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dural arteriovenous fistulas are not rare in japan , especially examples involving the cavernous sinus .
however , reports of large series are few , and the clinical entity is not widely known .
the classical triad [ 2 , 3 ] of pulsating exophthalmos , conjunctival chemosis , and pulsatile - innitus are well - established clinical symptoms of the disease but are not usually present in the majority of the patients as early indicators . the d - ccf may therefore be overlooked , especially the bilateral type .
the purpose of this paper is to clarify the clinical course with a pallet of symptoms in patients with d - ccf , with especial attention to results of radiological studies .
142 patients with dural arteriovenous fistulas were consecutively experienced in our institute from october 1985 to june 2010 , all of whom were defined by angiography .
seventy - six ( 54% ) demonstrating involvement of the cavernous sinus ( cs ) are the subjects of this paper .
sixty - one ( 80% ) were female and 15 ( 20% ) were male , with ages ranging from 40 to 77 years , with an average of 64 years .
radiological findings with bilateral carotid angiography and clinical courses of symptoms were evaluated in detail for each case .
data were analysed by the student t - test ( two - tailed ) for paired values , using the statistical program stat mate iii for windows version 3.19 .
five of the others were classified as barrow type b , and the remaining three demonstrated slow and mild inflow into the cavernous sinus .
all these conservative cases showed no aggressive symptoms like decrease of visual acuity , severe retro - orbital pain , or cranial nerve palsies .
endovascular surgery was performed for the other 65 patients , transarterial embolization ( tae ) for inflowing meningeal branches from the external carotid arteries being the initial approach in all cases . until 1996 ,
polyvinyl alcohol particles ranging from 150 to 200 microns in diameter were mainly used as embolic material . since 1997 , platinum particles of 100200 microns and platinum coils were employed . to avoid complications , provocative testing using 2% xylocaine raging from 30 mg to 60 mg
if the catheter could not be advanced , a platinum coil was used as embolic material .
eighteen patients required transvenous embolization ( tve ) following tae because of residual cortical venous drainage or aggressive symptoms .
one of 2 who received stereotactic radiosurgery ( srs ) in addition to tae and tve has already been reported .
the other received srs after tae without tve because of no available transvenous access routes .
the most common initial symptom was diplopia , evident in 47 patients ( 62% ) ( table 1 ) . in three cases it was transient , and in the others it persisted until admission .
unilateral third cranial nerve palsy was the most frequent , found in 25 , followed by unilateral sixth cranial nerve palsy in 14 .
one of the classical triad , at least was the next most frequent symptom , encountered in 27 .
retro - orbital or / and forehead pain was recognized in 24 , this being unilateral in 22 . in all except 5 , this was transient in the range of 2 hours to 1 month ( average 12 days ) .
two demonstrated persistence for 1 month and the other three for a comparatively long periods with repeated remission and aggravation for 5 m , 5 m , and 7 m until admission . in the majority of patients with cranial nerve palsies , the initial diagnoses were aneurysm , unknown origin and diabetes mellitus . in those with conjunctival chemosis , the initial diagnosis was conjunctivitis . in patients with bilateral conjunctival chemosis and proptosis ,
retro - orbital or / and forehead pain was initially diagnosed as trigeminal neuralgia and migraine . with cranial nerve palsies combined with retro - orbital pain , the initial diagnosis was the tolosa - hunt syndrome .
bilateral carotid angiography was conducted for all 76 patients ( table 2 ) . in fifty seven patients ( 75% ) , branches from the bilateral carotid arteries
bilateral type d by barrow 's classification was most frequent in 38 of 57 .
bilateral cs was noted in 30 ( 39% ) , with the intercavernous sinus ( ics ) affected in 20 ( 26% ) .
ipsilateral cs only was evident in 26 ( 34% ) overall and in 12 of 57 of the bilateral type .
the total 12 with bilateral clinical symptoms comprised 4 with bilateral cranial nerve palsies , 1 with bilateral classical triad , 3 with bilateral cranial nerve palsies and the bilateral classical triad , and three with the unilateral classical triad and contralateral cranial nerve palsy .
nine were classified as bilateral type d by barrow , two as type db , and the other one as type d. in seven , bilateral cs and ics were affected , one with bilateral cs , two with ipsilateral cs and ics .
multiple venous drainage routes were common radiological findings in all of 4 cases presenting with bilateral cranial nerve palsies .
the superior ophthalmic vein ( sov ) was the drainage route in three of these , but pulsating exophthalmos and conjunctival chemosis were not shown .
the clinical symptoms and the time course before arrival at hospital were divided into four groups , as shown in table 3 : type i : cranial nerve palsies only on admission ; type ii : cranial nerve palsies preceded by classical symptoms ; type iii : classical symptoms only on admission ; type iv : classical symptoms preceded by cranial nerve palsies .
the most common symptoms on arrival were type ii , in 27 of 76 patients , all except 6 of whom presented more than 3 months after the initial signs . the next was type i in 25 cases , all but two arriving less than 3 months after development of the initial symptom .
the time until admission in type i ranged from 4 days to 6 months ( mean : 6.7 w 6.0 ) , which was significantly short ( p < 0.01 , g test : ) as compared with type ii ( mean : 25.1 w 23.5 ) . the symptoms on arrival were limited to classical triad ( type iii ) in only 13 patients .
eleven patients initially developed the classical triad followed by cranial nerve palsies ( type iv ) .
in type i , drainage routes ranged from one to eight ( average : 2.6 1.3 ) .
cortical venous drainage was present in 9 of the 25 ( 33% ) . in 22 ( 88% ) ,
sov was the venous drainage route in 11 ( 44% ) and the pterigoid plexus ( pp ) in 8 out of the 9 . in type ii , drainage routes ranged from one to six ( mean : 2.3 1.4 ) .
pp did not attribute as a venous drainage route . in the 13 type iii cases , drainage routes ranged from 1 to three ( mean : 1.7 0.9 ) . all except four had only one venous drainage route , the sov .
cortical venous drainage was only present in one ( 7.7% ) . in the 11 of type iv , drainage routes ranged from one to six ( mean : 2.2 2.0 ) .
sov and/or inferior ophthalmic vein ( iov ) participated as venous drainage routes in all and ips in 4 .
the numbers with venous drainage and cortical vein involvement among types i~iv did not show statistically significant variation .
unfortunately , one patient died of lung infarction caused by a deep venous thrombosis from the lower limb . in only one patient , the sixth cranial nerve palsy persisted despite complete disappearance of the dural arteriovenous fistula after tae , as defined by long - term follow - up magnetic resonance angiography ( mra ) .
one other patient with an isolated sinus having only the petrosal vein via the superior petrosal sinus venous drainage route , who rejected additional open surgery , developed subarachnoid hemorrhage followed by srs .
this patient only showed disturbance of consciousness as a severe disability ( sd ) on the glasgow - outcome scale 4/9 .
in 4 of eighteen patients treated with additional tve , the sixth cranial nerve palsy developed between two and four days after the tve treatment . in three patients
this resolved completely 2 months , 3 months , and 11 months later . in 9 of
11 patients conservatively followed - up , all except one of fistulas were completely occluded on mra after 1 month to 13 years 5 months ( average : 5 years 3 months ) .
one patient still exhibited residual fistulas on follow - up angiography 1 year after the initial symptoms .
another was complicated with central retinal thrombosis during the follow - up period and the other 2 patients were lost to follow up . in 47 patients treated with only tae , all but two were followed by mra . in 43 ( 90% ) of 45 , complete obliteration was established on mra after periods ranging from 1 month to 13 years 8 months ( average : 4 years 6 months ) .
the remaining two patients had residual fistulas after 6 year 2 months and 2 year .
both of the patients refused additional treatment because of the lack of symptoms . in all 18 patients treated with tae followed by tve ,
fistulas were completely occluded on follow - up mra after 1 month to 3 years 6 months ( average : 2 years 3 months ) after the treatment . in one patient receiving tae , tve , and srs ,
follow - up mra 10 years and 8 months after the treatment showed complete disappearance of the fistula .
the clinical symptoms pulsating exophthalmos , bruit , and conjunctival chemosis are well known to be a classical triad indicative of carotid cavernous fistula ( ccf ) [ 2 , 3 ] , related to venous drainage routes .
walker and allegre in fact reported that lack of exophthalmos is very rare , while taniguchi et al .
however , we have already presented evidence that dural ccf patients usually do not show the classical triad , sometimes presenting only with cranial nerve palsies , with many venous drainage routes including the cortical . in the present dural ccf series ,
cranial nerve palsies were the most common initial symptoms , the majority being persistent in contrast to an earlier report .
on the other hand , at least one of the classical triad was the initial symptom in only 36% of cases .
newton and hoyt reported unilateral head pain to be the commonest early symptom usually passed off as an unusual migraine attack . in this series also , unilateral headache was frequently recognized mostly transient with a duration of less than 1 month ) .
cranial nerve palsy followed by classical symptoms ( type ii ) was most frequent followed by cranial nerve palsy alone .
multiple venous drainages route may be attributable for symptoms limited to cranial nerve palsies because dispersion of the arterial flow will conceal the classical triad .
in type ii , instead of pp , the sov contributed the major venous drainage route presumably related to occlusion of the pp over time .
the mechanism is obscure but may involve thrombi . with type iii and type iv ,
the sov and/or the iov were the main venous drainage routes without any cortical contribution perhaps because of secondary occlusion of the latter .
serious complications such as intracerebral hemorrhage are very rare with dural avfs of the cavernous sinus , the majority of patients exhibiting an extremely benign clinical course [ 10 , 11 ] . exceptionally , some may feature decrease of visual acuity including central retinal thrombosis and cortical venous reflux evident on angiography .
spontaneous regression in dural ccf is not uncommon and was noted in 5 of 11 cases reported by newton and hoyt , 19 of 26 by sasaki et al . , and three of 18 by vinuela et al . .
recently , tve has been proposed as a more appropriate curative treatment than transarterial embolization ( tae ) , but it may result in serious outcomes like embolic stroke as reported by halbach et al .
reported complication occurring in 7 of 16 patients undergoing tve one featuring epidural extravasation from perforation of the inferior petrosal sinus and the other 6 transient aggravation of symptoms ( chemosis and sixth / third cranial nerve palsy in three each ) .
araki et al . reported extravasation from the uncal vein during tve for sov and iov via the ips and emphasized the importance of obliterating cortical venous drainage as early as possible , even when the reflux is small .
watanabe et al . described a dural ccf patient in whom the cavernous sinus received normal cortical drainage from the insular vein .
post - embolization mri showed cerebral infarction caused by congestion in the area of the posterior insular vein because the anticipated drainage of the insular vein via the uncal vein had not occurred .
nakamura et al . emphasized preservation of sylvian venous flow on finding the affected cavernous sinus to receive not only the shunted flow but also the sylvian venous drainage in three cases ( 12% ) of 26 dural ccfs treated . in all of our series treated with tae , complications fortunately did not occur .
however , in 4 ( 22% ) of eighteen undergoing additional tve , sixth cranial nerve palsy developed between two and four days after the treatment . while it fortunately resolved completely after less than 3 months in two cases and 11 months in one , it improved and persisted in the other .
reported deterioration of oculo - motor dysfunction in two of 9 patients with dural avf involving the cavernous sinus after tve due to two different causes .
high intrasinus pressure caused by blockage of the drainage pathway resulted in the cranial nerve palsy in one . implanted coils directly compressed the cranial nerve in the other . in our series
, sixth cranial nerve palsy developed after several days due to thrombosis around the platinum coils inserted into the posteromedial part of the cavernous sinus .
targeting tve with a minimum of coils may be optimal treatment , but only after tae to reduce the arterial inflow and the size of the affected lesions in our series , bilateral type d by barrow 's classification accounted for half ( 50% ) .
takahashi et al . reported ics involvement in 5 of 8 consecutive patients , all of which could be successfully treated by the whole cavernous sinus packing via the tve .
however , they mentioned difficulty with target embolization via the ips in cases with fistulas at the bilateral cavernous sinuses or posterior intercavernous sinus . in dural ccf patients
, bilateral angiography would appear to be essential given our findings for bilateral cavernous sinuses and also ics .
in dural ccf patients with wide involvement of the sinuses ( type of bilateral cs and bilateral cs and ics ) , especially with a cortical venous reflux , tae first for reduction of inflow initially has been recommended as a reasonable treatment strategy to avoid serious complication because of the comparatively nonaggressive nature of the disease .
described complications occurring in 5 of 38 dural ccf cases undergoing tae , but ibca treatment was an additional factor in four of these .
particles may be safer than liquid emboli and in our series of tae using only particles , no complications occurred . repeated provocative testing and care of dangerous anastomosis are also important .
comparative long follow - up mra ( average : 4 years 6 months ) showed complete obliteration in 93% but additional tve is needed for residual fistulas , especially in cases still having cortical venous drainage .
this can be curative as evidenced by complete occlusion of fistulas in all 18 of our patients treated with tae followed by tve .
in our series of dural ccf patients , the most common initial symptom was cranial nerve palsy , most featuring multiple , including cortical , venous drainage .
bilateral carotid arteries often inflow into cavernous and intercavernous sinuses , which should be taken into account in choice of therapeutic strategy . | introduction .
the purpose of this paper is to clarify the clinical course , with the dural carotid cavernous fistula ( ccf ) , featuring a pallet of symptoms , paying special attention to radiological findings
. methods .
seventy - six consecutive patients with dural ccfs were investigated in detail , all of whom were defined by angiography . results .
the most common initial symptom was diplopia in 47 patients ( 62% ) and the most frequently observed on arrival were type ii , featuring cranial nerve palsies followed by the classical triad in 27 , and then type i only with cranial nerve palsies .
the time until admission with type i ( mean : 6.7 w 6.0 ) was significantly shorter than that with type ii ( mean : 25.1 w 23.5 )
. branches from bilateral carotid arteries widely inflowing into bilateral carotid cavernous sinus were present in 30 ( 39% ) , 20 ( 26% ) of which also demonstrated direct inflow into the intercavernous sinus .
type i and ii had more multiple venous drainage routes as compared with type iii ( classical triad only on arrival ) and iv ( initial development of the classical triad followed by cranial nerve palsy ) .
conclusion . in our series of dural ccf patients ,
the most common initial symptom was cranial nerve palsy , mostly featuring multiple venous drainage including cortical drainage .
such palsies should be added to the classical triad as indicative symptoms .
bilateral carotid arteries often inflow into cavernous and intercavernous sinuses , which should be taken into account in choice of therapeutic strategy . |
disease mapping contains a set of statistical methods , which leads to generate accurate maps based on estimations of incidence , prevalence , and mortality of diseases .
the oldest example of disease mapping is the address of cholera victims based on distance from water resources , which was presented in 1854 by john snow . due to importance of geographic distribution of disease rates in determination of risk factors ,
the most important aims of disease mapping contains describing spatial variation in prevalence of diseases in order to formulate hypothesis about causes of diseases , specification of high risk areas which needed increased notice and interference , generating an accurate map of disease risk in area for the purpose of better resource allocation , and risk estimation as well as producing disease atlase .
there are different models and methods to develop maps of diseases including simple statistic illustration , informal methods , basic models , bayesian models , multilevel models , etc .
bayesian approach to disease mapping consists of prior information about variation in disease rates , in addition to observed events in each area .
it also could consider spatial pattern of disease in which closer geographic areas have more similar disease rates .
bayesian models in disease mapping comprise variously wide range with every one of them has its own formulation , characteristics , advantages , and disadvantages .
several studies were conducted about comparison of these models . however , except a few and limited studies , disease mapping models were not compared , and their general condition was not investigated .
clayton and kaldor made comparisons about a limited set of models ( empirical bayesian and full bayesian models ) .
they concluded that in spite of some differences among relative risk estimations in all methods , the order of relative risk is similar in the whole mapping area .
other efforts have been carried out to find the sensitivity analysis of full bayesian models that are confined to a limited range of bayesian models .
regarding the importance of empirical and full bayesian models , this paper describes and compares three bayesian models in study of relative risk estimation of suicide in counties of ilam province during 18 months from march of 2007 till october of 2008 .
it can begin from safe and preventable behavior and end up to sever and unpreventable ones . according to the world health organization report ,
the worldwide number of suicide attempts leading to death was one million people annually , and the number of suicide commitment was 20 - 30 times more than this .
it is also estimated that around 1,530,000 million people will die due to suicide , and 10 - 20 times more than this figure will commit suicide by the year2020 .
it means that , averagely , one death due to suicide per 20 seconds and one suicide commitment per one to two second will happen by the year 2020 .
lithuania and russia have the first ranks of suicide in the world . in these countries
46 and 41 suicidesper 100,000 ( one hundred thousand ) people have been committed suicide in these countries respectively .
suicide as a social anomaly has a high prevalence in iran too . according to reports from welfare organization of iran ,
international statistics indicates that suicide rate in iran was 9 per 100,000 people , which contributed 1% of total number of deaths .
recently , among all provinces of iran , ilam province has shown one of the highest suicide rates .
this province located in the west of iran contains eight counties , and its population has been 545,787 people in year 2006 .
there are some differences among its counties according to weather , cultural , and social conditions .
in addition , some of the counties near the border of iraq have more post - war problems of 8-year iraq - iran war for a long time . therefore , because of importance of suicide as a complex psychological phenomenon , influenced by individual and environmental factors , investigation and comparison of suicide rate in all counties of ilam province seems necessary and worthwhile to study . concerning the importance of suicide and its high rate in ilam province and existence of only some descriptive reports about suicide incidence , it is essential to investigate geographical variation of suicide incidence rates among counties of ilam province using advanced statistical models .
in this applied ecological study , suicide data of ilam province collected by ilam university of medical science during 18 months from march of 2007 till october of 2008 were analyzed .
relative risk of a disease is calculated by use of proportion of having a disease in a group with exposure to a risk factor to a group without this risk factor . in this study
, we consider relative risk as proportion of the cases committed suicide to expected numbers .
data analysis was carried out using gamma- poisson , log - normal , and bym bayesian models in winbugs software .
estimates from each model were compared with one another as well as with standardized mortality ratio as a classic non - bayesian model .
the results of bayesian models were compared by use of deviance information criterion ( dic ) .
first of all , regarding the importance of these methods , specific properties , and some differences among them , it is essential to explain briefly theoretical principles , advantages , disadvantages , and characterization of each model . the first step to evaluate a disease incidence within an area is to estimate the expected rate of incidence in that area .
the proportion of observed cases to expected one is called standardized mortality ratio ( smr ) . in studies related to diseases incidence , we use the proportion of observed to expected disease incidence , which is called standardized incidence ratio ( sir ) .
this ratio is an estimation of relative risk within each area . in order to utilize this method ,
the map should be divided into n non - overlapped adjacent areas ( i = 1 , 2 , ,
oi and ei are the number of observed and expected events in i area , respectively . during the study ,
ei was assumed constant and known and calculated by multiplying the overall crude incidence rate by the area population or its estimation .
, it is assumed that the number of events between areas are independent and follow poisson distribution with mean eii .
therefore , the likelihood for oi is calculated by : also , logarithm of likelihood function is : so , the estimation of maximum likelihood is : although use of smr and sir is so common , there is some substantial shortcoming related to these estimations . to overcome these difficulties ,
hierarchical bayesian models play an important role in modeling the complexity of data structure in spatial epidemiology and can overcome smrs shortcomings .
in addition to observed numbers , bayesian approach in disease mapping contains prior information about geographical variation of disease rates in whole map . it can be also considered disease spatial correlation ( the tendency of nearer areas to the same rates of disease ) .
this model assumes that the number of deaths in areas follow a poisson distribution with mean eii . the prior distribution for relative risks is also considered gamma ( a , b ) .
to obtain posterior distribution , gamma prior distribution for relative risks is combined with poisson likelihood .
so , relative risk have posterior distribution gamma(a + oi , b + ei ) with mean which .
thus , posterior mean for area i is weighted average of smr for area i and overall relative risk that weights are conversely related to smr variance .
if the observed and expected counts are high , the estimator tends to smr , but when they are low , it tends to prior mean .
2 . even if the real distribution of relative risks is gamma , this model can estimate mean and variance using maximum likelihood method , which is more valuable than moment method .
although gamma prior distribution for relative risk is proper from mathematical point of view , it could be limiting because of difficulty with entering covariates .
log - normal model is more flexible than gamma- poisson model for relative risks of disease : in this model , vi is included in order to consider spatial correlation and similarity of adjacent areas .
this model was introduced by clayton and kaldor and extended by besag , york , and mollie , and its formulation is described as : log(i ) = + ui + vi in this model , relative risk parameter is splitted into three components :
: trend component that is overall level of relative risk.ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks .
to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model .
for this component , spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al .
is : that , and if i and j were adjacent , wij = 1 , otherwise wij = 0.vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi )
the prior distribution of this parameter is : vi ~ n ( 0 , v )
: trend component that is overall level of relative risk .
ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component ,
spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al .
is : if i and j were adjacent , wij = 1 , otherwise wij = 0 .
vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi )
the prior distribution of this parameter is : vi ~ n ( 0 , v ) both u and v parameters control variability of u and v. to analysis of full bayesian , the prior distributions should be determined for these parameters .
the first step to evaluate a disease incidence within an area is to estimate the expected rate of incidence in that area .
the proportion of observed cases to expected one is called standardized mortality ratio ( smr ) . in studies related to diseases incidence
, we use the proportion of observed to expected disease incidence , which is called standardized incidence ratio ( sir ) .
this ratio is an estimation of relative risk within each area . in order to utilize this method ,
the map should be divided into n non - overlapped adjacent areas ( i = 1 , 2 , ,
oi and ei are the number of observed and expected events in i area , respectively . during the study , ei was assumed constant and known and calculated by multiplying the overall crude incidence rate by the area population or its estimation
, it is assumed that the number of events between areas are independent and follow poisson distribution with mean eii .
therefore , the likelihood for oi is calculated by : also , logarithm of likelihood function is : so , the estimation of maximum likelihood is : although use of smr and sir is so common , there is some substantial shortcoming related to these estimations . to overcome these difficulties ,
hierarchical bayesian models play an important role in modeling the complexity of data structure in spatial epidemiology and can overcome smrs shortcomings .
in addition to observed numbers , bayesian approach in disease mapping contains prior information about geographical variation of disease rates in whole map . it can be also considered disease spatial correlation ( the tendency of nearer areas to the same rates of disease ) .
this model assumes that the number of deaths in areas follow a poisson distribution with mean eii . the prior distribution for relative risks is also considered gamma ( a , b ) .
to obtain posterior distribution , gamma prior distribution for relative risks is combined with poisson likelihood .
so , relative risk have posterior distribution gamma(a + oi , b + ei ) with mean which .
thus , posterior mean for area i is weighted average of smr for area i and overall relative risk that weights are conversely related to smr variance .
if the observed and expected counts are high , the estimator tends to smr , but when they are low , it tends to prior mean .
the advantages of this method are : 1 . this model estimates full posterior , which allows hypothesis testing and calculates confidence intervals .
2 . even if the real distribution of relative risks is gamma , this model can estimate mean and variance using maximum likelihood method , which is more valuable than moment method .
although gamma prior distribution for relative risk is proper from mathematical point of view , it could be limiting because of difficulty with entering covariates .
log - normal model is more flexible than gamma- poisson model for relative risks of disease : in this model , vi is included in order to consider spatial correlation and similarity of adjacent areas .
this model was introduced by clayton and kaldor and extended by besag , york , and mollie , and its formulation is described as : log(i ) = + ui + vi in this model , relative risk parameter is splitted into three components :
: trend component that is overall level of relative risk.ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account
, the random variable ui which is uncorrelated with other { uj } sis included in the model .
for this component , spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al .
is : that , and if i and j were adjacent , wij = 1 , otherwise wij = 0.vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi )
the prior distribution of this parameter is : vi ~ n ( 0 , v )
: trend component that is overall level of relative risk .
ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component ,
spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al .
is : if i and j were adjacent , wij = 1 , otherwise wij = 0 .
vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi )
the prior distribution of this parameter is : vi ~ n ( 0 , v ) both u and v parameters control variability of u and v. to analysis of full bayesian , the prior distributions should be determined for these parameters .
the first step to evaluate a disease incidence within an area is to estimate the expected rate of incidence in that area .
the proportion of observed cases to expected one is called standardized mortality ratio ( smr ) . in studies related to diseases incidence
, we use the proportion of observed to expected disease incidence , which is called standardized incidence ratio ( sir ) .
this ratio is an estimation of relative risk within each area . in order to utilize this method ,
the map should be divided into n non - overlapped adjacent areas ( i = 1 , 2 , ,
oi and ei are the number of observed and expected events in i area , respectively . during the study , ei was assumed constant and known and calculated by multiplying the overall crude incidence rate by the area population or its estimation
, it is assumed that the number of events between areas are independent and follow poisson distribution with mean eii .
therefore , the likelihood for oi is calculated by : also , logarithm of likelihood function is : so , the estimation of maximum likelihood is : although use of smr and sir is so common , there is some substantial shortcoming related to these estimations . to overcome these difficulties ,
hierarchical bayesian models play an important role in modeling the complexity of data structure in spatial epidemiology and can overcome smrs shortcomings .
in addition to observed numbers , bayesian approach in disease mapping contains prior information about geographical variation of disease rates in whole map . it can be also considered disease spatial correlation ( the tendency of nearer areas to the same rates of disease ) .
this model assumes that the number of deaths in areas follow a poisson distribution with mean eii . the prior distribution for relative risks is also considered gamma ( a , b ) .
to obtain posterior distribution , gamma prior distribution for relative risks is combined with poisson likelihood .
so , relative risk have posterior distribution gamma(a + oi , b + ei ) with mean which .
thus , posterior mean for area i is weighted average of smr for area i and overall relative risk that weights are conversely related to smr variance .
if the observed and expected counts are high , the estimator tends to smr , but when they are low , it tends to prior mean .
the advantages of this method are : 1 . this model estimates full posterior , which allows hypothesis testing and calculates confidence intervals .
2 . even if the real distribution of relative risks is gamma , this model can estimate mean and variance using maximum likelihood method , which is more valuable than moment method .
although gamma prior distribution for relative risk is proper from mathematical point of view , it could be limiting because of difficulty with entering covariates .
log - normal model is more flexible than gamma- poisson model for relative risks of disease : in this model , vi is included in order to consider spatial correlation and similarity of adjacent areas .
this model was introduced by clayton and kaldor and extended by besag , york , and mollie , and its formulation is described as : log(i ) = + ui + vi in this model , relative risk parameter is splitted into three components :
: trend component that is overall level of relative risk.ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component ,
spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al .
is : that , and if i and j were adjacent , wij = 1 , otherwise wij = 0.vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) is introduced that is an uncorrelated overdispersion parameter .
the prior distribution of this parameter is : vi ~ n ( 0 , v )
: trend component that is overall level of relative risk .
ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component ,
spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al .
is : if i and j were adjacent , wij = 1 , otherwise wij = 0 .
vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi )
the prior distribution of this parameter is : vi ~ n ( 0 , v ) both u and v parameters control variability of u and v. to analysis of full bayesian , the prior distributions should be determined for these parameters .
information about ilam counties population , suicide numbers based on 2006 national census conducted by statistical center of iran and registered suicide cases in each county during 18-month study period from march 2007 till october 2008 is shown in table 1 .
population of counties based on national census in year 2006 , the number of suicide cases , and its proportion to each county population , from march 2007 till october 2008 the expected number of suicide cases in each county is calculated by multiplying the provincial crude incidence rate by the county population or its estimation ( third column multiplied by fifth column in table 1 ) .
expected number of suicide from march 2007 till october 2008 for estimating relative risk of suicide , at first , estimation based on standardized incidence ratio as classical non - bayesian model was calculated . according to this estimation , the relative risks in counties from highest to lowest ones are dare - shahr ( 2.311 ) , ilam ( 1.158 ) , dehloran ( 1.074 ) , eyvan ( 0.839 ) , abdanan ( 0.677 ) , mehran ( 0.471 ) , shirvan - cherdavel ( 0.302 ) , and malekshahi ( 0.293 ) .
then , in order to apply and compare bayesian models in estimating relative risk of suicide in counties of ilam province , dates were fitted to gamma- poisson ( gp ) , log - normal ( ln ) , and full bayesian ( bym ) models by use of winbugs software .
estimation of relative risk for suicide in each county was calculated and shown in tables 35 along with standard deviation and confidence interval .
using these results , in addition to compare counties relative risks , we can compare three mentioned models . according to estimations made by fitting these models ,
relative risk estimations of this county using gamma- poisson , log - normal , and bym model were 2.243 , 2.275 , and 2.279 , respectively .
estimation relative risk of suicide in ilam counties using gamma - poisson model estimation relative risk of suicide in ilam counties using log - normal model estimation relative risk of suicide in ilam counties using bym model estimation of relative risk for this county based on three mentioned models were 0.321 , 0.321 , and 0.319 respectively that indicates the lower relative risk than expected and the average rate in this province . regarding
the confidence intervals resulted from the three models , dare - shahr and ilam counties shown to be significantly higher risk rather than average province ( relative risk more than 1 ) and shirvan - cherdavel , malekshahi , and mehran were found to be lower risk rather than the average amount in the province ( relative risk lower than 1 ) .
moreover , the three models were compared by use of dic criterion [ table 6 ] .
this criterion is obtained by summing two other criteria , which represents goodness of fit and model complexity respectively .
although the estimations obtained from gamma - poisson , log - normal , and bym model represented some differences of standardized incidence ratios according to either amounts and counties incidence ranks , they showed approximately similar estimations for suicide relative risk .
these results are in accordance with clayton and kaldor 's findings in comparing bayesian models .
they showed that in spite of some differences among relative risk estimations , the ranks for each area are same in all three models .
in all three models , the highest standard deviation belonged to dare - shahr county , which has the most number of suicides , and the lowest one was seen in shirvan - cherdavel county with the least ones .
one justification for similarities between results of the models could be related to the low number of study areas and registered incidence cases .
if the counts in lower levels such as districts or villages are available , their comparisons will be more valuable than recent results . according to
the estimations resulted from fitting the models , the ranks of ilam province counties from highest to lowest relative risk of suicide are , in turn : dare - shahr , ilam , dehloran , eyvan , abdanan , mehran , malekshahi , and shirvan - cherdavel .
this result is accordance with the only study of geographical variation of suicide incidence in ilam province counties during 1994 - 2001 , which showed that dare - shahr is the highest risk county .
. it could be due to different data registry systems , underestimation in various data resources , and the differences in study periods .
with regard to the importance of suicide incidence and its remarkable mortality ratio in ilam province , and also for the reason of having various suicide registry systems such as ilam university of medical science , welfare organization , forensic medicine , and the police , it is recommended to conduct more comprehensive survey covering the maximum recorded cases , repeat the study in longer period and in more valuable level of counties like districts or villages .
additionally , it 's better to add time term to the study in order to investigate temporal variation along with spatial variation . | introduction : disease mapping includes a set of statistical techniques that provides maps based on estimates of diseases rates .
bayesian ones are the most important models in this field .
they consider prior information on changes in the disease rates in overall map and spatial pattern of the disease .
these include a broad range of models with their own formulation , characteristics , strengths , and weaknesses . in the present study ,
we explain and compare three important and widely - used bayesian models in the study of evaluating relative risk of suicide in ilam province.materials and methods : in this applied - ecological research , suicide incidence in ilam province in 2008 and 2009 was analyzed by use of gamma - poisson , log - normal , and bym bayesian models .
models were fitted to data using winbugs software.results:fitting the three models showed that darehshahr and shirvan - chrdavol had the highest and the lowest relative risk of suicide , respectively ( relative risks based on gamma - poisson , log - normal , and bym models were 2.243 , 2.275 , and 2.279 for dareshahr and 0.321 , 0.321 , and 0.319 for shirvan - chrdavol , respectively).conclusion : despite some differences in estimates , the ranks of relative risks in counties in all three models are the same .
the counties based on the relative risks of suicide from the most to the least are : darehshahr , ilam , dehloran , eyvan , abdanan , mehran , malekshahi , and shirvan - chrdavol . |
a few studies have found that abnormal findings on diffusion - weighted magnetic resonance imaging ( mri ) are useful for diagnosing cerebral fat embolism in the acute stage .
we applied serial mri to a case of cerebral fat embolism with cognitive impairment lasting for 2 months .
although marked resolution of the previous abnormal findings was demonstrated , t2 * -weighted gradient - echo mri revealed multiple tiny lesions .
we suggest that t2 * -weighted gradient - echo mri is useful in defining the clinical severity of patients with cerebral fat embolism .
although such embolisms are diagnosed on the basis of clinical manifestations accompanied by hypoxemia , neurologic dysfunction , and petechiae , their neurologic symptoms are variable and often nonspecific .
there are several reports of magnetic resonance imaging ( mri ) being a useful diagnostic tool for assessing cerebral fat embolism,1,2 with a few reports emphasizing that abnormal findings on diffusion - weighted mri ( dwi ) have significant diagnostic value in acute cerebral fat embolism.3,4 the pathogenesis of disruption of the blood - brain barrier by cerebral fat embolism is still unclear , but it is thought that fat globules occlude the microvasculature , producing necrosis and hemorrhage in the surrounding parenchyma.5 local inflammatory and toxic reactions by free fatty acid causes breakdown of the blood - brain barrier.6 considering that the pathologic findings in cerebral fat embolism are characterized by multiple petechiae and purpura , and that t2 * -weighted gradient - echo mri is a sensitive method for detecting residual hemorrhage , this mri sequence might aid the diagnosis of cerebral fat embolism . to the best of our knowledge , no previous study has emphasized the diagnostic usefulness of gradient - echo mri in patients with cerebral fat embolism .
this report presents a patient with cerebral fat embolism documented by the presence of multiple low signals on t2 * -weighted gradient - echo mri , in whom there was marked resolution of the previous abnormal findings .
a 54-year - old male suffered from bilateral femoral fractures due to a traffic accident .
he was alert and well oriented on admission , but became stuporous with extensor posturing 36 hours after the accident .
the findings of basic blood tests , including arterial blood gas , were normal , and the findings of chest x - rays were also unremarkable .
brain computer tomography ( ct ) performed 3 hours after the deterioration showed normal findings .
mri was performed 10 hours later , with dwi , t2-weighted imaging ( t2wi ) , and fluid - attenuated inversion recovery ( flair ) images showing multiple high - signal lesions in the bilateral hemisphere .
other lesions were observed in the thalamus , basal ganglia , corpus callosum , and occipital cortex ( fig .
three days later , multiple petechiae were detected on the skin of the anterior chest .
his severe cognitive impairment , which included disorientation , apraxia , acalculia , and memory impairment , lasted for 2 months .
follow - up mri performed 50 days after the onset of the cognitive impairment showed marked resolution of previous abnormal high - signal lesions and a few subtle remaining white - matter lesions ( fig .
in addition , t2 * -weighted gradient - echo mri revealed multiple tiny low - signal lesions distributed over the areas where the previous abnormal lesions had been located .
fat embolism syndrome has nonspecific and variable cerebral manifestations , including headache , lethargy , irritability , convulsions , and coma . despite the marked neurologic dysfunction in our patient
there are several reports of isolated neurologic disorder occurring after cerebral fat embolism.7 the history of traumatic bone fractures , delayed onset of a nonfocal neurologic dysfunction , the appearance of petechiae over the anterior chest , and marked abnormal signals over conspicuous sites on mri supported the diagnosis of cerebral fat embolism in this patient .
small , scattered , hyperintense lesions on t2wi and flair imaging have been found previously.2 - 4 these lesions typically appear in the periventricular , subcortical , and deep white matter , as well as in the deep gray matter , including the corpus callosum.8 - 10 the lesions are usually not detected by ct and are more prominent on dwi than t2wi and flair images , especially during the acute stage . these abnormalities in the signal intensity on dwi presumably reflect the foci of cytotoxic edema .
suggested that focal parenchymal hemorrhage is not a predominant feature in t2 * -weighted gradient - echo mri.8,9 however , their cases might not accurately reflect the full spectrum of patients with cerebral fat embolism because they showed relatively good clinical improvement .
the number of white - matter lesions on mri is correlated with the patient 's score on the glasgow coma scale.10 simon et al .
considered that gradient - echo imaging might be useful in determining the severity of cerebral fat embolism , although hemorrhage was not a predominant feature of the multiple white - matter lesions in their reported case.9 neuropsychological testing has revealed obvious cognitive deficits in patients with stenosis of the internal carotid artery and lesions on the periventricular , subcortical , and deep white matter.11 periventricular and subcortical white - matter lesions have been associated with worse performance on all cognitive measures tested.12 the patient in the present study exhibited continuous neurologic impairment , and t2 * -weighted gradient - echo mri showed that the lesions were distributed throughout the white matter of the cerebrum .
reports of serial mri and gradient - echo mri in cases of cerebral fat embolism are rare .
previous studies of this condition found improvement of the neurologic impairment and the gradually disappearance of lesions on mri within a few weeks to a few months .
these findings might reflect immediate cytotoxic edema secondary to ischemia and subsequent vasogenic edema developing at a later stage.4 the patient in the present study presented with persistent cognitive impairment during the follow - up period .
follow - up mri ( t2wi and flair ) showed that some lesions disappeared , with only subtle ill - defined signal changes remaining .
a t2 * -weighted gradient - echo mri sequence is sensitive to hemosiderin deposits in the brain parenchyma and is very useful for assessing the presence of old and new hemorrhage.13,14 gradient - echo mri sequences were not obtained during the initial mri examination in this patient .
however , t2 * -weighted gradient - echo mri in the subacute and chronic stages of the follow - up mri study in this patient revealed prominent multiple low signals , which might explain the residual neurologic impairment .
the findings of the present study suggest that t2 * -weighted gradient - echo mri can be used to analyze the neurologic impairment in patients with cerebral fat embolism , with the findings also closely reflecting the clinical severity .
gradient - echo imaging is useful for detecting the petechial hemorrhage that is the characteristic pathologic finding of cerebral fat embolism .
gradient - echo mri might be useful in defining the clinical severity of cerebral fat embolism with diffuse petechial lesions in the subacute or chronic stage , especially when abnormal signals are not detected in other forms of mri . in conclusion
, mri can be used for the early diagnosis of cerebral fat embolism with high sensitivity .
we suggest that t2 * -weighted gradient - echo mri provides useful information for determining the clinical severity of patients with cerebral fat embolism . | backgrounda few studies have found that abnormal findings on diffusion - weighted magnetic resonance imaging ( mri ) are useful for diagnosing cerebral fat embolism in the acute stage.case reportwe applied serial mri to a case of cerebral fat embolism with cognitive impairment lasting for 2 months .
although marked resolution of the previous abnormal findings was demonstrated , t2 * -weighted gradient - echo mri revealed multiple tiny lesions.conclusionswe suggest that t2 * -weighted gradient - echo mri is useful in defining the clinical severity of patients with cerebral fat embolism . |
dermatitis herpetiformis ( dh ) is an autoimmune skin disorder frequently associated with gastrointestinal disease characterized by intensely itchy , chronic papulo - vesicles , distributed symmetrically on extensor surfaces .
it has also been found associated with a number of other autoimmune illnesses including autoimmune thyroid disease .
pituitary mass mimicking as pituitary macroadenoma has also been reported in cases of primary hypothyroidism.[36 ] we report here a case which had a combination of primary hypothyroidism , pituitary mass mimicking macroadenoma and dh .
a 15-year - old girl , resident of meerut , uttar pradesh , presented with complaints of diminution of vision of left eye since may 2008 and insidious onset , throbbing , progressively increasing headache on the left side since july 2008 . computed tomography of brain in august 2008 revealed a pituitary mass reported as microadenoma . with the advice to get a magnetic resonance imaging ( mri ) for the same , she reported to the neurosurgeon in a tertiary care super - specialty hospital in new delhi in september 2008 .
referral to ophthalmologist revealed distant vision of left eye of 6/60 with pale optic disc suggestive of optic atrophy .
hormonal assay revealed level of t3 of 0.3 ng / ml ( n 0.52 - 1.85 ) , t4 3.0 g / dl ( n 4.82 - 11.60 ) and thyroid stimulating hormone ( tsh ) > 100 lu / ml ( n 0.39 - 6.16 ) suggestive of hypothyroidism .
fsh ( 3.5 u / l ) , lh ( 1.2 u / l ) , gh ( o.1 ng / ml ) and cortisol ( 9.3 mcg / dl ) levels were found to be normal .
patient was diagnosed as a case of primary hypothyroidism and started on tablet thyroxin 150 g / day .
mri of sella revealed well - defined , uniformly enhancing enlargement of the anterior lobe of pituitary gland .
intravenous contrast showed heterogeneous enhancement , the lesion measured 13 ( cc ) 7 ( ml ) 6 ( ap ) mm .
laterally the lesion was encroaching the duramater [ figure 1 ] , opined as pituitary macroadenoma . in the meantime patient also had two episodes of projectile vomiting , hence the endocrinologist opined that ideally the patient should be made euthyroid but in view of the signs of raised intracranial tension she can be taken up for surgery .
the intravenous contrast view below showed heterogeneous enhancement with size of the pituitary being 13 ( cc ) 7 ( ml ) 6 ( ap ) mm the child was again reviewed by the neurosurgeon in october and advised admission for transcranial excision of the pituitary adenoma .
ten days prior to her date of surgery , she developed mildly itchy vesicobullous rash on left side of her neck [ figure 2 ] , gradually spread to involve scalp , face , trunk and limbs with sparing of palms and soles .
tense vesico - bullous rash on erythematous background on left side of the neck in addition , she had attained her menarche at the age of 13 .
the periods were regular though painful initially , became irregular since september 2007 and subsequently she developed secondary amenorrhea since january 2008 .
dermatological examination revealed tense vesicles and bullae over neck , face , trunk and extremities .
few of the lesions had ruptured spontaneously to form superficial erosions , crusted lesions and few healed with hypopigmentation .
histopathology of the bulla revealed split at dermoepidermal junction with collection of neutrophils in the cavity [ figure 3 ] .
immunoflourescence revealed deposits of iga antibodies in granular pattern at the dermoepidermal junction with accentuation at dermal papilla , consistent with the diagnosis of dh .
histopathology of bullous lesion showing split at dermoepidermal junction with collection of neutrophils in the bulla fluid ( h and e stain , 400 ) patient was started on tab prednisolone 10 mg thrice daily for 4 weeks and then gradually tapered over next 2 weeks .
tablet dapsone 100 mg daily was added after 2 weeks of steroids and she was continuing on tab thyroxin . by mid february 2009
repeat mri scans in mid february revealed total regression of pituitary mass [ figure 5 ] .
t3 ( 0.6 ng / ml ) and t4 ( 5.0 g / dl ) levels had normalized , although tsh levels were still high ( 60 iu / ml ) requiring adjustment of dose of thyroxin by the endocrinologist .
clearance of skin rash after treatment mri scans of the brain showing regression of pituitary mass
idiopathic hypothyroidism , triggered by autoimmunity , most commonly affects women in the age group of 30 - 70 .
the trigger or the initiating agent of this autoimmune process is not known . in our case
the primary event was primary hypothyroidism with its onset at the age of probably 14 years , when she had started having her menstrual irregularity in september 2007 .
it was detected only after cd8-mediated damage to the thyroid tissue had destroyed substantial amount of the tissue and it was not able to produce adequate quantities of t3 and t4 hormones .
this was inspite of increase stimulation of hypothalamus and anterior lobe of the pituitary gland producing high levels of tsh through the feedback mechanism .
increased secretion of thyrotropin - releasing hormone ( trh ) by the hypothalamus led to hyperplasia of the anterior lobe of the pituitary gland to such an extent that it mimicked a macroadenoma .
this pituitary mass because of its pressure affect on the optic chiasma causing impaired blood supply led to optic atrophy on the left side , which gave the presenting symptom of diminished vision .
later as the pituitary mass kept increasing in size the symptoms of headache and projectile vomiting appeared .
primary hypothyroidism was detected on routine screening in a case of pituitary macroadenoma by the endocrinologist .
the child was started on thyroxin therapy and endocrinologist gave a go ahead to the neurosurgeon to remove the macroadenoma in view of the signs of increased intracranial tension .
the surgery was planned twice and she was lucky both times . on the first occasion she did not report for surgery due to domestic compulsions and second time when she developed bullous rash just prior to the surgery requiring dermatological referral .
although the patient was put on thyroxin replacement therapy , the autoimmune process was continuing as was manifested by the appearance of rash and demonstration of iga autoantibodies in the lesions of dh .
