the study population esap did differentiate a statically significant difference in the population for ahi ( p < 0.001 ) and bmi ( p both ahi and bmi were significantly greater on average for esap positive subjects as compared to esap negative subjects . between the esap positive and negative groups there is no statistical difference for gender ( p = 0.49 ) or age ( p = 0.66 ) [ table 2 ] . a comparison of esap negative and positive patients for bmi , ahi , age , and gender the sensitivity and specificity of esap were 68.3% and 100% , respectively , with regard to an ahi of 5 . the positive predictive power was 100% and the negative predictive power was 31.6% for an ahi of 5 . using bmi to predict ahi of 5 revealed weaker prediction . the ppvs for bmi 30 and bmi 35 were 92% and 90% , respectively , at an ahi of 5 . the ppvs of esap remained similar when stratified by bmi > 30 and bmi > 35 for three levels of ahi ( 5 , > 10 , and > 15 ) [ table 3 ] . the field of sleep medicine is working to improve the diagnosis and treatment of osa in a cost - effective manner ; it is estimated that 80% of the people with osa are undiagnosed . incorporating novel methods will be required to successfully meet these demands . over the past decade , many screening algorithms and questionnaires have been developed such as stop - bang and the berlin questionnaire . obesity and a large neck have long been established as risk factors for osa . to our knowledge , this is the first time that this well - recognized fact has been leveraged in a simple neck grasp maneuver , as a diagnostic tool for osa . our retrospective pilot data support our hypothesis that a positive esap , the inability to fit hands around the neck , strongly correlates with osa in a general sleep clinic population and this correlation is superior to bmi alone . esap 's usefulness is consistent with the known fact that neck size predicts osa and that proportionate data , corrected for body size , are more valuable than absolute measurements . esap is more easily applicable than linear measure , and it does not require the adjustments for gender required with linear measure . first , we recognize that our threshold for presence of osa was low ( ahi of 5 ) . however , in a symptomatic sleep clinic population , this is arguably an appropriate set point for consideration of treatment . when the threshold for osa was increased to ahi of > 15 , our data showed that the ppv of esap to remain clinically useful at 86% . we did not evaluate esap combined with any other parameter or test such as snoring , observed apnea , presences of hypertension , or epworth sleepiness scale . this raises the second point of note that the high pretest probability of this preselected referral population improved esap predictive power . this does not reduce the value of the esap test when applied to this population . the final point of comment on the 100% ppv pertains to an issue that needs to be clarified for the entire field of sleep medicine , not just our esap validity ; that is the definition of sleep disordered breathing events , namely hypopneas . our retrospective the more recent 2014 definition lowers the threshold for diagnosing osa and hence increases the ppv of esap . this does not stand to negate esap positive value , but needs to be recognized . collop ; we are not attempting to bring any additional clarity to the definitions other than to say that the esap data stands in the midst of this change in terminology . the most obvious and important limitation of the esap test is the rate of false negatives , npv of 31.6% . the lack of value of a negative esap needs recognition when the esap is applied . the standard testing ( psg or hst ) is still required if clinically indicated in an esap negative patient . other factors , such as craniofacial anatomy and upper airway anatomy , contribute to osa regardless of neck size and esap negativity . that point understood , the positive esap remains valuable at identifying the 80% of patients with undiagnosed osa , the majority of whom are obese and likely esap positive . the equally objective measure of bmi was inferior [ table 2 ] to esap over a range of ahi . esap is arguably the simplest of all objective tests , requiring only seconds of clinical time and no device . in unique populations , such as commercial drivers , this objective screen might prove invaluable to highway safety ; however , to date this population has not be evaluated . first , the general economic burden to society of untreated osa remains high despite three decades of recognition of the syndrome . second , the health - care expenditure for untreated osa is estimated at $ 3.4 billion in the us . the initial step in addressing this economic problem is improving diagnosis , which esap appears every effective in the sleep clinic population . lastly , esap may result in a cost reduction in the area of diagnostic testing to identify osa by reducing reliance on psg . as we hypothesized , esap positive ( inability to span neck ) was predictive of osa . esap shows promise for ease of clinical use to predict the presence of osa in a general sleep clinic population . further research to evaluate this simple stand - alone diagnostic maneuver is warranted to confirm the high predictive value in this and other populations . the process of disease recognition of osa remains a challenge and esap may prove to be the easiest and least expensive tool available . the authors have no conflicts of interest including financial support , or involvement with organizations that have a vested financial interest . the authors have no conflicts of interest including financial support , or involvement with organizations that have a vested financial interest . | background : considering the high estimates of undiagnosed and untreated obstructive sleep apnea ( osa ) , there is a need for simple and accurate diagnostic tests .
neck circumference has long been correlated with osa , but its usefulness as a diagnostic tool has been limited.aims:we proposed to evaluate the value of a simple neck grasp test to help identify osa .
we hypothesized that the inability of a patient in a sleep clinic to fit their hands around their neck is predictive of osa.materials and methods : a retrospective review of medical records of patients evaluated in a general sleep clinic was performed .
easy sleep apnea predictor ( esap ) positive was defined as the inability to place the hands around the neck with digits touching in the anterior and posterior .
esap negative was the ability to place hands around the neck .
positive for osa in this symptomatic sleep clinic population was defined as an apnea hypopnea index ( ahi ) of 5.results : a total of 47 subjects ( 36% female ) had esap data available , which were reviewed .
the mean age was 51.6 years ( sd 14.4 , range 29 - 81 years ) .
the mean body mass index ( bmi ) was 38.8 ( sd 9.9 , range 20.4 - 69.5 ) .
review showed 87.2% ( n = 41 ) tested positive for osa by ahi of 5 .
the sensitivity and specificity of esap were 68.3% and 100% , respectively .
the positive predictive power was 100% and the negative predictive power was 31.6%.conclusion : as we hypothesized , esap positive ( inability to span neck ) was predictive of osa in a population of sleep clinic patients .
an esap positive test was 100% predictive of the presence of osa ( ahi of 5 ) .
esap shows promise for ease of clinical use to predict the presence of osa in a general sleep clinic population . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the incidence and prevalence of patients with chronic kidney disease ( ckd ) is increasing worldwide . in brazil , the brazilian society of nephrology has collected information annually on patients with end - stage renal disease ( esrd ) since 1999 and made it available on the society 's website.1 in 2008 , the estimated number of patients in dialysis amounted to 87,044 , of whom 90% were on hemodialysis and 10% on peritoneal dialysis . some studies have evaluated the quality of life ( qol ) of patients undergoing dialysis , but there is limited information available on the qol of patients on conservative treatment of ckd and the relationship between qol and the early stages of the disease . the qol of these patients seems to be poorer than that of the general population , but better than for patients on dialysis.2,3 certain factors such as anemia , associated diseases and early treatment by a nephrologist appear to have an impact on the qol of these patients.35 the objectives of this cross - sectional study were to assess the qol of patients with stages 15 ckd on conservative treatment in order to identify a possible association between qol and progression of kidney insufficiency . a total of 202 patients were randomly selected , including 165 with non - dialytic ckd followed up at the ckd outpatient clinic of the division of nephrology , federal university of so paulo , so paulo , brazil , and 37 patients undergoing hemodialysis at the institution . a sample of 30% of all patients under follow - up was selected using a systematic random sampling method . initially , a list of all patients with ckd ( and another for those on hemodialysis ) was made , and a consecutive number was given to each one . then , a starting point was chosen at random and every fifth record ( this interval was prespecified ) on the list was selected . the subjects were interviewed prior to the medical visits or after the hemodialysis session in a separate room by two trained interviewers . included patients should be on hemodialysis treatment for > 6 months and < 6 years and were submitted to a conventional in - center hemodialysis regimen consisting of three weekly hemodialysis sessions lasting 4 hours each . patients were excluded if they did not consent to participate , were younger than 18 years or had hearing , speech or cognitive deficits that would impair understanding of the questions . the protocol was approved by the ethics committee of the institution , and all subjects gave written informed consent to participate in the study . to assess the qol , we used the medical outcomes study short form 36-item health survey ( sf-36 ) , a generic instrument translated and validated in brazilian patients with esrd.6,7 this instrument is divided into 8 dimensions : physical functioning , physical role functioning , pain , general health , vitality , social role functioning , emotional role functioning , mental health . the results of each scale vary from 0 to 100 ( worse to best possible status ) . the physical and mental components of the 8 scales were combined into a physical component summary ( pcs ) and a mental component summary ( mcs ) . the 2 summary measures were standardized so as to have a mean value of 50 and a standard deviation ( sd ) of 10 in the general population.7 the karnofsky performance status ( kps)8 was used to assess self - sufficiency and functional capacity , which determines functional impairment in the performance of activities of daily living , using a score ranging from 100 ( indicating no evident disease ) to 0 ( indicating death ) . the socioeconomic level of the patients was evaluated according to the criteria of the brazilian association of research companies , which are recognized in brazil and divide the population into social classes a , b , c , d and e , with class a corresponding to the highest socioeconomic level.9 other sociodemographic and clinical variables were also analyzed in an attempt to identify a possible association with qol . glomerular filtration rate ( gfr ) was estimated by the creatinine clearance according to the formula of cockcroft and gault10 adjusted to 1.73 m of body surface area , using the latest serum creatinine measurement available for each patient . the stages of ckd were classified as suggested by the kidney disease outcomes quality initiative11 and are presented in table 1 . for the purpose of analysis , ckd stages 1 and 2 and stages 4 and 5 were combined , because of the small number of patients in each group , to increase the power of statistical comparisons between groups . - square tests were performed to compare categorical variables . for the continuous variables , we used student 's unpaired t - test to compare two groups , and anova to compare more than two groups . whenever the anova test result was significant , we then compared the groups two by two . pearson 's correlation test was employed to correlate the pcs and the mcs and kps results with the other continuous variables . statistical significance was set at p<0.05 , and all tests performed were two - tailed . chi - square tests were performed to compare categorical variables . for the continuous variables , we used student 's unpaired t - test to compare two groups , and anova to compare more than two groups . whenever the anova test result was significant , we then compared the groups two by two . pearson 's correlation test was employed to correlate the pcs and the mcs and kps results with the other continuous variables . statistical significance was set at p<0.05 , and all tests performed were two - tailed . of the 191 patients who participated in the study , 155 were predialysis ckd patients and 36 were on hd . the total sample of predialysis ckd patients was divided into five stages according to gfr and subsequently grouped into three groups ( stages 1 + 2 , 3 and 4 + 5 ) . the patients in the three groups of ckd stage and those on hemodialysis were similar with regard to their sociodemographic characteristics ( table 2 ) . with respect to laboratory variables , the groups differed in some characteristics , as shown in table 3 . qol , as evaluated by the means of sf-36 domains , was low for most dimensions in all stages and was not statistically significant different among the patients in the three groups of ckd stage or on hemodialysis . the dimensions showing lower values in stages 1 and 2 were emotional role functioning and general health ; in stage 3 , physical role functioning and vitality ; and in stages 4 and 5 and hemodialysis , physical role functioning and general health . no difference was observed among the groups regarding the pcs , the mcs or on the kps ( table 4 ) . evaluating sociodemographic data , patients who had a higher educational level performed better than the others in mean pcs . as for the patients ' mean mcs scores , males and asians showed better results and those with no individual monthly income had lower values than those with some income . the kps scores showed no statistically significant differences related to demographic data , except for occupation : those who were professionally active performed better ( table 5 ) . no significantly difference was found between the mean pcs and mcs and the kps scores when the patients were divided by the etiology of ckd and the occurrence of hospitalization . with regard to age , there was a negative correlation with the mean pcs and a positive correlation with the mean mcs scores . hemoglobin level was positively correlated with pcs and kps , whereas the serum phosphorus levels showed a negative correlation with mcs ( table 6 ) . evaluating patients for the number of comorbidities , we found that those with three or more were older , retired and diabetic . low levels of calcium and high levels of urea were also found in patients with more comorbidities . with respect to the qol , patients with more comorbidities had worse rates in the assessment of functional capacity ( assessed by both sf-36 and the kps ) , physical role functioning and pcs . our results indicate low qol scores in the early stages of ckd , although we have not demonstrated a significant decrease in qol progressively in the different stages of renal disease . not only were mean values of the physical components reduced as early as stages 13 of ckd but mental health also seemed to be compromised , based on the mean values of the sf-36 scores , which were below 70 in most dimensions . few studies have evaluated the qol of patients in the early stages of ckd , especially in the first two stages , or sought to relate qol and gfr.3,12 in these studies , a significant reduction in qol was also not identified according to the progression of renal dysfunction . what is reported more frequently in the literature is a decrease in the physical domains of qol in the advanced stages of ckd , which was also identified in our study.1316 in our study , the hemoglobin levels showed a correlation with better pcs and kps scores . in the literature , the impact of anemia on qol in ckd is well described from the predialysis ckd phases through esrd.17,18 we observed that a greater number of comorbidities were associated with older age , diabetes and unemployment or retired status . the presence of three or more comorbidities had a negative impact on the domains physical functioning , physical role functioning and pcs , and on kps . some reports have suggested that the presence of comorbidities is a major determinant of a decline in qol.19,20 diabetes mellitus has also been associated with low qol.21 it is known , however , that the subjective assessment of qol is multifactorial , and therefore the progression of renal dysfunction may not be the only determinant in its deterioration . in our study , more sociodemographic factors ( age , ethnicity , gender , professional activity , education , income ) were associated with decreased qol than physical factors . added to this , it is possible that subjective factors such as adaptation to disease and treatment , satisfaction with the medical staff and social support , among others , may interfere directly in the assessment of qol , but were not evaluated in this study . the influence of these different factors on the assessment of qol may explain the difficulty in establishing a linear relation with the gfr . some limitations of the present study are the relatively small sample size to detect significant differences between the stages of ckd and the difficulties we encountered in recruiting subjects in the initial stages of the disease . the cross - sectional design of the study only permitted us to determine associations between variables and not causal relationships . thus , longitudinal studies that take into account qualitative assessments should be conducted to seek a better understanding of the influence of the progression of ckd on qol . in conclusion , in this study , we observed a negative impact on the qol of patients in the early stages of ckd , although we were not able to detect a significant association between the stages of the disease and the sf-36 domains . however , it was possible to establish sociodemographic , clinical and laboratory risk factors for a worse qol in this population ( educational level , gender , individual income , professional activity , age , hemoglobin levels , serum phosphorus levels , diabetes and comorbidities ) . although several of the variables that were associated with alterations in the qol can not be changed ( e.g. , age , gender , ethnicity ) , efforts should be made to decrease the effects of those factors that can be changed , such as improving the hemoglobin levels and adequately managing the comorbidities . the health professionals responsible for the care provided to this population should ideally be familiar with and trained in the application of the qol assessment tools , which may be valuable in the global assistance of these patients , even in the earlier stages of disease , and allow timely health care interventions in the course of the disease . this study was supported by grants from capes ( coordenao de aperfeioamento de pessoal de nvel superior ) . | aim : to compare the dimensions of quality of life in the stages of chronic kidney disease and the influence of sociodemographic , clinical and laboratory data.introduction:the information available on the quality of life of patients on conservative treatment and the relationship between the quality of life and glomerular filtration rate is limited.methods:155 patients in stages 15 of chronic kidney disease and 36 in hemodialysis were studied .
quality of life was rated by the medical outcomes study short form 36-item ( sf-36 ) and functional status by the karnofsky performance scale .
clinical , laboratory and sociodemographic variables were investigated.results:quality of life decreased in all stages of kidney disease . a reduction in physical functioning , physical role functioning and in the physical component summary was observed progressively in the different stages of kidney disease .
individuals with higher educational level who were professionally active displayed higher physical component summary values , whereas men and those with a higher income presented better mental component summary values .
older patients performed worse on the physical component summary and better on the mental component summary .
hemoglobin levels correlated with higher physical component summary values and the karnofsky scale .
three or more comorbidities had an impact on the physical dimension.conclusion:quality of life is decreased in renal patients in the early stages of disease .
no association was detected between the stages of the disease and the quality of life .
it was possible to establish sociodemographic , clinical and laboratory risk factors for a worse quality of life in this population . |
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in the previous issue of critical care , torgersen and coworkers present data about careful evaluation of hemodynamic monitoring of patients with cardiogenic shock ( cs ) in the intensive care unit as continuous variables during the initial 24-hour period . the authors stated that hourly time integrals of the cardiac index ( ci ) ( cardiac output per body surface area ) and the cardiac power index ( cpi ) ( cardiac power output per body surface area ) were the most important hemodynamic variables designating mortality as continuous parameters . in addition , instead of intermittent measures of the ci and the cpi , the authors analyzed the area under a given level divided into hourly intervals , named hourly time integrals . these results have the potential to provide a paradigm shift in the setting of cs . two take - home messages can be extracted from the study of torgersen and coworkers . first is the emphasis on the importance of continuous monitorization in patients with cs , and the second message is providing data for the importance of the interpretation of the cpi as a continuous variable in an acute setting . cs is traditionally defined as a state of severe tissue hypoperfusion secondary to cardiac dysfunction . therefore , it is vital to identify signals driving the cs prognosis . for decades , hemodynamic monitoring has been desired for patients with acute pathologies with the aim of guiding therapy and risk - stratification . many parameters derived from hemodynamics - such as the cardiac output , systemic blood pressure , systemic vascular resistance , stroke volume , and pulmonary capillary wedge pressure - have so far been investigated to designate prognosis with unequivocal results . among these hemodynamic parameters , the cardiac power output and the cpi serve as interesting markers showing cardiovascular coupling at one glance , in contrast to other parameters that provide information about either the cardiac system or the vascular system . a critically low cpi might be a result of unresponsiveness to therapeutic interventions rather than a causative factor for death . one should mention that although the hourly time integrals of drops in the ci and cpi predicted short - term mortality , this might not mean that the ci and the cpi are targets for treatment . intra - aortic balloon pump ( iabp ) use is known to increase cardiac power output , and hence the cpi . adjustment should have been performed for the use of an iabp ( 37.8% of patients ) . adjustment for gender was also needed , as the cardiac power output is also lower in females . in a very recent meta - analysis , it was shown that a percutaneous left ventricular assist device yielded higher ci and higher mean arterial pressure compared with the iabp . this could be translated as a higher cpi with the left ventricular assist device compared with that under iabp use . the 30-day mortality was similar in both interventions , however , despite better early hemodynamic status with the left ventricular assist device . torgersen and coworkers brought us some novel thresholds , however , which should be tested in multicenter trials . the present paper emphasizes the continuous effort that should be made to risk - stratify patients with cs . hemodynamic variables are important to consider in the setting of cs , particularly those parameters integrating generation of cardiac energy with spreading of blood flow , such as the cpi . since it was shown that almost one - half of nonsurvivors of cs die with a normal ci , this finding changed the paradigm of cs recently from being only a cardiac problem into a disease of the entire circulatory system . data tell us the importance of systemic inflammatory response upon release of inflammatory mediators and neurohormones yielding alterations in tissue microvasculature , which may result in multi - organ dysfunction syndrome in cs . we therefore agree that hemodynamic signals combining cardiac function with tissue perfusion such as the cpi may be optimal markers of outcome . on the contrary , it might be as important to consider any invasive hemodynamic parameter as a continuous ( and not intermittent ) marker , as presented by torgersen and coworkers , instead of considering them per piece . ci : cardiac index ; cpi : cardiac power index ; cs : cardiogenic shock ; iabp : intra - aortic balloon pump . | cardiogenic shock is a lethal condition .
physicians are searching for hemodynamic markers which could help risk - stratification of patients in this picture .
torgersen and coworkers present an hourly time integral of the cardiac power index and cardiac index drops to predict outcomes in the setting of cardiogenic shock . continuous monitoring of hemodynamic markers may have a role in prediction of outcomes . |
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cerebral white matter injury ( wmi ) is recognized as the most common form of injury to the developing brain of neonates , especially survivors of preterm birth1 ) . although multiple factors are involved in brain injury , both infection and inflammation are well - established risks factors for wmi in the preterm population234 ) . the major period of vulnerability for wmi occurs prior to the onset of myelination ( 2332 gestational weeks)5 ) . preterm neonates are at high risk of severe and/or multiple bacterial infections between birth and hospital discharge6 ) . for these reasons , several studies have sought to demonstrate a relationship between infection / inflammation and wmi in preterm neonates as well as to elucidate the underlying mechanisms47 ) . however , infection / inflammation - associated brain injury does not occur exclusively in preterm neonates but is also seen in term neonates . the risk of cerebral palsy is increased by 2 to 12 fold in term neonates with antenatal choriomanionitis8910 ) . nevertheless , the principal anatomic substrate of infection / inflammation - associated cerebral injury in term neonates is not known . in this regard , recent reports of neonatal seizures associated with rotavirus infection in term neonates suggest several new and surprising perspectives111213 ) . first , infection / inflammation might induce wmi in term - neonates as well as preterm neonates . second , wmi might be caused not only by bacterial but also by viral infections . third , the developing brain can be damaged by afebrile infections outside the central nervous system ( cns ) . although further studies are needed to establish the relationship between rotavirus infection and wmi in term neonates , these recent reports111213 ) underscore the need for a paradigm shift recognizing the following : ( 1 ) that common viral infections can induce wmi in term neonates without direct viral invasion , and ( 2 ) that rotavirus is a common cause of neonatal seizures in term neonates . in this paper we then discuss the clinical characteristics and prognosis of term neonates with rotavirus - associated wmi and compare both with the features of 2 other viral infections , parechovirus and enterovirus . finally , we suggest further directions of research into rotavirus - associated wmi in the developing brain . rotavirus is the most common cause of gastroenteritis - associated seizures in infants and young children14 ) . although neonates are vulnerable to seizures as well as rotavirus infection , the connection between the two in this age group has been overlooked for many years . only a few studies have examined the association between rotavirus infection and neurological symptoms in newborns15161718 ) . rotavirus infection has been suggested as possible cause of ' fifth day fits ' in 1990s by herrmann et al.15 ) . they15 ) observed an epidemic occurrence of 21 cases of ' fifth day fits ' . after a coincidental detection of rotavirus in the stools of several cases of fifth day fits , they tested the stools of 19 fifth - day fitters and 30 matched controls15 ) . rotavirus was detected in 18 of the 19 neonates ( 95% ) with ' fifth day fits ' compared with only 12 of the 30 matched controls ( 40% ) ( p<0.01)15 ) . before proceeding with the main topic of this review , rotavirus - associated wmi in newborns , we first need to consider the entity of ' fifth day fits ' . the term was introduced in the 1980s to describe an epidemic of neonatal seizures that occurred during the 1970s1920 ) . ' fifth day fits ' are defined as the onset of seizures between the fourth and sixth days of life in otherwise apparently healthy full - term infants20 ) . the newborns are neurologically normal at the onset of the convulsion but become drowsy and hypotonic for several days thereafter19 ) . although the long - term prognosis was not defined in the original reports , these patients were assessed as normal at the time of their discharge from the hospital21 ) . acute zinc deficiency and feeding type were suggested as causes of fifth day fits , but neither was convincingly confirmed2223 ) . environmental factors were also proposed as the etiology because the prevalence of this syndrome was periodically variable . in newborns , they became an important issue following an outbreak in a neonatal care unit in mid-1970s242526 ) . the contemporary , epidemic - like occurrence of ' fifth day fits ' and rotavirus infection in newborns lent support to the suggestion of herrmann et al.15 ) however , the association between rotavirus and ' fifth day fits ' has not been replicated . around the same time with herrmann et al.15 ) , an association between rotavirus infection and bradycardia - apnea episodes has been reported.16 ) the incidence of bradycardia - apnea episodes was higher in neonates with than without rotavirus infection ( 33% vs. 8% , p<0.05)16 ) . bradycardia - apnea episodes were significantly more common in term than in preterm newborns16 ) . however , unfortunately , they16 ) did not report when symptom onset occurred nor did they address the numerous central and peripheral causes that may induce apnea in newborns . a few years later , a study in south africa17 ) yielded results that conflicted with the previous 2 studies1516 ) . retrospective and prospective analyses found no association between rotavirus infection and the neurological symptoms of newborns17 ) . however , the demographic characteristics of the patients were unclear , and significant selection bias was suspected17 ) . however , recent studies11121318 ) that included neuroimaging again suggested rotavirus as a cause of neonatal seizures or wmi . recent studies11121318 ) of wmi in neonatal seizures and the potential involvement of rotavirus infection are summarized in table 1 . verboon - maciolek et al.18 ) firstly suggested rotavirus as a cause of cerebral injury in newborns based on the cranial ultrasonography and magnetic resonance imaging ( mri ) findings of five preterm and three term infants who developed seizures or apnea during rotavirus infection . clinical signs during the first week of life were noted in 2 of the 3 full - term infants18 ) . in preterm infants , the symptoms became evident during the late preterm period ( 3436 weeks of postgestation)18 ) . cranial ultrasonography demonstrated mild to severe periventricular echogenicity coinciding with illness related to rotavirus infection and subsequent cystic evolution in 6 patients18 ) . among these 6 patients , diffuse high signal intensity attributable to a cystic lesion in the white matter was seen on mri in four preterm newborns18 ) . the other 2 term infants showed focal diffusion - weighted mri changes at disease onset and focal white matter cysts on repeat mri18 ) . systemic infection was proven in only 2 patients based on a positive polymerase chain reaction test of the cerebrospinal fluid ( csf ) and/or blood18 ) . neuroimaging was normal in preterm newborns in weekly cranial ultrasonography before rotavirus infection , and no other causes for seizures / wmi ( including human parechovirus , enterovirus , and herpes simplex virus ) were detected18 ) . the researchers18 ) therefore suggested an association between rotavirus enteritis and wmi indistinguishable from that in late onset cystic - periventricular leukomalacia ( pvl ) . the most impressive finding of verboon - maciolek et al.18 ) was that rotavirus apparently induces diffuse wmi with cystic evolution in the developing brain without direct invasion of the cns . the adverse neurological sequelae of cystic pvl highlight the need for additional studies to confirm their findings . three recent reports from korea111213 ) lend support to the role of rotavirus infection in wmi . the main findings of these three studies111213 ) were similar and can be summarized as follows : ( 1 ) neonates presenting with seizures characterized by a distinctive diffusion - weighted imaging ( dwi ) pattern had a high positive rate of rotavirus infection . ( 2 ) patients with this dwi pattern were apparently healthy term infants who presented with similar clinical features and suffered seizures between days 4 and 6 of life . ( 4 ) rotavirus was detected only in stool specimens , not in blood and csf specimens . ( 5 ) the presence of other viral pathogens , especially human parechovirus and enterovirus , was not proven . ( 7 ) cystic evolution and neurological sequelae were observed in some of the patients . the pattern of wmi was evident on dwi , which showed extensive and symmetrical restricted diffusion in the periventricular white matter and along entire fiber tracts , including the corpus callosum ( fig . this pattern was also seen in the full - term infants evaluated by verboon - maciolek et al.18 ) . most findings of verboon - maciolek et al.18 ) were similar to those reported from korea111213 ) , but there were also several differences between the studies . first , preterm newborns were excluded from the three korean studies111213 ) , perhaps because it is difficult to perform mri in preterm neonates at the time of illness for reasons such as the clinical condition of the patients or technical limitations at the evaluating centers . according to verboon - maciolek et al.18 ) , outcomes were worse in preterm than in full - term neonates , but this remains to be confirmed . second , the sample size was larger in each of the three studies from korea111213 ) than in the study of verboon - maciolek et al.18 ) . a 5-year single center retrospective study from korea by yeom et al.11 ) showed that rotavirus infection was identified in 17 neonates with the aforementioned dwi pattern . other 2 studies1213 ) from same single tertiary center reported that total 32 infants with this dwi pattern had rotavirus in their stool specimens for 5 consecutive years . by contrast , verboon - maciolek et al.18 ) reported on only eight patients from four hospitals for 6 years . the difference in patient number may be explained by the different patient criteria for testing rotavirus and the sensitivity of dwi in the detection of acute edema . all eight patients in the study of verboon - maciolek et al.18 ) had symptoms compatible with rotavirus gastroenteritis . however , in all three korean studies111213 ) , a screening test for rotavirus was routinely performed and almost all patients were found to be asymptomatic for rotavirus infection . thus , if only patients with gastroenteritis symptoms had been tested for rotavirus , a relationship between the two would not have been found111213 ) . verboon - maciolek et al.18 ) used ultrasound in their evaluations of preterm neonates . however , the sensitivity of dwi to the acute phase of edema is superior to that of ultrasound and conventional mri , especially in the immature brain27 ) . conventional mri techniques are insensitive to acute edema in the newborn brain due to the lack of myelination in neonates28 ) . dwi with corresponding apparent diffusion coefficient maps can detect acute edema earlier and more clearly28 ) . according to yeom et al.11 ) , if dwi had not been performed , the unique pattern of wmi might not have been recognized because only subtle signal changes were detected in most of their patients evaluated by conventional mri techniques ( fig . although the long - term neurological sequelae have yet to be defined , rotavirus - associated wmi may not always be benign . cystic evolution was observed in 27%75% of patients with rotavirus - associated wmi ( fig . verboon - maciolek et al.18 ) reported adverse outcomes , including epilepsy and cerebral palsy , in four of their 8 patients . lee et al.12 ) detected mild neurological deficits , including speech or motor delay , in four of the 13 patients included in their study . the factors associated with poor prognosis have yet to be identified ; however , they may include rotavirus infection or coinfection with enterovirus in preterm infants1318 ) . the clinical features of the patients with rotavirus - associated wmi who were described in recent reports11121318 ) are surprisingly similar to those of patients with the largely forgotten clinical entity of ' fifth day fits ' . in an examination at median 4.6 years , developmental abnormalities were observed in more than half of the patients with ' fifth day fits'21 ) . the pattern of wmi seen in rotavirus infections is not unique and is , in fact , strikingly similar to that seen in neonatal enterovirus and parechovirus encephalitis11293031323334 ) . in all 3 infections , the major findings on cerebral imaging are diffuse high signal intensity in the white matter on t2-weighted sequences and restricted diffusion in the periventricular white matter , corpus callosum , and deep white matter . however , the signal changes seen on conventional mri sequence are more subtle in the wmi of rotavirus infections than in the wmi related to enterovirus and parechovirus infections3034 ) . a predilection for cystic evolution in the frontal lobes is another common feature1112183031 ) , as is the timing of wmi susceptibility following viral infection . despite the broad range in the onset of these viral illnesses , thus , it seems that the white matter is susceptible to viral infection during the late preterm to full - term period and the final common pathway to virus - induced wmi may not differ among rotavirus , enterovirus , and parechovirus . however , there are differences in the clinical characteristics of the patients infected with the different viruses . as mentioned above , rotavirus - associated wmi characteristically occurs around the fifth day of life in full - term neonates . by contrast , the onset of illness is more heterogeneous in full - term neonates with enterovirus and parechovirus infections : 213 days and 149 days , respectively30313235 ) . the most notable difference between infections with rotavirus vs. the other viruses is the clinical presentation of the respective patients . sepsis - like illness , including fever and rash , is observed in most cases of enteroviral and parechviral meningoencephalitis303132 ) , whereas neither fever / rash nor symptoms of gastroenteritis are seen in most neonatal patients with rotavirus - associated wmi1113 ) . in cases of rotavirus infection , virus was not detected in the csf111213 ) , whereas it is usually detected in the csf of neonates infected with enterovirus and parechovirus3031333435 ) . in addition , csf pleocytosis was not observed in rotavirus infection but was present in some cases of enterovirus infection1130 ) . the similarities and differences in wmi induced by the three viruses are summarized in table 2 . the activation of brain microglia is the principal initiating event in systemic infection / inflammation leading to wmi36 ) . the activated microglia release compounds such as reactive oxygen and nitrogen species and cytokines , all of which are toxic to the premyelinating oligodendrocytes ( pre - ols ) in white matter36 ) . pre - ols are highly vulnerable to free radical attack and to glutamate - induced excitotoxicity36 ) . this is the mechanism underlying pre - ol injury in systemic infection / inflammation , and it results in the acute loss of pre - ol , pre - ol maturation arrest , and/or abnormal myelination36 ) . in wmi caused by enterovirus and parechovirus , the single - stranded ribonucleic acid of enterovirus and parechovirus may activate the microglia via the activation of intracellular toll - like receptors ( tlr ) 7 and 8 , resulting in wmi37 ) . in addition , tlr8 localizes to neurons and axons , and its activation results in the inhibition of axonal outgrowth and neuronal apoptosis without cns inflammation38 ) . the latter hypothesis could explain the rare finding of csf pleocytosis in cases of wmi induced by parechovirus37 ) . rotavirus , by contrast , is unlikely to penetrate the brain in cases of wmi in newborns1118 ) . thus , at least in these patients , wmi caused by rotavirus can not be explained by either of the 2 aforementioned mechanisms , and how rotavirus leads to wmi in neonates remains unclear . because rotavirus infection is common in neonatal care units , recent studies11121318 ) demonstrating rotavirus - induced wmi would be of great concern to pediatricians . according to these studies111213 ) , rotavirus is one of the most common causes of neonatal seizures . furthermore , they11121318 ) suggest a new paradigm of viral infection : infection with a common virus can induce wmi in term neonates even without direct invasion . however , the causal link between rotavirus infection and wmi in neonates remains to be validated in prospective cohort studies . if rotavirus infection indeed induces wmi in neonates , the long - term prognosis of these patients determined . despite the lack of evidence , an enterotoxin of rotavirus ( nsp4 ) has been considered as cause of the neurological complications of rotavirus39 ) . variability in nsp4 across rotavirus strains may be a key determinant of pathogenicity29 ) . or neonates who had suffered from antenatal subclinical chorioamnionitis may be vulnerable to wmi by postnatal rotavirus infection . the relationship between rotavirus infection and wmi strongly suggests the use of sequential neuroimaging in all rotavirus - infected newborns with neurological manifestations . neonates with neurological symptoms should be tested for rotavirus infection and evaluated using mri including dwi . however , it might be a new aspect of ' fifth day fits ' , what we knew but forgot for long time . | that rotavirus infection can cause neurological symptoms in young children has been well established .
however , it is surprising why rotavirus infection has been overlooked as a cause of neonatal seizures for many years , despite significant research interest in neonatal rotavirus infection .
neonates are the age group most vulnerable to seizures , which are typically attributed to a wide range of causes . by contrast , because rotavirus infection is usually asymptomatic , it has been difficult to identify an association between this virus and neonatal seizures .
the conventional wisdom has been that , although neonates are commonly infected with rotavirus , neurological complications are rare in this age . however , recent studies using diffusion - weighted imaging ( dwi ) have suggested a connection between rotavirus infection and neonatal seizures and that rotavirus infection can induce diffuse white matter injury without direct invasion of the central nervous system .
the clinical features of white matter injury in rotavirus - infected neonates include the onset of seizures at days 46 of life in apparently healthy term infants .
the recent findings seem to contradict the conventional wisdom .
however , white matter injury might not be a completely new aspect of rotavirus infection in neonates , considering the forgotten clinical entity of neonatal seizures , ' fifth day fits ' . with increased use of dwi in neonatal seizures
, we are just starting to understand connection between viral infection and white matter injury in neonates . in this review
, we discuss the historical aspects of rotavirus infection and neonatal seizures . we also present the clinical features of white matter injury in neonatal rotavirus infection . |
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the primary muscles for trunk stabilization are the abdominal muscles , gluteal muscles ,
diaphragm , and pelvic floor muscles . these muscles serve as a muscular corset that works as
a unit to stabilize the trunk , and therefore affects limb movement and functional
activities1 . the muscles required for
trunk stabilization include global muscles , which directly control thoracic and pelvic
movement , and local muscles , which stabilize the lumbar region . trunk stabilization
exercises that employ harmonious movement of global and local muscles improve balance and
functional movements2 . in doing so , these
compound movements reduce low back pain ( lbp ) by increasing spinal stability through
improvements in the motor control and strength of the trunk muscles3 . in addition , stabilization exercises reduce pain ,
disability , and kinesiophobia in patients with menstrual lbp4 . common exercises for trunk stabilization include the pelvic tilt , the quadruped position ,
abdominal hollowing , and bridge exercises with hip extension in a supine crook - lying
position5 . bridge exercises make
patients feel more comfortable by reducing pain and retraining the global and local muscles
to ensure they are coordinated in an appropriate manner6 . these are useful for increasing hip extensor strength and motor
control of the pelvis and lumbar region7 . according to previous studies , the angle of the knee joint during bridge exercise affects
the activity of the hip extensor muscle8 . furthermore , bridge exercises have been associated with increased gluteus maximus activity
and decreased pelvic anterior tilt as the knee abduction angle is increased9 . prior research has indicated that the knee
joint angle affects trunk muscle activity during bridge exercises . kim et al . reported that
abdominal muscle activity decreased with reductions in the knee joint flexion angle10 ; however , kim et al . reported that
abdominal muscle activity increased with a corresponding decrease in the knee joint flexion
angle11 . similar studies have indicated
that changes in knee and hip joint angles affect abdominal and hip muscle activity . in most
of these studies , unilateral muscle activity was investigated , and the knee angle was the
same on both sides . thus , the investigated changes in the activity of abdominal and hip
extensor muscles on both sides during bridging exercises when just one knee joint angle is
varied were investigated . the subjects were 22 healthy males in their 20s and 30s , who had sufficient muscle
strength , range of motion , and balance to perform the bridge exercise . their average age ,
height , and weight were 30.07 4.18 years , 174.13 1.27 cm , and 73.13 7.32 kg ,
respectively . prior to participation , all subjects were required to provide written informed
consent , in accordance with the ethical principles of the declaration of helsinki . the
protocol for this study was approved by the local ethics committee of the catholic
university of pusan ( cupirb-2016 - 001 ) . those with disorders of the nervous system ,
cardiopulmonary system , or musculoskeletal system of the trunk and the lower extremities
were excluded from the study . the position of the bridge exercise was as follows : the subjects were asked to cross their
arms and place them on their chest and to place their feet shoulder - width distance apart . the bridging exercise in this study included 4 variations ; in each case , the non - dominant
knee was at 90 flexion , and the dominant knee varied among 120 , 90 , 60 , and 0 flexion
when the participants lifted their hips from the table . next , the trunk and lower
extremities were elevated to form a straight line , with hip flexion at 0. using a universal
manual goniometer , the investigator measured the hip and knee joint angles to place the
subjects in the bridge position . at a dominant knee joint flexion angle of 0 , participants
were instructed to lift the dominant leg until the bilateral thighs were parallel . to
control for the angle of the pelvis in every exercise , a bar was set at a fixed height above
the anterior superior iliac spine bilaterally . to prevent excessive lumbar hyperlordosis
during the bridge exercise , the exercise was practiced after maintaining a lumbar neutral
position following a pelvic tilt . all trials were repeated 3 times , and the trial order was
randomized using a table of random sampling numbers . each exercise was performed for 7
seconds . excluding 2 seconds at the start and end , the muscle activity data for 3 seconds in
the middle of the exercise were used for analysis . a surface electromyography system ( telemyo 2400t - g2 dynamic emg , noraxon , usa ) was
used to measure activity in the external oblique ( eo ) , internal oblique ( io ) , gluteus
maximus ( gm ) , and semitendinosus ( st ) muscles bilaterally . the sampling rate was set to
1,024 hz . a band - pass filter between 10 and 350 hz was used . the emg data were processed
into the root mean square ( rms ) value , which was calculated from 150-ms data windows . if a statistically significant difference was found , a bonferroni correction was
employed , setting a significance level of 0.0083 ( 0.05/6 ) . bilateral eo and io muscle activity was significantly higher with knee flexion at 0 than
at 120 , 90 , and 60. bilateral gm muscle activity was significantly different for 0
compared with that at 120 , 90 , and 60 knee flexion . ipsilateral st muscle activity was
significantly increased at 90 and 60 knee flexion compared with 120 , and significantly
decreased at 0 of knee flexion compared with 120 , 90 , and 60. contralateral st muscle
activity was significantly higher at 60 of knee flexion than at 120 and significantly
higher at 0 of knee flexion than at 120 , 90 , and 60 ( table 1table 1.comparison of muscle activation according to the various bridge exercise
positions ( v)muscleside12090600eoipsilateral4.21 1.534.45 1.155.22 1.7215.20 5.79contralateral5.30 2.105.20 1.605.77 1.9612.71 6.10ioipsilateral4.27 2.424.77 3.305.38 3.9014.68 7.39contralateral4.17 1.244.04 0.935.01 1.6911.09 5.53gmipsilateral19.16 10.1718.95 10.7016.62 11.865.85 1.87contralateral17.64 10.0318.71 9.0421.68 11.1259.22 28.45stipsilateral18.55 11.6456.97 31.2979.31 32.134.03 1.66contralateral30.25 20.0353.15 33.6664.09 28.10123.20 47.39mean sd , eo : external oblique ; io : internal oblique ; gm : gluteus maximus ; st :
semitendinosus . 120 : 120 of knee flexion ; 90 : 90 of knee flexion ; 60 : 60 of knee
flexion ; 0 : 0 of knee flexion . mean sd , eo : external oblique ; io : internal oblique ; gm : gluteus maximus ; st :
semitendinosus . 120 : 120 of knee flexion ; 90 : 90 of knee flexion ; 60 : 60 of knee
flexion ; 0 : 0 of knee flexion . bridge exercises have an important relationship with functional movements , such as
movements in bed , use of the toilet , removal of pressure from the lower back , dressing ,
using the lower extremities , and gait - related pelvic movements . furthermore , bridge
exercises also increase postural control when moving from a sitting to a standing position ,
and strengthen the extensor muscles of the lower spine and hip joints in preparation for the
stance phase of gait7 . according to
previous studies , changes in bilateral knee joint angles during bridge exercises affect
muscle activity ; however , only trunk muscles were examined . in addition , few studies have
examined the effect of bridge exercises on hip extensor activity6 , 12 , 13 . there is limited evidence for the use of one - sided
muscular training for hemiplegic patients or patients with one - sided musculoskeletal
disorders . therefore , an attempted was made to identify the effects of asymmetric knee
flexion on the activation of the abdominal ( eo , io ) and hip extensor ( gm , st ) muscles . bilateral eo and io muscle activity was significantly higher at 0 knee flexion than at
120 , 90 , and 60. the significant difference during unilateral exercise could be explained
by the requirement to counteract the force of gravity to maintain the trunk and pelvis in a
bridged position . furthermore , the unilateral conditions may have induced rotational forces
around the trunk s longitudinal axis due to loss of support , which requires bilateral
co - contracted abdominal muscle activity14 . kim et al . argued that the activation of the io and eo muscles is significantly higher at
60 than at 120 of knee flexion because of the increased abdominal pressure required for
core stabilization and the increase in the distance of the feet from the trunk11 . however , this study showed no significant
difference because only the flexion angle of the dominant knee was changed , while the
non - dominant knee was held constant at 90 flexion . at 90 and 60 knee flexion , ipsilateral st activity was significantly higher compared with
that at 120 because of muscle length - tension relationships during isometric contraction . st
activity was significantly decreased when the st length was shortened and knee flexion
angles increased15 . although contralateral st activity was not associated with a change in length under all
conditions , it was significantly higher at 60 ipsilateral knee flexion than at 120. at 60
knee flexion , intrinsic muscle activity of the foot may be decreased because the ipsilateral
forefoot sometimes did not contact the floor . ipsilateral st was differently activated according to knee joint angle ( 120 , 90 , 60 ) ;
conversely , ipsilateral gm activity was not significantly affected when the knee joint angle
was varied ( 120 , 90 , 60 ) , as the gm did not change its length at different angles because
it only crosses one joint17 . there was a significantly greater difference bilaterally in the abdominal muscles and in
the contralateral st and gm with the unilateral bridge exercise ( 0 ) compared with bridge
exercises performed at 120 , 90 , or 60 knee flexion . this increased activation was due , in
part , to participants having to balance against gravity when using one foot for a
unilateral bridge exercise18 . first , the results of the present study can not be
generalized to other populations because all of the study subjects were healthy males 2040
years old . second , the rectus abdominis and erector spinae muscles maintain a neutral
position during pelvic tilt and lifting of the pelvis ; however , their activities were not
measured in this study . in conclusion , both eo and io activity were significantly higher when the unilateral bridge
exercise was performed at 0 knee flexion than at 120 , 90 , and 60 flexion of the dominant
knee . contralateral gm activity was significantly higher at 0 of knee flexion than at 120 ,
90 , and 60. the st , a muscle that crosses 2 joints , had higher activity with knee flexion
angles less than 90. thus , bridge exercises with unilateral knee flexion angles of less
than 90 are recommended to train the ipsilateral st . the unilateral bridge exercise with
one foot on the floor is recommended for training both the eo and io abdominal muscles , and
the contralateral hip extensor muscles . | [ purpose ] this study compared abdominal and hip extensor muscle activity during a bridge
exercise with various knee joint angles .
[ subjects and methods ] twenty - two healthy male
subjects performed a bridge exercise in which the knee joint angle was altered . while
subjects performed the bridge exercise , external oblique , internal oblique , gluteus
maximus , and semitendinosus muscle activity was measured using electromyography .
[ results ]
the bilateral external and internal oblique muscle activity was significantly higher at 0
knee flexion compared to 120 , 90 , and 60. the bilateral gluteus maximus muscle activity
was significantly different at 0 of knee flexion compared to 120 , 90 , and 60. the
ipsilateral semitendinosus muscle activity was significantly increased at 90 and 60 of
knee flexion compared to 120 , and significantly decreased at 0 knee flexion compared
with 120 , 90 , and 60. the contralateral semitendinosus muscle activity was
significantly higher at 60 of knee flexion than at 120 , and significantly higher at 0
of knee flexion than at 120 , 90 , and 60. [ conclusion ] bridge exercises performed with
knee flexion less than 90 may be used to train the ipsilateral semitendinosus .
furthermore , bridge exercise performed with one leg may be used to train abdominal and hip
extensor muscles . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the presence of h pylori results in reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) produced by the host in the gastric mucosa . although there are many cell types that can contribute to the production of ros / rns , including the epithelial cells , it is primarily the neutrophils that contribute the greatest amount . nicotinamide adenine dinucleotide phosphate ( nadph oxidase [ nox ] ) on the cell membrane catalyzes the ros production to kill bacteria.12 , 16 during this process , nox is activated to receive an electron from nadph , which is donated to oxygen to create superoxide ( o2 ) . . however , the separation between neutrophils in the tissue and bacteria in the lumen make it difficult to kill all of the h pylori present . consequently , the ongoing attempt to do so is thought to result in the chronic active inflammation and damage to the gastric mucosa during the course of the prolonged infection . the presence of h pylori results in the influx of phagocytic cells in an effort to clear the infection . macrophages and neutrophils phagocytize the bacteria in an attempt to kill the organism with ros / rns . in addition , the host neutrophils and epithelial cells also express a critical enzyme , the inducible nitric oxide synthase ( inos ) , which produces no . h pylori infection results in the formation of ros and rns by increasing the immune cell expression of nox and inos . patients infected with h pylori have increased levels of ros along with increased levels of no - derived metabolites , indicating the activation of inos.5 , 18 , 19 , 20 in vivo studies with inos - deficient mice show decreased gastric cancer incidence after infection with h pylori compared with wild - type mice . in addition to the phagocytic cells attempting to clear h pylori , there is recent evidence that gastric epithelial cells also express nox , however , the details remain unclear.22 , 23 the nadph subunit nox1 is expressed in gastric tissues and likely contributes to the ros production during h pylori infection . ros is produced at a much lower level in the epithelial cells compared with the phagocytic cells of the immune response and contributes to redox - sensitive signaling and may not directly kill h pylori . in addition , dual oxidases located on the gastric epithelial cells are known to produce h2o2 in response to infection , also contributing to the ros levels . the combination of the phagocytic and epithelial cell ros production creates an oxidative stress environment that contributes to the gastric carcinogenesis . h pylori strains contain multiple virulence factors that may contribute to the host s production of oxidative stress . the presence of caga in a strain results in an increased risk of gastric carcinogenesis compared with individuals infected with caga - negative strains . increased hydrogen peroxide levels and oxidative dna damage are seen with caga - positive strains.27 , 28 in addition , there is an increase in tumor necrosis factor- and il8 , which are inflammatory and oxidative stress markers . although the precise mechanism caga uses for carcinogenesis has not yet been defined , it is clear that these actions can contribute to the development of gastric cancer . another virulence factor that may increase the chance for the development of gastric cancer is vaca . vaca is capable of inducing an influx of ca and the generation of ros that results in the activation of nuclear factor-b , thereby increasing proinflammatory immune response . h pylori has the ability to both recruit neutrophils and protect itself from oxidative bursts with the aid of virulence factors urease , neutrophil activating factor a ( napa ) , and the enzyme catalase . urease and napa recruit neutrophils to the site of infection and induce the oxidative burst from the neutrophils once they arrive . contributing to the survival of h pylori while creating a chronic inflammatory state , the neutrophils are less likely to undergo apoptosis , and h pylori located in the lumen is protected from the oxy - radicals released by napa and catalase . baba - positive strains induce a strong il8 and weak il33 cytokine response.33 , 34 this immune response drives a proinflammatory response without eventually killing the bacteria . also important another adhesion is sialic acid binding adhesion , which induces oxidative bursts in granulocytes . -glutamyl transferase is a virulence factor that contributes to production of il8 and activation of nuclear factor-b while stimulating the production of h2o2 from the gastric epithelium . it also is known that treatment of primary gastric cells and the ags cancer cell line with -glutamyl transferase results in dna damage from oxidative stress . the multiple ways of inducing the host immune response combined with the damage resulting from the oxidative stress response can initiate the steps toward carcinogenesis . moreover , h pylori also is able to protect itself from the host immune response by inducing apoptosis of macrophages . in vitro macrophages stimulated by the lipopolysaccharide of h pylori produce polyamine , which suppress their inos and induces apoptosis . within the gastric epithelial cells , h pylori also is thought to produce o2 , which is moderately cytotoxic and likely originates from the mitochondrial respiratory chain of electrons . although o2 is harmful , the reaction of h2o2 and metals is much more potent . h pylori is capable of inducing a host response and then manipulating it to create a tolerant , prosurvival environment for the bacteria , which produces a chronic inflammatory environment that is harmful to the host . the long lag time between the initial infection and carcinogenesis combined with the late - stage diagnosis results in a low 5-year survival rate . as previously mentioned , h pylori is capable of inducing a prolonged inflammatory state that contributes to carcinogenesis . caga - positive strains are capable of inducing an oxidative stress response in vitro and these strains are associated more frequently with gastric cancer . in vitro studies also have shown an increase in oxidative damage and apoptosis.42 , 43 however , studies have shown some cells with dna damage are less likely to undergo apoptosis , thus increasing the potential for cancer to arise from these cells . . in vitro studies have shown cells with deficient dna repair mechanisms that are infected with h pylori result in more oxidative stress and dna damage.44 , 45 in vivo work with mice deficient in part of the base excision repair mechanism also showed severe gastric lesions after h pylori infection . the ability of h pylori to induce dna strand breaks likely contributes to genomic instability and may facilitate the carcinogenesis . studies have shown an increase of phosphohistone h2ax , a marker of repair for double - strand dna breaks , after h pylori infection . we propose ( figure 1 ) that ros causes dna damage subsequent to 8-hydroxy-2deoxyguanosine accumulation . the loss of a base after damage would result in an abasic site that could lead to a single - strand break in the dna . the lack of repair or continued damage may induce double - strand breaks in the dna , although dna strands can be induced by other means . if a cell fails to repair too many breaks , it may result in a neoplastic precursor . inhibition of the base excision repair and mismatch repair systems during infection allows for cellular transformation to occur . ags cells express decreased messenger rna expression of apurinic / apyrimidinic endonuclease 1 ( ape1 ) , which makes the cells less able to repair dna mutations and may result in increased genomic instability . ape1 is a multifunctional molecule that repairs damage dna via its carboxy - terminus while its amino - terminus regulates transcription . initial cloning experiments discovered ape1 as the mammalian ortholog of escherichia coli xth and a dna repair enzyme.50 , 51 these early studies identified ape1 as a molecule to evaluate genomic instability . shortly after identification as a dna repair enzyme , ape1 also was determined to be a redox protein . a recent study showed the ability of ape1 to regulate epithelial ros via rac1 and nox1 after h pylori infection . ape1 was shown to decrease the expression of nox1 and interact with rac1 to prevent the formation of the nadph oxidase complex , thus limiting ros production . if this multifunctional molecule is impaired , both the feedback loop to control ros and dna repair are unable to contain the negative effects of the h pylori infection that may contribute to gastric cancer . further studies , especially in vivo , are needed to evaluate the protecting effects of ape1 from dna damage because the ability of the cells to maintain genomic integrity is critical to preventing carcinogenesis . another source of damage within the epithelial cells during h pylori infection is spermine oxidase , which is an enzyme in the pathway to produce spermidine . during this process , h2o2 also is produced , which results in the depolarization of the mitochondrial membrane , thereby activating caspase - mediated apoptosis.43 , 54 studies have shown an increase in spermine oxidase is correlated with increased dna damage . in addition , the increased apoptosis can result in an increase in proliferation in the localized area that also can contribute to gastric carcinogenesis . an in - depth review can be found by chaturvedi et al . transforming growth factor-1 ( tgf-1 ) is a multifunctional cytokine that is known to regulate proliferation and cell differentiation , among other cellular processes , and is involved in the regulation of the immune response . studies have shown that the severity of gastritis can be correlated with increased expression of tgf-1 and that gastric mucosal biopsy specimens infected with h pylori have higher tgf-1 gene expression compared with uninfected samples.56 , 57 although overexpression of tgf- can be correlated with an increased immune response , underexpression also is harmful in h pylori infection . when tgf- is suppressed , it is unable to prevent the h2o2 release from macrophages , which results in an uncontrolled respiratory burst . in addition , tgf- stimulates the induction of foxp3 treg cells that inhibit lymphocyte activation and favors persistent h pylori infection and the harmful results.59 , 60 a recent study showed that tgf-1 induced by h pylori infection results in activation of the epithelial mesenchymal transition pathway and the development of gastric cancer stem cells . a better understanding of the interactions between ros and tgf- will help clarify its contributions to carcinogenesis . animal models can be useful to evaluate infection in the complexity of a living organism . previous studies have used the big blue mouse model to assess the dna damage from infection with helicobacter because this models allows for the removal of a lambda vector to measure the mutations . these studies demonstrated increased genetic point mutations indicating oxidative stress occurring as early as 6 months post infection . infection also was correlated with hyperplasia , neutrophil infiltration , and mutated p53 status.62 , 63 an additional study also showed the increased point mutations from oxidative stress along with gastric lesions and a proinflammatory immune response after infection . these studies suggested that long - term infection with helicobacter can result in a proinflammatory immune response along with oxidative stress , which may contribute to gastric neoplasia . the chronic oxidative stress produced by cells in an attempt to eradicate the bacteria results in a harmful microenvironment for the host rather than an effective means to eliminate the pathogen . continued host efforts to clear the bacteria merely result in an increased chance of carcinogenesis . the oxidative stress produced results not only in dna damage , but also prevents dna repair mechanisms from functioning properly ( figure 2 ) . this is in addition to the increased apoptosis and subsequent cell proliferation also resulting from oxidative stress along with the development of cancer stem cells . continued study of this process and the resulting steps to cancer are required to fully understand the mechanisms at work and , perhaps , develop an effective gastric cancer prevention or early treatment . | helicobacter pylori is a gram - negative , microaerophilic bacterium that infects the stomach and can lead to , among other disorders , the development of gastric cancer .
the inability of the host to clear the infection results in a chronic inflammatory state with continued oxidative stress within the tissue .
reactive oxygen species and reactive nitrogen species produced by the immune and epithelial cells damage the host cells and can result in dna damage .
h pylori has evolved to evoke this damaging response while blunting the host s efforts to kill the bacteria .
this long - lasting state with inflammation and oxidative stress can result in gastric carcinogenesis . continued efforts to better understand the bacterium and
the host response will serve to prevent or provide improved early diagnosis and treatment of gastric cancer . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
chronic recurrent non - specific parotitis is characterized by recurrent episodes of swelling and pain of unknown etiology in the parotid gland . sialography is a hallmark in the diagnosis of salivary gland disorders ; newer imaging modalities like ct - sialography , sialoendoscopy and mri can be used . various treatment modalities have been tried , from conservative approach to surgical excision depending on the recurrence rate and severity of the condition . although symptomatic treatment with antibiotics and analgesic , injection of intraductal medicament , aggressive treatment like duct ligation or excision of gland are some of the treatment modalities , there is no established algorithm as to which treatment method should be opted in such clinical situation . a 20 years old male patient reported with pain and salty taste in the mouth that had began before a week . examination revealed an elevated right parotid papilla ; ropy , cloudy appearing saliva was oozing out on milking the gland . sialography as a treatment showed a good response with no recurrence after two years of follow - up . recurrent attacks significantly affect the quality of life and also lead to progressive gland destruction . hence , conventional sialography is useful in the diagnosis and also effective as a therapeutic aid in recurrent parotitis . acute refers to sudden onset of pain and swelling in parotid gland whereas chronic recurrent parotitis ( crp ) is characterized by intermittent , painful and swelling of the gland which may or may not be associated with food intake ( 1 ) . the term chronic recurrent non - specific parotitisis used in cases where no definite etiology is identified ( 2 ) . researchers have suggested that recurrent parotitis arises due to retrograde infections eventual to stasis of saliva , allergic , immune deficiency , genetic and hereditary factors ( 2 , 3 ) . however , none of these factors have been proven as an actual cause for the disease . there is lot of disagreement regarding the treatment of recurrent parotitis due to ambiguity about its etiology . treatment of acute phase is aimed at relieving symptoms and prevention of damage to gland parenchyma ( 4 ) . proposed treatments for crp include steroids , tetracycline and 1% methyl violet as intra - ductal medicament ( 5 , 6 ) . conventional sialography can also be used as a therapeutic aid in cases of recurrent infection as it helps in salivary gland lavage , removal of small calculi and mucous plugs within the ducts ( 7 , 8) . our primary aim in this case was to relieve the pain and reduce the frequency of recurrence . to achieve this history : a 20 years old male patient presented with pain in front of the right ear and salty taste on having food that had began before a week . pain in the right parotid gland region was sudden , intermittent , throbbing in quality and initiated while the patient was chewing food and increased in intensity especially on taking citrus food . the patient gave history of at least two episodes of recurrent swelling and pain per year on the right side of the face since two - three years . physical examination : on general physical examination , the patient was febrile and lymph nodes were palpable in the submandibular region bilaterally which was single , firm , freely mobile and tender . on extra oral examination , the right parotid gland was tender on palpation and firm in consistency whereas the left parotid gland was normal . intra - oral examination revealed an elevated right parotid papilla ( figure 2 ) . on milking the gland a thick , ropy , cloudy appearing saliva was oozing out of the duct extra oral view showing the bilaterally symmetrical face intra oral view showing elevated right parotid papilla investigation : although antibiotic sensitivity test was the norm of investigation , it was not advised as the patient was already taking antibiotic which was prescribed by a private practitioner . salivary flow rate was assessed using drooling method . before unstimulated whole saliva was collected , the patient was instructed to refrain from eating and drinking for 90 minutes to avoid any salivary stimulation . later , the patient was asked to drool the saliva in a vial at every one minute for five minutes . for stimulated method , 2% citric acid was placed on the tongue at every 30 seconds for five minutes and the patient asked to drool the saliva in a vial . salivary flow rate of unstimulated and stimulated saliva was 0.3ml / min and 1ml / min respectively suggesting normal salivary flow rate . sialography was performed with 2ml of sodium diatrizoate contrast media which was slowly injected into the gland until some resistance was felt , and the patient reported slight pain in the gland area . digital opg showed uniform normal course and caliber of stensen 's duct measuring about 23 mm in diameter from opening till the periphery of the gland . terminal ductules showed areas of blobs and dots of contrast media indicating sialectasis ( figure 3 ) . fifty percent excretion of the dye was observed in digital opg after one minute ( figure 4 ) , and complete excretion of dye was observed in five minutes suggesting normal functioning of the gland . the patient was instructed to use secretogogues ( lime juice ) for three days to clear the debris and to stimulate the salivation . sialograph shows the duct which is normal in course and caliber with dots and blobs appearance at the terminal ductules approximately 50% excretion of contrast medium after 1 diagnosis and treatment : based on patient history , clinical finding , investigation and sialographic appearance a final diagnosis of chronic recurrent non - specific parotitis was made as we were not able to find out any specific etiologic factor . sialography was performed not only for the diagnostic purpose but also for gland lavage which helped in clearing the mucus plug or cellular debris . the patient was advised to continue antibiotics and analgesic for seven days and was kept under regular follow - up once in six months for a two years ' duration . the treatment seemed to be effective as there was no recurrence during the two years ' follow - up . crp patients suffer from recurrent swelling and tenderness of the involved gland which gradually leads to the destruction of the gland . reducing the frequency of recurrence and improving the quality of life are the objective of treatment . key to the successful treatment of crp is the complete removal of cellular debris and precipitated serum proteins from the ductal lumen . this can be skillful achieved with sialography , ductal dilation with lacrimal probe and gland lavage ( 9 ) . elevated parotid papilla , reduced salivary flow and secretion are viscous and milky in appearance with clumps of material interspersed ( 10 ) . the etiology of the disease is multifactorial .there are various theories to explain the pathogenesis , one theory postulates that reduced salivary flow results in decreased mechanical cleansing , allowing bacteria to colonize and invade the duct . whereas the other proposes that repeated episodes of acute infection may lead to mucus metaplasia of ductal epithelium resulting in increased mucus content of secretions , stasis and further episodes of inflammation . secretory disorders like difference in the secretion and excretion of fluid are also considered as having an important role in the pathogenesis(10 ) . studies have reported that the ultrasonagraphy and sialography appearance for recurrent parotitis is characterized by sialectasis with strictures and dilatation of the major duct ( 9 ) . sailoendoscopy revealed white wall and lack of vascularity in the ductal layer in 75% of crp cases and multiple fibrinous debris and mucous plug in 45% of juvenile recurrent parotitis ( 11 ) . in our case although sialography is primarily used for diagnostic purpose , it can also be used as treatment for recurrent parotitis and obstructive disorders ( 12 ) . sialography improves patency of duct during cannulation by flushing action of irrigant which helps in removing any epithelial debris and mucous plug . the iodine content of the contrast medium acts as an antiseptic agent , thus reducing symptoms and preventing recurrence . this treatment should be repeated once in every two days along with sialogouges until the swelling is subsided and saliva is clear . in our case , sialography was done only once and the patient showed no recurrence during the two years ' follow - up , similar with many other reported cases ( 5,7,8,13 ) . other intracanal medicaments have been tried by many authors - bowling etal reported intraductal tetracycline instillation causing acinar atrophy in rats ( 6 ) . mandel and kaynar ( 1995 ) stated that although steroid reduces swelling and inflammation , it is not effective in preventing recurrence ( 5).nahileli et al . ( 2004 ) stated that use of sailoendoscopy facilitates direct visualization of the intraglandular structures and combination of steroid lavage with ductal dilatation will help in reducing the symptoms as well as recurrence(14 ) . however , the success of the treatment depends on intraductal lavage of the affected gland rather than the type of intraductal medicament used as various studies showed no difference in the frequency of recurrence rate . watkin and hobsely found that 56% of adult and 64% of children showed good response to conservative treatment in a five years ' follow - up study ( 15 ) . bilateral sailoendoscopy and lavage with intraductal hydrocortisone resulted in 92% recurrence free rate upto 36 months in juvenile recurrent parotitis cases ( 16 ) . few case reports showed sialography as alternative treatment for this condition as it is minimally invasive procedure with favorable outcome in juvenile recurrent parotitis ( 58 ) . however , if the symptom persists or worsens , then an aggressive treatment should be opted such as duct ligation , parotidectomy and tympanic neurectomy . in conclusion , as the cause for crp is multifactorial , patients should be educated to take higher liquid content in the diet , to do self - massaging of the gland and to maintain oral hygiene so as to avoid retrograde infection . in spite of various advanced imaging techniques , there is no established algorithm as to which imaging modality should be done in a given clinical situation . in our case we preferred sialography as it is simple to perform and cost - effective . it also has added therapeutic effect , especially in conditions where it can prevent recurrence and provide maximum benefit to the patient . | backgroundchronic recurrent non - specific parotitis is characterized by recurrent episodes of swelling and pain of unknown etiology in the parotid gland .
sialography is a hallmark in the diagnosis of salivary gland disorders ; newer imaging modalities like ct - sialography , sialoendoscopy and mri can be used .
various treatment modalities have been tried , from conservative approach to surgical excision depending on the recurrence rate and severity of the condition .
although symptomatic treatment with antibiotics and analgesic , injection of intraductal medicament , aggressive treatment like duct ligation or excision of gland are some of the treatment modalities , there is no established algorithm as to which treatment method should be opted in such clinical situation.case detaila 20 years old male patient reported with pain and salty taste in the mouth that had began before a week .
examination revealed an elevated right parotid papilla ; ropy , cloudy appearing saliva was oozing out on milking the gland .
unstimulated and stimulated whole salivary flow rate was assessed using drooling method .
sialography was used as a diagnostic and a therapeutic aid . in our case ,
sialography as a treatment showed a good response with no recurrence after two years of follow - up .
we highlighted the role of sialography as a therapeutic aid.conclusionrecurrent attacks significantly affect the quality of life and also lead to progressive gland destruction . preventing or reducing the frequency of recurrence remains the goal of therapeutic procedure .
hence , conventional sialography is useful in the diagnosis and also effective as a therapeutic aid in recurrent parotitis . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
acetylcholine is a neurotransmitter and neuromodulator in the cns that has a salient role in cognition ( hasselmo and sarter , 2011 ; picciotto et al . , 2012 ) . these are likely to be instrumental in mediating hippocampal neural network activity , such as theta - rhythm oscillations that are necessary for memory acquisition ( buzski , 2002 ) . moreover , altered acetylcholine receptor activity and cholinergic fiber lesions lower the threshold for hippocampal long - term potentiation , the cellular correlate of learning and memory ( hasselmo and sarter , 2011 ; picciotto et al . , 2012 ) . hence , ascertaining how acetylcholine affects hippocampal neuronal excitability is vital for understanding cognitive processing . the hippocampal dentate gyrus ( dg ) plays a fundamental role in processes such as memory encoding and storage ( acsdy and kli , 2007 ; pelkey and mcbain , 2008 ) . granule cells , the dg principal neurons , receive the primary input to the hippocampus from the cortex , which is relayed to hippocampal ca3 neurons via their axons ( mossy fibers ) ( acsdy and kli , 2007 ; pelkey and mcbain , 2008 ) . this , coupled with the unique granule cell somato - dendritic membrane properties ( krueppel et al . , 2011 ; lbke et al . , 1998 ; perna - andrade and jonas , 2014 ) , results in them having a very low mean action potential firing frequency rate in vivo ( henze et al . thus , only granule cells that receive a strong glutamatergic drive will participate in information transfer to the ca3 region ( acsdy and kli , 2007 ) . a high density of cholinergic fibers , though , also innervates the dg ( aznavour et al . , 2005 ) . interestingly , granule cells display action potential bursts during exploration - associated theta rhythms ( skaggs et al . , 1996 ) however , relatively little is known about how cholinergic activity impacts dg cell function ( hasselmo and sarter , 2011 ; picciotto et al . , 2012 ) . the cellular mechanisms by which acetylcholine exerts its effects are complex and are likely to depend on the timing of the release of acetylcholine as well as the state of neurons ( picciotto et al . , 2012 ) . cholinergic receptor modulation of hippocampal pyramidal cell somato - dendritic excitability and synaptic plasticity has been extensively explored ( hasselmo and sarter , 2011 ; picciotto et al . , 2012 ) . cholinergic fibers , however , innervate hippocampal cell axons too ( aznavour et al . , 2005 ) . understanding this is crucial as altered axonal information processing will impact neural firing patterns and synaptic release and , thereby , neural network excitability ( bean , 2007 ; debanne et al . , 2011 ; kole and stuart , 2012 ) . here , we show that cholinergic fiber stimulation results in a persistent reduction of the granule cell action potential threshold and increased propensity to elicit action potentials . these effects are due to muscarinic receptor - induced sustained axonal ca influx via t - type ca channels , which then causes an enduring decrease in axonal kv7 k channel function , reducing the spike threshold . hence , our results show that cholinergic afferent discharge primes granule cell axons to more readily elicit action potentials . this represents a unique means by which cholinergic afferent input enhances neuronal information processing and possibly influences memory formation . to investigate how endogenous acetylcholine affects granule cell intrinsic activity , we made patch - clamp recordings from mature cells in brain slices before and after extracellular stimulation of afferents in the stratum moleculare ( figure 1a ) . these cells had input resistances ( rn ) of 297.94 23.9 m ( n = 117 ) and complex dendritic trees ( figure 1a ) as revealed by post hoc morphological analysis . experiments were performed in the presence of glutamatergic and gabaergic ionotropic and metabotropic receptor inhibitors , unless otherwise stated . cholinergic fibers are most active during paradoxical sleep in vivo , firing in high - frequency bursts ( average frequency of 1629 spikes / s ) ( brazhnik and fox , 1999 ; lee et al . , 2005 ) . we stimulated cholinergic fibers eight to ten times at an intra - burst frequency of 40 hz and inter - burst interval of 125 ms ( 3 hz ; near theta frequency ; average frequency = 27.8 hz ; figure 1a ) . the stimulus strength was adjusted to yield single slow cholinergic synaptic potentials with amplitudes of 0.56 0.1 mv ( n = 15 ) and summation ratios of 2.49 0.4 ( n = 15 ; figure 1a ) . this high - frequency ( hf ) stimulation paradigm was repeated ten times at 0.1 hz . hf stimulation significantly lowered the action potential ( spike ) threshold , without affecting other intrinsic membrane properties ( figure 1b , tables s1 and s2 ) . consequently , granule cells generated more action potentials with depolarization ( figure 1b ) . similar results were also obtained if the superfusate contained 1.3 mm ca concentrations ( spike threshold decrease immediately and 30 min post - stimulation = 4.16 0.6 mv and 5.00 0.6 mv [ n = 5 ] ; tables s1 and s2 ) instead of 2 mm ca ( figure 1b ) . cholinergic neurons , though , fire at lower frequencies in vivo during slow - wave sleep ( average frequency of 0.81.9 spikes / s ) ( lee et al . , 2005 ; simon et al . , to mimic this , eight stimuli were delivered at 325 ms intervals ( 3.08 hz ) during 10 s ( average frequency = 0.8 hz ; figure s1a ) and repeated ten times at 0.1 hz . the initial synaptic potential amplitude ( 0.88 0.2 mv , n = 6 ) and summation ratio ( 2.39 0.3 , this lf stimulation also reduced the action potential threshold and enhanced granule cell firing for at least 30 min , with other spike parameters or intrinsic membrane properties not affected ( figure s1 and tables s1 and s2 ) . similar effects were observed if cholinergic fibers expressing channelrhodopsin and the fluorescent marker , eyfp , were optically stimulated at 0.8 hz ( protocol similar to lf electrical stimulation ) with or without glutamate and gaba receptor blockers ( figure s2 ) . since lf optical or electrical stimulation effects were comparable to those produced by hf electrical stimulation , it implies that activation of a small population of acetylcholine receptors is sufficient to cause action potential threshold changes . the granule cell action potential is initiated at the axon initial segment ( ais ) ( schmidt - hieber and bischofberger , 2010 ) , implying that cholinergic fiber stimulation caused axonal changes . this differed from previous reports indicating that cholinergic fibers affect somato - dendritic excitability in other hippocampal neurons ( buchanan et al . , 2010 ; cole and nicoll , 1983 ; gu and yakel , 2011 ) . one explanation might be that the released acetylcholine was degraded rapidly . to test this , we performed hf stimulation in the presence of the acetylcholinesterase inhibitor , neostigmine ( 3 m ) . the cholinergic synaptic potential summation ratio ( 10.3 3.6 , n = 10 ) and the spike threshold decrease were significantly greater with neostigmine than in its absence ( figure s3 ) . additionally , neostigmine caused neuronal depolarization immediately after cholinergic afferent stimulation , which recovered within 10 min ( figure s3 ) . notwithstanding this , the primary effect of cholinergic afferent stimulation was to induce a long - term spike threshold reduction . we next asked whether the cholinergic afferent stimulation - induced spike threshold decrease might enhance excitatory synaptic potential - spike ( e - s ) coupling and spontaneous action potential firing . to elicit 50 hz glutamatergic excitatory postsynaptic potential ( epsp ) trains , we stimulated afferents onto granule cell dendrites before and after hf cholinergic fiber stimulation ( figure 1b ) in the presence of gaba receptor inhibitors only . the stimulation strength was adjusted so that the epsp summation was subthreshold ( average first epsp amplitude = 5.01 1.4 mv , n = 6 ; figure 1b ) . following hf cholinergic afferent stimulation , the initial epsp amplitude was comparable ( 5.24 1.5 mv , n = 6 ) but 50 hz epsp trains now resulted in spikes ( figure 1b ) . this was not due to alterations in pre - synaptic function as subthreshold epsps generated using an injected -waveform yielded spikes following hf cholinergic afferent stimulation ( figure 1b ) . loose cell - attached mode was significantly enhanced for at least 25 min following hf cholinergic fiber stimulation ( figure 1b ) . hence , cholinergic afferent stimulation enhances information processing in hippocampal granule cells by persistently reducing the spike threshold . next , we investigated whether muscarinic or nicotinic cholinergic receptors might cause the spike threshold changes . the slow synaptic potentials induced by hf and lf stimulation paradigms as well as the associated spike threshold reductions were inhibited by the muscarinic receptor antagonist , atropine ( 3 m , figure 1 , figure s1 ) but not by co - application of methyllycaconitine ( an 7 receptor antagonist , 10 nm ) ( alkondon et al . , 1992 ) and hexamethonium ( 200 m , a nicotinic receptor antagonist ) ( papke et al . , 2010 ) ( spike threshold change with nicotinic receptor inhibitors = 5.58 0.5 mv [ n = 6 ] ; table s2 ) . if atropine was applied following hf cholinergic fiber stimulation ( initial epsp amplitude = 0.54 0.2 mv [ n = 6 ] ; summation ratio = 5.87 0.7 ) , the long - lasting spike threshold decrease and increased granule cell firing still occurred ( spike threshold decrease immediately and 25 min post - stimulation = 4.28 0.4 mv and 4.78 0.4 mv [ n = 6 ] ; tables s1 and s2 ) . thus , muscarinic receptor activation was required for the initiation but not the maintenance of the acetylcholine - mediated spike threshold plasticity . further , the muscarinic receptor agonist , oxotremorine - m ( oxo - m ; 1 m ) replicated the effects of cholinergic fiber stimulation . oxo - m enhanced action potential firing and decreased spike threshold , which persisted for up to 40 min following oxo - m washout ( figures 2a and 2f ) . however , since oxo - m was bath applied , it had added global effects on granule cell excitability . thus , oxo - m , like neostigmine , produced several epiphenomena : rmp depolarization , enhanced rn , and an afterdepolarization ( adp ) after the action potentials ( figure 2a , table s2 ) . these ancillary effects fully reversed upon washout and so did not contribute to the persistent increase in spiking induced by oxo - m . is the acetylcholine - induced long - lasting spike threshold decrease axonal ? to test this , we patched onto neurons situated near the slice surface whose axons were severed , as shown by post hoc morphological analysis ( residual axon length = 6.11 1.2 m sholl analysis showed that the dendritic trees of neurons with intact axons and axonless cells were comparable ( sholl , 1953 ) ( figure s4 ) . the intrinsic membrane properties of neurons with and without an axon were also similar , indicating that the somato - dendritic conductance properties were unchanged by axon cutting ( table s3 ) . unlike in long axon neurons ( figure 2a ) , 1 m oxo - m application onto axonless neurons did not alter the spike threshold or granule cell firing ( figures 2b and 2f ) . oxo - m , though , still depolarized the rmp , enhanced rn , and induced an adp following action potential firing in a reversible manner , to an extent comparable to that observed in neurons with axons ( figure 2b , table s2 ) . this supports the concept that these auxiliary effects are due to changes in somato - dendritic conductances . to further test whether the effects of acetylcholine were axonal , we focally applied 1 m oxo - m for 20 ms onto axons 2530 m from the soma or stimulated cholinergic afferents in the ca3a region ( i.e. , those targeting distal mossy fibers ) . both stimuli lowered the spike threshold and enhanced granule cell firing for at least 30 min but did not alter other intrinsic membrane properties ( figures 2c , 2e , and 2f ) . synaptic potential - like events , which occurred if the stimulation electrode was placed near proximal dendrites ( figure 1 ) , were not observed either . in contrast , a local puff of 1 m oxo - m onto proximal granule cell dendrites 2530 m from the soma resulted in a robust excitatory synaptic potential - like event ( amplitude = 5.88 2.2 mv , n = 5 ) , greater than that with proximal dendrite cholinergic fiber stimulation ( figure 1a ) , but had little effect on action potential threshold ( figures 2d and 2f ) . instead , the rmp depolarized , rn was enhanced , and a small adp was induced , effects that reversed fully within 10 min of washout ( figure 2d , table s2 ) . hence , axonal and somato - dendritic muscarinic receptors have differential effects : somato - dendritic receptor activation leads to synaptic potential - like events , rmp and rn alterations , and an adp , while axonal muscarinic receptor stimulation solely causes sustained action potential threshold changes . since cholinergic fiber stimulation results in a long - lasting spike threshold change only , these data strongly suggest that synaptically released acetylcholine alters granule cell axonal function by preferentially activating axonal muscarinic receptors . to test whether muscarinic receptors are axonal , we performed immunogold labeling using selective m1 and m3 receptor antibodies ( yamasaki et al . , 2010 ) . no discernible labeling was observed with the m3 receptor antibody . however , under experimental conditions that did not yield immunolabelling in m1 receptor null tissue ( yamasaki et al . , 2010 ) , m1 receptor antibody labeling was detected in granule cell processes , most strongly in dendrites and spines but also along the entire length of mossy fibers ( figure 2 g ) . to ascertain whether cholinergic fibers innervate granule cell axons , we performed choline acetyltransferase ( chat ) labeling . interestingly , chat - immunoreactive fibers and boutons were abundant in close proximity of aiss ( identified by 1v spectrin labeling ; figure s5 , movie s1 ) . thus , local acetylcholine release is likely to activate axonal m1 receptors to induce persistent spike threshold reductions . since action potentials in granule cells initiate at the ais ( schmidt - hieber and bischofberger , 2010 ) , this suggests that ais properties are altered following muscarinic receptor activation . the effects that we observed could be due to ( 1 ) altered ais position or ( 2 ) modified ais ion channel expression and/or biophysical properties ( grubb and burrone , 2010 ) . we first determined whether ais localization was altered by performing immunohistochemical analysis in control and oxo - m - treated slices . electrophysiological recordings before fixation confirmed that oxo - m caused spike threshold plasticity ( figure 2a ) . there was no difference in iv spectrin , nav subunits , and ankyrin g labeling between control and oxo - m - treated slices . the labeling intensity of these peaked 10 m from the soma ( figure s6 ) . the average ais lengths in control and oxo - m - treated slices were 28.84 1.05 m ( n = 57 , 3 slices ) and 26.58 3.14 m ( n = 59 , 3 slices , p = 0.54 ) , respectively . this indicates that acute muscarinic receptor activation does not alter the ais localization or length . action potential initiation and upstroke in granule cells are dependent upon na channels ( schmidt - hieber and bischofberger , 2010 ) . as the action potential amplitude and width were not altered by endogenous acetylcholine or external application of oxo - m ( table s1 ) , na channel properties were unlikely to have been affected . in support , the ais nav subunit antibody labeling distribution and intensity in control and oxo - m - treated slices was similar ( figure s6 ) . mossy fibers express kv7 , kv7.2 and kv7.3 subunits too ( cooper et al . , 2001 ) . kv7 channels underlie a non - inactivating k current , the m current ( brown and passmore , 2009 ) , that is inhibited by muscarinic receptors and regulates the action potential threshold in many central neurons ( battefeld et al . , 2014 ; shah et al . , 2008 immunohistochemistry showed that kv7.2 and kv7.3 subunits were located in granule cell distal ais ( approximately 20 m from the soma ; figure s6 ) . oxo - m treatment did not affect their expression and distribution ( figure s6 ) . application of the specific kv7 channel inhibitor , xe991 ( 3 m ; brown and passmore , 2009 ) , enhanced action potential firing in granule cells with intact axons but not in axonless cells ( figure 3 ) . this effect was predominantly due to reduced action potential threshold , rmp and rn being unaffected ( figure 3 , table s4 ) . similar results were obtained if axonal kv7 channel function was selectively disrupted by intracellular ankyrin g binding peptide ( abp , yiaegesdtd ; 8 mm ) ( shah et al . , 2008 ) , but not scrambled abp ( tseydaedig ; 8 mm ; figure 3 ) . additional xe991 application onto neurons dialysed with abp , but not those containing sabp , had no further effect ( figure 3 ) . these findings together with kv7.2/7.3 antibody labeling ( figure s6 ) strongly suggest that kv7 channels are present only in granule cell axons and that their axonal location serves to regulate the action potential threshold . in agreement , we found that the kv7 current measured using voltage clamp could be recorded solely from neurons with long axons but not from axonless neurons ( figure s7 ) . to discern whether altered kv7 channel function underlies the muscarinic receptor activation - induced persistent action potential threshold reduction , we pretreated granule cells with xe991 ( 3 m ) and applied either hf cholinergic afferent stimulation or oxo - m . neurons treated with xe991 had a lower spike threshold and enhanced excitability than non - treated neurons ( figure 4 ) . for hf stimulation , the initial epsp amplitude ( 0.44 0.1 mv , n = 5 ) and summation ratio ( 3.04 1.3 mv , n = 5 ) were comparable to that in non - xe991-treated neurons . however , neither hf cholinergic afferent stimulation nor oxo - m application in the presence of xe991 altered the spike threshold or granule cell firing ( figure 4a ) . oxo - m subsidiary effects ( rmp and rn changes and adp induction ) still occurred and recovered within 25 min of washout ( figure 4a , table s2 ) , further supporting the notion that kv7 channels do not underlie these somato - dendritic effects in granule cells . the above results suggest that axonal kv7 channels are inhibited in a long - lasting manner by brief muscarinic receptor activation . indeed , a 10 min oxo - m application abolished the kv7 current , an effect that did not reverse with washout for at least 20 min ( figure 4b ) . , muscarinic receptor activation induces long - lasting spike threshold plasticity by persistently inhibiting the axonal kv7 current . the persistent effect of muscarinic receptor activation on axonal kv7 currents ( figure 4b ) was unexpected as cholinergic suppression of the somatic kv7 current in other neurons is fully reversible . it is typically due to membrane phosphatidylinositol-4.5-bisphosphate ( pip2 ) level depletion ( brown and passmore , 2009 ; gamper and shapiro , 2007 ; shen et al . , 2005 ) . upon cholinergic agonist washout , pip2 levels are replenished , leading to kv7 current recovery ( brown and passmore , 2009 ; gamper and shapiro , 2007 ) . our results imply that pip2 levels may not recover rapidly at the ais , leading to prolonged axonal kv7 channel inhibition . to test this , we incorporated the water - soluble analog dioctanoyl ( dic8)-pip2 ( 125 m [ lukacs et al . , 2013 ] ) in the patch pipette to boost pip2 levels . interestingly , oxo - m still persistently lowered the spike threshold ( figure 5a ) . the oxo - m - induced adp , though , was reduced considerably with dic8-pip2 ( figure 5a , table s2 ) , suggesting that somato - dendritic pip2 levels were increased . it should be noted , though , that the ais is effectively a surface diffusion barrier ( leterrier and dargent , 2014 ) and , therefore , artificially altering pip2 levels might not affect intrinsic ais lipid levels . nonetheless , it suggests that the signaling mechanisms underlying muscarinic - receptor suppression of axonal kv7 currents may differ from that described at neuronal somata . intracellular ca ( [ ca]i ) can also suppress the kv7 current ( gamper and shapiro , 2007 ; selyanko and brown , 1996 ) . muscarinic receptor stimulation in hippocampal neurons might raise [ ca]i by ca release from intracellular stores ( gamper and shapiro , 2007 ) or augmenting voltage - gated ca channel ( vgcc ) opening ( park and spruston , 2012 ) . we tested these possibilities by incorporating 20 mm bapta into the patch pipette or applying the vgcc inhibitor , cadmium chloride ( cdcl2 ; 200 m ) . oxo - m treatment then no longer persistently altered the action potential firing or threshold ( figure 5b ) . secondary alterations in rmp and rn still occurred ( figure 5b , table s2 ) , though the oxo - m - induced adp was abolished ( table s2 ) . hence , muscarinic receptor stimulation elevates [ ca]i , via vgccs to cause spike threshold changes . since the spike threshold effects occur at rest ( figure 1b ) , the vgccs involved are likely to be low - threshold channels such as t- , r- , and l - type ca channels ( catterall , 2011 ) . interestingly , in ca1 neurons , muscarinic receptor stimulation enhances r - type ca channel activity ( park and spruston , 2012 ) . in granule cells , though , the t - type ca channel inhibitors , nicl2 ( 50 m ) and tta - p2 ( 500 nm ) ( dreyfus et al . , 2010 ) , but not the r - and l - type ca channel inhibitors , snx482 ( 500 nm ) and nifedipine ( 10 m ) , prevented the oxo - m - induced sustained action potential firing and threshold change ( figure 6 ) . all auxiliary effects of oxo - m ( depolarized rmp , enhanced rn , and adp generation ) still occurred , leading to a reversible increase in action potential firing ( figure 6a , table s2 ) . tta - p2 also prevented the effects of hf cholinergic afferent stimulation on granule cell excitability ( figure 6 , table s2 ) but had little effect on cholinergic epsp amplitude or summation ( summation ratio = 2.50 0.36 , n = 5 ) . the above results suggest that the persistent effects of muscarinic activation on granule cell axons required ca entry via t - type ca channels . accordingly , oxo - m effects on the kv7 current recorded from long axon granule cells were prevented by tta - p2 ( 500 nm ; figure 6d ) . tta - p2 itself did not affect the kv7 current and the current was still blocked by 3 m xe991 . three subunits , cav3.13.3 , encode for t - type ca channels ( catterall , 2011 ) . cav3.2 antibody immunogold particles were detected on wild - type axons , dendrites , and somata spines ( figure 7a ) . the granule cell axon cav3.2 labeling , although less than in dendrites and spines , was significantly greater than that in cav3.2 null granule cell axons ( figure 7a ) , indicating that cav3.2 subunits are located in mossy fibers . do muscarinic receptors activate axonal t - type ca channels ? to test this , we imaged ca in granule cell axons using two - photon microscopy . oxo - m application depolarized somata by 6.29 0.7 mv ( n = 8) . simultaneously , the axonal basal [ ca]i was increased significantly ( figure 7b ) . interestingly , the axonal basal [ ca]i augmentation only partly recovered following washout of oxo - m for up to 40 min and reached a new baseline level significantly above the control , despite the rmp fully recovering ( figure 7b ) . the axonal [ ca]i increase was not due to a somatic rmp change because a 6 mv somatic depolarization caused a substantially smaller axonal [ ca]i rise than that observed with oxo - m and fully reversed on rmp restoration ( data not shown ) . the oxo - m - induced basal axonal [ ca]i escalation was abolished by tta - p2 ( 500 nm ; figure 7b ) . hence , muscarinic receptor activation increases basal t - type ca channel function in granule cells resulting in a long - term rise in basal axonal [ ca]i . previous studies have indicated that axonal t - type ca currents can regulate the action potential threshold ( bender et al . , 2010 ; ohkuma et al . , 2013 ) . t - type ca channel inhibitors , though , did not affect the granule cell spike threshold ( figure 6b ) . in support , we found the action potential - associated [ ca]i rise was similar under control conditions , following 10 min oxo - m treatment and after 25 min washout ( figure 7c ) . this reinforces the notion that oxo - m - augmented t - type ca channel activity predominantly enhances basal axonal [ ca]i levels . one reason might be that the biophysical properties of axonal t - type ca channels are altered . we thus recorded the t - type ca current from long axon and axonless neuronal somata . t - type ca currents were stable for up to 40 min , activated above 80 mv ( figure s8 ) , and were inhibited by tta - p2 ( 500 nm , n = 16 ) and cdcl2 ( 200 m , n = 3 ) but not xe991 ( 3 m , n = 6 ; data not shown ) . although the half - activation potentials in long axon ( v1/2 = 61.2 1.3 mv , n = 8) and axonless ( v1/2 = 59.6 1.4 mv , n = 6 ) neurons were comparable , the current amplitudes were significantly smaller in axonless neurons than long axon neurons ( maximal amplitude at 40 mv in long axon and axonless neurons = 112.5 11.0 pa [ n = 8 ] and 70.7 18.8 pa [ n = 6 ] , p < 0.05 ) . oxo - m shifted the t - type ca current activation curve in long axon neurons , but not axonless neurons , to the left without affecting the slope of the curve ( figure s8 ) . the currents at 80 mv and 70 mv were substantially enhanced by oxo - m in long axon neurons but the peak current amplitudes were no different ( figure s8 ) . these oxo - m effects on t - type ca currents in long axon neurons were maintained for at least 25 min following washout ( figure s8 ) . hence , these results strongly support the idea that muscarinic receptor activation leads to a persistent rise in axonal basal [ ca]i by enhancing t - type ca channel function . if the persistent increase in t - type ca channel activity at rest is the cause of the muscarinic receptor - induced action potential threshold plasticity , then applying tta - p2 after oxo - m should rescue this . indeed , we found that inclusion of tta - p2 ( 500 nm ) during washout after oxo - m treatment of granule cells restored the spike firing and threshold to that observed under control conditions ( figure 8) . moreover , since t - type ca channels are not active at 90 mv ( figure s8 ) , muscarinic receptor activation at this potential would not be predicted to induce action potential threshold or firing alterations . in support , 1 m oxo - m application and washout had little effect on spike threshold or firing at 90 mv ( control and oxo - m spike threshold difference = 0.55 0.4 mv [ n = 6 ] ) . hence , our findings strongly suggest that muscarinic receptor activation in granule cells leads to a long - term enhanced t - type ca channel openings at rest , which results in sustained elevated [ ca]i and persistently reduced axonal kv7 channel function . we found that synaptically released acetylcholine preferentially lowered the action potential threshold in granule cells , an effect that persisted at least 30 min ( figure 1 , figures s1s3 ) . consequently , there was a greater propensity for granule cells to elicit action potentials with depolarizing stimuli and e - s coupling and spontaneous spike frequency were enhanced ( figure 1 ) . these effects were due to axonal muscarinic receptor - induced sustained enhancement of axonal t - type ca channel function at rest ( figures 2 , 6 , and 7 , figure s8 ) such that axonal basal [ ca]i was elevated ( figure 7 ) . this inhibited kv7 channel function ( figure 5 ) , and as kv7 channels regulate the spike threshold in granule cells ( figure 3 ) , there was a prolonged reduction in this . indeed , the long - lasting decrease in spike threshold could be reversed if t - type ca channel activity was inhibited following the muscarinic receptor stimulation ( figure 8) . given that increased granule cell spike frequency will result in more effective discharge of post - synaptic hippocampal ca3 neurons ( henze et al . , 2002 ) , our results suggest that acetylcholine is likely to boost granule cell information processing by augmenting axonal signaling . as granule cell activity is normally sparse in vivo ( henze et al . , 2002 ; perna - andrade and jonas , 2014 ) , this is likely to be a critical mechanism for enhancing their excitability to allow efficient information transfer to post - synaptic neurons . chat labeling showed that cholinergic fibers project to dg molecular , granular , and hilus regions where granule cell dendrites , somata , and axons are located ( figure s5 ) ( aznavour et al . , 2005 ) . remarkably , in these neurons electrical or optical stimulation paradigms , with or without glutamate and gaba receptor inhibitors , resulted in endogenous acetylcholine release that affected the action potential threshold only ( figure 1 , figures s1 and s2 ) . other spike characteristics or intrinsic membrane properties were unaltered . oxo - m local axonal application or cholinergic afferent stimulation to axons replicated these findings ( figure 2 ) . muscarinic receptors were located on mossy fibers but their density was lower than in dendrites and spines ( figure 2 g ) . these findings suggest that the effects of endogenous acetylcholine release are highly specific and constrained to defined targets and cholinergic fiber stimulation preferentially activates axonal muscarinic receptors , even though they are present at a lower density in axons . this is in line with previous studies using cell lines that show that a small fraction of receptors adequately causes the appropriate downstream effects ( falkenburger et al . , 2013 ) . one reason for the preferential axonal response might be that granule cell dendritic intrinsic properties are such that signals received by these are strongly attenuated ( krueppel et al . , 2011 ) . synaptically released acetylcholine may have also stimulated dendritic muscarinic receptors because this , like focal application of oxo - m to dendrites , produced synaptic potential - like events ( figure 1 ) . this dendritic muscarinic receptor activation , though , was not sufficient to cause intrinsic membrane property alterations that are associated with somato - dendritic receptor activation ( figure 2 ) . nonetheless , under some conditions , cholinergic afferent inputs might robustly activate dendritic muscarinic receptors , leading to altered somato - dendritic membrane properties , as in hippocampal pyramidal neurons ( buchanan et al . , 2010 ; indeed , in the presence of neostigmine , a significantly greater reduction in spike threshold together with a transient somatic rmp depolarization occurred ( figure s3 ) . the rmp depolarization is likely to be due to increased somato - dendritic muscarinic receptor recruitment from enhanced volume transmission of synaptically released acetylcholine . this indicates that the cholinergic fiber stimulation strength coupled , perhaps , with the pattern of stimulation in vivo might dictate axonal versus dendritic effects of acetylcholine . the preferential axonal function alteration by cholinergic afferent stimulation is of considerable significance for granule cells . most glutamatergic synaptic inputs are onto granule cell dendrites but , as dendritic signals in granule cells are significantly attenuated , only strong inputs will impact their excitability ( krueppel et al . , 2011 ) . our results suggest that the cholinergic fiber stimulation dynamically boosts granule cell activity so that even weak synaptic information received by their dendrites can be efficiently transferred to post - synaptic neurons . indeed , e - s potentiation and spontaneous action potential frequency were enhanced following cholinergic afferent stimulation ( figure 1b ) . consequently , mossy fiber transmission may be enhanced , leading to enhanced synaptic potentiation at these synapses . we show that that muscarinic receptor stimulation led to a persistent axonal basal [ ca]i enhancement by increasing t- ( cav3.2 ) type ca channel activity at rest ( figures 6 and 7 ) . this was due to a shift in the axonal t - type ca activation curve by muscarinic receptors ( figure s8 ) . as cav3.2 subunits are expressed globally in granule cells ( figure 7 ) , this suggests that the coupling of muscarinic receptors to cav3.2 channels might be weaker in somata and dendrites compared with axons . accordingly , oxo - m did not alter somato - dendritic t - type currents in axonless neurons ( figure s8 ) . although this was unexpected , a selective axonal , but not dendritic , t - type ca channel inhibition by dopamine occurs in cochlear interneurons ( bender et al . , cholinergic receptor - induced t - type ca channel activation is both necessary and sufficient for the enhanced excitability as t - type ca channel antagonists prevented and reversed the spike threshold long - lasting plasticity ( figures 6 and 8) . unlike in some other neurons ( bender et al . , 2010 ; ohkuma et al . , 2013 ) , the t - type ca channels per se did not affect the action potential threshold ( figure 6 ) . further , though t - type ca channel activity at potentials near the granule cell rmp were enhanced by oxo - m , the current at voltages near the spike threshold were similar ( figure s8 ) . in support , the spike - associated [ ca]i rise was similar before and after oxo - m treatment ( figure 7 ) . the axonal basal [ ca]i rise by oxo - m then suppressed axonal kv7 current , which regulates the spike threshold ( figures 3 and 5 ) while it is known that [ ca]i 100 nm inhibits kv7/m channels ( selyanko and brown , 1996 ) , the muscarinic receptor - induced sustained ca entry via t - type ca channels causing kv7 channel plasticity is a novel , distinct phenomenon . how might brief muscarinic receptor activation lead to a persistent shift in t - type ca activation curve ( figure s8 ) ? native and expressed cav3.2 currents ( including those in some interneuron aiss [ bender et al . , 2010 ] ) are modified by kinases such as protein kinase c ( bender et al . since muscarinic receptor stimulation alters the activity of multiple kinases , there is a strong possibility that the muscarinic receptor - induced axonal t - type ca channel long - term plasticity is kinase mediated too . in summary , our findings show that cholinergic fiber activation reduces the action potential threshold persistently such that granule cells will respond with greater fidelity to additional excitatory inputs ( figure 1 ) . this is , as yet , an unreported action of acetylcholine that might influence cognitive function . while , we conducted our study in the dentate gyrus , the findings may also be applicable to other hippocampal regions . in fact , cholinergic fibers innervate the stratum oriens where hippocampal ca1 and ca3 neuron axons reside ( aznavour et al . , 2005 ) . in these neurons , kv7 subunits are axonal ( devaux et al . , 2004 ) and also play a significant role in setting the action potential threshold ( shah et al . , 2008 ) . hence , the acetylcholine - induced action potential threshold long - lasting plasticity might be a common cellular mechanism that plays a fundamental role in neuronal information processing and storage . 22- to 27-day - old male sprague dawley rat pups and 5- to 6-week - old b6 mice expressing channelrhodopsin 2 ( chr2 ) and eyfp in cholinergic fibers ( kind gift from prof . d. kullman , ucl institute of neurology ) were decapitated and the brain removed and submerged in ice - cold solution containing the following : 87 mm nacl , 25 mm nahco3 , 10 mm glucose , 75 mm sucrose , 2.5 mm kcl , 1.25 mm nah2po4 , 0.5 mm cacl2 , 7 mm mgcl2 ( ph 7.3 ) , 325 mosm / l . the brain was hemi - sected and a cut parallel to the dorsal part of the brain made . the ventral side brain halves were glued onto a slice holder and 350 m slices made ( leica vt1200s , leica ) . slices were incubated in the cutting solution for 10 min at 35c and then stored at room temperature . slices were transferred to a submerged chamber containing external solution containing the following : 125 mm nacl , 25 mm nahco3 , 25 mm glucose , 2.5 mm kcl , 1.25 mm nah2po4 , 2 mm cacl2 , 1 mm mgcl2 , 0.05 mm cnqx , 0.05 mm dl - ap5 , 0.01 mm bicuculline , 0.001 mm cgp 55845 ( ph 7.3 ) , 32c36c . the internal pipette solution contained the following : 120 mm kmeso4 , 15 mm kcl , 10 mm hepes , 2 mm mgcl2 , 0.2 mm egta , 2 mm na2atp , 0.3 mm tris - gtp , and 14 mm tris - phosphocreatinine ( ph 7.3 ) with koh , 295300 mosm / l . in some experiments , 8 mm abp or sabp was added to the internal solution . when 20 mm k4bapta was added to the pipette solution , kmeso4 was reduced to 60 mm and osmolarity adjusted by adding n - methyl - d - glucamine ( nmdg ) . pipettes had resistances of 58 m. in all experiments , neurobiotin ( 0.2% w / v ) was included in the intracellular pipette solution . slices were fixed in 4% paraformaldehyde and stained with streptavidin alexa fluor 488 conjugate 24 hr later ( huang et al . , 2012 ) electrophysiological recordings were made using a multiclamp 700b amplifier ( molecular devices ) . series resistance was in the order of 1020 m. recordings were discarded if the series resistance increased by more than 20% . all reagents were purchased from sigma - aldrich apart from tetrodotoxin , bicuculline , cgp 55845 , dl - ap5 , and xe991 , which were obtained from abcam . neurobiotin was acquired from vector laboratories and streptavidin alexa fluor 488 was procured from life technologies . unless otherwise stated , the following was added to the whole - cell external recording solution : 0.1 mm ( rs)--methylserine - o - phosphate ( msop ) and 1 mm ( rs)--methyl-4-carboxyphenylglycine ( mcpg ) disodium salt . cholinergic afferents were electrically stimulated using tungsten electrodes ( a - m systems ) or by activating channelrhodpsin with a 488 nm laser ( cairn research ) . 810 , 0.1 ms , 50200 a pulses or 8 , 10 ms , 9 mw light pulses were delivered . glutamatergic epsps were evoked using tungsten electrodes placed in stratum moleculare 100150 m from granule cell bodies . simulated epsps were generated by injecting the waveform : a=(t/)exp(1(t/))where a is the injected current amplitude and is the rise time constant . loose seals of 236.8 52 m were made with 35 m patch pipettes filled with 150 mm nacl . 23 m patch pipettes filled with oxo - m were placed in close proximity to identified axons or dendrites 2530 m from the somata . 20 ms , 10 psi pressure was applied using a picospritzer iii ( intracel ) . the puff encompassed an area of approximately 30 m , indicating that somatic muscarinic receptors were unlikely to be affected . the external solution was supplemented with 0.001 mm tetrodotoxin ( ttx ) and 0.1 mm 4-aminopyridine ( 4-ap ) . series resistance was between 20 and 40 m and was 70% compensated . a de - activation protocol ( shah et al . , 2008 ) was applied in the absence and presence of the kv7 channel blocker , xe991 ( 3 m ) . the external solution was supplemented with 0.001 mm ttx , 2 mm cscl2 , 10 mm tea , 0.1 mm 4-ap , 0.005 mm nifedipine , 0.0001 mm -agatoxin iva , 0.0001 mm -conotoxin gvia , and 0.0002 mm snx482 . the internal pipette solution was 120 mm cscl2 , 1 mm cacl2 , 5 mm mgcl2 , 10 mm egta , 10 mm hepes , 2 mm na2atp , 0.3 mm tris - gtp , and 14 mm tris - phosphocreatinine ( ph 7.3 ) with koh , 295300 mosm / l . a 1 s pre - pulse to 100 mv followed by 1 s pulses ranging from 90 mv to 30 mv in 10 mv increments were applied . leak currents were obtained with 1 s , 10 mv hyperpolarizing steps from 100 mv . all recordings were leak subtracted and tta - p2 ( 500 nm ) applied at the end . clampfit ( v10.0 ) was used . the threshold of single action potentials elicited by 30 ms current steps from 70 mv was measured by differentiating the spike voltage with respect to time ( dv / dt ) . the threshold was defined as the voltage at the point of deflection for dv / dt to be greater than zero . action potential height was measured from threshold to the peak , whereas action potential width was the breadth at half the height . to calculate rn , we divided the difference in steady - state voltage in the last 25 ms elicited by a 100 pa 400 ms hyperpolarizing step at 70 mv by the applied current . action potentials elicited by 400 ms depolarizing steps were counted . the peak and area under adp , if evoked , were also measured . cholinergic epsp summation ratios were calculated as the amplitude of the last epsp in the train divided by the amplitude of the first epsp . the numbers of spontaneous action potentials generated in 15 min during loose cell - attached recordings were counted and the frequency calculated . for kv7/m - current voltage - clamp data , the traces obtained in the presence of the xe991 ( 3 m ) were subtracted from those in the absence . the subtracted traces were fitted with the following to obtain the decay time constants : a1e(-t/1)+a2e(-t/2)where 1 and 2 represent time constants of the initial and late phase of the kv7 current . the kv7 conductance values were generated from the normalized amplitudes of the subtracted currents ( shah et al . , 2008 ) and were plotted against the voltage . the curves were fitted using the boltzmann equation : y = a2+(a1a2)/(1+exp((xx0/dx)))where a1 and a2 are the initial and maximum values , x0 is the half - activation voltage and dx is the slope of the curve . ca currents obtained in the presence of tta - p2 ( 500 nm ) were subtracted from those in the absence of the current to obtain the t - type ca current . the peak current amplitude was plotted against the voltage to obtain the activation curves ( dreyfus et al . , 2010 ) . curves were fitted using the above boltzmann equation . a similar method to that described by huang et al . briefly , confocal ( zeiss lsm 710 ) images of stained neurons were acquired . in image j ( nih ) , 10 m concentric circles were generated around the somata ( sholl , 1953 ) , the number of dendrites crossing each circle counted . a prairie ultima multi - photon microscopy system ( prairie technologies ) was used . the mai tai laser ( spectra - physics ) the internal solution was supplemented with a red fluorophore ( 50 m alexa fluor 594 ; invitrogen ) and the ca - sensitive green fluorophore , oregon green bapta-1 ( 200 m ; invitrogen ) for basal [ ca]i measurements . when measuring [ ca]i during action potential waveforms , the ca - sensitive green fluorophore , fluo-5f ( 250 m ; invitrogen ) together with alexa fluor 594 were utilized ( bender et al . cells were filled with dyes for at least 20 min before images were acquired . at least three cross - sectional line scans were made at 667 hz in the distal axon initial segment ( 2530 m from soma ; figure 7b ) and averaged to obtain the signal . / r=(fgreenfdarkgreen)/(fredidark , red).fgreen and fred are the green and red fluorescence signals and fdark green and idark , red are the background currents in the green and red channels , respectively . three adult rats and cav3.2 null mice were deeply anesthetized by intraperitoneal injection of ketamine - xylazine 1:1 ( 0.1 ml / kg ) and transcardially perfused with ice - cold fixative containing 4% paraformaldehyde , 0.05% glutaraldehyde , and 15% saturated picric acid in 0.1 m phosphate buffer ( pb , ph 7.4 ) . after perfusion , brains were removed and immersed in the fixative for 2 hr or overnight at 4c . free - floating sections were incubated in 10% ngs diluted in tbs , then with the primary antibodies ( 25 g / ml ) , followed by goat anti - rabbit or goat anti - mouse igg coupled to 1.4 nm gold ( nanoprobes ) . sections were post - fixed in 1% glutaraldehyde and washed in double - distilled water , followed by silver enhancement of the gold particles with an hq silver kit ( nanoprobes ) . m pb ) , block stained with uranyl acetate , dehydrated in graded series of ethanol , and flat embedded on glass slides in durcupan ( fluka ) resin . regions of interest ( rois ) were cut at 7090 nm on an ultramicrotome ( reichert ultracut e , leica ) and collected on 200-mesh copper grids . staining was performed on drops of 1% aqueous uranyl acetate followed by reynolds s lead citrate . affinity - purified rabbit anti - m1 was characterized previously ( yamasaki et al . , 2010 ) . the monoclonal antibody against cav3.2 ( clone n55/10 ) no labeling was observed if the primary antibody was either omitted or replaced with 5% ( v / v ) normal serum . all procedures were approved by the animal care and use committee of baylor college of medicine . three postnatal days 2229 sprague - dawley rats were deeply anesthetized and perfused with pbs containing 4% paraformaldehyde and 0.1% glutaraldehyde . brains were removed , kept at 4c overnight in the fixative , and then stored in pbs . 40 m floating sections were cut ( vt1200s , leica ) , rinsed in pbs , blocked , and permeabilized for 1 hr at room temperature and incubated with primary antibodies overnight ( mouse -chat , chemicon ; rabbit -iv - spectrin [ ogawa et al . , 2006 ] ) . sections were washed extensively , incubated with secondary antibodies ( goat -mouse igg1 1 555 , invitrogen ; donkey -rabbit dylight 488 , jackson ) , washed again , and coverslipped ( prolong gold with dapi , invitrogen ) . image stacks were acquired using a c2 laser - scanning confocal microscope ( nikon ) . maximal intensity projections of z stacks 3d views , and movies were created using nis - elements . hippocampal slices were treated with either vehicle ( control ) or oxo - m ( 1 m ) , irradiated in sodium citrate - edta buffer ( ph 8.5 ) using a microwave oven ( sears ) , then washed in pbs , blocked for 1 hr at room temperature , and incubated with primary antibodies for 48 hr ( mouse -pannav igg1 , sigma ; rabbit -kcnq2n ; guinea pig -kcnq3n ) followed by incubation with secondary antibodies ( donkey -mouse - cy3 , jackson ; donkey -rabbit - dylight 488 , jackson ; donkey -guinea pig - cy5 , jackson ) . four regions of interest ( rois ) spanning the dentate granule cell layer were analyzed . roi dimensions were 92.3 m 46.1 m 20.3 m deep . five aiss in each roi were analyzed for length and labeling intensity . using maximal projection and 3d views in nis - elements , we drew a 3d polyline along the axon , from soma to beyond the ais . this was converted to a 2d ( xy versus z ) plot and labeling at each pixel along the trajectory for each fluorophore was read . raw pixel intensities , without background subtraction , were binned in 2 m lengths and averaged per bin for each group . statistical significance was determined using either paired or unpaired student s t tests as appropriate . statistical significance of differences at p < 0.05 is indicated as asterisks ( ) in all figures . | summaryacetylcholine critically influences hippocampal - dependent learning .
cholinergic fibers innervate hippocampal neuron axons , dendrites , and somata .
the effects of acetylcholine on axonal information processing , though , remain unknown . by stimulating cholinergic fibers and making electrophysiological recordings from hippocampal dentate gyrus granule cells ,
we show that synaptically released acetylcholine preferentially lowered the action potential threshold , enhancing intrinsic excitability and synaptic potential - spike coupling .
these effects persisted for at least 30 min after the stimulation paradigm and were due to muscarinic receptor activation .
this caused sustained elevation of axonal intracellular ca2 + via t - type ca2 + channels , as indicated by two - photon imaging .
the enhanced ca2 + levels inhibited an axonal kv7/m current , decreasing the spike threshold . in support , immunohistochemistry revealed muscarinic m1 receptor , cav3.2 , and kv7.2/7.3 subunit localization in granule cell axons .
since alterations in axonal signaling affect neuronal firing patterns and neurotransmitter release , this is an unreported cellular mechanism by which acetylcholine might , at least partly , enhance cognitive processing . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
recently , in this journal , dr undurti das suggested that treatment of sepsis , septic shock and burns should also include infusion of glucose insulin potassium ( gik ) , with the objective of the infusion being to achieve plasma glucose concentrations below 6.1 mmol / l ( < 110 mg / dl ) . that conclusion was based on the combined results of a number of studies . in our opinion , however , no single clinical trial has yet been reported that supports this conclusion . it has been shown that achieving and maintaining euglycaemia ( i.e. plasma glucose concentrations of 4.46.1 mmol / l ) in patients admitted to surgical intensive care units leads to marked reductions in morbidity and mortality . however , in that study the investigators did not use a gik infusion ; rather , meticulous regulation of serum glucose was achieved by combining intensive insulin treatment with intravenous glucose ( 200300 g/24 hours ) . the following day the infusion was replaced by parenteral nutrition , combined parenteral and enteral nutrition , or enteral nutrition , according to a set scheme . potassium was only supplemented when a check - up showed that hypokalaemia was either imminent or present ( g van den berghe , icc van der horst , personal correspondence ) . no studies are available in cases of sepsis , septic shock and burn patients in which euglycaemia was pursued with the aid of gik infusion and that assessed mortality . there are few data pertaining to the haemodynamic effects of gik in sepsis . in 15 patients with septic shock , this effect was also shown in 14 patients with peritonitis and signs of hypovolemic shock , in spite of positive fluid balance and cathecholamine treatment . in burns victims this appears to clarify the roles of the individual components of gik , in particular with regard to haemodynamic support of the patient by insulin . in the study conducted in a surgical intensive care unit , the fact that there were four times as many patients that died from established sepsis in the conventionally treated group as compared with the intensively treated group supports the value of intervention in glucose metabolism during sepsis . a problem that should be anticipated is obtaining and maintaining euglycaemia ; after all , one of the features of sepsis is the presence of hyperglycaemia as well as hypoglycaemia . the scheme used in the above - mentioned study has not been validated in septic patients . it is to be expected that serum glucose values will have to be checked more frequently than once every 24 hours . the diabetes insulin glucose infusion acute myocardial infarction ( digami ) study , which included 620 patients , is the only study that aimed at meticulous regulation of serum glucose concentrations by infusing glucose and insulin . glucose infusion for 24 hours , followed by a minimum of 3 months of intensive insulin therapy , or to conventional treatment . the objective was to maintain serum glucose concentrations between 7.0 and 10.0 mmol / l , which is well above concentration range of 4.46.1 after 1 year , those investigators found that , in particular , the group of patients who had had no previous insulin treatment and were not known to have risk factors such as a history of myocardial infarction gained most from the glucose metabolism intervention ( mortality was 8.6% versus 18.0% in the control group ) . for the study population as a whole , the absolute reduction in mortality was 7.5% ( p = 0.0273 ) . however , a definite place for gik infusion in the treatment of acute myocardial infarction has not yet been settled . in 1997 , a meta - analysis of studies of gik in the treatment of acute myocardial infarction was published . that meta - analysis included nine studies and 1932 patients , and showed that gik treatment resulted in a reduction in 30-day mortality from 21% to 16.1% ( p = 0.004 ) . in the four studies in which gik was administered at high doses ( n = 228 ) the difference appeared to be even larger : a reduction from 12% to 6.5% ( not significant ) . there were substantial differences between the nine studies , in particular with regard to the time at which the first symptoms appeared and the start of treatment , and regarding the composition of the gik cocktail and the duration of treatment . moreover , only 17 patients were treated by adding gik to reperfusion , which is the present standard treatment . the estudios cardiologicos latinoamerica ( ecla ) pilot trial , including 490 patients , showed that 30-day mortality in the group of patients randomly assigned to gik was lower than that in the control group ( 6.7% versus 11.5% ; not significant ) . as mentioned in the commentary of das , the effect was most pronounced in patients in whom gik had been combined with reperfusion treatment , mostly thrombolysis ( 5.1% versus 15.1% ; p = 0.01 ) . however , mortality was very high in the control group , even higher than in the control group of patients that had not been treated with reperfusion ( 11.5% ) . moreover , the polish gik ( pol - gik ) trial , which was published 1 year later , could not confirm the results of the ecla pilot trial . in a group of 954 patients , the mortality of 8.9% in the gik group was even significantly higher than that in the control group ( 4.8% ; p = 0.01 ) . mortality due to a cardiovascular incident was not significantly different , and as such the cause of the difference remained obscure . the guidelines of the american college of cardiology/ american heart association state that gik in the treatment of acute myocardial infarction is very promising , but that it will take a large randomized study to determine its effectiveness once and for all . recently , the glucose insulin potassium study ( gips ) reached its conclusion . in that study 940 patients were randomly assigned to primary coronary angioplasty with gik ( glucose 20% with 80 mmol potassium at a rate of 3 ml / kg body weight / hour and 50 iu insulin in 50 ml water administered according to serum glucose concentrations ) or to angioplasty without gik . although the mortality reduction in the overall population did not reach significance , the results showed that in 856 patients without signs of heart failure ( i.e. killip class i ) the mortality reduction was 3.0% ( p = 0.01 ) . it is to be expected that in the future the role of gik , with or without meticulous glucose regulation , will be established in the treatment of various conditions . at present clinical studies only support its effectiveness in patients admitted to a surgical intensive care unit and treated with meticulous glucose regulation combined with nutrition , and in patients with diabetes mellitus and acute myocardial infarction who are treated with an insulin new clinical studies will show whether experimentally obtained results , such as the effect of insulin on immune function and apoptosis , can effectively be translated into routine practice , particularly in sepsis , septic shock and burns patients . the research of icc van der horst was supported by a generous grant from the netherlands heart foundation ( 99.028 ) . | there is no hard evidence yet for a positive effect of glucose insulin potassium infusion in sepsis , septic shock or burn patients .
each individual element of the glucose insulin potassium regimen , and eventually euglycaemia , should theoretically be beneficial . at present
, evidence exists only for reduced mortality with strict metabolic treatment ( i.e. blood glucose levels of 4.46.1
mmol / l ) in critically ill patients admitted to surgical intensive care units , and for better metabolic regulation ( i.e. blood glucose levels of 7.010.0
mmol / l ) in patients with hyperglycaemia and/or diabetes mellitus , and in patients without signs of heart failure ( i.e. killip class i ) during acute myocardial infarction . |
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most eukaryotic cells have an impressive capacity to apportion a fragment of their plasma membrane for invagination into enclosed structures that are then pinched off to form intracellular vesicles . the internalized membrane and cargo is then delivered to a number of different intracellular destinations such as lysosomes and the golgi or recycled back to the plasma membrane . this process is generally termed endocytosis and is required for processes such as nutrient uptake and degradation , down - regulation of activated receptor signalling and antigen processing in the immune response . spatial organization of the endomembrane system and directed transport on endocytic pathways are both controlled by the cell cytoskeleton that typically controls centripetal movement to a perinuclear region that also contains lyso - somes , recycling endosomes , the microtubule organizing centre , centrosome and the golgi apparatus [ 2 , 3 ] . it is now known that a number of different internalization mechanisms exist , including uptake via clathrin coated pits , caveolae and other poorly characterized processes that do not seem to rely on either clathrin or caveolae [ 48 ] ( fig . a , caveolae delivering to caveosomes ; b , clathrin independent endocytosis , c , clathrin coated uptake d , macropinocytosis showing macropinosome formation via fusion of ruffles with each other or the plasma membrane , e , phagocytosis . the contribution of a common early endosome in linking these pathways and partitioning membranes , proteins and cargo , over delivery to distinct classes of sorting endosomes as depicted by the dashed lines is unknown . phagocytosis is confined to specific cell types such as macrophages and dendritic cells and the same is true about macropinocytosis , a process that shares many features with phagocytosis see below . phagocytosis has been extensively characterized at morphological and molecular levels but the lack of recent reviews detailing macropinocytosis is a reflection of our poor understanding of unique molecular features that may govern this event , and in many cell types , its exact physiological role over other endocytic processes [ 1012 ] . given the fact that pathogens are master highjackers of other endocytic pathways including phagocytosis , it is not surprising that macropinocytosis is also utilized by microorganisms to gain access to the cells . examples include salmonella , listeria and adenoviruses and macropinocytosis inducing toxins have also been described [ 1317 ] . in dictyostelium , macropinocytosis , demonstrated by the formation of circular ruffles or crowns , accounts for most of fluid phase uptake and scanning electron microscopy ( sem ) images of the projecting circular ruffles demonstrate the extensive reorganization of the plasma membrane that is required to form macropinosomes in this organism and in mammalian cells [ 1820 ] . the distinct structures of these circular ruffles compared with other types of regulated and constitutive ruffling suggest macropinocytosis represents several distinct processes controlled via common and specific mediators . a general feature of macropinocytosis and phago - cytosis is that the active portion of the plasma membrane is initially not involved in invagination but rather in an actin - dependent protrusion of the plasma membrane to the external milieu . there is presumably no regulation regarding the size or morphology of the enclosed macropinosome but in the case of phago - cytosis , as previously noted , this is largely pre - determined by the shape of the enveloped entity . common to both , however , is that the formed macropinosome or phagosome may be several micrometers in diameter ; significantly larger than structures formed from other endocytic invaginations that rarely exceed 150 nm . macropinocytosis , as a form of pinocytosis , was initially described by warren lewis in 1931 . motion picture account of the appearance of waving sheets , macropinosome formation , traffic and subsequent shrinkage in rat macrophages . much later , the prominence of macropinocytosis was shown to be greatly enhanced by the addition of growth factors and a large portion of what is now known about this process in macrophages was gained from experiments performed in cells incubated with macrophage - colony stimulating factor ( m - csf ) . more recently , macropinocytosis has been shown to be prominent in cell types that do not phagocytose , but this is mostly a feature of their response to growth factor stimulation . these include human epidermoid carcinoma ( a431 ) cells after stimulation with epidermal growth factor ( egf ) and polarized madin darby canine kidney epithelial cells following hepatocyte growth factor / scatter factor ( hgf / sf ) stimulation . constitutive macropinocytosis has been observed in dendritic cells [ 23 , 24 ] and nih3t3 fibroblasts expressing a ruffling kinase but whether this is a required cellular activity in non - phagocytic cells is unclear . the membrane ruffling and macropinocytosis that immediately follows the addition of growth factors is accompanied by a rapid increase in uptake of markers for fluid phase endocytosis . presumably this is because of the engulfment of relatively large volumes of extracellular material into these enlarged macropinosomes . markers such as fluorescent dextrans for fluorescent microscopy and horseradish peroxidase for electron microscopy are shown to be contained in relatively few numbers of large , non - coated structures whose intracellular fate is discussed below . cells infected with adenovirus type 2 for 10 min show extensive leading - edge ruffling ; prominent also are filopodia in close proximity to these ruffles and the uptake of fluid phase markers , as opposed to clathrin coated vesicles is increased . similarly , treatment of the same cell line with egf results in an extremely rapid ( 30 s ) increase in fluid phase uptake accompanying extensive membrane ruffling [ 26 , 27 ] ; the resulting increase in fluid phase uptake is often used as a defining feature of this process . within minutes of growth factor activation , cells boast the appearance of ruffles that cover the entire surface of the cell including the leading edge where they manifest as lamellipodia [ 28 , 29 ] . the appearance and morphology of these is quite different to the circular ruffles , prominent in dictyostelium ( phacocytic cups ) and hgf - sf stimulated epithelial cells [ 19 , 20 , 30 , 31 ] . there is no consensus as to the proportion of membrane ruffles that end up as macropinosomes , but the formation of an enclosed macropinosome is more than likely a result of fusion of the protruding structures or back - fusion of a protrusion with an unruffled section of the plasma membrane ( fig . 1 ) . the resulting macropinosomes are , predictably , highly heterogeneous in size and morphology . a requirement for actin and the actin polymerization machinery on ruffling and phagocytosis including rho family members and their upstream effectors such as ras , plc and pi 3-kinase , activated themselves by receptor tyrosine kinases , suggest that both processes are similarly organized . there is also considerable overlap relating to the requirements of proteins regulating these two processes with those shown to be involved in promoting cell motility . a number of studies have shown , in different cell types , that activation of the non - receptor kinase v - src oncogene promotes macropinocytosis [ 3337 ] . the original demonstration of v - src effects showed that its kinase activity led to a constitutive formation of macropinosomes ( in the absence of external growth factor ) , a 2-fold increase in fluid phase uptake and a less significant , 1.3-fold , increase in transferrin uptake . this small increase in transferrin uptake compared with much higher stimulation of fluid phase endocytosis , up to tenfold , is common to many studies and suggests that transferrin receptors may be sequestered away from the forming macropinosome . there is also some recent evidence for this from experiments in dictyostelium , showing that this organism excluded some but not all plasma membrane proteins away from the forming macropinocytic cup . these studies suggest that the composition of the macropinocytic cup and nascent macropinosome is regulated , defining the later as a unique endocytic organelle rather than just an enclosed fragment of the original plasma membrane . as mentioned earlier , once growth of the protrusion is finished there will be a requirement for a membrane fusion event to close leading edges to fashion the macropinosome ( fig . the identities of several proteins and lipids that mediate membrane fusion on the endocytic and secretory pathway are now known but to date there have been no reports on the specific requirements for a ruffle ruffle or ruffle plasma membrane fusion protein . the fungal metabolite wortmannin inhibits lipid kinases that phosphorylate phosphatidylinositol ( pi ) at the 3 position;it is a prominent inhibitor of signalling emanating from the canonical pi 3-kinase that generates pi ( 3 , 4 , 5 ) p3 from pi(4 , 5)p2 . nanomolar concentrations of wortmannin inhibit fluid phase uptake and the homotypic fusion of early endosomes [ 39 , 40 ] and subsequent studies revealed that a number of pi 3-kinase variants were involved in endocytic traffic with the class iii variant hvps34 , that specifically phosphorylates pi , being prominent on endosomal membranes and microdomains that are subsequently enriched in its product pi(3)p [ 4143 ] . in macrophages , wortmannin also affected macropinocytosis and phagocytosis but the inhibitory step was in the closure of ruffles or phagocytic cups rather than the formation of membrane protrusions that were still clearly observed by sem . whether this was solely due to the known effects of pi 3-kinase activity on the actin cytoskeleton or whether there is a separate requirement for a pi 3-kinase in a fusogenic event to generate an enclosed macropinosome is currently unknown . a second class iii pi 3-kinase inhibitor , 3-methyladenine , does not impede macropinosome formation or internalization in egf - stimulated a431 cells , but inhibited homotypic macropinosome macropinosome fusion , suggesting that endosome fusion and macropinosome fusion share a requirement for pi(3)p . approximately twelve rab members have now been located on endocytic structures and several have been implicated in regulating the dynamics of distinct endocytic processes [ 45 , 46 ] . one of these is rab5 and represents one the most studied rab variants that controls several endocytic processes including invagination at the plasma membrane , endosomal fusion , motility and signalling . transfection of cells with a constitutively active rab5 leads to the formation of swollen endosomes not dissimilar in appearance to early macropinosomes , and increased expression of rab5 together with an active form of ras promotes the formation of circular ruffles . in agreement for a rab5 role in circular ruffling , transfection of cells with the dominant negative rab5 mutant ( s34n ) inhibited circular but not cell edge ruffling . rab5 involvement in macropinocytosis was further demonstrated when one of its effectors , rabankyrin-5 , was found to promote macropinocytosis and partial silencing of rabankyrin-5 diminished egf - stimulated fluid phase uptake . before assigning a role for rab5 in ruffling and macropinocytosis it is noted that transfection of cells with rab5 s34n has effects on numerous endocytic pathways thus similar effects may be observed in cells expressing other early endocytic variants such as rab21 and rab22a that give similar endocytic defects when their gtp - binding mutants are over - expressed . thus these effects on ruffling may point towards downstream effects of perturbation of the endocytic pathways in general rather than pointing to rab5 as being absolutely required for ruffling . sirna analysis and silencing all three rab5 variants would point further to an absolute requirement for this protein in macropinosome formation and traffic . a second rab protein , rab34 , has also been implicated in the formation of ruffles and macropinocytosis ; similarly , the effects of this protein were shown in over - expressed systems or cells expressing rab34 mutants . adp - ribosylation factors ( arfs ) are also small gtpases that function in membrane traffic . one variant , arf6 , is localized to the plasma membrane and in conjunction with the actin cytoskeleton and its exchange factors and activators , acts as a prominent ruffling factor regulating macropinocytosis , cell adhesion and migration [ 52 , 53 ] . the impressive capacity of the cell to internalize its plasma membrane is somewhat overshadowed by its ability to later sort components on the endocytic pathway to a number of cellular destinations . plasma membrane derived vesicles rapidly fuse with sorting endosomes ( often termed early endosomes ) that represent a tuboreticular network ; indeed the entire endocytic pathway can be similarly defined . it is currently unknown how many types of sorting endosomes exists within a single cell or the extent of mixing of membrane and cargo that then occurs between different pathways . there is now evidence to suggest that molecules entering via clathrin coated vesicles may be sorted before fusing with early endosomes or even prior to completion of the budding step to generate the clathrin coated vesicle [ 5557 ] . the fate of a protein entering these sorting compartments is in part determined by both the nature of its lipid environment and by sorting signals it possesses on its cytoplasmic and transmembrane domain(s ) . it is in the interest of the cell to ensure that some proteins such as activated receptors are trafficked to the degradative lysosomes and that others such as the transferrin receptor are recycled back to the plasma membrane for another round of function . to fulfil this requirement , it organizes on endosomal membranes the formation of retrieval and sorting multiprotein complexes such as endosomal sorting complexes required for transport ( escrt ) and retromer , that either interact with the cytoplasmic portions of endocytosed molecules , with distinct lipids on the endosomal membrane or with endosomal located regulatory proteins . the reader is referred to these reviews on specific aspects of the regulation of sorting and retrieval on the endocytic pathway [ 5862 ] . fluorescence and electron microscopy studies show early macropinosomes as large , uncoated vacuole like structures identified most commonly with an internalized fluid phase tracer such as dextran or horseradish peroxidase . numerous groups have attempted to map the route the macropinosome and/or its associated cargo traverses downstream of the plasma membrane and from data review it is unlikely that they mature , in all cell types , via a common pathway to a single pre - defined location . the intracellular dynamics of macropinosomes was first extensively studied in egf stimulated a431 fibroblasts [ 22 , 63 ] and macrophages [ 6466 ] and later reviewed . in macrophages , the macropinosomes followed a somewhat conventional centripetal route to tubular lysosomes;as determined by acquisition and loss of classical marker proteins such as the transferrin receptor and rab7 , identifying early and late endosomes , respectively . there were many similarities to this process when macropinosomes were studies in dicyostelium or in mammalian cells transfected with gfp - c - src . in contrast to observations in macrophages , there was little evidence of co - localization of egf - induced a341 macropinosomes with other endocytic markers such as transferrin , neither were macropinocytic cargo delivered to lysosomes in these cells . in a separate study , the transferrin receptor was found to be enriched , via exocytosis , on plasma membrane ruffles of stimulated cells thus suggesting that membrane for ruffles and possibly macropinosome formation was provided in part from the endosomal pathway . early macropinosomes are however often noted to be devoid of transferrin and/or the transferrin receptor , strengthening the idea that sorting is occurring at the plasma membrane during ruffling and macropinosome formation , and that distinct lipid domains contribute to generate ruffles and macropinosomes . more recent experiments in a431 cells demonstrated a higher degree of co - localization of egf , and transferrin in macropinosomes but the fraction of egf on enlarged structures or ruffles seemed to be significantly higher than transferrin , that was predominantly localized to more widely distributed smaller vesicles . evidence for macropinosomes being , at least at some stage , unique organelles comes from tracer co - localization experiments showing homotypic , i.e. macropinosome - macropinosome fusion but there was little evidence of macropinosomes fusing with other endocytic structures . in dictyostelium , the situation was somewhat different as much smaller endosomes appeared to contribute further to internalized macropinosomes suggestive of heterotypoic fusion ; homotypic fusion was also prominent . the use of alternative markers of endocytic pathways has recently enhanced our knowledge of the trafficking of macropinosomes though , as expected there is some discrepancy as to whether they progress as distinct entities . antibodies recognizing the early endosomal autoantigen 1 protein eea1 are now commonly used to label early endosomes in mammalian cells . on these structures , eea1 binds to pi(3)p via its fyve domain , falls off the endoso - mal membrane in the presence of wortmannin , binds other regulatory proteins such as rab5 and regulates early endosome fusion and sorting [ 7072 ] . expression of rabankyrin-5 in a431 cells resulted in the formation of swollen vesicles that were later defined as macropinosomes and egf stimulated cells also sequestered endogenous rabankyrin-5 on these structures . the larger macropinosomes also harboured rab5 but did not contain eea1 , however smaller structures that appeared to be more mature macropinosomes contained all three proteins . earlier studies showed that eea1 transiently associated with macropinosomes as 5 min macropinosomes were essentially eea1 negative and then over a 25 min period eea1 labelling increased and then decreased as the macropinosomes evolved ; in agreement with previous studies there was no evidence of the progression of macropinosomes to typical late endosomes and lysosomes . in dendritic cells , early macropinosomes ( < 4min ) were depleted of early endocytic markers but then acquired eea1 and the transferrin receptor , however , the macropinocytic cargo did not then co - localize with markers of late endosomes and lysosomes . in a separate study , also in a431 cells , immature macropinosomes were labelled with early endocytic markers eea1 and rab5 and also rab7 , a classical marker of late endo - somes and lysosomes . these studies were however performed in rab7 over - expressing cells , that in the absence of stimulus , often display swollen structures , reminiscent of macropinosomes . this suggests that macropinosomes rapidly develop classical early endosome characteristics before developing into late endocytic structures or diminishing in size and loosing their identity via membrane retrieval . a member of the sorting nexin ( snx ) family of proteins was recently shown be involved in macropinosome maturation . snxs comprise a family of approximately 30 peripheral membrane proteins that are characterized by having a snx phox homology ( px ) domain that bind most notably to pi(3)p [ 77 , 78 ] ; a number have therefore been located on endosomal structures . a least seven members also contain a bin / amphiphysin / rvs ( bar ) domain that is thought to aid in the binding of the px domain to membrane but also to sense membrane curvature . snx5 was shown to co - localize with eea1 on microdomains of macropinosomes but was also apparent in tubular extensions that seemed to emanate from these structures ; tubulation of macropinosomes was noted earlier in mammalian and amoeba cells [ 22 , 68 ] . this suggest that tubulation may be one mechanism that helps retrieve membrane back to the plasma membrane , resulting in the shrinkage of the macropinosome that was noted in the first demonstration of macropinocytosis . snx5 and other sorting proteins may be working in conjunction with microtubules that ran in parallel to the tubular membranes and also appeared to organize themselves as a mesh around the macropinosome . similar conclusions come from research in dictyostelium showing that microtubules were closely associated with macropinocytic vacuoles and tubular extensions emanating from them ; macropinosome fusion was also dependent on microtubules . there may therefore be a requirement for alternate protein complexes akin to escrt and retromer to retrieve and sort macropinosome membrane and cargo . whether the formed tubules recycle back to the plasma membrane to replete lost membrane is unknown but studies in dendritic cells suggest that retrieval may rather be a function of the regulated exocytosis of structures , enlargeosomes , that fuse with the plasma membrane in a calcium - dependent fashion . the enlargeosome was initially described as being a unique vesicular compartment , showing no co - localization with conventional endo - lysosomal markers but containing the protein desmoyokin - ahnak , that was recruited , with the enlargeosome , to the plasma membrane . this exo - cytic response to macropinocytosis in dendritic cells is therefore quite different to that described in egf - stimulated cells where transferrin positive vesicles served the function of enlargeosomes . there is also some evidence for the involvement of the endo - plasmic reticulum in the traffic of material entering via macropinocytosis , or in providing membrane for macropinosome formation . our understanding of disease processes will grow exponentially now that the human genome is unravelled and that tools are available to more easily study differences in expression and sequence of genes and proteins . in the wake of this is an increasing demand for the design of therapeutic entities to reverse disease - causing events at the intracellular level . this calls for targeting organs and cells , and third - order targeting of intracellular targets in the cytosol or in membrane bound compartments . in the realm of gene delivery the target is most often the nucleus but it is becoming apparent that a number of other organelles are potential drug delivery targets . endocytic pathways provide highly efficient routes that enable drug delivery researchers the opportunity to introduce a macromolecular entity across the plasma membrane , however the likelihood exists that a significant fraction , perhaps all , of the therapeutic payload will be recycled or will end up degraded in lysosomes . holy grails in drug delivery research are molecules that somehow promote the escape of therapeutic entities such as genes and proteins from early stages of the endocytic pathway . a wide variety of strategies have been designed including the use of bioresponsive agents that modify themselves and the endosome environment in the decreasing ph that accompanies transit in the endo - lysosomal system , through to the use of domains of proteins , often from pathogens , that have been shown to translocate through biological membranes . these include the hundereds of amino acid sequences that are now classified as cell penetrating peptides ( cpp ) or protein transduction domains and the reader is referred here to reviews in an edition of advanced drug delivery reviews dedicated to protein and peptide - mediated translocation . the most intensely studied cpps include those derived from the transduction domains of the hiv transcription factor tat ( hiv - tat peptide ) or the drosophila melanogaster homeobox protein antennapedia ( penetratin ) , together with synthetic oligomers of arginine ( r4-r16 ) . the sequences of a few of these are shown in table 1 and common to many , but not to all , is the relatively high number of positive amino acids lysine and arginine over other amino acids . in view of the heterogeneity of their sequences and properties they are often further subclassified to arginine - rich , amphipathic , non - basic and more specific criteria [ 84 , 85 ] . the charge on the cationic peptides allows them to interact strongly with negative charges on the plasma membrane and this interaction is reduced in cell lines with defects in heparan sulphate and glycosaminoglycan synthesis [ 8688 ] . beyond interacting with cells , a number of cpps traverse biological membranes , be it alone or attached to much larger cargo ranging from small molecule drugs to antisense oligonucleotides , sirna , plasmids , peptides and proteins [ 8992 ] . they do not appear to be cell - type specific and their incorporation into targeting and intracellular delivery complexes show they hold great promise as agents for enhancing translocation across biological barriers . their uptake mechanisms have therefore been studied in great detail with a view to the subsequent design of more efficient delivery and translocating systems . sequences of the most studied cpps with respect to endocytic uptake initially there was agreement that a number of cpps entered cells in a temperature independent manner and once inside they rapidly entered the nucleus . later , a large part of their interaction with cells and nuclear delivery was revealed to be a product of fixation , and that a significant fraction of what was termed cell associated peptide was confined to the plasma membrane [ 93 , 94 ] . all microscopy and cytometry experiments with these entities are now performed in live cells , and trypsinization and heparin washing is now commonly used to reduce plasma membrane binding prior to quantifying intracellular peptide . confocal microscopy highlights the fact that the peptides enter vesicular structures in a number of different cell types including the hela and a549 cells shown in figure 2 . but despite extensive study there is no consensus regarding the uptake mechanism and whether there is a preferred entry route . as previously mentioned , hundereds of cpps have now been described and as there is no consensual sequence , it is unlikely that a common mechanism of entry exists and this is highlighted in a number of studies [ 87 , 9597 ] . equally , any cargo that they are attached to could also influence cellular interaction , endocytosis and intracellular dynamics [ 96 , 98 , 99 ] . focus here will be on experiments performed with fluorescent variants of hiv - tat , oligoarginines and penetratin ( table 1 ) . hela ( a ) and a549 ( b ) cells were incubated for 1 hr at 37c with 2 m of the d - enantiomerof r8-alexa488 peptide ( rrrrrrrrgc - alexa488 ) and immediately analysed by scanning confocal fluorescence microscopy combined with scanning direct interference contrast . shown are superimposed images from 25 to 30 sections through the z - axis . the peptide - labelled vesicles in these cells are concentrated in a perinuclear region but it remains to be seen whether any are , or are derived from , macropinosomes . previous studies in hela cells show a significant fraction of these to be late endosomes and lysosmes . in egf stimulated a431 cells the increased fluid phase uptake that accompanies macropinocytosis was found to be inhibited by the ion - exchange inhibitor amiloride , however , endocytosis of the transferrin receptor was unaffected by this drug . similarly , the enhanced fluid phase uptake , driven by adenovirus - induced macropinocytosis was inhibited by 5-(n - ethyl - n - isopropyl ) amiloride ( eipa ) , a more potent analogue . consequently , these agents are regularly used to define uptake via macropinocytosis , but amiloride and eipa also strongly inhibit receptor - mediated uptake of albumin in renal proximal tubule - derived epithelial cells [ 100102 ] . here , it is unlikely that uptake is via macropinocytosis and albumin has been shown to enter cells via clathrin coated pits and caveolae [ 103106 ] . to various extents , eipa also inhibits the uptake of hiv - tat peptide and oligoarginine peptides raising the possibility of a link between macropinocytosis and uptake of cpps [ 87 , 107 , 108 ] , and supporting previous studies showing amiloride inhibition on the uptake of a tat peptide - cre protein conjugate . the microscopy studies to date show the peptides , as single fluorescent conjugates , labelling typical pinocytic vesicles and tubules ( fig . 2 ) as opposed to macropinocytic vacuoles but careful analyses of their location immediately after addition to cells , when macropinosomes may be much more pronounced , has yet to be performed . one reason for this is that their extensive binding to the plasma membrane makes interpretation at early time points quite difficult , especially when the amount of internalized peptide is low and thus the fluorescent signal weak . an exception to this rule are leukaemic cells such as k562 and kg1a that intriguingly show minimal plasma membrane binding of hiv - tat or octaarginine peptides [ 110112 ] . there is strong evidence that these peptides promote undefined actin rearrangements and this like membrane ruffling may be a rac - dependent activity as both tat peptide and octaarginine ( r8 ) rapidly increased the levels of activated rac and also induced the formation of lamellipodia . in other studies cpps hiv - tat and nona - arginine induced the rapid internalization of the egf- and tumour necrosis factor receptor and it will be interesting to determine whether there is a coincidental increase in fluid phase endocytosis in these cells . support for this comes from observations that the tat cre conjugate and tat peptide conjugated to a fusogenic peptide , enhanced fluid phase uptake of 70 kd dextran . a number of studies have now shown that a fraction ( up to 90% ) of hiv - tat and r8 peptide uptake at 37 c occurs independently of inhibition by ion - exchange inhibitors eipa and amiloride [ 87 , 108 , 110 , 111 ] . uptake of r8 and hiv - tat in leukaemia cells was relatively insentitive to eipa but at concentrations higher than 50 m a much higher fraction of the peptide was found in the cytosol . in a separate study , the cytosolic delivery of oligoarginine and uptake of a cpp - immunogenic antigen chimera was unaffected by amiloride treatment [ 114 , 115 ] . amiloride and amiloride analogues have additional quite dramatic effects on the actin cytoskeleton and cell morphology , and their capacity to inhibit macropinocytosis may be mediated through actin . the sub - cellular distribution of conventional markers for early and late endo - somes , eea1 and lamp1 , were also highly sensitive to amiloride and eipa treatment and of specific note for endocytosis studies is that inhibition of the na / h exchanger will also affect cellular ph levels . these agents have a plethora of other effects on cells suggesting they should be used with great caution if forming the sole criteria for macropinocytosis . studies proposing that uptake is via macropinocytosis are also contradictory to others that demonstrate peptide inhibition by agents that disrupt uptake by clathrin coated vesicles . downstream of the plasma membrane , hiv - tat peptide and octaarginine co - localize with markers of early endosomes [ 94 , 108 , 118 ] but there is some controversy regarding the subsequent fate of the peptides;and this aspect of their intracellular dynamics is , as noted , is extremely important for drug delivery applications . analysis using confocal microscopy is often difficult to interpret as the possibility exists that a proportion of the signal is emanating from partially degraded peptide , and if the ph - sensitive fluorophore fluorescein isothiocyanate is used as a label , then its emission will be significantly diminished within endosomes and especially lysosomes . our studies suggest that a major fraction of octaarginine and hiv - tat peptide in leukaemia and hela cells , is rapidly delivered to lysosomes , though other studies suggest that lysosomes are bypassed by a penetratin peptide that was trafficked from endosomes to the golgi . these endocytosis studies must also be reviewed in parallel with those that clearly show energy independent uptake of cpps through the plasma membrane [ 97 , 98 , 108 , 110 , 112 ] . recent studies in leukaemia cells suggest that r8 enters cells efficiently at 4c but at temperatures 12c the same cell may internalize the peptide by endocytosis and direct plasma membrane translocation ; the relative contribution of each is dependent on temperature and the peptide concentration . this raises the possibility that entry into the cytosol from the extracellular media or the endo - lysosome lumen occurs via a similar concentration dependent mechanism ( fig . model membrane systems have also been utilized to investigate the binding and translocation capacities of a number of cpps and highlight the dependency of peptide sequence and lipid composition of the membranes . proposed models include those suggesting that cpps are driven via a potential difference following membrane destabilization , that they induce the formation of inverted micelles or that they themselves mediate pore formation . in view of the significant differences in sequences and properties of peptides included in the cpp family , there are likely to be more than one mechanism by which they traverse lipid barriers . proposed model for the cellular entry of cationic cell penetrating peptides hiv - tat and r8 . uptake of the peptides occurs by at least two temperature and concentration dependent mechanisms . at 4c , entry can only occur via plasma membrane translocation . at 37c and at high , threshold , extracellular concentration the peptide may enter the cell via a number of endocytic pathways and via direct plasma membrane translocation . a number of endocytic pathways including macropinocytosis caused by peptide induced plasma membrane ruffling is likely to be utilized . escape from undefined endosomal compartments ( ? ) could conceivably be the result of the accumulation of the peptide to a similar threshold concentration that promotes translocation across the plasma membrane . further studies of macropinocytosis in different cell types will undoubtedly shed more light on this poorly understood endocytic mechanism . the hunt will continue for unique markers or regulators of this process and more specific inhibitory drugs , and these will then allow for a better understanding of the factors governing the formation and maturation of the nascent macropinosome and its fate once released from the plasma membrane . the jury is clearly still out with regards to whether the interaction of cpps with cells induces a specific form of macropinocytosis and enhances internalization by one or more forms of endocytosis . at the relatively high concentrations ( number of peptide molecules per cell ) typically used to study these peptides by fluorescent microscopy ( typically 110 m with 1 105 10 cells ) it is likely that the peptides enter through multiple pathways in the fluid phase and via non - specific adsorptive endocytosis . cpps will undoubtedly pose many more fascinating questions as their intracellular dynamics is further studied . a more daunting challenge in drug delivery research is to identify the route of translocation of the active moiety , as opposed to what is seen through a microscope lens , that then mediates a biological response . | abstractmacropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized .
the process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation .
internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size , morphological heterogeneity and lack of coat structures .
a paucity of information is available on other distinguishing features for macropinocytosis such as specific marker proteins and drugs that interfere with its mechanism over other endocytic processes .
this has hampered efforts to characterize the dynamics of this pathway and to identify regulatory proteins that are expressed in order to allow it to proceed .
upon internalization , macropinosomes acquire regulatory proteins common to other endocytic pathways , suggesting that their identities as unique structures are short - lived .
there is however less consensus regarding the overall fate of the macropinosome cargo or its limiting membrane and processes such as fusion , tubulation , recycling and regulated exocytosis have all been implicated in shaping the macropinosome and directing cargo traffic .
macropinocytosis has also been implicated in the internalization of cell penetrating peptides that are of significant interest to researchers aiming to utilize their translocation abilities to deliver therapeutic entities such as genes and proteins into cells .
this review focuses on recent findings on the regulation of macropinocytosis , the intracellular fate of the macropinosome and discusses evidence for the role of this pathway as a mechanism of entry for cell penetrating peptides . |
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among the different entities causing rhinoliquorrhea in closest proximity to the sphenoid bone , trigeminal meningoceles ( tms ) , lateral sphenoidal meningoceles ( lsms ) , and the persistence of the lateral craniopharyngeal canal ( sternberg cruveilhier canal , sc ) have been described . tms have only been reported several times in the literature , and nomenclature is still heterogeneous . in general , reports cover various mass lesions in the meckel cave , ranging from lipoma and meningioma to schwannoma . only a few deal with meningoceles in the pterygopalatine region , such as in patients with typical stigmata of neurofibromatosis type 1 ( nf 1).1
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tms are localized in the lateral sphenoid wing lateral to v2 and the foramen rotundum underneath the semilunar ganglion , reaching to the superior orbital fissure , pterygopalatine fossa , and medial orbital space , mostly emerging from an enlarged meckel cave and giving rise to osseous erosion of the sphenoid wing . as a different and more frequent reason for rhinoliquorrhea , lsms have been described . they lie medially to v2 near the base of the sphenoidal bone3
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8 and can be distinguished from tms by the absence of orbital or pterygopalatine fossa involvement . the third dura leak associated pathology of this region is the developmental anomaly of a persistent sc embryonal canal ( lateral craniopharyngeal canal ) . still controversially discussed in literature , it has been found in some cases as an underlying condition for lsms.9
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11 the canal lies medially to the foramen rotundum and extends from the maxillary nerve root ( v2 ) to a recess of the lateral sphenoid wall , opening into the sphenoid sinus . from an anatomical point of view , there are several weak spots in the embryonic development of the sphenoidal bone , which can explain the occurrence of meningoceles or spontaneous rhinoliquorrhea , especially in the area of the trigeminal ganglion and the carotid artery . the cartilage precursor of the skull base has to form around the cranial nerves , arteries , and veins while respecting their lumen . this can lead to thinned cranial base structures that might be eroded during a lifetime by inflammatory processes or increased intracranial pressure.12
the bone formation itself is inhomogeneous . the collision zone between endochondral ossification ( lesser wing ) and intramembranous ossification ( greater wing ) lies laterally to the foramen rotundum , extending to the region of the foramen ovale ( fig . study , 25 patients with lateral sphenoidal cerebrospinal fluid ( csf ) leaks were all shown to have the bony defects in this area , lateral to the foramen rotundum.8
cranial base in a 6-month - old embryo shown from above ( modified from fenart and landouzy ) depicting the four main parts forming the sphenoid bone . cranial nerves : ii , v1 , v2 , v3 , vii , ix , x , xi , xii . alisph , alisphenoid ; boc , basioccipitale ; bsph , basisphenoid ; fm , foramen magnum ; fz , fusion zone between ( lateral ) and endochondral ( medial ) ossification ; orbsph , orbitosphenoid ; petr , petrous bone ; prsph , presphenoid ; scc , site of the sternberg cruveilhier canal . the sutures between the ossification centers can leave small basal clefts , such as the lateral craniopharyngeal canal ( sc ) . the canal represents a remnant of the fusion zone between the alisphenoid ( greater wing ) and the basisphenoid ( fig . 1).13
development of the sphenoid bone is crucial for the whole cranial base.14
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16 distorted midline structure development can result in encephaloceles and palate cleft malformations,17
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19 and midline basal encephaloceles are known as very rare malformations.11
20 lateral sphenoid malformations give rise to lateral sphenoid meningoceles , as well as to meningoceles of the trigeminal nerve . the meningoceles seem to be associated with sphenoid bone malformations , increased intracranial csf pressure , and accompanying erosive processes . as shown in the following case description , tms can result in rhinoliquorrhea and csf leakage to the pterygopalatine fossa and periorbital fatty tissue , followed by exophthalmos and conjunctivitis together with conjunctival edema . in literature , many different terms have been employed to describe the pathology of a tm . arachnoid cyst of meckel 's cavity appears to be imprecise , because lsms also arise from it . the terms transalar sphenoid meningocele and transsphenoidal and transethmoidal meningoceles have been used synonymously in literature.19
20
we suggest the term lateral sphenoidal meningocele to describe pathologies medial to v2 and the term trigeminal meningoceles as anatomically more precise for lesions lateral to the foramen rotundum and v2 . she also reported a left - sided orbital swelling , especially when bending forward or in prone position . her first meningitis had been at the age of 17 years , a second occurred a few years later , and both had been treated successfully with antibiotics . a former traumatic head injury was denied and basal cranial fractures had been ruled out with multiple imaging techniques . extended neuroradiological imaging included cisternographic magnetic resonance ( mr ) scan after intrathecal gadolinium - application in prone position . it revealed a left - sided csf leak along a csf - containing enlargement of the temporal fossa that extended into the pterygopalatine fossa . origin of the fistula was suspected in the temporopolar parasellar region in close proximity to the cavernous sinus and to the meckel cavity . the clivus , sellar area , and ethmoidal cells appeared to be anatomically altered in the ct - scan ( fig . ( a ) preoperative magnetic resonance ( mr ) t1-weighted axial images enhanced by cisternography . ( b ) oblique reconstruction parallel to the optic canal , ( c ) axial and ( d ) coronal reconstruction . the surgical strategy was discussed in the multidisciplinary skull - base board and under suspicion of a temporomedial meningocele of the maxillary nerve , a pterional craniotomy with transsylvian approach and closure of the parasellar entry point of the meningocele was advised . in a first operation , the suspected entry point of the fistula the patient then decided to be treated in another neurosurgical department , where a second pterional operation was performed without relieving the symptoms . about 2 years after first surgery , the patient decided to restart treatment in our institution . intrathecal contrast - enhanced ct revealed the refilled fistula and an enlarged , csf - containing space in the paraclival region , close to the maxillar nerve and the meckel cave . a third pterional exploration was proposed , but the patient opted for conservative therapy . only after increasing orbital swelling and reappearance of rhinoliquorrhea fusion of the ct dataset with neuronavigation pictures allowed identification of the entrance of the meckel cavity . the wall of the cavity showed a cisternlike arachnoid covering in which the nerve fibers crossing the cavity were partly adhering to the basal arachnoid layer of the cyst and were spread apart . two walls of the cavity were found , corresponding to arachnoid cystic structures and dural tissue , thus displaying typical criteria for meningoceles . to close the fistula , abdominal fat and muscle tissue were harvested and the periarachnoidal space of the meckel cave was filled in proximal and distal direction . hereafter , fat tissue was positioned in the remaining arachnoid space rostrally . between fat and muscle tissue , liquid dura glue ( duraseal xact , covidien , mansfield , massachusetts , usa ) the opening of the cavity was sealed with tachosil ( takeda pharmaceuticals ; zurich , switzerland ) and surgicel ( ethicon , somerville , new jersey , usa ) . after this intervention , rhinoliquorrhea and orbital swelling disappeared and the third nerve palsy nearly completely recovered within 3 months . however , about 4 months postoperatively the patient experienced an intermittent csf leak . shortly thereafter , orbital swelling and conjunctivitis reoccurred . t2- and ciss 3d t2-scans revealed a partly occluded , yet csf - containing , meningocele . the patient refused any further interventions , especially implantation of a ventriculoperitoneal ( vp ) shunt to reduce the intracranial csf pressure , and she was lost to follow - up . review of the literature was performed in the medline database using the search terms trigeminal , encephalocele , meckel 's cave , sphenoid , pterygopalatine , csf fistula , and sternberg in all combinations of two keywords . search resulted in 38 relevant cases from 23 reports , ranging from 1888 to 2011 ( table 1 ) . within these 38 reports , patients presented with rhinoliquorrhea in 92.1% ( 35 patients ) , headaches in 34.2% ( 13 patients ) , and meningitis in 15.7% ( 6 patients ) . abbreviations : csf , cerebrospinal fluid ; lsm , lateral sphenoid meningocele ; sc , sternberg cruveilhier canal ; sof , superior orbital fissure ; tm , trigeminal meningocele . out of 38 patients , 6 had a tm , whereas a persistent sc or lsm was found in 32 patients . a total of 29 patients underwent endoscopic surgery for lsms , whereas in 3 cases the transcranial approach was preferred . in two cases4 transcranial reoperation was performed after endoscopic surgery due to recurrence of csf leaks.4 recurrence rate was 13.7% ( 4 of 29 cases ) . from the six tms , three were treated by an endoscopic transsphenoidal approach , with recurrence of csf leaks in two of them ( 66% ) . one of three patients undergoing craniotomy and open repair experienced recurrent therapy - resistant csf leakage ( present case , 33% ) . apart from persistent rhinoliquorrhea , the most common complications in the endoscopic group were meningitis ( 6.3% ) and maxillary nerve irritation ( 3.1% ) . in the craniotomy group , the case presented here illustrates a rare tm extending from an enlarged meckel cave into the medial cranial and pterygopalatine fossa , causing therapy - resistant csf leakage and orbital affection with an unusual collection of csf in the temporal muscle ( fig . although the predominant symptoms of csf leaks such as rhinoliquorrhea , headache , and meningitis do not help in localizing the dural defect , clinical appearance may vary with the localization of the specific arachnoidal cyst , meningocele , or encephalocele . for example , arachnoid cysts of the region of the meckel cavity often become symptomatic with facial numbness or pain due to their relation to the trigeminal nerve.21
the therapeutic difficulties of that unusual entity have not been solved . in contrast to the far lateral tms , lsms and persistent sc can be treated successfully with a transsphenoidal approach . this may be caused by their more medial localization , medially to the foramen rotundum and v2 at the sphenoid base . patients with tms are likely to have a greater benefit from a transcranial approach , which provides better access to the lateral aspects of the meckel cave and the semilunar ganglion . this is underlined by the higher recurrence rate when using the transsphenoidal approach ( table 1 ) . alternatively , transmaxillar transpterygoid approaches may be employed for tms with a limited pterygoid fossa leakage.1
csf leaks associated with tms can be assumed to be of idiopathic origin with an elevated intracranial pressure ( icp ) as an additional factor . thus , icp recording and icp normalization should be taken into account , and routine use of lumbar drainage as a diagnostic and therapeutic measure has been proposed in lateral sphenoid csf leaks.22
in light of the reviewed literature , together with the presented therapy - resistant case , alternative strategies such as implantation of a vp shunt or temporary lumbar drainage to reduce csf pressure have to be discussed . the occurrence of the spontaneous csf leak as part of an idiopathic hydrocephalus syndrome should be considered , with acetazolamide or furosemide being a treatment option.22 however , the one case treated conservatively experienced persistence of symptoms , similar to our patient , who experienced recurrence after deciding for nonsurgical follow - up ( table 1 ) . midfacial degloving has been suggested as approach to the pterygopalatine fossa meningoceles and remains an option for recurrence.21
a staged treatment algorithm using lumbar csf drainage , icp recording , and invasive location of the skull base defect with intrathecal administration of contrast medium seems to be most promising . depending on the location and anatomical shape of the tm , open craniotomy or endoscopic transpterygoid approach can be selected.6
7
8
22 whereas for medial pathologies ( sc , lsm ) endoscopic transsphenoidal approaches appear to be advantageous , tms should be primarily considered for an open craniotomy . alternatively to transcranial approaches , transfacial or transmaxillary / transpterygoid approaches have to be considered as treatment for recurrent , extended tms involving the pterygopalatine fossa , in which transsphenoidal endoscopic techniques have limited success rates . in case of therapy - refractory csf leaks and marked elevation of icp , vp shunt placement and/or medical treatment should be considered before reintervention.22 compared with the spontaneous rhinoliquorrhea caused by a persistent sc or lsms , the point of leakage in tms lies laterally to v2 and can not always clearly be visualized , even by sophisticated neuroradiological techniques . this can result in considerable technical difficulties and therefore therapeutic decisions should follow the advice of a multidisciplinary skull base team after taking into account the individual anatomic situation of a patient , preceding operations , and specific risk factors for elevated icp . | trigeminal meningoceles , lateral to the maxillary nerve ( v2 ) , have seldom been reported as underlying pathology for spontaneous rhinoliquorrhea .
in contrast to sphenoid meningoceles arising from a persistent lateral craniopharyngeal canal ( sternberg cruveilhier , medial to v2 ) , their occurrence seems to be generated by addition of erosive processes to the constitutively thin bony shell underneath the semilunar ganglion , lateral to the round foramen ( and v2).the developmental and anatomical relationships of trigeminal meningoceles to the sphenoid bone are depicted , and in a review of the literature we present the different surgical approaches employed for sealing the dura leak . in view of these techniques
we discuss an unusual case of therapy - resistant rhinoliquorrhea with left - sided trigeminal meningocele involving the meckel cave at the lateral sphenoid and reaching the superior orbital fissure and the medial orbital space.in contrast to patients who have lateral sphenoidal meningoceles with a persistent lateral craniopharyngeal canal ( sternberg cruveilhier ) , who can be treated successfully using an endoscopic transsphenoidal approach ( recurrence rate 13.7% ) , the recurrence rate of cerebrospinal fluid ( csf ) efflux for trigeminal meningoceles lies much higher ( endoscopically 66% , open craniotomy 33% ) .
the surgical strategy thus has to be chosen individually , taking into account specific anatomical situations and eventually preceding operations . |
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the study protocol was reviewed and approved by the ethical boards of all participating centers . the study was conducted during january 2004december 2007 in clinical centers that offered primary healthcare to illegal migrants in northern ( brescia ) , central ( rome ) , and southern ( palermo ) italy . european union country who registered for a visit at each center were asked to participate in the study . participants were divided into 4 groups according to their risk for hiv infection : 1 ) commercial sex workers , 2 ) persons reporting unsafe sex ( occasional not - for - money homosexual or heterosexual contacts with persons other than their regular partner ) , 3 ) persons with other risk factors ( intravenous drug use or blood transfusion in their country of origin , and 4 ) persons with no risk factors identified . participants were tested for antibodies against hiv types 1 and 2 and for p24 antigen by using a commercial microparticle enzyme immunoassay ( axsym hiv ag / ab combo ; abbott laboratories , abbott park , il , usa ) . hiv - positive serum samples were further tested for hiv antibody avidity by using the axsym hiv 1/2go assay ( abbott laboratories ) as reported ( 9 ) , to ascertain likely time of infection . an avidity index < 0.80 indicated that infection was acquired recently ( within the past 6 months ) ; a higher index indicated that infection was acquired earlier ( > 6 months ago ) . a cutoff value of 0.80 was used and validated as having 93.0% sensitivity , 98.5% specificity ( 10 ) , and > 90.0% reproducibility . avidity results were cross - checked with reported time of migration to assess likely place of exposure . a total of 4,078 persons were invited to participate in the study . of 3,976 ( 97.5% ) who agreed to participate , 3,003 ( 73.6% ) underwent hiv testing ( 2,815 in brescia , 48 in rome , and 140 in palermo ) . in terms of hiv risk for 2,853 respondents , 191 were commercial sex workers , 1,246 practiced unsafe sex , 47 reported other risks , and 1,494 reported no risk factors ( total = 2,978 because multiple risk factors were reported by some persons ) . demographic characteristics for the participants are shown in table 1 . * except where indicated , values are no . n = 2,997 hiv-1 infection was detected for 29 ( 0.97% ) of 3,003 participants ( 95% confidence interval [ ci ] 0.90%1.2% ) ; no participants were infected with hiv-2 . univariate analysis showed that sociodemographic and behavior factors associated with hiv infection were christian religion ( p = 0.029 , odds ratio [ or ] 3.07 , 95% ci 1.068.83 ) , migration from sub - saharan africa ( p = 0.001 , or 11.94 , 95% ci 1.6188.81 ) , commercial sex ( p = 0.0001 , or 18.2 , 95% ci 6.2552.97 ) , and unsafe sex ( p = 0.016 , or 3.43 , 95% ci 1.229.66 ) . multiple logistic regression showed that factors independently associated with increased risk for hiv infection were migration from sub - saharan africa ( p = 0.0001 , or 3.7 , 95% ci 2.29.4 ) , commercial sex ( p = 0.025 , or 18.4 , 95% ci 4.948.5 ) , and unsafe sex ( p = 0.01 , or 2.3 , 95% ci 1.78.6 ) . place of infection could not be determined for 17 ( 63.0% ) of 27 persons ; 6 ( 22.2% ) of 27 were presumably recently infected in italy , and 4 ( 14.8% ) of 27 presumably acquired their infection in their country of origin before emigration . sociodemographic characteristics of our population did not differ from those reported nationwide ( 1 ) . our data confirm that many illegal migrants practice unsafe sex ( low rate of condom use ) . these findings are worrisome if one considers poor knowledge of hiv transmission reported in our study population ( 11 ) . consequently , migrants are particularly vulnerable to stis , as shown by the high prevalence rate ( 0.97% ) for the adult migrant population , which is higher than the estimated 0.4% prevalence rate for the national population in italy ( 12 ) . the higher hiv prevalence rate for persons from an area ( sub - saharan africa ) in which hiv is highly endemic might reflect exposure in the country of origin or new infections in the host country . a total of 6 ( 22.2% ) of the 27 hiv infections for which avidity index data were available were probably acquired in italy by migrants from sub - saharan africa ( n = 3 ) , eastern europe ( n = 2 ) , and latin america ( n = 1 ) . conversely , all 4 ( 14.8% ) persons who acquired infection before migration to italy were originally from sub - saharan africa . however , migrants are unevenly distributed in italy , with the largest communities in northern italy . second , the study acceptance rate was only 73.6% . however , this acceptability rate is similar to rates in other studies in europe ( 13,14 ) . furthermore , sociodemographic characteristics of our sample were homogeneous with those of other studies from the same centers and nationwide ( 1,15 ) , and we did not anticipate any differences between participants and those who did not participate . our data show high hiv prevalence for an illegal migrant population in italy . for persons originally from sub - saharan africa and , as for the native population in italy , practicing commercial or unsafe sex these data indicate the prominent role of social determinants of hiv infection , including marital status and living conditions . health education and free access to hiv testing and care for the illegal migrant population in western countries is needed , particularly for persons from sub - saharan africa . | to determine hiv prevalence and place of exposure for illegal migrants in italy , we tested 3,003 illegal adult migrants for hiv ; 29 ( 0.97% ) were hiv positive .
antibody avidity index results ( indicators of time of infection ) were available for 27 of those persons and showed that 6 ( 22.2% ) presumably acquired their infection after migration . |
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intentional ingestion of foreign bodies is common in psychiatric patients and prison inmates [ 1 , 2 ] . various types of foreign bodies ingested are toothbrushes , pens , plastic spoons , pencils , batteries , food bolus , paper clips , razor blades , coins , bones , pins and pills [ 1 , 2 , 3 ] . foreign bodies in children are usually coins . once in the stomach , most ingested foreign bodies pass without any intervention . foreign bodies can get lodged in the esophagus , stomach , small bowel or rectum . generally , this occurs in narrow areas ( i.e. pylorus and ileocecal valve ) . the inability to swallow saliva , dysphagia and neck tenderness are common clinical features in foreign bodies of the esophagus . abdominal pain , bowel obstruction or perforation can occur with sharp objects in the esophagus , stomach , small bowel or colon . few recent studies have shown that in the setting of intentional ingestion , the rate of endoscopic intervention may be 6376% and the need for surgical intervention ranges from 12 to 16% [ 1 , 4 ] . in a study of predominantly intentional ingestions , a success rate of 90% with endoscopic extraction was observed . all sharp and pointed foreign bodies should be removed before they pass from the stomach because of the risk of intestinal perforation . timing of endoscopy for ingested foreign bodies varies and depend on patient age , clinical condition , type , size , shape , content and anatomic location of the foreign body in the gastrointestinal tract ( table 1 ) . rat tooth forceps and snares are the most commonly used accessory devices to remove foreign bodies . an overtube can be used while removing the foreign body to prevent accidental slippage of the foreign body into the trachea [ 1 , 9 ] . drug packets have a risk of rupture and leakage of contents can be fatal , so endoscopic removal should not be attempted . a 26-year - old man with a past medical history of bipolar disorder , schizophrenia and diabetes mellitus was brought from a correctional facility after confessing to have swallowed a few shower curtain hooks . he complained of chest pain , shortness of breath and abdominal pain with nausea and vomiting . outpatient medications included metformin , metoprolol , sublingual nitroglycerin , percocet , baby aspirin , risperdal and trazodone . abdominal x - ray done in the emergency room revealed multiple foreign bodies in the stomach ( fig . 1a , yellow arrow ) . labs revealed normal metabolic panel , hemoglobin 14 g / dl , white blood cells 8,100/mm , platelets 213,000/mm , mean corpuscular volume 85 fl and international normalized ratio 1.01 . an upper endoscopy was done in emergency room , with the use of an overtube , which showed the metal pieces as well as retained food in the stomach and gastritis . all other foreign bodies which were present on the abdominal x - ray could not be visualized initially as there was retained food in the stomach . then a rat tooth forceps was passed through the accessory channel and fishing for foreign bodies was attempted in the retained food material . after multiple attempts , four other foreign bodies were found and each one was caught by the forceps and then the scope was removed with the forceps holding the foreign body . though three were removed easily , the last foreign body kept slipping back into the esophagus when the endoscope was pulled into the overtube . so finally , when the last foreign body , which was held by forceps , was pulled into the overtube it was removed in total with the endoscope and the rat tooth forceps holding the foreign body . the result of which was not available at the time of endoscopy , there was an additional foreign body in the right mainstem bronchus ( fig . 1b , yellow arrow ) . pulmonary and subsequently cardiothoracic surgery were consulted for a rigid bronchoscopy and possible retrieval of the foreign body . on hospital day 1 , a repeat chest x - ray was done , which failed to show the foreign body which was originally present in the right mainstem bronchus . a computed tomography ( ct ) scan of the chest and abdomen was done for evaluation , which showed the foreign body in the cecum . the patient had coughed up the foreign body and swallowed it into the gastrointestinal tract . the patient was monitored for 3 days with serial abdominal x - rays ( fig . 1c and d , yellow arrows ) , but the foreign body did not pass . he was ultimately given golytely for bowel preparation and a colonoscopy was planned to remove the foreign body . colonoscopy was done and the foreign body was caught by the snare with the long axis of the foreign body oriented in parallel to the long axis of the colon , and the colonoscope with the snare holding the foreign body was pulled out slowly and safely from the patient 's rectum without any complications . this case report describes the rare scenario of a patient ingesting six shower curtain hooks of which five were found in the stomach and one in the right mainstem bronchus . the presence of food material in the stomach can pose a challenge for the endoscopist trying to remove the foreign body safely . radiologic studies such as x - rays or ct scans are helpful in localization of the foreign body in the gastrointestinal tract , in assessing the shape and size of the foreign body and in planning management appropriately , if retrieval is warranted . illegal drugs wrapped in packets can usually be seen radiographically , and ct scanning may be helpful , although false - negative scan results have been reported . blunt objects in the stomach pass spontaneously within 46 days . with sharp objects in the stomach , complications can occur in up to 1535% of cases [ 5 , 11 ] and hence every attempt has to be made to remove them endoscopically . long objects ( 610 cm ) are unlikely to pass into the duodenal sweep and should be removed . a hood can also be used while removing the foreign body to prevent accidental slippage . once the foreign body is pulled into the overtube with an endoscope , all three can be removed together from the patient 's mouth in one single motion to avoid losing grasp of the object within the overtube . we used this technique in removing the last foreign body from the stomach . upon careful review of the initial abdominal x - ray a small tip of the foreign body in the bronchus can be viewed ( fig . the presence of an additional foreign body in the right mainstem bronchus prompted surgical consultation for rigid bronchoscopy , but before the patient was taken to the operating room , he coughed up the foreign body and swallowed it . to our knowledge , this is the first case report of a patient intentionally transferring a foreign body from one organ system to another . the foreign body was later identified in the cecum on abdominal ct . a foreign body which has crossed the ileocecal valve therefore , the patient was observed to see whether the object was progressing to the distal rectum . as the foreign body was in the colon for 3 days without any rectal passage , endoscopic retrieval was performed to shorten the waiting time to natural expulsion , given the patient was from a correctional facility and might have re - ingested the foreign body if left to his own devices . in conclusion , the current case illustrates the unusual probability of simultaneous foreign bodies in the airway and gastrointestinal tract and the success of endoscopy in safely retrieving the foreign bodies from the stomach and colon . | intentional ingestion of foreign bodies is common in psychiatric patients and prison inmates .
timing of endoscopy for ingested foreign bodies varies and depends on the type and location of the foreign body in the gastrointestinal tract .
we present the case of a 26-year - old man who was brought from a correctional facility after confessing to have swallowed a few shower curtain hooks .
abdominal x - ray done in the emergency room revealed multiple foreign bodies in the stomach .
an upper endoscopy was done in the emergency room with the use of an overtube .
the first metal piece was caught by a snare and removed with the endoscope .
all other foreign bodies which were present on the abdominal x - ray could not be visualized initially as there was retained food in the stomach .
after multiple attempts , four other foreign bodies were found and each one was caught by the forceps and then the scope was removed with the forceps holding the foreign body .
there was an additional foreign body in the right mainstem bronchus .
the patient had coughed up the foreign body and swallowed it into the gastrointestinal tract .
a computed tomography scan of chest and abdomen was done for evaluation , which showed the foreign body in the cecum . to our knowledge , this is the first case report of a patient intentionally transferring a foreign body from one organ system to another .
colonoscopy was done and the foreign body was removed rectally with a snare without any complications . |
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from 87 veterinary clinics solicited in 2007 , we selected 47 on the basis of their size and geographic distribution . veterinarians were instructed to obtain samples from all dogs routinely tested for heartworm . in northern areas , where heartworm is rarely tested for , they recorded each dog s age , town of residence , lyme disease vaccination status ( ever or never vaccinated ) , and the test results . each dog owner completed a form ( 99.6% response rate ) to describe the dog , its function , history of unexplained lameness , travel history ( town , state , visited within the past year ) , history of tick infestation , and use of tick control products ( yes or no ) . we used spearman rank correlation tests to examine associations between canine seropositivity , human lyme disease cases reported to the maine center for disease control and prevention ( augusta , me , usa ) ( 4 ) , and the number of deer ticks submitted to our laboratory in 2007 . we used b. burgdorferi and a. phagocytophilum test results and questionnaire responses to cross - tabulate responses and calculate the likelihood ( odds ratios ) of b. burgdorferi and a. phagocytophilum positivity as a function of risk factors by using tests of association . analyses were conducted by using sas version 9.2 for windows ( sas , cary , nc , usa ) . of 1,087 dogs tested across maine s 16 counties , 12.7% were b. burgdorferi positive and 7.1% were a. phagocytophilum the distribution of all dogs seropositive for either pathogen is shown by town in figure 1 . at the county level , canine b. burgdorferi seropositivity among unvaccinated dogs correlated positively with the number of human lyme disease cases reported for 2007(spearman = 0.84 ; p<0.0001 ) and the number of deer ticks submitted to our laboratory for identification ( spearman = 0.63 ; p = 0.009 ) . in figure 2 , which shows statewide distributions by county north to south , only unvaccinated dogs dogs had been exposed to a. phagocytophilum in all but 2 northern counties . at the town level , remarkably higher levels of canine a. phagocytophilum seropositivity were found in southern coastal cape elizabeth ( cumberland county ) ( 76.5% , n = 17 ) and york ( york county ) ( 58.0% , n = 19 ) than in towns in their immediate vicinity . * tested by using snap 4dx test kit ( idexx laboratories , westbrook , me , usa ) . towns where dogs were tested for seropositivity to borrelia burgdorferi ( a ) and anaplasma phagocytophilum ( b ) in a statewide serosurvey of domestic dogs , maine , usa , 2007 . a ) canine seroprevalence for anaplasma phagocytophilum and , in dogs never vaccinated against lyme disease , for borrelia burgdorferi in maine counties arranged north to south , 2007 . b ) maine counties , with the 10 tick - abundant counties used in the analyses shaded in gray . counties : 1 , aroostook ; 2 , piscataquis ; 3 , somerset ; 4 , penobscot ; 5 , franklin ; 6 , washington ; 7 , hancock ; 8 , oxford ; 9 , waldo ; 10 , kennebec ; 11 , knox ; 12 , lincoln ; 13 , androscoggin ; 14 , sagadahoc ; 15 , cumberland ; 16 , york . never - vaccinated dogs were 1.5 as likely to be seropositive for b. burgdorferi than were vaccinated dogs ( 15.3% vs. 9.9% ; p = 0.008 ) ( table 2 ) . vaccine use was higher in 10 southern counties where lyme disease has become endemic ( figure 2 , panel b ) than in the 6 northern counties where it is emerging ( 63.9% vs. 42.7% ; p<0.0001 ) and correlated positively with the number of deer ticks submitted to our laboratory for identification in 2007 ( n = 16 counties , spearman = 0.63 ; p = 0.009 ) . two thirds of respondents said that their dogs had traveled out of town ; however , no associations were found between b. burgdorferi or a. phagocytophilum seropositivity and the dog s travel history . three of 59 dogs in the northernmost county of maine were b. burgdorferi positive , 1 of which had never traveled beyond its home town . eighty - three percent of owners reported using acaricides . despite the effective protection reported for topical acaricides ( 6,7 ) , no difference in seropositivity between treated and untreated dogs was evident on the basis of their reported use ( table 2 ) . unexplained lameness was 1.5 more likely in dogs that were only a. phagocytophilum positive than in those only b. burgdorferi positive ( 40.0% vs. 26.5% ; p<0.06 ) . * a total of 1,087 dogs were tested . or , odds ratio ; ci , confidence interval ; ns , not significant . this study demonstrates that risk of contracting lyme disease has reached northernmost maine and that anaplasmosis is now being transmitted to dogs throughout the lower half of the state . the study expands on nationwide snap 4dx data documenting b. burgdorferi and a. phagocytophilum positivity in the southern half of the state ( 8) . in southern coastal maine , overabundant white - tail deer , appropriate habitat , and maritime climate all contribute to high densities of i. scapularis ticks ( 3 ) and consequent disease transmission ; thus , the remarkably high level of a. phagocytophilum seroreactivity observed in the southern coastal towns of cape elizabeth and york calls for further work to understand the dynamics of the intense local emergence of this pathogen . the higher level of unexplained lameness in a. phagocytophilum positive dogs than in b. burgdorferi positive dogs is consistent with findings by beall et al . ( 9 ) , who reported a 2.6 greater incidence of a. phagocytophilum seroreactivity than b. burgdorferi seroreactivity among 32 lame , non co - infected dogs in minnesota who were suspected of having either disease . the lameness also reflects the high percentage of b. burgdorferi positivity among asymptomatic dogs ( 10 ) . that b. burgdorferi and a. phagocytophilum seropositivity rates were essentially identical between dogs who had a history of travel and those who did not lessens concern about travel as a confounding variable , an exposure difficult to interpret in any event , given the spotty distribution of ticks even where lyme disease is endemic ( 11 ) . in a recent study , hamer et al . ( 12 ) reported that a serosurvey of canines for b. burgdorferi is ineffective in a region that includes areas with little b. burgdorferi transmission , and less informative than analysis of ticks removed from dogs . our inability to detect an effect of topical acaricides may reflect their ubiquitous use for flea control and a lack of information on the frequency of their use . although the widespread use of protective measures now complicates interpretation of serosurveys of canines , in selected dogs the availability of a reliable , multitarget test that is used routinely nationwide ( 8) remains a valuable and cost - effective method for documenting transmission of the agents of lyme borreliosis and anaplasmosis , particularly in areas where disease is emerging . | to determine if the range of deer ticks in maine had expanded , we conducted a multitarget serosurvey of domestic dogs ( canis lupus familiaris ) in 2007 .
an extension of exposure to borrelia burgdorferi to the northern border and local transmission of anaplasma phagocytophilum throughout southern areas was found . |
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cerebral palsy ( cp ) describes set of permanent , mutable , motor development disorders that
originate from a primary brain lesion and cause secondary musculoskeletal problems and
limitations with regard to activities of daily living1 . despite the consensus regarding the occurrence of sensory , motor ,
and functional impairments in children with cp2 , 3 , spasticity , muscle
weakness , and insufficient motor control can give rise to secondary musculoskeletal
complications , such as contractures and deformities , which result in limited movements7 . thus , the identification of factors that
lead to functional impairment is of fundamental importance to clinical decision making and
the evaluation of the effects of therapeutic strategies4 . a study by bae et al.8 showed that as the
tissue compliance of spastic muscles at relaxation increases , muscle tone decreases and
muscle activity increases , and spasticity leads to a lower moment - angle9 . a number of treatments performed in either the home or
school setting are proposed to improve function10 . however , the effectiveness of such therapies depends on
well - conducted functional evaluation and patient fitness11 , 12 . methods and tools
developed for the evaluation of function have been used in individuals with cp , such as the
house scale13 , the pediatric evaluation
of disability inventory14 , the melbourne
assessment15 , the pediatric outcome d
collection instrument16 , the assisting
hand assessment17 , abilhand - kids18 , and the shriners hospital for children
upper extremity evaluation19 . while some
of these measures have been validated , no consensus has been reached as to the best
evaluation method for identifying improvements in upper limb function in individuals with
cp20 . yu et al.21 believes that detailed and diverse investigations should
be performed by considering the number and characteristics of subjects . the aim of the present study was to perform a systematic review of the literature on the
scales and methods most often used for the evaluation of upper limb function in individuals
with cp . searches were conducted in the medline , pedro , lilacs , scielo , and pubmed databases using
combinations of the following key words : cerebral palsy , upper limb / extremity , and
functional scales . the following inclusion criteria were used for the selection of articles : randomized
controlled study , evaluation of upper limb function in individuals with cp , and publication
between 2006 and 2014 . the methodological quality of the articles was evaluated using the physiotherapy evidence
database ( pedro ) scale . the pedro scale has 11 items , each of which receives a score of
either 0 or 1 , except item 1 , which is not scored . this scale is used to evaluate the methodological quality of randomized , controlled ,
clinical trials with regard to two important factors as follows : whether the study has
internal validity ( whether the results offer sufficient information ) , and whether the study
has both clinical and statistical relevance for a clear interpretation of the results and
reproduction by other researchers . any divergence in opinion between the two researchers was
discussed until a consensus was reached on the score of the study in question . the database search resulted in the retrieval of nine articles , four of which failed to
meet the inclusion criteria ( fig . 1fig . the five articles included in the present review had pedro scores of 6 to 9 points
( demonstrating methodological adequacy ) and addressed the use of upper limb evaluation
measures for individuals with cp ( tables
1table 1.articles included in the literature reviewarticleauthor and year ofpublicationpedrotype of study1koman et al . , 2008266/10clinical trial and 2table 2.scores of the articles included in the literature reviewpedro12345eligibilitynyyyyrandomized allocationyyyyyconfidential allocationyyyyysimilar prognosisnyynyblinded subjectsyynnnblinded therapistsnnnnnblinded evaluatorsyyyynkey resultsyyyyncomparison between groupsyyyyyprecision and variabilityyyyyyscore7/109/108/107/106/10y : yes ; n : no ) . the five studies involved 296 male and female individuals aged 2 to 18 years
who were diagnosed with cp . all of the studies used one or more measures to evaluate upper
limb function . ofsubjectscharacteristics of samplemethodsoutcomes171spastic hemiparesiseg : 36- botulinum toxincg : 35- placebo injectionthe eg achieved a better wrist extension result in the
melbourne assessment of unilateral upper limb function than the cg.2105spastic hemiparesiseg1 : 34- intensive two - hand trainingeg2 : 33- modified citcg : 33-
standard treatmentthe modified cit group achieved better movement quality in the
quest than in the other groups and exhibited better quality of life , as measured
by the besta scale.375spastic hemiparesiseg1 : 25- citeg2 : 24- cit + electrical stimulationcg : 26-
occupational therapyamong all the groups , eg2 demonstrated the best results for the
upper extremity functional test and grasping subtest of the peabody developmental
motor scales.422spastic hemiparesiseg : 11- citcg : 11- control interventionthe eg achieved better results in the pmds-2 , botmp , and pmal than
in the cg.523spastic hemiparesiseg : 12- botulinum toxincg : 11- placebo injectionno statistically significant differences were found
between the groups . intraclass concordance was found for daily activities ,
speaking , and communication on the pediatric quality of life inventory.eg : experimental group ; cg : control group ; cit : constraint - induced therapy ; quest :
quality upper extremity skill test ; pmds-2 : peabody motor developmental scales ii ;
botmp : bruininks - oseretsky test of motor proficiency ; pmal : pediatric motor activity
log displays the general characteristics ( sample size , sample characteristics , and
methods ) and outcomes of the studies analyzed . flowchart of the studies included in the literature review eg : experimental group ; cg : control group ; cit : constraint - induced therapy ; quest :
quality upper extremity skill test ; pmds-2 : peabody motor developmental scales ii ;
botmp : bruininks - oseretsky test of motor proficiency ; pmal : pediatric motor activity
log among the upper limb function evaluation measures available in the literature , few are
specific to individuals with cp , as most scales are standardized for use on adult stroke
survivors . despite the similarities between the two types of brain lesions , the articles analyzed
in the present systematic review demonstrate the scarcity of studies on upper limb function
in individuals with cp , especially with regard to evaluation measures . according to koman et al.22 , the
melbourne assessment of unilateral upper limb function scale provides objective measures of
upper limb function , allows the assessment of the quality of upper limb movements , and
demonstrates moderate to high consistency as an evaluation method . all of the studies analyzed emphasized the evaluation of upper limb function associated
with a functional therapeutic method or neurolytic block . fedrizzi et al.23 applied the quality upper extremity skill
test ( quest ) and the besta scale , which demonstrated good performance and applicability . the
quest allows an assessment of the quality of one- and two - hand movements in individuals with
cp but does not allow an assessment of quality of life . the besta scale is used for the
assessment of quality of life , as well as functional capacity and movement performance . xu et al.24 used the upper extremity
functional test to assess function , dexterity , and movement efficiency , and the grasping
subtest of peabody developmental motor scales , which is also known as the peabody
developmental motor scales 2 or pdms 2 , for the two - hand evaluation . in the literature , the
pdms-2 is used less than the upper extremity functional test . however , the authors do not
state whether one of the two scales is more applicable than the other . lin et al.25 also used the pdms-2 and
the bruininks - oseretsky test of motor proficiency ( botmp ) for the assessment of range of
motion and the pediatric motor activity log ( pmal ) to quantify functional capacity , along
with the caregiver functional use survey for the evaluation of caregivers . improvements in
unilateral and bilateral skills were demonstrated by the pmal , but not the botmp . the study reports the use of
scales that allows the evaluation of functional capacity in children with cp but does not
suggest that any particular scale is more favorable in clinical practice due to its greater
applicability or the greater reliability of its results . redman et al.26 analyzed the use of the
pediatric quality of life ( pedsql ) scale . although the authors demonstrated the
applicability of this scale for individuals with cp , the pedsql is not sufficiently
sensitive for the detection of small but clinically important changes and has no subscales
for the evaluation of upper limb function . few studies have addressed the use of assessment measures for upper limb function in
individuals with cp . the studies analyzed in the present systematic review used different
measures , and no consensus has been reached on the most appropriate scale or which has ideal
clinical applicability in this population . therefore , further studies on this issue are
needed to allow the evaluation of upper limb function in individuals with cp by using
well - defined methods that provide reliable information . | [ purpose ] the aim of the present study was to perform a systematic review of the
literature on the scales and methods most often used for the evaluation of upper limb
function in individuals with cerebral palsy .