autoimmune thyroiditis is a cd8 t - cell - mediated disease and these stimulated t - cells may as well be stimulating b - cells to secrete other autoantibodies including the ones responsible for dh . with the introduction of corticosteroids
the autoimmune process was brought under control by decreasing cd8 cell - mediated damage to the thyroid tissueand decrease in autoantibodies by decreased b - cell stimulation , thus both autoimmune thyroiditis and dh improved .
this would have led to some increase in endogenous production of thyroid hormones from the salvaged thyroid acini and along with exogenous thyroxin led to decrease levels of trh from the hypothalamus causing regression of pituitary mass . thus an unnecessary surgery could be avoided .
the presence of pituitary mass mimicking macroadenoma in a case of primary hypothyroidism responding to thyroxin therapy has been reported earlier.[37 ] our case presented with pituitary mass , was found to have primary hypothyroidism and later developed dh , successfully treated with thyroxin , corticosteroids and dapsone . | we report a case of 15-year - old girl who was diagnosed as a case of pituitary macroadenoma on computed tomography and magnetic resonance imaging ( mri ) scans for her complains of diminished vision in the left eye and headache . on investigation
she was found to have optic atrophy of left eye and primary hypothyroidism .
she was started on thyroxin therapy in october 2008 and planned for transcranial excision of the tumor . just before the date for planned surgery she developed mildly itchy vesico - bullous rash and the surgery was postponed .
on histopathology and immunoflourescence studies it was confirmed to be the rash of dermatitis herpetiformis ( dh ) and treatment was started with corticosteroids in the last week of december 2008 and dapsone was added from mid january 2009 .
the lesions responded dramatically and within 6 weeks of starting treatment , she had become asymptomatic except for persisting diminution of vision .
repeat mri scans in mid - february 2009 revealed total regression of pituitary mass .
the case is being reported for interesting association of primary hypothyroidism , pituitary mass mimicking macroadenoma and dh ; its successful outcome with medical management in the form of total regression of pituitary mass , thereby avoiding an unnecessary surgery . |
until recently , surgical management of breast cancer ( bc ) has focused on two main choices : tumor resection using breast conserving therapy ( bct ) and mastectomy with optional tissue displacement by breast reconstruction . from 2003
, the techniques that combine the skill of resection and reconstruction in one procedure were introduced that can be named as the third approach , oncoplastic breast surgery ( obs ) .
this approach involves reconstruction of the resection defects by volume displacement using adjacent breast tissue .
mastectomy includes excision of the breast tissue and is divided into subtypes according to the resection of lymph nodes and muscles .
traditionally , it is employed when the risk of local recurrence is increased by tumor size of greater than 5 cm , presence of lymphovascular invasion , presence of more than 4 suspected nodes , and involved or closed margins .
bct is composed of lumpectomy or wide local excision and axillary dissection with or without radiotherapy .
it was accepted as a surgical option after thorough evaluation in six international prospective randomized trials for early stages of bc ( stages i and ii ) [ 3 , 4 ] .
traditional contraindication to perform bct includes large tumor size ( > 5 cm ) , skin or chest wall involvement , multicentric tumors , or anticipated poor cosmetic outcome , and whenever radiation therapy is contraindicated .
they range from simple reshaping to more complicated techniques involving concomitant contralateral breast symmetrisation procedures . as in many solid tumors , when facing bc , patients and surgeons are focused on three concerns : survival , local control , and quality of life . in this review article
we compare mastectomy , bct , and obs to elucidate the current literature consent from four points of view , including ( 1 ) oncological result , ( 2 ) cosmetic outcome , ( 3 ) quality of life ( qol ) , and ( 4 ) health economy .
a computerized medline search was performed for english language abstracts and english and french articles published between january 2000 and june 2013 on mastectomy , breast conserving therapy , and oncoplastic breast surgery .
the headings were combined with the words including oncological result , local recurrence , survival , cosmetic outcome , quality of life , and health economy .
of note , studies comparing qol between mastectomy and bct that were published before 2008 have been reviewed by pockaj et al .
previously . finally a narrative review was constructed to summarize the comparisons of the three modalities in the four identified perspectives by the expert authors .
it sounds that the oncological result is the most important part of any procedure done for the bc treatment .
oncological results are mostly investigated in two fields : local recurrence and survival . although radical and modified radical mastectomies are both effective , they accompanied a huge psychological burden .
studies demonstrate that the overall disease - free survival from bc is equivalent for mastectomy and bct along with postoperative radiotherapy ; especially in women with early stage bc ( stages i and ii ) .
next we discuss the local recurrence and the survival status associated with each of the bc surgical modalities . in mastectomized patients , a 210% chest wall recurrence has been reported .
so , the national institute of health recognized the equivalency of medical outcome for these two procedures in a consensus development conference statement in 1990 and recommended bct as an appropriate method of primary therapy for most women with early stage bc [ 3 , 8 , 11 ] .
there are factors associated with local recurrence after bct such as young age , positive resection margins ( for both mastectomy and bct ) , multicentric disease , vascular invasion , high - grade tumor , necrosis tumor , infralymphatic extension , and extensive intraductal component .
there are controversial opinions on the optimal extent of resection to obtain tumor - free margins including 2 mm negative microscopic margin versus 1 - 2 cm macroscopic uninvolved tissue .
it seems , that with breast irradiation , as a part of bct , wide tumor - free margins have little practical importance . on the other hand ,
some studies show that the surgical margin significantly affects the 10-year disease - free survival rate and is the most important factor to reduce the risk for local recurrence .
many factors contribute to this decision such as patient 's desire ; as she should undergo daily outpatient radiotherapy over 5 - 6 weeks to a total dose of 50 gy .
other factors include size of the breast ( small breasts are not good candidate for bct ) and tumor size of less than 2.55 cm ( tumors larger than 5 cm are consistent with likelihood of local recurrence ) .
also , there are few cases in which bct is contraindicated for concerns of toxicity or marked risk of local recurrence .
multifocal disease ( two or more gross lesions in different quadrants or diffuse microcalcification on mammography ) , prior radiation therapy , and rheumatologic disorders are relative contraindications to bct .
many experts believe that oncological result is the priority which shall not be exchanged with morphological symmetry and cosmetic outcomes . local recurrence rate after obs has not thoroughly studied yet .
most of the available studies on obs have shortcomings in their methodology which undercuts their conclusive robustness .
however , a number of studies showed 07% local recurrence rate after up to 54 months of followup [ 1431 ] .
studies on the long - term outcome after obs are not available , but the local recurrence rate of 01.8% per year for obs on intermediate followup suggests that these techniques are associated with low local recurrence rates .
new studies have recommended obs as a probable approach for all women undergoing mastectomy for bc .
there are no significant differences between bct with radiotherapy and mastectomy regarding the overall survival rates ( 4095% in five years ) . until now ,
no prospective randomized or retrospective trial study , comparing the survival rate between bct and obs , has been performed .
nevertheless , authors of noncomparative studies have reported that obs results in the same survival rate .
we should consider that obs techniques commonly render the possibility of a simple further excision .
the difficulty of the procedure leaves many patients with involved margins who need further mastectomy , while mastectomy can be avoided in patients with bct .
aesthetic surgery of healthy breasts has become a major industry over the past half century . some of the indications have medical basis , but much of the demand has arisen from more complex psychosocial , economic , and reproductive pressure of human male to human female .
developments in aesthetic breast surgery have encouraged considerable experimentation in immediate or delayed symmetrisation at cancer surgery .
cosmetic reconstruction should not follow geometric patterns but should emphasize on perceived contour and normal clothing lines .
one goal of breast reconstruction is to restore the breast as normally and as attractively while minimizing visible scars .
the understandable advantages of bct are equivalent local and distant control rates as compared with mastectomy .
but the key to a successful bct is achieving a cosmetic outcome that is acceptable to the patient and the physician .
cosmetic outcome is the end result of a range of factors which come together under a broad head of surgery , radiotherapy , chemotherapy , and hormonal treatment . on the other hand , feeling of disfigurement , mutilation , and insult to femininity
are the matter to be feared in modified radical mastectomy [ 37 , 38 ] .
it is obvious that the comparison between mastectomy without reconstruction and the other two methods ( bct and obs ) is not meaningful .
various criteria have been employed in different studies to compare the cosmetic outcome . in breast cancer treatment and outcome
survey ( bctos ) , a 22-item survey instrument was designed to measure the perceived aesthetic and functional status after bct and radiotherapy .
for instance , aesthetic triangle was defined by the sternal notch - nipple ( sn - n ) and nipple - nipple parameters .
numbers of innovating grading systems were introduced by panels of surgeons and nonmedical personnel [ 15 , 37 ] .
briefly , a good aesthetic result nearly means little or no residual asymmetry , minor postradiotherapy after - effects and no dimpling .
investigators had classified breast asymmetry risk factors in bct into two groups : patient - related and treatment - related factors .
patient - related risk factors include young age ( since breast and nipple areolar complex lay progressively infero - lateral ) , high bmi , large tumor size , and resection of superior - medial and inferior - lateral tumors .
the allowed volume reduction is 5% in medial , in comparison to 15% in lateral .
treatment - related risk factors for asymmetry contain reexcision lumpectomy , postoperative seroma and radiation therapy .
extremely small sized breasts and tumor sizes greater than 5 cm may have an unacceptable cosmetic result .
our findings show that the cosmetic result of bct is not always satisfactory to the patients or the surgeons and obs seems to provide a more promising future [ 15 , 37 , 38 , 40 ] .
cosmetic failure rates after bct are approximately 30% ; while this rate is 018% for obs [ 1419 , 2131 , 40 ] .
selecting the appropriate patient according to the known risk factors can prevent frankly poor results in 2530% of bcts .
this includes absolute indications for mastectomy , containing widespread dcis , multifocal tumor , and recurrence . by progressions in the treatment of bc ,
patients prefer to be treated well even by mastectomy rather than choosing a pure aesthetical satisfactory bct .
given that different techniques of obs have been introduced in recent years , the investigations on aesthetic outcomes after obs are very few .
according to these few studies it seems that there are some important advantages for obs in comparison with the other traditional surgeries .
first of all , these techniques have reduced the worries of incomplete excision for surgeons .
a very large excision can be taken with obs along with a good cosmetic result and least deformity .
the effect of radiation therapy as an insult for cosmetic outcome gets the minimal level .
timing of radiotherapy , beam intensity , boost area , volume , and dose are all important in the cosmetic outcome .
the last but not the least important point refers to the fact that the amount of safe margin and the need for reoperation have been reduced in patients operated by these newer techniques .
the limitation of these surveys is that direct comparison between these studies is difficult and also the methods vary both in regard to the surgery and to the cosmetic assessment . role of plastic surgery in surgical treatment of bc has increased during the last two decades and this trend is likely to continue during the next decade [ 11 , 36 ] .
therefore , we recommend a multicentral , longitudinal , and prospective study to standardize a cosmetic outcome assessment and to clarify the indication of each technique .
bc is a common disease with a relatively good prognosis especially when detected in early stages .
improvement in qol can motivate more women to follow screening programs for early diagnosis of the disease .
vast medical literature is available on the qol of patients who underwent mastectomy and bct .
qol is a subjective tool to measure common perception of patients regarding a number of aspects including body image , satisfaction , attractiveness , feeling whole , subjective cosmetic results , appearance , scar , insecurity , sexual problems , postsurgical complications such as lymphedema , arm swelling , limitation in range of motion , psychiatric distress , anxiety , depression , and worrying about the future .
these aspects are associated with other factors like chemotherapy , medical condition , social support , income , education , and so on [ 4 , 7 , 4146 ] . for such purposes
, studies inquired recognized questionnaires like european organization for research and treatment of cancer ( eortc ) , cancer rehabilitation evaluation system short form ( cares - sf ) , short form questionnaire ( sf-30 ) , quality of life questionnaire ( qlq - c30 ) , and hospital anxiety and depression scale ( hads ) . in some articles
randomized clinical trials ( rcts ) have shown no difference in prognosis and survival between bct and mastectomy [ 42 , 47 , 48 ] .
two of the six international trials reported their 20-year followups confirming equal long - term survival and local control with mastectomy when compared to bct [ 3 , 47 , 49 ] . in this regard pockaj and
colleagues comprehensively reviewed medical literature to investigate the qol outcome and factors that influence these outcomes in women undergoing various bc surgical procedures .
summing the results of various studies , we found that qol assessment is divided into two groups : short term and long - term after the procedure .
it seems that long - term evaluation of qol with famous questionnaires is more validated [ 4 , 7 , 42 ] .
it is a common belief that if surgical effect could be established with less - extensive surgery , cosmetic outcome would be improved and the qol would be maintained with minimal attenuation in functional aspects of the operated areas .
many small sample - sized rct surveys showed that the initially expected results of excellent qol in bct patients have not been definitely shown except for body image ( attractiveness , appearance , feeling whole , cosmetic outcome , scar , and insecurity ) in comparison with mastectomy or even breast reconstruction after mastectomy [ 4 , 7 , 43 , 50 , 51 ] .
however , few studies which had long - term evaluations after the operation showed either a statistically significant different or no different results [ 53 , 54 ] . in one of these studies , although there was no statistically significant difference in qol between the two groups , among the patients who were younger than 50 years at the time of diagnosis , bct appeared to be protective against psychiatric distress when compared with total mastectomy .
patients of 50 years of age or older at the time of diagnosis who received bct had higher psychological stress level about fearing of recurrence and deficit of treatment .
some nonrandomized clinical trials declared that being a younger patient with superior socioeconomic status , higher educational background , stable marital relationship , and using bct is linked to a better qol [ 4 , 43 ] and sexuality . on the other hand , most studies showed that mastectomized patients scored constantly worse in the variables associated with body image and were less satisfied with their cosmetic outcome and were more likely to choose a different surgical treatment if they had a chance to do it over again . of note , they had more physical symptoms and discomfort around the surgical site [ 4 , 7 ] .
so we propose that bct carries a lower complication rate , a shorter operative time , an improved body image , and decreased insecurities in intimate relations in comparison with mastectomy . actually , the increasing rate of bct in all age groups is of the major quality indicators as well [ 3 , 55 ] .
the percept that immediate breast reconstruction improves health - related qol underlines the increasing practice of breast reconstruction , internationally .
however , we should take into consideration that the longer time of surgery , the more fat necrosis in obese patients in some type of reduction mammoplasty , and the poor cosmetic outcome in 514% of obss necessitates more rct studies in this field .
nevertheless , newer studies indicate obs as a safe and effective method even as a delayed surgery .
we did not find any study directly comparing mastectomy , bct , and obs from the view of health economy .
previous studies insist on the restricted access of low income women to radiotherapy after bct .
this fact places them at an increased risk of having worse outcome in comparison to those treated with standard mastectomy [ 60 , 61 ] . besides
, lower proportion of bct and obs is performed among the patients without insurance or medicaid support .
it sounds as if plastic surgery all over the world is an expensive operation , but , in comparison with mastectomy and bct , one should consider all the after effects before declaring the most economically advantageous method .
we note a prompt need for economic evaluation studies to be performed in this field .
aesthetic outcome is one of the major concerns of patients and physicians in bc surgery .
high patient satisfaction and proven oncological safety are the advantages of obs when compared to bct and mastectomy .
when performed by trained specialists , obs accompanies a favourable outcome even in settings with inferior resources .
however , the superiority of patient 's administration of cosmetic outcome and qol may decline in long followup .
the long - term results of obs are yet to be understood and the traditional procedures may be more promising in their oncological outcome .
proper patient selection and careful planning are of great importance to achieve an acceptable result [ 56 , 59 ] . | surgical management of breast cancer has evolved considerably over the last two decades
. there has been a major shift toward less - invasive local treatments , from radical mastectomy to breast - conserving therapy ( bct ) and oncoplastic breast surgery ( obs ) . in order to investigate the efficacy of each of the three abovementioned methods , a literature review was conducted for measurable outcomes including local recurrence , survival , cosmetic outcome , quality of life ( qol ) , and health economy . from the point of view of oncological result , there is no difference between mastectomy and bct in local recurrence rate and survival .
long - term results for obs are not available . the items assessed in the qol sound a better score for obs in comparison with mastectomy or bct .
obs is also associated with a better cosmetic outcome .
although having low income seems to be associated with lower bct and obs utilization , prognosis of breast cancer is worse in these women as well .
thus , health economy is the matter that should be studied seriously .
obs is an innovative , progressive , and complicated subspeciality that lacks published randomized clinical trials comparing surgical techniques and objective measures of outcome , especially from oncologic and health economy points of view . |
cardiovascular disease is one of the major causes of morbidity and mortality worldwide , especially among patients of chronic kidney disease ( ckd ) [ 1 , 2 ] .
there is an increasing evidence of cardiometabolic syndrome ( cms ) involvement in ckd [ 3 , 4 ] , but a causal relationship has not been proven .
cardiometabolic syndrome is a cluster of metabolic abnormalities combining obesity with 2 - 3 risk factors which include insulin resistance , hypertension , and high triglyceride or low high density lipoprotein ( hdl ) serum levels
. it also increases the risk of cardiovascular disease ( cvd ) and type 2 diabetes .
it is believed that atherosclerotic changes in blood vessels in chronic renal diseases contribute significantly to the high cardiovascular morbidity and mortality .
morphological and functional abnormalities of the endothelium are considered as prodromal stage of atherosclerosis and early marker of cvd that facilitate the progress of atherosclerosis [ 7 , 8 ] and contribute to the development of hypertension through the enhancement of vascular resistance . on the other hand ,
there is a strong evidence supporting the view that atherosclerosis is a disease characterized by low - level vascular inflammation [ 911 ] .
inflammation , inflammatory action of local stimuli such as products of the oxidation process and glycation end products , oxidative stress , and degradation of extracellular matrix ( ecm ) change vasculature in terms of development of atherosclerosis .
renal disease is associated with a graded increase in oxidative stress ( os ) markers even in early ckd .
some studies have documented that peritoneal dialysis is associated with decreased levels of oxidative stress and inflammatory markers compared to haemodialysis
. a small number of trials have been carried in order to determine associations between oxidative stress with vascular structure and function with equivocal results [ 14 , 15 ] . on
the other hand , uncontrolled expression of matrix metalloproteinases ( mmps ) , enzymes that degrade extracellular matrix ( ecm ) , can result in tissue damage and the development of a number of destructive diseases such as arthritis , atherosclerotic plaque rupture , aortic aneurysm , and progression of tumors [ 16 , 17 ] .
however , the relevance of malondialdehyde as a marker of oxidative stress which is generated by peroxidation of unsaturated fats as well as the role of matrix metalloproteinase-9 in atherosclerosis progression in patients ( pts ) with chronic kidney disease ( ckd ) not yet on dialysis compared to patients on peritoneal dialysis is less known , particularly with respect to cardiometabolic syndrome .
the aim of this paper was to examine whether the serum concentration of malondialdehyde and matrix metalloproteinase-9 is involved in the process of atherosclerosis in patients with ckd not yet dialysis - dependent and those on peritoneal dialysis ( pd ) with signs of cms .
this cross - section study was conducted at the clinic for nephrology , clinical center university in sarajevo , from june 2014 through december 2014 .
fifty - two adult patients with cms and chronic kidney disease were included in the study .
the subjects were divided into ckd patients not yet dialysis - dependent ( 30 pts ; egfr < 60 > 15 ml / min/1.73 m ) and patients on peritoneal dialysis for > 6 months ( 22 pts ) .
all pd patients underwent 4 to 5 dialysis changes with 2 liters of dialysis solution .
subjects who had an episode of peritonitis within the previous 3 months and patients with evidence of malignancy , autoimmune disease or chronic liver disease , active infection , history of cardiovascular or peripheral vascular disease , and diabetes and recent treatment with iron were excluded from the study .
depending on the degree of severity of atherosclerotic changes in the carotid arteries all observed group patients were divided into three subgroups ( without atherosclerosis ( as0 ) , moderate atherosclerosis ( as1/2 ) , and severe atherosclerosis ( as3 ) ) .
estimated glomerular filtration rate was performed using the mdrd ( abbreviated modification of diet in renal disease equation gfr ) .
body mass index ( bmi ) was calculated as the ratio of body weight in kilograms and the square of height in meters ( bmi = kg / m ) .
body weight for bmi in pd patients is measured with dry abdomen ( without dialysate solution ) . the blood pressure ( bp )
was measured with mercury sphygmomanometer after 15 minutes of rest , according to recommendation by the british hypertension society .
hypertension was defined as systolic bp 140 mmhg and diastolic bp 90 mmhg or use of antihypertensive medications .
informed consent was obtained from all participants and the local ethics committee approved the study .
the concentration of malondialdehyde ( mda ) was analyzed at the center for cytogenetics and molecular medicine at the medical faculty in sarajevo using a competitive enzyme immunoassay test ( elisa ) , which was performed with commercial kit for the assessment of the overall level of mda ( manufacturer : uscn life science inc . , us - cea597ge ) .
reading of the results is carried out at 450 nm on a plate reader stat fax 2100 , usa .
the measurement concentration of mad was expressed in nanograms per milliliter ( ng / ml ) .
the concentration of the enzyme matrix metalloproteinase-9 ( mmp-9 ) in serum was quantified by elisa at the clinic for immunology university clinical centre sarajevo , according to manufacturer 's instructions ( r&d systems , inc .
reading of the results is carried out by spectrophotometry at 450 nm ( reader biotek elx50 ) , with the correction wavelength at 540 nm or 570 nm .
the measurement concentration of mmp-9 was expressed in nanograms per milliliter ( ng / ml ) .
the severity of carotid artery atherosclerosis was evaluated using the mean common carotid artery ( ca ) , intima media thickness ( imt ) , and plaque score ( ps ) .
high - resolution b mode and color doppler and pulse doppler ultrasonography of both carotid arteries were performed with an ultrasound scanner ( wall - track system : w - t , maastricht , the netherlands ) equipped with a 7.5-mhz linear array transducer .
measurement was done by the same angiologist who was not familiar with the clinical status of the study patients .
after the carotid arteries were located by transverse scans , the probe was rotated 90 to obtain and record a longitudinal image of the anterior and posterior walls .
the high - resolution images of the walls of the bilateral ca , internal carotid arteries ( ica ) , and carotid bulbs were examined according to recommendations of the american society of echocardiography carotid intima media thickness task force .
the imt was defined as the distance between the leading edge of the lumen - intima echo and the leading edge of the media - adventitia echo in plaque - free area .
at least three measurements ( a , b , and c ) were taken over one centimeter length of each wall segment ca , and these measurements on both sides were collected and divided to obtain the mean imt .
the ps was calculated by adding the maximal thickness in millimeters of plaques in each segment on both sides ( a + b + c + thickness of the contralateral carotid artery plaques ) .
the presence of atherosclerosis in cca is estimated as recommended by the mannheim consensus about atherosclerosis : ( 1 ) without atherosclerosis ( as0 ) : imt less than 80% of the reference interval ( ri ) , age- and gender - adjusted , with ri values obtained from previously published studies of monitoring , using the same ultrasound procedures;(2 ) mild atherosclerosis ( as1 ) : imt > 80% of the ri;(3 ) moderate atherosclerosis ( as2 ) : the presence of carotid plaque , with no significant stenosis ( psv < 125 cm / s);(4 ) severe atherosclerosis ( as3 ) : the presence of carotid plaque with threatening stenosis ( psv > 125 cm / s ) . without atherosclerosis ( as0 ) : imt less than 80% of the reference interval ( ri ) , age- and gender - adjusted , with ri values obtained from previously published studies of monitoring , using the same ultrasound procedures ; mild atherosclerosis ( as1 ) : imt > 80% of the ri ; moderate atherosclerosis ( as2 ) : the presence of carotid plaque , with no significant stenosis ( psv < 125 cm / s ) ; severe atherosclerosis ( as3 ) : the presence of carotid plaque with threatening stenosis ( psv > 125 cm / s ) .
all data were expressed as the mean sd or as median and interquartile range .
the distribution of variables was tested by the kolmogorov - smirnov and/or shapiro - wilk test .
student 's t - test was used to compare the means of variable with normal distribution .
the difference in median with interquartile range between two groups was analyzed by the mann - whitney test .
pearson 's test was used to correlate data with normal distribution and spearman 's test for data with a skewed distribution .
a multiple regression analysis was applied to define the independent connection serum concentration of mda and mmp-9 with ultrasound parameters of ca .
the significant independent variables were determined according to their standardized effect , defined as regression coefficient / standard error of the regression ( ) .
all statistical calculations were performed with the spss 16 software ( version 16.0 , spss inc . ,
the average age of respondents was 59.84 5.16 years and was not different from the age of the control group .
there was also no difference in age and smoking in cms patients with ckd without the need for dialysis treatment and those on peritoneal dialysis .
all monitored ckd and pd patients were overweight , while bmi of control group patients was within the limits that are considered healthy ( between 20.1 and 24.5 ) . in our study , all observed ckd patients , dependent or not dependent on dialysis treatment , were hypertensive ( bp 140/90 mm hg ) , but significantly higher values of both systolic and diastolic blood pressure were registered in the group of patients on pd treatment compared to other ckd patients .
although there were no significant intergroup differences in serum triglyceride levels , all groups of patients had an elevated level of serum triglycerides ( above the recommended value of 1.7
basal characteristics of the anthropometric profile , blood pressure values , metabolic pattern , and serum concentration of mda and mmp-9 in the whole group of ckd and pd subjects with cms and in two subgroups of ckd with and without dialysis ( pd ) are presented in table 1 .
depending on the stages of atherosclerosis our data showed significant changes in serum concentration of mda and mmp-9 between the different subgroups of ckd nondialysis - dependent patients , as well as in patients on peritoneal dialysis ( table 2 ) .
the level of serum mda through all the stages of atherosclerosis was significantly lower in pd patients compared to observed ckd nondialysis - dependent patients ( in as1/2 35.6 ( 28.339.1 ) versus 40.3 ( 31.945.6 ) ng / ml ; p
< 0.05 and in as3 53.9 ( 49.863.2 ) versus 74.3 ( 68.276.3 ) ng / ml ; p < 0.05 ) .
there were no significant differences in serum concentration of mda in the monitored pd and ckd patients without expressed atherosclerotic changes ( as0 20.6 ( 10.431.3 ) versus 24.1 ( 18.827 ) ng / ml ; p > 0.05 ) ( figure 1 ) .
the level of serum concentration of mmp-9 was significantly different in pd patients compared to ckd nondialysis - dependent patients .
significantly lower level of this biomarker is registered in the stage as1/2 of atherosclerosis in pd patients ( 374.8 ( 346.4397.7 ) ng / ml ) compared to the ckd patients treated conservatively ( 425.2 ( 405.5439.5 ) ng / ml ) ( p < 0.05 ) , whereas in stage as3 serum mmp-9 concentration was significantly higher in pd patients in comparison with other ckd patients ( p < 0.05 ) .
such a relationship but lower concentration of mmp-9 in the serum was present in stage as0 in both groups ( figure 2 ) .
significant correlation was confirmed between serum concentration of mda and imt - ca in all observed patients ( r = 0.859 ; p < 0.01 ) ( figure 3 ) , as well as between serum concentration of mda and value of plaque score ( rho = 0.869 ; p < 0.01 ) ( figure 4 ) .
significant relation was also confirmed between serum concentration of mmp-9 and imt - ca ( r = 0.762 ; p < 0.01 ) ( figure 5 ) and between serum concentration of mmp-9 and value of plaque score in all observed ckd and pd patients ( r = 0.785 ; p < 0.01 ) ( figure 6 ) . in multiple regression model serum concentrations of mda and
mmp-9 were significant predictors of imt - ca in all monitored ckd and pd patients ( p < 0.05 ) , as well as plaque score on carotid arteries in these populations ( p < 0.05 ) .
this model is able to explain 70% of the variance in the results of imt ( r = 0.765 ) and about 79% ( r = 0.786 ) of the variation that occurs with the plaque score on carotid arteries in chronic kidney disease ( table 3 ) .
the degree of intracellular and extracellular oxidative stress is related to the severity of renal failure .
it was also noted that tissue damage caused by lipid peroxidation plays an important role in the developmentof various diseases including atherosclerosis , which is associated with high cardiovascular morbidity and mortality in ckd [ 23 , 24 ] . in recent years
a few studies were published dealing with the status of oxidative stress in relation to various disorders that accompany chronic renal disease and renal replacement therapy [ 2529 ] . in the present paper we demonstrated that the serum concentration of mda did not statistically differ in ckd patients with cms treated conservatively and patients undergoing peritoneal dialysis .
however , it was observed that serum concentration of mda progressively increases with severity of atherosclerotic changes in the carotid arteries within both groups of patients . on the other hand
, we have also shown significant changes in serum concentration of mda between the different subgroups of ckd and pd patients according to the status of atherosclerotic changes in the carotid arteries .
the highest levels of serum mda were in stage as3 in both groups of patients .
significant correlation was confirmed between serum concentration of mda with imt - ca and plaque score in all study patients .
in addition , our results indicate that the level of serum mda through all the stages of atherosclerosis was significantly lower in pd patients in comparison with observed ckd patients treated conservatively , which can be attributed to the influence of preserved residual renal function ( rrf ) and the absence of trusted signs of inflammation .
since peritoneal dialysis is intrinsic type of dialysis through the peritoneum as biocompatible membrane , this result also suggests the possible influence of peritoneal dialysis on oxidative stress .
a few studies reported increased os among pd patients , but other prooxidants ( tbars , tac ) were measured , associated with hypoalbuminemic state or loss of residual renal function ( rrf ) , and without comparing it with nondialysis ckd patients [ 25 , 27 , 30 ] .
. found that pd patients have markedly impaired endothelial function as documented by impaired flow mediated dilatation of brachial artery with higher serum concentration of os markers .
furthermore , raju et al . confirmed significant increase in serum mda in hemodialysis patients , compared with the same patients before they had started the hemodialysis treatment , which could be attributed to a bioincompatibility of dialysis membrane and diffusion of hydrophilic compounds to the dialysate and influx of endotoxin from the dialysate .
these factors lead to activation of macrophages and production of reactive oxygen species ( ros ) and also a loss of antioxidants during hemodialysis sessions .
all the above factors cause an increase in production of free radicals , peroxidation of lipids , and further rise in serum mda level after episodes of dialysis .
our results suggest that oxidative stress is one of the factors that mediate the relationship between ckd , atherosclerosis , and cms .
activity of mmp-9 is highly associated with the progression of ckd , diabetes , and coronary arterial disease [ 33 , 34 ] .
this mmp is secreted by inflammatory cells in the adventitia or smooth muscle cells in the media .
the degraded elastic fiber induces calcium deposition , which in conjunction with altered vascular structure is associated with vessel stiffening [ 35 , 36 ] . in ckd arterial
stiffening increases cardiac afterload , left ventricular hypertrophy , reduces coronary artery perfusion and myocardial ischaemia , and increases pulse pressure that promotes atheroma formation and vascular remodeling . in our study
, no significant difference was found in the serum concentration of mmp-9 between ckd patients treated conservatively and pd patients , but the significant increase was found in the value of this biomarker within both groups with progression of atherosclerotic process .
these findings are in line with the findings of addabbo and associates , who have demonstrated that mmp-9 levels strongly correlated with carotid atherosclerosis burden irrespective of other factors in early , moderate , and advanced ckd .
our data also showed the decreased serum concentrations of mmp-9 in the stage as1/2 of atherosclerosis in pd patients compared with ckd patients without dialysis treatment .
however , it is our belief that the decrease in mmp-9 concentration in as1/2 stage of pd patients might occur due to the impact of peritoneal dialysis on mmp-9 serum concentration .
mmp-9 was significantly higher in stage as3 in pd patients compared with ckd patients treated conservatively .
to our knowledge , the impact of peritoneal dialysis on the concentration mmp-9 has not yet been clarified .
some authors have demonstrated that the hemodialysis process leads to a reduction in plasma concentration of mmp-9 in some patients .
a significant correlation of mmp-9 serum concentration with ca - imt and plaque score was found in all monitored patients in this study . also , in multiple regression model we confirmed a significant independent association of mda and mmp-9 with these ultrasonography parameters characterizing arterial wall and atherosclerotic changes of carotid arteries .
the need for identification of risk factors and serum markers of atherosclerosis in the process of early detection and prediction of risk for cardiovascular disease has attracted a lot of attention in recent years .
the significantly lower levels of mda in pd - cms patients in comparison with ckd - cms patients not yet dialysis - dependent and its increase with atherosclerosis progression , as well as obtained higher values of mmp-9 during progression of atherosclerosis especially in pd patients , could be a new contributing factor of our study .
however , this study has certain limitations , such as the small number of respondents and a cross - sectional design of study .
a question on the periodontal disease status of those patients was not included , although periodontal disease and kidney disease are highly associated and periodontal disease is alone associated with increases in mmp-9 . whether the longitudinal profile of mda and mmp-9 in ckd nondialysis - dependent patients and pd patients , both with signs of cms , provides additional information on the predictive power of these biomarkers for the progression of atherosclerosis remains a question for testing in future longitudinal studies with a larger number of patients .
the results suggest that mda and mmp-9 could be mediators of ckd - related vascular remodeling in cms . the study data also suggest that that factors mediating relationship between cardiometabolic syndrome , chronic kidney disease , and atherosclerosis can include malondialdehyde and mmp-9 . | objective was to assess whether the concentration of malondialdehyde ( mda ) as a marker of lipid peroxidation and serum concentration of matrix metalloproteinase-9 ( mmp-9 ) are involved in the process of atherosclerosis in chronic kidney disease ( ckd ) patients nondialysis - dependent and those on peritoneal dialysis ( pd ) , both with signs of cardiometabolic syndrome ( cms ) .
thirty ckd and 22 pd patients were included in a study . all
observed patients were divided into three subgroups depending on the degree of atherosclerotic changes in the carotid arteries ( ca ) .
severity of atherosclerotic changes in the ca was evaluated by ultrasonography .
we confirmed significantly lower level of serum mda throughout all the stages of atherosclerosis in pd patients compared with observed ckd patients ( p < 0.05 ) and increased serum concentration of mda and mmp-9 with the progression of severity atherosclerotic changes in both groups of patients .
the multiple regression analysis revealed that mda and mmp-9 are significant predictors of changes in imt - ca ckd patients ( p < 0.05 ) and plaque score on ca in these patients ( p < 0.05 ) .
the results suggest that mda and mmp-9 could be mediators of ckd - related vascular remodeling in cms . |
the most common disorders are depression , substance abuse , disturbance of attention , and eating disorders .
it is defined by who as a state of well - being in which every individual realizes his or her own potential , can cope with the normal stresses of life , can work productively and fruitfully , and is able to make a contribution to her or his community .
positive mental health is seen as a resource which is essential to general well - being .
the promotion of mental health is a more extensive concept than merely preventing mental health problems .
mental health promotion strives to find and enhance factors and processes that protect mental health and reduce factors harmful to it .
mental health promotion can improve people 's survival skills and ability to feel empathy and , consequently , protect their mental health and improve their ability to support other members of their community with mental health problems .
the promotion of mental health not only consists of the support of the mental health of individuals and provision of mental health services but also includes activities at the community and society levels [ 1014 ] .
actions to promote mental health include national mental health programmes , laws and policies , development of mentally healthy communities and physical environments and providing of opportunities for leisure activities . as mental health
is an integral part of health , mental health promotion is an integral part of overall health promotion .
an environment that promotes or hinders schoolchildren 's mental health involves the person 's entire mental , physical , and social environment , especially the school , home , and friends .
there is evidence of how mental health promotion in schools has positive effects on the age group .
taking into account the whole of the school community especially , environment and the families of the pupils as well as focusing on positive mental health and adequate lengths of the treatments are factors which contribute to the success of mental health promotion .
preventing mental health problems and doing mental health promotion have an effect in one 's success in school and well - being in life in general .
the mental health and behavioural problems of children and adolescents can often be seen as difficulties in later phases of their lives [ 2125 ] .
barry and jenkins made an overview of the evidence from systematic reviews and several mental health promoting programmes .
these are high - quality implementation , evaluation and sustainability and such things as adopting a whole - school and a social competence approach , performing interventions over multiple years and having a strong theoretical base .
used theoretical foundations are mostly psychology theories such like child development theory or cognitive behavioural therapy but in many programs there is no theory basis at all [ 10 , 26 ] .
cooperation and the participation of all community members in mental health promotion have been found to be significant factors for the success of planned mental health programs [ 4 , 10 , 27 , 28 ] .
self - expression and self - ruling are key factors for well - being ; therefore , just being heard affects mental health and well - being [ 2729 ] . who european ministerial conference
also focuses in its mental health declaration fostering awareness of importance of mental well - being ( priority 1 ) and recognizing knowledge and experiences of service users and carers ( family member , friend , or another informal caregiver ) as an important basis on planning and developing mental health services ( priority 5 ) .
schoolchildren are the main target of mental health promotion , which makes them key elements in the discussion on promoting mental health at school .
it is the primary , most important , and most influential system to which the child belongs .
schoolchildren and their families represent service users in this study material . despite the large number of projects and recommendations on the promotion of schoolchildren 's mental health ,
the literature does not offer a comprehensive theoretical description of what mental health work with schoolchildren is as a whole [ 10 , 26 ] .
having a theoretical description of mental health promotion work helps in the development of facilities [ 10 , 26 , 28 ] .
the employees , the schoolchildren , and their families as well as previous knowledge of the subject .
this study is a part of a more extensive research to produce a theory of mental health promotion in the upper level of comprehensive school . in these schools ,
this study aims at describing the concepts referring to the promotion of mental health from the schoolchildren 's and their families ' perspective and , consequently , facilitating the development of the practices of mental health promotion . in this study , families ' perspective was studied by interviewing mothers of schoolchildren . in this study
, it was intended to especially highlight the perspective of the schoolchildren and their families .
therefore , the analysis was left on the level of axial coding to produce and describe the concepts .
after that , it is easier to develop practices and support their self - ruling ability .
schoolchildren are the main targets of mental health promotion , which makes them key elements in the discussion on promoting mental health at school .
self - expression and self - rule are key factors for well - being ; therefore , just being heard affects mental health and well - being [ 2729 ] .
the purpose of this study was to find the key aspects and requirements for promoting mental health in the way the pupils and their families see them .
this study was conducted in a finnish upper - level comprehensive school with 446 pupils in 20052007 .
this is the only upper - level comprehensive school in the quite small city where the school is located . in finland ,
the last three grades of comprehensive school attended from age of 12 to age of 16are usually , and also in this case , located in a bigger school further away from the homes .
the school offers an excellent environment to reach the whole age group because of the overarching school system . in finland ,
almost every child ( 99.7% ) completes the nine - year - long basic education .
the first type consisted of material gathered from the interviews with school pupils and their parents .
the parents who agreed to be interviewed were all mothers , but they were asked to speak from the viewpoint of the whole family .
the second type consisted of the pupils ' written replies to the well - being survey conducted by their school .
the school well - being profile is a tool that is used to measure well - being in school communities .
what is the best feature in your school ? and what things in your school should especially be improved ? .
because positive mental health is an intregral part of general well - being , it was assumed that statements regarding mental health could be found in this study 's survey material . in this study
the responses to these two questions were incorporated into the other data sets and the statements drawn from them were analysed together with the rest of the data .
a sufficient amount of data was considered having been collected when the analysis no longer resulted in statements that did not fit into the categories .
the interviews with the schoolchildren and mothers produced 54 pages of text and the verbal answers to the school well - being profile produced 436 statements that were also added to the research material .
a total of 1523 statements were written up as a result of the interviews and the survey questions .
the themes of the interviews were as follows : the conceptions of the interviewees of mental health work in general and in this school;school as a working environment;particular concerns , such as bullying and absences , and actions to prevent them;getting help in mental health problem situations ; cooperation between the home of the pupil and the school;cooperation between primary health care , social services , and special health care;the most crucial development needs in mental health promotion of schoolchildren .
the conceptions of the interviewees of mental health work in general and in this school ; school as a working environment ; particular concerns , such as bullying and absences , and actions to prevent them ; getting help in mental health problem situations ; cooperation between the home of the pupil and the school ; cooperation between primary health care , social services , and special health care ; the most crucial development needs in mental health promotion of schoolchildren .
the interviews were conducted by the researcher and they were carried out in the school during the school day .
the principal was aware of the aims and methods of the study and of the fact that there was a need for interviewees who have things to say through their own experience and who represent the schoolchildren comprehensively .
she approached a group of parents and these four were chosen to be the respondents due to their willingness to participate .
the criteria for selecting the pupils to be interviewed were that the pupils represented different grades and were of both sexes .
the relatively open interview themes helped to see the world from the perspective of participants and to develop deeper understanding of their viewpoint . in the interview material of the parents ,
for the schoolchildren , it seemed to be easier to produce statements anonymously in the survey than it was in the interview . with the survey material , it was possible to reach many schoolchildren and get their opinions on their school .
the open questions in the survey also provided a way to find aspects the schoolchildren themselves found important . in an interview situation ,
the school well - being profile survey was completed by 423 out of 426 pupils present in school on the day of the survey .
it aims at creating a substantive theory , including concepts and assumptions on the relationships among them [ 32 , 37 , 38 ] .
grounded theory was selected because it is analysing method that takes account of people and their experiences . in grounded theory , the researcher aims to come close to the actor 's perspective and tries to capture his or her viewpoint .
this perspective of schoolchildren and families is needed to understand the mental health promotion in school .
to understand experience , it must be located within the larger events in a social , political , cultural , ethnic and gender related , informational , and technological framework .
developing knowledge helps to develop practice [ 32 , 40 ] . the theoretical background of the grounded theory method lies in symbolic interactionism and pragmatism .
symbolic interactionism brings attention to human behaviour and interaction by focusing on the significance of events and things in people 's everyday lives .
it is useful for conceptualising complex situations , understanding unsolved social problems , and understanding the impact of new ideologies .
the objective of grounded theory is to generate basic models as explanations of social life in general .
one , the purely inductive method represented by glaser , develops theory purely on the basis of the material .
the other , favoured by strauss and applied in this study , inspects the existing theoretical knowledge and combines it with the data gained through research [ 32 , 37 , 39 ] . because of the relatively large number of articles about some aspects of mental health promotion at school , it is reasonable to examine and use previous articles about the promotion of schoolchildren 's mental health while producing a comprehensive theoretical description
this is why the strauss method was selected for this study [ 32 , 37 , 39 ] .
the schoolchildren 's and families ' perspective that comes from the material of this study can be compared to the existing knowledge and viewpoints [ 37 , 39 ] .
some points should be taken into account when under - age respondents are used in a study .
, respondents aged 12 to 16 are considered old enough to participate in some cases even without permission from their parents .
such cases are situations where sensitive questions are not asked and it is seen that the questions do not convulse the youngsters . however , in this study , it was still ensured that the pupils ' parents were aware of the interviews and gave their consent .
letters were sent to the homes of the interviewed pupils to inform their parents of the interviews .
consent forms were sent with these letter and the parents were asked to give their child their consent to participate in the interview by signing the form . before the interview
, all respondents gave their written consent to the use of the interview as research material .
the participating pupils and parents received a brief introduction to the study and its objectives .
the questions were designed in such a way that the objectives were approached with as familiar situations and concepts as possible .
the pupils were also informed of the use of the well - being profile responses in the promotion of mental health and well - being at school .
the study was endorsed by permissions from the joint municipal authority for primary healthcare , the social welfare board , the board of education , and also the ethics committee of the local hospital district . grounded theory focuses on concept formation .
they express time , place , people , and people 's patterns of action [ 32 , 42 ] .
, the researcher also spent a lot of time discussing with the school personnel to do so . getting
familiar with the school context and with the literature also enhanced the researcher 's preunderstanding about the subject [ 32 , 38 ] . both materials , the transcribed interview material and the written answers of the survey , were analysed together .
statements the informants used to illustrate mental health promotion were picked from the text [ 32 , 38 ] .
then the researcher analysed the collected material with the constant comparative method to identify similarities and differences between the statements .
the questions that were used to support the categorisation of the material included the following :
the process of grouping concepts which seem to pertain to the same phenomena is called categorizing . as a result of the regrouping and comparison ,
all of the statements were placed in a category of statements pertaining to the same theme and a shared concept illustrating the phenomenon was identified .
table 1 presents an example of how the statements were placed into categories and subcategories and shows the concepts used to name the categories .
data analysis occurred simultaneously with data collection . during the data collection categories developed in terms of their properties and dimensions .
the codes produced by the analysis determined the direction of the data collection . following the principle of saturation ,
the amount of data was considered sufficient when no new utterances emerged from the data collected but the same themes began to recur .
sufficient sampling was determined to occur when categories offered considerable depth and breadth of understanding about the phenomenon and relationships to other categories were clear . in the next phase , the axial coding ,
the data was examined to identify structural factors , contextual factors , and factors related to the phenomenon , circumstances , and action .
this way , the five key concepts which the parents and the schoolchildren used in describing the promotion of mental health in the upper - level comprehensive school emerged [ 32 , 40 ] . at this stage of the research
therefore , the analysis was left on the level of axial coding . on this level
it is possible to form causal conditions , context , phenomena , and factors related to action and interaction from the material .
finally , the generated concepts were compared with the existing research information on the topic .
with regard to mental health promotion , five key concepts were discovered in the material : school environment , school friends and teachers , cooperation , actions to promote well - being and mental health , and getting help with problems .
table 2 shows the key concepts related to promoting mental health and properties and describes them from the schoolchildren 's and their parents ' perspective .
school environment comprises the physical conditions and educational equipment in the school including resources , teaching , rules , and services for pupils , such as school lunch and their impacts on the pupils .
this concept also comprises the requirements of the teaching , the flexibility and individuality of the teaching , and the characteristics of the pupils themselves .
they were pleased with the high - quality teaching tools and spacious classrooms but thought that the recesses should offer more activity opportunities ; just standing around is not enough .
on the other hand , the rules governing recess - time activities and school practices aroused divergent opinions : some were against these rules and some wanted them to be adhered to and controlled more actively . also , the parents considered that the school environment influences children 's well - being and they had made efforts to improve the school premises , for example , via the parents ' association.the schoolyard could use some improving , there has been some opinions about that . about that something should be done there there ( sic).the atmosphere is good.needs to be improved : rules the schoolyard could use some improving , there has been some opinions about that .
needs to be improved : rules learning was considered to be a positive thing .
the pupils were happy with the idea of optional subjects , but , on the other hand , they wanted to have more nontheoretical subjects such as physical exercise , music , and home economics .
also the parents wished the curricula to be more favourable for pupils who are more inclined towards artistic performance than theoretical learning .
the pupils and parents wanted to add individual aspects and flexibility to the teaching and teaching methods with an increased degree of student participation .
individualism was considered to be a mental health - promoting factor in the form of promoting the self - esteem and feelings of success.it's that supporting of growth and finding those positive capabilities.the best thing in this school are ( sic ) the electives .
learning difficulties , vandalism , and absences were further issues associated with the school environment although to a lesser extent . in general , factors related to the pupils ' age and personal properties were essential in this respect .
the available human and financial resources were brought up as potential obstacles to detecting and resolving problems.well , the curator - situation has been inadequate.the school nurse is nevertheless always available every day .
well , the curator - situation has been inadequate . the school nurse is nevertheless always available every day .
school friends and teachers include social relationships in school and the whole school community .
friends were also considered to be important and to make the school feel like a comfortable place .
breaking up groups of friends when transferring from primary school to the upper - level comprehensive school is an issue that aroused much thought , especially in the transfer phase .
safety in the school community was also considered important.i think it 's that the teachers treat the pupils equally.nice school friends
the class teacher was expected to notice pupils ' needs for support and respond to them .
the pupils often complimented their teachers as being nice , skilled , and good teachers .
however , there were also comments about too high demands and dictator - like and unfair behaviour.teachers have the expertise to see in my opinion.i wished remedial education for my son in some of the subjects and the teacher did carry my wish forward then.and the teachers are quite nice .
i wished remedial education for my son in some of the subjects and the teacher did carry my wish forward then . and the teachers are quite nice .
co - operation and communication between parents and employees were things that the parents particularly expected .
this concept also includes cooperation between employees and between children and adults and sufficient amount of information .
the parents considered that their task is to take an interest in the child 's life , monitor the child 's health and motivation , and ensure sufficient rest and nutrition .
child upbringing was seen as a joint project in which parents and teachers function as partners , both relying on their own expertise ; parents know the child 's personality and background , whereas teachers are competent at issues related to learning.i ( a mother ) myself try to look after resting , bedtimes .
i ( a mother ) myself try to look after resting , bedtimes .
parents are not professionals of education . according to the pupils , contacts between their homes and the school were scarce , but they believed that information will be relayed whenever necessary .
the parents said that they get information on the school work from bulletins and parents ' meetings .
one specific channel for information is the parents ' association which gathers information on the school and tries to contribute to the school environment 's development by , for example , arranging fund - raising events.there's not much contacting from home , but the school , however , contacts even for smaller matters.when it 's needed , everything is told .
like , when something comes up that needs to be told , the school is informed of it . there 's not much contacting from home , but the school , however , contacts even for smaller matters .
like , when something comes up that needs to be told , the school is informed of it .
actions to promote well - being and mental health are related to having an effect on enhancing the school 's conditions and social environment . when talking about the factors that influence mental health , both the parents and the pupils mentioned friendship and stress .
furthermore , the parents believed that learning experiences influence adolescent 's self - confidence and , consequently , mental health .
the results indicated that the school had paid attention to the development of friendships and community spirit , as well as preparing for other changes related to the transition from lower - level to upper - level comprehensive school.in spring , they ( the new students getting to know their future school ) already visited this place more than once .
and then they had their future class together and they , like , already got to know each other . in spring , they ( the new students getting to know their future school ) already visited this place more than once .
and then they had their future class together and they , like , already got to know each other . with regard to well - being and feeling well in general , there was a lot of discussion related to the above - mentioned school environment factors , resources , and social relationships . ensuring a sensible eating routine and a sufficient amount of sleep , improving the safety and cosiness of the school environment , fair treatment and possibilities to influence school matters
, these things can be influenced by the parents and the pupils , for example , via the pupils ' council.well this is pretty good , you know , because there is a well arranged pupils ' council in here .
it has improved the cosiness . well this is pretty good , you know , because there is a well arranged pupils ' council in here .
the pupils said they had received some information about mental health - related issues from their teachers .
health education lessons were mentioned as one channel in learning to take care of your well - being .
the parents stated that they got information about the school 's practices and services in parents ' meetings.in parents ' meetings , it 's told how they do act in these situations which , for example , concern bullying .
in parents ' meetings , it 's told how they do act in these situations which , for example , concern bullying . getting help with problems includes skills to notice mental health problems and possibilities to get proper and timely help .
the respondents had a little knowledge of instances that offer actual mental health care services .
the school health care was seen as the primary instance to contact such matters and it was considered to be easy to access .
some respondents were aware of the existence of the pupil welfare group but not of its activities or members .
the respondents believed that the main responsibility for seeking help was with the individual concerned .
they did not believe that the chances of detecting problems were great , especially with quiet , socially withdrawn pupils .
the respondents ' views on mental health work were mainly based on experienced problem situations , such as problems and negative feelings related to learning difficulties and experiences of offering help in stressful situations .
cooperation and communication between the home and the school were considered important and preferable factors with regard to mental health problems.well , we have a school nurse who 's spezialised ( sic ) in mental health .
like , i was immediately asked whether i would like to go there to talk to him / her because i worry a lot about the exams.well , in my opinion , it works well now that people contact each other immediately .
like , the form teacher contacts the parents when there 's something that 's puzzling him or her , and the parents do the same if they are puzzled .
well , we have a school nurse who 's spezialised ( sic ) in mental health .
like , i was immediately asked whether i would like to go there to talk to him / her because i worry a lot about the exams .
well , in my opinion , it works well now that people contact each other immediately .
like , the form teacher contacts the parents when there 's something that 's puzzling him or her , and the parents do the same if they are puzzled .
in grounded theory , previous literature is used to support and to validate the findings .
the founded literature did not provide a comprehensive description of mental health promotion in school , but there was a good amount of studies and articles related to the individual categories .
previous studies were searched with such terms as mental health promotion and school , children , or adolescents in several databases .
several studies argued about the same kind of aspects being important as these study results did .
this supports the conclusion that concepts discovered from study material are actually things that are essential in promoting mental health .
school environment and school friends and teachers especially were mentioned in many studies .
konu and lintonen , and goodman et al . wrote about the importance of developing an environment that supports the well - being of the pupils . in their studies on mental health promotion projects and reviews thereof , they found strong evidence of the social competence approach , which brings focus on the promotion of resourcefulness and generic coping and competence skills . also
family members should help the nursing staff to gain a clearer picture of the depth and the diversity of the family health and support the resources that promote family health .
cooperation in mental health projects is also a key element in poole 's project on preventive mental health work , in which families , schools , municipalities , and authorities worked in extensive cooperation to develop a model of partnership between the school and the families .
adelman and taylor [ 46 , 47 ] named the collaborative work of school staff , health professionals , and the community resources as one very important part of successful mental health promotion work in their reviews of school mental health promotion . according to deschesnes et al .
cooperation between the family , school , and community was a significant factor in the promotion of schoolchildren 's mental health . in this study
this is why it was important to include the adolescents ' and families ' viewpoint .
the influence of the entire social school environment , including the teachers and their major role , is stated in the results and enhancing these elements is seen important in many studies [ 10 , 29 , 35 , 43 ] .
barry and jenkins wrote about the significance of social and problem - solving skills for well - being .
in the light of barry 's results , it is easy to understand how important role the schoolchildren and their parents give to human relations and co - operation .
the results of getting help with problems especially reflect the viewpoints of the pupils and their families . in previous studies
anyway , previous results of co - operation [ 31 , 44 , 45 , 48 ] , mental health and social skills training and being heard [ 49 , 50 ] are things that also support this finding . the importance of being heard and possibilities to have influence in one 's own school matters can be seen in the results of this study .
. wrote in their study on children 's viewpoints on child psychiatric care that relationships with other children seem to be of extreme importance . according to the unicef 's convention on the rights of the child
, children have a right to express their opinions and to have their views taken seriously and given due weight .
children also have a responsibility to respect the rights of others , especially those of their parents .
the convention refers to the family as the fundamental group of society and the natural environment for the growth and well - being of children . in qualitative research , validity and reliability
data generated using the grounded theory method can be assessed in terms of credibility , conformability , and truth value [ 38 , 40 ] . the views and opinions of the parents and youngsters , on which matters are important , can be considered credible .
this study features interviews with four youngsters and a survey among a large group of schoolchildren on their opinions of their school .
the material from the interviews with parents was rich and versatile and described the parents ' thoughts on their children 's schooling from various angles .
combining several types of data proved to be a helpful way to find more information and a richer description about the studied phenomenon .
for example , using the open questions from the survey supplemented the interview material remarkably .
as the material collection proceeded , certain statements became more frequent , which means that the material itself can be considered in order to confirm its own credibility .
the impression of the promotion of schoolchildren 's mental health gained from the material provides a versatile insight into the youngsters ' and parents ' viewpoints from various angles .
a particular objective of this study was to bring out human experiences , which was well achieved with the interview material .
the schoolchildren 's views on mental health promotion were clearly visible [ 32 , 40 ] . a substantive theory produced by grounded theory
one concern is whether the mothers ' viewpoints can be extrapolated to the general family , even though they were told to represent the whole family .
no fathers agreed to be interviewed , but mothers were asked to speak from the viewpoint of the whole family . at least their viewpoint can be seen to be a remarkable part of family viewpoints .
however , a quality theory will inevitably identify a basic process that is also relevant more generally .
details related to the school 's practices and human resources vary between schools and countries , which is why detailed practices , such as group - formation on transition to the upper - level school , can not be generalised into a description of mental health promotion practices in schools as a whole . instead
, the process of collecting and analysing the pupils ' viewpoints used in this study , as well as the founded key aspects of mental health promotion in school , can be considered to apply to a wider context and internationally .
grounded theory proved to be an effective mean of eliciting people 's personal viewpoints . when theory is developed from the personal descriptions of the actors and the objects of action , it is easily translatable into a development tool .
pupils and their families are the key targets of mental health promotion work in schools and bringing up their viewpoints is important and forms a significant research result .
the founded viewpoints help the school personnel develop their work and select the focus areas of development , even in schools with different human resources and practices [ 10 , 26 , 51 ] .
schools are one of the most important settings for promoting mental health of young people . according to previous literature , the term mental health is often misunderstood and misused .
pupils and parents , as well , need knowledge of mental health promotion and problem solving in school .
this study , by giving names and contents to different fields of mental health promotion , helps the whole school community to find key development areas .
this should lead to services being better tailored to people 's needs and better used .
the results indicate that the pupils find teaching and teachers to be very important factors at school . with these factors in mind , it is important to pay attention to the opportunities to increase flexibility and individuality .
finding competent teachers would also make the school work seem more worthwhile for the pupils .
the school staff needs guidance in mental health promotion and theory - based means for the practical implementation thereof .
it is often the case that the operators are not aware of the underlying theory behind development programmes , which makes the practical application more difficult . through the perspectives of different actors ,
it is easier to find an understandable and practical description of the action . with the help of the produced concepts , the mental health promotion of schoolchildren
can thus be observed and promoted , which is the central benefit of this study .
however , co - operation between the families and the school staff , as well as increasing the pupils ' possibilities to influence , could help in this respect as well .
the whole school community must be taken into consideration . combining viewpoints of children , families , and professionals and
then discussing and developing mental health promotion activities in cooperation would not only increase awareness but also enable development that takes all viewpoints into account .
the next phase of the research is to combine these different viewpoints into a substantive theory on mental health promotion in school .
the theory which will be built will help the whole school community to develop mental health promoting work by giving a framework and helping to find key development areas . | while developing mental health work in schools , it is very important to consider the viewpoint of pupils .
parents can also give remarkable information on their children 's viewpoint .
the purpose of this study was to produce a description of the concepts used by schoolchildren aged 1216 years and their families associated with promoting mental health in schools .
the research material comprised interviews with schoolchildren and mothers , and verbal answers from the school well - being profile survey ( n = 426 ) .
the analysis was conducted by applying the grounded theory method as introduced by strauss .
the study was conducted in a finnish comprehensive school . |
integrins are heterodimeric membrane receptors that mediate cell adhesion to ecm or to another cell ( hynes , 2002 ) .
ligand binding provides transmembrane mechanical links to transmit forces from extracellular contacts to intracellular structures ( e.g. , the cytoskeleton ) and signals for a wide variety of cellular processes .
for example , binding of integrin 51 to fibronectin ( fn ) plays an important role in fibroblast spreading and motility ( akiyama et al . , 1989 ) , t cell migration ( shimizu et al . , 1990 ) , osteoblastic and myogenic proliferation , and differentiation ( garcia et al . , 1999 ) .
integrins are often expressed on cells in an inactive , low affinity state with slow on rate and/or fast off rate for ligand binding , but they can be activated to high affinity states with fast on rates and/or slow off rates ( hynes , 2002 ; luo et al . , 2007 ) .
activation can also be triggered by divalent cations and by binding of activating mabs to cell surface or purified integrins ( humphries , 2000 ) .
affinity regulation in integrins is thought to be allosteric ( hynes , 2002 ; luo et al . , 2007 ) .
the overall shape of the integrin molecule is that of a large head region ( the headpiece ) supported on two long legs . in the low affinity state , integrin legs
were observed to have bent knees , which were straightened upon activation ( xiong et al . , 2001 ; takagi et al . ,
therefore , it seems reasonable to speculate that tensile force applied via a bound ligand may induce unbending of the knees , thereby converting the integrin from a low affinity state with short bond lifetimes to a high affinity state with long bond lifetimes ( chigaev et al .
, 2003 ; zhu et al . , 2005 ; alon and dustin , 2007 ; luo et al . , 2007 ; mcever and zhu , 2007 ) .
indeed , recent studies have provided experimental support for force - enhanced integrin function ( astrof et al . , 2006 ; woolf et al .
, 2007 ; friedland et al . , 2009 ) . also , steered molecular dynamics simulations have suggested how force activation of integrin might occur ( jin et al .
, 2004 ; puklin - faucher et al . , 2006 ) . the counterintuitive behavior in which force prolongs bond lifetimes is called catch bonds , which is in contrast to the ordinary slip bond behavior where force shortens bond lifetimes ( dembo et al . , 1988 ) .
catch bonds have been demonstrated in interactions between selectins and ligands ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) ,
actin and myosin ( guo and guilford , 2006 ) , fimh receptor and mannose ( yakovenko et al . , 2008 ) , and glycoprotein ib ( gpib ) and von willebrand factor ( vwf ;
however , loading rate - dependent rupture force measurements with atomic force microscopy ( afm ) force - ramp experiments and dynamic force spectroscopy analysis have not revealed catch bonds for integrin ligand interactions ( li et al .
, 2004 ) . using afm force - clamp experiments , we measured lifetimes of single fn51 bonds at forces as low as 4 pn . catch bonds were observed in < 30 pn .
changing divalent cations altered lifetimes in the same force range and binding of a mab that reports the extended conformation of 51 , but the catch bonds remained . truncating the 51 leg regions further prolonged bond lifetimes and abolished the response of the lifetime versus force curve to divalent cations
; nevertheless , catch bonds were still observed , showing that leg extension is not required for catch bonds .
two activating mabs that bind the headpiece shift catch bonds to a lower force range , indicating that force - induced activation of the headpiece is involved in catch bond formation .
thus , catch bond formation appears to involve force - assisted activation of the headpiece but not integrin extension .
using afm ( fig . 1 a ) , we measured interactions between an fn fragment ( fniii710 [ fn fragment encompassing the 710th type iii repeats ] ) and a recombinant 51 consisting of either the full extracellular portion ( 51-fc ) or only the headpiece ( truncated 51 [ tr51]-fc ) fused with fc ( coe et al . , 2001 ;
we have shown previously that the recombinant proteins closely mimic the function and conformational regulation of the native integrin ( coe et al . , 2001 ; mould et al .
, 2002 , 2003a , b , 2005 ) . to avoid uncontrolled adsorption of integrin via different domains , which could produce variable results and restrict conformational changes ,
tr51-fc was captured by fab of an anti - fc mab ( gg-7 ) preadsorbed on a polystyrene petri dish ( fig .
the petri dish was driven by a piezoelectric translator ( pzt ) to contact fniii710 adsorbed on a cantilever tip ( fig .
1 d ) of adhesion was detected upon retraction of the pzt , which was then clamped at a desired force for lifetime measurement ( fig .
a laser is focused on the back of cantilever end and bounced onto a photodiode to measure force on the tip that bends the cantilever .
a petri dish is mounted on a pzt with an integrated capacitive sensor to allow for distance control with subnanometer precision .
molecules depicted represent a composite of several adsorbed or captured on the cantilever tip or the petri dish .
extended and bent 51-fc , depicted as heterodimers of an ( light blue ) and a ( red ) subunit fused to an fc ( yellow ) , and tr51-fc , consisting of the propeller ( teal ) and thigh ( dark green ) domains of the subunit as well as the a ( pink ) , hybrid ( dark blue ) , and plexin / semaphorin / integrin ( tan ) domains of the subunit fused to an fc , were captured by an anti - fc mab ( gg-7 ) fab ( blue ) preadsorbed on the petri dish . in some experiments ,
these were replaced by an anti - fn mab ( hfn7.1 ; brown ) captured by an anti mouse fc antibody ( orange ) preadsorbed on the petri dish .
fniii710 ( purple ) was adsorbed on the cantilever tip . in some control experiments ,
the petri dish was moved up by the pzt to contact the cantilever tip ( blue trace ) , immediately retracted to a small distance ( green trace ) from the tip to reduce nonspecific adhesion , held at this distance for 0.5 s to allow for bond formation ( brown trace ) , and retracted away from the tip to detect adhesion ( red trace ) .
the trace illustrates a contact cycle without binding where the retraction curve returned to zero force upon petri dish retraction .
the color codes are the same as those in c , which illustrates a contact cycle with binding and lifetime measurement .
once the pulling force reached a preset value ( indicated ) , a feedback loop was triggered to keep the cantilever deflection at the set point .
the lifetime at that force ( indicated ) was measured until bond failure , signified by the springing back of the cantilever to the level of zero mean force .
the bending configurations of the cantilever are depicted with colors matching the corresponding colors of the traces in c and d. experiments were performed in 1 mm ca plus 1 mm mg ( ca / mg ) , 2 mm mg plus 2 mm egta ( mg / egta ) , or 2 mm mn in the absence or presence of blocking or activating mabs or a competing peptide . at the same gg-7coating concentrations , binding of 51-fc ( fig .
2 c ) to fniii710 became progressively more frequent when the cation condition was changed from ca / mg to mg / egta and to mn .
this was corroborated by flow cytometry data showing that mg / egta and mn increased the staining of 51-fc coated beads by a mab that reports unbending of the knees ( 9eg7 ; fig .
binding was specific , as it was abolished by addition to the solution of mab hfn7.1 , which blocked the integrin - binding site in fniii710 or an rgd - containing peptide cyclo(-grgdsp ) , which competed with the rgd loop in fniii710 for 51 binding ( fig .
( < 20% ) , a requirement for most adhesion events to be mediated by single bonds , progressively lower gg-7coating concentrations were used for experiments in ca / mg , mg / egta , and mn to compensate for the progressively higher binding affinities of tr51-fc ( fig .
binding was abrogated when the cantilever tips coated with fniii710 were switched to those coated with bsa or when the petri dishes functionalized with integrins were switched to those that were not functionalized ( fig .
frequencies of adhesion between 10 g / ml fniii710 adsorbed on cantilever tips and 10 g / ml 51-fc ( a ) or tr51-fc ( c ) captured by 15 or 100 g / ml gg-7 fab precoated on petri dishes in buffer containing the indicated cations without or with cyclo(-grgdsp ) or hfn7.1 in solution were enumerated in 100 tests for each spot and presented as mean sem of 35 spots .
frequencies ( measured with the same protocol as in a and c ) of adhesion between 10 g / ml fniii710 coated on cantilever tips and 10 g / ml 51-fc ( b ) or tr51-fc ( d ) captured by gg-7 fab preadsorbed on petri dishes with indicated concentrations ( closed bars ) are compared with those between bsa - coated tips and tr51-fc functionalized petri dishes ( open bars ) and with those between fniii710-coated tips and gg-7coated petri dishes without incubating with tr51-fc ( hatched bars ) in the indicated cation conditions .
mechanical regulation of fn51 dissociation was quantified by the force dependence of mean lifetimes of mostly single fniii71051 bonds measured in each of the three cation conditions ( fig .
3 , a c , circles ) . as force increased , lifetime first decreased to a minimum , then increased to a maximum , and decreased again , exhibiting a triphasic transition from slip bonds to catch bonds and then to slip bonds again .
the first slip bond regimen was most clearly observed in ca / mg ( fig .
the much less frequent nonspecific binding between bsa - coated cantilevers and polystyrene petri dishes functionalized with 51-fc contributed negligibly to these lifetime versus force curves , as most of these events were ruptured at forces < 20 pn ( fig .
s2 a ) , and those that survived the ramping had very short lifetimes ( fig .
( a c ) plots of lifetime versus force of 51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips ( circles ) in ca / mg ( a ) , mg / egta ( b ) , and mn ( c ) .
( d ) a qualitatively similar plot ( triangles ) of m51 reconstituted into lipid bilayer dissociating from fniii710-coated cantilever tips in mg / egta confirmed the catch bond observation . also plotted in a
c is the lifetime versus force curve of fc dissociating from gg-7 ( squares ) . for b and c
, the black and gray curves overlap at forces > 30 pn , indicating that measured lifetimes beyond 30 pn were caused by fc
also shown in c is a lifetime versus force plot ( diamonds ) of 51-fc functionalized petri dish dissociating from bsa - coated cantilever tips measured in mn .
( e ) schematic of the molecular arrangement indicating possible dissociation loci between fniii710 and 51-fc or between 51-fc and gg-7 .
( f ) schematic of the molecular arrangement for experiments that measured the capturing strength of the fc
error bars indicate mean sem . to control for potential artifacts of the chimeric integrin that fuses the extracellular portions of the 5 and 1 chains with a human igg fc region ( coe et al . , 2001
s2 a ) of fniii710 to membrane 51 ( m51 ) purified from human smooth muscle cells reconstituted in glass - supported lipid bilayers in mg / egta .
although the lifetimes were shorter , the force - dependent curve of m51 exhibited the same triphasic pattern ( fig .
thus , catch bonds were observed at forces ranging from 1030 pn for fn interacting with 51-fc and m51 . because 51-fc was captured by preadsorbed gg-7 , rupture of the molecular complex might result from dissociation of the fn51 or fc gg-7 bond ( fig .
we neglect potential detachment of fniii710 from the cantilever tip or of gg-7 from the petri dish because physioadsorption of proteins is generally much stronger than specific protein protein interactions ( rief et al . , 1997 ) .
to determine the rupture loci , we overlaid the force - dependent lifetimes of directly adsorbed 51-fc interacting with gg-7 ( fig . 3 f ) on each panel of fig .
3 ( a c , squares ) . slip bonds were observed over the entire force range tested .
the mean lifetimes at forces < 25 pn were much longer than those of fniii710 interacting with captured 51-fc , indicating that the observed catch bonds were characteristic of the fn51 bond rather than the fc
however , at forces > 30 pn , the black circle curves in fig . 3 ( b and c ) became indistinguishable from the gray square curve , suggesting that the second slip bonds of the former curves result from dissociation of the fc
gg-7 bond rather than the fn51 bond . because the dissociation rate of two bonds in series equals to the sum of the two individual dissociation rates ,
the lifetimes of these two bonds in series would have been significantly shorter than either bond measured separately if lifetime of the fn51-fc bond were comparable with the fc
the fact that the lifetime versus force curves of the fn51-fc gg-7 and 51-fc
gg-7 bonds were indistinguishable shows that the fn51 bond lifetimes in mg / egta or mn were substantially longer than the fc
they might continue to behave as catch bonds at forces > 30 pn before ( and if ) it transitioned to slip bonds .
however , the real differences of fn51 bond lifetimes between ca / mg and mg / egta or mn conditions may be much greater than those apparent in fig .
3 ( a c ) . for comparison , we also measured force - dependent lifetimes of 51 and fniii710 bound to their respective blocking mabs , p1d6 and hfn7.1 ( fig .
4 , schematics ) , which were also specific to the antigen antibody interactions ( fig . s2 , c and d ) . similar to the fc gg-7 interaction and consistent with other antibody antigen interactions characterized previously ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) , slip bonds were observed in both cases ( fig .
4 ) as the mean lifetimes decreased monotonically with increasing force in the same range where the triphasic transitions between slip and catch bonds were observed for 51 interacting with its physiological ligand ( fig .
4 , compare a with b ) , revealing different interaction characteristics of the different antibody
lifetimes of antibody antigen bonds . ( a and b ) plots of lifetime ( mean sem ) versus force of interactions between 51-fc functionalized petri dish and p1d6-coated cantilever tips ( a ) and between hfn7.1 captured by anti
a large percentage of 51-fc appeared to exist in a bent conformation in ca / mg , but more of the integrin became extended in mg / egta and mn ( fig .
yet in all three cation conditions , lifetimes at low forces ( < 10 pn ) were similarly short ( < 2 s ) .
as force increased from 1030 pn , catch bonds were observed , but lifetimes were prolonged less by force in ca / mg ( fig .
these data indicate that catch bonds may not result from a force - induced unbending of the integrin . to obtain more definitive evidence
3 ( a c ) using tr51-fc that contains only a five - domain headpiece of the integrin fused with fc ( coe et al . , 2001 ; mould et al . , 2002 ) .
as anticipated , catch bonds were still observed , confirming that the integrin legs and the unbending conformational change were not required for the fn51 catch bonds ( fig . 5 , a c ) .
interestingly , in the 1025-pn range , truncating the integrin leg regions enabled force to prolong lifetimes to a greater extent than the integrin with legs ( fig . 5 ,
the lifetime versus force curves in the catch bond regimen were quite similar for all three cation conditions ( compare fig . 5 , a c )
however , similar to the fniii71051 case , the force where the fniii710tr51 catch bonds might transition to slip bonds could not be determined , for the mean lifetimes of the fniii710tr51-fc
gg-7 bond at a force > 25 pn in all three cation conditions ( fig . 5 , a
3 ) , indicating that these represented lifetimes of the fc gg-7 bond rather than the fniii710tr51-fc bond .
a c ) plots of lifetime ( mean sem ) versus force of tr51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips ( circles ) in ca / mg ( a ) , mg / egta ( b ) , and mn ( c ) . data from fig .
the lifetime versus force curves of 51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips are shown as triangles , and 51-fc coated cantilever tips dissociating from gg-7functionalized petri dish are shown as squares .
( d ) schematic of the molecular arrangement indicating possible dissociation loci between fniii710 and tr51-fc or between tr51-fc and gg-7 . to explore the structural mechanism of the observed catch bonds , we measured fniii71051-fc bond lifetimes in mn in the presence of ts2/16 ( fig . 6 a ) or 12g10 ( fig . 6 b ) .
6 d , schematic ) to further activate integrins by shifting or stabilizing the position of the 1 helix known to be critical for integrin activation ( mould et al .
both mabs left shifted the catch bond region , but ts2/16 yielded longer lifetimes at low forces ( fig .
interestingly , ts2/16 also left shifted the fniii710tr51-fc catch bond region but did not prolong lifetimes as much as it did for the fniii71051-fc case ( fig .
these data emphasize the importance of the a domain 1 helix and link the fniii710tr51 catch bonds to 51 activation .
( a c ) lifetime ( mean sem ) versus force plots ( circles ) of 51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips in mn and 10 g / ml ts2/16 ( a ) or 12g10 ( b ) or tr51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips in mn and 10 g / ml ts2/16 ( c ) . for comparison , data from fig .
3 c are replotted in a and b , and some of the data from fig .
5 c are replotted in c , where the lifetime versus force curves of tr51-fc functionalized petri dish dissociating from fniii710-coated cantilever tips are shown as triangles , and 51-fc coated cantilever tips dissociating from gg-7functionalized petri dish are shown as squares .
( d ) schematic of the molecular arrangement indicating binding sites for ts2/16 and 12g10 .
catch bonds represent unusual , counterintuitive behaviors that have recently been observed in selectin ligand ( marshall et al . , 2003 ; sarangapani et al . , 2004 ) ,
actin myosin ( guo and guilford , 2006 ) , fimh mannose ( yakovenko et al . , 2008 ) , and gpibvwf ( yago et al . , 2008 ) interactions . based on the integrin structures and their conformational change models ,
it has been speculated that integrin ligand interactions may also behave as catch bonds ( chigaev et al . , 2003 ; zhu et al . , 2005 ; alon and dustin , 2007 ; luo et al . , 2007
ligand catch bonds ( zhang et al . , 2002 , 2004 ; li et al . , 2003 ) .
in the previous work , force - ramp experiments were used to measure ramp rate - dependent rupture forces , which were analyzed by dynamic force spectroscopy , assuming that dissociation occurs along a single pathway and off rate increases exponentially with increasing force as modeled by bell ( 1978 ) , which precluded catch bonds . using afm force - clamp experiments , we observed triphasic force - dependent bond lifetimes that deviate from the bell model , demonstrating fn51 catch bonds ( figs . 3 , 5 , and 6 ) .
it is known that integrin - mediated adhesion can be strengthened by integrin clustering or binding of multiple integrins cooperatively , which can be induced by force ( galbraith et al . ,
however , this is unlikely the mechanism for the observed catch bonds for the following reasons .
first , ( tr)51-fc molecules were captured by gg-7 fab , which was unlikely to adsorb on petri dishes as clusters . nor was the fniii710 likely to adsorb on afm tips as clusters . without preclustering , without active cellular mechanisms to cluster the molecules , and without lateral mobility , it is unlikely that integrins bind in clusters in our experiments .
second , using the same afm setup in the control experiments , we observed only slip bonds for the 51-fc
gg-7 interaction was monomeric ( because fab was used ; fig . 3 f ) ,
3 a , schematic ) , and fniii710hfn7.1anti mouse fc interaction might be multimeric ( because whole hfn7.1 and anti mouse fc were used ; fig . 3 b , schematic ) .
third , we examined the correlation of ( or the lack thereof ) bond lifetime with molecular stiffness ( fig .
we have previously shown that the stiffness of multiple bonds is multiple times of that of a single bond ( sarangapani , 2005 ) , which predicts a positive correlation between lifetime and stiffness if the longer lifetimes were caused by greater bond multiplicity . however , such correlation was not observed ( fig .
s3 , a and b ) . to the contrary , the mean stiffness values for molecular complexes that had lifetimes clustered around 0.3 , 2.8 , and 10
these data ruled out higher multiplicity of bonds as the cause for longer lifetimes at high forces than the lifetimes at low forces in the catch bond regime .
finally , we have previously shown that formation of dimeric bonds can not generate catch bonds if the corresponding monomeric interaction is a slip bond .
the effects of dimeric bonds are to shift the lifetime versus force curve of the monomeric bond rightward toward doubling the force and upward toward doubling the lifetime ( marshall et al . , 2003 ) .
our flow cytometry data suggest that more 51-fc became extended in mg / egta and mn than in ca / mg ( fig .
s1 ) , which is consistent with previous flow cytometric ( humphries , 2000 ) , crystallographic ( xiong et al . , 2001 ) , and electron microscopic ( takagi et al . , 2002 ; zhu et al . ,
the affinity of fn51 binding was low in ca / mg but high in mg / egta or mn ( mould et al . , 1995 ) , which was manifested as different adhesion frequencies ( fig .
2 , a and c ) . at low forces ( < 10 pn ) , fn51 bonds dissociated rapidly ( lifetimes < 2 s ) in all three cation conditions ( fig .
c ) , suggesting that mg / egta or mn increased the on rate for association but did not significantly impact the off rate for dissociation .
these are consistent with our previous kinetic measurements of integrin l2 interacting from intercellular adhesion molecule 1 under these cations , which showed fast force - free off rates and a much greater responsiveness to cation conditions of on rates than off rates ( zhang et al . , 2005 ) .
in previous tension - free studies , mn has often been considered to be able to activate integrin to a high affinity state for ligand binding ( mould et al . , 1995 ;
takagi et al . , 2002 ) , which corresponds to the bond state at low forces in our experiment .
then , force is able to further strengthen the bond , inducing new bond states previously not identified ( friedland et al . , 2009 ) .
it has been suggested that force applied to a bent integrin may straighten it to an extended integrin , thereby giving rise to catch bonds ( chigaev et al .
2005 ; alon and dustin , 2007 ; luo et al . , 2007 ; mcever and zhu , 2007 ) .
however , catch bonds were observed not only for fn interacting with 51 ( fig .
c ) in three different cation conditions but also for fn interacting with tr51 that lack the leg regions ( fig . 5 , a
c ) , which indicates that force - induced unbending is not a required conformational change for fn51 catch bonds .
an essential feature of integrin activation is a swing of the subunit hybrid domain away from the subunit ( takagi et al . , 2002 ;
, it has been proposed that , without a lateral force to separate the integrin legs , pulling on an extended integrin would prevent the hybrid domain from swinging outwards , thereby stabilizing the inactive conformation of the headpiece ( zhu et al . ,
2008 ) . however , in our experiments , no lateral force was applied to separate the integrin legs , which were restrained . yet , pulling on 51 via a bound fn prolonged bond lifetimes at low forces , indicating that force activates rather than deactivates 51 .
it is possible that , at least in our system , the bond strengthening caused by pulling on the 1 helix connection may outweigh the effects of force on hybrid domain movement .
similar to the recombinant 51-fc fusion protein , native 51 purified from cell membrane and reconstituted into glass - supported lipid bilayers also formed catch bonds with fn in the same force range , although the apparent lifetimes were shorter ( fig .
for the majority of the adhesion events , the shorter lifetimes may not be caused by extraction of 51 from the lipid bilayer because the running adhesion frequency remained stable in a large number of repeated contacts ( fig .
s4 b ) , even when the same cantilever tip was used to contact the same lipid bilayer location , indicating that neither the tip nor the bilayer lost functionality over time ( chesla et al . ,
1998 ) . however , it is possible that some very long lifetimes ( > 10 s ) could be cut short by extrapolating of integrin from the bilayer .
in addition , more 51-fc might be in an extended conformation than m51 in the same cation condition , resulting in longer lifetimes for the catch bonds of fn with 51-fc than with m51 ( mould et al . , 2005 ) . as discussed in the preceding paragraph , in the bent conformation ,
although catch bonds were observed for both interactions of fn with 51-fc and m51 , the causes of their lifetime differences remain unclear and require further studies .
binding of ts2/16 or 12g10 left shifted the fn tr51-fc catch bond region by prolonging lifetimes at low forces ( fig .
both mabs bind at or near to the 1 helix of the a domain ( fig .
6 d ; mould et al . , 2002 ) , suggesting that this region could be important in regulating the catch bond behavior .
in addition to the rgd - binding site at the a domain of the 1 subunit , the propeller of the 5 subunit may bind the synergy site at the fniii9 domain to strengthen the fn51 interaction ( garcia et al .
, 2002 ; mould et al . , 2003b ; friedland et al . , 2009 ) .
it has recently been proposed that the fn51 bond can be switched from a relaxed to the tensioned states in response to mechanical force through engaging the synergy site in fn ( friedland et al . , 2009 ) .
multiple sites have also been proposed for ligand binding to integrin headpieces ( hynes , 2002 ; liddington and ginsberg , 2002 ; friedland et al .
thus , fn51 catch bonds could also involve force - induced binding of multiple sites that strengthen the molecular complex .
interestingly , the natural log ( number of measurements with a lifetime > t ) versus t plots for the fn51-fc bond exhibited multiple line segments ( fig .
c ) , which are in contrast to the more linear plots of the 51-fc
. the negative slope of a line reflects the off rate of the subpopulation of dissociation events associated with that line ( marshall et al . , 2003 ) .
multiple line segments suggest multiple states of single fn51-fc bonds rather than multiple fn51-fc bonds because 51-fc were captured by gg-7 fab randomly and sparsely adsorbed and because the lifetimes were measured from single rupture events .
similar multiple - bond states were observed for fimh mannose catch bonds ( thomas et al . , 2006 ) .
these are different from the selectin ligand catch bonds that show single lines in such plots ( marshall et al . , 2003 ; sarangapani et al . , 2004 )
instead of the aforementioned allosteric , multisite , and multistate models , a sliding rebinding model was proposed for selectin ligand ( lou et al . , 2006 ; lou and zhu , 2007 ) and gpibvwf ( yago et al . , 2008 )
these mechanisms are not mutually exclusive and may work together to give rise to integrin ligand catch bonds .
they also transduce signals bidirectionally from inside out and from outside in across the cell membrane .
such a dual role makes integrins prime candidates for force - sensing molecules in mechanotransduction ( schwartz and desimone , 2008 ) .
catch bonds provide a physical mechanism for force sensing if different bond lifetimes correspond to different activation states that transduce distinct signals .
the ability of integrin ligand bonds to be strengthened by force may be of great importance not only for leukocyte trafficking , which occurs under hydrodynamic forces , but also for the migration of many other cell types , which involves cyclic adhesion and detachment between the cell and the ecm .
catch bonds may provide a mechanical mechanism for the cell to regulate adhesion by applying different forces at different times and locations when and where different adhesion strengths are desired .
recombinant 51-fc and tr51-fc chimeras were generated by chol761h cells that were transiently transfected with cdna constructs encoding the human 5 ( fitzgerald et al . , 1987 ) and 1 ( coe et al . , 2001 ) subunits or tr5 and tr1 subunits ( coe et al . , 2001 ) .
fniii710 with a biotin tag at the n terminus was produced using standard recombinant dna techniques ( petrie et al . , 2006 ) .
anti-51blocking ( p1d6 ) and activating ( ts2/16 ) mabs were obtained from millipore , reporting mab ( 9eg7 ) was obtained from bd , and another activating mab ( 12g10 ) was obtained from abcam .
the anti - fn mab ( hfn7.1 ) was obtained from developmental studies hybridoma bank .
the anti human fc - capturing mab ( gg-7 ) was obtained from sigma - aldrich .
the afm is a modification of a previously described system ( marshall et al . , 2003 ; sarangapani et al . , 2004 )
it consists of a pzt on which a petri dish is directly mounted ( fig .
1 a ) . the pzt has a capacitive feedback control that gives subnanometer position resolution .
a laser ( oz optics ) focused on the end of the cantilever ( tm microscopes ) is deflected onto a photodiode ( hamamatsu photonics ) to measure cantilever deflection , which is converted to force using the cantilever spring constant .
spring constant ( 312 pn / nm ) for each cantilever was calibrated in situ using thermal fluctuation analysis ( hutter and bechhoefer , 1993 ) . a personal computer with a data acquisition board ( national instruments )
was used to control movements of the pzt and to collect signals from the photodiode .
labview ( national instruments ) was used as the interface between the user and the data acquisition board . to measure the interaction of 51-fc or tr51-fc with fniii710 ( fig
4 a , schematic ) , cantilever tips were incubated with 1020 g / ml fniii710 or p1d6 overnight at 4c ( fig .
1 b ) . after rinsing with tbs ( 25 mm tris - hcl and 150 mm nacl , ph 7.4 )
, the cantilevers were incubated for 15 min at room temperature in tbs containing 1% bsa to block nonspecific adhesion .
gg-7 was cleaved into fab and fc fragments by using the fab preparation kit following the manufacturer 's instructions ( thermo fisher scientific ) .
2 ) was adsorbed on a small spot on a petri dish by overnight incubation at 4c . to capture tr51-fc ,
the gg-7precoated petri dish was rinsed three times with tbs and incubated with 10 g / ml tr51-fc at the desired cation condition ( ca / mg , mg / egta , or mn ) for 30 min .
the petri dish was again rinsed three times with tbs and filled with 5 ml tbs plus 1% bsa and the indicated cations . in some experiments ,
10 g / ml hfn7.1 , 1 mg / ml cyclo(-grgdsp ) ( anaspec , inc . ) , or 10 g / ml ts2/16 or 12g10 ( fig .
6 d ) were added to the buffer . to measure the interaction of fniii710 with hfn7.1 ( fig .
4 b , schematic ) or 51-fc with gg-7 fab ( fig . 3 f ) ,
fniii710 or 51-fc was adsorbed on cantilevers and treated as described in the previous paragraph . 10 g / ml goat anti
mouse fc polyclonal antibody or 20 g / ml fragmented gg-7 was adsorbed on a labeled spot on a petri dish overnight at 4c . to capture hfn7.1 ,
the petri dish was rinsed three times with tbs and incubated with 10 g / ml hfn7.1 for 30 min .
the petri dish was again rinsed three times with tbs and filled with 5 ml tbs plus 1% bsa and the indicated cations . to measure the interaction of fniii710 with m51
, cantilevers were coated with streptavidin at 50 g / ml overnight at 4c and further functionalized by incubation with 10 l of 1 g / ml fniii710 for 15 min at room temperature .
( 1993 ) . in brief , vesicles were formed by hydrating a dried lipid film of 1,2-dimyristoyl - sn - glycero-3-phosphocholine and 1,2-dimyristoyl - sn - glycero-3-[phospho - rac-(1-glycerol ) ] ( avanti polar lipids , inc . ) in a 50:50 ratio with 0.1% triton x-100 ( thermo fisher scientific ) in tbs and mixed with m51 , resulting in a final concentration of 0.27 mg / ml ( 0.4 mm ) of lipid and 0.1 mg / ml of integrin .
triton x-100 was removed by adsorption to biobeads sm-2 ( bio - rad laboratories , inc . ) at 37c for 4 h. the resulting lipid vesicle solution was stored under argon at 4c and used within several months .
coverslips of 40 mm in diameter ( bioptechs ) were cleaned with a mixture of 70% 12 n sulfuric acid and 30% hydrogen peroxide by volume at 100c for 45 min , rinsed extensively with deionized water , and dried completely under an argon stream .
the cleaned coverslip , which was used immediately , was placed in a petri dish , and a 4-l drop of m51-incorporated lipid vesicle solution was placed on the coverslip surface .
after 20 min of incubation under a damp paper towel , the petri dish was filled with 5 ml tbs with 2 mm mg , 2 mm egta , and 1% bsa .
the m51 bilayers that formed had low molecular densities to ensure their infrequent binding to the fniii710-coated cantilever tips , as required for measuring single bonds .
the afm force - clamp experiments were performed by repeatedly bringing the petri dish in contact with the cantilever tip , then immediately retracting a small distance ( 05 nm ) , holding at that distance for 0.5 s to allow bond formation , and retracting again at a speed of 200 nm / s . by preventing the cantilever tip from compressing the petri dish during the time for molecular association , the nonspecific binding was greatly suppressed ( fig . 2 ; and fig . s2 , b
the presence of adhesion was detected from the force - scan curves ( fig . 1 , c and d ) .
a feedback system was used to clamp the force at a desired level to enable measurement of bond lifetime at constant force .
approximately 50 lifetimes were measured in the full - force range ( 570 pn ) using a cantilever and a petri dish in each experiment .
gg-7 interaction in 1 mm ca / mg condition , 13 experiments were performed to acquire 846 lifetimes at forces ranging from 570 pn .
these were segregated into 12 force bins , each of which spanned 56 pn and contained at least 50 lifetimes .
plots of natural log ( number of events with a lifetime > t ) versus t were exemplified for the fn51-fc
125 g ( 50 l ) streptavidin - coated magnetic beads ( thermo fisher scientific ) were incubated with gg-7 biotinylated using biotintag micro biotinylation kit ( sigma - aldrich ) in 1 ml pbs at a concentration of 5 g / ml for 30 min .