[ materials and methods ] searches were
conducted in the medline , pedro , lilacs , scielo , and pubmed databases .
the following
inclusion criteria were used for the selection of articles : randomized controlled study ,
evaluation of upper limb function in individuals with cerebral palsy , and publication
between 2006 and 2014 .
the methodological quality of the articles was evaluated using the
pedro evidence scale .
[ results ] five articles met the inclusion criteria and achieved 6
points or higher on the pedro scale of methodological quality . [ conclusion ] the studies
analyzed used different evaluation scales , but no consensus has been reached thus far on
which scale is the most appropriate .
thus , further studies are needed to establish an
adequate method for the evaluation of upper limb function in individuals with cerebral
palsy . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
systemic lupus erythematosus ( sle ) is a complex autoimmune disease of unknown origin affecting virtually every organ in the human body . enhanced apoptosis in conjunction with defective clearance of apoptotic cells results in occurrence of high levels of autoantibodies . inflammatory cytokines , like type i and type ii interferons and interleukin-6 ( il-6 ) , il-1 , and tumor necrosis factor - alpha ( tnf- ) as well as immunomodulatory cytokines like il-10 and tgf- , have been identified as important players in sle . apart from those il-21 and il-17 have been lately identified to play a relevant role in autoimmunity , while recent findings regarding il-2 brought this cytokine back in focus of sle research . beside interferons this paper will highlight some recent advances of il-6 , il-21 , il-17 , and il-2 research with regard to sle . type i interferons ( ifns ) are important cytokines , whose most prominent function is to mediate the early immune response to viral infections . viral rna and dna are recognized by toll - like receptors ( tlrs ) and trigger ifn release of leukocytes . although all leukocytes produce ifn , plasmacytoid dendritic cells ( pdcs ) are the main producer . only 0,20,8% of peripheral mononuclear cells ( pbmcs ) are pdcs , but their capacity to produce ifn is unique and 100200 times enhanced compared to any other cell type [ 3 , 4 ] . the ability to release such high amounts of ifn might be caused by the fact that pdcs constitutively express toll - like receptor 7 ( tlr7 ) and toll - like receptor 9 ( tlr9 ) . after secretion ifn binds its heteromeric type i ifn receptor on target cells , transduces signals mainly via jak / stat pathways , and initiates gene transcription of so - called interferon - stimulated genes . microarray analysis detected > 300 genes induced by interferon . by activation of genes which are responsible for antimicrobial responses , antigen processing , and inflammation , ifns exert several immunomodulatory effects and are therefore supposed to be key cytokines not only in the innate immune system but also in adaptive immune responses . many of the symptoms that sle patients develop are congruent with symptoms of patients suffering from influenza or as a side effect of interferon - alpha ( ifn- ) therapy . sle patients often show enhanced ifn- serum levels , and the ifn levels correlate with anti - dsdna production and disease activity . furthermore , ifn- therapy may lead to autoantibody production and an sle - like syndrome [ 11 , 12 ] . genetic association studies of patients with sle identified several genes , amongst which components of the upstream and downstream pathways of type i interferon are the most frequently found including signal transducer and activator of transcription 4 ( stat4 ) and interferon regulatory factor 5 ( irf5 ) [ 1416 ] . in fact the irf5 risk haplotype in sle patients is associated with high serum ifn- activity . these genetic association studies are in accordance with the fundamental observations identified by gene expression profiling of sle pbmcs in the group of virginia pascual . these experiments demonstrate a significant upregulation of interferon - regulated gene transcripts in adult and paediatric sle pbmcs [ 18 , 19 ] . this characteristic is referred to as the interferon signature and assessed as a new biomarker for disease activity . these observations raised the questions of how the ifn signature in sle patients develops and how ifns are involved in pathogenesis of sle . one cause of immune complex formation is an increased apoptosis and defective clearance of apoptotic material on the one hand and high occurrence of autoantibodies on the other hand . in 1998 cederblad et al . observed the production of ifn- by pbmcs when serum samples from sle patients were used as culture supplement . further studies showed that immune complexes induce ifn- production by pdcs [ 2124 ] . immune complexes are internalized after binding fc gamma riia on the surface of pdcs and activate tlr9 and tlr7 in the endosomal compartment , which induces secretion of ifn- . indeed pdc are reduced in sle blood , but this reduction might be related to enhanced recruitment to tissues [ 26 , 27 ] . the overproduction of ifns in sle exerts wide effects , which result in the above - mentioned ifn signature . we would like to accent a few of these effects which were intensively observed and papered by obermoser and pascual . first ifn- promotes feedback loops by induction of tlr7 in pdcs , mdcs , and monocytes which enhance synthesis of ifn . secondly ifns contribute to disruption of peripheral tolerance by promoting dc maturation ( mdc ) and thereby reducing numbers of immature dcs . immature dcs are important to keep up immune tolerance by induction and maintenance of regulatory t cells . in addition immature dcs promote anergy and deletion of self - reactive t cells by presenting self - peptide mhc complexes in the absence of costimulatory signals to self - reactive t cells . thirdly mdcs can also directly enhance selection and survival of autoreative b cells by producing b - cell activating factor ( baff ) . this cytokine belongs to the family of b - lymphocyte stimulators ( blyss ) and contributes to survival of b cells . finally ifn- drives disease activity by enhancing cytotoxicity of cd8 t cells and also directly increases numbers of autoreactive cd4 t cells by upregulation of the costimulatory molecules cd80 and cd86 on antigen - presenting cells ( apcs ) . therefore , activation of the ifn system by ics as endogenous ifn inducers in sle patients generates a self - reinforcing trial which rnnblom and alm describe as a vicious circle ( figure 1 ) . the widely used and old drug resochin changes the ph of the endosomes and therefore the affinity of tlr7 and tlr9 towards ics . antibodies to block ifn - alpha ( sifalimumab , rontalizumab ) are currently being tested in clinical trials . in a phase i trial treatment of sle patients with an anti - ifn- monoclonal antibody influenced interferon signature and skin lesions of these patients . il-6 is produced in many cell types , like monocytes , fibroblasts , endothelial cells , and also t and b lymphocytes and has a range of biological activities on various target cells . differentiation of b cells in plasma cells and induction of igg production is induced by il-6 as well as differentiation and proliferation of t cells and macrophages . further effects of il-6 are bone marrow stem cell maturation , activation of neutrophils , and stimulation of the production of platelets from megacaryocytes and osteoclast differentiation . il-6 signaling occurs via its heteromeric receptor complex , which consists of two glycoproteins , an il-6-specific binding chain ( il-6r ) and a signal transducing chain ( gp130 ) . binding of il-6 on il-6r triggers dimerisation of gp130 , which results in activation of jak1 and tyrosine phosphorylation of gp130 . il-6r expression is limited to several cells , but a so - called gp130 transsignaling occurs when il-6 binds a soluble il-6r form and then interacts with the more unique expressed gp130 ( figure 2 ) . murine lupus models indicate the involvement of il-6 in b - cell hyperactivation and onset of autoimmune disease . in mrl / lpr mice il-6 and soluble il-6r serum levels are increased related to age [ 45 , 46 ] . il-6-deficient mrl / lpr mice show a delayed onset of lupus nephritis and prolonged survival . il-6 receptor blockade suppressed igg antibody production in nzb / w f1 mice and development of autoimmune disease [ 48 , 49 ] , whereas exogenous administration of il-6 accelerates glomerulonephritis in nzb / w f1 mice . recent investigations suggest that il-6 blockade not only targets autoreactive b cells but also inhibits autoreactive t cells in nzb / w f1 mice . next to its effects on b cells il-6 is a key cytokine that determines t - cell differentiation of nave t cells into so - called regulatory t cells with a suppressive phenotype or into t cells with a proinflammatory th17 phenotype . since il-6r blockade in mouse model of arthritis inhibited the differentiation of th17 cells , effects of il-6 blockade on t - cell responses and therefore benefits for autoimmune diseases should also be taken into consideration . patients with active sle have increased il-6 serum levels [ 53 , 54 ] which in some studies correlated with disease activity or anti - dna levels [ 40 , 54 ] . elevated il-6 levels are associated with b - cell hyperactivity and autoantibody production and secretion of igg anti - dna antibodies were reduced by neutralizing il-6 and restored by adding exogenous il-6 in vitro [ 40 , 53 ] . in addition to its systemic effects il-6 also has a role in local inflammation , for example , in lupus nephritis and is supposed to be involved in mesangial cell proliferation , one of the hallmarks of proliferative lupus nephritis . patients with active lupus nephritis show elevated urinary il-6 secretion [ 55 , 56 ] , and the expression of il-6 is increased along glomerular and tubulus tissue in lupus nephritis kidneys in situ . il-6 is increased during cardiopulmonary complications of sle , and sle patients with neuropsychiatric syndromes show elevated il-6 levels in the cerebrospinal fluid . as il-6 exerts systemic effects and also mediates local inflammation , il-6 targeting therapy , which has been shown to be efficacious in inflammatory autoimmune diseases , might also be promising in the treatment of sle patients . tocilizumab is a humanized monoclonal antibody , which inhibits il-6 signaling by binding il-6r and soluble il-6 receptors . it was recently tested in an open label - phase 1 dosage escalation study in sle patients . the results are promising regarding decreased levels of anti - dsdna antibodies and of acute - phase reactants in tocilizumab treated patients . interferon - gamma ( ifn- ) activates macrophages at the site of inflammation , contributes to cytotoxic t - cell activity , has antiviral capacities , and is strongly associated with th1 responses . it induces differentiation of nave t cells into th1 cells and triggers th1 differentiation in an autocrine manner . ifn- signaling induces phosphorylation of stat1 which leads to expression of the th1-lineage - specific transcription factor t - bet and subsequent expression of ifn- . due to the fact that th1-mediated effects can explain many features of autoimmune diseases , ifn- became an archetypical inducer of organ - specific autoimmunity . ifn- might contribute to autoimmune disease by inducing production of igg2a and igg3 isotype antibodies that activate complement and furthermore by activating macrophages and promoting tissue inflammation . however , in autoimmune models like experimental autoimmune encephalomyelitis and collagen - induced arthritis [ 64 , 66 ] , ifn--deficient mice are more susceptible ; therefore , the role of ifn- is not proinflammatory per se . recent studies detected diverse mechanisms via which ifn- might counteract inflammatory pathways ( review in ) . one important mechanism might be that ifn- inhibits the development of autoimmune - related th17 cells [ 67 , 68 ] . t - helper cells expressing ifn- correlate with age and development of disease in nzb / w f1 mice . additionally treatment of nzb / w f1 mice with recombinant ifn- accelerated development of disease , while administration of monoclonal antibodies against ifn- resulted in remission of disease . furthermore , ifn-r - deficient nzb / w f1 mice show reduced glomerulonephritis and reduced serum concentration of anti - dsdna antibodies . ifn--deficient mrl / lpr mice are prevented from early death and have reduced lymphadenopathy and reduced glomerulonephritis . however , treatment of mrl / lpr mice with recombinant ifn- leads to dichotomic effects . while treatment at an early age proves to be protective , treatment later in life accelerates disease manifestations . in a pristine - induced lupus model several studies on lupus models suggest that an imbalance towards th1 dominance plays a role in acceleration of disease [ 7678 ] . in human patients with sle a disbalance in mechanisms that regulate th1 and th17 cells with an enhanced expression of th17 cells recent studies detected unusual ifn- and il-17 double - positive t cells which indicates a quite complex and not yet understood plasticity of th1 and th17 cells . the complex role of ifn- in sle is underscored by contradictory clinical studies that find a correlation between serum ifn- level and disease activity and a correlation between ifn- expression and severity of lupus nephritis while others show decreased ifn- levels in lupus nephritis [ 83 , 84 ] . nevertheless , amg-811 , a human monoclonal antibody to ifn- , is under investigation in a phase ib study in sle patients . il-2 production is induced after t - cell receptor ( tcr ) activation , induces itself in paracrine and autocrine loops , and also upregulates surface expression of its receptor . il-2 was initially discovered as a cytokine which drives clonal expansion of t cells , but the phenotypes of il-2-deficient or il-2-receptor- ( il-2r- ) deficient mice expand the tasks and impact of il-2 . mice with il-2 or il-2r deficiency show an enlargement of peripheral lymphoid organs ( lymphadenopathy and splenomegaly ) and impaired activation - induced cell death ( aicd ) and develop autoimmune disorders [ 86 , 87 ] . in addition to this , a defective production of il-2 is observed in several murine models of autoimmune diseases including three well - established lupus models . in all of these models the production of il-2 is reduced once disease starts to appear [ 8991 ] . these observations are somewhat inconsistent with the view of il-2 as growth factor for t cells and raise the question of how loss of il-2 is connected with loss of immunotolerance . interestingly , il-2 deficiency in mouse models is paralleled by reduced levels of regulatory t cells ( tregs ) . therefore , the uncontrolled activation of b and t cells in the absence of il-2 might be caused by deficiencies of regulatory t cells in these mice . direct evidence that regulatory t cells depend on il-2 comes from experiments which show that il-2 is required for homeostatic maintenance of regulatory t cells as well as for their thymic development and il-2 also directly affects suppressive function of regulatory t cells . in addition to its effect on regulatory t cells , it was very recently discovered that il-2 also affects th17 cells . il-2 limits production of il-17 in vivo and in vitro , and low levels of il-2 favour occurrence of th17 cells . il-2-deficient mice show enhanced serum levels of il-17 and a higher number of il-17 producing t cells in peripheral lymph nodes . laurence et al . showed by adoptive transfer experiments that the il-17 overproduction is not caused by a secondary manifestation of disease , but directly due to deficiency of il-2 . it is therefore currently accepted that il-2 , beyond its role as a growth factor , is important to maintain functionality and homeostasis of regulatory t cells on the one hand and to inhibit production of il-17 on the other hand . as a consequence il-2 appears to be a crucial cytokine to prevent formation of autoimmunity . in accordance with this sle t cells show reduced il-2 production [ 9698 ] and il-2 deficiency is also paralleled by low numbers of regulatory t cells in sle patients . the molecular mechanism of the il-2 defect in sle is caused amongst others by overexpression of camp response element modulator alpha ( crem ) , a transcription factor which binds to the il-2 promoter and inhibits il-2 transcription . anti tcr / cd3 antibodies present in sle sera induce expression of crem , which leads to an increased crem binding to the il-2 promoter and decreased il-2 production . we recently showed that increased crem expression is the result of enhanced crem promoter activity in sle t cells and crem promoter activity correlates with disease activity . interestingly the defective il-2 production of sle t cells can be restored by introducing a plasmid encoding antisense crem into these cells . the il-2 activating transcription factor cre - binding protein ( creb ) shares the same binding site on the il-2 promotor and is displaced by crem in sle cells possibly because of high levels of crem . furthermore , diminished activity of creb , caused by increased levels of the serine / threonine phosphatase pp2a , the phosphatase that is responsible for dephosphorylation of creb , contributes to reduced production of il-2 . it is not clear whether lower il-2 levels in sle also contribute to enhanced il-17 levels , but the ratio of treg to th17 cells in sle patients with active disease is significantly lower than that in healthy controls and inversely correlates with the severity of active sle . aicd is a controlled apoptotic mechanism by which excess effector cells are eliminated and it is regulated by cd95 and tnfr1 [ 106108 ] . this process is affected in sle patients , in whom t cells are more resistant to aicd [ 109 , 110 ] resulting in persistence of autoreactive t cells . furthermore , il-2 is also important for the development of cd8 t - cell cytoxicity . cytotoxic t cells ( ctl ) destroy virus - infected t cells and are important to defend infections . some sle patients develop cytotoxic defects , while a lot of sle patients suffer from increased mortality and morbidity during infections . altogether defective il-2 production in sle t cells seems to contribute to several immune alterations including reduced numbers and function of regulatory t cells , decreased aicd , decreased ctl responses and to upregulation of il-17 production . this raised the question whether compensation of low il-2 levels by adding exogenous il-2 would result in lower disease activity . treated mice the homeostatic balance of treg and t effector cells was re - established and impeded disease progression . however , the half live of exogenous cytokines in vivo is quite short , while il-2 in complexes with an antibody is more functional . furthermore in vivo expansion of regulatory t cells with il-2/il-2mab complexes induces resistance to experimental autoimmune encephalomyelitis . therefore il-2 seems to have a therapeutic potential to treat autoimmune diseases , but the activity of il-2 as growth factor bears a risk . il-2 has been used as adjuvant for treatment of patients with renal cancer albeit with considerable side effects . the effect of il-2 seems to depend on the administered dose , it is possible that low doses favour tregs , while high doses favor memory / effector cell function . further expand the impact of il-2 to a cytokine that in addition to its influence on regulatory t cell and th17 cells broadly regulates t helper cell differentiation . further investigations is needed to understand the several and sometimes ambivalent roles of il-2 . it should be taken into consideration to therapeutically influence mechanisms upstream from il-2 , which are responsible for reduced il-2 expression in sle . il-21 is produced by a range of differentiated cd4 t cell subsets and natural killer ( nk)t cells . il-21 signals through a heterodimeric receptor , which is formed by common gamma chain ( shared with il-2 , il-4 , il-7 , il-9 , il-13 and il-15 receptors ) and an il-21 specific receptor ( il-21r ) [ 118 , 119 ] . since il-21r is expressed on cd4 , cd8 t cells , b cells , nk cells , dendritic cells , macrophages and keratinocytes , il-21 acts on a range of lymphoid lineages and exerts pleiotropic effects il-7 it promotes cd8 t cell expansion [ 117 , 120 , 121 ] . il-21 is induced by il-6 and rort and stabilizes and maintains th17 cells by upregulating its own expression and the expression of il-23r [ 117 , 121 ] . induced regulatory t cells furthermore il-21 counteracts suppressive effects of tregs , however it is not known if il-21 acts on tregs or cd4 t cells in this circumstances . furthermore il-21 plays a role in follicular t helper cell ( tfh ) development and is necessary for germinal center ( gc ) formation [ 124 , 125 ] . gcs can be the origin of autoantibodies and abnormalities in gcs can lead to aberrant selection of autoreactive b cells and might contribute to autoimmunity . il-21 has a role in b cell activation and differentiation of plasma cells that produce igg , but also induces apoptosis of resting and activated b cells . il-21 without antigen or in the presence of a non - specific polyclonal signal induces deletion of autoreactive b cells . il-21 in context of a specific antigen and t cell interaction leads to expansion of responding cells . il-21 can also act anti - inflammatory , it inhibits dendritic cell maturation and stimulates il-10 production [ 129 , 130 ] . sle patients have higher serum levels of il-21 , while il-21 and il-21r polymorphisms are associated with susceptibility to sle [ 131 , 132 ] . a subset of patients with sle shows increased numbers of circulating cd4 cxcr5 cells ( tfh cells ) . the sanroque mouse bears a mutation in a gene that negatively regulates tfh cell development . these mice develop lupus - like symptoms , paralleled by an overproduction of il-21 and increased levels of tfh cells . mrl / lpr mice show increasing numbers of tfh cells and extrafollicular t helper cells with age and disease development . mrl / lpr mice treated with il-21r / fc to block il-21 signaling displayed reduced level of autoantibodies and sle - like symptoms . the lupus mouse bxsb.b6-yaa+ shows increased il-21 mrna levels compared to wildtype mice and disease was prevented by genetic deletion of il-21r in these mice . notably treatment of bxsb.b6-yaa+ mice with an il-21r / fc fragment negatively influenced survival early on and positively influenced survival at later stages of disease . because of these pleiotropic effects , it remains debatable if il-21 blockade might be useful to treat sle . il-17 is produced by several t - cell subsets including t helper cells ( cd4 t cells ) , cytotoxic t cells ( cd8 t cells ) , double - negative ( cd4cd8cd3 ) t cells , gamma - delta t cells but also by natural killer ( nk ) cells and neutrophils . a new cd4 t - cell subset , which preferentially produces il-17 but not il-4 or ifn- , is termed th17 cells . beyond il-17a and important factors for the differentiation of murine as well as human th17 cells include il-6 , il-21 , and il-1 together with tgf- [ 122 , 140146 ] . in addition to these cytokines , il-23 is crucial for expansion and maintenance of th17 cells . th17 cells are involved in the immune response against bacteria , like citrobacter , klebsiella pneumoniae , and borrelia burgdoerferi and against fungi like candida albicans . some of these infections can not be cleared by th1 or th2 cells . beyond these protective roles , il-17 and th17 cells il-17 receptors are broadly expressed not only on immune cells but also on epithelial and endothelial cells [ 139 , 149151 ] . il-17 signaling through these receptors increases production of chemokines ( interleukin-8 ( il-8 ) , monocyte chemoattractant protein-1 , growth - related oncogene protein - alpha ) , which leads to recruitment of monocytes and neutrophils into the inflamed tissue [ 152154 ] . moreover , il-17 also induces t - cell infiltration by upregulating the expression of intercellular adhesion molecule 1 ( icam-1 ) . il-17 induces secretion of many proinflammatory proteins , among them prostaglandin e2 , granulocyte - macrophage colony - stimulating factor ( gm - csf ) , and granulocyte colony stimulating factor [ 155157 ] , and also cytokines which induce a positive feedback loop and lead to further il-17 production like interleukin-6 ( il-6 ) , il-1 ( interleukin-1 beta ) and il-21 ( interleukin-21 ) . recent experiments provide evidence that il-17 alone or in synergy with baff also promotes b - cell differentiation and autoantibody production [ 158 , 159 ] . sle patients have raised serum levels of il-17 . enhanced percentages of il-17 producing cells one source of il-17 in sle patients is double - negative t cells ( dnts ) . sle patients have expanded numbers of double - negative t cells ( dnts ) compared to healthy individuals . il-17 producing cells infiltrate skin , lung , and kidneys of sle and lupus nephritis ( ln ) patients [ 160 , 165167 ] and most likely contribute to organ damage by exerting - above - mentioned effects . evidence that il-17 also contributes to b cell activation in ln comes from in vitro experiments with pbmcs . these experiments document that il-17 induces induction of igg and anti - dsdna production . in the last years the mrl / lpr mice model provided some evidence for the functional contribution of il-17 to disease progression and organ damage . mrl / lpr mice have increased numbers of double - negative t cells ( dnts ) , which produce high amounts of il-17 and expression of il-17 , and il-23 receptor ( il-23r ) increases with disease progression . lymphoid cells from mrl / lpr mice can induce nephritis in nonautoimmune species after il-23 in vitro treatment . after ischemic reperfusion of the gut , enhanced il-17-mediated tissue injury was observed in mrl / lpr mice . splenocytes from snf1 ( new zealand black x swr f1 ) mice secrete higher levels of il-17 than nonautoimmune b6 mice . in congruence with observation from the mrl / lpr model il-17-producing t cells bxd2 mice express high levels of il-17 in serum and increased numbers of il-17 cells in the spleen , which form spontaneous autoreactive germinal centers in concert with il-17r expressing b cells . these features could be blocked by inhibition or deletion of the il-17 receptor . although these data indicate that il-17 plays a role in pathogenesis of autoimmune diseases , it is not clear whether targeting il-17 is suited to treat sle . next to th17 cell other t - cell subsets like th1 cells crossregulate each other . in a graft - versus - host - disease model the absence of donor th17 cells leads to an exacerbated disease by augmented th1 differentiation . recent studies showed that increases in th17 cells are directly correlated with the depletion of treg cells during sle flares . it is therefore suggested to consider possibilities to recover the balance between th17 and regulatory t cells to treat sle and other autoimmune diseases [ 148 , 175 ] . tgf - beta induces the differentiation of treg cells from nave t cells ; however , the addition of il-6 or il-21 results in th17 differentiation [ 140 , 176 , 177 ] . the lineage transcription factors of th17 and treg cells , rort / ror and foxp3 , respectively , bind each other and inhibit each other 's function [ 178 , 179 ] . il-2 is an indispensable growth factor for tregs but inhibits th17 differentiation [ 94 , 95 ] , and il-21 promotes th17 differentiation and inhibits the induction of regulatory t cells . finally tregs treated with il-6 can produce il-17 [ 180182 ] and can convert into il-17 producing autoimmune effector cells . the balance of th17 and treg cells is regulated by several transcription factors which are activated in a context - dependent manner depending on external cytokines . low levels of il-2 , enhanced production of il-21 and il-6 [ 53 , 185 ] might lead to enhanced il-17 levels . we do not know if tregs lose expression of foxp3 and become il-17-producing cells during sle flares . future investigations might shed light on the question whether il-17 blockade or blockade of cytokines or transcription factors that regulate th17-treg homeostasis will be useful to treat sle . cytokines are important mediators of intercellular communication and orchestrate the interaction of immune cells during immune responses . in sle several cytokines are involved in general immune dysregulation and also in local inflammation which leads to tissue injury and organ damage . here we summarized recent advances in the studies of some cytokines , which contribute to sle pathogenesis . it is widely accepted that interferons have a crucial role in the pathogenesis of sle . the therapeutic blockade of the ifn driven vicious circle might be one of the most promising anti - cytokine therapies in the future . furthermore , an aberrant sle t - cell phenotype which is characterized by a dysregulated production of il-17 and il-21 and low production of il-2 also aggravates disease pathology ( figure 3 ) . | systemic lupus erythematosus ( sle ) is an autoimmune disease of unknown origin affecting virtually all organ systems . beyond genetic and environmental factors ,
cytokine imbalances contribute to immune dysfunction , trigger inflammation , and induce organ damage .
the key cytokine that is involved in sle pathogenesis is interferon alpha .
interferon secretion is induced by immune complexes and leads to upregulation of several inflammatory proteins , which account for the so - called ifn signature that can be found in the majority of sle pbmcs .
additionally il-6 and ifn - y as well as t - cell - derived cytokines like il-17 , il-21 , and il-2 are dysregulated in sle .
the latter induce a t - cell phenotype that is characterized by enhanced b - cell help and enhanced secretion of proinflammatory cytokines but reduced induction of suppressive t cells and activation - induced cell death .
this paper will focus on these cytokines and highlights pathophysiological approaches and therapeutic potential . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
systemic hypertension ( sh ) is considered an important risk factor for coronary heart
disease and ischemic as well as hemorrhagic stroke1 . in accord to world health organization , the sh cause 7.5 million
deaths , about 12.8% of the total of all deaths2 . studies have demonstrated an association between body weight , diet
composition , dietary fat , physical inactivity and high blood pressure levels . among these
risk factors , physical
activity has been considered an important lifestyle modification that may aid in the
prevention of hypertension3 . several
studies have found that aerobic training reduced both systolic blood pressure ( sbp ) and
diastolic blood pressure ( dbp)4 , 5 . furthermore , physical exercise have been a strategy to
reduce the medicine consumption and doses used in hypertensive subjects6 . in addition to the importance of physical activity in the control of hypertension , same
variables , such as heart rate ( hr ) at rest and distance walked in the 6 minute walk test
( 6mwt ) have been used as marker of physical conditioning . a lower hr at rest was found after
aerobic training with consequent reduction of blood pressure in hypertensive subjects7 . moreover , these effects may be associated
with a better performance in the 6mwt8 . however , during the physiotherapy assessment , same factors can interfere with the hr and
6mwt values , such as the use of beta blockers drugs , hypotireoidism , and orthopedic
problems . thus , the evaluation of respiratory muscle strength may be a tool to investigation
of physical conditioning gain and of influence of this conditioning on the blood pressure
levels in hypertensive subjects . respiratory muscle strength is a marker related to the evolution of several pathologies ,
mainly of pulmonary origins9 , 10 . respiratory muscle strength is assessed by maximal
inspiratory pressure ( mip ) , which evaluates the inspiratory muscle strength , and maximum
expiratory pressure ( mep ) , which evaluates the strength of the expiratory muscles . mip and
mep represent the highest pressure that can be generated during maximum inspiration and
expiration , respectively , against a closed airway11 . a study evaluating male and female swimmers found that mip and mep
can be used as a marker of an athlete s performance12 . these authors demonstrated that mip and mep values after the
physical training were higher that baseline values . furthermore , roceto et al.13
demonstrated that an aerobic exercise program once a week for 12 weeks increased the maximum
respiratory pressure in patients with obstructive pulmonary disease . additionally , a higher
levels of respiratory muscle strength were associated with a slower rate of mobility decline
in older persons14 , and some of these
elderly were hypertensive . despite studies that have demonstrated that the aerobic training promotes reduction in
blood pressure in hypertensive subjects and consequently improves physical conditioning15 , no study has associated these effects
with an increase in respiratory muscle strength . thus , the present study aimed to associate
the increase of mip and mep with the blood pressure levels in hypertensive subjects that
performed an aerobic training program . ninety - six subjects with sh diagnosis , referred by cardiologists for cardiac rehabilitation
program of the unifal , between the dates of march 2014 to november 2015 . initially were 96
subjects evaluated , the 6 were excluded for abandoning the training program . the
participants were of both sexes ( 43 females and 27 males , intervention group ; 17 females and
3 males , control group ) with a mean age 59.65 13.21 ( intervention group ) and 56.10 8.75
( control group ) years . they were divided in two groups : intervention group , composed by
exercised subjects ( n=70 ) and the control group , composed by non - exercised subjects ( n=20 ) .
among the subjects studied , all had arterial hypertension controlled by medicine , in the
intervention group 30% were diabetic , 14% had had heart surgery , 37% reported sleep
disorders , 60% lung disease and , in the control group , 45% were diabetic , 0.2% had had heart
surgery , 65% reported sleep disorders and 41.42% lung disease . furthermore , in the
intervention group , 47.1% of participants were classified as obese , 32.9% were classified as
overweight , 14.3% classified as having a healthy weight and 5.7% underweight ; in the
control , 60.0% of participants were classified as obese , 35.0% were classified as overweight
and 5.0% were classified as having a healthy weight . the inclusion criteria were the
following : sedentary participants and have no history of psychiatry or psychological
disorders or abnormalities , aged30 years with chronic mild - to - moderate and stable
( > 1 year duration ) hypertension . subjects excluded from the study included those with
grade iii or iv heart failure , recent acute myocardial infarction , unstable angina , acute
pericarditis , pulmonary hypertension or severe untreated blood , ventricular tachycardia at
rest , acute infections , iii or iv peripheral arterial obstruction , and uncontrolled
diabetes . this was a controlled clinical study that evaluated the comparison between body
mass , ( bmi ) , 6mwt , sbp , dbp , mip and mep inter and intra - groups and the correlation between
mip , mep , 6mwt with the sbp and dbp levels and the correlation between 6mwt with mip and mep
values intra - groups , before and after 8 weeks . the study was performed in the physiotherapy
clinic at the federal university of alfenas ( unifal ) , brazil . written informed consent was
obtained from all volunteers before participation , and the study was approved by ethics
committee of the unifal . the interview concentrated
on the time of hypertension onset , symptoms , life quality , pharmacotherapy , physical
activity , and work . in addition , an anthropometric measurement was performed , which measured
the subjects physical characteristics ( weight [ kg ] and height[m ] ) and body composition
( body mass index [ bmi ] ( kg / m ) . furthermore , we did the measurements of mip and mep for the evaluation of respiratory
muscle strength . mip and mep were measured with respiratory pressure meter at the beginning
and at the end of the eight - weeks . the measurement is non - invasive , is well tolerated by
patients , and is recommended by the american thoracic society and the european respiratory
society as a test of respiratory muscle strength16 . in addition , 6mwt was used to evaluate the cardiovascular and
physical conditioning gain17 . hr , arterial blood pressure ,
borg s rate of perceived exertion ( rpe ) , and arterial oxygen saturation ( sa02 ) were measured
at rest continuously every 2 minutes during walking and after the test17 . in the training program , the patients underwent to aerobic exercise on a treadmill
( movement rt250- manaus , am , brazil ) 2 times a week for 8 weeks . exercise sessions started
with a 10-minute warm - up period ( stretching and slow walking ) followed by 30 minutes of
aerobic activity on a treadmill and ended with an appropriate cooling - off period of 10
minutes . patients exercised at 60 to 70% hr , calculated according to the karvonen formula ,
which was measured continuously with a portable hr monitor ( polar rs200 , woodbury , ny , usa ) . blood pressure was monitored periodically during each workout to ensure that it was within
safe limits ( sbp<220 mmhg , dbp<110 mmhg ) . the workload was adjusted to maintain the
target hr and blood pressure within the prescribed limits throughout the exercise session . arterial blood pressure , hr , and rpe were
measured before , during ( every 10 minutes ) , and after the training program . data normality was verified
through the kolmogorov - smirnov test . for comparison between the intragroup evaluations , the
paired t - test and the wilcoxon test . to comparison intergroup , was used t - test independent ,
mann - whitney and one - way anova . in the correlation between the data we used the spearman
correlation test . a significance level of 5% statistical analyses were performed using spss
for windows version 20.0 ( spss , chicago , il , usa ) . table 1table 1.comparison of intragroup and intergroup variablesvariablesintervention group ( n=70)control group ( n=20)intergrouppre - intervention ci ( 95%)post - intervention ci ( 95%)d powerpre - intervention ci ( 95%)post - intervention ci ( 95%)d powerpre d powerpost d powerage ( years)59.6 13.256.1 8.70.353.465.852.060.10.4height ( cm)159.9 9.9158.6 9.20.1155.2164.5154.2162.90.08body mass ( kg)84.5 15.184.1 15.00.0279.5 12.179.6 11.90.0050.10.177.491.577.191.10.0573.885.274.085.10.050.30.3bmi ( kg / m)32.4 7.032.0 7.1 0.0631.7 4.831.7 4.910.0040.060.0229.235.728.635.30.0929.433.929.434.00.050.090.056mwt ( m)373.4 96.4537.0 81.1***1.8420.7 85.7412.2 83.9 * 0.10.20.6328.2418.5499.0575.01.0380.6460.8372.9451.50.10.50.9sbp ( mmhg)131.2 13.1121.0 10.2***0.8133.5 11.8136.5 11.8***0.20.070.6125.0137.4116.2125.71.0127.9139.0130.5142.00.60.10.9dbp ( mmhg)83.0 15.573.5 8.7***0.785.5 9.986.0 12.7***0.080.10.675.790.269.477.50.980.890.180.091.90.10.10.9mip ( mmh2o)94.3 27.4106.2 20.9***0.495.0 23.795.5 24.80.020.010.282.0107.796.4116.00.983.8106.183.8107.10.050.050.5mep ( mmh2o)100.7 20.5109.6 17.4***0.494.0 22.490.4 21.10.10.10.491.0110.3101.4117.80.983.5104.580.5100.20.30.20.9independente t test ; mann - whitney test ; paired t
test ; wilcoxon test ; anova one way test ; * p<0.05 ,
* * * p<0.001 , power>80% ; d : effect size ; bmi : body mass index ; 6mwt :
six - minute walk test ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ;
mip : maximum inspiratory pressure ; mep : maximum expiratory pressure ; : mean standard deviation ;
ci : confidence interval shows the comparison of intragroup and intergroup variables . we can verify
that there were no statistical difference of participants main anthropometric
characteristics , such as age , height , body weight and bmi , before and after 8 weeks . independente t test ; mann - whitney test ; paired t
test ; wilcoxon test ; anova one way test ; * p<0.05 ,
* * * p<0.001 , power>80% ; d : effect size ; bmi : body mass index ; 6mwt :
six - minute walk test ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ;
mip : maximum inspiratory pressure ; mep : maximum expiratory pressure ; : mean standard deviation ;
ci : confidence interval although no significant differences occurred in the anthropometric characteristics
assessed , participants walked a greater distance ( 373.43 96.44 m 537.07 81.15 m ,
p<0.001 ) in the 6mwt after the aerobic training program , indicating an improvement in the
physical conditioning ( table 1 ) . furthermore ,
after the aerobic training program , the levels of sbp and dbp were significantly reduced ,
respectively ( 131.25 13.16 mmhg 120.00 10.20 mmhg , p<0.001 and 83.00 15.59 mmhg
73.50 8.75 mmhg , p<0.001 ) , in the intervention group ( table 1 ) . table 1 also
shows that after the aerobic training program , respiratory muscle strength was significantly
increased , represented by mip ( 94.30 27.48 cmh2o 106.25 20.95
cmh2o , p<0.001 ) and mep ( 100.70 20.51 cmh2o 109.65 17.45
cmh2o , p<0.001 ) , respectively . however , there was no correlation of the results provided by mip , mep and 6mwt with sbp and
dbp levels , after the aerobic training program ( table
2table 2.correlation between respiratory pressure with blood arterial pressure and
six - minute walk test for both groupsvariablesintervention group ( n=70)control group ( n=20)systolic blood pressurediastolic blood pressuresystolic blood pressurediastolic blood pressurepre - interventionpost - interventionpre - interventionpost - interventionpre - interventionpost - interventionpre - interventionpost - interventionmaximum inspiratory pressurepre - intervention0.007 ( r)0.09 ( r)0.1 ( r)0.4 ( r)post - intervention0.02 ( r)0.007 ( r)0.05 ( r)0.3 ( r)maximum expiratory pressurepre - intervention0.08 ( r)0.05 ( r)0.1 ( r)0.4 ( r)post - intervention0.09 ( r)0.2 ( r)0.07 ( r)0.06 ( r)six - minute walk test pre - intervention0.08 ( r)0.03 ( r)0.3 ( r)0.4 ( r)post - intervention0.1 ( r)0.1 ( r)0.08 ( r)0.1 ( r)maximum inspiratory pressuremaximum expiratory pressuremaximum inspiratory pressuremaximum expiratory pressurepre - interventionpost - interventionpre - interventionpost - interventionpre - interventionpost - interventionpre - interventionpost - interventionsix - minute walk test pre - intervention0.2 ( r)0.2 ( r)**0.2 ( r)0.2 ( r)post - intervention0.3 ( r)**0.3 ( r)**0.2 ( r)0.5 ( r)**spearman test . * * p<0.01 . ) . nonetheless , there was a correlation between the distance walked in the 6mwt
with the mip and mep values ( 6mwt and mip , p<0.01 ; 6mwt and mep , p<0.02 ) ( table 2 ) . physical activity has been recognized in the literature as a method of preventing
hypertension . over the years , it has been found that for optimal bp , pharmacological drugs
alone are not sufficient for preventing hypertension and , the physical activity is a crucial
aspect of treatment as well . thus , the american college of sports medicine recommends that
hypertensive people start a regular exercise program with moderate intensity18 . in the present study , we found that aerobic exercise 2 times a week for 8 weeks was
efficient in promoting a reduction in arterial blood pressure levels in hypertensive
patients . another study found similar results using the same training period19 , reinforcing the hypothesis that 8 weeks
of aerobic exercise are efficient in reducing arterial blood pressure . despite the control of hypertension , in our study , the aerobic training did not produce
changes in body weight and bmi , mainly reduction as demonstrated in similar studies . a
reduction of body weight and bmi and blood pressure was found in women with postmenopausal
hypertension after 8 weeks of low - impact aerobic training20 . an explanation for the
lack of reduction of bw in this study could be the frequency of exercise ( 2 times a week ) ,
but martin et al.21 using a protocol of a
30-minute aerobic training program held 4 times a week for 10 weeks , also found no
differences in the weight of participants after the training . thus , we suggest that despite
the fact that the participants received information in relation to the importance of a
balanced diet , they lacked the accompaniment of a nutritionist to assess them with greater
efficiency . in addition , education on how to modify lifestyle and gain more self - control over
one s eating has demonstrated a reduction in the weight and bmi of obese adults22 . thus , we suggest that to physical
exercise be effective in controlling body weight , it must be associated with a food
education , especially with the accompaniment of a specialist . although the present found no improvement in terms of weight lost and bmi after the aerobic
training program , it was demonstrated by the 6mwt that after the training , participants
could walk a greater distance , suggesting a gain in physical and cardiovascular conditioning
through the exercise protocol used . the 6mwt is commonly used to assess the fitness level of
healthy adults and of older adults with disabilities and pathologies , such as stroke ,
chronic obstructive pulmonary disease , pulmonary arterial hypertension , pulmonary fibrosis ,
and heart failure23,24,25 . the 6mwt is highly reproducible and is a
strong marker of prognosis of several treatments . however , orthopedic problems , medicines ,
labyrinthitis , etc . can influence the response to the test . despite the present study
suggest that aerobic exercise program produced an increase in the 6mwt , we not found a
correlation this finding with the reductions of sbp and dbp levels . a large number of
patients were taking medications to sh control and this may have influenced on this
correlation . however , the increase of distance walked in the 6mwt was correlated with the
improvement of mip and mep after aerobic training . we suggesting that the physical and
cardiovascular conditioning gain produced by aerobic exercise program increased the
respiratory muscle strength of hypertensive subjects . thus , the measurement of mip and mep
can be an important tool to evaluated physical conditioning gain . although the improvement
of mip and mep produced by aerobic exercise program not correlated with the sbp and dbp
levels , future studies using a larger number of patients will find this correlation . most studies have shown that physical activity can reduce high levels of arterial blood
pressure in hypertensive subjects3 , but
these studies have not associated this effect with mip and mep values . in addition , a study
demonstrated that swimmers present a greater respiratory muscle strength compared a
non - swimmers26 , indicating that aerobic
conditioning promotes an increase in respiratory muscle strength . another study , evaluating
the elderly , demonstrated that respiratory muscle strength is associated with mobility
decline14 . in addition , our study
showed for the first time this correlation with hypertensive subjects . watson et al.27 found that the reduction
in respiratory muscle strength , by mip and mep , in patients with heart failure is related to
poor cardiac output . nevertheless , this muscle weakness was not limited by exercise capacity
in these patients . however , mip and mep were found to be reduced , with a consequent
reduction in the handgrip force of patients with congestive heart failure28 . in some of these pathologies previously described , the authors suggest that decreased mep
and mip is related with generalized skeletal muscle weakness in pulmonary hypertension ,
possibly related to decreased systemic oxygen transport , leading to a variety of
consequences . these include muscle atrophy , a relative increase in the easily fatigable
type - iib fibers , decreased oxidative enzymes and mitochondria , abnormal intracellular
calcium profiles , and decreased phosphocreatine - all of which have been previously reported
in heart failure29 . in addition , all of
these consequences are related to a sedentary lifestyle and consequently with arterial blood
hypertension . thus , the present study suggests that the mip and mep values are associated with arterial
blood pressure levels since a reduction of respiratory muscle strength was associated with
high arterial blood pressure . furthermore , we demonstrated that an aerobic exercise training
program of 8 weeks was efficient in promoting a gain in cardiovascular and physical
conditioning and an increase of respiratory muscle strength with a consequent reduction in
arterial blood pressure in hypertensive subjects . | [ purpose ] the purpose of present study was associate the increase of respiratory muscle
strength with blood pressure levels in hypertensive subjects who underwent an aerobic
exercise program .
[ subjects and methods ] 90 hypertensive subjects were divided in two
groups : intervention and control .
all participants had an interview with a physiotherapist
and were evaluated by 6-minute walk test , maximal inspiratory pressure , maximal expiratory
pressure , heart rate , systolic blood pressure and diastolic blood pressure , before and
after the 8 weeks . in the intervention group , the subjects underwent aerobic exercise
program , 2 times a week for 8 weeks [ results ] after the program , the levels of blood
pressure were significantly reduced and the distance walked in the 6-minute walk test and
the respiratory muscle strength were increased , compared to pre intervention and control
group values . however , there was no correlation between the results provided by 6-minute
walk test , maximal inspiratory pressure and maximal expiratory pressure with systolic
arterial blood pressure levels . nonetheless , the distance walked correlated with
respiratory muscle strength values , in the intervention group .
[ conclusion ] the present
study demonstrated that the aerobic training was effective in reducing the arterial blood
pressure in hypertensive subjects associated with an improvement of physical conditioning
and respiratory muscle strength . |
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appropriate rotational alignment of the femoral component is essential for successful total knee arthroplasty ( tka ) as well as long - term survival of the implant itself [ 1 , 3 ] . this is because the rotational alignment of the femoral component not only affects tracking of the patellar component but also determines the flexion gap of the femoral component [ 2 , 13 ] . previous studies suggested using the posterior condylar axis ( pca ) , whiteside s line or the transepicondylar axis ( tea ) [ 9 , 11 , 14 , 20 ] as a reference axis for determining rotational alignment of the femoral component , and there have been studies on the angles created between these reference axes [ 8 , 12 , 15 , 16 , 18 ] . however , it is not always easy to apply such traditional references in the operative field because arthritic changes such as deformities , bony defects and osteophytes not only make it difficult to identify these references but also distort them [ 3 , 5 ] . recently , researchers proposed two reference axes in the anterior femur as alternatives when conventional reference axes are ill - defined or distorted . the trochlear anterior line ( tal ) is the line which connects the anterior points of greatest protrusion of the femoral medial and lateral condyles [ 6 , 8 , 19 ] , whereas the femoral anterior tangent line ( fat ) is a line parallel to the anterior surface just proximal to the point where the femoral trochlea ends [ 15 , 16 ] . both reference axes can be used to determine the rotational alignment of the femoral component and have been regarded as useful indices [ 8 , 1517 , 19 ] . they are located in the anterior aspect of the femur and anatomically close hence determining the relative spatial relationship is relatively facilitating . studies regarding the relative position of the two reference axes not only provide valuable supplemental information for determining rotational alignment of the femoral component but also can serve as key anthropometric data of the anterior distal femur and provide useful information when designing implants . despite the potential significance , the purpose of this study was to determine the relative spatial correlation between the tal and the fat and to elucidate which reference axis might be more reliable by comparing variances between the two lines . seventy - six consecutive korean patients that received tka from october 2011 to april 2012 for osteoarthritis of the knee at our institution were selected . patients were excluded if they had had a previous bony surgery or replacement that might have changed femoral geometry . the average age was 70.3 6.0 years ( range 5085 ) . the average preoperative mechanical axis deviation ( mad ) was 10.5 5.3. these were all women . on both knees prior to the operation , 2-mm sliced computed tomography ( ct ) ( siemens ltd . the images were scanned centring the knee joint using a 512 512 pixel matrix at a thickness of 2 mm for a length of 200 mm , obtaining more than 100 sequential images in total , and these were exported to a software program ( xelis software , version 1.0.2.2 ; infinitt , seoul , korea ) to create three - dimensional images . the tibia , patella , as well as osteophytes from the images were omitted to facilitate simulation and observe anatomical indices . this computer software allowed us to create a three - dimensional model from two - dimensional images and depict lines and dots on specific areas of the model , which could be transposed back onto the two - dimensional images . as described in previous methods , the tal was defined as a line connecting the anterior aspects of greatest protrusion of the femoral medial and lateral condyles ( fig . the fat was defined as a line parallel to the anterior surface of the distal femur in the axial plane where the femoral trochlea begins ( fig . the surgical tea was defined as the line connecting the most prominent lateral epicondylar projection and medial epicondylar groove , the ap axis as the line connecting the deepest point of the patellar groove and the point of the intercondylar notch , and the pca as the line connecting points between the articular cartilages of both femoral posterior condyles [ 14 , 18 , 20 ] . all of these lines could be superimposed in any axial plane , and the angle between these axes could be measured by using the functions embedded in the software . the angle between the tal and the tea was defined as tal / tea and that between the fat and tea as fat / tea . we also measured the angle between the tal and fat ( tal / fat ) , pca and tea ( pca / tea ) , and whiteside s line and tea ( ap / tea ) . two independent observers ( hmj and dsj ) measured all angles , and one observer ( hmj ) evaluated 4 weeks apart to assess the inter - observer and intra - observer reproducibility . the inter - observer reproducibility was 0.832 , 0.875 , 0.845 , 0.864 and 0.858 , respectively for tal / tea , fat / tea , tal / fat , pca / tea and ap / tea . the intra - observer variability was 0.902 , 0.921 , 0.893 , 0.917 and 0.897 , respectively . we tried to decide whether any correlation existed between the tal / fat and the preoperative mad and the age of the patients prior to the operation . the correlation between the tal / tea and the fat / tea was calculated , and their variance was compared to determine which angle had smaller variance.fig . 1traditional and additional alternative references for femoral component rotation are depicted . a 3d - reconstructed distal femur seen from below . tea transepicondylar axis , ap anteroposterior , pca posterior condylar axis , tal anterior trochlear line and fat femoral anterior tangent line traditional and additional alternative references for femoral component rotation are depicted . a 3d - reconstructed distal femur seen from below . tea transepicondylar axis , ap anteroposterior , pca posterior condylar axis , tal anterior trochlear line and fat femoral anterior tangent line this retrospective study was approved by the institutional review board of our hospital ( irb approval , ajou university hospital , med - mdb-14 - 173 ) . all numbers were calculated to the second decimal place and presented to the first after raising the second . a sample size of 75 patients with ct scan would provide sufficient power ( > 80 % ) to show differences of variances between the tal and fat > 10 % as statistically significant ( two - tailed = 0.05 ) . all demographic data and measured angles were shown to fall into a normal distribution , and all statistical values were illustrated as average and standard deviation . correlation analysis performed using pearson s correlation coefficient , which is in general subcategorized as poor ( 0.000.20 ) , fair ( 0.210.40 ) , moderate ( 0.410.60 ) , good ( 610.80 ) and perfect ( 0.811.00 ) . the pearson s correlation coefficient was also used to evaluate the inter - observer and intra - observer reproducibility of all measurements . the variances between the angles tal / tea and fat / tea were compared by using f - test . all numbers were calculated to the second decimal place and presented to the first after raising the second . a sample size of 75 patients with ct scan would provide sufficient power ( > 80 % ) to show differences of variances between the tal and fat > 10 % as statistically significant ( two - tailed = 0.05 ) . all demographic data and measured angles were shown to fall into a normal distribution , and all statistical values were illustrated as average and standard deviation . correlation analysis performed using pearson s correlation coefficient , which is in general subcategorized as poor ( 0.000.20 ) , fair ( 0.210.40 ) , moderate ( 0.410.60 ) , good ( 610.80 ) and perfect ( 0.811.00 ) . the pearson s correlation coefficient was also used to evaluate the inter - observer and intra - observer reproducibility of all measurements . the variances between the angles tal / tea and fat / tea were compared by using f - test . the tal was internally rotated with respect to the tea by 6.1 2.5 , whereas the fat by 9.5 3.8. the fat was internally rotated by 3.4 3.3 with respect to the tal . the pca was internally rotated by 2.7 1.2 with respect to the tea , and the line perpendicular to the ap axis was externally rotated by 1.3 3.8 with respect to the tea . there was no significant correlation between fat / tal and the mad and age [ mad : r = 0.053 ( n.s . ) , age : r = 0.136 ( n.s . ) ] . correlation between tal / tea and fat / tea was moderate ( r = 0.520 ; p < 3 ) . when comparing variances between the tal / tea and fat / tea , the value was 0.002 , demonstrating that the variance of the tal / tea was significantly smaller than that of the fat / tea and hence signifying that the tal / tea had a relatively more homogenous distribution.fig . the angle between the tal and surgical tea is observed to correlate with the tal and fat ( r = 0.52 ; p < 0.001)fig . 3distribution of the angles between the tal and surgical tea ( tal / tea ) and between the fat and surgical tea ( fat / tea ) . the variability of the fat line and tal line to the clinical tea was different ( = 0.002 ) relationships between tal / tea and fat / tea . the angle between the tal and surgical tea is observed to correlate with the tal and fat ( r = 0.52 ; p < 0.001 ) distribution of the angles between the tal and surgical tea ( tal / tea ) and between the fat and surgical tea ( fat / tea ) . the variability of the fat line and tal line to the clinical tea was different ( = 0.002 ) the most important finding of the present study was that tal has a relatively more homogenous distribution than fat based on comparison of two novel anatomical references located on anterior femoral cortex for femoral rotation during tka in female patients . fat is internally rotated by 3.4 3.3 with respect to the tal . this study is to our knowledge unprecedented in reporting the relative spatial correlation between the fat and the tal . a number of authors agreed that the tea is the anatomical reference axis for the rotational alignment of the femoral component [ 2 , 3 , 5 , 12 ] , and biomechanical studies have elucidated that the surgical tea falls on the centre of knee rotation . the fat , as in our study , has been reported to be internally rotated by 12.2 3.6 with respect to the clinical tea in previous ct - based studies . in this study , authors did not report the angle between the surgical and clinical tea . however , the condylar twist angle , which is the angle formed by the pca and the clinical tea , was 5.7 2.8 and considering the angle between pca and surgical tea to be around 3 the fat / tea should be 9.2 3.6. this is quite close to what we have obtained in this study , which was 9.5 3.8. the tal was reported to be internally rotated by 7.3 1.8 with respect to the tea in healthy knee joints , which is about 1 more internally rotated than what was obtained in our study ; however , in patients with arthritis , this has been reported to be 5.6 2.3 , which is comparable to our results . the values of fat / tal were fairly homogenously distributed without showing any correlation with either age or the degree of preoperative varus deformity of the knee joint . as confirmed in previous studies , both the fat and the tal showed relatively consistent distribution , providing evidence that these are reliable reference axes that could be applied intraoperatively irrespective of preoperative mad [ 8 , 16 ] . there was a moderate correlation between the angles of tal / tea and fat / tea . this finding reveals key points with regard to anatomy of the anterior aspect of the distal femur ; although the size of the medial and lateral anterior condyle is increased distally , the degree of internal rotation of the anterior surface of the distal femur with respect to the tea remains relatively constant . depending on the rotation of anterior cortical surface where the fat is measured the degree of protrusion of the anterior forefront of the medial and lateral condyles determined . when comparing variances between the fat and the tal , the latter demonstrated a more consistent distribution . the anterior protrusion of the lateral condyle is universally more anterior and bigger than that of the medial condyle , so the distribution of the tal / tea is uniform . however , the shape of the fat tends to be more variable ; the shape of the cortical bone is internally rotated where the fat lies in general with respect to the tea , but recent cadaveric study showed the median surface of the cortical bone may be depressed or protruded , resulting in negative values of the fat with respect to the tea . this may be why the variance of the fat / tea tends to be larger than that of the tal / tea , implying that the tal is more reliable as an indicator of rotational alignment . researchers showed surgeons can use fat as a reliable alternative reference line with a simple apparatus . first of all , as all the individuals involved in this study were korean patients , clinicians should consider ethnic differences . secondly , as this study was based on 3d graphical representation , we were able to find accurate anatomical indices by using axial planes and three - dimensional images with computer simulation . this is due to the fact that the most anteriorly protruding points may change depending on the surgeon s level of view and the degree of knee joint flexion . as all previous studies dealing with tal , as well as ours , were based on ct images [ 16 , 19 ] , it should be validated whether the tal can be measured in a reproducible manner intraoperatively in future studies . thirdly , current study carried limitations as only elderly female patients with varus deformity were included causing restrictions when applying our results to the general population . one recent study showed medial condyles of asian females are relatively larger than those of males . , surgeons can rely on these alternative anatomical references when posterior condylar surface and trochlear groove are worn and distorted . surgeons usually compare the whiteside line , the pca and the tea for more accurate rotational alignment of the femoral component . when one or more of these conventional references are hard to recognize , anterior references such as the fat or the tal can be evaluated . as the tal is located more close to the articular surface , it is more easily accessible than the fat . more soft tissue stripping is required to access the fat , and the result of this study suggests that such additional exposure is not always necessary because the tal is more uniformly distributed and reliable . the fat was internally rotated by 3.4 3.3 with respect to the tal , irrespective of the preoperative mad , and the tal / tea values were more homogenously distributed than those of the fat / tea in elderly female patients . therefore , tal is more reliable as an alternative reference for femoral rotation than fat . such information can be useful for determining rotational alignment of the femoral component when conventional reference axes such as the pca or the ap axis are unclear or largely differ . | purposeaccurate rotational alignment of the femoral component is of vital importance for successful total knee arthroplasty ( tka ) .
two anatomical references located on the anterior femur were recently introduced . to determine which is more reliable reference axis for the femoral component rotation in female patients receiving tka ,
the trochlear anterior line was compared with the femoral anterior tangent line.materials and methodspreoperative computed tomography in 76 patients receiving tka for varus deformity was performed , and the images were reconstructed into three - dimensional models .
the trochlear anterior line was defined as the line connecting the most anterior portion of the lateral and medial femoral condyles and the femoral anterior tangent line as the line parallel to distal anterior femoral surface .
the two angles between these reference axes and the surgical transepicondylar axis ( tea ) in three - dimensional images ( trochlear anterior line / tea , femoral anterior tangent line / tea ) were measured .
the correlation between these two angles was computed .
we investigated to see whether a significant difference in variance existed.resultsthe trochlear anterior line was internally rotated by 6.1 2.5 with respect to tea , whereas the femoral anterior tangent line by 9.5 3.8. the trochlear anterior line was externally rotated by 3.4 3.3 with respect to the femoral anterior tangent line .
there was a significant correlation between the trochlear anterior line / tea and the femoral anterior tangent line / tea.conclusionsthe variance of the trochlear anterior line / tea was significantly smaller than that of the femoral anterior tangent line / tea demonstrating a more consistent distribution . when conventional reference axes such as the posterior condylar axis or the anteroposterior axis are unclear or differ , surgeons can rely on these alternative references .
when trochlear anterior line and femoral anterior tangent line contradicts , the former might be more reliable for the rotational alignment of the femoral component in female patients .
level of evidencecase series with no comparison group , level iv . |
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variable number of hrthle ( oxyphilic ) cells in thyroid aspirates is seen in various non - neoplastic and neoplastic lesions of the thyroid . hyperplastic nodules in the setting of multinodular goiter and hashimoto 's thyroiditis varies in the extent of hrthle cell metaplasia . occasionally thyroid aspirates in these conditions consist exclusively of hrthle cells . in such aspirates , we report one such case of an elderly woman who had clinical features of hyperthyroidism and presented with goiter . a 60-year - old female came to the surgical outpatient unit with complaint of painless midline neck swelling of 3 months duration . she gave history of weight loss , heat intolerance , tremors and palpitation since 15 days . thyroid hormone evaluation revealed hyperthyroid function viz : t3 280 mg / dl , t4 16 g / dl and thyroid - stimulating hormone 0.21m / ml . ultrasonography of the neck showed uneven enlargement of both the lobes of thyroid gland and showed isoechoic to hypoechoic texture . left lobe showed well - defined nodule ( 2.8 cm 1.7 cm ) with mixed echogenicity and without any cavitations or calcification . fine needle aspiration ( fna ) smears from the left thyroid lobe were cellular , hemorrhagic and comprised of hrthle cells arranged in flat sheets and lying singly . hrthle cells showed moderate nuclear pleomorphism , abundant granular cytoplasm and well defined cytoplasmic borders . occasional lymphocyte was seen in hrthle cell sheets [ figure 1a and b , arrows ] . repeat aspiration from right thyroid lobe had low cellular yield and showed small cluster of thyroid follicular cells infiltrated by lymphocytes . elevated anti - thyroid peroxidase antibody titer ( 21 iu / ml ) supported the diagnosis . ( a ) hrthle cells showed moderate nuclear pleomorphism , and few cells showed prominent nucleoli , ( b ) occasional lymphocyte in hrthle cell sheets ( arrow ) , ( leishman stain 400 ) hrthle cell in thyroid aspirates is seen in variety of lesions such as hashimoto 's thyroiditis , multinodular goitre , hrthle cell neoplasms ( adenoma / carcinoma ) , head and neck irradiation , systemic chemotherapy , oncocytic variant of medullary , papillary carcinoma and long - standing graves disease . several studies have reached contradictory conclusions regarding the value of specific cytologic findings in these lesions . a mixture of hrthle cells and normal follicular epithelial cells is more consistent with a hyperplastic nodule . nuclei tend to be more uniform in size in hrthle cell tumors than hashimoto 's thyroiditis . in our case , hrthle cells showed moderate nuclear pleomorphism but normal thyroid follicular cells were not seen in the aspirate . correlated cytology features of thyroid nodules showing predominant hrthle cell component with corresponding histological features in 139 cases . the following 14 cytological features were studied : overall cellularity , cytoarchitecture , percentage of hrthle cells , percentage of single hrthle cells , percentage of follicular cells observed as naked hrthle cell nuclei , background colloid , chronic inflammation , cystic change , transgressing blood vessels , intracytoplasmic lumina , multinucleated hrthle cells , nuclear to cytoplasmic ratio , nuclear pleomorphism / atypia and nucleolar prominence . out of the 14 features , non - macrofollicular architecture , absence of background colloid , absence of chronic inflammation and presence of transgressing blood vessels were statistically significant in predicting hrthle cell neoplasm in 86% of hrthle cell lesions . they found > 90% hrthle cells and > 10% single hrthle cells in cytology smears of hrthle cell neoplasms . in our case , after careful search , occasional lymphocyte was seen lying in hrthle cell sheets which made us to think of hashimoto 's thyroiditis . hyperthyroid function in our case can be explained by the fact that the patient had hashitoxicosis which is a transient hyperthyroid phase of hashimoto 's thyroiditis . observed that some cases of non - neoplastic hrthle cell proliferations in hashimoto 's thyroiditis mimic suspicious / follicular neoplasm of hrthle cell type on cytology . they found the discordance between the predicted and actual risk of malignancy , associated with suspicious or follicular neoplasm of hrthle cell type pattern in patients with hashimoto 's thyroiditis . failure to demonstrate lymphocytes and to appreciate non - neoplastic nature of hrthle cells in cytology smears , often results in misdiagnosis of hrthle cell neoplasm . disorganised and poorly cohesive masses of oxyphilic cells with prominent nucleoli are more indicative of a neoplastic lesion . although prominent nucleoli are commonly seen in hrthle cell neoplasms , they may be seen in non - neoplastic hrthle cell lesions . similar findings were seen in our case . in spite of all these criteria , in many instances , making a clear distinction between neoplastic and nonneoplastic hrthle cell lesions may be difficult . in our case , occasional lymphocyte in hrthle cell sheets and repeat aspiration from the other lobe of the thyroid established the correct diagnosis of hashimoto 's thyroiditis . focal nodular hyperplasia of the hrthle cells in hashimoto 's thyroiditis varies in the extent of hrthle cell metaplasia . careful search of lymphocytes in hrthle cell sheets on fna smears , multiple aspirates , associated clinical findings and ancillary techniques , reduce the diagnostic pitfall and avoides unnecessary surgery . | hrthle cells are seen in a variety of nonneoplastic and neoplastic thyroid gland lesions . number and morphology of hrthle cell vary in thyroid aspirate .
occasionally , thyroid aspirate in focal nodular hrthle cell hyperplasia in hashimoto 's thyroiditis exclusively comprise of hrthle cells and mimics hrthle cell neoplasm .
fine needle aspiration ( fna ) diagnosis in such cases is challenging . a 60-year - old female presented with goiter and clinical features of hyperthyroidism .
fna smears showed hrthle cells arranged in flat sheets and lying singly with occasional lymphocytes in hrthle cell sheets .
repeat aspiration from other site showed lymphocytes , infiltrating the thyroid follicular cells .
we conclude that a careful search of lymphocytes in hrthle cell sheets in cytology smears , multiple aspirates , associated clinical findings and ancillary techniques reduce the diagnostic pitfall and avoid unnecessary surgery . |
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leprosy as a neglected disease has both resulted in , and caused poverty since ancient time . disability and stigma induced by leprosy , which widens the gap between the rich and the poor , is a clear evidence of inequality in health aspect . this inequality is of course a lot more tangible and its stigma is more severe in countries where the disease is less prevalent . despite one hundred years of academic and scientific debates , complete discovery of the causes and ways of transmission mycobacterium lepra is known as one of the nine skin pathogenic mycobacteria with neuro - degenerative complications , which are seen in all continents and races worldwide . according to the who classification , this disease is divided into two main groups of multi bacillary ( mb ) and pauci bacillary ( pb ) . delay in diagnosis , treatment and care as well as disease pathogen and immunological reactions lead to destruction of peripheral nerves and disability . leprosy is known as one of the most important causes of permanent disability in the world , which is mainly due to skin damage , paralysis of face , and hand muscles ( secondary to motor neuron nerve damage ) and damages and scars in the legs ( anesthetic ) , not only results in secondary lesions as wound , shortening , finger erosion and clubbing , keratitis and blindness ( impairment ) , but also it affects individual skills ( disability ) , causes stigma and prohibits social activities ( losing educational and job opportunities , disturbed social relations , physical dependence and poverty ) of the afflicted ( handicap ) . consequently , the patient has to inevitably abandon his place of residence and anonymously start a miserable life with other afflicted ones deprived of shelter and food security ( destitution ) . this dramatic start is an end to an absolute right of a normal , which is withheld from these patients . despite the relationship between leprosy and poverty in the individual , social and national level , there is no association between the level of gdp and human development indices with the new cases of leprosy in different countries , and all types of poverty including starvation , homelessness , more severe disease and death , inaccessibility to treatment and education services , job deprivation and fear from future are seen in a patient came down with leprosy . despite introduction of free of charge multi - drug therapy treatment ( mdt ) in 1981 , the global campaign to eliminate leprosy in 1991 , who 's announcement on elimination of the disease in 2001 and 94% reduction in prevalence of the disease , it still has a high - rate of incidence and international efforts have had the least effect on reducing disability and stigmatization caused by leprosy . having failed to control the disease , the who decided to change leprosy elimination strategy to reducing burden of the disease ( 2000 - 2011 ) and continued controlling activities via operational plans and programs ( 2011 - 2015 ) and validated the strategy of reducing burden of the disease by defining the index of equality and social justice as well as necessitating worldwide reduction of grade 2 disability ( g2d ) by at least 35% until 2015 . once the act ( 2008 ) of human right committee of the un makes governments committed to reduce stigmatization of leprosy and preserve dignity of patients and their families , the 2011 - 2015 operational plan would be of paramount importance . the main problem of recovered cases of the disease , in particular the newly affected ones is disability and stigma induced by the disease . this disability is mainly due to delayed diagnosis , which originates from different individual , social and cultural factors as well as accessibility to quality health . in other words , early diagnosis , treatment and appropriate family support help treat and improve a large number of patients without disability . several studies in china , bangladesh and ethiopia have revealed that grade of disability is associated with delayed diagnosis . unequal distribution of disability in these patients is indicative of inequality of this outcome among patients affected with leprosy . although underlying determinants of health are multi factorial , they are unfair and undesirable and except for biological variations , the remaining ones are avoidable and amendable . to bridge the gap between different social groups which have avoidable difference in health outcomes , a systemic approach should be taken to amend and reduce them . to reduce health inequality , numerous researches have been carried out on clinical , basic , health care , disability , rehabilitation and leprosy stigma worldwide . but very few research studies have been designed to investigate health inequality regarding gender , place of residence and disease stigma . in these studies novel inequality indices , which have been introduced and applied by the world bank over the past 10 years , were not used very well . this study is intended to assess inequality of disability in patients with leprosy in the province of west azerbaijan located in the north west of iran , by extended concentration index method and achievement index method which are introduced by mr . adam wagstaff to determine effective factors on incidence of this inequality ( decomposition approach ) . this was an observational cross - sectional study ( 2006 - 2007 ) on patients with leprosy ( under treatment and terminated period of treatment ) in the province of west azerbaijan in the west north of iran . these patients were examined according to a set of instructions by four trained general practitioners and their individual and health history , clinical examination ( visual , sensory and motor neuron ) , evaluation of peripheral nerves and grade of damage and deformities as well as socio - economic determinants were checked and recorded . in this study , cases of leprosy , which were either under treatment or terminated treatment had been registered in health care system of provincial health centers , which were under full coverage due to disease diagnosis system and exclusiveness of medication for leprosy . the outcome was to consider presence or absence of g2d based on the who classifications . according to the who definition , any type of observable deformity in one of the upper or lower limbs and impaired vision , including loss of vision less than 6/10 or inability to count fingers in 6 m distance or lagophthalmos independent variables in this study consist of socio - economic status ( ses ) , age , gender , education , method of diagnosis , grade of the disease , history of bcg vaccine , history of mdt , and place of residence . having considered initial arrangements and permission , data of the study were collected in the cases living place or work place ( rarely ) . of 23 considered variables the followings were used to develop ses index in principle component analysis ( pca ) : level of education , housing tenure ( ownership , rental , ownership transfer ) and condition , number of family in a household , number of bed rooms , number of family members per room , fuel supply , light and electricity supply , sanitary toilet and bathroom , receiving donations from others and charity foundations . then each case was classified according to the 5 groups to be utilized in the model . concentration index ( ci ) was applied to evaluate and measure socio - economic inequality in g2d of patients with leprosy . this is a popular approach to evaluate health inequalities , which due to demonstrating aspects of socio - economic inequality , evaluating inequality in all socio - economic groups and its sensitivity to manner of population distribution in social groups of community is unanimously accepted and approved . the estimated grade of disability by ci was not a real number , just an ordinal scale , based on which it is not possible to compare difference or relevance between two values of the index . for instance , ci interpretation in case of a dentist visit in austria is 0.015 , but in portugal it is 0.259 . the only thing to be said here is that inequality in austria is less than that in portugal , but it can not be concluded that inequality in the latter is 20 times more than the former . with regard to changing approach from elimination and eradication to controlling or reducing health hazards , extended ci was introduced and applied first by adam wag staff in 2002 in order to resolve interpretation limitations and provide the possibility of transparent and clear judgment based on ci index . inequality aversion and its numeric value depend on the manner of considered variable distribution . giving an equal and balanced weight to individuals regardless of their ses is indicative of neutral attitude towards inequality , while giving more weight to individuals in lower ses is suggestive of aversion attitude towards inequality . the most important and practical features of extended ci are as follows : possibility of numerical comparison of health inequality in different conditions regarding time and place is provided . the obscure and vague judgment and interpretation is converted to a clear and transparent one , which is completely numerical and helps modify authorities attitude towards the issue of inequality . scale of inequality aversion per different values for v ( inequality aversion coefficient ) can be estimated and possibility of accurate monitoring and observation is provided . according to inevitability of inequality in majority of health outcomes due to environmental reasons and individual features , possibility of reducing inequality with regard to supplies and resources is provided and it helps prioritize health programs to reduce inequalities in general . decomposing the ci into its determinant factors ( decomposing approach ) : despite the use of ci and expanded ci indices in evaluation of socio - economic inequality and performance of socio - economic systems regarding distribution of health related variables along with designing managerial interventions and determining the gap between the rich and the poor and modification of health system in general , developing operational interventions and objectives necessitates taking measures to identify determinants of the existing inequality . decomposition is based on identifying factors , which cause inequality among different socio - economic sectors in terms of the outcome variable ( leprosy - induced disability ) . to identify important and influential factors on incidence of disability ( outcome ) , the ci is decomposed and different effective factors which play a role in causing inequality are determined and share of each is estimated , then manner of distribution of the very factor or its effect on disability is evaluated , based on which by prioritizing effective factors , objective interventions would be designed and developed . although , decomposition in the field of health was first applied for continuous outcome variable by use of regression ( ordinary least square [ ols ] ) , due to the widespread use of binary variables in health studies and inconsistency between ols and binary data , the probit model was introduced and later with some simple modifications another model was introduced and practiced by dr . hossein pour et al . , which is called logit model . in this study , linear regression model is applied , which links the considered health variable ( y ) to a group of independent variables , which is also recommended by wag staff . to determine proportion of each effective factor in disability inequality induced by leprosy , first correlation of each factor with the variable of outcome should be calculated by use of regression models , and then they obtained regression equation is substituted in the main equation for ci . first , the ci is calculated for each independent variable and residuals and their equivalences are inserted in the regression equation , the result will be the overall regression equation . methodological stages of decomposition of socio - economic inequality of health outcome into its determinants :
to regress the certain outcome versus deter - minants to determine independent variables coefficient by use of a proper regression modelto estimate the mean of the outcome and determinantsto calculate the ci of the outcome and determinants ( c , ck)to calculate elasticity ( multiplying the mean of each determinant by the relevant coefficient and dividing it by the mean of the total outcome)to determine proportion of each determinant entered the model by use of the formulato calculate percentage of proportion of each determinant by dividing the proportion of each determinant by the total ci . to regress the certain outcome versus deter - minants to determine independent variables coefficient by use of a proper regression model to estimate the mean of the outcome and determinants to calculate the ci of the outcome and determinants ( c , ck ) to calculate elasticity ( multiplying the mean of each determinant by the relevant coefficient and dividing it by the mean of the total outcome ) to determine proportion of each determinant entered the model by use of the formula to calculate percentage of proportion of each determinant by dividing the proportion of each determinant by the total ci . it is noteworthy that the ci of the dependent variable , which is obtained in the linear regression model , will be different from the numerical value of the ci of the study due to the predictive nature of regression . in case it is used , the error term is indicative of presence of other unknown factors . calculation of the achievement index : to evaluate effectiveness of health care systems , mean of health level had been applied until the year 2000 . since a health care system may have a successful performance in one aspect but a weak one in another , the overall attainment index was vaguely used in the global health report in 2000 , but in 2 year - time ( 2002 ) its calculation approach was introduced and has been applied since then . this overall attainment index consists of 5 overall health criteria and its distribution , overall level of responsiveness and its distribution , distribution of cooperation in supply of financial resources and combination of goodness ( the best attainable mean level ) and fairness ( the least degree of difference among individuals and groups ) . this index calculates mean and inequality in a certain level ( v ) of health in the form of a combined assessment with giving a balanced weight to the mean of health outcome . concentration index ( ci ) was applied to evaluate and measure socio - economic inequality in g2d of patients with leprosy . this is a popular approach to evaluate health inequalities , which due to demonstrating aspects of socio - economic inequality , evaluating inequality in all socio - economic groups and its sensitivity to manner of population distribution in social groups of community is unanimously accepted and approved . the estimated grade of disability by ci was not a real number , just an ordinal scale , based on which it is not possible to compare difference or relevance between two values of the index . for instance , ci interpretation in case of a dentist visit in austria is 0.015 , but in portugal it is 0.259 . the only thing to be said here is that inequality in austria is less than that in portugal , but it can not be concluded that inequality in the latter is 20 times more than the former . with regard to changing approach from elimination and eradication to controlling or reducing health hazards , extended ci was introduced and applied first by adam wag staff in 2002 in order to resolve interpretation limitations and provide the possibility of transparent and clear judgment based on ci index . inequality aversion and its numeric value depend on the manner of considered variable distribution . giving an equal and balanced weight to individuals regardless of their ses is indicative of neutral attitude towards inequality , while giving more weight to individuals in lower ses is suggestive of aversion attitude towards inequality . the most important and practical features of extended ci are as follows : possibility of numerical comparison of health inequality in different conditions regarding time and place is provided . the obscure and vague judgment and interpretation is converted to a clear and transparent one , which is completely numerical and helps modify authorities attitude towards the issue of inequality . scale of inequality aversion per different values for v ( inequality aversion coefficient ) can be estimated and possibility of accurate monitoring and observation is provided . according to inevitability of inequality in majority of health outcomes due to environmental reasons and individual features , possibility of reducing inequality with regard to supplies and resources is provided and it helps prioritize health programs to reduce inequalities in general . decomposing the ci into its determinant factors ( decomposing approach ) : despite the use of ci and expanded ci indices in evaluation of socio - economic inequality and performance of socio - economic systems regarding distribution of health related variables along with designing managerial interventions and determining the gap between the rich and the poor and modification of health system in general , developing operational interventions and objectives necessitates taking measures to identify determinants of the existing inequality . decomposition is based on identifying factors , which cause inequality among different socio - economic sectors in terms of the outcome variable ( leprosy - induced disability ) . to identify important and influential factors on incidence of disability ( outcome ) , the ci is decomposed and different effective factors which play a role in causing inequality are determined and share of each is estimated , then manner of distribution of the very factor or its effect on disability is evaluated , based on which by prioritizing effective factors , objective interventions would be designed and developed . although , decomposition in the field of health was first applied for continuous outcome variable by use of regression ( ordinary least square [ ols ] ) , due to the widespread use of binary variables in health studies and inconsistency between ols and binary data , the probit model was introduced and later with some simple modifications another model was introduced and practiced by dr . in this study , linear regression model is applied , which links the considered health variable ( y ) to a group of independent variables , which is also recommended by wag staff . to determine proportion of each effective factor in disability inequality induced by leprosy , first correlation of each factor with the variable of outcome should be calculated by use of regression models , and then they obtained regression equation is substituted in the main equation for ci . first , the ci is calculated for each independent variable and residuals and their equivalences are inserted in the regression equation , the result will be the overall regression equation . methodological stages of decomposition of socio - economic inequality of health outcome into its determinants :
to regress the certain outcome versus deter - minants to determine independent variables coefficient by use of a proper regression modelto estimate the mean of the outcome and determinantsto calculate the ci of the outcome and determinants ( c , ck)to calculate elasticity ( multiplying the mean of each determinant by the relevant coefficient and dividing it by the mean of the total outcome)to determine proportion of each determinant entered the model by use of the formulato calculate percentage of proportion of each determinant by dividing the proportion of each determinant by the total ci . to regress the certain outcome versus deter - minants to determine independent variables coefficient by use of a proper regression model to estimate the mean of the outcome and determinants to calculate the ci of the outcome and determinants ( c , ck ) to calculate elasticity ( multiplying the mean of each determinant by the relevant coefficient and dividing it by the mean of the total outcome ) to determine proportion of each determinant entered the model by use of the formula to calculate percentage of proportion of each determinant by dividing the proportion of each determinant by the total ci . it is noteworthy that the ci of the dependent variable , which is obtained in the linear regression model , will be different from the numerical value of the ci of the study due to the predictive nature of regression . in case it is used , the error term is indicative of presence of other unknown factors . calculation of the achievement index : to evaluate effectiveness of health care systems , mean of health level had been applied until the year 2000 . since a health care system may have a successful performance in one aspect but a weak one in another , the overall attainment index was vaguely used in the global health report in 2000 , but in 2 year - time ( 2002 ) its calculation approach was introduced and has been applied since then . this overall attainment index consists of 5 overall health criteria and its distribution , overall level of responsiveness and its distribution , distribution of cooperation in supply of financial resources and combination of goodness ( the best attainable mean level ) and fairness ( the least degree of difference among individuals and groups ) . this index calculates mean and inequality in a certain level ( v ) of health in the form of a combined assessment with giving a balanced weight to the mean of health outcome . as it is shown in table 1 , majority of patients were male ( 65.3% ) and 67% were affected by the mb type . the g2d in limbs , eyes and overall g2d is 58% , 36.3% and 65.3% respectively . almost half of the male patients ( 45.7% ) had lost their jobs due to disability and about half of all patients ( 46.5% ) are financially dependent on donations . table 2 depicts the association between g2d and its risk factors by two modes of crude and adjusted analyses ( univariate analysis ) and ( multivariate analysis ) in logit model ( ci : 95% ) . in this study a significant association was found between age at disease onset , education , bcg vaccine , the interval from clinical manifestation to diagnosis and g2d . the estimated ci in table 3 shows that socio - economic inequality of g2d in leprosy affected patients was 0.0782 , which is indicative of pro - poor inequality . extended ci was applied in order to gain a better insight into the real nature of inequality . increased value of v from 2 ( standard ci ) to 5 results in extension of ci of g2d from 0.0782 to 0.163 , which is suggestive of the difference of the g2d between the most deprived and the poorest stratum more than two times in comparison with other socio - economic strata ( the more negative the number , the more weight given to the most deprived and the poorest strata ) . leprosy affected patients demographic and clinical characteristics ( current situation ) , w. azerbaijan province in iran 2007 demographic and epidemiologic risk factors for disability grade ii in leprosy affected in west azerbaijan province in iran 2007 summary statistics of extended concentration index and achievement index for different degree of inequality aversion in leprosy affected in west azerbaijan province in iran 2007 achievement index is also included in table 3 . coefficient one ( of these indexes ) is indicative of the mean of the numerical value of g2d which by increase of the number of coefficient to 5 , it leads to a 16% rise in the mean value . in other words , mean value of disability rises among them by giving more weight to the poor , which is an evident indication of pro - poor inequality and somewhat rise in dis achievement index . the result of decomposition of the existing inequality in leprosy g2d was indicative of unequal and unfair determinant distribution of dependent variable . this analysis revealed that on the one hand , some determinants were unequally and unfairly distributed ( ci for each explanatory variable ) , including urbanization , mdt history , mb leprosy affected , education , female gender , bcg vaccine , prolonged and consistent period of treatment with dapsone drug , on the other hand , some of the determinants such as receiving monotherapy , education , urbanization , and bcg vaccination had stronger association with disability ( the last two columns of table 4 ) . very low proportion and effect of gender in disability ci was suggestive of relative independence of disability from gender in this investigation and this unequal distribution is an evident reason for defective health care system in leprosy affected patient detection among women . type of patient detection not only had a low cik , but also smaller proportion in the total ci disability . also elasticity of each factor in relation with the proportion of that factor had an important effect on the total ci . in other words , the most significant and the largest proportion of inequality concerns with having education or not and receiving bcg vaccine or not . the objective of this study was to measure inequality of g2d in leprosy affected patients . this assessment first of all revealed presence of inequality in leprosy induced disability and second , objective measurement of the numerical value of inequality was made possible through this approach . besides , result of this assessment as a proxy for quality of health care in leprosy showed that there is a problem with fair accessibility to leprosy health care services in the health network . moreover , decomposing approach provided the possibility to relatively measure the source of inequality of leprosy induced disability and determine share of each numerically . results of this study added new evidences to the previously existing data regarding the role of risk factors of leprosy induced disability . most of the current studies evaluated effective factors in incidence of disability , and they provide descriptive epidemiologic and sometimes analytical information regarding the disease , disability and some risk factors along with preventive measures . besides , they highly advise early diagnosis , treatment and drug - induced complication treatment / treating adverse effects of drugs in all patients ( all age groups and both genders ) in order to prevent disability . as a result , outcomes of such studies do not provide health authorities with clear and straightforward objectives and strategies to be developed and planned . therefore , the result of these studies ( above ) does not help authorities to prioritize strategies and measures based on the existing resources , in order to detect different social strata and susceptible patients and develop necessary interventions , nor estimate the rate of success in accomplishing each goal and objective they have already set . moreover , drawing necessary comparisons between different countries or in one region during several years , based on defined indices , has not been made possible by these current investigations . the applied approach in this study could numerically clarify and assess all of these components . results of this study on the one hand , guide health authorities from their previous superficial attitudes regarding health care management ( e.g. , mean values ) towards deeper and more sophisticated levels and bridge the gap between the poor and the rich by means of objective management of resources and on the other hand , guarantee fulfilling equity indices in health issues , in particular lowering disparity in this regard . this study showed that low ses is proportional to illiteracy , female gender , living in rural area , affected spouse and risk of the disease in higher age groups and it is directly related with g2d . despite clear evidences regarding inequality in leprosy among socio - economic strata due to different reasons , results of this study reveal that among evaluated leprosy affected patients ( prevalence ) mean of g2d is much higher ( 65% ) in comparison with the who index ( 10% ) , the emro index ( 22% ) and even the national mean ( 55% ) . decomposition approach could estimate source of existing inequality in the form of demographic , socio - economic determinants , risk - factors and effective factors in health system . as it is expected , these factors do not play an equal role in inequality , since both prevalence of determinants in incidence of disability and degree of association ( between determinants ) are different . rather small sample size , recall bias due to the gap between the study and incidence , diagnosis and treatment of the disease and studying cases of prevalence . since there was no non - response in this study , we did not face bias selection either and due to use of patients recorded files , recall bias reached the lowest . effective factors in disability , which are identified in decomposition analysis , are the ones whose roles in majority of studies are evaluated and examined . in this present study , proportion of gender , living place and type of the disease in disability were not very tangible , each of which will be explained in detail . although it seems that male cases of mb leprosy are two times more than women and cases of pb are equal in number between the two , considering socio - economic , cultural and physiological reasons women have less access to and utilize social services ( as health ) , in different studies and evaluations the ratio of detected male patients to female ( m / f ) for instance in yemen is more than three time and g2d is 40/6 ( 41% ) and in nigeria and brazil this ratio is quite similar between males and females and g2d in males ( 23% ) is one and a half times more than that in females . in this study , ratio of patients and their disability is 1.9 ( male to female ) , which could be due to women 's relative access to health services and better coverage of health services in their case . higher prevalence in urban areas ( in contrast with higher rate of incidence in rural areas ) and accommodation in slum areas lead to polarization and consistent risk of disease transmission due to higher population density and increased exposure in family colonies or wanderers . therefore , besides increased rate of poverty in urban areas , accessibility to health care services is influenced and present inequalities become more widespread.[16
171819202122232425262728293031323334353637383940 ] similarly , cases of leprosy in iran are seen more in rural areas , but at present more than 71% of cases of this study are located in margins of urban places ( slums ) and in the studied geographic area no significant difference is seen between urban and rural living places , which could be indicative of low - quality health care in both rural and urban regions ( in contrast with what is expected ) . passive case detection in conducted studies determines low - rate of disability . in this study , although more than 30% of diagnosis is estimated to be passive ( with possibility of error ) , most of the patients were detected actively and also via periodical case detection and close contact , which itself explains high - rate of disability . inequality in social affairs is undesirable , which not only weakens social solidarity but also has an adverse effect on individuals efficiency . however , consequences of inequality are more important and influential in the field of health in comparison with others . nowadays , inequality in health has become a global challenge within and between countries and health authorities and policy makers along with the international organizations including the who insist on reducing health inequalities , which is also officially accepted by governments . moreover , governments are committed to explore causes and routes of health inequality and take effective measures and develop essential plans to reduce it accordingly . leprosy induced disability is a sign of inequality in the field of health , which has a route both in public health care system and effective social determinants of health . this disease affects social life of patients , in particular in non - endemic countries where self - stigma , shame , and secrecy is higher , which in fact leads to patients deprivation of their social rights and ultimately isolates them from the society and causes delayed diagnosis and treatment , which eventually results in increased incidence of disability and consistent disease transmission . the arisen misunderstanding among managers of health systems in majority of countries regarding the concept of elimination has resulted in suspension of controlling measures and supplies are shifted into other neglected tropical diseases , and even this misunderstanding has led to inequality in scientific research on leprosy . the assessment method applied in this study provides new opportunities for the health system to control leprosy in order to take effective measures and develop consistent strategies to determine numerical mean of g2d and the possible gap between the rich and the poor , presence or absence of inequality and determine its numerical value ( ci ) , estimate the present gap between the rich and the poor ( expanded ci ) , determine effective elements in incidence of g2d in socio - economic strata ( decomposition ) which differs from country to country . hence , determining the scale or value of inequality aversion based on existing resources , monitoring and accurate observation will be made possible and the reduced scale of g2d inequality will be classified . once similar studies and projects are implemented in different countries during several years , drawing comparison and evaluating success or failure would be realized . it is recommended that data of the study be collected simultaneously with diagnosis , onset of treatment and care ( longitudinal studies ) in order to carry out more accurate studies and evaluations . as a result of this , more novel knowledge will be generated regarding disease management which will also provide the possibility for international evaluations . this disease has initially and originally been in the stage of elimination and majority of patients are detected in the northern part , especially the east and the west north of the country . given the iranian epidemiologic characteristics of the disease , we can refer to smaller number of female patients , reduced female case detection over the past years and variable percentage of detected patients in relation with their living place . cases of g2d have risen from 20% in 2000 to more than 55% during the past 10 years ) . | background : despite significant reduction in global disease prevalence , leprosy still has a high rate of disability while its determinants are unfair and many of them are amendable .
the objective of this study was to measure inequality of disability in leprosy in iran.methods:this was a cross - sectional study ( 2006 - 2007 ) on all living people affected by leprosy registered in w. azerbaijan province health center , western north of iran .
the outcome of the study was the socio - economic inequality considering presence or absence of grade 2 disability ( g2d ) based on the who classifications .
an extended concentration index decomposition approach was used for analysis.results:among 452 cases , 65.3% were male and 67% were affected by the multi bacillary type .
overall g2d was 65.3% .
the estimated concentration index was 0.0782 , showing presence of pro - poor socio - economic inequality of g2d , while extended ci estimation ( = 5 ) was 0.163 .
achievement index with coefficient ( = 5 ) revealed that g2d mean was 16% more than classic mean in the poorest group .
the result of decomposition of the existing inequality revealed that , some of the determinants such as receiving mono - therapy , education , urbanization , and bacillus calmette guerin ( bcg ) vaccination had shared contribution ( 67.4% , 61.8% , 59.2% , and 57.5% respectively).conclusions : this study provided new perspective for the health system to leprosy control considering the significant gap between rich and poor ( inequality ) regarding g2d disability , and its effective elements in socio - economic strata .
some effective actions can be considered to reduce the scale of existing inequality . |
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diabetes mellitus ( dm ) is a hormonal disease characterized by changes in carbohydrate , protein , and lipid metabolisms . the main feature of diabetes is an increase in blood glucose levels ( hyperglycemia ) , which results from either a defect in insulin secretion from the pancreas , change in insulin action , or both . type 1 dm includes diabetes resulting primarily from destruction of the beta cells in the islets of langerhans of the pancreas which often leads to absolute insulin deficiency . the onset of the disease is often abrupt , and patients with this type of diabetes are more prone to ketoacidosis with wide fluctuations in plasma glucose levels . the causes of type 2 dm range from insulin resistance accompanied by relative insulin deficiency to a predominantly secretory defect with insulin resistance . its onset is generally more gradual than for type 1 , and this condition is often associated with obesity . type 2 diabetes also carries a strong genetic component , with the disease being more common in north americans of african descent , hispanics , and aboriginal people . gestational diabetes mellitus ( gdm ) is a condition in which glucose intolerance begins during pregnancy . the children of mothers with gdm are at greater risk of experiencing obesity and diabetes as young adults as well , there is a greater risk of the mother of developing type 2 diabetes in the future . all the forms of dm are associated with hyperglycemia , hyperlipidemia , and associated complications . the five classic major complications of diabetes include microangiopathy , nephropathy , neuropathy , macrovascular disease , and delayed wound healing . periodontal disease is a chronic inflammatory disease which represents a primarily anaerobic gram - negative oral infection that results in gingival inflammation , loss of attachment , bone destruction , and eventually the loss of teeth in severe cases . certain organisms within the microbial flora of dental plaque are the major etiologic agents of periodontitis which produce endotoxins in the form of lipopolysaccharides ( lps ) that are instrumental in generating a host - mediated tissue destructive immune response . recent studies have warranted a change in the traditional paradigm that periodontitis is an oral disease and that the tissue destructive response remains localized within the periodontium , limiting effects of the disease to oral tissues supporting the teeth . these studies have indicated that periodontitis may produce a number of alterations in systemic health , hence proving its association with various systemic diseases or conditions including diabetes . diabetes has many adverse effects on the periodontium , including decreased collagen turnover , impaired neutrophil function , and increased periodontal destruction . with respect to the periodontal microflora , no appreciable differences in the sites of periodontal disease have been found between diabetic and non - diabetic subjects . a great deal of attention has been directed to potential differences in the immunomodulatory responses to bacteria between diabetic and non - diabetic subjects . neutrophil chemotaxis and phagocytic activities are compromised in diabetic patients , which can lead to reduced bacterial killing and enhanced periodontal destruction . various studies have revealed levels of the acute - phase reactants like fibrinogen and c - reactive protein ( crp ) to be higher in people with insulin resistance and obesity . a majority of clinical and epidemiological studies give evidence to demonstrate that individuals with diabetes ( type 1 and type 2 ) tend to have a higher prevalence and more severe / rapidly progressing forms of periodontitis than non - diabetics . traditionally , research concerning the relationship between diabetes and periodontitis had focused on vascular changes in the periodontal tissues ( gingival microangiopathy ) , granulocyte hypo function , increased tissue labiality resulting from reduced collagen production / enhanced gingival collagenase activity , and changes in oral microflora . these studies although provided useful information concerning basic changes at the local level , did not reflect on the possible primary systemic relationships between diabetes and periodontitis . latest investigations performed at the cellular / molecular level demonstrate common changes in systemic physiology and have thus provided preliminary evidence of potential mechanisms responsible for the witnessed associations [ figure 1 ] . these include diabetes - induced alterations of immune cell phenotype and elevation of serum proinflammatory cytokine / lipid levels . in recent times , some studies have demonstrated that periodontitis itself can produce these same alterations , and in the presence of diabetes , produces exacerbation of these detrimental changes . potential mechanistic links in the bidirectional interrelationship between diabetes and periodontal disease a potential mechanistic link involves the broad axis of inflammation , specifically immune cell phenotype , serum lipid levels , and tissue homeostasis . obesity is a leading cause of insulin resistance and serves as a medium leading to deterioration of both conditions of diabetes and periodontitis . adipocytes , once assumed to be just fat storage cells , are metabolically actively involved in the production of a wide range of molecules called adipokines . these adipocytes modulate levels of insulin and insulin sensitivity , any alteration of the same results in clinical conditions of diabetes . two types of adipokines exist , one which are synthesized by adipocytes in increased levels during metabolic disorders ( e.g. , tumor necrosis factor- , interleukin-6 , crp ) , better known as cytokines with well - defined roles in inflammation and immunity . others ( e.g. , leptin , adiponectin , resistin , visfatin ) which are involved in regulation of energy expenditure . leptin and adiponectin have an insulin - sensitizing effect and are protective against type 2 diabetes . adiponectin inhibits tumor necrosis factor- ( tnf- ) and inhibits transformation of monocytes to foam cells , and causes downregulation of proinflammatory cytokines and upregulates the synthesis of interleukin-10 ( il-10 ) . crp , an acute phase protein is secreted in small amounts from human adipocytes , upregulates proinflammatory action of other inflammatory mediators such as plasminogen activator inhibitor-1 , and also directly contributes to atheroma formation . diabetes - induced changes in immune cell function produce an upregulation of proinflammatory cytokines from monocytes / polymorphnuclear leukocytes ( pmn ) and downregulation of growth factors from macrophages . this predisposes to chronic inflammation , progressive tissue breakdown , and diminished tissue repair capacity . periodontal tissues frequently manifest these tissue breakdown changes as they are constantly injured by endotoxins originating from bacterial biofilms . diabetic patients being prone to hyperglycemia , thus hyperlipidemia is of high significance , as recent studies demonstrate that hyperlipidemia may be one of the factors associated with diabetes - induced immune cell alterations which results in further deterioration of periodontal conditions in these patients . evidence now proves that tissue destruction associated with periodontitis is due to release of proinflammatory cytokines and immune cell response to lipopolysaccharide and other metabolites of the local bacterial flora . the most convincing evidence of destruction by proinflammatory cytokines implicates the role of interleukin-1 beta ( il-1 ) and tnf-. it is believed that il-1 recruits inflammatory cells , facilitates polymorphonuclear leukocyte preparing / degranulation , increases synthesis of inflammatory mediators ( prostaglandins)/matrix metalloproteinases ( mmp ) , inhibits collagen synthesis , and activates both t and b lymphocytes . tnf- is a major signal for cellular apoptosis , bone resorption , mmp secretion , intercellular adhesion molecule ( icam ) expression , and interleukin-6 ( il-6 ) production . il-6 , once produced , stimulates formation of osteoclasts , promotes osteoclastic bone resorption , and facilitates t - cell differentiation . these cytokines are believed to exert a further effect on lipid metabolism by influencing the production of other cytokines , altering hemodynamics / amino acid utilization of various tissues involved in lipid metabolism or modifying hypothalamic - pituitary - adrenal axis , increasing plasma production of adrenocorticotropic hormone , cortisol , adrenaline , noradrenaline , and glucagon . in both type l and type 2 diabetes , the hyperlipidemia often manifests marked elevations of low - density lipoprotein ( ldl)/triglycerides ( trg ) and omega-6 free fatty acids . these serum lipid abnormalities result due to disruption of fatty acid metabolism and accumulation of omega-6 polyunsaturated fatty acids that contribute to formation of ldl / trg . the conversion of omega-6 polyunsaturated essential fatty acids to active metabolites , which are key components of cell membrane structure , is impaired . increasing evidence suggests that lipid composition of membranes is a critical factor influencing cellular function . the physical / chemical properties of membranes are largely determined by the nature of fatty acids within the phospholipid bilayer affecting receptor responses and operation of membrane - bound enzyme systems . it has been confirmed that diabetes - induced changes in membrane fluidity modulate function of membrane proteins potentially impairing cellular function / homeostasis . thus , in contrast to previous dogma concerning hyperglycemia , abnormal fatty acid metabolism and hyperlipidemia are also thought to be responsible for impairments in a variety of cell types and development of some diabetic complications . the function of inflammatory cells , such as neutrophils , monocytes , and macrophages , is altered in diabetic patients . this hyperresponsive monocytic phenotype is not associated with hyperglycemia can exist independently of periodontitis and may be related to hyperlipidemia . others postulate a genetic basis in the hla - dr and hla - dq gene regions and/or polymorphisms in the promoter regions of cytokine genes . using an animal study , sakallioglu et al . stated increased levels of monocyte chemoattractant protein ( mcp)-1 in gingival tissues of diabetic rats without periodontitis as compared to non - diabetic rats with periodontitis . monocytes play a significant part in periodontal tissue breakdown and are present in a greater concentration in patients with periodontitis . these cells exhibit enhanced mcp-1 expression in periodontal tissues , and raised levels of mcp-1 levels have been reported in diabetic patients compared with healthy controls . local and systemic hyper - responsiveness of these monocytes leads to increased tnf- levels in gingival crevicular fluid ( gcf ) . the pentose phosphate pathway is contributory in the formation of nicotinamide adenine dinucleotide phosphatase ( nadph ) and ribose-5-phosphate for fatty acid , and nucleotide synthesis , respectively . nadph is important for nadph oxidase activity and for the rejuvenation of glutathione in neutrophils , and activation of nadph oxidase results in a respiratory burst in neutrophils during the process of phagocytosis . there is a body of evidence suggesting that nadph oxidases play a major role in the pathogenesis of inflammation , hypertrophy , endothelial dysfunction , apoptosis , migration , and remodeling in hypertension , angiogenesis , and type 2 dm . in diabetic patients , glucose-6-phosphate dehydrogenase ( g6pdh ) converts glucose-6-phosphate into 6-phosphoglucono--lactone and is the rate - limiting enzyme in the pentose phosphate pathway . g6pdh activity has been found to be considerably decreased in neutrophils , macrophages , and lymphocytes isolated from diabetic rats . these findings suggest that the pentose phosphate pathway is downregulated in neutrophils from diabetic rats . in neutrophils in which g6pdh activity is deficient , phagocytosis , bactericidal ability , and superoxide production are impaired . glutamine is a cellular energy source next to glucose and both are necessary for lymphocyte function . glutamine is involved in protein , lipid , and nucleotide syntheses , as well as in nadph oxidase activity . glutamine increases bacterial killing activity in vitro , as well as the rate of reactive oxygen species ( ros ) production by neutrophils and inhibits spontaneous neutrophil apoptosis . therefore , decreased glutamine utilization may contribute to impaired neutrophil function in diabetes as a result of increased apoptosis . glutamine oxidation and glutaminase activity glutamine is also necessary for the provision of glutamate for glutathione synthesis , which is an antioxidant involved in preventing damage to important cellular components caused by reactive oxygen species such as free radicals and peroxides . in healthy subjects , glucose intake results in increased intranuclear nuclear factor ( nf)-b binding , decreased ib levels , increased ib kinase ( ikk ) activity , increased expression of ikk and ikk enzymes , and increased tnf- mrna expression in mononuclear cells ( mncs ) . these changes are harmonious with an increase in oxidative load in the mncs after glucose intake and thus trigger pro - inflammatory changes in the mncs . many cross - sectional studies have demonstrated hyper - reactivity of peripheral blood neutrophils in chronic periodontitis . other ros and reactive nitrogen species ( rns ) arise from superoxide and are termed secondary ros and rns . these free radicals derived from the mitochondrial cellular membrane , nucleus , lysosomes , peroxisomes , endoplasmic reticulum , and cytoplasm are unstable , either donating unpaired electrons to other cellular molecules or extracting electrons from other molecules in order to achieve a stable milieu . in low to moderate concentrations , they serve an important homeostatic function but in high concentrations , they are harmful and may contribute to the pathogenesis of chronic inflammatory diseases . both ros and rns have been reported to be involved in the etiopathogenesis of type 2 dm . evidence proves that oxidative stress is an important factor responsible for local tissue damage in chronic periodontitis . in hyperglycemic individuals , oxidation of circulating ldl leads to increased oxidant stress within the vasculature inducing chemotaxsis of macrophages and monocytes to the vessel wall . this oxidation results in cellular adhesion and increased production of cytokines and growth factors , resulting in stimulation of smooth muscle cell proliferation and causing an increase in the vessel thickness . other changes like increased atheroma formation and microthrombi in large blood vessels , alteration of vascular permeability , and endothelial cell functions in small vessels have also been observed . altered wound healing is one of the most common complications of dm . in a glucose - rich environment , the reparative capacity of periodontal tissues is compromised . the production of collagen and glycosaminoglycans is significantly reduced in high - glucose environments . in diabetic patients , the formation of age begins when glucose attaches to the amino groups on proteins to form an unstable glycated protein ( schiff base ) . eventually , after chemical rearrangement , these glycated proteins are converted to a more stable , yet still reversible , glucose protein complex known as the amadori product . however , if hyperglycemia is sustained , the amadori products become highly stable and form age . thus , even if hyperglycemia is corrected at this stage , the age in the affected tissues does not return to normal . the age thus formed accumulates in the periodontium , causing changes in the cells and extracellular matrix ( ecm ) components . collagen produced by fibroblasts under these conditions is susceptible to rapid degradation by matrix metalloproteinase ( mmp ) enzymes , such as collagenase , the production of which is significantly higher in dm . tissue collagenase is present in an active form in diabetics whereas the latent form is seen in non - diabetic subjects . in poorly controlled diabetic patients , collagen becomes cross - linked , resulting in a marked reduction of solubility . at the ultrastructural level , collagen homeostasis and turnover is altered . age has an adverse effect on bone collagen at the cellular level and this may result in alterations in bone metabolism . some studies have reported significant levels of osteoclasts and increased osteoclast activity in diabetic patients , whereas others have reported decreased bone resorption under similar conditions . circulating ldl becomes cross - linked to this age - modified collagen and contributes to atheroma formation in the diabetic macrovasculature . in central and peripheral arteries , this enhances the macrovascular complications of diabetes . in smaller vessels , collagen in the vessels can lead to increased basement membrane thickness and compromised transport of nutrients across the membrane . the surface of smooth muscle cells , endothelial cells , neurons , macrophages , and monocytes expresses the receptor for age ( rage ) . ages can then bind to rage , leading to further complications such as the development of vascular lesions , increased vascular permeability , increased expression of adhesion molecules , and increased migration and activation of monocytes . these activated monocytes adhere to vascular endothelium via adhesion molecules like intercellular adhesion molecule ( icam-1 ) , endothelial leukocyte adhesion molecule ( elam-1 ) , and vascular cell adhesion molecule ( vcam-1 ) . these monocytes then penetrate the endothelium and migrate under intima layer where they ingest ldl in an oxidized state and become foam cells which are characteristic of atheromatous plaque . once within the arterial media , these monocytes transform to macrophages releasing an array of proinflammatory cytokines and mitogenic factors causing muscle and collagen proliferation leading to thickening of the vessel walls . the effect on the endothelial cells is an increase in vascular permeability and thrombus formation . the age - rage interaction on smooth muscle cells results in cellular proliferation within the arterial wall . as ages are chemotactic for monocytes , age - rage interaction induces increased cellular oxidant stress and activates the transcription factor nuclear factor kappa - beta ( nfkb ) on monocytes . this then alters the phenotype of the monocyte / macrophage and results in increased production of proinflammatory cytokines and growth factors such as interleukin-1 ( il - i ) , tnf- , platelet - derived growth factor ( pdgf ) , and insulin - like growth factor ( igf ) . all these cytokines and growth factors have been shown to contribute to the chronic inflammatory process in the formation of atheromatous lesions . in addition , oxidized ldl , elevated in many diabetic patients , also activates nfkb and may result in similar processes . thus , alterations in lipid and protein metabolisms induced by the sustained hyperglycemia characteristic of diabetes may play a major role and provide a common link between all the classic complications of this disease . in addition , age can stimulate increased production of vascular endothelial growth factor ( vegf ) , a multifunctional cytokine that has an important role in neovascularization . vegf levels have been reported in the serum and all microvascular tissues of diabetic patients . furthermore , elevated vegf expression has been noted in the periodontium , similar to that in other end organs , in diabetics . recent studies have highlighted the important role of cell apoptosis in the development of diabetic complications . in diabetic patients , there is increased production of pro - apoptotic factors , such as ros , tnf- , and age 's . chronic periodontitis can lead to exacerbation of insulin resistance , with subsequent deterioration of glycemic control . periodontal therapy eliminates the inflammation and helps to counteract insulin resistance . every cell ( except brain cells ) when there is an increase in energy requirement or increase in blood glucose levels , the excess glucose is loaded on the expressed insulin receptors and transported intracellularly . thus , excess glucose from circulation is removed and stored intracellularly mostly in adipose tissue . when the cells become resistant to action of insulin , there is an increase in insulin production by the pancreas to attempt and force glucose in the cells . this state of reduced responsiveness to normal circulating levels of insulin is insulin resistance and results in hyperinsulinemia . increased insulin causes direct damage to the arteries causing atheroma formation and abnormalities in lipid metabolism resulting in increased levels of trg and high - density lipoprotein ( hdl ) . this results in hyperlipidemia , increased cholesterol ( ch ) , and trg as seen in individuals with insulin resistance . an increase in circulating lipids leads to excessive lipid oxidation , deposits of these oxidized fractions on vessel wall , and atherosclerosis . it is believed that bacterial lps have a significant effect on insulin sensitivity although the pathogenesis is poorly understood . the release of il - i and tnf- in response to bacteremia / endotoxemia has numerous metabolic effects in addition to hyperlipidemia . elevated levels of ll-1 are thought to play a role in the development of type i diabetes . it has been demonstrated that il - i facilitates protein kinase c activation leading to pancreatic -cell destruction through apoptotic mechanisms . additionally , il - i has been shown to be cytotoxic to cells in culture and in animal models through depletion of cellular energy stores and production of nitric oxide . tnf- has been implicated as a causative factor in insulin resistance and type 2 diabetes in animal models and in human studies . elevated levels of tnf- alter intracellular insulin signaling ( inhibiting tyrosine kinase activity of the insulin receptor ) and reduce synthesis of the insulin - responsive glucose transporter , creating an insulin resistance syndrome similar to the insulin resistance that characterizes type 2 diabetes . additionally , tnf- has been implicated in the development of macrophage - dependent cytotoxicity of pancreatic islets in diabetes . thus , infection - induced insulin resistance syndromes , if longstanding or chronic , are considered to be precursors to active diabetes due to the pancreatic -cell destruction that results from sustained elevations of il-1/tnf-. in fact , some investigators suggest that a proinflammatory imbalance created by excess il - l/tnf- is one of the most critical determinants of -cell loss in diabetic patients . all these findings suggest that proinflammatory cytokines , such as ll-1 and tnf- , produced as a systemic response to periodontal infection , are responsible for insulin resistance and subsequent poor glycemic control in periodontitis patients . treatment modalities include the potential therapeutic interventions which alter the mechanistic interrelationships between diabetes and periodontitis . these can be achieved by ;
reduction in the level of serum cytokines levels by the use of drugs ( monoclonal antibodies directed against il - i/tnf- or receptor antagonists targeted specifically to the il - i/tnf- receptors)reduction in the level of serum lipid levels by the use of drugs like fibrates and statins , or through dietary modulation . the reduction of serum lipid levels within the physiologic range utilizing lipid - lowering drugs in vivo actually caused significant increases in macrophage growth factor production . there has been recent interest in the use of dietary interventions targeted to alteration of fatty acid or absolute lipid content for the amelioration of diabetes - induced complications including periodontitis . a recent study has linked the severity of periodontitis to an imbalance between omega-6 and omega-3 free fatty acids and suggested that alveolar bone loss may be reduced by a diet rich in omega-3 fatty acids . it has been shown that high - fat diets and obesity are linked to increased systemic inflammatory cytokine production , while fat - restricted diets can effectively reduce the inflammatory response . indeed , some investigators have suggested that a low - fat diet and/or lipid - lowering drugs may be effective for the prevention or adjunctive treatment of periodontitis in diabetic and even non - diabetic patients . reduction in the level of serum cytokines levels by the use of drugs ( monoclonal antibodies directed against il - i/tnf- or receptor antagonists targeted specifically to the il - i/tnf- receptors ) reduction in the level of serum lipid levels by the use of drugs like fibrates and statins , or through dietary modulation . the reduction of serum lipid levels within the physiologic range utilizing lipid - lowering drugs in vivo actually caused significant increases in macrophage growth factor production . there has been recent interest in the use of dietary interventions targeted to alteration of fatty acid or absolute lipid content for the amelioration of diabetes - induced complications including periodontitis . a recent study has linked the severity of periodontitis to an imbalance between omega-6 and omega-3 free fatty acids and suggested that alveolar bone loss may be reduced by a diet rich in omega-3 fatty acids . it has been shown that high - fat diets and obesity are linked to increased systemic inflammatory cytokine production , while fat - restricted diets can effectively reduce the inflammatory response . indeed , some investigators have suggested that a low - fat diet and/or lipid - lowering drugs may be effective for the prevention or adjunctive treatment of periodontitis in diabetic and even non - diabetic patients . diet - induced reduction of serum ldl / trg may have an advantage over drug therapies designed to reduce or eliminate serum il - i/tnf- because :
reduction of ldl / trg is widely considered to be beneficial for many aspects of general and cardiovascular healthdietary interventions that manipulate serum lipids appear to be associated with fewer and less dangerous side effects compared to drug therapies that target il-1/tnf-reduction of il-/tnf- is likely to have many biological effects that are systemic in nature and can not be easily localized or targeted to specific desirable outcomes . reduction of ldl / trg is widely considered to be beneficial for many aspects of general and cardiovascular health dietary interventions that manipulate serum lipids appear to be associated with fewer and less dangerous side effects compared to drug therapies that target il-1/tnf- reduction of il-/tnf- is likely to have many biological effects that are systemic in nature and can not be easily localized or targeted to specific desirable outcomes . dietary interventions that reduce or alter serum lipid profiles have proved to be effective for the treatment of many diabetic complications . the ability of these therapies to reduce serum lipid / proinflammatory cytokine levels , reverse pathological changes in immune cell phenotype , and decrease the severity of chronic inflammatory diseases has been documented . the most common dietary approaches involve fat - restricted intake and supplementation using fish / plant oils or alteration of omega-3/omega-6 fatty acids . the effectiveness of these lipid - lowering therapies for amelioration of diabetic complications and reduction of the severity of chronic inflammatory diseases / conditions suggests that they could be used as preventive or adjunctive approaches for periodontitis in diabetic and non - diabetic patients . it is possible that the reduction of serum lipids in diabetic patients will provide some protection against lipid - induced alterations of immune cell phenotype responsible for increased serum proinflammatory cytokines and impaired local tissue response . additionally , in diabetic patients with periodontitis , reduction of serum lipids may improve the response to traditional periodontal therapy . the aim of the traditional approach of periodontal therapy with scaling and root planing is to reduce the number of pathogens from the infected periodontium and disruption of the microbial colonies conducive for bacterial growth several studies have shown that scaling and root planing combined with the systemic administration of doxycycline can improve glycemic control . a study by kiran et al . has reported a mean reduction in glycated hemoglobin ( hba1c ) in diabetic patients from 7.3% to 6.5% with only scaling and root planing compared with a slight but non - significant increase in hba1c levels in a diabetic control group that did not receive any treatment . singh et al . demonstrated a significant decrease in hba1c values in patients undergoing non - surgical periodontal treatment with systemic doxycycline therapy compared with controls . the aim of the traditional approach of periodontal therapy with scaling and root planing is to reduce the number of pathogens from the infected periodontium and disruption of the microbial colonies conducive for bacterial growth . several studies have shown that scaling and root planing combined with the systemic administration of doxycycline can improve glycemic control . a study by kiran et al . has reported a mean reduction in glycated hemoglobin ( hba1c ) in diabetic patients from 7.3% to 6.5% with only scaling and root planing compared with a slight but non - significant increase in hba1c levels in a diabetic control group that did not receive any treatment . singh et al . demonstrated a significant decrease in hba1c values in patients undergoing non - surgical periodontal treatment with systemic doxycycline therapy compared with controls . diabetes has many adverse effects on the periodontium , including decreased collagen turnover , impaired neutrophil function , and increased periodontal destruction . , no appreciable differences in the sites of periodontal disease have been found between diabetic and non - diabetic subjects . a great deal of attention has been directed to potential differences in the immunomodulatory responses to bacteria between diabetic and non - diabetic subjects . neutrophil chemotaxis and phagocytic activities are compromised in diabetic patients , which can lead to reduced bacterial killing and enhanced periodontal destruction . various studies have revealed levels of the acute - phase reactants like fibrinogen and c - reactive protein ( crp ) to be higher in people with insulin resistance and obesity . a majority of clinical and epidemiological studies give evidence to demonstrate that individuals with diabetes ( type 1 and type 2 ) tend to have a higher prevalence and more severe / rapidly progressing forms of periodontitis than non - diabetics . traditionally , research concerning the relationship between diabetes and periodontitis had focused on vascular changes in the periodontal tissues ( gingival microangiopathy ) , granulocyte hypo function , increased tissue labiality resulting from reduced collagen production / enhanced gingival collagenase activity , and changes in oral microflora . these studies although provided useful information concerning basic changes at the local level , did not reflect on the possible primary systemic relationships between diabetes and periodontitis . latest investigations performed at the cellular / molecular level demonstrate common changes in systemic physiology and have thus provided preliminary evidence of potential mechanisms responsible for the witnessed associations [ figure 1 ] . these include diabetes - induced alterations of immune cell phenotype and elevation of serum proinflammatory cytokine / lipid levels . in recent times , some studies have demonstrated that periodontitis itself can produce these same alterations , and in the presence of diabetes , produces exacerbation of these detrimental changes . a potential mechanistic link involves the broad axis of inflammation , specifically immune cell phenotype , serum lipid levels , and tissue homeostasis . thus , inflammation links insulin resistance , obesity , and diabetes . obesity is a leading cause of insulin resistance and serves as a medium leading to deterioration of both conditions of diabetes and periodontitis . adipocytes , once assumed to be just fat storage cells , are metabolically actively involved in the production of a wide range of molecules called adipokines . these adipocytes modulate levels of insulin and insulin sensitivity , any alteration of the same results in clinical conditions of diabetes . two types of adipokines exist , one which are synthesized by adipocytes in increased levels during metabolic disorders ( e.g. , tumor necrosis factor- , interleukin-6 , crp ) , better known as cytokines with well - defined roles in inflammation and immunity . others ( e.g. , leptin , adiponectin , resistin , visfatin ) which are involved in regulation of energy expenditure . leptin and adiponectin have an insulin - sensitizing effect and are protective against type 2 diabetes . adiponectin inhibits tumor necrosis factor- ( tnf- ) and inhibits transformation of monocytes to foam cells , and causes downregulation of proinflammatory cytokines and upregulates the synthesis of interleukin-10 ( il-10 ) . crp , an acute phase protein is secreted in small amounts from human adipocytes , upregulates proinflammatory action of other inflammatory mediators such as plasminogen activator inhibitor-1 , and also directly contributes to atheroma formation . diabetes - induced changes in immune cell function produce an upregulation of proinflammatory cytokines from monocytes / polymorphnuclear leukocytes ( pmn ) and downregulation of growth factors from macrophages . this predisposes to chronic inflammation , progressive tissue breakdown , and diminished tissue repair capacity . periodontal tissues frequently manifest these tissue breakdown changes as they are constantly injured by endotoxins originating from bacterial biofilms . diabetic patients being prone to hyperglycemia , thus hyperlipidemia is of high significance , as recent studies demonstrate that hyperlipidemia may be one of the factors associated with diabetes - induced immune cell alterations which results in further deterioration of periodontal conditions in these patients . evidence now proves that tissue destruction associated with periodontitis is due to release of proinflammatory cytokines and immune cell response to lipopolysaccharide and other metabolites of the local bacterial flora . the most convincing evidence of destruction by proinflammatory cytokines implicates the role of interleukin-1 beta ( il-1 ) and tnf-. it is believed that il-1 recruits inflammatory cells , facilitates polymorphonuclear leukocyte preparing / degranulation , increases synthesis of inflammatory mediators ( prostaglandins)/matrix metalloproteinases ( mmp ) , inhibits collagen synthesis , and activates both t and b lymphocytes . tnf- is a major signal for cellular apoptosis , bone resorption , mmp secretion , intercellular adhesion molecule ( icam ) expression , and interleukin-6 ( il-6 ) production . il-6 , once produced , stimulates formation of osteoclasts , promotes osteoclastic bone resorption , and facilitates t - cell differentiation . these cytokines are believed to exert a further effect on lipid metabolism by influencing the production of other cytokines , altering hemodynamics / amino acid utilization of various tissues involved in lipid metabolism or modifying hypothalamic - pituitary - adrenal axis , increasing plasma production of adrenocorticotropic hormone , cortisol , adrenaline , noradrenaline , and glucagon . in both type l and type 2 diabetes , the hyperlipidemia often manifests marked elevations of low - density lipoprotein ( ldl)/triglycerides ( trg ) and omega-6 free fatty acids . these serum lipid abnormalities result due to disruption of fatty acid metabolism and accumulation of omega-6 polyunsaturated fatty acids that contribute to formation of ldl / trg . the conversion of omega-6 polyunsaturated essential fatty acids to active metabolites , which are key components of cell membrane structure , is impaired . increasing evidence suggests that lipid composition of membranes is a critical factor influencing cellular function . the physical / chemical properties of membranes are largely determined by the nature of fatty acids within the phospholipid bilayer affecting receptor responses and operation of membrane - bound enzyme systems . it has been confirmed that diabetes - induced changes in membrane fluidity modulate function of membrane proteins potentially impairing cellular function / homeostasis . thus , in contrast to previous dogma concerning hyperglycemia , abnormal fatty acid metabolism and hyperlipidemia are also thought to be responsible for impairments in a variety of cell types and development of some diabetic complications . the function of inflammatory cells , such as neutrophils , monocytes , and macrophages , is altered in diabetic patients . this hyperresponsive monocytic phenotype is not associated with hyperglycemia can exist independently of periodontitis and may be related to hyperlipidemia . others postulate a genetic basis in the hla - dr and hla - dq gene regions and/or polymorphisms in the promoter regions of cytokine genes . using an animal study , sakallioglu et al . stated increased levels of monocyte chemoattractant protein ( mcp)-1 in gingival tissues of diabetic rats without periodontitis as compared to non - diabetic rats with periodontitis . monocytes play a significant part in periodontal tissue breakdown and are present in a greater concentration in patients with periodontitis . these cells exhibit enhanced mcp-1 expression in periodontal tissues , and raised levels of mcp-1 levels have been reported in diabetic patients compared with healthy controls . local and systemic hyper - responsiveness of these monocytes leads to increased tnf- levels in gingival crevicular fluid ( gcf ) . the pentose phosphate pathway is contributory in the formation of nicotinamide adenine dinucleotide phosphatase ( nadph ) and ribose-5-phosphate for fatty acid , and nucleotide synthesis , respectively . nadph is important for nadph oxidase activity and for the rejuvenation of glutathione in neutrophils , and activation of nadph oxidase results in a respiratory burst in neutrophils during the process of phagocytosis . there is a body of evidence suggesting that nadph oxidases play a major role in the pathogenesis of inflammation , hypertrophy , endothelial dysfunction , apoptosis , migration , and remodeling in hypertension , angiogenesis , and type 2 dm . in diabetic patients , glucose-6-phosphate dehydrogenase ( g6pdh ) converts glucose-6-phosphate into 6-phosphoglucono--lactone and is the rate - limiting enzyme in the pentose phosphate pathway . g6pdh activity has been found to be considerably decreased in neutrophils , macrophages , and lymphocytes isolated from diabetic rats . these findings suggest that the pentose phosphate pathway is downregulated in neutrophils from diabetic rats . in neutrophils in which g6pdh activity is deficient , phagocytosis , bactericidal ability , and superoxide production are impaired . glutamine is a cellular energy source next to glucose and both are necessary for lymphocyte function . glutamine is involved in protein , lipid , and nucleotide syntheses , as well as in nadph oxidase activity . glutamine increases bacterial killing activity in vitro , as well as the rate of reactive oxygen species ( ros ) production by neutrophils and inhibits spontaneous neutrophil apoptosis . therefore , decreased glutamine utilization may contribute to impaired neutrophil function in diabetes as a result of increased apoptosis . glutamine is also necessary for the provision of glutamate for glutathione synthesis , which is an antioxidant involved in preventing damage to important cellular components caused by reactive oxygen species such as free radicals and peroxides . in healthy subjects , glucose intake results in increased intranuclear nuclear factor ( nf)-b binding , decreased ib levels , increased ib kinase ( ikk ) activity , increased expression of ikk and ikk enzymes , and increased tnf- mrna expression in mononuclear cells ( mncs ) . these changes are harmonious with an increase in oxidative load in the mncs after glucose intake and thus trigger pro - inflammatory changes in the mncs . many cross - sectional studies have demonstrated hyper - reactivity of peripheral blood neutrophils in chronic periodontitis . other ros and reactive nitrogen species ( rns ) arise from superoxide and are termed secondary ros and rns . these free radicals derived from the mitochondrial cellular membrane , nucleus , lysosomes , peroxisomes , endoplasmic reticulum , and cytoplasm are unstable , either donating unpaired electrons to other cellular molecules or extracting electrons from other molecules in order to achieve a stable milieu . in low to moderate concentrations , they serve an important homeostatic function but in high concentrations , they are harmful and may contribute to the pathogenesis of chronic inflammatory diseases . both ros and rns have been reported to be involved in the etiopathogenesis of type 2 dm . evidence proves that oxidative stress is an important factor responsible for local tissue damage in chronic periodontitis . in hyperglycemic individuals , oxidation of circulating ldl leads to increased oxidant stress within the vasculature inducing chemotaxsis of macrophages and monocytes to the vessel wall . this oxidation results in cellular adhesion and increased production of cytokines and growth factors , resulting in stimulation of smooth muscle cell proliferation and causing an increase in the vessel thickness . other changes like increased atheroma formation and microthrombi in large blood vessels , alteration of vascular permeability , and endothelial cell functions in small vessels have also been observed . altered wound healing is one of the most common complications of dm . in a glucose - rich environment , the reparative capacity of periodontal tissues is compromised . the production of collagen and glycosaminoglycans is significantly reduced in high - glucose environments . in diabetic patients , the formation of age begins when glucose attaches to the amino groups on proteins to form an unstable glycated protein ( schiff base ) . eventually , after chemical rearrangement , these glycated proteins are converted to a more stable , yet still reversible , glucose protein complex known as the amadori product . however , if hyperglycemia is sustained , the amadori products become highly stable and form age . thus , even if hyperglycemia is corrected at this stage , the age in the affected tissues does not return to normal . the age thus formed accumulates in the periodontium , causing changes in the cells and extracellular matrix ( ecm ) components . collagen produced by fibroblasts under these conditions is susceptible to rapid degradation by matrix metalloproteinase ( mmp ) enzymes , such as collagenase , the production of which is significantly higher in dm . tissue collagenase is present in an active form in diabetics whereas the latent form is seen in non - diabetic subjects . in poorly controlled diabetic patients , collagen becomes cross - linked , resulting in a marked reduction of solubility . at the ultrastructural level , collagen homeostasis and turnover is altered . age has an adverse effect on bone collagen at the cellular level and this may result in alterations in bone metabolism . some studies have reported significant levels of osteoclasts and increased osteoclast activity in diabetic patients , whereas others have reported decreased bone resorption under similar conditions . age - modified collagen accumulates in blood vessel walls , narrowing the lumen . circulating ldl becomes cross - linked to this age - modified collagen and contributes to atheroma formation in the diabetic macrovasculature . in central and peripheral arteries , this enhances the macrovascular complications of diabetes . in smaller vessels , collagen in the vessels can lead to increased basement membrane thickness and compromised transport of nutrients across the membrane . the surface of smooth muscle cells , endothelial cells , neurons , macrophages , and monocytes expresses the receptor for age ( rage ) . ages can then bind to rage , leading to further complications such as the development of vascular lesions , increased vascular permeability , increased expression of adhesion molecules , and increased migration and activation of monocytes . these activated monocytes adhere to vascular endothelium via adhesion molecules like intercellular adhesion molecule ( icam-1 ) , endothelial leukocyte adhesion molecule ( elam-1 ) , and vascular cell adhesion molecule ( vcam-1 ) . these monocytes then penetrate the endothelium and migrate under intima layer where they ingest ldl in an oxidized state and become foam cells which are characteristic of atheromatous plaque . once within the arterial media , these monocytes transform to macrophages releasing an array of proinflammatory cytokines and mitogenic factors causing muscle and collagen proliferation leading to thickening of the vessel walls . the effect on the endothelial cells is an increase in vascular permeability and thrombus formation . the age - rage interaction on smooth muscle cells results in cellular proliferation within the arterial wall . as ages are chemotactic for monocytes , age - rage interaction induces increased cellular oxidant stress and activates the transcription factor nuclear factor kappa - beta ( nfkb ) on monocytes . this then alters the phenotype of the monocyte / macrophage and results in increased production of proinflammatory cytokines and growth factors such as interleukin-1 ( il - i ) , tnf- , platelet - derived growth factor ( pdgf ) , and insulin - like growth factor ( igf ) . all these cytokines and growth factors have been shown to contribute to the chronic inflammatory process in the formation of atheromatous lesions . in addition , oxidized ldl , elevated in many diabetic patients , also activates nfkb and may result in similar processes . thus , alterations in lipid and protein metabolisms induced by the sustained hyperglycemia characteristic of diabetes may play a major role and provide a common link between all the classic complications of this disease . in addition , age can stimulate increased production of vascular endothelial growth factor ( vegf ) , a multifunctional cytokine that has an important role in neovascularization . vegf levels have been reported in the serum and all microvascular tissues of diabetic patients . furthermore , elevated vegf expression has been noted in the periodontium , similar to that in other end organs , in diabetics . recent studies have highlighted the important role of cell apoptosis in the development of diabetic complications . in diabetic patients , there is increased production of pro - apoptotic factors , such as ros , tnf- , and age 's . chronic periodontitis can lead to exacerbation of insulin resistance , with subsequent deterioration of glycemic control . periodontal therapy eliminates the inflammation and helps to counteract insulin resistance . every cell ( except brain cells ) when there is an increase in energy requirement or increase in blood glucose levels , the excess glucose is loaded on the expressed insulin receptors and transported intracellularly . thus , excess glucose from circulation is removed and stored intracellularly mostly in adipose tissue . when the cells become resistant to action of insulin , there is an increase in insulin production by the pancreas to attempt and force glucose in the cells . this state of reduced responsiveness to normal circulating levels of insulin is insulin resistance and results in hyperinsulinemia . increased insulin causes direct damage to the arteries causing atheroma formation and abnormalities in lipid metabolism resulting in increased levels of trg and high - density lipoprotein ( hdl ) . this results in hyperlipidemia , increased cholesterol ( ch ) , and trg as seen in individuals with insulin resistance . an increase in circulating lipids leads to excessive lipid oxidation , deposits of these oxidized fractions on vessel wall , and atherosclerosis . it is believed that bacterial lps have a significant effect on insulin sensitivity although the pathogenesis is poorly understood . the release of il - i and tnf- in response to bacteremia / endotoxemia has numerous metabolic effects in addition to hyperlipidemia . elevated levels of ll-1 are thought to play a role in the development of type i diabetes . it has been demonstrated that il - i facilitates protein kinase c activation leading to pancreatic -cell destruction through apoptotic mechanisms . additionally , il - i has been shown to be cytotoxic to cells in culture and in animal models through depletion of cellular energy stores and production of nitric oxide . tnf- has been implicated as a causative factor in insulin resistance and type 2 diabetes in animal models and in human studies . elevated levels of tnf- alter intracellular insulin signaling ( inhibiting tyrosine kinase activity of the insulin receptor ) and reduce synthesis of the insulin - responsive glucose transporter , creating an insulin resistance syndrome similar to the insulin resistance that characterizes type 2 diabetes . additionally , tnf- has been implicated in the development of macrophage - dependent cytotoxicity of pancreatic islets in diabetes . thus , infection - induced insulin resistance syndromes , if longstanding or chronic , are considered to be precursors to active diabetes due to the pancreatic -cell destruction that results from sustained elevations of il-1/tnf-. in fact , some investigators suggest that a proinflammatory imbalance created by excess il - l/tnf- is one of the most critical determinants of -cell loss in diabetic patients . all these findings suggest that proinflammatory cytokines , such as ll-1 and tnf- , produced as a systemic response to periodontal infection , are responsible for insulin resistance and subsequent poor glycemic control in periodontitis patients . a potential mechanistic link involves the broad axis of inflammation , specifically immune cell phenotype , serum lipid levels , and tissue homeostasis . thus , inflammation links insulin resistance , obesity , and diabetes . obesity is a leading cause of insulin resistance and serves as a medium leading to deterioration of both conditions of diabetes and periodontitis . adipocytes , once assumed to be just fat storage cells , are metabolically actively involved in the production of a wide range of molecules called adipokines . these adipocytes modulate levels of insulin and insulin sensitivity , any alteration of the same results in clinical conditions of diabetes . two types of adipokines exist , one which are synthesized by adipocytes in increased levels during metabolic disorders ( e.g. , tumor necrosis factor- , interleukin-6 , crp ) , better known as cytokines with well - defined roles in inflammation and immunity . others ( e.g. , leptin , adiponectin , resistin , visfatin ) which are involved in regulation of energy expenditure . leptin and adiponectin have an insulin - sensitizing effect and are protective against type 2 diabetes . adiponectin inhibits tumor necrosis factor- ( tnf- ) and inhibits transformation of monocytes to foam cells , and causes downregulation of proinflammatory cytokines and upregulates the synthesis of interleukin-10 ( il-10 ) . crp , an acute phase protein is secreted in small amounts from human adipocytes , upregulates proinflammatory action of other inflammatory mediators such as plasminogen activator inhibitor-1 , and also directly contributes to atheroma formation . diabetes - induced changes in immune cell function produce an upregulation of proinflammatory cytokines from monocytes / polymorphnuclear leukocytes ( pmn ) and downregulation of growth factors from macrophages . this predisposes to chronic inflammation , progressive tissue breakdown , and diminished tissue repair capacity . periodontal tissues frequently manifest these tissue breakdown changes as they are constantly injured by endotoxins originating from bacterial biofilms . diabetic patients being prone to hyperglycemia , thus hyperlipidemia is of high significance , as recent studies demonstrate that hyperlipidemia may be one of the factors associated with diabetes - induced immune cell alterations which results in further deterioration of periodontal conditions in these patients . evidence now proves that tissue destruction associated with periodontitis is due to release of proinflammatory cytokines and immune cell response to lipopolysaccharide and other metabolites of the local bacterial flora . the most convincing evidence of destruction by proinflammatory cytokines implicates the role of interleukin-1 beta ( il-1 ) and tnf-. it is believed that il-1 recruits inflammatory cells , facilitates polymorphonuclear leukocyte preparing / degranulation , increases synthesis of inflammatory mediators ( prostaglandins)/matrix metalloproteinases ( mmp ) , inhibits collagen synthesis , and activates both t and b lymphocytes . tnf- is a major signal for cellular apoptosis , bone resorption , mmp secretion , intercellular adhesion molecule ( icam ) expression , and interleukin-6 ( il-6 ) production . il-6 , once produced , stimulates formation of osteoclasts , promotes osteoclastic bone resorption , and facilitates t - cell differentiation . these cytokines are believed to exert a further effect on lipid metabolism by influencing the production of other cytokines , altering hemodynamics / amino acid utilization of various tissues involved in lipid metabolism or modifying hypothalamic - pituitary - adrenal axis , increasing plasma production of adrenocorticotropic hormone , cortisol , adrenaline , noradrenaline , and glucagon . in both type l and type 2 diabetes , the hyperlipidemia often manifests marked elevations of low - density lipoprotein ( ldl)/triglycerides ( trg ) and omega-6 free fatty acids . these serum lipid abnormalities result due to disruption of fatty acid metabolism and accumulation of omega-6 polyunsaturated fatty acids that contribute to formation of ldl / trg . the conversion of omega-6 polyunsaturated essential fatty acids to active metabolites , which are key components of cell membrane structure , is impaired . increasing evidence suggests that lipid composition of membranes is a critical factor influencing cellular function . the physical / chemical properties of membranes are largely determined by the nature of fatty acids within the phospholipid bilayer affecting receptor responses and operation of membrane - bound enzyme systems . it has been confirmed that diabetes - induced changes in membrane fluidity modulate function of membrane proteins potentially impairing cellular function / homeostasis . thus , in contrast to previous dogma concerning hyperglycemia , abnormal fatty acid metabolism and hyperlipidemia are also thought to be responsible for impairments in a variety of cell types and development of some diabetic complications . the function of inflammatory cells , such as neutrophils , monocytes , and macrophages , is altered in diabetic patients . this hyperresponsive monocytic phenotype is not associated with hyperglycemia can exist independently of periodontitis and may be related to hyperlipidemia . others postulate a genetic basis in the hla - dr and hla - dq gene regions and/or polymorphisms in the promoter regions of cytokine genes . using an animal study , sakallioglu et al . stated increased levels of monocyte chemoattractant protein ( mcp)-1 in gingival tissues of diabetic rats without periodontitis as compared to non - diabetic rats with periodontitis . monocytes play a significant part in periodontal tissue breakdown and are present in a greater concentration in patients with periodontitis . these cells exhibit enhanced mcp-1 expression in periodontal tissues , and raised levels of mcp-1 levels have been reported in diabetic patients compared with healthy controls . local and systemic hyper - responsiveness of these monocytes leads to increased tnf- levels in gingival crevicular fluid ( gcf ) . the pentose phosphate pathway is contributory in the formation of nicotinamide adenine dinucleotide phosphatase ( nadph ) and ribose-5-phosphate for fatty acid , and nucleotide synthesis , respectively . nadph is important for nadph oxidase activity and for the rejuvenation of glutathione in neutrophils , and activation of nadph oxidase results in a respiratory burst in neutrophils during the process of phagocytosis . there is a body of evidence suggesting that nadph oxidases play a major role in the pathogenesis of inflammation , hypertrophy , endothelial dysfunction , apoptosis , migration , and remodeling in hypertension , angiogenesis , and type 2 dm . in diabetic patients , glucose-6-phosphate dehydrogenase ( g6pdh ) converts glucose-6-phosphate into 6-phosphoglucono--lactone and is the rate - limiting enzyme in the pentose phosphate pathway . g6pdh activity has been found to be considerably decreased in neutrophils , macrophages , and lymphocytes isolated from diabetic rats . these findings suggest that the pentose phosphate pathway is downregulated in neutrophils from diabetic rats . in neutrophils in which g6pdh activity is deficient , phagocytosis , bactericidal ability , and superoxide production are impaired . glutamine is a cellular energy source next to glucose and both are necessary for lymphocyte function . glutamine is involved in protein , lipid , and nucleotide syntheses , as well as in nadph oxidase activity . glutamine increases bacterial killing activity in vitro , as well as the rate of reactive oxygen species ( ros ) production by neutrophils and inhibits spontaneous neutrophil apoptosis . therefore , decreased glutamine utilization may contribute to impaired neutrophil function in diabetes as a result of increased apoptosis . glutamine is also necessary for the provision of glutamate for glutathione synthesis , which is an antioxidant involved in preventing damage to important cellular components caused by reactive oxygen species such as free radicals and peroxides . in healthy subjects , glucose intake results in increased intranuclear nuclear factor ( nf)-b binding , decreased ib levels , increased ib kinase ( ikk ) activity , increased expression of ikk and ikk enzymes , and increased tnf- mrna expression in mononuclear cells ( mncs ) . these changes are harmonious with an increase in oxidative load in the mncs after glucose intake and thus trigger pro - inflammatory changes in the mncs . many cross - sectional studies have demonstrated hyper - reactivity of peripheral blood neutrophils in chronic periodontitis . other ros and reactive nitrogen species ( rns ) arise from superoxide and are termed secondary ros and rns . these free radicals derived from the mitochondrial cellular membrane , nucleus , lysosomes , peroxisomes , endoplasmic reticulum , and cytoplasm are unstable , either donating unpaired electrons to other cellular molecules or extracting electrons from other molecules in order to achieve a stable milieu . in low to moderate concentrations , they serve an important homeostatic function but in high concentrations , they are harmful and may contribute to the pathogenesis of chronic inflammatory diseases . both ros and rns have been reported to be involved in the etiopathogenesis of type 2 dm . evidence proves that oxidative stress is an important factor responsible for local tissue damage in chronic periodontitis . in hyperglycemic individuals , oxidation of circulating ldl leads to increased oxidant stress within the vasculature inducing chemotaxsis of macrophages and monocytes to the vessel wall . this oxidation results in cellular adhesion and increased production of cytokines and growth factors , resulting in stimulation of smooth muscle cell proliferation and causing an increase in the vessel thickness . other changes like increased atheroma formation and microthrombi in large blood vessels , alteration of vascular permeability , and endothelial cell functions in small vessels have also been observed . altered wound healing is one of the most common complications of dm . in a glucose - rich environment , the production of collagen and glycosaminoglycans is significantly reduced in high - glucose environments . in diabetic patients , the formation of age begins when glucose attaches to the amino groups on proteins to form an unstable glycated protein ( schiff base ) . eventually , after chemical rearrangement , these glycated proteins are converted to a more stable , yet still reversible , glucose protein complex known as the amadori product . however , if hyperglycemia is sustained , the amadori products become highly stable and form age . thus , even if hyperglycemia is corrected at this stage , the age in the affected tissues does not return to normal . the age thus formed accumulates in the periodontium , causing changes in the cells and extracellular matrix ( ecm ) components . collagen produced by fibroblasts under these conditions is susceptible to rapid degradation by matrix metalloproteinase ( mmp ) enzymes , such as collagenase , the production of which is significantly higher in dm . tissue collagenase is present in an active form in diabetics whereas the latent form is seen in non - diabetic subjects . in poorly controlled diabetic patients , collagen becomes cross - linked , resulting in a marked reduction of solubility . at the ultrastructural level , collagen homeostasis and turnover is altered . age has an adverse effect on bone collagen at the cellular level and this may result in alterations in bone metabolism . some studies have reported significant levels of osteoclasts and increased osteoclast activity in diabetic patients , whereas others have reported decreased bone resorption under similar conditions . circulating ldl becomes cross - linked to this age - modified collagen and contributes to atheroma formation in the diabetic macrovasculature . in central and peripheral arteries , this enhances the macrovascular complications of diabetes . in smaller vessels , collagen in the vessels can lead to increased basement membrane thickness and compromised transport of nutrients across the membrane . the surface of smooth muscle cells , endothelial cells , neurons , macrophages , and monocytes expresses the receptor for age ( rage ) . ages can then bind to rage , leading to further complications such as the development of vascular lesions , increased vascular permeability , increased expression of adhesion molecules , and increased migration and activation of monocytes . these activated monocytes adhere to vascular endothelium via adhesion molecules like intercellular adhesion molecule ( icam-1 ) , endothelial leukocyte adhesion molecule ( elam-1 ) , and vascular cell adhesion molecule ( vcam-1 ) . these monocytes then penetrate the endothelium and migrate under intima layer where they ingest ldl in an oxidized state and become foam cells which are characteristic of atheromatous plaque . once within the arterial media , these monocytes transform to macrophages releasing an array of proinflammatory cytokines and mitogenic factors causing muscle and collagen proliferation leading to thickening of the vessel walls . the effect on the endothelial cells is an increase in vascular permeability and thrombus formation . the age - rage interaction on smooth muscle cells results in cellular proliferation within the arterial wall . as ages are chemotactic for monocytes , age - rage interaction induces increased cellular oxidant stress and activates the transcription factor nuclear factor kappa - beta ( nfkb ) on monocytes . this then alters the phenotype of the monocyte / macrophage and results in increased production of proinflammatory cytokines and growth factors such as interleukin-1 ( il - i ) , tnf- , platelet - derived growth factor ( pdgf ) , and insulin - like growth factor ( igf ) . all these cytokines and growth factors have been shown to contribute to the chronic inflammatory process in the formation of atheromatous lesions . in addition , oxidized ldl , elevated in many diabetic patients , also activates nfkb and may result in similar processes . thus , alterations in lipid and protein metabolisms induced by the sustained hyperglycemia characteristic of diabetes may play a major role and provide a common link between all the classic complications of this disease . in addition , age can stimulate increased production of vascular endothelial growth factor ( vegf ) , a multifunctional cytokine that has an important role in neovascularization . vegf levels have been reported in the serum and all microvascular tissues of diabetic patients . furthermore , elevated vegf expression has been noted in the periodontium , similar to that in other end organs , in diabetics . recent studies have highlighted the important role of cell apoptosis in the development of diabetic complications . in diabetic patients , there is increased production of pro - apoptotic factors , such as ros , tnf- , and age 's . chronic periodontitis can lead to exacerbation of insulin resistance , with subsequent deterioration of glycemic control . when there is an increase in energy requirement or increase in blood glucose levels , the excess glucose is loaded on the expressed insulin receptors and transported intracellularly . thus , excess glucose from circulation is removed and stored intracellularly mostly in adipose tissue . when the cells become resistant to action of insulin , there is an increase in insulin production by the pancreas to attempt and force glucose in the cells . this state of reduced responsiveness to normal circulating levels of insulin is insulin resistance and results in hyperinsulinemia . increased insulin causes direct damage to the arteries causing atheroma formation and abnormalities in lipid metabolism resulting in increased levels of trg and high - density lipoprotein ( hdl ) . this results in hyperlipidemia , increased cholesterol ( ch ) , and trg as seen in individuals with insulin resistance . an increase in circulating lipids leads to excessive lipid oxidation , deposits of these oxidized fractions on vessel wall , and atherosclerosis . it is believed that bacterial lps have a significant effect on insulin sensitivity although the pathogenesis is poorly understood . the release of il - i and tnf- in response to bacteremia / endotoxemia has numerous metabolic effects in addition to hyperlipidemia . elevated levels of ll-1 are thought to play a role in the development of type i diabetes . it has been demonstrated that il - i facilitates protein kinase c activation leading to pancreatic -cell destruction through apoptotic mechanisms . additionally , il - i has been shown to be cytotoxic to cells in culture and in animal models through depletion of cellular energy stores and production of nitric oxide . tnf- has been implicated as a causative factor in insulin resistance and type 2 diabetes in animal models and in human studies . elevated levels of tnf- alter intracellular insulin signaling ( inhibiting tyrosine kinase activity of the insulin receptor ) and reduce synthesis of the insulin - responsive glucose transporter , creating an insulin resistance syndrome similar to the insulin resistance that characterizes type 2 diabetes . additionally , tnf- has been implicated in the development of macrophage - dependent cytotoxicity of pancreatic islets in diabetes . thus , infection - induced insulin resistance syndromes , if longstanding or chronic , are considered to be precursors to active diabetes due to the pancreatic -cell destruction that results from sustained elevations of il-1/tnf-. in fact , some investigators suggest that a proinflammatory imbalance created by excess il - l/tnf- is one of the most critical determinants of -cell loss in diabetic patients . all these findings suggest that proinflammatory cytokines , such as ll-1 and tnf- , produced as a systemic response to periodontal infection , are responsible for insulin resistance and subsequent poor glycemic control in periodontitis patients . treatment modalities include the potential therapeutic interventions which alter the mechanistic interrelationships between diabetes and periodontitis . these can be achieved by ;
reduction in the level of serum cytokines levels by the use of drugs ( monoclonal antibodies directed against il - i/tnf- or receptor antagonists targeted specifically to the il - i/tnf- receptors)reduction in the level of serum lipid levels by the use of drugs like fibrates and statins , or through dietary modulation . the reduction of serum lipid levels within the physiologic range utilizing lipid - lowering drugs in vivo actually caused significant increases in macrophage growth factor production . there has been recent interest in the use of dietary interventions targeted to alteration of fatty acid or absolute lipid content for the amelioration of diabetes - induced complications including periodontitis . a recent study has linked the severity of periodontitis to an imbalance between omega-6 and omega-3 free fatty acids and suggested that alveolar bone loss may be reduced by a diet rich in omega-3 fatty acids . it has been shown that high - fat diets and obesity are linked to increased systemic inflammatory cytokine production , while fat - restricted diets can effectively reduce the inflammatory response . indeed , some investigators have suggested that a low - fat diet and/or lipid - lowering drugs may be effective for the prevention or adjunctive treatment of periodontitis in diabetic and even non - diabetic patients . reduction in the level of serum cytokines levels by the use of drugs ( monoclonal antibodies directed against il - i/tnf- or receptor antagonists targeted specifically to the il - i/tnf- receptors ) reduction in the level of serum lipid levels by the use of drugs like fibrates and statins , or through dietary modulation . the reduction of serum lipid levels within the physiologic range utilizing lipid - lowering drugs in vivo actually caused significant increases in macrophage growth factor production . there has been recent interest in the use of dietary interventions targeted to alteration of fatty acid or absolute lipid content for the amelioration of diabetes - induced complications including periodontitis . a recent study has linked the severity of periodontitis to an imbalance between omega-6 and omega-3 free fatty acids and suggested that alveolar bone loss may be reduced by a diet rich in omega-3 fatty acids . it has been shown that high - fat diets and obesity are linked to increased systemic inflammatory cytokine production , while fat - restricted diets can effectively reduce the inflammatory response . indeed , some investigators have suggested that a low - fat diet and/or lipid - lowering drugs may be effective for the prevention or adjunctive treatment of periodontitis in diabetic and even non - diabetic patients . diet - induced reduction of serum ldl / trg may have an advantage over drug therapies designed to reduce or eliminate serum il - i/tnf- because :
reduction of ldl / trg is widely considered to be beneficial for many aspects of general and cardiovascular healthdietary interventions that manipulate serum lipids appear to be associated with fewer and less dangerous side effects compared to drug therapies that target il-1/tnf-reduction of il-/tnf- is likely to have many biological effects that are systemic in nature and can not be easily localized or targeted to specific desirable outcomes . reduction of ldl / trg is widely considered to be beneficial for many aspects of general and cardiovascular health dietary interventions that manipulate serum lipids appear to be associated with fewer and less dangerous side effects compared to drug therapies that target il-1/tnf- reduction of il-/tnf- is likely to have many biological effects that are systemic in nature and can not be easily localized or targeted to specific desirable outcomes . dietary interventions that reduce or alter serum lipid profiles have proved to be effective for the treatment of many diabetic complications . the ability of these therapies to reduce serum lipid / proinflammatory cytokine levels , reverse pathological changes in immune cell phenotype , and decrease the severity of chronic inflammatory diseases has been documented . the most common dietary approaches involve fat - restricted intake and supplementation using fish / plant oils or alteration of omega-3/omega-6 fatty acids . the effectiveness of these lipid - lowering therapies for amelioration of diabetic complications and reduction of the severity of chronic inflammatory diseases / conditions suggests that they could be used as preventive or adjunctive approaches for periodontitis in diabetic and non - diabetic patients . it is possible that the reduction of serum lipids in diabetic patients will provide some protection against lipid - induced alterations of immune cell phenotype responsible for increased serum proinflammatory cytokines and impaired local tissue response . additionally , in diabetic patients with periodontitis , reduction of serum lipids may improve the response to traditional periodontal therapy . the aim of the traditional approach of periodontal therapy with scaling and root planing is to reduce the number of pathogens from the infected periodontium and disruption of the microbial colonies conducive for bacterial growth several studies have shown that scaling and root planing combined with the systemic administration of doxycycline can improve glycemic control . a study by kiran et al . has reported a mean reduction in glycated hemoglobin ( hba1c ) in diabetic patients from 7.3% to 6.5% with only scaling and root planing compared with a slight but non - significant increase in hba1c levels in a diabetic control group that did not receive any treatment . singh et al . demonstrated a significant decrease in hba1c values in patients undergoing non - surgical periodontal treatment with systemic doxycycline therapy compared with controls . the aim of the traditional approach of periodontal therapy with scaling and root planing is to reduce the number of pathogens from the infected periodontium and disruption of the microbial colonies conducive for bacterial growth . several studies have shown that scaling and root planing combined with the systemic administration of doxycycline can improve glycemic control . a study by kiran et al . has reported a mean reduction in glycated hemoglobin ( hba1c ) in diabetic patients from 7.3% to 6.5% with only scaling and root planing compared with a slight but non - significant increase in hba1c levels in a diabetic control group that did not receive any treatment . singh et al . demonstrated a significant decrease in hba1c values in patients undergoing non - surgical periodontal treatment with systemic doxycycline therapy compared with controls . , many studies have reported that the presence of diabetes mellitus increases the incidence and severity of periodontal disease . there appears to be a relationship between the two processes , whereby the consequences of diabetes mellitus serve as modifiers of the expression of periodontal pathology . , it has not been clarified whether good metabolic control influences the success of periodontal treatment or vice versa . likewise , it remains to be determined whether the mechanisms involved are the same in both type 1 and type 2 diabetes mellitus . for proper treatment of diabetic patients with periodontitis , the medical and dental professions should work together the signs , symptoms , and clinical presentation of periodontitis need to be recognized by physicians so that diabetic patients are promptly referred to dentists for treatment and similarly dentists should understand the parameters of glycemia that are used to establish a diagnosis of diabetes and the methods used in diabetic care , thus potentially preventing further complications . | both diabetes and periodontitis are chronic diseases .
diabetes has many adverse effects on the periodontium , and conversely periodontitis may have deleterious effects further aggravating the condition in diabetics .
the potential common pathophysiologic pathways include those associated with inflammation , altered host responses , altered tissue homeostasis , and insulin resistance .