40 l beads were washed three times in tbs , incubated with 20 g / ml 51-fc in 200 ml tbs with desired cations for 30 min , again washed three times in tbs with appropriate cations , and incubated with 9eg7 at 10 g / ml for 30 min .
9eg7 was preconjugated with allophycocyanin using the lightning - link apc - xl conjugation kit following the manufacturer 's instructions ( innova bioscience ) . after washing three times with tbs containing the appropriate cations ,
s1 shows flow cytometry analysis of the expression of 9eg7 epitope by 51-fc in different cation conditions . fig . | binding of integrins to ligands provides anchorage and signals for the cell , making them prime candidates for mechanosensing molecules .
how force regulates integrin ligand dissociation is unclear .
we used atomic force microscopy to measure the force - dependent lifetimes of single bonds between a fibronectin fragment and an integrin 51-fc fusion protein or membrane 51 .
force prolonged bond lifetimes in the 1030-pn range , a counterintuitive behavior called catch bonds . changing cations from ca2+/mg2 + to mg2+/egta and to mn2 + caused longer lifetime in the same 1030-pn catch bond region .
a truncated 51 construct containing the headpiece but not the legs formed longer - lived catch bonds that were not affected by cation changes at forces < 30 pn .
binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range .
thus , catch bond formation appears to involve force - assisted activation of the headpiece but not integrin extension . |
herpes simplex virus ( hsv ) is the most common cause of infectious esophagitis after candidiasis .
herpes simplex esophagitis ( hse ) in immunocompromised hosts is well documented in the literature , particularly among those diagnosed with human immunodeficiency virus ( hiv ) infection .
however , rare cases have been reported in immunocompetent individuals . in a search of medline until october 2012
we present the first case of hse in a healthy 36-year - old female at 27 weeks gestation who recovered without antiviral therapy .
a healthy 36-year - old ( gravida 3 , para 3 ) female presented to the infectious diseases department at 27 weeks gestation with a six day history of fever , epigastric pain and dysphagia .
physical examination revealed gingivostomatitis , ulcerations and erythema of the gingiva , buccal mucosa and tongue .
upper endoscopy revealed several white patches and exudates throughout the esophagus with an edematous friable mucosa suggestive of severe candidal esophagitis . except for mild gastric mucosal erythema ,
histological examination of the esophageal biopsies revealed multinucleated giant cells with intranuclear inclusion bodies , typical for hse ( figure 1 ) .
her symptoms resolved completely within five days following the administration of intravenous hydration and a high dose proton - pump inhibitor .
she delivered a healthy daughter that weighed 3100 g , whose apgar scores were 9 ( one minute ) and 10 ( five minutes ) .
after 14 months of follow - up , the patient and her baby remained in good health .
histologic section of an esophageal biopsy obtained from a 36-year - old pregnant female showing multinucleated giant cells with intranuclear inclusion bodies , typical for herpes simplex esophagitis ( hse ) .
it has been initially described as an incidental observation during post - mortem examination with an incidence of 1.8% .
hse is an opportunistic infection in immunosuppressed patients with hiv , in cases of underlying malignancies , organ transplantations , inflammatory bowel diseases , and in patients who are prescribed corticosteroids , other immunosuppressive therapy , and radiation therapy .
the reasons for not considering this diagnosis in healthy adults may be due to its spontaneous remission and the inability to perform an esophagoscopy due to the presence of severe dysphagia .
hse may represent the reactivation of a latent infection , however it is more often due to a primary infection , with local spread of the virus from an orolabial or pharyngeal focus .
prior exposure to a family member with possible hsv lesions has been reported in about 20% of the cases .
trauma resulting from gastroesophageal reflux , esophageal instrumentation , nasogastric drainage or ingestion of caustics may predispose an immunocompetent individual to hse.10-
12 our patient had no history suggestive of gastroesophageal reflux or other predisposing factors .
the most common mode of transmission of an hsv infection is via direct contact of the fetus with infected vaginal secretions during delivery .
hse usually affects young males and is typically manifested by the acute onset of odynophagia , dysphagia , retrosternal pain , and fever .
gingivostomatitis with bilateral oropharyngeal lesions , ulcerations and erythema of the gingiva , buccal mucosa and tongue , lymphadenopathy and fever are usual manifestations of primary infection , while unilateral oropharyngeal lesions and cold sores suggest secondary infection .
the disease predominantly affects the distal or mid - esophagus , but can affect the entire esophagus , and even the stomach .
the gross appearance may vary depending on the timing of endoscopy . in the early stage , vesicles are seen , which then slough to form discrete , circumscribed ulcers with raised edges ; the mucosa is friable.9 ,
16 these lesions may be punched - out and volcano - like in appearance , or may coalesce to exhibit a cobblestone or shaggy ulcerative appearance .
exudate is present in the majority of cases16 and mucosal necrosis may be seen later .
specimens that have been obtained from the base of the ulcer are frequently devoid of epithelial cells .
the most effective diagnostic method for hse is histology .
the characteristic histologic appearance is the presence of multinucleated giant cells with eosinophilic intranuclear inclusions , called cowdry type a intranuclear inclusions and nuclear chromatin with a groundglass appearance .
pcr techniques for the detection of viral genome have demonstrated very high sensitivity and specificity in the diagnosis of a herpetic infection , even greater than those of the viral culture .
serologic tests have limited value , because of the high prevalence of hsv antibodies in individuals , but may be useful in diagnosing primary infections .
in contrast to immunocompromised patients , hse is usually a self - limited disease that has a favorable outcome in immunocompetent individuals . in these cases
, hse resolves spontaneously within 1 to 2 weeks , and exceptionally may be complicated by gastrointestinal bleeding or esophageal perforation.18-
20
acyclovir is a well established treatment for hse in the immunocompromised host but its efficacy in immunocompetent adults and adolescents is controversial .
therapy with acyclovir in immunocompetent patients may shorten the duration of symptoms although a controlled study has not been performed and may not be feasible because of the rarity of the disease .
acyclovir is safe in pregnant women and infants , with no congenital malformations or infant toxicity.23 ,
24 pregnant women are relatively immunocompromised ; thus antiviral therapy may be indicated in cases of hsv esophagitis .
our patient recovered promptly without antiviral therapy .
in conclusion , hse in pregnant woman
hse should be suspected even in healthy patients who present with the triad of symptoms of odynophagia , fever and retrosternal pain .
an upper endoscopy with biopsy for histopathology and viral culture should be performed to confirm the diagnosis .
| herpes simplex esophagitis ( hse ) has rarely been reported in immunocompetent individuals . in a search of medline until october 2012 , we found only one case of hse in a pregnant female .
we present the first case of hse in a healthy 36-year - old female at 27 weeks gestation who recovered without antiviral therapy . |
families are regarded as a crucial part of specialized childcare during illness ( 1 ) .
the concept of family - centered care includes the values of individuality , flexibility , cultural competence , and partnership with families ( 2 ) .
the involvement of family members carries particular weight in pediatric care , because children are directly dependent on family members for their care ( 3 ) .
in addition , it is important to consider that , in later years , family members request that professionals consider their views and practical knowledge and regard them as resources , mainly within the field of psychiatry ( 4 ) .
parents participation in the care of hospitalized children can be a challenging experience for both the parents and the members of the medical staff ( 5 ) .
this type of parental care has many advantages , including the parents skills and helping to maintain family relationships , as well as instilling in the parents a feeling of confidence that they can provide appropriate care after the child is discharged .
it also has also many advantages for the child , such as reduced stress and enhanced feelings of safety , which can have positive effects on the child s emotional wellbeing and behavioral disorders ( 6 ) .
the views of nurses who work in pediatric care concerning the influence of families are critical in this study , because its focus is rooted in the conceptual frameworks of the theory of planned behavior ( tpb ) model by ajzen ( 7 ) .
the tpb was first proposed by ajzen in 1985 as an extension to the theory of reasoned action , which included attitudes as variables that influenced intentions and behaviors ( 8) .
the variable of attitude refers to an individual s perceptions about the advantages and disadvantages of nurses performing a particular behavior to influence family nursing practice ( 9 ) .
shifting nurses perceptions about a proposed change in behavior is likely to be critically important in the eventual successful implementation of changes in conventional interventions ( 10 ) . in accordance with this model ,
the positive attitude of nurses towards family - centered care ( fcc ) is fundamentally important in facilitating changes in behavior that can lead to healing and shorter hospital stays for children .
therefore , it is essential to determine the attitudes of pediatric nurses towards the importance of families in providing appropriate care for children ( 11 ) .
in addition , it is important for professionals to recognize and appreciate family members practical knowledge to avoid family members having feelings of detachment in their child s care , which reinforces how vital it is for families to participate in the care of their children ( 2 ) . in a study conducted in sweden (
n = 634 ) of randomly selected nurses , it was found that more experienced nurses were more likely to respect the care of the family than younger and unskilled nurses .
however , the main finding was that , in general , nurses have a positive view towards families ( 12 , 13 ) .
the purpose of this study was to determine the attitudes of nurses towards the importance of family - centered care in pediatric wards before and after an educational intervention program that focused on applying the theory of planned behavior model . based on theoretical framework of the theory of planned behavior model
the following hypothesis was tested : nurses attitudes toward the provision of family - centered care are different before and after the educational intervention ( three months ) .
families are regarded as a crucial part of specialized childcare during illness ( 1 ) .
the concept of family - centered care includes the values of individuality , flexibility , cultural competence , and partnership with families ( 2 ) .
the involvement of family members carries particular weight in pediatric care , because children are directly dependent on family members for their care ( 3 ) .
in addition , it is important to consider that , in later years , family members request that professionals consider their views and practical knowledge and regard them as resources , mainly within the field of psychiatry ( 4 ) .
parents participation in the care of hospitalized children can be a challenging experience for both the parents and the members of the medical staff ( 5 ) .
this type of parental care has many advantages , including the parents skills and helping to maintain family relationships , as well as instilling in the parents a feeling of confidence that they can provide appropriate care after the child is discharged .
it also has also many advantages for the child , such as reduced stress and enhanced feelings of safety , which can have positive effects on the child s emotional wellbeing and behavioral disorders ( 6 ) .
the views of nurses who work in pediatric care concerning the influence of families are critical in this study , because its focus is rooted in the conceptual frameworks of the theory of planned behavior ( tpb ) model by ajzen ( 7 ) .
the tpb was first proposed by ajzen in 1985 as an extension to the theory of reasoned action , which included attitudes as variables that influenced intentions and behaviors ( 8) .
the variable of attitude refers to an individual s perceptions about the advantages and disadvantages of nurses performing a particular behavior to influence family nursing practice ( 9 ) .
shifting nurses perceptions about a proposed change in behavior is likely to be critically important in the eventual successful implementation of changes in conventional interventions ( 10 ) . in accordance with this model ,
the positive attitude of nurses towards family - centered care ( fcc ) is fundamentally important in facilitating changes in behavior that can lead to healing and shorter hospital stays for children .
therefore , it is essential to determine the attitudes of pediatric nurses towards the importance of families in providing appropriate care for children ( 11 ) .
in addition , it is important for professionals to recognize and appreciate family members practical knowledge to avoid family members having feelings of detachment in their child s care , which reinforces how vital it is for families to participate in the care of their children ( 2 ) . in a study conducted in sweden (
n = 634 ) of randomly selected nurses , it was found that more experienced nurses were more likely to respect the care of the family than younger and unskilled nurses .
however , the main finding was that , in general , nurses have a positive view towards families ( 12 , 13 ) .
the purpose of this study was to determine the attitudes of nurses towards the importance of family - centered care in pediatric wards before and after an educational intervention program that focused on applying the theory of planned behavior model . based on theoretical framework of the theory of planned behavior model
the following hypothesis was tested : nurses attitudes toward the provision of family - centered care are different before and after the educational intervention ( three months ) .
this experimental study was focused on the effectiveness of educational intervention concerning family - centered care in changing the attitudes of nurses in the pediatric wards of the hospitals affiliated with shahid beheshti university of medical sciences in tehran in 2015 .
the original sample size of nurses was 158 after 10% had dropped out , so , based on the pilot study data we had collected , we calculated that the final sample size of nurses to be 174 , with 87 randomly assigned to the control group and 87 randomly assigned to the intervention group . thus , we decided to have 100 nurses in each group .
multistage random sampling methods were used to select the participants . according to the information that was obtained from the ministry of health
, we chose the eligible hospitals that had the required sample sizes of nurses for both groups .
the first digit of the first random number was matched with corresponding matching number on the list of hospitals .
after picking the first hospital to be included in the sample , we proceeded in order in the table of random numbers from top to bottom and left to right to select the next hospitals to be included in the sample .
the inclusion criteria were : 1 ) full - time employment , 2 ) a bachelor s degree in nursing or higher , 3 ) currently working in a pediatric ward or at least one year of experience work doing so , and 4 ) between the ages of 2555 .
the exclusion criteria were : 1 ) foreign nurses and 2 ) working part - time or on contract .
based on the inclusion and exclusion criteria , 100 participants were chosen from the three intervention hospitals and 100 participants were chosen from the one control hospital . in the intervention group ,
one participant dropped out for the follow - up intervention assessment ( after three months ) and three participants dropped out for the follow - up in the control group ( after three months ) .
a post - test was conducted immediately , and follow - up was conducted in both groups after three months .
there were 200 participants during the study period at baseline ( 100 in intervention and 100 in control ) , and there were 196 participants at three months ( 99 in the intervention group and 97 in the control group ) who completed the three - month , post - intervention assessment .
the participants in the two groups answered the questions in the same questionnaire that was used at the beginning and after three months .
to determine the most commonly - held nurses attitudes , we conducted an elicitation study on a sample of 15 nurses who worked in the pediatric wards . in this stage , each interview lasted for 3575 minutes . in every part of the interview , we used the probing method to get additional information and to get clarification of the information ( table 1 ) .
all interviews were conducted by researchers , and the interviews were continued until the researchers felt that the maximum amount of information had been obtained .
four more interviews were conducted to ensure that a total of 15 interviews was conducted . in this research
the interviews were transcribed verbatim , and they were analyzed simultaneously with the data collection process .
after each interview , the information was recorded in the shortest possible time , and the information was reviewed two or three times .
the nurses sentences , each typically in the form of an expression of the meaning of a word and their implications were extracted from the interviews . to facilitate the detection of the thoughts they had expressed during the interviews , relevant clauses were inserted .
then , the information gathered from the interviews was classified into relevant categories , and we used the findings of the qualitative research ( semi - structured interviews with nurses and children ) .
thirty - one items were collected and evaluated on a 5-point likert scale ( strongly agree , agree , neutral , disagree , strongly disagree ) .
then , we conducted a pilot study to assess the reliability of the questionnaire and gathered additional comments from the population about the items on the questionnaire to prepare the final draft of the questionnaire for the main study . since the socio - demographic characteristics of subjects can affect the correlations among the variables in the proposed model , we included in the pilot questionnaire the participants ages , levels of education , work experience in the hospital , and work experience in pediatric wards . in the pilot study
, the questionnaire was tested with 30 pediatric nurses in hospitals affiliated with shahid beheshti university to provide a brief introduction to the study and so the researcher could explain the importance of their participation in the study .
then , the participants were asked to follow the hyperlink at the end of the invitation . in keeping with the requirements of research ethics , the first page of the research project
provided an introduction to the study and its objectives and indicated the requirements for participation in the study . to assess the participants attitudes concerning the study ,
we incorporated related statements , such as the mother s participation helps a hospitalized child feel secure and the mother s participation shortens the duration of hospitalization . fcc based on the theory of planned behavior survey that was used in this study consisted of two sections .
section one included questions about demographic variables , and section two consisted of a tool to measure pediatric nurses attitudes toward fcc .
some questions had negative scores ( att15-att20 and att24-att26 ) , we conducted data analysis after recoding
. validity is defined as whether a scale yields meaningful information about the behavior / construct in which the researcher is interested .
the measures in this study were evaluated by the researcher , her faculty adviser , and a pilot group of experts .
in addition , the validity of the content of each of the scales was assessed by manuals that provided specific instructions for creating each subscale , to clearly represent the necessary components of the constructs in the tpb model ( 1416 ) .
the specific wording of each item was consistent with an accurate reflection of the construct of perceived behavioral control in the tpb ( 16 ) . this same level of instruction was used for each domain . given that the guidelines from theory developer ( ajzen ) in 2005 were followed explicitly to create these measures , they were esteemed to adequately reflect the constructs from his theory of planned behavior .
the tool was validated by the content validity index ( cvi ) and face validity methods . to validate the content
, we sent the questionnaire to two faculty members at university putra malaysia ( upm ) who were experts in pediatric nursing and to five faculty members at shahid beheshti university and tarbiat moddaress university in tehran who were experts in pediatric nursing for corrections , suggestions and feedbacks .
expert panels , the members of which were selected based on their experience , certified that the content of the instrument was valid .
they were asked to review the items on the questionnaire for clarity and to determine whether they fit the subscale label and definition .
the professionals were nursing faculty members , supervisors , and members of the nurses staffs .
measurements of attitude , subjective norms , perceived behavior control , intention , and behavior of family - centered care were obtained through questionnaires based on theory of planned behavior by francis in 2004 ( 16 ) .
the reliability ( internal consistency ) of each of the scales was tested using cronbach s alpha reliability coefficients .
the results of the reliability tests indicated that , for the control group , the values of chronbach s alpha for all related items were almost 0.8 or above , which showed that the instrument had adequate consistency and reliability across the study .
descriptive statistics were used to describe the demographics of the sample and background variables . to evaluate the main research hypotheses of the current research
, we used the inferential method , including two - way repeated measure analysis of variance ( rmanova ) , followed by the bonferroni test for mean comparison between the control group and the experimental group at three steps , i.e. , pre - test , post - test , and follow - up test .
this study was approved by the university of putra malaysia , the ministry of health sciences , and the ethics committee of the faculty of medicine and health sciences of shaheed beheshti university in iran . written informed - consent forms were obtained from the nurses before were allowed to participate in the study .
this experimental study was focused on the effectiveness of educational intervention concerning family - centered care in changing the attitudes of nurses in the pediatric wards of the hospitals affiliated with shahid beheshti university of medical sciences in tehran in 2015 .
the original sample size of nurses was 158 after 10% had dropped out , so , based on the pilot study data we had collected , we calculated that the final sample size of nurses to be 174 , with 87 randomly assigned to the control group and 87 randomly assigned to the intervention group .
multistage random sampling methods were used to select the participants . according to the information that was obtained from the ministry of health
, we chose the eligible hospitals that had the required sample sizes of nurses for both groups .
the first digit of the first random number was matched with corresponding matching number on the list of hospitals .
after picking the first hospital to be included in the sample , we proceeded in order in the table of random numbers from top to bottom and left to right to select the next hospitals to be included in the sample .
the inclusion criteria were : 1 ) full - time employment , 2 ) a bachelor s degree in nursing or higher , 3 ) currently working in a pediatric ward or at least one year of experience work doing so , and 4 ) between the ages of 2555 .
the exclusion criteria were : 1 ) foreign nurses and 2 ) working part - time or on contract .
based on the inclusion and exclusion criteria , 100 participants were chosen from the three intervention hospitals and 100 participants were chosen from the one control hospital . in the intervention group ,
one participant dropped out for the follow - up intervention assessment ( after three months ) and three participants dropped out for the follow - up in the control group ( after three months ) .
a post - test was conducted immediately , and follow - up was conducted in both groups after three months .
there were 200 participants during the study period at baseline ( 100 in intervention and 100 in control ) , and there were 196 participants at three months ( 99 in the intervention group and 97 in the control group ) who completed the three - month , post - intervention assessment .
the participants in the two groups answered the questions in the same questionnaire that was used at the beginning and after three months .
to determine the most commonly - held nurses attitudes , we conducted an elicitation study on a sample of 15 nurses who worked in the pediatric wards . in this stage , each interview lasted for 3575 minutes . in every part of the interview , we used the probing method to get additional information and to get clarification of the information ( table 1 ) .
all interviews were conducted by researchers , and the interviews were continued until the researchers felt that the maximum amount of information had been obtained .
four more interviews were conducted to ensure that a total of 15 interviews was conducted . in this research
the interviews were transcribed verbatim , and they were analyzed simultaneously with the data collection process .
after each interview , the information was recorded in the shortest possible time , and the information was reviewed two or three times .
the nurses sentences , each typically in the form of an expression of the meaning of a word and their implications were extracted from the interviews . to facilitate the detection of the thoughts they had expressed during the interviews , relevant clauses were inserted .
then , the information gathered from the interviews was classified into relevant categories , and we used the findings of the qualitative research ( semi - structured interviews with nurses and children ) .
thirty - one items were collected and evaluated on a 5-point likert scale ( strongly agree , agree , neutral , disagree , strongly disagree ) .
then , we conducted a pilot study to assess the reliability of the questionnaire and gathered additional comments from the population about the items on the questionnaire to prepare the final draft of the questionnaire for the main study . since the socio - demographic characteristics of subjects can affect the correlations among the variables in the proposed model , we included in the pilot questionnaire the participants ages , levels of education , work experience in the hospital , and work experience in pediatric wards . in the pilot study ,
the questionnaire was tested with 30 pediatric nurses in hospitals affiliated with shahid beheshti university to provide a brief introduction to the study and so the researcher could explain the importance of their participation in the study .
then , the participants were asked to follow the hyperlink at the end of the invitation . in keeping with the requirements of research ethics , the first page of the research project provided an introduction to the study and its objectives and indicated the requirements for participation in the study . to assess the participants attitudes concerning the study , we incorporated related statements , such as the mother s participation helps a hospitalized child feel secure and the mother s participation shortens the duration of hospitalization .
fcc based on the theory of planned behavior survey that was used in this study consisted of two sections .
section one included questions about demographic variables , and section two consisted of a tool to measure pediatric nurses attitudes toward fcc .
some questions had negative scores ( att15-att20 and att24-att26 ) , we conducted data analysis after recoding . validity is defined as whether a scale yields meaningful information about the behavior / construct in which the researcher is interested .
the measures in this study were evaluated by the researcher , her faculty adviser , and a pilot group of experts .
in addition , the validity of the content of each of the scales was assessed by manuals that provided specific instructions for creating each subscale , to clearly represent the necessary components of the constructs in the tpb model ( 1416 ) .
the specific wording of each item was consistent with an accurate reflection of the construct of perceived behavioral control in the tpb ( 16 ) .
given that the guidelines from theory developer ( ajzen ) in 2005 were followed explicitly to create these measures , they were esteemed to adequately reflect the constructs from his theory of planned behavior .
the tool was validated by the content validity index ( cvi ) and face validity methods . to validate the content
, we sent the questionnaire to two faculty members at university putra malaysia ( upm ) who were experts in pediatric nursing and to five faculty members at shahid beheshti university and tarbiat moddaress university in tehran who were experts in pediatric nursing for corrections , suggestions and feedbacks .
expert panels , the members of which were selected based on their experience , certified that the content of the instrument was valid .
they were asked to review the items on the questionnaire for clarity and to determine whether they fit the subscale label and definition .
the professionals were nursing faculty members , supervisors , and members of the nurses staffs .
measurements of attitude , subjective norms , perceived behavior control , intention , and behavior of family - centered care were obtained through questionnaires based on theory of planned behavior by francis in 2004 ( 16 ) .
the reliability ( internal consistency ) of each of the scales was tested using cronbach s alpha reliability coefficients .
the results of the reliability tests indicated that , for the control group , the values of chronbach s alpha for all related items were almost 0.8 or above , which showed that the instrument had adequate consistency and reliability across the study .
descriptive statistics were used to describe the demographics of the sample and background variables . to evaluate the main research hypotheses of the current research
, we used the inferential method , including two - way repeated measure analysis of variance ( rmanova ) , followed by the bonferroni test for mean comparison between the control group and the experimental group at three steps , i.e. , pre - test , post - test , and follow - up test .
this study was approved by the university of putra malaysia , the ministry of health sciences , and the ethics committee of the faculty of medicine and health sciences of shaheed beheshti university in iran . written informed - consent forms were obtained from the nurses before were allowed to participate in the study .
the sample consisted of 200 nurses who responded to the attitude questionnaire before participating in the educational intervention concerning fcc .
the results of the chi - squared test indicated that there was no significant difference between the ages of the nurses in the intervention and control groups ( x = 0.166 , p = 0.920 ) .
these results also indicated that 2% of the respondents in the intervention group and 2.1% of the respondents in the control group had incomes between 300 to 500 k tomans ( table 2 ) .
all related items of attitude in the control group in the pre - test mother participation secures hospitalized child feeling . had the highest mean ( m= 4.14 , s.d= 0.59 ) followed by
mother knowledge can be increase the quality of care . ( m= 4.12 , s.d= 0.56 ) . the lowest mean related to mother can be present when cardiopulmonary resuscitation ( cpr ) is being done on the child if they wish .
also the highest mean observed for same items ( m= 4.06 , s.d= 0.58 ) and item 27 ( m= 4.07 , s.d= 0.54 ) respectively .
all related items of attitude in intervention group in pre - test the mother s participation helps a hospitalized child feel secure had the highest mean ( m = 3.99 , s.d .
= 0.51 ) , and it was followed by the mother s participation comforts the child and takes care of her or his physical needs ( m = 3.96 , s.d .
the lowest mean value was related to mother can be present , if she wishes , when cpr is being done on her child
the highest means were observed for the mother s presence is adequate , and her participation in providing care for her child is not required
= 0.52 ) and the mother s participation helps a hospitalized child feel secure (
the lowest mean was observed for the mother s participation shortens the length of the hospital stay ( m = 2.76 , s.d .
= 1.08 ) , and the same results were observed in the follow - up test . to determine how the respondents socio - demographic characteristics and attitudes concerning the provision of family - centered care were correlated , spearman correlation coefficient analyses were used to identify the demographic characteristics that had a significant relationship with attitude in the pre - test , post - test , and follow - up test .
table 3 shows that no significant relationship existed between age and attitude and the provision of family - centered care ( p > 0.05 ) . in order to evaluate the differences in the mean of attitude scores within the three stages of pre - test , post - test , and follow - up test for both groups ( i.e.
, the intervention and control groups ) , a two - way , repeated anova measure was used to assess whether the two groups had differences in their attitudes in the tests .
the sphericity assumption states that the difference scores of the paired levels of the repeated measures factor have identical population variance . in the same way as the other anova assumptions of normality and homogeneity of variance , it is critical to mention that the sphericity assumption refers to population parameters rather than sample statistics .
mauchly s sphericity test was used to evaluate the sphericity assumption , and the result showed that the sphericity assumption for attitude was violated ( x = 103.1 , p < 0.01 ) , therefore , the f - value was adjusted by a greenhouse - geisser correction .
the results of repeated anova measure of attitude score showed that the interactions between the group and the tests were statistically significant ( f(1.42 , 275 ) = 168.474 , p < 0.05 , = 0.465 ) , therefore , to test the related hypothesis , a post - hoc test ( bonferroni test ) was used to compare the mean scores ( table 4 ) . to show the efficacy of intervention , we compared the pre - test , post - test , and follow - up test in both the intervention and control groups separately .
the result of post - hoc test ( bonferroni test ) indicated that the difference between the pre - test and post - test in attitude score in intervention group was significant ( p < 0.05 ) .
these results indicated that there were no significant differences between any of the tests for the control group ( p > 0.05 ) ( table 5 ) .
it was evident that , in the intervention group , the mean attitude score increased after intervention , but it was consistent over time in the control group .
attitude toward performance behavior reflects an individual s global positive or negative evaluations of performing a practical behavior ; that is , attitude is determined by the individual s perception about the value of a given outcome of a behavior ( 7 , 9 ) . in this study
, it was found that the intervention group recorded a significant positive change in the mean attitude score ( p < 0.005 ) , and the increase in the proportion of pediatric nurses who had moderate attitude scores had increased to higher levels at the three - month follow - up .
compared to the baseline , a significant increase in attitude level was observed immediately in the intervention group , and also at three months after intervention , but no such increase was observed in the control group .
this difference could be attributed to the availability of information obtained from the educational process that was conducted for the intervention group .
a number of factors could have contributed to the success of the intervention , such as our sample s being comprised of well - educated , motivated nurses .
also , the attitudes of the nurses in the two groups ( intervention and control ) concerning the provision of fcc at three different times ( i.e. , pre - test , post - test , and follow - up test ) indicated that there was a significant association between the nurses attitudes , because the interaction between groups and the tests were statistically significant . also , the mean attitude score between the control and intervention groups in the pre - test was not statistically significant ( p > 0.05 ) , while the differences between the intervention and control groups were significant for attitude in the post - test ( p < 0.05 ) and in the follow - up test ( p < 0.05 ) , while the difference between the pre - test and post - test in attitude score in the case group was significant .
it was apparent that intervention to encourage nurses to stop doing some things and start doing other things was an important step toward creating behavioral changes .
the results of this study support the effectiveness of multi - component education in promoting attitude changes in pediatric nurses in iran .
therefore , the results of the study indicated that hypothesis education is one of the most effective factors to improve attitude , because 93.9% of the population has no education about fcc .
the mean for attitude increased in the case group , but there were no significant differences in any of the tests for the control group .
this finding indicated that it is possible to achieve a significant positive change in pediatric nurses attitudes with educational intervention .
similarly , the findings of a previous study indicated that it is possible to increase nurses knowledge and attitudes with the help of brief , interactive workshops .
the findings of that study were based only on positive changes in rns attitudes and knowledge after four weeks of workshops ( 18 ) .
in addition , intervention was effective in that it increased new mothers intentions to breast feed their babies expanded their knowledge about caring for their babies , and produced better attitudes and subjective norms ( 19 ) .
so , the findings of our study were in good agreement with them concerning the importance and benefits of providing educational opportunities .
based on the results of a study in shiraz , enhancing the treatment team s knowledge and attitudes concerning the positive effects of fcc treatment produced improvements in patients healthcare , qualitative improvements in healthcare services , the nurses job satisfaction ( 20 ) .
in addition , it was found that this approach had a sustainable impact that lasted for an undetermined time after the intervention .
it certainly was apparent at the time of the three - month follow - up test . if educational programs are based on assessments nurses needs and are conducted purposefully using different methods , such as lectures , pamphlets , posters , and booklets , nurses attitudes subjective norms , perceived behaviors , behavioral intentions , and actual behaviors can be affected in a notably positive manner ( 21 ) .
also , the results of a study indicated that the mean level of nurses attitudes toward obtaining special skills were significantly different after the educational intervention ( 22 ) .
comprehensive educational programs that emphasize personal educational needs reduce the cost of providing care , and improve the quality of care ( 23 ) .
therefore , continuing education is a basic need to ensure that pediatric nurses can adapt to the progression of knowledge and the rapid changes in scientific approaches and people s behaviors .
as evidence of this , a notable increase occurred in the mean scores of nurses knowledge immediately and at three months after the educational process ( 25 ) . in that study ,
the mean scores of nurses knowledge increased from 59.214.8 before their education training to 88.68.4 directly after the training and 719.8 three months after the training ( p < 0.016 ) .
the results of our study were contrary to the findings of their study in that there were no significant differences in the nurses attitudes for the three times they were evaluated .
the key purpose of the tpb is to provide an explanation for the att- behavior relationship ( 26 ) .
tpb will be rejected if att does not predict intention , and studies that have measured att , intention , and behavior consistently have demonstrated that the more favorable an individual s attitude is toward a particular behavior , the stronger that person s intention to express that behavior will be ( 7 , 2729 ) . in 115 studies that used tpb in different areas in which the att - intention relationship was measured , att explained approximately 24% of the variance in intention ( 27 ) .
. one of the most important and common problems among nurses and parents is their lack of communication and the lack of a good relationship .
parental participation , which was introduced as a moral issue , may be driven now by tpb factors .
this study provided evidence to support the efficacy of tpb in the design and evaluation of educational intervention to promote positive attitudes concerning the implementation of fcc .
the findings supported various types of intervention , including educational material and pamphlets , posters , powerpoint presentations , lectures , and training , all of which are effective strategies in promoting fcc behaviors among nurses .
the findings also indicated that , while nurses endorse the concept of fcc , its practical implementation is more problematic .
also , the results of this study are being used as the basis for assisting pediatric nurses to apply the concepts of family - centered care more consistently in their practice .
thus , an important target of study for future research is to follow the relationship between nurses extent of training and the importance they give fcc in practice .
such research has the potential to identify the factors that lead nurses to maintain some distance between themselves and the family members of sick children . | introduction : family - centered care sustains the unity of the child s and the family s health . the aim of this study was to determine nurses attitudes toward parents participation in the care of their hospitalized children in iran in 2015.methods:in this experimental study , 200 pediatric nurses from hospitals affiliated with the shaheed beheshti university of medical sciences in tehran were selected using the multi - stage , random - sampling method .
data were gathered using a questionnaire that covered demographic information and nurses attitudes . the questionnaire consisted of 31 items and was completed by the nurses in three stages : 1 ) before intervention ( pre - test ) ,
2 ) immediately after intervention ( post - test ) , and 3 ) three months after intervention ( follow - up ) .
the data were analyzed via spss software and using descriptive and analytical methods .
descriptive statistics , the spearman correlation coefficient , and repeated measure analysis ( the bonferroni method ) were used to assess the data.results:the results indicated that there was a significant increase in the mean score of attitude after intervention [ m ( pre ) = 3.35% , m ( post ) = 3.97% , p < 0.001 ) ] .
most of subjects had neutral attitudes toward family participation in their children s care .
there were no significant relationship between the nurses socio - demographic characteristics and their attitudes.conclusion:the nurses attitudes toward the family s participation in the care of their hospitalized children were moderate .
the nurses attitudes should be improved by taking part in continuous training programs . |
these patients can have associated urethrovaginal or vesicovaginal fistula , requiring early or delayed surgical repair .
spontaneous closure of the fistula associated with obstetric or gynecological injury has been described in the literature . to the best of our knowledge ,
a 45-year - old female sustained pelvic fracture with urethral injury due to a road traffic accident . initial resuscitation , orthopedic management with internal fixation of the pubic bone , and suprapubic urinary diversion were done elsewhere .
she consulted us after 3 months of the initial injury with history of continuous leakage of the urine per vaginum . on pelvic examination
, urine was seen emanating from the vagina and a scar was felt in the proximal vagina .
she came after 15 days with acute urinary retention and cessation of the urinary leak per vaginum .
on filling the bladder with methylene blue through the suprapubic catheter , no leak was demonstrable through the vagina .
antegrade cystoscopy revealed normal bladder wall along with complete obstruction and scarring at the level of bladder neck .
a mitrofanoff procedure was performed using the tapered ileal segment as intraoperatively , the appendix was found to be very short .
she was discharged in satisfactory condition and is doing well till the last follow - up .
female urethral injury during pelvic fracture is rare because of the short length of the relatively mobile urethra which lies behind and is protected by the pubic bone .
orkin et al . , have reported 6% incidence of urethral injury in a review of 2,000 cases of pelvic fracture .
children outnumber the adults in such injuries perhaps because of greater compressibility of the less ossified bones . there can be a urethral contusion , longitudinal urethral laceration or urethral transaction in such cases . in cases of female urethral disruption related to pelvic fracture ,
immediate repair of the pelvic fracture with the urethral and vaginal injuries is advocated since suprapubic drainage with the spontaneous evolution of these lesions usually leads to complete obliteration of the urethra , urethrovaginal fistula , and various degrees of vaginal stenosis as was seen in our patient .
primary endoscopic realignment of the separated urethral ends over a catheter , avoiding tissue dissection or sutures in the traumatized area is an alternative approach recommended by others . however , with realignment , the likelihood of development of a urethral or bladder neck stricture has been reported to be quite high , needing delayed surgical reconstruction .
the reconstructive surgery is also more extensive , often requiring a bladder neck construction through transpubic approach .
our patient presented late with a completely obliterated urethra with an initial urethrovaginal fistula which later spontaneously got obliterated .
the extent of the injury precluded reconstructive surgery and required a catheterizable channel in the bladder . in the cases of fistulas formed due to obstetric or gynecological injuries , spontaneous healing is described in the literature , especially in those cases where the fistula is small and early , with prolonged bladder drainage is provided .
our case does bring out a fact that is less emphasized in the medical teaching that while persistence of fistula is often attributed to the distal obstruction , closure of fistula does not necessarily mean restoration of normal anatomy but could be a development of proximal obstruction . to the best of our knowledge
, we are describing the first case of spontaneous closure of the urethrovaginal fistula associated with pelvic fracture .
the possible explanation for the spontaneous closure in this case is probably because of progressive scarring leading to entrapment of the fistula and encasement of the bladder neck in the dense scar .
this can also be a marker of extensive urethral injury and the likelihood of complete urethral obliteration as opposed to a short segment stricture .
in female urethral injury due to pelvic fracture , early primary repair should be attempted to avoid subsequent devastating complications like urethrovaginal fistula or urethral obliteration . delayed repair in expert hands
however , complete obliteration of the urethra extending up to the bladder neck often precludes reconstructive surgery and may require a catheterizable channel in the bladder .
spontaneous closure of the urethrovaginal fistula can occur if there is extensive and dense scarring around the fistulous area . | female urethral injury following pelvic fracture is a rare entity . due to the absence of large series , management guidelines are still not standardized .
patients can have associated urethrovaginal or vesicovaginal fistula , management of which poses a major challenge to the reconstructive urologist .
spontaneous closure of fistula produced by gynecological or obstetrical injuries have been described in the literature .
spontaneous closure of fistula caused due to pelvic fracture has not been described in the literature . |
nephrotic syndrome is a heterogeneous group of childhood disorders associated with proteinuria , hypoalbuminaemia , oedema and failure to thrive , and is usually managed by glucocorticoid treatment .
between 10% and 20% of the patients fail to respond to this therapy and are diagnosed with steroid - resistant nephrotic syndrome ( srns).the prognosis for srns is usually poor , due to the increased risk of developing end - stage renal disease .
defects in several genes result in srns : nphs1 , nphs2 , wt1 , actn4 and cd2ap [ 15 ] .
mutations in the wt1 gene are the second most common cause of sporadic srns following defects in nphs2 .
wt1 encodes a zinc - finger transcription factor with multiple isoforms , which has critical role for development of the genitourinary tract and maintenance of podocyte differentiation .
dominant wt1 mutations have been implicated in a large number of disorders such as frasier , denys drash and wagr syndromes .
srns alone is found to be associated with defects in exons 8 and 9 of the gene . here
, we report on the case of a female srns patient with onset of the disease at 22 months of age and frequent relapses .
the renal pathology in this child is due to a novel missense mutation affecting the dna - binding zinc - finger domain of wt1 .
the patient was born to parents with no known family history of kidney disease , at the end of 28th gestational week , weighing 950 g with signs of respiratory distress syndrome and anaemia .
g / l per 24 h ) at 22 months of age . ultrasound examination revealed hyperechogenic parenchyma .
the female sex of the child was confirmed by str analysis . due to lack of response to steroid treatment ,
g / l per 24 h , blood pressure 115200 mmhg systolic and 70100 mmhg diastolic ) .
an alternate regimen of immunosuppressant ( cyclosporine and mycophenolate ) , ace inhibitor and corticosteroid was established . at present , the child is 6 years old , with levels of proteinuria 1.11
g / l per 24 h. genetic testing for mutations in the most commonly affected genes , nphs2 and wt1 , was carried out .
while no defects were found in the podocin gene , a previously unknown nucleotide substitution , c > a in position 1184 , was identified in wt1 . on the amino acid level , this results in replacement of serine with tyrosine in position 395 .
the substitution was found neither in the parents of the patient nor in any of the 120 unrelated control samples from individuals with no known kidney disorder .
a clustalw2 multiple species alignment of the sequence surrounding serine 395 indicated that the affected residue is conserved among organisms as diverse as xenopus , mouse , chimpanzee and human ( figure 1 ) .
, ser395 is located at the first turn of the -helix in zinc - finger 3 ( zf3 ) . to investigate the effect of s395y on wt1
analysis of the predicted mutant model revealed that the tyrosine s bulky side chain would clash with dna in two of three possible rotamers , while in the third rotamer , there would be a collision with phe383 of the protein ( figure 2 ) .
the -helix of wt1 zinc - finger 3 is shown as secondary structure model with the three possible rotamers of ser395 mutated to tyr ( designated as s395ya , s395yb and s395yc , respectively ) as well as the neighbouring residue phe383 .
we identified a novel heterozygous nucleotide change , c1184a , in exon 9 of wt1 in a srns patient . on the protein level
the absence of the mutation in both parents indicates a de novo origin . according to the data in the human gene mutation database ( hgmd ) , missense mutations are the most common cause of wt1-associated diseases .
this poses difficulties in elucidating the genotype phenotype correlations since the effect of a substitution on the protein level may be difficult to predict .
the absence of the respective variant in healthy individuals and the evolutionary conservation of the affected residues are often reliable indications for the pathogenic character .
it was found to be absent among healthy controls and affects an evolutionarily conserved residue in a functionally important domain of the protein .
however , in order to understand the correlation between genetic defect and pathology , one could use molecular models based on the crystal structure data for predicting the effect on the protein level .
a study of the molecular structure of wt1 reveals the important role of serine 395 for correct folding of the zf3 and localization of the -helix in the major groove of dna .
the residue is conserved in all wt1 zfs and also in the homologous domain of the zif268 protein .
the structure of the wt1 complex with the 14-bp dna duplex showed that zf3 along with zf2 and zf4 makes base - specific contacts in the major groove . by introducing ser395tyr in the model , we could show that the bulky tyr side chain would either prevent the proper folding of the protein or interfere with the dna binding ; hence , the mutation would have a negative impact on the function of wt1 ( figure 2 ) . in summary , we have identified a new nucleotide change in wt1 , which results in serine to tyrosine substitution in position 395 .
ser395tyr affects an important , evolutionarily conserved part of the transcription factor responsible for dna binding .
molecular modelling indicated that the serine residue is essential for the proper functioning of wt1 .
taken together , these results allow us to conclude that c1184a is pathological by nature . | we report the case of a paediatric patient with steroid - resistant nephrotic syndrome due to a novel dominant wilms tumour 1 mutation .
the nucleotide change c1184a , identified in exon 9 , results in amino acid substitution ser395tyr .
genotyping of parents and healthy controls indicated that this is a de novo mutation not present in healthy individuals .
the affected amino acid is evolutionarily conserved and is located in a functionally important domain of the protein involved in dna binding .
molecular modelling based on crystallography data indicated that the substitution would have a deleterious effect on the protein function . |
the nonenzymatic replication
and transmission of genetic information
may have played an important role in the transition from prebiotic
chemistry to cellular life by enabling the evolution of selectively
advantageous ribozyme catalysts prior to the emergence of ribozyme
rna polymerases .
the chemistry of nonenzymatic
oligonucleotide replication was extensively explored by orgel and
his students and colleagues .
their most successful
model system involved the use of 2-methylimidazole - activated mononucleotides
to nonenzymatically copy short c - rich oligonucleotide templates .
considerable progress has since been achieved
in demonstrating template - directed primer extension in the presence
of activated nucleotide derivatives .
however , several challenges still need to
be addressed to show that nonenzymatic rna replication could be sufficiently
fast and accurate to allow for the evolution of new rna - encoded functions
within replicating protocells .
foremost among these problems is the
slow rate of nonenzymatic primer extension on templates that contain
a and u residues .
the rate of primer extension varies by more than
2 orders of magnitude depending on the specific nucleotide being added
to the growing chain .
2-methylimidazole - activated amp or ump ( 2-meimpa
or 2-meimpu ) are added to the 3-end
of a primer ( annealed to a complementary template ) at a much slower
rate than 2-meimpg or 2-meimpc .
the rate of primer extension
with 2-meimpu is so slow as to be comparable to the rate of 2-meimpu
hydrolysis .
a second major problem is
that g : u and a : c wobble pairing lead to a very high error rate in
nonenzymatic template copying , potentially
precluding the emergence of functional rnas such as ribozymes from
diverse sequence populations .
we sought to address these challenges
by replacing u with su , a nucleobase known to significantly
stabilize base pairing
with a while modestly destabilizing wobble pairing with g. remarkably , the thione - mediated stabilization of the su : a base pair within an rna duplex is achieved without detectable
structural perturbation , suggesting that
the effect may be due at least in part to preorganization of the su - containing single strand . in
addition the larger and more diffuse electron cloud of sulfur vs oxygen
makes it more polarizable and hence may contribute to better stacking . replacing thymine with 2-thiothymine in dna is also stabilizing , and in rna
we have therefore also explored the use of 2-thiothymine ribonucleotides
( st ) in rna copying reactions .
in addition to the
simple thermodynamic considerations , several
independent arguments support the possibility that su
or st might lead to improved nonenzymatic rna copying .
nonenzymatic polymerization is most effective in the context of an
a - form helix .
su substitution
in the anticodon loop of trna has been shown to increase the 3-endo
( n ) conformer abundance of neighboring nucleotide sugars , and st stabilizes trnas from extreme
thermophiles , most likely through stabilization
of the 3-endo sugar conformation .
we have previously shown
that monomers that are in the 2-endo conformation in solution
switch to the 3-endo state upon binding to an rna template .
since the su mononucleotide has
been reported to exist in solution predominantly in the 3-endo
conformation , this preference should favor
binding to the template .
finally , we have recently reported that nonenzymatic
primer extension in another system , in which primer , template , and
monomers are 3-amino-2,3-dideoxynucleotides ,
exhibits enhanced rates and fidelity upon replacement of t with st .
we report the effects of su and st substitutions on both the rate and fidelity of nonenzymatic
primer extension reactions in an all rna system .
we show that these
substitutions in the activated monomer , but not in the template , contribute
to an enhanced ability to copy mixed sequence templates and that the
fidelity of copying is greatly increased .
to examine
the effect of 2-thio substitution on nonenzymatic rna
primer extension , we measured rates of primer extension using both
2-thio substituted u and ribo - t monomers and templates .
we used two
thiolated - nucleobase activated monomers , 2-thiouridine-5-phosphor-(2-methyl)imidazolide
( 2-meimpsu ) and 2-thio-5-methyluridine-5-phosphor-(2-methyl)imidazolide
( 2-meimpst ) , in nonenzymatic template - directed primer
extension reactions with fluorophore - tagged rna primers ( figure 1 ) .
we used two types of templates for this study ,
one type with six - nucleotide homopolymeric template regions u6 and a6 , and a second type with a single u or a
followed by five c residues , uc5 and ac5 ( figure 2 ; see also supporting information
section 1 for a complete list of oligonucleotides used in this
study ) . for pseudo - first - order rate determinations ,
primer extension
reactions were studied for a maximum of 50 min , much less than the
25 day half - time of hydrolysis of the activated nucleotides .
pseudo - first - order rates and monomer template dissociation
constants were determined from plots of the fraction of unreacted
primer as a function of time , for a series of activated nucleotide
concentrations ( table 1 ; figure 2 ; see also supporting information figure
1 ) .
2-meimpx analogues form watson crick base pairs on
a complementary template and participate in template - directed primer
extension .
( b ) structure of thiolated uracil and thymine nucleobases
in 2-methylimidazole - activated nucleotides .
we first considered the homopolymeric templates a6 ,
u6 , su6 , and st6 . while primer extension with 2-meimpu on the a6 template was essentially undetectable at monomer concentrations
up to 175 mm ( kmax < 0.01 h )
, 2-meimpsu and 2-meimpst resulted in maximal
rates of primer extension ( kmax ) of 0.56
0.069 and 2.3 0.71 h , respectively
( table1 ; figure 2a ;
see also supporting information figure 1b(1 ) ) .
it is notable that the single - atom oxygen to sulfur substitution
resulted in readily observable primer extension ; furthermore , methylation
at the 5-position of 2-thiouracil increased the rate of primer extension
an additional 4-fold .
in contrast , primer extension with 2-meimpa
on u6 , su6 , and st6 templates approached the lower bounds of experimental measurement
by this assay ( kmax = 0.027 0.0036 ,
0.067 0.0081 , and 0.071 0.014 h ,
respectively ) ( table 1 ; figure 2b ; see also supporting information figure
1b(1 ) ) .
these low rates and the modest effects of 2-thiolation
and 5-methylation may reflect the poor stacking of u and modified
u monomers and thus poor preorganization of templates consisting of
multiple u ( or su or st ) residues in a row
into an a - type helical conformation . activated monomer and template
nucleotides are indicated at left .
200 mm hepes ph 7.0 , 0.5 m
p1 , 1.5 m template , on ice and ( 1 ) 1.0 m nacl , 200 mm mgcl2 ; ( 2 ) 1.0 m nacl , 200 mm mgcl2 , 40 mm 2-meimpg ;
( 3 ) 100 mm mgcl2 , 40 mm 2-meimpg . kinetic studies of primer extension reactions .
pseudo - first - order
rates were determined from the extent of primer disappearance as a
function of time , and the resultant observed rates were determined
as a function of activated nucleotide concentration to give kmax .
reaction conditions : 200 mm hepes ph 7.0 ,
0.5 m primer p1 , 1.5 m template , on ice . buffer 1 ( blue ) :
1.0 m nacl , 200 mm mgcl2 .
( a ) primer extension reaction on template t1 ( a6 ) with 2-meimpu * ( u * = u , su , or st ) .
( b )
primer extension reaction on templates t2 ( u6 ) , t3 ( su6 ) , or t4 ( st6 ) with 2-meimpa .
( c ) primer extension reaction on template t8 ( ac5 ) with
2-meimpu * ( u * = u , su , and st ) and 40 mm 2-meimpg .
( d ) primer extension reaction on template t5 ( uc5 ) , t6
( suc5 ) , and t7 ( stc5 ) with 2-meimpa and 40 mm 2-meimpg .
although homopolymeric templates have been traditionally
used to
assess the reactivity of individual activated nucleotides , they are
not directly relevant to potentially functional sequences , which are
more likely to contain all four nucleotides .
we therefore used a set
of mixed sequence templates to examine primer extension in a more
realistic setting .
the template sequences followed the pattern 5-c5n-(primer binding sequence)-3 where n was a , u , su , or st . with the 5-c5a-(primer
binding sequence)-3 template ,
the corresponding rates of monomer
addition followed a similar pattern to those of the a6 template .
the incorporation of either 2-meimpu , 2-meimpsu or 2-meimpst in the presence of 2-meimpg led to kmax values of 0.26 0.063 , 0.96 0.11 , and 4.4
0.45 h , respectively ( figure 2c , buffer 1 containing 200 mm hepes ph 7.0 , 200 mm mgcl2 and 1 m nacl ; also see supporting information
figure 1b(2 ) ) .
similarly , we found that the incorporation of
2-meimpa in the presence of 2-meimpg was approximately an order of
magnitude faster on the single - u , su or st
templates than on the corresponding homopolymeric templates , with
observed rates of 0.83 0.045 , 0.38 0.019 , and 0.78
0.024 h , respectively ( figure 2d , buffer 1 ; see also supporting information
figure 1b(2 ) ) .
however , as with the homopolymer templates ,
thiolation of u or t in the template had little effect on the rate
of primer extension . on the basis of the fact that base stacking plays
a role in determining the melting temperature of complementary oligonucleotides , we suggest that the increased rates on the single - u
or a templates , compared to the homopolymer templates , are best explained
by improved stacking interactions of the incoming monomer ( adjacent
to the primer ) with downstream g monomers base paired to the c5 portion of the template .
the above experiments were
carried out using a high salt buffer
( buffer 1 , containing 200 mm mgcl2 and 1 m nacl ) that promotes
base pairing by masking the interstrand repulsion of negatively charged
phosphates . however , molar salt concentrations are incompatible with
primitive fatty acid based vesicles , and
we therefore examined primer extension without added nacl and with
a lower mgcl2 concentration .
we observed that in buffer
2 ( 200 mm hepes ph 7.0 and 100 mm mgcl2 ) , kmax and kd values were modestly
reduced , by less than 2-fold in most cases ( table 1 ; figure 2c , d ; see also supporting information figure 1b(3 ) ) .
we used next generation sequencing
( ngs ) to assess the fidelity
of the primer extension products obtained on mixed sequence templates ,
in the presence of competing activated monomers .
we generated libraries
of extended primers flanked by the necessary adaptor sequences at
both the 5 and 3 ends , as illustrated in figure 3 .
briefly , nonenzymatic primer extension was carried
out with a primer ( p2 ) that included the 5-adaptor sequence
necessary for on - bead amplification during sequencing , and also included
a 5-biotin so that the template strand could be removed by
a convenient bind and wash procedure using magnetic streptavidin beads .
following template removal , the nonenzymatically extended primer molecules
were released from the streptavidin beads .
an adaptor was then ligated
to the 3-end of each primer , followed by reverse - transcription
and pcr to yield the library for sequence analysis .
( a ) top : primer - extension was carried out on 2.0 m template
t9 ( agagag ) in the presence of 40 mm 2-meimpc and 50 mm 2-meimpu *
( u * = u , su , or st ) on ice for 7 days .
bottom :
products were sequenced , and the sequence reads binned according to
the number of nucleotides added to the primer ; y = c or u*. reaction
conditions : 2.0 m primer p2 , 200 mm hepes ph 7.0 , 100 mm mgcl2 .
( b ) top : primer - extension was carried out on 2.0 m
template t5 ( uc5 ) , t6 ( suc5 ) , or
t7 ( stc5 ) in the presence of 40 mm 2-meimpg
and 50 mm 2-meimpa on ice for 7 days .
bottom : products were sequenced
and the sequence reads binned according to the number of nucleotides
added to the primer ; r = a or g. we compared primer extension across a 5-gagaga-(primer
binding sequence)-3 template ( t9 ) with u , su ,
and st as activated monomers ( figure 4a ; see also supporting information table
1a ) in the presence of 2-meimpc and sequenced the extended
primers as described above to assess the extent and fidelity of the
template copying reaction .
primer extension with 2-meimpu and 2-meimpc
yielded largely products extended by only two or three nucleotides
( figure 4a , left panel ) . however , in an otherwise
identical reaction with 2-meimpsu and 2-meimpc we observed
a wider distribution of product lengths including full - length product ,
i.e. , primer + six nucleotides ( figure 4a ,
middle panel ) .
this effect was further enhanced when 2-meimpst replaced 2-meimpu ( figure 4a , right
panel ) .
sequence reads obtained
from the products of primer - extension on template t9 ( agagag ) , as
described in figure 4 , were sorted into bins
according to the number and identity of nucleotides added to the primer .
( a ) left : products extended by at least two nucleotides , with correct
incorporation of u * at position 1 , were sorted according to whether
the correct nucleotide c or the incorrect nucleotide u * was incorporated
at position 2 .
right : products extended by at least two nucleotides ,
with incorrect incorporation of c at position 1 , were sorted according
to whether the correct nucleotide c or the incorrect nucleotide u *
was incorporated at position 2 .
( b ) left : products extended by at
least three nucleotides , with correct incorporation at positions 1
and 2 , were sorted according to whether the correct nucleotide u *
or the incorrect nucleotide c was incorporated at position 3 .
right :
products extended by at least three nucleotides , with incorrect incorporation
of u * at position 2 , were sorted according to whether the correct
nucleotide u*or the incorrect nucleotide c was incorporated at position
3 .
examination of the sequences of the products of
primer extension
on the 5-gagaga-(primer binding sequence)-3 template
revealed enhanced fidelity with the 2-meimpsu and 2-meimpst monomers , compared to the standard 2-meimpu monomer . in
order to examine the effect of the modified nucleotides on g
: u wobble
mispairing , we examined all products in which the primer had extended
by two or more bases and where the first nucleotide added was the
correct u ( or su or st ) .
correct primer extension
would then result in incorporation of a c at position 2 , while g : u
mispairing would result in the incorporation of a u ( or su or st ) .
we found that g : u mispairing at the second
position was diminished from 4.3% in the case of 2-meimpu to 1.6%
with 2-meimpsu and 2.0% with 2-meimpst ( figure 5a ; see also supporting information
table 2 ) , corresponding to a 23 fold improvement in
fidelity with the 2-thiolated u or t monomers .
because primer extension
with 2-meimpu was so inefficient , we were unable to compare the frequency
of watson
crick vs wobble pairing at the next g residue in
the template , which is at position 4 .
surprisingly , we discovered
that a : c mispairing was also diminished
when u was replaced with su or st .
the expected
product of primer extension on the 5-gagaga-(primer binding
sequence)-3 template at positions 1 , 2 , and 3 was primer - u*cu*.
when we examined sequences where the first two nucleotides were correct ,
we found that an incorrect c was incorporated at position 3 over 19%
of the time , following primer extension with 2-meimpu and 2-meimpc .
we noted a 56 fold drop in the amount of primer - ucc when 2-thiolated
u - derivatives were used , with only 4.3% and 2.9% c incorporation at
position 3 when primer extension was carried out with 2-meimpsu and 2-meimpsu , respectively ( figure 5b ; see also supporting information
table 2 ) .
finally , we observed that both g : u and a : c
misincorporation largely
terminated further primer extension and , furthermore , that the small
amount of continued primer extension was highly error prone .
we first
consider the case of a g : u mismatch at position 2 , corresponding to
primer - uu products ( see supporting information
table 2 ) .
primer extension in the presence of 2-meimpu and
2-meimpc resulted in 2394 such sequences , but only 21 sequences corresponding
to the addition of one more nucleotide to the growing primer , i.e. ,
less than 1% continued extension .
in contrast , we observed 53 134
sequences corresponding to the correct 2-nucleotide extension product
primer - uc , and 13 077 sequences corresponding to continued
primer extension by at least one more nucleotide , i.e. , about 20%
continued primer extension .
remarkably , primer extension following
a g : u mismatch was highly error prone , with an error rate of almost
40% . replacing 2-meimpu with either 2-meimpsu or 2-meimpst did not significantly increase the fidelity of post mismatch
synthesis ( 2550% error rate in both cases ) .
examination of
sequences corresponding to an a : c mismatch at position + 1 reveals
similar poor fidelity at the following position . to assess the
capacity of su and st in
the template to mitigate g : u mispairing , we performed nonenzymatic
primer extension reactions with 5-c5u*-3
templates ( where u * = u , su , or st ) in the
presence of both 2-meimpa and 2-meimpg .
interestingly , there was insignificant
differentiation between u , su , or st when
they were templating primer extension in this context , and the distributions
of product length were comparable in all three cases ( figure 4b ; see also supporting information
table 1b ) .
a single - atom substitution
of oxygen by sulfur at the 2-position
of the activated uridine nucleotide 2-meimpu significantly improves
the rate and fidelity of nonenzymatic template - directed primer extension .
under different template and buffer conditions ,
the rate of primer
extension consistently followed the order st > su > u. surprisingly , this enhanced rate and fidelity of
template
copying was only observed with 2-thio - u or -t as the activated monomer ,
and no significant effect was observed when these modified nucleotides
were present in the template strand .
although the reasons for this
striking difference are not entirely clear , we suggest the following
speculative explanation . we consider first an incoming u ( or su or st ) monomer . although an su : a
base pair at an internal position in a duplex stem is much more stable
than a standard u : a base pair , little if any stabilization is seen
for an su : a base pair at the end of a helix .
this is consistent with the fact that we do
not see a statistically significant decrease in the kd of the 2-meimpsu or 2-meimpst monomers relative to 2-meimpu ( although the kd values for the very slow reactions on the a6 template
have large standard errors ) .
we do however see an increased maximal
rate of reaction ( at saturating monomer concentration ) for the 2-thiolated
monomers .
we suggest that this stems from preorganization of the 2-thionucleotides
in the 3-endo conformation , which could
help to properly position and orient the phosphate group of the incoming
monomer for reaction with the attacking 3-hydroxyl of the
primer .
weaker hydrogen bonding between the sulfur of su(t ) and the imino proton of g would weaken wobble pairing , favoring
correct binding of c , and that together with a further distorted geometry
may account for the decreased formation of u : g mismatches observed
with su and st .
we attribute the decreased
frequency of a : c mismatches to the faster reaction rate of su or st ( relative to u ) when paired with a ; effectively
su and st outcompete c so that a : c mismatches
do not have time to form .
we now consider the substitution of u in
the template with su or st . in this case ,
the binding of an incoming a monomer to the primer
template
duplex is facilitated by the stronger stacking interactions of the
purine nucleobase with flanking nucleotides , i.e. , the 3-nucleotide
of the primer , and downstream monomers . as a result ,
the kd of activated a is lower than that of the activated u
monomers . in addition , template preorganization by an internal su or st may contribute to enhanced binding of
2-meimpa , as observed for the 5-cccccu * templates
. however ,
once the activated a monomer is bound to the template , it has the
same reactivity whether the template base is u , su , or
st , and as a result , the rate at saturation ( i.e. , kmax ) does not change .
accurate direct measurements
of binding affinities and geometries may allow for experimental testing
of the above hypotheses .
the stalling of primer extension following
a mismatch has previously
been shown , under certain circumstances , to lead to an enhanced effective
fidelity of replication , because the first template copies to be completed
tend to be the most accurate .
however ,
this effect comes at the cost of significantly slowed overall rate
of replication .
our data suggest that the formation of both u : g and
c : a wobble pairs during nonenzymatic template - directed primer extension
leads to a very strong stalling effect , i.e. , a greatly decreased
rate of primer extension following a wobble mismatch . the magnitude
of this effect ( ca . 20-fold , figure 5 ) is surprising
and should be examined in additional sequence contexts .
nevertheless ,
we now expect that replacing u with su or st should improve the overall rate of primer extension not only because
of the increased rate at which the 2-thio monomers are incorporated
but also because the increased accuracy of primer extension will lead
to less stalling after mismatches .
in addition , we note that the strongly
decreased fidelity that we observed for primer extension following
a mismatch is consistent with previous proposals that errors introduced
during nonenzymatic rna copying may be dominated by multiple sequential
errors , as opposed to isolated single mutations .
such clustered mutations may speed the exploration of sequence
space and the optimization of functions under selection .
enhancing
the ability of rna to make large jumps through sequence space may
be particularly important in facilitating the emergence of novel rna - coded
functions .
finally , our results raise the question of the prebiotic
availability
of su or st .
it is conceivable that these
2-thio nucleotides could be generated spontaneously in a sulfur - rich
early earth environment through a route analogous to that proposed
for the synthesis of the canonical pyrimidine nucleotides .
the experimental demonstration of an efficient
pathway for the prebiotic synthesis of either su or st nucleotides would support their proposed role in facilitating
nonenzymatic rna replication during the origin of life .
representative
reaction
protocol : 4.0 l of 1.0 m hepes ph 7.0 buffer , 5.0 l
of nuclease - free water , 1.0 l of primer p1 , and 3.0 l
of template t5 were combined in a thin - walled pcr microtube .
after
being mixed well by pipetting up and down multiple times , the oligonucleotides
were incubated at 90 c for 5 min and annealed at 25 c
for 5 min .
a 2.0 l amount of 1.0 m mgcl2 was then
added to the solution and mixed well . to initiate primer extension ,
1.0 l of 1.0 m 2-meimpa and 4.0 l of 200 mm 2-meimpg
were added to the lid of the microtube cap .
the reaction was initiated
when the nucleotides were spun down into the buffered primer / template
solution .
the solution was mixed well and incubated in a metal block
in an ice bath for the duration of the experiment .
aliquots ( 4.0 l
each ) were removed at 10 , 20 , 30 , 40 , and 50 min and were immediately
quenched by addition to 26 l of precipitation buffer [ 3.0 l
of 3.0 m naoac ph 5.5 , 2.0 l of 500 mm edta ph 8.0 , 1.0 l
of 5 mg / ml glycogen , 2.0 l of 10x tbe ( 1.0 m tris , 1.0 m boric
acid , and 20 mm edta ph 8.0 ) , and 18.0 l 8.0 m urea ] .
after
the mixture was briefly vortexed to mix the contents , 75 l
of pure ethanol was added .
the sample was mixed and kept at 25
c for a minimum of 30 min and was then centrifuged at 15 000
rcf in an eppendorf 5424r centrifuge at 4 c .
the rna pellets
were then taken up in 5.0 l of 8.0 m urea in 1x tbe and incubated
at 90 c for 5 min before the rna products were separated by
20% ( 19:1 ) denaturing page .
the gel was scanned with a typhoon 9410
variable mode imager , and the bands were quantified using the accompanying
imagequant tl software package .
nonenzymatic template - directed
primer extension reactions were performed as described above except
that 2.0 m primer p2 and 2.0 m template were used .
ten millimolar tris - hcl ( ph 7.5 ) ,
10 mm edta ( ph 8.0 ) , 2.0 m nacl ; buffer a : 100 mm naoh , 5 mm nacl ;
buffer b : 10 mm nacl ; elution buffer : 95% formamide , 10 mm edta ( ph
8.0 ) ; ssc buffer : 150 mm nacl , 15 mm na citrate , ph 7.0 .
a 100 l amount of streptavidin
myone c1 dynabeads ( 10 mg / ml ; invitrogen ) was separately decanted
for each library assembly reaction . gentle draw / expel pipetting was
used as a bead mixing technique ; vortexing was avoided .
the beads
were mixed and washed 3 with 200 l bind and wash
buffer and were allowed to sit on a magnetic rack for 1 min following
each wash step .
beads were then washed 2 200
l buffer a , followed by 2 200 l buffer b. the
rna pellet from an extension reaction using primer p2 was diluted
with 200 l of nuclease - free water and 200 l of bind
and wash buffer .
the rna solution was added to the washed
and decanted beads and then briefly mixed to suspend the beads in
the rna solution .
after the mixture was rested on the magnetic rack for 1 min , the supernatant
was drawn off .
beads with
bound primer / template complex were mixed with 250 l of ssc
buffer and decanted after resting on the magnetic rack for 1 min .
the beads were suspended in 100 l of 150 mm naoh and incubated
at 25 c for 10 min .
the tube was placed on the magnetic rack
for 1 min , and the supernatant was drawn off .
the beads were then
mixed with 250 l of 100 mm naoh and immediately placed on the
magnetic rack for 1 min followed by removal of the supernatant .
the
biotinylated oligonucleotides were immediately eluted from the beads
by mixing with 250 l of elution buffer at 65 c for 5
min followed by 1 min on the magnetic rack and removal of the supernatant .
the rna from this final supernatant was precipitated upon addition
of 1120 l of ethanol and 30 l of 3.0 m naoac ph 5.5
and incubation for 30 min at 25 c .
a 20.0 l
amount of 100 m 3-adaptor ( 5-phosphate - aga
tcg gaa gag cac acg tct3-3-t-5 ; dna ) and 6.0
l of nuclease - free water were added to the biotinylated rna
pellet and placed in a thin - walled pcr microtube .
after dissolution ,
the mixture was incubated at 65 c for 2 min then placed on ice ,
and 4.0 l of 10x t4 rna ligase i buffer , 4.0 l of 50%
peg 8000 , 4.0 l of dmso ( molecular biology grade ) , and 2.0
l of t4 rna ligase i ( 10,000 u / ml ) were added . after mixing ,
the solution was incubated at 37 c for 2 h. the samples were
cleaned up with a qiaquick pcr purification kit ( eluted with h2o not eb buffer from kit ; qiagen ) to remove excess 3-adaptor
( 22-mer ) and retain the ligated product ( 92-mer ) .
the samples were
then lyophilized to dryness and taken up in 15 l of nuclease - free
water .
the following sequences
used a combination of materials from superscript iii first - strand
synthesis supermix ( invitrogen ) and nebnext multiplex small rna library
prep set 1 for illumina ( neb ) .
a 1.0 l amount of sr rt primer
for illumina ( diluted 1:2 ; neb ) and 2.0 l of annealing buffer
( invitrogen ) were added to the 15 l solution of rna .
the mixture
was incubated in a thin - walled pcr microtube at 75 c using a
thermal cycler for 5 min , 37 c for 15 min , and finally 25 c
for 15 min . at 25 c , 20 l of 2x first - strand reaction
mix ( invitrogen ) and 2.0 l of superscript iii / rnaseout enzyme
mix ( invitrogen )
were added to the rna solution , mixed , and incubated
at 50 c for 1 h and 85 c for 5 min and then kept on ice .
a 2.5 l amount of sr primer for illumina ( neb )
, 2.5 l
of indexed prime ( neb ) , 5.0 l of nuclease - free water , and 50
l of longamp taq 2x master mix ( neb ) were
added to the reverse - transcribed mixture and were cycled 12 times
( 30 s of initial denaturation , 94 c ; 15 s , 94 c ; 30 s ,
62 c ; 15 s , 70 c ; 5 min of final extension at 70 c ;
hold at 4 c ) .
the desalted and
enzyme - free dna was purified on a 20% tbe precast gel cassette ( invitrogen )
with quick - load pbr322 dna - mspi digest ( neb ) as a marker .
the target
band of 140 bp was sliced out , crushed with a disposable plastic rnase - free
pestle ( fisher ) , and eluted with 250 l of dna gel elution buffer
( neb ) .
the dsdna was desalted with a qiaquick pcr purification kit
and quantified by qpcr with a kapa sybr fast universal qpcr kit for
illumina ( kapa biosystems ) .
samples were prepared as per the
manufacturer s recommendations with a 30% phix spike to bring
an appropriate level of initial sequence diversity to the libraries .
a multiplexed paired - end protocol was used : 25 nucleotides for read-1 ,
index read , 25 nucleotides for read-2 . upon completion of sequencing
the
script takes two fastq files , one for the forward read and one for
the reverse read , and filters out reads that either lack the exact
adapter sequence in the reverse read , lack the adapter in the forward
read ( with a maximum edit distance tolerance of 1 ) , or have forward
and reverse reads that are not identical in the region of nonenzymatic
primer extension .
it then outputs all retained sequences and their
corresponding total read counts , as well as the number of reads discarded
for the reasons mentioned above . | the
nonenzymatic replication of rna oligonucleotides is thought
to have played a key role in the origin of life prior to the evolution
of ribozyme - catalyzed rna replication .
although the copying of oligo - c
templates by 2-methylimidazole - activated g monomers can be quite efficient ,
the copying of mixed sequence templates , especially those containing
a and u , is particularly slow and error - prone .
the greater thermodynamic
stability of the 2-thio - u(s2u):a base pair , relative to
the canonical u : a base pair , suggests that replacing u with s2u might enhance the rate and fidelity of the nonenzymatic
copying of rna templates .
here we report that this single atom substitution
in the activated monomer improves both the kinetics and the fidelity
of nonenzymatic primer extension on mixed - sequence rna templates .
in addition , the mean lengths of primer extension products obtained
with s2u is greater than those obtained with u , augmenting
the potential for nonenzymatic replication of heritable function - rich
sequences .
we suggest that noncanonical nucleotides such as s2u may have played a role during the infancy of the rna world
by facilitating the nonenzymatic replication of genomic rna oligonucleotides . |
cell behavior is regulated by numerous distinct cues that impinge on them in vivo . alongside chemical gradients
geard and willadsen , 2009 ; niehrs , 2010 ; ben - zvi et al . , 2011 ; gershenson , 2012 ) and physical forces ( beloussov and grabovsky , 2006 ; beloussov , 2008 ; nelson , 2009 ; von dassow and davidson , 2011 ; davidson , 2012 ) , cell activity is orchestrated toward the creation and repair of high - order anatomical structures by a set of bioelectrical cues ( levin , 2012a , b ; levin and stevenson , 2012 ) .
here bioelectricity refers to endogenous electrical signaling via ion channels and pumps at the plasma membrane ; specifically excluded due to length constraints is the rich literature on external electromagnetic fields ( funk et al . , 2009 ;
cifra et al . , 2011 ; hronik - tupaj and kaplan , 2012 ) , ultraweak photon emission ( farhadi et al . , 2007 ; fels , 2009 ; sun et al .
, 2010 ; beloussov , 2011 ) , and subcellular organelle potentials ( bustamante et al . , 1995 ;
the importance of bioelectricity for cells beyond excitable nerve and muscle was realized long ago , and solid functional data implicate steady ion currents in embryogenesis and wound healing ( burr and northrop , 1935 ; lund , 1947 ; jaffe and nuccitelli , 1977 ; nuccitelli et al . , 1986 ; borgens et al .
, 1989 ; hotary and robinson , 1992 ) . by tracking developmental currents and applying physiological - strength electric fields
, it was shown that transepithelial electric fields regulate cell migration , orientation , and nerve growth ( jaffe and poo , 1979 ; patel and poo , 1982 ; borgens et al .
, 2005 ; nishiyama et al . , 2008 ; cao et al .
, 2011 , 2013 ; ozkucur et al . , 2011 ; pullar , 2011 ; reid et al . , 2011b ; vieira et al . ,
2011 ; pan and borgens , 2012 ; zhao et al . , 2012 ; yamashita , 2013 ) .
however , recent advances and development of molecular - level techniques ( adams , 2008 ; adams and levin , 2013 ; levin , 2013 ; tseng and levin , 2013 ) have identified a new aspect of bioelectricity that regulates individual cell function and helps coordinate the embryogenesis and regenerative repair of complex structures .
this review focuses on the instructive cues mediated by spatiotemporal patterns of voltage potentials across the membranes ( vmem ; figure 1a ) of nonneural cells and the roles these play in coordinating cell behavior during regeneration , development , and cancer .
bioelectrical signaling at the cell and organism levels , at the level of single cells , bioelectrical signals are produced by ion channel proteins , transduced into second - messenger responses , and alter key aspects of cell behavior .
( a ) the voltage potential ( vmem ) at the cell membrane is produced by the movement of ions through across a cell membrane .
ions move via many different ion channels and pumps , under the control of concentration and electric gradients .
( b ) change of vmem is transduced into cellular effector cascades by a range of mechanisms , including voltage - sensitive phosphatases , voltage - gated calcium channels , and voltage - sensitive transporters of signaling molecules such as serotonin and butyrate .
( diagram modified , with permission , from figure 1b of levin , 2007 . ) ( c ) bioelectrical signals feed into epigenetic and transcriptional cascades and thus trigger changes in cell properties such as proliferation , differentiation , migration , shape change , and programmed cell death .
( d ) voltage reporter dye reveals gradients of vmem across the anterior - posterior axis of planarian flatworms .
( taken , with permission , from figure 2b of beane et al . , 2013 . )
( e ) in amputated worms , a circuit composed of proton and potassium conductances sets the voltage states at each blastema , which in turn determines the anatomical identity of each end of a regenerating fragment . ( diagram taken , with permission , from figure 7c of beane et al . , 2011 .
) ( f ) manipulating this circuit in amputated planaria using pharmacological or genetic techniques that target ion flux allows the programming of stem cell mediated morphogenesis to specific anatomical outcomes , such as the creation of two - head animals shown here .
in general , terminally differentiated , quiescent cells tend to be strongly polarized ( bearing a more - negative resting potential ) , whereas embryonic , stem , and tumor cells tend to be depolarized ( closer to zero ; binggeli and weinstein , 1986 ) .
the picture is complicated by two still poorly understood factors : the relationship of overall vmem state to the cell cycle dependent ( sinusoidally varying ) changes in voltage potential ( arcangeli et al . , 1995 ;
higashimori and sontheimer , 2007 ; aprea and calegari , 2012 ) and the fact that many cells in fact do not have a single vmem but bear a set of distinct voltage domains over their surface ( o'connell and tamkun , 2005 ; o'connell et al . , 2006 ; levin , 2012a ) .
crucially , vmem is not simply a readout but is also a functional determinant of cell behavior , such as proliferative state and plasticity ( table 1 ) , due to a number of mechanisms that functionally couple voltage potential changes to downstream cascades ( figure 1 , b and c ) .
these data derive from genetic experiments , as well as pharmacological screens designed to identify compounds that regulate stem cell differentiation or cancer progression ( alves et al . , 2011 ; sun et al . , 2013 )
differentiation and proliferation are controlled by changes in vmem , as shown in human mesenchymal stem cells ( sundelacruz et al . , 2008 , 2013 ;
cardiomyocytes ( lan et al . , 2014 ) , inhibitory postsynaptic currents ( jiang et al . , 2009 ) , vascular muscle ( jia et al . , 2013 ) , embryonic stem cells ( ng et al . , 2010 ; du et al . , 2013 ) , myoblasts ( in which hyperpolarization driven by the kir2.1 channel plays a key role ; hinard et al . , 2008 ; li et al . , 2010
) , the specification of neurotransmitter types ( root et al . , 2008 ) , and the control of precursor differentiation ( van vliet et al . , 2010 ; yasuda and adams , 2010 ; lange et al . , 2011 ; liebau et al . , 2011 ; ring et al . , 2012 ;
given the known roles of vmem in regulating normal migration , differentiation , and proliferation ( aprea and calegari , 2012 ; ding et al .
, 2012 ; zhang et al . , 2012 ; cao et al . , 2013 ; yamashita , 2013 ) , it is not surprising that control of ion flux ( park et al . , 2008 ; house et al . , 2010 ) and membrane voltage ( morokuma et al . , 2008a ; blackiston et al . , 2011 ; chernet and levin , 2013a , 2013b ; yang and brackenbury , 2013 ) are also increasingly implicated in the cell dysregulation of cancer ( table 2 ) .
the differential activation of voltage - responsive transduction mechanisms on opposite sides of a cell allows bioelectric signals to regulate cell polarity .
this was long ago shown in the symmetry breaking and control of outgrowth point in the algae fucus ( jaffe , 1966 , 1968 ) and has been recently shown using high - resolution imaging and genetic techniques in yeast ( minc and chang , 2010 ) and pollen tubes ( certal et al . , 2008 ; michard et al . ,
the cytoskeleton is one target of such signaling ( chifflet et al . , 2003 ;
, 2006 ; sekulic et al . , 2011 ; campetelli et al . ,
positional information can likewise be dictated by voltage properties of cells ( baglioni et al . ,
2012 ) and their neighbors ( shi and borgens , 1995 ) . studies of embryonic left
right patterning of the xenopus embryo have revealed how bioelectrical processes link individual cell dynamics to axial patterning of the entire body plan ( levin and palmer , 2007 ; aw and levin , 2009 ) : cytoskeletal chirality within the fertilized egg drives asymmetric distribution of ion transporter proteins in the early blastomeres , and the resulting gradient drives unidirectional ( preneural ) serotonin flow through cell fields , eventually triggering differential gene expression on the left versus right sides of the body ( levin , 2006 ; levin et al . , 2006 ; aw et al . , 2008 ;
lobikin et al . , 2012b ; vandenberg et al . , 2012 , 2013 ) .
the dissection and synthesis of such systems at the genetic and physiological levels is beginning to reveal the properties of biophysical pathways by which individual cell polarity is integrated into large - scale patterning outcomes ( marshall , 2011 ) .
the first step in analyzing a bioelectric signal is the characterization of the spatiotemporal distributions of ionic parameters and a determination of how they correlate with patterning events .
vmem in cells can be quantified using several approaches ; unlike mrna and protein levels revealed by sequencing or immunohistochemistry , bioelectric properties are only ascertainable in vivo and can not be analyzed in fixed tissue .
voltage gradients can now be visualized continuously in situ using fluorescent reporters of transmembrane potential ( adams and levin , 2012a , b ; figure 1d ) and more exotic nanoscale materials ( tyner et al . , 2007 ) suitable for use in any optically accessible tissue ( steinberg et al . , 2007 ; yun et al
these are a significant improvement on physiological impalement of single cells : far less invasive , and able to report multiple vmem values across tissues and even within cell membrane subdomains ( lechleiter et al . , 1991 ; adams and levin , 2013 ) .
reagents include cell - permeant dyes such as cc2-dmpe and disbac2(3 ) ( adams et al . , 2006 ; adams and levin , 2012b ; oviedo et al . , 2008 ; ozkucur et al . , 2010 ) and genetically encoded protein reporters ( tsutsui et al . , 2008 ;
additional tools for the characterization of bioelectrical events include highly sensitive ion - selective extracellular electrode probes ( reid et al . , 2007 ; smith et al . , 2007 ) that reveal ion flux , microelectrode arrays ( aryasomayajula et al . , 2010 ; schonecker et al . ,
2014 ) , and reporters of individual ion species such as protons ( tantama et al . , 2011 ) and sodium ( tseng et al . , 2010 ; dubach et al .
significant opportunities exist for the development of specific , bright , ratiometric dyes that localize exclusively to the desired subcellular locale ( e.g. , plasma membrane or nucleus ) .
especially exciting will be the use of multiple physiological dyes in fluorescence - activated cell sorting experiments to identify subpopulations of pure stem and other cell types that differ in key bioelectric properties ( mello de queiroz et al . , 2008 ) , as has been observed for human endothelial cells ( yu et al . , 2002 ) .
of importance , such experiments on dissociated cells will clearly highlight properties that are cell autonomous versus those physiological conditions that can only be maintained within a group context .
the mechanistic investigation of bioelectric cues and their interactions with canonical biochemical pathways has been enriched by several new functional techniques ( adams and levin , 2006b , 2013 ; reid et al .
the comprehensive workflow for probing developmental bioelectricity can be illustrated by two examples . in the first , a tiered pharmacological screen ( adams and levin , 2006a ) implicated a proton pump and two channels as specifically required for tail regeneration but not for wound healing or development of the primary tail ( adams et al . , 2007 ) .
these loss - of - function data were confirmed using reagents with molecular specificity by misexpression of a dominant - negative form of a v - atpase subunit protein .
marker analysis was used to show why tails failed to regenerate in v - atpase inhibited tails ( loss of regeneration - specific gene up - regulation , lack of the obligate increase of mitosis near the wound , and abrogation of innervation into the regenerate ) . fluorescent dye imaging provided physiomic
profiling of the changes of vmem during the stages of regeneration and confirmed that the unique voltage changes characteristic of the regenerating state were blocked by v - atpase inhibition and were absent during stages at which tadpoles normally are not competent to regenerate their tails . on the basis of these findings , to develop a gain - of - function application , a yeast p - type proton pump was misexpressed in regeneration - incompetent animals , leading to restoration of mitosis , gene expression ( msx-1 , notch ) , innervation , and morphological regeneration of a complete tail .
additional rescue experiments using net - electroneutral proton exchangers allowed the independent testing of ph versus voltage signaling .
one key result was that the anatomical outcome ( regeneration rescue ) can be induced by a completely heterologous hyperpolarizing pump , which has no sequence or structural homology to the native xenopus protein endogenously driving regeneration .
this demonstrated that the necessary and sufficient trigger for regeneration is not a specific gene product ( v - atpase ) , but a bioelectrical state , which can be implemented using a variety of different reagents .
this finding facilitated development of a purely pharmacological method of modulating ion flows in the wound to induce tail ( tseng et al .
, 2010 ) and leg ( tseng and levin , 2013 ) regeneration without the need for gene therapy .
the available tools enable a multistep strategy that combines pharmacological screening , physiological imaging , and molecular - genetic tools to generate loss- and gain - of - function data showing how a bioelectric pathway normally works and how it can be exploited to trigger pattern formation .
a similar approach was taken with an initial gain - of - function screen , misexpressing ion channels in frog embryogenesis .
one of the outcomes was the finding that a specific vmem range was necessary and sufficient to trigger ectopic eye development ( pai et al . , 2012 ) .
dye imaging data showed that the location of the endogenous eyes is demarcated by a prepattern of vmem states in the anterior neurectoderm and that experimental alteration of this prepattern results in abnormal craniofacial gene expression and eye and facial malformations ( vandenberg , 2011 ; pai et al . , 2012 ) . to complement the data showing that bioelectric states are an endogenous component of eye development
, it was then shown that driving eye - specific vmem states in other body regions ( by misexpression of ion channels ) was sufficient to induce anatomically complete ( well - formed ) ectopic eyes ( figure 2a ) .
marker analysis revealed that this occurs via establishment of a positive feedback loop between hyperpolarization and rx1/pax6 expression , whereas a suppression screen of transduction mechanisms implicated voltage - gated calcium signaling as the transduction mechanism .
eye genes such as pax6 do not produce eyes outside the head in vertebrates ( chow et al .
moreover , as with the tail , individual cell types appropriate to the eye did not have to be specified .
together these data revealed the unique properties of bioelectric triggers to reprogram body regions at the level of organ identity and overcome lineage specification limits observed with biochemical inducers .
( a ) targeted vmem change , via misexpression of ion channels in the frog embryo , induces the formation of ectopic structures such as complete eyes , even in regions normally not competent to form eyes ( such as on the gut ) .
( used , with permission , from figure 3 g of pai et al . ,
( b ) tracking the ion channel expression using a lineage marker reveals that the effect is not cell - autonomous : in a lens created in the tail of a tadpole by ion channel expression , only about half of the ectopic cells express the heterologous ion channel ( revealed by blue lacz staining ) ; the other half of the induced structure consists of host cells recruited to participate in making the appropriate shape but not themselves targeted by the vmem - altering reagent .
( c ) melanocytes seen in a cross section of a xenopus tadpole are normally few in number , round , and confined to their normal locations .
( d ) depolarization induced by ion channel modulation induces these cells to overproliferate , acquire an elongated shape , and invade many organs ( red arrow ) . of importance
, this effect is also not cell autonomous , as seen in the melanocyte phenotype , which results when cells ( marked by ion channel expression construct lineage label in blue ) are depolarized at a considerable distance from the melanocytes .
( taken , with permission , from figure 6a of chernet and levin , 2013b . )
( e ) a normal planarian has a head and tail and regenerates each at the appropriate end of an amputated fragment .
when it is cut into thirds and the middle fragment is briefly exposed to octanol , which temporarily blocks long - range bioelectrical signaling between the wound and mature tissues , a two - headed worm results ( f ) . remarkably , upon further rounds of cutting in plain water ( long after the octanol has left the tissues , as confirmed by hplc ) , the two - headed form results ( h , i ; images of two - headed worms provided by fallon durant , tufts university , medford , ma ) .
this change in the animal 's target morphology ( the shape to which it regenerates upon damage ) appears to be permanent and persists across the animal 's normal reproductive mode ( fissioning ) , despite the fact that the genomic sequence has not been altered .
chromatin modifications alone do not explain this , because the posterior wound cells , which could have been epigenetically reprogrammed to a head fate , are discarded at each cut : the information encoding a bipolar two - head animal is present even in the normal gut fragment it is distributed throughout the body .
we propose that this information is a kind of memory , encoded in electrical networks of somatic cells coupled by gap junctions , and is stored at the level of bioelectrical dynamics .