this review examines the relationship that exists between periodontal diseases and diabetes mellitus with a focus on potential common pathophysiologic mechanisms . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
a 72-year - old female was admitted to hospital with deep vein thrombosis ( dvt ) . the patient was recently diagnosed with persistent nonvalvular atrial fibrillation , and it was decided that she would benefit from a warfarin slow - start regimen for stroke prophylaxis . warfarin was prescribed at 2 mg daily as a slow loading dose . on day 3 , the international normalized ratio ( inr ) was at 0.9 ( range = 0.881.12 ) . the primary care physician increased the dose of warfarin to a 5 mg daily dose . after receiving the second 5 mg dose , the patient woke up with pain and swelling in the left leg and upper thigh . the patient s past medical and family history was noncontributory for a history of thrombophilia or other major thrombotic risk factors . laboratory levels were as follows : protein c activity = 13% ( range = 70%140% ) ; protein s activity = 28% ( range = 70%140% ) , inr = 1 ( range = 0.881.12 ) ; prothrombin time = 10.5 seconds ( range = 9.512.0 seconds ) ; and activated partial thromboplastin time = 30.5 seconds ( range = 23.435.4 seconds ) . these values indicated that the patient was found to be reactive to initial anticoagulant therapy with warfarin . laboratory tests including factor v leiden , lupus screen , antinuclear antibody , and anticardiolipin , were negative for an inherited or acquired prothrombotic state . a plan to rule out protein c and protein s deficiencies and inadequate antithrombin 3 was scheduled to take place after the cessation of warfarin therapy in 3 months time ( prove to be negative ) . she was discharged on 3 mg of warfarin per day , with instructions to continue the use of compression stockings . the use of unopposed warfarin as monotherapy ( without an initial heparin overlap ) as advocated by the slow - start regimen is often regarded as safe and achieves therapeutic anticoagulation in the majority of patients within 34 weeks.1,2 this regimen is suitable for use in both the secondary and primary care setting , and it allows for the induction of anticoagulation therapy requiring only weekly monitoring.2 this appears to avoid over - anticoagulation and bleeding associated with rapid loading . oates et al1 developed an algorithm for safe and efficient prophylactic anticoagulation that can be used in outpatient settings . in their study , the inr range of 23 was maintained in half of the patients tested during the observation period . no age limits were set for entry into the study , and there were no exclusion criteria at entry.1 tait and sefcick2 studied 200 patients who were started on 3 mg of warfarin daily for 1 week , and subsequent doses were determined by weekly inr measurement . current guidelines advise treating doctors to start warfarin at 2 mg / day for 1 week , then to repeat the inr . if the inr is < 2 , then the patient should continue with warfarin at 2 mg / day , and the inr should be repeated at the end of the second week . at that stage , the treating doctors can adjust the warfarin dose , as per nomogram , with the aim of achieving a target inr of 2.5 . warfarin loading doses may paradoxically result in a hypercoagulable state and potential clot formation because of significant reductions in protein c and protein s.3,4 a precipitous reduction in the concentration of protein c and protein s ( with an approximate half - life of 8 hours ) occurs during the first 36 hours of warfarin therapy , which is when warfarin has a very limited effect on prothrombin ( which has an approximate half - life of 50 hours).5 consequently , because of the potential risk of thromboembolism during the initiation of warfarin therapy , the use of concurrent heparin is extremely important.6 warfarin - induced skin necrosis is a recognized adverse event during the loading of warfarin therapy . similarly , it begins on day 36 after initiating warfarin.7 it is also thought that the precipitous reduction in the concentration of protein c and protein s creates a hypercoagulable state , which cause thrombotic occlusions of the microvasculature with resulting necrosis of the subcutaneous fat.7,8 the use of a small loading dose of warfarin ( 2 mg / day ) in the slow - start regimen is basically designed to combat the precipitous reduction in protein c and protein s , and then to obviate the need to use concurrent heparin therapy.1 in an attempt to achieve rapid anticoagulation , doctors might depart from the standard protocol of the slow - start regimen and use a larger unopposed loading dose of warfarin , which could result in the development of dvt with potentially life - threatening complications.5 with the growing popularity of using the slow - start unopposed warfarin regimen in outpatient and community settings , there is an increased risk of warfarin - related adverse events when treating doctors fail to adhere to protocols . the desire of some doctors to achieve rapid anticoagulation by initiating or escalating warfarin monotherapy could be catastrophic . the incidence of warfarin unopposed therapy causing dvt is expected to rise with increase adoption of the slow - start regimen . although the development of dvt after giving a higher loading dose in our patient could be a pure coincidence , we felt that the temporal relationship between the onset of the dvt timed with the increase in the warfarin loading dose is compelling . also , the paucity of obvious clinical risk factors for a hypercoagulable state increased our suspicion . the naranjo score was at + 2 , which renders the development of dvt as a possible adverse drug reaction to warfarin.9 this case study illustrates the importance of considering dvt as a possible complication of unopposed warfarin therapy . warfarin - induced skin necrosis is a well - established adverse event of a warfarin loading dose.7 the hypercoagulable state of the blood caused by warfarin is regarded as the main cause for this adverse event . this case is the first ever reported case of a possible iatrogenic warfarin - induced dvt . understanding the pharmacokinetics and pharmacodynamics of warfarin would help ensure safe and effective prescription of the drug . a high index of suspicion may allow for the rapid reversal of the warfarin effect with therapeutic heparinization before the processes of adverse procoagulation begin . widvt for the adverse event of warfarin - induced dvt in order to help physicians become more alert to and aware of the consequences of initiating an unopposed warfarin loading dose . | we are presenting a 72-year - old female who was admitted to hospital with deep vein thrombosis ( dvt ) .
she was known to have atrial fibrillation and was initiated on warfarin for stroke prophylaxis 3 days earlier .
she was given warfarin therapy without low molecular weight heparin cover as per slow - start regimen
protocol .
the warfarin dose was increased after 3 days to achieve rapid anticoagulation , resulting in dvt in the left leg .
we propose that the higher unopposed warfarin dose utilized in this case resulted in dvt .
warfarin loading doses may paradoxically result in a hypercoagulable state and potential clot formation because of significant reductions in protein c and protein s levels . |
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there are currently in excess of 150 radiotherapy centres worldwide using co-60 sources in modern high dose - rate ( hdr ) brachytherapy treatment units . these new systems utilise miniaturised co-60 sources , rather than traditional ir-192 sources and becoming very popular due to longer source replacement intervals , lower operating costs and a reduced frequency of movement of radioactive sources between countries , compared to ir-192 . whilst the availability of miniaturised high specific - activity co-60 sources for high dose - rate brachytherapy is a recent development , the use of physically larger co-60 sources in low dose - rate applications has a long history dating back to the 1960s and 1970s with the cathetron , ralstron and selectron treatment units , reported in 1964 by henschke . by the 1980s , ir-192 had become the most popular isotope for hdr brachytherapy due to its smaller physical size . the application of hdr brachytherapy in carcinoma of the cervix is a well established treatment technique and work by dale , has shown there is negligible clinical significance in the biological response of tissues of varying density to the different energy spectra from co-60 and ir-192 . there are several publications on the clinical use of co-60 hdr and on comparisons of physical dose differences with much more common ir-192 sources . bocharova have evaluated acute toxicity with co-60 in gynaecological cancer and shown this to be a tolerable isotope for hdr treatments , reporting the results to be comparable to ir-192 . the dose deposition differences around single co-60 and ir-192 sources ( anisotropy , radial dose function and isodose curves ) have been reviewed by strohmaier , finding no advantage or disadvantage for co-60 sources compared to ir-192 . however , this review is based on the available work by venselaar , richter et al . and park et al . , who confined their analysis to point dose and qualitative isodose comparisons only . park et al . considered cervix treatment plans and compared only the icru reference point doses for two ir-192 sources and a co-60 source using treatment plans based on 2d orthogonal radiographs , finding rectal doses to be higher ( average + 0.8% ) and bladder doses lower ( average 1.1% ) for co-60 compared to ir-192 . have also only considered differences in dose distributions produced by treatment plans with identical source dwell loading patterns . while this provides valuable information on the inherent physical differences between the sources , what is also required is information on any residual differences in typical clinical treatment plans , where non - identical dwell - times are allowed with the two isotopes ; i.e. comparing actual typical treatment plans that may be achieved with co-60 and ir-192 . whilst prescription to manchester system point a is still common , an evaluation of the differences between the two isotopes under the improved dosimetry recommendations from gec - estro is required : based on 3d treatment planning and dvh parameter reporting , in order to fully and comprehensively evaluate differences in treatment plan dose distributions between co-60 and ir-192 sources for hdr gynaecological brachytherapy . the current work builds on previous comparisons of physical characteristics of the two isotopes and simple planning comparisons , by investigating co-60 and ir-192 brachytherapy treatment plans in terms of dose - volume histogram ( dvh ) reporting metrics , with both standard identical loading patterns prescribed to point a and optimised plans prescribed to the high - risk clinical target volume ( hr - ctv ) . typical treatment times and source replacement costs the comparison between co-60 and ir-192 isotopes was undertaken by considering the source types manufactured by eckert & ziegler bebig gmbh , germany ( in future termed ez bebig ) , models co0.a86 and ir.a85 - 2 , respectively . these sources are geometrically identical and were therefore ideal to compare the effects of isotope choice . both sources consist of an active source core of length 3.5 mm and diameter 0.6 mm , surrounded by a cylindrical steel jacket with a length of 5 mm and an outer diameter of 1.0 mm . monte carlo derived dosimetric parameters for the two source types were utilised [ 1416 ] , alongside published data on the physical properties of the two isotopes . a consecutive series of eight cervix cancer patients were planned for hdr brachytherapy using the hdrplus treatment planning system ( version 2.5 ) , supplied by ez bebig , on ct image data using tg43 calculation algorithm . pujades - claumarchirant et al . have shown the tg43 algorithm approach will provide accuracy within 2% for both co-60 and ir-192 sources . a diagnostic mri scan acquired post - external beam radiotherapy was additionally available in order to improve soft tissue outlining , although this was not acquired with applicators in situ and not directly fused with the ct data set . an experienced consultant clinical oncologist outlined hr - ctv and organs at risk ( oar ) : rectum , sigmoid and bladder on each data set . given this is a planning comparison , it was not considered necessary to have these volumes independently reviewed . this applicator consists of an intrauterine tube ( iu ) and a two - channel - ring . the possible source dwell positions are physically similar to a fletcher - style iu and ovoids with the lateral straight channels being physically housed within a plastic ring of outer diameters 36 to 45 mm . are actually in a straight line rather than curved geometry , as would be anticipated from the physical ring appearance . the planning system library - applicators were used in all treatment plans and the applicators were not moved between the co-60 and ir-192 planning for each patient , to eliminate any uncertainty due to applicator reconstruction . standard loading patterns ( positions and times of source dwells within the applicators ) based on the traditional manchester system were utilised , with relative total dwell - time loadings of iu and ring of typically 1.25 : 1 ( ranging between 1 : 1 and 1.56 : 1 , depending on the length of iu and diameter of ring combination ) , producing typical pear - shaped isodose curves . treatment plans were produced for each patient using co-60 source and then again using ir-192 source in two conditions : ( a ) traditional approach with fixed dwell distribution normalised to 100% of prescription dose at point a and ( b ) optimised dwell patterns prescribed to hr - ctv with manual adjustment of dwell times , but using the same dwell positions and no inverse planning . in the first set of comparisons , plans were produced with identical dwell positions and relative times ( scaled for dose - rate difference ) for the two isotopes , to provide a direct comparison of the dose deposition between co-60 and ir-192 sources , due to inherent differences in the physical properties between the two sources . in the second comparison set , normal treatment planning approaches were applied to evaluate any residual differences in dosimetry in normal clinical use , i.e. optimisation of dwell times and distributions were applied to reduce dose to the rectum ( aim for icru rectal reference point < 67% of prescription dose ) using the the level of optimisation may unintentionally have been different between plans produced with the two isotopes to achieve the desired treatment planning aims : coverage of hr - ctv and minimised dose to oar . to mitigate against this , a single experienced treatment planner produced all treatment plans to ensure the level of plan optimisation and all other parameters were identical between the plans . all patients were actually treated with an optimised co-60 plan ; the work presented here is a retrospective planning study on the comparison of co-60 and ir-192 source delivery . the following quantitative dose - volume parameters were calculated for each plan : hr - ctv d90 ( the dose to 90% of the hr - ctv ) , v100% ( the hr - ctv volume covered by the prescription dose ) , v150% , v200% and v400% ( the hr - ctv volumes covered by the stated percentage of prescription dose ) , and d2cc ( maximum dose to 2 cm ) for each oar . the data was further analysed by comparing a sparing factor , evaluated as the ratio of d2cc for each oar and d90 for the hr - ctv , to make an assessment of the clinical quality of the plan . where appropriate , a paired two - sided t - test was used to assess the statistical significance of the differences between the co-60 and ir-192 plans . the physical source parameters that relate to the clinical use of co-60 and ir-192 hdr sources were compiled . this data was used to evaluate the equivalent source strength of co-60 compared to ir-192 to deliver the same dose , the variation in clinical treatment irradiation times to be expected when using the two isotopes and the recommended source change frequencies . the current cost of co-60 and ir-192 sources from ez bebig were also obtained to evaluate the differing cumulative source replacement costs of using the two isotopes for hdr brachytherapy . a consecutive series of eight cervix cancer patients were planned for hdr brachytherapy using the hdrplus treatment planning system ( version 2.5 ) , supplied by ez bebig , on ct image data using tg43 calculation algorithm . pujades - claumarchirant et al . have shown the tg43 algorithm approach will provide accuracy within 2% for both co-60 and ir-192 sources . a diagnostic mri scan acquired post - external beam radiotherapy was additionally available in order to improve soft tissue outlining , although this was not acquired with applicators in situ and not directly fused with the ct data set . an experienced consultant clinical oncologist outlined hr - ctv and organs at risk ( oar ) : rectum , sigmoid and bladder on each data set . given this is a planning comparison , it was not considered necessary to have these volumes independently reviewed . this applicator consists of an intrauterine tube ( iu ) and a two - channel - ring . the possible source dwell positions are physically similar to a fletcher - style iu and ovoids with the lateral straight channels being physically housed within a plastic ring of outer diameters 36 to 45 mm . are actually in a straight line rather than curved geometry , as would be anticipated from the physical ring appearance . the planning system library - applicators were used in all treatment plans and the applicators were not moved between the co-60 and ir-192 planning for each patient , to eliminate any uncertainty due to applicator reconstruction . standard loading patterns ( positions and times of source dwells within the applicators ) based on the traditional manchester system were utilised , with relative total dwell - time loadings of iu and ring of typically 1.25 : 1 ( ranging between 1 : 1 and 1.56 : 1 , depending on the length of iu and diameter of ring combination ) , producing typical pear - shaped isodose curves . treatment plans were produced for each patient using co-60 source and then again using ir-192 source in two conditions : ( a ) traditional approach with fixed dwell distribution normalised to 100% of prescription dose at point a and ( b ) optimised dwell patterns prescribed to hr - ctv with manual adjustment of dwell times , but using the same dwell positions and no inverse planning . in the first set of comparisons , plans were produced with identical dwell positions and relative times ( scaled for dose - rate difference ) for the two isotopes , to provide a direct comparison of the dose deposition between co-60 and ir-192 sources , due to inherent differences in the physical properties between the two sources . in the second comparison set , normal treatment planning approaches were applied to evaluate any residual differences in dosimetry in normal clinical use , i.e. optimisation of dwell times and distributions were applied to reduce dose to the rectum ( aim for icru rectal reference point < 67% of prescription dose ) using the local isodose shaping the level of optimisation may unintentionally have been different between plans produced with the two isotopes to achieve the desired treatment planning aims : coverage of hr - ctv and minimised dose to oar . to mitigate against this , a single experienced treatment planner produced all treatment plans to ensure the level of plan optimisation and all other parameters were identical between the plans . all patients were actually treated with an optimised co-60 plan ; the work presented here is a retrospective planning study on the comparison of co-60 and ir-192 source delivery . the following quantitative dose - volume parameters were calculated for each plan : hr - ctv d90 ( the dose to 90% of the hr - ctv ) , v100% ( the hr - ctv volume covered by the prescription dose ) , v150% , v200% and v400% ( the hr - ctv volumes covered by the stated percentage of prescription dose ) , and d2cc ( maximum dose to 2 cm ) for each oar . the icru reference point doses to oars and point a were also calculated . a qualitative visual assessment of the variation in isodose line positions was also undertaken . the data was further analysed by comparing a sparing factor , evaluated as the ratio of d2cc for each oar and d90 for the hr - ctv , to make an assessment of the clinical quality of the plan . where appropriate , a paired two - sided t - test was used to assess the statistical significance of the differences between the co-60 and ir-192 plans . the physical source parameters that relate to the clinical use of co-60 and ir-192 hdr sources were compiled . this data was used to evaluate the equivalent source strength of co-60 compared to ir-192 to deliver the same dose , the variation in clinical treatment irradiation times to be expected when using the two isotopes and the recommended source change frequencies . the current cost of co-60 and ir-192 sources from ez bebig were also obtained to evaluate the differing cumulative source replacement costs of using the two isotopes for hdr brachytherapy . figure 1 illustrates a simple comparison of the isodose lines calculated when using co-60 and ir-192 sources for a typical hdr cervix treatment , with ez bebig two - channel split ring and iu applicator , with identical loading for the two sources , prescribed to point a ( condition [ a ] in method ) . the figure shows the effect of the inherent differences in the two isotopes on the resulting dose distributions . differentiation between the plans was observed in isodose shifts of up to 4.0 mm superior to the intrauterine applicator and up to 2.0 mm posterior to the ring applicator , both with co-60 isodoses further from the applicator than the equivalent ir-192 lines . similar dose enhancements were observed along the applicator axes inferior and posterior , although of smaller magnitude . in all other regions the isodose lines for ir-192 were found to be further from the applicator than co-60 lines , by several mm , with divergence increasing with escalating distance from the applicators ; average of 1.0 mm at 30% isodose , approximately 20 mm to 30 mm from the applicator and 3.0 mm at 10% isodose level , approximately 40 mm to 50 mm from the applicator ( from fig . 2 , discussed later , it can be seen that the radial dose function , g(r ) , is larger for ir-192 than for co-60 in these regions ) . comparison of isodose lines ( 5 , 10 , 20 , 30 , 50 , 100 and 200% of prescription dose ) for co-60 source ( solid lines ) and ir-192 source ( dashed lines ) for a standard hdr cervix treatment , with identical dwell positions and relative times , based on manchester loading , normalised to 100% of prescription dose at point a , shown in sagittal projection . ( condition [ a ] in method ) dose distribution around the ir-192 and co-60 sources in a uniform unit - density medium . dose rate variation as a function of distance from the sources , normalised at 2.0 cm , ( a ) perpendicular to the source axis and ( b ) along the source axis . ( c ) anisotropy function variation with polar angle around the sources , at 2.0 cm from the source centre , with 0 degrees at the distal source tip as a result of these differences , in all treatment plans considered , co-60 plan produced higher - dose lobes along the extension of the applicator axes , delivering locally up to 10% greater dose within the rectum compared to ir-192 plan . consistently lower doses were delivered from co-60 plans to regions more distant from the applicators , including distant portions of the bladder , rectum outside the high - dose lobe and sigmoid , by up to 1.5% compared to ir-192 plans . quantitative analysis of the difference in point - dose ( icru ) and dose - volume parameters ( gec - estro ) between co-60 and ir-192 plans is given in table 1 : ( a ) for plans prescribed to point a with identical loading and ( b ) prescribed to hr - ctv with clinically optimised loading . in all plans , optimisation of the dwell times reduces the dose differences apparent between co-60 and ir-192 plans in the identical loading case . comparison of point and dose - volume parameters for the treatment plans of eight patients produced using co-60 and ir-192 sources , ( a ) prescribed to point a with identical dwell distribution , and ( b ) prescribed to the hr - ctv with typical clinical optimisation . ( d2cc is expressed as the physical brachytherapy dose ) for the plans prescribed to point a with identical loading , table 1 ( a ) there were statistically significantly differences ( p < 0.01 ) in all of the volume parameters for hr - ctv between co-60 and ir-192 plans , with co-60 delivering higher doses in each case , with a mean of 2.4% for v100% , 5.9% for v150 , increasing to 22.1% for v400% . there was also no significant difference in the bladder d2cc or icru bladder reference point nor the sigmoid d2cc , between co-60 and ir-192 plans . however , the rectum d2cc and icru rectal reference point both show statistically significant increased doses from co-60 compared to ir-192 plans ( + 3.3% ( p < 0.01 ) and + 2.2% ( p = 0.03 ) , respectively ) . for the plans prescribed to the hr - ctv with clinically optimised loading , table 1 ( b ) there was no significant difference in the hr - ctv v100% and d90gy nor any oar d2cc . there was significant difference in the higher - percentage dose volume parameters , with co-60 delivering larger dose than ir-192 for by 4.4% at v150% , increasing to 11.6% at v400% . figure 3 presents the bladder , rectum and sigmoid sparing factors ( evaluated as d2cc / d90 ) for co-60 and ir-192 plans for the eight patients , with all plans prescribed to hr - ctv with clinical dwell - time optimisation ( condition [ b ] in method ) . the p - value of a paired t - test was just below statistical significance for a comparison of co-60 and ir-192 sparing factors for the rectum ( p = 0.058 ) , with means of 0.88 and 0.85 , respectively . there was a statistically significant difference for the sigmoid sparing factor ( p = 0.05 ) , but with a small magnitude difference in the mean values , 0.62 and 0.63 for co-60 and ir-192 , respectively . care must be taken in interpretation of this statistical analysis due to the relatively small sample size ( n = 8) . comparison of sparing factors calculated for the bladder , rectum and sigmoid in eight cervix cancer patient plans , prescribed to hr - ctv , using co-60 and ir-192 hdr sources . ( condition [ b ] in method )
table 2 provides a compilation of the physical source parameters relevant to the clinical use of co-60 and ir-192 hdr sources . the equivalent source strength of co-60 to deliver the same dose as ir-192 was evaluated using simple calculations with the above data : according to the tg43 formulism , the absorbed dose is proportional to the source strength multiplied by the dose rate constant multiplied by time . when considering source activity , the dose is proportional to activity multiplied by air kerma rate constant , multiplied by dose rate constant , multiplied by time . hence , using the data in table 2 , for hdr brachytherapy , 1 gbq of a co-60 source delivers the same dose as 2.77 gbq of an ir-192 source ( although source strength should be used , nominal activity is still often quoted and hence referred to here for completeness ) . further , the required irradiation treatment time for a new co-60 source is larger by a factor of 1.8 compared to a new ir-192 source , based on the typical initial source activities given in table 2 . the required irradiation time for brachytherapy treatments increases as the sources decay . due to the different decay half - lives ( t1/2 ) and the different initial conditions , the variation in required irradiation time with time for the two sources 4 , where co-60 source is replaced at five years and ir-192 sources are changed every four months . for these source change frequencies , the average irradiation times per patient and similarly the total cumulative hdr irradiation time are around 25% greater for co-60 than ir-192 . however , if the co-60 source is replaced at four years , the average increase in treatment time compared to ir-192 reduces to 15% . there may also be a radiobiological effect that favours replacement at four years rather than five . indicated that cell repair of sub - lethal damage starts at around 30 minutes after irradiation . hence , longer treatment times may decrease the effect of radiation damage due to the initiation of repair during exposure . if a co-60 source is changed every four years , the total treatment time never exceeds that required by an ir-192 source , as shown in fig . the differing energy of gamma emissions from the two isotopes affects the dose distribution around the sources . the radial dose function , which accounts for the effects of absorption and scatter in the medium , for ir-192 source provides higher values than for co-60 source within the region of interest in hdr brachytherapy , as shown in fig . 2a , up to approximately 25 cm from the sources ( the radial dose function evaluation depends on the size of the phantom used in monte carlo simulations , which were equivalent in the data presented ) . this is due to increased scatter of ir-192 emissions compared to co-60 , due to their lower energy . however , the variation in radial dose function of the two sources is dominated by the geometric factor , which includes inverse - square law dose reduction ( and effects of line source activity distribution ) . these physical properties therefore result in negligible differences between the two sources in dose - rate with distance from the source , perpendicular to the source axis , as shown in fig . 2a . the variation in dose - rate with distance , along the source axis , 2b . the co-60 and ir-192 sources have a different magnitude of variation of dose - rate with polar angle around their line source geometries , due to relatively more self - absorption within the source for the lower energy emissions of ir-192 compared to co-60 . 2c , with a 40% reduction in anisotropy function along the source axis ( 0 degree polar angle ) , at 2.0 cm from the source , for ir-192 compared to co-60 . comparison of total treatment irradiation times for co-60 and ir-192 sources for a typical cervix hdr treatment , as a function of time over a six year period , with co-60 source change at five years and ir-192 source change at four months selected physical properties of ir-192 and co-60 isotopes and hdr sources : mean energy ( e , mean ) and energy range ( e , range ) of gamma radiation , half - life time ( t1/2 ) , dose rate constant ( . ) , air kerma rate constant ( k ) , typical initial source activity of hdr sources ( a ) , and first tenth - value layer in concrete ( tvlconcrete ) . ( from national nuclear data centre ) the cost of a co-60 hdr source is approximately 5 times greater than the cost of an equivalent ir-192 source ( ez bebig , at 2011 ) . if typical source replacement frequencies are used , changing co-60 source at five years and ir-192 source every four months , an hdr brachytherapy treatment unit would require fifteen ir-192 sources for each co-60 source . the total cost of these sources is 275% greater for ir-192 than for co-60 , an increase of approximately forty - five thousand euros over five years . the additional costs of transportation and engineer - installation of the sources increase the differential total costs by in excess of five thousand euros over five years . figure 1 illustrates a simple comparison of the isodose lines calculated when using co-60 and ir-192 sources for a typical hdr cervix treatment , with ez bebig two - channel split ring and iu applicator , with identical loading for the two sources , prescribed to point a ( condition [ a ] in method ) . the figure shows the effect of the inherent differences in the two isotopes on the resulting dose distributions . differentiation between the plans was observed in isodose shifts of up to 4.0 mm superior to the intrauterine applicator and up to 2.0 mm posterior to the ring applicator , both with co-60 isodoses further from the applicator than the equivalent ir-192 lines . similar dose enhancements were observed along the applicator axes inferior and posterior , although of smaller magnitude . in all other regions the isodose lines for ir-192 were found to be further from the applicator than co-60 lines , by several mm , with divergence increasing with escalating distance from the applicators ; average of 1.0 mm at 30% isodose , approximately 20 mm to 30 mm from the applicator and 3.0 mm at 10% isodose level , approximately 40 mm to 50 mm from the applicator ( from fig . 2 , discussed later , it can be seen that the radial dose function , g(r ) , is larger for ir-192 than for co-60 in these regions ) . comparison of isodose lines ( 5 , 10 , 20 , 30 , 50 , 100 and 200% of prescription dose ) for co-60 source ( solid lines ) and ir-192 source ( dashed lines ) for a standard hdr cervix treatment , with identical dwell positions and relative times , based on manchester loading , normalised to 100% of prescription dose at point a , shown in sagittal projection . ( condition [ a ] in method ) dose distribution around the ir-192 and co-60 sources in a uniform unit - density medium . dose rate variation as a function of distance from the sources , normalised at 2.0 cm , ( a ) perpendicular to the source axis and ( b ) along the source axis . ( c ) anisotropy function variation with polar angle around the sources , at 2.0 cm from the source centre , with 0 degrees at the distal source tip as a result of these differences , in all treatment plans considered , co-60 plan produced higher - dose lobes along the extension of the applicator axes , delivering locally up to 10% greater dose within the rectum compared to ir-192 plan . consistently lower doses were delivered from co-60 plans to regions more distant from the applicators , including distant portions of the bladder , rectum outside the high - dose lobe and sigmoid , by up to 1.5% compared to ir-192 plans . quantitative analysis of the difference in point - dose ( icru ) and dose - volume parameters ( gec - estro ) between co-60 and ir-192 plans is given in table 1 : ( a ) for plans prescribed to point a with identical loading and ( b ) prescribed to hr - ctv with clinically optimised loading . in all plans , optimisation of the dwell times reduces the dose differences apparent between co-60 and ir-192 plans in the identical loading case . comparison of point and dose - volume parameters for the treatment plans of eight patients produced using co-60 and ir-192 sources , ( a ) prescribed to point a with identical dwell distribution , and ( b ) prescribed to the hr - ctv with typical clinical optimisation . ( d2cc is expressed as the physical brachytherapy dose ) for the plans prescribed to point a with identical loading , table 1 ( a ) there were statistically significantly differences ( p < 0.01 ) in all of the volume parameters for hr - ctv between co-60 and ir-192 plans , with co-60 delivering higher doses in each case , with a mean of 2.4% for v100% , 5.9% for v150 , increasing to 22.1% for v400% . there was also no significant difference in the bladder d2cc or icru bladder reference point nor the sigmoid d2cc , between co-60 and ir-192 plans . however , the rectum d2cc and icru rectal reference point both show statistically significant increased doses from co-60 compared to ir-192 plans ( + 3.3% ( p < 0.01 ) and + 2.2% ( p = 0.03 ) , respectively ) . for the plans prescribed to the hr - ctv with clinically optimised loading , table 1 ( b ) there was no significant difference in the hr - ctv v100% and d90gy nor any oar d2cc . there was significant difference in the higher - percentage dose volume parameters , with co-60 delivering larger dose than ir-192 for by 4.4% at v150% , increasing to 11.6% at v400% . figure 3 presents the bladder , rectum and sigmoid sparing factors ( evaluated as d2cc / d90 ) for co-60 and ir-192 plans for the eight patients , with all plans prescribed to hr - ctv with clinical dwell - time optimisation ( condition [ b ] in method ) . the p - value of a paired t - test was just below statistical significance for a comparison of co-60 and ir-192 sparing factors for the rectum ( p = 0.058 ) , with means of 0.88 and 0.85 , respectively . there was a statistically significant difference for the sigmoid sparing factor ( p = 0.05 ) , but with a small magnitude difference in the mean values , 0.62 and 0.63 for co-60 and ir-192 , respectively . care must be taken in interpretation of this statistical analysis due to the relatively small sample size ( n = 8) . comparison of sparing factors calculated for the bladder , rectum and sigmoid in eight cervix cancer patient plans , prescribed to hr - ctv , using co-60 and ir-192 hdr sources . table 2 provides a compilation of the physical source parameters relevant to the clinical use of co-60 and ir-192 hdr sources . the equivalent source strength of co-60 to deliver the same dose as ir-192 was evaluated using simple calculations with the above data : according to the tg43 formulism , the absorbed dose is proportional to the source strength multiplied by the dose rate constant multiplied by time . when considering source activity , the dose is proportional to activity multiplied by air kerma rate constant , multiplied by dose rate constant , multiplied by time . hence , using the data in table 2 , for hdr brachytherapy , 1 gbq of a co-60 source delivers the same dose as 2.77 gbq of an ir-192 source ( although source strength should be used , nominal activity is still often quoted and hence referred to here for completeness ) . further , the required irradiation treatment time for a new co-60 source is larger by a factor of 1.8 compared to a new ir-192 source , based on the typical initial source activities given in table 2 . the required irradiation time for brachytherapy treatments increases as the sources decay . due to the different decay half - lives ( t1/2 ) and the different initial conditions , the variation in required irradiation time with time for the two sources 4 , where co-60 source is replaced at five years and ir-192 sources are changed every four months . for these source change frequencies , the average irradiation times per patient and similarly the total cumulative hdr irradiation time are around 25% greater for co-60 than ir-192 . however , if the co-60 source is replaced at four years , the average increase in treatment time compared to ir-192 reduces to 15% . there may also be a radiobiological effect that favours replacement at four years rather than five . indicated that cell repair of sub - lethal damage starts at around 30 minutes after irradiation . hence , longer treatment times may decrease the effect of radiation damage due to the initiation of repair during exposure . if a co-60 source is changed every four years , the total treatment time never exceeds that required by an ir-192 source , as shown in fig . 4 and any potential effect can be mitigated . the differing energy of gamma emissions from the two isotopes affects the dose distribution around the sources . the radial dose function , which accounts for the effects of absorption and scatter in the medium , for ir-192 source provides higher values than for co-60 source within the region of interest in hdr brachytherapy , as shown in fig . 2a , up to approximately 25 cm from the sources ( the radial dose function evaluation depends on the size of the phantom used in monte carlo simulations , which were equivalent in the data presented ) . this is due to increased scatter of ir-192 emissions compared to co-60 , due to their lower energy . however , the variation in radial dose function of the two sources is dominated by the geometric factor , which includes inverse - square law dose reduction ( and effects of line source activity distribution ) . these physical properties therefore result in negligible differences between the two sources in dose - rate with distance from the source , perpendicular to the source axis , as shown in fig . 2a . the variation in dose - rate with distance , along the source axis , the co-60 and ir-192 sources have a different magnitude of variation of dose - rate with polar angle around their line source geometries , due to relatively more self - absorption within the source for the lower energy emissions of ir-192 compared to co-60 . 2c , with a 40% reduction in anisotropy function along the source axis ( 0 degree polar angle ) , at 2.0 cm from the source , for ir-192 compared to co-60 . comparison of total treatment irradiation times for co-60 and ir-192 sources for a typical cervix hdr treatment , as a function of time over a six year period , with co-60 source change at five years and ir-192 source change at four months selected physical properties of ir-192 and co-60 isotopes and hdr sources : mean energy ( e , mean ) and energy range ( e , range ) of gamma radiation , half - life time ( t1/2 ) , dose rate constant ( . ) , air kerma rate constant ( k ) , typical initial source activity of hdr sources ( a ) , and first tenth - value layer in concrete ( tvlconcrete ) . ( from national nuclear data centre ) the cost of a co-60 hdr source is approximately 5 times greater than the cost of an equivalent ir-192 source ( ez bebig , at 2011 ) . if typical source replacement frequencies are used , changing co-60 source at five years and ir-192 source every four months , an hdr brachytherapy treatment unit would require fifteen ir-192 sources for each co-60 source . the total cost of these sources is 275% greater for ir-192 than for co-60 , an increase of approximately forty - five thousand euros over five years . the additional costs of transportation and engineer - installation of the sources increase the differential total costs by in excess of five thousand euros over five years . from the analysis of eight patients treatment plans , using a 3d image - based approach , only small differences in dose distribution were observed when using either co-60 or ir-192 isotopes . variations were seen along the extension of the applicator axes , with co-60 delivering several percent higher doses than ir-192 , due to reduced anisotropy variations compared to ir-192 , because of less self- absorption within the line source at the higher gamma emission energies . when treatment plans were prescribed to point a and identical manchester - based loading patterns were used , there was a small , but statistically significant increase in volume of hr - ctv covered by the prescription dose , as well as a small increase to the rectum oar , when using co-60 rather than ir-192 . when treatment plans were prescribed to the hr - ctv and dwell times were optimised , there was no statistically significant difference in any oar parameter nor in the hr - ctv v100% coverage between the two isotopes . the small , but statistically significant dose distribution differences seen when comparing identically - loaded treatment plans for co-60 and ir-192 , which are a function of the inherent physical differences of the sources , are reduced to being not statistically significant in typical clinical treatment planning , when dwell - time optimisation is applied . in comparing co-60 ( ez bebig ) and ir-192 ( nucletron ) hdr sources using a 2d image - based approach and identical loading patterns , park et al . reported an average decrease of 1.140.61% to the icru bladder reference point and an average increase of 0.831.48% to the icru rectal reference point , when using co-60 rather than ir-192 . these findings are consistent with the work presented here in terms of point a prescribing , icru reference points and identical source loading . however , although of technical interest , the dose differences for identical loading and dwell - time patterns , such as that reported by park et al . and in this work under condition a are not of particular clinical significance for modern treatment planning . of more significance are the clinical treatment plans that can be produced when optimisation of source dwells is permitted , which is a standard practice for modern brachytherapy , as reported in this work under condition b. differences between co-60 and ir-192 were then reduced to 0.4% ( sd = 2.6 ) for bladder d2cc and 0.9% ( sd = 3.3 ) for rectal d2cc , neither of which were statistically significant . the generally quoted quality metrics of hr - ctv d90gy , v100% and oar d2cc do not show significant differences between the two isotopes . however , in all plans , the hr - ctv volume covered by 150% , 200% and 400% was significantly different , with co-60 always delivering greater coverage . there is some debate on the clinical significance of over - dose regions , indeed prabhakar has discussed several studies in which an increase in high dose regions may be a disadvantage . there was no significant difference in the bladder or rectum sparing factors , evaluated as d2cc / d90 , between the two isotopes for any of the considered plans . even when rectal doses were a few per cent higher with co-60 compared to ir-192 , for the identical dwell - loading plans , the increase in rectal dose were consistent with increased dose to the hr - ctv ; the sparing factor ratio being unchanged . co-60 may deliver a small increased dose to the rectum and small enhancement of the target overdose volume compared to ir-192 , depending on planning technique employed . however , the differences in volume and point doses in the treatment plans for co-60 and ir-192 were judged to be clinically not significant , especially in context of more significant effect of other variables that can be modified in hdr brachytherapy treatment planning , including prescribing method , loading patterns , extent of dwell - time optimisation , application of image - guidance techniques , choice of applicator , use of supplementary interstitial needles , etc . strohmaier also concluded there to be insignificant clinical differences between the two isotopes based on analyses using icru point doses and with 2d image - based treatment planning . it is apparent that the inherent differences between the two isotopes , which itself lead to small differences in dose distribution , can be overcome by application of treatment planning optimisation techniques . the key physical parameters for co-60 and ir-192 hdr sources have been presented and analysed to deduce clinically relevant information for use in brachytherapy treatments . it is essential that this data is fully understood to mitigate the risk of any treatment error in moving from ir-192 to co-60 . co-60 and ir-192 sources of identical shape and construction show practically identical dose distributions despite definite differences in the key physical characteristics of the isotopes , particularly emission spectra . this is because of the overwhelming effect of geometry in brachytherapy and small differences in radial dose distributions and anisotropy . in terms of economics , there is significant financial saving in co-60 source replacements every five years compared to ir-192 replacements every four months , amounting to more than fifty thousand euros over five years ( at 2011 ) . the reduced frequency of source changes also reduces the physics calibration and quality control time , reducing the overall equipment down - time and required support time by approximately 40% . however , the initial cost of environmental radiation shielding is greater for co-60 than ir-192 , due to the higher energy emissions . the significantly longer operating life of the co-60 source requires enhanced mechanical reliability over an equivalent ir-192 source . this may have implications for a full - circle single - channel ring applicator due to increased mechanical wear on the source cable when traversing a small radius ring . the irradiation time per patient , over five year use of a co-60 source , is on average 25% greater than for ir-192 sources . however , if patient set - up time and ancillary activities are included in the total patient treatment time , which are of course independent of source type , the percentage increase of total patient - time for co-60 compared to ir-192 is reduced to approximately 10% . based on the 3d dosimetric analysis of patient plans considered in this study , we have shown that co-60 can be used as an effective substitute for the more common ir-192 sources in hdr brachytherapy for cervix cancer , with no clinically significant differences evident in the resulting treatment plan dose distributions , especially when treatment planning dwell - time optimisation is utilised . the choice of isotope is certainly less significant than other variables in brachytherapy , including choice of prescribing method , extent of image - guidance or dose optimisation techniques . the inherent physical differences between the sources , leads to reduced anisotropy deviations with co-60 compared to ir-192 at high polar angles . this causes small increases in dose deposition along the extension of the source axis with co-60 , which may increase rectal doses for identical source loading patterns compared to ir-192 . co-60 treatment plans delivered small increases in target coverage ( v100 ) ( p < 0.01 ) for standard loading and equivalent target coverage for optimised plans , compared to ir-192 . there was no statistically significant difference in d90 between the two isotopes ( p 0.4 ) . increases in the overdose volume ( v150 to v400 ) ( p = 0.03 to < 0.01 ) were observed for co-60 compared to ir-192 for both standard loading and optimised plans . there are significant logistical and financial benefits of using the longer half - life co-60 source . for the same dose - rate , a co-60 source requires only 36% of the activity of an ir-192 source . for typically supplied source strengths , the total treatment irradiation times will be on average 25% greater duration with co-60 than ir-192 , over the lifetime of the source , however the overall patient attendance time will be approximately 10% greater due to ancillary activities independent of actual irradiation . | purposeto evaluate whether co-60 is equivalent to ir-192 for hdr cervical brachytherapy , through 3d - dvh dose comparisons in standard and optimised plans .
previous studies have only considered 2d dosimetry , point dose comparisons or identical loading .
typical treatment times and economics are considered.material and methodsplans were produced for eight cervix patients using co-60 and ir-192 sources , ct imaging and iu / two - channel - ring applicator ( eckert ziegler bebig ) .
the comparison was made under two conditions : ( a ) identical dwell positions and loading , prescribed to point a and ( b ) optimised source dwells , prescribed to hr - ctv .
this provided a direct comparison of inherent differences and residual differences under typical clinical plan optimisation .
the dvh ( target and oar ) , icru reference points and isodose distributions were compared .
typical treatment times and source replacement costs were compared.resultssmall differences ( p < 0.01 ) in 3d dosimetry exist when using co-60 compared to ir-192 , prescribed to point a with identical loading patterns , particularly 3.3% increase in rectum d2cc . no significant difference was observed in this parameter when prescribing to the hr - ctv using dwell - time optimisation .
there was no statistically significant difference in d90 between the two isotopes .
co-60 plans delivered consistently higher v150% ( mean + 4.4% , p = 0.03 ) and v400% ( mean + 11.6% , p
< 0.01 ) compared to ir-192 in optimised plans .
differences in physical source properties were overwhelmed by geometric effects.conclusionsco-60 may be used as an effective alternative to ir-192 for hdr cervix brachytherapy , producing similar plans of equivalent d90 , but with logistical benefits . there is a small dose increase along the extension of the source axis when using co-60 compared to ir-192 , leading to small rectal dose increases for identical loading patterns .
this can be eliminated by planning optimisation techniques .
such optimisation may also be associated with increases in the overdose volume ( v150-v400 ) with co-60 compared to ir-192 . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
neurocognitive and concussion like sickness behaviour is a cluster of signs and symptoms following a traumatic brain injury or systemic infections . these signs and symptoms such as headaches , dizziness , neuropsychiatric manifestations , and cognitive impairment are usually deficits in somatic and behavioural domains . literature shows that impairment in mental functions following traumatic event has a common biological origin in the form of neuroinflammation , which triggers a complex cascade of events such as activation of inflammatory cells and proteins and expression of cytokines [ 1 , 3 ] . these inflammatory events lead to unavoidable brain damage such as alteration in hippocampal cholinergic function , which mediate changes in cognition and behaviour . the circumstantial data suggests that proinflammatory cytokines may play a role in instigating long - term cognitive and depressive like behaviour by infiltrating neurological tissue in individuals [ 4 , 5 ] . cytokines have been studied to assess neurologic injury in various surgeries , traumas , infections , strokes , neuropsychiatric disorders , and autoimmune diseases such as multiple sclerosis [ 6 , 7 ] . postoperative cognitive deficits such as impairment of recent memory , concentration , language comprehension , and social integration have been reported in 25.8% of patients one week after the surgery and in 9.9% of patients three months after the surgery . [ 9 , 10 ] reported that neurocognitive decline ( ncd ) is a common complication with a prevalence up to 50% . the postoperative cognitive deficits also depend on type of surgery , medications , and preexisting medical conditions . the cognitive deficits after trauma and after operation are associated with significant decline in patient 's quality of life , prolonged hospitalization , and increased overall morbidity and mortality . in our opinion the overexpression of proinflammatory cytokines in muscular trauma directly influences the hippocampal dependent long - term potentiation and memory , that is , spatial memory , attention , executive function , object recognition , and contextual fear conditioning and synaptic plasticity . the higher cognitive processes rely heavily on learning and memory processes but their relationship with cytokines remains poorly understood.in this review we proposed cytokines and neuroinflammatory model of neurocognitive symptoms in trauma situation . the main research question of the current study is whether there is an association of muscular il-6 , il-1 , tnf , and other inflammatory mediators with neurocognitive impairment when released as result of the trauma or perioperatively . our main hypothesis is that il-6 , tnf , il-1 , and other inflammatory mediators released in muscular , orthopedic trauma or perioperative conditions are associated with neurocognitive impairment and concussion like illness and are not merely the result of anesthesia or medications . the purpose of this review is to systematically evaluate the literature and to clarify this entrenched belief . in our opinion , this hypothesis has implications for the pathogenesis and treatment of cognitive psychosomatic deficits in the trauma and postoperatively . the mc master university database using ovid / medline database was searched for articles published between 1946 and july 2013 using the following combination of the terms : cognitive impairment , traumatic brain injury , cytokines ( il-1 , il-6 , il-8 , and tnf - a ) , neuroinflammation , concussion like symptoms , blood brain barrier , systemic inflammatory response , and polytrauma . all articles were published in peer - reviewed journals , reporting original data on cytokines and systemic inflammatory response . all the key words were used by using mesh words and were initially combined by using or . the words in each category such as neuroinflammation , cytokines , and cognition our initial data search showed gave us 303447 in neurocognitive domains , 396588 in muscle and peripheral injury group , and 715522 in neuroinflammation category . on combing with and the selection was further limited to human and english language , which gave us 172 published articles . for articles review , i followed the prisma and second chapter of 3rd edition of clinical epidemiology in clinical research . we also excluded articles that studied cytokines response in nontraumatic causes such as stroke , sah , hiv , or infections , cancers , and immunotherapy . out of those 172 published articles , 9 articles were selected to support the systemic effects of the cytokines , 23 articles to support the cognitive and behavioural symptoms that can be explained secondary to cytokines , and 21 articles to support the evidence that cytokines are related to peripheral trauma . clinical studies of peripheral blood or autopsy specimens show elevated increases in cytokines tnf- , il-1 , and il-6 in serum and cerebrospinal fluid ( csf ) of patients with mild to moderate late - onset alzheimer disease ( ad ) [ 11 , 12 ] . simultaneously , age - associated changes in glial reactivity may predispose individuals to exacerbated neuroinflammatory cytokine responses that are permissive to cognitive and behavioural complications . it has been suggested that surgical tissue trauma and stress response induce perioperative nonspecific inflammatory response . il-6 response to injury is robust , being demonstrated across many types of injuries including muscle , skin , bone , lung , and fat . elevated il-6 , in a lumbar decompression surgery , in the first 24 hours is associated with cognitive deficits and prolonged hospital stay . hogevold et al reported that chemical mediators particularly il-6 , ck , tnf - alpha and il-1 are strongly correlated with muscular injury response and surgery , which supports our opinion . found that only elevations in il-6 , s100b , were associated with cognitive impairment and delirium , following hip fracture surgery [ 17 , 18 ] . perioperative increase in crp and inflammatory cytokines such as il-1 and -10 is associated with neurocognitive deficits ( ncd ) in patients after cardiopulmonary bypass [ 6 , 19 ] . reported elevated levels of inflammatory biomarkers in csf as predictor of cognitive decline in coronary artery bypass surgery . haas has found that professional amateur sports athletes , whiplash , and polytrauma patients show neurocognitive weakness in the absence of brain injuries , gram negative pathogenesis , infections , cerebrovascular disease , and neurodegeneration . elevated serum il-6 has been shown to correlate with multiorgan failure and death in polytrauma patients [ 22 , 23 ] . , alexander , gunstad and suhr , and iverson and lange reported increased serum concentrations of proinflammatory cytokines , including il-6 , in patients with multiple injuries and a high prevalence of the neurocognitive and behavioural symptoms ( see table 1 ) . in various diseases states , and behavioral syndromes , inflammatory biomarkers have been found to be positively correlated with fatigue [ 4650 , 61 , 62 ] , sleep disturbances and irritability [ 5254 , 63 ] , and irritability . negative changes in mood and impaired learning and memory are significantly correlated with increases in il-6 , tnf- , and il-1 [ 17 , 29 ] . on contrary to the above , there are studies that show that a decrease in peripheral systemic inflammation reduces the neuroinflammation , thus decreasing the sickness induction behaviour and improving cognitive functions . acute and chronic exercises have anti - inflammatory effects , reducing levels of proinflammatory cytokines and crp . exercise as a therapy for pcs seems to be supported by the fact that young athletic individuals have evidence of anti - inflammatory mediators that oppose the actions of il-6 and tnf- . nonsteroidal anti - inflammatory drug ( nsaids ) user has a lower risk and progression of ad and nsaids are already being explored as a treatment for depression . acetylsalicylic acid has already been shown to accelerate remission in individuals who are not responsive to ssris . interestingly , a recent report from tobinick and gross shows a rapid cognitive improvement following perispinal etanercept ( a potent tnf- antagonist ) administration in an alzheimer patient . animal inflammatory models suggest focal dysregulation in cerebrovascular flow in areas important to memory , such as the hippocampus . animals treated with cytokines , such as il-1 or tnf- or ifn-1 , exhibit sickness behaviours , including reduced locomotor activity , diminished social interactions , and diminished consummatory behaviours . transgenic mice expressing il-6 in glial cells show ataxia , seizures , and extensive neurodegeneration . tnf uptake into the hypothalamus is 9 times faster than into the parietal cortex and is not taken up by the striatum or the midbrain . differences also exist between brain and spinal cord transport rates ; for example , the transport rate of il-1 into the spinal cord is about 80% of the brain . variations also occurred among the regions of the spinal cord . the cervical spinal cord was the region with the fastest transport rate for both interferon ( inf ) and tnf . reported that ibuprofen restored learning ability in rats with hepatic encephalopathy induced by portacaval shunts . the posttraumatic blood levels and pharmacological therapy aimed at enhancing the protective cytokines and inhibiting the damaging cytokines have shown improved survival rates in experimental animals . trauma initiates immune reaction , circulating t cells activation , proliferation , and cytokines expression . once activated , cytokines exert inflammatory and compensatory anti - inflammatory process and wound repair and healing mechanisms [ 7375 ] . however their dysregulation and prolonged and excessive inflammatory response to pathophysiological insults lead to secondary injury , immune alteration , and multiorgan failure . a person is more prone to develop cognitive decline following a major muscular injury or surgery as compared to minor injury or laparoscopic surgery [ 77 , 78 ] . after the major trauma or surgery , immune cells such as cd+4 t lymphocytes , peripheral blood mononuclear cells ( pmbc ) , and macrophages are activated [ 79 , 80 ] . cytokines are derived from type i and ii cd+4 helper t cells ( ht ) . type 1 cd+4 ht promote cytokines il-1-alpha and -beta , il-2 , ifn- , and tnf . type ii cd+4 ( ht ) secrete cytokines such as il-4 , il-6 , and il-10 . a balance between the cd+4 ( i and ii ) cells is critical to maintain immune homeostasis ; however , imbalance between the two cell lineages is responsible for antigen presentation to peripheral blood mononuclear cells ( pmbc ) , increase in expression of tnf , il-1 , and il6 , and alteration in antigen presentation and mitogen in trauma patients [ 78 , 82 , 83 ] . these systemic cytokines are involved in cell - to - cell communication and are partially propagated by systemic immune response system ( sirs ) and multiorgan dysfunction ( mods ) . the brain is an immunologically active organ and is in direct communication with the immune and endocrine systems . human brain has il-1 , il-2 , il-6 , and tnf - cytokine receptors at frontal cortex , hippocampus , hypothalamus , cerebellums , and cerebrovascular endothelium . the hippocampal formation and the dentate hilar region are differentially sensitive to injury relative to other regions of the brain , even in the absence of hypoxia or elevated intracranial pressure or without actual neuronal cell death . cytokines alert human brain through immunoneuropsychiatric ( inp ) cascade , secondary to peripheral inflammatory process due to injury . in brain , the peripheral cytokines act as a second messenger and activate calcium , which triggers the blood brain barrier ( bbb ) damage and destruction of tight junctions [ 77 , 85 ] . first is direct or active transport of cytokines across the blood brain barrier through cytokines receptors . second is indirect transport diffusion at the circumventricular region ( cvo ) , where the bbb is incomplete . another indirect mechanism of peripheral cytokines transport is via vagal nerve stimulation of nucleus tractus solitarius ( nts ) in brain stem and then preoptic area of hypothalamus [ 12 , 86 , 87 ] ( figure 1 ) . once in brain , the peripheral cytokines particularly il-1 stimulate the microglia , which further stimulates the endogenous or central cytokines ( il-1 , il-6 , and tnf- production ) . cytokines are pleotropic mediators and exert their effects through complex immune cascades , interaction with complement system , altered excitotoxic glutamate transmission , abnormal neurotransmission , oxidative stress , and nitric oxide production , leading to apoptotic neurodegeneration [ 8890 ] . neuroinflammation influences neuronal and axonal survival [ 9195 ] and alters the central noradrenergic , dopaminergic , tryptophan , and serotonergic neurotransmission in the hypothalamus hippocampus . the cell debris and central stress further induce the central expression of il-1 , il-6 , and tnf in a vicious cycle [ 97 , 98 ] . cytokine known as mediator of physical and psychological stress also alters the hypothalamic - pituitary - adrenal axis and cortisol regulation . peripheral cytokines also exert indirect effects on the cognitions such as disrupting sleep regulation , micronutrient deficiency by appetite suppression , and endocrine interactions . on the other hand as a mediator of bidirectional communication between cns and the peripheral immune system , systemic inflammatory reactions can influence brain function and conversely cns processes may affect distant organs ( figures 2 and 3 ) . the most important and well - studied cytokines after peripheral trauma are il-6 , tumour necrosis factor - alpha ( tnf- ) , il-1 , il-2 , il-8 , and il-4 and recently il-18 , il-12 , and ifn- [ 15 , 30 ] . in the periphery , il-1 , il-6 , and tnf are typically considered proinflammatory , whereas il-4 , il-10 , and il-13 are typically considered anti - inflammatory . the proinflammatory cytokines particularly il-6 are the acute - phase response proteins that contribute to the development and resolution of signs and symptoms of acute and chronic inflammation after soft tissue injury [ 93 , 101 ] ; see table 2 . peripheral il-1 plays important role in neuroendocrine modulation , proliferation , and expression of microglia . peripheral il-1 also alters central release and turnover of norepinephrine , serotonin , dopamine , and cholinergic neurotransmission . il-1 also affects hippocampal neurons and the synaptic plasticity [ 103106 ] , thus inhibiting the long - term potentiation ( ltp ) . peripheral il-6 is acute - phase protein , increases vascular permeability , and induces lymphocytic activation . il-6 once in brain induces microglia and astrocyte activation , which further triggers the release of proinflammatory cytokines . il-6 alters the neuroendocrine neurotransmission , hypothalamic - pituitary - adrenal ( hpa ) axis , and acth release . il-6 impacts cognitive function via effects on synaptic plasticity [ 17 , 110 ] , decline in learning , memory , and long - term potentiation ( ltp ) . it exerts its effects by activating caspases [ 112 , 113 ] , which activate the death signalling pathway via glutamate excitation . tnf alters the synaptic efficacy by upregulating surface expression of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic ( ampa ) receptors and phosphatidylinositol 3 ( pi3 ) kinase - dependent processes , thus causing a decrease in the synaptic inhibition and cognitive impairment . crp is associated with inflammation , impaired endothelial function , and cerebral amyloid deposits in areas important to memory , such as the hippocampus . crp is associated with memory , visuospatial impairment , and disruption of frontal subcortical pathways . il-2 alters dopaminergic transmission and impairs the spatial memory performance via hippocampal neurodegeneration and suppression of long - term potentiation . immunotherapy , using il-2 , induces a depressive like state that may be attenuated by antidepressant treatment . ifn-. ifn - gamma is associated with more general cognitive dysfunction , confusion , psychomotor slowing , parasthesia , visual disorientation anxiety , and depression . ifn induces il-1 , tnf secretion , and depletion of serotonin , which contributes to cognitive and behavioural effects . as previously mentioned , brain regions particularly dorsolateral frontal cortex , hippocampus , hypothalamus , cerebellums , and cerebrovascular endothelium are susceptible to cytokines and neuroinflammation . these brain regions are involved in memory , learning , executive functions , and personality . the manifestations of sickness behaviour include increased somatic complaints , lethargy , sleep disruption , reduced social activity , reduced mobility , anhedonia , decreased learning , anorexia , decreased libido , and neuropsychiatric side effects including depressed mood , poor motivation , and impaired thought processing ; see table 3 . almost all the signs and symptoms of disease , including altered behaviour and neuropsychiatric phenomena , can be accounted for by the actions of immune cell and peripheral cytokines secretions . these profound discoveries have recently been applied to psychosocial disease , schizophrenia [ 35 , 121 ] , and depression [ 121 , 122 ] yielding completely new models for the etiology of these unexplained diseases . there is abundant evidence that peripheral inflammation can worsen or cause the axonal injury and exacerbate preexisting psychiatric disorders as well as the new onset of mood disorders ( depression and mania ) , anxiety disorders , and psychotic disorders . overall , based on the above literature reviews , the peripheral inflammatory response interferes with cognitive function as evidenced by abnormal memory , learning , and inability to develop long - term potentiation in hippocampus . given that similar symptoms may be seen in such diverse situations as perioperatively , after orthopedic or general trauma , perhaps it is time to consider pcs as merely one of many states in which there is an elevation of inflammatory cytokines . this hypothesis certainly opens a room to develop or determine inflammatory markers that might be helpful to address the cognitive weakness in postoperative and trauma patients and to study potential prediction of postsurgical or posttrauma risks and complications . currently , pcs or sickness behaviour such as neurocognitive and behavioural deficits is treated based on the specific symptoms primarily supportive to the individual and as such not treating the underlying cause . despite ongoing research , little progress has been achieved in terms of prevention or management of this problem , largely because of an incomplete understanding of the pathophysiology of cognitive impairment , which is essential to improve outcomes . in our opinion , some aggressive anti - inflammatory measures ( including inflammatory cytokines antagonists or nsaids ) may improve cognitive function in cognitive deficient subjects , particularly in trauma and perioperative patient . in our opinion the same will also be true for the pcs patients and may prevent the long - term neurologic sequelae of tbi , systemic inflammation , including cognitive impairment . on the basis of the above overview , we believe there is sufficient clinical and research evidence to suggest clearly that cognitive impairment is not only limited to concussion , systemic infections and neurodegeneration . furthermore , most pcs symptoms can be explained with current evidence by increased levels of cytokines such as il-1 , il-6 , tnf- , and ifn- , which are all important cytokines after trauma . peripheral cytokines as those due to muscular injury or orthopedic trauma can influence neurotransmission and cause cognitive deficits . there is abundant evidence that there are cytokine - mediated interactions between neurons and glial cells , subserving cognition ( e.g. , cholinergic and dopaminergic pathways ) , and can modulate neuronal and glial cell function to facilitate neuronal regeneration and contribute to cognitive impairment . | besides brain injury and systemic infection , cognitive and concussion like sickness behaviour is associated with muscular trauma and perioperative patients , which represents a major obstacle to daily activities and rehabilitation .
the neuroinflammatory response triggers glial activation and consequently the release of proinflammatory cytokines within the hippocampus .
we review clinical studies that have investigated neurocognitive and psychosomatic symptoms related to muscular trauma and in perioperative conditions . these include impaired attention and executive and general cognitive functioning .
the purpose of this literature review is to focus on the systemic inflammation and the role of proinflammatory cytokines il1 , il6,and tnf and other inflammatory mediators which mediates the cognitive impairment and induces sickness behaviour .
moreover , this review will also help to determine if some patients could have long - term cognitive changes associated with musculoskeletal injuries or as a consequence of surgery and thereby will lead to efforts in reducing that risk . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
in recent years , valence - to - core
x - ray emission spectroscopy ( vtc xes ) has emerged as a powerful probe
of transition - metal active site structure in both biological and chemical
catalysis . the sensitivity of this method to ligand identity has enabled vtc
xes to reveal the presence of bridging oxo ligands in the mn4ca cluster of photosystem ii and to establish
the presence of a central carbide in the femoco cluster of nitrogenase . vtc xes utilizes a high - energy x - ray beam
to create a 1s core hole on a transition - metal photoabsorber followed
by detection of the fluorescence that occurs when valence electrons
refill the core hole . the spectral features that result are the so - called
k and k2,5 peaks ( figure 1 ) , which generally correspond to ligand ns to metal 1s and ligand np to metal 1s
transitions , respectively . originating from orbitals that are dominantly
ligand in character , these features gain intensity through small amounts
of metal np mixing ; hence , vtc xes serves as a probe
of the filled , ligand - localized valence orbitals
of a metal complex . conversely , the related
techniques of xas and exafs probe the unoccupied orbitals and metrics
of nearest neighbor atoms , respectively , providing information that
is complementary to but less inherently ligand selective than that
of xes ( e.g. , exafs can not distinguish among c , n , and o scatterers ) . recently , the ligand sensitivity of vtc spectra has been applied to
assess such factors as the number of co or n2 ligands bound
to a metal center and to quantify the degree of ligand bond activation . schematic of a first - row transition metal k vtc
xes spectrum with the mo origin of the transitions at right . because the k feature
energetics derive principally from ligand ns orbitals ,
the k feature allows ligands to be readily identified
by differences in ionization potentials : shifts of more than 8 ev
occur among c , n , o , and f. this ionization
potential sensitivity also enables these spectra to detect more subtle
structural changes such as protonation events , with clear ( 2 ev ) shifts observed between
oxo and hydroxo species . sensitivity
to protonation is especially important in bioinorganic chemistry ,
in large part because of the ubiquity of proton transfer and the corresponding
challenges associated with identification of single protonation events . in addition to the energy , the intensity of valence - to - core transitions
also carries valuable information . for some time it has been known
that the intensity of these features has an exponential dependence
on metal ligand bond length . as the metal ligand
distance decreases , the amount of metal np mixing
into the valence molecular orbitals increases , thus imparting more
dipole - allowed character to these transitions . this results in intense
k features which are normally rather weak for
complexes with terminal oxo or nitrido ligands . the differences in valence - to - core intensities
have also been used to distinguish between high- and low - spin complexes ,
with the latter having considerably shorter bond lengths and hence
higher valence - to - core intensities . the
sensitivity of valence - to - core xes to ligand identity ( including protonation
state and ionization potential ) and metal ligand bond length
suggests it may be an ideal tool for the identification of dimeric
oxygenated intermediates in biological systems . previous studies have
shown the utility of vtc xes for identifying single protonation events
in a series of structurally related mn dimers . herein , we take a broader view and examine a series of
eight dimeric oxo - bridged fe complexes in which the nature of both
the bridging and supporting ligand framework is varied ( figure 2 ) . our goal is to investigate the generality of
k spectral interpretations on the basis of simple ionization
potential and bond length considerations . the present study verifies
previous observations of the contribution of ligand ionization potential
to the k energies . we have found , however , that the
observed k intensities can not be rationalized in terms
of fe o bond lengths alone . in order to explain the observed
spectral trends , we find that the fe o fe bond angle
must also be considered . this can be understood empirically by invoking
a simple walsh - type diagram for the mixing of the
fe np orbitals with the ligand 2s orbital . these results demonstrate ,
to the best of our knowledge , the first observation of a bond angle
dependence on the intensity of k valence - to - core features . the implications of these results for the identification of oxygenated
intermediates in binuclear iron enzymes are discussed . compounds 18 , shown in figure 2 , were all synthesized according to literature procedures and were characterized by optical absorption spectroscopy . samples for x - ray emission experiments were prepared by grinding the
solid to a fine powder , packing into a 1 mm think aluminum cell , and
sealing with 38 m kapton tape . for temperature - sensitive samples , the above procedure was modified to allow for preparation at reduced
temperatures . in brief , sample packing occurred on an aluminum block
that was partially immersed in a dry ice / acetone bath . after preparation ,
samples were immediately transferred to a liquid n2 filled
dewar for storage . x - ray emission spectra were collected at the c - line
of the cornell high energy synchrotron source with ring conditions
of 5.3 gev and 200 ma . the incident beam energy was set to 9 kev using
upstream multilayers ( with an 90 ev band - pass ) and was calibrated
by setting the first inflection point of a copper foil scan to 8979
ev , providing 3 10 photons / s in a 1
2 mm spot . the sample was positioned at 45
relative to the incident beam and was maintained below 77 k in an
ars helium displex cryostat . emitted x - rays were energy selected using
three to five spherically bent ge(620 ) crystals arranged in rowland
geometry and were detected using a silicon drift detector with a 3
mm vertical slit . data were normalized with respect to the incident
flux using a nitrogen - filled ion chamber just upstream of the sample . a helium - filled flight path was used between the sample , crystal analyzers ,
and detector to minimize signal attenuation . radiation - induced damage
was assessed by collecting successive scans on a single sample spot ,
and multiple spots were used when needed . in the case of 8 , the second scans showed slight evidence
of damage ( figure s1 , supporting information ) ; however , a combined average of both the first and second scans
was not statistically different from the average of only first scans . scans of the k mainline
and valence - to - core region were collected and averaged separately
using pymca before being spliced together . the important quantity to compare between these spectra is the intensity
of the k feature above the background signal ; hence ,
absolute intensities are not reported . energy calibration was achieved
using a reference spectrum of fe2o3 . because
these spectra were collected over several experimental runs , a larger
than normal uncertainty in the calibrated energies exists ( the typical
experimental precision is 0.10.2 ev ) . the estimated error in reported energies is < 0.5 ev . however ,
we note that for the present study the focus is on the intensities
rather than the energies . all dft calculations
were performed using the orca 2.9 or 3.0 quantum chemical suite . geometry optimizations on the compounds studied
were begun from crystal structure coordinates or , where these were
not available , from manually modified structures of closely related
complexes . hypothetical model
complexes featuring identical tacn supporting ligands and variable
fe o fe bond angles were manually constructed from crystallographic
( me3tacn)fe fragments ( webcsd code abawuv ) ; the bond lengths and angles of the fe o fe
cores for these models were fixed during optimization , and all other
atoms were allowed to optimize freely . optimizations were performed
using the bp86 functional and the def2-tzvp basis
set with solvation modeled using the
continuum solvation model ( cosmo ) with
an infinite dielectric constant . an expanded cp(ppp ) basis set was used on fe with an increased integration
accuracy ( grid7 ) . when necessary , the broken symmetry formalism was
used to account for antiferromagnetic coupling between fe centers . xes spectra were calculated according to published procedures ; the energies of the transitions were calculated by taking the differences
between the fe 1s and valence orbital energies , while intensities
were determined by summing the electric dipole , magnetic dipole , and
electric quadrupole contributions to the total oscillator strength . a scalar energy shift of 182.5 ev was applied to all orca - calculated spectra to
align with experiment . the calculated spectra for 18 were broadened using a matlab script that applies a modulated
broadening across the vtc region ( 5 ev hwhm at low energies to 1.25
ev at higher energies ) ; further explanation and details of this modulation
can be found in the supporting information . the spectra for hypothetical compounds were broadened in orca using
a constant gaussian of 2.5 ev . figure 3a presents the vtc xes data for compounds 18 , revealing significant differences in both the k
and k2,5 regions for this series of compounds . given
the wide variation in coordinating ligands and metal ligand
bond lengths , this observation is not surprising , especially since
contributions from both ligand ns and np orbitals are present in the k2,5 region . the
k peak is , however , a relatively pure transition that
originates from the bridging o 2s orbital and thus provides a useful
spectroscopic handle for comparing the fe o(x)fe cores
of these compounds . panel
a shows the experimental spectra and panel b is a zoomed - in view of
the k features ( black background lines have been added
to aid in peak identification ) , while panel c shows the corresponding
calculated spectra . the spectra have been vertically offset for clarity
and arranged according to increasing average fe obridging bond length with shorter distances shown at the top . as discussed in the introduction , the intensity of the k feature
is modulated by the amount of metal np mixing into
the ligand - centered molecular orbitals and the energy of this feature
is governed by the ligand ionization potential . most simply ,
the compounds featuring mono--oxo bridges with short fe o
bonds ( 37 ) would be expected to
possess a k peak at 7092 ev that is dominated
by o 2s contributions . contributions from the supporting ligands would
be significantly less than that from the oxo ligand due to the relatively
longer fe l bond lengths and because the n / o 2s orbitals are
delocalized over the supporting ligands ( figure s3 , supporting information ) . the
intensity is also expected to depend on the number of short fe o
bonds , with the bis--oxo core of 8 giving rise
to a k feature of roughly double the intensity of the
mono--oxo species . in contrast , for compounds possessing
bridging ox ligands ( x = ph , h for 1 and 2 , respectively ) the k feature would be expected to
occur at lower energy ( 7090 ev ) and to have significantly
reduced intensity . both of these changes stem from delocalization
of the o 2s orbital due to intraligand covalent bonding . bonding redirects
the o 2s electron density away from the metal centers and thus reduces
fe np mixing , lowering k intensity . additionally , the resulting bonding orbital is stabilized relative
to that of an isolated oxo ligand , lowering the energy of the transition . the combination of these effects is likely to completely obscure the
k feature beneath the tail of the k mainline . the k regions of the experimental xes spectra , shown
in figure 3b , only partially fulfill these
predictions . the compounds with bridging ox ligands do indeed have
very low ( 2 ) to nonexistent k features
( 1 ) , while 8 with two bridging oxos has
the greatest intensity . for the mono--oxos ( 37 ) , however , deviations from the simple expectations occur . as discussed above , all compounds with a bridging oxo are expected
to have k peaks with intensities that increase as the
bond length decreases ( i.e. , from bottom to top of figure 3 ) . compounds 57 conform to this prediction , but surprisingly , 3 and 4 do not . indeed , these latter compounds have no discernible
k feature at all , despite the fact that they possess
some of the shortest fe obridging bond lengths measured .
arranging the compounds according to average fe o or fe ligand
bond length does not solve this problem , even when controlling for
the number of o atom donors . clearly other factors must be contributing
to the observed spectra beyond bond length and number of donors . for help with understanding these observations , we performed dft
calculations to simulate the experimental spectra ( figure 3c ) , since these calculations have been well - established
as being able to effectively reproduce experiment . these
calculations are in acceptable agreement with experiment with respect
to the number of observed features and their relative energies . for
some compounds , the intensity ratio of the features within k2,5 deviates from experiment ( e.g. , compound 3 ) , which is likely the result of effects not captured in these one - electron
dft calculations , such as charge transfer contributions . furthermore ,
the calculations are known to overestimate the intensity of the k
feature relative to that of k2,5 , and this remains true here . for example , in compound 1 , no k feature is seen experimentally ( in
reality it is likely present but is too low in intensity to be seen
above the tail of the k mainline ) , though a small calculated
feature is present . given this limitation coupled to the very
different coordination spheres of these compounds little additional
information may be extracted from these calculated spectra . the observations seen for the k features of these
compounds can not be rationalized on the basis of the existing understanding
of vtc spectra ; thus , they warrant more systematic investigation .
inspection of table 1 reveals that 3 and 4 have fe o fe core bond angles that
are significantly greater than those for the other mono--oxo
species , leading to the possibility that the bond angle exerts some
control over these spectra . it is well - known that the intensity of
vtc features is governed by the amount of metal np mixing into the ligand orbitals ; therefore , if the bond angle were
to affect these intensities , there should be a molecular orbital based
mechanism which modulates the mixing . to picture this conceptually ,
a walsh - type diagram was employed ( figure 4 ) . from this simple picture , it can be seen that , in a linear complex ,
the o 2s orbital can favorably interact with the fe pz orbital ; however , it lies at a node of the fe px orbital , prohibiting mixing . as the angle
is reduced , however , a new , additional interaction with the fe px orbital also becomes possible , resulting
in greater total fe np character in the o 2s orbital . this is of course a very simplified picture that neglects factors
such as varying ligand field strength and charge donation from the
ligands , but it provides a viable explanation for the observations
seen in the data . the distance quoted here is the average bond distance for every
fe o bond from all ligands as taken from the crystal structures . the two distinct o and oh groups are disordered
in the crystal structure ; therefore , this value is an average over
both . as no crystal structure
is available for this compound , as the fe o fe bond angle decreases
from 180 , additional fe np mixing into the
o 2s becomes possible , thus explaining the low k intensity
seen for 3 and 4 relative to that for the
other mono--oxo species . to test the validity of this walsh diagram and the predictions
it engenders , we turned to dft calculations for a series of hypothetical
mono--oxo compounds that vary only in fe o fe
angle ( figure 5 ) . unlike the compounds studied
experimentally , these simple models vary only in
fe o fe bond angles , allowing for a controlled investigation
of the effects of bond angle without competing changes from the supporting
ligands . fixed core angles of 90 , 120 , 150 , and 180 were chosen ,
along with a bis--oxo species having 90 core bond angles ,
and all fe o bond lengths were set to 1.85 ; all other
atoms were geometry optimized . in this manner , the influence of the
fe o fe bond angle could be deconvoluted from other
geometric changes . calculated xes spectra for these complexes are
shown in figure 6 , and numerical data can be
found in table 2 . structures of the hypothetical
mono--oxo ( top , 150 fe o fe bond angle )
and bis--oxo ( bottom , 90 fe o fe angles )
complexes used to systematically investigate the effect of bond angle
on k intensity . the other mono--oxo species
are identical with that shown except for their fe o fe
angles . o bonds , panel b shows the analogous spectra
for the models with 1.95 bonds , and panel c is an overlay of
all calculated spectra . importantly , there is overlap in the integrated
intensities between these two groups , indicating that area alone can not
be used to judge the bond length or number of bridging oxo ligands
for fe o fe dimers when the precise geometry of these
complexes is unknown . the spin orbitals were not used due to more
extensive orbital mixing that prevented identification of an isolated
o 2s for some compounds . total oscillator strengths were calculated by summing all k
transitions from the spin orbitals to each fe . the contribution to the oscillator strength
due to o was calculated by weighting the individual transition fosc values by the amount of o character present
in the donor mo . from these
calculated spectra it can easily be seen that , even at constant
fe o bond length , the k intensity increases
with decreasing fe o fe bond angle . with a linear complex
as the starting point , this effect is initially small but grows in
magnitude as the angle becomes progressively smaller , with nearly
a 15% increase in k intensity seen for the 90
complex relative to the linear species . perhaps not surprisingly ,
the bis--oxo compound has roughly double the intensity of its
90 mono--oxo congener . this observed increase in intensity
is accompanied by the expected increase in fe np
character mixed into the o 2s orbitals ; thus , decreasing the fe o fe
bond angle allows for more fe np character to mix
into the orbitals of the bridging atoms . importantly , the change in
total oscillator strength is due entirely to changes occurring in
the o transitions , ruling out any influence from changes in bonding
to the supporting ligand . furthermore , inspection of the x , y , and z components
of the calculated emission reveals that , as the oxo translates along
the x axis ( e.g. , to smaller bond angles ) , the x component of the emission increases dramatically while
the z component slightly decreases ( figure 7 ) , leading to an overall increase in k
intensity . such a deconvolution of the px , py , and pz components has been experimentally accomplished for a manganese
nitride complex , whereby the axial nitride was shown to interact dominantly
with the manganese pz orbital . taken together , these computational results
validate the walsh - type picture from figure 4 and support the rationalization that compounds 3 and 4 have k intensities lower than those for the
other mono--oxo species due to their larger fe o fe
bond angles . deconvolutions of the x , y , and z components of the calculated xes spectra
for the 180 ( solid lines ) and 90 ( dashed lines ) models . the x component increases significantly upon bond
angle contraction , while the intensity of the z component
is slightly reduced . finally , to demonstrate the competing influences of bond
length and bond angle on k intensity , we also calculated
vtc spectra for a series of compounds analogous to those in figure 5 but with 1.95 fe o bond lengths . these spectra , along with a comparison to those from the 1.85
compounds , are also found in figure 6 ( panels
b and c , respectively ) . the spectra for these models follow the same
trend as is seen for those with 1.85 fe moreover , the intensities
of these two series overlap , with compounds possessing small angles
and long bond lengths giving spectra with k features
similar to those with large angles but shorter bonds . care must therefore
be taken when attempting to infer structural information from the
intensities of these features . as valence - to - core xes matures as a probe of geometric and electronic
structure , it is finding increasing applications to bioinorganic systems . it
has already been used to distinguish between competing structural
proposals for metalloenzyme active sites : for example , with the identification
of a carbide as the interstitial atom of femoco . beyond mere atom identification , model studies also indicate
that valence - to - core xes shows promise for probing attributes such
as ligand protonation state and , in some cases ,
bond activation . one can expect that , as the information
content of these spectra is further developed , xes will find application
toward an increasing number of bioinorganic systems . one such
target for study by xes is soluble methane monooxygenase ( smmo ) . this
enzyme is responsible for the biological oxidation of methane to methanol ,
and modeling its reactivity has been the subject of significant synthetic
effort . intermediate
q the species that putatively attacks methane has , however , eluded definitive structural characterization . exafs experiments , with their ability to distinguish both first - shell
light atoms and intermetallic distances , have favored a closed , bis--oxo
diamond core ( figure 8) on
the basis of an observed short 2.46 fe fe separation . this assignment is not without some controversy ,
however , as model studies have generally favored an open core bearing
a mono--oxo bridge and a second , terminal oxo ligand ( figure 8) . later exafs measurements on a
similar intermediate in rnr - x have also called into question the bis--oxo
assignment of these structures . clearly ,
additional , complementary probes of these systems are desirable to
help resolve this uncertainty . metrical parameters and computational
models for closed bis--oxo ( left ) and open mono--oxo
( right ) mmo q cores . at first glance , xes , with its sensitivity to ligand environment ,
might be expected to be able to differentiate between these two proposals . however , a closer inspection in light of the current results reveals
that , while the bis--oxo core has longer average bond lengths
than the open core , it also has a significantly smaller fe o fe
angle . as these structural attributes are known to have opposite effects
on k intensity , one can not , a priori , predict what
to expect experimentally . to explore the feasibility of these
experiments , computational models were constructed featuring the proposed
core metrics ( figure 8) . as was done above , h3tacn was used as a supporting ligand
so as not to interfere with the oxygen k features this
is , of course , an idealized situation that would not be present for
the protein itself . the calculated valence - to - core xes spectra are
shown in figure 9 . calculated valence - to - core
xes spectra for the mmo model compounds . as can be seen , apart from a small ( 0.2 ev ) energy
shift , there is very little difference between the k
features of these models . indeed , these calculated spectra are very
similar across the entire valence - to - core region . the small differences
that are seen are unlikely to enable experimental discrimination between
the proposed species , especially given that other o atom donors are
present in the protein . it thus appears that the competing effects
of increasing bond length and decreasing bond angle nearly exactly
cancel out in this case . in this instance , detailed studies of xas
pre - edge structure are more likely to yield fruitful results than
would analysis of k features . in the present study , we have demonstrated that , in addition to
bond length , the bond angle is another structural attribute to which
valence - to - core xes is sensitive . hence , these results indicate that
caution must be used when attempting to infer structural information
from the intensities of valence - to - core features , since bond length
is not the sole determining factor . spectra for compounds with short
bonds and large angles will be very similar to those with long bonds
and small angles ( figure 6 ) . if the experimental
data presented here ( figure 3 ) were used solely
to judge bond lengths , one would arrive at an incorrect conclusion .
rather than detract from these spectra , this new observation instead
adds to the rich information that may be extracted from this spectral
region . | transition - metal
k x - ray emission spectroscopy ( xes ) is a developing technique
that probes the occupied molecular orbitals of a metal complex . as
an element - specific probe of metal centers , k xes is finding
increasing applications in catalytic and , in particular , bioinorganic
systems . for the continued development of xes as a probe of these
complex systems ,
however , the full range of factors which contribute
to xes spectral modulations must be explored . in this report
, an investigation
of a series of oxo - bridged iron dimers reveals that the intensity
of valence - to - core features is sensitive to the fe o fe
bond angle .
the intensity of these features has a well - known dependence
on metal ligand bond distance , but a dependence upon bond angle
has not previously been documented .
herein , we explore the angular
dependence of valence - to - core xes features both experimentally and
computationally . taken together , these results show that , as the fe o fe
angle decreases , the intensity of the k feature increases
and that this effect is modulated by increasing amounts of fe np mixing into the o 2s orbital at smaller bond angles .
the relevance of these findings to the identification of oxygenated
intermediates in bioinorganic systems is highlighted , with special
emphasis given to the case of soluble methane monooxygenase . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
almost
40 years after the first report of their existence , intense
activity aimed at the chemical synthesis of the chlorosulfolipids
( 15 , figure 1 ) began independently and essentially simultaneously in at least
four research groups around the world . apparently purely coincidental ,
this confluence of research might well have stemmed from the fact
that truly novel and unstudied classes of natural product targets
are extremely rare in current times and make very attractive research
problems . since 2009 , the groups of carreira , yoshimitsu , matsuda , and our own have contributed
syntheses of chlorosulfolipids and , in so doing , have taken
what once looked like intractable problems for synthesis and found
multiple creative ways for their construction . with the exception
of carreira s tour de force synthesis of the proposed structure of mytilipin c ( 5 ) that determined
the incorrectness of that structure , all
of the published work to date has been focused on the three structurally
similar chlorosulfolipids mytilipin a ( 3 ) , danicalipin a ( 1 ) , and malhamensilipin
a ( 2 ) . at the outset of
our work , we sought a general strategy toward these three targets ;
however , the unknown relative configuration of danicalipin a and malhamensilipin
a prevented the development of such an approach at the time . our productive
collaboration with the gerwick group unveiled the relative and absolute
configuration of these two lipids and revealed
that a central stereotriad was conserved among
the three lipids ( 13 ) , but that
there were important differences at other centers . indeed , the difference
at c16 between danicalipin a and malhamensilipin a precluded
the direct translation of our successful synthesis of the former to
the latter . the approaches adopted for mytilipin a by carreira and for danicalipin a and malhamensilipin
a by our
group took advantage of alkene oxidation reactions
for the introduction of all of the polar atoms in the stereochemically
rich regions of these targets . however , since shortly after our interest
in the chlorosulfolipids began , we have been keenly interested
in an approach involving diastereoselective carbonyl additions
to ,-dichloroaldehydes . conceptually , this approach
was attractive because these starting materials are easy to access at
least in racemic form and additions to the aldehyde should
be highly stereocontrolled . however ,
the poor stability of the these aldehydes , which eliminate hcl easily ,
prevented our early attempts to use this tactic . to our knowledge ,
only yoshimitsu and co - workers had successfully added nucleophiles
to ,-dichloroaldehydes prior to the work that we describe here . it was that significant
challenge in implementation that led us instead to the alkene oxidation
approach that permitted the first syntheses of danicalipin a and malhamensilipin
a. although effective , our first - generation
syntheses of 1 and 2 were fraught with the
several problems : ( 1 ) the
critical convergent wittig reaction was both poorly stereocontrolled
and somewhat erratic in terms of reproducibility ; ( 2 ) the enantioselective
route to malhamensilipin a could not be applied to danicalipin
a because of a stereochemical difference in the targets ; and ( 3 ) the
routes were longer than we had hoped . in this article , we describe
the evolution of our second - generation strategy that is applicable
to chlorosulfolipids 13 in enantioenriched
forms by virtue of an interesting kinetic resolution of chlorinated cis - vinyl epoxides . this approach also obviates the troublesome
wittig reaction , which is replaced by a convergent z - selective alkene cross metathesis reaction . the results are ( 1 )
for danicalipin a , eight steps racemic , nine steps enantioselective
( previous best 12 steps racemic or 13
steps enantioselective ) ; ( 2 ) for mytilipin
a , seven steps racemic , eight steps enantioselective ( previous best
10 steps racemic or 19 steps enantioselective ) ; and ( 3 ) for malhamensilipin a , 11 steps
formal enantioselective ( previous best was our previous 12-step route ,
which was the only prior synthesis ) . to put the second - generation approach into perspective ,
our first
synthesis of racemic danicalipin a is shown in scheme 1a . as alluded to above , we were aiming for a shorter synthesis
that could be generalized to targets 13 that obviates the troublesome wittig olefination and that takes
advantage of the common stereotriad highlighted in figure 1 . stereospecific anti - dichlorination of either an ( e)- or
a ( z)-allylic alcohol will lead to anti- or syn - dichloroalcohol products , respectively .
assuming high levels of 1,2-stereoinduction , a
haloallylation reaction would afford either syn - halohydrin 15 or cis - vinyl epoxide 16 , depending upon workup conditions . either of these intermediates
could be productive substrates for z - selective alkene
cross metathesis as a replacement for the wittig olefination ; the
products that result would intersect with the late stages of our previous
syntheses . the major impediments to the implementation of this plan
were : ( 1 ) it was not certain that an efficient and stereoselective carbonyl addition to dichloroaldehydes would
be possible ; ( 2 ) there was no obvious way to render the synthesis
enantioselective ; and ( 3 ) z - selective alkene cross
metathesis was , at the time we began this work , very much in its infancy
and was not certain to work on such unusual and potentially reactive
substrates . because of our familiarity with
its late - stage chemistry , we aimed
to first apply our new strategy to an enantioselective synthesis of
danicalipin a. as a result , in the following sections , we will first
discuss the solutions to the three key unknowns described above in
the context of this target . we will then demonstrate the generality
of the approach with the syntheses of all three targets in subsequent
sections . for our new synthetic route , it was necessary to develop conditions
for mild and highly diastereoselective haloallylation
of ,-dichloroaldehydes to establish an efficient
route toward the requisite cis - vinyl epoxide of type 16 . in our earliest studies , attempted grignard , organolithium ,
or alkali metal enolate additions to these aldehydes were met with
failure , as were lewis acid - catalyzed addition of -nucleophiles . while the yoshimitsu group had some success in this area , carreira alludes to similar problems in their disclosure of the
mytilipin a synthesis . on the other hand ,
additions to -chloroaldehydes were generally quite efficient
and often stereoselective ; these outcomes were not surprising given
the lack of elimination pathways and the rather well - known stereocontrol
imparted by -acceptor groups on carbonyl additions . for example , the chloroallylation of -chloroisovaleraldehyde
( 19 ) with ( z)--chloroallylstannane 20(9 ) in the presence of bf3oet2 provided undesired syn , syn-21 with high diastereoselectivity
( scheme 2a ) . not surprisingly , this reaction
type was not successfully extended to electrophiles with -chlorides . in contrast , 19 could be converted to desired anti , syn-21 by chloroallylation
with ( z)--chloroallylborane 22 . however , the base - promoted
epoxide formation surprisingly proceeded with poor site selectivity
to give a mixture of constitutional isomers 23 and 24 . nonetheless , this haloallylborane reactivity could
be extended to ,-dichloroaldehydes ( see below ) ,
and this outcome was the first hint that this type of electrophile
tends to survive the milder conditions associated with closed transition
structure allylations and related reactions . these observations were
important in the eventual discovery that bromoallylaluminum
reagents of type 28(9 ) were
optimal from the perspectives of efficiency , stereoselectivity , and
ease of preparation . an attractive sequence resulted : after dichlorination
of ( e)-2-nonen-1-ol ( 25 ) and careful
oxidation with the dess martin periodinane , bromoallylation
followed by basic workup afforded vinyl epoxide 30 as
a single regioisomer in high yield and with essentially perfect diastereoselectivity
consistent with both the felkin anh and cornforth models ( scheme 2b ) this sequence could produce racemic 30 in multigram scale in about 70% yield from the commercially available
allylic alcohol precursor . because we were clearly
beholden to starting our synthesis from
,-dichloroaldehydes , we required either enantioselective
access to these key intermediates , or a means to resolve them , if
we were to render our synthesis enantioselective . although technology
for asymmetric alkene chlorination is improving , with nicolaou s
recent enantioselective dichlorination of allylic alcohols being particularly noteworthy , there is not
currently a method that would prove economical enough in the preparation
of highly enantioenriched dichloroalcohols to service a natural
product synthesis endeavor of this type . certainly , we spent
some time trying to develop just such a reaction , but with no success . attempts to obtain enantioenriched material via enantioselective dichlorination
with cinchona alkaloid - derived chiral
variants of mioskowski s reagent
( et4ncl3 ) or resolution
of dichlorinated primary alcohols by peptide - catalyzed or enzymatic means were unsuccessful . of course ,
highly effective examples of enzymatic resolution of chiral primary alcohols are few . clearly , either resolution methods of later stage intermediates
or yoshimitsu s elegant stereospecific dichlorodeoxygenation
reactions of epoxides were the most promising
ways to access enantioenriched intermediates . owing to the single
additional step involved in resolutions compared with the multiple
steps involved in the epoxide - based strategy , we took the former approach
to solve our problem . the carreira group developed an asymmetric
variant of their synthesis
of mytilipin a ( 3 ) via ( parallel ) resolution of racemic
dichloride 31 ( eq 1 ) . sharpless asymmetric epoxidation of the allylic
alcohol functional group afforded epoxide 32 in 1.3:1
dr ; the enantiopurity of the desired diastereomer was moderate at
89:11 er.1 we undertook an extensive investigation into
asymmetric carbonyl
additions to ,-dichloroaldehydes using chiral
reagents or catalysts . the kinetic resolutions of racemic ,-dichloroaldehyde
( )-27 via enantioselective haloallylation with
chiral oehlschlager / brown haloallylborane reagents ( 33/34 ) proceeded with poor
enantioselectivity ( scheme 3a ) . according to
the enantioselective chloroallylation procedure of kobayashi , the chiral zinc catalyst derived from the bipyridine
ligand 38 afforded the product ( )-35 in moderate enantiopurity , and useful selectivity factors however , the resolved starting
material , which is always easier to obtain in higher enantiopurity
via kinetic resolution , was unstable
to the reaction conditions and could not be isolated , leaving only
partially resolved product 35 . furthermore , as we showed
in scheme 2a , epoxide formation from ,-dichloroalcohols
of type 35 was not selective , and the bromoallylation
corresponding to that shown in scheme 3b was
never successfully implemented . we next considered resolving racemic vinyl epoxide 30 derived from diastereoselective haloallylation / epoxide
formation
of the ,-dichloroaldehyde ( scheme 2b ) . this type of vinyl epoxide was readily prepared on multigram
scale and should be easily recovered after kinetic resolution . furthermore ,
it has a strong bias for regiocontrol of ring opening ; clearly the
allylic terminus is activated while the other epoxide carbon is deactivated
by the proximal chlorides . therefore , we postulated that some of the
many available enantioselective meso - epoxide desymmetrization
protocols should be plausibly extended to kinetic resolution of substrates
of type 30 . we began with jacobsen s epoxide
opening chemistry , using
highly reactive ( r , r)-(oligosalen)co
catalysts . to the best of our knowledge , resolutions of internal epoxides with the jacobsen system
have not been reported ; however , we felt that the cis - vinyl epoxide might be a close structural mimic of competent cyclic meso - epoxides that are frequently desymmetrized using jacobsen chemistry . surprisingly , substrate 30 proved unreactive toward nucleophiles such as water , phenol , or
benzyl alcohol under published conditions for desymmetrization of meso - epoxides . for reasons that we do not understand , denmark s
catalytic system for desymmetrization of meso - epoxides
via ring - opening chlorinolysis , using the
lewis base activation of lewis acids concept , proved much more successful . in the original
denmark group study , meso - stilbene oxide ( 39 ) was effectively desymmetrized in the presence of a chiral phosphoramide
lewis base catalyst ( r)-40 and sicl4 , a weak lewis acid , to afford the syn-1,2-chlorohydrin
( 1s,2s)-42 in high
enantiopurity ( scheme 4a ) . later , it was found that the dimeric phosphoramide lewis
base ( r , r)-41 , which
is typically more selective for other sicl4-mediated enantioselective
transformations , provided ( 1s,2s)-42 with notably diminished enantiopurity . the stereochemical outcome of desymmetrization
of meso - epoxide suggested that the ( r)-binam - derived phosphoramde lewis base catalysts would enrich our cis - vinyl epoxide reactants in the desired enantiomer by
selectively catalyzing ring - opening chlorinolysis of the undesired
enantiomer . under denmark s conditions ,
the cis - vinyl
epoxide ( )-30 was found to be less reactive than meso - epoxides , probably because of the more sterically congested
environment presented by the proximal chlorine bearing carbons . consequently ,
the kinetic resolution with ( r)-40 was
carried out at slightly elevated temperature ( 50 c )
with higher catalyst loading ( 20 mol% ) ( scheme 4b ) . unfortunately , the resolution with ( r)-40 proceeded with poor selectivity ( selectivity factor , s = 4 ) . surprisingly , in contrast to denmark s result ,
the dimeric chiral lewis base ( r , r)-41 was more selective ( s = 14 ) for
our kinetic resolution than the monomeric chiral lewis base ( r)-40 . interestingly and unexpectedly , the
resolved vinyl epoxide from the kinetic resolutions with ( r)-40 and ( r , r)-41 were enriched in the opposite enantiomers . other
chiral lewis bases such as trans - cyclohexanediamine - derived
phosphoramide ( r , r)-43 and ( r)-binapo ( ( r)-44 ) were also tested . these lewis bases were more reactive than ( r)-40 or ( r , r)-41 but virtually unselective ( s < clearly , we had a good lead with catalyst ( r , r)-41 at this point . during the optimization
of the kinetic resolution , it was found
that the selectivity is highly dependent on the reaction temperature . when ( )-30 was resolved with 10 mol% of ( r , r)-41 at 78 c ,
a substantially improved selectivity factor of 33 was obtained ( table 1 , entry 1 ) . however , the reaction was even more
sluggish and proceeded to only 24% conversion after 24 h. even with
higher catalyst loading ( 20 mol% ) and extended reaction time ( 48 h ) ,
the conversion was improved to only 42% and the reaction became increasingly
slower as the reaction progressed ( entry 2 ) . the amounts of sicl4 and i - pr2net seem to have little
effect on the conversion and selectivity . because it is reasonable
to postulate that the rate of chlorinolysis would be increased at
higher concentration of chloride nucleophile , the kinetic resolution
was conducted in the presence of exogenous soluble chloride ( entry
3 ) . although the conversion was improved to 61% in the presence of
1 equiv of et4ncl , the selectivity factor diminished significantly . the kinetic resolution could be efficiently carried
out at 0.2 m with little attenuation of selectivity ( entry 4 ) . the
reaction could be further accelerated by further increasing the concentration ;
however , the selectivity factor decreased substantially ( entries 5
and 6 ) . on the other hand , the selectivity factor could be improved
by performing the reaction in more diluted condition . a selectivity
factor of 61 was obtained at 0.05 m even though the reaction was too
slow to be practical ( entry 7 ) . unfortunately , the mechanistic origin
for the drastic effects of the exogenous chloride and the reaction
concentration on the selectivity was unclear . eventually , an ideal
53% conversion was achieved with 20 mol% of ( s , s)-41 after 24 h at 0.2 m , and the desired
enantiomer of unreacted vinyl epoxide ( )-30 was
isolated in 43% yield with 97.3:2.7 er on a preparative scale ( entry
8 and eq 2 ) . the catalyst could be fully recovered
after reaction.table 1optimization of kinetic resolution
of ( )-30aentryconcentration ( m)time
( h)conversionb ( % ) er ( reactant)cer ( product)ds10.1242466.0:34.03.9:96.1332e0.1484284.5:15.55.3:94.7373f0.1486193.1:6.922.2:77.8940.2484183.9:16.15.8:94.23350.4244483.7:16.310.0:90.01861.0246688.7:11.331.9:68.1570.05723272.4:27.62.5:97.5618eg0.224532.7:97.388.1:11.927aall reactions employed
1.0 equiv
of sicl4 and 0.1 equiv of i - pr2net on 0.12.0 mmol scale.bdetermined by h nmr
analysis.cdetermined by
csp-gc.ddetermined by csp - sfc
after 2,4-dinitrobenzoylation.e20 mol% of catalyst.f1.0 equiv of et4ncl.gpreparative scale , using ( s , s)-41 , to preferentially
recover ( )-30.2 all reactions employed
1.0 equiv
of sicl4 and 0.1 equiv of i - pr2net on 0.12.0 mmol scale . determined by h nmr
analysis . determined by
csp - gc . preparative scale , using ( s , s)-41 , to preferentially
recover ( )-30 . the optimized kinetic resolution conditions
were next applied to
a substrate that was destined for the enantioselective synthesis of
mytilipin a. the cis - vinyl epoxide ( )-49 was prepared in a similar manner to that used to make ( )-30 ( scheme 5 ) . ( e)-crotyl
alcohol ( 45 ) was treated with molecular chlorine in the
presence of et4ncl to give the anti-1,2-dichloride
( )-46 . martin periodinane
followed by a careful workup afforded the sensitive and volatile ,-dichloroaldehyde
( )-47 in crude form , which was immediately converted
to volatile cis - vinyl epoxide ( )-49 via bromoallylalumination and epoxide formation , again
with near perfect diastereocontrol . the moderate yield in this
case can be attributed to volatility of the intermediate aldehyde
and the vinyl epoxide product . surprisingly , the kinetic resolution of ( )-49 , which differs only by alkyl chain length compared to ( )-30 , was only moderately efficient with catalyst ( r , r)-41 . under the optimized conditions
developed for ( )-30 , a selectivity factor of only
6 although
it was possible to recover ( + ) -49 with an improved enantiopurity
at higher conversion ( entry 2 ) , a more practical level of selectivity
was desired . similarly to the case of ( )-30 , higher
selectivity could be achieved at lower concentration . consequently ,
the selectivity factor was improved to 8 at 0.15 m concentration ( entry
3 ) . furthermore , a selectivity factor of 13 at 57% conversion was
realized at 0.1 m concentration , and enantioenriched ( + ) -49 was isolated in 93.4:6.6 er and 43% yield ( entry 4 and eq 3 ) . the monomeric phosphoramide ( r)-40 and ( r)-binapo ( ( r)-44 ) were even less selective ( s =
4 , not shown ) , although ( r)-binapo was more
reactive than dimeric phosphoramide catalyst ( r , r)-41 . curiously , it was difficult to reliably
analyze the enantiopurity of the chlorohydrin product ( + ) -50 because of apparently facile selective sublimation of the major
enantiomer under vacuum , which resulted in enantiodepletion
of the sample ( eq 4).table 2optimization of kinetic resolution
of ( )-49aentryconcentration ( m)time ( h)conversionb ( % ) er ( reactant)cs10.2245684.6:16.4620.2366692.4:7.6d630.15726594.8:5.2840.1725793.4:6.613aall reactions employed 1.0 equiv
of sicl4 and 0.1 equiv of i - pr2net and were conducted on 0.250.54 mmol scale.bdetermined by h nmr
analysis.cdetermined by
csp-gc.ddetermined by csp - sfc
after benzoylation.3 all reactions employed 1.0 equiv
of sicl4 and 0.1 equiv of i - pr2net and were conducted on 0.250.54 mmol scale . determined by h nmr
analysis . determined by
csp - gc . determined by csp - sfc
after benzoylation . the same kinetic resolution strategy was also examined for
the
enantioselective synthesis of ( + ) -malhamensilipin a. the corresponding cis - vinyl epoxide ( )-55 , which differs
from substrates 30 and 49 by virtue of its syn-1,2-dichloride moiety , was prepared from ( z)-2-undecen-1-ol ( 51 ) via the same sequence used for
previous substrates ( scheme 6a ) . unfortunately ,
( )-55 was considerably less reactive toward chlorinolysis
than the other vinyl epoxide substrates , and the enantioselectivity
was very poor ( scheme 6b ) . under the general
conditions with 20 mol% of ( r , r)-41 , the resolution proceeded to only 15% conversion even after
72 h and afforded a selectivity factor of only 4 . modified reaction
conditions with higher concentrations , higher temperatures , or addition
of exogenous chloride , while likely to accelerate the reaction , would
equally likely attenuate the already very low enantioselectivity ,
as we had previously observed in the study of ( )-30 . when ( )-55 was resolved with the typically more
reactive ( s)-binapo ( ( s)-44 ) , a much higher reaction rate was indeed observed . contrary to previous
cases , the selectivity factor was also improved , although it was still
moderate ( s = 89 ) . from a preparative scale reaction , the resolved vinyl epoxide ( )-55 was
obtained in 3.4:96.6 er and 34% yield . several related chiral bis(phosphine
oxide)s such as ( s)-tol - binapo , ( s)-h8-binapo , and ( r)-segphos
dioxide were also tested , but the selectivity was not improved . concurrent with the development
of an effective kinetic resolution
method , the key convergent metathesis step was investigated with ( )-30 , a potential precursor to danicalipin a. at the time of
conception of our metathesis - based second - generation approach , only
the first hint that z - selective alkene cross metathesis
was a viable reaction had appeared in the literature . moreover , whereas a z - configured alkene
is required en route to malhamensilipin a and mytilipin a so
that stereospecific anti - dichlorination would afford
the correct relative syn - configuration ( at c11/c12
and c9/c10 , respectively ) , it was not obviously a necessity for danicalipin
a because of the unchlorinated carbon at c12 . therefore , the feasibility
of the alkene cross metathesis approach was initially evaluated with
normal alkene metathesis catalysts . two different orders of
operations were considered for alkene cross metathesis and ring - opening
chlorinolysis ( scheme 7 ) . the left - hand sequence
involves the alkene cross metathesis of a vinyl epoxide ( 16 ) followed by chlorinolysis of the resulting internal alkenyl epoxide ,
which might be plagued by double inversion at the allylic center and
sn2 side reactions , as seen in previous studies . on the other hand , the right - hand sequence is initiated with chlorinolysis
of the terminal vinyl epoxide , which might proceed as a clean sn2 reaction under certain conditions , for example , the racemic
version of the vinyl epoxide chlorinolysis resolution using
sicl4 and an achiral lewis base catalyst such as hmpa . the resulting allylic chlorohydrin 15 would then be a
potential substrate for subsequent alkene cross metathesis to deliver 58 . with the latter sequence , the enantioenriched 1,2-chlorohydrin
product from the kinetic resolution electron - poor allylic
chloride ( )-35 underwent
alkene cross metathesis with 1-decene in the presence of 10 mol% of
the grubbs second - generation catalyst ( g ii ) at room
temperature to afford the ( e)-alkene product 59 in 43% yield along with dimeric side products ( scheme 8) ; because of the relatively complex crude reaction
mixture , it was difficult to determine the inherent e / z selectivity of this reaction . grubbs
second - generation catalyst ( hg ii ) promoted slower but
cleaner alkene cross metathesis to give an 84:16 e : z mixture of alkene isomers in 85% conversion and
60% yield of isomerically pure 59 . unfortunately , iodochlorination
of 59 with icl provided a complex crude mixture ( not
shown ) , in contrast to the case of the corresponding z - isomer that had been iodochlorinated with high efficiency
in our first - generation approach , although with low diastereoselectivity . attempts to directly hydrochlorinate the unactivated alkene under
iron - mediated radical hydrofunctionalization conditions recently
reported by boger was also unsuccessful ,
probably because of the low reactivity of the electron - deficient alkene . to generate the more desirable ( z)-alkene isomer , z - selective alkene cross metathesis
of allylic chloride 35 with various terminal alkene partners
in the presence of
recently developed grubbs cycloadamantyl catalyst 60 , which was generously provided first
by the grubbs group and later by materia , was investigated . however ,
not only 35 but also hydroxy - protected substrates and
the less chlorinated substrate 62 exhibited no reactivity
( scheme 9 ) . ruthenium metathesis catalysts
are clearly able to execute cross metatheses of allylic chlorides ;
at this stage , we have no reasonable understanding of the apparent
limitation of the z - selective catalysts toward allylic
chlorides , nor do we know if it is a truly general limitation . alternatively , the corresponding cis - vinyl epoxide
was examined as a substrate for alkene cross metathesis . similarly
to the corresponding allylic chloride , cis - vinyl
epoxide ( )-30 underwent alkene cross metathesis
with 1-decene in the presence of 10 mol% of g ii to afford 64 with moderate 82:18 e : z - selectivity ( scheme 10 ) . unlike the case
of chlorohydrin substrates , it was difficult to separate the internal
alkenyl epoxide product from the unreacted terminal vinyl epoxide
reactant . these compounds were isolated as a mixture ( estimated yields
of the product and the recovered reactant : 57 and 10% , respectively
based on nmr integration ) . in contrast , a complex mixture was obtained
from the similar reaction with hg ii . while the desired
product was not detected , one of the major components in the crude
mixture was identified as the unsaturated chloroaldehyde 65 , which implies the formation of ,-dichloroaldehyde 27 ( scheme 2b ) under the reaction conditions . additionally , the presence of chlorohydrin 66 as a minor
component in the crude mixture further suggests the formation of 27 followed by elimination of hcl , which is presumably responsible
for epoxide chlorinolysis of a small amount of desired alkene cross
metathesis product . the formation of 65 was confirmed
from the reactions between 30 and hg ii ( 10
and 100 mol% ) in the absence of other metathesis partners . at this
stage , we can not put forth a reasonable mechanism for this interesting
three - carbon degradation of vinyl epoxides . gratifyingly , cis - vinyl epoxide 30 turned out to be a competent
substrate for z - selective
alkene cross metathesis . in the presence of 1 mol% of catalyst 60 , 30 underwent alkene cross metathesis with
an excess of 1-hexene to 12% conversion at 35 c in 1 h ( scheme 11 ) . such exceptionally
high z - selectivity had only been rarely observed
with this catalyst . other solvents such
as toluene or dichloromethane had no significant impact on the
conversion and selectivity . the catalytic activity was typically lost
within a few hours , and the reactions would proceed no further . the
conversion could be improved to about 5060% ( nmr estimate )
with higher loading of catalyst ( 10 mol% ) , and the use of chlorinated
solvents such as dichloromethane or 1,2-dichloroethane
proved beneficial because of the poor solubility of 60 in other solvents . however , the decomposition of the starting vinyl
epoxide was a serious side reaction , and significant amounts of an
as yet unidentified decomposition product were formed . with this preliminary
success in hand , we turned to the use of
the relevant alkene 71 as the metathesis partner , which
was made from known aldehyde 68(24 ) via a slight modification of yoshimitsu s procedure as shown in scheme 12a . this high molecular weight compound could not be used in as large
excess as the model alkenes owing to effects on reaction concentration ;
initial reactions suffered from very low efficiencies , and the decomposition
of the starting vinyl epoxide remained problematic . the related alkene 70 , with a free hydroxyl group and attendant lower molecular
weight that could potentially be used in greater excess , was unreactive
( scheme 12b ) . to achieve higher conversion and suppress the decomposition ,
an
extensive optimization of the reaction conditions was conducted . a
variety of reaction solvents including tetrahydrofuran , diethyl ether , t - butyl methyl ether , toluene , chlorobenzene , hexafluorobenzene , ,,-trifluorotoluene , and octafluorotoluene , as well as neat conditions were employed , but
the reaction efficiency was not improved . the reaction was even slower
at room temperature , and performing the reaction at higher temperature
( 60 c ) only resulted in greater decomposition . a wide
range of additives were also evaluated . amine bases such
as i - pr2net and di - tert - butylpyridine promoted decomposition . 1,4-benzoquinone ,
known to scavenge ruthenium hydride species that might be formed during reaction and cause decomposition , only
attenuated the catalytic reactivity of 60 . the reaction
became slightly cleaner in the presence of 3 or 4 molecular
sieves , but substrate decomposition could not be completely avoided . ti(oi - pr)4 and hexachloroethane , which
have been used to improve the reactivity of other alkene cross metathesis
reactions , had no influence on the reaction . we hoped that removal of ethylene from the reaction mixture
would shift the cross metathesis equilibrium and drive these reactions
to higher conversion . therefore , the reaction was carried out under
static vacuum , continuous vacuum , and in an open vessel inside a glovebox ,
but to no avail . more rigorous removal of ethylene was attempted by
vigorously bubbling argon through the reaction mixture , and gratifyingly ,
the formation of the unknown was finally prevented . under optimized
conditions , with 10 mol% of 60 , ( )-10 was obtained in 19% yield along with 74% recovered starting material
( scheme 12b ) . it was more challenging to suppress
the decomposition with higher catalyst loadings , and the mass balance
was poorer . the decomposition could be minimized by slowing down the
reaction rate via a portionwise addition of catalyst , giving the product 10 in 29% yield with 40% recovered starting material using
30 mol% of 60 . although we were unable to achieve more
than the equivalent of a single turnover , this sequence still stands
as a marked improvement over the previous wittig - based route . access
to enantioenriched 10 now requires only five steps , compared
with our previous eight - step approach that afforded racemic material .
as a result , this moderate success completed a much shorter , enantioselective
formal synthesis of danicalipin a because of the interception of intermediate 10 from our first - generation synthesis . however , more improvements
in the end - game were still possible ( see below ) . convergent z - selective alkene cross metathesis
for mytilipin a with the corresponding cis - vinyl
epoxide 49 proceeded similarly to the corresponding reaction
for danicalipin a. alkene metathesis partner 74 was obtained
in two steps from 8-bromo-1-octene ( 73 ) via formylation
of grignard reagent followed by takai - utimoto chloroolefination ( eq 5 ) . the convergent metathesis reactions were carried
out with vigorous bubbling of argon to prevent the decomposition of
starting vinyl epoxide , and the desired alkene ( )-(z)-75 was produced as a single geometrical isomer . again ,
we were unable to achieve more than a single turnover with 1030
mol% of catalyst 60 ( table 3 ,
entries 1 and 2 ) . the use of fluorinated solvents such as ,,-trifluorotoluene did not result in any improvement ( entry 3 ) . unfortunately , higher loading of catalyst only resulted in significant
loss of mass balance and the yield of product was only marginally
improved ( entries 4 and 5 ) . despite the low efficiency of the z - selective alkene cross metathesis , the direct incorporation
of the vinyl chloride is a marked improvement over previous syntheses
because it eliminates at least three postconvergence steps . cross metathesis partner 74 might appear upon cursory
analysis to be poised for side reactivity because as a 1,9-diene cyclooctene
formation could occur via ring - closing metathesis . vinyl
chlorides are relatively slow to react in metathesis processes , and
cyclo - octene formation can also be a sluggish reaction . almost certainly ,
however , the high kinetic selectivity of catalyst 60 for
( z)-alkenes is presumably the most important factor
that prevents reaction with the ( e)-vinyl chloride
in either rcm or cross metathesis events.5table 3z - selective alkene
cross metathesis for the synthesis of mytilipin aaentry60 ( mol%)solventtime ( h)yield
( % ) 110dce / ch2cl2210230dce / ch2cl2332330phcf3/ch2cl2333450phcf3/ch2cl24345b100dce339aall reactions were carried out on
0.150.25 mmol scale.b5 equiv of 74 was employed . unfortunately , the convergent z - selective alkene
cross metathesis was even less efficient for malhamensilipin
a. the metathesis product ( + ) -76 was isolated only in
19% yield from the reaction of ( )-55 with 71 under the analogous conditions to those used for danicalipin
a ( eq 6 ) . for reasons explained below , the
improvement of this convergent step was not a priority.6 while admittedly not as efficient as desired , the convergent z - selective alkene cross metathesis is noteworthy for its
complete diastereoselectivity in all cases examined . to see
if the extremely high selectivity we observed was general for cis - vinyl epoxides , as well as to investigate the low catalytic
activity of 60 with respect to the specific chlorinated
cases relevant to the chlorosulfolipids , we tested the
reactivity of unchlorinated cis - vinyl epoxide 79 with 1-decene ( scheme 13 ) . in the
presence of 10 mol% of 60 , complete conversion to ( z)-vinyl epoxide 80 was observed ( 83% isolated
yield , > 20:1 z : e ) . even with
only
1 mol% of catalyst , the reaction proceeded to 46% conversion and therefore ,
it appears that cis - vinyl epoxides are subject to
highly z - selective alkene cross metathesis with 60 , and that the poor efficiency observed in the convergent
steps for chlorosulfolipids is likely specific to chlorinated
substrates . recently , the grubbs group also demonstrated that vinyl
epoxides are excellent substrates for z - selective
metathesis using these catalysts . completion of the synthesis of ( + ) -danicalipin
a took advantage
of a similar reaction sequence to that previously developed in the
context of our first - generation approach ( scheme 1a ) . lewis acid - mediated chlorinolysis of the internal alkenyl
epoxide 10 typically afforded a diastereomeric mixture
of the desired sn2 product 11 and the double
inversion product 81 as well as the constitutional
isomer 82 formed via sn2 substitution
( scheme 14a ) . the extent of side product formation
was highly dependent on the choice of lewis acid and the concentration
of chloride anion . because exclusive sn2 reactivity was
observed from the reaction of terminal cis - vinyl
epoxide with sicl4 in the presence of hmpa , a combination
of sicl4 and a number of lewis base activators including
pyridine , dmap , pyridine n - oxide , hmpa , dmpu , dmi ,
and tmu was evaluated with or without et4ncl . in all cases ,
a variable amount of side products were produced and a useful level
of selectivity was not accomplished ( 90:1031:69 dr , 250%
sn2 ) . both undesired pathways were reasonably attenuated
when the epoxide was opened using dry hcl ; however , high selectivity
was desired specifically for the exclusion of double inversion product 81 , which is more difficult to separate from the desired product . double inversion could be completely overcome by employing bf3oet2 at 78 c with a high concentration
of et4ncl . despite the presence of a rather large amount
of sn2 product 82 , the desired isomer 11 a major problem of our first - generation synthesis was the poorly diastereoselective
iodochlorination reaction of 11 ( 1.8:1
dr ) , which was compounded further by the very painstaking separation
of diastereomers at that stage or after deiodination . we found that
transient introduction of a trimethylsilyl group on the c14 hydroxyl
permitted high diastereocontrol ( 95:5 dr ) in the iodochlorination ,
and because the silyl group could be introduced and removed in the
same pot , this result had a significantly positive impact on the synthesis . overall , the new approach facilitated a nine - step synthesis of enantioenriched
( + ) -danicalipin a ( 4.6% overall yield ) , which is a significant improvement
over our 12-step racemic first - generation synthesis . completion of the synthesis of mytilipin a required only
three
postconvergence steps ( scheme 14b ) . bf3oet2-mediated vinyl epoxide chlorinolysis
with inversion of configuration proceeded with exclusive diastereoselectivity
and delivered diene 85 . dichlorination of the electron - deficient
allylic chloride afforded hexachloride 86 in 86% yield
with high diastereoselectivity ( 93:7 dr of crude product , purified
to 97:3 ) and complete chemoselectivity with respect to the isolated
vinyl chloride . sulfation of the secondary alcohol according to carreira s
conditions completed the synthesis of
mytilipin a. in this way , racemic chlorosulfolipid could
be accessed in 8.6% yield over the seven linear steps sequence , and
enantioenriched mytilipin a is available via a longest linear sequence
of eight steps ( 3.7% overall yield ) . it is indeed fortuitous
that we chose to first pursue danicalipin
a with this new approach . the choice of malhamensilipin a as
a first target could easily have discouraged us from pursuing this
strategy . although , as described above , this strategy led to much
improved syntheses of mytilipin a and danicalipin a , there was ultimately
little improvement in the synthesis of malhamensilipin a , for
which we had already established an enantioselective synthesis , via
the same number of steps , and for which the wittig reaction was not
improved upon with the metathesis option . therefore , while we are
pleased to claim a formal enantioselective synthesis of malhamensilipin
a as part of this second - generation , general strategy for chlorosulfolipid
synthesis , we would suggest that our first enantioselective synthesis
of this single target would likely be the preferred method to access
samples of this natural product . however , if new catalysts become
available that can better effect these challenging z - selective cross metatheses , and if a truly effective method for
asymmetric dichlorination of allylic alcohols is discovered , the strategy
described here would be hard to beat for any of these three chlorosulfolipid
targets . indeed , this approach has the distinct advantage that it
can be rendered enantioselective without recourse to resolution once
asymmetric catalysis technology is developed for allylic alcohol dichlorination . we have developed a concise and general approach
for the enantioselective
synthesis of three chlorosulfolipid targets that takes strategic
advantage of a common stereotriad . diastereoselective carbonyl
addition to sensitive ,-dichloroaldehydes , z - selective alkene cross metatheses , and kinetic resolution
of chlorinated vinyl epoxides are key advances that permitted success
in this second - generation approach . enantioenriched danicalipin a ,
mytilipin a , and malhamensilipin a are accessed in nine , eight ,
and 11 steps , respectively . given the paucity of efforts toward
this class of natural products
until about five years ago , it is remarkable that so many effective
solutions to these targets from multiple research groups have appeared
in such short order . certainly , polychlorinated natural products
are not to be feared as objectives for chemical synthesis and are
rather well - behaved in the contexts of many different reaction types . all reactions were performed
in oven - dried ( 140 c ) or flame - dried glassware under an atmosphere
of dry argon unless otherwise noted . reaction solvents including dichloromethane ,
toluene , n , n - dimethylformamide ,
and tetrahydrofuran were dried by percolation through a column packed
with neutral alumina and a column packed with q5 reactant , a supported
copper catalyst for scavenging oxygen , under a positive pressure of
argon . dichloroethane ( dce ) was heated to reflux over cah2 for 3 h , distilled under argon , and stored over 3 molecular
sieves prior to use . column chromatography was performed using 60
( 0.0400.063 mm ) mesh silica gel ( sio2 ) . the following reagents were distilled from the indicated drying agents
under argon prior to use : 2,2,6,6-tetramethylpiperidine
( na ) , allyl bromide ( cah2 ) , triethylamine ( cah2 ) , n , n - diisopropylethylamine
( cah2 ) , trimethylsilyl chloride ( tmscl , cah2 ) , and ethylene diamine ( cah2 ) . silicon tetrachloride
was heated at reflux for 2 h under a flow of argon and then distilled
prior to use . z - selective grubbs cycloadamantyl
catalyst ( 60 , materia ) was stored in the glovebox and
used as received . dimeric denmark catalysts ( ( r , r)-41 and ( s , s)-41 , obiter ) were used as received and recovered by
recrystallization from boiling benzene . ( e)-2-nonen-1-ol
( 25 ) , boron trifluoride diethyl etherate , and tri - n - butyltin hydride were distilled prior to use . martin
periodinane , diethylaluminum chloride , n - butyllithium ,
imidazole , iodine monochloride ( 1.0 m in ch2cl2 ) , camphorsulfonic acid ( csa ) , triethylborane ( 1.0 m
in thf ) , chlorosulfonic acid , nickel(ii ) acetate tetrahydrate ,
sodium borohydride , magnesium ( 20100 mesh ) , 1,2-dibromoethane ,
1-bromo-10-undecene , n - chlorosuccinimide , t - butyldimethylsilyl chloride , and paraformaldehyde
were used without further purification . h and c spectra were referenced to residual
solvent ( cdcl3 : 7.26 ppm , h , 77.00 ppm , c ; cd3od : 3.31 ppm , h , 49.00 ppm , c ) . chemical shifts are reported in parts per million , and
multiplicities are indicated by s ( singlet ) , d ( doublet ) , t ( triplet ) ,
q ( quartet ) , m ( multiplet ) , br s ( broad singlet ) , and app ( apparent ) . infrared
( ir ) spectra were recorded on an ft - ir instrument on nacl plates ,
and peaks are reported in cm . high - resolution
mass spectra ( hrms ) data are reported in the form of ( m / z ) . visualization of analytical thin - layer
chromatography was accomplished with uv(254 ) and potassium permanganate
( kmno4 ) or p - anisaldehyde staining solutions . optical rotation data were obtained on a digital polarimeter and are
reported as follows : concentration ( c = g/100 ml )
and solvent . analytical gas chromatography ( csp - gc ) was performed
on a gas chromatograph equipped with a flame ionization detector and
a dimethylated -cyclodextrin ( b - dm , 30 m ) capillary column .
the injector temperature and the detector temperature were 200 c
with a split ratio of approximately 100:1 . to
a stirred solution of et4ncl ( 6.63 g , 40.0 mmol ) and ( e)-2-nonen-1-ol ( 2.84
g , 20.0 mmol ) in ch2cl2 ( 60 ml ) was bubbled
cl2 at 0 c until the reaction mixture turned yellow
( 2 min ) . the resulting colorless solution was diluted with
ch2cl2 ( 50 ml ) and shaken with a mixture of
saturated aqueous nahco3 solution ( 50 ml ) and saturated
aqueous na2s2o3 solution ( 50 ml ) . the organic layer was separated , and the aqueous layer was extracted
with ch2cl2 ( 50 ml ) . the combined organic extracts
were shaken with saturated aqueous nacl solution ( 100 ml ) . the organic
layer was separated , and the aqueous layer was extracted with ch2cl2 ( 50 ml ) . the combined organic extracts were
dried over na2so4 , filtered , and concentrated
in vacuo ( 25 mmhg ) . the residue was purified by bulb - to - bulb distillation
under reduced pressure ( 0.25 mmhg , abt 123126 c ) to
afford ( )-26 ( 4.04 g , 95% , contained 1.5%
of 1,3-dichloro-2-nonanol ) as a colorless oil . data for ( )-26 : h nmr ( 600 mhz , cdcl3 )
4.12 ( app td , j = 8.7 , 8.7 , 2.7 hz , 1h ) , 4.094.06
( m , 1h ) , 4.024 ( d , j = 6.6 hz , 1h ) , 4.017 ( d , j = 6.6 hz , 1h ) , 2.112.02 ( m , 1h ) , 1.97 ( app t , j = 6.9 , 6.9 hz , 1h ) , 1.821.75 ( m , 1h ) , 1.641.54
( m , 1h ) , 1.481.39 ( m , 1h ) , 1.391.23 ( m , 6h ) , 0.89
( dd , j = 6.8 , 6.8 hz , 3h ) ; c nmr ( 126
mhz , cdcl3 ) 66.4 , 64.5 , 61.8 , 34.9 , 31.6 , 28.6 ,
25.5 , 22.5 , 14.0 ; ir ( thin film ) 3390 , 2924 , 2858 , 1463 , 1455 , 1434 ,
1379 , 1066 , 725 , 655 cm ; hrms ( ci - tof ) m / z calcd for c9h18cl2onh4 [ m + nh4 ] 230.1078 , found 230.1071 . to a stirred suspension of ( )-26 ( 2.13 g , 10.0 mmol ) and nahco3 ( 2.52 g , 30.0
mmol ) in ch2cl2 ( 10 ml , saturated with h2o ) martin periodinane ( 6.36 g , 15.0
mmol ) slowly over 1 min at 0 c under air . after stirring for
10 min , the ice bath was removed and the reaction mixture was stirred
at rt for 30 min prior to the addition of n - pentane
( 100 ml ) . the resulting mixture was filtered , washed with saturated
aqueous nahco3 ( 50 ml ) , dried over na2so4 , filtered , and concentrated in vacuo ( 25 mmhg ) to give 27 ( 1.99 g ) as a pale yellow oil . the crude material was used
directly for the next reaction without further purification ( 3%
2-chloro-2-nonenal ) . data for ( )-27 : h nmr ( 600 mhz , cdcl3 )
9.43 ( d , j = 3.1 hz , 1h ) , 4.25 ( dd , j = 7.4 , 3.1 hz , 1h ) , 4.244.21 ( m , 1h ) , 2.021.97 ( m ,
1h ) , 1.841.77 ( m , 1h ) , 1.631.54 ( m , 1h ) , 1.481.39
( m , 1h ) , 1.391.27 ( m , 6h ) , 0.90 ( dd , j =
6.9 , 6.9 hz , 3h ) ; c nmr ( 126 mhz , cdcl3 )
191.4 , 64.9 , 59.8 , 34.0 , 31.5 , 28.5 , 25.5 , 22.5 , 14.0 ; ir ( thin film )
2926 , 2858 , 1734 , 1458 cm ; hrms ( ci - tof ) m / z calcd for c9h15clonh4 [ m hcl + nh4 ] 192.1155 , found 192.1158 . to a stirred solution of tmp ( 3.71
ml , 22.0 mmol ) in thf ( 50 ml ) was added n - buli ( 2.50
m in hexanes , 8.40 ml , 21.0 mmol ) at 78 c . after being
stirred for 30 min , the litmp solution was cannulated into a solution
of allyl bromide ( 1.82 ml , 21.0 mmol ) and et2alcl ( 1.0
m in hexanes , 40.0 ml , 40.0 mmol ) in thf ( 100 ml ) at 78 c
over 5 min . the resulting solution was stored at 78 c ,
while ( )-27 was prepared ( see above ) . a solution
of ( )-27 in thf ( 10 ml + rinsed with 5 ml
2 ) was added dropwise over 15 min . after being stirred at 78
c for 4 h , the reaction mixture was poured into an ice - cold
5 m aq naoh solution ( 200 ml ) . the biphasic mixture was vigorously
stirred at rt for 1 h prior to the dilution with n - pentane ( 100 ml ) and filtration . the organic layer was separated ,
and the aqueous layer was extracted with n - pentane
( 100 ml 2 ) . the combined organic extracts were washed with
saturated aqueous nh4cl solution ( 200 ml 2 ) , dried
over na2so4 , filtered , and concentrated in vacuo
( 25 mmhg ) . the residue was purified by column chromatography ( sio2 , = 5.0 cm , l = 13.5 cm , n - pentane / ch2cl2 , 9/1 , rf = 0.29 , p - anisaldehyde )
and bulb - to - bulb distillation under reduced pressure ( 0.25 mmhg , abt
123127 c ) to give ( )-30 ( 1.89 g ,
75% from ( )-26 , 98:2 dr ) as a colorless oil . data
for ( )-30 : h nmr ( 600 mhz , cdcl3 ) 5.82 ( ddd , j = 17.1 , 10.6 , 5.6
hz , 1h ) , 5.52 ( d , j = 17.1 hz , 1h ) , 5.45 ( d , j = 10.7 hz , 1h ) , 4.21 ( ddd , j = 9.4 , 4.6 ,
4.1 hz , 1h ) , 3.76 ( dd , j = 9.0 , 4.2 hz , 1h ) , 3.57
( app t , j = 4.9 , 4.9 hz , 1h ) , 3.46 ( dd , j = 9.0 , 4.3 hz , 1h ) , 1.981.87 ( m , 2h ) , 1.651.59 ( m ,
1h ) , 1.471.39 ( m , 1h ) , 1.361.26 ( m , 6h ) , 0.89 ( dd , j = 6.9 , 6.9 hz , 3h ) ; c nmr ( 126 mhz , cdcl3 ) 130.4 , 121.2 , 65.2 , 60.6 , 57.7 , 56.0 , 34.5 , 31.6 ,
28.6 , 26.5 , 22.5 , 14.0 ; ir ( thin film ) 2956 , 2928 , 2858 , 1463 , 1455 ,
1250 , 981 , 934 , 783 , 668 , 597 cm ; hrms ( ci - tof ) m / z calcd for c12h20cl2onh4 [ m + nh4 ] 268.1235 , found 268.1236 . to a stirred solution of ( )-30 ( 126 mg , 0.502 mmol ) and ( s , s)-41 ( 84 mg , 0.10 mmol ) in ch2cl2 ( 2.5 ml ) were added i - pr2net ( 9 l ,
0.05 mmol ) and sicl4 ( 57 l , 0.50 mmol ) at 78
c . after 24 h , a solution of ch3oh / et3n / ch2cl2 ( 1/1/5 , 4 ml ) was added quickly at
78 c . the resulting solution was vigorously stirred
with a saturated aqueous nahco3 solution ( 20 ml ) at rt
for 2 h prior to filtration . the organic layer was separated , and
the aqueous layer was extracted with ch2cl2 ( 10
ml ) . the combined organic extracts were dried over na2so4 , filtered , and concentrated in vacuo ( 25 mmhg ) . ( s , s)-41 was recovered from
the residue by column chromatography ( sio2 , = 2.2
cm , l = 7 cm , ch2cl2/i - proh , 10/1 , rf = 0.37 , uv ) . the fractions that contained 30 and 35 were combined and purified by column chromatography ( sio2 , = 2.2 cm , l = 11 cm , n - pentane / ch2cl2 , 8/1 to 4/1 , p - anisaldehyde ) to give ( )-35 ( 70 mg , 49% , rf = 0.12 in 8/1 , 88.1:11.9
er ) as a colorless oil and ( )-30 as a colorless
oil , which was purified again by column chromatography ( 54 mg , 43% , rf = 0.30 in 8/1 , 2.7:97.3 er ) .
data for ( )-30 : [ ]d = 29.9 ( c 1.00 , chcl3 ) ; gc ( b - dm ,
30 psi , 145 c ) tr 15.5 min ( 2.7% ) ,
16.5 min ( 97.3% ) . data for ( )-35 : [ ]d = 60.6 ( c 1.00 , chcl3 ) ; gc ( b - dm , 30 psi , 165 c ) tr 18.5 min ( 88.1% ) , 19.1 min ( 11.9% ) ; h nmr ( 500 mhz ,
cdcl3 ) 6.03 ( ddd , j = 16.9 , 10.2 ,
7.7 hz , 1h ) , 5.49 ( d , j = 16.9 hz , 1h ) , 5.35 ( d , j = 10.2 hz , 1h ) , 5.07 ( d , j = 7.6 hz ,
1h ) , 4.51 ( app dt , j = 10.5 , 2.7 , 2.7 hz , 1h ) , 4.31
( dd , j = 9.4 , 2.7 hz , 1h ) , 3.89 ( app td , j = 9.8 , 9.8 , 1.3 hz , 1h ) , 2.23 ( d , j =
9.9 hz , 1h ) , 1.921.83 ( m , 1h ) , 1.831.74 ( m , 1h ) , 1.681.58
( m , 1h ) , 1.461.23 ( m , 7h ) , 0.89 ( app t , j = 6.6 , 6.6 hz , 3h ) ; c nmr ( 126 mhz , cdcl3 ) 134.4 , 119.6 , 74.5 , 66.5 , 65.0 , 62.5 , 32.4 , 31.6 , 28.6 ,
26.5 , 22.6 , 14.0 ; ir ( thin film ) 3540 , 2956 , 2927 , 2857 , 1465 , 1379 ,
1265 , 1096 , 1069 , 987 , 935 cm ; hrms ( ci - tof ) m / z calcd for c12h21cl3onh4 [ m + nh4 ] 304.1002 , found 304.1000 . the
solvents were bubbled with argon for 15 min before use . to a stirred
solution of ( )-30 ( 52 mg , 0.21 mmol ) and 71 ( 152 mg , 0.414 mmol ) in dce ( 210 l ) in a test tube
( 12 mm 75 mm ) was added a solution of 60 ( 39 mg ,
0.062 mmol ) in ch2cl2 ( 210 l ) in three
portions ( 0 , 0.5 , 1.0 h ) at 35 c while the reaction mixture
was vigorously bubbled with argon ( saturated with dce ) . after being stirred at 35 c with argon
bubbling for an additional 2 h , the reaction mixture was cooled to
rt , filtered through silica gel ( = 2.2 cm , l = 9 cm , ch2cl2 , 40 ml ) , and concentrated in
vacuo ( 25 mmhg ) . the residue was purified by column chromatography
( sio2 , = 3.8 cm , l = 15 cm , n - pentane / ch2cl2 , 8/1 , rf = 0.24 , p - anisaldehyde )
to give ( + ) -10 ( 35 mg , 29% , > 20:1 = z : e ) as a colorless oil . data for ( + ) -10 : [ ]d = + 14.2 ( c 1.00 ,
chcl3 ) ; h nmr ( 600 mhz , cdcl3 )
5.86 ( app dt , j = 10.9 , 7.6 hz , 7.6 , 1h ) , 5.265.20
( m , 1h ) , 4.21 ( ddd , j = 9.6 , 4.4 , 4.0 hz , 1h ) , 3.92
( s , 2h ) , 3.76 ( dd , j = 9.1 , 4.0 hz , 1h ) , 3.74 ( dd , j = 7.9 , 4.3 hz , 1h ) , 3.44 ( dd , j = 9.1 ,
4.2 hz , 1h ) , 2.272.19 ( m , 2h ) , 2.192.14 ( m , 2h ) , 1.991.86
( m , 2h ) , 1.661.55 ( m , 3h ) , 1.461.39 ( m , 3h ) , 1.391.26
( m , 14h ) , 0.91 ( s , 9h ) , 0.89 ( app t , j = 6.9 , 6.9
hz , 3h ) , 0.11 ( s , 6h ) ; c nmr ( 126 mhz , cdcl3 ) 139.6 , 121.5 , 93.5 , 72.1 , 65.2 , 61.5 , 57.4 , 52.5 , 43.5 ,
34.3 , 31.6 , 29.29 , 29.27 , 29.26 , 29.1 , 29.0 , 28.6 , 28.1 , 26.5 , 25.7 ,
24.7 , 22.5 , 18.3 , 14.0 , 5.4 . to
a stirred solution of ( + ) -10 ( 35 mg , 0.059 mmol ) and
et4ncl ( 30 mg , 0.18 mmol ) in ch2cl2 ( 240 l ) was added bf3oet2 ( 15
l , 0.12 mmol ) at 78 c . after being stirred for
1 h , the reaction mixture was poured into an ice - cold saturated aqueous
nahco3 solution ( 10 ml ) . to the biphasic mixture the
organic layer was separated , and the aqueous layer was extracted with
ch2cl2 ( 10 ml 2 ) . the combined organic
extracts were dried over na2so4 , filtered , and
concentrated in vacuo ( 25 mmhg ) . the residue was purified by column
chromatography ( sio2 , = 1.5 cm , l = 9 cm , n - pentane / ch2cl2 ,
5/1 to 3/1 to 1/1 , p - anisaldehyde ) to give ( + ) -11 ( 27 mg , 73% , rf = 0.25 in 3/1 , > 20:1 dr ) as a colorless oil and sn2
product 82 ( 9.6 mg , 26% , rf = 0.33 and 0.24 in 1/1 , 6:4 dr ) as a colorless oil . data for ( + ) -11 : [ ]d =
+ 62.5 ( c 1.00 , chcl3 ) ; h nmr
( 600 mhz , cdcl3 ) 5.73 ( app t , j = 10.3 , 10.3 hz , 1h ) , 5.66 ( app dt , j = 10.7 , 7.4 ,
7.4 hz , 1h ) , 5.38 ( dd , j = 9.9 , 1.7 hz , 1h ) , 4.49
( app dt , j = 10.3 , 2.9 , 2.9 hz , 1h ) , 4.29 ( dd , j = 9.0 , 3.1 hz , 1h ) , 3.92 ( s , 2h ) , 3.833.78 ( m ,
1h ) , 2.34 ( d , j = 10.5 hz , 1h ) , 2.212.09
( m , 4h ) , 1.911.83 ( m , 1h ) , 1.831.76 ( m , 1h ) , 1.671.62
( m , 1h ) , 1.611.56 ( m , 2h ) , 1.461.36 ( m , 3h ) , 1.361.25
( m , 14h ) , 0.91 ( s , 9h ) , 0.89 ( app t , j = 6.8 , 6.8
hz , 3h ) , 0.11 ( s , 6h ) ; c nmr ( 126 mhz , cdcl3 ) 135.7 , 125.8 , 93.5 , 75.1 , 66.8 , 62.4 , 60.1 , 43.5 , 32.5 ,
31.6 , 29.3 , 29.2 , 29.1 , 29.0 ( 2c ) , 28.6 , 27.6 , 26.5 , 25.7 , 24.7 , 22.6 ,
18.3 , 14.0 , 5.4 . data for 82 ( a 6:4 mixture of
diastereomers ) : h nmr ( 600 mhz , cdcl3 )
5.975.90 ( m , 1h ) , 5.79 ( ddd , j = 15.2 , 13.9 ,
6.8 , 1h ) , 4.77 ( app td , j = 7.3 , 7.3 , 3.9 hz , 0.4h ) ,
4.72 ( app td , j = 7.2 , 7.2 , 4.3 hz , 0.6h ) , 4.39 ( app
quintet , j = 7.4 , 7.4 , 7.4 , 7.4 hz , 1h ) , 4.18 ( ddd , j = 8.8 , 6.7 , 4.1 hz , 1h ) , 3.92 ( s , 2h ) , 3.913.88
( m , 1h ) , 2.202.14 ( m , 2h ) , 2.132.04 ( m , 2h ) , 1.891.73
( m , 3h ) , 1.651.51 ( m , 3h ) , 1.501.37 ( m , 3h ) , 1.371.23
( m , 14h ) , 0.91 ( s , 9h ) , 0.89 ( app t , j = 6.8 , 6.8
hz , 3h ) , 0.11 ( s , 6h ) ; c nmr ( 126 mhz , cdcl3 ) 136.0 , 135.7 , 128.22 , 128.17 , 93.5 , 72.1 , 72.0 , 71.7 , 69.4 ,
69.1 , 61.9 , 61.7 , 61.6 , 43.5 , 38.4 , 38.3 , 34.7 , 34.5 , 31.6 , 29.30 ,
29.26 , 29.0 , 28.9 , 28.61 , 28.60 , 26.43 , 26.35 , 25.7 , 25.3 , 25.2 , 24.7 ,
22.5 , 18.3 , 14.1 , 5.4 ; ir ( thin film ) 3403 , 2929 , 2857 , 1463 ,
1256 , 1153 , 1120 , 970 , 840 , 780 cm . ; hrms ( esi - tof ) m / z calcd for c28h53cl5o2sina [ m + na ] 647.2155 , found 647.2143 . to
a stirred solution of ( + ) -11 ( 27 mg , 0.043 mmol ) and
imidazole ( 8.8 mg , 0.13 mmol ) in ch2cl2 ( 430
l ) was added tmscl ( 11 l , 0.086 mmol ) at rt . after being
stirred for 10 min , the reaction mixture was cooled to 78
c and icl ( 1.0 m in ch2cl2 , 215 l ,
0.215 mmol ) was added . after being stirred for 20 min at 78
c , a solution of csa ( 100 mg , 0.43 mmol ) in ch3oh
( 645 l ) was added and the cold bath was removed . after being
stirred for 30 min , the brown solution was poured into a stirred mixture
of saturated aqueous nahco3 solution ( 5 ml ) and saturated
aqueous na2s2o3 solution ( 5 ml ) . the resulting colorless biphasic mixture was diluted with ch2cl2 ( 10 ml ) and h2o ( 10 ml ) . the organic layer
was separated , and the aqueous layer was extracted with ch2cl2 ( 10 ml 2 ) . the combined organic extracts were
washed with saturated aqueous nh4cl solution ( 20 ml ) , and
the aqueous layer was extracted with ch2cl2 ( 10
ml 2 ) . the combined organic extracts were dried over na2so4 , filtered , and concentrated in vacuo ( 25 mmhg ) . the residue was purified by column chromatography ( sio2 , = 1.1 cm , l = 5.5 cm , n - pentane / ch2cl2 , 5/1 to 3/1 , rf = 0.29 in 3/1 , p - anisaldehyde )
to give ( )-83 ( 28 mg , 82% , 95:5 dr ) as a colorless
oil . data for ( )-83 : [ ]d = 7.6 ( c 1.00 , chcl3 ) ; h nmr ( 600 mhz , cdcl3 ) 4.98 ( d , j = 10.9 hz , 1h ) , 4.72 ( app t , j = 10.6 ,
10.6 hz , 1h ) , 4.56 ( dd , j = 10.9 , 1.8 hz , 1h ) , 4.48
( app dt , j = 10.5 , 2.6 , 2.6 hz , 1h ) , 4.38 ( dd , j = 9.8 , 2.4 hz , 1h ) , 3.92 ( s , 2h ) , 3.753.71 ( m ,
1h ) , 2.192.16 ( m , 2h ) , 2.14 ( d , j = 11.3
hz , 1h ) , 2.041.97 ( m , 1h ) , 1.971.88 ( m , 1h ) , 1.831.78
( m , 1h ) , 1.771.71 ( m , 1h ) , 1.691.62 ( m , 1h ) , 1.621.56
( m , 2h ) , 1.531.46 ( m , 1h ) , 1.461.38 ( m , 3h ) , 1.381.27
( m , 13h ) , 0.92 ( s , 9h ) , 0.90 ( app t , j = 6.8 , 6.8
hz , 3h ) , 0.11 ( s , 6h ) ; c nmr ( 126 mhz , cdcl3 ) 93.5 , 74.6 , 72.1 , 66.3 , 66.0 , 62.9 , 62.8 , 43.5 , 42.6 , 40.6 ,
32.8 , 31.6 , 29.2 ( 2c ) , 29.0 , 28.9 , 28.6 , 26.6 , 26.2 , 25.7 , 24.7 , 22.6 ,
18.3 , 14.1 , 5.3 . toluene
was bubbled with argon for 20 min before use . to a stirred solution
of ( )-83 ( 28 mg , 0.035 mmol ) in toluene ( 355
l ) were added n - bu3snh ( 11 l ,
0.041 mmol , 99% pure by h nmr in c6d6 ) and et3b ( 1.0 m in thf ,
7 l , 0.007 mmol ) at 78 c . after being stirred
for 2 h at 78 c , n - pentane ( 3.55 ml ) was added and the resulting solution was concentrated in vacuo ( 25
mmhg ) . the residue was purified by column chromatography ( sio2 , = 1.1 cm , l = 5.5 cm , n - pentane / ch2cl2 , 1/0 to 4/1 , rf = 0.25 in 4/1 , p - anisaldehyde )
to give ( + ) -84 ( 21.5 mg , 91% ) as a colorless oil . data
for ( + ) -84 : [ ]d = + 34.3
( c 1.00 , chcl3 ) ; h nmr ( 600
mhz , cdcl3 ) 4.96 ( d , j = 10.3
hz , 1h ) , 4.51 ( app dt , j = 10.6 , 2.4 , 2.4 hz , 1h ) ,
4.30 ( dd , j = 9.7 , 2.4 hz , 1h ) , 4.174.13
( m , 1h ) , 3.92 ( s , 2h ) , 3.77 ( app t , j = 10.7 , 10.7
hz , 1h ) , 2.352.28 ( m , 1h ) , 2.202.16 ( m , 2h ) , 2.16
( d , j = 11.6 hz , 1h ) , 2.021.95 ( m , 1h ) , 1.941.85
( m , 1h ) , 1.831.73 ( m , 3h ) , 1.681.62 ( m , 1h ) , 1.621.57
( m , 2h ) , 1.571.49 ( m , 1h ) , 1.491.39 ( m , 2h ) , 1.391.26
( m , 14h ) , 0.91 ( s , 9h ) , 0.89 ( app t , j = 6.8 , 6.8
hz , 3h ) , 0.11 ( s , 6h ) ; c nmr ( 126 mhz , cdcl3 ) 93.5 , 75.1 , 72.1 , 66.5 , 63.0 , 62.7 , 60.4 , 44.3 , 43.5 , 38.7 ,
32.4 , 31.6 , 29.3 , 29.2 , 29.0 ( 2c ) , 28.6 , 26.6 , 26.2 , 25.7 , 24.7 , 22.6 ,
18.3 , 14.0 , 5.3 . to a stirred solution of ( + ) -84 ( 21.5 mg ,
0.0324 mmol ) in ch2cl2 ( 650 l ) was added
clso3h ( 5 drops ) via a pasteur pipet at rt under air . after
being stirred for 10 min , the reaction mixture was slowly poured into
a vigorously stirred mixture of a saturated aqueous nahco3 solution ( 6.5 ml ) and solid nahco3 ( 650 mg ) . the resulting
heterogeneous mixture was diluted with etoh ( 26 ml ) , filtered , and
concentrated in vacuo ( 30 mmhg ) . the residue was suspended in thf
( 20 ml ) , filtered , and concentrated in vacuo ( 25 mmhg ) . the residue
purified by column chromatography ( sio2 , = 2.2
cm , l = 14.5 cm , ch2cl2/ch3oh , 3/1 , rf =
0.38 , p - anisaldehyde ) to give ( + ) -1 ( 23.4
mg , 96% ) as a colorless amorphous solid . data for ( + ) -1 : [ ]d = + 34.2 ( c 2.34 ,
ch3oh ) ( lit . [ ]d + 33.0 ( c 0.40 , ch3oh ) , [ ]d + 31.5 ( c 0.25 , ch3oh ) , [ ]d + 12.8 ( c 0.2 , ch3oh ) ) ; h nmr ( 600 mhz , cd3od )
4.89 ( d , j = 11.2 hz , 1h ) , 4.75 ( d , j = 10.7 hz , 1h ) , 4.55 ( d , j = 10.2 hz , 1h ) , 4.45
( dd , j = 10.2 , 1.5 hz , 1h ) , 4.31 ( s , 2h ) , 4.234.19
( m , 1h ) , 2.562.49 ( m , 1h ) , 2.272.24 ( m , 2h ) , 2.152.06
( m , 1h ) , 1.991.92 ( m , 1h ) , 1.851.76 ( m , 2h ) , 1.761.69
( m , 1h ) , 1.691.62 ( m , 2h ) , 1.611.52 ( m , 2h ) , 1.511.42
( m , 2h ) , 1.421.27 ( m , 14h ) , 0.90 ( app t , j = 6.9 , 6.9 hz , 3h ) ; c nmr ( 126 mhz , cd3od ,
313 k ) 91.3 , 80.9 , 75.6 , 68.4 , 63.3 , 62.4 , 62.2 , 45.5 , 45.1 ,
39.9 , 33.5 , 32.9 , 30.4 , 30.3 , 30.07 , 30.05 , 30.0 , 27.6 , 27.4 , 25.8 ,
23.6 , 14.4 . the analytical data for ( + ) -1 were in agreement
with the data given in refs ( 2k ) , ( 4c ) , ( 5 ) , and ( 6b ) . to a stirred solution of ni(oac)24h2o ( 9.12 g , 36.7 mmol ) in ch3oh ( 500 ml ) was added
nabh4 ( 1.38 g , 36.7 mmol ) portionwise over 5 min at 0 c . the blue solution immediately turned black upon addition of nabh4 . after being stirred for an additional 5 min , the ice bath
was removed and ethylene diamine ( 4.90 ml , 36.7 mmol ) was added . after
being stirred for 5 min , a solution of undec-2-yn-1-ol ( 24.7 g , 147 mmol ) in ch3oh ( 230
ml ) was added . the reaction mixture was quickly purged with h2 three times and stirred overnight under a balloon of h2 prior to the dilution with h2o ( 100 ml ) and n - pentane ( 100 ml ) . after filtration through celite , the
organic layer was separated and the aqueous layer was extracted with n - pentane ( 100 ml 3 ) . the combined organic extracts
were washed with h2o ( 50 ml ) and saturated aqueous nacl
solution ( 50 ml ) , dried over mgso4 , filtered , and concentrated
in vacuo ( 5 mmhg ) to afford 51 ( 25.0 g , 98% ) as a colorless
oil . data for 51 : h nmr
( 600 mhz , cdcl3 ) 5.625.52 ( m , 2h ) , 4.19
( d , j = 6.4 hz , 2h ) , 2.07 ( q , j =
7.2 hz , 2h ) , 1.381.32 ( m , 2h ) , 1.321.23 ( m , 10h ) ,
0.88 ( t , j = 6.9 hz , 3h ) ; c nmr ( 126
mhz , cdcl3 ) 133.5 , 128.4 , 58.8 , 32.0 , 29.8 , 29.6 ,
29.42 , 29.38 , 27.6 , 22.8 , 14.3 ; ir ( thin film ) 3347 , 3938 , 3857 , 1015
cm ; hrms ( ci - tof ) m / z calcd for c11h22onh4 [ m + nh4 ] 188.2014 , found 188.2023 . to a stirred solution of ( 4.96
g , 29.1 mmol ) in ch2cl2 ( 70 ml ) was added et4ncl3 ( 13.8 g , 58.3 mmol ) portionwise over 5 min
at rt . after the yellow color disappeared over the course of 10 min ,
another portion of et4ncl3 ( 6.89 g , 29.1 mmol )
was added portionwise over 3 min . after being stirred for 30 min , the reaction mixture was poured into a mixture of saturated aqueous
nahco3 solution ( 15 ml ) and saturated aqueous na2s2o3 solution ( 15 ml ) . the organic layer was
separated , and the aqueous layer was extracted with hexanes ( 30 ml
3 ) . the combined organic extracts were dried over mgso4 , filtered , and concentrated in vacuo ( 5 mmhg ) . the residue
was purified by column chromatography ( 150 ml of sio2 ,
10% etoac / hexanes , rf = 0.6 in 30% etoac / hexanes , kmno4 ) to give ( )-52 ( 5.79 g , 82% ) as a colorless oil . data for ( )-52 : h nmr ( 500 mhz , cdcl3 )
4.22 ( ddd , j = 8.1 , 5.4 , 2.6 hz , 1h ) , 4.18 ( m , 1h ) ,
3.95 ( ddd , j = 11.9 , 7.7 , 6.0 hz , 1h ) , 3.89 ( ddd , j = 12.1 , 7.5 , 5.6 hz , 1h ) , 1.94 ( dd , j = 7.7 , 5.4 hz , 1h ) , 1.901.84 ( m , 2h ) , 1.571.51 ( m ,
1h ) , 1.441.22 ( m , 11h ) , 0.88 ( app t , j =
6.6 , 6.6 hz , 3h ) ; c ( 126 mhz , cdcl3 )
65.6 , 64.7 , 62.2 , 35.3 , 32.0 , 29.5 , 29.3 , 29.1 , 26.7 , 22.8 , 14.3 ;
ir ( thin film ) 3363 , 3923 , 2855 , 1455 , 1041 cm ; hrms ( ci - tof ) m / z calcd for c11h22cl2onh4 [ m
+ nh4 ] 258.1392 , found 258.1401 . to a stirred suspension of ( )-52 ( 2.20 g , 9.12 mmol ) and nahco3 ( 2.30 g , 27.4
mmol ) in ch2cl2 ( 46 ml , saturated with h2o ) martin periodinane ( 5.80 g , 13.7
mmol ) portionwise over 1 min at 0 c under air . after being stirred
for 5 min , the ice bath was removed and the reaction mixture was stirred
at rt for 25 min prior to the addition of hexanes ( 20 ml ) and saturated
aqueous nahco3 solution ( 100 ml ) . the organic layer was
separated , and the aqueous layer was extracted with hexanes ( 50 ml
3 ) . the combined organic extracts were filtered , dried over
mgso4 , filtered , and concentrated in vacuo ( 5 mmhg ) to
give ( )-53 as a pale yellow oil . the crude material
was generally used directly for the next reaction within 30 min and
without further purification ( it was often contaminated with up to
5% 2-chloro-2-undecenal ) . data for ( )-53 : h nmr ( 500 mhz , cdcl3 ) 9.55 ( s , 1h ) , 4.40
( app s , 2h ) , 1.921.86 ( m , 2h ) , 1.551.48 ( m , 1h ) , 1.401.22
( m , 11h ) , 0.88 ( t , j = 7.0 hz , 3h ) ; c nmr ( 126 mhz , cdcl3 ) 194.8 , 67.0 , 60.1 , 35.1 ,
31.8 , 29.3 , 29.1 , 28.8 , 26.1 , 22.6 , 14.1 ; ir ( thin film ) 2927 , 2856 ,
1736 , 1465 cm ; hrms ( esi - tof ) m / z calcd for c11h19clona [ m hcl + na ] 225.1022 , found 225.1013 . to a stirred solution of tmp ( 3.39
ml , 20.1 mmol ) in thf ( 46 ml ) was added n - buli ( 2.47
m in hexanes , 7.75 ml , 19.2 mmol ) at 78 c . after being
stirred for 15 min , the litmp solution was cannulated into a solution
of allyl bromide ( 1.66 ml , 19.2 mmol ) and et2alcl ( 1.0
m in hexanes , 36.5 ml , 36.5 mmol ) in thf ( 46 ml ) at 78 c
over 15 min . the resulting solution was stored at 78 c ,
while ( )-53 was prepared ( see above ) . a solution
of ( )-53 in thf ( 10 ml + rinsed with 8 ml
2 ) was added dropwise down the side of the flask . after being stirred
at 78 c for 5 h , the cooling bath was removed and a
6 m aq naoh solution ( 100 ml ) was added . after stirring vigorously
for 1 h , the biphasic mixture was diluted with hexanes ( 100 ml ) and
shaken in a separatory funnel . the organic layer was separated , and
the aqueous layer was extracted with hexanes ( 100 ml 3 ) . the
combined organic extracts were washed with saturated aqueous nacl
solution ( 50 ml 3 ) , filtered through silica gel ( ch2cl2 , 300 ml ) , and concentrated in vacuo ( 5 mmhg ) . the
residue was purified by column chromatography ( 500 ml of sio2 , 5% ch2cl2/hexanes , rf = 0.2 , kmno4 ) and bulb - to - bulb
distillation under reduced pressure ( 0.1 mmhg , abt 150 c ) to
give ( )-55 as a colorless oil ( 1.83 g , 72% from
( )-52 ) . data for ( )-55 : h nmr ( 500 mhz , cdcl3 ) 5.82 ( ddd , j = 17.0 , 10.6 , 5.3 hz , 1h ) , 5.49 ( d , j = 17.2 hz , 1h ) , 5.45 ( d , j = 10.7 hz , 1h ) , 4.26
( ddd , j = 8.3 , 4.9 , 2.7 hz , 1h ) , 3.67 ( dd , j = 8.9 , 2.7 hz , 1h ) , 3.64 ( app t , j =
4.7 hz , 1h ) , 3.54 ( dd , j = 9.7 , 4.7 hz , 1h ) , 1.94
( app dtd , j = 14.2 , 9.5 , 4.8 hz , 1h ) , 1.83 , ( app
ddt , j = 14.0 , 10.1 , 5.4 hz , 1h ) , 1.581.50 ,
( m , 1h ) , 1.431.35 ( m , 1h ) , 1.351.22 ( m , 10h ) , 0.88
( app t , j = 6.6 hz , 3h ) ; c nmr ( 126
mhz , cdcl3 ) 130.5 , 121.3 , 63.5 , 60.4 , 58.6 , 57.4 ,
35.8 , 32.0 , 29.5 , 29.3 , 29.1 , 26.5 , 22.8 , 14.3 ; ir ( thin film ) 2926 ,
2855 , 932 cm ; hrms ( ci - tof ) m / z calcd for c14h24cl2onh4 [ m + nh4 ] 296.1548 ,
found 296.1560 . to a stirred solution of ( )-55 ( 500 mg , 1.79 mmol ) and ( s)-binapo ( 234
mg , 0.358 mmol ) in ch2cl2 ( 36 ml ) were added i - pr2net ( 31.0 l , 0.179 mmol ) and sicl4 ( 144 l , 1.25 mmol ) slowly at 78 c . after
39 h , a solution of ch3oh / et3n / ch2cl2 ( 1/1/5 , 5 ml ) was added quickly at 78 c . the resulting solution was vigorously stirred with a saturated aqueous
nahco3 solution ( 20 ml ) at rt for 2 h. the organic layer
was separated , and the aqueous layer was extracted with ch2cl2 ( 20 ml 3 ) . the combined organic layers were
dried over mgso4 , filtered , and concentrated in vacuo ( 5
mmhg ) . the residue was purified by column chromatography ( sio2 , 5% etoac / hexanes , kmno4 ) to give ( )-55 ( 165 mg , 33% , 3.4:96.6 er , rf = 0.7 in 10% etoac / hexanes ) as a colorless oil and
( + ) -56 ( 367 mg , 65% , 71.4:28.6 er , rf = 0.5 in 10% etoac / hexanes ) as pale
yellow crystals . the enantiopurity of the recovered reactant ( )-55 was measured after ring - opening chlorinolysis to form ( )-56 . data for ( )-55 : [ ]d = 23.5 ( c 1.74 , chcl3 ) ; gc ( b - dm , 30 psi , 180 c ) tr 23.9
min ( 2.6% ) , 25.1 min ( 97.4% ) . data for ( + ) -56 : mp 34.036.0
c ; [ ]d = + 1.6 ( c 2.01 , chcl3 ) ; gc ( b - dm , 30 psi , 180 c ) tr 23.7 min ( 71.4% ) , 25.3 min ( 28.6% ) ; h nmr ( 600 mhz , cdcl3 ) 6.07 ( ddd , j = 17.1 , 10.3 , 7.4 hz , 1h ) , 5.49 ( dd , j = 16.1 ,
1.0 hz , 1h ) , 5.35 ( dd , j = 10.3 , 0.8 hz , 1h ) , 5.10
( dd , j = 7.4 , 1.0 hz , 1h ) , 4.56 ( ddd , j = 9.0 , 5.2 , 1.4 hz , 1h ) , 4.10 ( dd , j = 9.4 , 1.5
hz , 1h ) , 4.05 ( dd , j = 9.1 , 1.1 hz , 1h ) , 2.18 ( d , j = 8.8 hz , 1h ) , 2.00 ( app dtd , j = 14.0 ,
10.0 , 4.7 hz , 1h ) , 1.80 ( app ddt , j = 15.5 , 10.7 ,
5.5 hz , 1h ) , 1.581.51 ( m , 1h ) , 1.451.38 ( m , 1h ) , 1.361.24
( m , 10h ) , 0.89 ( app t , j = 6.9 hz , 3h ) ; c nmr ( 126 mhz , cdcl3 ) 134.9 , 119.5 , 74.5 , 64.7 ,
64.5 , 61.7 , 36.5 , 32.0 , 29.5 , 29.3 , 29.2 , 26.7 , 22.8 , 14.3 ; ir ( thin
film ) 3390 , 2925 , 2855 , 933 cm ; hrms ( esi - tof ) m / z calcd for c14h25cl4o [ m + cl ] 349.0659 ,
found 349.0665 . the solvents were bubbled with
argon for 15 min before use . to a stirred solution of ( )-55 ( 134 mg , 0.481 mmol ) and 71 ( 530 mg , 1.44
mmol ) in dce ( 480 l ) was added a solution of 60 ( 91.3 mg , 0.144 mmol ) in ch2cl2 ( 600 l )
in six portions ( 0 , 15 , 30 , 45 , 60 , 75 min ) at 35 c while the
reaction mixture was vigorously bubbled with argon ( saturated with
dce ) . after being stirred at 35 c
with argon bubbling for an additional 105 min , the reaction mixture was cooled to rt , filtered through a plug of silica gel ( ch2cl2 , 10 ml ) , and concentrated in vacuo ( 5 mmhg ) . the residue
was purified via column chromatography ( 140 ml of sio2 ,
5% ch2cl2/hexanes , rf = 0.23 in 10% ch2cl2/hexanes )
to give ( + ) -76 ( 57.1 mg , 19% , > 20:1 = z : e ) as a colorless oil . data for ( + ) -76 : [ ]d = + 0.088 ( c 2.65 , chcl3 ) ; h nmr ( 499 mhz , cdcl3 ) 5.86 ( app td , j = 8.9 , 8.4 hz , 1h ) , 5.22
( app t , j = 8.7 hz , 1h ) , 4.294.24 ( m , 1h ) ,
3.92 ( s , 2h ) , 3.823.77 ( m , 1h ) , 3.65 ( d , j = 8.9 hz , 1h ) , 3.52 ( dd , j = 8.0 , 2.2 hz , 1h ) ,
2.22 ( app q , j = 7.2 hz , 2h ) , 2.192.15 ( m ,
2h ) , 1.981.89 ( m , 1h ) , 1.861.78 ( m , 1h ) , 1.621.52
( m , 3h ) , 1.451.39 ( m , 3h ) , 1.371.23 ( m , 18h ) , 0.91
( s , 9h ) , 0.88 ( app t , j = 6.3 hz , 3h ) , 0.11 ( s , 6h ) ; c nmr ( 126 mhz , cdcl3 ) 139.8 , 121.6 , 93.7 ,
72.3 , 63.5 , 31.3 , 58.3 , 54.0 , 43.7 , 35.9 , 32.0 , 29.5 , 29.45 , 29.43
( 2c ) , 29.32 , 29.28 , 29.18 , 29.12 , 28.4 , 26.5 , 25.9 , 24.9 , 22.8 , 18.4 ,
14.2 , 5.2 ; ir ( thin film ) 2927 , 2855 , 1119 , 939 , 779 cm ; hrms ( esi - tof ) m / z calcd for c30h56o2cl4sina [ m + na ] 639.2701 , found 639.2719 . to a flask containing magnesium
( 3.43 g , 141 mmol )
in thf ( 10 ml ) was added 1,2-dibromoethane ( 275 l , 3.19
mmol ) slowly . after dilution with additional thf ( 70 ml ) , a solution of
1-bromo-10-undecene ( 10.0 ml , 45.6 mmol ) in thf ( 20 ml ) was added
over 1 h via a syringe pump . after being stirred for 1 h , the mixture
was cooled to 0 c and allowed to settle . the liquid phase was
transferred via a cannula to a rapidly stirred solution of dmf ( 53
ml , 684 mmol ) and thf ( 53 ml ) at 0 c . after being stirred for
20 min at rt , the reaction mixture was diluted with hexanes ( 200 ml )
and poured into 1 m aq hcl ( 200 ml ) . the organic layer was separated ,
and the aqueous layer was extracted with hexanes ( 200 ml 3 ) . the combined organic extracts were washed with brine ( 100 ml ) , dried
over mgso4 , filtered , and concentrated in vacuo ( 5 mmhg ) . the residue was purified by column chromatography ( 300 ml of sio2 , 5% etoac in hexanes ) to afford 68 ( 6.33 g ,
76% ) as a colorless oil . data for 68 : h nmr
( 500 mhz , cdcl3 ) 9.76 ( s , 1h ) , 5.81 ( ddt , j = 17.0 , 10.1 , 6.7 hz , 1h ) , 4.99 ( dd , j = 17.0 , 1.4 hz , 1h ) , 4.92 ( dd , j = 10.2 , 0.8 hz ,
1h ) , 2.41 ( td , j = 7.6 , 1.7 hz , 2h ) , 2.03 ( app q , j = 7.1 hz , 2h ) , 1.62 ( tt , j = 7.3 , 6.6
hz , 2h ) , 1.391.35 ( m , 2h ) , 1.331.25 ( m , 10h ) ; c nmr ( 126 mhz , cdcl3 ) 203.0 , 139.2 , 114.1 ,
43.9 , 33.8 , 29.4 , 29.34 , 29.31 , 29.13 , 29.07 , 28.9 , 22.1 ; ir ( thin
film ) 2926 , 2854 , 2715 , 1727 cm ; hrms ( esi - tof ) m / z calcd for c12h22ona [ m + na ] 205.1568 , found 205.1561 . to
a flask containing t - butylamine ( 634 l , 6.03
mmol ) was added 11-dodecenal ( 68 ) ( 1.00 g , 5.49 mmol )
dropwise at 0 c . after being
stirred at rt for 45 min , the cloudy reaction mixture was dried over
k2co3 ( 3.79 g , 27.4 mmol ) , filtered , and concentrated
in vacuo ( 25 mmhg ) . the residue was purified by bulb - to - bulb distillation
under reduced pressure ( 0.25 mmhg , abt 128135 c ) to
give the corresponding t - butylimine ( 1.21 g , 92:8 imine : aldehyde ) as a colorless oil . the t - butylimine was dissolved in ch2cl2 ( 15 ml ) , and n - chlorosuccinimide ( 2.04
g , 15.3 mmol ) was added at rt under air . after being stirred for 24
h , the organic layer was separated ,
and the aqueous layer was extracted with ch2cl2 . the combined organic extracts were washed with saturated aqueous
nacl solution , dried over na2so4 , filtered ,
and concentrated in vacuo ( 25 mmhg ) . the residue was diluted with
hexanes , filtered , and concentrated in vacuo ( 25 mmhg ) to give the
corresponding ,-dichloro - t - butylimine as a yellow oil ( 1.54 g , 94:6 dichloride : monochloride ) . the crude material was dissolved in thf ( 10 ml ) , and 6 m aq hcl ( 10
ml ) was added at rt . the organic layer was separated ,
and the aqueous layer was extracted with et2o . the combined
organic extracts were washed with saturated aqueous nahco3 solution , dried over mgso4 , filtered , and concentrated
in vacuo ( 25 mmhg ) . the residue was purified by column chromatography
( sio2 , = 2.2 cm , l = 7 cm , n - pentane / ch2cl2 , 2/1 , rf = 0.20 , streaky , kmno4 ) and bulb - to - bulb distillation under reduced pressure ( 0.25 mmhg ,
abt 129135 c ) to give 69 ( 1.01 g , 73% over
three steps , 94% pure ) as a colorless oil . data for 69 : h nmr ( 500 mhz , cdcl3 )
9.25 ( s , 1h ) , 5.81 ( ddt , j = 16.9 , 10.2 , 6.7 hz ,
1h ) , 4.99 ( dd , j = 17.1 , 1.9 hz , 1h ) , 4.93 ( dd , j = 10.2 , 1.0 hz , 1h ) , 2.312.23 ( m , 2h ) , 2.04 ( dd , j = 14.4 , 6.9 hz , 2h ) , 1.661.58 ( m , 2h ) , 1.431.24
( m , 10h ) . to a stirred solution of 69 ( 1.00 g , 3.98
mmol ) in ethanol ( 12 ml ) was added nabh4 ( 151 mg , 3.98
mmol ) at 0 c under air . after being stirred for 30 min at rt ,
1 m aq hcl ( 12 ml ) was added . the combined organic
extracts were dried over na2so4 , filtered , and
concentrated in vacuo ( 25 mmhg ) . the residue was purified by column
chromatography ( sio2 , = 2.2 cm , l = 13 cm , n - pentane / ch2cl2 , 1/1 , rf = 0.29 , p - anisaldehyde ) to give 70 ( 934 mg , 93% ) as
a colorless oil . data for 70 : h nmr ( 500
mhz , cdcl3 ) 5.81 ( ddt , j = 16.9 ,
10.2 , 6.7 hz , 1h ) , 4.99 ( dd , j = 17.1 , 1.5 hz , 1h ) ,
4.93 ( d , j = 10.2 hz , 1h ) , 3.90 ( d , j = 7.6 hz , 2h ) , 2.29 ( t , j = 7.6 hz , 1h ) , 2.242.18
( m , 2h ) , 2.04 ( dd , j = 14.3 , 6.9 hz , 2h ) , 1.681.59
( m , 2h ) , 1.421.26 ( m , 10h ) ; c nmr ( 126 mhz , cdcl3 ) 139.1 , 114.2 , 94.7 , 72.1 , 43.5 , 33.8 , 29.28 , 29.27 ,
29.02 , 28.98 , 28.8 , 24.8 ; ir ( thin film ) 3484 , 2926 , 2854 cm ; hrms ( ci - tof ) m / z calcd for c12h22ocl2nh4 [ m
+ nh4 ] 270.1392 , found 270.1390 . to
a stirred solution of 70 ( 348 mg , 1.37 mmol ) and imidazole
( 187 mg , 2.75 mmol ) in ch2cl2 ( 2 ml ) was added
tbscl ( 228 mg , 1.51 mmol ) at rt . after being stirred for 48 h , the
reaction mixture was diluted with ch2cl2 ( 5
ml ) and shaken with saturated aqueous nahco3 ( 5 ml ) . the
organic layer was separated , and the aqueous layer was extracted with
hexanes ( 10 ml 3 ) . the combined organic extracts were washed
with saturated aqueous nacl ( 3 ml ) , concentrated in vacuo ( 5 mmhg ) ,
and passed through a pad of silica gel ( 5% etoac in hexanes , 10 ml ) . the residue was purified by bulb - to - bulb distillation under reduced
pressure ( 0.05 mmhg , abt 170180 c ) to afford 71 ( 453 mg , 90% ) as a colorless oil . data for 71 : h nmr ( 500 mhz , cdcl3 ) 5.81 ( ddt , j = 17.0 , 10.1 , 6.7 hz , 1h ) , 4.99 ( dd , j = 17.3 , 1.7 hz , 1h ) , 4.93 ( dd , j = 10.2 , 1.0 hz ,
1h ) , 3.92 ( s , 2h ) , 2.152.19 ( m , 2h ) , 2.04 ( app q , j = 7.2 hz , 2h ) , 1.551.62 ( m , 2h ) , 1.271.40
( m , 10h ) , 0.91 ( s , 9h ) , 0.11 ( s , 6h ) ; c nmr ( 126 mhz ,
cdcl3 ) 139.2 , 114.1 , 93.5 , 72.1 , 43.5 , 33.8 , 29.30 ,
29.29 , 29.1 , 29.0 , 28.9 , 25.7 , 24.7 , 18.3 , 5.4 ; ir ( thin film )
2928 , 2856 , 1118 , 838 cm ; hrms ( ci - tof ) m / z calcd for c18h36cl2osinh4 [ m + nh4 ] 384.2256 , found 384.2257 . | a second - generation synthesis of
three structurally related chlorosulfolipids
has been developed .