( e i taken , with permission , from figure 2 of levin , 2014 ; photographs of planaria taken by taisaku nogi , children 's health research institute , canada , and fallon durant . ) of interest , many forward genetic approaches have identified ion channel genes responsible for patterning phenotypes , as have unbiased transcriptional network analyses in development ( langlois and martyniuk , 2013 ) and cancer ( house et al . , 2010 ) .
these include patterning of the face , limb , brain , and viscera in a range of model systems and a number of channelopathies that form an important class of human birth defects ( table 3 ) .
thus upstream of endogenous bioelectrical signaling lie a set of ion channel and pump proteins that establish resting potential and alter it in response to physiological , transcriptional , and mechanical signals .
such data often come from studies that , unlike the previously discussed two examples , did not set out to investigate bioelectricity , and the overall structure of developmental bioelectric signaling is starting to emerge from the synthesis of bioelectric projects investigating molecular mechanisms and molecular biology efforts that implicate ion channel activity in instructive roles .
downstream of voltage change lie two types of endpoints at the mrna and chromatin modification levels .
transcriptional responses to depolarization include genes such as notch , bmp , sox10 , nurr1 , slug , fos , jun , npy , and wnt ( bartel et al . , 1989 ; higuchi et al . , 1990 ; raya et al . , 2004 ; morokuma et al . ,
2008a ; he et al . , 2011 ; lange et al . , 2011 ; tseng et al . , 2011 ; dahal et al . , 2012 ; swapna and borodinsky , 2012 ; adams et al . ,
epigenetic responses are triggered by movement of butyrate through an ion - dependent transporter , slc5a8 ; butyrate is an hdac1 inhibitor , and this allows voltage change to regulate chromatin acetylation ( davie , 2003 ; tong et al . , 2004 ;
this is believed to mediate control of tumorigenesis by depolarization and is also implicated in bioelectrical signaling during tail regeneration in xenopus ( tseng et al . , 2011 ; chernet and levin , 2013a , 2014 ) .
a set of transduction mechanisms has been identified by which changes of resting potential affect events at the nucleus ( figure 1 , b and c , and table 4 ) .
one involves voltage - gated calcium channels , which convert voltage change into signaling via this versatile second - messenger molecule ( nilius et al . , 1993 ; dolmetsch et al . , 1998 ;
this mode has been implicated in control of growth - cone turning ( nishiyama et al . ,
2008 ) , eye patterning ( pai et al . , 2012 ) , and flatworm regeneration ( nogi et al . , 2009 ; beane et al .
another uses the voltage gradients among cells to move small signaling molecules such as serotonin through gap junction coupled cell fields , as occurs in
right patterning ( fukumoto et al . , 2005b ; adams et al . , 2006 ) and control of neuronal pathfinding ( blackiston et al . , 2015 ) .
finally , voltage - sensitive phosphatases couple vmem change to the plethora of events regulated by pten phosphatases ( murata et al . , 2005 ; okamura and dixon , 2011 ) .
known transduction mechanisms by which ion flows affects cell behavior . of interest , when they conflict , bioelectrical cues tend to trump chemical signals .
one example is the guidance of cell motility : if a chemical gradient and an electric field are set up in opposite directions , the bioelectric vector trumps the chemical cue in directing cell movement ( zhao , 2009 ; cao et al . , 2011 ) .
another example is the differentiation of human mesenchymal stem cells ( hmscs ) , which normally hyperpolarize as they differentiate ; despite the presence of potent chemical inducers , hmscs will not differentiate if kept artificially depolarized ( sundelacruz et al . , 2008 ) .
indeed , the voltage state can even partially reverse the differentiation state , inducing plasticity in differentiated hmscs ( sundelacruz et al . ,
2013 ) . by identifying the specific ion channel genes that set vmem states , the transduction mechanisms that sense vmem change , and the downstream transcriptional or epigenetic targets ( which include ion channels themselves ) , recent
work has established the causal chain integrating bioelectrical cues with chemical pathways ( table 5 ) .
neither signaling mode is entirely upstream of the other cellular processes are regulated by the continuous cyclical interplay between transcriptional control of ion channel profiles within cells and the regulation of transcription by voltage dynamics . future work will identify new ion channel genes important for specific functions , additional transduction mechanisms by which cells sense their depolarization and hyperpolarization , and genome - wide ( next - generation sequencing [ ngs ] or microarray ) profiles of transcriptional programs triggered by specific vmem change .
cells can sense the voltage states of their neighbors through gap junctions ( gjs)versatile ( and themselves voltage - sensitive ) channels allowing the direct sharing of current and other small molecules between cells ( palacios - prado and bukauskas , 2009 ; pereda et al .
the importance of gj - mediated cues for cellular decision making has been shown , for example , in the development of the neocortex ( sutor and hagerty , 2005 ) and more broadly in setting up the patterns of chemical synapses ( anava et al .
cells can also read the bioelectrical state of distant regions via the chemical molecules redistributed ( and transported or diffused ) across long distances by bioelectric state change .
this was long ago suggested by burr , who used voltage readings at remote locations of the body to detect transplanted or induced tumors ( burr et al .
recent data in the frog model implicate long - range signaling via bioelectrical control of butyrate ( chernet and levin , 2014 ) and serotonin ( blackiston et al . , 2011 ; lobikin et al . , 2012a ) in tumorigenesis and metastatic induction .
additional modes for nonlocal bioelectrical signaling include tunneling nanotubes ( chinnery et al . , 2008 ; wittig et al . , 2012 ) and exosomes , which contain numerous ion channels ( lotvall and valadi , 2007 ; valadi et al . , 2007 ; wahlgren et al . , 2012
) and could regulate bioelectric states of cells that incorporate them . because bioelectrical gradients mediate signaling beyond the single - cell level , they form a versatile medium for carrying information .
spatiotemporal gradients of vmem among cells in vivo are now known to regulate organ identity , positional information , size control , and polarity of anatomical axes .
much like hox genes , whose combinatorial patterns of gene expression encode specific body regions during development , it has recently been shown that bioelectric prepatterns in the developing face of the frog and planarian models regulate the gene expression , size , and shape of craniofacial components ( vandenberg et al . , 2011 ;
in the frog , for example , patterns of hyperpolarization in the nascent face reveal the prospective locations of the eyes and other structures ; experimental perturbation of these distributions alters the boundaries of expression of face patterning genes such as frizzled , with the expected effects on craniofacial anatomy .
right axis in frog and chick embryos ( levin et al . , 2002 ; adams et al . , 2006 )
and set the size of regenerating structures in segmented worms and regenerating zebrafish tails ( kurtz and schrank , 1955 ; beane et al . , 2013 ; perathoner et al . ,
ion transporters , such as the v - atpase , are required for normal left
right patterning in several vertebrate models ( adams et al . , 2006 ) , zebrafish fin regeneration ( monteiro et al . ,
2014 ) , and zebrafish eye development ( nuckels et al . , 2009 ) .
these examples illustrate that bioelectric patterns can be necessary aspects of development because , when they are specifically disrupted , predictable and coherent changes in morphogenesis occur . of importance , many of these data sets used distinct ion species ( potassium , sodium , chloride , or protons ) to show that the necessary parameter is indeed the voltage potential , not any one channel gene ( which could have had scaffold or binding roles ) or even any one ion type ( which could have had chemical , not electrical , roles ) .
as with the gain - of - function examples discussed later , the voltage is what matters for the outcome , not which ion or channel was used to set it .
in addition to specifying directly the pattern of subsequent anatomy , some bioelectric signals seem to trigger whole developmental modules . in the case of tail regeneration in xenopus
, genetic , optogenetic , and pharmacological experiments have been used to recapitulate a regeneration - specific bioelectric state in nonregenerative animals and induce complete regrowth of this complex neuromuscular appendage ( adams et al .
not only could appropriate vmem state overcome physiological , chemical , and age - dependent blockade of regenerative capacity , but it was seen that a very simple ( low information content ) stimulus , such as pump protons , could be sufficient to trigger a complete and self - limiting cascade of events that rebuilt the appendage ( tseng and levin , 2013 ) , in essence providing a build whatever normally goes here signal .
these examples reveal that bioelectric state can function as a sufficient signal or master regulator ; this bodes well for the use of this approach in regenerative medicine , as we may not need to micromanage the morphogenesis of complex structures but instead rely on patterning subroutines already present in the host .
f ) can be directed to make heads or tails by appropriate modulation of resting potential ( beane et al . , 2011 , 2013 ) .
in vertebrates , whole - eye formation can be induced ectopically , far outside the head , even in mesoderm or endoderm ( figure 2a ) by misexpression of specific ion channels in vivo ( pai et al . , 2012 ) ; this process is mediated by a feedback loop between hyperpolarization and expression of eye - specific genes such as rx1 and pax6 , which in its absence can not initiate eye formation outside of the head .
it is also interesting that this signaling is not cell autonomous : cells with unique voltage characteristics serve as organizers , recruiting wild - type host tissues to participate in the ectopic morphogenesis ( figure 2b ) .
these examples illustrate the fact that bioelectric state provides instructive information to patterning processes and reveal that cell groups can be programmed at the level of complex organs , not only at the level of specifying individual cell types .
understanding in detail the mapping between bioelectric states and the anatomical outcomes quantitatively cracking the bioelectric code is a major open direction in this field .
possibilities for the parameters that functionally determine distinct organ types include spatial distribution of absolute vmem values within a cell group , relative differences in vmem across cell borders , and/or time - dependent changes of vmem within cells .
one technology that is likely to be instrumental in testing hypotheses about the bioelectric code is optogenetics ( knopfel et al . , 2010 ; liu and tonegawa , 2010 ) , which will facilitate the reading and writing of bioelectric patterning information in vivo .
the first steps have been taken , showing regulation of stem cells via optogenetic signaling ( stroh et al . , 2010 ;
wang et al . , 2014 ) , and a recent report showed the induction of tail regeneration by optical modulation of bioelectric state after amputation ( adams et al . ,
the information - bearing signal ( the necessary and sufficient trigger ) for events such as eye induction , head determination , and tail regeneration via vmem change is a physiological state , not a gene product ( levin , 2013 ; tseng and levin , 2013 ) .
studies reveal that the exact identity of the channel or pump used to trigger such morphological changes is often irrelevant
many sodium , potassium , chloride , or proton conductances can be used , as long as the appropriate vmem state is reached .
this means that the actual cause of the given morphological change can be a bioelectrical property not necessarily in 1:1 correspondence with any genetic locus . because channels and pumps can open and close posttranslationally
, two cells expressing precisely the same mrna and protein can be in very different bioelectrical states .
thus rich patterns of bioelectrical gradients can exist in a transcriptionally homogeneous tissue and be completely invisible to protein and mrna profiling until they trigger distinct downstream transcriptional targets .
conversely , cells with very different channel and pump complements may have the same vmem , since resting potential is an ensemble state that is a function of many different ion flows .
the implication is that mrna and protein profiling approaches are insufficient to detect and characterize important biophysical determinants of morphogenesis , and knockout screens may completely miss bioelectric pathways , since knockouts of single ion channels will be subject to compensation and redundancy by other channels contributing to vmem .
one context in which bioelectric and genetic state information can diverge is cancer ( yang and brackenbury , 2013 ; chernet and levin , 2013b ) .
dependent invasion of body tissues , and drastic arborization ) can be induced in genetically normal melanocytes by depolarization of somatic cells ( blackiston et al . , 2011 ;
this effect is not cell autonomous ( figure 2 , c and d ) , showing that the bioelectric state of cells at considerable distance can trigger metastatic behavior .
conversely , the formation of tumors by human oncogenes such as p53 and kras mutations can be suppressed , despite the strong presence of oncogene protein within the cells , by artificially preventing the depolarization that occurs during oncogenic transformation ( chernet and levin , 2013a ) .
these examples reveal the potential dissociation between genetic state and disease outcome ; an implication of these data is that the neoplastic state can not always be predicted from examination of the genome , transcriptomes , or proteome , although in some cases , ion channel expression is altered ( onkal and djamgoz , 2009 ; becchetti , 2011 ; lang and stournaras , 2014 ) . on the other hand ,
the functionally determinative voltage states can not be seen in fixed tissue , stressing the importance of gathering real - time in vivo bioelectric information over and above analysis of mutations , mrna profiles , and protein levels .
another implication for cancer biology is that although expression of some ion channel might be a useful marker ( wang , 2004 ; fraser et al .
2006 ) , there will also be many cases in which the transcriptional profile reveals nothing ( because of signaling via posttranslational gating of channel state ) , and drugs targeting one specific channel type ( arcangeli et al .
, 2009 , 2012 ) may have no effect ( due to compensation and redundancy of channel types ) .
if indeed cancer is augmented or induced by a depolarized bioelectric state ( binggeli and weinstein , 1986 ; olivotto et al .
, 1996 ; yang and brackenbury , 2013 ) , we will have to think less about individual ion channels as oncogenes ( pillozzi et al .
, 2004 ; lallet - daher et al . , 2013 ; than et al . , 2013 ) and
focus instead on the way in which many channels contribute to a bioelectrical oncostate , to develop strategies for dominating the resting potential irrespective of native channel identity ( sharmeen et al .
bioelectric patterns are clearly important drivers of cell behavior and pattern formation , but how do these patterns originate ?
diverse resting potentials across a tissue can arise from preexisting differences in ion channel transcription , but that is not the only way ( justet et al . , 2013 ) .
such regionalized patterns of vmem can also form de novo in transcriptionally and proteomically identical cells because cells coupled by gap junctions ( electrical synapses ) form a ( slow ) electrically excitable medium ; this is a particularly interesting aspect because such media are known to have powerful computational capabilities ( fenton et al .
, 1999 ; gorgcki and gorgcka , 2007 ; adamatzky et al . , 2011 ) .
positive feedback loops implemented by elements such as voltage - gated ion channels , which both set and respond to vmem changes , can drive spontaneous symmetry breaking and amplification of physiological noise .
considerable self - organization dynamics can take place without a need for preexisting chemical prepattern ( toko et al .
, 1987 ; schiffmann , 1991 , 1997 ; palacios - prado and bukauskas , 2009 ) or transcriptional activity ; for example , human red blood cells have a physiological , not genetic , circadian clock rhythm driven by a slow ionic oscillation ( chakravarty and rizvi , 2011 ; o'neill and reddy , 2011 ) .
such dynamics has been studied in nerve and muscle ( zykov , 1990 ; chen et al . , 1997 ;
, 2009 ; boettiger and oster , 2009 ) , and turing - type self - organization has long been appreciated in chemical signaling ( takagi and kaneko , 2005 ; muller et al . , 2012 ; sheth et al . , 2012
however , capabilities and properties of self - organization of voltage patterns in groups of nonneural cells remain to be formally analyzed
. quantitative analysis of in silico models of bioelectric dynamics will need to be integrated with deep new data sets from appropriate physiomic technologies to fully understand and control developmental patterning in vivo .
one unexpected recent finding illustrates the storage of patterning information in physiological networks and has significant implications for evolution .
planarian flatworms have the remarkable ability to regenerate completely from partial body fragments ( reddien and sanchez alvarado , 2004 ; salo et al . , 2009 ; lobo et al . ,
the cells at one edge make a tail , whereas those at the other edge make a head , revealing that the adult stem cells that implement regeneration are not locally controlled ( since the cells were direct neighbors until the scalpel separated them ) but must communicate with the remaining tissue to decide what anatomical structures must be formed .
it was shown that this long - range communication occurs via gap junction mediated electrical synapses ( scemes et al .
2013 ) , and works together with a bioelectric circuit that determines head versus tail identity in each end 's blastema ( beane et al . , 2011 , 2013 ) .
brief inhibition of this gap junction mediated communication results in worms developing heads at both ends ( nogi and levin , 2005 ; oviedo et al . , 2010 ) .
i ) is that weeks later , when these two - headed animals have their heads and tails amputated again ( in just water , with no further perturbation ) , the same two - headed phenotype results , and this is repeated upon subsequent amputations .
thus a transient perturbation of physiological cell : cell communication stably changes the pattern to which the animal regenerates upon damage , despite normal genomic sequence .
this again illustrates the potential divergence of genetic versus physiological information , especially since the phenotype is stable across fission ( this animal 's most frequent reproductive mode ) , and thus could have significant implications for evolution .
although epigenetic processes may be involved , chromatin modification mechanisms alone are not a sufficient explanation , since the ectopic heads ( tissue that might be suggested to have been epigenetically reprogrammed into a head state from its original tail identity ) are thrown away at each generation of cutting .
what remains is a gut fragment , which somehow knows that it is to form two heads , not one , upon further cutting ; the information about basic anatomical polarity and body organization must be stored in a distributed form throughout the animal .
quantitative , field - like models of this circuit remain to be developed to understand precisely how information guiding specific shape outcomes is encoded in ( represented by ) bioelectric states among cells .
major open questions for future progress include the mechanisms by which cells compare bioelectric state across distances , additional molecular details of the interactions of bioelectrical signals with chemical gradients and physical forces , and the development of quantitative models of bioelectric circuits that store stable patterning information during morphogenesis .
expansions of the toolkit of synthetic biology will soon allow the rational top - down programming of bioelectric circuits , which will have important implications for regenerative medicine , cancer biology , and bioengineering ( reid et al .
optogenetics , once expanded to facilitate the control of stable vmem in large , nonexcitable cell groups , will play a large part , and there is significant room for advances in better voltage reporters and techniques for in vivo modulation of bioelectric state .
one hypothesis for the development of deep , quantitative theory in this field is that pattering information may be stored within nonneural bioelectric cell networks using the same molecular mechanisms and information - processing algorithms that underlie behavioral memory in the nervous system .
it is thus possible that the techniques such as those now used to extract mental imagery from electrical measurements of living human brains ( nishimoto et al . , 2011 )
may shed crucial light on the encoding of anatomical pattern in the electrical circuits of somatic cells ; conversely , the cracking of the bioelectric code in development and regeneration may have important benefits for the understanding of the semantics of electric states in the brain .
in practical terms , the molecular biologist needs to consider not only transcriptional and protein profiles when working to understand regulation of single - cell behavior and pattern formation .
significant instructive information is generated at the level of bioelectricity ; ion channels and gap junctions are the molecular elements of such circuits , but bioelectrical signaling has its own unique dynamics that will become increasingly tractable with development of new technology specifically targeting stable vmem states . the existence of bioelectric signaling among most cell types , not only neurons , suggests that the field of applicability of electroceuticals ( famm et al . , 2013 ; sinha , 2013 ; birmingham et al . , 2014 )
more broadly , to the extent that the data of developmental bioelectricity are erasing artificial distinctions between neural and nonneural cell types , the insights of computational neuroscience and cognitive science will become relevant to cell and developmental biology .
it is possible that the most effective ways to understand high - order ( anatomical - level ) outcomes will involve not only bottom - up models of molecular pathways but also top - down models in which information and control theory concepts play central roles . in this way
, molecular bioelectricity may be revealing a mechanistic path toward understanding the intelligence exhibited by cell behavior and harnessing it toward transformative advances in biomedicine and the information sciences ( albrecht - buehler , 1985 ; rubenstein et al . | in addition to biochemical gradients and transcriptional networks , cell behavior is regulated by endogenous bioelectrical cues originating in the activity of ion channels and pumps , operating in a wide variety of cell types .
instructive signals mediated by changes in resting potential control proliferation , differentiation , cell shape , and apoptosis of stem , progenitor , and somatic cells . of importance , however , cells are regulated not only by their own vmem but also by the vmem of their neighbors , forming networks via electrical synapses known as gap junctions .
spatiotemporal changes in vmem distribution among nonneural somatic tissues regulate pattern formation and serve as signals that trigger limb regeneration , induce eye formation , set polarity of whole - body anatomical axes , and orchestrate craniofacial patterning .
new tools for tracking and functionally altering vmem gradients in vivo have identified novel roles for bioelectrical signaling and revealed the molecular pathways by which vmem changes are transduced into cascades of downstream gene expression .
because channels and gap junctions are gated posttranslationally , bioelectrical networks have their own characteristic dynamics that do not reduce to molecular profiling of channel expression ( although they couple functionally to transcriptional networks ) .
the recent data provide an exciting opportunity to crack the bioelectric code , and learn to program cellular activity at the level of organs , not only cell types .
the understanding of how patterning information is encoded in bioelectrical networks , which may require concepts from computational neuroscience , will have transformative implications for embryogenesis , regeneration , cancer , and synthetic bioengineering . |
type 2 diabetes mellitus ( t2 dm ) is the most common endocrine disorder and its incidence
is increasing worldwide .
this condition is a serious public health concern due to the
need for lifelong care , premature death and the fact that it remains incurable .
insulin resistance , whereby target tissues do
not respond to this hormone , is a characteristic of the disease 's first phase , when
there is often a corresponding hyperinsulinemia .
this resistance comes together with inflammatory processes in the
development of diabetes chronic complications . among these complications , periodontal diseases ( pd )
are very
common and immune modulating factors are necessary for pathogen clearance , but also
contribute to host tissues damage , as those seen in pd .
clues to the involvement of inflammation in diabetes date back to more than a century
ago .
proinflammatory molecules as tumor necroses factor ( tnf)- , leptin , interleukin
( il)-6 , resistin , monocyte chemoattractant protein-1 ( mcp-1 ) and visfatin , among others ,
are expressed at high levels in activated macrophages and/or other cells .
proinflammatory cytokines such as tnf- and il-1 activate jnk and ikk/nf-b through
classical receptor - mediated mechanisms which are also activated by pattern recognition
receptors , bound to substances as lipopolissacharide ( lps ) from gram negative bacteria .
these include the toll like receptors ( tlrs ) and the receptor for advanced glycation end
products ( rage ) .
prolonged hyperglycemia and the accompanying production of excess
amounts of advanced glycation end products ( ages ) can activate nf-b .
jnk promotes
insulin resistance through the phosphorylation of serine residues in insulin receptor
signaling ( irs)-1 that negatively regulates normal signaling through the insulin
receptor / irs-1 axis .
nf-b induces insulin resistance by promoting the expression of
numerous target genes as those for tnf- , il-6 , il-8 , mcp-1 , mip-1 , mip-2 , resistin ,
icam-1 , vcam-1 .
the inflammation associated with pd , characterized by elevated pro - inflammatory
cytokines , innate immune receptor expression , and cellular infiltrate is exacerbated in
patients with t2 dm where tlr-4 and rages play a significant role and contribute to
induce responses by oral epithelial cells .
the local production of cytokines in response to periodontal bacteria and their products
results in higher serum concentrations of pro - inflammatory biomarkers and a poor glycemic control in t2 dm
patients is associated with a loss of mucosal barrier integrity and accumulation of
innate immune receptor ligands resulting in an exacerbation of ongoing
inflammation .
thus , the adverse effect of periodontitis in t2 dm may be explained by an increase in
systemic inflammation , which contributes to insulin resistance .
the interrelation between more severe pd and dm is established and there are
evidences on the importance of cytokine analysis in t2 dm evolution and therapy blockade . as chronic or recurring inflammation contributes to an aberrant continuation of acute
phase response and
may also lead to further diabetes complications , such as micro and
macroangiopathy and impaired healing , it is suggested that periodontal
disease with increased inflammatory response at local and systemic levels may collaborate to insulin resistance
present on t2 dm pathogenesis .
however , scientific evidence on the effects of chronic
periodontitis on diabetes mellitus remains inadequate and inconclusive .
furthermore , whether periodontal therapy
may help to control serum levels of inflammatory cytokines still remains
controversial .
while some
studies showed an effective improvement in periodontal parameters , circulating
inflammatory markers and glycemic control after periodontal treatment in patients with
t2 dm , others have shown that
these responses were inconsistent across individuals and not sustainable over
time .
the elevated levels of systemic inflammatory mediators , such as il-6 , in obesity or
metabolic syndrome enhance the host response to periodontal pathogens , hence increasing
the chance of periodontal destruction .
il-6 is a multifunctional cytokine produced by a
variety of cells including macrophages , neutrophils , and endothelial cells .
the double
edge effects ( i.e. , pro- and anti - inflammatory ) of this molecule entail complexity in
investigating its role and , until now , no evidence from animal or human studies supports
the hypothetical model in which metabolic syndrome - induced il-6 increases the risk of
destructive periodontal disease .
interleukin 8 ( il-8 ) is a chemokine important for recruiting neutrophils during healing
and its levels were shown to be tightly linked to increased susceptibility to
periodontitis .
monocyte chemoattractant protein 1 ( mcp-1 ) is also thought to play an important role in
inflammatory processes .
it has been implicated as a key factor in recruitment and
activation of peripheral blood leukocytes in atherosclerotic lesions and adipose tissue .
elevated levels of circulating mcp-1 have been found in patients with t2 dm , and
experimental data have shown that this protein increases macrophage infiltration ,
inflammation and insulin resistance in transgenic mice .
some studies have evaluated
markers such as interleukin-6 ( il-6 ) , tumor necrosis factor - a ( tnf- ) , c - reactive
protein ( crp ) , and il-1 and related them to insulin resistance and glycemic control ; however , their results are still
conflicting . based on the evidences that il-6 , il-8 and mcp-1 are important key markers of immune
response , which have been considered as critical mediators of initiation , progression
and/or suppression of chronic periodontitis , and that no previous study had analyzed
this combination of mediators in groups with and without diabetes and periodontal
disease , this study aimed to evaluate serum levels of inflammatory markers , il-8 , il-6
and mcp-1 , in type 2 diabetic patients in the presence of chronic periodontitis .
this study was conducted between january 2012 and march 2013 in full accordance with the
helsinki declaration of 1975 , as revised in 2000 .
the study protocol and the informed
consent form were reviewed and approved by the biomedical sciences institute ethics
committee ( # 011/cep ) from the university of so paulo ( so paulo , brazil ) .
t2 dm
individuals were recruited from the diabetes center at the federal university of so
paulo .
non - diabetic patients were recruited from the school of dentistry , university of
so paulo .
all
volunteers received full mouth periodontal clinical examination performed at six
sites per tooth ( excluding third molars ) from a calibrated examiner
( hpca ) .
the
presence of supragingival biofilm was recorded as plaque index ( pl ) , marginal gingival
bleeding was recorded as gingival bleeding ( gb ) ; bleeding on probing ( bp ) , probing depth
( pd ) and attachment level ( al ) were also evaluated . the probe used was north caroline
( unc-15 , hu - friedy , chicago , il , usa ) .
pl , gb and bp were recorded as absent
( 0 ) or present ( 1 ) .
inclusion criteria were : age 35 years , confirmed diagnosis of type
2 diabetes over 3 years ( for individuals with t2 dm ) , generalized moderate to severe
chronic periodontitis ( american academy of periodontology , 1998 ) ( 30% of sites with pd
> 4 mm and bleeding on probing ) and 15 remaining teeth .
pregnant women , smokers ,
patients with body mass index ( bmi ) > 40 kg / m or who received periodontal
therapy , antibiotic or antiseptic therapy 6 months prior to the study were excluded .
blood samples were obtained and glycated hemoglobin ( hba1c ) levels were determined for
all individuals at the clinical laboratory of the university of so paulo hospital .
approximately 5 ml of blood were collected by venipuncture in untreated tubes ( bd
vacutainer rapid serum tube , becton dickinson co. , so paulo , sp , brazil ) .
serum was
obtained by centrifugation at 2,500 g for 2 minutes , aliquoted and
stored at -80c until further analysis .
quantitative measurement of il-6 , il-8 and mcp-1
was assayed by enzyme - linked immunosorbent assay ( human standard elisa development kit ,
peprotech inc
. , rocky hill , ct , usa ) and read out using a micro plate reader ( model 680 ,
bio - rad laboratories inc .
serum from each patient was tested in
triplicate , and a calibration curve was added to each plate .
patients with t2 dm and periodontitis
were divided in two groups , as adequate glycemic control when hba1c < 8.0% ( dma+p , n =
10 ) and inadequate glycemic control when hba1c 8.0% ( dmi + p , n=10 ) .
the other 3 groups
were composed of t2 dm individuals without periodontitis ( dm , n = 10 ) , non - diabetic with
periodontitis ( p , n=6 ) and non - diabetic without periodontitis ( h , n=6 ) .
periodontal clinical data were analyzed using appropriate statistical software ( spss
version 17.0 for windows , spss inc . ,
data were analyzed by non - parametric statistical methods since data were
not normally distributed .
tests were based on median values with variability
measures ( 25% and 75% quartiles ) .
bop data , percentages of positive sites were obtained
per patient and , thereafter , median values were calculated for the groups .
for pd and al , measured by millimeters
, percent frequency was first obtained per patient
and , thereafter , as a median value for the group .
data from elisa were analyzed by kruskal - wallis and dunn 's multiple comparison test
( prism 5.0 project , graph pad software inc . , la jolla , ca , usa ) .
values were obtained
per patient and a median value was calculated for each group .
a total of 546 patients were assessed for eligibility , and only 42 fulfilled the
inclusion criteria ( figure 1 ) .
flow diagram of patients included in the study demographic and hba1c data from each group are described in table 1 .
demographic characteristics and glycated level ( meanstandard deviation ) for
participants in each group dm = type 2 diabetes mellitus ; p = periodontitis ; bmi = body mass index .
groups : h
( dm - p- ) , p ( dm - p+ ) , dm ( dm+p- ) , dma+p ( dm+with adequate glycemic control p+ ) ,
dmi+p ( dm+with inadequate glycemic control p+ )
table 2 provides periodontal clinical parameters
for each group with periodontitis : dmi+p , dma+p and p. there were significant
differences between diabetic ( dmi+p and dma+p ) and non - diabetic ( p ) for pd ( p=0.04 ) and
al ( p = 0.01 ) values indicating a more severe disease among the diabetic patients .
intergroup clinical
parameters comparison for dmi + p and dma+p showed no significant differences ( data not
shown ) .
medians and quartiles ( 25 - 75% ) of clinical parameters for the dma+p , dmi+p and p
groups statistically significance difference between dma+p;dmi+p and p
( kruskall - wallis test and tukey test ; =5% ) .
gb= gingival bleeding index ;
pl = visible plaque index ; bop = bleeding on probing ; pd = probing depth ;
al = attachment level there were no differences between groups for il-6 ( p=0.6351 ) , il-8 ( p=0.9460 ) and mcp-1
( p=0.2987 ) sera leveis ( figure 2 ) .
il-6 ( a ) , il-8 ( b ) and mcp-1 ( c ) serum levels in patients without periodontitis
and t2 dm ( h ) , patients with periodontitis and without t2 dm ( p ) , patients with t2 dm
and without periodontitis ( dm ) , patients with t2 dm with adequate glycemic control
and periodontitis ( dma+p ) and patients with t2 dm with inadequate glycemic control
and periodontitis ( dmi+p ) .
mcp-1=monocyte chemoattractant protein-1 ;
il-6=interleukin 6 ; il-8=interleukin 8 ; t2dm = type 2 diabetes mellitus .
the present study evaluated clinical and inflammatory parameters among groups with and
without t2 dm and/or periodontitis . t2
dm patients presented significantly higher pd and al values when compared to group p ,
without diabetes and with periodontitis .
this result is consistent with a number of
cross - sectional and longitudinal studies which had shown that the increased severity of pd in
t2 dm patients is due to an interorgan crosstalk under inflammatory conditions .
the positive correlation between glycemic control and severity of periodontal disease
has been reported in the literature .
thus , we had expected to find a significant relationship between
hba1c levels ( representing adequate - hba1c < 8.0% - and inadequate - hba1c 8.0% -
glycemic control ) and periodontitis severity ( in terms of increased pd and bop ) .
however , the periodontal clinical parameters data did not differ according to the
diabetes control , corresponding to dma+p and dmi+p groups , respectively .
we can
speculate that , in order to observe correlation , we would need to consider a stricter
glycemic control of individuals with t2 dm , that is , hba1c between 6.5 and 7.0% , which
would hamper even more the group selection .
besides , hba1c refers to glycemic control in
the last 2 or 3 months and we do not know how the chronic glycemic control in these
individuals was .
we also have to point out that hyperglycemia is only one factor of the
multifactorial process of periodontal disease .
the role of
locally produced il-6 in the periodontitis pathogenesis has been demonstrated , since high levels of this cytokine were detected in symptomatic large
lesions rather than in asymptomatic small lesions .
this cytokine is overexpressed in sites with pd mainly in
patients without diabetes and patients with well - controlled t2 dm when compared with
patients with poorly controlled diabetes .
the effect of periodontal treatment on il-6 levels showed
conflicting data , yielding a tendency toward a decrease or increase in
serum levels in t2 dm patients .
furthermore , a controversial study suggested an increase or no change in serum il-6
levels in obesity and metabolic syndrome .
it should be noticed that t2 dm individuals evaluated in the present
study were under treatment with insulin , which may have induced low levels of
inflammatory biomarkers such as il-6 , counteracting the high levels induced
by periodontal inflammation . in the present study
, there were no significant differences in the sera levels of the
studied inflammatory markers , mcp-1 and il-8 , among the groups , although previous data
demonstrated that both were overexpressed in the gingival tissue with chronic
periodontitis .
a search in the
literature revealed no study in humans regarding t2 dm and/ or periodontitis and mcp-1
serum or gingival fluid levels . in rats
experimental models , mcp-1 gingival crevicular
fluid concentrations did not significantly differ between groups with diabetes and
periodontitis and its levels were higher than in control healthy group .
high levels of il-8 were shown in the gingival fluid of subjects with periodontal
disease .
however , subjects with t2 dm presented significantly lower levels of il-8 in the
gcf when compared to healthy ones and there was no correlation between il-8 gingival
levels and hba1c level .
furthermore , il-8 levels in serum
tended to decrease after periodontal therapy in t2 dm patients . due to the use of restrictive inclusion and exclusion criteria , in an attempt to
minimize the occurrence of confounding factors ,
however , it is interesting to consider that some studies evaluated only
specific dental sites through gingival crevicular fluid analysis , whereas the serum
analysis provides a more valuable analysis of the inflammatory markers in the subject .
to the best of our knowledge
, there have been no studies evaluating these serum markers
under all different conditions analyzed in this study .
nevertheless , this study confirms the higher severity of periodontal disease in
individuals with diabetes , highlighting the need for special oral healthcare for these
patients .
this study pointed out the great variability of the serum inflammatory
biomarkers under all systemic and periodontal conditions studied .
we can suggest that
follow - up studies with stricter glycemic control and larger samples should be conducted
in order to investigate the cumulative influence of periodontal conditions on serum
inflammatory biomarkers in individuals with t2 dm .
although periodontitis was more severe in patients with diabetes , the serum levels of
the investigated inflammatory markers were not different among the periodontitis
patients . | diabetes has been associated with periodontitis , but the mechanisms through which
periodontal diseases affect the metabolic control remain unclear.objective this study aimed to evaluate serum leveis of inflammatory markers , il-8 , il-6 and
monocyte chemoattractant protein 1 ( mcp-1 ) , in type 2 diabetic patients in the
presence of chronic periodontitis .
material and methods forty two individuals were enrolled in this study and assigned to one of five
groups : diabetes mellitus with inadequate glycemic control and periodontitis
( dmi+p , n = 10 ) , diabetes mellitus with adequate glycemic control and
periodontitis ( dma+p , n = 10 ) , diabetes mellitus without periodontitis ( dm , n =
10 ) , periodontitis without diabetes ( p , n=6 ) , and neither diabetes nor
periodontitis ( h , n = 6 ) .
periodontal clinical examination included visible plaque
index ( pl ) , gingival bleeding index ( gb ) , probing depth ( pd ) , attachment level
( al ) and bleeding on probing ( bp ) .
glycemic control was evaluated by serum
concentration of glycated hemoglobin ( hbalc ) .
inflammatory serum markers il-8 ,
il-6 and ( mcp-1 ) were measured by elisa.resultsdmi+p and dma+p groups presented higher pd ( p=0.025 ) and al ( p=0.003 ) values when
compared to the p group .
there were no significant differences among groups for
il-6 , il-8 and mcp-1 serum levels.conclusions although periodontitis was more severe in diabetic patients , the serum levels of
the investigated inflammatory markers did not differ among the groups . |
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\begin{document}$$ \mathcal{r}({\rm mode } ) = \frac{\mathcal{b}(b^{+ } \to{\rm mode}\to p\bar{p } k^{+})}{\mathcal{b}(b^{+}\to j/\psi k^{+}\to p\bar{p } k^{+ } ) } , $ $ \end{document } where mode corresponds to the intermediate c(1s ) , (2s ) , c(2s ) , c0(1p ) , hc(1p ) , x(3872 ) or x(3915 ) states , together with a kaon .
the lhcb detector is a single - arm forward spectrometer covering the pseudorapidity range 2<<5 , designed for the study of particles containing b or c quarks .
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\begin{document}$4{\rm\,tm}$\end{document } , and three stations of silicon - strip detectors and straw drift - tubes placed downstream .
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\begin{document}$\delta\ln\mathcal{l}_{\alpha\beta}$\end{document } under particle hypotheses and , respectively .
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\begin{document}$\delta \ln\mathcal{l}_{p\pi}>-5$\end{document}. the reconstructed b candidates are required to have an invariant mass in the range 50795579 mev / c . the asymmetric invariant mass range around the nominal b mass is designed to select also \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p \bar{p } \pi^{+}$\end{document } candidates without any requirement on the pid of the kaon .
the pv associated to each b candidate is defined to be the one for which the b candidate has the smallest ip . the b candidate is required to have a vertex fit with a \documentclass[12pt]{minimal }
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\begin{document}$\chi^{2}/{\rm ndf}<12$\end{document } and a distance greater than 3 mm , a for the flight distance greater than 500 , and an ip <10 with respect to the associated pv .
the maximum distance of closest approach between daughter tracks has to be less than 0.2 mm .
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\begin{document}$\cos\theta _ { \rm fl}>0.99998$\end{document}. the reconstructed candidates that meet the above criteria are filtered using a boosted decision tree ( bdt ) algorithm .
the bdt is trained with a sample of simulated \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p \bar{p } k^{+}$\end{document } signal candidates and a background sample of data candidates taken from the invariant mass sidebands in the ranges 50805220 mev / c and 53405480 mev / c .
the variables used by the bdt to discriminate between signal and background candidates are : the \documentclass[12pt]{minimal }
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\begin{document}$p_{\rm t}$\end{document } of each reconstructed track ; the sum of the daughters pt ; the sum of the ip of the three daughter tracks with respect to the primary vertex ; the ip of the daughter , with the highest \documentclass[12pt]{minimal }
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\begin{document}$p_{\rm t } > 900~\text { gev}/c$\end{document } ; the maximum distance of closest approach between any two of the b daughter particles ; the ip of the b candidate with respect to the primary vertex ; the distance between primary and secondary vertices ; the \documentclass[12pt]{minimal }
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\begin{document}$\frac{p\sin\theta}{p\sin\theta+ \sum_{i } p_{\rm t , i}}$\end{document } , where p is the total momentum of the three - particle final state , is the angle between the direction of the sum of the daughter s momentum and the direction of the flight distance of the b and \documentclass[12pt]{minimal }
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\begin{document}$\sum_{i } p_{{\rm t},i}$\end{document } is the sum of the transverse momenta of the daughters ; and the log likelihood difference for each daughter between the assumed pid hypothesis and the pion hypothesis .
1 ) is chosen in order to have a signal to background ratio of the order of unity .
the efficiency of the bdt selection is greater than 92 % with a background rejection greater than 86 % .
1distribution of the bdt algorithm response evaluated for background candidates from the data sidebands ( red hatched area ) , and signal candidates from simulation ( blue filled area ) .
the dotted line ( black ) indicates the chosen bdt response value ( color figure online ) distribution of the bdt algorithm response evaluated for background candidates from the data sidebands ( red hatched area ) , and signal candidates from simulation ( blue filled area ) .
the dotted line ( black ) indicates the chosen bdt response value ( color figure online )
the signal yield is determined from an unbinned extended maximum likelihood fit to the invariant mass of selected \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p \bar{p } k^{+}$\end{document } candidates , shown in fig .
is parametrized as the sum of two gaussian functions with the same mean and different widths .
the signal yield of the charmless component is determined by performing the same fit described above to the sample of \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p \bar{p } k^{+}$\end{document } candidates with \documentclass[12pt]{minimal }
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\begin{document}$m_{p\bar{p } } < 2.85~\text { gev}/c^{2}$\end{document } , shown in fig .
the b mass and widths , evaluated with the invariant mass fits to all of the \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p \bar{p } k^{+}$\end{document } candidates , are compatible with the values obtained for the charmless component .
2invariant mass distribution of ( a ) all selected \documentclass[12pt]{minimal }
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\begin{document}$m_{p \bar { p } } < 2.85~\text { gev}/c^{2}$\end{document}. the points with error bars are the data and the solid lines are the result of the fit .
the dotted lines represent the two gaussian functions ( red ) and the dashed line the linear function ( green ) used to parametrize the signal and the background , respectively .
the two plots below the mass distributions show the pulls ( color figure online ) invariant mass distribution of ( a ) all selected \documentclass[12pt]{minimal }
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\begin{document}$m_{p \bar { p } } < 2.85~\text { gev}/c^{2}$\end{document}. the points with error bars are the data and the solid lines are the result of the fit .
the dotted lines represent the two gaussian functions ( red ) and the dashed line the linear function ( green ) used to parametrize the signal and the background , respectively .
the two plots below the mass distributions show the pulls ( color figure online ) the signal yields for the charmonium contributions , \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p\bar{p } k^{+}$\end{document } candidates within the b mass signal window , \documentclass[12pt]{minimal }
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\begin{document}$\vert m_{p\bar{p } k^{+ } } - m_{b^{+}}\vert <
50~\text { mev}/c^{2}$\end{document}. simulations show that no narrow structures are induced in the \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p \bar{\varlambda } \to p \bar{p } k^{+}$\end{document } intermediate states .
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\begin{document}$p\bar{p}$\end{document } invariant mass distribution , shown in fig .
the signal components of the narrow resonances j/ , (2s ) , hc(1p ) , and x(3872 ) , whose natural widths are much smaller than the \documentclass[12pt]{minimal }
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\begin{document}$p\bar{p}$\end{document } invariant mass resolution , are parametrized by gaussian functions .
the signal components for the c(1s ) , c0(1p ) , c(2s ) , and x(3915 ) are parametrized by voigtian functions.2 since the \documentclass[12pt]{minimal }
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\begin{document}$p\bar{p}$\end{document } invariant mass resolution is approximately constant in the explored range , the resolution parameters for all resonances , except the (2s ) , are fixed to the j/ value ( j/=8.90.2 mev / c ) .
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\begin{document}$f(m)=e^{c_{1}m+ c_{2}m^{2}}$\end{document } where c1 and c2 are fit parameters .
the j/ and (2s ) resolution parameters , the mass values of the c(1s ) , j/ , and (2s ) states , and the c(1s ) natural width are left free in the fit .
the masses and widths for the other signal components are fixed to the corresponding world averages .
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\begin{document}$p\bar{p}$\end{document } invariant mass resolution , determined by the fit to the (2s ) is (2s)=7.91.7 mev / c .
3invariant mass distribution of the \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p\bar{p } k^{+}$\end{document } candidates within the b
mass signal window , \documentclass[12pt]{minimal }
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\begin{document}$\vert m(p\bar{p } k^{+ } ) - m_{b^{+}}\vert < 50~\text { mev}/c^{2}$\end{document}. the dotted lines represent the gaussian and voigtian functions ( red ) and the dashed line the smooth function ( green ) used to parametrize the signal and the background , respectively .
the bottom plot shows the pulls ( color figure online ) invariant mass distribution of the \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p\bar{p } k^{+}$\end{document } candidates within the b
mass signal window , \documentclass[12pt]{minimal }
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\begin{document}$\vert m(p\bar{p } k^{+ } ) - m_{b^{+}}\vert <
50~\text { mev}/c^{2}$\end{document}. the dotted lines represent the gaussian and voigtian functions ( red ) and the dashed line the smooth function ( green ) used to parametrize the signal and the background , respectively . the bottom plot shows the pulls ( color figure online ) the fit result is shown in fig .
3 . figures 4 and 5 show the details of the fit result in the regions around the c(1s ) and j/ , c(2s ) and (2s ) , c0(1p ) and hc(1p ) , and x(3872 ) and x(3915 ) resonances .
any bias introduced by the inaccurate description of the tails of the c(1s ) , j/ and (2s ) resonances is taken into account in the systematic uncertainty evaluation .
4invariant mass distribution of the \documentclass[12pt]{minimal }
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\begin{document}$p \bar{p}$\end{document } system in the regions around ( a ) the
c(1s ) and j/ and ( b ) the
c(2s ) and (2s ) states .
the dotted lines represent the gaussian and the voigtian functions ( red ) and the dashed line the smooth function ( green ) used to parametrize the signal and the background , respectively .
the two plots below the mass distribution show the pulls ( color figure online)fig .
5invariant mass distribution of the \documentclass[12pt]{minimal }
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\begin{document}$p \bar{p}$\end{document } system in the regions around ( a ) the
c0(1p ) and h
c and ( b ) the x(3872 ) and x(3915 ) states .
the dotted lines represent the gaussian and voigitian functions ( red ) and the dashed line the smooth function ( green ) used to parametrize the signal and the background , respectively .
the two plots below the mass distribution show the pulls ( color figure online ) invariant mass distribution of the \documentclass[12pt]{minimal }
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\begin{document}$p \bar{p}$\end{document } system in the regions around ( a ) the
c(1s ) and j/ and
the dotted lines represent the gaussian and the voigtian functions ( red ) and the dashed line the smooth function ( green ) used to parametrize the signal and the background , respectively .
the two plots below the mass distribution show the pulls ( color figure online ) invariant mass distribution of the \documentclass[12pt]{minimal }
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\begin{document}$p \bar{p}$\end{document } system in the regions around ( a ) the
c0(1p ) and h
c and ( b ) the x(3872 ) and x(3915 ) states .
the dotted lines represent the gaussian and voigitian functions ( red ) and the dashed line the smooth function ( green ) used to parametrize the signal and the background , respectively .
the two plots below the mass distribution show the pulls ( color figure online ) the contribution of \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p \bar{p } k^{+}$\end{document } decays , denoted as non - signal , is estimated from a fit to the \documentclass[12pt]{minimal }
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\begin{document}$p \bar{p}$\end{document } mass in the b mass sidebands 51305180 and 53805430 mev / c . except for the j/ mode , no evidence of a non - signal contribution
the j/ signal yield in the b mass window is 4311 candidates and is subtracted from the number of j/ signal candidates .
the signal yields , corrected for the non - signal contribution , are reported in table 1 . for the intermediate charmonium states c(2s ) , c0(1p ) ,
hc(1p ) , x(3872 ) and x(3915 ) , there is no evidence of signal .
the 95 % cl upper limits on the number of candidates are shown in table 1 and are determined from the likelihood profile integrating over the nuisance parameters . since for the x(3872 ) the fitted signal yield is negative , the upper limit has been calculated integrating the likelihood only in the physical region of a signal yield greater than zero . table 1signal yields for the different channels and corresponding 95 % cl upper limits for modes with less than 3 statistical significance . for the j/ mode ,
uncertainties are statistical only
b
decay modesignal yieldupper limit ( 95 % cl )
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\begin{document}$p\bar{p } k^{+}$\end{document } [ total]6951 176
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\begin{document}$p\bar{p } k^{+}\ [ m_{p \bar { p } } < 2.85~\text { gev}/c^{2}]$\end{document }
3238 122
j/k
1458 42
c(1s)k
856 46
(2s)k
107 16
c(2s)k
39 15<65.4
c0(1p)k
15 13<38.1
h
c(1p)k
21 11<40.2
x(3872)k
9 8<10.3
x(3915)k
13 17<42.1 signal yields for the different channels and corresponding 95 % cl upper limits for modes with less than 3 statistical significance . for the j/ mode ,
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\begin{document } $ $ \begin{aligned } \mathcal{r}({\rm mode } ) = & \frac{\mathcal{b}(b^{+ } \to{\rm mode}\to p\bar{p } k^{+})}{\mathcal{b}(b^{+}\to j/\psi k^{+}\to p\bar{p } k^{+ } ) } \\= & \frac { n_{\rm mode}}{n _ { j/\psi } } \times \frac{\epsilon _ { j/\psi } } { \epsilon_{\rm mode } } , \end{aligned}$$ \end{document } where \documentclass[12pt]{minimal }
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\begin{document}$n_{\rm mode}$\end{document } and nj/ are the signal yields for the given mode and the reference mode , \documentclass[12pt]{minimal }
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\begin{document}$\epsilon_{\rm mode}/\epsilon _
the efficiency is the product of the reconstruction , trigger , and selection efficiencies , and is estimated using simulated data samples . since the track multiplicity distribution for simulated events differs from that observed in data ,
simulated candidates are assigned a weight so that the weighted distribution reproduces the observed multiplicity distribution .
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\begin{document}$\delta\ln\mathcal{l}_{p\pi}$\end{document } for kaons and protons in data are obtained in bins of momentum , pseudorapidity and number of tracks from control samples of dd(k) decays for kaons and p decays for protons , which are then used on a track - by - track basis to correct the simulation .
the efficiency as a function of \documentclass[12pt]{minimal }
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\begin{document}$m_{p \bar{p}}$\end{document } is shown in fig
a linear fit to the efficiency distribution is performed and the efficiency ratios are determined based on the fit result .
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\begin{document}$b^{+ } \to p \bar{p } k^{+}$\end{document } decays .
the solid line represents the linear fit to the efficiency distribution ; the dashed line is the point - by - point interpolation used to estimate the systematic uncertainty efficiency as a function of \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p \bar{p } k^{+}$\end{document } decays .
the solid line represents the linear fit to the efficiency distribution ; the dashed line is the point - by - point interpolation used to estimate the systematic uncertainty
the measurements of the relative branching fractions depend on the ratios of signal yields and efficiencies with respect to the reference mode .
since the final state is the same in all cases , most of the systematic uncertainties cancel .
the systematic uncertainty on the efficiency ratio , in each region of \documentclass[12pt]{minimal }
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\begin{document}$p \bar{p}$\end{document } invariant mass , is determined from the difference between the efficiency ratios calculated using the solid fitted line and the dashed point - by - point interpolation shown in fig .
the uncertainty associated with the evaluation of the b signal yield has been determined by varying the fit range by 30 mev / c , using a single gaussian instead of a double gaussian function to model the signal pdf , and using an exponential function to model the background . for each charmonium resonance
the systematic uncertainty on the signal yield has been investigated by varying the b mass signal window by 10 mev / c , the signal and background shape parametrization and the subtraction of the \documentclass[12pt]{minimal }
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\begin{document}$c\bar{c}$\end{document } contribution from the continuum .
the systematic uncertainty associated with the parametrization of the signal tails of the j/ , c(1s ) and (2s ) resonances is taken into account by taking the difference between the number of candidates in the observed distribution and the number of candidates calculated from the integral of the fit function in the range 6 to 2.5. the systematic uncertainty associated with the selection procedure is estimated by changing the value of the bdt selection to 0.03 , which retains 85 % of the signal with a 30 % background , and is found to be negligible .
the contributions to the systematic uncertainties from the different sources are listed in table 2 .
table 2relative systematic uncertainties ( in % ) on the relative branching fractions from different sources .
the total systematic uncertainty is determined by adding the individual contributions in quadraturesource
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\begin{document}$\mathcal{r}({\rm total})$\end{document }
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\begin{document}$\mathcal{r}(m_{p \bar { p } } < 2.85~\text { gev}/c^{2 } ) $ \end{document }
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\begin{document}$\mathcal{r } ( \eta _ { c } ( 1s))$\end{document }
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\begin{document}$\mathcal{r}(\psi(2s))$\end{document }
efficiency ratio0.210.53.34.8
b
mass fit range0.160.5sig . and bkg .
shape2.53.61.86.5
b
mass window0.60.60.93.8non - signal component0.45.1signal tail param.1.01.01.24.3total2.83.84.111.3source
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\begin{document}$\mathcal{r}(\chi_{c0}(1p))$\end{document }
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\begin{document}$\mathcal{r}(x(3872))$\end{document }
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efficiency ratio4.42.53.46.57.0
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mass fit rangesig . and bkg .
shape3.93.314.35.610.1
b
mass window11.323.623.617.57.5non - signal componentsignal tail param.1.01.01.01.01.0total12.824.027.819.515.5 relative systematic uncertainties ( in % ) on the relative branching fractions from different sources .
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\begin{document}$\mathcal{b}(b^{+ } \to j/\psi k^{+ } ) = ( 1.013\pm0.034)\times10^{-3}$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$\mathcal{b}(j/\psi\to p\bar{p } ) = ( 2.17\pm0.07)\times10^{-3}$\end{document } are listed in table 4 .
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\begin{document}$\mathcal{b}(b^{+ } \to\chi_{c0}(1p ) k^{+ } \to p \bar{p } k^{+})$\end{document } is compatible with the world average \documentclass[12pt]{minimal }
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\begin{document}$\mathcal{b}(b^{+ } \to\chi _ { c0}(1p ) k^{+ } ) \times\mathcal{b}(\chi_{c0}(1p ) \to p \bar{p } ) = ( 0.030 \pm0.004 ) \times 10^{-6}$\end{document } .
we combine our upper limit for x(3872 ) with the known value for \documentclass[12pt]{minimal }
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\begin{document}$\mathcal{b } ( b^{+ } \to x(3872 ) k^{+ } ) \times\mathcal{b } ( x(3872 ) \to j/\psi\pi^{+ } \pi^{-})= ( 8.6 \pm0.8 ) \times10^{-6}$\end{document } to obtain the limit \documentclass[12pt]{minimal }
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\begin{document}$$ \frac{\mathcal{b } ( x(3872 ) \to p \bar{p})}{\mathcal{b } ( x(3872 ) \to j/\psi\pi^{+ } \pi^{- } ) } < 2.0\times10^{-3}. $ $ \end{document } this limit challenges some of the predictions for the molecular interpretations of the x(3872 ) state and is approaching the range of predictions for a conventional c1(2p ) state [ 17 , 18 ] . using our result and the c(2s ) branching fraction \documentclass[12pt]{minimal }
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\begin{document}$\mathcal{b } ( b^{+ } \to\eta_{c}(2s ) k^{+})\times \mathcal{b } ( \eta_{c}(2s ) \to k \bar{k } \pi ) = ( 3.4\ , ^{+2.3}_{-1.6 } ) \times10^{-6}$\end{document } , a limit of \documentclass[12pt]{minimal }
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\begin{document}$$ \frac{\mathcal{b } ( \eta_{c}(2s ) \to p \bar{p})}{\mathcal{b } ( \eta_{c}(2s ) \to k \bar{k } \pi ) } < 3.1 \times10^{-2 } $ $ \end{document } is obtained .
table 3signal yields , efficiency ratios , ratios of branching fractions and corresponding upper limits
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\begin{document}$b^{+}\to({\rm mode})$\end{document }
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\begin{document}$\to p\bar{p } k^{+}$\end{document }
yield stat syst
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\begin{document}$\epsilon_{\rm mode}/\epsilon _ { j/\psi } $ \end{document }
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stat systupper limit 95 % cl
j/k
145842241total69511761710.9700.0024.910.190.14
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\begin{document}${m_{p \bar { p } } < 2.85~\text { gev}/c^{2}}$\end{document }
32381221211.0970.0062.020.100.08
c(1s)k
85646191.0160.0340.5780.0350.026
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10716130.9210.0440.0800.0120.009
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391550.9270.0410.0290.0110.004<0.048
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151340.9570.0240.0110.0090.003<0.028
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211150.9430.0320.0150.0080.004<0.029
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9820.8960.0580.0070.0060.002<0.008
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131750.8900.0620.0100.0130.002<0.032table 4branching fractions for \documentclass[12pt]{minimal }
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\begin{document}$\mathcal{b } ( j/\psi \to p\bar{p})$\end{document } branching fractions . for the charmonium modes we compare our values to the product of the independently measured branching fractions .
the first uncertainties are statistical , the second systematic in the present measurement , and the third systematic from the uncertainty on the j/ branching fraction
b
decay mode
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\begin{document}$\mathcal{b}(b^{+}\to({\rm mode})\to p\bar{p } k^{+})$\end{document } ( 10)ul ( 95 % cl ) ( 10)previous measurements ( 10 ) [ 4 , 5]total10.810.420.300.49
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\begin{document}$10.76^{+0.36}_{-0.33 } \pm0.70$\end{document }
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4.460.210.180.205.120.31
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1.270.080.050.061.540.16
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0.1750.0270.0200.0080.1760.012
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0.0630.0250.0090.003<0.106
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0.0340.0180.0080.002<0.064
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0.0220.0290.0040.001<0.071 signal yields , efficiency ratios , ratios of branching fractions and corresponding upper limits branching fractions for \documentclass[12pt]{minimal }
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\begin{document}$\mathcal{b } ( j/\psi \to p\bar{p})$\end{document } branching fractions . for the charmonium modes we compare our values to the product of the independently measured branching fractions .
the first uncertainties are statistical , the second systematic in the present measurement , and the third systematic from the uncertainty on the j/ branching fraction
based on a sample of 6951176 \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p \bar{p } k^{+}$\end{document } decays reconstructed in a data sample , corresponding to an integrated luminosity of 1.0 fb , collected with the lhcb detector , the following relative branching fractions are measured an upper limit on the ratio \documentclass[12pt]{minimal }
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\begin{document}$\frac{\mathcal{b } ( b^{+ } \to x(3872 ) k^{+ } \to p \bar{p } k^{+})}{\mathcal{b}(b^{+ } \to j/\psi k^{+ } \to p \bar{p } k^{+ } ) } < 0.017$\end{document } is obtained , from which a limit of \documentclass[12pt]{minimal }
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\begin{document}$$ \frac{\mathcal{b } ( x(3872 ) \to p \bar{p})}{\mathcal{b } ( x(3872 ) \to j/\psi\pi^{+ } \pi^{- } ) } < 2.0\times10^{-3 } $ $ \end{document } is derived . | the branching fractions of the decay \documentclass[12pt]{minimal }
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\begin{document}$b^{+ } \to p \bar{p } k^{+}$\end{document } for different intermediate states are measured using data , corresponding to an integrated luminosity of 1.0 fb1 , collected by the lhcb experiment . the total branching fraction ,
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\begin{document}$(m_{p\bar{p}}<2.85~\text { gev}/c^{2})$\end{document } and the branching fractions via the resonant \documentclass[12pt]{minimal }
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\begin{document}$c\bar{c}$\end{document } states c(1s ) and (2s ) relative to the decay via a j/ intermediate state are upper limits on the b+ branching fractions into the c(2s ) meson and into the charmonium - like states x(3872 ) and x(3915 ) are also obtained . |
a 28-year - old man presented with swelling and redness on the right inguinal and buttock area of two weeks duration .
initial laboratory findings showed elevated esr ( 66 mm / hr ) and crp ( 15.4 mg / dl ) levels .
mri was performed to identify the infection focus and guide treatment planning , and showed a thin - walled fluid collection between the obturator internus tendon and the posterior surface of the ischium ( fig .
however , diffuse enhancement of soft tissue was noted in the right inguinal area , the lesser pelvis , and along the sciatic nerve .
diffuse pelvic infection with reactive bursitis of an obturator internus bursa was diagnosed based on clinical and mr imaging features . after eight days on antibiotics
, his laboratory findings returned to the normal range , and follow - up mr 12 days later showed complete disappearance of the fluid pocket and of the soft tissue enhancement in the right pelvic area ( fig .
a 33-year - old man was presented with a right buttock pain of two days duration , and had been febrile for two days , though this had been well controlled by antipyretics .
a physical examination revealed tenderness , swelling , and local heating on the posterior region of the right buttock and paresthesia on the posterolateral aspect of his right leg .
pyriformis and patrick tests were positive , and laboratory findings showed elevated esr ( 27 mm / hr ) and crp ( 12.6 mg / dl ) with mild leukocytosis ( 10,600/ui ) .
mri was performed under the impression of septic arthritis of the hip , and revealed a boomerang - shaped fluid collection over the posterior surface of the ischium , and diffuse enhancement of the obturator internus tendon and inferior gemellus muscle ( figs .
an accompanying multidetector ct ( mdct ) scan showed calcification along the obturator internus tendon ( fig .
a 28-year - old man presented with swelling and redness on the right inguinal and buttock area of two weeks duration .
initial laboratory findings showed elevated esr ( 66 mm / hr ) and crp ( 15.4 mg / dl ) levels .
mri was performed to identify the infection focus and guide treatment planning , and showed a thin - walled fluid collection between the obturator internus tendon and the posterior surface of the ischium ( fig .
however , diffuse enhancement of soft tissue was noted in the right inguinal area , the lesser pelvis , and along the sciatic nerve .
diffuse pelvic infection with reactive bursitis of an obturator internus bursa was diagnosed based on clinical and mr imaging features . after eight days on antibiotics
, his laboratory findings returned to the normal range , and follow - up mr 12 days later showed complete disappearance of the fluid pocket and of the soft tissue enhancement in the right pelvic area ( fig .
a 33-year - old man was presented with a right buttock pain of two days duration , and had been febrile for two days , though this had been well controlled by antipyretics .
a physical examination revealed tenderness , swelling , and local heating on the posterior region of the right buttock and paresthesia on the posterolateral aspect of his right leg .
pyriformis and patrick tests were positive , and laboratory findings showed elevated esr ( 27 mm / hr ) and crp ( 12.6 mg / dl ) with mild leukocytosis ( 10,600/ui ) .
mri was performed under the impression of septic arthritis of the hip , and revealed a boomerang - shaped fluid collection over the posterior surface of the ischium , and diffuse enhancement of the obturator internus tendon and inferior gemellus muscle ( figs .
an accompanying multidetector ct ( mdct ) scan showed calcification along the obturator internus tendon ( fig .
a bursa is a sac lined with synovial cells that typically forms in an area of tendon friction .
recently , the mr imaging features of a number of uncommon bursae , such as , obturator externus bursa , subgluteus medius bursa , and subgluteus minimus bursa , have been described ( 4 , 6 ) .
the obturator internus muscle originates widely from the margin of the obturator foramen , obturator membrane , iliac bone , and the base of the ischial spine , and covers most of the lateral pelvic wall ( 7 ) .
it then passes through the lesser sciatic foramen , and finally , its fibers converge to form a tendon that inserts into the medial aspect of the greater trochanter of the femur , together with the tendons of the superior and inferior gemelli muscles ( 1 ) . in more detail ,
the obturator internus tendon makes a right - angled bend over the grooved surface of the ischium , between the ischial spine and tuberosity , and then passes horizontally across the posterior to the hip joint before it inserts into the greater trochanter .
moreover , the obturator internus bursa is located between the obturator internus tendon and the grooved surface of the ischium ( 1 ) .
a number of pathologic conditions involving the obturator internus muscle , tendon , or bursa have been described in the orthopedic literature ( 8 - 10 ) , although no imaging findings of obturator internus bursitis have been described .
in fact , swezey ( 8) commented that obturator internus bursitis has been overlooked as a focus of myofascial irritability with lower back pain . because patients with obturator internus bursitis have vague symptoms , such as , nonspecific myofacial irritability , fever , and buttock swelling , clinical diagnosis is made by direct palpation over the anatomical locus of the obturator internus bursa .
when a patient is lying on one side with the affected knee drawn toward the chest , the obturator mternus bursa may be palpated superiorly from the ischial tuberosity .
several papers have described the clinical findings of obturator internus muscle abscesses in children , and haveemphasized the use of mri and ct to confirm the diagnosis ( 9 ) .
moreover , obturator internus muscle abscesses are frequently mistaken for septic arthritis of the hip joint , because of their similar clinical presentations .
the mri findings of obturator internus muscle abscesses are ; muscle swelling and a rim - enhanced fluid pocket in or adjacent to the obturator internus muscle .
these abscesses usually have a thick enhancing wall and a larger cavity than infected obturator internus bursa . in our cases
, there was no evidence of septic arthritis , i.e. , of joint effusion , synovial enhancement , or bone marrow signal changes in the hip joint .
in one case report concerning obturator internus tendinitis , the patient was treated with a ct - guided injection of steroid and local anesthetic into the tendon sheath ( 10 ) . normally , the obturator internus bursa is in a collapsed state , and is only distended and visualized by mri when it is inflamed or infected .
fluid collection in the obturator internus bursa is caused by primary bursal infection or is associated with a pelvic infection . in the described cases , we were able to characterize the locations and shapes of obturator internal bursae on mr images .
these bursae were visualized as narrow and elongated thin - walled fluid pockets resembling " boomerangs " .
the transverse plane provided best visualization , because the bursae were located parallel to the curved portion of the obturator internus tendon posterior to the ischium .
furthermore , fat - suppressed gadolinium - enhanced t1-weighted mr images nicely demonstrate these lesions . in our cases , fluids within bursae were not drained . nevertheless , the clinical and radiological improvements achieved by antibiotic treatment confirmed in both cases a diagnosis of obturator internus bursitis .
the majority of patients with obturator internus bursitis or obturator internus muscle abscesses have been reported to respond adequately to antibiotics without surgical drainage ( 8 , 9 ) .
thus , knowledge of the mri features of obturator internus bursitis can avoid unnecessary surgery . in conclusion ,
a diagnosis of obturator internus bursitis should be considered when the " boomerang "- shaped fluid collection is observed between the obturator internus tendon and the posterior grooved surface of the ischium on mr images . | the authors report two cases with distension of the obturator internus bursa identified on mr images , and describe the location and characteristic features of obturator internus bursitis ; the " boomerang "- shaped fluid distension between the obturator internus tendon and the posterior grooved surface of the ischium . |
a pandemic caused by the new influenza a ( h1n1/09 ) virus resurged in march 2009 .
this new virus is a quadruple - reassortant influenza a virus composed of human , swine and avian strains .
clinical disease generally appears mild , but complications can occur , especially in those with underlying disease , pregnancy or patients on immunosuppression . our aim was to evaluate the clinical course in renal transplant recipients , as they are at increased risk of more severe h1n1 infection and mortality .
the virus appears to have a predilection for infection of the lower respiratory tract , in some cases producing a severe pneumonia , acute respiratory distress syndrome or even multisystem organ failure . in germany , no influenza ( h1n1 ) strains circulating before december 2009 were reported to be resistant to neuraminidase inhibitors .
we describe here three renal transplant patients with proven h1n1 infection . a 48-year - old woman with her third renal transplantation in 2009 experienced severe surgical complications post - transplantation with an application of a colostomy and a split skin graft .
she was under double immunosuppression with tacrolimus and prednisolone at the time of admission , presenting at reduced clinical condition with headache and dysuria .
creatinine was at 1.3 mg / dl ( mdrd egfr i.e. estimated gfr by utilizing the modification of diet in renal disease ( mdrd ) study equation 45 ml / min ) .
antibiotic treatment was started and additional chest radiography on day 2 after admission revealed an interstitial pneumonia , but simultaneous testing of h1n1 in nasopharyngeal swabs was negative .
the bronchoalveolar lavage fluid ( day 4 ) revealed the presence of cytomegalovirus , and intravenous ganciclovir was initiated . since day 4
, fever peaked to 39c and c - reactive protein ( crp ) to 4 mg / dl ( figure 1 ) , and she developed a productive cough .
oseltamivir ( 75 mg b.i.d . ) led to a complete resolution of symptoms within 5 days .
mdrd egfr was at 37 mg / dl ( creatinine 1.5 mg / dl ) .
run of the curve of temperature and crp measures of patient 1 , patient 2 and patient 3 .
arrows indicate positive and negative h1n1 pcr results . a 65-year - old woman transplanted in 2009 , with triple immunosuppression with tacrolimus , prednisolone and mycophenolic acid [ in reduced dosage ( 500 mg / b.i.d .
) because of recurrent urinary tract infections ] developed mild pulmonary symptoms with a non - productive cough 14 days prior to another urinary tract infection .
renal function was stable ( creatinine 1.5 mg / dl , mdrd egfr 36 ml / min ) .
the initial chest x - ray was negative , but she was admitted to the hospital for intravenous therapy of the urinary pseudomonas spp .
another chest x - ray ( day 5 ) showed an infiltrate , while crp peaked to 14.8 mg / dl ( figure 1 ) .
after positive nasopharyngeal swab h1n1 pcr ( day 6 ) , oseltamivir ( 75 mg / b.i.d . ) was initiated .
creatinine increased to 1.8 mg / dl ( mdrd egfr 27 ml / min , day 15 ) .
a 61-year - old male , transplanted in 1999 and suffering from chronic hepatitis c infection , received double immunosuppression consisting of tacrolimus and prednisolone . in july 2009 , he underwent nephrectomy of the left polycystic kidney due to a complicated cyst with ensuing multi - month hospital treatment because of postoperative complications with sepsis , an infected fistula of the pancreas and secondary wound healing with an application of a vacuum pump and consecutive antibiotic treatment ( vancomycin , ciprofloxacin ) . in november , the patient , still under antibiotic treatment , showed stable clinical condition and transplant function ( creatinine 2.4 mg / dl , mdrd egfr 28 ml / min ) .
temperature then suddenly peaked to 39c ; crp was at 3.4 mg / dl ( figure 1 ) as he developed respiratory symptoms with cough and rhinitis .
the chest x - ray revealed no infiltration , but an h1n1 pcr from nasopharyngeal swab was positive .
antiviral therapy with oseltamivir 75 mg / b.i.d . led to a resolution of symptoms and a negative pcr control 1 week later ( figure 1 ) .
the renal function remained stable ( creatinine 2.3 mg / dl , mdrd egfr 29 ml / min ) , without tacrolimus adjustment .
a 48-year - old woman with her third renal transplantation in 2009 experienced severe surgical complications post - transplantation with an application of a colostomy and a split skin graft .
she was under double immunosuppression with tacrolimus and prednisolone at the time of admission , presenting at reduced clinical condition with headache and dysuria .
creatinine was at 1.3 mg / dl ( mdrd egfr i.e. estimated gfr by utilizing the modification of diet in renal disease ( mdrd ) study equation 45 ml / min ) .
antibiotic treatment was started and additional chest radiography on day 2 after admission revealed an interstitial pneumonia , but simultaneous testing of h1n1 in nasopharyngeal swabs was negative .
the bronchoalveolar lavage fluid ( day 4 ) revealed the presence of cytomegalovirus , and intravenous ganciclovir was initiated . since day 4
, fever peaked to 39c and c - reactive protein ( crp ) to 4 mg / dl ( figure 1 ) , and she developed a productive cough .
oseltamivir ( 75 mg b.i.d . ) led to a complete resolution of symptoms within 5 days .
mdrd egfr was at 37 mg / dl ( creatinine 1.5 mg / dl ) .
run of the curve of temperature and crp measures of patient 1 , patient 2 and patient 3 .
a 65-year - old woman transplanted in 2009 , with triple immunosuppression with tacrolimus , prednisolone and mycophenolic acid [ in reduced dosage ( 500 mg / b.i.d . ) because of recurrent urinary tract infections ] developed mild pulmonary symptoms with a non - productive cough 14 days prior to another urinary tract infection .
renal function was stable ( creatinine 1.5 mg / dl , mdrd egfr 36 ml / min ) .
the initial chest x - ray was negative , but she was admitted to the hospital for intravenous therapy of the urinary pseudomonas spp .
another chest x - ray ( day 5 ) showed an infiltrate , while crp peaked to 14.8 mg / dl ( figure 1 ) .
after positive nasopharyngeal swab h1n1 pcr ( day 6 ) , oseltamivir ( 75 mg / b.i.d . ) was initiated .
creatinine increased to 1.8 mg / dl ( mdrd egfr 27 ml / min , day 15 ) .
a 61-year - old male , transplanted in 1999 and suffering from chronic hepatitis c infection , received double immunosuppression consisting of tacrolimus and prednisolone . in july 2009
, he underwent nephrectomy of the left polycystic kidney due to a complicated cyst with ensuing multi - month hospital treatment because of postoperative complications with sepsis , an infected fistula of the pancreas and secondary wound healing with an application of a vacuum pump and consecutive antibiotic treatment ( vancomycin , ciprofloxacin ) . in november , the patient , still under antibiotic treatment , showed stable clinical condition and transplant function ( creatinine 2.4 mg / dl , mdrd egfr 28 ml / min ) .
temperature then suddenly peaked to 39c ; crp was at 3.4 mg / dl ( figure 1 ) as he developed respiratory symptoms with cough and rhinitis .
the chest x - ray revealed no infiltration , but an h1n1 pcr from nasopharyngeal swab was positive .
antiviral therapy with oseltamivir 75 mg / b.i.d . led to a resolution of symptoms and a negative pcr control 1 week later ( figure 1 ) .
the renal function remained stable ( creatinine 2.3 mg / dl , mdrd egfr 29 ml / min ) , without tacrolimus adjustment .
influenza after solid organ transplantation appears to be more common among lung transplant recipients but also causes significant morbidity among renal , liver and heart transplantation patients .
influenza has also been associated with allograft rejection . to our knowledge , this is the first report on infections of renal transplant recipients with the new influenza a ( h1n1/09 ) virus .
the three cases differ with respect to time after transplantation and to the immunosuppressive regimen .
the first two cases are characterized by short time after grafting and reduced immunosuppression due to infectious complications .
the third case had a longer time span after grafting with reduced immunosuppression and multiple infectious / surgical complications .
the patients had not been vaccinated against the new influenza a. they were treated with oseltamivir in a standard dosage ( 75 mg / b.i.d . ) .
onset of respiratory symptoms was fulminant in case 1 , but antiviral treatment was delayed and led to a rapid resolution of symptoms and a negative pcr result 4 days later .
the second case exhibited mild respiratory symptoms for nearly 20 days before h1n1 pcr testing and antiviral therapy , which was effective within 1 week , despite fungal co - infection of the lung .
both cases underline the benefit of antiviral treatment in the immunocompromised patient even when started late after infection . in the third case ,
fast symptom relief with clearance of the virus after oseltamivir treatment is concordant with other reports , but this observation is noteworthy and reassuring in immunosuppressed patients .
overall , h1n1 infection showed an unexpectedly mild clinical course , similar to the general population .
renal transplant function remained stable , while gfr decline in case 3 was most probably induced by drug interactions .
the immunosuppressed renal transplant patient is prone to a severe h1n1 infection , but the cases described here showed a surprisingly mild clinical course despite respiratory co - infections .
it is essential to test for influenza a ( h1n1/09 ) , as early administration of antiviral agents is recommended .
our cases show that early antiviral therapy contributes to a fast resolution of respiratory symptoms and clearance of the virus .
in addition , in our three cases , even delayed therapy was associated with a fast symptom relief .
furthermore , oseltamivir in a standard dose proved effective without deteriorating transplant function or leading to adverse effects . | the pandemic new influenza a ( h1n1/09 ) virus may be especially threatening for immunosuppressed renal transplant patients as they are at increased risk for complications , prolonged infection and mortality .
this is the first case report of renal transplant patients with pcr - confirmed h1n1 respiratory tract infection .
they showed a surprisingly mild clinical course despite respiratory fungal or viral co - infections in two cases .
treatment with oseltamivir in standard dosage was immediately started after diagnosis and proved to be rapidly beneficial with respect to clinical outcome and virus shedding without deteriorating renal transplant function . |
the trypanosomatids trypanosoma cruzi , trypanosoma brucei , and leishmania major are the causative agents of chagas disease , african sleeping sickness and leishmaniasis , respectively .
these protozoan pathogens affect over 27 million people , primarily in developing countries within tropical and subtropical regions .
there are no vaccines for these diseases and only a few drugs , which are largely ineffective due to toxicity and resistance .
these three pathogens ( herein collectively referred to as tritryps ) share many general characteristics , especially the presence of the unique mitochondrion , which contains a dense region named as kinetoplast .
this mitochondrial region is composed by a network of several thousand minicircles and a few dozen maxicircles that form the kinetoplast dna ( kdna ) .
minicircles encode guide rnas that modify maxicircle transcripts by rna editing while maxicircles are correspondent to the mitochondrial dna in higher eukaryotes that encodes rrnas and the subunits of respiratory complexes .
actually , kdna replication always takes place earlier than mitosis , indicating that the kdna may be needed for cell division , either by signaling a successful replication or by affecting the structure .
furthermore , the trypanosome mitochondrion may hold vital metabolic pathways besides a possible role in ca+2 homeostasis , fatty acid metabolism , and apoptosis .
in fact , kdna function and integrity may play a crucial role in the survival of some stages of tritryps lifecycles [ 35 ] .
however , the kdna is subjected to large amounts of endogenous oxidative damage generated by oxidative phosphorylation .
thus , an efficient kdna maintenance mechanism is necessary to repair and avoid oxidative lesions in the mitochondrial dna .
the draft genome sequences of the tritryps , released in 2005 , have allowed a better understanding of the genetic and evolutionary characteristics of these parasites [ 69 ] .
a comparison of gene content and genome architecture of t. cruzi , t. brucei , and l. major revealed large syntenic polycistronic gene clusters .
in addition , many species - specific genes , such as large surface antigen families , occur at nonsyntenic chromosome - internal and subtelomeric regions .
syntenic discontinuities are associated with retroelements , structural rnas , and gene family expansion . along with these factors , gene divergence , acquisition and loss , and rearrangement within the syntenic regions help to shape the genome of each parasite .
expansion of gene families by tandem duplication is a potential mechanism by which parasites can increase expression levels to compensate for a general lack of transcriptional control due to polycistronic structure and the absence of general transcription factors . concerning the individual features of each parasite , which reflect differences in their lifecycles , t. brucei has large subtelomeric arrays that contain variant surface glycoprotein ( vsg ) genes used by the parasite to evade the mammalian immune system .
meanwhile , over 50% of the t. cruzi genome consists of repeated sequences , such as genes for large families of surface molecules , which might function in immune evasion and adaptation to an intracellular environment .
leishmania spp . has a simpler genome but also has the ability to amplify genomic regions .
this genus contains genes for the synthesis of complex surface glycoconjugates that are likely to enhance survival in the macrophage phagolysosome .
analyses of the tritryps genomes have identified differences in the dna maintenance mechanisms ( nuclear and mitochondrial ) between tritryps and other eukaryotes .
dna repair systems are responsible for preserving the genome stability via correcting dna lesions caused by damaging agents both from the environment and endogenous metabolic processes [ 1014 ] .
this system embraces several distinct pathways : ( 1 ) sanitization of the nucleotide pool , ( 2 ) direct reversal of the base modifications by demethylation processes , by the action of photolyases or dioxigenases , or ( 3 ) excision of ( i ) oxidized , methylated , or misincorporated bases by base excision repair ( ber ) , ( ii ) bulky damage by nucleotide excision repair ( ner ) , and ( iii ) misincorporated bases in the newly replicated dna strand by mismatch repair ( mmr ) .
dna is also susceptible to single - strand breaks ( ssbs ) and double - strand breaks ( dsbs ) , which can be repaired by homologous recombination ( hr ) and nonhomologous end joining ( nhej ) . even though these mechanisms repair the majority of dna lesions , some of the damage remains , leading to mutations or block of the dna replication .
alternative dna polymerases can bypass these lesions in an error - free or error - prone fashion using a tolerance process known as translesion synthesis ( tls ) .
basic knowledge of dna damage repair and tolerance processes is crucial to understanding how and why the genome is affected during the organism lifespan and how the cells will deal with it .
t. cruzi , t. brucei , and l. major appear to be able to catalyze most of the dna repair pathways [ 69 ] . here , we briefly review the current information on dna repair mechanisms in tritryps with an emphasis on experimentally characterized genes ( table 1 ) .
we highlight the main features of the major dna repair pathways and report the presence or absence of key genes in tritryps .
most of the genes were previously identified by their genome projects [ 69 , 15 ] , and few of them were identified through similarity screening and domain analysis .
the gene absence could truly represent a nonoccurence of the gene ( whose function could be compensated or not by another gene ) , a large sequence divergence , or even an annotation error , which made the search for a homolog difficult .
two mechanisms of direct repair are present in tritryps : alkylation reversal and oxidative damage repair [ 69 ] .
single homologs of o-6 methylguanine alkyltransferase ( mgmt ) can be found in the three genomes .
this enzyme catalyzes the repair of o - meg , a critical mutagenic lesion that yields g : c to a : t transitions .
alkb , an iron - dependent dioxygenase that reverses dna lesions ( 1-mea and 3-mec ) in single - strand dna ( ssdna ) or rna , is also present in tritryps .
the third mechanism of direct repair utilizes photolyases , which catalyze the splitting of pyrimidine dimers into the constituent monomers , a process called photoreactivation .
t. cruzi does not contain a clear photolyase homolog although t. brucei and l. major are thought to perform photoreactivation because they have a gene that contains an n - terminal photolyase domain [ 69 ] .
the absence of photoreactivation as a repair mechanism for pirimidine dimers in t. cruzi could be associated with the availability of transcription - coupled repair ( tcr ) , which would efficiently deal with such lesions .
ber is the predominant pathway for dealing with a wide range of lesions that modify individual bases without large effects on the double helix structure .
such modifications on dna bases can arise as a result of oxidation , alkylation , and/or deamination .
the ber pathway consists of modified base recognition and removal by a dna glycosylase , cleavage of the sugar - phosphate backbone , and excision of the abasic ( apurinic - apyrimidinic , ap ) site by a dna ap endonuclease , followed by dna synthesis and ligation steps .
the gapfilling and rejoining steps can occur by either of two subpathways : short - patch ber or long - patch ber . in the short - patch ber subpathway ,
only one nucleotide is replaced by dna pol and the nick is sealed by lig3 , all steps being coordinated by xrcc1 . in long - patch ber , 213 nucleotides are replaced with the involvement of the replicative polymerases ( pol ) or ( pol ) .
structure that is removed by fen1 through a single - stranded break for subsequent nick ligation by ligase 1 ( lig1 ) .
the long - patch mechanism also involves pcna , which interacts and coordinates the enzymes involved , and poly adp - ribose polymerase ( parp ) , that binds to dna ssbs preventing dsbs and facilitates access for the long - patch machinery .
the primary components of the ber pathway have been identified in t. cruzi , t. brucei and l. major genomes [ 69 ] and are organized in the tritrypdb database .
however , it is not clear whether they can perform short - patch and long - patch ber since the homologs of lig3 and xrcc1 , which are supposedly essential for the short - patch mechanism [ 46 , 49 , 50 ] , have not yet been identified in the three organisms .
however , these ber components are also absent in plants , and crdoba - caero et al
. recently demonstrated that ber of uracil and abasic sites occurs in arabidopsis thaliana whole - cell extracts by both single - nucleotide insertion and long - patch dna synthesis .
in contrast to the other tritryps , the l. major genome allegedly does not encode for the parp enzyme , which could play a role in the long - patch subpathway .
different dna glycosylases involved in the removal of modified bases from dna have been characterized in tritryps .
the uracyl - dna glycosylase from t. cruzi ( tcung ) was the first one to be characterized by frez - vidal and coworkers .
they demonstrated that the enzyme activity was enhanced by the addition of an ap endonuclease from l. major , suggesting that there could be a functional interaction between the two enzymes .
recently , pea - dias and colleagues reported that tcung is able to complement e. coli ung mutants , and that the trypanosome enzyme has a catalytic activity similar to human ung .
surprisingly , their results indicated that tcung is able to excise uracil in dna via short - patch ber using a polymerase that follows a pol-like pattern of inhibition .
the characterization of the tcung protein sequence suggested that it has a probable pcna - binding motif and could be directed either to the mitochondrion or nucleus .
another glycosylase found in tritryps is 8-oxog - dna glycosylase ( ogg1 ) , an enzyme that removes the oxidative lesion 7,8-dihydro-8-oxoguanine ( also known as 8-oxoguanine or 8-oxog ) when it is paired with cytosine . among the dna damage caused by reactive oxygen species ( ros ) ,
this lesion has the ability to mimic thymine functionally , forming a stable 8-oxog : a base pair .
this conformation allows the replicative dna polymerases to efficiently bypass 8-oxog failing to detect this damaged dna base .
a functional homolog of ogg1 in t. cruzi has been studied in vivo by furtado and colleagues ( unpublished data ) .
this gene is able to complement yeast ogg1 mutants , reducing the mutation rate of these cells .
the expression of ogg1-gfp fusion protein in t. cruzi revealed that the intracellular localization of ogg1 is both nuclear and mitochondrial .
in fact , overexpression of the ogg1 in t. cruzi diminishes the levels of 8-oxog within the nucleus and mitochondrion after hydrogen peroxide ( h2o2 ) treatment .
the unusual localization of ogg1 in the mitochondrion could indicate the importance of the maintenance of the kdna integrity in this parasite .
in addition to ogg1 glycosylase , mutt and muty also contribute to counteract the mutagenesis effects of 8-oxog .
mutt degrades 8-oxo - dgtp from the nucleotide pool to 8-oxo - dgmp , preventing mutations that arise from the misincorporation of this oxidized form of dgtp . on the other hand ,
when 8-oxo - dgtp is misincorporated opposite adenine in template dna , muty can fix an a : t to c : g mutation because it removes the correct adenine from the a : 8oxog pair .
therefore , when muty is present , the action of mutt is crucial because oxidized nucleotides must be eliminated from the nucleotide pool . at the time the genome sequence was released ,
homologs of 8-oxoguanine hydrolase mutt were not encountered in the tritryps genome [ 69 ] . a more accurate search of the tritryps genomes revealed that mutt homologs are present in t. brucei , l. major , and possibly in t. cruzi .
indeed , a t. cruzi muty homolog has been characterized ( kunrath - lima , unpublished data ) .
this gene is able to complement muty - deficient bacteria , diminishing its mutation rates .
moreover , the t. cruzi muty recombinant protein removes the adenine paired with 8-oxog in vitro from a 30 mer fluorescent substrate .
the ap endonucleases 1 from t. cruzi and l. major have also been characterized [ 1820 ] . both were able to efficiently complement ap endonuclease - deficient e. coli , conferring resistance to alkylating and oxidizing agents .
the l. major ap endonuclease was more extensively studied , and the purified protein exhibited endonuclease and high 3 phosphodiesterase activities on ap dna in vitro .
moreover , leishmania parasites overexpressing the ap endonuclease showed increased h2o2 and methotrexate resistance as well as reduced dna fragmentation .
the structural characteristics of the l. major enzyme exhibited similarities with previously characterized homologs . among the polymerases ,
pol from t. cruzi and t. brucei have already been characterized [ 21 , 22 ] .
the tcpol localizes to the parasite kinetoplast and exhibits dna polymerization and 5drp lyase activity .
similarly , the tbpol characterization also showed that , in addition to a mitochondrial localization , it is active as a dna polymerase and as a lyase .
the cellular localization of these polymerases highlights an important feature of the tritryps : the presence of kdna .
the kdna structure is so complex that it requires an unusual replication mechanism , which differs from higher eukaryotes [ 55 , 56 ] .
this complexity is reflected in the dna repair and replication machinery that can be localized to this organelle .
pol is an example of a polymerase that shows a nuclear localization in higher eukaryotes but is addressed to the kinetoplast in the tritryps [ 21 , 22 ] .
the l. major pol has not yet been experimentally characterized ; however , a pol from l. infantum was shown to have a nuclear localization , which could indicate that the l. major polymerase is also nuclear , as their primary protein sequences showed 100% identity .
the possibility that l. major possesses a nuclear pol , combined with the fact that this parasite does not have the parp enzyme [ 7 , 15 ] , suggests that short - patch ber could play an important role in nuclear dna repair for this organism . as leishmania proliferates inside macrophage phagolysosomes , a well - coordinated nuclear short - patch ber is essential to combat oxidative dna damage during parasite nuclear dna replication .
the tritryps genomes apparently do not encode for the other x - family polymerases , dna polymerase lambda ( pol ) , and mu ( pol ) [ 7;15 ] ; thus , l. major may be the only tritryps parasite that has an x - family polymerase in the nucleus , reinforcing the importance of short - patch ber in this organelle .
parp from t. cruzi , another enzyme that is involved in long - patch ber pathway , has also been characterized .
the activity of this enzyme has been shown to be dependent on the presence of dna and was enhanced by ssb in dna in a concentration - dependent manner . moreover
, it was demonstrated that dna - damaging agents , such as h2o2 and -lapachone , induced par synthesis in the parasite nucleus , indicating that this enzyme could be involved in the signaling of this phenomenon .
nucleotide excision repair is one of the most versatile dna repair mechanisms , responsible for repairing lesions that alter the tridimensional dna conformation , such as cisplatin adducts and uv - induced lesions ( pyrimidine dimers and pyrimidine photoproducts ) .
this mechanism can be divided into two major pathways : global genome repair ( ggr ) , which operates in the noncoding parts of the genome and in the nontranscribed strand of active genes , and tcr , which is activated when a lesion appears in a gene that is being transcribed , ensuring that the transcribed strand of active genes has a higher priority for being repaired than the rest of the genome . the ggr - ner mechanism comprises several steps : ( i ) distortion detection , performed by xpc and hr23b or alternatively by the complex ddb1/xpe - ddb2 ; ( ii ) double - strand opening by the tfiih complex via its xpb and xpd helicase subunits ; ( iii ) recruitment of xpa complexed with the three heterotrimeric replication protein a ( rpa ) subunits ; ( iv ) dna incision by the xpg endonuclease ( 3 side of lesion ) and by the xpf - errc1 heterodimer ( 5 side of the lesion ) ; ( v ) gap filling by the replicative polymerases and associated with pcna [ 68 , 69 ] ; ( vi ) nick sealing by ligase iii together with xrcc1 ( in quiescent cells ) or at a lower level by ligase i ( in actively replicative cells ) .
tcr - ner has a mechanism similar to ggr , but it differs in the initial steps because it lacks the xpc and ddb1 complexes .
tcr - ner is triggered by the stalling of rna polymerase ii , which subsequently recruits csa , csb , and xab2 .