key advances include highly stereocontrolled
additions to ,-dichloroaldehydes , kinetic resolutions
of complex chlorinated vinyl epoxide intermediates , and z - selective alkene cross metatheses of cis - vinyl
epoxides .
this strategy facilitated the synthesis of enantioenriched
danicalipin a , mytilipin a , and malhamensilipin a in nine , eight ,
and 11 steps , respectively . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
does hippocampal activity embody the cognitive map ? one should expect the neural instantiation
of tolman 's cognitive map to contain units ( neurons ) that are fully
allocentric , that is , identify places in the environment independent of the
subject 's perspective ( egocentric direction ) and ongoing behavior . furthermore ,
one should expect that the neural ensemble composed of these units would be
holistic ; that is , all the neuronal representations should be tied to one
another and change together between environments . and , if the map is to suit
the purpose tolman proposed in guiding behavior according to expectancies , the
map should signal the locations of current goals . initially , hippocampal place cells seemed to satisfy key criteria for elements of tolman 's
cognitive map . the first complete study characterized place cells as signaling
an animal 's location in the environment independent of egocentric direction and
ongoing behavior , as would be expected of the units in an allocentric representation
. an expansive literature followed on the initial observations ,
and many interpreted the results as support for the claim that the neural
substrate for the cognitive map lies in the circuitry of the hippocampus
[ 39 ] . location related hippocampal neuronal
activity tells us only where the animal is , not where it plans to go , as is
tolman 's intended function of a cognitive map . succeeding studies
directly refuted the idea that the hippocampal network contains purely
allocentric representations and a holistic map . inconsistent with a holistic
representation , simultaneously recorded place cells respond differently and
independently to changes in environmental cues or task demands ( e.g. , [ 1114 ] ) . furthermore , inconsistent with allocentric representation ,
the activity of most hippocampal neurons is dependent on egocentric spatial
parameters , including the direction and speed of the animal 's movements
. indeed , place cells reliably provide an allocentric
signal only under highly constrained conditions where all perceptual cues ,
behaviors , and cognitive demands are held constant . in addition , hippocampal
neuronal activity has been associated with a variety of nonspatial cues ,
behaviors , and task demands [ 1627 ] , consistent with additional findings showing a critical role for
the hippocampus in nonspatial as well as spatial learning and memory [ 2830 ] . also , several recent studies have
provided compelling evidence that so - called place cells are strongly influenced
by nonspatial cognitive demands in animals performing spatial memory tasks
[ 3137 ] , and thus signal where
the animal is only in particular circumstances associated with a behaviorally
salient task . in sum , place
cells do identify where the animal is when important things happen . but place
cells do not carry a reliable allocentric signal , and populations of place
cells do not operate as a holistic representation of space or anticipate the
locations of goals . therefore , hippocampal neurons do not have the requisite
properties to support tolman 's cognitive map . by contrast , the findings
indicate that hippocampal neurons represent events in the places where they
occur , consistent with current views of hippocampal involvement in episodic
memory ( e.g. , [ 38 , 39 ] ) . the recent
discovery of spatial firing patterns in the cortex immediately adjacent to the
hippocampus has refocused the search for the cognitive map to a zone within the
medial entorhinal area [ 36 , 4043 ] . a majority of the data describes the spatial
firing patterns of principal neurons in the medial entorhinal cortex , and more
specifically how a proportion of these neurons , the so - called grid cells ,
exhibit an intriguing and unique spatial firing pattern with several interesting
properties . first , the relative angles and densities of peaks within grids of
neighboring cells remain invariant both across environments and in response to
changes in local cues . second , while grid fields of
medial entorhinal neurons remain stable in response to modest environmental
manipulation , hippocampal ca3 neurons change their rate of firing ( rate
remapping , ) . in response to more significant environmental
change , grid fields of local ensembles of medial entorhinal neurons rotate
while maintaining relative geometric consistency , whereas ca3 neurons fire in a
different location ( global remapping ) , . thus , in response
to environmental manipulation , changes in medial entorhinal activity are more
systematic and predictable than corresponding hippocampal ca3 responses , and
consequently more stable as sensory inputs change . third , while lacking any
obvious topographic organization of space , the relative size of medial
entorhinal grid fields changes systematically along a dorsal - ventral axis
. although medial entorhinal cells are influenced by
egocentric parameters of head direction and velocity , ,
these findings modestly suggest that some version of the cognitive map may
reside within the medial entorhinal cortex , rather than the immediately
adjacent hippocampus . however , here we will suggest an alternate view
driven by recent data that includes our own experiment wherein sensory cues
were held constant throughout the experiment that spatial representations observed in
medial entorhinal cortex may make a specific contribution to episodic memory . the hippocampus is
strongly implicated in spatial memory and navigation as evidenced by both
behavioral and physiological studies . at the same time , a convergent stream of
behavioral , physiological , and computational modeling data indicate that
hippocampal processing is critical for episodic memory [ 4557 ] .
how can these two seemingly distinct lines of evidence be reconciled ? current
conceptions of episodic memory emphasize the temporal organization of sequences
of events as they unfold over time and space . representations of
events are composed as associations between specific objects , actions , and the
locations where they occur . recent experiments have revealed a critical role
of the hippocampus in memory for sequences of events that compose unique
episodes [ 47 , 59 ] . in addition , episodic memory
also relies on the capacity to distinguish event sequences that share common
elements . this property of episodic memory is
especially evident in spatial memories , for example , we are usually very good
at remembering unique events that occur day by day as we take the same route to
work each day . computational models suggest that the ability to disambiguate
overlapping elements from multiple experiences may be a critical feature of
hippocampal function that contributes to episodic memory . consistent with this view , rats with hippocampal lesions fail on a sequence
disambiguation task that involved two series of events that contain overlapping
items . additional support
for sequencing and disambiguation of serial events by hippocampal networks
comes from analyses of hippocampal neuronal activity in animals performing
spatial memory tasks . in one study , rats were trained on the classic spatial
t - maze alternation task in which successful performance depends on
distinguishing left- and right - turn episodes to guide each subsequent choice
. if hippocampal neurons encode each sequential behavioral
event within one type of episode , then neuronal activity at locations that
overlap in left- and right - turn trials should vary according to trial type . indeed , virtually all cells that were active as the rat traversed these common
locations were differentially active on left- versus right - turn trials . despite
modest differences in the proportion of neurons that exhibit this pattern of
activity across studies similar results
have been observed in several versions of this task [ 3135 , 37 , 61 ] . these findings suggest a reconciliation of the
spatial and episodic memory views of hippocampal function : place cells
represent the series of places where events occur in sequences that compose
distinct episodic memories . in order to
correctly trigger a series of event representations within a particular
episode , the hippocampus requires a mechanism to bind its representations of
event sequences according to the appropriate episode they compose . one
suggestion is that sequences are bound by a shared temporal context [ 49 , 62 ] and that the mechanism for
contextual binding involves context sensitive neurons that fire for prolonged
periods to bridge sequences of events that occur within a particular context
. here , we review evidence suggesting that the context
sensitive neurons exist in the medial entorhinal cortex and serve a function
complementary to that of hippocampal place cells which encode discrete events . thus far , all
observations of grid field activity patterns in medial entorhinal cortex are
derived from animals foraging in random directions within an open field . in
fact , derdikman et al . report that the grid structure breaks down when animals
are constrained to make hairpin turns within the previously unconstrained open
field . this is notable because in the standard , random foraging experimental
protocol , spatial cues provide the only regularities and constraints . in
contrast , what differed between the hairpin turn maze and the open field
condition was the imposition of behavioral constraints ; spatial cues were held
constant . importantly , it is only under the unconstrained open field condition
that hippocampal cells display purely allocentric spatial firing patterns . perhaps where stimulus or behavioral regularities are imposed , the activity of
neurons in medial entorhinal cortex , like neurons in the hippocampus , might
reflect the corresponding regularities embedded in the task protocol . in a recent study ,
we adopted the same spatial memory task used previously to
compare the activity of hippocampal and medial entorhinal neurons in animals
performing a continuous spatial alternation on a t - maze in which hippocampal
neurons encode sequences of locations traversed and disambiguate overlapping
routes . first , we were explicitly interested in comparing how medial entorhinal
and hippocampal neurons uniquely represent aspects of the continuous spatial
alternation , rather than in an analysis of grid cell properties . our
interpretation of our data therefore addresses the contribution of medial
entorhinal cortex to episodic memory , not whether a grid field forms on a
t - maze . second , just as the expansive place cell literature relies almost
exclusively on observations of hippocampal activity in situations that neither
require any manner of hippocampal processing nor impose any memory demands , our
experimental design exploited the capacity of medial entorhinal neurons to
encode spatial information . whether the task is hippocampal or entorhinal
dependent is not relevant to our interpretations . insofar as the continuous
spatial alternation is not a hippocampal dependent - task , it is worth noting
that hippocampal dependence is neither an operational definition of , nor a
pre - requisite for , memory . we trained rats to
perform the spatial alternation task on a t - maze that included return arms that
connected the end of each goal arm to the starting end of the central stem
( figure 1 ) . a left - turn trial began as the animal departed the right goal area ,
ran down the return arm to the central stem , traversed the central stem , and
made a left - turn into the left goal area to retrieve a water reward . similarly ,
a right - turn trial began when the animal departed the left goal area , returned
to and traversed the central stem , and made a right turn into the right goal
area . drawing on the model of episodic memory noted above , each left- or
right - turn trial can be considered a unique episode , constructed by connecting
sequential behavioral events identified by a series of loci along the maze . areas that lie along the central stem constitute overlapping elements of both
types of episodes , and are indeed represented differently by hippocampal
neurons depending on the ongoing episode ( figure 2 , [ 36 , 37 ] ) . furthermore , consistent with previous reports [ 40 , 65 ] , activity of neurons in medial entorhinal cortex also
signals an animal 's position along the maze . though we did not witness the
development of a grid - like firing pattern on the t - maze , a proportion of our
medial entorhinal neurons did exhibit a high degree of spatial specificity
. for example , the medial entorhinal cell shown in figure 3
fired predominantly at the proximal end of the central stem during both left-
and right - turn trials , while remaining largely silent through other regions of
the maze . many of our medial
entorhinal neurons that exhibited spatial specificity also exhibited
differential firing along the central stem of the maze during left- and
right - turn trials , similar to hippocampal neurons . the
patterns of neuronal activity illustrated in figure 4 represent typical
trial - type specific activity exhibited by medial entorhinal neurons . some
medial entorhinal cells fired selectively during the trial and distinguished
left - turn and right - turn trials . for example , the cell shown in figure 4(a ) was
selectively active when the rat was near the end of the central stem and fired
at a higher rate during right - turn compared to left - turn trials . however , most
medial entorhinal neurons showed only crude spatial specificity . for example ,
the cell shown in figure 4(b ) fired somewhat indiscriminately through different
regions of the maze , and although active along the entire central stem , was
significantly more active on left - turn trials . this pattern of activity was an
exclusive feature of medial entorhinal neurons , such that we observed no
hippocampal units with poorly localized , trial - type specific firing that
extended the length of the central stem . we used a two - way
anova and log - likelihood estimation to quantitatively compare the incidence and
robustness of trial - type disambiguation in medial entorhinal and hippocampal
neurons . dividing the central stem into seven equal segments , we used a two - way
anova to compare the spatial firing patterns on segments of the central stem
between left - turn and right - turn trial types for each cell . we considered that a significant main effect of trial type or a trial type by
segment interaction qualified a cell as differentiating left- from right - turn
trials . a significant main effect of segment without a significant main effect
of trial type or trial type by segment interaction denoted location - specific
activity only . the log - likelihood ratio , on the other
hand , represented the degree to which firing patterns on left - turn and
right - turn trials differed , and thus allowed us to measure the difference in
the firing patterns across trial types , rather than knowing simply that they
differed . | l , x]/p[r | r , x ] , where p[r | l , x ] is the probability density
function of left - turn ( l ) trials at position x , evaluated at the observed
firing rate r , and p[r | r , x ] is the equivalent function for right - turn ( r ) trials . for each cell , log - likelihood ratios were summed over all central
stem bins for each trial . where the log - likelihood sum is greater than zero ,
maximum likelihood analysis predicts that the data came from a left - turn trial ;
otherwise , a right - turn trial is predicted . we calculated the average absolute
value of the summed log - likelihood ratio , such that larger values of this term
indicate firing - rate patterns that are statistically more distinct ( for a more
detailed description , see ) . using the two - way
anova , we identified neurons in the hippocampus and medial entorhinal area that
distinguished trial type as animals traversed the central stem . based on the
two - way anova , 56% of medial entorhinal neurons ( 23/41 with place fields on the
central stem ) were significantly more active on either right- or left - turn
trials , whereas 33% ( 16/48 ) of hippocampal neurons exhibited differential
firing on the central stem . moreover , the log - likelihood estimation revealed
that medial entorhinal neurons more robustly distinguished left- from
right - turn trials than did hippocampal neurons , such that the average
log - likelihood ratio for medial entorhinal neurons was significantly greater
than for hippocampal neurons ( mec , 2.82 ; hippocampus , 1.7 ; wilcoxon rank - sum test , p < .003 ) . in other words , firing patterns of medial entorhinal neurons
were on average more distinct on either left- or right - turn trials than were
hippocampal neurons . two additional
measures were applied to describe how the patterns of activity in hippocampal
and medial entorhinal neurons differed along the central stem during left- and
right - turn trials . the first measure , pcorrect , is based on a maximum - likelihood guess
performed for each trial compared against the actual outcome of that trial , and
thus describes how accurate the log - likelihood estimate is for each trial type .
to calculate pcorrect , we
performed a maximum likelihood analysis using the conditional density functions
as described above . pcorrect represents the number of times that prediction was correct , divided by the
total number of trials . therefore , pcorrect is the average trial - by - trial probability that the log - likelihood analysis
gives the correct answer for each trial type : a population that more
consistently and significantly differentiates left- from right - turn trials will
have a higher pcorrect . the second measure , pchance ,
is the average probability that firing patterns across left- and right - turn
trials arose by chance , given a pcorrect of 0.5 ( i.e. , the firing rate contains no trial - specific information , which is
necessary to avoid biasing the calculation ) . to calculate pchance , we evaluated the following formula : pnk = n!/k!/(n k)!(0.5)0.5 , where n is the number of trials , and k is the number of apparently correct answers from maximal likelihood
analysis . to get pchance ,
we summed pnk for all values of k greater
than or equal to the number associated with our measured value of pcorrect . again ,
medial entorhinal neurons had a significantly higher mean pcorrect than hippocampal neurons ( mec , 70% ;
hippocampus , 63% ; wilcoxon rank - sum test , p < .0001 ) , indicating that
the activity of medial entorhinal neurons along the central stem more
successfully predicted trial type than hippocampal neurons . correspondingly ,
the difference in firing among left- and right - turn trials of medial entorhinal
neurons was on average less likely to have occurred by chance than that of hippocampal
neurons ( pchance equal to
or less than 0.05 : 90% mec ; 50% hippocampus ) . while the anova and log - likelihood estimation did not always agree on specific units ,
together they converged on the same conclusion ; just as hippocampal units did
not exclusively encode information about space , medial entorhinal neurons
likewise exhibited location - related firing modulated by mnemonic demands .
furthermore , medial entorhinal neurons performed better than hippocampal
neurons at distinguishing trial type on our version of the continuous spatial
alternation . conversely , hippocampal neurons
showed greater spatial specificity than medial entorhinal neurons , as is
evident by directly comparing the spatial firing patterns displayed in figures
3 and 4 . performed on all
hippocampal and medial entorhinal units with a firing field somewhere on the
maze meant to assess
spatial selectivity : place field size , spatial tuning , and spatial information
rate . on all three measures hippocampal and medial entorhinal activity differed
significantly for example , average hippocampal place field size for all units
with location related activity on the maze was significantly smaller than that
of medial entorhinal neurons ( 256.8 cm versus 330.8 cm ,
resp . ; wilcoxon rank - sum test , p < .0003 ) . the degree of spatial
tuning , or the ratio of firing inside versus outside a place field , for
hippocampal neurons was on average significantly higher than for medial
entorhinal neurons ( 11.5 versus 3.0 , resp . ; the amount of spatial information
conveyed by hippocampal neurons also was significantly greater than that of
medial entorhinal neurons ( 2.02 bits / second versus 0.89 bits / second , resp . ;
wilcoxon rank - sum test , p < .00001 ) . together the results of this study
suggest that disambiguation of overlapping experiences occurs prior to the
hippocampus , and that hippocampal and medial entorhinal circuits play distinct and
complementary roles in the continuous spatial alternation . medial entorhinal
neurons more successfully distinguished task related episodes in the context of
left- versus right - turn trial type , whereas hippocampal neurons provided a
greater degree of spatial specificity . together both regions supply requisite
elements of a neural code for particular events as they occur within unique
episodes . since the neural circuitry among
these brain regions constitutes a series of loops , it is difficult
to positively attribute specific functions to individual brain regions .
however , very recent evidence from observations of ca1 neuronal activity in
animals with lesions to layer iii of medial entorhinal cortex offers crucial
support . the results demonstrate that precise spatial coding
of ca1 neurons is more dependent on this direct entorhinal input than on
projections from ca3 which provide indirect input from layer ii of medial
entorhinal cortex , indicating that the manner of spatial
information processing most commonly observed in ca1 is the result of a clear
progression from medial entorhinal cortex to hippocampus . however , as
noted above , most of these neurons do not fire at discrete locations associated
with particular trial events , as do hippocampal neurons . instead , many medial
entorhinal cells show strong context sensitivity , outperforming hippocampal
neurons in distinguishing left - turn and right - turn trials . furthermore , the
prolonged firing periods of medial entorhinal cells are consistent with the
characterization of context sensitive neurons that could bind a series of
hippocampal representations of punctate events . a growing body of evidence
supports the notion that the medial entorhinal area is part of the
parahippocampal region that processes contextual representations . this evidence
is derived from knowledge about the anatomical pathways of the hippocampal
system and from recent functional imaging studies . inputs to the hippocampus arrive via the surrounding cortical areas that
compose the parahippocampal region . this region can be
subdivided into the perirhinal cortex , the parahippocampal cortex ( called
postrhinal cortex in rodents ) , and the entorhinal cortex . most neocortical
inputs to the perirhinal cortex are derived from association areas that process
unimodal sensory information about qualities of objects ( i.e. , what information ) , whereas most of the neocortical inputs to the parahippocampal
cortex ( called postrhinal cortex in rats ) originate in areas that process polymodal
spatial ( where ) information . what
and where streams of processing remain largely segregated as the perirhinal
cortex projects primarily to the lateral entorhinal area , whereas the
parahippocampal cortex projects mainly to the medial entorhinal area . while
there are also some connections between the perirhinal and parahippocampal
cortices and between the entorhinal areas , the hippocampal efferents back to the cortex
involve feedback connections from the hippocampus successively back to the
parahippocampal region and thence to neocortical areas from which the inputs
originated . information carried in the perirhinal - lateral entorhinal stream is combined
with where information carried in the parahippocampal - medial entorhinal
stream and the hippocampus associates items and their spatial context . when an
item is subsequently presented as a memory cue , the hippocampus completes the
full pattern and mediates a recovery of the contextual representation in the
parahippocampal cortex and medial entorhinal area , and the recovery of context
constitutes the experience of episodic recollection . in support of this model , evidence from functional imaging studies in humans indicates that the
parahippocampal cortex component of the where stream represents spatial
context . one line of evidence comes from the work of kanwisher and colleagues ,
showing that the parahippocampal region is activated when people view spatial
scenes and not objects or faces . the other line
of evidence comes from work of bar and colleagues , showing that the
parahippocampal cortex is activated when people view objects that have strong
spatial contextual associations ( e.g. , a refrigerator , a roulette wheel , ) . similarly , a cellular ( fos ) imaging study indicates that
the postrhinal cortex also is activated in rats by novel spatial arrangements
of cues . in addition , aminoff et al . reported that
adjacent components of the parahippocampal cortex are activated by spatial
context , and that this activity emerges as people view abstract patterns that
were elements of newly learned spatial patterns or simply temporally
associated . these findings extend the potential role of the parahippocampal
cortex to temporal contextual representations as well as spatial context . such
a view is consistent with the frequent observation that the parahippocampal
region is activated when humans recollect items in the context in which they
were learned ( reviewed in ) . we lack studies that compare response properties of the parahippocampal cortex and the medial
entorhinal area . however , the combined data from functional imaging of the
parahippocampal cortex in humans and animals and our recent study of spatial
firing properties of medial entorhinal neurons suggest that both the
parahippocampal cortex and medial entorhinal area components of the where
pathway may be specialized for the processing of spatial and temporal context
in humans and animals . much work remains to be done to test this hypothesis .
however , we believe there is sufficient evidence to consider the medial
entorhinal area as part of a contextual representation system rather than the
embodiment of a cognitive map that guides spatial navigation . | spatial mapping and navigation are figured prominently in the extant literature that describes hippocampal function .
the medial entorhinal cortex is likewise attracting increasing interest , insofar as evidence accumulates that this area also contributes to spatial information processing . here , we discuss recent electrophysiological findings that offer an alternate view of hippocampal and medial entorhinal function .
these findings suggest complementary contributions of the hippocampus and medial entorhinal cortex in support of episodic memory , wherein hippocampal networks encode sequences of events that compose temporally and spatially extended episodes , whereas medial entorhinal networks disambiguate overlapping episodes by binding sequential events into distinct memories . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
hrthle cell carcinoma ( hcc ) is a distinct and rare subtype of well - differentiated thyroid cancers . the aggressive nature of hcc stems from the higher propensity for multifocality , lymph node ( ln ) metastasis and distant metastasis compared to the other types of well - differentiated thyroid cancer . unlike the more common papillary thyroid cancer , hcc can not be diagnosed on fine - needle aspiration ( fna ) . rather , fna typically yields the diagnosis of hrthle cell neoplasm . the definition of a neoplasm differs in the literature , with some recommending the presence of greater than 50% hrthle cells 1 , 2 , while others use greater than 75% 3 , 4 as the cutoff . regardless of the percent cellularity , the presence of capsular and/or vascular invasion is required to make the diagnosis of hcc on histopathology . attempts have been made to identify clinical parameters that can accurately predict the malignant potential of hrthle cell neoplasms.2 however , tumor size has been shown to be predictive of malignancy in several studies.1 , 5 - 7 our group previously demonstrated that the risk of malignancy in a hrthle cell neoplasm was 55% for tumors > 4 cm and only 13% for tumors 4 cm.5 this finding is corroborated in other studies that found that between 44%8 and 65%1 of neoplasms > 4 cm were malignant . determining the malignant potential of hrthle cell neoplasm is important because it can change the initial surgical approach from a lobectomy to a total thyroidectomy . the aim of this study was to determine if tumor size correlated with ln metastasis in patients diagnosed with hcc . we retrospectively analyzed the records of all patients who underwent a thyroid operation at the university of california , san francisco from february 1997 to september 2008 . patients who were diagnosed with hrthle cell carcinoma on final pathology were used for analysis . patient demographics , preoperative imaging results , intraoperative findings , and postoperative outcome data were collected . the institutional review board at the university of california , san francisco , approved the study . a fine needle aspiration ( fna ) of suspicious thyroid nodules and/or lymph nodes was performed for diagnosis . an ultrasound ( us ) of the neck was performed to evaluate the thyroid gland and lymph node basins . the type of surgical procedure was dictated by clinical parameters : a thyroid lobectomy was performed on patients with a thyroid neoplasm and no contralateral thyroid nodules or suspicious cervical lymphadenopathy , with a completion thyroid lobectomy if final pathology revealed hcc ; a total thyroidectomy was performed on patients with a thyroid neoplasm and contralateral thyroid nodules or suspicious cervical lymphadenopathy ; a total thyroidectomy and cervical lymph node dissection ( central and/or lateral ) was performed when suspicious lymphadenopathy was found preoperatively on ultrasound , or during the operation . during the study period , 39 patients were diagnosed with hrthle cell carcinoma . the majority of patients with hcc were women and the mean age at diagnosis of all patients was 58.7 years ( table 1 ) . the preoperative diagnosis included 31 ( 79% ) patients with hrthle cell neoplasm , 3 ( 7% ) with thyroid goiter , 2 ( 5% ) with hcc , 2 ( 5% ) with follicular cell neoplasm and 1 ( 2% ) with graves ' disease . four patients had risk factors for thyroid cancer ; 1 had a family history of thyroid cancer , and 3 patients had a history of radiation exposure . at the initial operation , 22 ( 56% ) patients underwent a thyroid lobectomy , 14 ( 36% ) total thyroidectomy , and 3 ( 8% ) total thyroidectomy and lymph node dissection . patients who underwent an initial thyroid lobectomy underwent a subsequent operation for a completion thyroidectomy . of the 3 patients who received a lymph node dissection , 1 had ipsilateral central ln metastases identified intraoperatively and underwent a central ln dissection , and the other 2 had lateral ln metastasis identified on preoperative ultrasound and underwent an ipsilateral central and lateral ln dissection . though there was no gender difference between the two groups , patients with ln metastasis were older than those without ( figure 1 : similarly , patients with ln metastasis had larger tumors than those without ( figure 2 : mean size 6 and 4 cm , respectively ) . tumor size was predictive of lymph node metastasis as no patient with tumor < 5 cm presented with lymph node involvement ( 3/15 with > 5 cm cancer had node metastasis , 0/24 with < 5 cm cancer had node metastasis ) . all patients with lymph node metastasis had widely invasive tumors with capsular invasion , vascular invasion and multifocal disease . of the 36 tumors without ln metastasis , 9 ( 25% ) were moderately - to - widely invasive , 14 ( 39% ) had capsular invasion , 20 ( 56% ) had vascular invasion , and 4 ( 11% ) were multifocal our group previously showed that the overall rate of malignancy in fna proven hrthle cell neoplasms was 21% and that 55% of these tumors for example , it has been suggested that a total thyroidectomy , rather than a lobectomy , be considered for older patients with tumors larger than 4cm.8 in this series , we sought to determine if the size of the hcc tumor could predict the presence of ln metastasis . in our patient cohort , this is similar to other reported rates of 3% to 13%.3 , 9 others , however , have report higher rates of 18% to 25%.10 , 11 despite the associated risk of ln metastasis in patients with hcc , no studies have shown that hcc size predicts ln involvement . patients who presented with ln metastasis had larger tumors than those who presented without ln metastasis . interestingly , no patients with cancers < 5 cm presented with ln metastasis . on the other hand , 20% of patients with cancers this study also demonstrates that all patients with ln metastasis had widely invasive and multifocal tumors . this finding has been corroborated in another study that demonstrated that all seven patients with ln metastasis had widely invasive hcc.9 the presence of capsular invasion or vascular invasion , however , did not predict ln involvement . though hcc commonly affects women more often than men , gender was not an associated risk factor for developing ln metastasis . older age is considered a risk factor for malignancy in patients with hrthle cell neoplasms.8 similarly , we found that advanced age was a risk for the presence of ln metastasis in patients with hcc . specifically , all of our patients who presented with ln metastasis were octogenarians ( figure 1 ) . this finding suggests that older patients with large hcc are at risk for developing lymph node metastasis . in summary , as size has been shown to predict the malignant potential of hrthle cell neoplasms and this study demonstrate that size and age are also predictive of ln metastasis in patients with hcc . our data suggest that consideration should be given to performing a prophylactic ipsilateral central neck dissection at the time of their initial operation in older patients with large tumors . | introduction : hrthle cell carcinoma ( hcc ) is a rare tumor that tends to metastasize to the lymph nodes .
some studies have correlated size of hrthle cell tumors with the risk of malignancy .
whether the size of hcc correlates with the risk of lymph node ( ln ) metastases , to our knowledge has not been addressed.methods : a retrospective analysis was performed on all patients diagnosed with hcc on final pathology between 1997 and 2008 . the tumor size and lymph node status
was obtained for each patient .
the data were analyzed utilizing student 's t - test and the fisher 's exact test to calculate the two - tailed p-value.results : out of 39 patients diagnosed with hcc 3(8% ) had ln metastases ; 1 had ipsilateral central ln metastasis and 2 had ipsilateral central and lateral ln metastasis .
ln dissection was performed in patients with known metastasis ( 2 were evident on preoperative ultrasound and 1 intraoperatively ) .
patients with ln metastasis were older than those without ( mean age : 86.7 and 56.4 years , respectively ) , had larger tumors ( mean size : 6 and 4 cm ) and were commonly male ( 2 of 3 ) .
no tumor < 5 cm presented with lymph node involvement ( 3/15 with > 5 cm cancer had node metastasis , 0/24 with < 5 cm cancer had node metastasis).conclusions : similar to what has been found in patients with papillary thyroid cancer , older male patients with hrthle cell carcinomas greater than 5 cm are more likely to have lymph node metastasis .
our data suggest that these patients may benefit from a prophylactic ipsilateral central neck dissection at the time of their initial operation . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
implant therapy based on the principle of osseointegration has seen a remarkable expansion of its application in dentistry , in recent years . in the last decade , dental implants have become a reliable procedure for the treatment of partially or completely edentulous jaws . bony defect and several techniques have been proposed to promote defect fill with newly formed bone . one of the most popular procedures is guided bone regeneration ( gbr ) , which involves placing a membrane over the defect to create a secluded space into which osteogenic cells can migrate and remain undisturbed over the exposed part of the implant . a challenge in the reconstruction of periodontal structures is the targeted delivery of growth promoting molecules to the tooth root surface . polypeptide growth factors are molecules identified in the periodontal tissues that have been implicated in the growth and differentiation of cells from the periodontal tissues . platelet rich fibrin , which is a second generation platelet concentrate , offers the surgeon an access to growth factors with a simple and available technology . these growth factors which are autologous , nontoxic and non immunogenic , enhance and accelerate the normal bone regeneration pathways . this article presents a case report in which a fenestration defect around an implant was treated by gbr with platelet rich fibrin ( prf)-enhanced bone graft and the prf smeared barrier membrane for guided bone regeneration . a 30-year - old male patient reported with the chief complaint of his discolored cantilever bridge in the upper front tooth region . intra oral examination revealed missing 21 and a cantilever bridge , with 11 as an abutment [ figures 1 and 2 ] . history revealed that the tooth was extracted two years back due to trauma . the treatment planned was to give delayed loading implant replacement and metal ceramic crown for the patient in the region 21 , and a separate metal ceramic crown for 11 . preparation of the patient included scaling and root planing of the entire dentition and oral hygiene instructions . a two piece implant ( zimmer ) of diameter 4.25 mm and length 13 mm was placed in the region , 21 and the flap was sutured . after suture removal , the cantilever bridge was modified and given as temporary restoration [ figures 35 ] . implant with cover screw post - operative radiograph when the patient was reviewed after one month , there was a concavity in the buccal aspect of the region , 21 [ figure 6 ] . the region was examined with a bone meter , which revealed a buccolingual width of 5 mm . treatment was planned to treat the fenestration defect with a combination of bone graft , prf and guided tissue regeneration ( gtr ) membrane . one month after implant placement a full thick mucoperiosteal flap was raised with two vertical releasing incisions , beyond the mucogingival junction , which revealed a class i fenestration defect [ figure 7 ] . fenestration defect around an implant the fenestration defect was planned to be treated by placement of bone graft and prf mixture and resorbable gtr and prf membrane over the defect . the patient 's blood samples were taken in the operating room during the surgery . immediately after the blood draw , the dried monovettes ( without anticoagulant ) were centrifuged at 2700 rpm for 12 minutes in a tabletop centrifuge [ remy laboratories ] . the resultant product consists of the following three layers [ figure 8 ] :
the topmost layer consisting of acellular platelet poor plasmaprf clot in the middlered blood cells ( rbcs ) at the bottom .
the topmost layer consisting of acellular platelet poor plasma prf clot in the middle red blood cells ( rbcs ) at the bottom . because of the absence of an anticoagulant , blood begins to coagulate as soon as it comes in contact with the glass surface . therefore , for successful preparation of prf , speedy blood collection and immediate centrifugation , before the clotting cascade is initiated is absolutely essential . the prf clots were recovered and used in two ways :
some were placed in sterile cups and cut in few millimeter fragments . the mixture obtained constituted an easy- to - use homogeneous graft material [ figures 911]others were packed tightly in two sterile compresses inorder to obtain resistant fibrin membranes which could be placed on the grafting material along with the resorbable membrane before wound closure [ figure 12 ] . the mixture obtained constituted an easy- to - use homogeneous graft material [ figures 911 ] others were packed tightly in two sterile compresses inorder to obtain resistant fibrin membranes which could be placed on the grafting material along with the resorbable membrane before wound closure [ figure 12 ] . bone graft mixed with prf bone graft and prf mixture placed over defect prf and gtr membrane placed over defect care was taken that the membrane extended 3 - 4 mm apically and mesiodistally . the patient was given antibiotics ( amoxycillin 500 mg tid for five days ) and analgesics ( ibuprofen 400 mg and paracetamol 500 mg twice daily for three days ) and post - operative instructions were given . the sutures were removed 10 days after the procedure . the surgical site was examined for uneventful healing . six months after the gbr treatment , intra - oral examination with the bone meter revealed adequate buccolingual width of the ridge of 7 mm . in order not to disturb the minimal amount of bone that would have formed over the fenestration defect , the implant was uncovered and a healing abutment was connected to allow emergence of the implant through the soft tissues , thus facilitating access to the implant from the oral cavity and final restoration was placed [ figure 14 ] . preparation of the patient included scaling and root planing of the entire dentition and oral hygiene instructions . a two piece implant ( zimmer ) of diameter 4.25 mm and length 13 mm was placed in the region , 21 and the flap was sutured . after suture removal , implant with cover screw post - operative radiograph when the patient was reviewed after one month , there was a concavity in the buccal aspect of the region , 21 [ figure 6 ] . the region was examined with a bone meter , which revealed a buccolingual width of 5 mm . treatment was planned to treat the fenestration defect with a combination of bone graft , prf and guided tissue regeneration ( gtr ) membrane . one month after implant placement a full thick mucoperiosteal flap was raised with two vertical releasing incisions , beyond the mucogingival junction , which revealed a class i fenestration defect [ figure 7 ] . fenestration defect around an implant the fenestration defect was planned to be treated by placement of bone graft and prf mixture and resorbable gtr and prf membrane over the defect . the patient 's blood samples were taken in the operating room during the surgery . immediately after the blood draw , the dried monovettes ( without anticoagulant ) were centrifuged at 2700 rpm for 12 minutes in a tabletop centrifuge [ remy laboratories ] . the resultant product consists of the following three layers [ figure 8 ] :
the topmost layer consisting of acellular platelet poor plasmaprf clot in the middlered blood cells ( rbcs ) at the bottom .
the topmost layer consisting of acellular platelet poor plasma prf clot in the middle red blood cells ( rbcs ) at the bottom . because of the absence of an anticoagulant , blood begins to coagulate as soon as it comes in contact with the glass surface . therefore , for successful preparation of prf , speedy blood collection and immediate centrifugation , before the clotting cascade is initiated is absolutely essential . the prf clots were recovered and used in two ways :
some were placed in sterile cups and cut in few millimeter fragments . the mixture obtained constituted an easy- to - use homogeneous graft material [ figures 911]others were packed tightly in two sterile compresses inorder to obtain resistant fibrin membranes which could be placed on the grafting material along with the resorbable membrane before wound closure [ figure 12 ] . the mixture obtained constituted an easy- to - use homogeneous graft material [ figures 911 ] others were packed tightly in two sterile compresses inorder to obtain resistant fibrin membranes which could be placed on the grafting material along with the resorbable membrane before wound closure [ figure 12 ] . bone graft mixed with prf bone graft and prf mixture placed over defect prf and gtr membrane placed over defect care was taken that the membrane extended 3 - 4 mm apically and mesiodistally . the patient was given antibiotics ( amoxycillin 500 mg tid for five days ) and analgesics ( ibuprofen 400 mg and paracetamol 500 mg twice daily for three days ) and post - operative instructions were given . six months after the gbr treatment , intra - oral examination with the bone meter revealed adequate buccolingual width of the ridge of 7 mm . in order not to disturb the minimal amount of bone that would have formed over the fenestration defect , the implant was uncovered and a healing abutment was connected to allow emergence of the implant through the soft tissues , thus facilitating access to the implant from the oral cavity and final restoration was placed [ figure 14 ] . gbr is based on the principle of guided tissue regeneration , and was first performed in an experimental dog study . improvements in this technique have led to its wide - scale clinical applications to augment deficient alveolar ridges , treat implant fenestration or dehiscences , and permit immediate implant placement in large alveolar sockets . a minimum buccolingual width of 6 mm is recommended for placement of implants without undesirable complications . the most common complication associated with the placement of implants in narrow alveolar ridges or in an ideal position for esthetics is a dehiscence or fenestration defect . a fenestration is a vestibular or linguopalatal defect as an expression of a bone thickness deficiency that creates partial exposure of an implant that is completely surrounded by bone . in class i fenestrations , the implant surface penetrates the wall of bone by an insignificant amount and is located within the envelope of bone . in class ii fenestrations , there is a convexity and a significant portion of the implant is exposed outside the envelope of bone for reasons of restorability . in this case - report , there was class i fenestration defect around the implant and to group ii of the classification given by daniel buser , 1994 , in which the prosthetically guided placement of an implant results in exposure of the buccal implant surface . at present , it can be stated that biodegradable membranes have the potential to support bone formation if they are supported by bone graft material to resist collapse and if they are long - lasting enough to maintain their barrier function for extended periods in small to moderate bone defects . the degradation and resorption kinetics of a membrane for use in gbr should be set such that it remains intact for at least 6 - 9 months in large volume defects and then should be completely metabolized after 12 - 15 months . in a recent systematic review , a reasonable comparison between bioresorbable and non - resorbable membranes could not be drawn due to lack of well designed studies . in this case report , we used gtr membrane ( healiguide ) , which is made of collagen and prf to treat the defect . this is a significant benefit to the patient and represents an important step in the development of gbr procedures . for successful outcomes with gbr , the factors as outlined by mellonig et al . recent clinical and histologic findings suggest that the use of platelet concentrates have technical benefits and may enhance bone regeneration when used in conjunction with bone grafts . the amplification of platelet derived growth factor ( pdgf ) and transforming growth factor ( tgf ) beta is seen as an available and practical tool for enhancing the rate of bone formation and the final quality of bone formed . it eliminates the redundant process of adding anticoagulant as well as the need to neutralize it . it has been shown from the literature that it increases the rate of clinical graft consolidation , and prf- enhanced grafts produce more mature and dense bone than do grafts without prf . prf is in the form of a platelet gel and can be used in conjunction with bone grafts , which offers several advantages including promoting wound healing , bone growth and maturation , graft stabilization , wound sealing and hemostasis and improving the handling properties of graft materials . in an experimental study which used osteoblast cell cultures to investigate the influence of prp and prf on proliferation and differentiation of osteoblasts , it was found that prf had a superior influence over prp . also , bone augmentation grafts may act as space - maintaining devices to allow coronal migration of periodontal progenitor cells . the technique of applying biomaterials to support bioresorbable membranes avoid the risks associated with harvesting autogenic bone . bioglass is preferred for its high bioactivity , because of which the reaction layers appear to form within minutes of its implantation , and the osteogenic cells freed by the surgery rapidly colonize the particles . use of bioactive glass results in more rapid filling of the defects , which may result from more rapid accumulation of bone morphogenic proteins and other growth factors on the surface of bioactive particles , due to its high bioactive index . however , the bioactive glass in this case has no role beyond a scaffold and its absence of use would have produced the same result . the development of biomaterials , ideally coupled with the incorporation of bone growth factors and bioactive peptides , represents an important line of research in this direction . recent systematic reviews regarding the survival rate of implants into sites with regenerated / augmented bone using barrier membranes varied between 79% and 100% with the majority of studies indicating more than 90% after at least one year of function . thus , in this case report , a fenestration defect was effectively treated by the application of growth factors both to the bone graft and gtr membrane . at present , large bone defects are regularly augmented with autogenous block grafts and membranes . the use of synthetic materials would result in lower surgical risks and lower morbidity in the augmentation procedure and would represent an important step forward in simplifying bone regeneration techniques . although a meta - analysis of studies in the regeneration of intrabony defects with bone grafts has been reported , no such analysis has been made till date in evaluating the use of platelet concentrates alone or with bone grafts in the treatment of bony defects . we hope that this case - report would become a part of meta - analysis in the future to help plan an evidence - based treatment . | guided bone regeneration ( gbr ) in implant therapy is especially useful for implant placement with dehiscence defects or fenestration defects . in alveolar ridges with marked facial / buccal depressions or in knifeedge alveolar crests , the position and direction of fixture placement is restricted .
improvement of alveolar ridge morphology becomes possible with gbr .
this article describes a case in which the fenestration defect around an implant was treated by the application of platelet rich fibrin , a second generation platelet concentrate along with bone graft , and guided tissue regeneration membrane . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
osteoarthritis ( oa ) is the most common form of arthritis and a growing burden on the public healthcare . evidence has emerged that patients with oa have increased cardiovascular ( cv ) morbidity and mortality [ 1 , 2 ] ; however , very little is known about the exact mechanism underlying this association . several potential mechanisms such as obesity , chronic systemic inflammation , balance of adipokine levels , limited physical activity , and use of nonsteroid anti - inflammatory drugs might be involved in the elevated risk of cv disease ( cvd ) in oa [ 3 , 4 ] . the adipokines are white - adipose - tissue derived cell signaling proteins that participate in multiple homeostasis maintaining functions . evidence is accumulating on multiple adipokines among which leptin and adiponectin have been most thoroughly investigated . leptin has been associated with cv events , mortality , and diabetes [ 6 , 7 ] . adiponectin has been shown to have anti - inflammatory , antiatherogenic , and cardiomyocyte protective properties in experimental settings . however , in human population this role seems to be far more complex as higher concentrations have been linked to increased cv morbidity and mortality . studies have shown that oa chondrocytes produce more leptin inducing thereby the production of cartilage degradation enzymes . adiponectin seems to possess a protective role in oa through downregulating inflammatory mediators and upregulating protease inhibitors . arterial stiffness has been shown to be an independent determinant of cv morbidity and mortality and has been proposed as a surrogate endpoint for cvd . arterial stiffness can be measured noninvasively and cf - pwv as a marker of aortic stiffness is considered the gold standard method . it is noteworthy that systemic inflammation and adipokines that have been associated with oa are also involved in the pathogenesis of arterial stiffness . aortic elastic properties have been found to be elevated in radiographically established osteoarthritis . at the same time , no association has been found between knee bone marrow lesions and cf - pwv . a better understanding of the associations between oa , adipokines , and cvd might help extend future management strategies of oa beyond the current focus on treating only chronic symptoms prior to total joint replacement . the aim of this study was to find out whether oa is associated with arterial stiffness and adipokine levels . this cross - sectional study included 70 patients with primary end - stage hip and knee oa , who presented for total joint arthroplasty , and 70 age and gender matched controls . the oa patients were elected from the traumatology and orthopaedics clinic of tartu university hospital prior to hip and knee replacement surgery . all patients were diagnosed according to the american college of rheumatology criteria for knee and hip oa [ 15 , 16 ] . patients with any acute or chronic inflammatory disease , diabetes , coronary artery disease , cardiac arrhythmias or known valve pathology , peripheral atherosclerotic disease , malignancies , or renal insufficiency ( egfr < 60 ml / min/1.73 m ) were excluded . age and gender matched apparently healthy controls were recruited through local family physicians in the same geographical region . the exclusion criteria for the control group were the following : any acute or chronic inflammatory disease , any persistent knee or hip joint pain , a visit to family practitioner because of hip or knee joint complaints , diabetes , hypertension , coronary artery disease , cardiac arrhythmia , cerebrovascular or peripheral artery disease , malignancies , renal insufficiency , and regular use of any medication . the study was conducted in accordance with the helsinki declaration and was approved by the ethics committee of the university of tartu . written informed consent was obtained from all participants . blood samples were collected between 07:00 and 11:00 after an overnight fast and abstinence from tobacco and alcohol . the height and weight and waist and hip circumference of the patients were recorded and joint - specific evaluation was performed . the harris hip score ( hhs ) was used for hip joint evaluation and the hospital for special surgery ( hss ) knee score was used for knee joint evaluation . both of these test results range from 0 to 100 , where 100 is the best possible outcome . after 1015 minutes of rest in a temperature controlled quiet room in a supine position , the participants ' blood pressure and pulse wave velocity were recorded and pulse wave analysis was made . all pulse wave parameters were measured at least in duplicate and the average was calculated . pulse wave analysis and velocity left radial artery waveforms were recorded with a high fidelity micromanometer ( spt-301bh , millar instruments , tx , usa ) . corresponding ascending aortic waveforms were then generated using a transfer function to calculate central hemodynamics and augmentation index ( aix ) . the cf - pwv and carotid - radial pulse wave velocity ( cr - pwv ) were measured by sequentially recording ecg - gated carotid and femoral or radial artery waveforms using a sphygmocor device ( sphygmocor , atcor medical , sydney , australia ) . the pulse waveform was recorded from the right radial artery with a cardiovascular profiling instrument ( hdi / pulsewave cr-2000 , hypertension diagnostics inc . , eagan , usa ) and large ( c1 ) and small ( c2 ) artery elasticity indices were recorded . venous blood samples were centrifuged at room temperature and the serum was stored at 70c until analysis . serum adiponectin levels were measured using an enzyme - linked immunosorbent assay ( elisa ) ( human total adiponectin / acrp30 immunoassay , r&d systems europe , abingdon , uk ) . human total mmp-3 immunoassay kits available from r&d systems a bio - techne brand ( catalogue number dmp 300 ) were used for quantitative determination of human active and promatrix metalloproteinase 3 ( total pro - mmp-3 ) concentrations . the level of serum leptin was determined using the evidence investigator ( metabolic syndrome array-1 , randox laboratories , crumlin , uk ) . the serum levels of triglycerides , total cholesterol , ldl - cholesterol , hdl - cholesterol , white blood cell ( wbc ) count , high - sensitivity c - reactive protein ( hs - crp ) , serum creatinine , and egfr were measured using standard laboratory methods in the local clinical laboratory with automated analyzers . osteoarthritis was assessed according to the kellgren - lawrence grading system , where 0 represents no changes ; 1 represents doubtful joint space narrowing ; 2 represents definite osteophytes and doubtful joint space narrowing ; 3 represents definite osteophytes , joint space narrowing , sclerosis , and possible deformity ; and 4 represents marked joint space narrowing , large osteophytes , severe sclerosis , and definite bone deformity . the radiographs were evaluated independently by two raters blinded to the clinical data and a consensus score was produced . intraclass correlation coefficient ( icc ) was used to assess the consistency among the raters . the icc was found to be 0.76 , 95% ci ( 0.610.85 ) , which is considered good . , chicago il , usa ) for windows . the mann - whitney u test was used for comparing means between two groups . since there was a significant difference of bmi and blood pressure between the study groups , the levels of pwv were adjusted for bmi and mean arterial pressure and the levels of adipokines were adjusted for bmi before analysis . spearman 's rho was used for determining correlation between variables . a chi - square test or fischer 's exact test a total of 140 subjects ( women 49% , aged 4979 , and mean 61 years ) participated in the study . the level of bmi and the w / h - ratio were significantly higher , and hhs and hss knee scores were lower for the oa group . peripheral and central blood pressure were higher in the oa group . since there was a discrepancy in bmi and blood pressure ( a determinant of pwv ) between the study groups , the corresponding hemodynamic and biochemical variables were adjusted for mean arterial pressure and bmi . the cf - pwv was higher and c2 was lower for the oa group compared to the controls . the levels of leptin , wbc , hs - crp , and urea were higher and adiponectin and hdl - cholesterol were lower for the patient group ( table 3 ) . however , after correcting for bmi , the difference in hs - crp and platelet count was no longer significant and of borderline significance for adiponectin . a significant positive association was found between oa radiographic kellgren - lawrence grade and cf - pwv ( r = 0.272 , p = 0.023 ) ( figure 1(a ) ) . the association was further analyzed separately in the hip and knee subgroups ( r = 0.396 , p = 0.011 and r = 0.351 , p = 0.062 , resp . ) . multiple linear regression analysis using a stepwise selection procedure was performed to study the association between oa grade and cf - pwv . in the hip oa group cf - pwv was set as the dependent variable , and oa grade , age , mean arterial pressure ( map ) , and hs - crp remained significant independent predictors ( table 4(a ) ) . in the knee oa group , after adjusting for w / h - ratio and age , oa grade was not an independent predictor of cf - pwv ( table 4(b ) ) . in addition , kellgren - lawrence grade correlated positively with mmp-3 ( r = 0.235 , p = 0.05 ) ( figure 1(b ) ) and negatively with leptin ( r = 0.246 , p = 0.040 ) . however , furthermore , we found that adiponectin was correlated with c1 and aix@75 ( r = 0.249 , p = 0.003 and r = 0.293 , p < 0.001 , resp . ) ( figure 2 ) . in the model where adiponectin was set as the dependent variable , neither c1 nor aix were independent predictors when potential confounders were included ( data not shown ) . our study describes associations between adipokines , mmp-3 , and parameters of arterial stiffness in older adults with hip and knee oa and compares them with the corresponding indicators for asymptomatic controls . to our knowledge , this study is the first to demonstrate independent correlation between radiographic oa grade and cf - pwv . these findings support the notion that arterial stiffening might be linked to the pathogenesis of oa . in addition , we demonstrate that adipokines are associated with oa and with arterial stiffness and might thus play a role in linking these two pathologies . recently , a study of 80 end - stage knee oa patients found decreased elastic properties of aorta compared to non - oa controls . in addition , the authors found that radiographic severity of oa was associated with aortic stiffness . however , another study of knee oa patients found no relationship between presence or size of bone marrow lesions ( a surrogate for oa ) and cf - pwv . hand oa has been linked to arterial stiffness , but the relationship was largely attributable to the confounding effect of age . so far the relevant evidence has been controversial and to the best of our knowledge data concerning hip oa and arterial stiffness is lacking . in the present study of knee and hip oa we demonstrated independent association between oa severity and cf - pwv ( figure 1 , table 4 ) . we studied patients with hip oa , which is considered a different entity of oa , and found its independent association with cf - pwv . the reason for failing to reveal significant association between knee oa severity and cf - pwv might be attributed to too small sample size . yet we established relationship between hip oa and arterial stiffness , which might contribute to the higher cvd risk among oa population . the adipokines are a growing family of white - adipose - tissue derived factors that have multiple functions through different pathways and are involved in inflammation and modulation of immunological response but also in glucose and lipid metabolism . leptin upregulates mmps and induces cartilage loss [ 2123 ] ; however , its anabolic effects have also been described . although leptin has been associated with oa severity , the pathophysiological pathways are not fully understood . in addition to bone and cartilage , leptin influences also the cardiovascular system . it is an independent predictor of myocardial infarction and stroke and has been associated with increased arterial stiffness through promoting inflammation and mmp upregulation . in accord with previous studies , we found an elevated expression of leptin in oa patients and , in addition , an inverse association with oa radiographic grade , which might indicate the anabolic effect of leptin . these findings point to the possible key role of leptin in the pathogenesis of oa and suggest leptin as a link between oa and vascular damage . adiponectin is prevalently synthesised in adipose tissue and circulates in the blood in large quantities . adiponectin has anti - inflammatory properties and hypoadiponectinemia has a detrimental effect on aortic stiffness . in the present study , adiponectin correlated inversely with large artery elasticity index and positively with aix@75 . to the best of our knowledge , findings similar to ours were reported in essential hypertension , but inverse association was recently found in juvenile idiopathic arthritis patients . our results suggest that adiponectin as well as leptin might link oa with increased arterial stiffness . these findings describe the pleiotropic role of adipokines in oa and need further research to determine their potential as therapeutic targets . the mmp-3 is a catabolic enzyme that not only has the ability to degrade the extracellular matrix but plays a central role in activating other members of the mmp family . we found higher level of mmp-3 in oa patients compared to controls and positive correlation between radiographic grade and serum level of mmp-3 in oa subjects . the activation and secretion of mmp-3 are driven by inflammatory cytokines , which in turn enhances the production of inflammatory mediators like interleukin-1 . this is in line with our results , according to which oa group also showed higher values of wbc , which indicates a systemic inflammatory state in these patients . furthermore , mmp-3 is likely to influence arterial stiffness and has been found to be elevated in atherosclerotic aorta [ 33 , 34 ] . in conclusion , mmp-3 might be another marker that is associated with oa and vascular pathology . first , because of the cross - sectional study design , causal associations could not be established . second , since x - rays for the control group were not available , some asymptomatic oa cases might be included in the controls . third , data concerning physical activity was not collected but has been shown to influence arterial stiffness and , therefore , is a confounding factor that was not accounted for in the present study . in conclusion , these relationships provide a potential link between biochemical and functional alterations and oa as well as elevated cv risk in end - stage oa . | objective . osteoarthritis ( oa ) is associated with increased cardiovascular comorbidity and mortality .
evidence is lacking about whether arterial stiffness is involved in oa .
the objective of our study was to find out associations between oa , arterial stiffness , and adipokines .
design .
seventy end - stage knee and hip oa patients ( age 62 7 years ) and 70 asymptomatic controls ( age 60 7 years ) were investigated using the applanation tonometry to determine their parameters of arterial stiffness .
serum adiponectin , leptin , and matrix metalloproteinase 3 ( mmp-3 ) levels were determined using the elisa method .
correlation between variables was determined using spearman 's rho .
multiple regression analysis with a stepwise selection procedure was employed .
results .
radiographic oa grade was positively associated with increased carotid - femoral pulse wave velocity ( cf - pwv ) ( r = 0.272 , p = 0.023 ) .
we found that oa grade was also associated with leptin and mmp-3 levels ( rho = 0.246 , p = 0.040 and rho = 0.235 , p = 0.050 , resp . ) .
in addition , serum adiponectin level was positively associated with augmentation index and inversely with large artery elasticity index ( rho = 0.293 , p = 0.006 and rho = 0.249 , p = 0.003 , resp . ) .
conclusions .
our results suggest that oa severity is independently associated with increased arterial stiffness and is correlated with expression of adipokines .
thus , increased arterial stiffness and adipokines might play an important role in elevated cardiovascular risk in end - stage oa . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the incidence of the complications after tka including arterial occlusion , arteriovenous fistula , arterial aneurysm , and arterial severance is reported from 0.03% to 0.17%.1 , 2 there are approximately 35 cases that have been reported in the orthopedic literature until 2006 . approximately 46 cases have been reported in the orthopedic literature until 2015.1 , 2 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 nonetheless , the infrequency of these complications may make the diagnosis and treatment extremely challenging to surgeons , and limb - threatening ischemia may occur subsequently.3 , 4 we report a case of acute vascular occlusion in the midpiece of femoral artery following tka while aggressive thrombolysis and thrombectomy could not restore arterial circulation of the lower limb , and discuss the possible etiology causes . the patient , female , 64 years old , had severe osteoarthritis for 12 years in the left knee . she had no peripheral vascular diseases or cardiovascular system diseases , no history of long - term lying in bed and usage of glucocorticosteroid or smoking . tka was initiated with a tourniquet which was inflated to a pressure of 270 mmhg . after we released tourniquet , obvious pulsations could be palpated on the dorsalis pedis artery . 40 min postoperatively , the on - call resident found the dorsalis pedis artery pulsations turned weaker and weaker . she was guided to flex and extend her left knee continually , and no substantive hemorrhage was found . 40 ml blood volume was aspirated in the drain bottle in 2 h. doppler examination showed no arterial signal below the midpiece of femoral artery . 6 h postoperatively , 10 mg rivaroxaban ( bayer , germany ) was taken for routinely anticoagulant therapy . the femoral artery angiogram demonstrated the complete occlusion with abrupt cutoff of the femoral artery above the adductor magnus tendon hiatus furcation ( fig . 1 ) , and there was no occlusion in inferior segment of the femoral artery and the popliteal artery ( fig . 2 ) . 0.2 m international unit ( iu ) urokinase was injected in the same session 9 h postoperatively , but no effect . 11 h after tka , thrombus aspiration was performed with 5-fogarty aspiration catheter which was introduced from distal to proximal direction . after aspiration of 5 cm fresh thrombus material and injection of 0.2 m iu urokinase , the circulation was not restored . disarticulation at the left knee was performed 4 days later due to ischemic necrosis . in the procedure , we examined the whole segment of popliteal artery , and found that there was no injury , no mural thrombosis formed in the lumens and the ectotheca of the popliteal artery was complete . we have reviewed the published reports associated with arterial occlusion , and summarized the features in table 1 . approximately 46 cases have been reported in the orthopedic literature until 2015.1 , 2 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 popliteal artery thrombosis is the most frequent one . according to the report of holmberg in 1996 , the case reported here is , to our knowledge , the first one in the literature that embolism segment was located at the midpiece of femoral artery , above the adductor magnus tendon hiatus furcation . it is impossible to directly injure the midpiece of femoral artery in tka , so the reason for arterial occlusion is incomprehensible . if we suppose that superficial femoral artery depressed by tourniquet resulted in thrombosis , and the thrombus flow to the distal femoral artery induced occlusion at the arterial branch , thrombolytic and the thrombectomy should be effective . besides the thrombosis , acute arterial occlusion can also be caused by embolization of disrupted atheromatous plaques.1 , 11 it has been suggested that the mechanical pressure of the tourniquet may traumatize atheromatous vessels , causing fractures and dislodgment of the plaque , especially for the old patients . the vessels are calcified and lost its elasticity , and intimal tear and plaque embolization could result from the fixation of the femoral artery by the tourniquet and the stretching of the distal artery during intraoperative manipulation.11 , 12 , 13 in the procedure of artery angiogram , we saw the catheter could be introduced in close proximity to the artery tunica intima and passed smoothly through the occlusion site ( fig . angiogram showed that there was no occlusion in the inferior segment of the femoral artery or the popliteal artery . all the signs indicated that the pathologic changes such as arteriosclerosis or atheromatous plaque preexisted at the occlusion artery segment . when we released the tourniquet after surgery , the rapid changes of arterial pressure and ischemical reperfusion injury might induce an intimal injury or atheromatous plaque disruption in the femoral artery . hemorrhage in such plaque tissue led to intimal bulging , which resulted in the arterial occlusion in the present case . the curative effect of artery occlusion following tka was poor , as evidenced by high rates as 70% of limb loss in previous reports . necessary surgical treatment is possible with bypass grafting and revascularization2 , 4 or thrombectomy using aspiration catheter7 , 8 including prior thrombolytic therapy.5 , 6 thrombectomy is the most frequently described treatment , whereas the result is complete recovery or amputation . calligaro reported the largest single - center experience with management of acute ischemic complications associated with tha and tka . he claimed that this complication was best managed by an aggressive protocol including arterial bypasses and emergency revascularization . arterial thrombectomy was successful in approximately one fourth ( 5 of 18 , 28% ) , only when acute thrombosis without associated intimal damage . in our case , the femoral artery intimal injury and atheromatous plaque disruption were the most probable reasons for occlusion , and no thrombosis existed . the key point of the failure was that we did not find the real etiopathogenisis for artery occlusion in time . arterial complications following tka often occur in patients with an existing history of peripheral vascular disease or high risk factor diseases - related chronic arteriosclerosis such as hypertension , diabetes , rheumatoid , thromboangitis obliteran , hyperlipidemia , prior smoking and obesity . but this patient did not have any such history . it is suggested that there may be other undetermined risk factors and careful vascular examination that should be performed for all patients , not only to those with vascular disease history . to our knowledge , the only risk factor for this patient 's acute arterial occlusion might be the time of using tourniquet and her age . it is important to fully consider that arteriosclerosis or atheromatous may induce intimal injury in the treatment of acute arterial occlusion with a tourniquet following tka . for old patients , as the time of using tourniquet become shorter , the risk of artery intimal injury and atheromatous plaque disruption will decrease . | acute arterial occlusion is a rare complication following total knee arthroplasty ( tka ) .
the incidence as reported previously is from 0.03% to 0.17% ; however , the sequelae can be disastrous because of its potential threat to limb loss .
we report a case of acute arterial occlusion in the midpiece of femoral artery following tka occurred 40 min postoperatively .
the occlusion site existed at the midpiece of femoral artery is uncommon .
arterial circulation of the lower limb could not be restored by the thrombolysis and thrombectomy treatments performed within 11 h after tka . in the end , amputation had to be carried out . in the treatment of acute arterial occlusion following tka with a tourniquet , it is important to fully consider that arteriosclerosis may induce atheromatous plaque disruption , which might be the reason for acute arterial occlusion . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the most critical difficulty with the concept of mci is that it is an arbitrary label on a continuum of cognitive changes that occur in people as they age . as a result the arbitrary nature of the label can be seen most explicitly in the neuropsychological criteria , which may specify the threshold for applying the terms ( one or one and a half standard deviations less than age - matched controls ) , the composition of the battery , and the norms . the criteria concerning preserved or relatively preserved activities of daily living also permit considerable variability as to where the line is drawn by different clinicians . how complex must an impaired instrumental activity of daily living be before the label mci is applied ? for that matter , how simple should the task be before the affected person is said to convert to alzheimer 's disease ( ad ) ? differences in an individual 's performance of life 's tasks create both patient and clinician variability in perceptions as well as cross - cultural challenges in multinational studies ( gaines a , whitehouse pj , unpublished data ) . the existence of a continuum of cognitive changes is illustrated by mci being bounded on one side by ad and on the other by labels such as age - associated memory impairment ( aami ) or age - related cognitive decline . the emergence of aami was also closely linked to attempts to develop medicines to treat this condition . the criteria for applying this label included demonstrating test performance one standard deviation below younger - age controls , thus creating a large number of older individuals who could be labeled with aami . yet whether mci is normal or not is at the heart of the conceptual and practical ambiguities associated with this concept . clinicians know logically that there is a time in the life course of a patient , who will eventually be diagnosed as having ad , when the symptoms are present , but not sufficiently severe to warrant the label dementia . any progressive medical condition must have a phase in which the symptoms are emerging , but not of sufficient intensity to warrant a disease label . in medicine , increasing attention is being paid to so - called preclinical states , such as in hypertension , depression , and parkinson 's disease . thus , it is not at all surprising that different variants of mci have been identified , including amnestic mci , mci with symptoms in several different cognitive domains , and mci with focal symptoms in an intellectual area other than memory . the mci associated with frontal lobe dementia and vascular dementia would more likely be predicted to be nonamnestic . the symptoms in mci are mild and perhaps more variable than in dementia ; therefore , it is not surprising that the outcomes of longitudinal follow - up studies and drug studies might also be more variable . the logically complete set of outcomes for a patient with mci includes no change over time , further deterioration or even improvement . all these outcomes can be demonstrated in epidemiological studies when patients are followed for several years . there are , however , some people who improve , at least for a period of time , suggesting either a benign course to their medical condition or a mislabeling of the individual in the first place , perhaps due to a bad testing day or mild depression . if one accepts the 15% annual conversion rate , one also has to ask what happens over a more extended period of time , such as that usually associated with epidemiological studies . at 15% a year , most people would have been expected to convert to ad within 10 years . this point returns us to the issue of mci as an arbitrary label on the continuum of cognitive aging and raises the unresolved question of whether all human beings would develop ad if they lived long enough . most studies of those in their 80s , 90s , and beyond suggest that the incidence of ad continues to increase with age . thus , the major conceptual challenge to the further development of drugs to treat mci is the ambiguity around definition and the relationship to normal aging . other challenges also exist in the development of trial designs to demonstrate the effectiveness and safety of drugs . most studies are classified as either trials to demonstrate symptomatic benefit or trials to demonstrate disease modification . most of the interest in mci surfaced because of the desire to develop medications to prevent ad . in order to conduct a primary prevention protocol , one needs to enter into the study individuals who do not currently suffer from dementia . one of the easiest ways to enrich the sample in a prevention study is to include people who already suffer from minor degrees of cognitive difficulties , as they are more likely to proceed to full dementia . of course , this begs the question as to whether this is primary prevention , or really secondary prevention in which the enrolled individuals were already suffering from a dementia at an early stage and the observed deterioration was the further progression of the already existing disease condition . the problem with conducting either primary or secondary prevention studies is that there are no agreedupon designs . survival analysis as promoted by the national institute of aging 's alzheimer 's disease cooperative study in their studies of vitamin e , for example , can not clearly differentiate a prolonged symptomatic benefit from a disease - modifying or neuroprotective effect . the staggered - start , staggered - stop design , elaborated most clearly by leber , has been used in a few studies . however , it has been difficult for regulators to interpret the complex slope changes necessary to make the claim that a drug is disease - modifying . although considerable effort has been placed in developing biological markers , particularly neuroimaging , no test can currently replace a clinical diagnostic process for mci . regulatory bodies will most likely not consider surrogate markers such as hippocampal atrophy unless they are clearly linked to clinical improvement . thus , from a regulatory perspective , we are puzzled to know what designs to use to demonstrate a disease - modifying process that prevents the conversion of mci to ad . attempts have also been made to demonstrate that medications provide symptomatic benefit for people with mci . such studies have been designed in a fashion parallel to those in ad , using outcome measures tailored to persons with less cognitive impairment . here , the conceptual challenges are less evident in developing the trial designs , but the practical implications of such studies are perhaps less clear . even if such studies show positive effects , what are the functional benefits to individuals and the pharmacoeconomic impacts on societies ? another area of conceptual and practical confusion that permeates the study of people with mild degrees of cognitive impairment is the overlap with the emerging field of cognitive enhancement . at what point on the continuum of cognitive aging is a drug not treating a disease , but rather enhancing a person 's normal ability to function intellectually . our pilot study of the use of donepezil in 53-year - old pilots flying in flight simulators suggests that cholinergic drugs may benefit individuals in their middle years who are performing complex cognitive tasks in society . studies of the biology of brain aging , particularly changes in neurotransmitter systems , support the idea that persons with ad and mci lie on the continuum of neuronal alterations that begin perhaps quite early in life . a related conceptual and practical problem in developing drugs to treat mci is the overlap between western scientific allopathic medicine and so - called complementary and alternative medicine ( whitehouse pj , juengst e , unpublished data ) . many individuals take herbal and nutraceutical products to try to improve their memories or slow the progression of age - related cognitive deterioration . such therapies to treat brain aging overlap with those designed to slow the aging process itself . practically , this means that decisions must be made about whether to enroll individuals in studies who are taking such products ( and at what doses ) . conceptually , and from a regulatory perspective , it raises issues of what products are to be regulated by the food and drug administration ( fda ) and other drug regulatory bodies or monitored through other means or by no means at all . in both the usa and europe , there is increasing concern about the lack of oversight of such complementary and alternative medicines . yet , as we have seen vitamin e and ginkgo are examples of biological products that have been sold as essentially unregulated products , but that are also being studied scientifically . a final major area of challenge to the development of more effective drugs from mci is ethics . given current science and resources , what would a drug profile look like that cured or prevented ad ? what are the implications of such a term for the individual labeled with it and for their partner and potential caregiver ( corner l , bond j , unpublished data ) ? if i have a label of mci , does that mean that i do not have ad , that i have a mild form of ad or another dementia , or that i may or will eventually get dementia ? moreover , we already noted that some persons with the label mci improve . the implications of the term mci for an individual patient and clinician are closely linked to the fear of ad itself . perhaps in our enthusiasm for creating new medications , we have also intensified the terror that people feel about the possibility of suffering from dementia . perhaps the greatest ethical issue facing the development of drugs for cognitive impairment has to do with conflict of interest between researchers , physicians , and the drug industry . one of the lessons of the introduction of drugs to treat erectile dysfunction is that the line between disease and normality is thin . moreover , the ability to enhance cognition already motivates many people to take complementary and alternative medical products . the concern about the use of serotonin reuptake blockers to treat depression in childhood is but one example . a major challenge to biological psychiatry , but also to neurology , is maintaining the trust of our research participants and patients . one important issue that surfaced around the treatment of depression is the suppression of negative trials . we need to ensure that trials in dementia are entered into an international database and that the trial results made available to the scientific community or that research subjects are appropriately compensated . academic experts for hire as authors of papers in which their contributions are limited is another example of a major problem . the pharmaceutical industry is amazingly effective at not only selling their drugs , but also at influencing the very way we think about health . the amount of money put into drug treatments limits our incentive to think about alternative ways of addressing social problems due to various age - related cognitive challenges . the fda held a one - day hearing in march 2001 to address some of the conceptual issues surrounding the development of drugs for mci . although no official statement was made concerning the acceptability of mci as a therapeutic target , many experts interpreted the fda 's position that mci is very early dementia , most likely ad . further elaboration of this issue will likely require submission by industry of drugs for mci to the fda for consideration for approval . the regulatory process in the usa is relatively open . whether or not experts believe that a diagnostic entity is an appropriate target for drug development influences the regulators in the evaluation of protocols . the european medicines evaluation agency ( emea ) has not held open hearings about the concept of mci . such presentations suggest that the attitude in europe is similar to that in the usa , ie , the regulators will wait for more development in the field and for the submission of actual trials . in the fall of 2004 , a group of investigators in canada will meet to examine the draft academic guidelines that were issued several years ago . regulators will be involved in the meeting , and the topic of mci and related conditions will be discussed . no regulatory bodies in asia have taken a stance on mci as a target condition . under the auspices of the international working group for the harmonization of dementia drug guidelines , organized for the first time 8 years ago by the author and currently by jean - marc orgogozo , three asian regional meetings have been held . from the first in singapore in 2001 , to the second in china , and now this year in thailand , there has been growing interest in the concept of mci among academic opinion leaders in asia . of course , this is largely influenced by the western experts expressing their enthusiasm for the concept . attitudes toward the elderly and to age - related cognitive changes are different in asia . the back - translation into english of the term , mci , by a leading opinion leader in china is labeling millions of chinese with this term has some interesting social implications and potentially profound effects on attitudes toward the elderly in china . the international working group for the harmonization of dementia drug guidelines has had regulatory issues at the heart of its mission to promote global discussion about designs to treat mci , ad , and other conditions like vascular dementia . currently under the direction of lon schneider with input from other experts , including the author , the international psychogeriatric association is also planning a meeting for this winter ( 2004 ) on mci , which will involve discussions about regulatory issues . the fda held a one - day hearing in march 2001 to address some of the conceptual issues surrounding the development of drugs for mci . although no official statement was made concerning the acceptability of mci as a therapeutic target , many experts interpreted the fda 's position that mci is very early dementia , most likely ad . further elaboration of this issue will likely require submission by industry of drugs for mci to the fda for consideration for approval . the regulatory process in the usa is relatively open . whether or not experts believe that a diagnostic entity is an appropriate target for drug development influences the regulators in the evaluation of protocols the european medicines evaluation agency ( emea ) has not held open hearings about the concept of mci . such presentations suggest that the attitude in europe is similar to that in the usa , ie , the regulators will wait for more development in the field and for the submission of actual trials . in the fall of 2004 , a group of investigators in canada will meet to examine the draft academic guidelines that were issued several years ago . regulators will be involved in the meeting , and the topic of mci and related conditions will be discussed . no regulatory bodies in asia have taken a stance on mci as a target condition . under the auspices of the international working group for the harmonization of dementia drug guidelines , organized for the first time 8 years ago by the author and currently by jean - marc orgogozo , three asian regional meetings have been held . from the first in singapore in 2001 , to the second in china , and now this year in thailand , there has been growing interest in the concept of mci among academic opinion leaders in asia . of course , this is largely influenced by the western experts expressing their enthusiasm for the concept . attitudes toward the elderly and to age - related cognitive changes are different in asia . the back - translation into english of the term , mci , by a leading opinion leader in china is labeling millions of chinese with this term has some interesting social implications and potentially profound effects on attitudes toward the elderly in china . the international working group for the harmonization of dementia drug guidelines has had regulatory issues at the heart of its mission to promote global discussion about designs to treat mci , ad , and other conditions like vascular dementia . currently under the direction of lon schneider with input from other experts , including the author , the international psychogeriatric association is also planning a meeting for this winter ( 2004 ) on mci , which will involve discussions about regulatory issues . he doubts that more conferences alone will lead to consensus , since there have been many such conferences and the differences of opinion remain . at the 9th conference on alzheimer 's disease and related disorders in philadelphia , pa , in july 2004 , this author received the impression of a growing split between the usa and europe . in fact , within the usa , the original mayo clinic concept of the mci ( perhaps to be renamed petersen 's syndrome ) is still meeting resistance . the main issue that remains is the need to address more seriously the continuum of aging . of course , such a reconsideration of the categories of age - related cognitive impairment would have implications for ad as well . despite the work in genetics and neuroimaging , we are having a harder time differentiating the various types of dementia from each other . this is most likely explained by the fact that the process of brain aging affects individuals in many different ways and our attempts to assign dementias into discrete categories are failing because of the overlap in biologies at work in our brain . neuronal loss occurs in multiple different systems to different degrees creating a wide spectrum of cognitive , affective , and motor symptoms . as i have suggested previously , it may be possible to treat cognitive impairment as a nonspecific symptom rather than a feature of different discrete conditions . the biological substrate for such a claim is that overall loss occurs continuously in various brain nuclei as we age . for example , loss of cells in the cholinergic basal forebrain occurs in a variety of dementias and with normal aging . cholinesterase inhibitors appear to work , albeit in modest ways , in a variety of dementias characterized by cholinergic pathology , including not only ad , but also parkinson 's disease , vascular dementia , and other overlapping conditions . psychosis , which may be considered a discrete category , exists in a variety of conditions ; agitation occurs on a continuum . drugs to treat agitation are being developed and submitted for approval based on finding positive effects in three or more conditions , like dementia and mental retardation . could we consider submitting a portfolio of results for a drug to treat cognitive impairment in several conditions and get a label for a general indication ? such an approach would not only avoid the problems of nosology in the field of dementia , which are only increasing , but allow the use of these medications to treat mild degrees of symptoms as well as more profound cognitive deficits . such an approach would greatly expand the market for potential therapies . the boundaries between what is a disease and what is normality would grow even more unclear with an approach that labels cognitive impairment on a continuum . the costs of drugs to our health care system would likely increase . as an advocate for the importance of pharmacoeconomic studies , especially studies of quality of life , i would urge that we stress the importance of such cost - utility approaches even in the current regulatory and reimbursement environment , and even if that would increase the size of the potential market . a focus on drug treatment for cognitive impairment first , we are constantly focusing on what is wrong with our cognition as we age . more emphasis on cognitive vitality and the potential for older people to further develop cognitively and gain wisdom would be helpful in society . moreover , a focus on drugs makes us think that the only answers to the challenges of cognitive aging lie in medicine and biology . clearly , there are many ways to prevent the deterioration that can occur in cognitive abilities as we age , besides waiting for a magic bullet . developing a sense of purpose , engaging in civic activities , and taking responsibility for one 's personal legacy are all activities that can contribute to a sense of cognitive vitality , even in persons who suffer from mci and ad . | the development of the concept of mild cognitive impairment ( mci ) and its practical application have been intimately tied to attempts to produce therapeutic agents . understanding the regulatory environment and determining the way in which it can be influenced are critical to the development of drugs and their eventual approval . in this article , we review some of the current challenges surrounding the concept of mci relevant to drug development , summarize activities in various regions of the world , and conclude with some suggested next steps and an alternative framing for approving drugs for mci and related conditions . |
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a drug drug interaction may be the pharmacologic or clinical response to the administration of a drug combination , different from that anticipated from the known effects of the two agents when given alone . clinically significant drug interactions , which pose potential harm to the patients , may result from changes in pharmaceutical , pharmacokinetic , or pharmacodynamic properties . patient factors that increase the risk for drug interactions include being critically - ill , polypharmacy , having impaired hepatic or renal function , hypoxemia , or metabolic disturbances , and being elderly . given the paucity of data on drug interactions in pediatric patients , children should also be considered at a special risk . the clinical result of a drug - drug interaction may manifest as antagonism ( 1 + 1 < 2 ) , synergism ( 1 + 1 > 2 ) , or idiosyncratic reaction ( a response unexpected from the known effects of either agent ) . most patients receiving potentially interacting drugs do not experience adverse drug events ; however , serious adverse events are known to occur.[24 ] evidence from epidemiologic studies suggest that drug interactions contribute to a small , but significant number of adverse events in hospitalized patients.[58 ] with multiple drugs often prescribed , the potential for adverse drug interactions is an important concern . no associated adverse drug events were identified , but the retrospective design of the study probably limited our ability to detect them . the purpose of the study is to verify the rate and profile of drug interactions in medical prescriptions to hospitalized pediatric patients . the data have been collected in a retrospective design from the files and drug list of children hospitalized in pulmonology nephrology and gastroenterology wards in the pediatric clinic , from july 1999 to 2004 . they are as follows :
( a)pharmacodynamic interactions(b)pharmacodynamic - pharmacokinetic interactions(c)pharmacokinetic interactions
pharmacodynamic interactions pharmacodynamic - pharmacokinetic interactions pharmacokinetic interactions to evaluate the clinical significance of drug interactions we analyzed the data of 148 patients according to the significance rate of interaction , onset of interactions , severity of interaction , and documentation of clinical manifestation of interaction , in an aim to evaluate the significance rate of interaction the data were divided in to five categories ( category 1 through 5 ) , according to severity of interaction and documentation . the methodology to assess the significance rate of interaction was based on editorial group 's assessment of the interaction 's severity and documentation . initially , we collected data from 148 cases from which 34 cases had drug interactions . we analyzed the cases with drug interaction by the type of interactions , significance of interaction , rate of onset and severity . of 34 cases of interaction , 1 ( 2.94% ) was pharmacodynamic , 20 ( 58.82% ) cases were unknown , and pharmacokinetic ( 38.24% ) were 13 cases . according to the rate of significance , 4 cases were of the first significance rate of interaction , 18 cases of the second significance rate , 1 case of the third significance rate , 4 cases of the fourth significance rate , and 7 cases of the fifth significance rate . according to onset of cases that were of delayed onset , and according to severity of interactions in 7 cases we have noticed major severity interaction , in 19 cases moderate severity , and in 8 cases minor severity . the case of pharmacodynamic interaction ( 1/34 ) is an interaction of the moderate significance , delayed onset , minor severity , and suspect documentation . pharmacokinetic interactions have shown greater variability according to the significance rating , severity , and their documentation . more frequent interactions were those of the second significance rating , delayed onset , moderate severity , and established documentation ( 5/34 or 14.71% ) , while 3 cases of interactions were of the first significance rate and other 3 cases of the fifth significance rate ( 8.87% ) . the first significance rate pharmacokinetic interactions were of delayed onset , major severity of interaction , and probable documentation , while the fifth significance rate were of delayed onset , minor severity of interaction , and probable documentation . the interaction was of delayed onset and major severity of interaction [ table 2 ] . 34 ) unknown type of interactions have shown greater variability according to the significance rate , onset , severity of interaction and their documentation , as well . more frequently , these interactions belonged to the second significance rate ( 13 or 38.23% ) of whom 9 cases ( 26.47% ) are of suspect documentation , while 4 cases ( 11.76% ) are of probable documentation [ table 3 ] . in our study , we encountered a variety of drug interactions at the pediatrics clinic , but what needs more attention are the cases of first significance rate interaction , rapid onset , and major severity of interaction . digoxin interacts with furosemide and other loop diuretics because of changes in the electrolyte concentration induced by diuretics . although loop diuretic drugs themselves do not alter the kinetics of digoxin excretion , they induce a dose - dependent loss of potassium from the body , resulting in a decreased serum potassium concentration . it is well known that hypokalemia is associated with sensitivity to digitalis and , thus , increases its toxicity but it is not well appreciated that when the serum potassium is as low as 2 to 3 meq / l , the tubular secretion of digoxin is nearly blocked . this reduced tubular secretion of digoxin results in a diminished plasma clearance of digoxin and thereby a prolonged elimination half - life of digoxin . thus , hypokalemia itself can result in an increase in the serum digoxin level as well as enhancing toxicity . such a electrolyte imbalance can precipitate or give contribution to arrhythmias , especially among patients with previous cardiac arrhythmias . the ability of macrolide antibiotics to interact with the biotransformation of some other drugs has been widely recognized , mostly with erythromycin and troleandomycin . terfenadine undergoes rapid first - pass metabolism in the liver , where cytochrome p450iiia enzyme converts it to the active form , terfenadine carboxylate . if metabolism is inhibited , then the unmetabolized terfenadine accumulates and cardiotoxicity may ensue . thus , co - administration of macrolides and terfenadine causes an increase in the serum level of terfenadine to cardiotoxicity levels . co - administration of methylprednisolone and erythromycin can also increase the pharmacologic and toxic effect of methylprednisone . therapeutic efficacy of paracetamol ( analgesic / antipyretic ) may be diminished by rifampicin which can even increase the toxicity of paracetamol . rifampicin can induce hepatic microsomal enzymes , which metabolizes paracetamol more quickly to metabolites , which are potentially hepatotoxic . the isoniazid serum level can be decreased by corticosteroids , by increasing the rate of hepatic acetylating in slow accelerations and by increasing renal clearance in slow as well as rapid accelerations . by decreasing renal clearance , isoniazid can potentiate benzodiazepine action , through inhibition of oxidative metabolism of benzodiazepines in liver . administration of amino - glycosides or of loop diuretics , even alone , can cause ototoxicity . experimental data show that the synergistic effect appears when amino - glycosides are parenterally combined with these diuretics . administration of amino - glycosides in combination with cephalosporin can increase nephrotoxicity through an unknown yet mechanism . these problems are even bigger when this happens in vitro or in a syringe where physicochemical incompatibility occurs . in vivo inactivation of amino- glycosides by penicillin depends on diminished renal function and that is why the patient should be carefully monitored . the dose of antimicrobials should be lowered , if the patient has moderate or high renal insufficiency . rifampicin can reduce therapeutic effects of nifedipine , potentially through increased metabolism of nifedipine on the bowel wall ( cytocrome p 450 3a4 ) induced by rifampicin . combination of penicillin and chloramphenicol may show synergism against certain microorganisms , but on animal studies it has been reported even for cases of antagonism . the presence of drug interactions is a permanent risk in hospitals especially in pediatric hospitals . then , we can conclude that continued education , serum drug concentration monitoring in children , and participation of clinical pharmacologist in the multidisciplinary team during the application of drug therapy , computer system prescriptions ( a computer software that provides a program that does not let entering two or more drugs that interfere with each other , and also that informs the doctor of the adverse effects and interactions of each drug prescribed ) are necessary for the prevention of drug interactions , for improvement of treatment of hospitalized children . | objective : to investigate the prevalence of the major drug interactions in children and verify the rate and profile of drug interactions in hospitalized pediatric patients.materials and methods : a retrospective study was designed and data collected from the files of hospitalized children in pulmonology , nephrology , and gastroenterology wards of a pediatric clinic , from july 1999 to 2004.results:from the analyzed material , we detected 34 cases of interactions , of which 1 was pharmacodynamics interaction , 13 were pharmacokinetic interactions , and 20 of unknown mechanisms . according to the rate of significance , 4 cases were categorized in the first significance rate of interaction , 18 cases in the second significance rate , 1 case of the third significance rate , 4 cases of the fourth significance rate , and 7 cases of the fifth significance rate . according to onset of cases ,
33 cases were of delayed onset , and according to severity of interactions , in 7 cases we noticed major severity interaction , in 19 cases moderate severity and in 8 cases minor severity.conclusions:the presence of drug interactions is a permanent risk in the pediatric clinic .
then , we can conclude that continued education , computer system for prescriptions , pharmacotherapy monitoring of patients , and the pharmacist participation in the multidisciplinary team are some manners of improving the treatment to hospitalized patients . |
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coronary artery ectasia ( cae ) has been defined as segmental or diffuse luminal dilatation of the coronary arteries in coronary angiography . cae is a rare finding among coronary artery anomalies and considered to be congenital in 1020% of the cases while the remaining are acquired in origin . the common coexistence of cae with coronary artery disease ( cad ) suggests that it may be a variant of atherosclerosis [ 2 , 3 ] . however it is not clear why some patients with obstructive cad develop cae whereas most do not . dendritic cells ( dcs ) are potent antigen presenting and immune modulating cells with the unique ability to initiate a primary immune response to certain antigens by the activation of t lymphocytes [ 4 , 5 ] . to acquire the ability to contact and activate t cells , dcs must undergo a maturation process with the upregulation of antigen presenting molecules including mhc class i and class ii , adhesion molecules ( cd11a , cd11b , cd54 , cd50 , cd58 ) and costimulatory molecules ( cd40 , cd80 , cd86 ) [ 46 ] . in addition to these relatively well - known cell surface molecules ; cd83 which is the hallmark of mature dcs and cd11c expressions were also reported to have functions in the regulation of antigen presentation and enhancement of t cell activation in the recent reports [ 7 , 8 ] . during the development of an adaptive response , t cells form direct contacts with dcs and respond to peptide antigen displayed on mhc class ii and class i molecules present on dc surfaces . in dc / t cell interactions , the presence of costimulatory molecules is required for t cell activation and differentiation into effector cells . so far the presence of dc was described in atherosclerotic plaques of carotid arteries , aortas , and stenotic aortacoronary vein bypass grafts . the colocalization of dc and t cells as well as the expression of mhc - ii and costimulatory molecules on dcs in atherosclerotic plaques suggest that dc initiate an antigen - spesific immune response contributing to the progression of atherosclerosis [ 911 ] . until now research on the pathogenesis of cae has also focused on chronic transmural inflammation recently we have shown increased expression of monocyte and lymphocyte adhesion molecules in isolated cae . given the pivotal function of dcs in initiating t lymphocyte responses in atherosclerosis and the knowledge that peripheral blood monocytes can be differentiated into dcs by exposure to inflammatory factors ; in the present study we investigated the expression levels of mhc class ii , cd54 , cd11b , cd11c , cd83 , and cd86 on the surface of monocyte - derived dcs ( mdcs ) in normal subjects and cad patients with or without cae . our aim was to evaluate the role of dcs in cae development . the study population was selected from a series of 256 consecutive patients who underwent coronary angiography in our hospital between april 2006 and september 2006 due to the presence of chest pain or positive or equivocal results of noninvasive screening tests for myocardial ischemia . out of 256 patients , 6 consecutive patients with obstructive cad and cae ( group 1 , 4 male , mean age : 50.1 4.0 years ) and accepted to participate our study after giving informed consent , were identified and compared with age and sex matched 6 consecutive subjects with obstructive cad alone ( group 2 , 4 male , mean age : 51.1 4.5 ) and 6 consecutive subjects who had angiographically shown normal coronary arteries ( group 3 , 4 male , mean age : 51.1 4.2 ) . obstructive cad was defined as the stenosis of epicardial coronary artery > 50% angiographically . exclusion criteria were the presence of previous myocardial infarction , acute coronary syndromes , any inflammatory or immunologic disease , active local or systemic infection , history of recent infection ( <3 months ) , left ventricular dysfunction , left ventricular hypertrophy , cardiomyopathies , congenital heart disease , valvular heart disease , any abnormality in thyroid function test , arrhythmias and statin use within the last 6 months . besides , leukocyte count , erythrocyte sedimentation rate and fibrinogen levels were normal in all patients and control subjects . we used iohexol ( omnipaque , nycomed ireland cork , ireland ) as contrast agent during coronary angiography in all patients and control subjects . coronary angiograms were analyzed by two blinded interventional cardiologists without knowledge of the clinical status and laboratory measurements of the subjects . the definition of cae was that used in the coronary artery surgery study ( cass ) . according to the angiographic definition of cass , a vessel is considered to be ectasic when its diameter is 1.5 times that of the adjacent normal segment in segmental ectasia . when there was no identifiable adjacent normal segment , the mean diameter of the corresponding coronary segment in the control group served as normal values . the number of epicardial coronary arteries with ectasia was analysed by use of a computerized quantitative coronary angiography analysis system ( philips bh 5000 , netherland ) . heparinized blood samples of the groups were drawn after coronary angiography from an antecubital vein with a 19-gauge needle without venous stasis in the fasting state . peripheral blood mononuclear cells were isolated by density gradient centrifugation with ficoll hypaque 1077 density ( paa laboratories gmbh , austria ) . monocytes were isolated from peripheral blood mononuclear cells by their adherence to plastic . during 9 days of cell culture in the presence of rpmi-1640 medium ( sigma chemical , germany ) the technique of flow cytometry involves the staining of blood cells with fluorescence tagged antibodies targeted to specific cell surface associated antigens . this is followed by quantification of fluorescence intensity in the cell population of interest as a measure of the specific antigen abundance and by determination of percentage of cells displaying fluorescence intensity beyond a threshold . dc were incubated with the mouse antihuman , fluorescein isothiocyanate ( fitc)-conjugated antibodies against cd14 , cd11b , cd11c , cd54 , cd83 , cd86 , and mhc class ii - phycoerythrin ( beckman coulter , ca , usa ) for 15 minutes at room temperature . after immunofluorescence staining , cells were analyzed by epics profile ii flow cytometer ( beckman coulter ) . appropriate isotype - matched immunoglobulins ( beckman coulter ) were used as negative controls . the mean fluorescence intensity ( mfi ) continuous variables were expressed as mean sd and categorical variables were expressed as percentage . comparison of the expression levels of activation markers on mdcs among the groups was performed using one - way anova test . tukey hsd multivariate analysis was used to determine among which groups the activation marker levels were different . the comparison of categorical variables between the groups were assessed by fisher 's exact or chi - square tests where appropriate . the association between the levels of activation markers and the number of ectasic vessels was calculated by spearman 's rho correlation coefficient . there was statistically no significant difference between the groups with respect to age , gender , body mass index , hypertension , diabetes mellitus , cigarette smoking and hyperlipidemia ( p > .05 ) . the distribution of obstructive cad was also comparable between cad patients with or without cae [ single vessel involvement 13.3% , two - vessel involvement 26.7% , and three vessel involvement 60% for both groups ] ( p > .05 for all ) . the patients in group 1 had diffuse cae involving the left anterior descending artery in 4 ( 66.7% ) , the left circumflex artery in 5 ( 83.3% ) and the right coronary artery in 4 patients ( 66.7% ) . one - vessel , two - vessel , and three - vessel ectasia were found to be present in 1 ( 16.7% ) , 2 ( 33.3% ) , and 3 ( 50% ) patients , respectively . therefore most of the patients ( 83.3% ) had multivessel cae . clinical and coronary angiographic characteristics of the study population were presented in table 1 . cad patients with cae were detected to have significantly higher levels of certain activation markers such as cd11b ( 44.5 5.0 versus 30.0 3.8 and 20.9 3.6 ) , cd11c , ( 96.3 10.9 versus 66.1 6.4 and 50.4 5.7 ) cd54 ( 45.6 6.7 versus 31.1 4.9 and 20.8 3.2 ) , cd83 ( 44.6 6.1 versus 30.8 2.4 and 25.6 2.8 ) , cd86 ( 50.7 5.0 versus 39.2 4.1 and 29.5 4.1 ) and mhc class ii ( 112.4 11.3 versus 73.1 9.5 and 54.5 4.5 ) molecules on the surface of mdcs in comparison to cad patients without cae and normal subjects with angiographically normal coronary arteries ( figure 1 ) . mfi of cd14 on mdcs did not significantly differ among group 1 ( 13.3 3.1 ) , group 2 ( 12.9 2.6 ) , and group 3 ( 14 2.9 ) ( p > .05 ) . furthermore we detected a significant positive correlation between the number of the vessels with cae and the levels of cd11c ( figure 2 ) , cd86 ( figure 3 ) , and mhc class ii molecules ( figure 4 ) . the main findings of the present study are ( 1 ) the expression of cd11b , cd11c , cd54 , cd83 , cd86 , and mhc class ii molecules in cad patients with cae were higher than control subjects with cad alone and normal coronary arteries ; ( 2 ) there was a correlation between the levels of cd11c , mhc class ii , cd86 , and the number of coronary vessels with cae . to our knowledge this is the first study that demonstrate the role of mdcs for cae development in patients with cad . cae has been defined as localized or diffuse nonobstructive lesions of the epicardial coronary arteries with a luminal dilatation exceeding the 1.5 fold of normal adjacent segment or vessel diameter . it has been suggested that the pathogenesis of abdominal aortic aneurysm and cae is similar that chronic transmural inflammation with destruction of medial layer of the vessel has a prominent role [ 2 , 14 ] . recently we have reported an increase in the plasma levels of tumor necrosis factor - alpha and interleukin-6 in patients with isolated cae indicating an inflammatory process in the coronary circulation . . showed that levels of soluble cams ; intercellular adhesion molecule-1 , vascular cell adhesion molecule-1 and e - selectin were increased in patients with isolated cae in comparison to patients with obstructive cad and suggested that a more extensive vascular wall inflammation may have a role in the development of isolated cae . although the role of inflammation was demonstrated in the pathogenesis of cae , since inflammation takes part both in cae and atherosclerosis development , it is still not clear why some patients with obstructive cad develop cae whereas most do not . dcs are a component of the proposed vessel - associated lymphoid tissue and are found in the intima and adventitia of susceptible arteries before atherosclerotic lesion development [ 6 , 17 ] . in atherosclerotic plaques , the number of dcs increase related to the activation of residing intimal dcs and invasion of adventitial dcs to the plaque . monocytes that infiltrate the intima from the very early stages of atherosclerosis may differentiate into dcs and contribute to an increased dc population as well [ 6 , 13 , 18 ] . recent findings suggest that dcs play a role in plaque destabilization through activation of t cells . yilmaz et al . found that up to70% of dcs in the shoulders of vulnerable carotid plaques express increased level of dc activation marker cd83 . reported that cd86 was upregulated in patients with unstable angina in comparison to healthy donors . antigen processing , presentation , and t cell priming efficacy were shown to be maintained in dcs with increased expression of cd11c under hypercholesterolemic conditions associated with atherosclerosis . although the role of dcs in atherosclerosis were evaluated in various studies , there is limited data about dcs in cae which has been suggested as a variant of atherosclerosis . in the present study we have found increased expression of adhesion , costimulatory and antigen presenting molecules on the surface of mdcs in cad patients with cae compared to patients with cad alone as well as normal subjects . activated dcs have been shown to exhibit large numbers of adhesion molecules such as cd11b , cd54 which contribute to their ability to adhere to injured endothelium , transmigrate and also interact with t lymphocytes [ 5 , 20 , 21 ] . this interaction is accompanied by the increase in the expression of mhc and costimulatory molecules and the production of cytokines leading to their enhanced ability to prime t lymphocytes [ 46 ] . therefore increased expression of the surface markers of mdcs with a positive correlation between cd11c , cd86 , and mhc class ii molecules and the number of ectasic vessels observed in the present study may support the concept that a more severe and extensive chronic inflammation modulated by dcs takes place in the coronary circulation of cad patients with cae in correlation with the widespread involvement of cae . numerous studies have demonstrated that aneurysm tissue contains high levels of matrix metalloproteinases ( mmp ) which causes degradation of the extracellular matrix leading to weakening and dilatation of the aortic wall . the histopathological examinations of the coronary arteries in small number of cases with cae were reported to include inflammatory cells in the medial layer comprising chiefly macrophages which were shown to have immunoreactivity against mmp [ 23 , 24 ] . recently we reported increased expression of monocyte adhesion molecules in patients with isolated cae that may be associated with the medial destruction of the vessel wall and cae formation in agreement with microscopic examinations . reported high plasma levels of mmp-3 , mmp-9 and interleukin-6 in cae patients and suggested that inflammation enhanced mmp secretion in the coronary circulation may cause cae formation . dcs were also shown to secrete mmp to the same extent as macrophages [ 26 , 27 ] . hence our finding of significantly increased dc activation in cad patients with cae compared to patients with cad alone may be associated with weakening of the coronary artery wall by mmp of which secretion may be enhanced by dcs . it is conceivable that the small number of patients limits the power of our observations . the small number of patients in the present study reflects the small incidence of patients with cae . the phenotypic characteristics of the most mature dcs have been suggested to be complete loss of cd14 , and increased expression of cd83 , cd86 , and mhc class ii receptor . dcs generated by our methods had low expression of cd14 , moderate expression of cd83 , cd86 , and mhc class ii and thus would be considered as relatively immature dcs compared to dcs generated by the other methods [ 4 , 28 , 29 ] . however since mdcs of the three groups were cultivated in the same environment under the same conditions , the significantly different activation marker levels on the surface of mdcs among the groups detected in our study may be of importance . further studies with larger samples using additional stimulators for improvement of dc maturation which also investigate the correlation between dc activation and mmp secretion are needed to firmly establish our results . the present study indicates that mdcs display an increased cell surface concentration of adhesion , costimulatory , and antigen presentation molecules with respect to their activation in cad patients with cae compared to patients with cad alone and normal subjects . dc activation may play an important role for cae development in patients with obstructive cad . | background . coronary artery ectasia ( cae ) is defined as localized or diffuse dilation of the coronary arteries .
there are scarce data about the role of dendritic cells in cae development . in this study we investigated the activation markers on the surface of monocyte - derived dendritic cells ( mdcs ) in coronary artery disease ( cad ) patients with or without cae
.
method .
the study consisted of 6 patients who had obstructive cad with cae , 6 cad patients without cae and 6 subjects with angiographically normal coronary arteries .
mdcs were cultivated from peripheral blood monocytes .
surface activation markers were detected by flow cytometry .
results .
cad patients with cae were detected to have significantly higher mean fluorescence intensities of cd11b , cd11c , cd54 , cd83 , cd86 and mhc class ii molecules on mdcs in comparison to cad patients without cae and normal controls ( p < .001 for all ) .
a significant positive correlation was found between the number of vessels with cae and the levels of cd11c , cd86 , and mhc class ii molecules .
conclusion .
mdcs display an increased cell surface concentration of activation molecules in cad patients with cae compared to patients with cad alone .
dc activation may play an important role for cae development in patients with cad . |
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the most important arbovirus disease in humans , dengue , annually affects 80 million individuals in many countries , leading to 550,000 hospitalizations and 20,000 thousand deaths . the main vector is the mosquito aedes aegypti , an arthropod with an extremely high capacity to adapt to urban areas . since 1982 , the reemergence of dengue has been reported in urban centers in all brazilian regions . the magnitude of this disease has led to high public federal , state , and municipal investments in vector control , epidemiological surveillance , and patient care . during the 1990s , the incidence of dengue increased greatly as a consequence of the dissemination of a. aegypti . dispersion of the vector was followed by the dissemination of dengue virus serotypes 1 and 2 in twenty of the 27 states of the country . between 1990 and 2000 , several epidemics occurred , mainly in the largest urban areas of the southeast and the northeast , where the majority of notified cases were concentrated . the first great dengue epidemic occurred in 1998 , with approximately 528,000 cases . in brazil , the increase in the incidence of dengue cases in 2002 and the emergence of a third serotype ( denv-3 ) led to a prediction of an increased risk of dengue epidemics and an increase of the cases of dengue hemorrhagic fever ( dhf ) . to face the expected risks for 2002 , the brazil ministry of health , in collaboration with the pan - american health organization , carried out an international seminar in june 2000 to evaluate the dengue epidemic and to prepare a national dengue control program ( pncd ) . however , the current epidemiological situation shows that these program measures have not achieved the expected results . epidemiological impact assessments of these interventions have shown that their effectiveness has been extremely limited . regardless of each local health system , even when these measures are well managed , their effectiveness is always low , given the intense viral circulation detected in the successive epidemics and the results of serological surveys conducted in several brazilian cities [ 4 , 5 ] . the first dengue epidemic in belo horizonte ( bh ) , the principal city of brazil 's third metropolitan area , occurred in 1996 , and different from the subsequent epidemics , the 1996 epidemic started in the southern hemisphere 's fall . however , by the end of 1997 , another epidemic of great intensity started , characterized by the simultaneous circulation of denv-1 and denv-2 . , denv-3 was identified for the first time in bh , and now the three serotypes coexist . the control measures , adopted in bh until the 1998 epidemic , had only a limited role , without much impact on the final numbers of cases . this situation was repeated in 1997 and only changed its stance in 1998 , before the largest epidemic in the city when denv-1 and denv-2 virus serotypes were both circulating . in 2002 , it was observed that the spread of serotype 3 from the state where it was originally detected presented a different pattern from that observed with serotypes 1 and 2 . previously , the expansion of the new serotype ( denv-3 ) occurred slowly and some years elapsed before autochthonous cases occurred in other states . during the first three months of 2002 , the presence of the new serotype was detected in ten other states . in bh , it would be theoretically possible to attribute these results to the control measures proposed by the brazil ministry of health in 1996 , the program of eradication of a. aegypti known as peaa which was only implemented in the municipality in 1998 . this program took into account the difficulties of the previous control strategy and proposed an even more complex objective , predicated on the assumption that the vector could be eradicated . when compared to other large urban areas in southeast brazil , the dengue epidemic cycle in bh this epidemic behavior was probably only interrupted when the resistance to the larvicidal agent being used was detected in bh in 2006 . currently , vector control is the only way to interrupt disease transmission , given that there is neither an effective vaccine nor specific therapy . vector control , however , is not a simple task , especially given the complexities of urban settings . the failure of dengue control programs has been pointed out by several authors [ 7 , 1113 ] . spatial analyses are powerful tools in public health diagnosis and surveillance , allowing the identification of critical areas for intervention and the variables associated with the modulation of disease dynamics [ 14 , 15 ] . dengue , whose pattern is well known to be clustered in certain areas , is a health - related event for which spatial analysis techniques may be useful . spatial analyses and statistics , such as spatial autocorrelation analysis , cluster analysis , and temporal analysis , are commonly used to highlight spatial patterns of dengue cases and to test whether there is a pattern of dengue incidence in a particular area [ 17 , 18 ] . a geographic information system ( gis ) can be used to identify and assess potential compositional and contextual risk factors associated to disease transmission such as socioeconomic , climatic , demographic , and physical environment . gis technologies have been applied in epidemiologic and public health studies for many years [ 19 , 20 ] , providing information useful for studying and modeling the spatial - temporal dynamics of dengue [ 2123 ] . this paper aims to evaluate dengue dissemination in space and time , determining possible outbreak waves of dengue cases correlated with climatic data and presence of the vector . this study may contribute to implement interventions aimed at vector control and patient care , minimizing the collective and individual burden of this disease . this ecological study was conducted in belo horizonte ( bh ) , the capital of the state of minas gerais , in the southeast region of brazil ( 1955s 4357w ) . occupying an area of 330.23 km with 2,375,151 inhabitants in approximately 600,000 households ( figure 1 ) , bh is brazil 's sixth most populous city . situated at altitudes ranging from 700 to 1,200 meters ( mean 858 meters ) , bh has a tropical wet and dry climate with an average annual temperature of approximately 21c . each one of 147 primary care units is responsible for a geographic area known as a health services catchment area ( hsca ) . the hscas are aggregated in nine sanitary districts ( sds ) named as north , northeast , northwest , east , south central , west , venda nova , pampulha , and barreiro . all dengue cases reported from 1996 to 2011 ( partial ) to the municipal surveillance system which in turn are forwarded to brazil 's national reporting system were used . the notification form contains , along with other information , each patient 's address and the date of onset of dengue symptoms . dengue larvae vectors foci data reported for years 1996 to 2011 ( partial ) and eggs collected in ovitraps from 2003 to 2010 were used in this study . the data was obtained from the municipality vector reporting system sczoo which contains the address for each larva focus and ovitrap and the dates of the survey . the ovitraps which cover a radius of 200 meters are installed every two weeks . the building larval index ( bli ) as proposed by connor and monroe measures the density of a. aegypti in urban areas and is estimated as the proportion of houses with a. aegypti larvae . rainfall ( mm ) and temperature ( degrees celsius ) for the years 20012010 were obtained from weather station of the 5th district of brazil 's meteorological institute ( inmet ) . depending on the analysis ( see below ) , dengue incidence was calculated on a monthly or annual basis from 1996 to 2011 . then the dengue incidence in a given year for each sanitary district from 2005 to 2011 was correlated to september - october vector data ( the mean number of eggs in the ovitraps of each sd and the bli in the larvae foci survey ) from the previous year . we used the pearson correlation coefficient to estimate the correlation between the monthly incidence of dengue and climate data for the years 2001 to 2010 . the vector data was geocoded using the address of the larvae foci building and the locations of the ovitraps . spatial statistical techniques used in this study included kernel 's estimation in order to determine the possible outbreaks of disease and specific patterns of distribution on the urban space . to find how dengue spread in space and time , we created map objects that change status with time . a hotspot moreover , cases clusters that occur randomly can also have an influence on the spread of an infectious disease . tabwin 3.5 was used to make brazil municipalities maps ( http://www.datasus.gov.br/ ) , and r ( r development core team ; http://www.r-project.org/ ) was used to calculate the pearson correlations and kernel 's estimation . mapiinfo 8.5 was used to make bh hotspots maps , and excel 2003 was used to generate tables and figures . in this series of annual incident dengue cases , five distinct periods were identified : ( 1 ) between april 1996 , the first epidemic in bh , and july 1998 , the most important epidemic ; ( 2 ) between august 1998 and december 2000 with incidence rates not exceeding 10 cases per 100,000 inhabitants ; ( 3 ) between january 2001 and august 2002 , during which two new epidemics occurred ; ( 4 ) between august 2002 and december 2005 again with low dengue incidence rates ; ( 5 ) the last period , between january 2006 and august 2010 , during which the incidence rate was progressively higher ( figure 2 ) . the dengue temporal distribution with highest incidence in the rainy season presented a similar pattern during the period ( figure 2 ) . characteristically , dengue outbreaks generally occurred during the second part of the rainy season , when humidity was higher than average . in the period from 2005 to 2011 , annual incidence rates of dengue showed a statistically significant correlation with the bli according to sanitary district ( r = 0.60 , p = 0.0000002 ) . for the mean values of eggs captured in the ovitraps , the correlation was also statistically significant ( r = 0.69 , p = 0.00000005 ) ( table 1 ) . rainfall ( rf ) and temperature ( temp ) begin to increase in october , with dengue outbreaks occurring during the months of january to may , the period of highest rainfall and humidity . the number of cases then fall through june , a period when rf and temp also decrease ( figure 2 ) . analyzing the climatic data for the years 2001 to 2010 , monthly dengue incidence rates showed a statistically significant correlation with the rf of the previous month ( r = 0.36 , p = 0.00006 ) and the monthly minimum temperature ( r = 0.29 , p = 0.001 ) . the maps that comprise figure 3 illustrate the spatial and temporal evolution of dengue in cities of brazil and are accompanied by a comparative graph of annual incidence rates from 2001 to 2011 for bh , brazil . figures 4 and 5 demonstrate the spatial correlation between dengue cases hotspots and the location of aedes aegypti larvae foci in bh . figure 6 shows the same observation among dengue cases hotspots and the areas with the greatest presence of aedes aegypti eggs . the hotspot analysis also found a higher risk of dengue in areas of the city that are at lower elevations ( figures 7 and 8) . monitoring and planning control measures for dengue epidemics are vital for preventing or minimizing disease outbreaks . information based on notified cases only , however , is insufficient , because many people who are infected may either be asymptomatic or do not become part of the official statistics even if they present symptoms . the use of information on dengue incidences rates , mapping their patterns and dynamics of spread using spatial autocorrelation analysis , can be a valuable tool to analyze the spatial patterns change over time . therefore , instead of aiming to achieve a complete understanding of the transmission process , it may be more efficient to improve the surveillance system and optimize disease control . the heterogeneous intraurban distribution of dengue incidence according to sanitary districts for the years 2001 to 2011 suggests the importance of analyzing transmission at the sd level . the degree of acquired immunity to the dengue virus may vary across different areas of the municipality based on the spatial distribution of previous outbreaks . our results indicate that continuous vector surveillance using ovitraps and larvae foci is necessary , so that a greater number of areas with potential transmission can be identified , permitting the prioritization and scheduling of vector control measures . certainly , the identification of high - risk areas , in a process of surveillance and control of the disease and the mosquito , is an important step towards optimizing resources . once such areas have been identified , interventions may provide better results in decreasing incidences rather than through the traditional approach of a uniform control strategy for the city as a whole . determining whether greater vector presence or coefficients of dengue incidence predominant in certain intraurban areas may be operationalized through the use of the concept of persistence . for each sd , the number of months of uninterrupted vector presence would be calculated , thereby determining whether greater persistence occurs in specific sds over the various periods of the year . temporal analysis of climatic factors ( rainfall , temperature , and humidity ) revealed that dengue generally occurs when average temperatures increase , when the rainy season has started , and when the humidity is higher . previously , a report from bh showed that rainfall and relative humidity data from fifteen days before ( t1 ) showed very high correlation with dengue vector incidence in time t . there are other studies in the literature reporting an important correlation between climate and dengue occurrences or dengue vector abundance [ 3537 ] . however , the occurrence of a residual vector population or the occurrence of dengue cases in distinct intraurban areas in the cold and dry months , with much lower dengue incidence than in january to may , should be taken into account for disease control . identifying locations and patterns of the vector population ( species , density , and vector - control indices ) should also be used to direct interventions with disease reduction as the preferred outcome measure demonstrating impact , and ovitraps index , house index , container index , and breteau index as proxy indicators of impact . with these strategies , information will be available in real time , which may uncover other aspects about the relationship between vector and the disease that could be revealed through spatial analyses [ 38 , 39 ] . other tools such as the industrial control chart proposed by rich and terry , and adopted in several survey vigilance systems when applied to dengue require several improvements related to presentation and interpretation in order to enhance its usefulness . the ability to demonstrate trends , analyzing only notified dengue cases at a potentially earlier time point , is limited . heterogeneous internet access limits the use of query - based surveillance web tools to identify disease and location outbreaks as candidates for interventions . although this proposal is intriguing , so far the identification of a given outbreak is usually too late for control measures . our findings show that the strategies used in this study can help public health officials to visualize and understand the geographic distribution and trends of disease patterns and to prepare warnings and awareness campaigns . dengue spatial and temporal spread patterns and hotspot detection may constitute useful information for public health officials to control and predict dengue dissemination from critical hotspot areas . this may save time and cost and make public health department actions more efficient . public health officers may employ the model to plan a strategy to control dengue by analyzing the information received on distribution and hotspots for various months . some ancillary findings of the study such as influence of climate , which is seasonal and thus temporal , also contribute to knowledge regarding its significance . the methodology is based on principles of spatial statistics and has the potential to be applied to other epidemics . in the future , it will be important to have regular daily statistics accumulated over several years to permit faster recognition of outbreak locations and be prepared to promptly implement appropriate public health interventions . | this study considers the dengue occurrence in the city of belo horizonte over the last fifteen years .
approximately 186,000 cases registered from 1996 to 2011 were analyzed .
the home address of individuals whose dengue case was notified was used as a proxy for exposure location . for determining possible outbreaks of disease and the specific patterns of dengue cases , spatial statistics
used included kernel 's estimation .
the occurrence of waves of dengue outbreaks was correlated with climatic and vector presence data .
outbreaks had different durations and intensities : case clustering , thinned out both spatially and temporally .
these findings may be useful for public health professionals responsible for fighting the disease providing some tools for improving evaluation of interventions such as vector control and patient care , minimizing the collective and individual burden of the disease . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
shoes can be selected on the basis of mobility and functionality , but more emphasis has
been placed in recent times on the aesthetic appeal of footwear . many women wear high heels
of various shapes and live their daily lives while wearing high heels1 . a total of 59% of females wear high heels eight hours a
day2 , and this puts many women in a
position of having to make difficult choices between balancing fashion and comfort when
selecting their shoes . wearing high heels is related to a deterioration in body alignment ,
and it can lead to ankle instability by changing the static and dynamic body alignment
characteristics . the increase in muscle activity of the plantar flexor resulting from
wearing very high heels can reduce stability due to muscle imbalances of the feet and ankle
joints . it can also decrease one s ability to respond to unexpected resistance by increasing
muscle fatigue and decreasing the muscle control ability of the ankle joint3 . driving posture is an important element to be considered in the ergonomic design of cars . when designing and manufacturing cars , in order to provide maximum comfort and safety , the
complex biomechanical elements of the human musculoskeletal system are taken into
consideration4 . pressing of the
accelerator and brake pedals , which are important elements of car control , can be restricted
by the user s shoes5 . in addition , wearing
high heels , while driving , can cause discomfort when operating the pedals and could also
prove to be hazardous . the majority of previous studies associated with high heels have focused on variables
related to gait6 , 7 , whereas most of the studies on the relationship between cars and
humans have considered the designs of seating positions that offer optimal comfort5 regardless of footwear . however , when one
considers the amount of time that modern women spend in cars , studies that have dealt with
the effects of wearing high heels while pressing on pedals have been lacking . therefore , the
purpose of this study was to determine the effects of wearing high heels while driving on
lower extremity muscle activation . the subjects of this experimental study were 14 healthy women in their 20s who wear shoes
with high heels more than three times a week . their average age , weight , and height were
23.78 3.22 years , 59.02 7.88 kg , and 162.655.29 cm , respectively . none of the subjects
had experienced any pathologic symptoms in the feet and legs for the last 6 months , and none
had any history of operation on the lower extremities . further , they also had no history of
neurological disease . before taking part , all of the participants were thoroughly briefed
about the experimental procedures and asked to read and sign a consent form . the subjects were then asked to select their shoe sizes , 23.0 cm , 23.5 cm , or 24.0 cm for
identical high - heeled shoes of two different heights ( 5 cm and 7 cm ) and flat shoes of
sizes , which had been prepared for this experiment . in order to measure the lower extremity
muscle activation occurring while pressing a pedal when wearing high heels , the subjects
were asked to sit in the driver s seat of a car ( elantra hd , hyundai motor company , south
korea ) . they were then allowed to adjust the seat - back angles and the pedal - to - seat distance
so that they were able to operate the accelerator pedal with ease . the subjects , after
finding comfortable driving positions , were asked to place the appropriate shoes on the
accelerator pedal with the heel touching the floor and then asked to press the pedal with as
much pressure as possible for 3 seconds before removing their feet from the pedal . a total
of 3 measurements were taken for each heel height , and the heel height was randomly
selected . in order to avoid muscle fatigue , electromyographic ( emg ) measurements were taken from the
moment the pedal was pressed to the moment when the pedal was released . to measure the lower extremity muscle activation , electrodes were attached to the rectus
femoris , tibialis anterior , gastrocnemius , soleus were selected . , montreal , qc , canada ) was employed
for measurement of the muscle activation of each muscle . a surface electrode ( triode surface
electrode , thought technology ltd . , montreal , qc , canada ) consisting of a tripolar electrode
( positive - ground - negative ) was used . the frequency range of the electromyogram signals was
set to 20 to 500 hz , and the sampling frequency was set to 1,024 hz . the root mean square of each muscle was measured for five seconds while the subjects sat in
the driver s seat . the relative muscle contraction was calculated referring to 100% of the
mean electromyogram signal measured for the three seconds in the middle , excluding the data
of the first one second and the last one second , and expressed as the muscle activation in
terams of the percentage of reference voluntary contraction ( % rvc ) for one instance of
pushing the accelerator . 19 ) was used for data analysis . in order to determine the difference
between wearing high heels and low heels , one - way analysis of variance test ( anova ) was
carried out , and tukey s test was used as a post hoc test to verify differences . the levels of muscle activity for the gastrocnemius muscle in the flat , 5 cm , and 7 cm
shoes were 180.8361.78% , 285.39122.25% , and 366.15193.73% , respectively , and there were
significant differences between groups . the post hoc test showed a significant difference
between the flat and 7 cm shoes . those for the soleus muscle were 477.28209.21% ,
718.77380.50% , and 882.35509.93% , respectively , and there were significant differences
between groups . the post hoc test showed a significant difference between the flat and 7 cm
shoes ( table 1table 1.average muscle activation for each heel height ( unit : % rvc)muscleflat shoes5 cm7 cmrectus femoris133.527.5163.571.2177.769.3tibialis anterior160.9104.8205.686.2213.374.1gastrocnemius*180.8361.7285.39122.2366.15193.7soleus*477.28209.2718.77380.5882.35509.9each value represents the meansd . the values with different superscripts in the same
column are significantly different ( p<0.05 ) by tukey s test . ) . the values with different superscripts in the same
column are significantly different ( p<0.05 ) by tukey s test . while an increase in heel height affects the stability and mobility of feet greatly1 , it has also been reported that the gait
when high heels are worn greatly affects the stability of the ankle joint by increasing the
plantar flexion of the ankle joint and readily inducing muscle fatigue8 . most of the previous studies have looked into the gait
resulting from the change in the body s center of gravity when wearing high heels and the
kinetic and kinematic properties that are required to maintain balance while standing9 , 10 . however , in this study , the intention was to determine the effects of wearing high heels
while in a sitting position , that is , when the majority of the body s center of gravity is
supported by all the muscles of the trunk . plantar flexion and dorsiflexion of the ankle joint are the movements associated with
pressing the accelerator and releasing the accelerator of a car . the results of the present
study showed that with an increase in heel height , the muscle activation increased in the
gastrocnemius and soleus ankle joint plantar flexion agonists , especially with a heel height
of 7 cm , which resulted in a statistically significant increase in muscle activation .
despite statistically insignificant data , it was found that as the heel height increased ,
the tibialis anterior muscle activation of ankle joint dorsiflexion agonist have also
increased . this appeared to be the result of an increased plantar flexion angle resulting
from the increase in heel height : the movement of pressing the accelerator pedal results in
ankle joint plantar flexion and dorsiflexion . in addition , although not a statistically
significant increase , the muscle activation of the rectus femoris tended to increase with an
increase in heel height , and this was the result of an increase in knee joint extension . this concurs with the results of a previous study , which found that the proximal part
required preceding contraction due to the instability of the distal part in the ankle joint
with an increase in heel height11 . to summarize the results of this study , it was found that female drivers require greater
lower extremity muscle activation when wearing high heels than when wearing low heels . furthermore , the instability and muscle fatigue of the ankle joint , which result from
wearing high heels on a daily basis , can also occur while driving . | [ purpose ] the purpose of this study was to determine the effects of wearing high heels
while driving on lower extremity muscle activation . [ subjects ]
the subjects of this
experimental study were 14 healthy women in their 20s who normally wear shoes with high
heels .
[ methods ] the subjects were asked to place their shoes on an accelerator pedal with
the heel touching the floor and then asked to press the pedal with as much pressure as
possible for 3 seconds before removing their feet from the pedal .
a total of 3
measurements were taken for each heel height ( flat , 5 cm , 7 cm ) , and the heel height was
randomly selected .
[ results ] the levels of muscle activity , indicated as the percentage of
reference voluntary contraction , for gastrocnemius muscle in the flat , 5 cm , and 7 cm
shoes were 180.861.8% , 285.4122.3% , and 366.2193.7% , respectively , and there were
significant differences between groups . those for the soleus muscle were 477.3209.2% ,
718.8380.5% , and 882.4509.9% , and there were significant differences between groups .
[ conclusion ] to summarize the results of this study , it was found that female drivers
require greater lower extremity muscle activation when wearing high heels than when
wearing low heels .
furthermore , instability and muscle fatigue of the ankle joint , which
results from wearing high heels on a daily basis , could also occur while driving . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the auditory pathway is known as one of the most susceptible parts of the central nervous system to noxious agents . severe neonatal hyperbilirubinemia ( hb ) is a common cause of sensorineural hearing loss ( snhl ) and auditory neuropathy ( an ) [ 14 ] . moreover , surviving infants are at high risk of neurological damage , which can manifest as cerebral palsy , epilepsy , snhl , or cognitive deficits [ 59 ] . some audiological studies in children with serum bilirubin levels > 20 mg / dl , have reported auditory dysfunction in 1787% of cases [ 1014 ] . the usual treatments for this neonatal disease are phototherapy and blood exchange transfusion ( et ) . phototherapy reduces serum bilirubin levels through luminous oxidation , while et is used primarily to maintain bilirubin levels below toxicity levels , eliminate antibodies , and correct hemolytic anemia . however , some adverse events associated with et are asymptomatic electrolyte and other blood abnormalities , which are treatable in the neonate . overall , 74% of et were associated with adverse events ; the most common events were thrombocytopenia ( 44% ) , hypocalcemia ( 29% ) , and metabolic acidosis ( 24% ) , of which 69% , 74% , and 44% , respectively , required treatment [ 15 , 16 ] . the joint committee on infant hearing of the american academy of pediatrics ( aap ) considers et a risk factor for snhl . snhl , is a severe sensory sequelae in young infants and its early diagnosis depends on systematic hearing screening . newborn hearing screening , mainly in high - risk infants , is the most effective way of early snhl detection . . therefore the main goal of this study was to determine the frequency of snhl , an , and neurological comorbidity in a group of children with a history of neonatal hb and et treated at a third - level hospital in mexico city . we designed a retrospective , case - control study , with the following inclusion criteria : having been born at the national institute of perinatology dr . isidro espinosa de los reyes ( inperier ) in mexico city , between january 1 , 2000 and december 30 , 2010 ; a history of et secondary to severe hb after rh hemolytic disease ; abo incompatibility or multifactorial hb , regardless of birth gestational age or associated morbidity during the neonatal period , and belonging to the pediatric followup clinic for high - risk newborns . severe hb was defined as a bilirubin increase > 0.5 mg / dl per hour in term infants , or > 0.3 mg / dl for preterm infants , requiring exchange transfusion . rh hemolytic disease was defined as different maternal - infant antigens and a positive direct coomb 's test . abo incompatibility was defined as an infant 's blood type a or b with a type o mother . multifactorial hb was defined as the same maternal - infant blood type and severe hb . phototherapy and et were performed according to aap guidelines [ 1821 ] : ( 1 ) total serum bilirubin level over threshold values for et according to gestational age and ( 2 ) increase of bilirubin > 0.5 mg / dl per hour in term infants and > 0.3 mg / dl for preterm infants regardless of phototherapy . et was performed with a double transfusion volume ( 160 ml / kg ) for term infants and ( 180 ml / kg ) for preterm infants using compatible reconstituted fresh whole blood units . two groups were defined in the followup based on their hearing status : ( 1 ) children with snhl and ( 2 ) a control group of children , consisting of eighty - seven children ( 85% ) , who showed bilateral normal hearing ( bnh ) with history of exchange transfusion for severe hyperbilirubinemia . neonatal variables and procedures were compared as follows : gestational age at birth in weeks belongs to a term ( birth age between 37 and 42 weeks ) or preterm ( < 37 weeks ) infant group ; birth weight , apgar score at one and five minutes , gender , days of endotracheal ventilation , length of hospital stay , and age at time of studies . risk variables for snhl documented in the neonatal period were peak serum indirect bilirubin level in mg / dl at the time of et ; days of phototherapy ; exposure to other potentially ototoxic drugs such as aminoglycosides and diuretics ; severe perinatal asphyxia ( apgar < 3 at one minute , ph < 7.25 , pao2 < 50 mmhg ) ; intraventricular hemorrhage ( determined by transfontanelar ultrasonography ) during their stay in the neonatal intensive care unit ( nicu ) classified according to papile et al . exclusion criteria were as follows : family history of hearing loss ; maternal / fetal infections in the first trimester of pregnancy ( toxoplasmosis , rubella , cytomegalovirus , herpes virus , syphilis , human immunodeficiency ) ; and congenital or metabolic diseases associated with hyperbilirubinemia such as : hereditary spherocytosis , thalassemia , gilbert 's syndrome , crigler - najjar disease , galactosemia , and glucose-6-phosphate dehydrogenase deficiency ( diagnosis of these entities was carried out with the support of the genetics and hematology services of the hospital ) . parents were informed of the importance of their child 's participation , the purpose of the study , and research benefits . causes for not participating in the pediatric followup were as follows : low economic resources , living far away from the hospital , and both parents working , among others reasons . signed informed consent was requested when infants were recruited for the follow - up study , before hearing examinations in accordance with the institute 's research committee and of the declaration of helsinki . all specimens were protected from light after they were drawn , and these were analyzed immediately . for the quantitative determination of serum bilirubin , we utilized a dichlorophenyl diazonium ( dpd ) reagent . measurements were performed using a beckman synchron cx-9 equipment ( fullerton , ca , usa ) . all infants included in the study underwent determination of conventional brainstem auditory evoked potentials ( baep ) at 3 and 6 months of chronological age with a nicolet viking quest ( nicolet biomedical inc . , the test was conducted in a soundproof room reserved for this purpose within the neurophysiology unit , with the child in physiological sleep in a regular bed . baep determinations were performed after skin cleaning with alcohol - acetone and to apply conductive gel , using the international 1020 system electrode placement with the following assembly a1-cz , a2-cz . the studied ear was ( ) , cz ( + ) , and the contralateral ear was the ground . stimulation was carried out with monaural clicks in rarefaction at an intensity of 80 decibels ( db ) of normal hearing level ( nhl ) . the contralateral ear was simultaneously masked with a white noise 40 db below the intensity of the stimulus . one thousand and five hundred clicks where administered for each sweep , decreasing in 20 db steps to search for the threshold level in each ear . the duration of the stimulus was 100 microseconds and the clicks were delivered through tdh-49p headphones ( telephonics co. , huntington , ny , usa ) . a normal peripheral auditory sensitivity was considered when the infant had a response to 40 db nhl , displaying a robust positive wave v. to rule out middle ear pathology , children were studied with a carl zeiss op - mi-9 f-125 oto - microscope ( jenna , germany ) . afterward , we used a grason - stadler gsi tympstar v.2 impedanciometer ( madison , wi ) , with ansi s3.6 - 1996 calibration . the test tone used in tympanometry was 226 hz , 85 at db , pressure range = 600 to 400 deca - pascals , compliance range of 0.1 to 5.0 ml , with an accuracy of 5% . children should have had a jerger type a curve , with pressure variation of 150 to 50 deca - pascals ( to ensure a proper audiological test of quantitative and normative function in the assessment of middle ear and eustachian tube ) . children > 3 years of age underwent audiometry by conditioning game technique . at 3 years the child must be able to react voluntarily to sounds , if given sufficient motivation . once the child accepted the placement of tdh-50p balanced headphones , he was conditioned to put a toy in a rack , inserting it only during the test tone stimulus ; this is repeated decreasing by 10 db steps each time until the child no longer hears the test sound . after this , the test tone was increased by steps of 5 db until it is perceived again , thus determining hearing thresholds for frequencies between 125 and 8,000 hz in octave steps for each ear . the decrement - increment approach is the most commonly used technique in clinical audiology for determining hearing thresholds . we used a modified hughson - westlake method for children from sound to silence in steps of 10 by 10 db and silence to sound in steps of 5 by 5 db . we used a two - channel grason - stadler gsi 61 clinical audiometer with ansi s3.43 - 1992 iso 389 calibration and a bone vibrator placed on the forehead . for the contralateral ear , auditory masking white noise was used with automatic synchronization 10 db below the level of the analyzed frequency . hearing was considered normal in conditioned audiometry when the threshold was 20 db in the frequencies analyzed . the criteria for snhl were considered when both the air and bone conduction thresholds were increased and overlapping with hearing thresholds 25 db in at least two of the frequencies tested . all subjects were studied , diagnosed , and followed up by a certified pediatric audiologist ( mccf ) . in order to document auditory neuropathy , automatic transient - evoked otoacoustic emissions ( teoae ) were performed in infants with abnormal baep result in both determinations . a madsen otoacoustic - emission - analyzer accuscreen gn otometrics equipment ( copenhagen , denmark ) was utilized with the following technique : the study was conducted in a soundproof room , placing the probe in each of the ear canals . pass is equivalent to normal function of outer hair cells of the cochlea in the explored ear . the criteria for diagnosing an consisted of two abnormal baep determinations ( flat line or only wave v at high intensity stimulation ) and pass otoacoustic emissions result [ 2 , 3 ] . normal binaural hearing was considered when the infant passed the first or second test of conventional baep study , or when the infant passed the evaluation in the audiology clinic ; these children formed the control group . snhl was identified when the infant presented two beap studies with thresholds > 45 db nhl and did not pass the behavioral auditory tests . hearing loss was classified in severity stages by averaging the hearing thresholds at 500 , 1,000 , and 2,000 hz frequencies after performing the audiometric measurement for each ear . subjects with audiometric threshold between 21 and 40 db were classified with mild hearing loss ; those between 41 and 70 db with moderate hearing loss ; children with thresholds of 7190 db were classified with severe hearing loss and > 90 db profound hearing loss . the presence of neurological sequelae ( pathologic condition resulting from a disease , once the offending agent is removed ) was documented by serial neurological examinations performed by a certified neuropediatrician with the help of brain imaging scans , neurophysiological recordings , laboratory studies , and with posterior appointments to the follow - up clinic to determine alterations such as cerebral palsy and/or epilepsy ( according to international classification of diseases tenth edition , categories g80 , and g40 resp . ) . continuous data were presented as means and standard deviations and were analyzed using one - way analysis of variance ( anova ) and the mann - whitney u test . odds ratios ( or ) were calculated for categorical variables for snhl risk , with a statistical significance level of p < 0.05 . for the data analysis we used the spss 17.0 for windows ( spss , chicago , il ) from a population of 7,219 children in the pediatric follow - up clinic for high - risk newborns , 336 ( 4.6% ) children had undergone et . one - hundred - two infants met inclusion criteria for this study , with a mean age of 5.5 years 3.9 ( range of 2 to 10 years ) , 234 children did not meet the inclusion criteria ( 182 were outside the study period , 34 did not have complete audiological evaluations , and 18 rejected the followup in the clinic ) . causes of et were distributed as follows : rh isoimmunization , n = 48 ( 47% ) ; abo incompatibility , n = 28 ( 27.5% ) ; and multifactorial hb , n = 26 ( 25.5% ) ; the high number is possibly because our hospital is a referral center for high - risk pregnancies . comparison of the mean values of indirect bilirubin and the frequency of snhl among these three groups showed no differences ( table 1 ) . thirty - five children ( 34% ) were born at term and 67 ( 66% ) were preterm ; we found fifteen patients ( 15% ) with snhl in our sample . we constructed a group of children with bilateral normal hearing ( bnh ) with 87 patients ( 85% ) for comparison purposes . clinical characteristics of children with snhl and bnh with et are presented in table 2 . the mean apgar score at one and five minutes was significantly lower for the group with snhl . baep results in children with snhl were as follows : three infants had hearing thresholds of 80 db nhl , two presented only wave v at 95 db nhl , and ten had no response to > 95 db nhl stimulation . teoae recordings were negative in all cases of snhl and thus , an was not documented in the sample . audiometric measurements showed severe snhl in 10 cases ( hearing threshold of 82 db ) with profound snhl in 3 cases ( hearing threshold of 99 db ) . in all cases the auditory alteration was bilateral and symmetrical . an increased frequency of cerebral palsy was documented for the group with snhl ( 20% ) when compared with results from those of children with bnh ( 3% ) ( or = 7.0 [ 1.238.7 ] , p = 0.01 ) . epilepsy also showed a significant increased frequency in the group of children with snhl ( 20% ) , when compared to children with bnh ( 5% ) ( or = 5.1 [ 1.026.0 ] , p = 0.02 ) . this paper demonstrated a higher frequency of snhl ( 15% ) in children with a history of et treated in a 3rd level hospital in mexico city . hearing alteration was produced despite the cause of severe hyperbilirubinemia and was associated to preterm birth and low gestational age , level of indirect bilirubin , and exposure to furosemide . comparison with other studies is limited because of the differences in methodology , severity of hyperbilirubinemia , et criteria , and snhl classification . it is unclear why some infants do not develop hearing loss or neurological injury with the serum bilirubin levels that other infants do . some researchers studied the effect of hb on the auditory pathway during its acute phase , with a short prospective design , assessing baep and otoacoustic emissions before and after phototherapy or et [ 31 , 32 ] ; they found an increased risk for auditory damage in children with severe hb . other studies have included only term infants with nonhemolytic jaundice , eliminating several risk factors and hb that cause hearing damage [ 3336 ] . some researchers have included the measurement of demographic or ethnic factors in their statistical analysis to weigh a multidimensional overview of the hearing damage after hb [ 3739 ] . however , overall , their results usually coincide with our data , showing a higher frequency of auditory pathway dysfunction in children with severe hb and reports of alterations ranging from 17 to 87% of hearing dysfunction . in this paper we analyzed the variable et under usual clinical conditions present in the nicu , where newborns with severe hb usually present other associated co - morbidities , therefore being difficult to document severe hb as single disease . for example , patra et al . documented in a group of infants with history of neonatal et that 62% also had other neonatal morbidities at the time of et . in our sample , 50% of our children had other neonatal problems at the time of et ; thus , their results are in line with our observation . severe hb that requires et for its treatment is a clinical variable that can not be accurate or measured objectively , since it is not easy to precisely define a serum bilirubin value that indicates the need for et or that is directly associated with neural or auditory damage . thus , et is a strong qualitative variable associated as a risk factor to snhl . this paper demonstrates the need to pay special attention to the increased risk of snhl among infants treated with et for severe hb . however , not all cases of severe neonatal hb with et result in hearing or neurological deficits , and the exact threshold in which bilirubin becomes dangerous is not uniform among populations . hb is more prevalent and severe in preterm infants , and its course is more prolonged than in term infants as a result of the red blood cells , liver , and gastrointestinal system immaturity . as consequence of these facts , loop diuretics cause snhl by inhibiting ion transport of within the stria vascularis , reducing the electrochemical gradients that create the endocochlear potential . more important is the fact that loop diuretics enhance the rate of permanent hearing loss induced by aminoglycosides . the mechanism for interaction between aminoglycosides and loop diuretics implies alterations in the blood labyrinth barrier , which facilitates aminoglycoside entry to the endolymphatic compartment . we do not know if this auditory lesion may include other ototoxic agents like indirect bilirubin . in this study we found that exposure to furosemide was associated with snhl . given the previous findings we suggest avoiding the use of aminoglycosides and furosemide combination in neonates undergoing et to minimize the risk of snhl . the incidence of an in infants with severe hb and et has been reported as high [ 2 , 12 ] . nonetheless , our study did not document cases of an , which could be due to the small number of children studied or to regional or ethnic considerations of the sample . the pathophysiology of snhl secondary to severe hb is not well defined , although its toxicity can affect cochlear hair cells and neurons of the basal nuclei and of the central auditory pathways . a recent report of 30 infants with hearing loss and exposure to severe hb suggests that damage to the outer hair cells of the cochlea is very common ; twenty - six infants ( 87% ) out of 30 had cochlear damage and in four cases ( 13% ) an an was documented . audiological findings in the present study using baep , otoacoustic emissions and audiometry documented bilateral severe and profound snhl with similar affection to both ears . audiometry suggests damage to inner hair cells in the cochlea with greater injury to the basal turn ( high tones ) and manifestations of loss of sensitivity to sound stimuli . abnormal results of otoacoustic emissions in our children with et suggest also damage of the outer hair cells . this locates the auditory damage at the cochlear level and specifically at both : inner and outer hair cells of the organ of corti . as a secondary result , one of the main manifestations in neurodevelopment in these children may be a delay in language acquisition . unfortunately the damage to the auditory pathway is not the only sequelae of severe hb . as we observed in our study , ogunlesi et al . reported cerebral palsy in 86.4% of 22 infants with bilirubin encephalopathy , seizures in 40.9% , and deafness in 36.4% . in our paper , we documented an increased risk of cerebral palsy and epilepsy for the snhl group . mechanisms for the alteration comprise the loss of neurons of the basal nuclei that result in motor disorders , death of cells , and formation of scars of the cerebral cortex manifested as seizures . unfortunately , children with snhl are frequently accompanied by other neurological or sensory deficits which may have a more difficult rehabilitation . the size of the sample was small , and therefore we must have caution in the interpretation of these results . in infants with hb treated with et we reported here a higher frequency of snhl and neurological comorbidity ; however , we were unable to find an . this fact merits more research in the future by our work team . these results deserve a continuous long - term pediatric followup of infants with greater number of patients . the frequency of snhl in children with a history of et treated at a 3rd level hospital in mexico city was high ( 15% ) . moreover , children with snhl and history of hb - et have an increased risk of cerebral palsy and epilepsy . thus , the early diagnosis and early intervention are very important actions for a better outcome of these patients . | the objective was to determine frequency of sensorineural hearing loss ( snhl ) , identified by abnormal threshold in evoked potentials , absence of otoacoustic emissions and behavioral responses , auditory neuropathy ( an ) ( absence of evoked potentials , with preservation of otoacoustic emissions ) , and neurological comorbidity in infants with hyperbilirubinemia ( hb ) treated with exchange - transfusion ( et ) . from a total of 7,219 infants , et was performed on 336 ( 4.6% ) .
inclusion criteria were fulfilled in 102 ; 234 children did not meet criteria ( 182 outside of the study period , 34 did not have complete audiological evaluation , and 18 rejected the followup ) .
thirty - five children ( 34% ) were born at - term and 67 ( 66% ) were preterm .
children had a mean age of 5.5 3.9 years .
main causes of et were rh isoimmunization in 48 ( 47% ) , abo incompatibility in 28 ( 27.5% ) , and multifactorial causes in 26 ( 25.5% ) .
fifteen ( 15% ) children presented with snhl .
preterm newborns presented more often with snhl .
indirect bilirubin level was higher in children with snhl ( 22.2 versus 18.7 mg / dl , p = 0.02 ) .
no cases of an were documented .
an increased risk of neurologic sequelae was observed in children with snhl . in conclusion
, we disclosed a high frequency of snhl in children with neonatal hb and et and neurological alterations .
no cases of an were observed . |
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statistical techniques are used not only by academics and clinicians directly involved in medical research but also by advocates of evidence - based medicine , who must synthesise results from many different sources to reach useful conclusions . because of this widespread use , it is important that all those involved in research or the management of patients have a sound grasp of at least the basics of statistical methods . unfortunately , in practice this is often not true , with many relying on distant memories of poorly understood lectures from undergraduate courses . in response to this , critical care is launching a series of articles aimed at providing a simple introduction and/or refresher to some of the more common tools and ideas used in medical statistics . the articles are aimed at a non - specialist audience and will keep algebra and technical language to a minimum . although some of the topics covered in this series will probably be familiar , it is hoped that there will still be useful lessons to be learned , for example the underlying assumptions of a hypothesis test that were not fully appreciated , or some previously unrecognised confusion between terms . the first article , presented in this issue , covers the presentation and summary of data . it is unlikely that the material covered by this article will be entirely new to any reader but it is included as a simple introduction to some of the ideas and philosophies that will be built upon in subsequent articles . topics to be covered in the series include : standard errors and confidence intervals ; hypothesis testing and errors ; power calculations ; measures of disease ; parametric and non - parametric tests ; simple regression ; and analysis of survival data . ideally the series will evolve to meet the needs of critical care readers , and you are encouraged to suggest additional topics that you would like to see covered in the future . it is vital that the quality of medical research continues to improve and that readers develop a critical eye when considering evidence from published reports . the conduct of badly designed , under - powered and inappropriately analysed studies is not only an indefensible waste of precious resources but is also highly unethical . unfortunately such research is all too common , and every effort should be made to prevent these situations from arising . statistical statements can enlighten or mislead depending on how well they are understood , and individuals have a responsibility to ensure that their knowledge is sufficient for their needs . it is hoped that this series will inform readers but also that it will stimulate more thought and investigation as to the most appropriate statistical methods to use and the theory and assumptions behind them . there are many useful introductory texts that cover the ideas presented in this series , and more , in considerably greater detail [ 1 - 4 ] . however , even these might frequently not be sufficient and it is vital that researchers recognise their own limitations and seek professional advice whenever it is needed , if only for reassurance . medical statistics is a scientific discipline in its own right and a medical statistician fully achieves that role only after years of training and practical experience . most academic departments , and also many clinical departments , include properly qualified medical statisticians and they should be consulted as early as possible in the research process . | statistics is increasingly used in all fields of medicine but is often poorly understood and incorrectly applied .
critical care is therefore launching a series of articles aimed at providing a simple introduction or refresher to some of the more commonly used statistical tools and ideas .
this series does not aim to be an exhaustive review of medical statistics but rather a starting point to inform readers and stimulate more thought and investigation as to the most appropriate statistical methods to use and the theory and assumptions behind them . |
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prostate - specific antigen ( psa ) is the most widely used marker for the early detection of prostate cancer ( pc ) . patients who have elevated psa levels or abnormal findings on a digital rectal examination ( dre ) but whose biopsy results are negative pose a problem in pc screening . when the result of a dre is normal and psa is lower than 10 ng / ml , prostate biopsies fail to detect pc in 80% of men . however , psa is not a cancer - specific marker , and various benign processes also affect psa concentrations . elevated psa due to benign conditions ( benign prostatic hyperplasia [ bph ] and prostatitis ) most directly underscores the difficulty in making a decision about repeat biopsy . the question remains as to the nature of the relationship between psa and subclinical prostatic inflammation . histological inflammation is a frequent finding in prostate biopsies that are performed on men without pc . several studies have investigated the relationship between psa levels and the morphology of prostatic inflammation . although it is known that acute prostatitis can contribute to lack of total psa ( tpsa ) specificity , major disagreement remains over the effect of asymptomatic inflammation on free psa ( fpsa ) and percentage of free psa ( f / tpsa ) values . it is essential to understand how factors other than pc , such as inflammation of the prostate , influence changes in psa values . we therefore investigated the correlation between the morphological parameters of inflammation in prostate biopsies and tpsa , fpsa , and f / tpsa values in patients in the so - called " gray zone " ( tpsa<10 ng / ml ) and without clinically detectable pc . this study , which was performed from november 2008 to december 2012 , included a series of 106 men with tpsa < 10 ng / ml and/or f / tpsa<18% and who had undergone prostate biopsy that was negative for pc and showed no signs of prostatitis . serum psa measurement ( immulite - dpc assay , siemens , erlangen , germany ) , dre , and transrectal ultrasound ( sonoline si 400 u / s , siemens ) were performed on each patient . transrectal ultrasonography was used to determine the prostate volume [ vp=/6 ( d1d2d3 ) ] and to guide the biopsies . each patient underwent eight - core biopsy with an 18-gauge needle fitted to a bard magnum gun . patients were also excluded from the study if they had a documented history or clinical signs of prostatitis , had psa greater than l0 ng / ml , had prostate volume measured by transrectal ultrasound ( trus ) greater than 40 cm , had previously undergone prostate surgery , had received 5-alpha - reductase inhibitors , or had an indwelling catheter or evidence of urinary tract infection ( uti ) . hematoxylin - eosin - stained slides taken from archival sections of biopsied prostatic tissue were analyzed by one pathologist . each core of prostatic tissue ( eight cores sampled from each patient ) was scored for the type of inflammation and was graded by using a 4-point scale as follows : 0 , no inflammatory cells ( fig . 1b ) ; 2 , mononuclear and polymorphonuclear cell infiltrate ( chronic active inflammation ; fig . the effects of these morphologic aspects of inflammation were then correlated with serum tpsa , fpsa , and f / tpsa values . in almost every set of biopsies , different types of histological inflammation were found . thus , we used regression factor analysis to create a reduced classification for inflammation type . factor analysis was applied according to the criteria of dimensionality reduction based on the number of derived variables . input variables were the eight variables describing type of inflammation in individual biopsy cores of the prostate by use of the previously described 4-point scale . the criterion was to obtain a derived variable , that is , one factor on which each respondent was assigned a specific factor score . instead of using four grades of inflammation for each biopsy core , we created two groups for statistical analysis . by use of regression analysis , each patient the median of the factor scores was then calculated ( itfs median , 0.053 ) . patients with factor scores lower than or equal to the median ( itfs0.053 ) were included in the " more chronic inflammation " group . similarly , those with factor scores higher than the median ( itfs>0.053 ) were included in the " more acute inflammation " group . descriptive statistics were used to characterize the age of patients , vp , tpsa , fpsa , and f / tpsa . comparisons of tpsa , fpsa , and f / tpsa values between groups were done by using mann - whitney u tests , and p<0.05 was considered statistically significant . correlations among age , vp , tpsa , fpsa , f / tpsa , and itfs were determined by using spearman 's rank correlation analysis . for correlation analysis , p<0.01 was considered statistically significant . the median ( range ) age of the patients was 65 years ( 47 to 83 years ) and the patients ' median ( range ) prostate volume ( vp ) was 31.5 cm ( 16 to 40 cm ) . the median ( range ) levels of tpsa , fpsa , and f / tpsa were 6.6 ng / ml ( 3.4 to 10 ng / ml ) , 0.9 ng / ml ( 0.14 to 3.1 ng / ml ) , and 13.45% ( 2.5% to 35% ) . all patients had a vp of less than 40 cmto minimize the effect of adenoma on psa values . after initial histological coding of each biopsy core ( eight cores per prostate biopsy ) for inflammatory type , different types of histological inflammation were found in almost every set of prostate biopsies . therefore , instead of having 4 grades of inflammation type for each biopsy core and for simplification purposes , we used regression analysis to create a reduced classification for inflammation type with two groups of patients for comparison : patients with more chronic inflammation ( n=61 , 57.5% ) and patients with more acute inflammation ( n=45 , 42.5% ) . the clinical characteristics and differences between the two groups according to grade of inflammation are shown in table 1 . the median ( range ) levels of tpsa , fpsa , and f / tpsa were 6.4 ng / ml ( 3.4 to 10 ng / ml ) , 1.09 ng / ml ( 0.28 to 3.1 ng / ml ) , and 15% ( 5.5% to 35% ) for the chronic inflammation group , and 7.3 ng / ml ( 4.1 to 10 ng / ml ) , 0.79 ng / ml ( 0.14 to 2.9 ng / ml ) , and 12% ( 2.5% to 29.6% ) for the acute inflammation group . a significant difference was found in fpsa ( p=0.003 ) and f / tpsa ( p<0.001 ) levels between groups , whereas the difference in tpsa levels was not significant ( p=0.200 ) . the fpsa and f / tpsa values were significantly decreased in the group of patients with more acute inflammation than in the patients with more chronic inflammation in the biopsy specimens . when we looked at the distribution of inflammation groups related to tpsa and f / tpsa values , patients with more acute inflammation tended to group below the horizontal line that represented the 18% f / tpsa cutoff for better discrimination between pc and benign conditions ( fig . more acute histological inflammation decreased the percentage of free psa , a tendency similar to that in pc . with the use of spearman 's analysis ( table 2 ) , there was a significant negative correlation between type of inflammation and fpsa ( r=-0.31 , p=0.001 ) and f / tpsa ( r=0.43 , p<0.001 ) psa values , in that a decrease in fpsa and f / tpsa values was found in patients with more acute inflammation . total psa values correlated significantly with fpsa ( r=0.4 , p<0.001 ) but not with type of inflammation ( r=0.12 , p>0.01 ) . as expected , no correlation emerged between vp and tpsa values ( r=0.12 , p=0.22 ) , because only patients with small prostates ( < 40 cm ) were included in the study . on the other hand , vp correlated with age ( r=0.29 , p=0.003 ) , fpsa ( r=0.32 , p=0.001 ) , and f / tpsa ( r=0.31 , p=0.001 ) . also , there was a significant negative correlation between vp and inflammation type ( r=-0.48 , p<0.001 ) . we are often confronted with the association of abnormal psa levels and biopsies that reveal no pc but only inflammation . should the abnormal psa levels be interpreted as a sign of missed pc , or can an inflammation explain the elevation of psa ? although the determination of psa is the best tool for the early detection of pc , the diagnostic accuracy of psa is limited because of limited sensitivity and specificity , particularly in men with tpsa levels up to 10 ng / ml . the ratio of free - to - total psa , calculated as percentage of fpsa , has been suggested as a useful tool for differentiating between pc and bph , because the ratio is lower in pc than in bph . it is known that prostatitis can contribute to a rise in psa , but disagreement exists about the effect of histological inflammation on psa and its fractions . recent results suggest that a decreased f / tpsa is not only characteristic of pc . it is suggested that subclinical inflammation could affect both tpsa and fpsa values . the best predictor of the serum psa in patients with bph but without pc is the size of the prostate . meyer at al . reported that the serum psa increased only in patients with benign prostates larger than 40 g. to minimize the effect of prostatic adenoma on psa values , we included only patients with vp up to 40 g. as expected , no correlation emerged between vp and tpsa values ( r=0.12 , p=0.220 ) . by contrast , we found a positive correlation between vp and fpsa values ( r=0.32 , p=0.001 ) . while some authors did not find any correlation between tpsa levels and subclinical inflammation , others found the correlation to be significant . brawer et al . found elevated tpsa levels only in patients with clinically acute prostatitis . found elevated tpsa values in 71% of patients with acute prostatitis , 15% of patients with chronic prostatitis , and 6% of patients with nonbacterial prostatitis . the results from our study agree with those of nickel et al . and kwak et al . , who found no correlation between tpsa levels and type or extent of inflammation . the present study was performed to draw attention to the effect of subclinical inflammation on fpsa and f / tpsa values . our results confirm our hypothesis and those of others that more acute histological inflammation of the prostate induces a significant decrease of fpsa and f / tpsa values , compared with more chronic inflammation , although the scattergram of f / tpsa related to tpsa values showed overlap among all data ( fig . patients with more acute inflammation tended to group below the horizontal line that represented the 18% f / tpsa cutoff for more efficient discrimination between pc and benign conditions . that cutoff was previously shown to be the limit with the highest efficiency to differentiate between bph and pc . these data emphasize that all benign prostatic diseases do not differ from pc in regard to f / tpsa . many studies confirm that it can cause a rise of tpsa , but there are major discrepancies in findings when we look at fpsa values . morote et al . studied patients without evidence of pc in trus - guided biopsies . in that study , the size of the prostate was the only significant contributor to the psa concentration . in a study performed by minardi et al . , inflammation associated with bph compared with bph only led to false - positive tpsa and f / tpsa levels , because 60% of these patients had an f / tpsa ratio below l6% . our results comply with those of jung et al . and meyer et al . , who reported reduced f / tpsa values in patients with pc and inflammation compared with controls or bph patients . on the other hand , our results differ from those of ornstein et al . , who showed that f / tpsa levels do not appear to be altered by inflammation . reported that histologically more acute inflammation induces an increase in the f / tpsa ratio , whereas chronic inflammation seems to induce lower f / tpsa ratios in patients without pc . because our results confirm that histological inflammation ( especially more acute ) is characterized by slightly elevated tpsa ( and decreased fpsa values ) , f / tpsa fails to differentiate between cancerous and noncancerous prostatic disease . it is assumed that inflammation increases psa concentrations by causing leakage of psa from acini . recent studies have demonstrated that acute , clinical inflammation produces a more intense or different process of psa release than does subclinical inflammation of the prostate . all our patients had different patterns of inflammation but had no signs of clinical prostatitis . changes in tpsa values between groups were minimal and were not statistically significant ( the acute inflammation group had slightly higher tpsa values ) , but patients with more acute histological inflammation had significantly decreased fpsa and f / tpsa values . the molecular reasons for these inflammation - related changes in the psa forms are still not defined . it is predominantly bound to l - antichymotrypsin with approximately 70% to 90% of tpsa in serum . the approximately 10% to 30% of tpsa that is not bound to proteins represents fpsa . thus , changes in f / tpsa reflect changes in fpsa or psa 1-antichymotripsin . as in pc , the changed f / tpsa in subclinical prostatitis similarly proves that psa 1-antichymotripsin is elevated compared with fpsa . . showed that 1-antichymotripsin is also synthesized in pc cells but not in bph cells . in that way , more psa is complexed in pc than in bph , leading to the changes observed in serum . similar changes can occur in prostatic inflammation , because increased 1-antichymotripsin production is a typical reaction to inflammation . another explanation was proposed by zackrisson et al . , who studied the evolution of tpsa and fpsa and their ratios during 1-year follow - up after febrile uti . most men in their study had a rise in fpsa and tpsa during the acute phase of the uti . after 1 month , fpsa rapidly decreased to the levels that were maintained during the rest of the follow - up period , but elevations of tpsa were sustained for up to 6 months after the acute phase of uti . these changes in tpsa and fpsa levels during the chronic phase of uti could falsely be interpreted as a sign of pc . it was previously shown that the fpsa molecule has a much shorter mean half - life than does tpsa . our results imply that the decreased level of serum fpsa and f / tpsa is not typical of pc alone , because these values are similarly affected by subclinical inflammation . despite the significant effect of subclinical inflammation on fpsa and f / tpsa levels , we can not deduce if the f / tpsa is a reliable discriminator between pc and prostatitis . neither clinical examination nor psa permits reliable discrimination of pc and prostatitis , especially in patients with psa levels up to 10 ng / ml . these patients should undergo prostate biopsy . on the other hand , when we find considerable inflammation in our biopsy report , but no evidence of pc , long - term antibiotic or anti - inflammatory therapy seems like a reasonable option . . showed that the use of antibiotics for 4 weeks normalized tpsa and fpsa levels in 30% of studied patients . in this way subclinical inflammation seems to have a significant influence on fpsa and f / tpsa values in patients with tpsa levels up to 10 ng / ml but without detectable pc . subclinical inflammation is not characterized by elevated tpsa concentrations alone but also by a decreased percentage of fpsa , a tendency similar to that in pc . in the assessment of biopsy results negative for pc , it might be helpful to evaluate the degree and extension of histological inflammation , especially in terms of the necessity for subsequent repeated biopsies . | purposewe are often confronted with patients in the " gray zone " ( prostate - specific antigen [ psa]<10 ng / ml ) whose biopsies reveal no malignancy but only inflammation .
we investigated the relationship between histological inflammation and total psa ( tpsa ) , free psa ( fpsa ) , and percentage of free psa ( f / tpsa ) levels in patients without prostate cancer ( pc).materials and methods we studied 106 men with tpsa<10 ng / ml who had undergone biopsy that was negative for pc and who had no clinical prostatitis .
inflammation observed at biopsies was scored for inflammation type in each biopsy core by use of a four - point scale and was then correlated with tpsa , fpsa , and f / tpsa.resultsdifferent patterns of inflammation were found in each set of biopsies .
regression factor analysis was used to form two groups according to inflammation type : more chronic and more acute .
median tpsa , fpsa , and f / tpsa levels in the more chronic and more acute inflammation groups were 6.4 ng / ml , 1.09 ng / ml , and 15% , and 7.3 ng / ml , 0.79 ng / ml , and l2% , respectively . a significant difference was found in fpsa ( p=0.003 ) and f / tpsa ( p<0.001 ) , whereas the difference in tpsa was not significant ( p=0.200 ) .
total psa correlated with fpsa ( r=0.4 , p<0.001 ) but not with inflammation type ( r=0.12 , p>0.010 ) .
a correlation existed between inflammation type and fpsa ( r=-0.31 , p=0.001 ) and f / tpsa ( r=-0.43 , p<0.001 ) in that the fpsa and f / tpsa were lower in the group with more acute inflammation.conclusionssubclinical inflammation has a significant influence on fpsa in patients with tpsa<10 ng / ml but without pc or clinical prostatitis .
subclinical inflammation is not characterized by elevated tpsa alone but also by a decreased fpsa , a tendency similar to that in pc . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the external structures of the adult drosophila head arise from a larval precursor structure known as the eye the eye antennal imaginal disc resolves into morphologically distinct eye and antennal portions during the second larval instar stage , with the anterior part fated to become the antenna , and the posterior part fated to give rise to the compound eye of the adult ; both the eye and antennal discs also contribute to the adult head capsule . during the first and second instar , the eye disc consists of undifferentiated , proliferating cells . at the onset of the third and final instar , a constriction known as the morphogenetic furrow forms at the posterior margin of the eye disc and then gradually sweeps across the eye disc toward the anterior margin . as the furrow advances , cells anterior to it undergo cell cycle arrest , followed by the onset of retinal differentiation as cells enter the furrow , . during late larval and subsequent pupal stages , cells become progressively recruited to become photoreceptors , lens - secreting cone cells , pigment cells , and bristles of the adult compound eye . the dynamic nature of retinal development is reflected in the expression pattern of sine oculis ( so ) , which is necessary for eye differentiation . antennal disc as early as the first larval instar . during the second instar , so expression is confined to the eye portion of the eye antennal disc , being strongest near the posterior margin . as the furrow progresses across the eye disc during the third instar , so continues to be expressed in a band of cells anterior to the furrow , as well as in the differentiating cells posterior to the furrow . antennal discs from wandering third instar larvae , a stage at which the majority of the cells in the eye disc have passed through the morphogenetic furrow and begun to differentiate . we used w drosophila melanogaster larvae , which are homozygous for a loss - of - function mutation in the white ( w ) gene . the w gene is required for pigmentation of the adult eye . aside from the w mutation , the larvae used were not known to have homozygous mutations in any genes affecting the eye , and the eyes of the adult flies of this strain are morphologically normal . antennal disc complexes including mouth hooks , but not brains , from wandering third instar larvae in phosphate buffered saline ( pbs ) . the antennal disc does not express so , and hence its inclusion in the chip sample would not be expected to influence the so chip - seq profile . the discs were transferred into 500 l s2 media on ice for < 30 min , fixed by adding 20.25 l 37% formaldehyde and incubating at room temperature for 15 min , and quenched with 25 l 2.5 m glycine , followed by 5 min incubation on ice . the discs were washed 3 in pbs and placed on ice . following the dissection and fixation , 200 disc pairs were combined in a 1.5 ml microcentrifuge tube with 600 l chip lysis buffer ( 50 mm k - hepes [ ph 7.8 adjusted with koh ] , 140 mm nacl , 1 mm egta , 1 mm edta , 1% triton x-100 , 0.1% na - deoxycholate ) with a mini edta - free protease tablet ( roche ) ( 1 tablet/10 ml buffer ) . the discs were ground with a nuclease - free pestle , and passed 10 through a 25-gauge needle and 10 through a 27-gauge needle . the homogenized discs were incubated 20 min at 4 c on a nutator . the resulting chromatin was sonicated with a branson digital sonifier 250 using the following settings : 15% amplitude , 15 s ( 0.9 s on/0.2 s off ) , 15 rounds , and 2 min rest between rounds . a 5 l aliquot of the chromatin was run on a 1% agarose gel in order to test successful shearing , indicated by a ~ 1.54 kb dna smear . the rest of the chromatin sample was centrifuged for 10 min , 13,200 rpm at 4 c to remove cell debris , and the supernatant from the two tubes ( containing chromatin from 200 disc pairs each ) was combined in a single siliconized tube . we set aside 10 l of the supernatant at 20 c as input sample . we precleared the remaining sample by incubating at 3 h at 4 c on a nutator with 40 l nprotein a sepharose 4 fast flow bead slurry ( ge healthcare , previously washed 3 with pbs and 1 with chip lysis buffer ) . following the incubation , the beads were removed by centrifuging for 1 min , 5000 rpm at 4 c , and the supernatant was split into two equal samples , experimental and control . 1:500 polyclonal guinea pig anti - so antibody ( gift from ilaria rebay ; ) was added to the experimental sample , and the two samples were incubated overnight at 4 c on a nutator . in parallel , 60 l washed bead slurry was incubated overnight at 4 c on a nutator in blocking solution ( 30 l 100 bovine serum albumin [ neb ] , 13 l 10 g/l denatured salmon sperm dna , 500 l chip lysis buffer ) . following the overnight incubation , the blocked beads were centrifuged to remove the supernatant , and resuspended in 30 l chip lysis buffer to make 1:1 bead : buffer slurry . 20 l bead slurry was added to each chromatin sample , followed by 34 h incubation at 4 c on a nutator . after a brief centrifugation to remove the supernatant , each bead sample was washed 3 with chip lysis buffer , 1 with high salt chip lysis buffer ( same composition as chip lysis buffer but with 500 mm nacl ) , and 1 with te ( each wash was 1 ml , for 5 min at 4 c on a nutator ) . we resuspended each bead sample in 150 l te / sds ( 10 mm tris ph 8.0 , 1 mm edta , 1% sds ) , and added 140 l te / sds to the 10 l input sample . the chromatin was eluted from the beads by a 10 min incubation in a 65 c water bath , with brief vortexing every 2 min . the samples were centrifuged for 1 min , 14,000 rpm at room temperature . the supernatant was transferred to new tubes that were sealed with parafilm and incubated 5 h to overnight in a 65 c water bath to reverse crosslinks . we extracted the dna with a qiaquick pcr purification kit ( qiagen ) , eluting the dna in 30 l elution buffer ( 10 mm tris the purified dna was tested using qpcr with primers flanking a previously identified so - binding site 3 of the atonal gene ( 3ato ) , as well as control primers flanking a site not expected to bind so in the eye . if qpcr showed > 4-fold enrichment in the experimental ( + anti - so ) sample relative to the negative control ( anti - so ) sample , we proceeded with illumina library preparation following manufacturer 's instructions . briefly , ~ 10 ng of dna was end - repaired using polynucleotide kinase and klenow . the 5 ends of the dna fragments were phosphorylated and a single adenine base was added to the 3 ends using klenow exonuclease . illumina y - shaped index adaptors were ligated to the repaired ends , and the dna fragments were pcr amplified for 21 cycles . the libraries were quantified using the picogreen fluorescence assay and their size distributions were determined by the agilent 2100 bioanalyzer . the library was tested again by qpcr to ensure > 4-fold enrichment prior to sequencing . the libraries of two biological replicates were sequenced using the illumina genome analyzer iix and a total of 21.9 million 35-bp single - end reads were generated , including 12.4 million from the first replicate and 9.5 million from the second replicate . in order to maximize our power in downstream data analysis , the reads from two biological replicates were combined , and the combined reads were mapped to the d. melanogaster reference genome ( dm5 ) using eland software . approximately 4.74 million reads were mapped to the dm5 genome . among them , about 3.4 million reads were uniquely mapped . there were a total of over 19 million reads for control sample . among them , 6.2 million reads were mapped to dm5 genome and 5.7 million reads were unique . peaks were called from the mapped reads using model - based analysis of chip - seq ( macs ) . as default settings , peaks with less than 3-fold enrichment or with p > 10 were filtered out . a total of 7566 peaks were then obtained and annotated using an in - house bioinformatics tool ( a perl script , available upon request ) . most ( 84.7% ) of the peaks fully or partially overlap an annotated drosophila gene , with 52.4% of all peaks being < 1 kb from an annotated transcription start site ( tss ) . the external structures of the adult drosophila head arise from a larval precursor structure known as the eye the eye antennal imaginal disc resolves into morphologically distinct eye and antennal portions during the second larval instar stage , with the anterior part fated to become the antenna , and the posterior part fated to give rise to the compound eye of the adult ; both the eye and antennal discs also contribute to the adult head capsule . during the first and second instar , the eye disc consists of undifferentiated , proliferating cells . at the onset of the third and final instar , a constriction known as the morphogenetic furrow forms at the posterior margin of the eye disc and then gradually sweeps across the eye disc toward the anterior margin . as the furrow advances , cells anterior to it undergo cell cycle arrest , followed by the onset of retinal differentiation as cells enter the furrow , . during late larval and subsequent pupal stages , cells become progressively recruited to become photoreceptors , lens - secreting cone cells , pigment cells , and bristles of the adult compound eye . the dynamic nature of retinal development is reflected in the expression pattern of sine oculis ( so ) , which is necessary for eye differentiation . antennal disc as early as the first larval instar . during the second instar , so expression is confined to the eye portion of the eye antennal disc , being strongest near the posterior margin . as the furrow progresses across the eye disc during the third instar , so continues to be expressed in a band of cells anterior to the furrow , as well as in the differentiating cells posterior to the furrow . antennal discs from wandering third instar larvae , a stage at which the majority of the cells in the eye disc have passed through the morphogenetic furrow and begun to differentiate . we used w drosophila melanogaster larvae , which are homozygous for a loss - of - function mutation in the white ( w ) gene . the w gene is required for pigmentation of the adult eye . aside from the w mutation , the larvae used were not known to have homozygous mutations in any genes affecting the eye , and the eyes of the adult flies of this strain are morphologically normal . antennal disc complexes including mouth hooks , but not brains , from wandering third instar larvae in phosphate buffered saline ( pbs ) . the antennal disc does not express so , and hence its inclusion in the chip sample would not be expected to influence the so chip - seq profile . the discs were transferred into 500 l s2 media on ice for < 30 min , fixed by adding 20.25 l 37% formaldehyde and incubating at room temperature for 15 min , and quenched with 25 l 2.5 m glycine , followed by 5 min incubation on ice . the discs were washed 3 in pbs and placed on ice . following the dissection and fixation , 200 disc pairs were combined in a 1.5 ml microcentrifuge tube with 600 l chip lysis buffer ( 50 mm k - hepes [ ph 7.8 adjusted with koh ] , 140 mm nacl , 1 mm egta , 1 mm edta , 1% triton x-100 , 0.1% na - deoxycholate ) with a mini edta - free protease tablet ( roche ) ( 1 tablet/10 ml buffer ) . the discs were ground with a nuclease - free pestle , and passed 10 through a 25-gauge needle and 10 through a 27-gauge needle . the homogenized discs were incubated 20 min at 4 c on a nutator . the resulting chromatin was sonicated with a branson digital sonifier 250 using the following settings : 15% amplitude , 15 s ( 0.9 s on/0.2 s off ) , 15 rounds , and 2 min rest between rounds . a 5 l aliquot of the chromatin was run on a 1% agarose gel in order to test successful shearing , indicated by a ~ 1.54 kb dna smear . the rest of the chromatin sample was centrifuged for 10 min , 13,200 rpm at 4 c to remove cell debris , and the supernatant from the two tubes ( containing chromatin from 200 disc pairs each ) was combined in a single siliconized tube . we set aside 10 l of the supernatant at 20 c as input sample . we precleared the remaining sample by incubating at 3 h at 4 c on a nutator with 40 l nprotein a sepharose 4 fast flow bead slurry ( ge healthcare , previously washed 3 with pbs and 1 with chip lysis buffer ) . following the incubation , the beads were removed by centrifuging for 1 min , 5000 rpm at 4 c , and the supernatant was split into two equal samples , experimental and control . 1:500 polyclonal guinea pig anti - so antibody ( gift from ilaria rebay ; ) was added to the experimental sample , and the two samples were incubated overnight at 4 c on a nutator . in parallel , 60 l washed bead slurry was incubated overnight at 4 c on a nutator in blocking solution ( 30 l 100 bovine serum albumin [ neb ] , 13 l 10 g/l denatured salmon sperm dna , 500 l chip lysis buffer ) . following the overnight incubation , the blocked beads were centrifuged to remove the supernatant , and resuspended in 30 l chip lysis buffer to make 1:1 bead : buffer slurry . 20 l bead slurry was added to each chromatin sample , followed by 34 h incubation at 4 c on a nutator . after a brief centrifugation to remove the supernatant , each bead sample was washed 3 with chip lysis buffer , 1 with high salt chip lysis buffer ( same composition as chip lysis buffer but with 500 mm nacl ) , and 1 with te ( each wash was 1 ml , for 5 min at 4 c on a nutator ) . we resuspended each bead sample in 150 l te / sds ( 10 mm tris ph 8.0 , 1 mm edta , 1% sds ) , and added 140 l te / sds to the 10 l input sample . the chromatin was eluted from the beads by a 10 min incubation in a 65 c water bath , with brief vortexing every 2 min . the samples were centrifuged for 1 min , 14,000 rpm at room temperature . the supernatant was transferred to new tubes that were sealed with parafilm and incubated 5 h to overnight in a 65 c water bath to reverse crosslinks . we extracted the dna with a qiaquick pcr purification kit ( qiagen ) , eluting the dna in 30 l elution buffer ( 10 mm tris the purified dna was tested using qpcr with primers flanking a previously identified so - binding site 3 of the atonal gene ( 3ato ) , as well as control primers flanking a site not expected to bind so in the eye . if qpcr showed > 4-fold enrichment in the experimental ( + anti - so ) sample relative to the negative control ( anti - so ) sample , we proceeded with illumina library preparation following manufacturer 's instructions . briefly , ~ 10 ng of dna was end - repaired using polynucleotide kinase and klenow . the 5 ends of the dna fragments were phosphorylated and a single adenine base was added to the 3 ends using klenow exonuclease . illumina y - shaped index adaptors were ligated to the repaired ends , and the dna fragments were pcr amplified for 21 cycles . the libraries were quantified using the picogreen fluorescence assay and their size distributions were determined by the agilent 2100 bioanalyzer . the library was tested again by qpcr to ensure > 4-fold enrichment prior to sequencing . the libraries of two biological replicates were sequenced using the illumina genome analyzer iix and a total of 21.9 million 35-bp single - end reads were generated , including 12.4 million from the first replicate and 9.5 million from the second replicate . in order to maximize our power in downstream data analysis , the reads from two biological replicates were combined , and the combined reads were mapped to the d. melanogaster reference genome ( dm5 ) using eland software . approximately 4.74 million reads were mapped to the dm5 genome . among them , about 3.4 million reads were uniquely mapped . there were a total of over 19 million reads for control sample . among them , 6.2 million reads were mapped to dm5 genome and 5.7 million reads were unique . peaks were called from the mapped reads using model - based analysis of chip - seq ( macs ) . as default settings , peaks with less than 3-fold enrichment or with p > 10 were filtered out . a total of 7566 peaks were then obtained and annotated using an in - house bioinformatics tool ( a perl script , available upon request ) . most ( 84.7% ) of the peaks fully or partially overlap an annotated drosophila gene , with 52.4% of all peaks being < 1 kb from an annotated transcription start site ( tss ) . the so transcription factor is a necessary regulator of drosophila eye development , and its homologs have been implicated in cancer and developmental disorders in human patients , , , , . we have recently presented a genome - wide profile of so binding to chromatin in developing drosophila eye discs . our data set shows so dna - binding enrichment at enhancers previously shown to require so - mediated regulation in the developing eye , , , , , as well as so binding to or near genes that function in multiple aspects of eye development . the data suggest that a broad spectrum of genes may be regulated by so during eye development and is expected to expand our understanding of the genetic basis of eye formation . | the eye of the fruit fly drosophila melanogaster provides a highly tractable genetic model system for the study of animal development , and many genes that regulate drosophila eye formation have homologs implicated in human development and disease . among these
is the homeobox gene sine oculis ( so ) , which encodes a homeodomain transcription factor ( tf ) that is both necessary for eye development and sufficient to reprogram a subset of cells outside the normal eye field toward an eye fate .
we have performed a genome - wide analysis of so binding to dna prepared from developing drosophila eye tissue in order to identify candidate direct targets of so - mediated transcriptional regulation , as described in our recent article [ 20 ] .
the data are available from ncbi gene expression omnibus ( geo ) with the accession number gse52943 . here
we describe the methods , data analysis , and quality control of our so chip - seq dataset . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the initial management of the neck in early oral squamous cell carcinoma ( oscc ) with clinically negative neck nodes ( clinical t stage [ ct ] 1 or 2 and clinical n stage [ cn ] 0 ) remains a controversy . before the 1990s , the traditional policy for management of the clinically negative neck in early oscc was generally a wait - and - see or observation ( obs ) policy , unless the neck was being opened for other reasons such as better access to the primary tumor , reconstruction requirements , or delaying neck dissection until cervical metastases was clinically evident . the incidence of occult regional lymph node metastasis of oscc varies from 6% to 46% , according to previous reports1 . once regional metastases have occurred , the 5-year survival rate for patients with oral cancer decreases by one - half relative to that of patients with early - stage disease23 . in view of the high incidence of nodal recurrence of the observed neck , the results of the few prospective randomized studies and a retrospective study on the benefit of prophylactic neck treatment have been inconclusive . the studies failed to find statistically significant differences in prognoses between the groups of patients under obs without initial neck dissection and those groups initially managed with neck dissection567 . the aim of this study was to retrospectively review and compare the outcomes between patients under obs and patients who underwent elective neck dissection ( end ) at initial surgery and to identify factors that affect locoregional control and survival . this retrospective study included 215 oscc patients who underwent surgical treatment at department of oral and maxillofacial surgery , yonsei university dental hospital ( seoul , korea ) from 1990 to 2012 , based on a screening of medical records . this study was approved by the regional ethical review board of yonsei university dental hospital institutional review board ( irb no . 2 - 2014 - 0032 ) . patients with recurred tumor , second primary tumor , metastatic tumor , and patients who underwent salvage surgery were excluded . patients with positive neck nodes on physical exam or imaging studies , including computed tomography ( ct ) , magnetic resonance imaging , positron emission tomography ( pet ) , pet - ct , and ultrasonography , were excluded . after the selection process , 79 patients were suitable for analysis . among them , 52 patients underwent end and 27 patients did not receive neck dissection and underwent obs only . following the policy of most institutions , obs was applied when there was no evidence of neck node metastasis , and end was applied to patients with suspicion of neck node metastasis despite negative results . in the obs group , occult cervical metastasis was defined as a neck recurrence during follow - up , without failure at the primary site89 . cervical metastases in patients with recurrent primary tumor were not considered occult metastases because the nodal spread may have occurred after the initial treatment89 . in the end group , occult disease was defined as the presence of microscopic disease on the histopathologic examination of neck dissection specimens89 . survival curves were plotted using the kaplan - meier method and compared using the log - rank test . for multivariable analyses to find independently related factors of recurrence and survival , the cox proportional hazard model was used . all statistical analyses were performed with pasw statistics software version 18.0 ( ibm co. , armonk , ny , usa ) . the patients were divided into two groups : obs ( n=27 , 34.2% ) and end ( n=52 , 65.8% ) . the median age of the entire group was 59 years . the obs period varied from 1.4 months to 285.6 months with a mean of 87.3 months . regarding the site of primary lesion , 46 ( 58.2% ) were from tongue , 13 ( 16.5% ) from buccal cheek , 8 ( 10.1% ) from floor of mouth , 6 ( 7.6% ) from mandibular alveolar gingiva , 5 ( 6.3% ) from retromolar trigone , and 1 ( 1.3% ) was from maxillary alveolar gingiva . the stages were ct1 and ct2 in 17 patients ( 63.0% ) and 10 patients ( 37.0% ) in the obs group , respectively , and 20 patients ( 38.5% ) and 32 patients ( 61.5% ) in the end group . in total , there were 37 patients ( 46.8% ) with ct1 stage and 42 patients ( 53.2% ) with ct2 stage . forty - two cases ( 42/52 , 80.8% ) were pathologically confirmed as negative for neck node metastasis ( pn0 ) . among the remaining 10 cases ( 10/52 , 19.2% ) , 8 ( 15.4% ) were pn1 , and 2 ( 3.8% ) were pn2 . forty - one patients ( 78.8% ) underwent ipsilateral selective neck dissection ( snd ) . six patients ( 11.5% ) underwent bilateral snd , and 5 patients ( 9.6% ) underwent modified radical neck dissection . the end group showed a statistically significant benefit in disease - free survival ( p=0.027 ; fig . 2 ) however , there was no statistically significant difference in regional recurrence - free survival or cancer - specific survival between obs and end groups . occult cervical nodal metastases were found in 13 cases ( 13/79 , 16.5% ) , including neck recurrence in 3 cases of the obs group ( 3/27 , 11.1% ) and pathologically positive metastases in 10 cases of the end group ( 10/52 , 19.2% ) . in univariable analysis , occult metastasis was related to higher odds of cancer - specific death ( odds ratio [ or]=6.25 , p<0.01 ) . to determine the factors related to overall recurrence , we performed univariable and multivariable analyses . in univariable analyses , the end group was correlated with lower overall recurrence ( or=0.27 , p=0.006).(table 2 ) cancer - specific death was related to higher odds of overall recurrence ( or=11.61 , p<0.01).(table 2 ) in multivariable analysis , poor histologic grade was independently related to overall recurrence ( or=9.65 , p<0.01).(table 3 ) in a multivariable analysis , advanced age ( or=6.3 , p=0.022 ) , higher clinical t stage ( or=15.2 , p=0.01 ) , and poorly differentiated histologic grade ( or=6.6 , p=0.025 ) were independently related to cancer - specific death.(table 4 ) the patients were divided into two groups : obs ( n=27 , 34.2% ) and end ( n=52 , 65.8% ) . the median age of the entire group was 59 years . the obs period varied from 1.4 months to 285.6 months with a mean of 87.3 months . regarding the site of primary lesion , 46 ( 58.2% ) were from tongue , 13 ( 16.5% ) from buccal cheek , 8 ( 10.1% ) from floor of mouth , 6 ( 7.6% ) from mandibular alveolar gingiva , 5 ( 6.3% ) from retromolar trigone , and 1 ( 1.3% ) was from maxillary alveolar gingiva . the stages were ct1 and ct2 in 17 patients ( 63.0% ) and 10 patients ( 37.0% ) in the obs group , respectively , and 20 patients ( 38.5% ) and 32 patients ( 61.5% ) in the end group . in total , there were 37 patients ( 46.8% ) with ct1 stage and 42 patients ( 53.2% ) with ct2 stage . forty - two cases ( 42/52 , 80.8% ) were pathologically confirmed as negative for neck node metastasis ( pn0 ) . among the remaining 10 cases ( 10/52 , 19.2% ) , 8 ( 15.4% ) were pn1 , and 2 ( 3.8% ) were pn2 . forty - one patients ( 78.8% ) underwent ipsilateral selective neck dissection ( snd ) . six patients ( 11.5% ) underwent bilateral snd , and 5 patients ( 9.6% ) underwent modified radical neck dissection . the end group showed a statistically significant benefit in disease - free survival ( p=0.027 ; fig . 2 ) however , there was no statistically significant difference in regional recurrence - free survival or cancer - specific survival between obs and end groups . occult cervical nodal metastases were found in 13 cases ( 13/79 , 16.5% ) , including neck recurrence in 3 cases of the obs group ( 3/27 , 11.1% ) and pathologically positive metastases in 10 cases of the end group ( 10/52 , 19.2% ) . in univariable analysis , occult metastasis was related to higher odds of cancer - specific death ( odds ratio [ or]=6.25 , p<0.01 ) . to determine the factors related to overall recurrence , we performed univariable and multivariable analyses . in univariable analyses , the end group was correlated with lower overall recurrence ( or=0.27 , p=0.006).(table 2 ) cancer - specific death was related to higher odds of overall recurrence ( or=11.61 , p<0.01).(table 2 ) in multivariable analysis , poor histologic grade was independently related to overall recurrence ( or=9.65 , p<0.01).(table 3 ) in a multivariable analysis , advanced age ( or=6.3 , p=0.022 ) , higher clinical t stage ( or=15.2 , p=0.01 ) , and poorly differentiated histologic grade ( or=6.6 , p=0.025 ) were independently related to cancer - specific death.(table 4 ) the presence of regional neck metastases is widely accepted as a major determinant of prognosis in patients with oscc1112 . if the probability of neck metastases is high , a neck dissection will decrease the risk of regional recurrence . however , if the probability of neck metastases is low , neck dissection constitutes overtreatment , where the morbidity of the neck procedure only decreases quality of life while increasing functional deficits . while the problem could be solved if it were possible to predict the risk of neck metastases , such prediction has been difficult to introduce and apply in clinical practice . finally , there is little published guidance about management of the n0 neck in oscc13 . patients with ct1n0 and ct2n0 oscc have been reported to have occult metastases in 13% to 33% and 37% to 53% of cases , respectively , at the time of diagnosis14 . in this study , the rates for ct1n0 and ct2n0 in this study were 8.1% ( 3/37 ) and 23.8% ( 10/42 ) , respectively . though the occult metastasis rate is relatively low in our study , the tendency toward comparably higher incidence of occult metastasis in t2 compared to t1 , as well as ipsilateral neck being the common site of recurrence , seem congruent with results from previous studies9 . an interesting result of our study is that the end group was related to lower recurrence , but did not seem related to better survival . of course , we should carefully interpret these results since this study was a retrospective analysis and not a double - blind prospective randomized control trial , so a selection bias ( i.e. , predilection of end for advanced t stages ) may have influenced the results . most studies have focused on the rate of occult metastasis and related factors to demonstrate the necessity of end . our results tentatively suggest that end may lower the rate of recurrence , but not necessarily improve survival , and that the characteristics of the tumor itself , such as clinical t stage and poor histologic grade , may be important for survival . these results may be helpful when determining the necessity of end for early stage oscc . previous studies have suggested other factors that influence final outcome and survival of cancer treatment . eicher et al.15 recommended end for patients with moderately or poorly differentiated scc , radiological or histological signs of bony invasion , and tumors in the mandibular symphyseal region16 . ogura et al.17 examined mandibular bony invasion using dental ct and found it unfavorable as a prognostic indicator of 5-year survival . feng et al.11 reported that the follow - up compliance of patient populations was the vital factor in adopting the obs strategy for the cn0 neck . they stated that early detection of regional recurrence led to a 100% cervical salvage rate irrespective of t stage , the salvage rate otherwise dropping as remarkably low as < 30.0%11 . they concluded that end should be recommended as first - line management for all intermediate and advanced stage patients , with the exception of patients with stage t1 tumors , who have a low risk of nodal metastasis and for whom obs may be an acceptable alternative to end if the patients strictly comply with a cancer surveillance protocol11 . for better survival of early oscc patients without clinically evident neck node metastasis , characteristics of the tumor itself , such as advanced t stage and poor histologic grade , may be equally or more important than the treatment modality of the neck . the follow - up compliance of the patients must be guaranteed to adopt the obs strategy for the cn0 neck . | objectivesto evaluate the results of elective neck dissection versus those of observation in the treatment of early stage oral squamous cell carcinoma and to identify factors related to recurrence and survival.materials and methodsthis was a retrospective study of 52 patients who underwent elective neck dissection and 27 who did not receive neck dissection.resultsin survival analyses , elective neck dissection showed a benefit in overall recurrence ( p=0.027 ) , especially in stage i patients ( p=0.024 ) . with regard to survival , the benefit was statistically insignificant ( p=0.990 ) . in multivariable analysis , overall recurrence was independently related to poor histologic grade ( odds ratio [ or]=9.65 , p=0.006 ) , and cancer - specific death was independently related to advanced age ( or=6.3 , p=0.022 ) , higher clinical t stage ( or=15.2 , p=0.01 ) , and poorly differentiated histologic grade ( or=6.6 , p=0.025).conclusionthough there was lower recurrence in the elective neck dissection group , there were no statistically significant results on survival .
the characteristics of the tumor itself , such as clinical t stage and poor histologic grade , may be more important in cancer - specific survival . |
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cancer is a systemic disease that directly affects the region of onset and can metastasize to other sites , causing a variety of complications and loss of progressive organ function . the development of the disease may be initially slow or rapidly evolving , unavoidably affecting nutritional status . malnutrition is a possible complication in patients with cancer and can be the first symptom to reveal the presence of the disease . even before starting anticancer treatment , patients can experience profound metabolic and physiological alterations with increased needs of macro- and micronutrients . data on the prevalence of malnutrition vary broadly depending on the evaluation criteria such as tumor type , site , and extension , as well as anticancer treatment . the prevalence of malnutrition among cancer patients has been estimated to range between 15% and 80% ; the main symptoms embrace weight loss and asthenia of different degrees . malnutrition can negatively affect the clinical decision to resect the tumor , which is the main and potentially curative step in the management of cancer . indeed , malnutrition can increase the incidence of postoperative complications , such as delayed wound healing , dehiscence of anastomosis , morbidity , and mortality . medical treatment of cancer patients usually focuses on the administration of cytotoxic agents and/or radiation therapy . these tools can potentially eradicate or reduce tumor size , but may have several toxic side effects that in turn can also weaken the patient , particularly by decreasing appetite or inducing nausea and vomiting , fatigue , and asthenia . when malnutrition establishes , it can be necessary to reduce the dose of cytotoxic agents and/or modify the radiation timing between temporary or definite cessation of treatment . direct associations have been reported between the necessity to stop or delay anticancer treatment and a reduction in the time of remission , in overall survival and in global response rates to radio / chemotherapy . malnutrition impairs the immune status and reduces the body s defense against infectious diseases . in light of these possible complications , malnutrition represents a poor prognostic factor and , as such , should be prevented or detected as early as possible . there are limited data in the literature on the effectiveness of nutritional screening and early treatment of malnutrition [ 8 , 9 ] . an interdisciplinary approach ( oncologist , nutritionist , nurse , dietitian , physical therapist , psychologist , etc . ) is necessary for patients who are experiencing loss of physiological or biological function , fatigue , malnutrition , psychological distress , and other symptoms as a result of cancer disease or its treatment . our manuscript aims to enhance attention of professional healthcare providers and to encourage further studies into this topic . weight loss in cancer patients is due to diverse factors , among them the production of inflammatory and catabolic mediators playing important roles . such markers include acute - phase proteins , interleukin-6 ( il-6 ) , or the ubiquitine proteasome complex whose activity may be increased by the neoplasm itself , particularly in some rapidly growing tumors such as those of the pancreas or the lungs . the neoplastic mass can represent a possible mechanical obstruction to the way in the digestive tract , causing dysphagia and impaired swallowing ( head - neck , esophageal cancer , or mediastinic masses ) , early satiety , nausea , vomiting ( gastric and small bowel tumors ) , abdominal pain for intestinal sub - occlusion or occlusion ( small and large bowel , peritoneal carcinomatosis ) . moreover , the tumor can interfere with organ function , for example , causing diarrhea ( pancreatic and biliary cancer ) as a result of the lack of digestive enzymes [ 1 , 11 , 12 ] . the presence of continuous or occasional pain during eating and digestion may represent another important factor limiting , quantitatively and qualitatively , oral intake [ 6 , 13 ] . furthermore , following surgery for cancer removal , gastrointestinal changes can affect the digestive processes causing , for example , early satiety , dumping syndrome ( gastric surgery ) , or diarrhea ( pancreatic and colonic resection ) . finally , anticancer treatments ( chemotherapy and radiation therapy ) can cause anorexia , early satiety , nausea , vomiting , oral and intestinal mucositis with dysphagia , diarrhea , hemorroids , anal fissures , and modifications in smell and taste senses . all these symptoms may affect food choices and contribute to inadequate meal intake and reduced quality of life [ 1 , 8 ] . in some cases , co - administered medications , taken to control symptoms or to treat adverse effects associated with anticancer treatments , the nutritional and inflammatory status appears to be correlated with an increased risk of severe hematological toxicity following anticancer chemotherapy . moreover , chemotherapy - induced dna damage might become more severe in normal tissues in the presence of alterations of the cellular immune response due to high protein catabolism and stimulation of the acute - phase response . malnutrition can influence the outcome of chemotherapy , radiation , and surgery for cancer due to changes in metabolism , pharmacokinetics , and healing dynamics . moreover , malnutrition could be responsible for alterations in absorption , protein binding , hepatic metabolism , and renal elimination of drugs and their metabolites [ 15 , 16 ] . in malnourished patients , a reduced concentration of plasma proteins may significantly increase the likelihood of toxicity by agents with high protein binding , such as prednisolone , etoposide , cisplatinum , paclitaxel , and irinotecan metabolites . malnutrition decreases the oxidative metabolism in the liver performed by cytochrome p-450 isoenzymes through a depletion of nicotinamide adenine dinucleotide phosphate reserves . other liver metabolic pathways can also be impaired , decreasing clearance and thus prolonging drug half - life . the effect of malnutrition on renal function is less clear , but measurements of inulin clearance in malnourished children showed a reduced glomerular filtration rate . the various complications caused by cancer can affect patients quality of life in many domains : physical ( pain , sleep disturbances , loss of appetite , fatigue , activities of daily living ) , social , psychological ( depression , anxiety ) , and work - related [ 17 , 18 ] . together with the nutritional intervention , a full and appropriate assessment of the patient s needs must be performed . a baseline assessment also highlights current physical and emotional problems that the patient may be experiencing [ 17 , 18 ] . the nutritional and inflammatory status appears to be correlated with an increased risk of severe hematological toxicity following anticancer chemotherapy . moreover , chemotherapy - induced dna damage might become more severe in normal tissues in the presence of alterations of the cellular immune response due to high protein catabolism and stimulation of the acute - phase response . malnutrition can influence the outcome of chemotherapy , radiation , and surgery for cancer due to changes in metabolism , pharmacokinetics , and healing dynamics . moreover , malnutrition could be responsible for alterations in absorption , protein binding , hepatic metabolism , and renal elimination of drugs and their metabolites [ 15 , 16 ] . in malnourished patients , a reduced concentration of plasma proteins may significantly increase the likelihood of toxicity by agents with high protein binding , such as prednisolone , etoposide , cisplatinum , paclitaxel , and irinotecan metabolites . malnutrition decreases the oxidative metabolism in the liver performed by cytochrome p-450 isoenzymes through a depletion of nicotinamide adenine dinucleotide phosphate reserves . other liver metabolic pathways can also be impaired , decreasing clearance and thus prolonging drug half - life . the effect of malnutrition on renal function is less clear , but measurements of inulin clearance in malnourished children showed a reduced glomerular filtration rate . the various complications caused by cancer can affect patients quality of life in many domains : physical ( pain , sleep disturbances , loss of appetite , fatigue , activities of daily living ) , social , psychological ( depression , anxiety ) , and work - related [ 17 , 18 ] . depression is a known factor in the pathophysiology of anorexia and other nutritional troubles . together with the nutritional intervention , a full and appropriate assessment of the patient s needs must be performed . a baseline assessment also highlights current physical and emotional problems that the patient may be experiencing [ 17 , 18 ] . malnutrition is a clinical condition of imbalance of energy , protein , and other nutrients which causes measurable adverse effects on tissue / body composition , function , and clinical outcome [ 20 , 21 ] . anorexia and reduced food intake are frequently neglected in patients with cancer and contribute to nutritional impairment . cachexia is a multifactorial syndrome characterized by severe body weight , fat and muscle loss , and increased protein catabolism due to underlying disease(s ) as cancer , aids , chronic obstructive lung disease , and congestive heart failure . it results from a complex interplay between underlying disease , disease - related metabolic alterations , and reduced availability of nutrients ( reduced intake , impaired absorption , and/or increased losses ) . once established , cachexia can not be reversed , neither nutritionally nor with other treatments , and increases patients morbidity and mortality . contributory factors to the onset of cachexia are anorexia and metabolic alterations , i.e. , inflammatory status , increased muscle proteolysis , impaired carbohydrate , and protein and lipid metabolism . inflammation plays a pivotal role in the pathogenesis of cachexia : an imbalance between pro - inflammatory ( e.g. , tumor necrosis factor- [ tnf- ] , il-1 , il-6 , interferon- [ ifn- ] ) and anti - inflammatory ( e.g. , il-4 , il-12 , il-15 ) cytokines is currently considered to contribute to cachexia development and progression . pre - cachexia immediately precedes cachexia and can be diagnosed in the presence of all the following criteria : ( a ) underlying chronic disease ; ( b ) unintentional weight loss > = 5% of usual body weight during the last 6 months ; ( c ) chronic or recurrent systemic inflammatory response ; and ( d ) anorexia or anorexia - related symptoms . progressive loss of skeletal muscle mass is the most clinically relevant phenotypic feature of cachexia and causes negative clinical consequences on muscle strength , respiratory function , functional status , and quality of life . muscle loss , with increased muscle protein degradation results from an imbalance between anabolic and catabolic rates . systemic inflammation is one of the main actors together with tumor - derived factors such as proteolysis - inducing factor , bed rest , and inadequate nutrient intake [ 24 , 25 ] . nutritional screening aims to identify patients at risk of malnutrition in a simple and non - invasive way . a full evaluation ( nutritional assessment ) to confirm and classify the degree of malnutrition will follow . these two steps allow to early identify and treat patients with malnutrition or periodically follow - up those at high nutritional risk , to face any nutritional challenges before significant weight loss or before other clinical / biological signs of malnutrition appear [ 26 , 27 ] . due to time restrictions by healthcare professionals , practical organization and costs , nutritional screening has necessarily to be feasible , speedy , inexpensive , and non - invasive . the full evaluation of the patients should include data collection about :
social demography ( gender , age , professional status , and living conditions)primary tumor ( site , disease stage at diagnosis ) and the management of the disease ( previous and on - going treatments)anthropometry ( actual weight , body mass index [ bmi , the weight in kilograms divided by the height in centimeters squared ] , usual healthy body weight , unintentional weight loss since the start of the illness , or appearance of the first symptoms ; weight loss during the last week , the last month , and over the last 6 months;clinical examination , subjective global assessment and karnofsky index ( evaluating functional capacity ) ; biochemical data : serum albumin , prealbumin , total lymphocyte count cholesterol , c reactive protein ( crp ) , pseudocholinesterase ( pche ) [ 3032 ] . social demography ( gender , age , professional status , and living conditions ) primary tumor ( site , disease stage at diagnosis ) and the management of the disease ( previous and on - going treatments ) anthropometry ( actual weight , body mass index [ bmi , the weight in kilograms divided by the height in centimeters squared ] , usual healthy body weight , unintentional weight loss since the start of the illness , or appearance of the first symptoms ; weight loss during the last week , the last month , and over the last 6 months ; clinical examination , subjective global assessment and karnofsky index ( evaluating functional capacity ) ; biochemical data : serum albumin , prealbumin , total lymphocyte count cholesterol , c reactive protein ( crp ) , pseudocholinesterase ( pche ) [ 3032 ] . careful clinical history record and meticulous symptom assessment are the first steps to become sympathetic with the patient and to understand his / her conditions in more detail . another major step is the compilation , with the help of a dietitian , of a detailed food record in the days between chemotherapy cycles ( when patients are feeling better ) and the days of treatment ; the compilation of questionnaires about patients daily activities ( ability to work , social life , autonomy in personal hygiene , time spent in bed ) should also be recommended . a nutritional screening should be performed at the time of the diagnosis , possibly before starting the specific anticancer treatments . therefore , for patients at risk of malnutrition , the clinical nutritionist evaluates the patient s conditions and jointly with the dietitian , gives strategic advices on dietary intakes and choices , according to the scheduled treatment . if the patient is already malnourished or at high risk of malnutrition , a nutritional support should be promptly prescribed . independent of whether patient is or is not malnourished , periodical follow - up should be established and timed on the schedules of the oncologic therapies ; an appropriate follow - up should be performed every 2 or 3 weeks [ 3034 ] . bmi alone is not a sensitive parameter to analyze nutritional status because it may still yield high or normal results in patients with ascites or edema . unintentional weight loss could instead be the best single parameter for detecting malnutrition [ 30 , 35 ] . serum albumin level is the most widely used clinical nutritional index , but for its relatively long half - life and correlation with stress and illness , it remains a non - specific parameter of nutritional status . indeed , pro - inflammatory tumor - derived mechanisms influence the acute - phase protein response in the liver and thus albumin synthesis . acute - phase reactants comprise a range of proteins that rapidly change in plasma concentration responding to inflammation or tissue injury . positive acute - phase proteins , such as crp , directly rise with inflammatory disease activity . negative acute - phase proteins ( albumin , prealbumin , and transferrin ) respond inversely : their levels decrease in response to injury and inflammation and increase during recovery from the same conditions . decreased serum levels of albumin , prealbumin , and transferrin do not necessarily reflect a state of malnutrition , but could be a consequence of the body s physiologic response to injury . under these conditions , resolution of inflammation , and not exogenous substrate ( protein and energy ) from nutritional support , restores normal hepatic protein metabolism and , eventually , serum levels of negative acute - phase proteins . recently , the role of plasma pche as nutritional index has been described : its levels were found to be decreased in malnourished patients with or without hepatic involvement . one of the possible mechanisms responsible for pche activity reduction in cancer patients could be anorexia , which accompanies malignancy , as happens in anorexia nervosa too . bozzetti et al . observed that pche , body weight , and nitrogen balance improved during nutritional support in cancer patients . to adequately assess nutritional status in patients with cancer and in order to avoid possible interferences by inflammation and tumor - derived factors , not a single parameter but a cluster of two or more parameters should be considered [ 12 , 35 ] . bmi alone is not a sensitive parameter to analyze nutritional status because it may still yield high or normal results in patients with ascites or edema . unintentional weight loss could instead be the best single parameter for detecting malnutrition [ 30 , 35 ] . serum albumin level is the most widely used clinical nutritional index , but for its relatively long half - life and correlation with stress and illness , it remains a non - specific parameter of nutritional status . indeed , pro - inflammatory tumor - derived mechanisms influence the acute - phase protein response in the liver and thus albumin synthesis . acute - phase reactants comprise a range of proteins that rapidly change in plasma concentration responding to inflammation or tissue injury . positive acute - phase proteins , such as crp , directly rise with inflammatory disease activity . negative acute - phase proteins ( albumin , prealbumin , and transferrin ) respond inversely : their levels decrease in response to injury and inflammation and increase during recovery from the same conditions . decreased serum levels of albumin , prealbumin , and transferrin do not necessarily reflect a state of malnutrition , but could be a consequence of the body s physiologic response to injury . under these conditions , resolution of inflammation , and not exogenous substrate ( protein and energy ) from nutritional support , restores normal hepatic protein metabolism and , eventually , serum levels of negative acute - phase proteins . recently , the role of plasma pche as nutritional index has been described : its levels were found to be decreased in malnourished patients with or without hepatic involvement . one of the possible mechanisms responsible for pche activity reduction in cancer patients could be anorexia , which accompanies malignancy , as happens in anorexia nervosa too . observed that pche , body weight , and nitrogen balance improved during nutritional support in cancer patients . to adequately assess nutritional status in patients with cancer and in order to avoid possible interferences by inflammation and tumor - derived factors , not a single parameter but a cluster of two or more parameters should be considered [ 12 , 35 ] . research in clinical oncology has focused mainly on defining the best practice to evaluate anticancer treatment cytotoxic drugs , radiation , and surgical procedures as well as to clarify the traditional endpoints of disease - free and overall survival . malnutrition is associated with negative outcomes including increased morbidity , poor prognosis and tolerance to treatment , decreased quality of life , and increased health care costs . patients with or at risk of malnutrition should receive the most appropriate nutritional support ( oral supplements or enteral / parenteral support ; fig . 1 ) . furthermore , such patients should be followed - up during the evolution of the disease . for each single patient , for the specific type of cancer and for the involved body areas , all variables that could negatively affect the nutritional status picc : peripherally inserted central venous catheter . en : enteral nutrition algorithm for nutritional evaluation . picc : peripherally inserted central venous catheter . en : enteral nutrition the nutritional support has to be personalized according to the nutritional status of the single patient , to the toxicity of the respective patient s therapy , and to the influence of symptoms on the daily eating requirements . for example , in case of nausea and vomiting in the 57 days following chemotherapy with strong impairment of eating in already malnourished patients , parenteral nutritional support can be considered . under the same conditions , for a well - nourished patient , a hydrating head / neck cancer patients generally experience a considerable weight loss and malnutrition for dysphagia , mucositis , and xerostomy during and following radiation therapy . reported risk factors associated with serious weight loss are : baseline karnofsky performance status less than 80 , combination with chemotherapy , and receiving a total dose of radiation of 60 gray or more . treatment toxicity may be exacerbated by poor nutritional status before and during therapy and may impair recovery time because of the effects of malnutrition on wound healing . malnutrition may compromise treatment efficacy and reduce quality of life , possibly even affect survival [ 4 , 7 ] . in patients with head / neck cancer on radiotherapy or combined chemo- and radiotherapy , weight loss generally commences in the second week of therapy . considering that the gastrointestinal tract distal of the tumor and the treatment field is usually functional , enteral rather than parenteral feeding yields safer and more physiological results . the positioning of a nasogastric tube ( ngt ) for enteral nutrition can be advisable when a temporary dysphagia is anticipated ( during and soon after radiation therapy ) . on the other hand , at stage 3 or 4 of the disease , the prophylactic placement of a percutaneous gastrostomy ( peg ) for early enteral nutrition can be a reasonable approach . while ngt positioning has generally been recommended when nutritional support is required for a short period of time ( less than 1 month ) , peg tubes are the better choice for prolonged nutritional support . in malnourished patients who undergo radiotherapy as single or concomitant therapy for gastrointestinal neoplasms , abdominal pain , nausea , vomiting , or diarrhea may not permit an adequate oral support and intestinal absorption . therefore , a parenteral nutritional therapy through peripheral veins or preferably through a pre - existing central venous catheter could be necessary . in patients requiring abdominal radiation , these patients need specific dietary recommendations , such as semiliquid , hyperproteic , fiber - free , or lactose - free diet [ 43 , 44 ] . dietary supplements and alternative foods have to be discussed and prescribed [ 45 , 46 ] . also , patients with a regular oral intake need to be followed - up closely to prevent or diagnose and treat malnutrition as early as possible . during inflammatory processes , tissue depletion results in protein and fat loss as a result of the actions of pro - inflammatory cytokines . besides the inflammatory condition of cancer , chemotherapy increases oxidative stress , thereby providing a further boost to the inflammatory process . hypercaloric / hyperproteic oral supplements could be indicated , due to the hypercatabolism induced by the tumor and chemotherapy [ 47 , 48 ] . many formulas are now disposable , with high or low fat content , enriched with anti - inflammatory and antioxidant agents . among nutrients acting on the inflammatory process eicosapentaenoic acid ( epa ) is an n-3 , 21-carbon atom , polyunsaturated fatty acid , with five double bonds , found in oily fish . it seems to attenuate weight loss , particularly loss of skeletal muscle mass by down - regulating the increased expression and activity of the ubiquitin since epa has no effects on protein synthesis in muscle , epa has been combined with nutritional supplements rich in protein and energy , such as branched chain amino acids . fearon s group has pioneered the use of fish oil in the treatment of pancreatic cancer , reporting the ability of n-3 pufa , in particular , epa and docosahexaenoic acid to reduce weight loss rate in such patients . glutamine acts as a source of glutamate and may provide one of the three amino acids ( glycine , cysteine , and glutamate ) required for the synthesis of the key antioxidant glutathione , indirectly reducing inflammatory stress in the patients . antioxidants represent one of the largest categories of dietary supplements , which could be protective against the adverse effects of chemotherapy . severely malnourished patients with decreased dietary carnitine uptake may develop carnitine deficiency when treated with cisplatin , as the drug increases renal carnitine excretion approximately tenfold . trace elements consist mostly of metal ions acting mainly as basic components of essential enzymatic systems or proteins , which play major roles in the physiology of the gastrointestinal tract . some studies suggest that trace elements serve as co - factors in several metabolic pathways and a decrease in their concentration may facilitate the malnutrition process . zinc regulates the function of cytochromes , stabilizes plasma membranes , reduces lipid peroxidation , and has a role in the detoxification of ammonia . supplementation of these trace elements can delay cachexia onset with its subsequent depression of the immune system , influencing the neoplastic process and the effect of chemotherapy [ 51 , 52 ] . early preventive and treatment attempts have thus suggested the use of appetite stimulants . the most widely prescribed is megestrol acetate , a synthetic progestin , which may stimulate appetite via neuropeptide y in the ventromedial hypothalamus and by down - regulating the synthesis and release of pro - inflammatory cytokines . a systematic review of 15 randomized clinical trials of high - dose progestin therapy has showed a statistically significant improvement in both appetite and body weight . however , body composition analysis of patients who gained weight showed that weight gain was due to increased fat and not lean body mass . the inability of these two progestins to increase lean body mass would explain why patients show no significant improvement in the karnovsky index ( performance score ) or in quality of life . moreover , attention should be paid on the prescription of progestins for the increased risk of thrombo - embolic phenomena and edema . thus , cyproheptadine , a histamine antagonist with antiserotonergic and appetite - stimulating effects , produces only a slight improvement in appetite and does not significantly prevent progressive weight loss in anorectic cancer patients . corticosteroids such as dexamethasone , prednisolone , and methylprednisolone are used to enhance appetite , sensation of well - being and performance , usually at the end - stage of cancer , because of their catabolic effect on skeletal muscle . however , despite possible improvement in quality of life , they have no other beneficial effects . several neuropeptides regulate appetite and are currently under evaluation to assess their efficacy in the treatment of cancer anorexia / cachexia . among these is ghrelin , a neuropeptide released from the stomach in response to fasting that stimulates food intake . the cytotoxic effects of chemotherapy and radiation therapy can cause an inflammatory response of mucosal epithelial cells called mucositis . all mucous membrane - covered surfaces , from mouth to rectum , may be affected . oral mucositis disrupts the function and integrity of the oral cavity , affecting a satisfying oral food intake and negatively influencing treatment outcomes and quality of life . it is associated with significant clinical morbidity , which may include pain , dysphagia , malnutrition , and local and systemic infections . the largest damage at the oral mucosa is seen in patients with head / neck cancer treated with a combination of radio- and chemotherapy . although present throughout the gastrointestinal tract , mucositis in the oral cavity has been better characterized in the literature because of the ease of assessment . oral care is widely considered the basis of mucosal health , integrity , and function . however , the specific components , methods , and frequency of this treatment remain in debate , partly because of the ethical considerations of withholding oral care in clinical trials . in physiological conditions , the human saliva has lubricating properties and antibacterial / antiviral actions ; disturbances of the salivary flow alter the protective role of oral microflora . oral care protocols could help to minimize the effects of oral mucositis in patients receiving treatment for cancer : it can reduce the amount of microbial flora , control pain and bleeding , and prevent infection . good oral health also reduces the risk of gingival and dental complications [ 58 , 59 ] . few data are available on the use of sodium - bicarbonate rinsing to alkalinize oral cavity , followed by the prophylactic use of an antiseptic ( chlorexidine - based ) and/or antifungine ( nystatin , itraconazole ) mouth washes . also doubtful is the benefit of using local hydrating agents containing jaluronic acid and other cicatrizing agents . research in clinical oncology has focused mainly on defining the best practice to evaluate anticancer treatment cytotoxic drugs , radiation , and surgical procedures as well as to clarify the traditional endpoints of disease - free and overall survival . malnutrition is associated with negative outcomes including increased morbidity , poor prognosis and tolerance to treatment , decreased quality of life , and increased health care costs . patients with or at risk of malnutrition should receive the most appropriate nutritional support ( oral supplements or enteral / parenteral support ; fig . 1 ) . furthermore , such patients should be followed - up during the evolution of the disease . for each single patient , for the specific type of cancer and for the involved body areas , all variables that could negatively affect the nutritional status picc : peripherally inserted central venous catheter . en : enteral nutrition algorithm for nutritional evaluation . picc : peripherally inserted central venous catheter . en : enteral nutrition the nutritional support has to be personalized according to the nutritional status of the single patient , to the toxicity of the respective patient s therapy , and to the influence of symptoms on the daily eating requirements . for example , in case of nausea and vomiting in the 57 days following chemotherapy with strong impairment of eating in already malnourished patients , parenteral nutritional support can be considered . under the same conditions , for a well - nourished patient , a hydrating head / neck cancer patients generally experience a considerable weight loss and malnutrition for dysphagia , mucositis , and xerostomy during and following radiation therapy . reported risk factors associated with serious weight loss are : baseline karnofsky performance status less than 80 , combination with chemotherapy , and receiving a total dose of radiation of 60 gray or more . treatment toxicity may be exacerbated by poor nutritional status before and during therapy and may impair recovery time because of the effects of malnutrition on wound healing . malnutrition may compromise treatment efficacy and reduce quality of life , possibly even affect survival [ 4 , 7 ] . in patients with head / neck cancer on radiotherapy or combined chemo- and radiotherapy , weight loss generally commences in the second week of therapy . considering that the gastrointestinal tract distal of the tumor and the treatment field is usually functional , enteral rather than parenteral feeding yields safer and more physiological results . the positioning of a nasogastric tube ( ngt ) for enteral nutrition can be advisable when a temporary dysphagia is anticipated ( during and soon after radiation therapy ) . on the other hand , at stage 3 or 4 of the disease , the prophylactic placement of a percutaneous gastrostomy ( peg ) for early enteral nutrition can be a reasonable approach . while ngt positioning has generally been recommended when nutritional support is required for a short period of time ( less than 1 month ) , peg tubes are the better choice for prolonged nutritional support . in malnourished patients who undergo radiotherapy as single or concomitant therapy for gastrointestinal neoplasms , abdominal pain , nausea , vomiting , or diarrhea may not permit an adequate oral support and intestinal absorption . therefore , a parenteral nutritional therapy through peripheral veins or preferably through a pre - existing central venous catheter could be necessary . in patients requiring abdominal radiation , these patients need specific dietary recommendations , such as semiliquid , hyperproteic , fiber - free , or lactose - free diet [ 43 , 44 ] . dietary supplements and alternative foods have to be discussed and prescribed [ 45 , 46 ] . also , patients with a regular oral intake need to be followed - up closely to prevent or diagnose and treat malnutrition as early as possible . during inflammatory processes , tissue depletion results in protein and fat loss as a result of the actions of pro - inflammatory cytokines . besides the inflammatory condition of cancer , chemotherapy increases oxidative stress , thereby providing a further boost to the inflammatory process . hypercaloric / hyperproteic oral supplements could be indicated , due to the hypercatabolism induced by the tumor and chemotherapy [ 47 , 48 ] . many formulas are now disposable , with high or low fat content , enriched with anti - inflammatory and antioxidant agents . among nutrients acting on the inflammatory process eicosapentaenoic acid ( epa ) is an n-3 , 21-carbon atom , polyunsaturated fatty acid , with five double bonds , found in oily fish . it seems to attenuate weight loss , particularly loss of skeletal muscle mass by down - regulating the increased expression and activity of the ubiquitin since epa has no effects on protein synthesis in muscle , epa has been combined with nutritional supplements rich in protein and energy , such as branched chain amino acids . fearon s group has pioneered the use of fish oil in the treatment of pancreatic cancer , reporting the ability of n-3 pufa , in particular , epa and docosahexaenoic acid to reduce weight loss rate in such patients . glutamine acts as a source of glutamate and may provide one of the three amino acids ( glycine , cysteine , and glutamate ) required for the synthesis of the key antioxidant glutathione , indirectly reducing inflammatory stress in the patients . antioxidants represent one of the largest categories of dietary supplements , which could be protective against the adverse effects of chemotherapy . severely malnourished patients with decreased dietary carnitine uptake may develop carnitine deficiency when treated with cisplatin , as the drug increases renal carnitine excretion approximately tenfold . trace elements consist mostly of metal ions acting mainly as basic components of essential enzymatic systems or proteins , which play major roles in the physiology of the gastrointestinal tract . some studies suggest that trace elements serve as co - factors in several metabolic pathways and a decrease in their concentration may facilitate the malnutrition process . zinc regulates the function of cytochromes , stabilizes plasma membranes , reduces lipid peroxidation , and has a role in the detoxification of ammonia . supplementation of these trace elements can delay cachexia onset with its subsequent depression of the immune system , influencing the neoplastic process and the effect of chemotherapy [ 51 , 52 ] . early preventive and treatment attempts have thus suggested the use of appetite stimulants . the most widely prescribed is megestrol acetate , a synthetic progestin , which may stimulate appetite via neuropeptide y in the ventromedial hypothalamus and by down - regulating the synthesis and release of pro - inflammatory cytokines . a systematic review of 15 randomized clinical trials of high - dose progestin therapy has showed a statistically significant improvement in both appetite and body weight . however , body composition analysis of patients who gained weight showed that weight gain was due to increased fat and not lean body mass . the inability of these two progestins to increase lean body mass would explain why patients show no significant improvement in the karnovsky index ( performance score ) or in quality of life . moreover , attention should be paid on the prescription of progestins for the increased risk of thrombo - embolic phenomena and edema . thus , cyproheptadine , a histamine antagonist with antiserotonergic and appetite - stimulating effects , produces only a slight improvement in appetite and does not significantly prevent progressive weight loss in anorectic cancer patients . corticosteroids such as dexamethasone , prednisolone , and methylprednisolone are used to enhance appetite , sensation of well - being and performance , usually at the end - stage of cancer , because of their catabolic effect on skeletal muscle . however , despite possible improvement in quality of life , they have no other beneficial effects . several neuropeptides regulate appetite and are currently under evaluation to assess their efficacy in the treatment of cancer anorexia / cachexia . among these is ghrelin , a neuropeptide released from the stomach in response to fasting that stimulates food intake . the cytotoxic effects of chemotherapy and radiation therapy can cause an inflammatory response of mucosal epithelial cells called mucositis . all mucous membrane - covered surfaces , from mouth to rectum , may be affected . oral mucositis disrupts the function and integrity of the oral cavity , affecting a satisfying oral food intake and negatively influencing treatment outcomes and quality of life . it is associated with significant clinical morbidity , which may include pain , dysphagia , malnutrition , and local and systemic infections . the largest damage at the oral mucosa is seen in patients with head / neck cancer treated with a combination of radio- and chemotherapy . although present throughout the gastrointestinal tract , mucositis in the oral cavity has been better characterized in the literature because of the ease of assessment . oral care is widely considered the basis of mucosal health , integrity , and function . however , the specific components , methods , and frequency of this treatment remain in debate , partly because of the ethical considerations of withholding oral care in clinical trials . in physiological conditions , the human saliva has lubricating properties and antibacterial / antiviral actions ; disturbances of the salivary flow alter the protective role of oral microflora . oral care protocols could help to minimize the effects of oral mucositis in patients receiving treatment for cancer : it can reduce the amount of microbial flora , control pain and bleeding , and prevent infection . good oral health also reduces the risk of gingival and dental complications [ 58 , 59 ] . few data are available on the use of sodium - bicarbonate rinsing to alkalinize oral cavity , followed by the prophylactic use of an antiseptic ( chlorexidine - based ) and/or antifungine ( nystatin , itraconazole ) mouth washes . also doubtful is the benefit of using local hydrating agents containing jaluronic acid and other cicatrizing agents . the clinical evaluation of patients with cancer has to include nutritional assessment [ 1 , 28 ] . depending on the needs of the patient and his / her family , members of the rehabilitation team may include any or all involved physicians , oncology nurses , dietitians , physiotherapists , and psychologists . the team has to address the potential rehabilitation needs of the individual from cancer diagnosis to rehabilitation , covering the following objectives : pain - killer therapy , nutritional and psychosocial support , and optimization of physical and social functioning [ 2 , 8 ] . the implementation of a nutritional surveillance could enable rapid treatment of symptoms ( anorexia , dysphagia , nausea , vomiting , constipation , tiredness , etc . ) , which could have a role in the malnutrition process . the first step of cancer rehabilitation is the primary support to the negative effects of cancer disease and its therapy [ 68 ] . oncologists have to be encouraged to share their work with other team specialists : anesthesiologists for pain therapy , nutritionists for nutritional support , and physical and rehabilitation medicine specialists . all these figures have to cooperate to symptoms management of cancer patients [ 32 , 33 ] . it is necessary to achieve early treatment of malnutrition to avoid a gradual progression to cachexia , an irreversible condition . patients have to be treated when an effective intervention is still possible , before cachexia manifests . it will be meaningless and unethical to treat the irreversible phase of cachexia , when the patient is almost at the end of life [ 61 , 62 ] . in that condition malnutrition and above all cachexia have to be prevented and/or followed - up from the first diagnosis of cancer . evaluating data reported in the literature , it appears that few patients receive early nutritional counseling and nutritional issues are often underestimated in the diagnostic and therapeutic course of cancer rehabilitation . this seems to be related to excessive workload and insufficient knowledge / skill among healthcare professionals expected to take on the task of screening . it is necessary to make all healthcare professionals aware of the opportunity to screen cancer patients and identify those at risk of malnutrition . it is a duty of any physician and the patients right to improve preventive actions and intervene early during cancer treatment and progression of the disease [ 4 , 6 ] . | backgroundmalnutrition is a frequent complication in patients with cancer and can negatively affect the outcome of treatments . on the other hand , side effects of anticancer therapies can also lead to inadequate nutrient intake and subsequent malnutrition .
the nutritional screening aims to identify patients at risk of malnutrition for prompt treatment and/or careful follow-up.methods and resultsthis manuscript highlights the need of an interdisciplinary approach ( oncologist , nutritionist , dietitian , psychologist , etc . ) to empower patients who are experiencing loss of physiological and biological function , fatigue , malnutrition , psychological distress , etc .
, as a result of cancer disease or its treatment , and maintain an acceptable quality of life.conclusionsit is necessary to make all healthcare professionals aware of the opportunity to identify cancer patients at risk of malnutrition early in order to plan the best possible intervention and follow - up during cancer treatment and progression . |
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the use of laparoscopic gastrectomy has recently increased , and its indications have expanded to proximal and total gastrectomy . however , laparoscopic gastrointestinal anastomosis involves a high degree of difficulty and complexity . in gastrointestinal surgery , careful intestinal anastomosis particularly in cases of esophagojejunal anastomosis is important , as anastomotic leakage may occasionally be fatal . stapled anastomosis is a fundamental step in all these methods , although it involves a hand - sewn technique . thus , anastomotic methods are not commonly used because of the cumbersome laparoscopic intracorporeal sutures and tying involved . however , various devices have been developed to simplify the placement of intracorporeal sutures , and barbed suture is one such device . v - loc 180 ( covidien , mansfield , ma , usa ) is a new device for wound closure , and consists of a unidirectional barbed absorbable thread . evenly spaced barbs have high tissue - adhesion ability , as they attach to tissue at numerous points . moreover , the loop - end design avoids the need for tying a surgical knot ( figure 1 ) . clinical efficacy and suitability of barbed sutures have been reported in dermal closure and orthopedic surgery , and their intracorporeal use has also been indicated in the fields of urology and gynecology . the effective length , comprising the barbed portion , is approximately 12 cm , and no barbs are present in a length of 12 cm from the loop - end . some reports have described the use of barbed sutures for digestive anastomosis , including gastric bypass surgery in obese patients . however , only a few have reported on the use of barbed sutures for reconstruction after radical gastrectomy in patients with cancer or for esophagojejunal anastomosis . although single - layer entire - thickness running sutures are convenient and very cost - effective , several reports describe the use of double - layer running sutures to ensure safety . herein , we describe the surgical procedure for intracorporeal single - layer entire - thickness running suturing using the v - loc 180 device in cases involving esophagojejunal anastomosis and report on the short - term surgical outcomes . the use of laparoscopic gastrectomy has recently increased , and its indications have expanded to proximal and total gastrectomy . however , laparoscopic gastrointestinal anastomosis involves a high degree of difficulty and complexity . in gastrointestinal surgery , careful intestinal anastomosis particularly in cases of esophagojejunal anastomosis is important , as anastomotic leakage may occasionally be fatal . stapled anastomosis is a fundamental step in all these methods , although it involves a hand - sewn technique . thus , anastomotic methods are not commonly used because of the cumbersome laparoscopic intracorporeal sutures and tying involved . however , various devices have been developed to simplify the placement of intracorporeal sutures , and barbed suture is one such device . v - loc 180 ( covidien , mansfield , ma , usa ) is a new device for wound closure , and consists of a unidirectional barbed absorbable thread . evenly spaced barbs have high tissue - adhesion ability , as they attach to tissue at numerous points . moreover , the loop - end design avoids the need for tying a surgical knot ( figure 1 ) . clinical efficacy and suitability of barbed sutures have been reported in dermal closure and orthopedic surgery , and their intracorporeal use has also been indicated in the fields of urology and gynecology . the effective length , comprising the barbed portion , is approximately 12 cm , and no barbs are present in a length of 12 cm from the loop - end . some reports have described the use of barbed sutures for digestive anastomosis , including gastric bypass surgery in obese patients . however , only a few have reported on the use of barbed sutures for reconstruction after radical gastrectomy in patients with cancer or for esophagojejunal anastomosis . although single - layer entire - thickness running sutures are convenient and very cost - effective , several reports describe the use of double - layer running sutures to ensure safety . herein , we describe the surgical procedure for intracorporeal single - layer entire - thickness running suturing using the v - loc 180 device in cases involving esophagojejunal anastomosis and report on the short - term surgical outcomes . in total , 38 patients ( 25 men and 13 women ) underwent laparoscopic intracorporeal suturing with the v - loc 180 device ( vlocl0604 ; taper point , 1/2 circle/26 mm ; size , 3 - 0 ; length , 15 cm ; covidien ) at toyama prefectural hospital between august 2012 and march 2014 . two patients who underwent proximal gastrectomy with intracorporeal esophagojejunal and gastrojejunal anastomoses were also included . the basic indication for laparoscopic gastrectomy in our department is ct1n0m0 cancer , and d1 + is the standard dissection range , based on the japanese classification of gastric carcinoma ( fourth edition ) and japanese gastric cancer treatment guidelines . the diagnosis was made based on the findings of preoperative examinations , including gastrointestinal endoscopy , upper gastrointestinal series , and abdominal computed tomography . the data were retrospectively collected from medical records , operative reports , and histopathologic reports . patient characteristics ( age , sex , body mass index , indications , and operating procedures ) , surgery - related factors ( operation time , blood loss , transition to laparotomy , intracorporeal suturing time , and additional suture ) , and operative factors ( postoperative duration of hospital stay and intra-/postoperative complications ) were analyzed . the clavien dindo classification was used to categorize the postoperative complications ; cases categorized as grade ii and higher were regarded as having complications . since 2012 , laparoscopic total gastrectomy ( ltg ) , laparoscopic proximal gastrectomy ( lpg ) , and laparoscopy and endoscopy cooperative surgery ( lecs ) have been introduced and are performed based on the primary disease and localization of the tumor . for these procedures , the patient is placed in a reverse trendelenburg position under general anesthesia . the first port is placed through the umbilicus by using an open method . a laparoscope is inserted via the umbilical port and 4 operating ports ( bilateral subcostal and bilateral midabdominal ) . , the duodenum is divided with a regular 60-mm linear stapler , with a camel cartridge ( tri - staple ; covidien ) , before the dissection of lymph node ( ln ) stations 7 , 8a , and 9 . after full mobilization of the stomach and completion of nodal dissection , the esophagogastric junction is divided with a regular 60-mm linear stapler , with a purple cartridge ( tri - staple ; covidien ) . thereafter , roux - en - y reconstruction is performed , including an esophagojejunal anastomosis according to the overlap method , with a 45-mm linear stapler , with a purple cartridge ( tri - staple ; covidien ) . single - layer entire - thickness running suturing with v - loc 180 ( covidien ) was used to close the intestinal hole . furthermore , a side - to - side jejunojejunostomy was performed , including transumbilical minilaparotomy ( usually 4 cm ) using a 45-mm linear stapler , with a camel cartridge ( tri - staple ; covidien ) , and hand - sewn sutures ( 3 - 0 vicryl ; ethicon , somerville , nj , usa ) . for lpg , the esophagus and stomach were divided with a 60-mm linear stapler , with a purple cartridge ( tri - staple ; covidien ) . thereafter , double - tract reconstruction was performed , and the jejunojejunal anastomosis and esophagojejunal anastomosis were performed in the same manner as for ltg . with regard to gastrojejunostomy , the decision on whether the anastomosis should be made transumbilically or intracorporeally was left to the surgeon 's discretion . a side - to - side gastrojejunostomy was performed with a 45-mm linear stapler , with a purple cartridge ( tri - staple ; covidien ) . only intracorporeal suturing with v - loc 180 ( covidien ) was used . the mesentery defect was closed using 3 - 0 vicryl sutures ( ethicon , somerville , nj , usa ) , whereas petersen 's defect was not closed in the ltg and lpg cases . for lecs , after the gastric wall was devascularized laparoscopically , we penetrated the entire stomach layer endoscopically . single - layer entire - thickness running suturing with the v - loc 180 ( covidien ) device was used to close the gastric wall defect . the air leak test , performed with a nasal gastric tube , was used in all cases . gastrointestinal hole closures were roughly divided into 2 groups : stapler insertion hole closure and gastric wall defect closure . single - layer entire - thickness running sutures were placed by using the following method in all cases . the first step in intestinal hole closure is to pass a needle through a loop ( figures 2a , 3a ) . the first stitch may become loose , as there are no barbs adjacent to the loop . therefore , at the start of the closure site , 1 or 2 additional bites may be needed ( figures 2b , 3b ) . thus , single - layer entire - thickness running sutures are placed , which tightens at each bite ( figure 2c , 3c ) . at the end of the closure site , 1 or 2 seromuscular bites are added after completing the intestinal hole closure , following which the suture is cut flush to the bowel without the necessity for tying a knot ( figures 2d , 3d ) . in cases with a broad suturing pitch , additional knotted sutures are made by using 3 - 0 monocryl ( ethicon ) based on the surgeon 's discretion . schematic outline of intestinal hole closure in esophagojejunostomy using v - loc 180 for single - layer entire - thickness running suturing . ( a ) after side - to - side anastomosis , intestinal hole closure is initiated by passing the needle through a loop . ( b ) at the start of the closure site , 1 or 2 bites are added before the edge of the intestinal hole is sutured . ( c ) single - layer entire - thickness running suturing is performed , which tightens at each bite . ( d ) at the end of the closure site , 1 or 2 seromuscular bites are added after the completion of intestinal hole closure , and the suture is cut flush at the bowel , without the need for a knot . ( a ) initiation of intestinal hole closure using v - loc . by barely attaching the loop to the intestinal wall , ( b ) one or 2 additional bites are needed before the edge of the intestinal hole is sutured ( white arrowheads ) . ( c ) single - layer entire - thickness running sutures are placed , which tighten at each bite . ( d ) one or 2 seromuscular bites are added after the completion of intestinal hole closure ( white arrowheads ) , and the suture is cut flush to the bowel without a knot . an abdominal drain , bladder catheter , and nasal gastric tube were routinely placed . moreover , a postoperative radiographic contrast study was performed in all the patients . a few sips of water , a semifluid diet , and a soft blended diet were given to patients on postoperative days 3 , 4 , and 6 , respectively . on the first and third postoperative days , the amylase levels in the drain fluid were measured for the detection of a postoperative pancreatic fistula . in total , 38 patients ( 25 men and 13 women ) underwent laparoscopic intracorporeal suturing with the v - loc 180 device ( vlocl0604 ; taper point , 1/2 circle/26 mm ; size , 3 - 0 ; length , 15 cm ; covidien ) at toyama prefectural hospital between august 2012 and march 2014 . two patients who underwent proximal gastrectomy with intracorporeal esophagojejunal and gastrojejunal anastomoses were also included . the basic indication for laparoscopic gastrectomy in our department is ct1n0m0 cancer , and d1 + is the standard dissection range , based on the japanese classification of gastric carcinoma ( fourth edition ) and japanese gastric cancer treatment guidelines . the diagnosis was made based on the findings of preoperative examinations , including gastrointestinal endoscopy , upper gastrointestinal series , and abdominal computed tomography . the data were retrospectively collected from medical records , operative reports , and histopathologic reports . patient characteristics ( age , sex , body mass index , indications , and operating procedures ) , surgery - related factors ( operation time , blood loss , transition to laparotomy , intracorporeal suturing time , and additional suture ) , and operative factors ( postoperative duration of hospital stay and intra-/postoperative complications ) were analyzed . the clavien dindo classification was used to categorize the postoperative complications ; cases categorized as grade ii and higher were regarded as having complications . since 2012 , laparoscopic total gastrectomy ( ltg ) , laparoscopic proximal gastrectomy ( lpg ) , and laparoscopy and endoscopy cooperative surgery ( lecs ) have been introduced and are performed based on the primary disease and localization of the tumor . for these procedures , the patient is placed in a reverse trendelenburg position under general anesthesia . the first port is placed through the umbilicus by using an open method . a laparoscope is inserted via the umbilical port and 4 operating ports ( bilateral subcostal and bilateral midabdominal ) . , the duodenum is divided with a regular 60-mm linear stapler , with a camel cartridge ( tri - staple ; covidien ) , before the dissection of lymph node ( ln ) stations 7 , 8a , and 9 . after full mobilization of the stomach and completion of nodal dissection , the esophagogastric junction is divided with a regular 60-mm linear stapler , with a purple cartridge ( tri - staple ; covidien ) . thereafter , roux - en - y reconstruction is performed , including an esophagojejunal anastomosis according to the overlap method , with a 45-mm linear stapler , with a purple cartridge ( tri - staple ; covidien ) . single - layer entire - thickness running suturing with v - loc 180 ( covidien ) was used to close the intestinal hole . furthermore , a side - to - side jejunojejunostomy was performed , including transumbilical minilaparotomy ( usually 4 cm ) using a 45-mm linear stapler , with a camel cartridge ( tri - staple ; covidien ) , and hand - sewn sutures ( 3 - 0 vicryl ; ethicon , somerville , nj , usa ) . for lpg , the esophagus and stomach were divided with a 60-mm linear stapler , with a purple cartridge ( tri - staple ; covidien ) . thereafter , double - tract reconstruction was performed , and the jejunojejunal anastomosis and esophagojejunal anastomosis were performed in the same manner as for ltg . with regard to gastrojejunostomy , the decision on whether the anastomosis should be made transumbilically or intracorporeally was left to the surgeon 's discretion . a side - to - side gastrojejunostomy was performed with a 45-mm linear stapler , with a purple cartridge ( tri - staple ; covidien ) . only intracorporeal suturing with v - loc 180 ( covidien ) the mesentery defect was closed using 3 - 0 vicryl sutures ( ethicon , somerville , nj , usa ) , whereas petersen 's defect was not closed in the ltg and lpg cases . for lecs , after the gastric wall was devascularized laparoscopically , we penetrated the entire stomach layer endoscopically . single - layer entire - thickness running suturing with the v - loc 180 ( covidien ) device was used to close the gastric wall defect . the air leak test , performed with a nasal gastric tube , was used in all cases . gastrointestinal hole closures were roughly divided into 2 groups : stapler insertion hole closure and gastric wall defect closure . single - layer entire - thickness running sutures were placed by using the following method in all cases . the first step in intestinal hole closure is to pass a needle through a loop ( figures 2a , 3a ) . the first stitch may become loose , as there are no barbs adjacent to the loop . therefore , at the start of the closure site , 1 or 2 additional bites may be needed ( figures 2b , 3b ) . thus , single - layer entire - thickness running sutures are placed , which tightens at each bite ( figure 2c , 3c ) . at the end of the closure site , 1 or 2 seromuscular bites are added after completing the intestinal hole closure , following which the suture is cut flush to the bowel without the necessity for tying a knot ( figures 2d , 3d ) . in cases with a broad suturing pitch , additional knotted sutures are made by using 3 - 0 monocryl ( ethicon ) based on the surgeon 's discretion . schematic outline of intestinal hole closure in esophagojejunostomy using v - loc 180 for single - layer entire - thickness running suturing . ( a ) after side - to - side anastomosis , intestinal hole closure is initiated by passing the needle through a loop . ( b ) at the start of the closure site , 1 or 2 bites are added before the edge of the intestinal hole is sutured . ( c ) single - layer entire - thickness running suturing is performed , which tightens at each bite . ( d ) at the end of the closure site , 1 or 2 seromuscular bites are added after the completion of intestinal hole closure , and the suture is cut flush at the bowel , without the need for a knot . ( a ) initiation of intestinal hole closure using v - loc . by barely attaching the loop to the intestinal wall , ( b ) one or 2 additional bites are needed before the edge of the intestinal hole is sutured ( white arrowheads ) . ( c ) single - layer entire - thickness running sutures are placed , which tighten at each bite . ( d ) one or 2 seromuscular bites are added after the completion of intestinal hole closure ( white arrowheads ) , and the suture is cut flush to the bowel without a knot . an abdominal drain , bladder catheter , and nasal gastric tube were routinely placed . moreover , a postoperative radiographic contrast study was performed in all the patients . a few sips of water , a semifluid diet , and a soft blended diet were given to patients on postoperative days 3 , 4 , and 6 , respectively . after the patient consumed the semifluid diet , the abdominal drainage tube was removed . on the first and third postoperative days , the amylase levels in the drain fluid were measured for the detection of a postoperative pancreatic fistula . these patients had a mean age of 68.8 11.8 y ( range , 3788 ) and a body mass index of 22.2 11.8 ( range , 15.028.1 ) . of those , 31 patients had gastric cancer ( 28 with early cancer and 3 with pyloric stenosis ) , and 7 had gastric submucosal tumor . in total , 12 underwent ltg with roux - en - y reconstruction , 2 underwent laparoscopic distal gastrectomy with roux - en y reconstruction , 14 underwent lpg with double - tract reconstructions , 3 underwent gastrojejunal bypass , and 7 underwent partial gastrectomy . the anastomoses performed included esophagojejunostomies in 26 , gastrojejunostomies in 7 , and simple closures of gastric defect in 7 ( table 1 ) . of the 33 cases with esophagojejunostomies and gastrojejunostomies , additional knotted sutures were used in 13 . background of the patients bmi , body mass index ; tg , total gastrectomy ; dg , distal gastrectomy ; pg , proximal gastrectomy . the mean operative time for gastrectomy was 236 min ( range , 155340 ) , and the estimated blood loss was 59 ml ( range , 0440 ) . for partial gastrectomy ( lecs ) and gastrojejunal bypass , the mean operative times were 136 min ( range , 72200 ) and 90 min ( range , 65135 ) , respectively . the mean duration of hospital stays for gastrectomy , lecs , and bypass were 14.1 , 9.1 , and 15 d , respectively ( table 2 ) . when considering only the suturing time involving v - loc , the mean suturing time was 8.7 min ( range , 4.813.8 ) . the mean suturing time with v - loc was 14.7 min ( range , 9.134.9 min ) . operative data of each procedure : surgery - related factors two cases with positive air leak test results ( 1 case of esophagojejunostomy and 1 case of simple closure of gastric defect ) were noted during surgery after v - loc suturing and were managed by adding knotted sutures . no gastrointestinal injuries and bleeding were noted during suturing . when postoperative factors were examined , no complications related to the anastomosis site and no surgery - related deaths were noted . dindo grade ii . furthermore , 2 cases of intestinal obstruction were noted . of these , 1 patient had postoperative intestinal paresis ( grade ii ) and was managed conservatively , and the other was found to have internal herniation originating from a cord - like adhesion between the jejunojejunal anastomosis and colon ( grade iiib ) and underwent repeat laparoscopic surgery ( table 3 ) . the barbed suture , which obviates the need for tying knots , has emerged as a new device for wound closure . it has recently attracted interest as being useful in laparoscopic suturing . in the present study , we used v - loc 180 , which is an absorbable copolymer composed of glycolic acid and trimethylene carbonate the same materials that are used in maxon ( covidien ) . the tensile strength is maintained at 50% at 21 d , and complete absorption is observed in 180 d. in the current cases , we used a thread length of 15 cm , and it had excellent operability and maneuverability in the abdominal cavity . the effective length , comprising the barbed portion , is 12 cm . although cases wherein a gastric wall defect can not be closed by a single thread due to a relatively short effective length have been noted , the use of a longer length of v - loc may not be cost effective . hence , we used a length of 15 cm due to the associated operability and to facilitate easy suturing . the barb and loop - end design of this device enables its adherence to tissues without the need for tying a knot . however , v - loc does not have any barbs within 1 cm from the loop - end ( figure 1 ) , which is disadvantageous , in that it does not permit tight tissue adhesion at the first bite . therefore , we initiated suturing from the seromuscular portion , and the third bite was placed at the start of the intestinal incision , to avoid loosening . as tying a knot is not recommended in the barbed portion , because the thread could be cut , the suturing was completed by adding 2 bites beyond the end of intestinal incision , after which the suture was cut without any knot . however , the presence of a long barbed suture outside of the bowel has been reported to cause intestinal obstruction , and therefore , cutting this suture flush at the bowel is important . in the present study , 1 case of grade iiib intestinal obstruction was noted , because of the thread between the colon and jejunojejunal anastomosis , and was not related to the use of v - loc sutures . concept is being widely accepted in surgical procedures in various body locations as well as in fragile tissue , such as in cases of peritoneum closure . for intestinal anastomosis , this procedure has been shown to yield a shorter suturing time and similar bursting pressure , as compared to common monofilament sutures in preliminary research and animal models . furthermore , its usefulness and safety have been described in several large - scale trials involving laparoscopic bypass in obese patients . in patients with gastric cancer , barbed sutures have been used as a device for intestinal hole closure after side - to - side intestinal stapled anastomosis . however , barbed sutures have primarily been used for gastrointestinal anastomosis as double - layer running sutures , and their safety has been described . nevertheless , the use of single - layer entire - thickness running sutures is more desirable , considering the shorter surgical time and ease of suturing . although the present study was not controlled , the suturing time was found to be relatively short , and no anastomotic leakage was observed . furthermore , we conclude that single - layer entire - thickness running sutures are acceptable and safe , as they offer tight closure by barbed sutures . in addition , no anastomotic stenosis was observed , although we believe that this result is attributable to the side - to - side stapled anastomosis , not to the v - loc device . an important limitation of v - loc use may be the development of anastomotic leakage due to seromuscular injury , when v - loc has been used for staple line reinforcement . however , single - layer entire - thickness running sutures with the v - loc device showed a higher pressure and tightness test value ( defined as the pressure that can be exerted by the thread until a balloon bursts ) and no ruptures or leaks in a cadaver study . these findings indicate that the unidirectional , evenly spaced barbs distribute the tension equally because they attach to the tissue at numerous points . therefore , it appears that intestinal injury is a result of technical laparoscopy - related factors , such as the hand - sewn technique under direct vision , rather than a result of v - loc itself . in this report we noted that surgeons encountered difficulty in suturing because the anastomotic hole became gradually smaller as suturing progressed . however , to obtain a more secure suture and to avoid a positive air leak test result , it is essential to ensure tight closure as suturing progresses . in addition , of all the 40 case series we consider the double - layer method to provide the most reliable anastomosis possible ; therefore , additional knotted sutures are added at the surgeon 's discretion depending on intraoperative findings . we do not recommend the use of barbed suture alone ; a reliable suturing method is important . single - layer entire - thickness running suturing with the v - loc 180 device after stapled side - to - side intestinal anastomosis is considered to be a safe and feasible option . however , a greater number of such cases and careful and longer follow - up are necessary . | background and objectives : laparoscopic anastomotic methods are not commonly used because of the cumbersome laparoscopic intracorporeal sutures and tying involved .
the barbed suture is one of the various devices developed to simplify the placement of intracorporeal sutures .
however , barbed sutures are not commonly used during reconstruction after radical gastrectomy in cancer patients or for single - layer entire - thickness running suturing for intestinal anastomoses .
we describe the procedure for using barbed sutures and report on the short - term surgical outcomes.methods:between august 2012 and march 2014 , 15-cm - long barbed sutures ( v - loc 180 ; covidien , mansfield , ma , usa ) were used for laparoscopic intestinal anastomoses , including intestinal hole closure for esophagojejunal and gastrojejunal anastomoses after mechanical anastomoses and gastric wall closure after partial resection.results:in total , 38 patients underwent 40 laparoscopic anastomoses ( esophagojejunostomies , 26 ; gastrojejunostomies , 7 ; and simple closure of gastric defect , 7 ) ; no cases required conversion to open surgery .
two cases exhibited positive air leak test results during surgery ( 1 case of esophagojejunostomy and 1 case of simple closure of gastric defect ) .
two cases of intestinal obstruction were noted ; of those , one patient with postoperative intestinal paresis ( grade ii ) was managed conservatively , and the other underwent repeat laparoscopic surgery ( grade iiib ) for internal herniation unrelated to v - loc use .
no postoperative complications at the anastomosis site and no surgery - related deaths were noted.conclusion:single-layer entire - thickness running suturing with the v - loc 180 barbed suture after stapled side - to - side intestinal anastomosis was found to be safe and feasible in the reported cases . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
gold and gold alloys , whose primary advantages are their resistance to corrosion and tarnishing and their biocompatibility , have long been used to fabricate dental prostheses . despite these advantages , base metal alloys such as nickel - chromium ( ni - cr ) and cobalt - chromium ( co - cr ) are commonly used instead of high noble and noble alloys because of cost . in addition , although base metal alloys have some advantages over high noble or noble alloys , such as high hardness ( exceeds 400 kg / mm if carbon is present ) , low density , high elastic moduli , high tensile strength , and the possibility of electrochemical etching for resin bonding , they can corrode in accordance with their elemental composition . all dental alloys have a tendency to corrode in under intraoral conditions because of exposure to substances such as saliva , food , and beverages . high noble and noble alloys are biocompatible because of their resistance to oxidation and corrosion , while the corrosion resistance of base metal alloys depends on the integrity of their passive oxide layer , which may be destroyed mechanically and chemically by acidic changes . typically , the corrosion process takes place locally , but over time , this local process transforms to a gap corrosion , which causes several problems , such as gray discoloration at the margins of the fixed restorations close to the gingival sulcus . recently , manufacturers have started adding some noble alloys to the composition of base metal alloys to improve their biocompatibility and minimize corrosion while still keeping their cost reasonably low . another method , which is not commonly used , is the gold - hard - plating ( ghp ) of base metal alloys , also known as galvanic gold coating , layering application or gold plating - electrodeposition of gold . the problem is that solid gold is expensive . to get the look of gold without the cost , gold plating has many applications , including uses in jewelry , scientific experiments , electronics , industry , and dentistry . it is a technique that has been around for centuries and continues because it is effective . the underlying metal is made negatively charged through chemical baths and/or electricity . once the metal has a charge gold does not oxidize when exposed to air , so its electrical conductivity wo n't diminish over time . gold plating acts as an anti - corrosive coating to the material to which it is applied . the ghp technique used in dentistry involves the depositions of a 6 m to 8 m 24 k gold layer directly onto the cr - co cast prosthesis framework . this method prevents oxidation of the metal surface , alters its conductivity and resistance , provides a surface seal , reduces surface hardness , and improves color . according to the manufacturers instructions , the ghp technique is used primarily to coat cr - co alloys for removable dental prosthesis frameworks . rogers studied cr - co alloy gold plating and reported of advantages of clasps and frameworks of removable partial dentures . it was also reported that the strength of the gold plating is determined by the hardness and thickness of the gold deposition on the surface . in addition , electroplating of gold was used to enhance porcelain to metal bond strength and to minimize the discoloration of enamel , which is frequently associated with perforated and etched - metal resin - bonded retainers alike . this article describes the use of the ghp technique to gold - coat cr - co alloy used for the fabrication of a metal ceramic restoration ( mcr ) fabricated between mandibular right second premolar and second molar . the use of such a technique has not been reported for fixed restorations in the literature . the aim of the use of this technique for fixed partial dentures is to minimize gray discoloration at the margins of the restorations close to the gingival sulcus and enhance the esthetics . fabricate the mcr with cr - co alloy ( heraenium p , heraeus kulzer gmbh , hanau , germany ) ; finalize the clinical and laboratory procedures ( glaze the porcelain and polish the metal ) [ figure 1].airborne - particle abrade ( basic classic ; renfert gmbh , hilzingen , germany ) the intaglio surface with 110 m aluminum oxide , when this surface is to be coated.clean the restoration in an ultrasonic cleaner ( emmevi spa , badia polesine , italy ) and then with a steam cleaner ( vap 6-a ; emmevi spa , badia polesine , italy ) . make sure that no debris is left on the restoration surfaces.attach contact ligature wire ( 0.25 mm ) to the metal surface of the mcr to be gold - plated . ensure all surfaces to be gold - plated have electric contact [ figure 2].use a ligature wire ( 0.25 mm ) attached to the surfaces to be coated to hang and stabilize the mcr on the hanging device ( gramm technic gmbh , ditzingen , germany ) . stabilize the mcr horizontally with the wire so that the intaglio is placed vertically and that gas bubbles will not be trapped inside the framework.hang the hanging device ( gramm technic gmbh , ditzingen , germany ) in the gauging tool ( gramm technic gmbh , ditzingen , germany ) to verify correct distance to the anode [ figure 3].use the contact check ( gramm technic gmbh , ditzingen , germany ) to verify if all metal surfaces of the mcr are properly connected with ligature wire ( 0.25 mm ) [ figure 4].use galvano - wax ( gramm technic gmbh , ditzingen , germany ) or insulation lacquer ( protection lacquer , gramm technic gmbh , ditzingen , germany ) to isolate , and protect the surfaces which are not going to be coated with the ghp method [ figure 5].connect the mcr to the large plating head ( gramm technic gmbh , ditzingen , germany ) with the hanging device ( gramm technic gmbh , ditzingen , germany).determine the amount of gold solution ( gramm technic gmbh , ditzingen , germany ) required to gold - plate the surfaces , taking the manufacturer recommendations into consideration [ figure 6].perform the ghp procedures in the electrolysis device following the manufacturer 's instructions ( gammat optimo 2 , gramm technic gmbh , ditzingen , germany ) . fabricate the mcr with cr - co alloy ( heraenium p , heraeus kulzer gmbh , hanau , germany ) ; finalize the clinical and laboratory procedures ( glaze the porcelain and polish the metal ) [ figure 1 ] . airborne - particle abrade ( basic classic ; renfert gmbh , hilzingen , germany ) the intaglio surface with 110 m aluminum oxide , when this surface is to be coated . clean the restoration in an ultrasonic cleaner ( emmevi spa , badia polesine , italy ) and then with a steam cleaner ( vap 6-a ; emmevi spa , badia polesine , italy ) . attach contact ligature wire ( 0.25 mm ) to the metal surface of the mcr to be gold - plated . ensure all surfaces to be gold - plated have electric contact [ figure 2 ] . use a ligature wire ( 0.25 mm ) attached to the surfaces to be coated to hang and stabilize the mcr on the hanging device ( gramm technic gmbh , ditzingen , germany ) . stabilize the mcr horizontally with the wire so that the intaglio is placed vertically and that gas bubbles will not be trapped inside the framework . hang the hanging device ( gramm technic gmbh , ditzingen , germany ) in the gauging tool ( gramm technic gmbh , ditzingen , germany ) to verify correct distance to the anode [ figure 3 ] . use the contact check ( gramm technic gmbh , ditzingen , germany ) to verify if all metal surfaces of the mcr are properly connected with ligature wire ( 0.25 mm ) [ figure 4 ] . use galvano - wax ( gramm technic gmbh , ditzingen , germany ) or insulation lacquer ( protection lacquer , gramm technic gmbh , ditzingen , germany ) to isolate , and protect the surfaces which are not going to be coated with the ghp method [ figure 5 ] . connect the mcr to the large plating head ( gramm technic gmbh , ditzingen , germany ) with the hanging device ( gramm technic gmbh , ditzingen , germany ) . determine the amount of gold solution ( gramm technic gmbh , ditzingen , germany ) required to gold - plate the surfaces , taking the manufacturer recommendations into consideration [ figure 6 ] . perform the ghp procedures in the electrolysis device following the manufacturer 's instructions ( gammat optimo 2 , gramm technic gmbh , ditzingen , germany ) . note intaglio surfaces of the restoration are facing upwards to prevent trapping air bubbles measurement of distance between plate and restoration attached to hanging device with gauging tool evaluation of electrical contacts with contact check restoration surfaces isolated with galvano wax restoration after gold - hard - plating there have been several studies reporting the long - term survival rates and success of mcrs . these reported restorations were mostly fabricated with noble alloys with the addition of base metal alloys for oxidation . corrosion and the release of metal ions over time might have been responsible for reported discoloration and gingival problems . this modified ghp technique for mcrs may minimize corrosion of base metals and the concomitant gray discoloration of the soft tissues . the outer , intaglio surfaces , as well as only metal bands of the mcrs can be coated with gold . livaditis and tate investigated the effect of gold - plating on cementation and esthetics of adhesive bridges . they recommended gold - plating technique as a viable solution for reducing the discoloration problem that results from the framework in etched - metal resin - bonded prostheses . it has been observed that the number of clinical studies related with long - term success of gold - plating technique is limited . according to rogers , the durability of the plating depends upon the hardness of the gold deposited and its thickness . a thickness of up to 0.0005 inch when applied to castings does not appear to have any effect on their fit . also , it was reported that gold - plating technique is suitable for gold - plating cobalt - chromium castings and other chrome - containing alloys by surface activation . disadvantages of the technique include additional equipment , need for experienced laboratory technicians , cost and the need for future research for long term predictability of the technique . in addition , the long term efficacy of this technique should be established with clinical studies . | metal ceramic partial fixed dental prostheses have been commonly used for the replacement of missing teeth for many years . because of an increase in the price of gold ,
base metal alloys have been the choice of alloy for the fabrication of metal ceramic restorations in many dental clinics .
some major disadvantages of base metals are their corrosion and the dark coloration they may cause at the crown margins .
this article describes a galvanic gold - plating technique , which is used to minimize corrosion and improve the esthetics of metal ceramic restorations fabricated with cr - co base metal alloys .
this technique involves the deposition of a 6 m to 8 m 24 k gold layer directly onto the cr - co cast prosthesis framework .
the technique improves metal surface properties , making them more biocompatible and usable , however , requires additional equipment and experienced laboratory technicians .
clinical studies should be performed to corroborate the long term success of this technique . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
extranodal lymphoma refers to lymphomatous involvement in sites other than the lymph nodes , tonsils , thymus and waldeyer lymphatic ring . it has an incidence of approximately 40% in lymphoma patients and can occur in any organ , however involvement of the peritoneal cavity is a rare clinical presentation . according to the published data , the gastrointestinal tract is involved in 1030% of patients with non - hodgkin lymphoma ( nhl ) and involvement of the solid organs varies from 20 to 40% in the spleen to 4% in the adrenal glands . an autopsy series of 322 patients with nhl aimed to demonstrate the incidence of omental involvement , and they found evidence of involvement in 64 patients ( 20% ) . lymphoma does not usually involve the omentum , which is a peritoneal fold , because it consists of fibrofatty tissue devoid of lymphoid tissue . the route of peritoneal dissemination is unclear , although in cases of intestinal lymphomas , spread is believed to be contiguous via the transverse mesocolon , the gastrocolic ligament and visceral peritoneal surfaces . the peritoneal surface may be secondarily affected by 3 cell lines : epithelial ( carcinomatosis ) , mesenchymal ( sarcomatosis ) and lymphoid ( lymphomatosis ) . although these entities share some similar radiologic features , there are some contributory findings that may support the final diagnosis of lymphoma that the radiologist should know ( fig . peritoneal lymphomatosis is treated non - surgically , and often shows dramatic improvement with chemotherapy , and therefore its early and precise diagnosis is of utmost importance . the purpose of this article is to review the computed tomography ( ct ) findings of peritoneal lymphomatosis , highlighting the use of [ f]fluorodeoxyglucose ( fdg)-positron emission tomography ( pet)/ct , illustrating common and uncommon subtypes of lymphoma associated with this entity , and how to differentiate it from peritoneal carcinomatosis and peritoneal sarcomatosis . lymphomas are broadly subdivided into hodgkin lymphoma and nhl , based on distinct clinical and histologic features . a detailed discussion of the histologic subtypes of lymphomas is beyond the scope of this article . besides the pathologic classification , nhl subtypes are also grouped according to their clinical behavior as indolent , aggressive and very aggressive . in the long term , the longevity of patients with the highly aggressive and aggressive groups is better than that of the indolent group , because cure is almost not achievable in the latter . examples of indolent lymphomas are follicular lymphoma , small lymphocytic lymphoma , mantle cell lymphoma , lymphoplasmatic lymphoma , and some subtypes of marginal zone lymphomas . aggressive lymphomas include diffuse large b - cell lymphoma , many natural killer cell lymphomas and most peripheral t - cell lymphomas . follicular lymphoma and diffuse large b - cell lymphoma account for more than half of the cases of nhl . in the literature , peritoneal lymphomatosis is most commonly reported in association with diffuse large b - cell lymphoma . unlike nhl , hodgkin lymphoma tends to spread by contiguous lymph node involvement instead of multifocal nodal involvement , and peritoneal lymphomatosis is an extremely rare manifestation of hodgkin lymphoma . omental caking may manifest as fine nodular , soft tissue studding or large confluent soft tissue masses within the omentum . the pattern most commonly found in peritoneal lymphomatosis is omental caking with bulky homogeneous masses ( fig . another characteristic imaging feature of peritoneal lymphomatosis is a homogeneous smooth thickening , diffusely infiltrating the peritoneum and the leaves of the mesentery ( fig . small omental nodules associated with fine infiltration of the omental fat producing a smudged appearance can also be encountered ( fig . a stellate appearance of the mesentery is frequently seen and represents the results of an infiltrating process , causing thickening and rigidity of the mesentery ( fig . a retrospective evaluation of a small group of patients with peritoneal lymphomatosis found that most of the patients had mild to moderate ascites . additional findings may include peritoneal enhancement and peritoneal thickening , whether linear or nodular ( fig . enlarged lymph nodes are commonly seen in association with the mesenteric disease and should be a clue to the diagnosis . associated lymphomatous involvement of the other intra - abdominal organs can also be present ( figs . figure 2a 60-year - old woman with recurrent , refractory , diffuse , large b - cell lymphoma who has failed multiple chemotherapy regimens . ( a ) reformatted coronal and axial contrast - enhanced ct images show bulky homogeneous masses ( arrows ) and soft tissue ( curved arrows ) infiltrating the greater omentum , associated with bulky retroperitoneal lymphadenopathy ( asterisks ) , enlarged mesenteric lymph nodes and moderate ascites . ( b ) reformatted coronal fused pet / ct images show intense fdg uptake in retroperitoneal lymphadenopathy ( asterisk ) and omental masses ( arrows ) . figure 3a 69-year - old - man with diffuse large b - cell lymphoma at the time of diagnosis . reformatted coronal ( a ) and axial ( b ) contrast - enhanced ct images show diffuse homogeneous soft tissue infiltration along leaves of mesentery , associated with peritoneal thickening ( arrowheads , a , b ) and mild ascites ( asterisks , a ) . coronal maximum intensity projection pet image and axial fused pet / ct images ( c ) show diffusely increased metabolic activity in the omentum , mesentery and peritoneal lining , consistent with extensive lymphomatous involvement . bowel involvement was suspected on conventional contrast - enhanced ct , however axial fused pet / ct images show no fdg uptake in the small or large bowel . note the fdg - avid cardiophrenic and epiphrenic enlarged lymph nodes ( arrows , c ) . ( a ) axial contrast - enhanced ct image shows multiple small peritoneal nodules and a smudged appearance caused by the infiltrated adjacent omental fat . ( b ) axial contrast - enhanced ct image shows additional retroperitoneal enlarged lymph nodes ( asterisk ) and lymphomatous involvement of the spleen ( arrow ) . figure 5a 71-year - old woman with a history of follicular lymphoma with transformation to a diffuse large b - cell lymphoma . ( a ) axial contrast - enhanced ct images shows prominent straightened vessels secondary to lymphomatous infiltration of the mesentery causing a stellate mesenteric appearance ( curved arrows ) associated with large volume ascites . ( b ) axial contrast - enhanced ct image shows bulky homogeneous peritoneal masses ( arrow ) within the pelvis associated with free fluid and peritoneal enhancement ( arrowhead ) . axial contrast - enhanced ct images ( a c ) show homogeneous soft tissue in the greater omentum ( curved arrows , a c ) and along the leaves of the mesentery ( curved arrow , b ) associated with marked peritoneal thickening ( arrowheads , c ) and mild ascites . moderate splenomegaly ( letter s , a ) and multiple enlarged retroperitoneal lymph nodes ( asterisks , a , b ) are also seen . axial contrast - enhanced ct images ( a c ) show lymphomatous infiltration in the periportal region ( arrowheads , a , b ) , with nodules and masses in the lesser sac and greater omentum ( asterisks , a , b ) . cecal wall thickening ( curved arrows , c ) is also noted , representing lymphomatous involvement . axial fused pet / ct images ( d f ) show intense fdg uptake by the soft tissue infiltrating the periportal region ( arrowhead , d ) , the peritoneal nodules ( asterisks , d , e ) , and the cecum ( curved arrow , f ) . a focal area of fdg uptake in the spleen is consistent with lymphoma involvement ( arrow , d ) and was not seen in the contrast - enhanced ct ( a ) . despite the intense fdg uptake , a biopsy did not show evidence of transformation to a diffuse large b - cell lymphoma . figure 8a 65-year - old man with burkitt lymphoma and complete response to treatment after 6 cycles of chemotherapy . axial ct images ( a , c ) and axial fused pet / ct ( b , d ) images show aneurysmal dilatation and thickening of a jejunal segment with intense fdg uptake ( arrowheads , a d ) associated with nodular fdg uptake in the omentum that corresponds to large omental nodules and masses ( asterisks , a axial ct image ( e ) and axial fused pet / ct image ( f ) show complete resolution of the jejunal thickening ( e ) and no evidence of residual fdg uptake ( f ) after treatment . a 60-year - old woman with recurrent , refractory , diffuse , large b - cell lymphoma who has failed multiple chemotherapy regimens . ( a ) reformatted coronal and axial contrast - enhanced ct images show bulky homogeneous masses ( arrows ) and soft tissue ( curved arrows ) infiltrating the greater omentum , associated with bulky retroperitoneal lymphadenopathy ( asterisks ) , enlarged mesenteric lymph nodes and moderate ascites . the peritoneum is thickened and shows enhancement ( arrowheads ) . ( b ) reformatted coronal fused pet / ct images show intense fdg uptake in retroperitoneal lymphadenopathy ( asterisk ) and omental masses ( arrows ) . a 69-year - old - man with diffuse large b - cell lymphoma at the time of diagnosis reformatted coronal ( a ) and axial ( b ) contrast - enhanced ct images show diffuse homogeneous soft tissue infiltration along leaves of mesentery , associated with peritoneal thickening ( arrowheads , a , b ) and mild ascites ( asterisks , a ) . coronal maximum intensity projection pet image and axial fused pet / ct images ( c ) show diffusely increased metabolic activity in the omentum , mesentery and peritoneal lining , consistent with extensive lymphomatous involvement . bowel involvement was suspected on conventional contrast - enhanced ct , however axial fused pet / ct images show no fdg uptake in the small or large bowel . note the fdg - avid cardiophrenic and epiphrenic enlarged lymph nodes ( arrows , c ) . ( a ) axial contrast - enhanced ct image shows multiple small peritoneal nodules and a smudged appearance caused by the infiltrated adjacent omental fat . ( b ) axial contrast - enhanced ct image shows additional retroperitoneal enlarged lymph nodes ( asterisk ) and lymphomatous involvement of the spleen ( arrow ) . a 71-year - old woman with a history of follicular lymphoma with transformation to a diffuse large b - cell lymphoma . ( a ) axial contrast - enhanced ct images shows prominent straightened vessels secondary to lymphomatous infiltration of the mesentery causing a stellate mesenteric appearance ( curved arrows ) associated with large volume ascites . ( b ) axial contrast - enhanced ct image shows bulky homogeneous peritoneal masses ( arrow ) within the pelvis associated with free fluid and peritoneal enhancement ( arrowhead ) . a 54-year - old woman with a refractory mantle cell lymphoma . axial contrast - enhanced ct images ( a c ) show homogeneous soft tissue in the greater omentum ( curved arrows , a c ) and along the leaves of the mesentery ( curved arrow , b ) associated with marked peritoneal thickening ( arrowheads , c ) and mild ascites . moderate splenomegaly ( letter s , a ) and multiple enlarged retroperitoneal lymph nodes ( asterisks , a , b ) are also seen . a 50-year - old man with refractory follicular lymphoma . axial contrast - enhanced ct images ( a c ) show lymphomatous infiltration in the periportal region ( arrowheads , a , b ) , with nodules and masses in the lesser sac and greater omentum ( asterisks , a , b ) . cecal wall thickening ( curved arrows , c ) is also noted , representing lymphomatous involvement . axial fused pet / ct images ( d f ) show intense fdg uptake by the soft tissue infiltrating the periportal region ( arrowhead , d ) , the peritoneal nodules ( asterisks , d , e ) , and the cecum ( curved arrow , f ) . a focal area of fdg uptake in the spleen is consistent with lymphoma involvement ( arrow , d ) and was not seen in the contrast - enhanced ct ( a ) . despite the intense fdg uptake , a biopsy did not show evidence of transformation to a diffuse large b - cell lymphoma . a 65-year - old man with burkitt lymphoma and complete response to treatment after 6 cycles of chemotherapy . axial ct images ( a , c ) and axial fused pet / ct ( b , d ) images show aneurysmal dilatation and thickening of a jejunal segment with intense fdg uptake ( arrowheads , a d ) associated with nodular fdg uptake in the omentum that corresponds to large omental nodules and masses ( asterisks , a d ) . axial ct image ( e ) and axial fused pet / ct image ( f ) show complete resolution of the jejunal thickening ( e ) and no evidence of residual fdg uptake ( f ) after treatment . 3 ) but it can also develop during the course of disease or even as a manifestation of transformation of an indolent subtype of lymphoma to a higher grade , most commonly diffuse large b - cell lymphoma ( figs . 5 and 9 ) . figure 9a 58-year - old man with an 8-year history of follicular lymphoma with transformation to a diffuse large b - cell lymphoma . axial ct images ( a , c ) and axial ( b , d ) and coronal ( e ) fused pet / ct images show mild diffuse thickening of the gastric wall with intense fdg avidity ( curved arrows , a , b ) associated with an adjacent homogeneous soft tissue mass in the greater omentum showing intense fdg uptake consistent with peritoneal lymphomatosis ( arrowheads , c e ) . there is also right perirenal tissue with intense fdg uptake consistent with lymphomatous involvement ( asterisks , a transformation was suspected secondary to intense fdg uptake , which was ultimately proven by biopsy of the perirenal tissue . a 58-year - old man with an 8-year history of follicular lymphoma with transformation to a diffuse large b - cell lymphoma . axial ct images ( a , c ) and axial ( b , d ) and coronal ( e ) fused pet / ct images show mild diffuse thickening of the gastric wall with intense fdg avidity ( curved arrows , a , b ) associated with an adjacent homogeneous soft tissue mass in the greater omentum showing intense fdg uptake consistent with peritoneal lymphomatosis ( arrowheads , c e ) . there is also right perirenal tissue with intense fdg uptake consistent with lymphomatous involvement ( asterisks , a e ) . transformation was suspected secondary to intense fdg uptake , which was ultimately proven by biopsy of the perirenal tissue . although ct is considered the most convenient imaging technique for the evaluation of patients with lymphoma , fdg - pet / ct has been shown to improve staging , evaluation of therapy response and earlier detection of recurrent disease . due to its improved usefulness in distinguishing residual active lymphoma and benign fibrosis , fdg - pet / ct has been found valuable in assessment of treatment response ( fig . 8) . fdg - pet / ct is also helpful in the selection of suitable sites for biopsy , because this imaging modality not only demonstrates sites that are accessible but also that the site of interest is metabolically active , increasing the likelihood of a diagnostic result ( fig . the international prognostic index used for nhl is based on both imaging and clinical findings , and the stage and the number of extranodal sites of disease are included . in hodgkin lymphoma , these parameters are also of critical importance for the management strategies . in peritoneal lymphomatosis , fdg - pet / ct not only has greater sensitivity than ct for depicting peritoneal involvement in subtle cases but it also has greater accuracy in identifying extranodal sites other than the peritoneum , even in the absence of ct findings ( fig . 7 ) . the fdg - pet / ct findings modify the treatment approach in approximately 25% of patients . published data have been encouraging and suggest that fdg - pet / ct has a higher sensitivity in comparison with multidetector ct to detect peritoneal malignancies in general . the findings from retrospective studies have demonstrated that fdg - pet / ct has a sensitivity as high as 100% and a specificity as high as 97% for the detection of peritoneal seeding , compared with a sensitivity of 88% and a specificity of 97% for multidetector ct . there are 2 distinct patterns of glucose metabolism on fdg - pet / ct in peritoneal lymphomatosis : a nodular ( figs . 7 and 8) and a diffuse ( figs . 2 and 3 ) metabolic pattern . aggressive and highly aggressive nhl and hodgkin lymphoma usually show high fdg avidity . however , indolent lymphomas have overall low - grade or no fdg uptake because of its low metabolic activity . in patients with fdg - avid lymphomas , a complete metabolic response after therapy is a good indicator that the patient will remain disease - free in the long term . fdg avidity after 24 cycles of chemotherapy has been related to poor clinical outcome . in indolent lymphomas , treatment response is usually measured taking symptom relief , overall survival and progression- and event - free survival into consideration . fdg - pet / ct is valuable in cases where transformation of an indolent lymphoma to a higher grade subtype is suspected , by depicting abnormal fdg avidity at sites of transformation , because indolent lymphomas usually have low - grade fdg uptake ( fig . some subtypes of lymphoma ( e.g. , extranodal marginal zone lymphoma ) show variable fdg uptake and a baseline scan for comparison is of utmost importance in the assessment of therapeutic response . in most cases , fdg - pet / ct should be performed 68 weeks after conclusion of therapy to minimize misleading fdg uptake due to post - treatment inflammation . the diagnosis of peritoneal lymphomatosis can be very challenging because it may simulate peritoneal carcinomatosis or peritoneal sarcomatosis . peritoneal and omental seeding are known sites of dissemination of metastatic carcinoma , most commonly arising from the ovary , colon and stomach . nevertheless , ct findings of omental caking with homogeneous bulky masses rather than a nodular pattern ( fig . 10 ) , in addition to a diffuse distribution of enlarged lymph nodes , are helpful signs of peritoneal lymphomatosis . in peritoneal carcinomatosis ascites is a frequent finding in carcinomatosis , and may be moderate to large volume . although bulky masses are also usually seen in peritoneal sarcomatosis , they are frequently heterogeneous , hypervascular and may be associated with hemoperitoneum ( fig . a prospective evaluation of a large group of patients with soft tissue sarcoma found a prevalence of 2.7% for lymph node metastasis . axial contrast - enhanced ct image shows small soft tissue nodules ( curved arrows ) within the greater omentum associated with large volume ascites . axial contrast - enhanced ct images ( a , b ) show bulky heterogeneous masses within the greater omentum ( asterisks , a , b ) with no associated ascites or enlarged lymph nodes . a 49-year - old woman with ovarian cancer and peritoneal carcinomatosis . axial contrast - enhanced ct image shows small soft tissue nodules ( curved arrows ) within the greater omentum associated with large volume ascites . there is no retroperitoneal lymphadenopathy . a 52-year - old man with a gastrointestinal stromal tumor and peritoneal sarcomatosis . axial contrast - enhanced ct images ( a , b ) show bulky heterogeneous masses within the greater omentum ( asterisks , a , b ) with no associated ascites or enlarged lymph nodes . the appearance of peritoneal lymphomatosis may overlap with carcinomatosis and sarcomatosis , however bulky homogeneous masses or smooth peritoneal soft tissue thickening , diffuse lymphadenopathy , in addition to imaging features of variable extranodal lymphomatous involvement are contributory findings supporting the lymphoma diagnosis . fdg - pet / ct is a valuable asset in the staging and evaluation of metabolic response to therapy , therefore optimal management of the disease requires that the radiologist be familiar with the role of fdg - pet / ct , because its findings may influence treatment decisions . | abstractperitoneal lymphomatosis is a rare manifestation of lymphoma , seen most frequently with non - hodgkin lymphoma , and it is important to be familiar with this condition , because early diagnosis directly affects the management of patients .
this review illustrates the spectrum of imaging findings in peritoneal lymphomatosis , highlighting the use of positron emission tomography / computed tomography , showing common and uncommon subtypes of lymphoma associated with this entity , and how to differentiate it from peritoneal carcinomatosis and peritoneal sarcomatosis . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
methylcyclopentadienyl manganese tricarbonyl ( mmt , a registered trademark of afton chemical corporation ) is an organometallic fuel additive that was developed by the ethyl corporation in the 1950s . mmt has been used in a variety of fuels , including leaded and unleaded gasoline , diesel and turbine fuel , and fuel oil to raise octane and improve combustion . manganese concentrations in unleaded gasoline typically range from 5 to 20 ppm when mmt is used . in the united states , mmt is approved for use up to 8.3 ppm in conventional unleaded gasoline . as a fuel additive , mmt falls under the regulatory domain of the united states environmental protection agency ( usepa ) , and mmt manufacturers and/or importers are subject to relevant provisions of the usa clean air act ( caa ) . this paper provides an overview of the novel caa alternative tier 2 test program for mmt designed to collect critical manganese pharmacokinetic data in animals ( all test reports and correspondence related to the alternative tier 2 testing for mmt can be found in the federal docket management system ( fdms ) at http://www.regulations.gov identified by docket number epa - hq - oar-2004 - 0074 . ) . follow - up efforts led to the development of a series of physiologically based pharmacokinetic ( pbpk ) models for manganese . this paper will briefly examine the toxicology of manganese , the regulatory history of mmt , the design and conduct of the alternative tier 2 program for mmt , key research findings derived from the health effects research program , and critical lessons learned that can be applied to other chemicals . the second paper in this two - part series describes the pbpk models in greater detail and provides a framework for their application to the risk assessment of manganese . manganese is an essential trace metal that is required for normal amino acid , lipid , protein , and carbohydrate metabolism . under certain high - dose exposure conditions or disease states ( e.g. , hepatobiliary dysfunction ) , however , manganese can induce adverse neurological , reproductive , and respiratory effects in humans . manganese toxicity is dependent on the dose to target tissue and develops after inhalation , oral , and parenteral exposure ; however , this paper will focus predominantly on inhalation . atmospheric sources of manganese include manmade and natural sources including wind erosion of dusts and soils . industries associated with manganese emissions include ferroalloy production , iron and steel foundries , and power plant and coke oven combustion emissions . ambient ( background ) levels of manganese in rural and urban air range from 0.005 to 0.07 g mn / m . neurological effects occur at lower dose levels than other adverse effects , so consideration of these effects drives the human health risk assessment of inhaled manganese [ 46 ] . the earliest manifestations of manganese neurotoxicity ( manganism ) include fatigue , headache , muscle cramps , loss of appetite , apathy , insomnia , and diminished libido . as manganese exposure continues and the disease progresses , patients may develop dystonia , bradykinesia , rigidity , gait disorders , postural instability , micrographia , and muscle tremors ( for review see ) . individuals with chronic manganese neurotoxicity resemble patients with parkinson 's disease ; however , these syndromes can be distinguished both clinically and with neuroimaging studies . although these syndromes are clinically distinct , some studies suggest that manganese overexposure may pose a risk factor for parkinson 's disease [ 7 , 8 ] . a variety of inhalation exposure scenarios exist for manganese from occupational settings with mid- to high - dose exposures to much lower exposures found in the general environment . forms of manganese include but are not limited to manganese dioxide ( mno2 ) and other oxides , manganese sulfate ( mnso4 ) , manganese phosphates ( mnpo4 ) , and organometallic manganese compounds . absent underlying hepatobiliary disease , frank manganese neurotoxicity has been observed in workers that have been chronically exposed to dusts or fumes that contain high levels ( > 1 mg mn / m ) of manganese . more subtle neurobehavioral effects have been reported in welders and other workers at lower ( ~0.2 mg mn / m ) exposure concentrations . this cross - sectional study of male workers was used by the usepa as their critical study for deriving their chronic inhalation reference concentration for manganese ( rfc ) of 0.05 g / m that was last updated in 1993 . according to usepa , an rfc is an estimate ( with uncertainty spanning perhaps an order of magnitude ) of a continuous inhalation exposure to the human population ( including sensitive subgroups ) that is likely to be without an appreciable risk of deleterious effects during a lifetime . assessed employees from a belgian alkaline battery production plant and controls from a polymer processing plant . personal air samplers in the battery plant indicated an 8 hr time weighted average ( twa ) exposure of 0.215 mg / m for respirable manganese . an average cumulative exposure of 1.2 mg / myears of respirable manganese was associated with decrements in some neurofunctional performances ( e.g. , mean reaction times , eye - hand coordination and muscles tremor scores ) between the exposed group and the control group . health canada current risk assessment relied on a study of italian ferroalloy workers performed by lucchini and coworkers for the derivation of their rfc for manganese . lucchini examined several cohorts of workers ( furnace , casting , and welding job functions ) between 1981 and 1997 . for example , the geometric mean manganese concentrations ( in total dust ) went from 167 to 54.7 g / m in the maintenance area where welding was performed . an exposure concentration of approximately 71 g mn / m was derived from an estimated cumulative exposure index . lucchini reported an association between occupational manganese exposure and some neurological effects including higher symptom reporting , increased tremor frequency , altered motor function , and impaired memory . bailey and colleagues published an alternative rfc value of 27 g mn / m that was based upon their use of epidemiological studies published after 1992 . the american conference of industrial hygienists ( acgih ) threshold limit value ( tlv ) for elemental manganese and related inorganic compounds is 0.2 mg mn / m . this standard is set as a time - weighted average for an 8 hr shift 5 days per week designed to protect workers in occupational settings . ambient manganese exposure data are needed to assess the potential public health impacts of mmt in fuel . several of the most significant studies were performed in canadian cities when mmt was widely used in the local fuel supply . for example , an extremely large effort using probabilistic sampling techniques measured manganese exposure levels in toronto residents when all fuel contained mmt . the results of this study and related analyses of data from personal samplers [ 15 , 16 ] suggest that the median annualized nonoccupational exposure concentration was 0.008 g / m with the highest long - term exposures at or near 0.022 g / m . as a respirable fraction ( pm2.5 ) . roadway measurement of soil manganese concentrations did not reveal a measurable increase in manganese levels along urban toronto highways . studies from australia have shed additional light on the impact of mmt on atmospheric manganese concentrations [ 18 , 19 ] . gulson and coworkers reported no significant changes in environmental samples or blood manganese concentrations in children following the introduction of mmt in sydney . concerns regarding the use of mmt as a gasoline fuel additive have also been influenced by the usa experience of tetraethyl lead in gasoline . concerns about automotive emissions of lead prompted the usepa in 1973 to phase out the use of lead in gasoline . the experience with tetraethyl lead created an environment of distrust between public health officials , environmental legislators , advocacy groups , and fuel manufacturers . when the usepa ordered the phasing out of leaded gasoline , mmt and other alternative octane enhancers were used in unleaded gasoline . in 1977 , a congressional amendment to the caa banned the use of all fuel additives not substantially similar to gasoline , including mmt , unless the usepa granted a waiver . the prohibition on the use of mmt in gasoline was largely based on concerns that mmt use could affect the first generation of automotive emissions - control systems . this and several subsequent waiver petitions submitted by ethyl corporation were denied because of usepa concerns regarding potential increases in exhaust hydrocarbon emissions resulting from mmt use .
as part of a later waiver application for mmt , the usepa conducted a risk assessment of exposure to inhaled manganese in 1993 , with the publication of an inhalation rfc of 0.05 g / m . the usepa used standard rfc development methodologies for a noncancer endpoint and based the rfc on the roels et al . study that evaluated neurobehavioral and motor movement impairments observed in workers exposed to manganese dioxide ( mno2 ) . the exposure concentration reported by roels was considered by the usepa to be a lowest - observable - adverse - effect level ( loael ) . the loael value was adjusted for continuous exposure durations , and several uncertainty factors were applied . these uncertainty factors included use of a loael instead of a no - observable - adverse - effect level ( noael ) , extrapolation from subchronic to chronic , protection of potential sensitive members of the human population , and a factor reflecting other uncertainties in the database , such as less - than chronic periods of exposure , inadequate information regarding developmental and reproductive toxicity , and uncertainty about the toxicity of various forms of manganese . although the nutritional essentiality of manganese in the diet was discussed in the documentation of the rfc , it played no practical role in the calculation of an rfc . the usepa also published a risk characterization that included exposure and dose - response assessments for mmt in gasoline [ 23 , 24 ] . after completing this evaluation , epa determined that use of mmt would not cause or contribute to the failure of vehicle emission control systems . epa was unable to determine , however , whether a risk to the public health occurred from use in gasoline . the agency stated [ a]lthough it is not possible based on the present information to conclude whether specific adverse health effects will be associated with manganese exposures in the vicinity of or exceeding the ( estimated safe level over a lifetime of exposure ) , neither is it possible to conclude that adverse health effects will not be associated with such exposures given the information that is available at present and the uncertainties discussed here , a reasonable basis exists for concern regarding potential public health risks , especially for sensitive subpopulations , if mmt were to be widely used in unleaded gasoline . the usepa also concluded that long - term animal testing and exposure research were needed to more accurately define the risk . coincidental to these activities , in july 1994 , the usepa administrator denied ethyl corporation newest waiver petition specifically because of concerns about potential risks to public health and refused to register mmt for use in the usa in 1995 , the ethyl corporation successfully challenged the denial of its petition based on public health concerns ( ethyl corporation v. browner , 51 f.3d 1053 ( d.c . 1995 ) ) , as well as usepa decision not to register mmt ( ethyl corporation v. browner , 67 f.3d 941 ( d.c . the usepa formally approved the use of mmt in conventional unleaded gasoline and registered it as a fuel additive under the caa , allowing for its domestic sale . provisions of the caa provide the usepa with the authority to require testing of fuels and fuel additives used in motor vehicles , including mmt , to help fill data gaps and provide information that potentially would result in a more definitive risk evaluation . these health testing requirements are addressed in sections 211(b)(2 ) and 211(e ) of the caa . section 211(b)(2 ) states for the purpose of registration of fuels and fuel additives , the administrator shall , on a regular basis , require the manufacturer of any fuel or fuel additive to conduct tier 2 tests to determine potential public health and environmental effects of the fuel or additive ( including carcinogenic , teratogenic , or mutagenic effects ) . these studies would be conducted using test procedures and protocols established by the usepa . moreover , the caa also provides usepa with the discretion to modify the standard tier 2 health effects testing requirements for a fuel or fuel additive by substituting , adding , or deleting testing requirements or changing the underlying vehicle / engine specifications ( 40 cfr 79.58(c ) ) . health effects testing for mmt fell under this so - called alternative tier 2 requirement , as the concern and subsequent testing was focused around the inorganic exhaust products of mmt and not mmt itself . in 2001 , the ethyl corporation was notified by the usepa of the alternative tier 2 provisions for mmt which fell within two general categories : pharmacokinetic testing of manganese compounds and characterization of manganese emissions from vehicles utilizing fuels containing mmt . a central objective of the mmt alternative tier 2 program was to generate data to support development of physiologically based pharmacokinetic ( pbpk ) models for manganese . as a result , pbpk model development became the subject of a series of studies funded by afton chemical corp . and handled in a way similar to other facets of the alternative tier 2 program . multiple pharmacokinetic studies were performed in response to the usepa mandate ( table 1 ) . studies that were required by the usepa were performed in accordance with the usepa good laboratory practice ( glp ) standards for inhalation exposure health effects testing ( 40 cfr part 79.60 ) . all studies were performed at the hamner institutes for health sciences ( hamner ) ( formerly the chemical industry institute of toxicology ( ciit ) ) . all required study protocols , protocol amendments , and draft final reports underwent independent scientific review by project specific technical advisory panels ( taps ) composed of individuals with expertise in inhalation toxicology , pharmacokinetics , and neurotoxicology ( figure 1 ) . members of the taps , which changed for the different facets of the test program , were chosen by the study sponsor with input from the testing laboratory and approved by the usepa . all study results underwent additional independent peer review during subsequent publication of the work in scientific journals . these concerned the form of manganese to be examined , animal species used , and endpoints of interest . this decision was influenced in part by the observation that mmt undergoes rapid photolysis when exposed to light , forming methylcyclopentadiene , cyclopentadiene , carbon monoxide , and a manganese carbonyl that is readily oxidized to trimanganese tetroxide . early studies performed by ter haar and coworkers showed that the primary combustion product from mmt was mn3o4 . this study used fuel spiked with large concentrations of mmt , an automobile without a catalytic converter , and x - ray diffraction methods for chemical speciation , resulting in an artifactual apparent enrichment of mn3o4 in the exhaust . zayed et al . , using scanning electron microscopy coupled with energy dispersive x - ray spectrometry , also suggested that the chemical form of emitted manganese was as an oxide but could not rule out sulfates and other manganese species . other studies , using lower treat rates representative of actual use patterns and more advanced analytical methods , including x - ray absorption near - edge structure ( xanes ) spectra and k - edge x - ray absorption fine structure ( xafs ) spectroscopy , showed the presence of three major manganese - containing components in tailpipe emissions : a divalent manganese phosphate , sulfate , and oxides ( most likely as mn3o4 ) [ 2931 ] . the percentage of each component varied somewhat depending on the driving cycle and vehicle but remained relatively constant with the sulfate and the phosphate forms being the major ( ~80% ) components . the usepa subsequently agreed to the use of commercially available manganese phosphate ( as hureaulite ) , mnso4 , and mn3o4 for the inhalation studies rather than rely on the use of a dynamometer system that used mmt in the fuel source to generate exhaust particulates . this decision also allowed the hamner research team to achieve the high exposure concentrations ( mg / m ) required to conduct the study . such high concentrations were necessary to ensure tissue accumulation so the data could be used to inform the subsequent pbpk models . similar aerosol concentrations and particle sizes were used in the hamner inhalation studies to facilitate direct comparison of tissue manganese burdens among these experiments . early studies used all three forms of manganese while later experiments relied upon the form of manganese ( mnso4 ) that produced the greatest increase in brain manganese concentrations , representing a worse - case scenario with regards to potential brain delivery . another unique feature of the mmt alternative tier 2 test program was the use of rhesus monkeys for certain studies . a substantial literature base describing species differences in neurological responses following high - dose manganese exposure existed at the time the alternative tier 2 program was launched . unlike rats , manganese - exposed monkeys develop distribution patterns for this metal within the brain that mimic those seen in heavily exposed people and develop similar neuropathology and behavioral responses [ 3234 ] . despite these pharmacodynamic differences in response between rats and humans , pharmacokinetic data obtained from rats remained valuable because rats and primates show similar overall pharmacokinetic responses to manganese exposure , including the induction of homeostatic control mechanisms . the rat data provided critical insights into the dose - response relationship for inhaled manganese , especially during different life stages . once the decision to use rhesus monkeys was reached , there was a concerted effort to maximize the data that could be obtained from this study . study endpoints included tissue manganese concentrations following mnso4 exposure , brain magnetic resonance imaging ( mri ) studies , respiratory tract pathology , catecholamine neurochemistry , biomarkers of neurotoxicity and oxidative stress , and metabolomic biomarkers of exposure . the usepa waived the glp requirements for certain study endpoints , including the mri study , neurochemistry measurements , and the assessment of biomarker endpoints . another challenge that faced the research team was conduct of the required gestational and lactational inhalational studies . inhalation developmental neurotoxicity and pharmacokinetic studies generally rely on maternal separation during exposure , resulting in pup stress that may alter development , thereby confounding study results . to overcome this potential confounding variable , vitarella and coworkers developed a unique single - animal exposure cylinder designed to house a rat dam and her litter . these investigators first tested concentrations of manganese phosphate within the exposure cylinder to verify that particle concentrations within this system were equivalent to those achieved within a stainless steel 1-m inhalation chamber . once developed , this system was then used to support the rat lactational exposure study required by the usepa . a number of significant discoveries emerged from this testing program ( table 2 ) . while these studies are described in detail in their individual publications , several significant findings are summarized here . solubilitythe hamner particle solubility studies showed that hureaulite and mn3o4 are relatively insoluble in simulated lung lining fluids , while mnso4 is considerably more soluble in biological fluids . these studies also showed that soluble manganese forms like the sulfate are more rapidly cleared from the rat lung and delivered to the rat brain following inhalation than are insoluble manganese oxide and phosphate particles . the hamner particle solubility studies showed that hureaulite and mn3o4 are relatively insoluble in simulated lung lining fluids , while mnso4 is considerably more soluble in biological fluids . these studies also showed that soluble manganese forms like the sulfate are more rapidly cleared from the rat lung and delivered to the rat brain following inhalation than are insoluble manganese oxide and phosphate particles . direct olfactory to deep brain transportone project goal was to determine whether inhaled manganese could be transported to the brain directly via the olfactory nerve . interest in this topic was sparked by the knowledge that the olfactory system forms a direct interface between the air and the brain . the hamner conducted studies in rats using short - term ( 90 min ) inhalation exposure to radiolabeled ( mn ) aerosols in rats with one occluded nostril , thus restricting olfactory transport of manganese to only one side of the rat brain . these novel studies dramatically demonstrated that the olfactory route contributes the vast majority ( > 90% ) of the mn found in the olfactory pathway of the rat brain up to 8 days following acute inhalation exposure . to our knowledge , this was the first study to demonstrate that an inhaled metal could be delivered to the olfactory brain regions directly via the olfactory nerve . although olfactory transport rapidly delivers manganese to brain structures in the olfactory pathway , it appears to be relatively slow ( and inefficient ) in delivering inhaled manganese to the rat striatum and other more distant brain structures . one project goal was to determine whether inhaled manganese could be transported to the brain directly via the olfactory nerve . interest in this topic was sparked by the knowledge that the olfactory system forms a direct interface between the air and the brain . the hamner conducted studies in rats using short - term ( 90 min ) inhalation exposure to radiolabeled ( mn ) aerosols in rats with one occluded nostril , thus restricting olfactory transport of manganese to only one side of the rat brain . these novel studies dramatically demonstrated that the olfactory route contributes the vast majority ( > 90% ) of the mn found in the olfactory pathway of the rat brain up to 8 days following acute inhalation exposure . to our knowledge , this was the first study to demonstrate that an inhaled metal could be delivered to the olfactory brain regions directly via the olfactory nerve . although olfactory transport rapidly delivers manganese to brain structures in the olfactory pathway , it appears to be relatively slow ( and inefficient ) in delivering inhaled manganese to the rat striatum and other more distant brain structures . addition of inhaled manganese to existing oral exposuresindividuals with either deficient or excessive manganese tissue burdens have been postulated to be at increased risk for manganese toxicity following inhalation exposure . two related 14-day inhalation studies conducted by the hamner demonstrated that manganese body burden does not influence brain manganese concentrations following inhalation [ 44 , 54 ] . these studies placed postnatal day ( pnd ) 10 rats on specially formulated diets that contained 2 , 10 , or 100 ppm manganese . the lowest and highest diets were chosen in order to provide the animals with a marginally deficient or high - normal level of manganese . once tissue manganese concentrations stabilized ( i.e. , after 2 months on the special diets ) , rats were exposed by whole - body inhalation for 6 hr / day on 14 consecutive days to mnso4 or mn3o4 at concentrations equivalent to 0 , 0.03 , or 0.3 mg mn / m . feeding the 2 ppm manganese diet was associated with a number of effects , including reduced body weight gain , decreased liver manganese concentrations , and reduced whole - body manganese clearance rates . although rats kept on this diet and then exposed to 0.3 mg mn / m developed increased manganese concentrations in some tissues , the studies did not demonstrate any statistically significant diet and inhalation interactions on brain manganese concentrations . individuals with either deficient or excessive manganese tissue burdens have been postulated to be at increased risk for manganese toxicity following inhalation exposure . two related 14-day inhalation studies conducted by the hamner demonstrated that manganese body burden does not influence brain manganese concentrations following inhalation [ 44 , 54 ] . these studies placed postnatal day ( pnd ) 10 rats on specially formulated diets that contained 2 , 10 , or 100 ppm manganese . the lowest and highest diets were chosen in order to provide the animals with a marginally deficient or high - normal level of manganese . once tissue manganese concentrations stabilized ( i.e. , after 2 months on the special diets ) , rats were exposed by whole - body inhalation for 6 hr / day on 14 consecutive days to mnso4 or mn3o4 at concentrations equivalent to 0 , 0.03 , or 0.3 mg mn / m . feeding the 2 ppm manganese diet was associated with a number of effects , including reduced body weight gain , decreased liver manganese concentrations , and reduced whole - body manganese clearance rates . although rats kept on this diet and then exposed to 0.3 mg mn / m developed increased manganese concentrations in some tissues , the studies did not demonstrate any statistically significant diet and inhalation interactions on brain manganese concentrations . neonatal exposuresconcerns have been raised regarding increased risk of neonates for manganese - induced neurotoxicity . the increased sensitivity of neonatal animals to manganese appears to be due in part to their ability to develop higher brain manganese levels than adults when faced with equivalent or lesser manganese exposures by high - dose oral gavage . factors influencing this increased susceptibility of neonatal animals may include enhanced manganese absorption from the gastrointestinal tract , an incompletely formed blood - brain barrier , and a greatly reduced basal biliary manganese excretory rate until weaning . however , information was more limited regarding the potential risks in neonates exposed to airborne manganese . to further assess this concern , the hamner exposed rat dams and their offspring to air or mnso4 ( 0.05 , 0.5 , or 1 mg mn / m ) for 6 hr / day , 7 days / week starting 28 days prior to breeding and from pnd 1 through 18 . the experimentally determined manganese concentration in neonatal striatum and the model - predicted auc for this brain region did not imply significantly higher exposures in the pups when compared to those in adults up to the inhaled dose of 1 mg / m [ 55 , 56 ] . despite the virtual absence of basal biliary excretion in neonatal rats , they appear to induce their biliary excretion when challenged with excess manganese through the oral route . this inducible excretion in neonates was shown to be applicable to inhaled manganese as well to a level comparable to adults . because neonates have an increased requirement for manganese for optimal brain development , further elaboration of the dose - response relationship between brain manganese levels and neurotoxicity may further elucidate the potential vulnerability of neonatal brain to manganese . the increased sensitivity of neonatal animals to manganese appears to be due in part to their ability to develop higher brain manganese levels than adults when faced with equivalent or lesser manganese exposures by high - dose oral gavage . factors influencing this increased susceptibility of neonatal animals may include enhanced manganese absorption from the gastrointestinal tract , an incompletely formed blood - brain barrier , and a greatly reduced basal biliary manganese excretory rate until weaning . however , information was more limited regarding the potential risks in neonates exposed to airborne manganese . to further assess this concern , the hamner exposed rat dams and their offspring to air or mnso4 ( 0.05 , 0.5 , or 1 mg mn / m ) for 6 hr / day , 7 days / week starting 28 days prior to breeding and from pnd 1 through 18 . the experimentally determined manganese concentration in neonatal striatum and the model - predicted auc for this brain region did not imply significantly higher exposures in the pups when compared to those in adults up to the inhaled dose of 1 mg / m [ 55 , 56 ] . despite the virtual absence of basal biliary excretion in neonatal rats , they appear to induce their biliary excretion when challenged with excess manganese through the oral route . this inducible excretion in neonates was shown to be applicable to inhaled manganese as well to a level comparable to adults . because neonates have an increased requirement for manganese for optimal brain development , further elaboration of the dose - response relationship between brain manganese levels and neurotoxicity may further elucidate the potential vulnerability of neonatal brain to manganese . studies in rhesus monkeysthe hamner inhalation study that exposed juvenile rhesus monkeys to mnso4 is amongst the most critical completed to date with manganese . in this study , one group of monkeys was exposed to either air or mnso4 ( 0.06 , 0.3 , or 1.5 mg mn / m ) for 65 exposure days ( 6 hr / day , 5 days / week ) before tissue analysis . additional monkeys were exposed to mnso4 at 1.5 mg mn / m for 15 or 33 exposure days and evaluated immediately thereafter or for 65 exposure days followed by a 45- or 90-day delay before evaluation . monkeys exposed to mnso4 at 0.06 mg mn / m developed increased manganese concentrations in the globus pallidus , putamen , olfactory epithelium , olfactory bulb , and cerebellum . absolute manganese concentrations in the mnso4-exposed monkeys demonstrated a decreasing peripheral - central concentration gradient within the olfactory system ( i.e. , olfactory epithelium olfactory bulb > olfactory tract > olfactory cortex ) . . increased pallidal manganese concentrations were evident by brain mri and further confirmed by atomic absorption spectrometry analysis of the tissues . mri changes seen in this monkey study were similar to those reported in welders that have had high manganese exposure and subsequently developed bilateral hyperintensity on t1-weighted images in the globus pallidus and other brain regions . signal hyperintensities could not be visualized by mri between the olfactory bulb and more distal sites , suggesting that direct translocation of manganese from the olfactory bulb to the globus pallidus did not occur in the manganese - exposed monkeys . metabolomic analysis of serum and chemical analysis of brain tissues from the mnso4-exposed monkeys revealed changes indicative of oxidative stress at higher exposure concentrations [ 39 , 40 ] . dorman and coworkers also reported that exposure of monkeys to mnso4 at 1.5 mg mn / m for 15 exposure days resulted in increased lung manganese concentrations , mild subacute bronchiolitis , alveolar duct inflammation , and proliferation of bronchus - associated lymphoid tissue . bronchiolitis and alveolar duct inflammatory changes were absent 45 days after exposure , suggesting that these lesions are reversible upon cessation of subchronic high - dose manganese exposure . the hamner inhalation study that exposed juvenile rhesus monkeys to mnso4 is amongst the most critical completed to date with manganese . in this study , one group of monkeys was exposed to either air or mnso4 ( 0.06 , 0.3 , or 1.5 mg mn / m ) for 65 exposure days ( 6 hr / day , 5 days / week ) before tissue analysis . additional monkeys were exposed to mnso4 at 1.5 mg mn / m for 15 or 33 exposure days and evaluated immediately thereafter or for 65 exposure days followed by a 45- or 90-day delay before evaluation . monkeys exposed to mnso4 at 0.06 mg mn / m developed increased manganese concentrations in the globus pallidus , putamen , olfactory epithelium , olfactory bulb , and cerebellum . absolute manganese concentrations in the mnso4-exposed monkeys demonstrated a decreasing peripheral - central concentration gradient within the olfactory system ( i.e. , olfactory epithelium olfactory bulb > olfactory tract > olfactory cortex ) . these data are consistent with direct olfactory transport of inhaled manganese . increased pallidal manganese concentrations were evident by brain mri and further confirmed by atomic absorption spectrometry analysis of the tissues . mri changes seen in this monkey study were similar to those reported in welders that have had high manganese exposure and subsequently developed bilateral hyperintensity on t1-weighted images in the globus pallidus and other brain regions . signal hyperintensities could not be visualized by mri between the olfactory bulb and more distal sites , suggesting that direct translocation of manganese from the olfactory bulb to the globus pallidus did not occur in the manganese - exposed monkeys . metabolomic analysis of serum and chemical analysis of brain tissues from the mnso4-exposed monkeys revealed changes indicative of oxidative stress at higher exposure concentrations [ 39 , 40 ] . dorman and coworkers also reported that exposure of monkeys to mnso4 at 1.5 mg mn / m for 15 exposure days resulted in increased lung manganese concentrations , mild subacute bronchiolitis , alveolar duct inflammation , and proliferation of bronchus - associated lymphoid tissue . bronchiolitis and alveolar duct inflammatory changes were absent 45 days after exposure , suggesting that these lesions are reversible upon cessation of subchronic high - dose manganese exposure . the work described herein represents the most extensive set of pharmacokinetic studies performed to date under the usepa alternative tier 2 requirements . the pharmacokinetic data that was collected through this testing program has dramatically improved our understanding of the health risks posed by manganese . these studies have led to an improved understanding of the exposure conditions that lead to increased concentrations of the metal within the adult and developing brain and other tissues . this work has also led to the development of predictive , pbpk models for inhaled manganese that relate lung , brain , and other tissue manganese concentrations to exposure concentrations . these pbpk models should lead to the development of human health risk assessments for inhaled manganese that will consider both its essentiality and neurotoxicity . this testing program also represents an example of productive industry / government cooperation despite the challenging regulatory climate under which it commenced . evidence of cooperation was manifested by the sponsor 's willingness to conduct additional studies that were outside of the scope of the required testing program . some examples included work to evaluate olfactory transport of manganese following inhalation , short - term ( 2 weeks ) pharmacokinetic studies , and experiments designed to examine the elimination kinetics of inhaled manganese ( see table 1 ) . many of these studies were needed to develop the framework for subsequent development of the pbpk models . in addition , the sponsor also voluntarily developed the human pbpk models , as only the animal models were required by usepa . all parties involved in this program shared a common desire to develop the most robust set of experiments possible . figure 1 shows the extensive review and oversight by epa and others that contributed to the development of robust experimental protocols . in addition , all parties involved recognized the value of the development and use of novel technologies ( e.g. , inhalation exposure systems for lactating rats ) and incorporation of a wide array of endpoints , including mri . there was also great value in conducting the work at a multidisciplinary independent research institute that could help facilitate discussions between the usepa and the research sponsor . incorporation of additional manganese taps composed of external experts also built on the hamner 's rich history of independent external peer review of their research programs . this testing program also benefited from multiple postdoctoral fellows and undergraduate researchers ; thus , this program also played a key role in training new scientists . finally , the mmt testing program is aligned with the national research council vision described in their report entitled the three main components of the nrc vision for the future of toxicity testing are chemical characterization , toxicity testing , and dose - response and extrapolation modeling . the ncr vision also describes a paradigm shift away from a focus on identifying adverse effects observed in experimental animals at high doses toward identifying and avoiding biologically significant perturbations of key toxicity pathways . the identification of transition points between normal function and exposures that lead to accumulation and effects of an agent , as well as a consideration of the adaptive changes that respond to initial perturbations and function to maintain homeostasis , is a key to the nrc vision ( see [ 58 , figure 2.2 ] ) . in our opinion , significant changes in tissue manganese concentrations represent a critical early step in the development of manganese neurotoxicity . importantly , this work has identified a dose - dependent transition in manganese kinetics , which is a point where there is a change in the relationship of tissue accumulation as a function of dose [ 60 , 61 ] . the pharmacokinetic data and pbpk modeling have shown that background tissue manganese levels are well maintained at low to moderate exposure levels , due to the existence of homeostatic control mechanisms , such as increased biliary manganese excretion , that serve to regulate tissue levels . only once those mechanisms are overwhelmed do tissue levels start to increase significantly . due to the existence of this dose - dependent transition , only roughly one order of magnitude separates the point of departures on which previous manganese risk assessments are based and a level of exposure at which no significant changes in mn tissue concentration is predicted to occur in target tissues . this may indicate that large uncertainty factors are not necessary when extrapolating high - dose occupational exposure levels to low environmental exposure levels for the general population for an essential element such as mn . the human pbpk models for mn that emerged from this program can be used to further analyze the relationship between exposure and target tissue concentration and provide a consistent dose - response relationship for the effects of mn regardless of exposure route and duration . the models can be used to extrapolate to lower exposures to determine a concentration at which no significant effect on brain concentrations would be expected compared to normal variation . the human pbpk model can consider all life stages ( fetuses , neonates , adults , and old age ) , both genders , pregnancy , and form of mn . this model will be a critical tool for the quantitative risk assessment of environmental and occupational exposures to mn . the model and these applications are discussed in more detail in part 2 of this series . | concerns have been raised regarding environmental manganese exposure since high exposures have been associated with neurological disorders .
the usa environmental protection agency most recent human health risk assessment of inhaled manganese conducted in 1993 identified specific areas of uncertainty regarding manganese pharmacokinetics .
this led to the development of a test rule under the usa clean air act that required the generation of pharmacokinetic information on the inorganic manganese combustion products of the organometallic fuel additive methylcyclopentadienyl manganese tricarbonyl ( mmt ) .
the alternative tier 2 testing program for mmt , described in this paper , has yielded substantial pharmacokinetic data and has enabled the generation of physiologically based pharmacokinetic ( pbpk ) models for manganese .
these models are capable of predicting tissue manganese concentrations across a variety of dose routes , levels , and durations while accounting for factors such as age , gender , and reproductive status , enabling the consideration of tissue dosimetry in future risk assessments . |
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takayasu 's arteritis ( ta ) is a chronic vasculitis of uncertain etiology affecting large and medium - sized arteries , primarily the aorta and its branches as well as the coronary and pulmonary arteries . this disease is not endemic to a particular area , but is developed mostly in young women of asia . it has been reported that about 150 new cases develop annually in japan,1 ) but in the united states and europe , 1 to 3 cases per 1000000 persons have occurred per year.2 ) ulcerative colitis ( uc ) is an idiopathic chronic inflammatory disease limited to the mucosa and submucosa of the colon . uc almost always involves the rectum and may extend to other parts of the colon . the incidence of uc has been reported to be high in northern europe , the united kingdom , and north america . the prevalence of uc has been estimated to range from 37 to 246 cases per 100000 persons in north america.3 ) the occurrence of ta in patients with uc has been reported rarely with only about 50 cases in the world and more than half of these cases have been identified in japan.4 ) a case of this type has not been reported in korea until now , to our knowledge . herein , we report a middle - aged korean man with both ta and uc . a 35-year - old man visited our outpatient clinic in june 2010 , presenting with pain and swelling of the right neck for 6 months . he was diagnosed with uc at his age of 25 years and had taken 2250 mg of mesalazine daily . his chest x - ray was normal , but thyroid sonogram showed severe thickening of both carotid intima and media , and ulceration at the mid - portion of the right carotid artery ( fig . laboratory data were as follows : white blood cell count , 9550/mm with 67.9% neutrophils ; hemoglobin , 8.73 g / dl ; mean corpuscular volume , 57.2 femtolitre ; mean corpuscular hemoglobin concentration 29.7% ; erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) were high at 69 mm / h and 107.5 mg / l , respectively . the ankle brachial pressure index was normal ( 1.13 for right , 1.15 for left ) . the brachial - ankle pulse wave velocity showed harder arterial stiffness compared with age standard values ( 1479 cm / s for right , 1529 cm / s for left ) . for further evaluation , he was admitted to our hospital in july 2010 . on admission , his resting blood pressure was 110/60 and 100/60 mm hg in the right and left upper limbs , respectively . however , arteriographic narrowing of the aortic branches was diffuse , especially in the right and left external carotid artery ( fig . chest ct angiography showed concentric wall thickening of the ascending thoracic aorta , aortic arch , proximal descending thoracic aorta , as well as the innominate artery , left common carotid , and both subclavian arteries ( fig . he was diagnosed as concurrent ta and uc based on these findings and his history . the patient received prednisolone ( 30 mg / day ) and ferrous sulfate ( 256 mg twice a day ) with mesalazine . since then , several follow - ups were carried out during the following months and the patient 's symptom included right neck swelling and improvement of pain . crp and esr levels declined to 24 mm / h and 14.2 mg / l , respectively . hemoglobin levels were normalized ( 14.6 g / dl ) . during this follow - up period , laboratory data showed : auto antibodies including antinuclear , anti - neutrophil cytoplasmic , anti - dna and antiphospholipid antibodies , which are associated with other vascular diseases , but not ta5 ) were negative . human leukocyte antigen ( hla ) studies showed b51 and b52 , which are usually seen in cases with coexisting uc and ta . there was moderate pancolitis and a polyp ( 7 mm ) in the ascending colon ( fig . a polypectomy was performed and a biopsy showed no dysplasia . with the patient 's improved state , takayasu 's arteritis is a chronic vasculitis of unknown etiology , and usually seen in young asian women . ta , which primarily involves the aorta and its major branches , often occurs initially in the left middle or in the proximal subclavian artery . during the development of disease , both of the common carotid and vertebral arteries , the brachiocephalic artery , the right middle or proximal subclavian artery , and the aorta may be involved . in about 50% of patients , the abdominal aorta and pulmonary arteries are affected . with regard to diagnosis , criteria for the classification of ta have been suggested in 1990 by the american college of rheumatology ( acr ) as : 1 ) onset at age less than or equal to 40 years . 4 ) greater than a 10 mm hg difference in systolic blood pressure between arms . 6 ) arteriographic evidence of narrowing or occlusion of the entire aorta , its primary branches , or large arteries in the proximal upper or lower extremities . the presence of 3 or more of these 6 criteria demonstrated a sensitivity of 90.5% and a specificity of 97.8%.2 ) in this case , the patient had an occasional right upper extremity claudication , but did not have abnormal blood pressure of the right arm . however , for this patient , disease onset occurred at 35 years and he had bruits of the left subclavian artery . additionally , the patient had tenderness of the carotid artery and had an elevated esr and anemia , which are important evidence of ta.2 ) ulcerative colitis , a form of inflammatory bowel disease ( ibd ) , is characterized by relapsing inflammation affecting mostly the mucosal layer and sometimes the submucosal layer of bowel . in general , uc occurs in north america and europe , but the incidence has increased in asia and in other countries , including japan , korea , singapore , and latin america , inferring that industrialization may affects the occurrence of uc.6 ) uc and crohn 's disease ( cd ) , the 2 major types of ibd are sometimes related with other autoimmune diseases and extraintestinal symptoms , including vascular manifestations.7 ) as seen above , the prevalent geographical areas of uc and ta are different , but an overlap of these 2 diseases has been reported in sri lanka , india , pakistan , turkey , and especially in japan . in korea , a case of cd associated with ta was reported,8 ) but this is the first case of coexistent uc and ta . with respect to an autoimmunologic association on the coexistence of uc and ta , hla b52 and dr2 have been found in many japanese cases in which the patient suffered from concurrent uc and ta.9 - 11 ) indeed , our patient was positive for hla b52 , which is the common haplotype seen in cases with both diseases . in recent years , successful treatment of a patient with refractory active ta complicated by uc using anti - interleukin-6 receptor antibody was reported.12 ) in patients with these 2 diseases , there is a tendency that uc is diagnosed earlier than ta . the cause of this tendency is unknown , but there are suggestions that bacterial or viral invasion through the intestinal mucosa may be associated with the pathogenesis of these cases.13 ) like this patient , there are a few cases presenting both diseases even though the possibility is very low from an epidemiological point of view . in this sense , it is possible that they are immunologically related.14 ) in other words , considering that anti - colon antibodies and anti - aortic antibodies have been demonstrated in uc and ta , we can not exclude a possibility that some immunological defect might exist simultaneously for both diseases,15 ) or , we can hypothesize that certain microorganisms trigger chronic autoimmune disease associated with the arterial wall and colonic mucosa of immunologically susceptible individuals.16 ) among these conjectures , this case could be an another instance where an immunological and genetic link might exist between these two diseases , and it could be important for a patient having uc in korea that careful assessment of extraintestinal manifestations should be done with a consideration of autoimmune and vascular diseases including ta . | a 35-year - old korean man with a 10-year history of ulcerative colitis ( uc ) presented with pain and swelling of the right neck .
the patient was diagnosed with takayasu 's arteritis ( ta ) and had human leukocyte antigen ( hla ) b-52 , which is frequently found in patients having both uc and takayasu 's disease concurrently on hla analysis .
this case is the first report of a patient with both ta and uc in korea , to the best of our knowledge . |
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a total of 97 participants participated in the study , 54 native left - to - right readers and 43 right - to - left readers ( table 1 ) . all participants were fluent in english , and seventy - five of the participants , including the 43 right - to - left readers , were bilingual . of the native left - to - right readers , 30 were male ( m = 25.8 years , sd = 5.5 years ) , and of the native right - to - left readers 20 were male ( m = 25.2 years , sd = 5.3 years ) . all procedures received ethical approval from the university of saskatchewan behavioural research ethics board and all participants were recruited from the university of saskatchewan through posters . all participants were paid for their participation in the study.table 1participant s native languageltor reading languagesnumber of participantsrtol reading languagesnumber of participantsenglish22urdu19hindi14persian12chinese6arabic8bengali3aramaic1korean2hebrew1malayalam2pashto1chichewa1punjabi1oriya1ukrainian1russian1vietnamese1the native languages of left - to - right ( ltor ) readers and the corresponding number of participants , as well as the native languages of right - to - left ( rtol ) readers and the corresponding number of participants participant s native language the native languages of left - to - right ( ltor ) readers and the corresponding number of participants , as well as the native languages of right - to - left ( rtol ) readers and the corresponding number of participants participants simultaneously viewed two greyscales stimuli ( fig . 1 ) on a 17 inch crt monitor . the horizontal midlines of the stimuli were aligned with the middle of the screen with a vertical distance of 100 pixels between the upper and lower stimulus . two reversed luminance gradients that changed in brightness from one end to the other were outlined by a thin black outline and shown against a grey background . the stimuli measured 79 pixels high and changed in brightness over 80 increments , creating stimuli changing from black at one end to white at the other . the vertical position of the pixels within each increment was randomized to create a smooth change in brightness and create slight differences in the stimuli . the rectangles were presented as mirror reversals one on top of the other , but were equiluminant at a global level . the stimuli were presented in six different lengths : 320 , 400 , 480 , 560 , 640 , and 720 pixels.fig . 1sample stimulus pairs with opposite orientations from the greyscales task . both a and b are 400 pixels long , but stimuli a is positioned with the upper stimulus dark on left and lower stimulus dark on right where as stimuli b is positioned with the upper stimulus dark on the right and lower stimulus dark on the left . a left response results from the participant choosing the stimulus with the darker feature on the left , irrespective of whether the stimulus is on the top or bottom sample stimulus pairs with opposite orientations from the greyscales task . both a and b are 400 pixels long , but stimuli a is positioned with the upper stimulus dark on left and lower stimulus dark on right where as stimuli b is positioned with the upper stimulus dark on the right and lower stimulus dark on the left . a left response results from the participant choosing the stimulus with the darker feature on the left , irrespective of whether the stimulus is on the top or bottom the task consisted of 96 trials that were presented in a pseudo - randomized order . the combinations of length ( 320 , 400 , 480 , 560 , 640 , and 720 pixels ) and stimulus orientation ( upper stimulus dark on left / lower stimulus dark on right and vice versa ) were repeated four times . participants were seated 500 mm from the computer with the center of the monitor located along their midline . participants were asked to determine which stimulus appeared darker overall , and responses were made using the numbers 8 and 2 on the number pad . a simple button press was used to limit the amount of motor movement evoked by the task ( mccourt and olafson 1997 ) . the participants responses were categorized based on which stimulus they selected as having the darker feature on the left or the right irrespective of whether it was on the top or bottom . a leftward response was indicated when the participant chose the stimulus with the darker feature on the left , irrespective of whether the stimulus was situated on the top or bottom , whereas a rightward response was indicated when the participant chose the stimulus with the darker feature on the right , irrespective of whether the stimulus was situated on the top or bottom . the response bias was calculated by subtracting the number of leftward responses from the number of rightward responses and could range from 96 to + 96 ; hence a negative score indicated a leftward bias . participants were initially seated in a windowless room with overhead lighting and gave written consent to participate in the study . prior to completing the greyscales task , participants completed a demographic questionnaire addressing sex , age , native language , visual or hearing impairments , and handedness and footedness ( elias et al . a total of 97 participants participated in the study , 54 native left - to - right readers and 43 right - to - left readers ( table 1 ) . all participants were fluent in english , and seventy - five of the participants , including the 43 right - to - left readers , were bilingual . of the native left - to - right readers , 30 were male ( m = 25.8 years , sd = 5.5 years ) , and of the native right - to - left readers 20 were male ( m = 25.2 years , sd = 5.3 years ) . all procedures received ethical approval from the university of saskatchewan behavioural research ethics board and all participants were recruited from the university of saskatchewan through posters . all participants were paid for their participation in the study.table 1participant s native languageltor reading languagesnumber of participantsrtol reading languagesnumber of participantsenglish22urdu19hindi14persian12chinese6arabic8bengali3aramaic1korean2hebrew1malayalam2pashto1chichewa1punjabi1oriya1ukrainian1russian1vietnamese1the native languages of left - to - right ( ltor ) readers and the corresponding number of participants , as well as the native languages of right - to - left ( rtol ) readers and the corresponding number of participants participant s native language the native languages of left - to - right ( ltor ) readers and the corresponding number of participants , as well as the native languages of right - to - left ( rtol ) readers and the corresponding number of participants participants simultaneously viewed two greyscales stimuli ( fig . 1 ) on a 17 inch crt monitor . the horizontal midlines of the stimuli were aligned with the middle of the screen with a vertical distance of 100 pixels between the upper and lower stimulus . two reversed luminance gradients that changed in brightness from one end to the other were outlined by a thin black outline and shown against a grey background . the stimuli measured 79 pixels high and changed in brightness over 80 increments , creating stimuli changing from black at one end to white at the other . the vertical position of the pixels within each increment was randomized to create a smooth change in brightness and create slight differences in the stimuli . the rectangles were presented as mirror reversals one on top of the other , but were equiluminant at a global level . the stimuli were presented in six different lengths : 320 , 400 , 480 , 560 , 640 , and 720 pixels.fig . 1sample stimulus pairs with opposite orientations from the greyscales task . both a and b are 400 pixels long , but stimuli a is positioned with the upper stimulus dark on left and lower stimulus dark on right where as stimuli b is positioned with the upper stimulus dark on the right and lower stimulus dark on the left . a left response results from the participant choosing the stimulus with the darker feature on the left , irrespective of whether the stimulus is on the top or bottom sample stimulus pairs with opposite orientations from the greyscales task . both a and b are 400 pixels long , but stimuli a is positioned with the upper stimulus dark on left and lower stimulus dark on right where as stimuli b is positioned with the upper stimulus dark on the right and lower stimulus dark on the left . a left response results from the participant choosing the stimulus with the darker feature on the left , irrespective of whether the stimulus is on the top or bottom the task consisted of 96 trials that were presented in a pseudo - randomized order . the combinations of length ( 320 , 400 , 480 , 560 , 640 , and 720 pixels ) and stimulus orientation ( upper stimulus dark on left / lower stimulus dark on right and vice versa ) were repeated four times . participants were seated 500 mm from the computer with the center of the monitor located along their midline . participants were asked to determine which stimulus appeared darker overall , and responses were made using the numbers 8 and 2 on the number pad . a simple button press was used to limit the amount of motor movement evoked by the task ( mccourt and olafson 1997 ) . the participants responses were categorized based on which stimulus they selected as having the darker feature on the left or the right irrespective of whether it was on the top or bottom . a leftward response was indicated when the participant chose the stimulus with the darker feature on the left , irrespective of whether the stimulus was situated on the top or bottom , whereas a rightward response was indicated when the participant chose the stimulus with the darker feature on the right , irrespective of whether the stimulus was situated on the top or bottom . the response bias was calculated by subtracting the number of leftward responses from the number of rightward responses and could range from 96 to + 96 ; hence a negative score indicated a leftward bias . participants were initially seated in a windowless room with overhead lighting and gave written consent to participate in the study . prior to completing the greyscales task , participants completed a demographic questionnaire addressing sex , age , native language , visual or hearing impairments , and handedness and footedness ( elias et al . the response bias was analyzed with a 2 ( reading direction [ left - to - right , right - to - left ] ) 2 ( sex [ male , female ] ) repeated - measures anova . there was a significant effect for reading direction , f(1 , 93 ) = 8.489 , p = .004 , 2 = .0894 , but no significant effect of sex f(1 , 93 ) = .026 , p = .872 , 2 = .0003 , and no interaction between the two factors , f(1 , 93 ) = .049 , p = .825 , 2 = .0005 . the native left - to - right readers demonstrated a larger response bias ( m = 34.6 , sd = 37.67 ) compared to the right - to - left readers ( m = .99 , sd = 42.06 ) ( fig . 2 ) . one - sample t tests , compared to the test - value of zero , were conducted to evaluate whether a significant side bias was observed ; left - to - right readers demonstrated a leftward response bias , t(53 ) = 5.097 , p < .001 , 95 % ci 48.28 , 21.46 , whereas right - to - left readers failed to demonstrate a significant bias t(42 ) = .109 , p = .914 , 95 % ci 19.50 , 17.45.fig . 2the mean response bias for the greyscales task demonstrated by native left - to - right ( ltor ) and native right - to - left ( rtol ) readers . a negative score indicates a preference for the darkest edge of the equiluminant gradient stimulus pair to be located on the left . error bars represent standard error of the mean the mean response bias for the greyscales task demonstrated by native left - to - right ( ltor ) and native right - to - left ( rtol ) readers . a negative score indicates a preference for the darkest edge of the equiluminant gradient stimulus pair to be located on the left . error bars represent standard error of the mean additionally , the mean reaction time for left and right responses was analyzed with a 2 ( reading direction [ left - to - right , right - to - left ] ) 2 ( response choice [ right , left ] ) mixed - measures anova ( fig . there was no significant difference between the reaction time of left - to - right and right - to - left readers responses , f(1,95 ) = .079 , p = .779 , 2 = .0008 , and no interaction between the two factors , f(1 , 95 ) = .091 , p = .763 , 2 = .0009 , however there was a significant difference between the reaction time for left and right choices , f(1 , 95 ) = 4.029 , p = .048 , 2 = .0424 , with leftward responses ( m = 2755.98 , sd = 1736.18 ) occurring faster than rightward responses ( m = 2939.12 , sd = 1841.54).fig . 3the mean reaction times for left - to - right ( ltor ) and right - to - left ( rtol ) readers left and right response choices . error bars represent standard error of the mean the mean reaction times for left - to - right ( ltor ) and right - to - left ( rtol ) readers left and right response choices . in accord with a converging body of research examining left - to - right and right - to - left readers performance on a variety of visuospatial tasks , the reading groups demonstrated different bias strengths for the greyscales task . consistent with previous research employing the greyscales task , native left - to - right readers in the present study demonstrated a statistically significant leftward bias ( mattingley et al . however , inconsistent with nicholls and roberts ( 2002 ) findings , native right - to - left readers failed to demonstrate a significant leftward bias . . the sample of hebrew readers examined by nicholls and roberts ( 2002 ) may have had increased exposure to left - to - right scanning order , leading to their null results . hebrew children have been found to display weaker right - to - left tendencies compared to arabic children ( kugelmass and lieblich 1979 ) , potentially resulting from the left - to - right notation of arithmetic , music , and writing of some individual letters in hebrew ( vaid and singh 1989 ) . increased exposure to left - to - right notation , especially if hebrew was not nicholls and roberts ( 2002 ) participants native language , would have influenced the participants scanning tendencies and eye movement exploration , increasing the likelihood that a leftward bias will be observed ( abed 1991 ; chokron and imbert 1993 ) . the fact that participants with contrasting native reading directions performed differently on the greyscales task , demonstrates that scanning tendencies influence eye movement exploration and orientation of attention . eye tracking experiments examining left - to - right readers have found reliable initial saccades at the beginning of visual exploration to the left side of a image followed by a longer and weaker bias to the right ( dickinson and intraub 2009 ; foulsham et al . 2014 ) , even in the presence of a right - sided target ( nuthmann and matthias 2013 ) . however , these initial saccades to the left during scene viewing and line bisection task can be manipulated by the shape of a gaze - contingent window ( foulsham et al . limited research has examined right - to - left readers visual exploration with eye tracking , however , smith ( 2013 ) explored eye movements of right - to - left and left - to - right readers during a target finding task and observed that left - to - right readers identified targets the quickest when located in the upper left quadrant , whereas right - to - left readers had near identical identification times for targets located in the upper - right and upper - left quadrants . thus native scanning habits and manipulation of viewing contingencies appear to influence initial fixation and location of attention . the influence of scanning habits on attention asymmetries observed in the current study is consistent with literature examining the influence of scanning habits on visuospatial tasks . left - to - right and right - to - left readers have demonstrated significantly different perceptual biases in line bisection tasks ( chokron et al . 1997 ; chokron and imbert 1993 ; chokron and de agostini 1995 ) ; aesthetic preference tasks ( chokron and de agostini 2000 ; friedrich et al . 2014 ; ishii et al . 2011 ; nachson et al . 1999 ; perez gonzalez 2012 ) , and lighting tasks ( smith and elias 2013 ) . in addition to natural scanning habits developed from reading direction , manipulation of scanning direction during line bisection tasks ( brodie and pettigrew 1996 ) , and face perception ( butler and harvey 2006 ) have also been found to influence the direction and magnitude of the perceptual bias . visuospatial tasks are perceptual , however , the strategies used to complete visuospatial tasks influence orientation of attention across the visual field ( thomas et al . 2014 ) . pesudoneglect is often explained by the preferential activation of the right hemisphere that distributes attention to the left visual field and increases the salience of features located in the left hemispace the activation - orienting hypothesis ( kinsbourne 1970 ) . manipulating orientation of attention with cues during visuospatial tasks systematically biases perception leading participants to commonly overestimate portions of space to which their attention is directed , and thus spatial distribution of attention and attentional asymmetries have been proposed to underlie and influence perceptual biases ( milner et al . furthermore , orientation influences perceptual biases in right unilateral spatial neglect patients , as cues located on the left of visual spatial tasks annul rightward displacement ( nichelli et al . 1989 ) . specifically , the typical leftward response bias observed during the greyscales task is reversed by attentional cues , and the leftward response bias is insensitive to changes in the presentation of the stimuli ( nicholls et al . 2004 ; nicholls et al . these converging results have lead researchers to suggest that the greyscales task is a robust and sensitive measure to attentional biases , which appear , based on the findings in the current experiment , influenced by scanning habits and reading direction . the underlying neurological mechanisms responsible for the increased salience of features located in the left hemispace is currently debated ( de schotten et al . however , right - to - left scanning habits developed from reading direction appears to interact with spatial attention that is oriented to the left hemispace , resulting in no perceptual bias . thus left - to - right scanning habits developed from reading direction strengthen the lateralized bias whereas right - to - left scanning habits weaken the bias . this pattern is evident in the current study as both reading groups responded faster to leftward choices resulting from increased attention to the left hemifield , but the salience of stimulus with the darkest feature located on the left was weakened when participants native language read from right - to - left . hence , the results of the current study lead us to argue that the strength of perceptual biases is influenced by behavioural biases , such as scanning habits , and neural and anatomical asymmetries in the right parietal and frontal cortices ( szczepanski and kastner 2013 ; de schotten et al . 2011 ) .
to further isolate the influence of reading direction and scanning habits on perceptual biases researchers should examine homogenous sample groups to ensure that cultural differences are not contributing to the different response biases found . ideally , research examining left - to - right and right - to - left reading groups should examine groups that have extralinguistic ( e.g. context of usage , direction of arithmetic and music ) and linguistic similarities ( e.g. phonology and grammar ) , as well as a common geographical location and cultural foundation to minimize the confounding effects of cultural influences on reading direction . based on our findings , examining two samples with similar cultural values , but who also differ in reading direction understanding how perceptual asymmetries manifest , regardless of their underlying cause , is essential when studying behaviour as a marker of cognitive processes . patients with right unilateral hemispheric damage have been found to demonstrate a stronger rightward bias during the greyscales task ( mattingley et al . 2004 ) , leading researchers to suggest that the greyscales task is a highly sensitive and efficient tool for assessing pathological perceptual and attentional biases ( mattingley et al . 1994 ) . however , the altered response bias for right - to - left readers compared to left - to - right readers questions the strength and reliability of the leftward bias displayed by healthy participants , as the leftward bias is not consistent and is affected by learned cultural factors such as reading direction . thus , clinicians should exhibit caution when using abnormal performance on the greyscales task to aid diagnosis of neglect , specifically when the patient s native language reads from right - to - left . furthermore , research should examine right - to - left reading populations to determine the normal range of bias scores for right - to - left readers to increase the clinical utility of the greyscales task . | reliable leftward attentional and perceptual biases demonstrated in a variety of visuospatial tasks have been found to deviate from the left in research examining the influence of scanning habits .
the aim of the current research was to examine the influence of native script direction on pseudoneglect during the greyscales task in a representative sample of native right - to - left readers .
fifty - four native left - to - right readers and 43 right - to - left readers completed the greyscales task , which required judging the darker of two left
right mirrored brightness gradients .
native left - to - right readers demonstrated a left response bias on the greyscales task , whereas right - to - left readers failed to demonstrate a bias , however , both groups responded more quickly when making leftward choices .
the research suggests that the strength of attentional biases are influenced by behavioural biases , such as scanning habits , and neural and anatomical asymmetries in the right parietal and frontal cortices .
thus , to improve the clinical utility of the greyscales task for diagnosing neglect , right - to - left readers should be examined to fully understand the normal range of biases displayed by neurologically healthy individuals . |
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s. pneumoniae is associated with a high degree of morbidity and mortality in many countries around the world and is considered the main cause of death of millions children in the transition countries ( 1 ) , which account for up to 70% of deaths ( 2 ) . it is also considered the leading cause of significant mortality and morbidity in children in developed countries , emphasizing the age of less than two years ( 3 ) , which resulted in placing of this bacteria in the unenviable first place within morbidity hierarchy in transition countries ( 1 ) . a growing proportion of s. pneumoniae in the etiology of morbidity and mortality especially within vulnerable groups ( children under 3 years , adults and immunocompromised patients ) is highly correlated with a high incidence of individual ( especially to penicillin ) and multiple antibiotic resistance . resistance of pneumococci leads to changes in the clinical presentation of diseases which in turn leads to more difficult diagnosis and treatment . in addition to resistance to an antibiotic , additional treatment problems are caused by the increase of multiple antimicrobial resistance of certain pneumococcus strains , as a consequence of failure to doctrinal positions therapy ( therapy ex juvantibus ) and the implementation of the same without susceptibility testing . also , great contribution to this is given by the massive , unjustified use of antibiotics . unfortunately , in our country we do not have relevant data on the status of resistance , as well as the morbidity and mortality caused by it . the resistance may result from several different mechanisms , such as the inactivation of the antibiotic by bacterial enzymes , low permeability to antibiotics bacteria , changes in the target protein , leading to reduced binding of the antibiotic , or overproduction of the target protein structure and bypassing the metabolic pathways of the target ( 4 ) . the intrinsic mechanisms are determined by naturally occurring genes found in the chromosomes of the host . acquired mechanisms include gene mutations and horizontal transfer of resistance determinants resulting in plasmids , bacteriophages , transposomima and other mobile genetic material , leading to genetic differences which help the micro - organism to adapt to environmental influences , including antibiotics ( 5 ) . this type of transfer takes place not only among the members of one strain , but also between evolutionary distant organisms such as gram - negative and gram - positive bacteria . however , the intrinsic mechanisms that are not associated with the mobile elements , such as an efflux pump that removes many antibiotics , are currently recognized as the most significant factors that contribute to bacterial resistance to multiple antibiotics ( 6 ) . the main resistance mechanism of the pneumococci toward protein synthesis inhibitors is based on a modification of the mediated ermb - coding methylase ; efflux pump , mutation of 23s rrna , point mutation of rifampin - binding region of rpob ( 6 ) . the mechanism of resistance to inhibitors of cell wall implies structural changes of penicillin - binding protein 1a , 2x and 2b ( 7 ) . dihydropteroate synthase gene mutation or dihydrofolate reductase is responsible for the occurrence of resistance to folate antagonists ( 6 ) . point mutation of topoisomerase iv ( parc2pare2 ) and dna gyrase ( gyra2gyrb2 ) is the basis for the development of pneumococcal resistance to quinolones ( 8) . the goal of this study was to determine the prevalence of susceptibility / resistance of s. pneumoniae isolates to antibiotics that act on the cell wall synthesis , protein synthesis , folate antagonists and quinolones , in order to achieve proper treatment of pneumococcal disease and reduce morbidity and mortality . this study is of prospective - retrospective and analytical nature conducted by the institute of public health of canton sarajevo in the period from july 1 , 2013 to april 15 , 2014 . samples were swabs of the nose , nasopharynx , eye and ear of outpatients with severe symptoms , but without the same when taking control swabs during enrollment of children in kindergarten or school . the swabs were immediately seeded on blood agar , then incubated for 24h at 37 c with 5% co2 . the isolates were identified by typical appearance of colonies , alpha hemolysis and inhibitory zone around optochin , and the final confirmation of pneumococcal isolates was performed by specific serum agglutination . if the laboratory finding was confirmed that the tested isolate is s. pneumoniae , susceptibility was made . routinely it is made by disk diffusion method of susceptibility testing , and for invasive isolates was also determined the value of the mic for penicillin . in 74.9% of isolated streptococcus pneumonia susceptibility was reported to the tested antimicrobials , while resistance was present in 25.06% figure 1 provides percentage display of susceptibility and resistance of s. pneumoniae to the groups of antibiotics . s. pneumoniae showed the highest frequency of susceptibility to inhibitors of protein synthesis in 44.63% , followed by cell wall synthesis inhibitors and quinolones , with representation of the susceptibility of 13.52% and 10.99% , respectively . the lowest frequency of the susceptibility s. pneumoniae showed to folate antagonists in 5.80% . the susceptibility and resistance of s. pneumoniae to tested groups of antibiotics . resistance of s. pneumoniae is the most prominent to inhibitors of protein synthesis ( 10.93% ) , little bit less to the cell wall synthesis inhibitors ( 8.70% ) , and folate antagonists ( 5.31% ) , and at least to the quinolone ( 0.12% ) . to calculate yates correction factor , in order to correlate the individual antibiotics groups , compared are data on the susceptibility and resistance to inhibitors of cell wall synthesis with data on the susceptibility and resistance to inhibitors of protein synthesis . the resulting value of yates factors for data on susceptibility and resistance to inhibitors of cell wall synthesis and data on susceptibility and resistance to inhibitors of protein synthesis , is 45.3853 ( df = 1 , p = 0.0001 ) , based on which we can conclude that there is an statistical correlation . the compared data are also obtained on the susceptibility and resistance to folate antagonists , the data obtained on the susceptibility and resistance to quinolones , and yates correction factor amounted 103.6112 ( df = 1 , p = 0.0001 ( s ) ) ; on what basis it was concluded that there is an extreme statistical correlation . from figure 2 it can be seen that from the overall susceptibility of s. pneumoniae to all tested antibiotics , inhibitors of cell wall synthesis had participation of 18.04% , while the inhibitors of protein synthesis at the same time had the highest participation in susceptibility or 59.56% . susceptibility to folate antagonists has been represented in the lowest percentage or 7.74% , while quinolones had a share of 14.66% . of the total resistance to all tested antibiotics , inhibitors of cell wall synthesis had participation of 34.73% , while the protein synthesis inhibitors also had a major presence in the resistance or 43.60% . resistance to antagonists of folate was present in 31.18% of the sample , while quinolones had the lowest participation of only 0.49% . figure 3 shows the relationship of one antibiotic susceptibility representation within the overall susceptibility , according to the representation of resistance within the overall resistance . the ratio of susceptibility / resistance of certain antibiotics as compared to total susceptibility / resistance . the representation of the susceptibility of penicillin - g , oxacillin , erythromycin , clindamycin , trimethoprim - sulfamethoxazole , chloramphenicol , ciprofloxacin , rifampin and tetracycline amounted to : 14.17% , 3.87% , 8.16% , 8.16% , 7.74% , 14.74% , 14.66% , 13.92% , 14.58% , respectively . representation of resistance of penicillin - g , oxacillin , erythromycin , clindamycin , trimethoprim - sulfamethoxazole , chloramphenicol , ciprofloxacin , rifampicin and tetracycline amounted to 2% , 32.80% , 20% , 20% , 21.20% , 0.20% , 0.50% , 2.70% , 0.70% , respectively . figure 4 shows the relationship of susceptibility , intermediate susceptibility and resistance of s. pneumoniae to penicillin g whose values are obtained by disk diffusion and e - test . disk diffusion test showed resistance of s. pneumoniae to penicillin - g in 4.44% of cases , with the susceptibility of 95.56% . e - test determined resistance in the 4.44% , intermediate susceptibility at 26.11% and 69.44% susceptibility . intermediate susceptibility to penicillin - g is important in therapeutic treatment for patients because the strains with mentioned type of susceptibility require higher doses and penicillin - g in comparison to the usual ones . the relationship of the results obtained by disk - diffusion method and e - test for penicillin g chi square test results are significantly higher than the critical value which indicates the existence of a statistical difference in the results obtained by the disk - diffusion and e - test . not very low representation of resistance ( 25.06% ) that was observed in this study may be due to inadequate prescription and consumption of medicines , ex juvantibus therapy , predisposed specific serotypes of s. pneumoniae in the development of resistance to antibiotics and their different geographical distribution . when we talk about groups of drugs , the highest susceptibility demonstrated protein synthesis inhibitors with 44.63% , and the lowest folate antagonists . s. pneumoniae showed the resistance to inhibitors of protein synthesis in the highest percentage ( 10.93% ) and the lowest to the quinolone ( 0.12% ) . these results could be used in the selection of empirical therapy if it is not possible to perform susceptibility testing . results of our study showed extremely low resistance of s. pneumoniae to penicillin g , and high on - sulfamethoxazole trimethoprim , clindamycin and erythromycin . this may be a result of the existence of barriers to the use of penicillin g in the treatment of pneumococcal disease , due to fear of the possible dramatic allergic reactions including anaphylactic shock and greater recourse to treatment trimethoprim - sulfamethoxazole infection , erythromycin and clindamycin . the accumulation in the bones , deformity and temporary inhibition of bone growth , permanent damage to the teeth and hepatotoxicity are serious side effects of tetracycline , which are reason for their lower application especially in children under 12 years , pregnant and nursing women , the results of this study suggest that low - level resistance of the aforementioned drug are not surprising ( 9 ) . furthermore , the observed very small resistance frequency of s. pneumoniae to chloramphenicol , the aforesaid and is probably the result of a very low rate of application of the same in the treatment of pneumococcal disease , highlighting the child age , due to significant side effects of heavy use of the drug as well as bone marrow depression ( 9 ) . by e - test is observed a difference in response to treatment of s. pneumoniae with various doses of penicillin - g . this means that strains exhibiting resistance to standard doses of penicillin - g become susceptible to the same drug increased dose in the non - toxic amount of the drug . comparing the results of this study with the results of research zdilar and associates in 2005 ( canton sarajevo ) , were observed variations in the prevalence of susceptibility and resistance of s. pneumoniae to certain drugs . there has been a significant decline in resistance to penicillin g ( from 58% to 4.44% ) , trimethoprim - sulfamethoxazole ( from 71% to 47.78% ) , tetracycline ( 37% to 1.67% ) , chloramphenicol ( with 25% to 0.56% ) . notable are not such a big increase in resistance to erythromycin ( from 37% to 45% ) and a significant increase in the clindamycin ( from 11% to 45% ) . study results showed 98.89% susceptibility of s. pneumoniae to ciprofloxacin , which shows no significant deviation ( in the earlier study susceptibility was in 100% of cases ) . results of this study show some deviations from the results of the study by protekt us conducted on patients in the united states . the same has shown growth of intermediate susceptibility from 12.5% in 2000 - 2001 to 20% in 2003 - 2004 to penicillin , which is significantly less than the rate of intermediate susceptibility obtained in this study , which is 69.44% . protekt us study showed a drop in penicillin resistance in the united states from 26.3% in 2000 - 2001 to 16.5% in 2003 - 2004 . according to this study the largest representation of resistance to penicillin was in south africa ( 87.4% ) , far east ( 63% ) and middle east ( 54% ) . the largest representation was found in france , greece and spain . in seven countries in latin america pnsp rates reached a global level which is 30% ( 10 ) significantly lower incidence of resistance to penicillin g have shown a study conducted in the period from march 2008 to december 2009 in 60 hospitals of 11 asian countries with the rate of resistance to penicillin of 0.7% in tested material ( 11 ) and studies in germany , with an incidence of resistance of 2 % ( 12 ) . a study in italy showed intermediate susceptibility of 30.4% and 0.54% of total resistance ( 13 ) . results of studies conducted in china , taiwan , vietnam and moscow have shown different rates of resistance to erythromycin represented in the following order : 96.4% , 84.95% , 80.7% ( 11 ) , and were significantly higher than those obtained in this study , which amounts to 45% . similar values as our results were observed in the study developed in italy , where the resistance to erythromycin was represented in 42.3% of the tested material ( 13 ) . in the period from 2004 to 2005 , the prevalence of quinolone resistance was the following : in the netherlands 4.4% , poland 4.4% , finland 6.6% and italy 7.2% ( 14 ) , which is significantly higher prevalence of resistance from that found in our study ( 1.11% ) the study conducted in asian countries during the period from march 2008 to december 2009 showed existence of resistance to ciprofloxacin in 1.5% of cases , which is similar to the values obtained in this study ( 11 ) . studies conducted in china , italy and america showed increased presence of pneumococcal resistance compared to our results , by tetracycline in the amount of : 94.3% ( 15 ) 36.9% ( 16 ) and 15.6% ( 17 ) . resistance to trimethoprim - sulfamethoxazole was observed in 55.1% in china ( 15 ) and the same does not differ significantly from our results , while the much lower rate of resistance was observed in america 25,45% ( 16 ) . in comparison with other studies are not noted greater deviations of s. pneumoniae resistance to chloramphenicol , in relation to this study , suggesting or insufficiently developed mechanisms for achieving resistance to the drug or decreasing the use of the same . previously presented results of studies conducted in developed and developing countries indicate that the antimicrobial resistance of s. pneumoniae is global problem . there have been countless variations in the occurrence of serious pneumococcal disease , geographical distribution of invasive serotypes , the representation of resistance of s. pneumoniae to antibiotics and efficacy of therapeutic treatment . studies done in other countries point to a complex relationship between antibiotic use and prevalence of resistance and suggest that serotype distribution which causes invasive infections varies depending on the age , the socio - economic standards and timing of antibiotics administration . unfortunately , there are no relevant data that would pointed to the prevalence and severity of antimicrobial resistance of s. pneumoniae in bosnia and herzegovina and that would allow the proper approach to solving this problem . however , these data indicate that the presence of s. pneumoniae strains resistant to antibiotics of different groups according to the mechanism of action , isolated from samples of nose and nasopharynx , eye and ear of outpatients is in direct correlation with the same resistance in the region . the highest frequency of resistance in isolated pneumococci is recorded with erythromycin ( 20% ) , clindamycin ( 20% ) and trimethoprim - sulphametoxazole ( 21.10% ) , while the lowest incidence of isolated resistant pneumococci is recorded at chloramphenicol ( 0.20% ) , ciprofloxacin ( 0.50% ) , tetracycline ( 0.70% ) , penicillin - g and ( 2% ) and rifampicin - a ( 2.70% ) . e - test differentiated large number of resistant pneumococci as intermediately sensitive , which has implications for further treatment . comparing representation of antibiotics groups in the overall susceptibility and their representation in the total resistance which show therapeutic adequacy of the same . this point was of great importance , because it approximately can serve as a guide in the selection of a therapeutic agent in the case of impossibility of making susceptibility testing or therapeutic coverage waiting period . for example , inhibitors of cell wall synthesis and folate antagonists have significantly greater representation in the overall resistance then representation in the overall susceptibility and are therapeutically inadequate . it is evident that the presence of inhibitors of protein synthesis in the overall susceptibility is very high , but that also has a much significant share in the overall resistance . in order to reduce mortality , morbidity and the presence of pneumococcal antimicrobial resistance in bosnia and herzegovina , it should be made typing of invasive and resistant strains of s. pneumoniae and according to the results make vaccines appropriate for our region . the success of the vaccination program will not only lead to a reduction in the incidence of pneumococcal disease , but will also result in reduced use of antibiotics , used as a primary treatment . it will also reduce the occurrence of antimicrobial resistance to penicillin , -lactams and macrolides , which are commonly used antibiotics in the last thirty years . the results obtained in this study indicate the existence of a great need for rational use of antibiotics and establishing adequate monitoring patterns of pneumococcal resistance . physicians should keep local and regional patterns of resistance at the consideration during the selection of empirical therapy for diseases caused by this agent . for further control of the resistance development necessary is a multidisciplinary approach that includes the clinicians , epidemiologists , microbiologists and pharmacists . | introduction : pneumococcal infections are a major cause of morbidity and mortality worldwide , whose treatment is threatened with an increase in the number of strains resistant to antibiotic therapy.goal:the main goal of this research was to investigate the presence of antimicrobial susceptibility / resistance of s. pneumoniae.material and methods : taken are swabs of the nose and nasopharynx , eye and ear . in vitro tests that were made in order to study the antimicrobial resistance of pneumococci
are : disk diffusion method and e-test.results:the resistance to inhibitors of cell wall synthesis was recorded at 39.17% , protein synthesis inhibitors 19.67% , folate antagonists 47.78% and quinolone in 1.11% .
s. pneumoniae has shown drug resistance to erythromycin in 45% , clindamycin in 45% , chloramphenicol0.56% , rifampicin6.11% , tetracycline4.67% , penicillin - g in 4.44% , oxacillin in 73.89% , ciprofloxacin in 1.11% and trimethoprim - sulfamethoxazole in 5.34% of cases.conclusion:the highest resistance pneumococcus showed to erythromycin , clindamycin and trimethoprim - sulfamethoxazole and these should be avoided in the treatment .
the least resistance pneumococcus showed to tetracycline , rifampicin , chloramphenicol , penicillin - g and ciprofloxacin . |
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according to the inflammatory hypothesis of ad , chronic cerebral inflammation results in injury to neurons , contributing over time to cognitive decline . neuronal injury is hypothesized to result from the direct effects of inflammatory effectors , such as cytokines or activated complement , or indirect effects , such as increased production of neurotoxic -ap in response to cytokines or other inflammatory stimuli . originally based on the presence of markers for inflammation in and around neuritic plaques , this hypothesis has generated a large volume of in vitro cellular and molecular data indicating a variety of possible mechanisms for inflammatory injury to the ad brain . further , a number of epidemiologic studies indicate that anti - inflammatory medications may protect against ad . the inflammatory hypothesis of ad has developed in parallel with the oxidative injury hypothesis of ad , which states that oxidation of macromolecules by oxygen free radicals in ad results in neuronal injury and death . good evidence now exists for oxidative damage to the ad brain . a corollary to the oxidative injury hypothesis is that nitric oxide ( no ) and/or its highly reactive derivative peroxynitrite also play a role in cell injury or death in ad . peroxynitrite is currently thought to be a principal means whereby no expression can result , in cytotoxicity . evidence for peroxynitrite - induced nitration of neuronal proteins has been found in the ad brain . reactive oxygen species ( ros ) and reactive nitrogen species are hypothesized in ad to be both extrinsic to neurons ( generated by glial cells ) and intrinsic ( generated by neurons themselves under conditions of oxidative stress , such as -ap toxicity ) . microglia , which are found in and around neuritic plaques in ad , have pivotal roles in the inflammatory , oxidative , and reactive nitrogen hypotheses of neuronal injury in ad . as intrinsic immune effector cells of the brain , in a variety of diseases or disease models microglia secrete and respond to inflammatory cytokines , present antigen , secrete complement and express complement receptors , are phagocytic , show a respiratory burst resulting in production of oxygen free radicals , produce large amounts of reactive nitrogen species , and have a variety of other immune -related functions . -ap is thought to be neurotoxic and to play a key role in the pathophysiology of ad . significantly , -ap induces cultured microglia to produce many agents with the potential to directly or indirectly injure neurons , including inflammatory and chemotactic cytokines , no , and ros . however , -ap - induced increases in microglial production of these factors have been disappointingly modest , on the order of only two to three times control levels . studies using microglial - neuronal cocultures suggest that microglial activity may be important in -ap - mediated neurotoxicity in ad , but data are conflicting as to the mechanism . endotoxin- , cytokine- , or phorbol - ester - stimulated rodent microglia have been convincingly shown to be neurotoxic through no or ros mechanisms . more relevant to ad , meda found that -ap 25 - 35 induced neurotoxicity in microglial - neuronal cocultures , which was attributed to microglial tnf- and reactive nitrogen intermediates . mcmillian used -ap - stimulated mixed astrocyte / microglial / neuronal cultures and found that a nonspecific nitric oxide synthase ( nos ) inhibitor blocked neurotoxicity ; ii et al obtained similar results . in contrast , giulian also induced neurotoxicity with -ap in microglial - neuronal cocultures , but found no evidence of involvement , of no or other free radicals . van muiswinkel found that -ap increased superoxide production by phorbol - esterprimed microglia , but had no effect on no production ( neurotoxicity was not tested ) . incomplete understanding of this basic pathophysiology hinders the development , of drugs targeting glial neurotoxicity . one reason for conflicting results may be that prior studies of -ap - induced microglial neurotoxicity largely ignored important costimulatory agents present in the ad brain . the extracellular environment surrounding neuritic plaques in the ad brain is rich in a variety of proinflammatory agents including cytokines , which are likely to augment the effects of -ap on microglia . it has been reported that interferon- , phorbol ester , and lipopolysaccharide ( lps ) all augment the effects of -ap on microglia and monocytic cells . however our group has focused on two cytokines known to be increased in the central nervous system ( cns ) in ad , macrophage colony - stimulating factor ( m - csf ) and interleukin-1 ( il-1 ) , both of which are microglial activators . m - csf ( produced by neurons , astrocytes , and endothelial cells ) induces proliferation , migration , and activation of microglia . after traumatic brain injury , microglial expression of the m - csf receptor ( c - fms ) is greatly increased . m - csf treatment of microglia induces increased expression of macrophage scavenger receptors ( msrs ) . microglial adhesion to -ap , internalization of -ap , and possibly -ap - induced microglial activation may be mediated by msr class a. -ap also interacts with neuronal receptors for advanced glycation end products ( rages ) to increase neuronal mcsf expression , which causes further microglial activation . neuropathologic studies show increased immunoreactivity for the m - csf receptor ( c - fms ) on microglia in ad brain , and m - csf - labeled neurons colocalize with -ap deposits . m - csf levels in ad cerebrospinal fluid are five times greater than in controls . we found that cerebrospinal fluid tau , a marker for neurodegeneration in ad , is positively correlated with cerebrospinal fluid m - csf in ad ( figure 1 ) . granulocyte - macrophage colonystimulating factor ( gm - csf ) , another astrocyte product , also induces proliferation of microglia . for example , gm - csf can paradoxically induce ramification of cultured microglia , whereas m - csf does not . the proinflammatory cytokine il-1 is thought to play a key role in neuronal injury in ad . il-1 is increased in the brain in ad , and is associated mainly with activated , phagocytic microglia near plaques . il-1 immunoreactive microglia are found near diffuse as well as neuritic plaques , suggesting that il-1 is important in the early stages of plaque formation . il-1 affects expression and processing of beta - amyloid precursor protein.- in the ad brain , the regional distribution of il-1 immunoreactivity strongly parallels -ap deposition . because il-1 ( principally from microglia in the cns ) increases 0ap , then -ap could induce additional il-1 expression via autocrine or paracrine effects , resulting in a positive feedback loop . il-1 potentiates -ap - induced inflammatory cytokine release by glial cells , and may potentiate -ap toxicity . although astrocytes have neuroprotective functions , extensive astrocytic proliferation can inhibit neurite growth , whereas microglial proliferation is associated with cytotoxic activity . finally , il-1 induces microglial inducible macrophage nitric oxide synthase ( inqs ) and the release of ros . because of these multiple pathophysiologic actions , il-1 is fundamental to the cerebral inflammatory state in ad . although under some conditions il-1 may be neuroprotective , existing evidence strongly suggests a negative role for il-1 in ad . we investigated the roles of m - csf and il-1 in -ap - induced activation of microglia and -ap neurotoxicity . treatment of bv-2 microglia with -ap 1 - 40 alone induces a small increase in the expression of il-1 by bv-2 microglia , as previously reported in primary microglia . however , cotreatment of bv-2 cells with -ap 1 - 40 and m - csf results in a dramatic increase in il-1 secretion by these cells ( almost 70 times greater than control ) . compare this with the 1.5 times increase in il-1 expression reported by araujo and cotman using -ap 1 - 42 alone at a similar concentration . m - csf also significantly augments -ap 1 - 40-induced no ( nitrite ) production and inos mrna expression by bv-2 cells . m - csf augmentation of -ap induction of il-6 , a cytokine that promotes astrogliosis and activates microglia , is even more dramatic : over 200 times control values . through proinflammatory effects our results suggest that -ap , m - csf , il-1 , and il-6 form a self - perpetuating neurotoxic cascade in ad . we hypothesize that in ad , -ap ( via microglial rage and msr class ii ) induces microglia to secrete small amounts of il-1 , as our results and the results of others indicate . il-1 then induces astrocytes to express mcsf , which augments ( via c - fins receptors on microglia ) -ap - induced expression of il-1 by microglia , resulting in further m - csf expression by astrocytes . neurons themselves may also secrete m - csf in response to -ap , which may further activate microglia . meanwhile , microglia activated by -ap and m - csf would continue to generate high levels of no and ros , injuring neurons . our results suggest , that m - csf and -ap also induce microglial il-6 production . increased il-6 found in the ad brain could come from either microglia or astrocytes , or both . as we have shown , -ap induces -ap secretion by microglia , so local levels of this stimulus would also increase , leading to further microglia secretion of il-1 , and to additional neuronal m - csf expression . in this way , a self - perpetuating pathophysiologic cascade is initiated . our results show that costimulation of bv-2 cells with -ap 1 - 40 and gm - csf , another colony - stimulating factor produced by astrocytes that activates microglia , does not augment il-1 or no ( nitrite ) production . , we are focusing on microglial production of no , il-1 , il-6 , and ros in response to -ap , il-1 , and m - csf stimulation , and on how these events affect neurons in organotypic hippocampal cultures . organotypic hippocampal cultures contain the full complement of cerebral cell types including neurons , astrocytes , and microglia . hence , they more closely model the intact , brain than do monotypic cultures of neurons or glia . using the reverse transcriptase polymerase chain reaction ( rt - pcr ) , we have found that treatment of organotypic hippocampal cultures with -ap ( 22 m , 24 hours ' treatment . ) and m - csf results in a larger increase in the mrna for il-1 and inos than either agent , alone . m - csf augmentation of -ap - induced il-1 expression can also be detected in conditioned media from organotypic cultures using enzyme - linked immunosorbent , assay ( elisa ) . note that there is no toxicity after 24 hours ' treatment , as assessed by lactic dehydrogenase ( ldh ) in conditioned media . we are currently using immunohistochemical techniques with organotypic cultures to identify the cell type(s ) that show increased synthesis of il-1 and no after treatment with -ap and m - csf . -ap at a dose of 47 m induces a significant increase in ldh in slice culture medium after 72 hours of treatment . we are also examining expression of m - csf and its receptor in transgenic animal models for ad . in these models , mutant human beta - amyloid precursor protein transgenes result in deposition of p - ap in the brain , and a robust glial reaction surrounding these deposits . our hypothesis is that increased -ap deposition in these animals should lead to increased expression of m - csf and possibly its receptor . if our hypothesis is correct , agents that block the effects of m - csf on microglial cells could represent important therapeutic tools for ad . there is substantial evidence that , the chronic inflammatory reaction in ad results in neuronal injury , ultimately leading to cognitive decline . microglia activated by -ap and cofactors such as m - csf are likely to play a major role in generating neurotoxic agents in and around the neuritic plaque lesion . many potential therapeutic agents that could ameliorate the inflammatory reaction in ad are available , including nos inhibitors , agents that block the actions of proinflammatorycytokines , and antioxidants . nos inhibitors with isoform specificity are currently under development and should soon be available for testing . likewise , many anticytokine reagents are currently available , including older agents such as glucocorticoids , nonspecific nonsteroidal agents , and cytokine receptor antagonists , as well as newer agents such as low - molecular - weight cytokine inhibitors , convertase inhibitors , and highly specific cyclooxygenase inhibitors . however , recent , evidence using -ap immunizations and transgenic animals indicates that , the inflammatory response may also have a beneficial response in ad , possibly through catabolism of -ap and other abnormal protein products . therapeutic approaches to attenuating inflammation in ad may need to be precisely targeted to disrupt , deleterious aspects of the inflammatory response , while preserving beneficial effects . | there is increasing evidence that a chronic inflammatory response in the brain in alzheimer 's disease ( ad ) ultimately leads to neuronal injury and cognitive decline .
microglia , the primary immune effector cells of the brain , are thought to be key to this process .
this paper discusses the evidence for inflammation in ad , and describes the mechanism whereby microglia generate neurotoxic cytokines , reactive oxygen species , and nitric oxide .
evidence that the cytokine macrophage colony - stimulating factor ( m - csf ) is an important cofactor in microglial activation in ad is presented .
ongoing work using organotypic hippocampal expiant cultures to model the inflammatory process in the ad brain is also discussed .
potential avenues for therapeutic intervention are outlined . |
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nshap involved a nationally representative probability sample of community - dwelling adults aged 5784 years ( at the time of screening ) , generated from u.s . of 4,017 eligible subjects in the sample , 3,005 ( 1,455 men and 1,550 women ) were interviewed at home between july 2005 and march 2006 , yielding a weighted response rate of 75.5% ( unweighted 74.8% ) . the protocol was approved by the university of chicago and norc institutional review boards ; all respondents gave written informed consent . sex was defined as any mutually voluntary activity with another person that involves sexual contact , whether or not intercourse or orgasm occurs . sexually active respondents were asked about the presence of sexual problems selected on the basis of diagnostic and clinical criteria for sexual dysfunction ( 3 ) . all respondents who had not had sex in the previous 3 months were asked to indicate why from a list of reasons ( 3 ) . a self - administered questionnaire completed during the in - home interview asked about the frequency of masturbation , defined as stimulating your genitals ( sex organs ) for sexual pleasure , not with a sex partner , and ascertained whether orgasm occurred with masturbation . questions about sexual activity and problems were refused by 27% of respondents ; 1213% declined to answer the questions regarding masturbation . individuals were classified as having diagnosed diabetes , regardless of their a1c value , if they responded that they had been told by a physician that they had diabetes or if they were using one or more diabetes medications . to identify individuals with undiagnosed diabetes , we used an a1c cut point of 6.0% based on the correlation of a1c with the traditional fasting glucose criterion in older individuals . this a1c cut point for identifying undiagnosed diabetes was selected based on a sensitivity / specificity analysis of data from the 19992004 u.s . national health and nutrition examination survey for individuals aged 5785 ( 8) . by comparing different a1c cut points with diagnosis of diabetes based on fasting glucose levels , an a1c cut point of 6.0% maximized specificity of the assay for detecting undiagnosed diabetes without compromising sensitivity for all cut points examined between 5.0 and 7.0% ( specificity 0.91 for men and 0.91 for women ; sensitivity 0.68 for men and 0.69 for women ) ( supplementary fig . if their a1c value was < 6.0% and were classified as having undiagnosed diabetes if their a1c value was 6.0% ( supplementary fig . in light of recent changes in international diabetes care guidelines , we also performed sensitivity analyses using the a1c 6.5% cut point criterion and summarize these in the discussion ( 9,10 ) . details of medication data collection by direct observation and medication classification and coding have been described previously ( 11 ) . sixteen percent of all individuals and 26% of those in the analytic sample ( 17 and 24% weighted , respectively ) were taking at least one medication classified as an antidiabetic agent on a regular schedule , like every day or every week . these agents ( and the weighted proportion of individuals in the analytic sample taking them ) included biguanides ( 13.9% ) , sulfonylureas ( 12.5% ) , thiazolidinediones ( 7.2% ) , insulin ( 5% ) , antidiabetes combinations ( 2.6% ) , meglitinides ( 0.7% ) , -glucosidase inhibitors ( 0.3% ) , and miscellaneous antidiabetes agents ( 0.1% ) . of all the individuals using one or more diabetes medications , 95.1% also reported a diabetes diagnosis . of individuals classified as having diagnosed diabetes , 3.9% were classified on the basis of medication data alone . fingerstick dried blood specimens were sought from a random two - thirds of study respondents ( n = 2,494 ) , with a cooperation rate of 84.4% ( n = 2,105 ) ( supplementary fig . a1c was obtained using well - validated dried blood spot methods described previously ( 12,13 ) . physical health was self - rated using the 5-point excellent , very good , good , fair , or comorbidities were assessed using the katz modification of the charlson index ( 14 ) ( diabetes excluded ) , and activities of daily living were assessed using the katz activities of daily living scale ( 15 ) . respondents were asked whether a medical doctor had ever told them they had any of several common diabetes - related complications ( table 1 ) . communication with a physician about sexual matters since age 50 was assessed as described previously ( 3 ) . depressive symptoms were assessed using the 11-item short form of the center for epidemiological studies - depression ( ces - d ) index ( 16 ) , with response options 03 ; a score 9 was considered indicative of a clinically significant level of depressive symptoms , consistent with a threshold of 16 on the 20-item scale ( 17 ) . a1c value was not used as a criterion for classifying individuals as diagnosed diabetes . undiagnosed diabetes : no self - report or diabetes medication but have a1c 6.0 % . no diabetes : no self - report or diabetes medication and a1c < 6.0 . do you consider yourself primarily white or caucasian , black or african american , american indian , asian , or something else ? and do you consider yourself hispanic or latino ? six respondents who reported being both hispanic and black or african american were included in the black group . # health status was self - reported in response to the question , would you say your health is excellent , very good , good , fair , or poor ? specific comorbid conditions were also self - reported in response to the question , has a medical doctor ever told you that you have any of the following conditions ? with the option to choose all that apply . * * the katz modification of the charlson index was used to assess comorbidities . conditions included myocardial infarction , congestive heart failure , peripheral vascular disease , connective tissue disease , ulcer disease , chronic pulmonary disease , cerebrovascular disease , dementia , liver disease , and renal disease . the 11-item iowa short form of the ces - d scale was used to assess depressive symptoms ( 18 ) . the analytic sample consisted of the 1,993 participants for whom diabetes status could be determined based on a1c , medication data , and/or self - report ( supplementary fig . demographic and clinical characteristics were estimated separately within each of the three diabetes status groups by gender . bivariate associations with diabetes status were tested using the rao and scott ( 18 ) correction to the test to account for the survey design . logistic regression was used to model associations between diabetes conditions and sexual activity , behavior , and problems separately by gender . all models were adjusted for age - group ( 5764 , 6574 , and 7585 years ) , depressive symptoms ( ces - d scores < 9 vs. 9 ) , and the modified charlson comorbidity index ( 0 , 12 , and 3 ) except for outcomes with too few individuals in either outcome category to support a fully adjusted model ( 19 ) . education and race were also evaluated as potential confounders of the effect of diabetes status . for outcomes with a small number of individuals in either outcome category , confounding effects of each covariate listed above were evaluated separately ; unadjusted models are presented for these outcomes unless confounding , defined as a change in the odds ratio of 10% , was identified . all analyses accounted for the survey sampling design through incorporation of sampling strata and clusters , as well as weights that adjusted for a differential probability of selection and differential nonresponse . all sex was defined as any mutually voluntary activity with another person that involves sexual contact , whether or not intercourse or orgasm occurs . sexually active respondents were asked about the presence of sexual problems selected on the basis of diagnostic and clinical criteria for sexual dysfunction ( 3 ) . all respondents who had not had sex in the previous 3 months were asked to indicate why from a list of reasons ( 3 ) . a self - administered questionnaire completed during the in - home interview asked about the frequency of masturbation , defined as stimulating your genitals ( sex organs ) for sexual pleasure , not with a sex partner , and ascertained whether orgasm occurred with masturbation . questions about sexual activity and problems were refused by 27% of respondents ; 1213% declined to answer the questions regarding masturbation . individuals were classified as having diagnosed diabetes , regardless of their a1c value , if they responded that they had been told by a physician that they had diabetes or if they were using one or more diabetes medications . to identify individuals with undiagnosed diabetes , we used an a1c cut point of 6.0% based on the correlation of a1c with the traditional fasting glucose criterion in older individuals . this a1c cut point for identifying undiagnosed diabetes was selected based on a sensitivity / specificity analysis of data from the 19992004 u.s . national health and nutrition examination survey for individuals aged 5785 ( 8) . by comparing different a1c cut points with diagnosis of diabetes based on fasting glucose levels , an a1c cut point of 6.0% maximized specificity of the assay for detecting undiagnosed diabetes without compromising sensitivity for all cut points examined between 5.0 and 7.0% ( specificity 0.91 for men and 0.91 for women ; sensitivity 0.68 for men and 0.69 for women ) ( supplementary fig . in light of recent changes in international diabetes care guidelines , we also performed sensitivity analyses using the a1c 6.5% cut point criterion and summarize these in the discussion ( 9,10 ) . details of medication data collection by direct observation and medication classification and coding have been described previously ( 11 ) . sixteen percent of all individuals and 26% of those in the analytic sample ( 17 and 24% weighted , respectively ) were taking at least one medication classified as an antidiabetic agent on a regular schedule , like every day or every week . these agents ( and the weighted proportion of individuals in the analytic sample taking them ) included biguanides ( 13.9% ) , sulfonylureas ( 12.5% ) , thiazolidinediones ( 7.2% ) , insulin ( 5% ) , antidiabetes combinations ( 2.6% ) , meglitinides ( 0.7% ) , -glucosidase inhibitors ( 0.3% ) , and miscellaneous antidiabetes agents ( 0.1% ) . of all the individuals using one or more diabetes medications , 95.1% also reported a diabetes diagnosis . of individuals classified as having diagnosed diabetes , 3.9% were classified on the basis of medication data alone . fingerstick dried blood specimens were sought from a random two - thirds of study respondents ( n = 2,494 ) , with a cooperation rate of 84.4% ( n = 2,105 ) ( supplementary fig . a1c was obtained using well - validated dried blood spot methods described previously ( 12,13 ) . physical health was self - rated using the 5-point excellent , very good , good , fair , or comorbidities were assessed using the katz modification of the charlson index ( 14 ) ( diabetes excluded ) , and activities of daily living were assessed using the katz activities of daily living scale ( 15 ) . respondents were asked whether a medical doctor had ever told them they had any of several common diabetes - related complications ( table 1 ) . communication with a physician about sexual matters since age 50 was assessed as described previously ( 3 ) . depressive symptoms were assessed using the 11-item short form of the center for epidemiological studies - depression ( ces - d ) index ( 16 ) , with response options 03 ; a score 9 was considered indicative of a clinically significant level of depressive symptoms , consistent with a threshold of 16 on the 20-item scale ( 17 ) . a1c value was not used as a criterion for classifying individuals as diagnosed diabetes . undiagnosed diabetes : no self - report or diabetes medication but have a1c 6.0 % . no diabetes : no self - report or diabetes medication and a1c < 6.0 . do you consider yourself primarily white or caucasian , black or african american , american indian , asian , or something else ? and do you consider yourself hispanic or latino ? six respondents who reported being both hispanic and black or african american were included in the black group . # health status was self - reported in response to the question , would you say your health is excellent , very good , good , fair , or poor ? specific comorbid conditions were also self - reported in response to the question , has a medical doctor ever told you that you have any of the following conditions ? with the option to choose all that apply . * conditions included myocardial infarction , congestive heart failure , peripheral vascular disease , connective tissue disease , ulcer disease , chronic pulmonary disease , cerebrovascular disease , dementia , liver disease , and renal disease . the 11-item iowa short form of the ces - d scale was used to assess depressive symptoms ( 18 ) . the analytic sample consisted of the 1,993 participants for whom diabetes status could be determined based on a1c , medication data , and/or self - report ( supplementary fig . demographic and clinical characteristics were estimated separately within each of the three diabetes status groups by gender . bivariate associations with diabetes status were tested using the rao and scott ( 18 ) correction to the test to account for the survey design . logistic regression was used to model associations between diabetes conditions and sexual activity , behavior , and problems separately by gender . all models were adjusted for age - group ( 5764 , 6574 , and 7585 years ) , depressive symptoms ( ces - d scores < 9 vs. 9 ) , and the modified charlson comorbidity index ( 0 , 12 , and 3 ) except for outcomes with too few individuals in either outcome category to support a fully adjusted model ( 19 ) . education and race were also evaluated as potential confounders of the effect of diabetes status . for outcomes with a small number of individuals in either outcome category , confounding effects of each covariate listed above were evaluated separately ; unadjusted models are presented for these outcomes unless confounding , defined as a change in the odds ratio of 10% , was identified . all analyses accounted for the survey sampling design through incorporation of sampling strata and clusters , as well as weights that adjusted for a differential probability of selection and differential nonresponse . all reported estimates are weighted . table 1 summarizes the demographic and health characteristics of the analytic sample , stratified by diabetes status . self - rated health and capacity for activities of daily living were consistently lower , and the prevalence of several diabetes complications and comorbidities were consistently higher for individuals with diagnosed diabetes compared with those with no diabetes , with intermediate results for those with undiagnosed diabetes . men , regardless of age or diabetes status , were more likely than women to be married or living with a partner ( table 1 ) and were significantly more likely than women to be currently sexually active ( table 2 ) . sixty - one percent of men ( 69% of partnered men ) and 33% of women ( 62% of partnered women ) with diagnosed diabetes were currently sexually active . women with diagnosed diabetes were less likely than men with diagnosed diabetes ( adjusted odds ratio [ aor ] 0.28 [ 95% ci 0.160.48 ] ) and other women ( 0.63 [ 0.450.87 ] ) to be sexually active . among sexually active individuals , the majority engaged in sexual activity at least two to three times per month and neither the frequency of sexual activity nor specific partnered sexual behaviors differed by diabetes status or gender . sexual activity and behavior in older men and women stratified by diabetes status * all odds ratios are adjusted for age - group , comorbidities , and depression unless otherwise noted . respondents were asked about this activity or behavior if they reported having sex in the previous 12 months . this question was asked of all respondents by means of a self - administered questionnaire . partnered sexual behaviors did not differ by gender or diabetes status ( table 2 ) . however , adults with diagnosed diabetes were less likely than others to masturbate ( aor 0.50 [ 95% ci 0.320.78 for women ] 0.66 [ 0.440.97 for men ] ) and to experience orgasm with masturbation ( table 3 ) . sexual problems associated with diabetes conditions in sexually active men and women * all odds ratios are adjusted for age - group , comorbidities , and depression unless otherwise noted . respondents were asked about this activity or behavior if they reported having sex in the previous 12 months this question was asked only of respondents who reported at least one sexual problem . # association with diabetes status was significant at p < 0.05 . * * this question was asked of all respondents by means of a self - administered questionnaire . it was asked of everyone who reported masturbation , not exclusively of those who were sexually active . sexual problems were ascertained only for individuals who were sexually active in the prior 12 months ( table 3 ) . men with diagnosed diabetes were more likely than other men to report lack of interest in sex ( aor 1.72 [ 95% ci 1.122.63 ] ) ; among women , interest in sex did not differ by diabetes status . the prevalence of orgasm problems ( inability to climax or climaxing too quickly ) was similarly elevated among men with diagnosed and undiagnosed diabetes compared with men without diabetes , but erectile difficulties were elevated only among men with a diabetes diagnosis ( 2.52 [ 1.534.14 ] ) . among all individuals who had not been sexually active for 3 , men with diagnosed diabetes were more likely than all other groups ( men and women ) to report that they had not had sex because of their own physical health problems ( 60.9 vs. 34.5% for men with undiagnosed diabetes and 39.4% for men with no diabetes , p < 0.001 ; 16.2% of women with diabetes vs. 8.5% with undiagnosed diabetes and 9.2% with no diabetes ) . among women , the common reasons for sexual inactivity were similar between those with diagnosed and no diabetes , but women with undiagnosed diabetes were more likely to report lack of interest as a reason for sexual inactivity ( 54.5% for undiagnosed diabetes vs. 44.9% for diagnosed diabetes and 38.0% for no diabetes , p < 0.05 ) . men with diagnosed diabetes were more than twice as likely ( 46.8% ) as women with diagnosed diabetes ( 18.8% ) to discuss sex with a physician compared with 28.0% of men and only 11.3% of women with undiagnosed diabetes ( supplementary fig . 3a and b , available in an online appendix ) . among those who had discussed sexual matters with a physician , 16.7% of men overall ( 10.0% of men with diagnosed diabetes ) compared with 30.5% of women overall ( 28.4% of women with diagnosed diabetes ) reported that the physician initiated the conversation . approximately one - third of sexually active men and women with sexual problems reported avoiding sex because of problems ( table 3 ) ; this number did not vary by diabetes status . table 1 summarizes the demographic and health characteristics of the analytic sample , stratified by diabetes status . self - rated health and capacity for activities of daily living were consistently lower , and the prevalence of several diabetes complications and comorbidities were consistently higher for individuals with diagnosed diabetes compared with those with no diabetes , with intermediate results for those with undiagnosed diabetes . men , regardless of age or diabetes status , were more likely than women to be married or living with a partner ( table 1 ) and were significantly more likely than women to be currently sexually active ( table 2 ) . sixty - one percent of men ( 69% of partnered men ) and 33% of women ( 62% of partnered women ) with diagnosed diabetes were currently sexually active . women with diagnosed diabetes were less likely than men with diagnosed diabetes ( adjusted odds ratio [ aor ] 0.28 [ 95% ci 0.160.48 ] ) and other women ( 0.63 [ 0.450.87 ] ) to be sexually active . among sexually active individuals , the majority engaged in sexual activity at least two to three times per month and neither the frequency of sexual activity nor specific partnered sexual behaviors differed by diabetes status or gender . sexual activity and behavior in older men and women stratified by diabetes status * all odds ratios are adjusted for age - group , comorbidities , and depression unless otherwise noted . respondents were asked about this activity or behavior if they reported having sex in the previous 12 months . this question was asked of all respondents by means of a self - administered questionnaire . among sexually active individuals , partnered sexual behaviors did not differ by gender or diabetes status ( table 2 ) . however , adults with diagnosed diabetes were less likely than others to masturbate ( aor 0.50 [ 95% ci 0.320.78 for women ] 0.66 [ 0.440.97 for men ] ) and to experience orgasm with masturbation ( table 3 ) . sexual problems associated with diabetes conditions in sexually active men and women * all odds ratios are adjusted for age - group , comorbidities , and depression unless otherwise noted . respondents were asked about this activity or behavior if they reported having sex in the previous 12 months this question was asked only of respondents who reported at least one sexual problem . # association with diabetes status was significant at p < 0.05 . * * this question was asked of all respondents by means of a self - administered questionnaire . it was asked of everyone who reported masturbation , not exclusively of those who were sexually active . sexual problems were ascertained only for individuals who were sexually active in the prior 12 months ( table 3 ) . men with diagnosed diabetes were more likely than other men to report lack of interest in sex ( aor 1.72 [ 95% ci 1.122.63 ] ) ; among women , interest in sex did not differ by diabetes status . the prevalence of orgasm problems ( inability to climax or climaxing too quickly ) was similarly elevated among men with diagnosed and undiagnosed diabetes compared with men without diabetes , but erectile difficulties were elevated only among men with a diabetes diagnosis ( 2.52 [ 1.534.14 ] ) . among all individuals who had not been sexually active for 3 , men with diagnosed diabetes were more likely than all other groups ( men and women ) to report that they had not had sex because of their own physical health problems ( 60.9 vs. 34.5% for men with undiagnosed diabetes and 39.4% for men with no diabetes , p < 0.001 ; 16.2% of women with diabetes vs. 8.5% with undiagnosed diabetes and 9.2% with no diabetes ) . among women , the common reasons for sexual inactivity were similar between those with diagnosed and no diabetes , but women with undiagnosed diabetes were more likely to report lack of interest as a reason for sexual inactivity ( 54.5% for undiagnosed diabetes vs. 44.9% for diagnosed diabetes and 38.0% for no diabetes , p < 0.05 ) . men with diagnosed diabetes were more than twice as likely ( 46.8% ) as women with diagnosed diabetes ( 18.8% ) to discuss sex with a physician compared with 28.0% of men and only 11.3% of women with undiagnosed diabetes ( supplementary fig . 3a and b , available in an online appendix ) . among those who had discussed sexual matters with a physician , 16.7% of men overall ( 10.0% of men with diagnosed diabetes ) compared with 30.5% of women overall ( 28.4% of women with diagnosed diabetes ) reported that the physician initiated the conversation . approximately one - third of sexually active men and women with sexual problems reported avoiding sex because of problems ( table 3 ) ; this number did not vary by diabetes status . data , indicate that two - thirds of men and approximately one - third of women aged 5785 years with diabetes were sexually active . although diabetes was associated with a higher rate of sexual inactivity , those who were active participated in partnered sexual behaviors and activity at a rate similar to that of those without diabetes . as a group , the majority of individuals with diabetes were married or living with a partner , although women with diabetes were more likely to be alone . sexually active adults with diabetes had a similar prevalence of sexual problems , and women were more likely than men to avoid sex because of these problems . however , fewer than one in five women with diagnosed diabetes compared with nearly half of men had discussed sex with a physician . individuals with undiagnosed diabetes , particularly women , were even less likely than others to have discussed sex with a physician . in this study , we combined self - report measures , medication , and biological measures from a population - based probability sample to stratify individuals as having diagnosed , undiagnosed , or no diabetes . there is not yet full agreement about using a1c to diagnose diabetes in older adults ( 9,10,20 ) , but our strategy generated estimated prevalences of diagnosed and undiagnosed diabetes comparable to 20052006 u.s . population estimates using fasting plasma glucose and/or oral glucose tolerance testing for community - residing individuals aged 60 years ( among those with a1c results , 20.5% [ 95% ci 17.524% ] of 901 women and 25% [ 2129% ] of 843 men had diagnosed diabetes , whereas 19% [ 1622% ] of women and 22% [ 1925% ] of men had undiagnosed diabetes ) ( 21 ) . repetition of the analyses shown here using a 6.5% glycosylated hemoglobin threshold for diabetes classification in this population yielded few qualitative differences in the outcomes of interest but did result in a far smaller undiagnosed diabetic group ( 4.7% in women and 5.6% in men ) than found by classification based on traditional diagnostic criteria . using either threshold , as a group , individuals with undiagnosed diabetes are different from those with diagnosed diabetes in two important ways . first , those with undiagnosed diabetes seemed to be earlier in the course of the disease . second , undiagnosed diabetes is a pathophysiological state that lacks the psychological burden and/or social stigma associated with having diagnosed diabetes ( 5 ) . the etiology of sexual problems associated with undiagnosed diabetes ( controlling for other physical and psychological factors known to be associated with sexual problems ) might reflect a predominant physiological mechanism whereas the etiology of sexual problems associated with diagnosed diabetes might be more likely to have an additional , diabetes - specific psychosocial component . although cross - sectional data can not determine the causal direction of such relationships , understanding the sexuality of individuals with undiagnosed compared with that of those with diagnosed and no diabetes can shed light on the pathological mechanisms and the natural history of both diabetes and sexual dysfunction in later life . in this study , aside from the expected higher prevalence of erectile dysfunction in men with diagnosed diabetes ( 55% ) ( 1 ) , the prevalence of many sexual problems did not differ significantly according to diabetes status . dropping out from sexual activity may partly explain the lack of a diabetes association with sexual problems , especially in women . this finding is suggested by the significantly higher prevalence of sexual inactivity among women with diagnosed diabetes compared with that for men and a greater lack of interest in sex among sexually inactive women with diabetes compared with those without . furthermore , women with diabetes ( diagnosed and undiagnosed ) were nearly half as likely as other women to report masturbating , suggesting a reduction in sexual drive that was independent of partner status and of knowledge of the disease . the prevalence of masturbation was also lower in men with diagnosed or undiagnosed diabetes compared with that in men without but was three times higher than in women with diabetes ( 45% ) . interestingly , the rate of erectile dysfunction was not markedly elevated in men with undiagnosed diabetes ( 36 vs. 32% in men without diabetes ) , but the inability to experience orgasm was high and comparable to that of men with diagnosed diabetes ( 29 vs. 16% in men without diabetes ) . this finding suggests that loss of orgasmic function may not only occur as a consequence of erectile dysfunction as described by others ( 22 ) but also may actually precede erectile dysfunction , at least as perceived by some men with diabetes . in women , inability to experience orgasm with masturbation was also significantly higher among those with diagnosed diabetes . physicians who do ask about sexual function tend to engage patients with partners ( 23 ) and focus on male erectile issues for which treatment is readily available . asking about orgasm function in relation to partnered sex and masturbation and expanding these discussions to include women may assist in prevention of downstream sexual problems and personal and interpersonal distress and in earlier diagnosis of diabetes in some individuals . although no pharmacological treatment is approved as a remedy for anorgasmia , interventions such as education to inform the patient that anorgasmia is known to occur for a substantial proportion of sexually active individuals with diabetes , directed masturbation , use of a clitoral pump in women , and discussion of ways to enhance sexual arousal and intimacy can be therapeutic . medications are another important iatrogenic etiology of later - life sexual problems ( 24 ) . glucose - lowering medications are largely thought to improve sexual function by mitigating glycemic - related microvascular damage , as seen in clinical studies of erectile function in men with diabetes ( 4 ) . the stratification strategy used in our study classifies all individuals taking glucose - lowering medications as having diagnosed diabetes . this strategy would tend to underestimate the association between diagnosed diabetes and sexual function , particularly for the subgroup with uncontrolled diabetes . however , other medications used to treat diabetes , including antihypertensive and cholesterol - lowering drugs , may have deleterious effects on sexual function ( 24 ) . because of sample size , this study is limited by an inability to account for the effects of other medications in estimating the association between diabetes status and aspects of sexuality . prospective clinical trials are needed to fully elucidate the effects and interactions of medications on sexual activity and function among middle - age and older adults with diabetes ; virtually nothing is documented about the effects of diabetes medications on sexuality in older women . the prevalence of specific sexual problems was only assessed for those who were sexually active in the prior 12 months , therefore underestimating the prevalence of sexual problems in this population . next , in addition to the expected differences in population prevalence estimates for undiagnosed diabetes , reanalysis using a glycosylated hemoglobin cut point criterion of 6.5% results in differences between the groups for some outcomes , in part because of loss of precision in estimates . for example , the rate of erectile dysfunction was still higher in men with undiagnosed diabetes ( 40.5% ) compared with that of men without diabetes ( 32.1% ) , but the aor comparing these two groups was no longer significant ( 0.63 [ 95% ci 0.251.58 ] ) . no substantive differences were found in diabetes status comparisons among sociodemographic , health , or communication variables . as with virtually all clinical and population - based research on human sexuality , these data were self - reported , although the interview methods are widely accepted as being valid ( 25 ) . use of a population - based probability sample adds to prior knowledge ( 1,2 ) about later - life diabetes and sexuality by disaggregating individuals with undiagnosed or preclinical diabetes from those with no diabetes . this study builds on prior work by filling a void of information about older women 's sexuality and gender differences in sexuality among middle - age and older adults with diabetes . further research should be powered to also look at age - group comparisons . in conclusion , many middle - aged and older adults with diabetes are sexually active . sexual problems are common but are infrequently discussed with physicians , especially among women . physician knowledge about sexuality in relation to diabetes should improve patient education and counseling , as well as the identification of symptoms that could signal undiagnosed disease or a high risk for disease . attention to potentially treatable sexual problems in middle - aged and older adults with diabetes should improve quality of life and enhance overall diabetes management . | objectiveto describe sexual activity , behavior , and problems among middle - age and older adults by diabetes status.research design and methodsthis was a substudy of 1,993 community - residing adults , aged 5785 years , from a cross - sectional , nationally representative sample ( n = 3,005 ) . in - home interviews ,
observed medications , and a1c were used to stratify by diagnosed diabetes , undiagnosed diabetes , or no diabetes .
logistic regression was used to model associations between diabetes conditions and sexual characteristics , separately by gender.resultsthe survey response rate was 75.5% .
more than 60% of partnered individuals with diagnosed diabetes were sexually active .
women with diagnosed diabetes were less likely than men with diagnosed diabetes ( adjusted odds ratio 0.28 [ 95% ci 0.160.49 ] ) and other women ( 0.63 [ 0.450.87 ] ) to be sexually active .
partnered sexual behaviors did not differ by gender or diabetes status .
the prevalence of orgasm problems was similarly elevated among men with diagnosed and undiagnosed diabetes compared with that for other men , but erectile difficulties were elevated only among men with diagnosed diabetes ( 2.51 [ 1.53 to 4.14 ] ) .
women with undiagnosed diabetes were less likely to have discussed sex with a physician ( 11% ) than women with diagnosed diabetes ( 19% ) and men with undiagnosed ( 28% ) or diagnosed ( 47% ) diabetes.conclusionsmany middle - age and older adults with diabetes are sexually active and engage in sexual behaviors similarly to individuals without diabetes .
women with diabetes were more likely than men to cease all sexual activity .
older women with diabetes are as likely to have sexual problems but are significantly less likely than men to discuss them . |
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some studies have shown that elevated triglyceride ( tg ) levels and low levels of high - density lipoprotein cholesterol ( hdl - c ) accelerate the pathogenesis of type 2 diabetes. recently , several studies have investigated the potential effect of genetic factors associated with risk of type 2 diabetes on plasma lipid levels . analyzed the association of polymorphisms in the glucokinase ( hexokinase 4 ) regulator ( gckr ) gene with type 2 diabetes in a case - control study and with fasting blood glucose and tg levels in the general population . the a allele of snp rs780094 was found to be associated with reduced risk of type 2 diabetes and lower levels of fasting plasma glucose , but higher levels of tg , in a japanese population . in the study reported by chen et al . , the authors found that subjects with minor alleles of snps rs2283228 and rs2237892 in the kqt - like subfamily member 1 ( kcnq1 ) gene , which were associated with type 2 diabetes , had higher levels of tg . this evidence suggests that genetic variants associated with diabetes risk may also be potential genetic determinants of plasma lipid levels . a genome - wide association study ( gwas ) conducted in a french case - control study identified a novel type 2 diabetes susceptibility locus on chromosome 10q23.33 , which is located in a gene cluster including an insulin - degrading enzyme ( ide ) , a kinesin - interacting factor 11 ( kif11 ) , and a hematopoietically expressed homeobox protein ( hhex ) . following this discovery , several studies have confirmed this association in british , finnish , japanese , and chinese populations. recently , we have also found that snps rs7923837 and rs1111875 in the ide - kif11-hhex locus at 10q23.33 were independently associated with risk of type 2 diabetes in a chinese population . however , the relationship between ide - kif11-hhex locus and lipid traits in different populations is not clear . therefore , in an effort to evaluate the influence of the polymorphisms in ide - kif11-hhex locus on plasma lipid concentrations , we performed a fine - mapping study by genotyping seven tagging snps at 10q23.33 in 3,281 han chinese subjects to examine the associations of these variants with plasma levels of total cholesterol ( tc ) , tg , hdl - c and low density lipoprotein cholesterol ( ldl - c ) . the subjects in the current study were selected from a community - based non - communicable diseases screening program comprised of more than 50,000 participants in jiangsu province during 2004 and 2008 . subjects were excluded from the study if they had a history of diabetes , hypertension , coronary heart disease or cancer , or fasting plasma glucose 5.6 mmol / l . after providing informed consent , all subjects were interviewed face - to - face using a standard questionnaire that included demographic characteristics , risk factors and disease history . subjects who smoked 1 cigarette per day for over 1 year were defined as smokers , and those who consumed 3 or more alcohol drinks a week for over 6 months were considered as alcohol drinkers . physical examinations , including measurements of height , weight and blood pressure , as well as laboratory tests to measure tc , tg , hdl - c and fasting plasma glucose concentrations , were performed for each participant . sitting blood pressure body mass index ( bmi ) was calculated as weight ( in kilograms ) divided by the square of height ( in meters ) . fasting blood samples for routine laboratory examinations were obtained in the early morning after an overnight fast . all biochemical parameters were measured enzymatically on an auto - analyzer ( hitachi 7180 biochemistry auto - analyzer , japan ) according to the manufacturer 's instructions . for subjects with tg levels < 4.52 mmol / l , ldl - c levels were estimated indirectly using the friedewald 's formula . the institutional review board of nanjing medical university approved the study . based on our previous study , we used a block - based tagging strategy to select tagging snps using haploview 4.2 software according to the hapmap database[http://www.hapmap.org/ , phaseii nov08 , on ncbi b36 assembly , dbsnpb126 ; population : han chinese population ( chb ) and japanese population ( jpt ) ] . the criteria included snps with minor allele frequency ( maf ) 0.10 , hardy - weinberg equilibrium p 0.05 and call rate 95% when using pairwise linkage disequilibrium ( r ) of 0.8 as the threshold for each block . seven tagging snps ( rs7923837 , rs2488075 , rs947591 , rs11187146 , rs5015480 , rs4646957 and rs1111875 ) associated with type 2 diabetes in the ide - kif11-hhex locus at 10q23.33 were included in the current study . genomic dna was isolated from leucocytes of venous blood by proteinase k digestion and phenol / chloroform extraction . genotyping was performed using the taqman openarray genotyping system ( life technologies , carlsbad , ca , usa ) and the iplex sequenom massarray platform ( sequenom , inc . ) . for quality control , associations between the genotypes and plasma lipid concentrations were determined by multiple linear regression analysis with adjustment for age , sex , smoking status , drinking status and bmi . the hardy - weinberg equilibrium was tested by a goodness - of - fit test to compare the observed genotype frequencies with the expected ones among the 3,281 subjects . all statistical analyses were performed using statistical analysis system software version 9.1.3 ( sas institute , cary , nc , usa ) . all tests were two - sided and the significance level was set at p < 0.05 . bmi : body mass index ; hdl : high - density lipoprotein cholesterol ; ldl : low density lipoprotein cholesterol . the subjects in the current study were selected from a community - based non - communicable diseases screening program comprised of more than 50,000 participants in jiangsu province during 2004 and 2008 . subjects were excluded from the study if they had a history of diabetes , hypertension , coronary heart disease or cancer , or fasting plasma glucose 5.6 mmol / l . after providing informed consent , all subjects were interviewed face - to - face using a standard questionnaire that included demographic characteristics , risk factors and disease history . subjects who smoked 1 cigarette per day for over 1 year were defined as smokers , and those who consumed 3 or more alcohol drinks a week for over 6 months were considered as alcohol drinkers . physical examinations , including measurements of height , weight and blood pressure , as well as laboratory tests to measure tc , tg , hdl - c and fasting plasma glucose concentrations , were performed for each participant . sitting blood pressure body mass index ( bmi ) was calculated as weight ( in kilograms ) divided by the square of height ( in meters ) . fasting blood samples for routine laboratory examinations were obtained in the early morning after an overnight fast . all biochemical parameters were measured enzymatically on an auto - analyzer ( hitachi 7180 biochemistry auto - analyzer , japan ) according to the manufacturer 's instructions . for subjects with tg levels < 4.52 mmol / l , ldl - c levels were estimated indirectly using the friedewald 's formula . based on our previous study , we used a block - based tagging strategy to select tagging snps using haploview 4.2 software according to the hapmap database[http://www.hapmap.org/ , phaseii nov08 , on ncbi b36 assembly , dbsnpb126 ; population : han chinese population ( chb ) and japanese population ( jpt ) ] . the criteria included snps with minor allele frequency ( maf ) 0.10 , hardy - weinberg equilibrium p 0.05 and call rate 95% when using pairwise linkage disequilibrium ( r ) of 0.8 as the threshold for each block . seven tagging snps ( rs7923837 , rs2488075 , rs947591 , rs11187146 , rs5015480 , rs4646957 and rs1111875 ) associated with type 2 diabetes in the ide - kif11-hhex locus at 10q23.33 were included in the current study . genomic dna was isolated from leucocytes of venous blood by proteinase k digestion and phenol / chloroform extraction . genotyping was performed using the taqman openarray genotyping system ( life technologies , carlsbad , ca , usa ) and the iplex sequenom massarray platform ( sequenom , inc . ) . for quality control , associations between the genotypes and plasma lipid concentrations were determined by multiple linear regression analysis with adjustment for age , sex , smoking status , drinking status and bmi . the hardy - weinberg equilibrium was tested by a goodness - of - fit test to compare the observed genotype frequencies with the expected ones among the 3,281 subjects . all statistical analyses were performed using statistical analysis system software version 9.1.3 ( sas institute , cary , nc , usa ) . all tests were two - sided and the significance level was set at p < 0.05 . bmi : body mass index ; hdl : high - density lipoprotein cholesterol ; ldl : low density lipoprotein cholesterol . the demographic and biochemical characteristics of the 3,281 subjects included in this study are shown in table 1 . the mean age was 56.58 ( 9.88 ) years and the mean bmi value was 22.12 ( 2.63 ) kg / m . the mean values of tc , hdl - c , ldl - c and tg were 4.40 ( 0.80 ) mmol / l , 1.62 ( 0.38 ) mmol / l , 2.29 ( 0.74 ) mmol / l and 1.09 ( 0.45 ) mmol / l , respectively . among these subjects , 827 subjects ( 25.38% ) were smokers and 635 subjects ( 19.51% ) were drinkers . the observed genotype frequencies for the seven snps were all consistent with the hardy - weinberg equilibrium among 3,281 subjects ( p > 0.05 ) ( table 2 ) . we examined the association between each snp and tc , tg , hdl - c or ldl - c levels , respectively , in an additive model using a linear regression model with adjustment for age , sex , smoking , drinking and bmi ( table 2 ) . we found significant associations between rs7923837 and tg ( p = 0.019 ) , between rs2488075 and rs947591 and tc ( p = 0.041 and 0.018 , respectively ) . as shown in table 3 , the g allele of rs7923837 was associated with a lower tg levels ( 0.031 mmol / l average decrease per g allele ) . similarly , the c allele of rs2488075 and the a allele of rs947591 were both associated with lower tc levels ( 0.058 mmol / l average decrease per c allele and 0.063 mmol / l average decrease per a allele , respectively ) . conditional analysis indicated that rs2488075 and rs947591 were not significant after adjustment with each other , as the two snps were in strong linkage equilibrium ( ld ) ( r=0.734 ) . however , no significant associations were observed between the other four snps ( rs11187146 , rs5015480 , rs4646957 and rs1111875 ) and blood lipid concentrations . we then conducted a stratification analysis for rs7923837 , rs2488075 and rs947591 by age , sex , bmi , smoking , and drinking status . as shown in table 4 , the associations between rs7923837 and tg levels were more evident among subjects of the low age group ( p = 0.009 ) , male subjects ( p = 0.003 ) , non - drinkers ( p = 0.019 ) , and subjects with low bmi ( p = 0.003 ) . the associations between rs2488075 , rs947591 and tc levels were more evident among subjects of low age group ( p = 0.021 and 0.013 , respectively ) , female subjects ( p = 0.048 and 0.032 , respectively ) , non - smokers ( p = 0.004 and 0.002 , respectively ) , non - drinkers ( p = 0.007 and 0.003 , respectively ) and subjects with low bmi ( p = 0.038 and 0.037 , respectively ) . to the best of our knowledge , this is the first study to investigate the association between ide - kif11-hhex polymorphisms and plasma lipid concentrations in a chinese population . of the seven tagging snps at the ide - kif11-hhex locus , we found that rs7923837 was associated with plasma concentrations of tg , and rs2488075 and rs947591 were associated with plasma concentrations of tc . hdl - c : high - density lipoprotein cholesterol ; ldl - c : low density lipoprotein cholesterol ; maf : minor allele frequency ; snp : single nucleotide polymorphism . p value derived from the multiple linear regression with adjustment for sex , age , smoking , drinking and bmi assuming an additive genetic model . rs7923837 is located in the 3-flanking region of the hhex gene , which encodes a transcription factor that is involved in wnt signaling and is critical for hepatic and pancreatic development , . in addition , hhex may regulate -cell development and/or function by activating hepatocyte nuclear factor 1. several studies reported that the association between rs7923837 and type 2 diabetes is mediated through decreased -cell secretory capacity or decreased -cell mass. thus , hhex is critical for insulin signaling and islet function . hhex may influence metabolic phenotypes such as tg and tc because insulin is necessary for the regulation of metabolic phenotypes . on chromosome 10q23.33 , genetic variants in hhex have been established as susceptibility loci for type 2 diabetes . in our previous fine - mapping study , we reported that rs7923837 and rs1111875 were independently associated with risk of type 2 diabetes in a chinese population . found that the type 2 diabetes risk - associated g allele of rs7923837 was associated with higher pediatric bmi in european american children . cruz et al . analyzed the association between the hhex rs5015480 and risk of metabolic syndrome ( ms ) in a case - control study from mexico city and found that rs5015480 was significantly associated with ms . taken together , this evidence suggests that genetic variants in the ide - kif11-hhex gene cluster at 10q23.33 may contribute to metabolism - related traits and diseases , including circulating lipid levels and diabetes risk . genetic variants associated with the risk of type 2 diabetes have been found to influence plasma tg levels . the variant rs780094 in gckr was associated with a decreased risk of type 2 diabetes , but with higher tg levels , in a gwas of european population . similarly , in the current study , we found that the risk allele of diabetes was associated with lower tg levels . considering that the subjects included in this study were from a healthy population , the lipid level - related variant might interpret the variation of lipid levels of baseline . the relationships between the same variant and diabetes risk and lipid levels implies that low lipid baseline levels in some subjects are , in part , genetically determined , causing them to be more susceptible to type 2 diabetes . this was also supported by the results from the stratification analysis , which showed that the associations between genetic variants at 10q23.33 and lipid levels were more evident among young subjects , non - smokers , non - drinkers and subjects with low bmi . however , the underlying mechanism remains unclear , and further studies are needed to elucidate the roles of genetic variants at 10q23.33 in circulating lipid levels . c / c , c / r and r / r represent homozygotes for the common allele , and heterozygotes and homozygotes for the rare allele , respectively . p value for the multiple linear regression with adjustment for sex , age , smoking , drinking and bmi assuming an additive genetic model p value for the multiple linear regression with adjustment for sex , age , smoking , drinking and bmi assuming an additive genetic model ( the stratified factor in each stratum was excluded ) . the study by hao et al . suggested that plasma cholesterol plays a direct role in pancreatic islet dysfunction and may be a key factor underlying the progression of type 2 diabetes . genetic variants that are associated with the risk of type 2 diabetes have also been found to influence plasma tc levels . revealed a significant association between rs10885409 in tcf7l2 with type 2 diabetes and tc levels in asian indians . chen et al . found rs2237895 in kcnq1 , which was thought to be a candidate gene of diabetes that influenced plasma tc levels in the han chinese population . they argued that this variant might result in an increased expression of kcnq1 and a subsequent increase in insulin secretion , which could stimulate lipid synthesis . in this study , we also found that rs2488075 and rs947591 were associated with plasma tc levels . rs2488075 and rs947591 are located downstream of hhex , which may affect metabolic phenotypes . however , the mechanism that allows the cc genotype in rs2488075 and the aa genotype in rs947591 to contribute to lower levels of tc is still unknown first , the number of subjects in our study was moderate , thus the statistical power was limited . second , the associations were not strong statistically ; none of them passed multiple correction . third , bias and reverse causation can not be completely excluded in this observational epidemiological study . a mendelian randomization approach that uses the random inheritance of genetic variants from parents to offspring may be of benefit in further studies . thus , larger , well - designed epidemiological studies with ethnically diverse populations are warranted to confirm our findings . in summary , the results in the current study indicate that genetic variants in the ide - kif11-hhex gene cluster at 10q23.33 are associated with plasma lipid levels in the chinese population . these findings highlight the important correlation between lipid levels and diabetes development at the genetic level . | plasma lipid abnormalities are implicated in the pathogenic process of type 2 diabetes .
the ide - kif11-hhex gene cluster on chromosome 10q23.33 has been identified as a susceptibility locus for type 2 diabetes .
we hypothesized that genetic variants at 10q23.33 may be associated with plasma lipid concentrations .
seven tagging single nucleotide polymorphisms ( snps : rs7923837 , rs2488075 , rs947591 , rs11187146 , rs5015480 , rs4646957 and rs1111875 ) at 10q23.33 were genotyped in 3,281 subjects from a han chinese population , using the taqman openarray and sequenom massarray platforms .
multiple linear regression analyses showed that snp rs7923837 in the 3-flanking region of hhex was significantly associated with triglyceride levels ( p = 0.019 , 0.031
mmol / l average decrease per minor g allele ) and that rs2488075 and rs947591 in the downstream region of hhex were significantly associated with total cholesterol levels ( p = 0.041 , 0.058
mmol / l average decrease per minor c allele and p = 0.018 , 0.063
mmol / l average decrease per minor a allele , respectively ) .
however , the other four snps ( rs11187146 , rs5015480 , rs4646957 and rs1111875 ) were not significantly associated with any plasma lipid concentrations in this chinese population .
our data suggest that genetic variants in the ide - kif11-hhex gene cluster at 10q23.33 may partially explain the variation of plasma lipid levels in the han chinese population .
further studies are required to confirm these findings in other populations . |
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recruitment to the whitehall ii study took place between 1985 and 1988 among all office staff , aged 3555 years , in 20 london - based civil service departments ( 22 ) . informed consent was obtained from all participants , and the university college london medical school committee on the ethics of human research approved the protocol . the target population for the current study was a sample of 5,932 participants ( 4,189 men and 1,743 women , mean age 54.6 years ) for whom data were collected in at least two of the following cycles : from 19911993 to 19971999 , from 19971999 to 20022004 , and from 20022004 to 20072009 . included participants had complete data on type 2 diabetes , psychological distress , frs ( 21 ) , and covariates ( age , sex , socioeconomic status [ ses ] , ethnicity , antidepressant use , smoking , and physical activity ) at baseline . those included in the analyses were free of diabetes at the baseline cycle(s ) and had data on their prediabetes status and frs ( fig . 1 ) . each participant could thus contribute to a minimum of one and a maximum of three cycles . the 5,932 eligible participants produced 13,207 person - observations ; mean follow - up time between baseline and follow - up for each cycle was 5.46 ( sd 0.51 ) years . , venous blood samples were taken from individuals who were fasting 8 h ( those whose clinic visit was in the afternoon had a light fat - free breakfast and they were asked to fast for 5 h ) before undergoing a standard 2-h 75-g oral glucose tolerance test ( ogtt ) ( 1 ) . mmol / l ( 2326 ) , self - reported doctor - diagnosed diabetes , or use of diabetes medication . blood samples were handled at all phases using similar standard protocols , and baseline cases were excluded from the prospective analyses . prediabetes was defined as impaired fasting glucose ( ifg ; a fasting glucose of 5.66.9 mmol / l and 2-h glucose < 7.8 mmol / l ) and/or impaired glucose tolerance ( a fasting glucose < 7 mmol / l and a 2-h postload glucose of 7.811.0 the frs is based on a previously published detailed algorithm ( 21 ) with the following components : ifg , overweight or obesity , low level of hdl , high level of triglycerides , elevated blood pressure or antihypertensive medication , and parental diabetes . , participants with impaired glucose tolerance received the same score as those with ifg ( 10 points in both cases ) . in the framingham offspring study , participants with > 19 points had an 8-year incidence of type 2 diabetes > 15% ( 21 ) ; thus we used this score to determine high risk status . participants with normoglycemia scored 018 in the frs and were further classified into the low - risk group ( 09 points ) and intermediate - risk group ( 1019 ) , the latter range corresponding to the lower ( i.e. , intermediate)-risk group ( 1019 points ) among participants with prediabetes . based on the baseline information on prediabetes status and the frs , participants were classified into four groups : 1 ) no prediabetes , low frs ( 09 ) ; 2 ) no prediabetes , intermediate frs ( 1019 ) ; 3 ) prediabetes , intermediate frs ( 1019 ) ; and 4 ) prediabetes , high frs ( > 19 ) . the 30-item general health questionnaire ( ghq-30 ) was used to assess psychological distress ( 27 ) . the ghq is a screening instrument designed to detect common psychological symptoms , such as depression and anxiety . it is widely used in population - based surveys and trials and has been validated in the whitehall ii study ( 28 ) . each questionnaire item inquires about a specific symptom ; response categories are scored as either 1 or 0 to indicate presence or absence of the symptom . a total score of 5 or more led to individuals being defined as ghq - symptom cases and scores 04 as noncases ( 28 ) . sociodemographic covariates included age , sex , ses ( based on the last known occupational grade and divided into high , intermediate , and low ) , and ethnicity ( white , south asian , or other ) and were all based on survey responses ( 22 ) . antidepressant use ( yes / no ) and smoking ( yes / no ) were based on self - reported information at the baseline survey of each cycle . physical activity was assessed at cycle 1 based on answers to questions about the frequency and duration of participation in mildly energetic ( e.g. , weeding , general housework , bicycle repair ) , moderately energetic ( e.g. , dancing , cycling , leisurely swimming ) , and vigorous physical activity ( e.g. , running , hard swimming , playing squash ) . at cycles 2 and 3 , the questionnaire included 20 items on frequency and duration of participation in different physical activities ( e.g. , walking , cycling , sports ) that were used to compute hours per week of each intensity level . participants were classified as active ( > 2.5 h / week of moderate physical activity or > 1 h / week of vigorous physical activity ) , inactive ( < 1 h / week of moderate physical activity and < 1 h / week of vigorous physical activity ) , or moderately active ( if not active or inactive ) ( 29 ) . after harmonization of data across cycles , we examined associations between psychological distress at baseline and incident type 2 diabetes at follow - up for each cycle . we used generalized estimation equations ( gees ) with a logistic link to control for intraindividual correlation between repeated measurements to estimate odds ratios ( ors ) and their 95% cis . gee analysis was used because repeated measurements were nested within participants ( i.e. , the same individuals could contribute more than one observation to the dataset ) , and the gee method takes into account nonindependence of the within - participant observations when estimating standard errors . we first analyzed the association between psychological distress and the incidence of type 2 diabetes in the total cohort . then we grouped the participants according to their baseline prediabetes status , frs , and psychological distress into eight groups as follows : 1 ) normoglycemia , frs 09 , no distress ( reference group ) ; 2 ) normoglycemia , score 09 , distress ; 3 ) normoglycemia , score 1019 , no distress ; 4 ) normoglycemia , score 1019 , distress ; 5 ) prediabetes , score 1019 , no distress ; 6 ) prediabetes , score 1019 , distress ; 7 ) prediabetes , score > 19 , no distress ; and 8) prediabetes , score > 19 , distress . models were adjusted for age , sex , ses , ethnicity , antidepressant use , smoking , and physical activity . sas version 9.2 ( sas , cary , nc ) was used for all analyses . recruitment to the whitehall ii study took place between 1985 and 1988 among all office staff , aged 3555 years , in 20 london - based civil service departments ( 22 ) . informed consent was obtained from all participants , and the university college london medical school committee on the ethics of human research approved the protocol . the target population for the current study was a sample of 5,932 participants ( 4,189 men and 1,743 women , mean age 54.6 years ) for whom data were collected in at least two of the following cycles : from 19911993 to 19971999 , from 19971999 to 20022004 , and from 20022004 to 20072009 . included participants had complete data on type 2 diabetes , psychological distress , frs ( 21 ) , and covariates ( age , sex , socioeconomic status [ ses ] , ethnicity , antidepressant use , smoking , and physical activity ) at baseline . those included in the analyses were free of diabetes at the baseline cycle(s ) and had data on their prediabetes status and frs ( fig . 1 ) . each participant could thus contribute to a minimum of one and a maximum of three cycles . the 5,932 eligible participants produced 13,207 person - observations ; mean follow - up time between baseline and follow - up for each cycle was 5.46 ( sd 0.51 ) years . at each clinical phase , venous blood samples were taken from individuals who were fasting 8 h ( those whose clinic visit was in the afternoon had a light fat - free breakfast and they were asked to fast for 5 h ) before undergoing a standard 2-h 75-g oral glucose tolerance test ( ogtt ) ( 1 ) . mmol / l ( 2326 ) , self - reported doctor - diagnosed diabetes , or use of diabetes medication . blood samples were handled at all phases using similar standard protocols , and baseline cases were excluded from the prospective analyses . prediabetes was defined as impaired fasting glucose ( ifg ; a fasting glucose of 5.66.9 mmol / l and 2-h glucose < 7.8 mmol / l ) and/or impaired glucose tolerance ( a fasting glucose < 7 mmol / l and a 2-h postload glucose of 7.811.0 mmol / l ) ( 23 ) . the frs is based on a previously published detailed algorithm ( 21 ) with the following components : ifg , overweight or obesity , low level of hdl , high level of triglycerides , elevated blood pressure or antihypertensive medication , and parental diabetes . , participants with impaired glucose tolerance received the same score as those with ifg ( 10 points in both cases ) . in the framingham offspring study , participants with > 19 points had an 8-year incidence of type 2 diabetes > 15% ( 21 ) ; thus we used this score to determine high risk status . participants with normoglycemia scored 018 in the frs and were further classified into the low - risk group ( 09 points ) and intermediate - risk group ( 1019 ) , the latter range corresponding to the lower ( i.e. , intermediate)-risk group ( 1019 points ) among participants with prediabetes . based on the baseline information on prediabetes status and the frs , participants were classified into four groups : 1 ) no prediabetes , low frs ( 09 ) ; 2 ) no prediabetes , intermediate frs ( 1019 ) ; 3 ) prediabetes , intermediate frs ( 1019 ) ; and 4 ) prediabetes , high frs ( > 19 ) . the 30-item general health questionnaire ( ghq-30 ) was used to assess psychological distress ( 27 ) . the ghq is a screening instrument designed to detect common psychological symptoms , such as depression and anxiety . it is widely used in population - based surveys and trials and has been validated in the whitehall ii study ( 28 ) . each questionnaire item inquires about a specific symptom ; response categories are scored as either 1 or 0 to indicate presence or absence of the symptom . a total score of 5 or more led to individuals being defined as ghq - symptom cases and scores 04 as noncases ( 28 ) . sociodemographic covariates included age , sex , ses ( based on the last known occupational grade and divided into high , intermediate , and low ) , and ethnicity ( white , south asian , or other ) and were all based on survey responses ( 22 ) . antidepressant use ( yes / no ) and smoking ( yes / no ) were based on self - reported information at the baseline survey of each cycle . physical activity was assessed at cycle 1 based on answers to questions about the frequency and duration of participation in mildly energetic ( e.g. , weeding , general housework , bicycle repair ) , moderately energetic ( e.g. , dancing , cycling , leisurely swimming ) , and vigorous physical activity ( e.g. , running , hard swimming , playing squash ) . at cycles 2 and 3 , the questionnaire included 20 items on frequency and duration of participation in different physical activities ( e.g. , walking , cycling , sports ) that were used to compute hours per week of each intensity level . participants were classified as active ( > 2.5 h / week of moderate physical activity or > 1 h / week of vigorous physical activity ) , inactive ( < 1 h / week of moderate physical activity and < 1 h / week of vigorous physical activity ) , or moderately active ( if not active or inactive ) ( 29 ) . after harmonization of data across cycles , we examined associations between psychological distress at baseline and incident type 2 diabetes at follow - up for each cycle . we used generalized estimation equations ( gees ) with a logistic link to control for intraindividual correlation between repeated measurements to estimate odds ratios ( ors ) and their 95% cis . gee analysis was used because repeated measurements were nested within participants ( i.e. , the same individuals could contribute more than one observation to the dataset ) , and the gee method takes into account nonindependence of the within - participant observations when estimating standard errors . we first analyzed the association between psychological distress and the incidence of type 2 diabetes in the total cohort . then we grouped the participants according to their baseline prediabetes status , frs , and psychological distress into eight groups as follows : 1 ) normoglycemia , frs 09 , no distress ( reference group ) ; 2 ) normoglycemia , score 09 , distress ; 3 ) normoglycemia , score 1019 , no distress ; 4 ) normoglycemia , score 1019 , distress ; 5 ) prediabetes , score 1019 , no distress ; 6 ) prediabetes , score 1019 , distress ; 7 ) prediabetes , score > 19 , no distress ; and 8) prediabetes , score > 19 , distress . models were adjusted for age , sex , ses , ethnicity , antidepressant use , smoking , and physical activity . sas version 9.2 ( sas , cary , nc ) was used for all analyses . table 1 shows the characteristics of participants at the baseline of each study cycle and in total . the proportion of participants who were male and white , had high ses , were without psychological distress , and had incident type 2 diabetes was greater at the last cycle than at the first . antidepressant use was more prevalent and smoking less prevalent and both high and low physical activity were more prevalent in the latter cycles . characteristics of the participants at the baseline of the three cycles figures are number ( % ) unless otherwise stated . * total n refers to the sum of participants ( n of person - observations ) in total and across the three study cycles ( one participant can contribute to one or more study cycle ) ; n in each study cycle refers to number of participants at that cycle . associations between baseline covariates for participants with normoglycemia and prediabetes by psychological distress are presented in supplementary table 1 . irrespective of the participant s prediabetes status , psychological distress was associated with younger age , female sex , intermediate ses , nonwhite ethnicity , antidepressant use , smoking , and low physical activity . in the total cohort , psychological distress did not predict type 2 diabetes after adjustment for age , sex , ses , and ethnicity ( or 1.16 [ 95% ci 0.941.42 ] ; data not shown ) . we found no interaction between sex and psychological distress ( p = 0.37 ) or between ethnicity and psychological distress ( p = 0.91 ) in the prediction of type 2 diabetes . we then examined whether combinations of prediabetes status , frs , and psychological distress predicted the incidence of type 2 diabetes . type 2 diabetes incidence was low among participants with normoglycemia and a low risk score , irrespective of the presence ( 1.9% ) or absence ( 1.6% ) of psychological distress . among the normoglycemic participants with an intermediate risk score of 1019 , 7.6% of distressed and 6.0% of nondistressed people had type 2 diabetes at follow - up ; the confidence intervals suggesting no association with psychological distress . similarly , among participants with prediabetes and an frs of 1019 , no difference was found in the incidence of type 2 diabetes between those with ( 15.6% ) and without psychological distress ( 15.0% ) . however , among participants with prediabetes and a high frs ( > 19 ) , 40.9% of those with psychological distress developed type 2 diabetes at follow - up compared with only 28.5% of those without psychological distress . unadjusted incidence ( 95% ci ) of type 2 diabetes among participants with normoglycemia and participants with prediabetes ; participants further stratified by the frs and psychological distress . results of the multivariable - adjusted logistic regression models confirm those from the unadjusted analysis by showing a strong dose - response association between prediabetes and frs status , and risk of incident type 2 diabetes ( table 2 ) . moreover , comparisons indicate no difference regarding the association between psychological distress and type 2 diabetes among normoglycemic participants or among those with prediabetes and a lower frs , whereas among participants with prediabetes and a high frs ( > 19 ) , psychological distress was associated with a doubling of the risk of type 2 diabetes . a statistically significant interaction ( p = 0.039 ) high frs group with the other groups combined , as regards the association between psychological distress and incident type 2 diabetes . incidence of type 2 diabetes at follow - up among participants with normoglycemia and participants with prediabetes at baseline ; participants further stratified by frs and psychological distress alternative reference groups are shown in comparisons 18 . models are adjusted for age , sex , ses , ethnicity , antidepressant use , smoking , and physical activity . all comparisons are based on the same data , but they have a different reference group . or , odds ratio . the findings were replicated in sensitivity analysis using an alternative , less stringent cut point of > 18 to define high frs ( supplementary table 2 ) . we examined whether the association between psychological distress and incident type 2 diabetes differed between populations at different baseline risk levels of type 2 diabetes as assessed by the presence of prediabetes and the level of frs . the frs is based on traditional type 2 diabetes risk factors : prediabetes , overweight or obesity , low hdl level , increased level of triglycerides , hypertension , and a history of parental diabetes . in the current study , our main finding was that psychological distress is associated with a doubling of diabetes risk in a high - risk populations . in the overall analysis , psychological distress was not significantly associated with type 2 diabetes . subsequent stratified analysis revealed no association between psychological distress and type 2 diabetes in normoglycemic participants irrespective of the risk score and in participants with prediabetes and a lower frs . however , in the group of participants with prediabetes and high risk score ( > 19 ) , the 5-year incidence of diabetes was 28.5% in those without psychological distress and 40.9% in those with psychological distress at baseline . in the multivariate - adjusted model , the or was twofold increased among those with psychological distress compared with those without . the findings were replicated using a lower cut point ( > 18 ) for defining a high frs . earlier literature suggests that the relationship between psychosocial factors and type 2 diabetes may be complex ( 16,17 ) . earlier findings on the association between psychological factors , such as general stress and work stress , and incident type 2 diabetes have been mixed , including both null and positive findings ( 1017 ) . inconsistencies in earlier research may be due not only to the use of different stress and distress indicators across studies but also , as our present findings suggest , to a failure to recognize that the nondiabetes group might be heterogeneous in terms of vulnerability to distress ; those at an advanced stage of prediabetes may be more affected by the adverse metabolic effects of psychological distress than those at lower risk levels ( 1,17 ) . indeed , the effects of psychological stress factors have been suggested to be synergistic ( 17 ) . rather than a general risk factor in all diabetes - free populations , it might be a stage - specific risk factor that has a much stronger effect among those at an advanced stage of progression toward manifest type 2 diabetes . in line with our results , an earlier report from the whitehall ii study showed that work stress predicted type 2 diabetes among obese , but not nonobese , women ( 14 ) . plausible pathways through which psychological distress may accelerate progression to type 2 diabetes among high - risk individuals include health risk behaviors and direct physiological pathways , such as long - term dysregulation of the hypothalamic - pituitary - adrenal axis , leading to increased levels of glucocorticoids , especially cortisol , and changes in immune system activity , leading to increased concentrations of proinflammatory cytokines ( 1,8,16,17 ) . there is some evidence supporting inflammation and lifestyle factors as mediators between depressive symptoms and incident type 2 diabetes ( 8) . healthcare implications of this study include the notion that psychological distress might hamper the outcomes of intensive lifestyle and other treatment interventions targeted at high - risk groups ( 2,3 ) . psychological distress symptoms , such as stress , anxiety , depression , feelings of hopelessness , and sleep disturbances , have previously been shown to hinder commitment to major , long - term lifestyle changes and reduce adherence to pharmacological treatments ( 30 ) . first , although the study had a high response rate in the successive data collection phases , loss to follow - up accumulated over time , as in most long - term cohort studies . however , large differences in missing data as a function of psychological distress and type 2 diabetes seem unlikely . second , participants of the whitehall ii study are from an occupational cohort that is likely to cover a healthier end of the variation in health status compared with the general population , which limits the generalizability of our findings . as this instrument was not designed to make a psychiatric diagnosis of depression or anxiety , we can not exclude the possibility of confounding by unmeasured depression or anxiety disorders . however , this is unlikely to be a major source of bias because the ghq-30 has been shown to be a valid screening instrument for mental disorders , particularly depression in the whitehall ii study ( 28 ) . the strengths of this study are its large sample size and use of accurate , repeat assessments of all examined variables , use of the frs based on clinical measurements , and ascertainment of diabetes based on the standard 75-g ogtt at each clinical study cycle ( 23,24 ) . however , part of the incident type 2 diabetes identification was based on self - report , although antihyperglycemic medication was confirmed by asking the participants to take their medications or list of medications to the study clinic . of those participants who had self - reported diagnosis of diabetes without evidence on the use of antihyperglycemic medications ( 33.4% of incident cases ) , a substantial proportion was confirmed by a repeat ogtt or by antihyperglycemic medication at the subsequent phase , leaving only 23.6% of all incident diabetes cases unconfirmed . in summary , this observational study suggests that psychological distress may be related to accelerated progression of late - stage prediabetes to clinical diabetes . further investigations are needed to examine mechanisms linking psychological distress to onset of type 2 diabetes in this group . current prevention guidelines do not generally consider psychological factors such as stress or depression ( 31 ) , although some recognize them as contributing factors to be taken into account in efforts to prevent type 2 diabetes ( 32 ) . given the high comorbidity between psychological problems and diabetes and the accumulation of evidence on the role of psychological distress as a predictor of type 2 diabetes , it is important to consider whether more attention should be paid to psychological status among high - risk populations in addition to lifestyle modifications . | objectivewe examined whether psychological distress predicts incident type 2 diabetes and if the association differs between populations at higher or lower risk of type 2 diabetes.research design and methodsthis was a prospective cohort of 5,932 diabetes - free adults ( 4,189 men and 1,743 women , mean age 54.6 years ) with three 5-year data cycles ( 19912009 ) : a total of 13,207 person - observations .
participants were classified into four groups according to their prediabetes status and framingham offspring type 2 diabetes risk score : normoglycemia with a risk score of 09 , normoglycemia with a risk score of 1019 , prediabetes with a risk score of 1019 , and prediabetes with a risk score of > 19 .
psychological distress was assessed by the general health questionnaire .
incident type 2 diabetes was ascertained by 2-h oral glucose tolerance test , doctor diagnosis , or use of antihyperglycemic medication at the 5-year follow - up for each data cycle . adjustments were made for age , sex , ethnicity , socioeconomic status , antidepressant use , smoking , and physical activity.resultsamong participants with normoglycemia and among those with prediabetes combined with a low risk score , psychological distress did not predict type 2 diabetes .
diabetes incidence in these groups varied between 1.6 and 15.6% . among participants with prediabetes and a high risk score ,
40.9% of those with psychological distress compared with 28.5% of those without distress developed diabetes during the follow - up .
the corresponding adjusted odds ratio for psychological distress was 2.07 ( 95% ci 1.193.62).conclusionsthese data suggest that psychological distress is associated with an accelerated progression to manifest diabetes in a subpopulation with advanced prediabetes . |
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all vertebrates share the same basic principle of lymphocyte differentiation along distinct t - cell - like and b - cell - like lineages ( boehm , 2011 , hirano et al . , 2013 ) , suggesting that the genetic program regulating the developmental pathways of lymphocytes must have already existed in a common ancestor for all vertebrates . taking advantage of the apparent evolutionary conservation of lymphocyte - based immunity , we conducted genetic screens in zebrafish aimed at identifying previously unknown regulators of t lymphocyte development . zebrafish is particularly suited for such an analysis , as t cell development already begins in the embryo during the third day after fertilization ( langenau and zon , 2005 ) . during these early stages of embryonic development , maternal factors are expected to buffer , at least partially , the phenotypic consequences of zygotic defects in mutant fish unless particular cell types ( t cells in the present case ) exhibit a specific requirement for the unimpaired activity of a certain gene(s ) . we therefore expected that this unique biological feature of t cell development in zebrafish would allow us to identify cell - type - specific functions of genes that are also required for stages of development prior to the onset of lymphopoiesis . a similar approach would not be feasible in mammals , because their lymphocytes develop at a considerably later point in embryogenesis ; as a consequence , in mice , for instance , embryonic lethality often masks subsequent lineage - specific functions of pleiotropically acting genes , for example gata3 ( pandolfi et al . , 1995 ) . in the mouse , specific networks of transcription factors have been shown to regulate the three major phases of t cell development . in the initial phase , t cell progenitors are generated and recruited to the thymus ; subsequently , they are induced to adopt a t cell fate ; finally , they become responsive to signals emanating from the t cell receptor ( yui and rothenberg , 2014 ) . hence , assuming that these regulatory circuits emerged at an early stage in vertebrate evolution , a comprehensive genetic screen of t cell development in zebrafish would be predicted to identify at least some of the factors governing these three phases . in keeping with this expectation , we identified mutations in genes encoding lymphoid lineage - specific transcription factors , and components of cytokine signaling and dna replication / repair pathways . quite unexpectedly , however , pre - mrna - processing factors were also found to play a specific role in t cell development . using genetic interaction analysis and transcriptome profiling , we established a functional network of certain components of the pre - mrna splicing machinery and demonstrated that the role of this network for t cell development is evolutionarily conserved . two genetic screens were conducted in zebrafish to identify recessive mutations affecting t lymphocyte development ( boehm et al . , 2003 , 2006 ) . to this end , the expression of rag1 was determined at 5 days post - fertilization ( dpf ) by rna in situ hybridization . the product of the rag1 gene is essential for t cell receptor assembly in developing t cells in the thymus , the first site of lymphopoiesis in zebrafish embryos . only mutant fish with no overt developmental abnormalities apart from impaired intrathymic t cell development were considered for further characterization . together with the tbingen 2000 screen consortium , we screened f3 clutches of 4,584 f2 families , representing 4,253 mutagenized haploid genomes ; so far , 42 lines carrying recessive mutations affecting rag1 expression levels could be established . the freiburg gynogenetic screen of 281 genomes led to the establishment of three lines , all of which were found to harbor recessive mutations . owing to the considerable efforts associated with isolating mutated genes by positional cloning , we conducted an interim analysis after the identification of more than one - third of affected genes . the pertinent features of the first 15 complementation groups , for which the affected genes were identified by linkage analysis and positional cloning ( in two cases , aided by whole - genome sequencing ) , are summarized in table 1 . the fact that , among the first 17 of the 45 mutant lines analyzed here , two genes ( ikzf1 and top3a ) were represented by two distinct alleles each suggests that our screen was near saturation . according to their known functions , it was possible to group the affected genes into three categories : regulators of hematopoiesis and lymphopoiesis ; regulators of dna replication , repair , and cell cycle ; and regulators of pre - mrna processing . the products of the five genes in the third functional group ( snapc3 [ small nuclear rna - activating protein complex protein 3 ] , lsm8 [ like - sm protein 8 ] , gemin5 [ gem nuclear organelle associated protein 5 ] , tnpo3 [ transportin 3 ] , and cstf3 [ cleavage stimulation factor subunit 3 ] ) are implicated in pre - mrna processing . the mutations are predicted to affect different aspects of this multi - layered process , such as transcription of small nuclear rnas ( snrnas ) ( snapc3 ) ( hernandez , 2001 ) , formation of small nuclear rna - containing ribonucleoprotein ( snrnp ) splicing complexes ( gemin5 ; lsm8 ) ( friesen and dreyfuss , 2000 ) , nuclear import of splice regulators ( tnpo3 ) ( kataoka et al . , 1999 ) , and polyadenylation ( cstf3 ) ( xiang et al . , 2014 ) . as our screen was focused on t cell development , the prevalence of this group of ubiquitously expressed genes was unexpected , particularly because , to the best of our knowledge , there is no precedence for their hematopoietic / lymphopoietic roles from studies in mammals . previous biochemical studies indicated that the number of spliceosome - associated factors is in the order of 170 ( wahl et al . , 2009 ) , whereas the gene ontology term rna processing is associated with about 500 genes . assuming a coding capacity of the zebrafish genome of about 26,000 genes ( kettleborough et al . , 2013 ) , and using a conservative estimate that a total of 2,000 genes encode the various components of pre - mrna - processing pathways , the degree of enrichment of genes in this functional category in our screen ( 5/15 ) is significant ( hypergeometric test , p = 0.0036 ) . the unexpected prevalence of mutations in genes encoding components of the pre - mrna - processing machinery in our screen prompted us to examine whether the cell - type - specific functions of the gene products of this group are mirrored in functional similarities , i.e. , whether they belong to the same genetic network and whether their functions are evolutionarily conserved . despite the fact that the identified mutant alleles of lsm8 , gemin5 , and snapc3 most likely encode non - functional variants , larval development and hematopoietic development initially proceed normally apart from a pronounced defect in t cell development ( figures s1s3 ) . we monitored the presence of developing t cells in the thymus of 5 dpf larvae by evaluating the signal emanating from rag1-expressing thymocytes in both thymic lobes ; on a per - cell basis , rag1 expression levels are the same in all genotypes analyzed , allowing us to use the rag1 hybridization signal as a proxy for the number of rag1-expressing thymocytes . the signal emanating from growth hormone ( gh ) gene - expressing cells in the hypophysis was found to be unchanged in mutant fish and thus subsequently used as an internal standard ( figure 1a ) . the extent of t cell development was then expressed as the rag1/gh ratio and referred to as the thymopoietic index . the double - probe rna in situ analyses indicate severe reductions of rag1 signals in the thymic lobes of homozygous lsm8 , gemin5 , and snapc3 mutants ; heterozygotes exhibit no detectable decrease in the thymopoietic indices ( figures 1b1d ) . for example , in lsm8 and gemin5 mutants , the development of lymphoid precursors visualized in the ikaros : egfp transgenic background ( hess and boehm , 2012)is initially not affected ( figures 1e and 2a ) . however , by 5 dpf , fewer lymphoid precursors are present in the thymus as identified by ikaros expression ( figures 1e and 2b ) , in line with the reduced rag1 signal ( figures 1b , 1c , and 2c ) . by 8 dpf , t cell development in gemin5 fish this defect is lymphocyte intrinsic , because the stromal microenvironment of the thymus appears to be normal . the mutant thymic anlage expresses foxn1 ( figure 2e ) , the gene encoding the master regulator of thymic epithelial cell differentiation ( nehls et al . , 1996 ) , and is colonized normally by transplanted wild - type lymphoid precursors ( figure 2f ) . in contrast to impaired t cell development , several other features indicate that gemin5 embryos and larvae initially develop normally . for instance , development of different types of neuronal tissues appears undisturbed , including the hypophysis ( exhibiting normal numbers of growth - hormone expressing cells [ figure 2c ] ) , the hindbrain ( visualized by ikaros - expressing neurons [ figure 2 g ] ) , and the retina ( exhibiting the characteristic multi - layered organization [ figure 2h ] ) . moreover , the swim bladder develops normally and the body size is indistinguishable from that of wild - type siblings [ figure 2i ] ) . collectively , these observations suggest a surprisingly tissue - restricted consequence of gemin5 mutation during early stages of zebrafish development , well beyond the stages during which maternally supplied protein and/or mrna could be expected to compensate for the loss of zygotic gemin5 function . likewise , fish mutant for lsm8 ( figures 1 and s1 ) and snapc3 ( figure s3 ) also exhibit tissue - restricted phenotypes . importantly , the phenotypes that are observed in pre - mrna - processing mutants can not be explained simply by impairment of the rapid cell proliferation occurring in developing zebrafish embryos and larvae , as this would also cause , for instance , severe perturbations in the development of cranio - facial and neuronal structures in addition to affecting t cell development . rather , our findings point to a particular sensitivity of developing thymocytes to aberrations in certain components of snrnps . in order to gain insight into the functional interrelationships between the three regulators of pre - mrna processing identified here , epistasis analysis was performed . the smn ( survival of motor neurons ) complex ( part of which is gemin5 ) contributes to the formation of u1 , u2 , u4 , and u5 snrnps , and to the formation of the spliceosomal u6 snrnp ( containing the lsm2 - 8 protein ring ) ( friesen and dreyfuss , 2000 ) . the phenotype of impaired intrathymic t cell development in gemin5 fish was considerably more severe when the fish originated from gemin5;lms8 double - heterozygous parents rather than from single heterozygous gemin5 ( that is , gemin5;lms8 ) parents ; by contrast , the phenotype of lsm8 fish was not affected by parental heterozygosity of gemin5 ( figure 3a ) . the more severe phenotype of gemin5 mutants arising from lsm8 heterozygosity has a maternal origin ( figure 3b ) , suggesting that reduced levels of lsm8 mrna and/or protein in the oocyte affect the function of gemin5 in the early stages of embryogenesis . by contrast , maternal contribution of gemin5 mrna and/or protein appears to be less important than that of lsm8 , at least with respect to t cell development , because the extent of t cell development in lsm8 fish was unaffected by parental gemin5 heterozygosity ( figure 3a ) . impaired translation of maternal and zygotic lsm8 mrna ( using antisense oligonucleotides directed against the translational start site ) phenocopies the genetic lsm8 defect and affects t cell development at later stages of development , whereas interference with splicing of zygotic lsm8 pre - mrnas alone ( using antisense oligonucleotides directed against the splice donor site of exon 3 ) , has no effect on the thymopoietic index ( figures s4a and s4b ) . collectively , these observations indicate that reduced levels of lsm8 in the early embryo have a long - lasting effect that becomes apparent at much later stages of development and specifically affects t cell differentiation . of note , the effect of maternal heterozygosity of lsm8 was not observed in combination with a null mutation of the il7r gene , encoding a component of the il7 cytokine receptor ( figures s4c and s4d ) , which by itself also affects t cell development ( table 1 ) ( iwanami et al . , 2011 ) . collectively , our observations support the notion that gemin5 and lsm8 function predominantly in separate pathways with redundant or complementary roles ( mani et al . , 2008 ) . epistasis analysis of snapc3 mutants was complicated by the fact that their thymopoietic index is very low ; hence , it proved to be difficult to establish by analysis of compound mutants the presence and the direction ( synthetic or alleviating ) of genetic interactions between snapc3 and lsm8 mutations and snapc3 and gemin5 ( data not shown ) . next , we determined the effect of snapc3 and lsm8 mutations on the global splicing patterns in 4 dpf whole larvae using rna sequencing ( rna - seq ) , as it is technically not feasible to isolate the few developing thymocytes for transcriptome analysis . the alterations of splicing patterns observed in both mutants are dominated by the occurrence of exon - skipping events ( figures 4a and 4b ; table s1 ) . however , in lsm8 mutants , a significant number of genes ( including tnpo3 ) were additionally affected by incomplete splicing events , resulting in the frequent occurrence of retained introns ( figure 4b ; table s2 ) . other types of splicing events , such as the aberrant usage of mutually exclusive exons and the use of alternative donor and acceptor sites , did not appreciably contribute to the global changes in either genotype ( figures 4a and 4b ) . intriguingly , we observed a considerable degree of overlap between the genes that are affected by splicing aberrations in the two mutants ( figures 4c and 4d ; tables s1 and s2 ) . interestingly , in snapc3 mutant fish , skipping of the first coding exon of the gemin8 gene ( ensdarg00000053496 ; ensdart00000142315 ; table s1 ; inclusion levels , 0.86 0.05 [ mean sem ] for wild type ; 0.46 0.02 [ mean sem ] for mutant ; p = 0.002 ; t test , two - tailed ) was detected , predicting the formation of an n - terminally truncated protein . because gemin8 is , like gemin5 , part of the smn complex ( battle et al . , 2006 , wahl et al . , 2009 ) , this observation supports the notion that snapc3 and gemin5 act in the same molecular pathway . to further address the functional consequences of the identified mutations , we also analyzed the transcriptomes of snapc3 and lsm8 mutants and their wild - type counterparts for changes in gene expression levels ( 2-fold ; false discovery rate [ fdr ] < 5% ) ( table s3 ) . in snapc3 mutants , 140 genes were significantly upregulated ( figure 4e ) , including 17 protein - coding genes with known functions in pre - mrna splicing ( table s4 ) . most notable was the increase in expression levels for genes encoding components of the sm ( snrpa1 , snrpd2 , snrpd3 , snrpe , snrpf ) and the lsm ( lsm7 , lsm8 ) heptameric complexes ( table s4 ) . in lsm8 mutants , the expression levels of 427 genes were increased ( figure 4e ) , including 35 protein - coding genes , the functions of which have been linked to pre - mrna splicing ( table s4 ) . as is the case in snapc3 mutants , genes encoding components of the sm ( snrpb , snrpd1 , snrpd2 , snrpd3 , snrpe , snrpf ) and lsm ( lsm6 , lsm7 , lsm8 ) ring structures are upregulated ( table s4 ) . of the 58 genes upregulated in both snapc3 and lsm8 mutants , 16 ( 28% ) encode splicing - related proteins ( figure 4f ) . this includes snapc4 , encoding one component of the transcriptional activation complex of snrna genes , and of genes ( snrpd2 , snrpd3 , snrpe , snrpf , lsm7 , lsm8 ) encoding 6 of the 14 components of the sm and lsm heptameric ring structures containing snrnas ( table s4 ) . in both lsm8 and snapc3 mutants , expression of the foxg1d gene is reduced ( table s3 ) ; the mouse homolog of this gene ( foxg1 ) is implicated in the regulation of thymic epithelial cell differentiation ( wei and condie , 2011 ) , possibly contributing to impaired t cell development . we then tested the generality of the presumed transcriptional feedback regulation among components of snrnps by examining transcriptional responses in gemin5 mutants , focusing on the expression levels of sm and lsm genes . in support of our hypothesis , the qpcr results indicate that snapc3 , lsm8 , and gemin5 mutations all result in strong upregulation of lsm7 and lsm8 mrnas ( figure 4 g ) , defining a core group of genes co - regulated by perturbation of snapc3 , lsm8 , and gemin5 functions . by contrast , expression levels of mrnas of genes encoding the eight known gemin protein family members are largely unaffected in the three mutants ( table s5 ) , supporting the notion of the specific nature of the transcriptional feedback regulation . collectively , the present genetic interaction studies and transcriptome analyses suggest the presence of a network connecting snapc3 , lsm8 , and gemin5 genes encoding key components of snrnps that appears to converge on the transcriptional regulation of lsm7 and lsm8 genes . tnpo3 has been shown to participate in the import of splice regulators to the nucleoplasm ( maertens et al . , 2014 ) . hence , in contrast to the gene products of snapc3 , lsm8 , and gemin5 , tnpo3 is predicted to indirectly affect pre - mrna processing . in zebrafish tnpo3 mutant larvae , the number of lymphoid precursors in hematopoietic tissues is not affected at 1 dpf when definitive hematopoiesis has begun in zebrafish ; at later stages of development , lymphoid progenitors are found in the thymus ( albeit in somewhat reduced numbers ) , indicating that the homing process is largely unaffected by tnpo3 deficiency ( figures 5a and s5 ) . however , only few of thymocytes in tnpo3 mutants express rag1 ( figures 5b and 5c ) , despite normal expression levels of a thymocyte marker , ccr9b ( figure 5b ) , suggesting that intrathymic defects contribute to impaired t cell development in tnpo3-deficient fish . at the time point of our analyses ( 5 dpf ) , only t cells are present in zebrafish larvae , because b cells develop considerably later in development ( danilova and steiner , 2002 ) . hence , we reasoned that the splicing pattern of ptprc ( encoding the zebrafish ortholog of the mammalian thymocyte maturation marker cd45 ) might provide further evidence for impaired intrathymic t cell differentiation in tnpo3-deficient larvae . indeed , the mutant splicing pattern is dominated by lower molecular weight isoforms ( figure 5d ) that are characteristically found in immature mouse thymocytes ( lefrancois and goodman , 1987 ) . at present , it is not possible to determine the cause - and - effect relationship of differential ptprc splicing in the absence of tnpo3 function . however , tnpo3-deficient zebrafish larvae exhibit aberrant splicing of the nck2b gene ( table s1 ) , encoding the zebrafish homolog of the essential adaptor protein nck required for proper tcr signaling in the mouse ( alarcn et al . , 2003 ) , suggesting that differential splicing of ptprc is an indirect effect of tnpo3 deficiency . tnpo3 appears to be functionally connected to the snpac3/lsm8/gemin5 network of splice regulators , as indicated by alleviating genetic interaction between tnpo3 and gemin5 mutations . in double - deficient fish , the thymopoietic index is lower than in each of the single mutants , yet 3-fold higher than expected if the two genes acted independently ( mani et al . , 2008 ) ( figure 5e ) , suggesting that tnpo3 and gemin5 are active in the same pathway . as expected , splicing defects were abundant in 4 dpf tnpo3 mutants possibly owing to faulty transport of splice regulators to the nucleoplasm ( maertens et al . , 2014 ) . we recorded a considerably greater number of exon - skipping events in mutant fish compared to their wild - type siblings , alongside other splicing changes , such as the presence of retained introns and the use of mutually exclusive exons or alternative splice donor and splice acceptor sites ( figures 5f and 5 g ; tables s1 and s2 ) . interestingly , we observed that both lsm8 and tnpo3 deficiency are associated with aberrant splicing of gemin5 and tnpo3 pre - mrna , and snapc3- and tnpo3-deficient larvae both exhibit aberrant splicing patterns of gemin8 ( table s1 ) , further emphasizing the functional interrelationships in the snpac3/lsm8/gemin5/tnpo3 network . comparatively few alterations were observed at the level of gene expression ( 2-fold ; fdr < 5% [ table s3 ] ) in tnpo3 mutants . thirty - eight genes are upregulated in tnpo3 mutants , however , with little overlap to genes upregulated in snapc3 and lsm8 mutants , indicating the presence of mechanistic differences with respect to the regulation of snrnp assembly among the three mutants ( figure 5h ) . the presence of pervasive autoregulation affecting the processing of pre - mrnas of splice regulator genes as a consequence of mutations in snpac3 , lsm8 , and tnpo3 is suggested by our finding that among the 32 genes commonly affected by exon - skipping events ( figure 5 g ) , two genes srsf3a and ptbp3encode known regulators of pre - mrna processing ; and of the four genes commonly affected by intron retention , two encode splice regulators ( srsf1b , prpf39 ) ( table s2 ) . when we examined transcriptomes for changes common to all three mutants and potentially involved in t cell / thymus development , we noted that splicing of the dnmt4 ( also known as dnmt3bb.1 ) gene , which is expressed in the hematopoietic lineage ( takayama et al . , 2014 ) , is similarly affected in all three mutants ; the splice pattern suggests the presence of different compositions of the n - terminal non - catalytic domains of the predicted protein ( figure 5 g ; table s6 ) . many of the 34 downregulated genes ( figure 5i ) are signature genes of the exocrine pancreas , identifying a second cell type exhibiting particular sensitivity to the malfunction of the pre - mrna splicing regulators identified here . the lower expression levels of elastase , trypsin , amylase , and carboxypeptidase , etc . ( ela3l , try , amy2a , cpa1 ) genes suggest that the downstream effects of snapc3 , lsm8 , and tnpo3 mutations converge on malfunction of the exocrine pancreas ( table s7 ) . this conclusion is supported by the fact that in all three mutants , aberrant splicing of the nr5a2 transcription factor also occurs ( table s6 ) , which is required for liver and exocrine pancreas development ( nissim et al . , 2016 ) . we also note that the gene encoding the brush - border membrane glycoprotein trehalase ( treh ) is affected by intron retention in all three mutants , adding to impaired absorption of carbohydrates . although more work is needed to assess the functional significance of these findings , we hypothesize that malfunction of the exocrine pancreas and the intestine at least partly underlies the phenotype of premature death in the mutant fish . these results also underscore the notion of cell - type - restricted functions of snapc3 , lsm8 , and tnpo3 genes . having established the role of the snpac3/lsm8/gemin5/tnpo3 network ( figure 5j ) in zebrafish t cell development , we next examined a possible evolutionary conservation of functions of these genes in mammalian t cell development ; we chose the mouse tnpo3 gene for our studies . to this end , we generated conditional knockout mice using a floxed allele of tnpo3 ( exon 7 is flanked by loxp sites ) and lck : cre ( orban et al . . under these conditions , complete inactivation of the tnpo3 gene was achieved ( figure s6 ) . the predicted tnpo3 mutant protein consisting of the first 291 amino acids closely resembles the mutant form identified in zebrafish ( 202 amino acids ) ( figure s6 ) ; notably , they both lack the region required for interaction with rs cargo proteins ( maertens et al . , 2014 ) . in the thymus , tnpo3 deficiency was accompanied by a moderate , but non - significant reduction of cd4/cd8-double - positive ( dp ) t cell progenitors ; by contrast , single - positive ( sp ) thymocytes were significantly reduced , cd4 cells by about 40% and cd8 cells by about 60% ( figures 6a and 6b ) . as expected , the number of t cells remained unchanged , because the lck : cre transgene is not expressed in this lineage ( figure s6 ) . reduced levels of cd5 staining of tnpo3-deficient thymocytes ( particularly of those with cd8 surface phenotype ) indicate that tcr signaling is impaired in mutant cells ( figure 6c ) ; this feature is also evident in peripheral t cells ( figure s6 ) . these findings suggest that loss of tnpo3 in thymocytes is associated with an incomplete maturation block affecting the dp - to - sp transition . because expression levels of rag genes are unchanged in mutant dp cells ( data not shown ) this finding is reminiscent of the features of impaired tcr signaling identified in the tnpo3-deficient zebrafish larvae . the loss of single - positive cells in mutant thymi does not appear to be due to increased apoptosis , because the fractions of tunel - positive and annexin v - positive thymocytes remained unchanged ( data not shown ) ; these findings call for further mechanistic studies addressing migration , survival , etc . , of mutant t cells in the periphery . as expected for the lymphopenic condition in tnpo3-deficient mice ( figure 6d ) , increased proliferation levels of peripheral t cells are observed ( figure 6e ) , compatible with the notion that tnpo3 deficiency per se does not impair cell proliferation . collectively , the present data suggest an evolutionarily conserved role of tnpo3 in t cell differentiation . our unbiased genetic screens identified an extended genetic network linking genes of several distinct functional categories ( hematopoietic differentiation ; dna replication / repair ; pre - mrna processing ) required for early stages of intrathymic t cell development in zebrafish . the present study has established additional animal models for mammalian ( particularly human ) immunodeficiency syndromes and presents previously unknown candidate genes whose mutations might underlie failing immune function . indeed , we note that some of the genes that we have identified are implicated in human immunodeficiency disorders , such as il7r ( puel et al . , 1998 ) , jak3 ( russell et al . , 1995 ) , and pole1 ( pachlopnik schmid et al . , 2012 ) ( table 1 ) . the value of forward genetic screens to identify candidate genes is underscored by our finding of an enrichment of genes encoding factors involved in pre - mrna processing . this outcome was unexpected , because mutations in this group of genes have so far been associated mostly with neuronal disease , such as spinal muscular atrophy or retinitis pigmentosa ( singh and cooper , 2012 ) . mutations in spliceosomal factors manifest themselves as hypomorphic alleles ( singh and cooper , 2012 ) , indicating that the resulting phenotypes most likely arise from subtle perturbations in the multi - component protein complexes regulating pre - mrna splicing . for example , insufficient levels of the smn complex are associated with impaired maturation of snrnps and aberrant splicing reactions in neuronal tissues ( zhang et al . , 2008 , zhang et al . , 2013 ) , whereas complete loss of smn function is lethal ( hsieh - li et al . , 2000 ) . however , because defects in early development are buffered by maternal factors , our results also illustrate how the unique biological features of the zebrafish model allow the identification of tissue - restricted functions of pleiotropic genes also in the case of null alleles , which in the mammalian system can only be studied by conditional mutagenesis . although the results of the present epistasis analysis are consistent with those in previously published molecular studies on snrnp assembly and function ( wahl et al . , 2009 ) , an unexpected finding in the in vivo studies described here is the presence of a feedback loop connecting impaired snapc3 , lsm8 , and gemin5 function with the transcriptional regulation of the lsm7 and lsm8 genes . in contrast to many other genes in zebrafish , lsm genes do not possess paralogs . hence , the transcriptional response in our mutants is unlikely to be part of a direct compensatory mechanism , but rather appears to be a general consequence of snrnp biogenesis perturbation , because expression levels of lsm7 and lsm8 are also upregulated in zebrafish larvae mutant for the p110 splicing regulator ( trede et al . , 2007 ) , which is required for recycling of the u4/u6 snrnp from singular u4 and u6 snrnps ( bell et al . , 2002 ) another notable feature of transcriptional changes emerging from our studies is the presence of pervasive autoregulatory loops affecting the splicing of pre - mrnas - encoding splice regulators , as illustrated here by , for instance , the srsf3a gene , a homolog of the mammalian srsf3 gene previously shown to be regulated by variable inclusion of so - called poison - cassette exons ( lareau et al . , 2007 ) . on a more general level , our findings provide evidence for the emerging notion of tissue - restricted roles played by ubiquitous components of basic cellular pathways . future work may thus reveal an rna splicing code of context- and cell - type - dependent functions of some general - purpose factors involved in pre - mrna processing ; our mutants provide an entry point into the identification of the precise mechanism(s ) by which pre - mrna splicing affects t cell development . with respect to the mechanism(s ) underlying failing t cell development in zebrafish larvae , our work indicates that , despite mechanistic differences , certain commonalities exist among the aberrations caused by mutations in the snapc3/lsm8/gemin5/tnpo3 network . one particularly notable observation is the apparent association with dna methylation , because the splicing of several genes encoding putative de novo dna methylases is affected by perturbations in this network ; most importantly , aberrant processing of the dnmt3bb.1 gene is a common feature of snapc3 , lsm8 , and tnpo3 mutants . we consider this mechanistic link to be particularly intriguing , because our screen has independently uncovered a mis - sense mutation in the gene encoding the maintenance dna methylase dnmt1 ( table 1 ) . given that the status of dna methylation affects the differentiation of hematopoietic stem cells ( challen et al . , 2014 ) and modulates immune functions including t cell differentiation ( jin et al . , 2008 ) , it will be interesting to examine the possible presence of common changes in gene - specific dna methylation patterns to identify those genes whose methylation signatures are crucial to normal t cell development . finally , an important aspect of our present work addresses the evolutionarily conserved function of the identified network of splice regulators with respect to t cell differentiation . we chose tnpo3 to verify that the observations made in the zebrafish model also hold for the mammalian system . our conditional mouse tnpo3 knockout model exhibits a phenotype of impaired t cell differentiation , resembling the phenotype observed in tnpo3-deficient zebrafish larvae . the comparative analysis of mutant zebrafish tnpo3 and mouse tnpo3 genes highlights the advantages of the zebrafish model to identify cell - type - restricted functions of pleiotropic regulators , because tnpo3-deficient mice exhibit early embryonic lethality . future work will have to be aimed at addressing the degree of functional conservation for the other members of the identified genetic networks and the precise molecular mechanism(s ) underlying the exquisite sensitivity of t cells to perturbed pre - mrna splicing . the zebrafish ( danio rerio ) strains ekkwill ( ekk ) , tpfel long fin ( tl ) , wild - type - in - kalkutta ( wik ) , ab , assam ( ass ) , and tbingen ( t ) are maintained in the animal facility of the max planck institute of immunobiology and epigenetics . the ikaros : egfp transgenic line was described previously ( hess and boehm , 2012 ) . all animal experiments were approved by the institute s review committee and conducted under licenses from the local government ( regierungsprsidium freiburg ) . a detailed description of the screen design , coverage , complementation analysis , and mutant identification by positional cloning and whole genome sequencing can be found in supplemental experimental procedures . the esc line epd0318_3_g02 ( genetic background : c57bl/6n agouti(a / a ) ; allele name : tnpo3 ) was obtained from the komp repository and used to derive chimeric mice using standard procedures . for rescue experiments , mrnas were injected into fertilized eggs and analysis carried out by gh and rag1 rna in situ hybridization at various time points after injection . egfp cells were sorted from single - cell suspensions of kidney marrow cells isolated from adult ikaros : egfp transgenic zebrafish and injected into the sinus venosus of embryos from heterozygous intercrosses at 2 dpf . gfp cells in the thymic region were counted using imager.z1 ( zeiss ) at 3 days post - injection . the procedures for live imaging using the ikaros : egfp transgenic background were described previously ( hess and boehm , 2012 ) . for analysis of tnpo3 mutant mice , analytical flow cytometry was carried out as described in caldern and boehm ( 2012 ) . procedures for rna in situ hybridization and probes were described previously ( schorpp et al . , 2006 ) . total rna was extracted using tri reagent ( sigma ) following the manufacturer s instructions . after treatment with cloned dnasei ( takara ) , rna extraction using tri reagent was repeated . superscript ii reverse transcriptase ( invitrogen ) and random hexamer primers were used for cdna synthesis from total rna . qpcr was carried out as described ( rode and boehm , 2012 ) ; primers are listed in table s8 . total rna was extracted from whole zebrafish larvae at 4 dpf and subjected to transcriptome analysis . the libraries were sequenced in paired - end 75-bp mode on 0.8 lanes per sample on an illumina hiseq 2500 instrument . the high - throughput rna sequencing analysis pipeline , version 0.4.2 , written by fabian kilpert ( https://github.com/kilpert/rna-seq-qc ) was applied as described in supplemental experimental procedures . rna - seq data are deposited in ncbi s geo ( edgar et al . , 2002 ) and are accessible through geo : gse77480 . for single comparisons of independent groups , student s t test or the mann - whitney test the statistical models applied to rna - seq data are described in supplemental experimental procedures . , f.m . , and m.s . identified and characterized zebrafish mutants by positional cloning . | summarylymphocytes represent basic components of vertebrate adaptive immune systems , suggesting the utility of non - mammalian models to define the molecular basis of their development and differentiation . our forward genetic screens in zebrafish for recessive mutations affecting early t cell development revealed several major genetic pathways .
the identification of lineage - specific transcription factors and specific components of cytokine signaling and dna replication and/or repair pathways known from studies of immunocompromised mammals provided an evolutionary cross - validation of the screen design .
unexpectedly , however , genes encoding proteins required for pre - mrna processing were enriched in the collection of mutants identified here . in both zebrafish and mice , deficiency of the splice regulator tnpo3 impairs intrathymic t cell differentiation , illustrating the evolutionarily conserved and cell - type - specific functions of certain pre - mrna - processing factors for t cell development . |
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antidepressants are widely used during gestation for the treatment of depression . in the united states , the prevalence of antidepressant medication use during pregnancy increased from 5.7% in 1999 to 13.3% in 20031 ; in canada , 4.5% of pregnant women reported using them between 2001 and 2006.2 it remains , however , that there is continued confusion regarding their appropriate use during this critical time period . gestational exposure to antidepressants has been associated with an increased risk of spontaneous abortion,3 major congenital malformations,4
5 prematurity,6
7 low birth weight,6
7 neonatal withdrawal,8 and pregnancy - induced hypertension.9 discontinuation of antidepressants during pregnancy in severely depressed women was associated with relapse of maternal depression in some studies.10
11 nevertheless , up to 20% of women who continue antidepressant use during pregnancy remain depressed,12 suggesting a lack of efficacy in some pregnant women . currently , few studies have investigated the effects of antidepressant use during pregnancy on the neurodevelopment of children , including autism spectrum disorders ( asd ) . given recent projections by the world health organization that depression will be the second leading cause of death by 2020,13 antidepressants are likely to remain widely used , including during pregnancy . therefore , a better understanding of their long - term neurodevelopmental effects on children , when used during gestation , is a public health priority . autism spectrum disorder ( asd ) is defined as a neurodevelopmental disorder which is characterized by pervasive impairment of communication , language , and social interaction , and by repetitive / restricted and stereotyped patterns of behavior.14 according to the diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) , this complex disorder includes five subgroups : autistic disorder , rett syndrome , childhood disintegrative disorder , pervasive developmental disordernot otherwise specified ( pdd nos ) , and asperger syndrome.15 the diagnosis of asd is made at 3 years of age on average . the estimated prevalence of asd has increased over time from 0.04% in 1966 to approximately 1% today16
17 in the united states . this observed rise in the prevalence of asd can be partly attributed to extrinsic factors , including changes in the diagnostic criteria and their overlap with other diagnoses , better screening of the overall population ( higher detection ) , and greater awareness of the general population.18
19 nevertheless , both genetic and environmental factors could also be important in the increase in asd prevalence over time.20 these factors may include de novo mutations,21 specific genes conferring susceptibility,15 advanced maternal age,22 maternal diseases such as diabetes and hypertension,23
24 and a maternal history of psychiatric disorder.25
26 the genetic etiology of asd was first reported in a twin study in 1977,27 and numerous twin studies have since been conducted . in particular , in denmark , a study including 21 pairs of twins ( 11 monozygotic [ mz ] ; 10 dizygotic [ dz ] ) showed that 91% of mz twins and 0% of dz twins28 were concordant with regard to the presence of asd . a british twin study suggested that 60% of mz pairs were concordant for autism versus 0% for dz pairs ; in the same study , 92% of mz pairs were concordant for a broader spectrum of related cognitive or social abnormalities versus 10% of dz pairs.29 this later study also suggested that genetic susceptibility contributed strongly to autism and that multiple genes were involved . it remains , however , that the influence of genetic factors on the development of asd is complex and the cause of asd is unknown in the majority of cases . on the other hand , family history studies have shown that the risk of asd recurrence in another child was higher than the general population prevalence ( 1%),30 and ranged from 3 to 14%.31
32 a recent study indicated an even higher probability of asd recurrence among siblings ( 18.7% ; 95% confidence interval [ ci ] : 13.3425.5).33 several biological mechanisms might underlie the association between prenatal selective serotonin reuptake inhibitor ( ssri ) exposure and the occurrence of neurodevelopmental disorders , including asd . serotonin plays a critical role in the development of the brain.34
35 studies have shown that serotonin modulates numerous pre- and postnatal processes , as well as developmental processes , including cell division , neuronal migration , cell differentiation , and synaptogenesis.36 furthermore , some studies have suggested that serotonin acts as a morphogen during embryonic development , influencing the maturation of the brain.37 experimental studies using rodent models have also indicated that transient inhibition of the serotonin transporter with fluoxetine hydrochloride , an ssri , during brain development has consequences for behavior in later life , indicating a critical role for serotonin in the maturation of the brain systems.38
39 brain imaging research has shown atypical development of the capacity for serotonin synthesis in the brains of children with asd,40
41 and abnormalities in serotonin receptor 2a binding in the cerebral cortex.42 an intrinsic feedback mechanism affects circulating serotonin levels and the morphological modification of serotonergic neurons . ssris block the 5-hydroxytryptamine transporter ( 5-htt ) , leading to increased levels of serotonin in the extracellular space , and it was reported that autistic individuals have elevated 5-ht levels in their blood platelets.43
44 studies of the phenomenon of elevated platelet serotonin levels , termed hyperserotonemia , in rodents have shown an association between blood 5-ht levels and autistic - like behaviors.45 furthermore , hyperserotonemia was observed in 30% of individuals with autism.46 the mechanism of hyperserotonemia is still unknown . some studies suggested an increase uptake of serotonin into platelet,47 diminishing release from platelets,48 and decreased catabolism of serotonin.49 hence , dysfunction of the serotonin system during pregnancy following ssri exposure could directly induce changes in fetal brain development.50 to date , seven published studies have explored the association between antidepressant use during pregnancy and the risk of asd . the results of these studies are presented in table 1 and are illustrated in figs . 1 and 2 . note : no data were available on the risk of asd associated with ad use during pregnancy in the studies by harrington et al ( 2014)55 and hviid et al ( 2013).54 * odds ratio ( or ) ; * * hazards ratio ( hr)/rate ratio ( rr ) . note : no data are available on the risk of asd associated with ssri use during pregnancy in the study by clements et al ( 2014).56 * odds ratio ( or ) ; * * hazards ratio ( hr)/rate ratio ( rr ) . abbreviations : ad , antidepressants ; asd , autism spectrum disorder ; ci , confidence interval ; daa , dual - acting antidepressant ; hr , hazard ratio ; or , odds ratio ; rr , rate ratio ; snri , serotonin norepinephrine reuptake inhibitor ; ssri , selective serotonin reuptake inhibitor ; tca , tricyclic antidepressant ; td , typical development . control study using data from the kaiser permanent medical care program in northern california ( kpnc ) . cases were defined as infants born at kpnc facilities between 1995 and 1999 who had at least one international classification of diseases , 9th revision ( icd-9 ) diagnosis of asd between 1995 and 2002 . children without asd were randomly selected from the remaining cohort of live births and were matched to cases on sex , birth year , and hospital of birth ( ratio of 5:1 ) . there were 298 cases of asd , 20 ( 6.7% ) of whom were exposed to an antidepressant , and 15 ( 5.0% ) were exposed to ssris ( 13 were exposed to ssris only and 2 were exposed to ssris in combination with other antidepressants ) . after adjusting for maternal age , race / ethnicity , education , birth year , sex , hospital of birth , and birth weight , investigators found that antidepressant use during pregnancy was associated with an increased risk of asd in the offspring ( adjusted odds ratio [ or ] = 2.0 ; 95% ci : 1.23.6 ) . authors also showed that cases of asd were more than twice as likely to have been exposed to an ssri in utero than the controls ( adjusted or = 2.1 ; 95% ci : 1.04.4 ) , after adjustment for maternal history of any mental disorders was made . the use of ssris during any trimester was also associated with an increased risk of asd , but the association was only statistically significant during the first trimester ( adjusted or = 3.5 ; 95% ci : 1.57.9 ) . in an analysis restricted to a subgroup of women with mental disorders in the year before delivery , the risk of asd was associated with any ssri use during pregnancy ( or = 1.6 ; 95% ci : 0.64.0 ) , but this association was not statistically significant . however , authors highlighted the possible misclassification of history of maternal mental disorders , which could have introduced bias in the stratified analyses . a second concern involved the small number of asd - exposed cases in the second ( n = 5 ) and third trimesters of pregnancy ( n = 6 ) , which limited statistical power for these analyses . although they have used icd9 codes for the identification of asd , a subset of 50 children underwent clinical evaluation with the autism diagnostic interview revised ( adi - r ) and the autism diagnostic observation schedule generic ( ados ) , and 94% ( 47/50 ) of them met the criteria for asd . although rates of asd diagnoses have increased markedly over the past 20 years , analyses were adjusted for birth year . in 2013 , rai et al52 ( table 1 , figs . 1 and 2 ) conducted a nested case control study within the stockholm youth cohort , including all young people ( aged 017 years ) registered between 2001 and 2007 . cases of asd were defined using icd-9 and icd-10 codes , or dsm - iv criteria , and a multisource case ascertainment method , with registers covering all pathways of autism diagnosis and care within stockholm county . each case of asd was matched to 10 controls without asd on date of birth ( month and year ) and sex . a parental history of depression was obtained using the stockholm county adult psychiatric outpatient register , which records the dates and diagnoses of individuals with visits to any outpatient psychiatric services in stockholm county , and the swedish national patient register , which contains the dates and discharge diagnoses of all inpatients and outpatients visits in sweden . in this cohort , after adjustment for potential confounders , including any maternal psychiatric disorders , rai et al52 reported a statistically significantly increased risk of asd associated with antidepressant use during pregnancy ( adjusted or = 1.90 ; 95% ci : 1.153.14 ) . they also found that the risk of asd was 1.65 ( 95% ci : 0.903.03 ) and 2.69 ( 95% ci : 1.046.96 ) for the use of ssris and nonselective monoamine reuptake inhibitors during pregnancy , respectively . a history of depression and antidepressant use in pregnant mothers was strongly associated with asd in their offspring ( adjusted or = 3.34 ; 95% ci : 1.507.47 ) . in contrast , an association between antidepressant use during pregnancy and asd was not seen among women reporting the use of these medications for reasons other than for depression . again analyses were underpowered , and given that the prevalence of asd has increased over time , detection bias could not be ruled out . however , authors attempted to control for this bias by matching cases and controls by date of birth . 1 and 2 ) conducted a cohort study of all children born in denmark between 1996 and 2006 , identified using the danish civil registration system . authors obtained information on maternal use of antidepressants during pregnancy from the danish national prescription registry . information on asd diagnoses and parental psychiatric disorders were obtained from the danish psychiatric central register . analyses were adjusted for parental age at conception , parental psychiatric history , gestational age at delivery , birth weight , sex of the newborn , and parity . overall , 655,615 children were included in the cohort , 5,437 of who had a diagnosis of asd ; 8,833 ( 1.3% ) were exposed to antidepressants in utero . when analyses were restricted to children of mothers with affective disorders , no statistically significant association was found between gestational use of antidepressants and the risk of asd ( adjusted hazard ratio [ hr ] = 1.2 ; 95% ci : 0.72.1 ) . similarly , no statistically significant association was found between ssri exposure during pregnancy and the risk of asd ( adjusted hr = 1.4 ; 95% ci : 0.82.4 ) . authors further restricted the cohort to include only families with at least two siblings and in which at least one had been diagnosed with asd . in this analysis , no statistically significant association was found between prenatal exposure to any antidepressant ( adjusted hr = 1.1 ; 95% ci : 0.52.3 ) or to ssris ( adjusted hr = 0.9 ; 95% ci : 0.42.0 ) and asd in children , suggesting that genetic predisposition is a stronger risk factor for asd than gestational antidepressant use . however , sibling pairs with no asd diagnosis were excluded from this analysis , resulting in partial adjustment for genetic predispositions . indeed , exclusion of sibling pairs without asd potentially led to overestimation of the association between family history of asd and the risk of asd in another child , and potentially underestimated the association between antidepressant use during pregnancy and asd . 2 ) conducted a large population - based prospective cohort study in denmark , using data from national registries ( medical birth registry , national patient register , national prescription registry , danish civil registration system , and danish psychiatric central register ) from 1996 to 2005 , with follow - up through 2009 . only singleton births were included , and newborns with conditions associated with an increased risk of asd , such as congenital rubella syndrome , were excluded . the authors took into account the effects of potential confounders , such as maternal age at pregnancy onset , smoking status during pregnancy , maternal psychiatric conditions , year of birth , use of medications other than ssris , and maternal level of education . children were prospectively followed up from birth until january 1 , 2010 , or until the child 's 10th birthday . a total of 626,875 children were included , 6,068 ( 0.97% ) of who were exposed to ssris in utero . exposure to ssris any time from 4 weeks before the beginning of pregnancy until delivery was associated with a 20% increased risk of asd ( adjusted rate ratio [ rr ] = 1.20 ; 95% ci : 0.901.61 ) , but this estimate was not statistically significant . results were similar when the analysis was restricted to ssri use during pregnancy ( adjusted rr = 1.40 ; 95% ci : 0.922.13 ) . however , when ssri exposure was limited to 6 to 24 months before pregnancy , the adjusted rr was 1.46 ( 95% ci : 1.171.81 ) , suggesting that the observed association may be partly due to confounding by indication . to control for detection bias , hviid et al54 adjusted for calendar year during which the follow - up assessment was made . control study to investigate the association between prenatal ssri exposure and the risk of asd . the participants were families enrolled in the childhood autism risks from genetics and the environment ( charge ) study between april 2003 and august 2010 . the population controls were identified using state birth files , and were matched to autism cases by age , sex , and regional center . the vineland adaptive behavior scales and the mullen scales of early learning were used to define developmental delay ( dd ) . maternal interviews were conducted to ascertain prenatal ssri use , maternal mental health , and sociodemographic status . child pairs were included : 492 with asd , 154 with dd , and 320 with typical development ; 48 ( 5% ) mothers were prenatally exposed to ssris . after adjustment for potential confounders ( regional center , child 's year of birth , and birthplace of mother ) , prenatal exposure to ssris was associated with a nonsignificant increased risk of asd ( adjusted or = 1.55 ; 95% ci : 0.594.08 ) ; the association was , however , statistically significant among male children ( adjusted or = 2.92 ; 95% ci : 1.077.93 ) . no significant associations were noted when restricting analyses to mothers with anxiety or mood disorder at any time before delivery . no adjustment was made for genetic predisposition to asd ; detection bias was taken into account by adjusting for the child 's year of birth . 1 ) published a case control study on prenatal antidepressant exposure and the risk of asd in a large health system database.56 data on maternal health , asd diagnosis , and antidepressant prescription were collected from electronic health records of a large health care system in massachusetts , and were linked to birth records in the massachusetts registry of vital records and statistics . children with an asd diagnosis were matched in a ratio of 1:3 with non - asd children ( controls ) according to year of birth , delivery hospital , sex , insurance type , race / ethnicity , and prematurity status . exposure to antidepressants was defined as ( 1 ) any time during pregnancy and ( 2 ) according to trimester of use . after adjustment for history of maternal depression , the risk of asd associated with antidepressant exposure during pregnancy was elevated but not statistically significant ( adjusted or in first trimester = 1.43 ; 95% ci : 0.852.38 ; adjusted or in second trimester = 1.34 ; 95% ci : 0.772.27 ) . considering the possibility of confounding by indication , the authors evaluated the association between the use of antidepressants before pregnancy ( any time before the last menstrual period ) and the risk of asd . they reported that the use of antidepressants before pregnancy was significantly associated with asd ( adjusted or = 1.62 ; 95% ci : 1.172.23 ) after adjustment for maternal history of depression . the authors stressed that a potential source of bias was the systematic misclassification of case and control status , which could have led to the nonsignificant estimates . 1 and 2 ) assessed the risk of asd in children exposed to gestational antidepressants , taking into account potential confounding factors and any family history of asd . a cohort study was conducted using data from the quebec pregnancy cohort , and included data on all pregnancies and children in quebec in 1998 to 2009 . antidepressant use during pregnancy was defined as having at least one prescription filled during gestation , and according to the trimester of use ; antidepressant classes were also studied ( table 2 ) . asd outcomes were defined as infants with at least one diagnosis of asd before december 31 , 2009 ( table 2 ) . the cohort included 145,456 live - born children in 1998 to 2009 ; 1,054 ( 0.72% ) children had a diagnosis of asd . the mean age of children at the end of follow - up was 6.24 years ( standard deviation [ sd ] = 3.19 ) . adjusting for potential confounders , overall antidepressant use during the second or third trimester of pregnancy was statistically significantly associated with asd ( adjusted hr = 1.87 ; 95% ci : 1.153.04 ) ; ssri use during the second or third trimester of pregnancy ( adjusted hr = 2.17 ; 95% ci : 1.203.93 ) and the combined use of more than one antidepressant class during the same period ( adjusted hr = 4.39 ; 95% ci : 1.4413.32 ) was associated with the risk of asd . to separate the effects of antidepressants from the underlying indication , a stratified analysis on maternal depression status was performed , resulting in a significantly increased risk of asd associated with antidepressant use during the second or third trimester among children of depressed mothers ( adjusted hr = 1.75 ; 95% ci : 1.032.97 ) ; no statistically significant association was found among nondepressed mothers ( adjusted hr = 1.36 ; 95% ci : 0.345.50 ; 2 exposed cases ) . genetic predispositions , defined as having a sibling with asd , were further taken into account . in the subgroup of children with no genetic predisposition to asd , second / third trimester use of antidepressants was increasing the risk of asd ( adjusted hr = 1.87 ; 95% ci : 1.143.06 ) ; having a child with asd increased the risk of having another child with the disorder but nonstatistically significant ( adjusted hr = 8.36 ; 95% ci : 0.31221.71 ) , which could be explained by the lack of power for analyses on this stratum . abbreviations : ad , antidepressants ; asd , autism spectrum disorder ; maoi , monoamine oxydase inhibitor ; snri , serotonin norepinephrine reuptake inhibitor ; ssri , selective serotonin reuptake inhibitor ; tca , tricyclic antidepressant . taken together , studies are suggestive of an increased risk of asd associated with antidepressant use during pregnancy . the hyperserotonemia in autistic patients provides a possible biological explanation for this association . although , some findings are not statistically significant , this could partly be explained by lack of statistical power . lack of statistical power should not be mistaken for lack of effect . presented studies51
52
53
54
55
56
57 used different approaches to control for the possible confounding effect of the indication ( i.e. , adjustment for maternal depression and other psychiatric disorders , sibling analysis , restricting the analysis to the children of mothers with affective disorders , and stratified analyses according to maternal depression status ) nevertheless , although confounding by indication was adjusted for in the majority of studies presented , we can not completely rule out residual confounding by indication given the observational nature of the data . none of the seven studies controlled for the severity of maternal depression per se ; however , boukhris et al57 adjusted for proxies for severity , such as use of antidepressants before pregnancy , use of other antipsychotics , maternal diagnoses of other psychiatric disorders such as schizophrenia , and personality disorders . it remains that given the strength of the reported associations , serotonin inhibition during gestation seems to play an important role in the occurrence of asd . further research is needed to specifically assess the risk of asd associated with antidepressant types and dosages during pregnancy . more research is also required to determine the most relevant temporal window of exposure during pregnancy . | antidepressants are widely used during pregnancy .
several studies have shown that the use of antidepressants during pregnancy is linked to adverse outcomes , including congenital malformations , prematurity , and low birth weight .
however , there is a knowledge gap regarding the potential association between gestational exposure to antidepressants and the risk of autism spectrum disorders ( asd ) .
the etiology of asd remains unclear , although studies have implicated genetic predispositions and environmental risk factors in the development of asd in children . in this review
, we describe the association between gestational use of antidepressants , specifically selective serotonin reuptake inhibitors , and the risk of asd . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
our understanding of how much is spent on health and disease - related research and development ( r&d ) , who is spending it , on what , and where , is very limited . this failure to accurately oversee financial flows for health research hinders our ability to make health research investments effectively address international public health priorities . this inability is especially critical where resources and the capacity to undertake research are low
. the global forum for health research has published estimates for global spending on health research for the past ten years based primarily on a biennial science and technology ( s&t ) spending survey conducted by the oecd
. however , teasing out what proportion of s&t investments actually go to health research is not easy , requires significant assumptions , and the resulting global numbers can not be disaggregated by disease or purpose of the research ( e.g. , biomedical versus health systems research ) . the interpretation of such data is therefore complex and difficult for strategists and policy makers . so the existence of comprehensive and accurate information on research expenditure across the globe would bring a range of benefits to individual researchers , funders , national governments , and to those involved in managing and directing research resources . better estimates of r&d resource flows would enable the comparison and benchmarking of what is being spent in for example malaria drug development versus drug delivery research , or biomedical versus health systems research , or how much is spent on cancer research in different countries . it would help research funders to make strategic investments , enable coordination and reduce duplication to increase the impact of the billions of dollars that are invested in health research every year . our current view of the global health research landscape is further obscured by the diverse classification systems and nomenclatures that the funders of research have adopted to define their portfolios and deliver against their own remits . a multitude of such classification systems for health , disease and the related research is currently in use across the globe . these systems typically combine a description of the health or disease topic , often using the international statistical classification of diseases and related health problems ( icd10 ) or medical subject headings developed by the us national library of medicine ( mesh ) , with a description of the objective , purpose or type of research . while the resulting outputs differ markedly the similarity in the approach to classification - with use of a disease code combined with a description of the research purpose - suggests there is a common understanding or principle of what a classification system should incorporate . an ideal improvement would be that all research investments are classified using an agreed set of standards and definitions . however , encouraging research funders to harmonize and align their individual classification systems to a common standard may be unwieldy , impractical and perhaps an unrealistic expectation . in addition , it is no small challenge for many countries to provide even the most basic data on their r&d resource flows over time . for example , in the uk biomedical field , uk research funders have classified their funding portfolios against the health research classification system ( hrcs ) developed by the uk clinical research collaboration ( ukcrc )
. the hrcs was first used in 2005 to classify the portfolio of a number of major biomedical funding bodies and allowed some of the first analysis of research foci across uk organisations ; a second wave of classification was repeated for 2010 resource flows across the same organisations to compare and to describe any trends that may have emerged over time . while there is interest in using the hrcs by other european research funders
, undertaking the actual classification requires considerable manual coding and in addition the integration of the classification system with multiple funders grants systems is not a simple process . this requirement for a manual coding step , common to the use of any classification system , is both costly and time consuming . today the revolution in text , data mining and semantic web analysis presents us with new opportunities to achieve automated and large scale translation efforts . instead of organizations being required to classify their investments using a common standard , it should be feasible to develop a system that can translate diverse research funding descriptions to a lingua franca that delivers systematic and comprehensive maps of resource flows for the first time . natural language processing ( nlp ) algorithms used by collexis to search and match related documents using free text rather than complicated boolean search terms and translational software systems used by the french multi - terminology indexer ( f - mti )
. online language translation , such as google translate or yahoo s babel fish , is improving and will get better with cloud technology . the use of translation tools is currently being explored to track the impact and outcomes of research
. g - finder has provided a recent practical demonstration of how a translation approach can work to produce insight into international research commitments . supported by the bill & melinda gates foundation , the g - finder survey aims to provide comprehensive data to help funders and product developers better understand where funding gaps lie and how their investments fit into the global picture . it has done so for one area of health research , that of product r&d for neglected diseases . g - finder now covers all major public , private and philanthropic funders in high - income countries , and major funders in some middle - income innovative developing countries
. the work involved in collating and reconciling the data provided by all the funders contributing to the g - finder survey is , however , substantial . the g - finder team has developed its own way of mapping existing classifications of neglected disease r&d against an agreed , central code frame , but this is currently a largely manual process . automation of mapping individual classification systems against a commonly agreed standard using new software tools would be a major breakthrough innovation . in our view , a feasibility study is needed to explore whether such a mapping and translation approach can deliver more accurate and insightful resource flow mapping for health and disease - related r&d . as part of this study , many practical issues must be resolved including how to build on what is already strong and familiar across existing classification systems that are now in use , and how to generate agreement on the classification standard . in addition , the mechanism of translation itself and the degree to which it could be automated will be a key element in the usefulness of the envisioned translation system . different options for such a mechanism should be explored , as well as how it would be maintained , curated and governed and how any reporting and analysis would be delivered . we propose that a number of principles guide the development of a translation system to improve its chances of success . in our view , a translation system should be : accurate , cost effective and sustainable ; flexible and able to evolve over time ; equitable i.e. it meets the needs of all ( or most ) global users ; not burdensome to users that supply information and/or interface with the system ; and able to generate output that is open and accessible to all . . however , longer term efficiencies that will be gained through our ability to track global , regional and national investments in health and disease r&d , coupled with improved coordination and more strategic investments , should far outweigh that initial investment . our ultimate goal is to develop a translation tool that is of value to as many stakeholders as possible , and that is sufficiently detailed to enable its use in resource flow mapping and strategy and priority setting . the premise of our paper is that there is a role for automation to improve the efficiency of r&d classification and , as a consequence increase the likelihood of gaining access to better data . such efforts will be essential if the desire for greater harmonization in global health r&d are ever to be realised
. the views and opinions expressed in this article are those of the authors and do not necessarily reflect those of the organizations they represent . | today we have an incomplete picture of how much the world is spending on health and disease - related research and development ( r&d ) . as such it is difficult to align , or even begin to coordinate , health r&d investments with international public health priorities.current efforts to track and map global health research investments are complex , resource - intensive , and caveat - laden .
an ideal situation would be for all research funding to be classified using a set of common standards and definitions .
however , the adoption of such a standard by everyone is not a realistic , pragmatic or even necessary goal.it is time for new thinking informed by the innovations in automated online translation - e.g. yahoo 's babel fish .
we propose a feasibility study to develop a system that can translate and map the diverse research classification systems into a common standard , allowing the targeting of scarce research investments to where they are needed most . |
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image - guided large core - needle biopsy ( lcnb ) or vacuum - assisted core - needle biopsy ( vacnb ) are accurate and safe methods to obtain a preoperative diagnosis of non - palpable breast lesions . in 2003 , a new intervention was added to the spectrum of percutaneous biopsy techniques : radiofrequency - assisted intact specimen biopsy ( rfib ) . rfib uses radiofrequency cutting and enables the radiologist to obtain an intact sample of the target lesion . because tissue integrity is maintained and a larger sample is harvested from the target region , rfib is expected to yield a better diagnostic performance compared with core - needle biopsy , by reducing underestimation rates of ductal carcinoma in situ ( dcis ) and atypical ductal hyperplasia . the first series has shown promising results , with dcis underestimation rates varying from 3.2 to 21% and complete excision rates for malignant lesions of 3366% . evaluation of newly introduced complex interventions , such as rfib , is challenging , due to uncertain factors such as the learning curve , unknown complications and technical failure . in 2009 , a 5-stage model for the evaluation of innovative interventional procedures was introduced by members of the balliol collaboration , in order to achieve safe and effective innovation : the ideal recommendations . these recommendations describe 5 distinct phases that have to be completed when introducing a new interventional procedure : idea , development , exploration , assessment and long - term study . the authors propagate detailed reporting of early results to enable optimal technical development of a new interventional procedure . for rfib , the pitfalls and learning curve in the introductory period have not yet been well described . in line with the ideal recommendations , we have evaluated the technical feasibility and periprocedural complications of rfib for tissue sampling of non - palpable suspicious breast lesions in clinical practice . this study includes the first 19 patients who underwent rfib of the breast at our institution between june and november 2010 . female patients with a suspicious mammographic lesion presenting as microcalcifications and classified as breast imaging reporting and data system ( bi - rads ) category iii , iv or v were selected . patients with an implanted electronic device , a distance between the lesion and skin or chest wall smaller than 6 mm under compression , or a total compression thickness of the breast smaller than 30 mm were not eligible for the procedure . diagnostic mammograms were assessed by an experienced breast radiologist to determine bi - rads classification , lesion size and breast tissue density . breast tissue density was categorized according to the 4-point scale of the bi - rads classification ( 1 , almost entirely fatty ; 2 , scattered fibroglandular densities ; 3 , heterogeneously dense ; 4 , extremely dense ) . as advocated in the ideal recommendations , the technical aspects of each rfib procedure were analyzed in detail by studying patient records in close collaboration with the radiologists who performed the procedure . rfib was performed using the intact breast lesion excision system ( bles ) ( intact medical corporation , natick , ma , usa ) . rfib comprises the removal of an intact breast tissue sample using a high - frequency electrosurgical cutting device . the intact bles device consists of a disposable biopsy probe connected to a controller with a radiofrequency ( rf ) power source and motor control unit . the probe contains an extendable basket that consists of 5 wire electrodes stretched between 5 expanding capture blades . during the extension process , the wires cut the breast tissue with an electrosurgical cutting current of approximately 350 khz and purse down to close the distal end of the basket , thus capturing a tissue sample ( fig . depending on the size of the breast , the size of the lesion and its location within the breast , disposable biopsy probes of 15 or 20 mm were used , with basket dimensions of 15 21 mm and 20 25 mm , respectively . figure 1the intact breast lesion excision system consists of a disposable biopsy probe connected to a controller with a radiofrequency ( rf ) power source and motor control unit ( a ) . the probe contains an extendable basket with an rf wire that excises and captures the tissue sample ( b ) . the biopsy system can be attached to a standard stereotactic table with a handle mount ( c ) . the intact breast lesion excision system consists of a disposable biopsy probe connected to a controller with a radiofrequency ( rf ) power source and motor control unit ( a ) . the probe contains an extendable basket with an rf wire that excises and captures the tissue sample ( b ) . the biopsy system can be attached to a standard stereotactic table with a handle mount ( c ) . the rfib procedure was performed by 2 breast radiologists with at least 2 years of experience in performing radiography - guided breast biopsies and who had been trained in performing rfib . patients were positioned in prone position on a dedicated stereotactic table ( lorad stereoguide , danbury , ct , usa ) . after locating the breast lesion on the mammogram , the biopsy site was disinfected and a total volume of 2030 ml lidocaine ( 1% ) was applied subcutaneously , around the lesion in 4 quadrants , and directly posterior to the lesion . a preprocedural scout image was obtained to check if the position of the breast lesion had changed by the local anesthetic volume and the settings of the stereotactic table were adjusted if necessary . subsequently the distal end of the biopsy probe was positioned within 5 mm of the target lesion by cold cutting through the breast tissue with the bladed tip of the probe . an radiographic image of the biopsy specimen was obtained to check if it contained the target lesion . after the biopsy , a radiopaque marker was inserted through the biopsy canal for future localization of the biopsy site . the incision was closed using adhesive skin closure strips and covered with a sterile bandage . the rfib specimens were fixed in formaldehyde and routinely processed to sections that were stained with hematoxylin and eosin to establish the tissue diagnosis . a dedicated breast pathologist established the histologic diagnosis and assessed the extent of thermal damage , by measuring the maximum diameter of the thermal artifact zone in the equator of the biopsy specimen and at the poles ( fig . 2 ) .
figure 2illustration of normal breast tissue ( a ) versus thermally damaged breast tissue ( b ) . the latter shows blurring of the nuclei of the ductal epithelial cells and homogenization of connective tissue fibers and increased stromal eosinophilia . thermal damage was assessed by measuring the maximum diameter of the thermal artifact zone at the equator and the poles of the biopsy specimen ( c ) . typically , thermal damage was more extensive towards the distal pole of the biopsy specimen , where the wire electrodes come together .
figure 3radical removal of dcis . the lesion presented on the mammogram as a 7-mm cluster of suspicious microcalcifications ( a , arrow ) and was classified as bi - rads iv . rfib was performed after correct positioning of the biopsy probe ( b ) , capturing the entire cluster of microcalcifications , as was confirmed by the specimen radiograph ( c ) and by the absence of microcalcifications on the post - biopsy scout image ( d ) . in the post - biopsy scout image , the rfib cavity ( arrow ) illustration of normal breast tissue ( a ) versus thermally damaged breast tissue ( b ) . the latter shows blurring of the nuclei of the ductal epithelial cells and homogenization of connective tissue fibers and increased stromal eosinophilia . thermal damage was assessed by measuring the maximum diameter of the thermal artifact zone at the equator and the poles of the biopsy specimen ( c ) . typically , thermal damage was more extensive towards the distal pole of the biopsy specimen , where the wire electrodes come together . the lesion presented on the mammogram as a 7-mm cluster of suspicious microcalcifications ( a , arrow ) and was classified as bi - rads iv . rfib was performed after correct positioning of the biopsy probe ( b ) , capturing the entire cluster of microcalcifications , as was confirmed by the specimen radiograph ( c ) and by the absence of microcalcifications on the post - biopsy scout image ( d ) . in the post - biopsy scout image , rfib was performed for 19 breast lesions in 19 female patients with a median age of 59 years ( table 1 ) . seven breast lesions ( 37% ) were classified as bi - rads iii , 9 ( 47% ) as bi - rads iv and 3 ( 16% ) as bi - rads v. median lesion size on mammography was 8 mm ( range 276 mm ) . a 20-mm probe was used in 11 patients and a 15-mm probe in 8 patients . final histologic assessment showed 11 benign lesions ( 63% ) , 4 dcis lesions ( 21% ) and 3 invasive ductal carcinomas ( 16% ) ( table 2 ) . median , years ( range)59 ( 3774)lesion size on mammography , median , mm ( range)8 ( 276)n ( % ) breast tissue density on mammography(1 ) almost entirely fatty8 ( 42)(2 ) scattered fibroglandular densities6 ( 32)(3 ) heterogeneously dense4 ( 21)(4 ) extremely dense1 ( 5)breast lesion classificationbi - rads iii7 ( 37)bi - rads iv9 ( 47)bi - rads v3 ( 16)location of the target lesion in the breastcentral position2 ( 11)upper outer quadrant6 ( 32)3 o'clock position1 ( 5)lower inner quadrant2 ( 11)6 o'clock position2 ( 11)upper inner quadrant2 ( 11)12 o'clock position4 ( 21)categorized according to the bi - rads lexicon . table 2histopathologic diagnosesbiopsy specimen , n ( % ) final diagnosis ( n)benign12 ( 63)1ductal carcinoma in situ4 ( 21)3invasive ductal carcinoma3 ( 16)3only diagnoses confirmed by surgical excision are listed . one dcis lesion , presenting as a 7-mm cluster of microcalcifications , was completely excised with rfib with a 20-mm probe . the specimen appeared to contain the whole cluster of microcalcifications on the specimen radiograph , which was confirmed by the absence of microcalcifications on the post - biopsy mammogram . among the other 3 patients with rfib - confirmed dcis , surgical excision confirmed the diagnosis ( i.e. no underestimation of dcis ) . pathologic assessment revealed a dcis lesion and microcalcifications in one rfib sample ( as well as in the subsequent vacnb samples ) , and normal breast tissue in the other rfib sample ; the subsequent vacnb samples showed microcalcifications and duct ectasia with signs of chronic inflammation . in a third case , the specimen contained a probably benign lesion , but a conclusive diagnosis could not be made due to extensive thermal damage . the bleeding stopped after 20 min of manual compression . a second patient presented with minimal wound leakage 7 days after the rfib procedure . a delayed hematoma was reported . a third patient developed an infection of the biopsy site which was treated with oral antibiotics . after 7 days the infection had subsided and the patient underwent breast conserving surgery the next day . assessment of thermal damage revealed that the maximum thickness of the thermal artifact zone in the equator of the biopsy specimen ranged from 0.4 to 1.5 mm ( median 1.1 mm ) . at the poles of the specimen , the thermal artifact zone could be assessed in 13/19 specimens , showing a median diameter of 1.2 mm ( range 0.71.9 mm ) . table 3adverse eventscase no.eventconsequenceprobe size , mmbreast densityhistologic diagnosiscase no . for radiologist2wound leakage 7 days after rfiber visit , no medical intervention required , hematoma > 14 days202sclerosing fibroadenoma1st5biopsy specimen not representative based on scout image and specimen radiograph2nd biopsy procedure ( vacnb)151dcis , poorly differentiated3rd6biopsy specimen not representative on specimen radiograph and confirmed by pathology2nd biopsy procedure ( vacnb)201dilated ducts , minimal ductal hyperplasia4th14arterial bleeding after inserting the radiopaque markermanual pressure needed for 20 min , hematoma > 14 days151dcis , poorly differentiated10th18infection of biopsy sitetreatment with oral antibiotics204idc and dcis13th19biopsy specimen not assessable due to thermal damagewire - guided surgical excision biopsy203sclerosing adenosis6thabbreviations : rfib , radiofrequency - assisted intact specimen biopsy ; er , emergency room ; vacnb , vacuum - assisted core - needle biopsy ; dcis , ductal carcinoma in situ ; idc , invasive ductal carcinoma.categorized according to the bi - rads lexicon.diagnosis based on the specimens from the 2nd biopsy procedure . abbreviations : rfib , radiofrequency - assisted intact specimen biopsy ; er , emergency room ; vacnb , vacuum - assisted core - needle biopsy ; dcis , ductal carcinoma in situ ; idc , invasive ductal carcinoma . categorized according to the bi - rads lexicon . in this study we provide a detailed report , according to the ideal recommendations , of our initial clinical experiences with radiography - guided rfib . rfib yielded an interpretable biopsy specimen containing the target lesion in 17/19 ( 89% ) cases . three complications ( 16% ) occurred , which seems to exceed the complication rate reported in previous studies . allen et al . and seror et al . both reported 1 hematoma in their prospective cohorts of 74 and 163 patients , respectively . among the larger retrospective cohorts of killebrew et al . and , only the latter contained 1 record of an infection that resolved with oral antibiotics , although it is possible that minor complications occurred without being recorded . the lower complication rates reported in previous studies might be explained by exclusion of procedures performed during the introductory period , as mentioned by sie et al . who excluded the first 15 patients in each research site from analyses . in contrast to previous research , we followed the ideal recommendations to assess the learning curve that precedes the implementation of rfib in routine practice . in contrast to previous studies , the pathology slides of our patients were reviewed for thorough assessment of thermal damage by an experienced breast pathologist . this revealed that the diameter of the thermal artifact zone in the middle of the specimen ranges from 0.4 mm to 1.5 mm ( median 1.1 mm ) . still , thermal damage interfered with obtaining a conclusive pathologic diagnosis in only 1 of our cases . the thermal artifact zone in the equator of this rfib specimen was 1.0 mm , which was below the median . because the target lesion was located at the distal pole of the specimen , where the thermal damage seems to be more extensive and the total tissue volume smaller , the specimen from that case could not be fully assessed . allen et al . reported thermal artifact zones were invariably less than 1 mm . seror et al . reported that pathologic assessment was hampered by thermal damage in 6/166 ( 4% ) cases . in 1 case , the managing surgeon refrained from subsequent breast conserving surgery and referred the patient directly for adjuvant radiotherapy . this case is illustrative of the therapeutic potential of rfib . in our opinion , the use of rfib as a minimally invasive procedure for percutaneous excision of small breast lesions ( benign as well as malignant ) should be further explored . the population of patients with a breast lesion presenting as microcalcifications on mammography might not be best suited for this , because cluster size on mammography does not correlate well with the tumor size on pathology . when tumor excision with clear margins is the primary goal , perhaps patients with small ( < 1 cm ) solid tumors without microcalcifications should preferentially be selected for rfib . however , a prospective study has shown that ultrasound outperforms mammography in correct estimation of lesion size . in addition , ultrasound guidance provides a better three - dimensional orientation than stereotaxis , as well as real - time imaging feedback . for these reasons , we believe that ultrasound guidance should be evaluated as an alternative approach to obtain a therapeutic excision by rfib . in conclusion , our results show that tissue sampling of suspicious breast lesions can be performed successfully using rfib , but technical problems and periprocedural complications can occur during the learning curve and should be evaluated at an early stage . the potential of rfib as a minimally invasive technique for removal of small breast lesions is an interesting focus for further research . | abstractradiofrequency - assisted intact specimen biopsy ( rfib ) has been introduced for percutaneous biopsy or removal of breast tumors .
using radiofrequency cutting , the system enables the radiologist to obtain an intact sample of the target lesion .
according to the ideal recommendations , we performed a critical evaluation of our initial experience with rfib . between june and november 2010
, radiography - guided rfib was performed in 19 female patients .
all patients presented with suspicious microcalcifications ( bi - rads iii - v ) on mammography .
biopsy specimen integrity , thermal damage and histologic diagnosis were assessed by an expert breast pathologist .
data on technical success , diagnostic and therapeutic accuracy and periprocedural complications were collected and analyzed .
the median age of the patients was 59 years .
median lesion diameter on mammography was 8 mm ( range 276 mm ) .
the procedure was successful in 16/19 ( 84% ) patients and unsuccessful in 3/19 ( 16% ) patients ( 2 non - representative samples , 1 sample with extensive thermal damage ) .
histologic analysis of the rfib specimen revealed 12/19 ( 63% ) benign lesions and 7/19 ( 37% ) malignancies ( 4 ductal carcinoma in situ ( dcis ) lesions and 3 invasive ductal carcinomas ) . in 1 patient ,
a dcis lesion was completely removed with rfib .
overall , 3 periprocedural complications occurred ( 1 wound leakage , 1 arterial hemorrhage and 1 infection requiring oral antibiotics ) . tissue sampling of suspicious breast lesions can be performed successfully with rfib . in 1 patient dcis
was radically excised with rfib , which illustrates its potential as a minimally invasive therapeutic procedure for removal of small breast tumors .
this is an interesting focus for further research when larger probe sizes become available . |
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coenzyme b12-dependent enzymes1 , 2 catalyse a range of reactions , including radical rearrangement3 and elimination4 , 5 chemistry , and reductive dehalogenation.6 in those enzymes catalysing radical - based chemistry , the unusual co = c bond to the upper axial ligand of coenzyme b12 ( 5-deoxyadenosylcobalamin ) breaks homolytically to form a 5-deoxyadenosyl radical , which then abstracts a hydrogen atom from the substrate.7 in the case of many lyase and mutase enzymes such as ethanolamine ammonia lyase ( eal ) , the resulting substrate radical undergoes an irreversible rearrangement , after which a hydrogen atom is returned from the 5-deoxyadenosine to the product radical ( scheme 1).8 simplified reaction mechanism of eal showing how the 5-deoxyadenosyl c of the intrinsic coenzyme b12 can become deuterated upon ( multiple ) turnover of the enzyme with deuterated ethanolamine.20 note that for simplicity , co = c homolysis and h abstraction from the substrate to 5-deoxyadenosyl radical are drawn here as a concerted step . kinetic isotope effects ( kies ) can uniquely probe the nature of an h - transfer reaction s transition state , and have been very effectively employed in the study of enzyme mechanism.9 inflated kies also remain the definitive experimental test for the occurrence of quantum mechanical h - tunnelling ( qmt)10 , 11 and have been used to probe the coupling of protein dynamics to chemistry ( for examples see references 1214 ) . a number of studies have identified apparently inflated primary ( 1 ) kies on the h - transfer step(s ) that follows co = c bond homolysis in a number of b12-dependent enzymes.1519 however , the interpretation and comparison of these experiments is difficult due to the kinetic complexity associated with these multistep , reversible reactions . initially , qmt was evoked in b12-dependent enzymatic h - transfer due to the apparent magnitude of primary ( 1 ) deuterium kies ( ca . 1040 ) estimated from simple exponential fitting of stopped - flow data.1519 the oxidation state change from cob(iii)alamin to cob(ii)alamin , which occurs during homolysis , is likely to be the only change that can be resolved in the uv visible region on the stopped - flow timescale.21 consequently , the observation of a kie on this signal suggests that there is a significant degree of kinetic coupling between co = c bond homolysis and subsequent h - abstraction step(s ) . however , the stopped - flow transients are further complicated by the fact that when enzymes such as glutamate mutase16 and eal19 are mixed with deuterated substrates , an unusual reaction transient is observed , with what appear to be a burst or lag phase hump at longer timescales . if h - abstraction is not the fully rate - limiting of the two coupled steps , there is scope for the intrinsic kies to be even larger than those reported . however , studies with glutamate mutase using rapid - quench hplc and mass spectrometry analysis revealed modest intrinsic 1 h / h kies of 2.4 for l - glutamate22 and 4.1 for methyl aspartate.23 the authors also suggested that the biphasic nature of stopped - flow transients may include contributions from multiple turnovers in the second , lag phase , and emphasized the need for caution when analysing such complex data.22 non - inflated intrinsic 1 kies ( h / h kie < ca . 7 ) do not preclude a significant qmt contribution to the h transfer,24 and related rapid - quench and mass spectrometry studies suggest a significant qmt contribution in the case of glutamate mutase.25 , 26 isotopic labelling of the 5-deoxyadenosyl 5-c with h gives a large , inverse secondary ( 2 ) kie on the coupled homolysis / h - abstraction reaction with protiated substrate ( kie=0.72).27 this 2 kie approaches unity with deuterated substrate ( khd / ktd=1.05 ; that is , on a deuterium background ) , consistent with motion of the secondary hydrogen atoms on the 5-deoxyadenosyl being coupled to that of the transferring hydrogen in a transition state that involves a significant qmt contribution.25 clearly there is some uncertainly in the precise kinetic mechanism of these b12-dependent enzymes . here , we show that by using various combinations of isotopically labelled ethanolamine substrate and eal - bound b12 , it is possible to investigate the coupling and control of co = c cleavage and h - abstraction steps during the eal - catalysed pre - steady state reaction using relatively simple stopped - flow spectroscopy . the 5-deoxyadenosyl group of holoeal ( eal - b12 ) was specifically deuterated by treating holo - eal ( apo - eal freshly reconstituted with b12 ) with a large excess of deuterated ethanolamine substrate ( [ d4]ea ; scheme 1 ) prior to a size - exclusion purification of the b12-deuterated eal ( [ d2]eal ) . the deuteration state of the 5-deoxyadenosine is fairly stable8 and the h does not appear to wash out if the sample is kept in the dark in the absence of substrate . stopped - flow experiments were performed by mixing an excess of ea or [ d4]ea with eal or [ d2]eal ( figure 1 ) . at 298 k the reaction occurs rapidly relative to the stopped - flow mixing time ( ca . 1.5 ms),19 so a significant portion of the reaction transient can be lost in the instrument dead - time . this was minimized here by performing all measurements at 277 k. the transients observed upon mixing eal with [ d4]ea are visually similar to previous reports of pre - steady state stopped - flow experiments with b12-dependent enzymes and deuterated substrates,16 , 19 displaying a prominent hump at longer ( ca . 30500 ms ) timescales . this feature is largely abolished in the [ d2]ea transients , suggesting that this approach may be a useful method for simplifying the analysis of stopped - flow transients used to measure substrate kies on b12-dependent enzymes.1 averaged stopped - flow transients showing the change in eal or [ d2]eal absorbance at 525 nm upon mixing with saturating concentrations of ea or [ d4]ea ( 0.5 mm post - mixing ) at 277 k. the vertical dotted line indicates the stopped - flow dead - time of about 1.5 ms and only data collected at 1.5 ms were used for fitting to a double exponential function ( dotted lines ) . initially , transients in figure 1 were fitted to a double - exponential function , which gives acceptable results except in the case of the long time - scale hump observed upon mixing eal with [ d4]ea . apparent rate constants and observed kies are given in table 1 and table 2 , respectively . there are significant , but modest kies observed on the faster rate constant ( kobs1 ) , but not the slower kobs2 ( data not shown ) . the magnitude of the kie observed on kobs1 is much smaller than previous reports,18 but is similar to the observed kie on the reaction with the slower substrate s-2-aminopropanol.181 , 2 observed rate constants obtained from the fitted data in figure 1.[a ] averaged transients are presented in figure 1 . 10 ) were each fitted to a double exponential function with the averages 1 standard deviation of rate constants presented here . the deuteration state of the 5-deoxyadenosyl c ( e ) and the substrate ( s ) . the relative fraction of the amplitude of kobs1 , that is , a1/(a1+a2 ) . observed kies on kobs1 values from table 1 . the same nomenclature as table 1 is used , for example , h , h / h proton inventory - style stopped - flow experiments were also performed by mixing [ d2]eal with mixtures of ea and [ d4]ea ( figure 2 ) . a linear correlation between the observed kie on kobs1 and [ d4]ea concentration was obtained , which may be indicative of a single proton involved in the rate - limiting step.28 however , both kobs1 and kobs2 are apparent rate constants , which are likely to be different convolutions of some / all chemical steps up to , and including , the first irreversible step ( on the timescale of the experiment ) after co = c cleavage . in order to better analyse these data and to estimate intrinsic kie(s ) , a more realistic kinetic model is required.2 proton inventory - style stopped - flow experiment performed by mixing [ d2]eal with various mixtures of ea and [ d4]ea ( 0.5 mm total post - mixing concentration ) at 277 k. the transients are fitted to a double - exponential function ( dotted lines ) and the arrow shows the direction of increasing n , the mole fraction of [ d4]ea . the observed kie on kobs1 ( inset ) versus fraction of deuterated ea ( n ) fitted to a linear ( solid line ) and quadratic ( dashed line ) equation . a proposed reaction mechanism for the steps observed during the stopped - flow experiments is shown in scheme 2 . as the transients are not affected by decreasing the substrate concentration to low m concentrations ( data not shown ) , substrate binding is likely to be faster and substrate release much slower than other chemical steps . consequently , for simplicity we will assume in the following analysis that the ealea complex is formed in the experimental dead - time and substrate dissociation and rebinding does not occur on the experimental timescale ( ca . while only the cob(iii)alamin to cob(ii)alamin transition is directly observed in uv / vis stopped - flow experiments ( e.g. , a to b or a to b in scheme 2),21 the resulting transients will be a convolution of the reversible co = c homolysis ( k1 ) and h atom abstraction ( k2 ) steps , as well as the following irreversible8 substrate radical rearrangement ( k3 ) . thus , in principle , some information can be inferred on the formation and decay of the 5-deoxyadenosyl radical ( e.g. , b and b ) and substrate radical ( c ) intermediates species despite our inability to directly observe these species in our stopped - flow experiment . proposed 3-step reaction mechanism for the pre - steady state reaction of eal or [ d2]eal with ea and/or [ d4]ea , showing co = c cleavage ( k1 ) , h abstraction ( k2 ) and subsequent irreversible substrate radical rearrangement ( k3 ) . in a 2-step mechanism , the horizontal dotted lines delineate groups of species that can not interconvert in our kinetic model and those species in boxes are starting species : e , s = h , h , h , d , d , h and d , d , respectively from top to bottom . a major complication to the reaction mechanism in scheme 2 is scrambling of the deuteration state of the 5-deoxyadenosine 5-c in species c and the equivalent species in the ea versus [ d2]eal experiments . as the rotation of the 5-methyl group is likely to be much faster than the pre - steady state chemistry , both deuterium and protium radicals can be transferred back from the 5-deoxyadenosine to the substrate radical ( via k2 or k2 ) when [ d4]ea and eal or ea and [ d2]eal react ( scheme 2 ) . this reaction branching will generate two different isotopologues of species a and b in each case . if substrate dissociation and rebinding are not kinetically relevant , further branching is restricted by the stereospecificity of the h abstraction , as only the pro - s hydrogen of ea is expected to transfer to / from the 5-deoxyadenosyl species.29 , 30 a kie will be observed on the branching as only one of the k2 steps involves a deuterium transfer . further , the apparent rate will be reduced by the fraction of reacting species : if methyl rotation is much faster than h transfer , then during a single turnover , 1/3 of the 5-deoxyadenosine c hydrogen atoms will be deuterium for the reaction of [ d4]ea versus eal or 2/3 for eal versus [ d2]eal . to investigate whether intrinsic rate constants can be estimated from the stopped - flow experiments , the combined data in figures 1 and 2 were reanalysed using a complete , 3-step kinetic model describing scheme 2 ( model details given in the experimental section ) . a 2-step model , where co = c cleavage and h abstraction from the substrate by 5-deoxyadenosyl are concerted , was also considered . the data were globally fit ( figure 3 and figure s1 in the supporting information ) with shared rate constants . global fitting of the resampled data taken from figures 1 and 2 to the 3-step mechanism described in scheme 2 . fitting parameters are given in table 3 and table s2 in the supporting information and more detail is given in the experimental section . the 2-step and 3-step models give fits that are essentially indistinguishable over the experimental timescale ( figure s1 in the supporting information ) , and tabulated fitting parameters are given in table 3 and table s2 in the supporting information . while it is not necessary to describe the stopped - flow transients using a ( 3-step ) model where the 5-deoxyadenosyl radical is a bone fide intermediate , our data do not rule this out and suggest that in either case there is tight kinetic coupling of co = c homolysis ( k1 ) and initial h abstraction ( k2 ) reactions . an experimental approach that can directly detect the 5-deoxyadenosyl and/or substrate radical ( e.g. , time - resolved epr)31 , 32 is likely required to definitely determine whether the adenosyl radical is a stable intermediate under these conditions.3 global fitting parameters from figure 3 . [ a ] full fitting parameters for the 2-step and 3-step models are given in table s2 in the supporting information . [ b ] all rates constants in s. [ c ] this kie is shared between the k2 and k2 rate constants . see the text for more details . during the global fitting of the data in figure 3 , it was possible to obtain good fits if the value of the kie on k2 and k2 is shared ( kie2 in table 3 ) . this approach ignores the effect of - and -secondary isotope effects arising from deuteration of the substrate and 5-deoxyadenosine , respectively . however , these are expected to be much smaller than the 1 kie and are not expected to be reliably resolved in our stopped - flow experiments . 4 ) determined using the 2-step and 3-step models and the value of this kie is in good agreement with that obtained by fitting the [ d4]eal versus [ d2]ea transients to a double - exponential function ( table 2 ) . the kie on the reverse h transfer ( kie2 ) is also of a similar magnitude ( ca . 24 ) to kie2 and it appears that the kies on h transfer between the 5-deoxyadenosyl and substrate radicals in eal are likely to be less than the semi - classical limit of about 7.10 consequently , there is no direct evidence from these values for qmt during the h atom transfer from substrate to the 5-deoxyadenosyl radical . the global fitting in figure 3 did not converge unless the kies on k2 and k2 were allowed to differ . the observed kie on k2 ( kie2 ) appears to be about 40 , whereas kie2 is 24 ( table 3 ) . it is quite likely that scheme 2 oversimplifies the scrambling reaction by describing a convolution of the return hydrogen transfer with slower active - site rearrangement ( and fast methyl rotation ) . indeed , we have previously shown that the active site glutamate287 residue may reorient during turnover to control the path of the 5-deoxyadenosyl radical before binding the substrate.3335 if the reorientation of e287 occurs on a slower timescale than h transfer ( during k2 and/or k2 ) then these protein dynamics could effectively gate the h radical transfer steps . as protein dynamics are expected to be isotope - insensitive , gating should reduce the magnitude of the apparent kie . due to these complications , we can not confidently determine the absolute magnitude of the kie on the return h atom transfer from substrate radical to 5-deoxyadenosine . however , the global fitting does give insight into the origin of the long timescale hump the formation and decay of the 5-deoxyadenosyl radical species is modelled using the best - fit parameters for the 3-step model ( table 3 and table s2 in the supporting information ) . the hump is seen to arise from the formation of the scrambled 5-deoxyadenosyl radical ( b and equivalent species in scheme 2 ) . in the 2-step model , the equivalent mechanism ( h or d atom return from substrate radical to 5-deoxyadeonine ) also reproduces this hump feature . consequently , the long timescale hump in transients observed upon mixing other b12-enzymes with deuterated substrates ( or vice versa ) is likely to arise from reversibility in the h - transfer from substrate to 5-deoxyadenosyl radical , rather than from multiple turnovers of the enzyme . deuteration of the b12 c prior to stopped - flow kie measurements minimises the effect of the scrambling as this can only occur upon mixing with ea , not [ d4]ea , and the extent of the branching to a and b is minimised by the kie on k2.4 apparent evolution of the 5-deoxyadenosyl radical concentration modelled from the best - fit parameters for the 3-step model fitting in figure 3 . transients are for : a ) ea versus eal , b ) [ d4]ea versus [ d2]eal , c ) [ d4]ea versus eal , and d ) ea versus [ d2]eal . in c and d the black transients are convolutions of the contributions from the initial h or d abstraction ( red ) and the scrambled partially deuterated 5-deoxyadenosyl radical ( blue ) . the vertical dotted line indicates the stopped - flow dead - time of about 1.5 ms . we have selectively deuterated the c of the 5-deoxyadenosyl moiety of the intrinsic coenzyme b12 in eal prior to stopped - flow kie measurements with [ d4]ea . this approach significantly simplifies the analysis of the resulting transients and appears to allow reliable 1 kies to be determined from simple ( multi)exponential fitting of the data . global fitting of stopped - flow transients to kinetic models allows the estimation of some intrinsic rate constants and kies on the reversible h - transfer steps preceding substrate radical rearrangement in eal . no direct evidence for qmt during h - transfer was found ( i.e. , the kies are not inflated ) . the data can also be described by a 3-step model where co = c bond homolysis and h - abstraction from the substrate occur sequentially , consistent with recent computational studies on a coenzyme b12-dependent mutase.36 this approach allows us to model the concentration of the 5-deoxyadenosyl radical over the course of a single enzyme turnover , and the hump observed in some stopped - flow transients is shown to arise from scrambling of the deuteration state of the 5-deoxyadenosyl moiety . all reagents were analytical grade and purchased from sigma aldrich ( gillingham , uk ) except for [ d4]ethanolamine , which was purchased from goss scientific instruments limited ( crewe , uk ) . recombinant eal from salmonella enterica was prepared as described previously.19 , 34 holo - eal was prepared freshly on the day of experiments by reconstituting eal with excess coenzyme b12 prior to removal of unbound b12 by size - exclusion chromatography using a 10 dg desalting column ( biorad , hemel hempstead , uk ) . [ d2]eal was prepared by treating 1020 m of holo - eal with 2 mm [ d4]ea for 10 min prior to separation of products and unreacted substrate with a 10 dg desalting column . [ d2]eal was then brought to the desired concentration for stopped - flow using 10 kda molecular weight cut - off centrifugal filter devices . all preparations were performed on ice in the dark , while all kinetic measurements were performed in 20 mm hepes ph 7.5 at 277 k. stopped - flow experiments were performed essentially as described previously19 using an applied photophysics ( leatherhead , uk ) sx.18 mv - r stopped - flow spectrophotometer with approximately 30 m eal and 500 m ethanolamine post - mixing . the absorbance change accompanying interconversion between cob(iii)alamin and cob(ii)alamin was monitored at 525 nm . the stopped - flow transients in figures 1 and 2 were globally fit to the kinetic model shown in scheme 2 . to ensure even weighting , transients for each experiment were averaged ( as shown in figures 1 and 2 ) and then linearly interpolated before resampling . for each transient , 200 data points were taken at equal distance over a logarithmic timescale between 1.6 and 525 ms using mathematica 9.0 ( wolfram research , inc . , the [ d4]ea versus eal reaction in scheme 2 is described by :
1 the other reactions were modelled similarly , and the ea versus eal and [ d4]ea versus [ d2]eal reactions are simplified by the lack of scrambling and thus do not contain a and b species . the global model contains four each a , b and c species and two each a and b species . a 2-step mechanism where homolysis and h abstraction are concerted is similarly described by :
2 in this case , there are four each a and c species and two a species . to globally fit the seven transients in figures 1 and 2 to either model , all species were treated locally while the rate constants were shared globally . three global kies were fitted for , as described in the main text . a single global extinction coefficient for the sum of all six a and a species in each experiment was fitted for , but the relative starting concentration of each species was allowed to vary between 0.71.3 across each experiment to allow for uncertainty in active enzyme concentration . the start time was also allowed to vary by 2 ms to allow for variation in mixing times . | coenzyme b12-dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics . by selectively deuterating the substrate ( ethanolamine ) and/or the -carbon of the 5-deoxyadenosyl moiety of the intrinsic coenzyme b12 , it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5-deoxyadenosyl radical and substrate during single - turnover stopped - flow measurements .
these data are interpreted within the context of a kinetic model where the 5-deoxyadenosyl radical intermediate may be quasi - stable and rearrangement of the substrate radical is essentially irreversible .
global fitting of these data allows estimation of the intrinsic rate constants associated with coc homolysis and initial h - abstraction steps .
in contrast to previous stopped - flow studies , the apparent kinetic isotope effects are found to be relatively small . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
a wound refers to a case where the continuity of a normal skin structure is destroyed by
physical damage to the skin , and the process of recovering such discontinuity is referred to
as wound healing1 . wound healing consists
of three phases inflammation , proliferation , and remodeling and wound healing progresses by
cells playing their unique roles in each phase2 . thus far , 13 types of collagens acting importantly in the wound healing process have been
discovered , and among them , type i accounts for 85% of the wound healing process3 . collagens engage in normal or pathological
bioprocesses such as cell combination , cell differentiation , wound healing , all kinds of
fibroses and inflammatory responses , and cancer transformation4 . collagen synthesis in fibroblasts is influenced by diverse cytokines including transforming
growth factor 1 ( tgf-1 ) , insulin - like growth factor 1 , interleukin 1 , and platelet - derived
growth factor , and in particular , tgf-1 is known to play a major in vivo role5 . tgf-1 , which is secreted from fibroblasts ,
macrophages , and platelets , triggers chemotaxis of fibroblasts , macrophages , and monocytes . stimulation of fibroblasts increases synthesis and secretion of collagen fibers and other
substrate components and inhibits secretion of fiber decomposition enzymes . tgf-1 also
stimulates endothelial blood vessel cells , thereby promoting formation of blood vessels6 , 7 . kloth noted that electrical stimulation applied to wounded skin tissues increased
angiogenesis and perfusion , which raised the activity of granulation tissues and
fibroblasts , thereby promoting wound healing speed9 . physical therapy approaches to wound healing include hvpcs ,
microcurrents , low - intensity laser , and ultrasound10,11,12,13 . hvpcs has a monophasic
twin peak pulse wave form and very short pulse duration and therefore can safely stimulate
deep tissues without damage14 . the
effects of hvpcs related to wound healing are a decrease in chronic decubitus ulcer ,
inhibition of staphylococcus aureus growth , an increase in perfusion around ischemic wounds ,
and improved microcirculation15,16,17,18 . doran et al . observed that hvpcs with the cathode as an
active electrode decreased the size of the inflammatory area19 . lee et al . applied hvpcs to the wound areas of rats for seven days
and observed that the experimental group s wound healing rate and fibroblast and collagen
density were significantly higher than those of the control group10 . jeong and song applied hvpcs to normal skin of rats for
seven days and noted that expression of collagen i and tgf-1 was promoted in the dermis
layer20 . however , most previous studies have concerned hvpcs with a non - visible contraction
intensity , and research on the effects of hvpcs with a visible contraction intensity on
wound healing is lacking . accordingly , the aim of this study was to compare the expressions
of tgf-1 and collagen i at each time point by applying hvpcs with a non - visible contraction
intensity to wound areas of white rats , thereby presenting a theoretical groundwork for its
clinical practice . thirty - six white adult male sprague - dawley rats ( ntacsam : sd , samtako bio korea , osan ,
republic of korea ) , which were six weeks old and weighed from 200 to 220 g , were used and
were equally and randomly assigned to experimental group i , experimental group ii , and the
control group ( n = 12 per group ) . hvpcs with a non - visible contraction intensity was applied
to experimental group i , hvpcs with a visible contraction intensity was applied to
experimental group ii , and placebo stimulation was applied to the control group under the
same condition as used for the experimental groups after wounds were triggered . for
histological sections , four rats from each group were sacrificed on the third , fifth , and
seventh days . during the experimental period , whether the rats had any trauma or disease was
checked , and stress factors were minimized by reducing environmental changes . the
temperature and humidity of the breeding room were maintained at 22 2 c and 55 10% ,
respectively . the light - dark cycle was set at 12 hours to maintain the constant conditions
in the breeding room during the experimental period . the rats were allowed to freely take
water and solid feed ( samyang co. , republic of korea ) . the institutional animal care and use
committee at dongshin university approved the protocols used in this study , and the animals
were cared for in accordance with the guidelines for animal experiments ( 2010 ) . the rats were subjected to general anesthesia using a respiratory anesthetic , sevoflurane ,
and the hair on their back area was removed using a depilatory cream ( veet depilatory cream ,
reckitt benckiser , france ) . after removing the hair , the skin was disinfected with 75%
alcohol so that no depilatory cream remained on the skin . a wound with a diameter of 10 mm 11 surgical blade at locations more than
15 mm laterally distant from the center . the panniculus carnosus was exposed to induce
wounds in a circular form , and the wound areas were dressed with sterilized gauze . the rats
rested for 24 hours after induction of the wounds without any manipulation in order to
minimize the stress and physiological response resulting from the wounds21 . the skin tissue of a rat has a panniculus
carnosus layer , and therefore induction of a circular wound only may induce a wound healing
speed similar to that of humans22 , 23 . the wounds were covered with sterilized gauze soaked with normal saline . an active
electrode ( 22 cm ) was fixed on them , and an inactive electrode of the same size was fixed
on the abdomen and connected to an hvpcs device ( endomed 482 , enraf- nonius , rotterdam ,
netherlands ) . the same pulse frequency and pulse duration were established for application
of hvpcs to each group . a non - visible contraction intensity of 25 to 65 v was applied to the
experimental group i , and a visible contraction intensity of 40 to 85 v was applied to the
experimental group ii thirty minutes per day , for six days . placebo stimulation was applied
to the control group under the same condition as the experimental groups . for the first three days ,
the cathode was applied as the active electrode , and from the fourth day , the anode was
applied as the active electrode20 . fibroblasts move to the cathode due to their electrotropism24 , and when the active electrode for hvpcs is the cathode , cellular
responses of fibroblasts are activated25 . the diameters of wounds were measured right after their induction , on the third day , on the
fifth day , and on the seventh day with the naked eyes using calipers ( mitutoyo corporation ,
kawasaki , japan ) capable of measuring length in 0.05 mm increments . in order to compare the
sizes of wounds , the sizes of the left and right wounds were added and divided by two ( fig . 1fig . wound diameter measured with calipers western blotting was conducted in order to examine the expression of tgf-1 after
application of hvpcs . the wound tissues treated according to each manipulation condition
were washed three times with phosphate - buffered saline at 4 c cut finely lysis buffer
( 300 mm nacl , 50 mm tris cl , ph 7.6 ) with a volume three times larger than that of the
tissues , 0.5% triton x100 , 2 mm phenylmethylsulfonyl fluoride , 2 l / ml aprotinin , and 2
l / ml leupeptin were added at 4 c to react for 40 minutes . thereafter , they were
centrifuged at 4 c and 14,000 rpm for 15 minutes to extract supernatant fluids . protein
quantification was performed with the supernatant fluids , and the extracted proteins were
distributed to 7.512% sodium dodecyl sulfate ( sds ) polyacrylamide gel for
electrophoresis26 . then blotting was
performed on a polyvinylidene difluoride membrane ( amersham , 0.2 m , pore size , usa)27 . the membrane was reacted for one hour at
4 c in 5% ( w / v ) nonfat dried milk containing triethanolamine - buffered saline ( tbs ,
phosphate - buffered saline containing 0.01% ( v / v ) tween 20 ) and washed with tbs . tgf-1
( sc-146 , 1:500 , santa cruz biotechnology , santa cruz , ca , usa ) was reacted for 24 hours at
4 c , and to verify whether there was a specific reaction and the degree of the reaction , a
goat anti - rabbit igg antibody ( ab6721 , abcam , cambridge , ma , usa ) was reacted as the
secondary antibody for one hour ; an enhanced chemical luminescence kit ( rpn 2106 , amersham
life science , inc . , usa ) was used to determine the results . to examine whether the same
amount of protein was loaded , monoclonal anti--actin ( a-5316 , 1:5,000 , sigma - aldrich , st ,
louis , mo , usa ) and goat anti - mouse igg ( bd bioscience , franklin lakes , nj , usa ) antibodies
were used for comparison . after application of hvpcs , immunohistochemical assessment was conducted in order to look
at expression of type i collagen . after the paraffin removal and hydration processes for the
produced slide , 30% tris - ethylenediaminetetraacetic acid ( edta ) was reacted for 40 minutes
at 90 c and cooled for 20 minutes at a room temperature in order to remove nonspecific
reactions for immunohistochemistry . then as a preprocessing step , 3% hydrogen peroxidase was
used to block the activity of endogenous peroxidase . thereafter , a pbs solution to which
blocking serum had been added was used so that the primary antibody solution would be well
absorbed into the tissue inside . the tissue was then cleaned with 0.01 m pbs several times ,
and the primary antibody for type 1 collagen ( sigma - aldrich , st . louis , mo , usa ) was diluted
1:100 , 1:200 , 1:500 , and 1:2000 with pbs . it was reacted over night at 4 c and then washed
with pbs . then , the universal antibody was diluted with blocking serum in pbs and reacted
for 60 minutes at a room temperature . again , the tissue was washed with 0.01 m pbs three
times , for five minutes each time and reacted with streptavidin for 30 minutes at room
temperature . after washing with pbs , color development was performed with dab
( 3,3-diaminobenzidine , d3939 , sigma - aldrich , st . then , it
was washed with pbs once for three minutes , counterstained with hematoxylin , washed twice
for three minutes in flowing water , and dehydrated with 70% , 80% , 90% , and 100% ethanol for
two minutes for each . the tissue was treated with 100% xylene twice for two minutes to
render it transparent and then sealed with canada balsam ( sigma - aldrich , st . louis , mo , usa )
to produce permanent samples . for statistical analysis of this study , pasw statistics , ver 18.0 , was used . in order to
verify the significance of each group s wound size and expression of tgf-1 according to
intensity of hvpcs , one - way analysis of variance ( anova ) was conducted to examine
differences among the groups at each measured time point , and duncan s test was performed as
a post hoc test . a paired t - test was carried out in order to look at each group s
differences in measured variables at each measured time point . immunochemical assessment of type i collagen was performed in a
semiquantitative manner and classified as follows : negative reaction , mild negative reaction
when stainability was weak , moderate positive reaction when stainability was moderate , and
strong positive reaction when stainability was strong28 . according to the results of comparing changes in wound size at each time point , the control
group s wound size was significantly decreased on the fifth and seventh days ( p<0.01 ) ,
while the wound sizes of experimental groups i and ii were significantly decreased at all
time points ( p<0.05 ) . according to the results of one - way anova for comparison among the
groups , there was significant difference on the fifth and seventh days , and therefore , the wound size of experimental group ii was the most
significantly decreased on the fifth and seventh days ( p<0.01 ) ( table 1table 1.comparison of changes in wound size ( unit : mm)day 0day 3day 5day 7control group ( n=12)10.5 0.39.9 1.08.6 1.03.74 0.3f2.14.019.422.0post hoca , b > ca > b > cvalues are presented as the meansd . tested by one - way anova ( p<0.05 ;
p<0.01 ) and duncan s multiple range test . tested by paired t - test
( * p<0.05 ; * * p<0.01 ) . a. control group : sham group ( wound induced ) . b.
experimental group i : 2565v , high voltage pulsed current ( hvpcs ) . c. experimental
group ii : 4085v , hvpcs . ) . values are presented as the meansd . tested by one - way anova ( p<0.05 ;
p<0.01 ) and duncan s multiple range test . tested by paired t - test
( * p<0.05 ; * * p<0.01 ) . b.
experimental group i : 2565v , high voltage pulsed current ( hvpcs ) . c. experimental
group ii : 4085v , hvpcs . according to the results of comparing the expressions of tgf-1 at each time point , the
expression of tgf-1 in the experimental groups was significantly increased on the fifth day
( p<0.05 ) . according to the results of one - way anova to compare the expression of tgf-1
among the groups , there were significant differences on the fifth day , and therefore ,
duncan s post hoc test was conducted . the expression of tgf-1 in experimental group ii was
highest the fifth day ( p<0.01 ) ( table
2table 2.comparison of changes in tgf -1 expression ( unit : % ) day 3 day 5day 7control group ( n=12)99.28.199.05.799.59.7experimental group i ( n=12)105.78.1130.212.1101.28.8experimental group ii ( n=12)105.77.9146.611.4 * 117.010.7f0.822.53.8post hocc > b > avalues are presented as the meansd . tested by one - way anova ( p<0.05 ;
p<0.01 ) and duncan 's multiple range test . tested by paired t - test
( * p<0.05 ; * * p<0.01 ) . a. control group : sham group ( wound induced ) . b.
experimental group i : 2565v , high voltage pulsed current ( hvpcs ) . c. experimental
group ii : 4085v , hvpcs . ) . values are presented as the meansd . tested by one - way anova ( p<0.05 ;
p<0.01 ) and duncan 's multiple range test . tested by paired t - test
( * p<0.05 ; * * p<0.01 ) . b.
experimental group i : 2565v , high voltage pulsed current ( hvpcs ) . c. experimental
group ii : 4085v , hvpcs . according to the results of observation of the immunoreactivity to type i collagen , which
is known to be a component making up most of the internal structure of a wound , the control
group and experimental group i showed a mild positive reaction ( + ) on the fifth day , and
experimental group ii showed a strong positive reaction ( + + + ) in the dermis on the fifth day
and a moderate positive reaction ( + + ) on the seventh day ( table 3table 3.immunohistochemical reaction of type i collagen of the skingroupperiod3 days5 days7 dayscontrol group -+-experimental group i-+-experimental group ii-+++++ and figs . 2fig . 2.immunohistochemical findings for the type i collagen reaction in skin from the
control group ( immunohistochemical stain , 100 ) , 3fig . 3.immunohistochemical findings for the type i collagen reaction in skin from
experimental group i ( immunohistochemical stain , 100 ) , 4fig . 4.immunohistochemical findings for the type i collagen reaction in skin from
experimental group ii ( immunohistochemical stain , 100 ) ) . immunohistochemical findings for the type i collagen reaction in skin from the
control group ( immunohistochemical stain , 100 ) immunohistochemical findings for the type i collagen reaction in skin from
experimental group i ( immunohistochemical stain , 100 ) immunohistochemical findings for the type i collagen reaction in skin from
experimental group ii ( immunohistochemical stain , 100 ) hvpcs has a monophasic twin peak pulse wave form , low voltage of 150 v or higher , low total
current ( 1.5 ma ) , and very short pulse duration29 . the effects of hvpcs include a decrease in chronic decubitus
ulcer , inhibition of staphylococcus aureus growth , an increase in perfusion around ischemic
wounds , and improved microcirculation15,16,17,18 . this study aimed to examine expression of type i collagen and tgf-1 by applying hvpcs with
a visible contraction intensity to wound - induced white rats . the same pulse frequency and
duration were set for application of hvpcs to each group . a non - visual contraction intensity
of 25 to 65 v was applied to experimental group i , and a visual contraction intensity of 40
to 85 v was applied to experimental group ii ; both groups received hvpcs for thirty minutes
per day for six days . according to the results of comparing changes in wound size at each
time point , the control group saw a significant decrease in its wound size on the fifth and
seventh days ( p<0.01 ) , while experimental groups i and ii saw a significant decrease in
their wound sizes at all time points ( p<0.05 ) . wound size was compared among the groups ,
and that of the experimental group ii was more significantly decreased than the other groups
on the fifth and seventh days ( p<0.01 ) jeong and song applied hvpcs
with a non - visible contraction intensity ( 140 s , 120 pps , and 3050 v ) to normal white rats
30 minutes per day for seven days and reported that expression of tgf-1 in the dermis
significantly increased relative to the control group20 . however , western blotting was conducted in the present study to
compare the expression of tgf-1 in each group at each time point . the expression of tgf- 1
in experimental group ii was significantly increased on the fifth day ( p<0.05 ) , which was
more or less different from previous study results . according to the results of comparing
the expression of tgf-1 among the groups at each time point , the expression of tgf-1 in
experimental group ii this result
is considered to have been caused by the relatively high stimulation intensity , which
triggers contraction of nerve roots as a result of the pumping action of the muscles and
increased local vasodilatation and oxygen supply to tissues32 , 33 , thereby stimulating
endothelial blood vessel cells and promoting formation of blood vessels , which in turns
induces stimulation of macrophages and platelets , resulting in increased expression of
tgf-17 . according to the results of the immunohistochemical reaction performed to examine the
expression of type i collagen , the control group and experimental group i showed a mild
positive reaction ( + ) on the fifth day , and experimental group ii showed a strong positive
reaction ( + + + ) in the dermis on the fifth day and a moderate positive reaction ( + + ) on the
seventh day . richard et al . observed that wounds were in the inflammatory phase on the third
day after injury and that they were in the remodeling phase on the sixth day , with the
inflammatory stage almost over and granulation tissue starting to form34 . this result shows that synthesis of type i collagen is
most active on the fifth day and that synthesis of type i collagen is promoted by hvpcs with
a visible contraction intensity . lee et al . also observed
that fibroblast expression induced collagen synthesis10 . in the present study as well , it is judged that tgf-1 increased
by hvpcs with a visible contraction intensity promoted type i collagen synthesis . comparison of the sizes of the wounds among the groups showed that the most significant
decrease in wound size was found in experimental group ii on the fifth and seventh days .
according to the result of comparison of the expression of tgf-1 in each group at each time
point , the expression of tgf-1 in experimental group ii was significantly increased on the
fifth day . according to the results of the immunohistochemical reaction performed to examine
the expression of type i collagen , the control group and experimental group i showed a mild
positive reaction ( + ) on the fifth day , and experimental group ii showed a strong positive
reaction ( + + + ) in the dermis on the fifth day and a moderate positive reaction ( + + ) on the
seventh day . the above results suggest that hvpcs with a visible contraction intensity
increased expression of tgf-1 and synthesis of type i collagen , promoting wound
healing . | [ purpose ] this study aimed to examine the expression of transforming growth factor 1
( tgf-1 ) and type i collagen by applying high voltage pulsed current stimulation ( hvpcs )
with a visible contraction intensity to white rats with induced wounds .
[ subjects ]
thirty - six white rats were used for this study .
hvpcs with a non - visible contraction
intensity was applied to experimental group i , and hvpcs with a visible contraction
intensity was applied to experimental group ii .
placebo stimulation was applied to the
control group .
[ methods ] after wounds were triggered , the intervention appropriate for
each group was applied .
changes in the size of their wounds and expression of tgf- 1 and
type i collagen were measured on the third , fifth , and seventh days .
[ results ] comparison
of the sizes of the wounds among the groups showed that the most significant decreases
were found in experimental group ii on the fifth and seventh days .
tgf-1 expression
comparison revealed that experimental group ii had the most expression on the fifth day .
[ conclusion ] hvpcs with a visible contraction intensity was effective in promoting wound
healing by increasing expression of tgf-1 and synthesis of type i collagen . |
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duchenne muscular dystrophy ( dmd ) and becker muscular dystrophy ( bmd ) are allelic disorders of the dystrophin gene at the xp21 locus1,2 ) . dystrophin plays an essential structural role in both cardiac and skeletal muscle , protecting the sarcolemma from mechanical stresses of muscle contraction3 ) . whereas dmd is characterized by the total absence or dysfunction of the dystrophin protein , bmd is characterized by reduced expression of the protein4 ) . while respiratory failure is the main cause of mortality ( mostly in the third decade of life ) in dmd4 ) , heart failure is the primary cause of death in bmd5 ) . in addition , the severity and the age of onset of cardiac involvement in bmd shows no correlation to skeletal muscle involvement . according to the previous reports , cardiac involvement in bmd patients is detectable at later ages with an average onset age of 28.77.1 years6 ) , and advanced cardiomyopathy is rare in bmd patients below 20 years5 ) . moreover , few cases of severe cardiomyopathy in bmd have been reported to date5,6 ) . herein , we report the case of a patient with bmd who presented with dilated cardiomyopathy ( dcmp ) at the age of 18 years , and we discuss the factors that may influence early manifestation of dcmp in bmd patients with a review of the literature . an 18-year - old boy was admitted to seoul national university children 's hospital ; he experienced chest discomfort , nausea and dyspnea at rest . the patient , with no known family history of significant muscle disease , was first examined at 3 years of age because of enlarged calves . at the time of the first examination , blood tests revealed elevated serum levels of creatine kinase ( 6,378 u / l ) . analysis of the dystrophin - encoding gene by multiplex polymerase chain reaction showed a deletion of exons 45 to 49 . the patient remained ambulant with mild proximal weakness and has been able to ascend stairs without holding the lap one by one at the age of 18 years . at the time of admission his blood pressure was 118/61 mmhg and his pulse rate was 105 per minute without irregularity , and no evident heart murmur or rale was audible . the manual muscle test revealed only minimal weakness ( 4/5 or more ) of the lower proximal muscles , and the patient had no subjective difficulties when standing from the squatting position . chest roentgenogram showed cardiomegaly ( cardiothoracic ratio=54% ) , and an electrocardiogram ( ecg ) exhibited an abnormal st - t wave , biatrial enlargement , and left ventricular hypertrophy ( fig . two - dimensional and m - mode echocardiograms showed a severely dilated left ventricular cavity with diffuse hypokinesis ( fig . systolic indices were reduced , including fractional shortening ( 9% ) and ejection fraction ( 19% ) . the patient 's left ventricular function deteriorated gradually in spite of intensive medical treatment for heart failure , and he died from congestive heart failure 5 months after initial cardiac symptoms at the age of 18 years and 4 months . we report a case in which the patient had a dystrophin gene deletion , of exons 45 to 49 , and died from early - onset dcmp with bmd at the age of 18 years . cases of early onset of cardiac failure are rare although there is evidence of frequent progressive cardiac involvement in bmd , characterized by the development of dcmp7,8 ) . in bmd , asymptomatic cardiac involvement develops in over 70% of cases , and these patients remain asymptomatic for a longer period9 ) . only up to one - third of . cardiac involvement of bmd was reported to be uncommon in patients under 16 years of age , whereas more than 70% of patients demonstrated pathologic cardiac findings by the age of 40 years12,13 ) . the median age of onset of fractional shortening less than 25% was reported to be 30 years14 ) . several reports that cardiac dysfunction is not clearly correlated with skeletal muscle severities in dmd / bmd patients have been described , and they suggested possible pathogenesis and genotypic correlations . jefferies et al.15 ) revealed an association between mutations involving exons 12 and 14 to 17 or 31 to 42 , and early onset of dcmp . in addition , mutations involving exons 51 or 52 appeared to decrease the risk of cardiac involvement ; however , because of the relatively short follow - up period , this could not be established with certainty . yoshida et al.16 ) also suggested that a deletion around exon 1 may severely damage the expression and/or function of dystrophin selectively in cardiac muscle , but not in skeletal muscle . involving exon 13 ramelli et al.18 ) reported the isolated deletion of exon 48 seems to be correlated with a mild course of the disease . magri et al.19 ) reported that bmd patients with duplications of dystrophin gene experienced milder cardiac and respiratory involvement than patients with deletions , as demonstrated by the later age of onset . kaspa et al.3 ) suggested reasonable correlations between specific regions of the dystrophin gene and the age of dcmp onset in bmd patients through analysis of a large series of dystrophinopathy patients . bmd patients with deletion mutations affecting any portion of the actin - binding amino - terminal domain of dystrophin ( exons 2 to 9 ) or exons 45 to 49 are at risk of developing dcm in their second and third decades of life , respectively3 ) . they also provided novel evidence of a strong association of dcmp with specific structural alterations of the dystrophin rod domain : dcmp with affecting exons 45 to 49 is more sensitive to altered phasing of the spectrin repeats rather than absence or presence of any individual exons . they suggested that the alterations caused by out - of - phase mutations extend beyond the spectrin repeats unit and may lead to a severely altered configuration of the rod domain , ultimately affecting the entire dystrophin ( skeletal and cardiac ) protein . in this case , the patient had a deletion affecting exons 45 to 49 , encoding spectrin repeats 17 to 19 , that preserves hinge 3 of the dystrophin protein showed later onset and slowly progressive skeletal muscle symptoms instead of earliest onset dcmp . this is in agreement with studies on dystrophin - null mdx mice expressing a mini - dystrophin construct that lacks the 45 to 49 region but has an intact hinge 3 domain . in these mice , only a partial restoration of cardiac function was achieved despite complete rescue of the skeletal muscle pathology20 ) , which also suggested that cardiac dystrophin may be particularly sensitive to structural disruptions of exons 45 to 49 compared with skeletal muscle dystrophin . thus , we can assume that the early - onset dcmp and advanced heart failure in our case are related to the deletion site of exons 45 to 49 in the dystrophin gene , which is a more sensitive region in cardiac muscle dystrophin compared to that of skeletal muscle . to clarify the genotype - phenotype correlations of cardiac function in dystrophinopathy , further longitudinal large - scale epidemiologic study with functional or tissue - specific dystrophin expression studies of skeletal muscle dysfunction and cardiac symptoms early cardiac evaluation and aggressive noninvasive imaging of young patients with muscular dystrophy can help identify those patients who are most likely to benefit from heart failure therapy15 ) . on the basis of the results of previous studies , all the subjects with bmd who underwent drug therapy ( a combination of digoxin and angiotensin - converting enzyme inhibitors ) ultimately achieved normalization of the left ventricular dimension and systolic function compared with the dmd group , which did not show as dramatic an improvement15 ) . bushby et al.21 ) also recommended that bmd patients should undergo cardiac evaluation ( ecg and echocardiogram ) at diagnosis and be screened for the development of cardiomyopathy at least every 5 years . considering that skeletal muscle weakness is not correlated with progression of cardiomyopathy in some of the bmd patients , alert and regular cardiac functional evaluation is necessary even in patients with mild and slowly progressive proximal weakness , particularly in cases with deletions affecting exons 45 to 49 as in our case , and patients with dystrophin mutations involving the amino terminal domains . in conclusion , we report here a bmd case with dystrophin deletion from exon 45 to 49 , who presented with early cardiomyopathy , and we also recommend careful and regular cardiac evaluation of bmd patients who have mutations affecting sensitive sites of dystrophin , to monitor cardiac involvement . | an 18-year - old boy was admitted with chest discomfort , nausea , and dyspnea at rest . at the age of 3 years
, he underwent muscle biopsy and dystrophin gene analysis owing to an enlarged calf muscle and elevated serum kinase level ( 6,378 u / l ) without overt weakness ; based on the results , becker muscular dystrophy ( bmd ) was diagnosed .
the dystrophin gene showed deletion of exons 45 to 49 .
he remained ambulant and could step upstairs without significant difficulties .
a chest roentgenogram showed cardiomegaly ( cardiothoracic ratio , 54% ) , and his electrocardiogram ( ecg ) showed abnormal st - t wave , biatrial enlargement , and left ventricular hypertrophy .
the 2-dimensional and m - mode ecgs showed a severely dilated left ventricular cavity with diffuse hypokinesis .
the systolic indices were reduced , including fractional shortening ( 9% ) and ejection fraction ( 19% ) . despite receiving intensive medical treatment , he died from congestive heart failure 5 months after the initial cardiac symptoms .
we report a case of bmd with early - onset dilated cardiomyopathy associated with deletion of exons 45 to 49 .
early cardiomyopathy can occur in bmd patients with certain genotypes ; therefore , careful follow - up is required even in patients with mild phenotypes of bmd . |
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in eukaryotic cells , hundreds of molecules are exchanged between the nucleus and the cytoplasm every minute . this process , called nucleocytoplasmic transport , is crucial not only for basic cellular activities but also for regulating various cellular events . based on the literatures and database information , we can estimate that as much as ~30% of the proteins expressed in cells are nuclear proteins , indicating that the nucleocytoplasmic transport is the major intracellular trafficking pathway in terms of the quantity and diversity of molecules that are transported . to enter and exit the nucleus , molecules must translocate through nuclear pore complexes ( npcs ) , which are large protein assemblies that are embedded in the nuclear envelope . npcs allow the passive diffusion of small molecules , such as ions and proteins smaller than ~30 kda . however , larger molecules must bind to a nucleocytoplasmic transport carrier ; these are typically hydrophobic because the transport channel of the npcs is hydrophobic . theses proteins are conserved from yeast to mammals and are considered to facilitate the nuclear transport of most proteins and many different rnas . since 1995 , when the first nuclear import carrier ( importin ) was identified , our understanding of the basic mechanism of nucleocytoplasmic transport has advanced significantly . one key feature of this transport is that cargoes can continue to accumulate in one compartment against a chemical concentration gradient , i.e. , from the cytoplasm to the nucleus or from the nucleus to the cytoplasm . to achieve this , carriers bind to cargo in one compartment , translocate through npcs and dissociate from the cargo in the target compartment . the gtpase cycle of the small gtpase ran is coupled with importin -mediated transport pathways and plays a crucial role in the cargo binding and release that occurs in the nucleus or in the cytoplasm . each nuclear import or export cycle consumes one gtp hydrolyzed by ran , which serves as a driving force of the transport . to date , studies of nuclear protein import or export have focused almost exclusively on the importin - ran system , and the different transport pathways have not been identified / investigated . recently , we identified a transport pathway that is mediated by a novel carrier protein , hikeshi , that becomes active during the thermal stress . hikeshi does not belong to the importin , and it is evolutionarily conserved from yeast to mammals . hikeshi - medited trasnport does not use the ran system , but likely uses the atpase cycle of the molecular chaperone hsp70 as a driving force . upon exposure to environmental stresses , cells respond by altering many aspects of cellular physiology to protect cells from stress damage . after release from stresses , cells must attenuate the stress response and restore normal activities to survive . a shift in the temperature from the physiological state ( heat shock ) causes protein misfolding , protein dysfunction or protein aggregation , and thus perturbs protein homeostasis . in response to heat shock , one prominent phenomenon observed in cells is the increase in the cellular level of molecular chaperones known as heat - shock proteins ( hsps ) , which play essential roles in maintaining protein homeostasis . in addition to heat shock , a large variety of environmental stresses are known to induce the expression of hsps . therefore , the heat - shock response is considered synonymous with the cellular stress response . in addition , among many other stresses , heat - shock stress is most susceptible to reversion from stress damage within a short time frame . heat - shock stress is therefore an excellent model system in which to study a cellular stress response , as well as the recovery of cells from stress . during stresses , normal transcription and translation little was known about the nuclear transport during stress ; however , several groups reported that stresses , such as heat - shock and oxidative stresses , induce nuclear retention and inhibition of the nuclear export of importin , an adaptor molecule that connects classical nuclear localization signals ( nlss ) to importin , perturbing the importin /importin pathway . furthermore , in yeast and in mammalian cells , different stresses induce the cytoplasmic localization of ran , implying perturbation of the ran gtpase cycle , which could negatively affect all pathways mediated by importin family members . on the other hand , it was known for nearly 30 y that the major molecular chaperone hsp70/hsc70 ( hsp70s ) strongly accumulates in the nucleus in response to heat shock . however studies using microinjection and reconstituted transport showed that the nuclear import activity of hsp70s is upregulated during thermal stress(fig . this evidence indicates that cellular stress significantly affects the nuclear transport system : the conventional nuclear transport pathway is downregulated , whereas stress - specific nuclear transport becomes active ( fig . heat - shock stress downregulates the conventional nuclear transport pathway but upregulates the nuclear import of hsp70s . ( a ) nuclear import of importin family cargoes or hsc70 was examined in living cells by microinjection ( left panels ) or in reconstituted transport assay using digitonin - permeabilized semi - intact cells ( right panels ) . in living cells , sv40 t - nls ( importin cargo ) in contrast , hsc70s accumulate efficiently in the nucleus whereas sv40 t - nls does not under the heat shock condition . results of 5min after cytoplasmic injection are shown . in reconstituted transport , sv40 t - nls and m9 ( transportin cargo ) accumulate efficiently in the nucleus of permeabilized cells whereas hsc70 do not in the presence of cytosol prepared from normal cells . in contrast , hsc70s accumulate efficiently in the nucleus of permeabilized cells whereas sv40 t - nls and m9 do not in the presence of cytosol prepared from heat shock treated cells . ( b ) illustration of nuclear transport under normal condition and heat shock stress condition . what mediates the nuclear import of hsp70s under stress conditions when importin family member - mediated nuclear transport is downregulated ? to answer this question , we initially reconstituted heat shock - induced nuclear import using a cell - free transport assay using digitonin - permeabilized semi - intact cells . because hsp70 is a sticky protein , we could not identify its interaction partner molecule(s ) required for its nuclear import via a simple binding assay ( e.g. , pull - down assays or immunoprecipitation experiments ) . in the reconstituted transport experiment , a combination of cytosol or permeabilized cells prepared from either normal or heat shock - stressed hela cells revealed that the nuclear import activity of hsp70s was present in the cytosol prepared from stressed cells . the biochemical fractionation of the cytosol prepared from stressed cells , followed by an examination of the nuclear import activity of hsc70 in the cell - free transport assay , identified a protein encoded by human chromosome 11 open reading frame 73 ( c11orf73 ) as a factor essential for the nuclear import of hsp70s . c11orf73 encodes a protein that is evolutionarily conserved from yeast to mammals , but its function was unknown . bacterially expressed recombinant c11orf73 protein supported the nuclear import of hsc70 in the cell - free transport assay , and the knockdown of c11orf73 using sirna inhibited the heat shock - induced nuclear import of hsp70s in living cells , demonstrating the essential role of c11orf73 in the nuclear import of hsp70s . although it was evident that hikeshi is essential for the heat shock - induced nuclear import of hsp70s , our primary question was whether hikeshi functions as a nuclear import carrier . if hikeshi is a nuclear import carrier , it must bind to npc components ( nucleoporins ) and be able to translocate through nuclear pore complexes , as all known nuclear transport carriers do . it was also important that the binding between hikeshi and hsp70s be regulated to allow the nuclear accumulation of hsp70s against a chemical concentration gradient . in the case of importin , the gtpase cycle of ran plays a crucial role in this active transport : cargoes bind to importin in the cytoplasm where the rangtp concentration is low but dissociate in the nucleus where rangtp concentration is high because rangtp binding to importin triggers the cargo release . we therefore examined whether hikeshi fulfills the above two criteria of a carrier : first , its ability to translocate through npcs through interactions with fg - repeat containing nucleoporins ( fg - nups ) , and second , the regulation of its binding to hsp70s . gfp - hikeshi , when expressed in living cells , localized diffusively throughout the cytoplasm and the nucleus . when incubated with digitonin - permeabilized semi - intact cells , gfp - hikeshi enters the nucleus in the absence of soluble factors or energy sources , showing that it is capable of translocating through npcs on its own . this translocation was inhibited by the addition of importin or wheat germ agglutinin ( wga ) , indicating that its nuclear migration occurs through a specific interaction with nucleoporins . although the details of npc translocation are currently not understood , it is now widely accepted that all known transport carriers translocate through the central channel of npcs , which are filled with hydrophobic fxfg- or glfg - repeats ( fg - repeats ) , and that translocation proceeds through interactions between the carriers and fg repeats of nucleoporins . when examined in a bead hallo assay , hikeshi bound to fg - repeats containing nucleoporins , and this binding was inhibited by importin , indicating that hikeshi translocate through npcs by interacting with the fg repeats of nucleoporins in a manner similar to that of importin . demonstrating the physical interaction of hikeshi and hsp70s was initially challenging . hikeshi bound to hsp70s in a pull - down assay from cell extract , and it mediated the nuclear import of hsc70s in the presence of cell extract . interestingly , the interaction of hikeshi with hsp70s in the crude cell extract and the hikeshi - dependent transport of hsp70 always depended on the presence of atp . however , hikeshi neither supported the nuclear import of hsc70 nor bound to hsp70s in the absence of cell extract , even in the presence of atp . these observations showed that hikeshi and hsp70s do associate with each other but suggest that their binding requires some soluble factor(s ) in conjunction with atp . to identify the soluble factor(s ) necessary for the hikeshi - hsp70 interaction , we again performed biochemical fractionation and followed the nuclear import of hsp70 in the presence of hikeshi in cell - free transport assay . hsp110 is a co - chaperone of hsp70 , which functions as a nucleotide exchange factor of hsp70s . in cells , hsp70s do not function alone , but always function with co - chaperones that promote the atpase cycle of hsp70s . for example , hsp70s nucleotide exchange factors , such as hsp110 , convert the adp - bound form of hsp70 to the atp - bound form , whereas j - domain proteins such as hsp40 convert the atp - bound form to the adp - bound form . to better characterize the involvement of the atpase cycle of hsp70s in hikeshi - binding / transport , we examined the effects of co - chaperones on the hikeshi - mediated nuclear import of hsc70 . hsc70 was first pre - incubated with hsp110 in the presence of atp and then subjected to the transport assay after the removal of hsp110 . we found that hikeshi was able to mediate the nuclear import of the pre - incubated hsc70 in the absence of soluble factors . furthermore , in the absence of other soluble factors , hikeshi mediated the transport of an atpase - deficient point mutated hsp70 ( atp - fixed form ) even without pre - incubation with hsp110 . conversely , the addition of hsp40 disrupted the binding of hikeshi and hsp70s and inhibited the nuclear import of hsc70 mediated by hikeshi . these results show that hikeshi binds to the atp - bound form of hsp70s but dissociate from the adp - bound form ( fig . if the binding occurs in the cytoplasm and dissociation occurs in the nucleoplasm , then this property could explain the directionality of the transport . ( a ) binding and release of hikeshi to hsp70s is regulated by co - chaperones that modulate the nucleotide form of hsp70s . hikeshi binds to the atp - bound form of hsp70s , but dissociates from the adp - bound form . ( b ) current working model of the hikeshi - mediated nuclear import of hsp70s . in the cytoplasm , hikeshi binds to the atp - bound form hsp70s , translocate through the npcs . in the nucleus , hikeshi dissociates from the adp - bound form hsp70s by action of j - domain - containing co - chaperones , such as the hsp40 family , allowing hsp70s to function as a molecular chaperone in the nucleus . taken together , these results show that hikeshi fulfills the criteria of a nuclear transport carrier . our current working model of hikeshi - mediated nuclear import pathway importantly , our transport model highlights the involvement of co - chaperones in the hikeshi - mediated nuclear import of hsp70s . it must be noted that there exist many different co - chaperones in cells that are involved in either nucleotide exchange or atp hydrolysis of hsp70s . it is possible that other groups of co - chaperones , besides co - chaperones used in this study , participate in driving import of hsp70s mediated by hikeshi . it is important to determine which co - chaperones ( or groups of co - chaperone ) that are involved in the transport during stress to verify our model through careful analysis of their behaviors and regulation of activities during normal condition and stress condition . in any case , the proposed transport pathway is different from any of the known transport pathway reported previously , not only because it is mediated by a novel carrier but also because its driving force appears to be the atpase cycle of hsp70s , which is unique among reported nuclear transport pathways . gfp - hikeshi , when expressed in living cells , localized diffusively throughout the cytoplasm and the nucleus . when incubated with digitonin - permeabilized semi - intact cells , gfp - hikeshi enters the nucleus in the absence of soluble factors or energy sources , showing that it is capable of translocating through npcs on its own . this translocation was inhibited by the addition of importin or wheat germ agglutinin ( wga ) , indicating that its nuclear migration occurs through a specific interaction with nucleoporins . although the details of npc translocation are currently not understood , it is now widely accepted that all known transport carriers translocate through the central channel of npcs , which are filled with hydrophobic fxfg- or glfg - repeats ( fg - repeats ) , and that translocation proceeds through interactions between the carriers and fg repeats of nucleoporins . when examined in a bead hallo assay , hikeshi bound to fg - repeats containing nucleoporins , and this binding was inhibited by importin , indicating that hikeshi translocate through npcs by interacting with the fg repeats of nucleoporins in a manner similar to that of importin . hikeshi bound to hsp70s in a pull - down assay from cell extract , and it mediated the nuclear import of hsc70s in the presence of cell extract . interestingly , the interaction of hikeshi with hsp70s in the crude cell extract and the hikeshi - dependent transport of hsp70 always depended on the presence of atp . however , hikeshi neither supported the nuclear import of hsc70 nor bound to hsp70s in the absence of cell extract , even in the presence of atp . these observations showed that hikeshi and hsp70s do associate with each other but suggest that their binding requires some soluble factor(s ) in conjunction with atp . to identify the soluble factor(s ) necessary for the hikeshi - hsp70 interaction , we again performed biochemical fractionation and followed the nuclear import of hsp70 in the presence of hikeshi in cell - free transport assay . hsp110 is a co - chaperone of hsp70 , which functions as a nucleotide exchange factor of hsp70s . in cells , hsp70s do not function alone , but always function with co - chaperones that promote the atpase cycle of hsp70s . for example , hsp70s nucleotide exchange factors , such as hsp110 , convert the adp - bound form of hsp70 to the atp - bound form , whereas j - domain proteins such as hsp40 convert the atp - bound form to the adp - bound form . to better characterize the involvement of the atpase cycle of hsp70s in hikeshi - binding / transport , we examined the effects of co - chaperones on the hikeshi - mediated nuclear import of hsc70 . hsc70 was first pre - incubated with hsp110 in the presence of atp and then subjected to the transport assay after the removal of hsp110 . we found that hikeshi was able to mediate the nuclear import of the pre - incubated hsc70 in the absence of soluble factors . furthermore , in the absence of other soluble factors , hikeshi mediated the transport of an atpase - deficient point mutated hsp70 ( atp - fixed form ) even without pre - incubation with hsp110 . conversely , the addition of hsp40 disrupted the binding of hikeshi and hsp70s and inhibited the nuclear import of hsc70 mediated by hikeshi . these results show that hikeshi binds to the atp - bound form of hsp70s but dissociate from the adp - bound form ( fig . if the binding occurs in the cytoplasm and dissociation occurs in the nucleoplasm , then this property could explain the directionality of the transport . ( a ) binding and release of hikeshi to hsp70s is regulated by co - chaperones that modulate the nucleotide form of hsp70s . hikeshi binds to the atp - bound form of hsp70s , but dissociates from the adp - bound form . ( b ) current working model of the hikeshi - mediated nuclear import of hsp70s . in the cytoplasm , hikeshi binds to the atp - bound form hsp70s , translocate through the npcs . in the nucleus , hikeshi dissociates from the adp - bound form hsp70s by action of j - domain - containing co - chaperones , such as the hsp40 family , allowing hsp70s to function as a molecular chaperone in the nucleus . taken together , these results show that hikeshi fulfills the criteria of a nuclear transport carrier . our current working model of hikeshi - mediated nuclear import pathway importantly , our transport model highlights the involvement of co - chaperones in the hikeshi - mediated nuclear import of hsp70s . it must be noted that there exist many different co - chaperones in cells that are involved in either nucleotide exchange or atp hydrolysis of hsp70s . it is possible that other groups of co - chaperones , besides co - chaperones used in this study , participate in driving import of hsp70s mediated by hikeshi . it is important to determine which co - chaperones ( or groups of co - chaperone ) that are involved in the transport during stress to verify our model through careful analysis of their behaviors and regulation of activities during normal condition and stress condition . in any case , the proposed transport pathway is different from any of the known transport pathway reported previously , not only because it is mediated by a novel carrier but also because its driving force appears to be the atpase cycle of hsp70s , which is unique among reported nuclear transport pathways . to examine the physiological significance of hikeshi - mediated nuclear import , we first examined the cellular effects of hikeshi depletion using sirnas . hikeshi is expressed in normal cells , and its expression level increases by two- to 3-fold during thermal stress in hela cells . hikeshi depletion apparently does not affect cell growth under normal condition : however , more than 70% of cells failed to grow after heat treatment when hikeshi is depleted ( fig . 3 ) . such growth defects were rescued , at least in part , by expressing conventional basic nls - tagged hsc70 , which is imported into the nucleus by the importin and importin pathways , just before the heat shock treatment . the results confirm for the first time in the 30 y since the initial report of the stress - induced nuclear accumulation of hsp70s , that the presence of hsp70s in the nucleus during thermal stress is indeed important for cells to survive after stress . ( a ) sirna - mediated hikeshi knockdown inhibits the heat shock - induced nuclear accumulation of hsp70s in living cells . ( b ) sirna - mediated hikeshi knockdown significantly reduces the cell viability after release from heat shock stress . time course experiments show that the hikeshi - depleted cells start dying several hours after the release from stress . t shows the time ( hr ) after the temperature shift - down to the physiological temperature . this effect of hikeshi - depletion was rescued for about 50% , by expressing conventional basic nls tagged hsc70 just before the heat - shock treatment . what causes cell death in the hikeshi - depleted cells ? in time course experiments , we noted that cells do not die immediately after exposure to stress but rather begin dying several hours after release from stress , indicating that the cells die during recovery from stress . consistent with this observation , in hikeshi - depleted cells , the cellular levels of hsp70 rapidly increases as in normal cells in response to heat shock , indicating that the hikeshi - depleted cells respond to heat shock normally . however , unlike normal cells , the expression of hsp70 does not decrease after the cells are returned to physiological temperature . in a parallel observation , nuclear structure called nuclear stress granules ( nsgs ) , which are considered to represent the activity of the heat shock factors ( hsfs ) that activate hsp70s expression , rapidly appear in both hikeshi - depleted cells and normal cells in response to heat shock , again showing that the hikeshi - depleted cells can respond to heat shock normally . however , nsgs , which rapidly disappear in normal cells soon after a temperature shift - down persist for much longer time in the hikeshi - depleted cells , which is consistent with the persistently high expression levels of hsp70 after temperature shift down . our results show that the heat - shock response can not be attenuated in hikeshi - depleted cells even after a release from stress . surprisingly , the effect of the hikeshi depletion was not restricted to the attenuation of hsf activity but also seemed to have broader effects on the reversion of the heat shock - induced nuclear phenotype . it is known that some nucleolar proteins are released from the nucleolus and dispersed into the nucleoplasm in response to heat shock , but they re - accumulate in the nucleolus after release from stress . all of the above observations show that hikeshi is required to protect cells from heat shock damage and is required for the attenuation and reversion of multiple heat shock - induced nuclear phenotypes . these functions are reminiscent of hikeshi , which is the traditional edo - era japanese compound word meaning firefighter , smokejumper or troubleshooter . our results provide evidence for the physiological significance of hikeshi - mediated nuclear import pathway activated during thermal stress . revealing a nuclear transport pathway that becomes active during thermal stress will raise fundamental new questions . for example , in spite of many reports demonstrating that stresses affect numerous nuclear events involving dna metabolism and rna biogenesis , which must be reverted upon release from stresses in order for cells to survive , the mechanisms underlying the reversion of stress - induced nuclear phenotypes have not been studied in depth to date . our findings clearly show that there are active mechanism(s ) for the reversion of the thermal stress - induced nuclear phenotype and that the activity of hsp70s inside the nucleus is crucial , at least in part for this reversion . because the depletion of hikeshi seems to affect the reversion of various nuclear phenotypes , it is possible that the hikeshi mediates nuclear import of proteins other than hsp70s that are also responsible for reverting stress - induced nuclear phenotypes . finding of hikeshi - mediated nuclear transport pathway will provide a new avenue for research to address questions regarding how cells recover from thermal stress damage . another intriguing question to address is the molecular mechanism underlying the activation of hikeshi - mediated nuclear import . preliminary analysis shows that , at least in a reconstituted system , posttranslational protein modifications such as the phosphorylation of hikeshi or hsp70s are not involved in the activation of hikeshi - mediated nuclear import . because the atpase cycle of hsp70s , which is regulated by co - chaperones , is likely involved in the hikeshi - mediated nuclear import , the import activation mechanism might involve the regulation of the chaperone system . further study of the nuclear transport switching mechanism should provide new insights into the regulation of both the nuclear transport system and the molecular chaperone system . in addition , because hikeshi is evolutionarily conserved protein from yeast to mammals , it is also important to analyze the functions of hikeshi in other organisms to know the conservation and divergence of nuclear transport system and molecular chaperone system from aspects of evolution . | cellular stresses significantly affect nuclear transport systems .
nuclear transport pathways mediated by importin -family members , which are active under normal conditions , are downregulated . during thermal stress ,
a nuclear import pathway mediated by a novel carrier , which we named hikeshi , becomes active .
hikeshi is not a member of the importin family and mediates the nuclear import of hsp70s . unlike importin family - mediated nuclear transport , the hikeshi - mediated nuclear import of hsp70s
is not coupled to the gtpase cycle of the small gtpase ran but rather is coupled with the atpase cycle of hsp70s .
hikeshi - mediated nuclear import is essential for the attenuation and reversal of the thermal stress response in human cells .
the mechanism and functions of this newly identified nuclear import pathway will be discussed . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
g - protein - coupled receptors
( gpcrs ) belong to the superfamily of
membrane proteins and play a crucial role in signal transduction . gpcrs share a similar structural motif consisting
of the seven helical transmembrane ( tm ) core connected by three extracellular
and three intracellular loops . gpcrs are
dynamic proteins with several functionally important conformations
ranging from the inactive state to the fully
active state . the dynamics of gpcr conformations pose a challenge for their purification and crystallization in various
functional states , and site directed mutagenesis that confers thermostability
to the receptor has been a successful strategy to stabilize these
proteins in various conformations in detergents . the adenosine
a2a receptor ( a2ar ) is a class a gpcr and an
emerging drug target for the treatment of parkinson s disease ,
inflammation , and cardiac ischemia . a2ar - star2 is the inactive state mutant with eight point
mutations , while a2ar - gl31 with
four point mutations is the thermostabilized mutant stabilized in
an active - like state . hereafter , we refer to the inactive state mutant as just star2 and
the mutant in the active - like state as gl31 . the eight point mutations
in star2 are a54l , t88a , r107a , k122a , l202a , l235a , v239a , and s277a . the four point
mutations in gl31 are l48a , a54l , t65a , and q89a . here we have used the ballesteros the first number in the superscript is the tm helix in
which the amino acid is present , and the second number is the position
of this residue with respect to the most conserved residue in that
helix which is numbered 50 . analysis of the crystal structures
of the inactive state and the active
state of the 2-adrenergic receptor with the g protein
bound , shows that the transmembrane helices
tm5 and tm6 move significantly
with respect to tm3 upon activation ( when bound to both agonist and
the g protein ) . the crystal structure of the agonist bound gl31 shows
a similar type of movement of tm6 with respect to tm3 but not as far
as observed in the active state of the 2-adrenergic
receptor . similar limited movement of tm6 has also been observed in
the crystal structure of the agonist bound wild type a2ar . hence , we call the conformation of
gl31 the active - like state henceforth in the paper . while the crystal structures of the inactive and the active - like
states of a2ar show conformational differences , they do
not provide the answers for two important questions : ( 1 ) how do the
mutations stabilize the receptor , and ( 2 ) what are the differences
in the dynamics of the activation mechanism of the thermostable mutant
compared to the wild type ? we have used atomic level molecular dynamics
( md ) simulations to answer these questions , since it is not straightforward
to answer using experimental techniques . while some of the mutations
in the thermostable a2ar confer stability , other mutations
specifically stabilize the agonist bound state compared to the inactive
state . the role of the mutations and
how certain mutations specifically stabilize the active - like state
compared to the inactive state is not well understood . the insight
provided by how certain mutations stabilize the active - like state
compared to the inactive state would enormously benefit future design
of thermostable mutants for other gpcrs . additionally , the difference
between the dynamics of the active - like state mutant compared to the
wild type would provide insight into how the mutations in gl31 limit
the receptor ability to activate g proteins although it is in the
active - like conformation . the starting conformations
of a2ar for the md simulations were taken from the crystal
structures of gl31 and star2 ( pdb i d : 2ydo for gl31 and 3pwh for star2 ) . hydrogens were added , the
structures were solvated in the explicit palmitoyloleoylphosphatidylcholine
( popc ) lipid and water , and the solvent was packed using the inflategro package in gromacs . two initial conformations of the wild type were generated by mutating
the residues in the crystal structure of the mutants back to the wild
type using maestro9.2 . we did not use
the crystal structures of the wild type a2ar , since those
structures were crystallized with t4l . however , we also performed
simulations starting from the wild type a2ar crystallized
with t4l . we found no significant difference
in the dynamics ( figure s1 , supporting information ) and hence pursued with the wild type receptors derived from the
thermostable mutant structures . residues within 5 of the sites
of mutation were minimized using macromodel with position restraints
on all backbone atoms and all residues further than 5 from
the site of mutation . the wild type generated from gl31 mutant ( gl31-wt ) is the active - like wild type conformation , and that generated from star2 ( star2-wt ) is the inactive state md simulations on a2ar in a popc lipid
bilayer with periodic boundary conditions were performed using the
gromacs package with the gromos96 force field with spc water molecules . the settle and lincs algorithms
were used for the bond and angle for water and all other bonds , allowing
2 fs of time step . for the analysis , a cutoff distance of 12 for nonbond interactions was
introduced , and the pme ( particle mesh ewald ) method was used for long - range vdw interactions . each of the eight systems were equilibrated by performing
200 ps of md at 310 k using a nvt ensemble followed by 5 ns of md
under npt conditions with a pressure of 1 bar . the protein and ligand
were kept in place during these equilibration steps using position
restraints . after equilibration to the expected temperature and pressure ,
a total of 10 production simulations of up to 100 ns were performed
for each initial conformation with different initial velocities using
the nvt ensemble . all trajectories
obtained from
the molecular dynamics simulations were analyzed using tools provided
by gromacs and python script . pymol and
vmd were used for the structural conformation
analysis of the trajectories . for hydrogen bond analysis using 3.9
and 30 for the cutoff distance and angle , respectively interhelical
hydrogen bond interactions were derived from the stable hydrogen bond
criteria having more than 50% occupancy ( population ) through 1 s
trajectories , which is normalized by setting the most densely populated
point to 1 . the change
in the free energy
for single point mutations was calculated using the thermodynamic
integration ( ti ) technique . we used the hamiltonian
shown below which is a function of a coupling parameter that
varies progressively from 0 to 1 to change the system interactions
from the initial state ( a ) to the final state which is the single
point mutation ( b ) . the difference in free energy between the molecular
systems a and b is then calculated usingthe simulations were done at a few discrete
points i along the pathway , and
the integral was calculated numerically . using the final snapshot
of wild type receptor after 1 s of md trajectory as initial
conformation , the free energy changes due to the mutations were calculated
for both the wild type state ( = 0 ) and the single mutant state
( = 1 ) by integrating the average enthalpic contribution from
each window . for the better convergence of the change in free energy ,
we used unequal window intervals : = 0.01 ( =
0.000.10 , = 0.901.00 ) and =
0.02 ( = 0.100.90 ) . since the error estimation of the
free energy generally is crucial , we monitored the standard deviation
of the total calculations , expressing low fluctuation in the free
energy change ( 1.5 kcal / mol ) . to obtain the best estimates of
the free energies , g ( wt mut ) , all
the enthalpic contributions from each window from the entire 60 ns
( 12 ns of equilibration + 48 ns of production ) were used . the free
energy differences have been calculated using a united atom forcefield
gromos , and this might affect the accuracy of the calculated free
energies . the residue - based stress ( forces )
was calculated using both bond and nonbonded force on each residue
coming from all residues within 3 of this residue except the
ones that are directly bonded to the target residue . the force computation
was performed using the gromos96 53a6 force field following the procedure
described by stacklies et al . the average
stress is the average residue - based stress over the entire md trajectory
for each system . to understand the most important
collective motion in the receptor within a few dominant modes , we
performed the principal component analysis ( pca ) over the entire 1
s md simulation trajectory . since the loops
are highly flexible , they were omitted from the pca analysis . we used
the g_covar module of gromacs to perform the pca
and to extract eigenvalues and eigenvectors . to investigate the crucial
dominant motions , conformational changes along the two principal components the analysis
presented in the results section was done by assembling
all 10 trajectories for each system into an ensemble . the crystal structures of both the antagonist - bound
and the agonist - bound a2ar are available for both the wild
type and thermostabilized receptors . to investigate the intrinsic
dynamic behavior and the energetics of the mutants compared to the
wild type a2ar , we performed md simulation studies of 1
s each in the following receptor systems : the crystal structures
of inactive state star2 and active - like state gl31 thermostable mutants
with and without their respective ligands and the wild type receptor
with and without ligands . the wild type receptor structures in the
inactive and active - like conformations were derived by mutating the
crystal structure of the thermostable mutants to the wild type sequence . details of all the simulation systems are shown in table s1 ( supporting information ) . figure 1 shows the calculated
enthalpy averaged over the md trajectories of the thermostable mutants
and the wild type receptor in both the inactive and active - like state
conformations . while the star2 mutant ( shown in red in figure 1 ) is stable in its inactive state compared to the
wild type in the inactive state , the gl31 mutant is stable in its
active - like state compared to the wild type in the active - like conformation . this finding is in contrast to our previous calculations on the thermostable
mutant of the inactive state of the 1-adrenergic
receptor that showed very little enthalpic stabilization of the mutant
compared to the wild type . md simulations
starting from the active - like conformation of the star2 amino acid
sequence showed a collapse of the active - like structure to the inactive
state within 1 s , implying the thermostabilizing mutations
in star2 bias the conformation to the inactive state ( see figure s2 , supporting information ) . these results show that
the mutations in the thermostable mutants stabilize specific receptor
conformations . ( a ) calculated enthalpy of the various thermostable mutant
and
wild type sequences in both the inactive ( left ) and active - like ( right )
conformations without ligands . the energies of the gl31 thermostable
active - like mutant are shown in black , the inactive state mutant star2
in red , and the wild type in blue . ( b ) the free energy change upon
mutations compared to the difference in the measured experimental
stability of star2 . ( c ) the measured tm values compared to the calculated free energy difference for the
wild type and thermostable mutants for gl31 . we calculated the second order entropy as described
in the methods section , and observed that
the entropic contribution to stability was insignificant ( the ts factor is about 20 times less
than the enthalpy ) compared to the enthalpic contribution ( see figure
s3 , supporting information ) . it is interesting
to note that the enthalpic contribution to stability in the 1-adrenergic receptor was not substantially different for the
thermostable mutants compared to wild type . calculated free
energy changes for single point mutations in gl31 and star2 correlate
with experimentally measured thermostability : to understand the thermodynamic
consequences of single point mutations , we have used the alchemical
free energy simulations - thermodynamic integration ( ti ) method ( detailed
in the methods section ) to calculate the free
energy change upon single point mutations and compare them to the
experimental stabilities . the positions of mutations in gl31 are l48a , a54l , t65a , and q89a , and in star2 , they are a54l , t88a , r107a , k122a , l202a , l235a , v239a , and s277a . the results for the convergence tests of each of these
simulations are described in the supporting information ( figure s4 ) . figure 1b shows the quantitative
correlation of the calculated change in free energy to the experimental
measured stabilities measured by a single - point binding assay using
[ h]-zm241385 . there is significant
correlation between the calculated free energy difference and the
measured stabilities except for the s277a mutation . t88a with the
highest measured thermostability has a 28.2 kcal / mol difference in
free energy . the structural basis for this stability by t88a is discussed
in the next section . figure 1c shows the correlation
of the calculated change in free energies for single mutants and experimental
stability tm . here tm is the temperature at which 50% of the solubilized receptor
can still bind the radiolabeled neca ( agonist ) after heating for 30
min . l48a mutation shows the highest
thermostabilization ( with tm =
+ 14 c ) and also has the largest change in free energy upon mutation . we observe a good correlation between the calculated free energies
and the experimentally measured stability . details of the convergence
of the free energy calculations for point mutations are shown in figure
s4 ( supporting information ) . energetic contributions
from interhelical hydrogen bonds and hydrophobic interactions are
important in determining the stability and the packing of the tm core
in gpcrs . therefore , to analyze the structural basis of the enthalpic stabilization
of the thermostable mutants shown in the previous section , we calculated
the difference in the number of interhelical hydrogen bond and van
der waals ( vdw ) interactions that are stably formed during the dynamics
of the thermostable mutants gl31 and star2 and their respective wild
type conformations . interhelical interaction networks for active - like and
inactive
mutants ( gl31 and star2 ) and wild type receptor without ligands . the black lines
with different thicknesses show the interhelical interactions between
each pair of transmembrane helices , and the number of such interactions
is shown on the lines . parts a d show the interhelical hydrogen
bond interaction , while parts e h indicate the interhelical
van der waals ( vdw ) interactions . we calculated
all possible interhelical hydrogen bonds ( backbone backbone ,
backbone side chain , and side chain side chain ) formed
by a2ar in both gl31 and star2 mutants and the wild type
that show a significant population ( see the methods section ) over the course of the md simulations . figure 2 shows the total number of interhelical hydrogen
bond and vdw interactions where each helix is represented as a circle . the total number of interhelical contacts for each system is shown
below each figure . the numbers of interhelical hydrogen bonds and
vdw contacts are higher in both thermostable mutants gl31 and star2
compared to their respective wild type conformations . this makes both
star2 and gl31 more stable enthalpically than the wild type in the
respective states . both gl31 and the corresponding wild type in the
active - like conformation show a greater number of interhelical hydrogen
bonds than their corresponding inactive state structures . this is
especially interesting , since the number of interhelical hydrogen
bonds is higher in the intracellular region of tm5tm6 and
tm6tm7 where the g protein couples with the receptor ( 8 hydrogen
bonds in gl31 and 4 hydrogen bonds in star2 ) . interestingly , the centrally
located tm3 helix shows a greater number of interhelical hydrogen
bonds with other helices in the inactive star2 ( 10 h - bonds ) mutant
showing tighter helical core packing in the inactive state . in gl31 ,
we observed better packing between tm5 and tm6 compared to star2 ,
which is known to move as a unit with respect to tm3 upon activation . mutation of
hydrophobic residues in the interhelical interface has shown that
these contacts contribute significantly to the thermal stabilization
of receptors . figure 2e h shows that both of the thermostable mutants have a greater
number of favorable interhelical vdw interactions compared to the
wild type . in contrast to the interhelical hydrogen bonds , the inactive
state mutant star2 and the corresponding wild type in the inactive
state show more interhelical vdw interactions than the active - like
state . the vdw packing centered around tm3 and also between tm3 and
tm5 is stronger in the inactive state mutant than in the active - like
gl31 ( star2 , 15 ; gl31 , 11 ) . in the gl31 active - like state this could facilitate
the outward motion of tm6 away from tm3 in the active - like conformation .
in summary , the number of interhelical interactions is higher in the
thermostable mutants compared to the wild type receptor , thus accounting
for the enthalpic stabilization of the receptor mutants . in this section , we have analyzed the structural basis of the thermostability due to
single point mutations that specifically favor stabilization of the
active - like state gl31 compared to the inactive state . the four mutations
in gl31 are clustered on tm2 and tm3 : l48a , a54l , t65a , and q89a . l48a and q89a are mutations that selectively
stabilize the agonist - bound state of a2ar . l48a shows a marked 14 c increase
in thermostability specifically to the agonist - bound receptor . we observed that the mutation of l48a led to
the formation of the interhelical hydrogen bond between the backbone
amide nitrogen of l48a and the side chain of s94 , as
shown in figure 3 , and this hydrogen bond is
not observed in the crystal structure of gl31 . interhelical interactions
of the agonist specific mutations l48a
in active - like gl31 . ( a ) population of the interhelical hydrogen bond
between the amide nitrogen of l48a mutant and the side chain of s94
in the gl31 ( black curve ) and its wild type ( red curve ) . three inset
structures represent the three maxima in the population density : ( i )
gl31 , ( ii ) one possible conformation of the wild type receptor within
the hydrogen bond , and ( iii ) the second populated conformation of
the wild type where this hydrogen bond is broken . ( b ) the inter - relationship
between the formation of the hydrogen bond and movement of the intracellular
region of tm6 away from tm3 . ( c ) the population distribution of the
receptor conformations ( gl31 in black and wild type in red ) that have
a tm3tm6 distance greater than 7.9 with the hydrogen
bond distance between l48 and s94 . this interhelical hydrogen bond is not present in the wild
type
receptor due to the steric hindrance from the side chain of l48 located between the tm2 and tm3 helices ( figure 3a , insets ii and iii ) . as seen in figure 3a , this hydrogen bond is well populated in the gl31
mutant , while it is weakly populated in the wild type . we further
examined if the formation of this hydrogen bond is correlated
to the stabilization of the active - like state of the receptor . we
observed that formation of the interhelical hydrogen bond between
the backbone of l48a and the side chain of s94 ( these two residues are located in the lower half of their
respective helices ) correlates with the increase in distance between
the intracellular half of tm3 and tm6 , as shown in figure 3b . the distance between the backbone atoms of the
last pair of residues in the intracellular half of tm3 and tm6 ( r102a231 )
is shown to increase above 7.8 ( which is the distance in the
crystal structure of gl31 ) when the probability of a hydrogen bond
between tm2 and tm3 is high . thus , when the intracellular part of
tm3 is pulled toward the backbone of tm2 , tm6 is free to move away
from tm3 and hence this mutation facilitates the stabilization of
the active - like state . this is illustrated in figure 3c , which shows the population distribution of the conformations
that have the tm3tm6 distance above 7.8 as a function
of the hydrogen bond distance between l48a and s94 . figure 3c shows that when the
hydrogen bond is formed between l48a and s94 the population of receptor conformations of gl31 ( shown in black
bars ) showing larger movement of tm6 is higher than wild type ( red
bars ) . the q89a mutation shows preferential stabilization
of the active - like state by 6 c but also shows destabilization
of the inactive state by 8 c . mutation
of the large q89 residue to a smaller ala residue also
results in formation of a backbone side chain hydrogen bond between
its neighbor v86 of tm3 and s132 on
tm4 , as seen from figure s5 in the supporting
information . this specific hydrogen bond called cho = c
type , which is weaker than the n in addition , the neighboring amphiphilic residue t88 shows interhelical vdw interaction with w246 of tm6
in gl31 which is insignificant in the wild type . this mutation also
leads to rearrangement in the intracellular part of tm3 and tm6 with
reduced vdw interactions between i106 and the aliphatic
chain of k227 . this weakened interaction could release
tm6 to move into the active - like state ( bottom part of figure s5 , supporting information ) . the population density
of the hydrogen bond v86s132 and the vdw interaction t88w246
in both the mutant and wild type are shown in figure s6 of the supporting information . there are eight mutations in star2 spread over
tm2 to tm7 : a54l , t88a , r107a , k122a , l202a , l235a , v239a , and s277a . here , a54l is common to both inactive star2 and active - like gl31
mutants . the mutations r107a and l202a are proximal and improve the vdw packing
between i200 , i106 , and a204 on the intracellular regions of tm3 and tm5 ( figure 4a ) . l202 is close to the conserved residue
y197 ( tm5 ) , which in turn makes contact with the backbone
oxygen atom of l235a ( figure 4b ) . this hydrogen bond is not possible in the wild type due to the
long side chain of l202 and l235 being in the way of this interhelical
hydrogen bond . mutation of l202a and v239a also improves the interhelical vdw packing between tm3 , tm5 , and
tm6 . thus , while bulkier hydrophobic side chains provide good interhelical
contact , mutation of such residues to ala provides a tighter packing
of the helical backbone by facilitating interhelical backbone side
chain hydrogen bonds and improved van der waals packing of the neighboring
residues . mutation of this residue to ala favors better packing
of the side chain of the neighboring i124 with f44 ( brown arrow , figure s7 , supporting
information ) . interhelical packing comparison between inactive star2
( right )
and wild type ( left ) in the tm3 , tm5 , and tm6 transmembrane helices . ( a ) enhanced vdw packing between residue pairs ( shown in sticks ) that
are near the mutation positions r107a and l202a ( shown in spheres ) . ( b ) the interhelical hydrogen bond
between conserved y197 on tm5 and backbone of l235a mutant on tm6 shown in dotted red line . residues that
are involved in interhelical interaction are shown as cyan sticks ,
while the vdw packing is shown as surface and the hydrogen bond as
red dotted line . enhanced interhelical
vdw packing of residue pairs ( shown in sticks )
due to mutations t88a and s277a mutants
in the inactive mutant star2 ( right ) and its wild type ( left ) . the
residue pairs with enhanced vdw interactions close to t88a and s277a mutants are highlighted in the orange and
cyan surface and double ended arrows , respectively , and the positions
of mutations are shown as spheres . the t88a mutation leads to a significant change
in measured stability . two main clusters of hydrophobic interactions
near t88a and s277a mutants facilitate
strong interhelical hydrophobic interactions between tm2 , tm3 , tm6 ,
and tm7 . t88a and its neighboring residues v84 and l85 interact with f62 , f242 , and w246 ( orange arrows , figure 5 ) . s277a and its spatial neighbor
t88a tighten the packing between w246 and l249 on tm6 and l276 on tm7 that
is close to s277a mutant ( cyan arrows ) . the distance
distributions of the residue pairs that show enhanced vdw interactions
are shown for the wild type and the mutants in figure s8 of the supporting information . the crystal structures of the inactive
and active - like thermostable mutants of a2ar show movement
of tm5 and tm6 away from tm3 upon binding of the agonist , adenosine . we calculated the dynamic range of the movement of tm5 and tm6 away
from tm3 for the thermostable mutants and the wild type receptors . the salt - bridge
interaction known as the ionic lock
between arg102 on tm3 and glu228 on
tm6 in the inactive mutant state . ( a ) superposition of x - ray crystal
structures of active - like gl31 ( cyan ) and inactive star2 ( orange ) . active - like gl31 shows the absence of the ionic lock owing to side
chain rotation of e228 and outward movement of tm6 . ( b ) population density of the ionic lock between the cationic guanidinium
group ( nh * ) of r102 and the anionic carboxylate ( oe * )
of e228 in gl31 dynamics with adenosine bound ( solid
black lines ) , star2 dynamics with antagonist zm241385 bound ( solid
red lines ) , and wild type receptors in the inactive ( with antagonist
zm241385 bound , dashed red lines ) and active - like states ( with adenosine
bound , dashed black lines ) . ( c ) the same color scheme as in part b
showing the distribution of the cc distance of r102 on tm3 and e228 on
tm6 . in part b and c , red and black vertical dotted lines show the
cc distances observed
in the respective crystal structures . a salt bridge is formed between the side chains of arg102 ( that is highly conserved in class a gpcrs ) and glu228 . this salt bridge , also called the ionic lock
shown
in figure 6a , has been observed in the inactive
state structures of rhodopsin , but it
appears to be more dynamic in other class a gpcrs and may be broken
even when an antagonist is bound . figure 6 shows the population density of the ionic
lock distance that characterizes the extent of movement of
tm6 away from tm3 . the ionic lock is formed in the inactive star2
crystal structure with the antagonist zm241385 bound and not in the
crystal structure of the active - like gl31 structure with the agonist
adenosine bound . in our md simulations , the ionic lock is dynamic
in the inactive states of both star2 and the wild type a2ar . there is a higher population of the ensemble forming a strong
ionic lock both in the inactive state star2 mutant and the wild type
in the inactive state ( red lines in figure 6b ) than in gl31 mutant and the wild type receptor in the active - like
state ( black lines in figure 6b ) . however ,
the c distances between the residues that make the
ionic lock are distributed uniformly about the distances in the crystal
structure of both star2 and gl31 ( shown in figure 6c ) . to analyze the extent
of structural dynamics shown by the wild type a2ar compared
to the thermostable mutants , we calculated the number of microstates
in the most populated conformational state from the md simulation
trajectories . this was done by clustering analysis based on root - mean - square
deviation in coordinates ( see the methods section
for details ) . figure s9a of the supporting information shows the population densities of various conformational states
sampled by the thermostable mutants and the wild type receptors . figure
s9b ( supporting information ) shows the
number of microstates within the most populated conformation in each
system . it is seen that the number of microstates for the wild type
receptor is higher than both gl31 and star2 thermostable mutant receptors ,
showing more flexibility than the thermostable mutants . the distribution of net internal force on each residue ,
known as stress , provides insight into the regions of high stress
in the wild type a2ar and the thermostable mutants , as
we had previously shown for the thermostable mutant of the 1-adrenergic receptor . we hypothesize
that high stress at the location of functionally important residues
leads to large scale conformational changes required to activate the
receptor . to examine this , we have calculated the stress on each residue
using both the bonded and nonbonded components , averaged over the md simulations for the thermostable mutants
and wild type a2ar ( figure s10 , supporting
information ) . both the inactive state and the active - like state
of the wild type a2ar show higher stress than their respective
thermostable mutants star2 and gl31 . figure 7 shows the positions of high stress calculated for the wild type
a2a receptor in the active - like state . the residues shown
in pink sticks in figure 7 we also calculated the root - mean - square deviations ( rmsds )
in coordinates of the corresponding residues in the fully active g - protein - coupled
state of the 2-adrenergic receptor ( pdb i d : 3sn6 ) and the inactive
state of the 2-adrenergic receptor ( pdb i d : 2rh1 ) . residue positions of
high stress in the wild type a2a receptor show maximum
movement upon activation . blue to red color
coding is the difference between the coordinates of the inactive state
to the active state receptor . the rmsd value ranging from low to high , blue to red in figure 7 , shows the extent of movement of each residue upon
activation . the residues such as t279 and n280 on tm6 and tm7 show large fluctuations upon activation ,
as well as the high stress . thus , we observe that residue positions
of high stress are required for movement of helices during activation
and reducing this stress could stabilize the receptor but could make
the receptor inefficient in g protein coupling or activation . tate
and co - workers observed that the stabilization of the thermostable
mutants by ligand binding ( either adenosine or zm241385 ) is required
for crystallization of the mutant a2a receptors . to understand the effect of ligand binding on
the thermostability , we calculated the enthalpic contributions due
to ligand binding for active - like gl31 and inactive star2 structures
and wild type a2ar in both the inactive and active - like
conformational states ( figure s11 , supporting
information ) . we observed that the binding of the agonist adenosine
stabilizes the active - like state mutant gl31 more than the stabilization
resulting from the binding of the antagonist zm241385 to the inactive
state star2 mutant . this is consistent with the experimental observation
that agonist binding was required to purify the gl31 mutant , whereas
the star2 mutant could be purified in the absence of antagonist . also ,
ligand binding leads to greater stability increase for the thermostable
mutants compared to the wild type . adenosine binding stabilizes the
gl31 mutant better than the wild type by 19 kcal / mol . similar stabilization
of the thermostable mutant star2 ( 16 kcal / mol ) was observed relative
to the wild type receptor by the antagonist zm241385 binding . crystal structures
show partial overlap of the binding sites of the
agonist adenosine and the antagonist zm241385 with some features that
are distinct to each ligand . in zm241385 , the furan group ( o25 ) forms
a hydrogen bond with n253 in tm6 , but the ribose ring
moiety in the adenosine agonist forms hydrogen bonds with s277 and h278 in tm7 ( figure s12a , supporting information ) . we have examined
the dynamics of the ligand receptor interaction during the md simulations . the populations of the hydrogen bonds between adenosine ligand and
s277 and h278 in gl31 were not stable
during the dynamics . instead , t88 on tm3 and n181 on tm5 showed direct hydrogen bonds with adenosine ( figure
s12b , supporting information ) . however ,
the ligand receptor interactions were well maintained during the dynamics
of the star2 mutant . the asn253 within the binding
pocket remained connected to the zm241385 ligand directly , as observed
in the crystal structure of the star2 mutant . analysis of the number
of water molecules in the ligand binding pocket showed that star2
has a few more waters than active - like gl31 ( average number of 10
in gl31 and 11 in star2 ( figure s12c , supporting
information ) ) . while gl31 and the wild type a2ar
in the active - like conformation have similar water densities around
the ligand , star2 mutant has less water in the ligand binding site
than the wild type in the inactive state ( average waters 11 and 13
in star2 and wild type in the inactive state ) . both adenosine and
zm241385 retain direct contact with the residues in their respective
binding sites in the mutants compared to the wild type , showing that
the ligand stabilizes the mutants more than the wild type . using long time scale molecular dynamics simulations
and the thermodynamic
integration method for calculating the free energy change upon mutation ,
we have provided insights into the distinct structural and energetic
characteristics that contribute to the stability of the active - like
( gl31 ) and inactive state ( star2 ) thermostable mutants of a2ar . we have shown that the wild type receptor is less stable even
when ligand is bound compared to both of the thermostable mutants
gl31 and star2 . while the star2 mutant is stable in the inactive state ,
gl31 is more stable in its active - like state than its inactive state . md simulations starting from the amino acid sequence of star2 in the
active - like conformation collapse to the inactive state , showing that
the star2 sequence is optimized to stabilize the inactive receptor
conformation . the entropic contributions
to stabilization are low , as reflected by the entropy calculated using
mutual information . additionally , the number of microstates present
in the ensemble of the thermostable mutants is less than that in wild
type . this is in contrast to the thermostable mutant m23 of the 1-adrenergic receptor , which is stabilized by increasing the
side chain entropy . schematic representation
of the extent of dynamic motion observed
in the md simulations for the wild type ( left ) , and gl31 mutant ( right ) . we observed the stress calculated on each residue is less
in the
thermostable mutants than in the corresponding conformations of the
wild type receptor . there are residue positions with high stress ( net
force ) in both the wild type a2ar and 1-adrenergic receptor receptors however , in both the 1-adrenergic receptor and a2ar systems , the stress
on each residue is reduced upon making the thermostable mutations . we observed that the high points of stress in the receptors are very
often the most conserved positions such as pro , pro , and pro . these proline residues play an
important part in activation of the receptor by enabling the movement
of the helices . reducing the stress at these high stress points by
mutating neighboring residues could also reduce the potential for
the receptor to get activated which is possibly why the thermostable
mutants show markedly reduced g protein activation . we observed that both gl31 and star2 are less dynamic than
their corresponding wild type conformations . the movement of tm6 with
respect to tm3 in gl31 is more restrictive than in the wild type ,
as represented in figure 8 . while this restricted
movement is advantageous in purification and crystallization of gl31
mutant receptor , it could be the reason why these mutants are not
efficient in activating the g protein . the alchemical free energy changes calculated for single point
mutants correlate well with the measured stabilities . most of the
thermostabilizing mutations in gl31 and star2 are mutating large residues
such as phe that show well - packed side chain conformations , to ala . we observed that , in large to small side chain mutations , the loss
of side chain packing is compensated by the main chain of the tm helices
forming a hydrogen bond with the side chain , or main chain of residues
in neighboring helices , thus providing a stronger packing interaction . mutations also lead to rearrangement in the side chain conformations
of nearby residues that improve hydrophobic packing with neighboring
helices . the two mutations that specifically stabilize the active - like
state ( l48a and q89a ) show a correlation
of an interhelical hydrogen bond formed as a result of these mutations
to the opening of tm3 and tm6 in the mutant that was not observed
in the dynamics of the wild type . | the adenosine a2a receptor
( a2ar ) belongs
to the superfamily of membrane proteins called the g - protein - coupled
receptors ( gpcrs ) that form one of the largest superfamilies of drug
targets .
deriving thermostable mutants has been one of the strategies
used for crystallization of a2ar in both the agonist and
antagonist bound conformational states .
the crystal structures do
not reveal differences in the activation mechanism of the mutant receptors
compared to the wild type receptor , that have been observed experimentally .
these differences stem from the dynamic behavior of the mutant receptors .
furthermore , it is not understood how the mutations confer thermostability .
since these details are difficult to obtain from experiments , we have
used atomic level simulations to elucidate the dynamic behavior of
the agonist and antagonist bound mutants as well the wild type a2ar .
we found that significant enthalpic contribution leads
to stabilization of both the inactive state ( star2 ) and active - like
state ( gl31 ) thermostable mutants of a2ar .
stabilization
resulting from mutations of bulky residues to alanine is due to the
formation of interhelical hydrogen bonds and van der waals packing
that improves the transmembrane domain packing .
the thermostable mutant
gl31 shows less movement of the transmembrane helix tm6 with respect
to tm3 than the wild type receptor .
while restricted dynamics of gl31
is advantageous in its purification and crystallization , it could
also be the reason why these mutants are not efficient in activating
the g proteins .
we observed that the calculated stress on each residue
is higher in the wild type receptor compared to the thermostable mutants ,
and this stress is required for activation to occur .
thus , reduced
dynamic behavior of the thermostable mutants leading to lowered activation
of these receptors originates from reduced stress on each residue .
finally , accurate calculation of the change in free energy for single
mutations shows good correlation with the change in the measured thermostability .
these results provide insights into the effect of mutations that can
be incorporated in deriving thermostable mutants for other gpcrs . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
breast cancer is the most common type of malignancy and the second leading cause of cancer death in women , after lung cancer [ 1 , 2 ] . the cause of breast cancer is still unknown ; however , epidemiological evidence considers three causes including endocrine , genetic , and environmental factors . adipokines including adiponectin and leptin which are secreted by adipose tissue have a role in cancer biology in obese people . leptin ( from greek leptos , meaning thin ) was discovered in 1994 following isolation of obesity gene . leptin ( lep ) is a 16 kd glycoprotein hormone mainly secreted by adipose tissue . this hormone binds to its receptor ( lepr ) in hypothalamus and plays key roles in regulating metabolism . leptin has three main effects : it increases calorie expenditure , decreases atp production , and reduces appetite . in the body , leptin acts through two types of receptors : a type of long receptors existing in some parts of the brain and hypothalamus which belongs to type i cytokine family and inhibits neuropeptide y in the hypothalamus after activation by leptin , resulting in suppressed appetite and increased metabolism through impact on thyroid and adrenal hormones [ 9 , 10 ] . the second form of leptin receptor exists in peripheral tissues such as muscle , fat , liver , and intestines and exerts many metabolic effects in tissues upon activation , regarding the energy balance and body weight [ 9 , 11 ] . these six isoforms of leptin receptor ( obr ) belong to type i cytokine receptors family which are converted to six different types as obra they consist of a long isoform ( obrb ) , four short isoforms , and one secretory isoform . leptin receptor is produced by a gene on human chromosome 1 and mouse chromosome 4 , and cd295 has been assigned to it in differentiation systems . long chain leptin receptor type b is responsible for most of the known effects of leptin which performs signaling in four different pathways : jak - stat ( janus kinase - signal transducer and activator of transcription ) , mapk ( mitogen - activated protein kinase ) , pi3k ( phosphatidylinositol 3-kinase ) , and ampk ( adenosine monophosphate activated kinase ) . leptin is essential for the growth of human mammary gland and recent studies have shown that it may be effective in breast cancer . leptin gene is expressed in normal breast tissue , breast cancer cell lines , and solid tumors . it is overexpressed in more than 92% of breast cancers , while this was not reported in any of the normal breast epithelial cells . in addition to stimulating cell division , leptin can transform breast cancer cells into malignant forms . leptin plays a role in cell growth , differentiation , and angiogenesis and can increase endothelial cells products such as no and overexpression of vegf , fgf2 , and vegfr/2 . leptin acts also as a mitogenic and migration factor in some cells such as normal epithelial cells and malignant cells . leptin can stimulate the growth of cancer cells through expression of aromatase and production and activation of estrogen in breast cancer epithelium , resulting in reduction or inhibition of inhibitory effects of antiestrogens in proliferation of breast cancer cells . studies have shown that expression of leptin and its receptor is significantly increased in primary breast cancer with metastases . various polymorphisms occur in leptin and its receptor gene including -2548 g / a and q223r . in -2548 g / a polymorphism , an adenine replaces guanine in nucleotides -2548 upstream from the atg start site at 5 region of leptin gene promoter . q223r polymorphism is the result of guanine to adenine substitution in nucleotide 668 of exon 6 from the start codon , resulting in replacement of positively charged arginine instead of neutral glutamine at amino acid 223 . this polymorphism occurs in a region which encodes the extracellular domain of leptin receptor , affects the function of the receptor , and impairs the ability of leptin to bind to its receptor . study of these polymorphisms in different populations may show the relationship between the frequency of these polymorphisms and the risk for cancer . in fact , different populations have been studied in this regard and different results have been obtained . the present study investigated these polymorphisms in patients with breast cancer in yasuj , iran . forty - five women with breast cancer and 41 healthy women were enrolled in this study . the patients included those women who were diagnosed according to pathology and the signs of the disease , while the controls were those with negative pathology results for breast cancer . all subjects were informed about the use of the results and voluntary participation in the study , and written consents were obtained from the patients and controls . 5 ml blood sample was collected for performing pcr - rflp and measuring leptin . clinical information about patients , such as cancer stage , estrogen and progesterone receptors status , her2 status , and age , was recorded . briefly according to the procedure of this kit , 200 l of whole human blood treated with edta was used for extracting of genomic dna . 20 l qiagen protease ( or proteinase k ) was transferred into the bottom of a 1.5 ml microtube and 200 l whole blood was added to the tube and then 200 l lysis buffer ( al buffer of the kit ) was added to the sample and mixed by pulse - vortexing for 15 s and incubated at 56c for 10 min . then 200 l ethanol ( 96100% ) was added to the sample and mixed again by pulse - vortexing for 15 s. carefully the mixture was applied to the qiaamp mini spin column ( in a 2 ml collection tube ) and centrifuged at 6000 g ( 8000 rpm ) for 1 min . the qiaamp mini spin column was placed in a clean 2 ml collection tube and the tube containing the filtrate was discarded . carefully the qiaamp mini spin column was opened and 500 l buffer aw1 ( included in the kit ) was added and then centrifuged at 6000 g ( 8000 rpm ) for 1 min . again the qiaamp mini spin column was placed in a clean 2 ml collection tube and the collection tube containing the filtrate was discarded , and 500 l buffer aw2 ( included in the kit ) was added and centrifuged at full speed ( 20,000 g ; 14,000 rpm ) for 3 min . the qiaamp mini spin column was placed in a clean 1.5 ml microtube and the collection tube containing the filtrate was discarded and 200 l buffer ae ( included in the kit ) added and then incubated at room temperature for 1 min and centrifuged at 6000 g ( 8000 rpm ) for 1 min . a second elution step with a further 200 the primers indicated in table 1 were used to amplify the regions for -2548 g / a and q223r polymorphisms in leptin and leptin receptor genes , respectively . pcr conditions for a 25 l reaction volume were as follows : 2.5 l pcr buffer , 1 l mgcl2 , 0.5 l dntp mix , 1 l of each primer , 3 l template dna , and 0.3 l taq dna polymerase ( go taqdna polymerase , invitrogen ) . pcr program for both polymorphisms was as follows : initial denaturation at 94c for 4 minutes ; 40 cycles including denaturation at 94c for 30 s , annealing at 58c for 30 s , and extension at 72c for 30 s ; and final extension at 72c for 10 min . to confirm amplification of the desired fragments , the samples were then digested with mspi enzyme ( new england biolabs ) for leptin receptor gene and hha1 enzyme for leptin gene for 16 h. after the incubation period , the products were electrophoresed again on 2% agarose . regarding q223r lepr polymorphism , the presence of only one band of 416 bp length indicated individuals with qq homozygous genotype , the presence of three bands of 416 bp , 291 bp , and 125 bp length indicated individuals with qr heterozygous genotype , and the presence of two bands of 125 bp and 291 bp length indicated individuals with rr homozygous genotype . regarding -2548 g / a lep polymorphism , the presence of only one band of 241 bp length indicated individuals with gg homozygous genotype , the presence of three bands of 241 bp , 181 bp , and 60 bp length indicated individuals with ga heterozygous genotype , and the presence of two bands of 181 bp and 60 bp length indicated individuals with aa homozygous genotype .
t - test was used to compare the means and anova and logistic regression were used to study the relationship between different genotype of -2548 g / a lep polymorphism and q223r lepr polymorphism with breast cancer and leptin level . in all calculations , this study consisted of 45 women with breast cancer ( mean age = 47.09 11.45 years ) and 41 normal subjects ( mean age = 48.37 8.80 years ) . the mean level of serum leptin was 33.22 21.35 ng / ml in the patient group and 29.49 23.27 ng / ml in the normal group . comparison of serum leptin levels in patients and controls revealed no significant difference in the serum level of this hormone between the healthy subjects and the patients ( p = 0.3 ) . distributions of the genotypes and frequencies of the alleles of -2548 g / a lep polymorphism and q223r lepr polymorphism in breast cancer patients and healthy controls are represented in table 2 . according to table 2 for leptin gene , the prevalence of allele a in the patient and control groups was 72.2% and 64.6% , respectively ( or = 1.35 ; p = 0.33 ) . no significant association was found between the risk of breast cancer and homozygous gg , homozygous aa ( or = 1.13 ; p = 0.8 ) , and heterozygous ga ( or = 0.41 ; p = 0.2 ) . regarding genotype distribution of q223r lepr polymorphism in normal subjects , 6 were homozygous rr ( 14.6% ) , 18 heterozygous qr ( 43.9% ) , and 17 homozygous qq ( 41.5% ) . the genotype distribution of q223r lepr polymorphism in patients was 1 homozygous rr ( 2.2% ) , 25 heterozygous qr ( 55.6% ) , and 19 homozygous qq ( 42.2% ) . according to table 2 for leptin receptor gene , the frequency of allele r in the patient and control groups was 30% and 36.6% , respectively ( or = 0.71 ; p = 0.33 ) . no significant association was found between homozygous qq with homozygous rr ( or = 6.7 ; p = 0.09 ) and with heterozygous qr ( or = 8.3 ; p = 0.06 ) and risk of breast cancer . the relationship between different genotypes of -2548 g / a lep and q223r lepr polymorphisms with plasma leptin levels was investigated ( table 3 ) and no significant association was found between the levels of the hormone , -2548 g / a lep polymorphism , and q223r lepr polymorphism and breast cancer ( p > 0.05 ) . in addition , the frequency of -2548 g / a lep and q223r lepr polymorphisms and pathologic parameters in breast cancer patients ( table 4 ) was not significantly associated ( p > 0.05 ) . the present study examined the relationship between -2548 g / a polymorphism in leptin gene and q223r polymorphism in leptin receptor gene with the risk for breast cancer in yasuj , iran . breast cancer is the most common type of malignancy and the second leading cause of cancer death in women , so that its mortality rate was estimated approximately 25% until april 2013 . adipokines including adiponectin and leptin which are secreted by adipose tissue have a role in cancer biology in obese people . mutations in leptin and leptin receptor can decrease the leptin receptor signals . according to the literature , mutations in this gene are associated with cancer in humans . it has been shown recently that leptin and its receptor are involved in the processes leading to the initiation and progression of breast cancer . several polymorphisms in leptin gene and its receptor gene have been related to breast cancer , such as -2548 g / a and q223r , because -2548 g / a polymorphism in the promoter of leptin gene can affect the expression of this gene and q223r polymorphism in leptin receptor gene is associated with impaired binding of leptin to its receptor , affecting its whole activity [ 30 , 31 ] . the effect of these polymorphisms on the risk of breast cancer has been studied in different populations . ( 2006 ) found no significant correlation between four polymorphisms in leptin receptor gene and the risk of breast cancer . the results of this study showed that the difference of mean serum leptin levels between various genotype groups of q223r polymorphism was not significant . in the present study also no significant correlation was found between q223r polymorphism and breast cancer risk ; in addition , no significant difference was observed in the mean serum leptin levels between patients and controls and the mean difference in serum leptin levels was not significant between different genotype groups of q223r polymorphism . consistent with results of the present study , no relationship was observed between serum leptin concentrations and the risk of breast cancer in studies by mantzoros et al . ( 2012 ) , in a comprehensive study performed as a meta - analysis , and liu et al . ( 2011 ) found no significant association between -2548 g / a lep polymorphism and q223r lepr polymorphism with the risk of breast cancer in most ethnic communities ; this finding is consistent with the results obtained in the present study [ 35 , 36 ] . ( 2006 ) showed a significant association between q223r polymorphism and -2548 g / a polymorphism with the risk of breast cancer . they also reported that allele a was more frequent in patients with larger tumor size , while in the present study , no significant correlation was observed between pathological indicators and allele frequency in -2548 g / a lep polymorphism and q223r lepr polymorphism . in other studies , anuradha et al . in india ( 2012 ) showed that q223r lepr polymorphism is associated with the risk of breast cancer ; this finding is in contradiction with the results obtained in the present study [ 26 , 37 ] . in 2013 , he et al . studied q223r lepr and -2548 g / a lep polymorphisms in patients with different cancers . their results showed that individuals with aa genotype in leptin gene are likely to develop various cancers , especially prostate cancer . in addition , no relationship was found between q223r lepr polymorphism and the risk of different types of cancer . also in this study , no significant differences were found between the expressions of mrna of leptin and its receptor in different genotypes and different ethnicities . the results of our study showed that -2548 g / a lep and q223r lepr polymorphisms were highly polymorphic in the study population . the frequency of allele r in q223r lepr polymorphism was 0.366 in our study population which was higher than the figures reported in pima indian ( 0.32 ) and arab ( 0.34 ) populations and lower than the figures reported in caucasian ( 0.45 ) and asian ( 0.85 ) populations . however , chung et al . reported a little difference in r allele frequency of q223r lepr polymorphism according to racial groups . it is believed that small sample size and ethnic composition in usa have affected this result . the frequency of the allele a in -2548 g / a lep polymorphism was 0.646 in our study population . in a study by fan and say , allele a was reported more frequent in asian populations , at least among indian , chinese , and malays / peninsular bumiputras . the differences in the findings of similar studies and the present study can be attributed to various genetic polymorphisms in similar metabolic pathways ( gene - gene interaction ) . other factors such as environmental factors ( e.g. , stress and changes in environmental temperature ) can affect leptin expression as confounders and alter the risk of breast cancer . also , this difference in various studies can be attributed to racial differences ; a factor in a region and in a specific race may be a predisposing factor for developing certain diseases , and it may be not decisive in the second race in a different geographical area . in other words , genetic correlation is population dependent and shows different results in different populations . in this study , no significant association was found between -2548 g / a lep polymorphism , q223r lepr polymorphism , and serum leptin concentrations with the risk of breast cancer in the study population . further studies are required to accept or reject the conclusion and to achieve more comprehensive results . in this | recent studies have shown that polymorphisms in leptin and leptin receptor genes are associated with increased risk for breast cancer .
this study aimed at investigating -2548 g / a polymorphism in leptin gene and q223r polymorphism in leptin receptor gene in patients with breast cancer .
the study included 45 women with breast cancer and 41 healthy women .
pcr - rflp was used to determine the genotype of the subjects in terms of -2548 g / a polymorphism in leptin gene and q223r polymorphism in leptin receptor gene .
serum levels of leptin were also measured by elisa . for -2548 g / a polymorphism , the genotypes were homozygous aa ( or = 1.13 ; p = 0.8 ) and heterozygous ga ( or = 0.41 ; p = 0.2 ) and for q223r polymorphism , the genotypes were homozygous rr ( or = 6.7 ; p = 0.09 ) and heterozygous qr ( or = 8.3 ; p = 0.06 ) . the mean serum level of leptin was 33.22 21.35 ng / ml in patients and 29.49 23.27 ng / ml in the normal participants ( p = 0.3 ) .
although , despite the magnitude of the associations , the results suggested no statistically significant contribution of -2548 g / a polymorphism ( in leptin gene ) , q223r polymorphism ( in leptin receptor gene ) , and serum leptin levels in predicting the risk of breast cancer , further studies with larger sample size are suggested . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the rates of obesity and diabetes have increased rapidly over the last 20 years in the us as well as globally . it is not surprising that the incidence of gestational diabetes mellitus ( gdm ) is also increasing in parallel to the overall rise in obesity and type-2 diabetes . the adoption of new diagnostic criteria based on the recent hapo study will increase the prevalence of gdm to approximately 18% of all pregnancies . in light of the fact that 80 - 90% of women with gdm can be managed with lifestyle therapy alone , universal screening for gdm is increasingly considered justified . gdm is a serious complication of pregnancy that can increase the risks of several maternal - fetal disorders , including macrosomia , shoulder dystocia or birth injury , premature delivery , and preeclampsia . in addition to the increased risk of complications associated with gestation and delivery , there are also serious post - natal complications of gdm . about 5 - 10% of women with gdm are found to have diabetes immediately after pregnancy , and women who had gdm have a 10-fold higher chance of developing diabetes within the next 10 - 20 years . it is now apparent that children of mothers with gdm have an 8-fold higher risk of developing type-2 diabetes mellitus in later life . thus , untreated gdm contributes to the overall diabetic population in both the short and long term . universal or even widespread gdm screening is hampered by the fact that the standard assessments of diabetes and pre - diabetes , such as fasting insulin / glucose and hba1c , are not recommended for screening of gdm . instead , the recommended parameter is an oral glucose tolerance test ( ogtt ) , which is expensive and invasive , requiring a hospital visit and multiple blood draws . therefore , improved methods and analytes for gdm screening are needed to increase diagnosis rates and prevent maternal and child risk of future diabetes . specifically , the development of minimally invasive testing with robust analyte combinations will greatly aid in the identification of gdm and the institution of appropriate interventions , especially in at - risk and under - served populations . study recruitment and methods were approved by the institutional ethics committee , and informed consent was obtained from each participant . specifically , 1463 consecutive women within the second and third trimesters of pregnancy underwent a 75-g ogtt followed by a 2-hour plasma glucose determination . gdm was diagnosed as a 2-hour plasma glucose 7.8 mmol / l ( 140 mg / dl ) , consistent with who criteria . the current study employed a case - control design , in which 14 non - diabetic and 15 gdm serum samples were randomly selected from the described population . serum samples were analyzed to obtain measures of sex - hormone binding globulin ( shbg ) , adiponectin , human chorionic gonadotropin ( hcg ) , placental lactogen , c - reactive protein ( crp ) , pregnancy - specific glycoprotein-1 ( psg-1 ) , and fibronectin , as well as specific glycosylated forms of fibronectin and psg-1 [ table 2 ] . two - dimensional differential in - gel electrophoresis ( 2d - dige ) and immunoassays ( elisa ) were performed as previously described . participant characteristics by gdm status differences in serum analyte levels between normal and women with gestational diabetes t - tests were used for analysis of normally distributed continuous variables and the wilcoxon nonparametric equivalent for variables with skewed distribution . parametric and wilcoxon nonparametric t - tests were used to test differences across study groups for variables with normal and skewed distributions , respectively . ratios of proteins were computed and tested across study groups using wilcoxon nonparametric t - tests . receiver operating characteristic ( roc ) curves generated from predicted probabilities from logistic regression modeling were used to evaluate the classification ability of individual and multiple analyte combinations . the area under the roc curve ( auroc ) was computed from simple logistic regression to describe the classification ability of each protein , ratio , and glycosylated protein individually . based on the auroc results , individual proteins , ratios , and glycosylated proteins were added sequentially to build a multi - analyte model for improved classification performance . all statistical analyses were performed using sas software version 9.22 ( sas institute inc . , cary , nc ) . we previously reported the discovery and validation of novel biomarkers for intra - amniotic infection , down syndrome , pre - term birth , preeclampsia , diabetic nephropathy , and type-2 diabetes in multiple body fluids using combinations of 2d - dige and tandem mass spectroscopy . this is illustrated in figure 1 , which shows a 2d - dige comparison of the total glycoprotein fraction of pooled control and gdm maternal serum , in which protein spots that were differentially abundant in control compared with gdm samples appear as green or red spots , while proteins present at similar levels appear as yellow . pooled samples were first adsorbed on multi - lectin columns to purify the total glycoprotein fraction , this fraction was then eluted and subjected to 2- d dige . differentially abundant proteins ( arrows ) appear as red or green spots depending on the extent of under- or over - abundance for the present study , we selected two specific maternal serum glycoproteins , fibronectin and psg-1 for assessment of potential changes in glycosylation status . lectin reactivity profiling revealed that fibronectin glycosylation associated with sambucus nigralectin ( sna ) binding and psg-1 glycosylation associated with aleuria aurantialectin ( aal ) binding were significantly elevated in gdm maternal serum compared with control serum . therefore , these two protein - lectin pairs , fibronectin - sna and psg - aal , were selected for inclusion in a multi - analyte panel with additional biomarkers previously demonstrated to exhibit differential abundance in gdm , including adiponectin , sex hormone binding globulin ( shbg ) , and crp as well as the ratio of hcg to placental lactogen . these analytes were evaluated singly and in combination in a set of control and gdm maternal serum samples from the cohort described in table 1 . the mean participant age and pre - pregnancy bmi were 24.4 3.5 years and 20.3 3.3 kg / m , respectively . glycated hemoglobin measures did not differ between non - diabetic ( 5.2% ; iqr : 5.0 - 5.4% ) and gdm participants ( 5.4% ; iqr : 5.1 - 5.8% ) . fasting plasma glucose measures were also similar between groups 80 mg / dl ( iqr : 77 - 85 mg / dl ) and 84 mg / dl ( iqr : 79 - 90 mg / dl ) ; p = 0.20 ] . in addition , no statistically significant difference was observed between study groups for any other clinical parameter that was measured . although fasting plasma glucose and glycated hba1c measures were not different between groups , the levels of psg - aal , fibronectin - sna and the hcg / placental lactogen ratio were significantly elevated in the gdm group ( p = 0.004 , p = 0.006 and p = 0.03 , respectively ) , as shown in table 2 . the difference in maternal serum crp levels demonstrated borderline significance ( p = 0.05 ) , with a median concentration of 2.1 mg / l in non - diabetics and 5.7 mg / l in gdm participants . roc curves utilizing fibronectin - sna and psg - aal and the combination of these two analytes are shown in figure 2 . while the ability to detect gdm using both analytes is good ( auroc : 0.85 ) , their use in conjunction with the other analytes described table 2 within a multi - analyte model [ figure 3 ] demonstrated clearly superior performance ( auroc : 0.97 ) . specifically , the combination of fibronectin - sna and psg - aal alone had a detection rate of 74% at a false positive rate of 6% , while the multi - analyte model had a marked increase in the detection rate ( 87% ) at a false positive rate < 1% [ figure 3 ] . alternately , a single marker test with fibronectin - sna ( auroc : 0.81 ) is likely to be cost - effective in preventing gdm and in reducing the increased costs associated with its complications . receiver operating characteristic ( roc ) curves illustrating the ability of fibronectin and psg glycosylation to distinguish pregnant women with and without gestational diabetes receiver operating characteristic ( roc ) curves illustrating the classification performance of each protein and protein glycosylation pattern as individual analytes and as a multi - analyte model . these studies demonstrate that a multi - analyte test profile , comprising individual proteins , their ratios , and specific protein glycosylation patterns in maternal serum , can identify gdm patients independently classified by ogtt . these analytes are all amenable to analysis in dried bloodspots , which will enable the development of a minimally invasive , convenient , and cost - efficient screening test for gdm that will be particularly useful for evaluation of underserved populations that suffer significant disparities in diabetes care . fibronectin - sna is an early predictor before clinical hyperglycemia sets in . for cost considerations , a single marker fibronectin - sna can also predict early gdm , and women positive for fibronectin - sna can monitor fasting blood glucose and follow medical nutritional intervention to prevent gdm . | the prevalence of gestational diabetes mellitus ( gdm ) is increasing because of the worldwide obesity / diabetes epidemic .
the complications of untreated gdm affect both the mother and baby and include complications during pregnancy as well as increased risk of subsequent type-2 diabetes in mothers and offspring .
standard tests for hyperglycemia in diabetes , such as fasting glucose and hemoglobin ( hba1c ) , are currently not recommended for gdm screening .
instead , an oral glucose tolerance test is specified , which is invasive , time - consuming , and not easily accessible to many at - risk populations . in this study
, we describe a multi - analyte maternal serum profile test that incorporates novel glycoprotein biomarkers and previously described gdm - associated markers . in screening for gdm by multi - analyte panel ,
the detection rate was 87% at a false - positive rate of 1% . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
from may 2004 to may 2005 , a study of acute gastroenteritis in children < 16 years of age was undertaken by the institut pasteur , antananarivo , madagascar . children with a diagnosis of acute dehydrating watery diarrhea who were seen at the rehydration clinics and hospitals of antananarivo were eligible for the study . the study was approved by the ethical review board of the institut pasteur , antananarivo , madagascar . fecal samples were collected from the children and stored at 80c until analysis was undertaken at the university of liverpool , uk . there , viral rna was extracted from 150 l of 10%20% fecal suspensions in phosphate - buffered saline by using a guanidine and silica method ( 11 ) . rt - pcr was performed in a 50-l reaction mixture with primers that targeted the capsid n terminus / shell gene . for gi , forward primer g1skf ( 5-ctgcccgaattygtaaatga-3 ) and reverse primer g1skr ( 5-ccaacccarccattrtaca-3 ) were used , yielding a pcr product of 330 bp . for gii , the primers used were forward primer g2skf ( 5-cntgggagggcgatcgcaa-3 ) and reverse primers g2skr ( 5-ccrccngcatrhccrttrtacat-3 ) and g2alskr ( 5-ccaccagcatatgaattgtacat-3 ) , yielding a 344-bp product ( 12 ) . the pcr was performed with an initial denaturation at 94c , followed by 40 cycles of 60-sec denaturation at 94c , 60-s primer annealing at 50c , an extension for 2 min at 72c , followed by a final extension stage of 15 min at 72c . amplification products were examined under ultraviolet light after electrophoresis through a 2% agarose gel with ethidium bromide staining . products were extracted by using the qiaquick pcr purification kit ( qiagen , basingstoke , uk ) and were sequenced by macrogen inc . phylogenetic relationships were examined by aligning sequences with the clustalw multiple alignment program ( european molecular biology laboratory , heidelberg , germany ) . a phylogenetic tree was constructed according to the neighbor - joining method by using clustalx ( version 1.83 ) and the alignment file obtained by analysis with clustalw . an unrooted phylogram of norovirus isolates from the present study and prototype strains was plotted in the phylip format ( http://evolution.genetics.washington.edu/phylip.html ) output by using treeview software , version 3.1 ( http://taxonomy.zoology.gla.ac.uk/rod/treeview.html ) . assignment of norovirus to genotype was made according to the scheme proposed by zheng et al . the nucleotide sequences of the malagasy strains have been deposited at genbank ( accession nos . norovirus - positive samples were screened for other viruses by negative - stain electron microsopy and by rt - pcr for rotavirus and astrovirus ( 13,14 ) . during this 12-month study , 258 children with acute gastroenteritis in madagascar were screened for norovirus infection . because 21 samples contained insufficient stool , 237 samples were analyzed ( 142 from boys and 95 from girls ) . overall , 85% of children were <3 years of age , 77% were < 2 years , 43% were < 1 year , and 3% were newborns . the median age of the study population was 20 months ( range 1 day to 16 years ) . infection rates did not differ between boys and girls ( 6.3% and 5.2% , respectively , p>0.1 ) . no coinfections with other viruses ( rotavirus , astrovirus , and adenovirus ) were detected . ten ( 71% ) of the noroviruses detected were identified as belonging to genogroup gii ; the remaining 4 ( 29% ) , to gi . gi noroviruses were further classified into 3 genotypes : gi.1 ( 1 isolate ) , gi.4 ( 1 isolate ) , and gi.3 ( 2 isolates ) . * em , electron microscopy , nov , norovirus ; rv , rotavirus ; av , astrovirus ; pos , positive ; neg , negative . all norovirus - positive samples were negative for av and rv when tested by reverse transcription pcr . phylogenetic tree of noroviruses based on the 330-bp region ( for gi ) and 344-bp region ( for gii ) of the capsid n terminus / shell gene . fourteen novel sequences were included , designated according to isolate code , place , and year ( e.g. , dr001-madag04 ) ; 25 sequences of reference norovirus strains ( 6 ) were included , designated according to genogroup - genotype , place , country , and year ( e.g.,gii-2/melksham - grb1994 ) . comparative strains are gi-1/nv - usa1968 ( norwalk , m87661 ) , gi-2/sov - gbr1993 ( southampton , l07418 ) , gi-3/dsv - usa1993 ( desert shield , u04469 ) , gi-4/chiba - jpn2000 ( ab042808 ) , gi-5/musgrov - gbr1989 ( musgrove , aj277614 ) , gi-6/hesse - deu1998 ( af093797 ) , gi-7/wnchest - gbr1994 ( winchester , aj277609 ) , gi-8/boxer - usa2001 ( af538679 ) , gii-1/hawai - usa1971 ( u07611 ) , gii-2/melksham - gbr1994 ( x81879 ) , gii-3/toronto - can1993 ( u02030 ) , gii-4/bristol - gbr1993 ( x76716 ) , gii-5/hillingd - gbr1990 ( hillingdon , aj277607 ) , gii-6/seacrof - gbr1990 ( seacroft , aj277620 ) , gii-7/leeds - gbr1990 ( aj277608 ) , gii-8/amstdam - nld1990 ( amsterdam , af195848 ) , gii-9/vabeach - usa1997 ( ay038599 ) , gii-10/erfurt - deu2000 ( af427118 ) , gii-11/sw918-jpn1997 ( ab074893 ) , gii-12/wortley - gbr1990 ( aj277618 ) , gii-13/faytvil - usa1998 ( fayetteville , ay113106 ) , gii-14/m7-usa1999 ( ay130761 ) , gii-15/j23-usa1999 ( ay130762 ) , gii-16/tiffin - usa1999 ( ay502010 ) , and gii-17/cse1-usa2002 ( ay502009 ) . the median age of children with norovirus infection was 18 months ( range 351 months ) . all infections with genogroup gi noroviruses were found in children < 24 months of age ( table 2 ) . noroviruses were detected throughout the year ; however , infections peaked during the wet season in madagascar . november and december were the months of major norovirus prevalence ( 35.7% each month ) . seasonality of gi and gii norovirus infections , antananarivo , madagascar , may 2004may 2005 . to our knowledge , ours is the first study that has used molecular detection methods to investigate the role of noroviruses in pediatric gastroenteritis in madagascar . we showed that infections with gi and gii noroviruses are relatively common . in a 1-year collection of stool samples , we detected noroviruses by rt - pcr in 6% of children with acute gastroenteritis in antananarivo . this rate establishes norovirus as the second most commonly detected enteric virus in this population , behind rotavirus ( 38% ) and followed by astrovirus ( 2.5% ) ( data not shown ) . the median age of children with norovirus infection ( 18 months ) was higher than previously reported ( 7 ) and higher than that of the rotavirus - infected group ( median 10 months , range 1 day to 48 months ) and that of the astrovirus - infected group ( median 10 months , range 520 months ) ( data not shown ) . noroviruses were detected throughout the year , but the number peaked in november and december . such seasonality in a tropical country is not really expected , as year - round circulation has been previously documented ( 15 ) . our findings confirm the finding of previous studies that gii is the predominant norovirus genogroup circulating in communities worldwide . considerable genetic diversity was observed among the norovirus gii isolates , and some were identified as belonging to a potentially novel cluster . the closest reference strain to the potentially novel cluster was the recombinant hu / nov / gii.1/hawaii/1971/us . in contrast , norovirus gi isolates were clustered with prototype strains ; hu / nov / gi.3/dsv395/1990/sa ( desert shield ) was predominant ( 2 strains ) , followed by hu / nov / gi.1/norwalk/1968/us ( norwalk ) and hu / nov / gi.4/chiba407/1987/jp ( 1 each ) . the sample size was small , and we examined samples collected over a 13-month period . longer , longitudinal studies are required to address issues such as norovirus seasonality and temporal genetic variability . in addition , we restricted our analysis to specimens collected from patients at rehydration clinics and hospitals , so prevalence of norovirus infections in the general population may have been underestimated . furthermore , the use of short conserved sequences , although successful for diagnosis of norovirus infection , should be used with caution for classification and phylogenetic analyses . further analysis by full capsid sequencing might be required . nevertheless , continued norovirus surveillance is needed to monitor the spread and persistence of the various genotypes infecting children in madagascar . | of 237 children with acute gastroenteritis in antananarivo , madagascar , during may 2004may 2005 , 14 ( 6% ) were infected with norovirus .
seasonality ( november december peak ) was detected .
reverse transcription
pcr identified gii as the most common genogroup .
gis belonged to gi.1 , gi.3 , and gi.4 .
noroviruses in madagascar show extensive genetic diversity . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
it has been shown that the services pharmacists offer in community pharmacies improve health outcomes ( 1 ) . there are many studies that show the important role pharmacists play in detecting medical errors , drug interactions and inappropriate prescribing ( 2 - 6 ) . a study conducted in 1993 indicated that the lack of proper remuneration methods for pharmacists services is a major impediment for providing such services ( 7 ) . pharmacists remuneration systems are defined as the methods by which pharmacists are compensated by those paying for the healthcare services they are providing ( 8) . the remuneration method for pharmacists services in community pharmacies depends on each country s health and pharmaceutical policies . the particular characteristics of the healthcare system , such as the health insurance level of coverage , the out - of - pocket payments made by the patients and the regulated or unregulated price levels of medicines can all affect the profitability of pharmacies and pharmacists ( 9 ) , and perhaps their incentives for providing services as well . in recent years , most countries have followed cost containment policies to prevent expansion in their health expenditures , and these have affected the payments made to pharmacists in a number of ways . in iran , cost containment strategies have been followed as an aim in national drug policy ( ndp ) including price control and generic - based policy ( 10 , 11 ) . recently some arguments have been raised in favor of eliminating the dispensing fee system , as a tool for reducing patients out - of - pocket payments . conversely , pharmacists and pharmacy owners claim that their survival in the business depends on the dispensing fee and that it would not be fair to eliminate it ( 12 ) . the third group believes that eliminating the dispensing fee , is the only incentive of pharmacists and the main source of their profit would probably substantially damage the healthcare system from a variety of aspects . in this study , we investigated both the remuneration models and the pharmaceutical and healthcare policies related to pharmacists profitability in a number of selected countries : france , canada , ireland and turkey , and compare their situations to iran s , to help resolve this dilemma . the required data were collected by an unstructured comprehensive literature review , using computerized databases . we searched google , google scholar , pubmed and scopus for published articles , reports and acts , using the keywords : pharmacists remuneration , payment to pharmacists , dispensing fee , pharmacist professional fee , pharmacy reimbursement , pharmaceutical policy , pharmaceutical pricing , medicine margin and mark up , from february to september 2011 . in addition , for data collection about iran we also contacted the iranian food and drug organization ( fdo ) and iranian pharmacists association . in selecting the other countries , we considered both developed and developing countries and tried to collect some diverse examples from across the world . therefore , the following countries were chosen for evaluation and comparison to iran : france , ireland , canada and turkey . the findings about the countries that were investigated are presented below ( tables 1 and 2 ) . the general information about pharmacy regulations note : na : not available the remuneration systems and related subjects in investigated countries note : na : not available in 2000 , there was one pharmacy per 2,578 people and one qualified pharmacist per 1,190 people in france ( 13 ) . in terms of their type of activity , pharmacists in france are classified into seven groups ( a to h ) . from these , group a includes pharmacists who own their pharmacies and group d contains assistant pharmacists who are not owners but are employed in pharmacies , insurance offices and hospitals , etc . in 2010 , the percentage of pharmacists in groups a and d was 38% and 36.1% , respectively ( 14 ) . according to the law that was passed in 2000 , the number of pharmacists in a pharmacy depends on the pharmacy s revenue . pharmacies with annual revenues between 823,000 and 1.64 million euros must have two full time pharmacists , and those with annual revenues between 1.64 and 2.47 million euros require three full time pharmacists . following that , they have to employ one additional pharmacist for every 823,000 euros more in revenue ( 13 ) . chain pharmacies and online pharmacies are not allowed in france , but people can buy medicine from foreign online pharmacies ( 15 ) . also , over the counter ( otc ) medicines can only be sold in pharmacies ( 13 ) . in terms of price regulation for medicines , the prices for reimbursable medicines are fixed and regulated , and set by the ministry of social security . the prices for otc medicines that are not reimbursable are not regulated ( 16 ) . the pricing method in france france s health insurance system is based on a compulsory coverage for all french residents . in 2006 , about 88.4% of the country s population also had a voluntary insurance , most of which is supplementary . many of these insurance coverages include co - payment or co - insurance ( 15 ) . the reimbursement rate ( as shown in table 3 ) is not the same for all medicines and is identified based on the effectiveness of medicine and the severity of the illness , as determined by a scientific commission ( 16 ) . however , for a list consisting of 30 chronic and expensive diseases , the reimbursement is 100% ( 15 ) . reimbursement rates in france the medicine margin in france is a regressive margin related to the price . the margin of medicines up to the retail price of 22.9 euros is 26.1% , and from 22.9 to 150 euros it is 10% . for medicines with a retail price of more than 150 euros , the margin is 6% , but with a cap of 53 euros per pack ( 16 ) . although there is no dispensing fee in france and patient would only pay difference between the retail and the reimbursed prices , a 0.53-euro fixed fee per pack would be added to the margin for reimbursable medicines ( 15 ) . this fixed fee per pack is defined at 0.84 euro for special medicines that require advice from the pharmacist ( 17 ) . since 2004 , the wholesaler discount and the direct discount by the manufacturers to the pharmacies ( for companies that sell directly to pharmacies ) have been organized and currently do not exceed 2.5% for reimbursable medicines . for generics , there are more than 1,500 pharmacies in ireland and about 420,000 people visit pharmacies each week ( 18 ) . chain pharmacies are allowed in ireland ( 19 ) , but generic substitution is not legal ( 17 ) and online pharmacies are not still allowed ( 20 ) . the average wholesale price for all medicines in ireland is no more than in finland , germany , denmark , the netherlands , belgium , the u.k . , or there are four main coverage schemes in ireland : the general medical services scheme ( gms ) , the drug payment scheme ( dps ) , the long term illness scheme ( lti ) , and the high - tech scheme . which is designed for low - income people , patients pay 50 cent per prescription item , with a cap of 10 euros per family , per month . under this scheme , the pharmacist would be paid a dispensing fee , but no mark up . with the dps , patients pay 120 euros per month and the pharmacist would be paid with a dispensing fee and a mark up . medicines for about 15 chronic diseases , such as multiple sclerosis , diabetes and epilepsy , would be free using the lti , with pharmacists being paid both a dispensing fee and mark up . with the high - tech scheme , which is for expensive medicines such as chemotherapy agents , patients have to pay 120 euros as a deductible cost per month . the pharmacist is paid a 10% mark up as well as a patient s care fee ( 21 , 22 ) . in 2006 , there were more than 55.18 million prescription items in the gms , a 4.6 million increase compared to 2005 ( 23 ) . to control these rising expenditures in the pharmaceutical sector , a cost containment strategy was implemented after 2006 and under an agreement between the health department , health services executive and the irish pharmaceutical healthcare association , the new pricing mechanism resulted in the reduction of the price level of medicines ( 24 ) . the dispensing fee for all medicines was 3.51 euros ( 22 ) , but this has also been revised based on the number of items dispensed . also , the pharmacy mark up with the dps was reduced from 50% to 20% in july 2009 ( 25 ) . there are about 8,000 pharmacies in canada and nearly 60,000 prescriptions per pharmacy are filled annually ( 26 ) . for example , in ontario , the price of a first generic medicine would be set up to 70% of its brand and the price of the next generics would be up to 90% of the first generic . however , in quebec , the first generic medicine would be priced at up to 60% of its brand and the next generics up to 54% of the brand price ( 28 ) . in british colombia , the pricing is based on internal reference pricing and on considering the cost - effectiveness of medicines in each therapeutic group ( 29 ) . there is almost a universal coverage in canada and is the same in different provinces , except for outpatients prescription drugs , which are not universally covered and the situation is different from one province to another ( 30 ) . the public health coverage schemes ( which cover seniors , veterans , social assistance recipients , institutionalized populations , indians ) consist of provincial ( or territorial ) and federal schemes . in addition , two - thirds of canadians have private insurance coverage for prescription drugs , either individually or through their employers ( 31 ) . nevertheless , some provinces , such as british colombia , manitoba and saskatchewan , cover all of their residents ( 32 ) and most provinces have appropriate plans for low - income groups to protect them against expensive medical services ( 33 ) . catastrophic cost coverage ( limitation on payment for each person ) varies from province to province ( 34 ) . some insurers prefer to pay passively , with the patient paying out of the pocket and receiving compensation later ; in some other plans the cost of medicines is paid directly to pharmacies and beneficiaries pay only the co - payment ( 35 ) . the professional fee is compensated by some of the private insurance companies ( 27 ) . however , most private insurance plans have a deductible of about 25 canadian dollars per individual or 50 dollars per family . about 29% of the private insurance coverage did not have a co - payment in 2000 ( 36 ) . the drug formulary is not the same in all of the provinces ( 37 ) , and the reimbursement systems through public schemes differ from one province to another ( see table 4 ) . examplesof reimbursement methods in canadian provinces none of the federal schemes have co - payment , and childhood prescriptions are covered by social assistance . in addition , there is often a cap for co - payment in public schemes that is not common in private schemes ( 32 ) . in most provinces , payment to pharmacists is based on a fee for service model and in the form of a dispensing ( professional ) fee ( 38 ) . for example , in 2001 the dispensing fee in ontario was 6.74 dollars , while in nova scotia it was 7.67 dollars ( 39 ) . in some provinces the dispensing fee is scaled regressively , depending on the price of the prescriptions ( 3 to 10 scales defined ) . in addition , in april 2007 , ontario implemented the medscheck system , which includes an annual 30 min meeting with a pharmacist for medical consultation to review the appropriate use of medicines , etc . this system is only for patients who are suffering from three or more chronic diseases . this service is free for patients and is financed by the public sector ( 41 ) . a number of provinces , such as quebec , allow a 20% rebate ; however , this is not the case in some other provinces . for example , under the ontario drug plan , all kinds of rebates are going to be eliminated by 2014 ( 42 ) . generally , based on the estimated average of revenue earned by a pharmacy in 2006 , a pharmacy earned 250,000 dollars from dispensing fees and mark up , and 250,000 dollars from rebates ( 43 ) . approximately 32% of the healthcare expenditures in turkey are allocated to pharmaceuticals ( 44 ) . the number of prescriptions per month is 21 million and , in 2008 , the average cost of the prescriptions was about 42 turkish lira ( 45 ) . the prices of these medicines are set according to the lowest ex - factory price in the following reference countries : portugal , spain , france , italy and greece . there is a regressive margin for pharmacies and wholesalers , which is shown in table 5 ( 46 ) . margin of pharmaceuticals in turkey note : ytl : turkish liras pharmacies must pay within three months of buying from wholesalers , although sometimes the wholesaler offers a longer period . the reimbursement time is about six months after a prescription is dispensed ( 45 ) . reimbursement decisions are made by the social security institution reimbursement commission , which considers criteria such as pharmacoeconomic studies , budget impact analysis and ethical issues , etc ( 24 ) . no valuable data were available about the turkish dispensing fee system in the literature . there are more than 8,000 pharmacies in iran ( 47 ) ; 95% of these are private pharmacies . iran has two types of pharmacies : the majorities are day - opened pharmacies and some are 24 h , seven - day pharmacies that are distributed throughout the cities , depending on the populations . generic substitution is legal in pharmacy practice law . according to iranian national law , the number of pharmacists in a pharmacy depends on the number of prescriptions the pharmacy dispenses ( 48 ) , but this law the generic scheme policy in iran has been implemented as a tool for improving accessibility and affordability , by producing low - price medicines ( 49 ) . the usage of medicines without prescription is common in iran ( 50 ) , which may be attributed to the low price of medicines . the pricing of pharmaceuticals in iran is regulated and controlled by the pharmaceutical pricing commission of the food and drug organization . the cost plus pricing method has been in use for many years , but recently a value - based new pricing method was introduced , based on some reference countries . based on this new method , either the price would be set at least 60% lower than the brand for new generics ( branded generics ) and at 40% for high tech medicines or orphan medicines , or it would be set based on the lowest price in identified reference countries or the country of origin ( whichever is lower ) . the defined reference countries are spain , greece , saudi arabia , algeria and turkey ( 51 ) . the margin for pharmacies is currently between 5% and 15% , based on a regressive model for imported medicines . based on the official statistics , nearly 90% of iran s population is covered by at least one governmental health insurance system ( 52 ) . iran s health insurance system is greatly segmented and there are numerous insurance organizations and funds . the three main organizations are the social security organization ( ssi ) , which covers around 42% of the population ( all formal workers and their families ) , the medical service insurance organization ( msio ) , which covers 49% ( government employees , rural residents , self - employed people , students and clerics , etc . ) and the armed forces medical services organization , which covers 6% of the population ( 53 ) . the decision - making regarding the positive and negative lists falls under the authority of the medical services insurance supreme council in the ministry of labor & social affairs ( 54 ) . recently , pharmacoeconomic studies have been accepted as important criteria for making decisions about new medicines . the co - payment for outpatients is different with every coverage scheme , but in the three organizations mentioned above it is 30% and 10% for inpatient care ( 55 ) . also , medicines for some identified special diseases are free . because of delays in insurers reimbursing pharmacies , they may prefer co - payment and out - of - pocket payments . there is a dispensing fee per prescription , which is scaled according to prescription price , between 5,000 and 9,500 iran rials ( 0.41- 0.78 us dollars ) , which is 10% higher during the night time and on holidays ( 56 ) . one argument is that , because of the low price of medicines , sometimes the dispensing fee is more than the price sum of prescription items . there are no defined pharmacists cognitive services and no remuneration system for these services in iran . in this study , we evaluated the different remuneration models and profitability sources for pharmacists services in iran and several other selected countries . in terms of remuneration for pharmacists cognitive services , we were unable to find any evidence of such models in the countries we investigated , except for in some canadian provinces . dispensing fees or professional fees are common in most of the countries we studied . in ireland , the new plan for dispensing fees will be related to the number of items . in canada , the fee is in some cases a fixed amount per prescription and in others a regressive scaled fee . the dispensing fee in iran is based on the price of the prescription in three main scales . a study of the payment models has shown that fee for service and capitation are the most common methods used for the remuneration of pharmacists ( 38 ) . one study has suggested that between 75% and 85% of pharmacists are paid only through a salary method ( 57 ) . another study has shown that the performance - related remuneration can indeed affect the amount of services a pharmacist provides to patients and that these services may prevent some future costs ( 58 ) . this could be one of the reasons that pharmacists in iran are indifferent with respect to providing consultative services as one study has indicated that there is no desirable job satisfaction between practical pharmacists in iran ( 59 ) . the price of the medicines is one of the important factors influencing the profitability of pharmacies . because of the cost plus method of pricing in iran , the medicines seem to be less expensive than in the other countries that were investigated , which mostly follows reference pricing . we found that , in addition to the higher pharmacy margin in these selected countries , the price of medicines per se can generate a good profit . although , based on the current pricing method in iran , policymakers have considered different aspects , such as medicine wastage costs and the margins of the manufacturers , distributors and pharmacies , but this mechanism for pharmaceutical pricing has threatened quality and competition in the market , as well as the profitability of pharmacies . although most of the countries lower the pharmaceutical prices as a strategy for containing costs , the profitability for the pharmacies has to be achieved with this goal . another issue that has a dramatic impact on the profitability of pharmacies is the reimbursement method . among the countries studied , the one with the most accurate pharmaceutical reimbursement method was france , which takes the benefit and severity of the disease into consideration . in france , ireland , turkey and some of the canadian provinces considering the delays that pharmacies in iran experience in receiving reimbursements from the insurance systems , pharmacies are very vulnerable and policymakers need to take this into account before implementing any changes in reimbursement methods . it has been shown that pharmacy margins and patient co - payments are very important incentives for pharmacists in convincing patients to use a generic or brand medicine ( 60 ) . although the pharmacy margin for medicines manufactured in iran is higher than for medicines manufactured outside the country , this higher margin may not offset the significantly higher price of imported medicines and could , therefore , reduce any incentive for pharmacies to sell iranian generics . studies have shown that there is a negative relationship between the number of prescriptions dispensed hourly and the number of prescription errors detected ( 61 ) . therefore , it is important to impose strict guidelines to address this problem because , in addition to detecting prescription errors , pharmacists need enough time to speak to patients who require advice . studies have also shown that computer - based prescription and reception systems can help to prevent medical errors ( 62 ) . so far , iran has not been able to put such systems into effect because of some infrastructural challenges . this study suggests that there is no appropriate conformity between the pharmaceutical price level , the margins and the dispensing fee in iran . the current setting is not able to meet the pharmacists expectations in standard practice and , therefore , pharmacists are involved in providing unrelated services . in addition , the lack of incentive for pharmacists that results from the inappropriate remuneration system ultimately causes patients to be unsatisfied with the services they receive . these include implementing the new pricing method as soon as possible , changing the current formulation for the dispensing fee to a more appropriate model , having insurers cover the dispensing fee and making consultative services voluntary for patients . in addition , the iranian system needs to define both a remuneration system for some cognitive services , similar to those such as meds check that some canadian provinces have implemented , and a new remuneration system that is not based on salary for non - owner pharmacists . a relationship between the services pharmacists provide and the money they receive must also be established , as well as implementing rules about the number of pharmacists required in a pharmacy , based on the number of prescriptions , and restricting the activities of public pharmacies . limitation : in this study , we were faced with some limitations to the accessibility of data for some of the countries investigated . because we searched only published articles and reports , we were not able to find the most recent information in some cases , such as turkey and ireland . | pharmacists are members of the healthcare teams that provide valuable services to society .
their incentive to deliver such services is influenced by remuneration methods . in this study , we aimed to review the remuneration models for pharmacists services and the factors affecting the profitability of pharmacies in some selected countries , including france , ireland , canada and turkey , and compared them to iran .
international data were collected by literature review on google , google scholar , pubmed and scopus .
in addition , domestic data were collected by contacting relevant organizations .
there is no payment for pharmacists cognitive services in iran and in the countries investigated , except for some canadian provinces .
the dispensing fee system in iran does not seem to be adequate , especially considering that most of the insurers do not cover these fees .
the pricing method in iran has resulted in a low price level , in comparison to the other countries , and this issue has dramatically affected the profitability of pharmacies in standard practice .
it could be concluded that changing the current formulation for the dispensing fee to a more appropriate one , defining a remuneration system for non - owner pharmacists other than salary and implementing the new pricing method are necessary in order to improve the services provided by pharmacies . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
cc has been demonstrated to detect changes in myocardial contractility across a variety of cardiac conditions including hypertensive or hypertrophic cardiomyopathy and duchenne muscular dystrophy before changes in left ventricular ejection fraction ( lvef ) are observed . cell therapy offers a promising approach for regeneration of damaged vascular and cardiac tissue after acute myocardial infarction ( mi ) [ 48 ] . the amorcyte trial evaluated effect of autologous bone marrow derived cd34 + cell therapy on lvef and myocardial perfusion . significant improvement in myocardial perfusion and a trend towards improvement in lv ejection fraction were reported . because the changes seen in systolic function are modest and by design regional , the more sensitive myocardial strain techniques offer an attractive option for analyzing these results . accordingly , we evaluated the feasibility of mri - derived segmental cc analysis in patients treated with cell therapy following primary intervention for st - segment elevation myocardial infarction ( stemi ) , using the feature tracking ( ft ) technique . the ft technique was previously validated for cc assessment against harmonic phase imaging ( harp ) and subsequently utilized for assessment of cardiac involvement in churg strauss disease and in dobutamine cardiac mri stress testing . one of the major advantages of this technique is that additional imaging is not needed , as analysis is performed on cine ( steady - state free precession , ssfp ) images and does not require tagging . subjects with stemi who were treated with intracoronary stent implantation within 3 days of infarction and an lvef 50% by echocardiography demonstrating regional wall motion abnormality in the distribution of the infarct - related artery 4 days after stenting were enrolled . briefly , the treatment groups patients were treated with escalating dose of stem cells ( 5 , 10 , and then 15 million cd34 + cells ) and compared with control . at 2448 hours after bone marrow harvest , cd34 + cells were infused into the infarct - related artery through an over - the - wire balloon catheter placed within the previously stented segment using a stop flow technique . cardiac functional imaging was performed with retrospective electrocardiogram ( ecg ) gating , utilizing a standard ssfp technique , lv end - systolic and end - diastolic volumes , lvef , and infarct size at baseline , 3 months , and 6 months were assessed as described previously . using diogenes cmr ft software ( tomtec imaging systems , munich , germany ) , global cc was measured in left ventricular short axis steady - state free precession ( ssfp ) views at base , mid , and apex for each patient at baseline , 3 months , and 6 months by 2 independent readers who were blinded to treatment assignment . in addition , segmental cc was measured for all individual mid - ventricular segments ( segments 13 through 18 ) , segmental strain analysis was not undertaken in apical and basal segments due to low reproducibility ( for apical segments ) and no infarcted tissue present ( basal segments ) . diogenes cmr ft software ( tomtec imaging systems , munich , germany ) is a vector - based analysis tool based on a hierarchical algorithm that operates at multiple levels using a combination of 1-dimensional and 2-dimensional tracking techniques . based on a contour manually drawn by an expert reader along the lv endocardial border frame , the software automatically propagated the contour and followed its features throughout the remainder of the cardiac cycle . features tracked in each voxel ( the 3-dimensional analogue of a pixel ) by the software include brightness gradient at the tissue - cavity interface in homogeneities of the tissue ( with respect to a 256-level gray scale ) , geometrical roughness of the tissue edges , and additional specific anatomical elements ( papillary muscles and septum ) . tracking is based on optical flow algorithms applied on sequentially reduced windows to ensure resolving large amplitude displacements and still discern local differences , and also implicitly accounts for spatial coherence and time - periodic motion . the tracking over all frames of a set of individual material points along the border permits to quantify their frame by frame displacement . the circumferential strain is defined as the instantaneous local border lengthening or shortening : if l(t ) indicates the distance between two neighboring points along the border , the strain is computed as st(t ) = ( l(t ) lr)/lr , where lr indicates such length at the ecg r wave . this represents the lagrangian strain and it is the most frequently used in cardiac imaging [ 1416 ] . strain values are computed at 48 points along the border , the entire border is then automatically subdivided into six segments of same length and segmental strain is computed as the average strain of all the points falling in that segment . the global strain analysis can be accomplished very quickly , similar to speckle tracking in echocardiography ( this reference includes video of actual ft procedure ) . global circumferential strain data were summarized as mean sd as well as the median . due to small sample size , comparisons within each group in change from baseline for the global circumferential strain at 3 months and 6 months were conducted using the wilcoxon signed - rank test . similarly , change from baseline in circumferential strain at mid - anterior wall at 3 months and 6 months was conducted using the wilcoxon signed - rank test . bland and altman analysis was used to test the interobserver and intraobserver agreement for global circumferential strain at base , mid , and apex . the test statistic for testing the readings agreement between observer 1 and observer 2 was based on t = r(k-2/1-r2 ) , where t follows the t distribution with k-2 degrees of freedom , r is the correlation between the average readings ( [ observer 1 + observer 2]/2 ) and the difference ( observer 1 observer 2 ) with zero or small r indicating an agreement between methods ( i.e. , no systematic bias between readers ) , and k is the paired sample size . infarct size as a function of dose and changes in mid anterior strain at baseline , month 3 , and month 6 was analyzed using analysis of variance ( anova ) . statistical analysis was performed using sas software version 9.2 ( sas institute , inc . , cary , nc ) . most patients ( 9/10 in the control group and 12/13 in the treatment group ) had evidence of anterior wall myocardial infarction by late gadolinium enhancement ( lge ) . there was one lateral infarct in the control group and one inferior in the treatment group . no difference was detected in the treatment or control groups in global cc at any level , except for the basal slice in the treatment group , the change from baseline and 3 months was statistically significant ( 18.4 14.4 versus 24.4 4.8 , p = 0.03 , table 1 ) . mid - anterior cc in the treatment group showed improvement between baseline 18.5 8.6 and 3 months 22.6 7.0 , p = 0.03 with no further improvement at 6 months 20.3 12.4 , p = 0.34 ( figure 1 ) . there was no statistically significant difference in mid - anterior segments of the control group ( 19.7 7.3 to 18.6 10.7 , p = 0.91 ) at 3 months and no changes were observed at 6 months ( 19.7 7.3 to 23.0 8.4 , p = 0.36 ) . no changes were noted in any other segments in the mid - ventricular short axis between any time points . bland and altman analyses showed an excellent interobserver agreement for global cc at basal , mid , and mid segmental with ( pearson 's correlation between average and difference readings r ) r = 0.03 , p = 0.83 , r = 0.10 , p = 0.44 , and r = 0.08 , p = 0.14 , respectively . however , there was a poor interobserver agreement on apex with r = 0.40 , p = 0.0009 . in addition , there was an excellent intraobserver agreement for global cc at basal , mid , and apex with r = 0.07 , p = 0.69 , r = 0.19 , p = 0.29 , and r = 0.26 , p = 0.14 , respectively . in the treatment group , anova analysis showed no significant difference between the doses of stem cell , size of infarct at baseline , and changes in mid - anterior cc at baseline , month 3 , and month 6 . as previously reported , there were no significant differences in the change in infarct size or lvef in treatment versus control groups after 3 or 6 months . the major finding of this substudy from the amorcyte - randomized trial was feasibility of detection of segmental cc changes using ft technique in patients treated with intracoronary bone - marrow - derived cd34 + cell infusion after acute st elevation mi . this is of importance as ft analysis does not require additional imaging ( such as tagging ) and it can be used retrospectively as cine sequences are obtained in all studies while tagging is typically not a part of the standard protocol . ft technique is specifically designed for mri analysis and differs from recently reported velocity vector imaging ( vvi ) software ( which was designed for echocardiographic speckle tracking ) . in addition , we demonstrated that patients who received cell therapy had a statistically significant improvement in the mid - anterior cc without significant changes in lvef at 3 but not at 6 months . as most of the analyzed cohort suffered anterior wall mi , the improvement in cc within this infarcted region was likely driven by the beneficial effects of cell therapy . finally , both inter- and intraobserver agreement , for strain analysis were excellent for mid and basal segments . interestingly , intraobserver ( but not interobserver ) variability was poor for apical segment ; possible explanation is presence of apical trabeculations , which have been defined as endocardium by one reader versus compact myocardium used by other reader . improvement in 2d echo - derived lv strain has been reported by herbots et al . who demonstrated significant improvement in the global longitudinal strain rate in patients undergoing cell therapy following acute mi . strain rate improved in infarcted areas and appeared to be a more sensitive marker of response to therapy than was change in global left ventricular ef . similarly , plewka et al . reported improvement in systolic strain measurements involving infarcted segments in patients who received cell therapy . reported improvement in longitudinal strain in patients with prior anterior wall mi following cell therapy in the absence of concomitant revascularization . the current study is the first , to our knowledge , to demonstrate the utility of mri ft - derived segmental cc in the settings of stem cell therapy . furthermore , the broad spectrum of data available from mri ( valve assessment , lvef , infarct mass by lge , and strain ) may obviate the need for echocardiography following cell therapy after mi . finally a recent study by hopp et al . which is a substudy of autologous stem cell transplantation in acute myocardial infarction trial ( astami ) explored global and regional myocardial function after intracoronary injection of autologous mononuclear bone marrow cells in acute anterior wall myocardial infarction treated with percutaneous coronary intervention using mri tagging . the authors found that intracoronary injection of autologous mononuclear bone marrow cells did not influence regional or global myocardial function in this substudy . segmental cc may be an attractive and sensitive surrogate endpoint for evaluation of benefits of stem cell therapy . a recent meta - analysis of postinfarct stem cell trials concluded that changes in lvef were very modest and strain analysis may be amore useful and sensitive endpoint . indeed , in the present analysis , infarcted ( and treated ) mid - anterior segmental cc improved significantly in the absence of an appreciable change in lvef . although the clinical significance of improvement in segmental cc in the absence of a more global increase in lv contractility is yet to be determined ; such changes may predict a beneficial response to therapy with respect to lv remodeling and the degree of infarct zone electrical heterogeneity ( which may be associated with increased risk for ventricular reentrant arrhythmias ) . further studies examining the relationship of cc to both global measures of lv volume or function over time and to meaningful clinical outcomes are clearly warranted . small study population also limited the evaluation of a relationship between infarct transmurality , heterogeneity , presence of microvascular obstruction , myocardial hemorrhage , and the subsequent likelihood of response to stem cell therapy . further improvement in ft software resolution will be needed to assess changes in cc in the peri - infarct zone ( gray zone ) which is more likely to provide a favorable environment for infused stem cells than the fully infarcted tissue . additionally , only mid - myocardial strain was analyzed ; it is possible that additional changes would be detected with analysis of endocardial strain . as this method relies on perfect alignment of analyzed ssfp images at baseline and followup , a small misalignment may miss the edge of infarct and affect the results . in addition , lge images need to be obtained at exactly the same level as ssfp images , in order to assess changes in contractility in the infarct and peri - infarct regions . in conclusion , ft method can detect regional changes in cc following intracoronary stem cell infusion after myocardial infarction . | background . magnetic resonance imaging ( mri ) strain analysis is a sensitive method to assess myocardial function .
our objective was to define the feasibility of mri circumferential strain ( cc ) analysis in assessing subtle changes in myocardial function following stem cell therapy .
methods and results .
patients in the amorcyte phase i trial were randomly assigned to treatment with either autologous bone - marrow - derived stem cells infused into the infarct - related artery 5 to 11 days following primary pci or control .
mri studies were obtained at baseline , 3 , and 6 months .
cc was measured in the short axis views at the base , mid and apical slices of the left ventricle ( lv ) for each patient ( 13 treatments and 10 controls ) .
mid - anterior lv cc improved between baseline 18.5 8.6 and 3 months 22.6 7.0 , p = 0.03 .
there were no significant changes in cc at 3 months and 6 months compared to baseline for other segments .
there was excellent intraobserver and interobserver agreement for basal and mid circumferential strain .
conclusion .
mri segmental strain analysis is feasible in assessment of regional myocardial function following cell therapy with excellent intra- and inter - observer variability 's . using this method ,
a modest interval change in segmental cc was detected in treatment group . |
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apolipoprotein e ( apoe ) was first discovered in 1970s and identified as a component of triglyceride - rich lipoprotein complexes . since then , apoe has been widely studied in lipid metabolism , cardiovascular diseases , and neurodegenerative disorders such as alzheimer 's disease ( ad ) and parkinson disease ( pd ) . in addition to its well - established role in lipid transportation and atherosclerostic pathogenesis , apoe bears immunomodulatory properties and is associated with multiple sclerosis ( ms ) and psoriasis . apoe can modulate the functions of macrophages , suppress the proliferation of t cells , maintain the integrity of blood - brain barrier ( bbb ) and blood - nerve barrier ( bnb ) , inhibit the proliferation of smooth muscle ( sm ) cells , upregulate the production of nitric oxide ( no ) of platelets , facilitate the presentation of lipid antigen by cd1 molecules to natural killer t ( nkt ) cells , and so forth , ( figure 1 ) . cytokines are crucial in human inflammatory and autoimmune disorders and apoe might affect these disorders through interacting with cytokines . apoe is produced by liver , brain , spleen , kidney , lung and muscle tissues , and so forth . hepatic parenchyma cells produce 2/3 to 3/4 of the apoe in plasma . in the nervous system , apoe mrna is present in astrocytes , microglia , schwann cells ( scs ) , neurons , and so forth , ( figure 2 ) . there are three major isoforms of human apoe ( apoe2 , apoe3 , apoe4 ) encoded by the 2 , 3 , and 4 alleles on chromosome 19q13 . differences in the amino acid residues ( positions 112/158 ) of apoe distinguish the apoe2 ( cys / cys ) , apoe3 ( cys / arg ) , and apoe4 ( arg / arg ) isoforms . the apoe alleles show a peculiar distribution throughout the world ; the apoe3 allele is the most frequent in populations with a long - established agricultural economy , whereas the apoe4 allele is the ancestral allele , with a higher frequency in pygmies , khoi san , papuans , lapps , and some native americans . the structural polymorphism in apoe influences its conformation and bindings to lipoprotein particles and cellular receptors . apoe4 preferentially binds to lower density lipoproteins and is associated with increased risk of atherosclerosis and neurodegenerative disorders , including ad . this binding preference is due to domain interaction ( the presence of arg-112 causes arg-61 in the amino - terminal domain to interact with glu-255 in the carboxyl - terminal domain ) ( table 1 ) . additionally , physiological properties of apoe , such as antioxidant , antiapoptotic , immunomodulatory , and atheroprotective capacities are significantly influenced by apoe polymorphism . apoe exerts its biological functions , especially lipid transportation , by binding to its receptors , the low - density lipoprotein receptor ( ldlr ) family . the ldlr family includes ldlr , ldlr - related protein 1 ( lrp1 ) , very low density lipoprotein receptor ( vldlr ) , apoe receptor ( apoer)2 , megalin ( also known as lrp2 ) and lrp1b , and so forth , . as the prototype of this family , ldlr is the main receptor for cholesterol metabolism , to which apoe3 and apoe4 bind with 50-fold greater affinity than apoe2 . the interaction of apoe and ldlr determines the removal of apoe - rich lipoproteins ( vldl , chylomicron remnants , intermediate density lipoproteins ) , and the homeostasis of cholesterol and triglycerides . besides direct signal transduction , the ldlr family functions as signal modulators and integrators in several signaling pathways . the distribution of lipids among different body compartments occurs by means of different lipoprotein particles in the blood circulation . host responses to infections involve changes in lipid levels and lipid metabolism in the plasma . these plasma lipid changes might be mediated by three cytokines interleukin ( il)-1 , il-6 , and tumor necrosis factor ( tnf)- , which induce elevated levels of triglycerides and vldl , decreased levels of cholesterol , hdl and ldl [ 23 , 24 ] or increased levels of cholesterol in the serum , depending on the nature of the infectious agents . lipoproteins and lipids present in the serum may contribute to the host innate immunity against pathogens . studies using apoe deficient mice confirmed the role of apoe in host susceptibility to endotoxemia and klebsiella pneumoniae infection , while transgenic ( tg ) mice expressing human apoe3 and apoe4 genes revealed an isoform - specific effect of apoe on the proinflammatory response to lipopolysaccharide ( lps ) . infection of apoe knockout ( ko ) mice with listeria monocytogenes or klebsiella pneumoniae leads to an increased susceptibility to death as well as increased serum levels of tnf- as compared with wild - type ( wt ) mice [ 29 , 30 ] . binding of bacterial endotoxin to either hdl , ldl , or vldl results in a redirection of endotoxin uptake from kupffer cells to parenchymal hepatocytes where the endotoxin is deactivated . apoe facilitated sepsis - induced mortality in a dose - dependent manner and increased natural killer t-(nkt)-cell proliferation in the spleen . apoe deficient mice were markedly more susceptible to tuberculosis , evidenced by 100% mortality within 4 weeks of infection with tuberculosis . apoe 4/4 genotype was associated with an aggravated disease course of acquired immunodeficiency syndrome ( aids ) , especially with accelerated progression to death . compared with apoe3 , apoe4 enhanced human immunodeficiency virus ( hiv ) fusion / cell entry of both r5 and x4 hiv strains in vitro . apoe4 competes less efficiently than the other isoforms for entry into neuronal cells through heparan sulphate proteoglycan proteoglycans , which are involved in hiv attachment and entry into cells . hiv produces a chronic viral infection in the central nervous system ( cns ) that elicits chronic glial activation and cytokines overproduction . showed that hiv - infected subjects with an apoe4 allele had excess peripheral neuropathy . as a well - known risk factor for ad , an 4 allele along with the presence of herpes simplex virus ( hsv ) 1 in the brain confers an increased risk of developing to ad [ 36 , 37 ] . the production of infectious hepatitis c virus ( hcv ) was significantly reduced by the knockdown of apoe . the genotypes carrying the apoe2 were associated with the equivalent of a 35-fold reduction in the risk of chronic hcv infection . another study , however , showed that the expression of the apoe2 resulted in poorer recovery of infectious hcv than did apoe3 and apoe4 isoforms . either subjects without apoe3 or with a single apoe3 with high serum cholesterol are prone to a better prognosis . apoe gene polymorphism was also associated with hepatitis b virus ( hbv ) infection , and the 2 allele , compared with the other alleles , showed positive correlation with different hbv infection models . apoe suppressed inflammation through vldlr or apoer2 in macrophages by converting proinflammatory m1 to the antiinflammatory m2 phenotype . exogenous apoe suppressed lps and polyinosine - polycytidylic acid ( pic , a double - stranded rna that serves as a viral mimetic and a toll - like receptor ( tlr)3 agonist ) induced secretion of il-6 , il-1 and tnf- by raw 264.7 cells via repressing tlr - agonist - induced phosphorylation of c - jun n - terminal kinase ( jnk ) and c - jun . apoe peptide ( 141155)2 inhibits lps- and pic - induced macrophage inflammatory responses in a similar manner . apoe also reduces inflammatory signaling in astrocytes and microglia in response to proinflammatory stimuli [ 44 , 45 ] . th precursor ( thp ) cells can differentiate into th1 , th2 , or th0 cells . th0 cells can differentiate to th1 and th2 subpopulations depending primarily on the cytokines provided exogenously or from dendritic cells . th1 cells are involved in cellular immunity and immunoglobulin class switching to the igg2a isotype , whereas th2 cells are mainly associated with humoral immunity and immunoglobulin class switching to igg1 and ige . moreover , th1 cells promote the induction of complement - fixing , opsonizing antibodies and of antibodies involved in antibody - dependent cell cytotoxicity , for example , igg2a in mice . th1 cytokines include interferon ( ifn)- , il-12 , and tnf-. they can activate macrophages to produce reactive oxygen intermediates and no , stimulate their phagocytic functions , and enhance their antigen presenting capacity by upregulating major histocompatibility complex ( mhc ) class molecules . th2 cytokines include il-4 , il-5 , il-10 , and il-13 , which provide potent help for b - cell activation , immunoglobulin class switching to ige and igg1 in the mouse , and downregulation of macrophage activation ( figure 3 ) . imbalance of the th1/th2 can result in autoimmune disorders . in experimental autoimmune neuritis ( ean ) , an animal model of human guillain - barr syndrome ( gbs ) , th1 cytokines predominate and mediate inflammatory damage to the peripheral nerves , whereas th2 cytokines are associated with recovery from the disease [ 4648 ] . proinflammatory cytokines such as il-1 , il-6 , il-12 , il-17 , il-18 , il-23 , tnf- , ifn- , and macrophage inhibitory factor ( mif ) significantly contribute to disease development of ean by recruiting effector cells to the peripheral nervous system ( pns ) and by enabling in situ release of other products toxic for scs and myelin such as free radicals , oxygen intermediates , and no [ 46 , 47 , 4951 ] . antiinflammatory cytokines from th2 cells such as il-4 and il-10 may suppress the disease by playing an important antiinflammatory role [ 52 , 53 ] . the balance of functionally distinct t - cell subsets between th1 and th2 has a direct relevance to autoimmune disorders . recently , the th1/th2 paradigm has been challenged , following the discovery of a third subset of effector th cells that produce il-17 ( termed th17 cells ) and exhibit effector functions distinct from th1 and th2 cells . th17 cells are potent inducers of tissue inflammation and have been associated with the pathogenesis of many autoimmune diseases and rheumatic disorders . il17-producing th17 cells have been found to play an important role in many autoimmune diseases including experimental autoimmune encephalomyelitis ( eae ) , an animal of ms , while thymic regulatory t ( treg ) cells have a suppressive role , although their roles in ean and gbs need further investigation . increased mrna level of il-1 , il-2 , il-6 , ifn- , intercellular adhesion molecule ( icam)-1 , vascular cell adhesion molecule ( vcam)-1 , monocyte chemoattractant protein ( mcp)-1 , nuclear factor ( nf)b ( p65 ) and inhibitory ( i)b- and decreased mrna level of il-4 , il-10 , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) in apoe ko mice compared with wt mice imply that lacking apoe promotes th1 immune responses by changing these cytokines . il-6 , il-12 , tnf- , and ifn- were upregulated to a significantly greater level in apoe - deficient mice than in wt mice at both the mrna and protein levels in response to lps administration . apoe selectively regulates tlr4- and tlr3-mediated signaling of il-12 production and apoe may suppress the th1 immune response by modulating il-12 production . macrophages from apoe - deficient mice stimulated by exogenous antigens are more effective in upregulating mhc class molecules and cd80 , with elevated secretion of ifn- in responding t cells . severe hypercholesterolemia can induce a switch of autoimmune responses from th1 to th2 effector type in atherosclerotic apoe ko mice . apoe - deficient mice injected with lps produced elevated levels of il-1 , il-6 , and tnf- , when compared with wt mice , and exogenous apoe could reverse this effect . apoe targets myeloid differentiation primary response gene ( myd)88 and interleukin-1 receptor - associated kinase ( irak)1 activation whereby interrupting il-1 and il-18 signaling in vascular smooth muscle cells ( vsmcs ) . as a consequence il-1 signaling intermediates nfb transactivation was inhibited by apoe in myd88- and irak1- but not in traf6-transfected cells . no is a principal effector of macrophage - mediated inflammatory / immune responses . in the mononuclear - phagocyte system , no is formed enzymatically from l - arginine by inducible nitric oxide synthase ( inos ) . treatment of microglia and peripheral macrophages with apoe increased no production stimulated by ifn- and lps . apoe per se , however , seems unable alone to induce the production of either inos ; it functions via alteration of arginine availability . furthermore , upon proinflammatory stimulation , peripheral macrophages from male apoe4 tg mice could produce significantly higher levels of no than from apoe3 tg mice . is coupled with an increased arginine uptake in male apoe4 tg mice and is dependent on p38 mitogen - activited protein kinase ( mapk ) , whereas it is not the case in female mice . macrophages from female apoe tg mice produce higher levels of no than male ones , without any isoform - dependent differences . scs can secrete cytokines like il-6 and il-10 and actively participate in immune responses as facultative antigen presenting cells in the pns . the antigen presenting properties of scs were enhanced in apoe ko mice , concomitant with downregulated production of intracellular il-6 . in the recovery stage of ean , no produced by scs is pivotal for the termination of local immune responses by inducing apoptosis of effector t cells in the pns . apoe might act as an inhibitor for ean by suppressing the th1 response and shifting the th1/th2 to the th2 direction . an increased susceptibility to ean after upregulation of the autoreactive t - cell response to peripheral nerve component with higher levels of ifn- , il-12 , and tnf- and lower levels of il-10 was seen in apoe ko mice than wt mice . significantly increased secretion of th17-related cytokines ( il-17 and il-6 ) and expression of transcription factor retinoid - related orphan receptor gamma ( ror)t levels and obviously decreased number in treg cells , secretion of treg - related cytokines ( tgf-1 ) and expression of transcription factor ( foxp3 ) levels were found in apoe ko mice . th17/treg functional imbalance exists in apoe ko mice , suggesting a role of th17/treg imbalance in apoe functioning . il-17a was also found to be elevated in the plasma and tissues of apoe null mice . cog133 , a mimetic of apoe protein , when binding to a protein transduction domain creates cog112 , an antennapedia - linked apoe - mimetic peptide , improves therapeutic effects on eae through decreasing demyelination and diminishing the number of peripheral cells infiltrating into the spinal cord , as well as other inflammatory processes [ 74 , 75 ] . increased mrna level of inos , tnf- , ifn- , and il-17 and decreased nuclear translocation of nf-b and ib kinase ( ikk ) activity in colitis models suggest that cog112 inhibits inflammation through the nfb pathway . furthermore , intravenous administration of a small apoe - mimetic peptide derived from the receptor - binding region of the apoe holoprotein ( apoe-(133149 ) ) similarly suppressed both systemic and local brain inflammatory responses in mice after lps administration . however , different genetic basis determines modified immunological processes . lrp is implicated to mediate the immunomodulatory effects of apoe , although there seems to be no difference in the binding affinity of apoe isoforms to lrp [ 79 , 80 ] . apoe suppressed the secretion of tnf- and il-1 in an isoform - specific fashion ( e2 > e3 > e4 ) . a markedly higher level of tnf- was observed in supernatant of cultured macrophage derived from adult male apoe4 tg mice compared with macrophages from apoe3 ones . in the presence of lps , apoe3-expressing cells produce less tnf- and il-6 than apoe2 and apoe4-expressing cells which might be related with the extracellular signal - regulated kinases ( erk)1/2 signaling pathways . pronouncedly affects the cellular immune response in stably transfected murine macrophages . in apoe4 versus apoe3 macrophage cell line cells higher levels of proinflammatory cytokines including tnf- appeared evident followed by lps stimulation . preincubation with human recombinant apoe blocked glial secretion of tnf- in mixed neuron - glial cultures in response to lps stimulation in a dose - dependent manner ( apoe3 > apoe4 ) . this effect was the greatest when apoe was preincubated for 24 hours and was not dependent on any direct effect of apoe on cell viability . twice as many apoe4 carriers were demented ( 30% versus 15% ) or had peripheral neuropathy as compared with non - apoe4 carriers in the hiv infection . if there are apoe isoform - dependent differences in the antiinflammatory function of apoe in vivo , such differences might in part explain the differential risk for ad caused by apoe isoforms . however , several studies demonstrated that exogenously applied apoe4 had robust proinflammatory activity in astrocytes and microglial cells [ 85 , 86 ] . in mice receiving the basal diet without quercetin supplementation , levels of tnf- in whole blood stimulated ex vivo with lps were higher in apoe3 than apoe4 tg mice . apoe displayed an isoform - specific effect on inflammation in primary adult microglia culture , with apoe4 the most potent to stimulate production of prostaglandin - e2 and il-1 . activation of primary astrocytes from apoe targeted replacement ( tr ) mice with lps led to genotype - dependent differences in cytokine secretion that were the greatest in apoe2 tr mice ( apoe2 > activation of primary astrocytes from apoe tr mice with lps led to apoe - dependent differences in cytokine secretion that were greatest in apoe2 tr mice and that were associated with differences in nfb subunit activity . transforming growth factor ( tgf)-1 contains similar structure with apoe and lipoproteins containing the apoe3 isoform have higher tgf- levels and bioactivity than those containing apoe4 . association of tgf- with different types of lipoproteins may be beneficial to diseases like atherosclerosis and ad . intraventricular administration of lps resulted in higher production of proinflammatory cytokines , along with greater activation of nfb - regulated genes in the apoe4 than in apoe3 tg mice . inflammatory responses of apoe4 tr mice following intravenous administration of lps expressing the human apoe4 allele had higher systemic and brain levels of the proinflammatory cytokines tnf- and il-6 than their apoe3 counterparts , suggesting an isoform - specific effect of the immunomodulatory properties of apoe . cog1410 , an apoe - mimetic peptide , was also associated with a reduction in tnf- , il-1 , il-6 , and il-12 levels in both apoe3 tr and apoe4 tr mice assessed at 24 hours in the caecal ligation and puncture model of peritonitis . peripheral blood mononuclear cells ( pbmcs ) from ad patients with apoe4 allele produced higher spontaneous and phorbol-12-myristate-13-acetate-(pma- ) induced levels of il-1 after controlling for confounders . apoe4 carriers showed higher concentrations of il-1 than their counterparts without apoe4 after pma - stimulation . the apoe4 allele was associated with lower serum levels of il-10 in both acute coronary syndrome and chronic stable angina patients . recombinant apoe suppresses chemokine ( c c motif ) ligand ( ccl)2 and vascular endothelial growth factor ( vegf ) expression in retinal pigment epithelium ( rpe ) cells in an isoform - dependent manner ( apoe4 > apoe3 ) . the exact molecular mechanisms by which apoe isoforms differentially alter the immune responses have been postulated to influence different signaling pathways . apoe isoforms might be in part responsible for the differential modulation of nfb and mapk pathways [ 89 , 91 , 96 ] . a significant interaction between apoe genotype and the response to 17-estradiol was observed for no and tnf- production from microglia activated by recombinant mouse ifn- plus either lps or pic . the antiinflammatory activity of 17-estradiol is pronouncedly reduced in apoe4 tr mice compared with apoe3 tr mice . apoe genotype may affect the neuroprotective role of 17-estradiol and of hormone replacement therapy on brain function when the apoe4 gene is expressed . classical activation of macrophages by proinflammatory cytokines such as ifn- and tnf- can downregulate apoe production [ 9799 ] . however , antiinflammatory stimuli such as tgf- and estrogen promote apoe synthesis and release . gm - csf stimulated macrophages had a 35-fold reduction in apoe secretion , comparable with that observed for ifn- , while tnf- and il-1 resulted in a 2-fold decrease . in contrast to the reduction in apoe secretion by these cytokines , tgf- stimulated macrophages secreted 3-fold greater amounts of apoe than controls [ 100103 ] . treatment of primary rat astrocyte and mixed glial cell cultures with tnf- markedly reduced extracellular apoe protein levels . the effects of apoe in adipose tissue on body lipid homeostasis and atherosclerosis have been widely studied . tnf- may activate the binding of nfb p50 to a binding site at -43 in the apoe promoter . treatment of adipocytes with tnf- led to increased binding of nfb p50 , decreased binding of p65 and sp1 to this region of the apoe promoter , and repression of adipocyte apoe gene expression . reduction of p50 expression using sirna completely eliminated tnf--mediated reduction of endogenous apoe gene expression in adipocytes . ifn- dramatically increased the degradation of intracellular apoe and inhibited the accumulation of apoe in the supernatants of human monocyte - derived macrophages and human acute monocytic leukemia cell line ( thp-1 ) cells via post - translational modification of apoe [ 97 , 108 ] . ifn-1b has inhibitory effects on the production of apoe mrna , cellular protein , and secreted protein in blood monocytes . treatment of primary mixed glial cell cultures with il-1 induced a marked increase of extracellular apoe protein . tgf- can induce the expression of apoe in the thp-1 cell line by activating jnk , mapk p38 and caseine kinase ( ck)2 pathways . classical activation of macrophages by proinflammatory cytokines can downregulate apoe production and antiinflammatory stimuli can promote apoe synthesis and release , indicating a key role of apoe and cytokines in immunomodulation . apoe suppressing proinflammatory signalings , and vice versa , indicate an intricate apoe - mediated feedback regulation of inflammatory and immune responses . | apolipoprotein e ( apoe ) is a multifunctional glycosylated protein characterized by its wide tissue distribution . despite its importance in lipid transport and atherosclerosis pathogenesis
, apoe is associated with neurodegenerative disorders such as alzheimer 's disease ( ad ) and parkinson disease , and autoimmune disorders such as multiple sclerosis and psoriasis . among others ,
the role of apoe in modulating inflammation and oxidation is crucial in elucidating the risk factors of the above diseases since the function of apoe is closely linked with both proinflammatory and antiinflammatory cytokines .
moreover , apoe modulates inflammatory and immune responses in an isoform - dependent manner .
correspondingly , inflammatory cytokines can either upregulate or downregulate the production of apoe in various tissue types .
however , studies on the interactions between apoe and cytokines occasionally yield conflicting results , highlighting the complex roles of apoe and cytokines in various disorders .
the present paper summarizes the current knowledge about the cross - talk between apoe and cytokines , with emphasis on the effects of apoe on the th1/th2 balance . |
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early childhood caries ( ecc ) is a particularly virulent form of dental caries that is characterized by an overwhelming infectious challenge and is associated with unusual dietary practices . ecc has been defined as the presence of any decayed ( cavitated or noncavitated ) , extracted ( due to caries ) , or filled teeth in any primary tooth in a child aged 71 months or younger . the most important risk factors related to ecc are probably high frequency intake of sugary snacks and drinks , sweetened feeding bottles particularly used during the night , and malnutrition . additionally , poor oral hygiene , insufficient exposure to fluoride , and general psychosocial stress are common . nutritional anemia is the most common form of malnutrition and includes a lack of nutrients such as iron , folic acid , copper and vitamins a , b , c , and e. iron deficiency anemia accounts for 90% of all types of anemia in the world . although the prevalence of iron deficiency has declined in recent years , it remains an important pediatric public health problem . iron deficiency can impede physical and mental development in children and also negatively affects cellular immunity . a child 's diet that is high in low - nutrient carbohydrate will likely be deficient in required nutrients . malnutrition , such as iron deficiency , often impairs salivary gland function causing reduced salivary secretion and low buffering capacity . however , iron supplements reduce bacterial colonization and biofilm formation ; also an animal study has shown that iron supplements can reverse the carious process , and reduce the incidence of caries , especially on the buccal surfaces , which are bathed readily by saliva . an earlier study in iran has reported the prevalence of iron deficiency anemia in children aged 6 months to 5 years to be 19.7% . another local study reported that 3.2% of 7-year - old children had low intake of iron . ferritin is an intracellular protein that stores iron and releases it in a controlled fashion . it is a marker of the general iron level and is associated with the inflammatory response . as the etiology of ecc is multifactorial , and the effect of iron on the development of dental caries and concentration of this metal in noncarious as well as carious teeth remains controversial , the aim of this study is to explore a possible association between ecc and serum levels of iron and ferritin in children aged 2471 months in rafsanjan , iran . ethical approval for this study was received from the research ethics board of the rafsanjan university of medical sciences . two hundred and four children ( 117 boys and 87 girls ) aged 2471 months were recruited with convenient sampling method for this double - blind , cross - sectional study . the children were in - patients at the ali - en - abetaleb hospital , rafsanjan , iran , hospitalized for uncomplicated medical problems , including mild viral infection and dehydration and simple bone fractures between september 2008 and june 2010 . all participants parents signed a consent form and responded to a structured questionnaire with items regarding as age , gender and general health conditions . the children were selected among children who did not have diseases and conditions and did not take medicines affecting the serum iron level . the inclusion criteria were assessed and eligibility for study participation determined through responses to the questionnaire , interview with children 's parents , and discussion with the treating pediatrician . blood samples ( approximately 2 ml ) were collected by an expert nurse in the morning and used to determine serum levels of iron and ferritin levels ( pishtaz teb co , tehran , iran ) . dental examinations were carried out according to the world health organization criteria by one of the authors ( ms ) . the examinations were performed at hospital bedside using a disposable plane mouth mirror ( atlas teb co , tehran , iran ) and a penlight . teeth with less than two - thirds of the crown erupted were excluded . to assess the caries experience , the decayed , extracted due to caries , and filled primary teeth ( deft ) index was used . a tooth was diagnosed as decayed ( d component ) if its color was changed and there was any evidence of cavitated or noncavitated dental primary caries or recurrent caries adjacent to already filled teeth . component included extracted teeth and decayed teeth indicated for extraction due to caries , and the f component included restored teeth without caries . the dental examiner was not aware of the serum iron or ferritin levels at the time of clinical examination . data analysis included pearson 's correlation coefficient and t - test using spss-16.0 ( spss inc . , the mean ( sd ) age was 40.7 ( 14.7 ) months with 41.7 ( 14.6 ) months for boys and 39.3 ( 14.8 ) for girls . the analyses showed that 73.5% of children had normal serum iron level ; only 8.8% had low serum iron and 17.6% had high serum iron . ninety - eight and a half percent had normal serum ferritin levels while no child tested had low serum ferritin , and 1.5% had high serum ferritin . the mean values of the deft index and levels of serum iron and ferritin were 2.4( 3.3 ) , 93.8( 29.0 ) g / dl and 63.1( 32.2)ng / ml , respectively [ table 1 ] . normal limits of serum iron and serum ferritin are 50120 g / dl and 7140 ng / ml , respectively . there were no significant differences between the deft index , serum iron , and serum ferritin levels between the genders . caries experience and serum levels of iron and ferritin by gender in 2471 months old children in rafsanjan , iran ( n = 204 ) the prevalence of caries - free children was 54.4% ( 48.7% boys , 62.1% girls ) and among those with normal serum iron and serum ferritin levels was 38.2% ( 38.5% boys , 37.9% girls ) and 52.9% ( 46.2% boys , 62.1% girls ) , respectively ; whereas among children with low serum iron content only 5.9% ( 5.1% boys , 6.9% girls ) were free of caries . statistical analyses using pearson 's correlation coefficient showed a statistically significant inverse association between caries experience ( deft index ) and serum iron levels ( p=0.001 ) [ figure 1 ] , while no association was observed with the serum ferritin levels ( p = ns ) [ figure 2 ] . caries experience by serum iron level in 2471 month old children ( n= 204 ) caries experience by serum ferritin level in 2471 month old children ( n= 204 ) ecc is the currently accepted term used to describe dental caries occurring in infants and toddlers under the age of 6 years . ecc is a multifactorial , transmissible , and dietobacterial disease , and diet plays a critical role in the development and clinical features of this infection . nutritional anemia and dental caries are still among the most prevalent diseases in some developing countries . in this study , 8.8% of children had serum iron content below the normal limits of 50120 g / dl . animal studies have demonstrated that iron supplements , such as iron sucrose that has been used for prevention of anemia , are able to reduce the incidence of caries in rats . the concept that two major public health problems could be alleviated by the addition of iron to sucrose is attractive . based on our results , there was an inverse significant association between deft index and serum iron levels . a similar study in iran showed a significant correlation between the dmft index and low iron intake ; the dmft index of children with iron deficiency was much higher than in those with adequate iron intake . in children , the presence of extensive dental caries will pose challenges for chewing and may negatively impact absorption of nutrients in the gut due to poorly masticated food . it is reasonable to assume that the same dietary factors that cause iron depletion ( high consumption of beverages and low meat intake ) will predict dental caries . dietary diversification involves promotion of a diet with a wider variety of naturally iron - containing foods , especially red meat , poultry , and fish . heme iron from meat is better absorbed by the body than nonheme iron , which is found in dairy products , vegetables , and fruits . there is evidence that children who eat little to no meat or poultry and drink large amounts of juice and milk or snack on cookies or candy are at risk for iron depletion , because their large calorie intake from these other sources prevents them from eating other foods for nutritional needs . it seems children with dental caries , particularly occlusoproximal lesions , avoid consumption of diets containing meat due to meat fibers being entrapped in the cavities and a sense of discomfort . therefore , ecc may be a risk marker for iron deficiency anemia , so assessment of serum iron levels in ecc patients is appropriate to avoid the harmful health effects of anemia in developing children . serum ferritin is an acute - phase protein ; its level is an indicator of body - iron stores and may be normal or elevated in infective , inflammatory , or malignant disease . a normal serum ferritin level does not entirely rule out iron deficiency , which could be present even with a normal concentration of serum ferritin ( normal limits ; 7140 ng / ml ) . our study has shown that there was no association between serum ferritin levels and deft index , which is similar to the findings of another study that showed no difference in serum ferritin levels between groups classed as having active dental caries and inactive dental caries . iron ions will be precipitated on the enamel surface as thin acid resistant coatings containing gels and crystals of hydrous iron oxides . in addition , by adsorbing salivary calcium and phosphate ions these iron compounds seem to be able to nucleate the formation of apatites , thus mediating a replacement of minerals , which have been dissolved during the acid phases of the caries process . the severity of ecc is directly related to the early establishment of s. mutans and iron could have a protecting effect in the human oral cavity from its pathogenicity in other words iron can be significant inhibitory effect on growth of this microorganism . clearly , ecc is a complex disease that requires careful and extensive investigation . nevertheless , it is a preventable and infectious disease and its control should be a priority . since iron deficiency has permanent effects on growth and development , pediatric and general dentists should recommend assessment of iron level in ecc patients . one of the main limitations of this study was that we could not take blood sample from healthy children in the community because of ethical reasons . but we forced ourselves to select hospitalized children who did not have diseases and conditions and did not take medicines affecting the serum iron level . in conclusion , our findings showed that there was an inverse association between the serum iron level and ecc . as our study was cross - sectional , whereas the concentration of serum iron is variable with time in children , further investigation of this association over time , using a longitudinal study design may be appropriate . an inverse , statistically significant association was demonstrated between serum iron levels and caries experience in a cohort of young children aged 2471 months . to avoid the harmful sequelae associated with iron deficiency anemia , particularly amongst developing children , testing for iron deficiency anemia should be considered for children affected by ecc . | background : early childhood caries ( ecc ) is a virulent form of dental caries that can destroy the primary dentition of preschool children .
the purpose of this study was to investigate a possible association between ecc with serum iron and serum ferritin levels.materials and methods : following the ethical approval , 204 children aged 2471 months were recruited for a double - blind , randomized cross - sectional study .
each child was examined clinically for dental caries using the world health organization criteria in rafsanjan , iran .
decayed , extracted , and filled primary teeth ( deft ) index was used to measure the dental caries . to determine serum iron and serum ferritin levels 2 ml blood
was collected from each child .
data were then analyzed by pearson 's correlation coefficient and t - test using spss-16.0 software.results:the mean values and their standard deviations of the deft index and levels of serum iron and ferritin were 2.4( 3.3 ) , 93.8( 29.0 ) g / dl and 63.1( 32.2 ) ng / ml , respectively , with the two latter within .
there was no significant difference between genders .
pearson 's correlation coefficient showed that there was a statistically significant inverse association between ecc and serum iron level ( p<0.05 ) ; but no association was found with the serum ferritin level.conclusion:the deft index decreased significantly with increasing serum iron levels , but there was no association between ecc experience and serum ferritin levels . |
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fluorescence resonance energy transfer ( fret ) is becoming increasingly popular as a tool to study membrane protein structure and to monitor conformational changes in membrane proteins ( corry et al . 2006 ) . unlike nuclear magnetic resonance ( nmr ) spectroscopy and x - ray crystallography , fret can not provide high - resolution structural information on membrane proteins . on the other hand , the technique has obvious advantages in that it is applicable over a wide range of experimental conditions and has superior sensitivity . however , to obtain distance constraints from fret experiments , it is in general necessary to use fluorescent labels that are foreign to the protein . the use of probes introduces uncertainties in the interpretation of the fret data , and in the extrapolation to the native structure of the protein . clearly , to fully exploit fret as a structural technique , it is vital that we understand the factors that govern the conformation and dynamics of fluorescent labels used in structural studies . only recently have fluorescent labels been taken into account explicitly in molecular modeling studies ( corry and jayatilaka 2008 ; gustiananda et al . similar approaches have been carried out for the simulation of electron spin resonance ( esr ) spectra of spin - labeled phospholipids ( hkansson et al . 2001 ) and proteins ( desensi et al . 2008 ; steinhoff et al . 2000 ) . the fluorescent probe studies demonstrate that it is crucial to take into account the orientation , dynamics , and conformational space of the fluorescent labels for the calculation of accurate energy transfer efficiencies . for this reason , it is important to learn what factors affect the orientation and conformation of the fluorescent labels used in fret experiments . this is especially true in the case of fluorescent labeled membrane proteins , where specific interactions of the fluorescent label with the phospholipid bilayer are an additional factor complicating the interpretation of fluorescence experiments . moreover , due to the low dielectric environment inside the lipid bilayer , the effect of neighboring residues on the behavior of a fluorescent label could be more pronounced in the case of membrane proteins . several molecular dynamics simulation studies have been performed to date that attempt to understand the relationship between the conformational behavior of a fluorescent label and the conformational features of a polymer on an atomic level ( see , for instance , kosovan et al . even though a number of modeling studies have taken fluorescent labels into account explicitly ( sparr et al . 2005 ) , no systematic study on the behavior of fluorescent labels covalently attached to membrane proteins has been carried out to date . here , we perform molecular dynamics simulations of a number of aedans - labeled single cysteine mutants of a small reference membrane protein , m13 major coat protein , covering 60% of its primary sequence . m13 major coat protein is a single membrane - spanning , -helical membrane protein with a relatively large water - exposed region in the n - terminus ( vos et al . we analyze the behavior of the aedans label and the native tryptophan , which were used as acceptor and donor in our previous fret experiments ( nazarov et al . the effect of lipids on the tryptophan and aedans conformational space is quantified and discussed . the effect of neighboring side - chains in a low - dielectric membrane environment is studied by comparison with simulations of a series of aedans - labeled polyalanine peptides . our data show that the effect of both lipids and of neighboring side - chains on the aedans conformational space is limited . thus aedans is an excellent fluorescent label to probe the direct chemical environment of membrane proteins , which is quite unhindered by neighboring amino acid side - chains , lipid or water molecules . as such , our approach could help to develop a general strategy to study membrane protein structure and function in the future using aedans as a probe in fret or other fluorescence experiments . a straight -helical conformation of m13 major coat protein , as proposed by vos et al . the -helix was constructed using the computer program swiss pdbviewer ( guex and peitsch 1997 ) . the aedans label was incorporated into the molecular model in an extended configuration with all chain dihedral angles at 180 using the computer program molmol ( koradi et al . proteins were inserted into dopc bilayers using the method of blowing up the bilayer on a grid and shrinking it again as described in kandt et al . the proteins were positioned with a tilt angle of 23 relative to the bilayer normal , according to the orientation as determined in the literature with the valine residue at position 29 positioned at the center of the bilayer with its side - chain pointing downwards ( koehorst et al . simulated systems contained the labeled protein , 126 dopc molecules ( 63 per leaflet ) , and approximately 8,840 simple point charge ( spc ) water molecules ( berendsen et al . 1981 ) . as a control for interactions between the protein side - chains and the aedans label , polyalanine helices ( 25 residues ) with the labeled cysteine at various positions ( 2 , 5 , 10 , 13 , 17 , 20 , 23 ) were incorporated parallel to the z - axis in additional simulations with the same dopc membrane . the rest of the parameters for the simulations of the polyalanine simulations were identical to the parameters used in the case of the coat protein . for all mutants a 10-ns molecular dynamics simulation was performed after energy minimization and a short protein - restrained run . all simulations were performed with gromacs 3.3.1 ( berendsen et al . 1995 ; lindahl et al . 2001 ) and the optimized potential for liquid simulations ( opls ) all - atom protein force field ( tieleman et al . ( 1997 ) , combined with the spc water model ( berendsen et al . the force field parameters for the aedans label were calculated using the jaguar software ( schrdinger 2000 ) and can be obtained from the authors on request . water , lipids , and protein were coupled separately to a berendsen thermostat at 1 atm with a coupling constant of 0.1 ps ( berendsen et al . the pressure was coupled semi - isotropically to a berendsen thermostat using a coupling constant of 1 ps and a compressibility of 4.5 10 atm in the xy plane and normal to the membrane . long - range electrostatic interactions were calculated using the particle - mesh ewald ( pme ) algorithm ( darden et al . 1997 ) , allowing for a 2-fs time step . to avoid biasing due to the starting configuration of the dihedral angles the first 2 ns of simulation time were discarded . the orientation factor and energy transfer efficiency e were calculated for each frame of the remaining 8 ns of the trajectory and averaged . the rest of the analyses was performed with vmd ( humphrey et al . 1996 ) . the energy transfer efficiency is related to the distance between the donor and the acceptor and to the orientation of the two chromophores via ( dale and eisinger 1976):1\documentclass[12pt]{minimal }
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\begin{document}$$ < e_{i } > = \left\langle { { \frac{{\kappa_{i}^ { 2 } } } { { c^ { - 1 } r_{i}^{6 } + \kappa_{i}^{2 } } } } } \right\rangle . $ $ \end{document } for simplicity , neglecting effects of boltzmann weighting , i runs over all possible conformations of the donor and the acceptor . parameter e is the energy transfer efficiency , and is the orientation factor as defined in eq . 2 . parameter c describes the refractive index of the medium , the spectral overlap integral characteristic for this donor acceptor pair , and the quantum yield of the donor in the absence of acceptors . it was determined to be 2.9 10 in our previous work ( vos et al . the distance between the donor and the acceptor , r , is taken as the distance between the middle of the central bond of the indole and middle of the central bond of the dansyl chromophore . the orientation factor is related to the orientation of the donor transition dipole moment and acceptor absorption dipole moment ( gustiananda et al . 2004):2\documentclass[12pt]{minimal }
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\begin{document}$$ \kappa^{2 } = ( \cos \theta_{\text{t } } - 3\cos \theta_{\text{d } } \cos \theta_{\text{a } } ) ^{2 } . $ $ \end{document } here , is the angle between the donor transition dipole moment and the acceptor absorption dipole moment , d is the angle between the donor transition dipole moment and donor acceptor interconnecting vector , and a is the angle between the absorption dipole moment and the donor acceptor interconnecting vector . the indole chromophore has two emitting states that are dependent on solvent polarity . in apolar solvents , the chromophore emits from the lb state , whereas in polar solvents , the chromophore emits from the la state ( albinsson and norden 1992 ; sobolewski and domcke 1999 ) . since in our case , tryptophan is buried inside the phospholipid membrane , being a hydrophobic environment , we will assume that the indole chromophore emits exclusively from the lb state . chemically , the tryptophan side - chain is very similar to 5-methylindole , having a carbon atom connected to the indole ring at the same position . therefore , we expect the electronic configuration in tryptophan to be similar to that in 5-methylindole , enabling us to use the transition dipole moment of the lb state of 5-methylindole as reported in the literature ( albinsson and norden 1992 ) in our calculations of . this results in vector d shown in fig . 1 . the value and orientation of the absorption dipole moment of the aedans dansyl chromophore ( vector a in fig . 1 ) was taken from the literature , using the absorption dipole moment of 1,5-accys - aedans at 330 nm ( van der heide et al . the calculation of the angles , d , and as well as the calculation of the distance between the donor and acceptor was done using a perl script.fig . 1vectors of the tryptophan emission dipole ( d ) , the aedans absorption dipole ( a ) , and the interconnecting vector ( t ) used for the calculation of the orientation factor vectors of the tryptophan emission dipole ( d ) , the aedans absorption dipole ( a ) , and the interconnecting vector ( t ) used for the calculation of the orientation factor a straight -helical conformation of m13 major coat protein , as proposed by vos et al . the -helix was constructed using the computer program swiss pdbviewer ( guex and peitsch 1997 ) . the aedans label was incorporated into the molecular model in an extended configuration with all chain dihedral angles at 180 using the computer program molmol ( koradi et al . proteins were inserted into dopc bilayers using the method of blowing up the bilayer on a grid and shrinking it again as described in kandt et al . the proteins were positioned with a tilt angle of 23 relative to the bilayer normal , according to the orientation as determined in the literature with the valine residue at position 29 positioned at the center of the bilayer with its side - chain pointing downwards ( koehorst et al . simulated systems contained the labeled protein , 126 dopc molecules ( 63 per leaflet ) , and approximately 8,840 simple point charge ( spc ) water molecules ( berendsen et al . 1981 ) . as a control for interactions between the protein side - chains and the aedans label , polyalanine helices ( 25 residues ) with the labeled cysteine at various positions ( 2 , 5 , 10 , 13 , 17 , 20 , 23 ) were incorporated parallel to the z - axis in additional simulations with the same dopc membrane . the rest of the parameters for the simulations of the polyalanine simulations were identical to the parameters used in the case of the coat protein . for all mutants a 10-ns molecular dynamics simulation was performed after energy minimization and a short protein - restrained run . all simulations were performed with gromacs 3.3.1 ( berendsen et al . 1995 ; lindahl et al . 2001 ) and the optimized potential for liquid simulations ( opls ) all - atom protein force field ( tieleman et al . ( 1997 ) , combined with the spc water model ( berendsen et al . the force field parameters for the aedans label were calculated using the jaguar software ( schrdinger 2000 ) and can be obtained from the authors on request . water , lipids , and protein were coupled separately to a berendsen thermostat at 1 atm with a coupling constant of 0.1 ps ( berendsen et al . the pressure was coupled semi - isotropically to a berendsen thermostat using a coupling constant of 1 ps and a compressibility of 4.5 10 atm in the xy plane and normal to the membrane . long - range electrostatic interactions were calculated using the particle - mesh ewald ( pme ) algorithm ( darden et al . 1997 ) , allowing for a 2-fs time step . to avoid biasing due to the starting configuration of the dihedral angles the first 2 ns of simulation time were discarded . the orientation factor and energy transfer efficiency e were calculated for each frame of the remaining 8 ns of the trajectory and averaged . the rest of the analyses was performed with vmd ( humphrey et al . 1996 ) . the energy transfer efficiency is related to the distance between the donor and the acceptor and to the orientation of the two chromophores via ( dale and eisinger 1976):1\documentclass[12pt]{minimal }
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\begin{document}$$ < e_{i } > = \left\langle { { \frac{{\kappa_{i}^ { 2 } } } { { c^ { - 1 } r_{i}^{6 } + \kappa_{i}^{2 } } } } } \right\rangle . $ $ \end{document } for simplicity , neglecting effects of boltzmann weighting , i runs over all possible conformations of the donor and the acceptor . parameter e is the energy transfer efficiency , and is the orientation factor as defined in eq . 2 . parameter c describes the refractive index of the medium , the spectral overlap integral characteristic for this donor acceptor pair , and the quantum yield of the donor in the absence of acceptors . it was determined to be 2.9 10 in our previous work ( vos et al . the distance between the donor and the acceptor , r , is taken as the distance between the middle of the central bond of the indole and middle of the central bond of the dansyl chromophore . the orientation factor is related to the orientation of the donor transition dipole moment and acceptor absorption dipole moment ( gustiananda et al . 2004):2\documentclass[12pt]{minimal }
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\begin{document}$$ \kappa^{2 } = ( \cos \theta_{\text{t } } - 3\cos \theta_{\text{d } } \cos \theta_{\text{a } } ) ^{2 } . $ $ \end{document } here , is the angle between the donor transition dipole moment and the acceptor absorption dipole moment , d is the angle between the donor transition dipole moment and donor acceptor interconnecting vector , and a is the angle between the absorption dipole moment and the donor acceptor interconnecting vector . the indole chromophore has two emitting states that are dependent on solvent polarity . in apolar solvents , the chromophore emits from the lb state , whereas in polar solvents , the chromophore emits from the la state ( albinsson and norden 1992 ; sobolewski and domcke 1999 ) . since in our case , tryptophan is buried inside the phospholipid membrane , being a hydrophobic environment , we will assume that the indole chromophore emits exclusively from the lb state . chemically , the tryptophan side - chain is very similar to 5-methylindole , having a carbon atom connected to the indole ring at the same position . therefore , we expect the electronic configuration in tryptophan to be similar to that in 5-methylindole , enabling us to use the transition dipole moment of the lb state of 5-methylindole as reported in the literature ( albinsson and norden 1992 ) in our calculations of . this results in vector d shown in fig . 1 . the value and orientation of the absorption dipole moment of the aedans dansyl chromophore ( vector a in fig . 1 ) was taken from the literature , using the absorption dipole moment of 1,5-accys - aedans at 330 nm ( van der heide et al . 1992 ) . the calculation of the angles , d , and as well as the calculation of the distance between the donor and acceptor was done using a perl script.fig . 1vectors of the tryptophan emission dipole ( d ) , the aedans absorption dipole ( a ) , and the interconnecting vector ( t ) used for the calculation of the orientation factor vectors of the tryptophan emission dipole ( d ) , the aedans absorption dipole ( a ) , and the interconnecting vector ( t ) used for the calculation of the orientation factor depending on the initial configuration of a molecular dynamics simulation the phospholipid bilayer can take considerable time to equilibrate . for this reason we took a pre - equilibrated phospholipid bilayer as a starting configuration in all our simulations , and we used the inflategro script ( kandt et al . 2007 ) to insert the proteins , thereby minimizing disturbances of the lipids . to evaluate equilibration of the phospholipids , the area per lipid was analyzed over an extended simulation time of 30 ns ( data not shown ) for the aedans - labeled a18c and a27c mutant . during the first 2 ns of the simulation , the area per lipid decreased from ~0.75 to ~0.65 nm for both mutants , in good agreement with values reported in the literature ( tieleman et al . this indicates that after ~2 ns the bilayer is sufficiently equilibrated for our purpose of evaluating the behavior of the aedans probe and the tryptophan side - chain . therefore , the rest of our analyses were performed over the timeframe 210 ns . both the tryptophan side - chain and the aedans label adopt different conformations depending on the position of the aedans - labeled cysteine residue for membrane - embedded m13 coat protein mutants . this is illustrated in fig . 2 , which shows a surface plot of the tryptophan side - chain ( blue ) and the aedans label ( red ) over the course of the entire simulation for three representative aedans label positions : a mutant in the n - terminus ( a16c ) , in the transmembrane segment ( v29c ) , and in the c - terminus ( s46c ) . in the case of the mutants a16c and s46c , the tryptophan surface plot appears more disc shaped , whereas the tryptophan surface plot for the v29c mutant is more spherical , indicative of different rotameric states during the course of the simulation . the aedans conformational space has a confined , disc - like shape in the case of the v29c mutant . also for the a16c mutant , the aedans conformational space is relatively confined , whereas the conformational space of the s46c mutant has a bilobal shape , indicative of two major rotameric states.fig . 2space - filling representation of the conformational space of the tryptophan side - chain ( blue ) and of the aedans - labeled cysteine residue ( red ) over the course of 8-ns simulation time for membrane - embedded aedans - labeled a16c , v29c , and s46c m13 coat protein mutants , respectively . the protein is represented as a grey ribbon space - filling representation of the conformational space of the tryptophan side - chain ( blue ) and of the aedans - labeled cysteine residue ( red ) over the course of 8-ns simulation time for membrane - embedded aedans - labeled a16c , v29c , and s46c m13 coat protein mutants , respectively . the protein is represented as a grey ribbon during the simulations , membrane - embedded m13 coat protein remains close to an -helix , enabling the use of the long axis of the helix as a reference to describe the orientation of the tryptophan and aedans side - chains . thus , to quantify the conformational space occupied by the tryptophan side - chain , the angle of the tryptophan emission dipole moment with the long axis of the helix , w , is depicted in fig . 3 for membrane - embedded wild - type m13 coat protein and the mutants a16c , v29c , and s46c . in the case of the wild - type protein , the angular distribution of the tryptophan dipole moment with helical axis is symmetrical , centered on a value of 70. the angular distributions of the mutants a16c and s46c are also symmetrical , centered on 65 and 70 , respectively . however , in the case of the v29c mutant , the angular distribution resembles two peaks , centered on values of 70 and 95 , respectively.fig . 3distribution of the angle between the tryptophan emission dipole moment and the helical axis for membrane - embedded wild - type m13 coat protein ( wt ) , and the mutants a16c , v29c , and s46c distribution of the angle between the tryptophan emission dipole moment and the helical axis for membrane - embedded wild - type m13 coat protein ( wt ) , and the mutants a16c , v29c , and s46c an overview of values of the center of the distributions describing the tryptophan conformational space for all mutants is given in fig . one peak is observed centered at a value of w around 60. some mutants show a second peak at a higher value of w , mostly centered on 85. an exception is observed for mutants a7c and a9c , where the value of w of the second peak is 115 and 120 , respectively . only in the case of mutant a18c are three peaks observed , giving a third value for w of 118. all values of w fall within the range from 50 to 120 , indicating that the tryptophan emission dipole has a tendency to adopt a perpendicular orientation with respect to the helical axis . throughout the aedans label position , furthermore , no large deviations from the value of the wild - type protein ( w = 70 ) are observed . these observations indicate that the effect of the aedans label on the tryptophan conformational space is limited.fig . 4center of the distribution of angles between the tryptophan dipole and helical axis of the m13 coat protein , w , max , for all different mutants as a function of aedans label position . the filled circle represents the case of three peaks ( mutant a18c ) . for well - resolved peaks , error margins were estimated based on the width of the peak at half height center of the distribution of angles between the tryptophan dipole and helical axis of the m13 coat protein , w , max , for all different mutants as a function of aedans label position . the filled circle represents the case of three peaks ( mutant a18c ) . for well - resolved peaks , error margins were estimated based on the width of the peak at half height in fig . 5 , the angle between the aedans absorption dipole and long axis of the protein helix , a , is depicted for membrane - embedded m13 coat protein mutants a16c , v29c , and s46c . in the case of mutant a16c , the angular distribution of the aedans absorption dipole with respect to the helical axis is centered on a value of 80. for mutant v29c , two peaks are observed in the angular distribution : one large peak centered on 50 and a smaller peak centered on 110. the angular distribution of mutant s46c shows two peaks , centered at 20 and 65.fig . 5distribution of the angle between the aedans excitation dipole moment and the helical axis for membrane - embedded m13 coat protein mutants a16c , v29c , and s46c distribution of the angle between the aedans excitation dipole moment and the helical axis for membrane - embedded m13 coat protein mutants a16c , v29c , and s46c in fig . 6 , the values of the center of the distributions describing the aedans conformational space are depicted . the values of a fall within a range from 25 to 125 , indicating a wider distribution of conformational space of the aedans label as compared with the tryptophan side - chain , probably due to the longer aedans linker . since the protein makes a tilt of 23 with respect to the membrane normal ( koehorst et al . 2004 ) , the smallest values of a imply that for certain mutants the aedans label is almost aligned with the lipid tails . similar as in the case of tryptophan ( fig . 4 ) , no clear trend can be observed in the values of a , although it is obvious that the aedans conformational space varies strongly among different positions of the cysteine point mutation.fig . 6center of the distribution of the angles between the aedans absorption dipole and the helical axis of the m13 coat protein , a , max , for all different mutants as a function of aedans label position . the filled circle represents the case of three peaks ( mutant 18 ) . for well - resolved peaks , error margins were estimated based on the width of the peak at half height center of the distribution of the angles between the aedans absorption dipole and the helical axis of the m13 coat protein , a , max , for all different mutants as a function of aedans label position . the filled circle represents the case of three peaks ( mutant 18 ) . for well - resolved peaks , error margins were estimated based on the width of the peak at half height for the membrane - embedded m13 coat protein , the behavior of the aedans label is expected to be determined by three main factors : ( 1 ) the local rotations of the aedans side - chain attached to the protein backbone , ( 2 ) the restrictions of the rotamers by the backbone and side - chains of the neighboring amino acid residues , and ( 3 ) the restrictions imposed by the surrounding lipids . to investigate these possibilities , an additional series of molecular dynamics simulations was carried out , using a 25-residue membrane - spanning -helical polyalanine with a single aedans - labeled cysteine point mutation at different positions in the helix . even though polyalanines do not form a transmembrane helix in vitro ( lewis et al . 2001 ) , polyalanines are frequently used in molecular dynamics simulations as model transmembrane -helices ( choma et al . since the alanine side - chain is relatively small , specific interactions between the polyalanine helix and the aedans label are expected to be minimal , making it an ideal model transmembrane helix for our purpose . therefore , in case of the polyalanine helix the conformational space of the aedans label is expected to be determined mainly by restrictions imposed by the surrounding lipids . the aedans conformational space varies strongly among different positions of the cysteine point mutation , as shown by the values of a in fig . all values of a fall within a range from 20 to 100 , indicating a slightly narrower distribution of conformational space of the aedans labels in polyalanine as compared with aedans labels attached to m13 coat protein ( fig . this might be related to the more homogeneous structure of the polyalanine - membrane system.fig . 7center of the distribution of the angles between the aedans absorption dipole and the helical axis , a , max , for all different mutants in the case of a membrane - embedded polyalanine helix . the open triangles represent the angles in case a second peak is observed . for well - resolved peaks , error margins were estimated based on the width of the peak at half height center of the distribution of the angles between the aedans absorption dipole and the helical axis , a , max , for all different mutants in the case of a membrane - embedded polyalanine helix . the open triangles represent the angles in case a second peak is observed . for well - resolved peaks , error margins were estimated based on the width of the peak at half height the goal of this work is to advance fluorescent techniques , in particular fret , as tools to study membrane protein structure through studying the effect of the lipids and neighboring side - chains on the conformational space of a fluorescent labels attached to a model membrane protein . to evaluate the implications of our findings with respect to the energy transfer efficiencies measured in our previous work ( vos et al . 8 . when the donor acceptor pair in the experimental ensemble has the same fixed orientation or the same extent of dynamic averaging it is valid to substitute a single or average value for in eq . 2 ( dale and eisinger 1979 ) . qualitatively , the energy transfer efficiencies calculated here are in good agreement with the experimental values for mutant positions 139 , which could suggest that sampling might be sufficient , even though it is not complete for all individual mutants . however , the quality of the fit is not improved much compared with the values calculated in our previous work based on the < > = 0.67 approximation . for mutant positions 4050 , 8 is therefore that the energy transfer efficiencies in the c - terminus of the protein ( mutants 4050 ) show a systematic deviation towards lower values . interestingly , recent fret studies by our group on the same protein reconstituted in bilayers of hydrophobic mismatch indicate that residues 3850 have a high propensity for helical deformation ( vos et al . it is therefore likely that the discrepancy between the theoretical efficiencies calculated in the work presented here and the previously measured experimental efficiencies are due to the -helical starting structure that was used in silico , whereas the helix would be more distorted in vitro . however , since the protein backbone is effectively rigid during the time span of the simulations , this will most likely not have an effect on the aedans or the tryptophan conformational space , which is the focus of the current work.fig . 8experimental ( filled diamonds ) and theoretical ( open triangles ) energy transfer efficiencies for the aedans - labeled mutants used in the molecular dynamics simulations experimental ( filled diamonds ) and theoretical ( open triangles ) energy transfer efficiencies for the aedans - labeled mutants used in the molecular dynamics simulations the surface plots in fig . 2 of the tryptophan side - chain and of the aedans label of mutant v29c suggest a direct interaction between the tryptophan side - chain and the aedans label . the tryptophan and the aedans chromophores appear to be oriented in parallel planes , suggesting that the two chromophores are strongly interacting . the tryptophan conformation is best described as a disc - like conformation , which is also visible in the case of the a16c and s46c mutants . in this disc - like conformation , which is observed in the simulation of all mutants , the tryptophan emission dipole makes an angle of ~70 with the helical axis and the tryptophan emission and aedans absorption dipoles are more likely to align . 4 shows that this conformation of w = 60 is present for most mutants . for a large number of mutants , a second conformation is observed , represented by a peak centered on a value w = 90. a third conformation , with w = 115 , is visible in the case of the a7c and a9c mutants . the first conformation , with a value w = 70 , could be the conformation with the lowest energy . the other two conformations are not always probed , although the second conformation , with a value w = 90 , shows up in ~50% of all mutants . the third conformation is the rarest , only showing up in 14% of all mutants . only in the case of the a18c mutant the fact that there is no apparent correlation between the position of the aedans label and the conformational space of the tryptophan suggests that the differences between the different mutants and simulations are caused by incomplete probing of the tryptophan conformational space rather than by specific interactions affecting the energy of the different tryptophan rotameric states . even though 10 ns of simulation time represents a considerable amount of computational time , such a simulation might not be sufficient for exhaustive probing of the tryptophan conformational space . to address this issue , the angle of tryptophan dipole with the local helix axis over the simulation time was evaluated for a selection of mutants : 7 , 15 , 23 , 31 , 36 , 41 , and 46 ( data not shown ) . the main implications of this analysis are summarized in table 1 . except for the mutant s46c , tryptophan shows many transitions between the different conformations , indicating that sampling is significant . the tryptophan conformational space is thus well described by three different rotameric states , even though not all conformational states appear in each simulation.table 1sampling analysis of the angles of the tryptophan and aedans dipoles with the local helix axis over the simulation time ( 10 ns ) for the aedans - labeled coat protein . the analysis was carried out for a selection of mutantsmutant positiontryptophanaedans720160 , many transitions0180 , many transitions1520160 , many transitions0180 , few transitions230180 , many transitions20150 , many transitions3110180 , many transitions50120 , many transitions360180 , many transitions0160 , many transitions4120120 , many transitions50120 , many transitions46few transitionsfew transitionsabout 100 transitions per nsabout 1 transition per ns sampling analysis of the angles of the tryptophan and aedans dipoles with the local helix axis over the simulation time ( 10 ns ) for the aedans - labeled coat protein . the analysis was carried out for a selection of mutants about 100 transitions per ns about 1 transition per ns compared with the tryptophan emission dipole moment , the values of the angular distribution of the aedans absorption dipole moment is wider . the maxima of the a values can not be categorized into groups with almost discrete values , possibly because the conformational space of the aedans group is much larger than that of the tryptophan side - chain due to the long linker between the aedans chromophore and the protein backbone . for comparison , the aedans group is linked to the protein backbone by seven bonds about which the chromophore can rotate freely , compared with two bonds with free rotation in the case of tryptophan , making the aedans conformational space significantly larger . however , even though the values for the maxima of the aedans angular values do not fall into neatly defined categories , it is worthwhile to analyze the angular distribution of the aedans dipole with the helical axis further . this is done by categorizing the values of the maxima depicted in fig . 5 into different groups : groups a , b , c , d , e , and f , corresponding to values of a of 1030 , 3050 , 5070 , 7090 , 90110 , and 110130 , respectively . groups a , e , and f , representing the most extreme values of a , are least densely populated . possibly , this reflects a purely statistical effect , indicating that aedans behaves like a relatively unperturbed vector , sweeping a conical surface . throughout all the molecular dynamics simulations , a ranges from 30 to 50 ( group b ) . even though the values of a throughout the different simulations do not form a peak themselves , the results presented here do suggest that values 30 a 90 ( groups b , c , and d ) have a higher probability , regardless of the mutant position , and that extreme a values have a lower probability . to evaluate whether sampling of the aedans conformational space is significant , the angle of the aedans dipole with the local helix axis over the simulation time was evaluated for a selection of mutants : 7 , 15 , 23 , 31 , 36 , 41 , and 46 ( data not shown ) . mutants 15 and 46 show only a few transitions , indicating that sampling is limited , while for the other mutants the angles sample a significant range with many transitions , indicating that sampling is significant in most cases.fig . 9probability of finding the aedans labels in various conformations averaged over all mutants for aedans - labeled polyalanine ( black bars ) and for aedans - labeled coat protein ( white bars ) . groups a , b , c , d , e , and f , correspond to values of a , max of 1030 , 3050 , 5070 , 7090 , 90110 , and 110130 , respectively probability of finding the aedans labels in various conformations averaged over all mutants for aedans - labeled polyalanine ( black bars ) and for aedans - labeled coat protein ( white bars ) . groups a , b , c , d , e , and f , correspond to values of a , max of 1030 , 3050 , 5070 , 7090 , 90110 , and 110130 , respectively to elucidate to what extent specific interactions with the side - chains or with the neighboring lipid and water molecules affect the aedans conformational space , an additional set of simulations of aedans - labeled polyalanine was analyzed in the same way . the angular distribution diagrams for both the aedans - labeled coat protein and the aedans - labeled polyalanine show a considerable spread , depending on the position of the mutation . since specific interactions between the aedans label and the amino acid side - chains are not very important in the case of the polyalanine helix , the spread in the maxima of the angular distribution functions which is observed in the simulations of aedans - labeled polyalanine is not caused by specific , position - dependent interactions with neighboring side - chains . this raises the question of what determines the spread in a values observed in fig . 7 , i.e. , whether specific interactions between the label and lipid and water molecules are responsible or whether the different simulations probe different parts of the aedans conformational space , which is , in fact , very similar for the various mutant positions . if the interactions between the label and the lipids change the aedans conformational space , a certain degree of symmetry would be expected in fig . 7 . for instance , the distance between the mutant positions 2 and 5 and the lipid head group region is roughly the same as the distance between mutant positions 23 and 20 and the opposite lipid head group region . this implies that the aedans groups are exposed to approximately the same lipid environment for mutants 2 and 5 as for mutants 23 and 20 , respectively . however , the a values for positions 2 and 23 are different , as are the a values for positions 5 and 20 . also for the other mutant positions , where the aedans label is buried deeper into the bilayer , no symmetry can be inferred from the a values . therefore , it is not likely that the interaction with the lipid and water molecules is the decisive factor that determines the spread in a values as observed in the polyalanine simulations . more likely , the spread in a values observed here is caused by the fact that different simulations probe different parts of the aedans conformational space , as is the case even for the much smaller tryptophan side - chain . in fact , in the case of aedans , the spread in a values is expected to be even bigger than the spread in w values in the case of the tryptophan side - chain , due to the larger mass of the aedans label and the increased magnitude of the aedans conformational space . the comparison between the a values in the case of aedans - labeled coat protein and aedans - labeled polyalanine indicates that specific interactions between the aedans label and neighboring side - chains do not have a large effect on the aedans conformational space . secondly , the fact that the distribution in a values in the case of the aedans - labeled polyalanine is essentially random suggests that the position spread in the aedans conformational space is not strongly affected by interactions with neighboring lipid and water molecules . more likely , the spread in a values between the different mutant positions is caused by incomplete probing of the conformational space in each individual simulation . to test the latter idea , it is worthwhile to make a comparison between the distribution of the a values in the case of the aedans - labeled coat protein and the aedans - labeled polyalanine , averaged over all mutants . if position - dependent specific interactions between the label and neighboring side - chains , and between the label and surrounding lipid and water molecules , do not have a large effect on the aedans conformational space , the conformational space as observed in the simulations of the polyalanine simulations would be similar to the conformational space as observed in the simulations of the coat protein simulations across all different mutants . the a values found across the different simulations of m13 coat protein and polyalanine are depicted in fig . clearly there are differences between the behavior of aedans when attached to a polyalanine helix and to m13 major coat protein . for instance , when attached to a polyalanine single helix the label is not found in group b , whereas the label is frequently present in group b when it is attached to coat protein . possibly , this difference is because the number of mutants for the polyalanine is only limited as compared with those for coat protein , which means that sampling is less exhaustive for the label attached to the polyalanine helix then for the label attached to coat protein . to illustrate this , we compare combined probabilities for groups a and b , c and d , and e and f , respectively , for the aedans - labeled polyalanine and aedans - labeled coat protein . the combined probabilities for a / b , c / d , and e / f are 0.3 , 0.6 , and 0.1 in the case of polyalanine . for coat protein , hence , for both the aedans - labeled polyalanine helix and the coat and for the aedans - labeled coat protein , the label is most likely to be found in groups c / d , although in the case of aedans - labeled polyalanine the label is equally likely to be found in groups a / b . for both aedans - labeled polyalanine and coat protein , this result suggests that the average conformational space for aedans is similar in the case of the polyalanine simulations as in the case of the m13 simulations , indicating that specific interactions between the label and neighboring side - chains do not affect the aedans conformational space significantly . in summary , our results indicate that neither specific interactions between the aedans label and the lipid or water molecules , nor interactions between the aedans label and neighboring side - chains , have a large effect on the aedans conformational space . interestingly , this finding advocates the use of aedans as a relatively inert environmental probe that can move relatively unhindered through the lipid membrane . an important factor when using fret as a spectroscopic tool is the mutual orientation of the donor and acceptor during energy transfer . for this reason , we evaluate the average value of the orientation factor for all different aedans - labeled major coat protein mutants over the course of the simulation , < > , depicted in fig . we note that this value represents a static average , and therefore the extrapolation to energy transfer efficiencies as measured in a fluorescence experiment is not straightforward . however , for our purpose of evaluating potential systematic trends in the mutual orientation of donor and acceptor dipoles , evaluation of < > suffices . for most mutants , the value of < > is lower than 0.67 ( the isotropic dynamic average that is frequently used to calculate distances from energy transfer experiments ) . in certain cases , < > is almost zero , indicating a perpendicular orientation between the tryptophan emission dipole and the aedans absorption dipole . notably , for mutant positions 2332 , < > is strongly increased to as high as 2.5 , approaching the theoretical maximum of 4 , indicating that the dipoles are almost aligned . for this reason it is worthwhile to evaluate the average value of the orientation factor for all different aedans - labeled major coat protein mutants over the course of the simulation , < > , as depicted in fig . the value < > is lower than 2/3 , which is the isotropic dynamic average that is frequently used to calculate distances from energy transfer experiments . in certain cases , < > is almost zero , indicating a perpendicular orientation between the tryptophan emission dipole and the aedans absorption dipole . notably , for mutant positions 2332 , the energy transfer for most mutants is strongly increased to as high as 2.5 , approaching the theoretical maximum of 4 , indicating that the tryptophan emission dipole and the aedans absorption dipole are almost aligned , at least through part of the simulation.fig . 10implications for the average orientation factor < > implications for the average orientation factor < > even though the effect of neighboring side - chains on the aedans and tryptophan conformational space is limited , the data presented in fig . 9 suggest that there is some interaction between the aedans and the tryptophan group , resulting in an increased < > value for mutants in the transmembrane region . this could suggest a small degree of stacking between the aedans and tryptophan chromophores , as illustrated in fig . 2 , increasing the probability of dipole alignment . 6 gives no indications of a systematic effect on the overall conformational space , suggesting that the increase in < > could be due to an indirect effect , with both tryptophan and aedans aligning along the lipid acyl chains . our computer simulations suggest that the conformational space of the aedans label is only slightly affected by the presence of amino acid side - chains or lipids . this finding advocates the use of aedans as a relatively inert environmental probe that is structurally unhindered by the membrane lipids . as such , aedans fluorescence spectroscopy and fret could contribute to the development of a general strategy to study membrane protein structure and function in the future . | computer simulations were carried out of a number of aedans - labeled single cysteine mutants of a small reference membrane protein , m13 major coat protein , covering 60% of its primary sequence .
m13 major coat protein is a single membrane - spanning , -helical membrane protein with a relatively large water - exposed region in the n - terminus . in 10-ns molecular dynamics simulations
, we analyze the behavior of the aedans label and the native tryptophan , which were used as acceptor and donor in previous fret experiments .
the results indicate that aedans is a relatively inert environmental probe that can move unhindered through the lipid membrane when attached to a membrane protein . |
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they were first described by jadassohn in 1895 and are most commonly seen on the head and neck whilst similar lesions elsewhere on the body are termed verrucous epidermal nevi . usually recognized at birth or in early childhood , sebaceous nevi remain small and hairless until puberty when they may become larger and verrucous . in late adult life we report a patient with nevus sebaceous with one benign and one malignant tumor simultaneously occurring in a single lesion- syringocystadenoma papilliferum and basal cell carcinoma on face . a 27-year - old male came to the department of dermatology with a lesion on the left cheek that had been present since birth . in the preceding 2 months he had a linear lesion on the left cheek that measured 5 cm in length with a maximum width of 3 cm . this was a well - defined brownish , plaque with a rough surface [ figure 1 ] . past medical history and general physical examination were unremarkable . under local infiltration of xylocain 2% with adrenalin the lesion was excised as an ellipse with a 2 mm clearance margin and the wound closed primarily . on histology epidermis revealed hyperkeratosis , hyperplasia , mild koilocytosis , papillomatosis and focal invagination in dermis lined by squamous cells . the invaginating epidermis into the dermis forms a cyst with numerous papillary projections [ figure 2 ] . the cross sections of these papillae were lined by two layers a luminal columnar epithelium with evidence of decapitation secretions and outer flattened cuboidal epithelium . careful examination showed a cluster of basaloid cells with peripheral pallisading of lesional cell nuclei [ figure 3 ] . brownish pigment melanin was also seen in few of these basaloid cells . on the basis of these pathological findings a diagnosis of nevus sebaceous with foci of basal cell carcinoma and syringocystadenoma papilliferum was made . ( a ) lesion of nevus sebaceous of jadassohn on the left side of the face . ( b ) scar of the lesion after 10 days of complete excision ( a ) h and e stain scanner view 4 showing nevus sebaceous and syringocystadenoma papilliferum . ( b ) h and e stain scanner 4 view showing basal cell carcinoma and syringocystadenoma papilliferum . ( c ) h and e stain , low power 10 view showing dermis having mature sebaceous glands and a hair follicle ( a ) h and e stain high power 40 view showing cluster of basaloid cells with peripheral pallisading of lesional cell nuclei and clefting artifact between the epithelium and stroma . ( b ) h and e stain , low power 10 view showing dermal cysts with numerous papillary projections lined by two layers of a luminal columnar epithelium with evidence of decapitation secretion and outer flattened cuboidal epithelium sebaceous nevi are uncommon hamartomatous lesions seen in 0.3% of neonates and 0.68% of skin biopsy specimens . up to 95% mehregan and pinkus described the natural history of the lesion in three stages . in the infantile stage histologically , there is a paucity of underdeveloped sebaceous glands and hair follicles . at the pubertal stage , growth of the lesion is accelerated and it becomes verrucous . light microscopy shows masses of hypertrophic sebaceous glands with papillomatosis and hyperkeratosis of the overlying epidermis . the third or neoplastic stage sees the development of secondary tumors and usually occurs in late adult life . the clinical signs suggesting neoplastic transformation include rapid enlargement or the development of nodularity or ulceration . benign tumors are the most common including syringocystadenoma papilliferum , apocrine cystadenoma , trichoblastoma , trichilemmal cysts and keratoacanthoma . occasionally , multiple tumors are observed within a pre - existing lesion . malignant degeneration , although less common , occurs with a lifetime risk of between 5% and 20% . the majority of these tumors are basal cell carcinomas but squamous cell , sebaceous and apocrine carcinomas are also recognized . most are of low - grade malignancy but more aggressive histological features and metastases are occasionally seen . it has been suggested that these tumors may arise from pluripotential epithelial germ cells as a result of dedifferentiation of sebaceous nevus cells , the nature of the tumor depending upon the degree of subsequent differentiation . the histological changes are characterized by the papillomatous hyperplasia of the epidermis to different degrees . there are many irregular ducts like structures and cystic spaces lined with a double - layered epithelium . basal cell carcinoma is the most common malignant neoplasm associated with nevus sebaceous , which derives from original epithelial germ cells . it is composed of stroma - dependent multipotent basaloid cells and differentiates toward the epidermis or adnexa . it has the same origin as the nevus sebaceous and has the characteristic of multidifferentiation of the skin stem cells , although many researchers have shown that those lesions were mostly trichoblastomas misinterpreted as basal cell carcinoma . there is little evidence in the literature upon which to base clinical guidelines on the management of sebaceous nevus , regarding either the age at which intervention should be planned or the best modality of treatment . the two main options are excision with primary reconstruction and histological examination around the time of puberty and the more conservative approach of excision if clinical signs of malignant transformation occur . an alternative is dermabrasion or dermablation with diathermy or laser but this does not provide tissue for histological examination and occult basal cell carcinoma has been found in lesions with no evidence of transformation either clinically or on incisional biopsy . mehregan and pinkus go further and hypothesize that superficial destruction of the lesion may provoke cellular transformation . in our case the lesion was excised as an ellipse with a 2 mm clear margin and primary closer was done . sebaceous nevi can change into tumor in a lifetime but appearance of two different type of tumor like one benign and one malignant tumor in a single nevus is rarest of one . surgical excision of nevus with a safe margin should be done in all cases of sebaceous nevi . | nevus sebaceus of jadassohn is a congenital cutaneous hamartoma comprising of multiple skin structures .
it has the potential to develop into variety of neoplasms of various epidermal adnexal origins . while multiple tumors may occasionally arise , it is unusual to develop two different types of tumor , benign and malignant , to arise simultaneously within a single sebaceus nevus . here in , we report a case of a 27-year - old male with two neoplastic proliferations including a syringocystadenoma papilliferum a benign tumor and basal cell carcinoma a malignant tumor arising in a long - standing nevus sebaceus on the face .
neoplastic changes are common in nevus sebaceous present on scalp but our case is unique due to the presence of two different types of neoplasm in a nevus sebaceous which was present on face . |
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calcifying odontogenic cyst ( coc ) was first described as a distinct clinicopathologic entity by gorlin et al . , in 1962 . the lesion shows extreme diversity in its clinical and histopathological features as well as in its biological behavior . initially , coc was considered as a non - neoplastic cystic lesion , but solid lesions with neoplastic nature were also seen . cocs are frequently associated with odontogenic tumors , a finding which is a rare event in other types of odontogenic cysts or tumors . the most common of these is odontoma , but rarely , ameloblastoma , adenomatoid odontogenic tumor ( aot ) , odontoameloblastoma , ameloblastic fibroma , ameloblastic fibro - odontoma and odontogenic myxofibroma have been identified . in this article , we report the first case of a hybrid odontogenic tumor composed of calcifying cystic odontogenic tumor ( ccot ) and plexiform ameloblastoma of the anterior mandible that occurred in a 17-year - old indian male is described . a 17-year - old male patient reported with a presenting complaint of swelling in the lower part of face on the side since 1 month . the increase in the size of swelling was associated with tooth mobility in the mandibular right quadrant . the swelling was not associated with any episode of pain or paresthesia and could not be linked to any history of previous trauma or dental infection . patient visited a private practitioner to seek treatment , but instead was referred to out - patient department . on extraoral examination , a diffuse swelling was evident in the chin region on the right side extending to the mandibular body to some extent [ figure 1 ] . lymph node examination revealed palpable single right submandibular lymph node which was firm and non - tender . extraoral view of the patient showing diffuse swelling at the chin region intraoral examination [ figure 2 ] revealed diffuse swelling with 41 , 42 , 43 region involving both labial and lingual aspect of size around 1.5 cm 1 cm each , which was soft to firm and non - tender on palpation . radiographic diagnosis [ figures 3 and 4 ] revealed a large ill - defined , non - corticated interradicular radiolucent lesion with 43 and 42 region causing pathologic drifting and external root resorption of 42 , 41 , 31 . an important finding was evidence of a small focus of calcification near the periapical region of 41 . intraoral view ; ( a ) labial vestibule showing diffuse swelling ; ( b ) lingual extension of the swelling intraoral radiographic investigation ; ( a ) anterior islands organic producers association and ( b ) anterior mandibular occlusal topography ; showing a bony defect between 42 and 43 with displacement of teeth , root resorption of 42 , 43 and a focus of calcification opg view showing the extension of an ill - defined radiolucent bony lesion with root resorption of the involved teeth under the clinic - radiographic diagnosis of calcifying epithelial odontogenic cyst , patient was advised for aspirational biopsy , which yielded clear fluid . the histopathological examination of the specimen showed a cystic cavity lined with tall , columnar odontogenic cells along with the presence of ghost cell keratinization and basophilic material with cystic cavity lined by tall columnar odontogenic cells , which is suggestive of ccot associated with plexiform ameloblastoma [ figure 5 ] . the patient has been followed - up for 6 month until date and shows no signs of recurrence . ( a ) under scanner view ( 4 ) ; odontogenic epithelium and connective tissue can be seen , ( b ) under low power view ( 10 ) ; cystic cavity lined by tall columnar odontogenic cells are seen cocs are rare odontogenic cystic tumors constituting 0.37 - 2.1% of all odontogenic tumors . due to their low prevalence , reports since the first description by gorlin et al . , have by necessity involved only a small number of cases . the who classified coc as ccot under benign neoplasm related to odontogenic apparatus and defined it as a cystic lesion in which the epithelial lining shows a well - defined basal layer of columnar cells , an overlying layer that is often many cells thick and that may resemble stellate reticulum and masses of ghost epithelial cells that may be in the epithelial cyst lining or in the fibrous capsule . the latest classification by praetorius et al . , 2006 categorizes the lesion into four categories and considers each one of them to be distinct in its architectural presentation and biological behavior , as follows
grade 1
simple cystcoc . the following combinations have been published
ccot associated with an odontomeccot associated with aotccot associated with ameloblastomaccot associated with ameloblastic fibromaccot associated with ameloblastic fibro - odontomaccot associated with odontoameloblastomaccot associated with odontogenic myxofibroma.group 3
solid benign odontogenic neoplasms with similar cell morphology to that in the coc and with dentinoid formationdentinogenic ghost cell tumor . group 4
malignant odontogenic neoplasms with features similar to those of the dentinogenic ghost cell tumorghost cell odontogenic carcinoma . the following combinations have been published
cysts associated with odontogenic hamartomas or benign neoplasms : ccot . the following combinations have been published ccot associated with an odontome ccot associated with aot ccot associated with ameloblastoma ccot associated with ameloblastic fibroma ccot associated with ameloblastic fibro - odontoma ccot associated with odontoameloblastoma ccot associated with odontogenic myxofibroma . solid benign odontogenic neoplasms with similar cell morphology to that in the coc and with dentinoid formationdentinogenic ghost cell tumor . solid benign odontogenic neoplasms with similar cell morphology to that in the coc and with dentinoid formation dentinogenic ghost cell tumor . malignant odontogenic neoplasms with features similar to those of the dentinogenic ghost cell tumorghost cell odontogenic carcinoma . malignant odontogenic neoplasms with features similar to those of the dentinogenic ghost cell tumor ghost cell odontogenic carcinoma . other lesions showing combined histopathological features with ccot include orthokeratinized odontogenic cyst , ameloblastoma , dontogenic myxofibroma , ameloblastic fibroma , ameloblastic fibro - odontoma and both ameloblastic fibro - odontoma and aot . , in this article , this is the first report of a ccot lesions associated with plexiform ameloblastoma in anterior mandible . the mechanism is unknown of such unique mixture but it is widely considered that the development of ccot , which possesses the features of other odontogenic tumors results in the development of these tumors secondarily , rather than that these tumors are themselves secondary phenomena of a preexisting odontogenic tumor . these lesions are observed in association with an unerupted tooth in 10 - 32% of the cases . radiographically , it appears as either a unilocular or multilocular radiolucent area with either well - circumscribed or poorly defined margin . some reports have indicated that computed tomography ( ct ) may be more successful than plain film radiography in depicting such calcification because such calcifications may be obscured in plain radiographs by superimposition of anatomic details . ct findings characteristic of coc include calcification along the outline of the bony cavity and have been reported previously . in this article , the microscopic feature of ccot is characterized by the lining of a well - defined ameloblastomatous epithelium with variable amounts of ghost cells . some studies suggested that the ghost cells might present the product of abortive enamel matrixin odontogenic epithelium because enamel proteins were immunohistochemically expressed in the ghost cells . however , other studies indicated that the ghost cells represented different stages of normal and aberrant keratin formation or the metaplastic transformation and coagulative necrosis of the odontogenic epithelium . surgical enucleations had been performed in all cases . in this case having adjacent teeth with resorbed root , these teeth were removed simultaneously with enucleation of the lesion . although , enucleation and excision appeared to cure the hybrid lesion , long - term follow - up data and additional cases are still needed to clarify the clinical significance of these lesions . , 2006 categorizes the lesion into four categories and considers each one of them to be distinct in its architectural presentation and biological behavior , as follows
grade 1
simple cystcoc . the following combinations have been published
ccot associated with an odontomeccot associated with aotccot associated with ameloblastomaccot associated with ameloblastic fibromaccot associated with ameloblastic fibro - odontomaccot associated with odontoameloblastomaccot associated with odontogenic myxofibroma.group 3
solid benign odontogenic neoplasms with similar cell morphology to that in the coc and with dentinoid formationdentinogenic ghost cell tumor . group 4
malignant odontogenic neoplasms with features similar to those of the dentinogenic ghost cell tumorghost cell odontogenic carcinoma . the following combinations have been published
cysts associated with odontogenic hamartomas or benign neoplasms : ccot . the following combinations have been published ccot associated with an odontome ccot associated with aot ccot associated with ameloblastoma ccot associated with ameloblastic fibroma ccot associated with ameloblastic fibro - odontoma ccot associated with odontoameloblastoma ccot associated with odontogenic myxofibroma . solid benign odontogenic neoplasms with similar cell morphology to that in the coc and with dentinoid formationdentinogenic ghost cell tumor .
solid benign odontogenic neoplasms with similar cell morphology to that in the coc and with dentinoid formation dentinogenic ghost cell tumor . malignant odontogenic neoplasms with features similar to those of the dentinogenic ghost cell tumorghost cell odontogenic carcinoma . malignant odontogenic neoplasms with features similar to those of the dentinogenic ghost cell tumor ghost cell odontogenic carcinoma other lesions showing combined histopathological features with ccot include orthokeratinized odontogenic cyst , ameloblastoma , dontogenic myxofibroma , ameloblastic fibroma , ameloblastic fibro - odontoma and both ameloblastic fibro - odontoma and aot . , in this article , this is the first report of a ccot lesions associated with plexiform ameloblastoma in anterior mandible . the mechanism is unknown of such unique mixture but it is widely considered that the development of ccot , which possesses the features of other odontogenic tumors results in the development of these tumors secondarily , rather than that these tumors are themselves secondary phenomena of a preexisting odontogenic tumor . these lesions are observed in association with an unerupted tooth in 10 - 32% of the cases . radiographically , it appears as either a unilocular or multilocular radiolucent area with either well - circumscribed or poorly defined margin . some reports have indicated that computed tomography ( ct ) may be more successful than plain film radiography in depicting such calcification because such calcifications may be obscured in plain radiographs by superimposition of anatomic details . ct findings characteristic of coc include calcification along the outline of the bony cavity and have been reported previously . in this article , the microscopic feature of ccot is characterized by the lining of a well - defined ameloblastomatous epithelium with variable amounts of ghost cells . some studies suggested that the ghost cells might present the product of abortive enamel matrixin odontogenic epithelium because enamel proteins were immunohistochemically expressed in the ghost cells . however , other studies indicated that the ghost cells represented different stages of normal and aberrant keratin formation or the metaplastic transformation and coagulative necrosis of the odontogenic epithelium . surgical enucleations had been performed in all cases . in this case having adjacent teeth with resorbed root , these teeth were removed simultaneously with enucleation of the lesion . although , enucleation and excision appeared to cure the hybrid lesion , long - term follow - up data and additional cases are still needed to clarify the clinical significance of these lesions . | a hybrid odontogenic tumor comprising two distinct lesions is extremely rare .
we presented a hybrid odontogenic tumor composed of a calcifying cystic odontogenic tumor ( ccot ) and a plexiform ameloblastoma .
this tumor was observed in the anterior area of the mandible of a 17-year - old indian male .
masses of ghost epithelial cells with the characteristics of ccot were seen in the lining of the cyst .
the odontogenic epithelia with the features of plexiform ameloblastoma were also observed . |
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cells : fhk - tcl3.1 [ 3 , 17 ] and 293 t cells were maintained in dulbecco s modified
eagle s medium ( dmem ; gibco , grand island , ny , u.s.a . ) supplemented with 10%
heat - inactivated fetal calf serum ( fcs ; jr scientific , woodland , ca , u.s.a . ) , 100
units / ml penicillin and 100 g / ml
streptomycin ( gibco ) at 37c under 5% co2 . the myeloma cell line p3u1 was
maintained in rpmi1640 medium ( gibco ) supplemented with 10% heat - inactivated fcs , 100
units / ml penicillin , 100 g / ml
streptomycin and 55 m of 2-mercaptoethanol ( 2-me ) ( gibco ) at 37c under 5%
co2 . viruses : ehv-1 strain 89c25 was isolated from a racehorse with respiratory
disease caused by ehv-1 respiratory infection . 89c25 was plaque - purified three times in primary fetal horse kidney ( fhk ) cells and
designated as 89c25p . ehv-4 th20p strain [ 11 , 14 ] was
propagated in fhk - tcl3.1 cells and was used for indirect immunofluorescence assay ( ifa ) . construction of expression plasmids : for expression of glycoproteins of
ehv-1 , each gene was amplified from the genome of ehv-1 89c25p by polymerase chain reaction
( pcr ) using primers . 2.1 ( takara , otsu ,
japan ) , and the amplified fragments were digested with restriction enzymes and were then
cloned into the expression plasmid pcaggs , which was kindly provided by dr . the nucleotide sequences of at least two constructed plasmids were determined
using a big dye terminator v3.1 kit ( applied biosystems , foster city , ca , u.s.a . ) . all
plasmids were purified by qiaprep spin miniprep kit ( qiagen , hilden , germany ) or qiafilter
plasmid midi kit ( qiagen ) . expression in 293 t cells : 293 t cells were transfected with purified
plasmids using polyethylenimine ( pei ) . briefly , 3.2 g of plasmid were
mixed with 8 l of pei ( 2 mg / ml ) and were then transfected
to 293 t cells in 6 wells plate ( sumitomo bakelite , tokyo , japan ) , as reported previously
. production of mabs : balb / c mice ( female , aged six weeks ) were
intraperitoneally inoculated with 1 10 pfu of ehv-1 three times at an interval
of three or four weeks . at 3 to 7 days after final immunization , splenocytes were collected
and fused with p3u1 myeloma cells using 50% polyethylene glycol solution
( hybri - max ; sigma , st . hybridoma cells were selected in
git media ( wako , osaka , japan ) containing hypoxanthine aminopterin thymidine supplement
( hat ) ( gibco ) , 10% bm - condimed h1 hybridoma cloning supplement ( roche diagnostics , mannheim ,
germany ) and 10% fcs for 7 days at 37c under 5% co2 . supernatants of hybridomas
were screened by virus - neutralization ( vn ) test and/or ifa . hybridoma cells secreting
ehv-1-specific mabs were cloned twice by limiting dilution method and were then inoculated
into balb / c mice pretreated with pristane ( sigma ) for preparation of ascites . vn test : in order to detect vn activity , supernatants of hybridoma or
diluted ascites were mixed with ehv-1 for 60 min at 37c , and then , the mixtures were
directly inoculated into fhk - tcl3.1 cells that were washed with dmem without fcs . after incubation for 60 min at 37c under 5%
co2 , cells were washed twice with dmem without fcs and overlaid with 0.8%
agarose ( seaplaque gtg agarose ; lonza , rockland , me , u.s.a . ) in dmem containing 10% fcs . plates were placed at 37c in 5% co2 for 3 days , and cells were fixed with 5%
buffered formaldehyde . the number of plaques was counted , and hybridoma or diluted ascites that reduced the
number of plaques by more than 50% in comparison with the mean number of plaques in control
wells was considered to be positive . ifa : virus - infected fhk - tcl3.1 cells or plasmid - transfected 293 t cells
were collected , washed with pbs three times and placed on 24-well microscope slides
( matsunami glass , osaka , japan ) . after fixation with cold acetone for 30 min , cells were
incubated with mabs for 60 min at 37c . cells were then washed three times in pbs and
incubated with goat anti - mouse ig(h+l)-fitc human - adsorbed ( southern biotech , birmingham ,
al , u.s.a . ) for 30 min at 37c . after washing three times , fluorescence was observed under a
nikon optiphot 2 efd3 fluorescence phase contrast microscope ( nikon , tokyo , japan ) . immunoblot analysis : virus - infected fhk - tcl3.1 cells or
plasmid - transfected 293 t cells were extracted with ripa [ 25 mm tris - hcl ( ph 7.6 ) , 150 mm
sodium chloride ( nacl ) , 1% sodium dodecyl sulfate ( sds ) , 1% sodium deoxycholate and 1%
triton x-100 ] and then dissolved in 2sds sample buffer ( 125 mm tris - hcl , 4% sds , 40%
glycerol and 0.002% bromophenol blue ) with or without 2-me or directly dissolved in 1sds
sample buffer with or without 5% 2-me . after boiling for 3 min , samples were loaded on
sds - polyacrylamide gel ( page ) , and electrophoresis was carried out in sds buffer ( 25 mm
tris , 192 mm glycine and 0.1% sds ) . then , proteins were transferred to polyvinylidene
difluoride ( pvdf ) membrane ( immobillon ; millipore , billerica , ma , u.s.a . ) by semi - dry
blotting apparatus ( biocraft , be-310 , tokyo , japan ) . after membranes were reacted with 3%
gelatin ( bio - rad , hercules , ca , u.s.a . ) in tbs ( 20 mm tris - hcl and150 mm nacl , ph 7.5 ) for
30 min at 37c , they were then washed three times with tbs containing 0.05% tween 20 . after
washing , the membrane was incubated with diluted horse sera or mabs for 1 hr at 37c as
primary antibody , followed by incubation with the peroxidase - conjugated f(ab)2
fragment of anti - horse igg(h&l ) goat ( rockland , gilbertsville , pa , u.s.a . ) or
peroxidase - conjugated goat affinity purified antibody against mouse imunoglobulins igg , iga
and igm ( cappel , solon , oh , u.s.a . ) for 30 min at 37c as secondary antibody . the reaction was visualized
using 0.03% diaminobenzidine ( wako ) and 0.009% h2o2 ( wako ) in tbs . sera : sera were collected from three foals that were experimentally
infected with ehv-1 89c25p . serum specific to
ehv-1 ge was collected from balb / c mice immunized with fusion protein of glutathione
s - transferase and ge(169 - 201 ) . immunoglobulin class and subclass : immunoglobulin class and subclass of
mabs were determined using mouse monoclonal antibody isotyping kit ( roche ) . sequence analysis of each glycoprotein of ehv-1 strain 89c25p : nucleotide
sequences of at least two cloned genes encoding each glycoprotein of 89c25p were determined
and compared with those of ehv-1 strain ab4 . the
results showed that the amino acid sequences of gc , gd , ge , gg , gh , gi , gk , gl and gn of
89c25p were identical to those of ab4 , but those of gp2 , gb and gm were different at
positions 95 ( serine to phenylalanine ) , 938 ( lysine to glutamic acid ) and 84 ( methionine to
lysine ) , respectively ( data not shown ) . these plasmids were designated as pcag - gp2 - 1
pcag - gb-1 , pcag - gc-1 , pcag - gd-1 , pcag - ge-1 pcag - gg-1 , pcag - gh-1 , pcag - gi-1 , pcag - gk-1 ,
pcag - gl-1 , pcag - gm-1 and pcag - gn-1 . expression of ehv-1 glycoproteins in 293 t cells : twelve expression
plasmids encoding each glycoprotein were transfected into 293 t cells , and expression was
examined by immunoblot analysis using horse sera . expressed gp2 , gb , gc , gd , gg and gi were
detected , but ge , gh , gk , gl , gm and gn were not . three bands with molecular masses of
approximately 230 , 65 and 42 kilo daltons ( kda ) were detected in pcag - gp2 - 1-transfected
cells , two bands of 148 and 135 kda were detected in pcag - gb-1-transfected cells , two bands
of 150 and 6875 kda were detected in pcag - gc-1-transfected cells , one band of 50 kda was
detected in pcag - gd-1-transfected cells , two bands of 100 and 50 kda were detected in
pcag - gg-1-transfected cells , and two bands of 140 and 65 kda were detected in
pcag - gi-1-transfected cells ( data not shown ) . establishment and characterization of mabs : in order to characterize ehv-1
glycoproteins , nine mabs against ehv-1 strain 89c25p were produced ( table 1table 1.characterization of mabs against ehv-1mabsisotypeproteinsvn titerifaimmunoblot analysis
ehv-1ehv-48h11igg2agp2<1:101:1,280<1:100 + 5f8igmgc1:801:1,600<1:100 + 1g10igmgc1:6401:800<1:100 + 8h4igmgc1:1,2801:800<1:100 + 8b2igmgc1:3,2001:1,600<1:1005f12igmgb<1:101:1,280<1:100 + 7e11igmgb<1:101:1,280<1:100 + 6f4igg2agb<1:101:20,4801:800 + 6g12igmgb<1:101:1,2801:800+a ) immunoblot analysis was carried out under non - reducing conditions . ) . four mabs , 5f8 , 1g10 , 8h4 and 8b2 , had vn activity against ehv-1 , and all
were specific to gc ( table 1 , fig 1.comparison of gp2 , gb and gc expressed in plasmid - transfected 293 t cells and
ehv-1-infected fhk - tcl3.1 cells . mabs , 8h11(a ) , 5f12 ( b ) and 1g10 ( c ) were used as primary
antibodies . ) . four mabs , 5f12 , 6f4 , 6g12 and 7e11 , were specific to gb ( table 1 , fig.1b ) , and
two , 6f4 and 6g12 , cross - reacted with ehv-4 ( table
1 ) . all mabs did not recognize
glycoproteins on immunoblot analysis under reducing conditions ( data not shown ) . in
addition , the molecular masses of gp2 , gb and gc expressed in 293 t cells were similar to
those in ehv-1-infected cells ( fig . interestingly , the molecular mass of gp2 detected by mab 8h11 was over 250 kda and was
different from those detected in horse sera , 230 , 65 and 42 kda ( figs . comparison of gp2 , gb and gc expressed in plasmid - transfected 293 t cells and
ehv-1-infected fhk - tcl3.1 cells . mabs , 8h11(a ) , 5f12 ( b ) and 1g10 ( c ) were used as primary
antibodies . co - expression of glycoproteins : ehv-1 ge and gi , gm and gn and gh and gl
were co - expressed in 293 t cells , meaning that co - expressed ge and gi , and gm and gn reacted
strongly with horse sera , but co - expressed gh and gl did not ( fig . plasmids pcag - ge-1 and pcag - gi-1 ( a ) , pcag - gm-1
and pcag - gn-1 ( b ) and pcag - gh-1 and pcag - gl-1 ( c ) were co - transfected into 293 t cells .
immunoblot analysis was carried out under non - reducing conditions . the amount of
plasmid dna transfected into 293 t cells in 35 mm dishes is noted under each
figure . ) . with co - expression of ge and gi , two additional bands other than gi - specific bands
with molecular masses of 8090 and 250 kda were seen ( figs . furthermore , mouse
antibody specific to ge also recognized two bands with molecular masses of 8090 and 250 kda
with co - expressed ge and gi , but not with individually expressed ge and gi ( fig . 3afig . 3.(a ) comparison of co - expressed ge and gi by immunoblot analysis using mouse sera
specific to ge ( 169 - 201 ) and horse sera specific to ehv-1 . closed arrowheads show
ge - specific bands and open arrowheads do gi - specific bands . ( b ) comparison of
molecular masses of gp2 detected by mab 8h11 and horse sera specific to ehv-1 . immunoblot analysis was carried out under non - reducing conditions . closed arrowheads
show gp2-specific bands . ) . with co - expression of gm and gn , one band with a molecular mass of over 250 kda was
observed ( fig.2b ) . plasmids pcag - ge-1 and pcag - gi-1 ( a ) , pcag - gm-1
and pcag - gn-1 ( b ) and pcag - gh-1 and pcag - gl-1 ( c ) were co - transfected into 293 t cells . the amount of
plasmid dna transfected into 293 t cells in 35 mm dishes is noted under each
figure . ( a ) comparison of co - expressed ge and gi by immunoblot analysis using mouse sera
specific to ge ( 169 - 201 ) and horse sera specific to ehv-1 . closed arrowheads show
ge - specific bands and open arrowheads do gi - specific bands . ( b ) comparison of
molecular masses of gp2 detected by mab 8h11 and horse sera specific to ehv-1 . immunoblot analysis was carried out under non - reducing conditions . closed arrowheads
show gp2-specific bands . in this study , twelve glycoproteins in ehv-1 were analyzed using horse sera against ehv-1
and established mabs . in expression systems for single glycoproteins , gp2 , gb , gc , gd , gg
and gi reacted strongly with horse sera ( data not shown ) . although high immunogenicity has
been reported for gp2 , gb , gc , gd and gg , it is
interesting that the immunogenicity of gi was also high . furthermore , gp2 , gb and gc
expressed in 293 t cells were similar to those expressed in ehv-1-infected cells ( fig . we reported that ehv-1 ge is able to induce antibodies in horses and that the 20 amino
acids of ge at positions 169 to188 were a useful antigen for elisa to differentiate ehv-1
and ehv-4 infections . however , ge expressed in
293 t cells was not recognized by horse sera ( figs . after co - expression with gi ,
ge strongly reacted with horse sera against ehv-1 and mouse antibody specific to ge ( fig . 3a ) , indicating that gi might be required for
efficient expression of ge . in ehv-4 , ge ( 95kda ) was co - precipitated with gi ( 75kda ) by
immunoprecipiration using anti - gi serum , but not by anti - ge serum . it is unknown whether two bands , 8090 and 250 kda , in co - expressed
ge and gi contain only ge or complex of ge and gi . further experiment will be required to
clarify the role of gi in expression of ge . as shown in fig . 2b , over 250 kda proteins in
co - expressed gm and gn strongly reacted with horse sera against ehv-1 , indicating that gm / gn
complex is important for antigenicity in horses . in a previous study , it was reported that
gn is necessary and sufficient for functional processing of gm . therefore , we speculated that immature gm and gn are expressed in a
single expression system and that coexpression of gm and gn induces maturation of gm and
enhances the antigenicity . however , it is still unknown whether over 250 kda protein
contains only gm or both gm and gn . in ehv-1 , it is reported that co - expression of gh and gl is important for efficient
expression and for induction of protective immunity [ 15 , 23 ] . therefore , coexpression of gh and
gl was examined , but reactions with horse sera were not observed ( fig . 2c ) . it is unknown whether antigenicity of ehv-1 gh and gl
against horse is low or whether another protein is required for maturation of ehv-1 gh and
gl . horse sera against ehv-1 and mab against gp2 recognized different molecular masses of gp2 ,
230 , 65 and 42 kda and over 250 kda , respectively ( figs . ehv-1 gp2 is rich in serine
and threonine residues and is a heavily o - glycosylated protein with a
molecular mass in the range of 192 to 400 kda [ 25 ,
26 , 29 ] . furthermore , it has been reported that gp2 is cleaved into a highly glycosylated
n - terminal subunit ( over 200 kda ) and c - terminal subunit
( 42 kda ) . therefore , horse sera against ehv-1
and mab 8h11 might recognize different positions of gp2 , the c - terminal
subunit and glycosylated n - terminal subunit , respectively . in conclusion , our results showed that efficient expression and/or maturation of two
glycoproteins , ge and gm , requires co - expression of gi and gn , respectively . in addition ,
these materials , expression plasmids and mabs , may be useful for further analysis of ehv-1
glycoproteins . | abstractin this study , we attempted to express twelve glycoproteins of equine herpesvirus-1
( ehv-1 ) in 293 t cells and to characterize these using monoclonal antibodies ( mabs ) and
horse sera against ehv-1 .
expression of glycoprotein b ( gb ) , gc , gd , gg , gi and gp2 was
recognized by immunoblot analysis using horse sera , but that of ge , gh , gk , gl , gm and gn
was not .
four mabs recognized gb , four recognized gc and one recognized gp2 .
two mabs
against gb cross - reacted with ehv-4 .
interestingly , coexpression of ge and gi and gm and
gn enhanced their antigenicity
. furthermore , immunoblot analysis of gp2 showed that
different molecular masses of gp2 were recognized by the mab against gp2 and horse sera
against ehv-1 . in this study , it was demonstrated that at least six glycoproteins were
immunogenic to horses , and coexpression of ge and gi and gm and gn was important for
enhancement of antigenicity . |
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neurenteric cysts are rare endodermally derived lesions . they are most commonly located within the spinal canal and are usually associated with bony malformations . uncommon locations include craniocervical region , cerebellopontine angle , fourth ventricle , and supratentorial parenchyma.[36 ] authors here report a rare case of neurenteric cyst located in the ventral cervicomedullary region which was excised via a lateral suboccipital approach . a 20-year - old male patient presented to our outpatient services with complaints of neck pain for 4 months and intermittent vomiting for 2 months duration . neurological examination revealed left upper limb weakness ( power 4+/5 , medical research council grading ) . gadolinium - enhanced magnetic resonance imaging ( mri ) revealed a well - defined lobulated lesion in the ventral cervicomedullary region from medulla to upper border of c2 vertebra . the lesion was heterogeneous in appearance ; the anterior part measured 2.8 1.6 cm and was hypointense and hyperintense on t1- and t2-weighted images , respectively ; while the 1.7 1.1 cm sized posterior part was iso to mildly hyperintense on t1 and heterogeneously hyper intense on t2-weighted sequences [ figure 1a and b ] . the anterior part of the lesion was isointense , whereas the posterior part was hyperintense on fluid attenuation and inversion recovery ( flair ) sequences . there was mild restriction on diffusion - weighted sequences . on post - contrast study , rim enhancement was evident in the posterior part of the lesion [ figure 1c ] . surgery was performed in prone position . a midline suboccipital craniectomy ( with bone removal more on left side ) , left - sided c1 posterior arch removal , and partial c2 hemilaminectomy were performed . intraoperatively , there was a well - defined , intradural , extra - medullary encapsulated , relatively avascular , lobulated lesion containing easily suckable , cheesy material located anterior to medulla extending up to c2 vertebral level . vertebral artery , lower cranial nerves , and posterior inferior cerebellar artery were well visualized and preserved . tumor decompression was followed by near total excision of the cyst wall in view of adherence of the cyst wall focally to surrounding vital structures . postoperative course was uneventful and he was discharged on 7 postoperative day and is currently on regular follow - up . at the last follow - up of 3 months , patient was well with no deficits . ( a ) sagittal t1-weighted magnetic resonance imaging showing the anterior hypointense and posterior hyperintense part of the lesion extending from medulla to upper border of c2 ( b ) sagittal t2-weighted magnetic resonance imaging showing the lesion to be hyperintense ( c ) sagittal t1-weighted post - contrast magnetic resonance imaging showing an isolated rim enhancement of posterior part of the lesion postoperative sagittal t1-weighted post - contrast magnetic resonance imaging showing a very small residual lesion in the lower brainstem hematoxylin and eosin photomicrographs showed epithelial lining thrown into folds along with irregular bundles of collagen . higher magnification images show a prominent columnar epithelial lining with goblet cells focally thrown into papillary formations ( 100 ) , pseudostratification , and absence of cilia ( 400 ) . overall features were consistent with an endodermal ( neurenteric ) cyst of hind - gut type [ figure 3a and b ] . hematoxylin and eosin section photomicrographs showing prominent columnar epithelial lining with goblet cells focally thrown into papillary formations ( 100 ) ( a ) pseudo - stratification and absence of cilia ( 400 ) , ( b ) overall features were consistent with an endodermal ( neurenteric ) cyst of hind - gut type hematoxylin and eosin photomicrographs showed epithelial lining thrown into folds along with irregular bundles of collagen . higher magnification images show a prominent columnar epithelial lining with goblet cells focally thrown into papillary formations ( 100 ) , pseudostratification , and absence of cilia ( 400 ) . overall features were consistent with an endodermal ( neurenteric ) cyst of hind - gut type [ figure 3a and b ] . hematoxylin and eosin section photomicrographs showing prominent columnar epithelial lining with goblet cells focally thrown into papillary formations ( 100 ) ( a ) pseudo - stratification and absence of cilia ( 400 ) , ( b ) overall features were consistent with an endodermal ( neurenteric ) cyst of hind - gut type neurenteric cysts are benign , endodermally derived , epithelial - lined benign cystic lesions of central nervous system and constitute around 0.01% of central nervous system tumors . described in 1934 by puusepp et al . as intestinoma , different terminologies have been used in the nomenclature including endodermal cyst , epithelial cyst , enterogenous cyst , enterogenic cyst , bronchogenic cyst , and enteric cyst among several others . as the term neurenteric is used in cases of endodermal type cysts of the neural axis , the term neurenteric cyst has gained increasing popularity . although many theories have been proposed to account for the embryogenesis of these lesions , the most widely accepted theory has been that proposed by bremer et al . ( 1952 ) , which postulates that these are due to abnormal persistence of neurenteric canal or formation of an accessory neurenteric canal with a split notochord and displacement of endodermal cells . neurenteric cysts associated with the spinal cord are the most common and are typically situated in the ventral intradural extramedullary location in the lower cervical and thoracic spines . approximately , 50% of them are associated with bony malformations including scoliosis , spina bifida , klippel fiel syndrome , syringomyelia , and others . intracranial neurenteric cysts are uncommon and very rarely they have been associated with bony malformations . the first case of intracranial neurenteric cyst was reported by small ( 1962 ) and around 81 cases of intracranial neurenteric cysts have been reported in the literature since then . among these , the most common locations include midline posterior fossa , anterior to the brainstem which account for about two - third of cases . craniocervical junction , cerebellopontine angle , cerebellum , and fourth ventricle are other reported locations in the posterior fossa.[35 ] supratentorial location is , however , very rare . in a review of intracranial neurenteric cysts by tucker bejjani et al , these lesions are known to occur in all ages , most commonly in the third and fourth decades . most reported reviews show a slight male predilection but recently , report by tucker et al . most patients present either with mass effects or with features of aseptic meningitis due to spill - over of cyst contents . major differential diagnosis at any site includes arachnoid cyst , dermoid cyst , and epidermoid cyst . lesions are commonly hypodense on computed tomography ( ct ) scan and hence , are often undetectable . on mri , these lesions have a variable appearance but the most common one is of a well - defined , lobulated , t1 hypointense , t2 hyperintense lesion with no perilesional edema . they are hyperintense , show mild restriction of diffusion images and on post - contrast images , show no / minimal rim enhancement . , there was a more heterogeneous appearance on all mri sequences with rim enhancement of the posterior component . other cystic lesions including arachnoid cyst , epidermoid cyst , dermoid cyst , and other endodermal lesions such as colloid cyst should be kept as differential diagnosis while interpreting these lesions on mri . according to the wilkins and odem classification of spinal neurenteric cysts which is based on histopathological findings , three different types can be described : types a , b , and c. of these , type a is the most common , whereas type c is the rarest and most complex variety . most reported intracranial neurenteric cysts are of type a or b. none of them has been a type c variety . on light microscopy , findings are of cyst lining composed of columnar epithelium with goblet cells . the contents of the cyst can also vary ranging from thin fluid to mucoid and cheesy contents . these cysts show positivity for epithelial membrane antigen ( ema ) , cytokeratin , carcinoma embryonic antigen ( cea ) , and ca 19 - 9 while they are uniformly negative for glial fibrillary acid protein ( gfap ) , vimentin , and s-100 markers . these help in differentiating them from arachnoid cysts , ependymal cysts , and cysts of neuroectodermal origin . as these lesions are benign and have minimal adherence to the surrounding structures , total surgical excision is the treatment of choice . however , in cases of adherence of the cyst wall , subtotal excision or marsupialization is advisable . long - term follow - up with radiological studies is necessary as delayed recurrences can occur . signs of aseptic meningitis and increasing levels of ca 19 - 9 in cerebrospinal fluid ( csf ) have been depicted as possible indicators of recurrence . no adjuvant treatment is effective and hence is not required . although considered benign , malignant transformation is possible , but very rare . lesions are commonly hypodense on computed tomography ( ct ) scan and hence , are often undetectable . on mri , these lesions have a variable appearance but the most common one is of a well - defined , lobulated , t1 hypointense , t2 hyperintense lesion with no perilesional edema . they are hyperintense , show mild restriction of diffusion images and on post - contrast images , show no / minimal rim enhancement . these lesions are avascular as documented by angiographic studies previously . in our case , there was a more heterogeneous appearance on all mri sequences with rim enhancement of the posterior component . other cystic lesions including arachnoid cyst , epidermoid cyst , dermoid cyst , and other endodermal lesions such as colloid cyst should be kept as differential diagnosis while interpreting these lesions on mri . according to the wilkins and odem classification of spinal neurenteric cysts which is based on histopathological findings , three different types can be described : types a , b , and c. of these , type a is the most common , whereas type c is the rarest and most complex variety . most reported intracranial neurenteric cysts are of type a or b. none of them has been a type c variety . on light microscopy , findings are of cyst lining composed of columnar epithelium with goblet cells . the contents of the cyst can also vary ranging from thin fluid to mucoid and cheesy contents . these cysts show positivity for epithelial membrane antigen ( ema ) , cytokeratin , carcinoma embryonic antigen ( cea ) , and ca 19 - 9 while they are uniformly negative for glial fibrillary acid protein ( gfap ) , vimentin , and s-100 markers . these help in differentiating them from arachnoid cysts , ependymal cysts , and cysts of neuroectodermal origin . as these lesions are benign and have minimal adherence to the surrounding structures , total surgical excision is the treatment of choice . however , in cases of adherence of the cyst wall , subtotal excision or marsupialization is advisable . long - term follow - up with radiological studies is necessary as delayed recurrences can occur . signs of aseptic meningitis and increasing levels of ca 19 - 9 in cerebrospinal fluid ( csf ) have been depicted as possible indicators of recurrence . no adjuvant treatment is effective and hence is not required . although considered benign , malignant transformation is possible , but very rare . craniocervical region is an uncommon location for neurenteric cyst . they need to be considered in the differential diagnoses when presented with the classical imaging appearance as described . subtotal excision is advisable in cases where cyst wall is adhered to the surrounding structures . | neurenteric cysts are rare , benign , endodermally derived tumors of the central nervous system .
intracranial neurenteric cysts are rare with posterior fossa being the most common location among them .
neurenteric cyst of the craniocervical region is very rare .
authors report a rare case of neurenteric cyst located in the ventral cervicomedullary region .
the pertinent literature is reviewed regarding this uncommon entity . |
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chronic hepatitis b virus ( hbv ) infection causing chronic hepatitis , cirrhosis , and hepatocellular carcinoma affects more than 350 million people worldwide [ 1 , 2 ] . antiviral therapies that contain interferon and nucleos(t)ide analogues are used in patients who are infected with hbv and are at a higher risk of developing cirrhosis and hepatocellular carcinoma [ 1 , 3 , 4 ] . the purpose of the treatment is to suppress hbv replication and to prevent mortality associated with disease progression to end - stage liver disease and major complications [ 4 , 5 ] . currently , 5 oral nucleos(t)ide analogues are approved for the treatment of liver disease related to chronic hbv infection in europe and usa , although only 3 nucleos(t)ide analogues , namely , lamivudine ( lam ) , entecavir ( etv ) , and adefovir dipivoxil ( adv ) , are approved for use in japan . lam , the first approved nucleoside analogue for chronic hbv infection - related liver disease , suppresses hbv replication and improves hepatic inflammation in most patients . however , more than 60% of patients with chronic hbv infection who receive long - term lam therapy become resistant to the agent within 4 years of starting the therapy . for patients with lam resistance , virological breakthrough due to development of adv - resistant mutations occurred in 21% of 14 patients within 18 months after changing to adv monotherapy ; etv - resistant mutations were found in 8% of 151 patients 2 years after changing to etv monotherapy ( 1 mg once per day ) . therefore , lam monotherapy has not been used for patients with chronic hbv infection since etv was approved . in patients with lam resistance , tenofovir ( tdf , not yet approved for use in japan ) or adv therapy is advised . the appearance of virological and biochemical breakthroughs during combination therapy with adv and lam is very rare in patients with lam resistance ; therefore , combination therapy is recommended in such cases [ 11 , 12 ] . at present , combination therapy with adv and lam must be continued for a long period of time , even though long - term use of adv is associated with a slight risk of renal toxicity [ 13 , 14 ] . although adv has a favorable risk / benefit profile with little or no evidence of renal toxicity after 48 weeks of low - dose treatment ( 10 mg per day ) [ 15 , 16 ] , some studies have reported development of severe osteomalacia caused by renal tubular dysfunction ( fanconi syndrome ) after long - term use of adv [ 17 , 18 ] . therefore , establishment of a protocol is required for early identification of osteomalacia caused by renal tubular dysfunction during the administration of adv . serum bone - specific alkaline phosphatase ( bap ) is the most commonly used bone disease marker in hemodialyzed patients . serum bap levels increase gradually in osteomalacia secondary to the administration of adv , without increase in serum creatinine concentration [ 17 , 18 ] . therefore , serum bap concentration may be a potentially useful marker for early detection of osteomalacia caused by renal impairment secondary to the administration of adv . to determine the significance of these indicators for the management of patients receiving long - term adv plus lam therapy , we evaluated bone metabolism markers , including serum and urinary phosphate concentrations and serum bap , in chronic hepatitis b ( chb ) patients receiving nucleos(t)ide analogues . this study complies with the standards of the 1975 declaration of helsinki and current ethical guidelines ; written informed consent was obtained from each patient . between august 2003 and january 2012 , 168 consecutive patients positive for hepatitis b surface antigen ( hbsag ) , who presented to the division of gastroenterology and hepatology of our institution , received nucleos(t)ide analogue therapy . among them , 144 consecutive patients who received adv or the other nucleoside analogues ( lam or etv ) for more than 1 year were enrolled . most patients had hbv genotype b or c , as seen in a previous japanese study . of these , 20 patients had received a combination of 100 mg of lam plus 10 mg of adv per day ( adv group ) , and 124 patients had received either 100 mg of lam per day or 0.5 mg of etv ( non - adv group ) . patients in the adv group received additional adv because of increase in serum hbv dna levels ( 1 log10 copies / ml ) during lam monotherapy . the non - adv group included 34 patients who started lam therapy ( 100 mg per day ) but had been subsequently switched to etv therapy ( 0.5 mg per day ) to avoid the appearance of lam - refractory hbv . this group also included 90 patients who had received 0.5 mg of etv per day as a first - line therapy . exclusion criteria were as follows : presence of antibodies to hepatitis c virus or hiv ; a current alcohol consumption of > 20 g / day ; and presence of hepatocellular carcinoma , other liver diseases , progressive decompensated liver cirrhosis , or renal dysfunction at the time of starting nucleos(t)ide analogue therapy ( serum creatinine : male , > 1.3 mg / dl ; female , > 1.1 mg / dl ) . patients who had hypertension and/or diabetes mellitus were also excluded from this study owing to the risk of renal impairment . in addition , patients who were receiving vitamin d were excluded from this study because this therapy may affect bone turnover . all blood samples were obtained from patients after fasting . to reduce the confounding effects of covariates , we used propensity scores [ 22 , 23 ] to match the adv group to the non - adv group during nucleos(t)ide analogue therapy based on the stage of liver disease at the time of enrollment and renal function at the time of receiving the nucleos(t)ide analogue . variables that may have influenced the treatment outcomes , including age , sex , the duration of nucleos(t)ide analogue therapy , platelet count , serum alanine aminotransferase ( alt ) , serum albumin at the time of enrollment ( nucleos(t)ide analogue therapy for > 1 year ) , and serum creatinine level at the time of receiving nucleos(t)ide analogues , were used to generate a propensity score ranging from 0 to 1 by logistic regression . the nearest available match on the estimated propensity score was used to select participants in the adv group and find participants in the non - adv group with the closest propensity scores . nineteen well - matched pairs of patients in the adv and non - adv groups were obtained . hbsag in patients ' sera was tested by enzyme immunoassay using commercially available kits ( dainabott , tokyo , japan ) . the serum hbv dna concentration was monitored using a polymerase chain reaction assay ( cobas amplicor hbv monitor test , roche diagnostics k. k. , tokyo , japan ; lower limit of detection , 2.6 log copies / ml ) before november 2007 and by another polymerase chain reaction assay ( cobas ampliprep - cobas taqman hbv test , roche diagnostics k. k. ; lower limit of detection , 2.1 log copies / ml ) after december 2007 . ymdd mutations were detected using a line probe assay ( inno - lipa hbv dr assay , innogenetics nv ) . serum bap was measured at the time of enrollment and before nucleos(t)ide analogue administration using a commercially available polyacrylamide - gel ( pag ) disk electrophoresis kit designed for use in humans ( alkphor system , jokoh co. ltd , tokyo , japan ) using serum stored at 30c . serum levels of intact parathyroid hormone ( pth ) were measured using an electrochemiluminescence immunoassay ( roche diagnostics k. k. ) , and urinary cross - linked n - telopeptide of type i collagen ( ntx ) was measured using an elisa ( osteomark , inverness medical innovations inc . , waltham , ma , usa ) at the time of enrollment . bap , a sensitive marker reflecting the changes in bone metabolism , was calculated as follows : serum bap concentration at the time of enrollment minus serum bap concentration before administration of nucleos(t)ide analogues . levels of phosphate and creatinine were examined at the time of enrollment and before nucleos(t)ide analogue administration using serum stored at 30c . the mann - whitney u test and wilcoxon rank - sum test were used to analyze continuous variables . all tests of significance were two tailed , and a p value of < 0.05 was considered significant . all statistical analyses were performed using statistica for windows version 6 ( statsoft , oklahoma , usa ) . the baseline characteristics of the 144 patients enrolled in the study are summarized in table 1 . there were no significant differences with respect to age , sex , duration of nucleos(t)ide analogue therapy , platelet count , serum alt , serum albumin , total bilirubin at the time of enrollment , and serum creatinine level at the time of nucleos(t)ide analogue administration between the adv and non - adv groups ( table 2 ) . there was no difference in serum creatinine concentration between the adv and non - adv groups at the time of enrollment . serum phosphate concentration tended to be lower in the adv group , although the difference was not statistically significant . in contrast , urinary phosphate concentration was significantly higher in the adv group than in the non - adv group ( p = 0.0424 ) . there was no significant difference between the adv and non - adv groups in the concentration of serum alkaline phosphatase ( alp ) isoenzyme 2 , which is liver specific ; however , serum bap concentration was significantly higher in the adv group than in the non - adv group ( p = 0.0228 ) ( figure 1 ) . there were no significant differences in serum intact pth , 25-hydroxyvitamin d , and urinary ntx concentrations between the adv and non - adv groups ( figure 2 ) . there were no significant changes in serum creatinine or serum phosphate concentration with the administration of adv or the other nucleoside analogues ( lam or etv ) ( figure 3 ) . serum alp2 concentrations were significantly lower at the time of enrollment than before drug administration in both adv group ( p = 0.0126 ) and non - adv group ( p = 0.0025 ) . serum bap was significantly higher at the time of enrollment than before nucleos(t)ide analogue administration in the adv group ( p = 0.0001 ) . there was no significant change in serum bap concentration in the non - adv group ( figure 4 ) . although there was no correlation between the length of nucleoside analogue therapy and bap in the non - adv group , there was a significant positive correlation between the period of adv therapy and bap in the adv group ( r
= 0.2959 ; p = 0.0160 , r : correlation coefficient ) ( figure 5 ) . in the present study , we found that serum bap concentration was significantly elevated after the administration of adv for more than 1 year . this finding was not observed after the administration of the other nucleoside analogues . because serum bap concentration reflects bone metabolism and is increased in osteomalacia , this finding suggests a tendency for subclinical osteomalacia in patients using adv . however , we did not observe significant differences between the adv and non - adv treatment groups with respect to intact serum pth concentration , 25-hydroxyvitamin d , and urinary ntx concentration , which are important markers of hyperparathyroidism , osteomalacia , and osteoporosis , respectively . this result may have been obtained because these bone turnover markers were examined in patients who did not develop symptomatic renal impairment or osteomalacia related to adv treatment in this study . a study evaluating long - term adv therapy reported that 5% of the patients who received therapy up to 5 years had a slight elevation in serum creatinine concentration . although renal impairment is one of the most important side effects of adv , we did not find a significant elevation in serum creatinine concentration among patients in that treatment group . however , we did observe that serum phosphate concentration tended to be lower and urinary phosphate concentration was significantly higher at the time of enrollment ( 1 year after starting adv therapy ) than before starting nucleos(t)ide analogue therapy . therefore , it was difficult to detect potential renal dysfunction on the basis of elevation in serum creatinine concentration alone . according to a recent report , the maximal reabsorption of phosphate in the renal tubules and serum phosphate concentration were low in several patients receiving adv ; however , after changing therapy to etv , serum phosphate concentration improved . in the present study , urinary phosphate concentration was significantly higher in the adv group than in the non - adv group , although there was no significant difference in serum phosphate concentration . these findings may be attributed to the increase in urinary phosphate that occurs before a decrease in serum phosphate . therefore , monitoring for an increase in urinary phosphate may be critical for earlier detection of osteomalacia caused by renal tubular impairment . given that bap is a bone turnover marker , we examined change in serum bap concentration to determine the potential risks of osteomalacia caused by renal tubular impairment . therefore , osteomalacia caused by renal tubular impairment may be a unique side effect of adv , especially after long - term use of adv . adv plus lam combination therapy is very useful in patients with lam - resistant chronic hbv infection , and it is recommended as the first - line therapy for lam - resistant disease in japan . however , because the criteria for discontinuation of nucleos(t)ide analogue therapy are not clear , long - term adv plus lam combination therapy is required in these patients to avoid risk of relapse . long - term adv plus lam combination therapy carries a potential risk of renal impairment and development of fanconi syndrome . however , in the present study , patients did not present with symptoms of fanconi syndrome ; this finding may indicate that adv plus lam combination therapy is fairly safe for almost all patients if appropriate monitoring is provided for adverse effects of adv . our results suggest that compared to serum creatinine and/or phosphate concentration , increases in concentrations of urinary phosphate and serum bap are more useful indicators for early identification of renal tubular impairment . further , serum bap concentration increased before serum phosphate decreased ; this finding may indicate that serum phosphate was being released from the bones , even though it was being lost through the kidneys . in addition , bap , a sensitive maker for detecting bone metabolism abnormalities , increased with the duration of adv plus lam therapy . therefore , the risk of osteomalacia may increase with long - term use of adv . a previous report suggested that dose reduction of adv to 10 mg every other day leads to improvement of renal function without compromising treatment efficacy . in our experience , serum creatinine concentration increased from 0.9 mg / dl to 1.3 mg / dl after the addition of adv ( 10 mg / day ) to lam monotherapy ( 100 mg / day ) in 1 patient ; subsequently , after 3 months of reducing the dose of adv to 10 mg every other day , serum creatinine concentration improved to 1.0 mg / dl . however , because there is no obvious standard procedure for dose reduction , further studies are required to establish when and how to reduce adv dose for patients whose serum alp and/or urinary phosphate concentrations increase after long - term administration of adv . furthermore , 2 adult patients with acquired immunodeficiency syndrome presented with severe bone pain associated with tdf , which is also used for the treatment of chronic hbv infection - related liver disease in japan and is associated with an increase in serum alp . although the relatively high rate of etv resistance is a concern , therapy with 1.0 mg of etv per day may be considered when adv and tdf are contraindicated in patients with lam resistance because of treatment - induced renal impairment and osteomalacia . nucleos(t)ide analogue therapies are very useful for the treatment of chronic hbv infection - related liver disease , have few adverse effects with subjective symptoms , and are often used in long - term treatment . however , patients receiving these therapies should be supervised and monitored carefully for the early detection of adverse effects . periodic measurement of serum bap may be helpful in the early detection of osteomalacia in the absence of elevation of serum creatinine concentration . in conclusion , examination of serum bap concentration is useful for predicting osteomalacia caused by renal impairment in the absence of subjective symptoms and is essential for the establishment of adequate measures for determining the continuation of nucleos(t)ide analogue therapy for chronic hbv infection - related liver disease . | of 168 patients with chronic hepatitis b virus ( hbv ) infection - related liver disease , 20 patients who had received 100 mg of lamivudine plus 10 mg / day of adefovir dipivoxil ( adv ) ( adv group ) and 124 patients who had received 0.5 mg / day of entecavir or 100 mg / day of lamivudine ( non - adv group ) for > 1 year were enrolled . for comparative analyses
, 19 well - matched pairs were obtained from the groups by propensity scores . at the time of enrollment ,
serum creatinine and phosphate concentrations were similar between the adv and non - adv groups ; however , urinary phosphate ( p = 0.0424 ) and serum bone - specific alkaline phosphatase ( bap ) ( p = 0.0228 ) concentrations were significantly higher in the adv group than in the non - adv group .
serum bap was significantly higher at the time of enrollment than before adv administration in the adv group ( p = 0.0001 ) , although there was no significant change in serum bap concentration in the non - adv group .
there was a significant positive correlation between the period of adv therapy and bap ( r
2 = 0.2959 , p = 0.0160 ) .
serum bap concentration increased before increase in serum creatinine concentration and was useful for early detection of adverse events and for developing adequate measures for continuing adv for chronic hbv infection - related liver disease . |
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kawasaki disease ( kd ) is a multisystemic severe vasculitis of medium- and small - sized vessels . this acute febrile illness might have several inflammatory manifestations , such as cervical adenitis .
of note , unilateral cervical adenitis measuring > 1.5 cm in diameter is the less frequently observed criteria in kd patients and it is usually presented in association with other symptoms at disease onset . the finding of fever and lymphadenitis with intense local signs of inflammation and phlegmon formation is rarely described as the initial manifestation of kd ,
and it was previously misdiagnosed as bacterial adenitis ( ba ) . we report a case of a 7-year - old boy who had cervical lymphadenitis with adjacent cellulitis and phlegmon mimicking ba as the first presentation of kd . a previously healthy 7-year - old caucasian boy was admitted to our service with fever ( 39 to 40c ) , cervical lymphadenitis with adjacent cellulitis ( figure 1 ) and severe headache . the next day , he was admitted to hospital israelita albert einstein to receive intravenous antibiotics ( oxacillin and ceftriaxone ) in consequence of the persistent fever with worsening of the surrounding pain and inflammation . laboratory findings showed hemoglobin ( hb ) 10.2g / dl , white blood cell count ( wbc ) 11,640/mm ( 77% neutrophils , 12% lymphocytes , 9% monocytes , 1% eosinophils , and 1% basophils ) , platelets 301,000/mm , c - reactive protein ( crp ) 217.4mg / dl ( normal 0 - 0.3 ) , aspartate aminotransferase 28u / l ( normal 15 - 40u / l ) , alanine aminotransferase 20u / l ( normal 10 - 35u / l ) , amylase 58u / l ( normal 0 - 110u / l ) and antistreptolysin o 143iu / ml ( normal 0 - 200iu / ml ) . cervical computed tomography ( ct ) revealed right cervical lymph nodes increased in number and size , measuring up to 2.1 cm , without signs of liquefaction , besides densification and thickening of the regional soft tissues on the face and neck , strongly suggestive of extensive cellulitis and phlegmon . cervical collections were not observed . on the fourth day , he had dry and scaly lips ( figure 2 ) , and oxacillin was replaced by clindamycin since the patient was still febrile with persistent inflammatory signs . on the ninth day , he presented non - exudative bilateral conjunctival injection ( figure 3 ) . after 10 days of febrile disease , a discrete maculopapular rash appeared on patient s trunk , hands and feet , which fulfilled criteria for kd . we administrated intravenous immunoglobulin ( 2mg / kg ) and an improvement of the inflammatory manifestations was seen . two days later , he was discharged , and we prescribed oral cefuroxime and aspirin ( 3.7mg / kg / day ) , corticosteroid and mydriatic eye drops . on the 14th day , he had lamellar desquamation of the fingers , which lasted for 10 days ( figure 4 ) . at that moment , laboratory tests revealed : hb 11.5g / dl , wbc 5,400cells / mm ( 51% neutrophils , 34% lymphocytes , 11% monocytes , 3% eosinophils , and 1% basophils ) , platelets 280.000/mm and crp 1.7mg / dl . echocardiogram remained normal between 10 and 45 days of follow - up after symptoms resolution . figure 1cervical lymphadenitis with adjacent cellulitis initially seen in the described kawasaki disease patient
figure 2dry and injected lips
figure 3non - exudative bilateral conjunctival injection
figure 4lamellar desquamation of the fingers to our knowledge , only one case of cervical adenitis with phlegmon formation , as the first manifestation of kd , has been described . kd is slightly more common in boys than in girls ( male - to - female ratio of 1.5 - 1.8:1 ) and the majority of the affected patients are aged 6 months to 5 years old .
only about 15% of the patients are older than 5 years ,
such as the present case . moreover , incomplete kd has been more frequently reported in patients older than 5 years or younger than 12 months old . the subsequent delay to begin proper treatment in these cases increases the risk of coronary involvement , since its efficacy is higher when used within the first 10 days of illness .
regarding ethnicity , although kd is remarkably more frequent in asian descent , which suggest the existence of a genetic predisposition , this disease is found worldwide in a small but rising incidence . the diagnosis of kd is based on clinical features , and there is no clinical or laboratory pathognomonic findings . cervical lymphadenopathy is considered the less common criteria found in patients with kd , which is described in about 50% of cases , while others signs such as non exudative conjunctival injection , oropharynx mucosa abnormalities , polymorphous rash and extremities changes occur in approximately 90% of classic kd . furthermore , cervical adenitis is normally seen associated with other acute phase symptoms . in a recent review of the literature , isolated cervical adenitis and fever were reported in 57 patients as the first finding of kd . except for transient nonspecific lip dryness , our patient had persistent febrile adenitis as the only involvement of kd up to the ninth day of disease . in addition , the presence of adjacent cellulitis in the neck reinforced the importance of disregard the possibility of infectious causes , such as bacterial and viral illnesses , toxoplasmosis and cat scratch disease . the presence of phlegmon is quite rare in kd and was found only in one patient among the cases described by kanegaye et al . despite headache be a common finding during febrile episodes in children , especially with higher temperatures , in our case this symptom might have been due to aseptic meningitis , what is seen in approximately one - third of kd patients . in children , it usually manifests as irritability . a question that could be raised is whether the lymphadenitis seen at presentation of this case was in fact a ba acting as a trigger of kd . reports in the literature describe that up to 33% of kd patients have at least one concurrent infection at disease onset , which could be considered as an environmental trigger of the illness . the similarities in clinical and biochemical presentation of both kd , staphylococcal and streptococcal toxin mediated illnesses suggest the possibility of bacterial involvement in the disease etiology in genetic susceptible subjects . a previous study performed cervical ct in 11 of 57 kd patients with lymphadenopathy as first presentation versus 39 of 78 patients with ba . solid nodes were found in 91% of the former group , whereas this finding was showed in only 28% of the second group ( p=0.003 ) . in contrast , when the authors investigated the existence of phlegmon , this inflammation was present in 14 ba subjects , but in only one kd patient .
in our case the presence of cervical adenitis without abscess formation unresponsive to a broad - spectrum antibiotic therapy alerts the need of seeking differential diagnoses . in our patient the diagnosis of kd was confirmed by further fulfillment of kd s criteria . in fact , the majority of previous studies with kd patients with lymphadenitis reported as their initial presentation the administration of antibacterial agents as the first line treatment , but with poor response .
in our case the cellulitis was completely resolved just after the infusion of intravenous immunoglobulin , which is considered the recommended treatment for primary kd . kawasaki disease should be considered as the differential diagnosis for febrile cervical lymphadenitis in children who are unresponsive to initial empiric antibiotics , even if they have adjacent cellulitis and phlegmon . a doena de kawasaki ( dk ) uma vasculite grave multissistmica dos vasos mdios e pequenos . importante mencionar que adenite cervical unilateral > 1,5 cm frequentemente menos observada em pacientes com dk e geralmente apresenta - se em o achado de febre e linfadenite com sinais de inflamao intensa no local e formao de flegmo raramente descrito como sinal inicial da manifestao da dk , e foi previamente diagnosticado incorretamente como adenite bacteriana ( ab ) . relatamos caso de garoto de 7 anos de idade que apresentou linfadenite cervical com celulite adjacente e flegmo mimetizando ab como primeira apresentao da dk . paciente caucasiano , anteriormente saudvel , 7 anos de idade , foi admitido em nosso servio com febre ( 39 a 40c ) , linfadenite cervical com celulite adjacente ( figura 1 ) e cefaleia grave . no dia seguinte , o paciente foi internado no hospital israelita albert einstein para administrao de antibiticos intravenosos ( oxacilina e ceftriaxona ) , devido febre persistente , com piora da dor nas reas subjacentes e inflamao . os exames laboratoriais mostraram hemoglobina ( hb ) 10,2g / dl , contagem de leuccitos ( cl ) 11.640/mm ( 77% de neutrfilos , 12% de linfcitos , 9% de moncitos , 1% de eosinfilos e 1% de basfilos ) , plaquetas 301.000/mm , protena c - reativa ( pcr ) 217,4mg / dl ( normal 0 - 0,3mg / dl ) , aspartato aminotransferase 28u / l ( normal 15 - 40u / l ) , alanina aminotransferase 20u / l ( normal 10 - 35u / l ) , amilase 58u / l ( norma l0 - 110u / l ) e antiestreptolisina o 143iu / ml ( normal 0 - 200iu / ml ) . os testes serolgicos foram negativos para citomegalovrus , toxoplasmose , mononucleose e bartonelose . a tomografia computadorizada ( tc ) da coluna cervical relevou linfonodos cervicais direitos aumentados e em maior nmero , medindo at 2,1 cm , sem sinais de liquefao , alm de densificao e engrossamento dos tecidos moles regionais na face e pescoo , altamente sugestiva de celulite extensiva e flegmo . . a administrao de oxacilina foi substituda por clindamicina , j que o paciente permanecia febril com sinais inflamatrios persistentes . no nono dia , o paciente apresentava congesto ocular bilateral no exsudativa ( figura 3 ) . aps 10 dias de doena febril , notou - se exantema maculopapular discreto no tronco , mos e ps do paciente , portanto preenchendo os critrios para dk.a imunoglobulina intravenosa ( 2mg / kg ) foi administrada com melhora das manifestaes inflamatrias . aps 2 dias , o paciente recebeu alta hospitalar com prescrio de cefuroxima e aspirina por via oral ( 3,7mg / kg / dia ) , juntamente de corticoides e colrio midritico . no 14 dia , o paciente apresentava descamao lamelar dos dedos , que durou 10 dias ( figura 4 ) . novos exames laboratoriais revelaram : hb 11,5g / dl , cl 5,400celuls / mm ( 51% neutrfilos , 34% leuccitos , 11 moncitos , 3% eosinfilos e 1 basfilos ) , plaquetas 280.000/mm e pcr 1,7mg / dl . o ecocardiograma permaneceu normal entre 10 e 45 dias de acompanhamento aps a resoluo dos sintomas .
figura 1linfadenite cervical com celulite adjacente inicialmente observada no paciente com doena de kawasaki
figura 2lbios ressecados e descamados
figura 3congesto ocular bilateral no exsudativa de nosso conhecimento , apenas um caso de adenite cervical com formao de flegmo como primeira manifestao de dk foi descrito . a dk tem prevalncia um pouco maior em garotos do que em garotas ( razo masculino / feminino de 1,5 - 1,8:1 ) ; alm disso , a maioria dos pacientes acometidos tem idade entre 6 meses e 5 anos . cerca de 15% dos pacientes tem mais do que 5 anos , assim como o paciente deste relato . a dk incompleta tem sido mais frequentemente relatada uma demora subsequente para iniciar o tratamento adequado nesses casos aumenta o risco de envolvimento coronrio , j que sua eficcia alta quando utilizada no 10 primeiros dias da doena . em relao etnia , apesar de a dk ser muito mais frequente em descentes asiticos , sugerindo a existncia de predisposio gentica , ela encontrada em todo o mundo em pequena , porm crescente , incidncia .
o diagnstico da dk realizado baseado nas caractersticas clnicas e no em achados clnicos e laboratoriais patognomnicos . a linfadenopatia cervical considerado um critrio menos comum em pacientes com dk , sendo descrito em cerca de 50% dos casos , enquanto outros sinais , como congesto ocular bilateral no exsudativa , anormalidades na mucosa orofarngea , rash polimorfo e alteraes nas extremidades ocorrem em aproximadamente 90% dos casos de dk clssica . alm disso , a adenite cervical normalmente observada juntamente de outros sintomas das fases agudas . em reviso recente da literatura , a adenite cervical isolada e a febre foram relatadas em 57 pacientes como o primeiro achado de dk . com exceo do ressecamento labial no especfico e transiente , nosso paciente persistiu com febre at o nono dia da doena com adenite febril e o nico envolvimento foi de dk . alm disso , a presena de celulite adjacente regio cervical reitera a importncia de desconsiderar a possibilidade de causas infeciosas , como afeces bacterianas ou virais toxoplasmoses e doena de arranhadura de gato . a presena de flegmo bem rara em dk e foi encontrada somente em um paciente entre um ponto interessante foi a presena de cefaleia grave relatada por nosso paciente , apesar de a cefaleia se trata de um achado incomum durante os episdios febris em crianas , especialmente com altas temperaturas . nesse relato , esse sintoma pode ser devido meningite assptica , o que visto em aproximadamente um tero dos pacientes com dk . em crianas , geralmente , a cefaleia se manifesta como irritabilidade . uma questo que pode ser levantada se a linfadenite observada na apresentao deste caso foi , na verdade , uma ao de ab como desencadeadora da dk . 33% dos pacientes com dk tm pelo menos uma infeco concorrente no incio da doena , que pode ser considerada a desencadeadora da doena . as similaridades na apresentao clnica e bioqumica de ambos , dk e doenas mediadas pela toxina staphylococcus e streptococcal , sugerem possibilidade de envolvimento bacteriano na etiologia da doena em indivduos geneticamente suscetveis . um estudo anterior conduziu tomografia computadorizada cervical em 11 de 57 pacientes com dk e linfadenopatia como primeira apresentao versus 39 de 78 pacientes com ab . ndulos slidos foram encontrados em 91% do grupo principal , enquanto esse achado foi mostrado em apenas 28% do segundo grupo ( p=0,003 ) . em contrapartida , quando os autores pesquisaram a existncia de flegmo , este estava presente em 14 dos indivduos com ab , porm em apenas um paciente com dk . de modo similar , neste caso , a mesma caracterstica rara foi confirmada na tomografia computadorizada cervical conduzida . importante ressaltar que a presena de adenite cervical sem formao de abcesso e sem resposta a terapia com antibiticos de amplo espectro alerta a necessidade de diagnstico diferencial . no paciente descrito , o diagnstico de dk foi confirmado devido ao preenchimento dos critrios do quadro de dk . na verdade , a maioria dos casos de dk relatados anteriormente , que apresentou linfadenite como sua manifestao inicial , recebeu agentes antibacterianos como tratamento de primeira linha , porm revelando resposta inadequada . em nosso caso , a celulite foi completamente resolvida logo aps a infuso de imunoglobulina intravenosa , que considerado o tratamento recomendado para dk primria . a doena de kawasaki deve ser considerada diagnstico diferencial para linfadenite cervical febril em crianas que no respondem ao tratamento emprico com antibiticos , mesmo se apresentarem celulite adjacente e flegmo . | cervical adenitis > 1.5 cm in diameter is the less frequently observed criteria in patients with kawasaki disease and it is usually found in association with other symptoms during the acute phase .
moreover , the finding of fever and lymphadenitis with intense local signs of inflammation and phlegmon is rarely seen as the initial manifestation of kawasaki disease .
we report the case of a 7-year - old boy who had cervical lymphadenitis with adjacent cellulitis and phlegmon mimicking bacterial adenitis as the first presentation of kawasaki disease . the patient had fever , cervical lymphadenitis with adjacent cellulitis , and severe headache .
cefadroxil was prescribed based on the clinical diagnosis of bacterial adenitis .
because he remained febrile and phlogistic signs worsened , after 1 day of hospitalization , antibiotics were administrated intravenously ( ceftriaxone and oxacillin ) .
the computed tomography of the neck showed primary infectious / inflammatory process . on the fourth day ,
the patient had dry and scaly lips , and treatment with oxacillin was replaced by clindamycin because the patient was still febrile .
on the ninth day , he presented non - exudative bilateral conjunctival injection . on the tenth day of febrile disease ,
a rash appeared on his trunk , hands and feet .
patient s symptoms resolved after intravenous administration of immunoglobulin ( 2g / kg / dose ) , and he was discharged 2 days later . on the 14th day , the patient had lamellar desquamation of fingers .
kawasaki disease should be considered as a differential diagnosis in children with febrile cervical lymphadenitis unresponsive to empiric antibiotics even if they have adjacent cellulitis and phlegmon . |
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according to the centers for disease control and prevention ( cdc ) , more than one - third of u.s . generally , obesity is associated with high levels of the circulating hormone leptin ( hyperleptinemia ) and low levels of adiponectin [ 13 ] . leptin and adiponectin are cytokines produced excessively by adipocytes , hence the name adipokines . leptin is thought to be responsible for several cardiovascular diseases associated with obesity , while adiponectin is considered to be cardioprotective . it is secreted by adipocytes and binds to the hypothalamic leptin receptor ( ob - r ) to enhance metabolism and reduce appetite , thereby increasing energy expenditure and decreasing energy intake . it is a product of the ob gene and is associated with obesity , since a higher adipose tissue mass results in elevated leptin levels . leptin is also produced by other cells besides adipocytes , such as cardiomyocytes and vascular smooth muscle cells ( vsmc ) [ 7 , 8 ] . several studies have shown that the functional leptin receptor is also found in a variety of organs such as the heart , liver , kidneys , and pancreas [ 913 ] . it is located on cardiomyocytes , vascular smooth muscle cells , endothelial cells , myometrium , and cerebral and coronary vessels [ 17 , 18 ] . therefore , this hormone has a wide range of pleiotropic effects , affecting the cardiovascular , nervous , immune , and reproductive systems [ 1921 ] . leptin circulates in the blood at a level of 5 to 15 ng / ml in lean individuals . this level may reach up to 50 ng / ml in obese individuals , due to their higher adipose tissue mass . glucocorticoids and insulin act on adipocytes to increase leptin expression , possibly explaining the reason for increased leptin levels observed in obesity .on the other hand , fasting , testosterone , and thyroid hormone lead to a reduction in leptin expression [ 23 , 24 ] . although well - known as a product of adipocytes , leptin is also produced by a variety of different tissues and has many functions other than being a satiety factor [ 8 , 14 , 25 ] . in the murine model , the leptin receptor ob - r has six isoforms , ob - ra to ob - rf , which are strongly related to class i cytokine receptor family . ob - re is a soluble receptor secreted in the blood that binds to circulating leptin in order to maintain the concentration of free leptin [ 17 , 26 , 27 ] . ob - ra , c , d , f are short isoforms . ob - rb is the long , functional isoform , responsible for the intracellular signaling effects of leptin . binding of leptin to the ob - rb receptor activates the janus - activated kinase ( jak ) signal transduction pathway , signal transducers and activators of transcription ( stat ) pathway , insulin receptor substrate , and mitogen - activated protein kinase ( mapk ) pathway . when leptin binds to ob - rb , this receptor undergoes homooligomerization [ 30 , 31 ] and then binds to jak2 . this leads to autophosphorylation of jak2 and the phosphorylation of tyr985 , tyr1077 , and tyr1138 on ob - rb [ 30 , 3235 ] . phosphorylation of tyr1138 residue on ob - rb recruits stat3 proteins to the ob - rb / jak2 complex and leads to tyrosine phosphorylation of stat3 proteins , which form dimers and translocate to the nucleus in order to activate transcription of target genes . one of these genes is a member of the suppressors of the cytokine signaling family ( socs3 ) [ 33 , 36 , 37 ] . socs3 binds to tyr985 and other sites within the ob - rb / jak2 complex which inhibits leptin signaling [ 38 , 39 ] . jak2 phosphorylates tyr985 and leads to the phosphorylation of the sh2 ( src homology 2 ) domain of the tyrosine phosphatase shp-2 ( src homology 2-containing tyrosine phosphatase ) , which in turn activates the extracellular signal - regulated kinase ( erk ) signal transduction pathway . jak2 autophosphorylation may also lead to phosphorylation of insulin receptor substrate proteins , which activate the pi3k signaling pathway [ 40 , 41 ] . in the heart , both the leptin - activated erk and pi3k pathways are crucial for proliferation of cardiomyocytes and for the protection of cardiac tissue from ischemia / reperfusion injury [ 42 , 43 ] . another major pathway activated by leptin binding to its ob - rb receptor is the mapk pathway . after agonist binding , homooligomerization of the receptor , jak2 recruitment , and autophosphorylation , the tyr985 on ob - rb is phosphorylated . this recruits shp-2 and grb-2 ( growth factor receptor - bound protein 2 ) to phosphorylate and activate erk1/2 of the mapk family . erk1/2 activation ultimately leads to the expression of specific target genes , such as c - fos and egr-1 , which promote proliferation and differentiation [ 28 , 38 , 44 ] . activation of erk1/2 may also occur independently of tyr985 . in this case , jak2 binds to the sh2 domain of shp-2 [ 33 , 45 ] . thus , both the short leptin receptor isoform ob - ra and the long receptor isoform ob - rb can activate mapk , but to a lesser extent by ob - ra [ 17 , 33 ] . another protein that contains sh2 domain and associates with grb-2 is shc , which has been shown to phosphorylate tyrosine after leptin agonist binding . leptin - induced phosphorylation of stat3 and erk1/2 has been studied in isolated adult c57bl/6 mouse cardiomyocytes , with maximal activation observed at 15 minutes after leptin treatment . leptin - deficient ob / ob mice treated with leptin for four weeks also exhibited elevated stat3 and erk1/2 phosphorylation in their cardiac tissue , but the same treatment in ob - rb - deficient db / db mice did not lead to stat3 and erk1/2 phosphorylation . the and isoforms of p38 mapk are widely distributed and found at relatively high levels in the heart . leptin - induced p38 mapk activation is associated with the onset of hypertrophy and programmed cell death in cardiomyocytes and rat vascular smooth muscle cells [ 14 , 42 , 49 ] . leptin and the rho pathway . under the hypertension - induced force of mechanical stretch , guanine nucleotide exchange factors exchange gdp for gtp on the guanine nucleotide ( gtp ) binding protein rhoa , thereby activating it . rhoa then activates rho kinases ( rock ) , which activates lim kinase ( limk ) . limk phosphorylates cofilin , inactivating this actin depolymerizing protein and leading to the accumulation of f - actin and depletion in g - actin . when present at normal physiological levels , g - actin attenuates hypertrophy by inhibiting transcription factors like serum response factor ( srf ) which upregulate hypertrophic gene expression [ 52 , 53 ] . thus , the activation of the rhoa / rock pathway leads to a reduction in g - actin levels , promoting vascular remodeling and hypertrophy . several studies have revealed numerous effects of leptin on the cardiovascular system [ 20 , 54 , 55 ] . in this review , we will discuss the effect of leptin on the cardiac and vascular system ( figure 1 ) , focusing on cardiac hypertrophy , angiogenesis , the vasoactive response , blood pressure , and atherosclerosis . since cardiomyocytes become terminally differentiated early in life , the increase in cardiac mass is due to hypertrophy of the myocytes rather than hyperplasia . in situations of pressure overload , such as conditions of hypertension or aortic stenosis , myocyte width increases due to the parallel addition of sarcomeres , which in turn increases wall thickness . this kind of remodeling is concentric hypertrophy ( increase in wall thickness / chamber dimension ratio ) . left ventricular hypertrophy ( lvh ) occurs when the myocardium of the left ventricle of the heart enlarges . the left ventricle is the chamber which pumps oxygenated blood into the aorta , which in turn carries and delivers blood to the various tissues and organs . left ventricular hypertrophy generally develops in response to factors like age , hypertension , aortic valve stenosis , and obesity . as the workload increases , this remodeling may lead to an increased risk of cardiovascular diseases , such as heart failure , arrhythmia , ischemic heart disease , or myocardial infarction [ 56 , 59 ] . the functional leptin receptor ob - rb is found in the myocardium , allowing leptin to exert its actions on the heart . the role of leptin in the development lvh has been examined , with some researchers believing that leptin promotes lvh , while others strongly believing that leptin attenuates it . they studied the development of lvh in the leptin - lacking ob / ob and functional leptin receptor - lacking db / db mice . these morbidly obese mice exhibited a significant increase in left ventricular ( lv ) mass and lv wall thickness by 6 months of age as seen by echocardiographic examination , indicating that they had developed lvh . since blood pressure can lead to lvh , they measured the systolic blood pressure , lv end - diastolic pressure , and heart rate in order to adjust for these factors in case they were dissimilar between these mice and their littermate controls ; they observed that they were not different . hence , hypertension was not the cause of the increase in lv wall thickness and lv mass . histological examinations were also made on the cardiomyocytes from ob / ob and db / db mice to visually evaluate hypertrophy . clear myocyte hypertrophy was seen in the hearts of the ob / ob and db / db mice compared to the myocytes of wild type mice , with larger cellular diameter and distorted nuclear architecture . however , no significant interstitial fibrosis , metabolic inclusions of the cytoplasm , or myocardial adipose infiltration were observed . since a lack of leptin or leptin signaling seemed to result in lvh , barouch et al . went on to replete these ob / ob mice with leptin to study whether leptin had antihypertrophic ability . they had 3 groups of mice : leptin - infused mice , pair - fed mice ( on a diet to lose weight ) , and control mice . after almost 6 weeks , the pair - fed mice had lost a significant amount of weight , as did the leptin - infused mice , attributable to leptin 's neurohormonal ability to decrease appetite and increase energy expenditure . interestingly , there was a full reversal of lvh in the leptin - infused mice as seen by both echocardiographic evaluation and histological analysis . although the pair - fed mice lost as much weight as the leptin - infused mice , they did not have a reduction in lv wall thickness and lv mass , a similar observation to the controls . these results indicated not only that the lvh seen in ob / ob mice was simply due to their obesity , but also that leptin depletion was a significant cause . concluded that leptin has a direct antihypertrophic role on the heart , independent of weight loss . the next step should be to measure lv mass , thickness , and other indicators of lvh in humans , preferably those born with a mutant gene for leptin , in which they can not produce this hormone . they were unable to produce the leptin hormone , and thus they were hyperphagic and obese . after examination , they were treated with leptin , thereby losing weight and significantly improving in overall health [ 62 , 63 ] . perhaps their lv mass and thickness before leptin administration should have been measured and compared to these corresponding parameters after leptin treatment . if lvh is reduced after leptin repletion , we can conclude with further confidence that leptin directly reverses lvh in the heart . another confusing insight in the suggestion that leptin contributes to the reversal of lvh is that hypertension is known to lead to lvh , but hypertension is associated with increased levels of leptin . also , treating leptin deficient ob / ob mice with leptin should elevate their blood pressure , which in turn would be a mechanical cause for the development of lvh . examined lv mass , thickness , and cardiomyocyte hypertrophy after 6 weeks of leptin repletion and observed that these factors were diminished as a result of leptin treatment . however , continuous leptin administration for a period longer than only 6 weeks could possibly lead to actions of leptin besides those of being an antihypertrophic factor , but rather perhaps a hypertrophic factor . a population - based cross - sectional study in support of the antihypertrophic effect of leptin was done by pladevall et al . in rural spain . they studied 410 overweight adults and focused on the correlations between plasma leptin levels and lv mass index ( lvmi ) and sum of wall thicknesses ( swt ) , both parameters of lvh . they adjusted for several factors like systolic blood pressure , body mass index , gender , insulin resistance , and age and used a multivariate linear regression model that showed that fasting leptin was inversely and significantly related to lvmi . leptin levels were also inversely associated with swt , but not significantly . among the participants , this study suggests an inverse association between leptin levels and lvh , using the parameters of lvmi and swt . this study showed a negative correlation between leptin levels and swt and lvmi in all body mass index strata , with the effect being most prominent in nonobese individuals . this implies that the higher leptin levels seen in obese individuals should result in lower swt and lvmi , but obesity is associated with lvh . the only explanation is leptin resistance , but this would mean a positive association between leptin levels and swt and lvmi in the group of obese individuals . the researchers of this study were unable to establish a temporal relationship between leptin levels and lvh due to the cross - sectional nature of this study , and so prospective cohort studies are required to portray the temporal sequence of this inverse relationship . moreover , the latest study examining the protective role of leptin against lvh was based on a multi - ethnic study of atherosclerosis ( mesa ) . this longitudinal cohort study of multiethnic groups used data collected from 1,464 participants who had baseline mri scans of the heart and leptin concentration data . after adjusting for age , weight , height , race , and gender , allison et al . performed multivariate linear regression modeling which revealed that a 1-sd increment in leptin was significantly inversely associated with lv mass , lv volume , and odds ratio for lvh incidence . thus , this recent cross - sectional study of a multiethnic group revealed that leptin was significantly associated with reduced lv mass , lv volume , and odds for the occurrence of lvh . this interesting study is strengthened by the large size of the sample and multiethnic nature of the group . allison et al . attributed the antihypertrophic effects of leptin to leptin - induced minimization of triglyceride deposition in the myocardium . they believe that leptin resistance in obesity would prevent the high levels of leptin from inhibiting deposition of triglycerides in the heart . however , this study was a cross - sectional study design and only few subjects belonging to the highest levels of obesity were recruited . moreover , the researchers used a novel method for measuring lv mass and volume by mri , which resulted in more artifact and signal - to - noise issues than other protocols . leptin as a prohypertrophic factor . since obesity is associated with lvh and leptin levels are significantly increased in obesity , many researchers believe that leptin actually contributes to lvh . in vitro studies done on neonatal rat ventricular myocytes exposed to 3.1 nmol / l of leptin for 24 hours significantly increased cell surface area by 42% . the leptin - induced hypertrophic response was mediated by phosphorylation and subsequent activation of the map kinases p38 and erk1/2 , with acute responses of peak stimulation after 510 minutes of leptin administration . in order to rule out the possibility of osmosis - induced hypertrophy , the researchers tested for protein synthesis by leucine incorporation . leptin also significantly increased expression of -skeletal actin and myosin light chain-2 ( mlc-2 ) , both upregulated in cardiac hypertrophy . this study underscores leptin 's ability to induce ventricular hypertrophy at concentrations well within those of obese individuals , proposing a potential direct link between obesity - associated hyperleptinemia and increased risk of cardiovascular diseases , particularly those associated with hypertrophy . leptin is thought to induce cardiomyocyte hypertrophy through its signaling via the pi3k - akt and map kinases such as erk1/2 and p38 [ 14 , 43 , 68 , 69 ] . transgenic mice with dominant - negative mutants of pi3k in their myocardia had smaller hearts than controls , while those expressing constitutively active forms of pi3k or akt had larger hearts than controls [ 68 , 69 ] . also , rapamycin , which interferes with pi3k signaling , has been shown to attenuate cardiac hypertrophy . inhibitors of erk 1/2 and p38 activation have also been shown to reduce leptin - induced cardiomyocyte hypertrophy [ 14 , 43 ] . a human study was done by perego et al . to examine leptin 's role in developing lvh by calculating lv mass in obese individuals undergoing bariatric surgery of laparoscopic adjustable gastric banding ( lagb ) . lagb is a safe , minimally invasive surgery that reduces the size of the stomach in order to induce weight loss . the patients they chose for this study were morbidly obese and normotensive , to rule out the possibility of hypertension - induced lvh . as expected , both leptin levels and lv mass were significantly higher in the obese individuals as compared to the lean controls . univariate regression analysis showed a significant association between lv mass and bmi , leptin concentration , insulin , and homa index . in addition , lv mass correlated with blood glucose and blood pressure as evaluated by electrocardiogram and electrocardiography , respectively . however , when each of these factors ' contribution to lvh was individually studied , only leptin was found to be a significant determinant of lv mass increase , independent of age , gender , bmi , homa index , insulin , and glucose . obese patients who underwent the lagb were reevaluated a year later , after their bmi had decreased by an average of around 20% . the lv mass of these patients decreased by about 12% , leptin by almost 47% , insulin by around 49% , and homa index by 56% . however , the reduction in lv mass only correlated with the decrease in leptin levels at simple regression analysis . at multiple regression analysis , was done using normotensive obese patients in order to rule out the possibility of hypertension - mediated lvh and to focus on an excess amount of leptin in developing lvh . another study by paolisso et al . was directed at examining the role of leptin in lvh development in insulin - resistant hypertensive patients who were not necessarily obese . chronic leptin administration increases blood pressure and heart rate through sympathetic nervous system stimulation [ 73 , 74 ] , which is involved in the pathogenesis of lvh . they found that fasting plasma leptin levels were significantly higher in hypertensive subjects than in normotensive controls , with no change in these results after adjustment for bmi . the lv mass was greater in hypertensive patients according to echocardiographic evaluation . using multiple linear stepwise regression analysis , plasma leptin concentration was significantly and independently associated with the increase lv wall thickness in hypertensive patients . lv wall thickness has been positively correlated with insulin resistance in hypertension , and leptin has been associated with insulin resistance . although the correlation between plasma leptin levels and lv wall thickness might be driven by insulin resistance , leptin remained independently associated with lv wall thickness in the multivariate model even after adjusting for insulin action . this suggests that leptin 's role in promoting lvh is at least partially independent of insulin action . attributed leptin 's hypertrophic actions to its ability to activate the sympathetic nervous system , which in turn can lead to lvh in hypertensive patients [ 75 , 78 ] . the involvement of leptin in promoting atherosclerosis is still controversial , with many researchers supporting leptin 's role as an atherogenic factor , while others studying its antiatherogenic properties . have shown that leptin is secreted as a result of the hypertension - mimicking mechanical stretch of the rat portal vein , suggesting that leptin expression is increased in hypertension . they also observed that leptin directly leads to hypertrophy of the rat portal vein wall . with respect to the molecular mechanisms underlying the leptin - induced vascular remodeling , different pathways and signaling cascades are involved , such as the rhoa / rock pathway , pi3k / akt pathway , and the map kinases [ 8 , 51 ] not only are leptin concentrations directly increased as a result of hypertension , leptin itself contributes to the development of hypertension . rodents exposed to intravenous infusion and intracerebroventricular leptin administration demonstrated increased arterial pressure and heart rate [ 74 , 79 ] , while blocking the adrenergic system diminished leptin - induced hypertension . other mechanisms , besides increased sympathetic activity associated with hyperleptinemia , may also be responsible for the development of obesity - related hypertension . for instance , leptin leads to the secretion of proinflammatory cytokines , such as tnf- and il-6 , and to the generation of reactive oxygen species ( ros ) in endothelial cells [ 81 , 82 ] , both promoters of hypertension . leptin has also been shown to augment the release of the vasoconstrictor endothelin-1 ( et-1 ) primarily in endothelial cells , but also in cardiomyocytes , fibroblasts , and macrophages , also leading to an elevation in blood pressure . during hypertension , ros production this in turn leads to oxidative stress and the growth , migration , and hypertrophy of vsmc . leptin is secreted in high concentrations as a result of the mechanical stretch associated with hypertension , and leptin alone is able to increase ros generation [ 81 , 86 ] . hence , under conditions of hypertension and obesity , leptin concentrations are augmented , leading to an increase in ros production and oxidative stress , which in turn lead to dysfunction and vascular remodeling through oxidative damage . another vascular effect of leptin is its ability to promote angiogenesis . for angiogenesis to occur leptin promotes this process in endothelial cells by upregulating expression of vascular endothelial growth factor ( vegf ) and inducing actin cytoskeleton reorganization . leptin also contributes to vascular smooth muscle cell proliferation and migration through upregulating the serine / threonine kinase akt , activating erk1/2 [ 92 , 93 ] , and promoting reorganization of the actin cytoskeleton through the rhoa / rock pathway . leptin also increases the expression of matrix metalloproteinases ( mmps ) , thereby inducing vascular basement membrane degradation and modification of the extracellular matrix , both key events in angiogenesis [ 94 , 95 ] . moreover , leptin - induced ros generation also contributes to angiogenesis through the ability of ros to promote lipoprotein lipase expression from macrophages and vegf secretion by endothelial cells and vsmc . , leptin has been implicated in the development of atherosclerosis due to the presence of leptin receptor in the different compartments of atherosclerotic lesions . these include endothelial cells , vascular smooth muscle cells , macrophages , and foam cells . atherosclerosis occurs with neointimal formation , which is the thickened layer of cells that have proliferated and migrated . leptin is believed to cause atherosclerosis by promoting the proliferation and migration of vascular smooth muscle and endothelial cells , thus inducing neointimal growth [ 90 , 100 , 101 ] . leptin induces proliferation of vascular smooth muscle cells by a mechanism involving stimulation of phosphatidylinositol 3-kinase ( pi3k ) activity , activation of mitogen - activated protein ( map ) kinases , and progression to s and g2/m phases . it promotes the migration of vascular smooth muscle cells by activating the rho / rock pathway which promotes reorganization of the actin cytoskeleton . leptin further leads to neointimal growth by stimulating platelet aggregation , activating monocytes , and regulating the immune response [ 106 , 107 ] . moreover , leptin promotes oxidative stress [ 81 , 86 ] , which in turn leads to vascular smooth muscle and endothelial damage . this leads to the deposition of lipids within blood vessel and adhesion of macrophages and lymphocytes [ 108110 ] . following atherogenic high fat diet , wild type mice exhibited significant neointimal thickening after carotid artery injury . on the other hand , ob / ob mice , although obese and hyperlipidemic , did not have a significant increase in neointimal thickness even after feeding on a high fat diet and being exposed to vascular injury . the wild type and ob / ob mice were then treated with leptin daily for 3 weeks , which lead to a dramatic increase in lesion size and the severity of luminal stenosis after arterial injury , regardless of whether the diet was normal chow or a high fat diet . also , the vascular lesions formed in response to injury showed strong expression for the leptin receptor mrna in the endothelial cells , vascular smooth muscle cells , and macrophages , indicating that leptin indeed was mediating its effect on these different components of the neointima . paradoxically , some researchers believe that leptin reduces atherosclerosis . in a study that involved ins2:apoe mice which developed type 1 diabetes , hypercholesteremia , and atherosclerosis spontaneously , severe leptin deficiency was seen compared to nondiabetic ins2:apoe mice . at 13 weeks of age leptin therapy significantly decreased plasma cholesterol concentrations by around 41% , mainly in ldl fractions . it also substantially reduced aortic atherosclerotic lesion area in the ins2:apoe mice by almost 62% . this study proposed that leptin treatment could improve dyslipidemia and thus attenuate atherosclerosis in cases of type 1 diabetes . however , it does not directly prove that leptin could attenuate atherosclerosis , in nondiabetics per se . adiponectin , also termed adipocyte complement - related protein of 30 kda ( acrp30 ) , adipoq , apm1 , or gbp28 , is an adipokine produced and secreted exclusively by both white adipose tissue ( wat ) and brown adipose tissue . it accounts for around 0.01% of the total plasma protein in humans . in healthy lean individuals adiponectin level negatively correlates with cardiovascular and metabolic disorders [ 112116 ] , indicating adiponectin 's important role in the cardiovascular system . in contrast to other adipokines such as leptin , the levels of adiponectin in the plasma correlate inversely with adiposity and directly with insulin sensitivity [ 112 , 113 , 117 , 118 ] . as such , high adiponectin concentrations in the plasma are needed to perform normal physiological actions in the cardiovascular system . adiponectin possesses an oligomeric form [ 119 , 120 ] which correlates with its physiological activities and consequently attracts further characterization and elaboration of its structure . the gene that encodes for human adiponectin is present on chromosome 3q27 , a locus that is associated with diabetes and other cvds [ 120 , 122 ] . adiponectin can form a wide range of multimer complexes and exists in three oligomeric forms : a low molecular weight ( lmw ) trimer , a middle molecular weight ( mmw ) hexamer , and a high molecular weight ( hmw ) multimer of 1218 monomers [ 123125 ] . the hmw complex is the functional unit and induces anti - inflammatory , antiatherogenic , and antidiabetic effects that protect against cardiovascular and metabolic disorders . adiponectin binds to a number of receptors , most importantly the adiponectin receptors ( adipor1 and adipor2 ) and t - cadherin . different mice models with targeted deletion of adipor1 or adipor2 genes are defective in exhibiting adiponectin actions , indicating the important role of adipor1 and adipor2 in mediating adiponectin signaling . adipor1 is ubiquitously expressed , including the cardiovascular system , whereas adipor2 expression is high in the liver . adipor1 is expressed highly in the heart compared to adipor2 . upon binding to its receptors , adiponectin activates several signaling pathways , such as ampk and ppar- , and modulates gluconeogenesis and fatty acid oxidation [ 127 , 128 ] . indeed , several preclinical studies demonstrated the important role of adipors in enabling adiponectin to carry out its physiological and metabolic functions [ 129131 ] . deletion of adipor1 blocked the adiponectin - mediated phosphorylation of ampk , while adipor2 gene deletion increased adiposity and glucose intolerance , presumably due to increased gluconeogenesis . adipor2-null mice demonstrated a hindered adiponectin - mediated ppar- activation and unlike adipor1-deficient mice showed resistance to diet - induced glucose intolerance . t - cadherin is present in adiponectin - targeted sites including the heart , vsmc , and endothelial cells . it has been hypothesized that both adipors and t - cadherin can act together to regulate adiponectin signaling in certain cells and tissues . t - cadherin has been found as a crucial factor for adiponectin mediated cardioprotection in preclinical mice studies . indeed , it was found that the expression of t - cadherin was abundant in the myocardium where it provided protection from pathological cardiac remodeling induced by stress . t - cadherin - null mice showed an increased adiponectin level in the blood due to the diminished binding of adiponectin to its receptor sites on the heart . data obtained from different studies using both animal models and in vitro studies have demonstrated the multiple beneficial effects of adiponectin on the cardiovascular system , through direct and indirect actions on both cardiac and vascular cells ( figure 2 ) . left ventricular hypertrophy and its progression result from structural and functional cardiac disorders impairing the heart 's ability to fill up with blood easily or to pump blood out efficiently ; in both cases the body does not receive enough blood to meet its demands , hence interfering with its function . several signaling pathways influence the cardiac hypertrophy manifestation including ros formation , mapk , and ampk pathway activation [ 135 , 136 ] . adiponectin plays an important role in protection against cardiac remodeling by attenuating myocardial hypertrophy . however , extensive knowledge about the mechanisms involving the relationship between low adiponectin levels and the development and progression of cardiac hypertrophy is still lacking and requires further examination . the heart usually responds to mi through cardiac remodeling by changing the shape , size , and function of the heart at the infarct site . treatment with exogenous adiponectin significantly reduced the mi size in mice hearts subjected to ischaemia / reperfusion . t - cadherin was shown to mediate the protective role of adiponectin against ischaemia / reperfusion cardiac injury . this protective action was linked to the attenuation of ros levels and tnf- and the activation of ampk and cox-2 . studies revealed that , under physiological condition , adiponectin exerts its beneficial effects via increased no production from enos . however , under pathological states , adiponectin inhibits inos and thus decreases no release and promotes cardiac injury . in addition to its effects on cardiomyocytes , many studies demonstrated that adiponectin acts directly on vascular system and has protective effects against different vascular disorders , such as endothelial dysfunction , atherosclerosis , and hypertension . indeed , through no , adiponectin was shown to exert many physiological actions on the vascular system , such as prevention of atherosclerosis , inhibition of vsmc proliferation , and regulation of vascular contraction and blood pressure . moreover , adiponectin selectively binds to different growth factors , such as heparin - binding epidermal growth factor - like growth factor and platelet - derived growth factor bb , thus attenuating their binding to their receptors [ 150 , 151 ] . adipors are expressed in platelets , and in vitro studies performed on human platelets showed that adiponectin inhibits platelet aggregation following collagen induction . another important function of adiponectin is its anti - inflammatory effect which is attributed to its ability to activate ampk and other non - ampk mechanisms . this leads to the inhibition of nfk and consequently reduces the expression of adhesion molecules and the release of il-8 following tnf- stimulation [ 116 , 152 , 153 ] . leptin is associated with obesity and is a potential contributor to many of the cardiovascular risks linked to obesity . it promotes hypertension , vascular remodeling , ros generation , angiogenesis , atherosclerosis , and sympathetic nervous system stimulation ( see figure 1 ) . studies have shown that this hormone can predict myocardial infarction independently of conventional risk factors , and it is an independent predictor of myocardial infarction in patients with arterial hypertension . on the other hand , leptin has been shown to have protective actions on the cardiovascular , renal , and gastric systems in ischemia / reperfusion injury , so labeling leptin as a strictly harmful hormone is not quite fair . obese individuals are resistant to leptin , so some might argue that the leptin - associated harm on the cardiovascular system is due to leptin 's inability to elicit its effects appropriately . hence , further studies need to be done on leptin and specifically leptin resistance , in order to better understand leptin 's function in obesity and promotion of cardiovascular diseases . while some researchers studied and demonstrated that leptin could possibly attenuate and reverse lvh , most studies have shown that leptin actually contributes to lvh progression . several factors of lvh have been attributed to leptin , such as activation of the pi3k - akt pathway and the map kinases erk 1/2 and p38 . leptin 's action on the sympathetic nervous system could also contribute to the development of lvh . further studies need to be done on leptin 's role in lvh in order to fully explain the pathophysiology of this form of myocardial remodeling . it is enticing to study whether leptin could play a therapeutic role in the myocardium in cases of heart failure and ischemia . studies have revealed that adiponectin preserves the normal physiology of the heart by protecting the heart and blood vessels against atherosclerosis , inflammatory , and oxidative stress . with further studies focusing on adiponectin 's beneficial actions , | leptin and adiponectin are differentially expressed adipokines in obesity and cardiovascular diseases .
leptin levels are directly associated with adipose tissue mass , while adiponectin levels are downregulated in obesity .
although significantly produced by adipocytes , leptin is also produced by vascular smooth muscle cells and cardiomyocytes .
plasma leptin concentrations are elevated in cases of cardiovascular diseases , such as hypertension , congestive heart failure , and myocardial infarction . as for the event of left ventricular hypertrophy ,
researchers have been stirring controversy about the role of leptin in this form of cardiac remodeling . in this review ,
we discuss how leptin has been shown to play an antihypertrophic role in the development of left ventricular hypertrophy through in vitro experiments , population - based cross - sectional studies , and longitudinal cohort studies .
conversely , we also examine how leptin may actually promote left ventricular hypertrophy using in vitro analysis and human - based univariate and multiple linear stepwise regression analysis . on the other hand , as opposed to leptin 's generally detrimental effects on the cardiovascular system , adiponectin is a cardioprotective hormone that reduces left ventricular and vascular hypertrophy , oxidative stress , and inflammation . in this review
, we also highlight adiponectin signaling and its protective actions on the cardiovascular system . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
lectins are carbohydrate - reactive ligands that occur commonly in nature [ 1 , 2 ] . these are proteins and glycoproteins with carbohydrate - specific ligand activity that are heat sensitive and dependant on divalent cations . lectins react with sugars in glycolipids , glycoproteins or oligosaccharides [ 13 ] , and agglutinate erythrocytes via cell surface glycoproteins and glycolipids [ 1 , 2 ] . agglutination by lectins in vitro can be specifically inhibited with complementary simple sugars or oligosaccharides [ 1 , 2 ] . lectin haemagglutinins have been described in various species in the family panuliridae ( lobsters ) . a haemagglutinin from the freshwater lobster pacifastacus leniusculus had high specific activity against trypsinized rabbit erythrocytes and was inhibited by sialoglycoproteins such as mucin , fetuin , and ovalbumin [ 46 ] . although glycolipids appear to be at low levels on the surface of rat erythrocytes , there is enhaced exposure of glycolipids after treatment with proteolytic enzymes . agglutination activity for many of these lectins is enhanced by proteolytic enzyme treatment , providing evidence that these lectins react preferentially with glycolipids rather than glycoproteins . there is also an example of a plant lectin that reacts more strongly with proteolytic enzyme - treated rat red cells ; the solanum tuberosum ( potato ) lectin . this lectin that reacts preferentially with rat erythrocytes is most efficiently inhibited by a small oligosaccharide , n n n n-tetra - n - acetylchitotetraose . this lectin is also a strong agglutinin for rat erythrocytes that have been treated with proteolytic enzymes [ 1 , 9 ] . when a suitable substrate is available , purification strategies based on affinity chromatography provide a convenient single - step purification . there have been a number of reports of isolation of invertebrate lectins by affinity chromatography with fixed erythrocytes as a substrate . ochoa and kristiansen reported a similar procedure , using of red cell stroma as an affinity adsorbent . more specific techniques have included the use sepharose - colominic acid affinity chromatography to purify a sialic acid - specific lectin ( lag1 ) . this report describes the characterisation of a lectin from panulirus cygnus with selective reactivity with human abo group a red cells and with rat red cells . the properties and sugar reactivity of lectin are similar to characterised antibacterial lectins and it hypothesised that in this animal , this lectin may contribute to antibacterial activity in the haemolymph . lobsters of the species panulirus cygnus ( george , 1962 ) were trapped in waters near perth , western australia . within 12 hours of capture , an 18-gauge needle was inserted into the haemocoel , and haemolymph withdrawn into a 50 ml syringe . the haemolymph was allowed to clot , the exudate collected and stored in 1 ml portions at 20c . human type a , b , and o erythrocytes collected into edta were obtained from the australian red cross blood transfusion service . erythrocytes from chicken , dog , cat , horse , sheep , rat , and guinea pig collected in edta by venipuncture or cardiac puncture of clinically normal animals were obtained from a diagnostic veterinary laboratory . erythrocytes were washed and made up as 2.5% ( v / v ) suspensions in 100 mmol / l phosphate - buffered saline ( pbs ) ph 7.2 , containing 100 mmol / l cacl2 , 20 mmol / l mgcl2 and 15 mmol / l bovine serum albumin . papain was obtained from csl ( parkville , victoria , australia ) and erythrocytes treated using standard procedures recommended by the manufacturer . briefly , erythrocytes were washed 3 times in pbs and 500 l of packed erythrocytes incubated for 15 min at 37c with 500 l of a 1% ( w / v ) papain solution in 200 mmol / l acetate buffer the papainised erythrocytes were diluted in 100 ml of pbs and used immediately for ha tests . serial 1 : 2 dilutions of lectin were made in 25 l of pbs and 25 l of a 0.5% erythrocyte suspension added to each dilution . after 30-minute incubation at room temperature ( 2025c ) , haemagglutination was observed and scored on a 1 + to 4 + scale : a 1 + reaction was scored when erythrocytes were in small clumps but all of the erythrocytes were involved . the titre was interpreted as the highest dilution which produced at least 1 + agglutination of the 25 l volume used in the test . arbutin , cellobiose , n - acetylgalactosamine , -methylgalactoside , n - acetylglucosamine , glucose , a - methylglucoside , lactose , maltose , mannose , -methyl - mannoside , melibiose , raffinose and sucrose , all in the d - configuration , and l - fucose were purchased from the sigma chemical company ( st . louis , usa ) were made up as 100 mmol / l solutions in pbs . inhibition of ha by sugars was determined in v - bottom 96-well plastic microtitre trays , by mixing 25 l of 1 : 2 serial dilutions of sugar with 5 rat erythrocyte - ha units of haemolymph or affinity - chromatography purified lectin in 25 l of pbs . after incubation at room temperature for 30 mins , 25 l of a 0.5% suspension of rat erythrocytes was added . the results reported were a mean of 3 determinations , for the concentration of the sugar at which complete inhibition of ha was observed . blood from 4 rats was pooled , washed , and made up as 5% suspension . five ml of this suspension were added to 3 ml of con a - sepharose ( pharmacia fine chemicals , uppsala , sweden ) and incubated for 2 hours at 4c on a rotating mixer . 20 ml of pbs were added to the suspension , which was centrifuged at 100 g for 5 min and the supernatant discarded . to lyse erythrocytes bound to the con a , 5 ml of a 1% solution of triton - x 100 ( bdh chemicals , poole , uk ) were added to the pellet and the suspension was shaken for 1 min . the triton - x was then removed by washing 5 times with 20 ml of pbs as described above . the rat erythrocyte membrane proteins were fixed to the con a - sepharose and the unreacted con a inactivated by incubation at room temperature for 15 min with 10 ml of 10% glutaraldehyde . the glutaraldehyde was then removed by washing 3 times with 20 ml of pbs as described above . this rat erythrocyte membrane- ( rem- ) sepharose immunosorbent was used to purify the panulirus cygnus lectin by affinity chromatography . two ml of the rem - sepharose were poured into a c 10/10 chromatography column ( pharmacia fine chemicals , uppsala , sweden ) and washed with 100 ml of pbs . for affinity chromatography 1 ml of panulirus cygnus haemolymph was passed through the column and the column then washed with 100 ml of pbs . in separate experiments , elution of lectin was attempted with a 100 mmol / l glycine buffer ph 3 , 500 mmol / l urea 125 mmol / l sucrose in pbs or 200 mmol / l solutions of the sugars , n - acetylglucosamine , b - methylglucoside , maltose , n - acetylgalactosamine , lactose , a - methylgalactoside or mannose , in pbs . one ml of each solution was applied to the column , incubated for 10 min at room temperature , the column was then eluted with 5 ml of the same solution at a flow rate of 10 ml per hour and 1 ml fractions collected . the eluted samples were dialysed overnight against 500 mmol / l urea : 125 mmol / l sucrose . portions of the dialysed fractions were diluted 1 : 10 for ha tests with rat erythrocytes , to dilute the urea - sucrose solution . recovery was determined by dividing the total ha activity applied to the column by the total ha recovered . fractions containing ha activity were pooled and stored at 4c in 125 mmol / l sucrose500 mmol / l urea or frozen at 70c and used as a source of purified lectin described in other experiments . a sepharose 4b ( pharmacia - lkb biotechnology , north ryde , nsw , australia ) column 50 cm in length and 2.5 cm in diameter with a flow rate of 8 ml / hour was used for gel filtration . one ml of fresh haemolymph was applied to the column and eluted with a buffer containing 100 mmol / l tris / hc1 ph 8 . fractions of 5 ml were collected , the ha titre with rat erythrocytes and a280 measured , and then equal volumes of 500 mmol / l urea : 125 mmol / l sucrose added to each fraction . the elution volume for molecular weight markers ( feline panleukopenia virus to determine the void volume ; human igm , 960 kilodaltons ; human igg , 150 kilodaltons ; and human serum albumin , 69 kilodaltons ) had been determined previously . molecular size was determined from a graph of v
e versus log10 molecular size . agarose gel electrophoresis of haemolymph was carried out on a gelman ( ann arbor , michigan , usa ) immunoelectrophoresis apparatus with gels prepared on glass slides 5 8 cm using 1% agar in 100 mmol / l barbiturate buffer , ph 8 , with a gel thickness 1.5 mm . a row of wells 1.5 mm in diameter and 2 mm apart were punched in the gel 1.5 cm from the edge of the gel and the wells filled with haemolymph . gels were electrophoresed at 30 milliamps for 1.5 hours using an lkb ( pharmacia - lkb biotechnology , north ryde , nsw , australia ) immunoelectrophoresis apparatus . at the completion of the run , the gels were cut into eight 1 cm slices and the slices ground in 1 ml of pbs with a mortar and pestle , centrifuged at 1000 g for 5 min , and the a280 and ha titre of the supernatant determined . haemolymph and purified lectin were electrophoresed on native and reducing polyacrylamide gels on a bio - rad ( bio - rad laboratories , sydney , nsw , australia ) system using techniques recommended by the manufacturer . gels were stained with coomassie blue and the intensity of bands quantified by scanning with an lkb laser densitometer ( pharmacia - lkb biotechnology , north ryde , nsw , australia ) . the ha titre for fresh haemolymph and purified lectin were determined after heating at 37c or at 56c for 30 min . serial 1 : 2 dilutions of haemolymph or purified lectin were made in 25 ml of both pbs with 1 mmol / l ethylenediaminetetraacetic acid ( edta ) without divalent cations and pbs containing 100 mmol / l cacl2 , 20 mmol / l mgcl2 , and 15 mmol / l bovine serum albumin . an equal volume ( 25 ml ) of a 0.5% suspension of rat erythrocytes , prepared in pbs without divalent cations , was added to each well . after incubation at room temperature for 30 min , ha titres were determined as previously described . five ml of affinity column purified lectin in pbs with ca and mg ions were stored at 4c for 5 days , and 1 ml of the same preparation was stored in pbs with 125 mmol / l sucrose : 500 mmol / l urea for 5 days at 4c . the stored lectins were then centrifuged ( 2,000 g , 15 min ) and the precipitate that was recovered dissolved in 200 ml of a 500 mmol / l sucrose : 2 mol / l urea solution . a 25 ml sample of the redissolved precipitate was diluted 1 : 100 in pbs and the ha activity with rat erythrocytes determined . agglutinated rat erythrocytes to a titre 128 times greater than that for sheep or human erythrocytes ( table 1 ) . after treatment with papain , the titre with rat erythrocytes increased 32 folds ( table 1 ) . chicken erythrocytes when untreated did not react but were agglutinated by p. cygnus lectin after papain treatment ( table 1 ) . there was no significant change in the titres after papain treatment for sheep erythrocytes or human erythrocytes ( table 1 ) . it has been noted in studies of the rat erythrocyte - reactive solanum tuberosum ( potato ) lectin rat erythrocytes that there is little n - acetylgalactosamine associated with surface glycoproteins on rat red cells ( 12 ) and the lectin most likely reacts through n - acetylglucosamine in a -linkage displayed on a glycolipid . the lectin was adsorbed to fixed rat red cells bound to a sepharose support maxtrix . lectin was eluted by n - acetylgalactosamine with a 60% recovery , n - acetylglucosamine with a 115% recovery , glycine buffer ph 3 with a 115% recovery , and 125 mmol / l sucrose : 500 mmol / l urea with 115% recovery ( table 2 ) . no lectin detectable by ha with rat erythrocytes was eluted from the rem - sepharose column by 0.1 mol / l solutions of maltose , -methylgalactoside , lactose , or mannose . a single protein of 67 kilodaltons molecular size was detected by page of affinity - purified lectin . when purified lectin was stored in pbs at 4c for 5 days , a white precipitate developed and complete loss of ha , 000 g , 15 min ) and redissolved in 200 ml of a 500 mmol / l sucrose : 2 mol / l urea solution , only 10% of the original ha activity with rat erythrocytes was recovered . mmol / l sucrose : 500 mmol / l urea , and after storage at 4c for 5 days there was no loss of activity . the ha activity of purified lectin with rat erythrocytes was inhibited by 6.2 mmol / l n - acetylglucosamine , 12.5 mmol / l n - acetylgalactosamine , and 50 mmol / l -methylgalactoside but was not inhibited by 100 mmol / l solutions of mannose , arbutin , cellobiose , fucose , lactose , maltose , melibiose , raffinose , or sucrose ( table 3 ) . the profile of sugars that inhibited ha activity of haemolymph and affinity - purified lectin by sugars was identical ( table 3 ) . lectin activity was detected when pbs with ca and mg ions was used as a diluent , no activity was detected in pbs containing 1 mmol / l edta . when purified lectin was heated at 56c for 30 min , no ha activity with rat erythrocytes remained . when haemolymph was subject to agarose electrophoresis , the major haemolymph proteins migrated in a region of -electrophoretic mobility while the lectin , representing 5.8% of the total protein , was detected in a region of electrophoretic mobility . when haemolymph was subjected to gel filtration on sepharose 4b , the ha activity eluted as a single peak that comprised 4% of the total protein , with an estimated molecular size of 1.8 megadaltons . this activity was well separated from a peak in the elution profile that represented 96% of the total protein , composed of proteins ranging between 100 and 300 kilodaltons molecular size . this was in contrast to that single 67,000 da band that was detected by laser densitometer scanning of polyacrylamide gels following electrophoresis of affinity - purified lectin , suggesting that in haemolymph the lectin existed as a multimeric macromolecule . for the p cyguns anti - a lectin characterised in this study , a 67,000 da subunit was detected under reducing conditons , with 1,800,000 da multimer observed by gel filtration . the purified lectin also autoprecipitated in pbs at 4c , suggesting that there was spontaneous aggregation unless it was stored under chatropic conditions ; in this study with sucrose - urea . the haemagglutinating lectin in p. cygnus haemolymph was heat sensitive and required divalent cations for activity , properties commonly observed for invertebrate lectins . the properties of the p. cygnus anti - a lectin were similar to those of the hemagglutinin from pacifastacus leniusculus . both lectins have been found to have a high specific activity against erythrocytes treated with proteolytic enzymes . the purified pacifastacus leniusculus hemagglutinin was 420,000 da . , which dissociated to monomeric glycoprotein subunits [ 46 ] . in a second report , an n - acetylgalactosamine reactive 11 s macromolecular lectin from homarus americanus ( lag-2 ) was reported to have properties similar to the p. cygnus lectin in that it dissociated subunits of 55,000 da [ 13 , 14 ] . the procedure for preparation of an affinity substrate , with gluteraldehyde as a fixative , was similar to those of desai and allen who reported the use of formalinised erythrocytes and reitherman et al . who reported the use of erythrocyte stroma . it may be possible to identify a more specific substrate using techniques similar to those reported for the lag1 sialic acid - specific lectin or for the potato lectin as it has a similar activity profile . detection of lectins that react with n - acetyl - substituted sugars is common in invertebrate species . a human a rbc agglutinin from marine prawn penaeus indicus , with reactivity enhanced by trypsinisation and a specificity for acetylated aminosugars , was inhibited by salmonella lipopolysaccharide . a lectin from the pearl oyster , pinctada fucata ( martensii ) also has properties similar to the p. cygnus lectin described in this report ; a sugar specificity for n - acetylgalactosamine and a subunit structure with subunits of 20,000 da in aggregates of 440,000 da in haemolymph . yeaton listed 40 invertebrate species with lectins with known sugar specificity and 16 of these were reactive with acetylated aminosugars such as n - acetyl galactosamine . antibacterial activity has also been described for n - acetyl - d - glucosamine or n - acetyl - d - galactosamine specific lectins - tachylectin-2 and tachylectin 3 from the japanese horseshoe crab , tachypleus tridentatus [ 18 , 19 ] . the hemagglutinating activity of tachylectin 3 was strongly inhibited by s - type lpss from escherichia coli o111:b4 and other s - type lipopolysaccharides ( lpss ) from gram - negative bacteria but not by r - type lpss lacking o - antigens . the authors suggest that tachylectin-3 specifically recognizes gram - negative bacteria through the unique structural units of o - antigens . it is not known how many of these also display antibacterial activity that would be expected from their pattern of sugar specificity . antibacterial activity in panulirus cygnus haemolymph , reported as nonspecific and mainly associated with haemocytes , was investigated as an indicator of lobster immune - system status and health condition . lobsters that survived a simulated live - shipment procedure exhibited significantly lower antibacterial activity in haemolymph than did those found dead or weak after holding in a tank , leading to a conclusion that handling stress was associated with high activity levels in lobster haemolymph . lectin reactivity with gram - negative bacteria has been consistently reported for marine invertebrates [ 21 , 22 ] . there are many similarities between the p. cygnus anti - a lectin reported here blood group reactive lectins from other invertebrates that play a role in defence against pathogens . a semiquantitative assay of the anti - a lectin levels by haemagglutination is a convenient rapid test that can be used to measure changes in this haemolymph protein in response to environmental conditions and to investigate whether the reported antibacterial activity is mediated by this lectin . | a lectin detected in haemolymph from the australian spiny lobster panulirus cygnus agglutinated human abo group a cells to a higher titre than group o or b. the lectin also agglutinated rat and sheep erythrocytes , with reactivity with rat erythrocytes strongly enhanced by treatment with the proteolytic enzyme papain , an observation consistent with reactivity via a glycolipid .
the lectin , purified by affinity chromatography on fixed rat - erythrocyte stroma , was inhibited equally by n - acetylglucosamine and n - acetylgalactosamine .
comparison of data from gel filtration of haemolymph ( behaving as a 1,800,000 da macromolecule ) , and polyacrylamide gel electrophoresis of purified lectin ( a single 67,000 da band ) , suggested that in haemolymph the lecin was a multimer .
the purified anti - a lectin autoprecipitated unless the storage solution contained chaotropic inhibitors ( 125 mmol / l sucrose : 500 mmol / l urea ) .
the properties of this anti - a lectin and other similar lectins are consistent with a role in innate immunity in these invertebrates . |
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