although the entire ner mechanism is well conserved in nature , there is no sequence homology between the ner proteins from bacteria and eukaryotes . despite the sequence conservation shared by the eukaryotic ner proteins ,
not all the genes that encode those proteins are found among distantly related phylogenetic groups .
the most remarkable examples are the lack of xpa in arabidopsis thaliana and the lack of xpa , xpc , and xpe in plasmodium falciparum , which suggest that the ner mechanism can have slight variations between different taxons .
the tritryps genomes contain the majority of the ner components [ 7 , 15 ] , but the biochemical mechanisms of this pathway may present some minor differences from the higher eukaryotes . some of the genes are duplicated .
for example , tritryps have two copies of xpb and ddb1 appear duplicated in t. cruzi .
it is also possible that the tritryps ligation step is different from the ligation step from higher eukaryotes .
the tritryps lack a recognizable ligase iii , which together with its partner xrcc1 plays a major role in this final step .
however , because their genomes encode ligase i , it might be possible that the ligation step is performed exclusively by this protein in those parasites .
ddb2 , which interacts with ddb1 and recognizes uv - induced lesions , and rpa3 , a component of the rpa heterotrimer , also could not be identified in the genomes of these trypanosomatids .
the tfiih complex shows some differences when compared to yeast and mammals because it does not contain the cyclin - activating kinase ( cak ) subcomplexes .
in addition to that , a recent study showed that t. brucei tfiih contains two trypanosomatid - specific subunits of tfiih ( tsp1 and tsp2 ) , which are indispensable for parasite viability and transcription of splice - leader gene .
these subunits are also present in the genomes of t. cruzi and l. major . protein - coding genes are constitutively transcribed in trypanosomatids .
this peculiarity implies that tcr could be one of the most crucial mechanisms involved in repairing dna damage in those parasites .
although the role of csa in tcr is not clear , recent evidence indicates that csa is involved in csb ubiquitination and degradation following uv irradiation , which would restore transcription at a normal rate after the repair .
the absence of an evident csa in tritryps implies that the trypanosomatid tcr differs from the standard tcr mechanism , either by the lack or divergence of this component , or by the presence of an alternative protein to perform this step .
in fact , overexpression of t. cruzi dna polymerase ( pol ) , involved in the translesion synthesis of pirimidine dimers , and overexpression or haploinsufficiency of rad51 , a key protein in hr , do not confer any protection against uv irradiation , which could suggest that the uv - induced lesions are fully repaired before the cell enters the s - phase ( , passos - silva et al . ,
in addition , results obtained by our group show that t. cruzi repairs cisplatin - induced lesions at an extremely high rate , with total lesion removal in less than an hour ( rajo , unpublished data ) . taken together , these results led us to hypothesize that , in t. cruzi , lesions that cause dna distortions are readily detected and repaired by tcr because the great majority of the protein - coding genes are transcribed constitutively . whether the csa absence or the presence of an alternative csa is an adaptation to this distinctive repair is a topic for future investigation .
when compared to other taxons , ggr - ner in trypanosomatids seems to be similar to the ggr pathway encountered in plants , as both groups of organisms share peculiarities regarding the presence and absence of some ner genes .
although plants encode all the tfiih subunits and csa , the plant genome , like tritryps , does not possess an identifiable xpa , rpa3 , or ligase 3 .
interestingly , these dna repair similarities found in tritryps and plants can also be observed in the mmr pathway , which could suggest that both groups might share some commonalities in their dna repair mechanisms .
postreplicative dna mismatch repair promotes genetic stability by repairing dna replication errors ( single base - base mismatches and insertion or deletion loops , idls ) , inhibiting recombination between nonidentical dna sequences and participating in responses to dna damage induced by genotoxic agents , such as h2o2 , cisplatin , and n - methyl - n-nitro - n - nitrosoguanidine ( mnng ) .
in essence , postreplicative mmr operates through ( i ) dna mismatch recognition by muts ( msh2-msh6 ) or muts ( msh2-msh3 ) , ( ii ) excision of the damaged dna section mainly by exoi , and ( iii ) dna resynthesis by dna pol and ligation .
steps after dna mismatch recognition are coordinated by mlh heterodimers that bind to msh proteins and probably recruit and assembly downstream repair complexes .
strand discontinuities associated with dna replication can serve as entry points for strand excision , conferring strand specificity to mmr .
each trypanosomatid encodes a set of mmr proteins , which suggests they are fully competent for mismatch recognition and repair [ 7 , 15 ] .
components of the mmr pathway are major players in processes known to generate genetic diversity , such as mutagenesis and dna recombination .
evidences suggest that differences in mmr efficiency could be an important source of genetic diversity in organisms [ 7679 ] .
t. cruzi has a highly heterogeneous population , composed of a pool of strains with distinct characteristics such as morphology , growth rate , virulence , and sensitivity to drugs . despite its broad genetic diversity , three major lineages , named t. cruzi i ,
studies with a number of molecular markers revealed that parasites belonging to the t. cruzi i lineage have lower genetic variability compared to t. cruzi ii , and iii [ 8284 ] .
the great genetic diversity observed in t. cruzi ( and more precisely , in t. cruzi ii strains ) may play an important role in pathogenesis and survival of the parasite within its different hosts .
it is conceivable that components of dna repair pathways participate in processes that resulted in increasing genetic variability within the parasite population .
msh2 , the core eukaryotic mismatch repair gene , has been characterized in t. cruzi [ 25 , 26 , 86 ] .
sequence analyses of tcmsh2 showed the existence of three distinct isoforms , named tcmsh2a , b , and c , encoded in the genome of t. cruzi i , iii , and ii strains , respectively .
it is possible that these isoforms have distinct protein activity , leading to variations in the efficiency of mmr .
in fact , parasites that have tcmsh2a show increased sensitivity to cisplatin and mnng , increased microsatellite instability , and greater resistance to h2o2 when compared to parasites expressing tcmsh2b or tcmsh2c ( , campos et al .
further studies are needed to determine if these variations in mmr efficiency have a broader impact on genetic variation and behavior in t. cruzi strains .
attempts to generate tcmsh2-null mutants indicate that , in addition to its role in mmr , tcmsh2 acts in the parasite response to oxidative dna damage in an mmr independent manner . in t. brucei , msh2 has been studied along with mlh1 .
mutations in both genes give rise to increased microsatellite instability and lead to increased tolerance to the alkylating agent mnng .
these results indicate that mmr in trypanosomatids is active in repairing errors that arise during replication and in response to chemical damage .
double mutants of msh2 and mlh1 show an increased frequency of homologous recombination , both between perfectly matched dna molecules and between dna molecules with divergent sequences
because mlh1-null mutants do not show this phenotype , tbmsh2 seems to have an additional role in dealing with oxidative damage , which may occur independently of mmr .
interestingly , the heterologous expression of msh2 from t. cruzi was able to counteract the increased sensitivity to h2o2 in the t. brucei msh2-null mutant .
however , it did not affect the classical mmr - deficient phenotypes , such as microsatellite instability and resistance to mnng .
this differential activity of msh2 has also been reported in colon adenocarcinoma cell lines where msh2 , but not mlh1 , has been implicated in the repair of 8-oxog .
in addition , helicobacter pylori , which is suggested to be mmr - defective due to the lack of muth and mutl homologs , presents a muts homolog that is involved in repairing oxidative damage .
four additional msh - like genes can be found in the trypanosomatids : msh3 , msh4 , msh5 , and msh6 [ 7 , 15 , 27 ] .
the predicted msh6 polypeptides in tritryps have n - terminal truncations relative to eukaryotic orthologues . in comparison ,
msh7 , unique to plants , bears similar truncations in the n - terminus along with the conserved mismatch interaction residues indicative of the msh6 subgrouping .
msh4 and msh5 predicted proteins that appear to lack an n - terminal mismatch interaction , indicating an absence of function in the mismatch repair and a possible role in meiotic recombination .
other mutl homologs , such as pms2 , mlh2 , and mlh3 , appear to be absent .
trypanosomatid mmr is therefore likely to involve only an mlh1-pms1 heterodimer whereas the functions performed by the dimers formed between mlh1 and its three other binding partners in yeast are either absent or fulfilled by mlh1-pms1 .
dsbs can arise when replication forks encounter blocking lesions , which leads to fork collapse , or can be induced by ionizing radiation and radiomimetic chemicals .
failure to accurately repair such damage can result in cell death or large - scale chromosome changes , including deletions , translocations , and chromosome fusions that enhance genome instability .
two distinct and evolutionarily conserved pathways for dsb repair exist : homologous recombination and nonhomologous end joining .
the two ends of the dsb are held together and religated , often following the loss of some sequence by nucleolytic degradation or addition by polymerization .
eukaryotic nhej is a multistep pathway beginning with limited end processing by the mre11/rad50/nbs1 ( mrn ) complex and initial recognition of dsbs through end binding by ku , a ring comprised of the ku70 , and ku80 subunits . in higher eukaryotes , the dna - dependent protein kinase catalytic subunit ( dna - pkcs )
is also recruited . in the final step , dna ligase iv with its binding partners
xrcc4 ( lifi in yeast ) and xlf ( also called cernunnos ) seals the break .
nhej seems to be absent in trypanosomatids . with the exception of mre11 , rad50 , ku70 and ku80 ,
no other factors implicated in nhej could be identified in these organisms . ku70 and ku80 have been identified in t. brucei , t. cruzi , and l. major .
studies in t. brucei have shown that these genes act in telomere maintenance [ 29 , 30 ] , a function they provide in addition to nhej .
however , the mutants of ku70 and ku80 did not display higher sensitivity to dna - damaging agents , suggesting that they play , at most , a minor role in dsbs repair possibly due to the absence of nhej in this organism .
these absences in tritryps suggest one of two possibilities : either nhej is absent from these organisms or its catalytic components have been modified beyond recognition , perhaps using a distinct dna ligase .
hr is required for dna dsbs repair and provides critical support for dna replication in the recovery of stalled or broken replication forks .
in addition , hr is involved in the repair of incomplete telomeres and in the correct segregation of homologous chromosomes during meiosis . the broad reaction scheme [ 93 , 94 ]
can be considered in three steps : initiation ( or presynapsis ) when the nucleolytic resection of dsbs occurs , generating single - stranded tails with 3-oh ends ; strand exchange ( synapsis ) , when the end(s ) of the dsb invades the intact dna molecule via regions of sequence homology ; resolution ( postsynapsis ) , when strand exchange intermediates are separated and the dsb is repaired .
essential components of this mechanism have been identified in the genome of t. cruzi , t. brucei , and l. major .
hr can contribute to different strategies evolved by trypanosomatids to create genetic variability that is needed for survival in their hosts .
antigenic variation is used by t. brucei to evade the host immune system through the switch of surface proteins ( vsgs ) .
this mechanism is regulated by hr , allowing the switch of one vsg at time to the expression site . meanwhile ,
t. cruzi displays a wide range of surface molecules that are highly polymorphic and may represent a useful arsenal to evade immune systems .
recent works have been suggesting that hr is responsible for creating mosaic genes of surface molecules through segmental gene conversion and for decreasing the divergence between duplicated regions such as surface multigenic families [ 83 , 96 ] .
in addition , experiments with genetic manipulation have shown that homologous recombination is the main mechanism for integration of transformed dna in these organisms [ 97100 ] .
the complex of proteins involved in the presynapsis step of hr can be found in tritryps , such as mre11 , rad50 , nsb1 , and rpa .
mutation of mre11 causes impairment in t. brucei homologous recombination , increases dna damage sensitivity , and leads to gross chromosomal rearrangements [ 31 , 32 ] .
mre11 does not contribute to recombination during antigenic variation , an important mechanism used by t. brucei to escape host immune response as mentioned before .
the core step of hr is the search for homology , homologous dna pairing , and strand exchange reaction that is mediated by recombinases , such as rad51 and dmc1 .
dmc1 , a putative meiosis - specific recombinase , has only been studied in t. brucei .
the lack of dmc1 does not affect hr repair or vsg switching in this parasite .
the presence of genes involved in meiosis is an intriguing feature of tritryps because they reproduce primarily through clonal reproduction .
even though the population structure of each parasite is largely clonal , evidence of genetic exchange in the wild - type populations of t. brucei , t. cruzi [ 103 , 104 ] , and l. major has been presented .
however , it is unclear whether or not the existence of meiotic recombination genes implies that the trypanosomatids use meiosis .
the expression of rad51 in t. cruzi and l. major is induced by dna - damaging agents [ 33 , 35 ] . moreover ,
the overexpression of rad51 in t. cruzi confers a faster recovery and a more efficient dna repair of dsbs formed after genotoxic treatment .
in addition , t. cruzi rad51 accumulates in the nucleus after exposure to gamma radiation ( passos - silva et al . ,
submitted ) . besides , the levels of rad51 in t. cruzi reflect its susceptibility to oxidative agents .
the overexpression of tcrad51 confers a greater resistance to h2o2 whereas the deletion of one of the tcrad51 alleles increases the sensitivity when compared to wild - type parasites ( passos - silva et al . ,
thus , rad51 seems to be involved in a greater resistance to oxidative damage in t. cruzi dna . an active response to
oxidative stress is an important feature of t. cruzi and l. major because they have an intracellular stage in the host that is subjected to a rigorous oxidizing environment . for t. brucei , rad51 , and
null mutants of rad51 led to impairments in vsg switch and dna transformation and a higher sensitivity to genotoxic agents .
however , an rad51-independent recombination pathway is also present , as evidenced by two mechanisms detected in t. brucei rad51 mutants : ( i ) antigenic variation by gene conversion and ( ii ) integration of transformed dna by homology - based recombination although the frequency of detection is low .
interchromosomal hr is the major pathway used by t. brucei to repair dsbs , as demonstrated by glover and colleagues .
after the generation of single dsb through scei endonuclease , rad51 accumulates into the foci and a g2 m checkpoint is activated .
in addition , tritryps show intriguing differences concerning gamma radiation treatment , which generates high levels of dsbs .
t. cruzi and l. major are highly resistant to gamma radiation when compared to other eukaryotes [ 33 , 108 , 109 ]
. however , this resistance is not seen in t. brucei ( unpublished data ) .
in fact , after gamma radiation treatment , the expression of rad51 in t. cruzi and l. major are induced [ 33 , 35 ] whereas the rad51 levels in t. brucei do not increase .
as mentioned before , these intriguing differences concerning the efficiency of recombination in tritryps could be due to the distinct mechanisms used by these organisms to create genetic variability and to evade the mammalian immune system .
the loading of recombinases in the ssdna is a rate - limiting process that is enhanced by recombination mediators .
brca2 , the rad51 paralogs , and rad54 are among the recombination mediators present in trypanosomatids .
whether or not this has a significant impact on recombination is unclear . unlike the yeast mutant ,
in which rad52 is a key protein for hr , mouse rad52 mutants display an exceedingly mild recombination defect and no ionizing radiation sensitivity .
it is unclear which proteins functionally replace the yeast rad52 protein in mammalian cells or trypanosomatids .
brca2 can interact with rad51 through the brc repeat motifs [ 111113 ] and unrelated sequences .
on the other hand , t. brucei brca2 has undergone an expansion in the number of brc repeats ( 15 brc repeats ) , and these elements are crucial for the efficiency of hr and rad51 localization .
four rad51 paralogs appear to be encoded by t. brucei and t. cruzi , one of which appears to be missing in l. major .
two of the t. brucei rad51-like proteins play a role in dna repair and recombination .
studies in t. brucei have been showing that hr in this organism is regulated by mmr through the rejection of hr between insufficiently homologous dna sequences .
this has been evidenced by experiments done with msh2 mutants which are able to recombine mismatched substrates more efficiently than wild type cells .
in contrast , the hr that occurs during vsg switching uses a short and divergent substrate such as the 70 bp repeats upstream of vsg genes .
thus , the vsg switching may happen through a specific recombination pathway that is independent of mmr or the suppression of mmr would be necessary [ 100 , 114 ] .
lesions in dna can block replicative dna polymerases ( pol and pol ) , causing the stall of the replication fork . this halt leads to pcna monoubiquitination by rad6/rad18 complex , promoting the switch from replicative dna polymerase to tls dna polymerase , which catalyses nucleotide insertion opposite the lesion .
tls dna polymerases contain a minimally stringent catalytic site , allowing for the accommodation of templates containing damaged bases .
moreover , this group of specialized dna polymerases has lost 3-5 proofreading activity , having a highly mutagenic character .
t. cruzi , t. brucei , and l. major genomes encode for a wide variety of translesion synthesis proteins .
these parasites show an expansion of pol gene , present in two , ten , and three copies in t. cruzi , t. brucei , and l. major genomes , respectively [ 69 ] . the gene duplication / amplification displayed by tritryps pol gene could result in an increment of pol gene expression level which would compensate the lack of pretranscriptional mechanisms in these organisms .
tcpol is able to complement yeast rad30 mutant ( pol-null mutant ) , increasing yeast resistance to uv radiation , which indicates that pol is able to bypass uv lesions .
this resistance could be associated with the ability of tcpol to bypass 8-oxog lesions in vitro , suggesting that this enzyme is able to incorporate nucleotides opposite oxidative lesions as well .
in contrast to the result seen in yeast , parasites overexpressing this nuclear polymerase do not show a higher resistance to uv radiation .
the lack of conferred resistance might be related to the number of lesions remaining during s phase because it is possible that the majority of uv lesions would be repaired by tcr - ner prior to dna replication , as the majority of the protein - coding genes are constitutively transcribed in this organism .
this result indicates that t. cruzi is the first organism described in the literature to contain one exclusively mitochondrial pol. mitochondrial tcpol bypasses 8-oxog in vitro , which correlates with the increase in h2o2 resistance observed in parasites overexpressing this protein .
this dna polymerase could also participate in the homologous recombination pathway in t. cruzi because it synthesizes dna within recombination intermediates . reinforcing this hypothesis
, tcpol overexpression confers higher resistance to gamma radiation and zeocin , which are agents known to cause dsbs .
recent results have shown that the other copy of tcpol has nuclear localization ( rajo , unpublished results ) .
tls deals with dna damage that blocks the replication fork , thus rescuing the cell from death .
this accounts for the survival increase displayed by tcpol-overexpressing parasites when treated with agents that cause dsbs .
in addition , tcpol- and tcpol-overexpressing cells also presented increased resistance to h2o2 treatment [ 39 , 40 ] .
the presence of tls dna polymerases that efficiently bypass oxidative lesions might be important during t. cruzi lifecycle , especially in the intracellular amastigote phase , when this organism deals with ros generated by the infected host cell . moreover , because tls can operate in an error - prone fashion , tls can generate dna punctual mutations in the parasite genome . this can be correlated with the generation of genetic variability in tritryps , notably in surface molecules .
in fact , mutation is considered one of the main driving forces that increase the divergence between genes from multigenic families in t. cruzi , in contrast to the genetic conversion , another main driving force that decreases this divergence [ 83 , 96 ] .
a variable repertoire of surface molecules is a key strategy for t. cruzi to achieve a successful rate of infection .
these proteins interact with different molecules on the host cell membranes and the extracellular matrix , increasing its chance to adapt to distinct cell types and hosts [ 83 , 96 ] . besides , the polymorphism of t. cruzi surface proteins contributes to evade cellular immune response of the mammalian host through the presentation of a broad range of possible target epitopes to cd8+t cells
. this can result in an inefficient activation of nave cd8+t cells , leading to a delayed protective immune response .
thus , tls can affect the general diversity in the organism , which is important for acquiring evolutionary novelty and for adaptation to the parasitic lifestyle .
the genome sequencing of t. cruzi , t. brucei , and l. major has provided insight into the dna maintenance mechanisms in these pathological protozoa [ 69 ] .
experimental data have revealed additional particular features of these systems in the tritryps , which presumably reflect the distinct aspects of the infectious cycle that shape the survival strategies of each protozoa pathogen . among these particularities ,
the unexpected mitochondrial localization of some typically nuclear proteins ( furtado , unpublished data ) [ 21 , 22 , 40 ] and the additional roles of a few proteins in response to oxidative treatment were highlighted in this paper ( passos - silva , submitted ) [ 26 , 39 , 40 ] .
the former is important during the replicative stages of the parasites when the metabolic activity is higher and the unique mitochondrion is then exposed to higher amounts of ros generated by oxidative phosphorylation [ 35 ] .
the later feature is particularly critical during the intracellular stage of the parasites l. major and t. cruzi when they are subjected to the immune response of mammalian host cells through oxidative stress [ 106 , 119 ] .
the dna damage repair and tolerance mechanisms of tritryps are also involved in the generation of genetic variability which raises the successful rate of infection through the increasing of surface molecules diversity in t. cruzi and the expanding of the repertoire of vsgs in t. brucei .
indeed , strains of t. cruzi that have a higher genetic variability ( possibly a reflection of a less efficient mmr campos et al . , unpublished data ) are frequently associated with human infection [ 120 , 121 ] . on the other hand
, hr regulates antigenic variation in t. brucei , the strategy used by this parasite to evade mammalian immune system .
a clear association between dna repair in l. major and evasion of the mammalian immunological response has not been established yet possibly due to a relatively narrow range of studies that investigate dna repair in this organism .
essentially , with the exception of nhej , the major dna repair pathways appear to be present in tritryps .
further studies are necessary to clarify the information about dna repair pathways in tritryps , specifically the differences in tritryps machinery from the typical eukaryotic machinery for dna repair , which could provide potential points of attack against the parasites . | a wide variety of dna lesions arise due to environmental agents , normal cellular metabolism , or intrinsic weaknesses in the chemical bonds of dna .
diverse cellular mechanisms have evolved to maintain genome stability , including mechanisms to repair damaged dna , to avoid the incorporation of modified nucleotides , and to tolerate lesions ( translesion synthesis ) .
studies of the mechanisms related to dna metabolism in trypanosomatids have been very limited .
together with recent experimental studies , the genome sequencing of trypanosoma brucei , trypanosoma cruzi , and leishmania major , three related pathogens with different life cycles and disease pathology , has revealed interesting features of the dna repair mechanism in these protozoan parasites , which will be reviewed here . |
verruca vulgaris is caused by a human papillomavirus ( hpv ) infection of the epidermis .
verruca in the external auditory canal ( eac ) has rarely been reported . a previous case report introduced surgical excision for verruca treatment because there was no available nonsurgical treatment for verruca in the eac .
we present a case of verruca in both eacs that was successfully treated with intralesional bleomycin injection .
a 32-year - old male patient presented with ear fullness and palpable lumps in both eacs .
the lumps were identified 18 months prior to the examination and the patient indicated that the lumps appeared to be getting larger .
otoscopic examination revealed multiple pinkish , papillomatous masses that filled both of his eacs ( fig .
however , we performed intralesional bleomycin injection , which is a simple and effective method for treatment of verruca vulgaris on the hands , feet or face .
bleomycin was diluted with 1% lidocaine to make a 1-u / ml bleomycin solution . a 1-ml syringe with a 30-gauge needle
the bleomycin solution was injected twice ; 0.5 ml at the first injection and 0.2 ml at the second injection one month later . one month after first injection , the verruca decreased to approximately one - third of the initial size ( fig .
two months after the second injections , the verruca was not observed and there was no scarring or necrosis ( fig .
eight months after the second injection , the patient confirmed that there was no recurrence .
verruca vulgaris is caused by an hpv infection , and there are over 100 human papilloma dna viruses that belong to the dna papovaviridae family .
histopathological examination is used for a definitive diagnosis , and revealed characteristic features of hyperkeratosis , papillomatosis , parakeratosis , acanthosis , and koilocytosis .
it is associated with low - risk hpv infections ( types 6 and 11 ) that often result in a benign clinical course with no recurrence or malignant transformation .
eac infections are thought to be associated with a history of repeated manipulation using the finger tips or ear picking tools .
in addition , it has been suggested that the hair follicles may serve as possible reservoirs .
eac verruca causes mechanical obstruction which can lead to pressure necrosis of the adjacent bone or conductive hearing impairments , although tympanic membrane involvement is seldom reported .
there are currently several methods to treat verruca , which include topical treatments , cryotherapy , immunotherapy , photodynamic therapy , laser therapy , curettage and cautery , radiation therapy , surgical excision and intralesional bleomycin injections .
a case report introduced surgical excision of verruca in the eac as the preferred treatment because of difficulty to apply nonsurgical treatment such cryotherapy , photodynamic therapy or laser therapy .
however , the most common complication associated with surgical excision is the possibility of scarring and subsequent eac stenosis .
intralesional bleomycin injections have been used to treat verruca on the hands , forearm , feet and face .
bleomycin was originally isolated from the fungus , streptomyces verticillus , and is frequently used as an antitumor agent .
bleomycin blocks the cell cycle at g2 , cleaves single - stranded and double - stranded dna and degrades cellular rnas ; it also forms a complex with metal ions , which reduce oxygen to free radicals .
other possible underlying mechanisms are endothelial damage , which induce tumor necrosis factor , hemorrhage of subepidermis and intraepidermis , and the appearance of apoptotic cells .
the cytotoxic effect and uptake of bleomycin is increased by mixing drug with local anesthetics because local anesthetics disrupt the cell membrane structure .
after bleomycin intralesional injection , verruca on the hands and feet has exhibited hemorrhagic black eschars which suggest adequate bleomycin infiltration .
eschar formation is thought to be caused by absolutely decreased blood flow , necrosis , and subsequent detachment of the lesion .
however , verruca on the face indicated that the verruca gradually shrank after injection , and disappeared without eschar formation .
we thought that the blood supply is decreased after injection but not completely interrupted because of rich and large dermal capillaries of the face .
incomplete blockage of blood flow may not cause hemorrhagic necrosis , eschar formation and consequential verruca detachment .
induction of endothelial and keratinocyte apoptosis by direct or indirect damage may be result in verruca shrinkage without necrosis or eschar .
its clinical course was similar to one of verruca on the face . in conclusion , we present a case of large and multiple verruca in both eacs that was successfully treated with twice intralesional bleomycin injections at one - month interval .
an intralesional bleomycin injection is simple , easy to apply without any difficulty and limitation , and had a high degree of patient satisfaction for treatment of verruca in the eac .
we propose that it may be an effective , first - line treatment of verruca in the eac . | verruca vulgaris is caused by human papillomavirus ( hpv ) infections .
verruca in the external auditory canal ( eac ) has rarely been reported . a previous case report introduced surgical excision as a treatment for verruca in the eac .
we present a case of verruca vulgaris in both eacs that was successfully treated with an intralesional bleomycin injection . a 32-year - old male patient presented with ear fullness and palpable lumps in both eacs .
both of his canals were filled with multiple pinkish , papillomatous masses .
verruca vulgaris was confirmed by skin biopsy .
an otolaryngologist referred this patient and recommended surgical excision .
however , we performed intralesional bleomycin injections for treatment .
twice intralesional bleomycin injections at one - month intervals had excellent results without recurrence , ulceration or scar formation .
this result indicates that bleomycin injections may prove to be an effective first - line treatment of verruca in the eac . |
italy has recently ratified the framework convention on tobacco control ( fctc ) , approved by world health assembly ( wha ) in 2003 , and was among first countries to promote a smoke free society implementing effective strategies for smoking prevention . in italy , as in all developed countries , cigarette smoking is the main avoidable cause of morbidity and mortality .
worldwide there are over a billion smokers , with italy having 11.2 million smokers ( about 22% of the population over age 15 ) . in italy , the number of smokers shows a decreasing trend in recent years ; in 2008 the reduction in prevalence was 1.5% from 2007 .
male italian smokers belong mainly to the 2544 age group , while females are in the 4564 range ; young smokers ( < 24 years ) number about 1.5 million .
these characteristics are different from what is observed in smoking treatments services in which patients are usually elderly .
a direct correlation between smoking ( including second - hand smoke ) and neoplastic , respiratory and cardiovascular diseases is widely demonstrated [ 68 ] .
tobacco use during pregnancy is a well - known risk factor for low birth weight or abortion ; exposure to second hand smoke is related to an increased risk of sudden infant death syndrome , otitis media , respiratory tract infections and asthma in children .
smoking - induced deaths in italy reach nearly 85 000 each year ( 25% in the age group 3565 ) , and about 15% of deaths are smoking related .
several studies have demonstrated that quitting smoking is related to a reduction of risk of illness and to an increased life span , especially in young people and also evident in the elderly .
smoking cessation measures have shown partial adherence in italy and one of the main goals of the local health prevention services is to reach maximum percentage of long lasting abstinent patients in quit smoking treatments .
therapeutic approaches are varied and evolving , therefore it is essential to enhance awareness about the factors affecting the success of such treatments .
this would enable to personalize therapies and , consequently , give a greater number of smokers the possibility of long - lasting cessation .
evaluate epidemiologic factors of patients attending stop smoking courses in a national health system ( ssn ) treatment service in rome , identifying determinants that influence cessation of cigarette smoking ; propose a suitable methodology for public health personnel to help them to improve treatment success rating .
the retrospective study ( figure 1 ) has included patients frequenting seven stop smoking courses organized from 2003 to 2005 by an ssn treatment service in the centre of rome , whose attendance was free of charge and accessible to all inhabitants who decided to participate in .
the stop smoking courses were based on a group therapy called gruppo di fumatori in trattamento treated smokers group , inspired to the five - day plan of mcfarland et al . .
this program was based on a cognitive - behavioural approach consisting of five steps ( preparedness , full immersion , maintenance , involvement , and further aid ) for a total of 10 meetings ; smokers were forced to stop abruptly ( cold turkey method ) at the third day of the course .
we included in the analysis all patients who did not withdraw before third day of the courses ; follow - up of patients was conducted directly or through the phone at the end of the courses and 3 , 6 , and 12 months later to verify smoking abstinence .
we decided to consider lost to follow - up subjects unreachable by phone after 3 attempts in different days . during former interviews socio - demographic characteristics ( gender , age , occupation , marital status , and education level ) , information on habits and smoke addiction ( sports activity , coffee use , alcohol consumption ; age of first use of cigarettes , number of cigarettes smoked per day , years of addiction , quit smoking attempts and principal reason to stop smoking ) ,
some clinical features ( weight , height , blood pressure and heart rate ) and other data ( living with other smokers or having smokers in the family , information sources about treatment services , illnesses and/or risk factors ) were collected .
furthermore two tests were administered to patients : fagerstrom test to evaluate nicotine dependence and self - efficacy test to estimate the belief in one 's own capability to stop smoking measured on a scale of 1 to 10 . the fagerstrom test based on a scale of 0 to 10
is directly related to dependence severity : 02 low , 3 - 4 medium , 5 - 6 high , 710 very high .
all anamnestic and follow - up data were collected in a microsoft excel database . a unique identifying alphanumeric code was assigned to every patient to preserve their privacy .
quit rates were evaluated 6 and 12 months after the end of the courses .
we considered early smokers patients who started smoking before the age of 15 and long lasting those who had smoked for more than 20 years .
education was divided into higher ( academic degree and high school ) and lower
body mass index ( bmi : weight / height ) was measured and patients were stratified in four groups : bmi < 20 , 20 bmi<25 , 25 bmi < 30 , and bmi 30 . the working group decided to consider confident in their capabilities to stop smoking subject with scores 7 in self - efficacy test and highly nicotine - addicted those with score 5 in fagerstrom test .
epi - info 3.3 software ( trademark of the centers for disease control and prevention ( cdc ) ) was used for statistical analysis .
student 's t - test for unpaired data was used to test statistically significant differences between heart rate averages measured at the beginning and end of the courses and differences in age between the sexes .
the influence of epidemiologic characteristics on smoking cessation in univariate and multivariate analysis was tested , calculating relative risks ( rr ) with confidence intervals of 95% ( c.i .
statistical threshold was set at.05 and evaluated with chi - square test ( with yates ' correction when needed ) .
we chose to enter into logistic regression model variables resulting in p < .15 in univariate analysis .
from a total of 147 subjects who have started frequenting stop smoking courses , 123 ( 83.7% ) attended all course meetings .
age range was between 29 and 76 years , and mean age was 53 ( 11 ) without statistical differences between the sexes .
socio - demographic and epidemiological characteristics are illustrated in table 1 , while smoking habits are shown in table 2 .
the average of courses frequency and self - efficacy test were 8/10 ( 1.8 ) and 7.4/10 ( 2.4 ) , respectively ; 64.4% of patients declared themselves
confident in quitting smoking . at the end of the courses , 81 patients ( 66% )
stopped smoking ; patients who did not abstain lowered their tobacco consumption about 40% ( 37.2% ) .
data about heart rate , measured at the beginning and end of the courses in 53 patients , showed a statistical significant mean reduction of 16 beats , tested with student 's t - test ( p < .0001 ) .
three months after the end of courses , 58% of patients were still abstinent ; after 6 and 12 months , this value decreased to 47% and 39% , respectively .
patients younger than 50 years statistically tended to continue smoking 6 months ( p = .02r.r .
95% : 1.022.52 ) after the end of the courses . a low self - confidence in the possibility of cessation of smoking ( self - efficacy test < 7 ) was significantly related to continuing tobacco consumption after 6 months ( p = .016 r.r .
95% : 1.142.99 ) ; this relation was not significant when tested after 12 months ( p = .058 ) .
low adherence to therapeutic program , defined as having attended less than 7/10 meetings , was statistically associated to maintenance of tobacco use at 6 months ( p = .006r.r .
failure in therapy was related although not significantly to being a heavy smoker ( at 6 months p = .07 r.r .
95% : 0.932.26 ) , having a smoking father ( at 12 months p = .08r.r .
analysis performed on logistic regression model confirmed the association between continuing smoking after 6 months and low frequency of courses ( p = .013 ) ( likelihood ratio 16,78p = .01 ) .
for low self - confidence , a relationship close to the upper limit for significance ( p = .052 ) was observed ; no association was confirmed at 12 months ( table 3 ) . an intermediate multivariate analysis performed to verify if course frequency effect could have had some correlations with other baseline information ( age and self efficacy ) showed no significant association . the influence of several variables on smoking cessation such as gender , occupation , age of first use of cigarettes , years of addiction , marital status , bmi , living with other smokers , education level , quit smoking attempts , sports activity , coffee use , principal reason to stop smoking , information sources about treatment services , illness and/or presence of risk factors and alcohol consumption were tested
although several studies confirm that cessation of smoking is possible even without any kind of therapeutical approach [ 16 , 17 ] , most authors have highlighted that the best rate of success in smoking cessation can be obtained through pharmacological and psychological therapies , especially when combined [ 3 , 18 , 19 ] .
remarkably , the higher percentage of abstinent patients 6 months after the end of the therapy seems attainable by administering nicotine replacement therapy together with group therapy [ 3 , 20 ] .
nevertheless these evidences , there is still a large number of patients receiving stop smoking therapies that are not able to free themselves from tobacco dependence .
for this reason in the last years factors influencing smoking cessation became the focus of many studies in order to improve success of such therapies .
our treatment service is the only national health system smoke cessation centre of a metropolitan area that counts about 127.324 inhabitants .
although this cohort is not representative of italian population , it shows the same general features of all patients attending smoking cessation services . unlike some recent reports [ 3 , 5 ] , the majority of people in our sample are women
; this peculiarity could be explained by the female preference for group therapy than other types of stop smoking approaches found also in other studies [ 20 , 22 , 23 ] . mean age of patients attending these stop smoking courses is higher than that reported by italian and global smokers statistics [ 3 , 20 , 24 ] ; this could be related to the demographic profile of the area in which the service is located , which presents an high percentage of elder ( over 65 years old ) inhabitants . regarding education level ,
official data show that the majority of italian smokers have a medium to low education level ; however patients attending italian stop smoking services usually have a higher one , as highlighted in our study [ 3 , 24 ] . in this cohort , the percentage of heavy smokers is almost twice than the general italian smoker population , but , however , overlaps other articles [ 3 , 20 ] ; the same ratio holds good with regard to the percentage of long lasting smokers ( in this cohort about 90% ) .
degree of nicotine dependence shown in this study is similar to the reports in literature [ 3 , 20 ] .
regarding patients who previously attempted to quit smoking , the evidences obtained from this study demonstrate a higher percentage versus the national data ( 67% versus 30% ) ; this corresponds to the results shown in an italian multicentric study . in our sample
the principal reason to attempt to stop smoking is related to health matters as reported by other authors .
considering percentage of abstinent patients , the results of the treatment of this study are higher than reported by other group therapy - based studies , both at 6 months [ 3 , 20 ] and 12 [ 20 , 24 ] months after the end of courses .
the lack of an objective measure to verify patients ' smoking cessation could represent a limitation ; however , in almost all the ssn smoking cessation services the standard procedure is to follow up patients by phone . on the other hand
it is important to stress the psychological approach of group therapy , that makes the smoker responsible for successful cessation and the person in charge of follow - up more confident of truthfulness of patient 's answers .
this study highlights a statistically significant risk of continuing smoking in subjects younger than 50 years .
this is confirmed by several authors [ 2729 ] , but not found by others ; a reasonable explanation could be the low importance that younger people give their health status .
the findings in literature , confirmed by the results obtained in this study , relate significantly low self - confidence in the possibility to stop smoking [ 3133 ] and low adherence to therapeutic program [ 3 , 24 , 34 ] with failure in therapy . in logistic analysis only the relation between failure in smoking treatment success and low attending frequency ( < 7 )
was confirmed ; we would like to highlight that relation with low self confidence reached a significance close to the upper limits , making us still considering it a deserving attention factor .
the differences observed in univariate and multivariate analysis results suggested us to perform an intermediate multivariate analysis in order to distinguish the importance of tested variables and to evaluate possible influence of age and self efficacy on course frequency effect .
the results of this analysis showed us a uniform effect of course frequency with respect to baseline information considered , confirming us the reliability of main results of our study .
self - reported cigarette consumption per day [ 3537 ] and degree of nicotine dependence [ 3840 ] do not show statistical correlations with the capability to stop smoking as found in other studies .
likewise , several articles have shown a direct relation between smoking cessation and gender [ 4143 ] , first cigarette use in post teenage years [ 41 , 42 , 44 ] , years of addiction , high bmi [ 34 , 45 , 46 ] , living with nonsmoker flatmates or having a smoking parent [ 4749 ] , education level [ 28 , 50 , 51 ] , previous attempt to stop smoking , marital status [ 3 , 28 , 52 ] and occupation [ 37 , 45 ] . in our country
there are few reliable data about the relation between alcohol and tobacco consumption . in our sample
this well - known association [ 48 , 53 ] is not confirmed , as reported in table 1 .
this founding is probably related to the setting in which the study was conducted ( in an area of the centre of italy ) : in fact although we observed in the last years an increase of alcohol consumption in general italian population , there are still geographical differences between northern and centre / southern regions in which we still found a lighter alcohol consumption . in the light of our results
there are some suggestions to better target stop smoking treatment services interventions : for instance , low self - confidence in the possibility to stop smoking and the low adherence to a therapeutic program could be improved enhancing psychological motivation . for
younger patients could be useful adding pharmacological therapy to the psychological one , obtaining a well known successful approach .
furthermore , information , education and communication campaigns , whose effectiveness was recently demonstrated in a study pointed on radio commercials and internet advertisements , could be focused on population of < 50 years old .
results obtained from this study highlight some factors , which should be considered when planning therapeutic approaches for smoking cessation . in the future , all smoking treatment services should investigate epidemiologic determinants affecting the cessation of cigarette smoking in patients attending their therapeutic programs to improve the effectiveness of interventions and implement the most suitable approach for the target population . | this retrospective study aims to evaluate epidemiologic characteristics of patients attending stop smoking courses , based on group therapy , testing their influence on smoking cessation in univariate and multivariate model .
a total of 123 patients were included in this study .
mean age was 53 ( 11 ) .
sixty - seven percent were women . at the end of the courses 66% of patients stopped smoking , after 12 months only 39% remained abstinent .
patients younger than 50 years statistically tended to continue smoking 6 months ( p = .02r.r .
= 1.49 , c.i .
95% : 1.062.44 ) and 12 months ( p = .03r.r . = 1.37 , c.i .
95% : 1.022.52 ) after the end of the courses . a low self - confidence in quitting smoking was significantly related to continuing tobacco consumption after 6 months ( p = .016r.r .
= 1.84 , c.i .
95% : 1.142.99 ) .
low adherence to therapeutic program was statistically associated to maintenance of tobacco use at 6 months ( p = .006r.r .
= 1.76 , c.i .
95% : 1.322.35 ) and 12 months ( p = .050r.r . = 1.45 , c.i .
95% : 1.111.88 ) .
this association was confirmed at 6 months in the analysis performed on logistic regression model ( p = .013 ) . |
the alterations in plasma lipids related to obesity that contribute to the higher incidence of cardiovascular disease in obese subjects mainly involve hypertriglyceridemia1,2 and low serum high - density lipoprotein ( hdl ) cholesterol .
increases in low - density lipoprotein ( ldl ) cholesterol levels are less frequent3,4 and have been less well investigated .
lipidic emulsions of defined composition that are made without protein can mimic the intravascular behavior of plasma lipoproteins . injected into the bloodstream , chylomicron - like or ldl - like
emulsions acquire circulating apolipoproteins that modulate the emulsion s metabolism and thus serve as a probe of the metabolic status of lipoproteins .
accumulation of ldl in the plasma leading to hypercholesterolemia is mostly due to reduced ldl plasma clearance by the ldl receptors rather than increased lipoprotein production . in a recent study that used ldl - like nanoemulsions to probe ldl metabolism , we observed that , in sedentary subjects , ldl removal from the plasma compartment was slower than in athletes , although there was no difference in ldl cholesterol between sedentary subjects and athletes.5 this suggests that the increased removal of ldl in athletes is compensated for by an increased input of lipoprotein into plasma , either by increased conversion of ldl from vldl or by increased direct ldl synthesis by the liver . to verify whether there are defects in ldl intravascular catabolism in obese subjects
, we tested the plasma clearance of an ldl - like nanoemulsion in subjects with morbid grade iii obesity ( body mass index ( bmi ) > 40 kg / m ) in comparison with control subjects ( 25 kg / m ) of normal weight .
ten obese ( three male and seven female ) and ten non - obese ( four male and six female ) subjects participated in the study . all participants were volunteers selected from the outpatient clinics of the institution .
none of them were addicted to alcohol or had diabetes mellitus , liver , renal , thyroid , inflammatory , or neoplastic disease , and none were pregnant .
the design and objectives of the study were explained to the participants , and written informed consent was obtained .
blood samples for the determination of plasma lipids and apolipoproteins were collected after a 12-h fast .
commercial enzymatic methods were used for the determination of total cholesterol ( boehinger - mannheim , penzberg , germany ) , triglycerides ( abbott laboratories ) and hdl cholesterol after chemical precipitation of apo - b - containing lipoproteins with magnesium phosphotungstate .
ldl cholesterol was calculated by the formula of friedewald.6 plasma apo ai and apo b were assayed by radial immunodiffusion ( lipo - partigen r - apoa - i and nor - partigen r - apob plates , behing , marburg , germany ) . the ldl - like nanoemulsion was prepared from a lipid mixture composed of 40 mg / mmol egg phosphatidylcholine , 20 mg /mmol cholesteryl oleate , 1 mg /mmol triolein , and 0.5 mg /mmol cholesterol purchased from sigma chemical co. ( st louis , mo ) , in addition to a mixture of [ c]cholesteryl oleate and [ h]-triglycerides purchased from amersham international ( amersham , uk ) .
emulsification of the lipids by prolonged ultrasonic irradiation in aqueous media and two - step ultracentrifugation of the crude emulsion with density adjustment by the addition of kbr in order to obtain the lde microemulsion was carried out by the method of ginsburg et al.7 as modified by maranho et al.8 the participants fasted for 12 h prior to the test at 9 am , but they were allowed two standard meals during the study at 12:30 pm and 6 pm .
the ldl - like nanoemulsion containing 70 kbq of [ c ] cholesteryl oleate and 121 kbq of [ h ] triacylglycerol at a total of 56 mg /mmol in a 100-l volume was intravenously injected in a bolus .
plasma samples were collected over 24 h ; the first sample was collected 5 minutes after the injection of the nanoemulsion , and , after that , at 1 , 2 , 4 , 6 , 8 , 12 , and 24 h after the injection .
the safety of the radioactive dose injected into the subjects was assured according to radioprotection regulations9 described elsewhere.10 the procedures for lipid extraction and the addition of scintillation solution to each blood sample for radioactive counting and determination of the plasma decay curves of the radiosotopic labels of the nanoemulsion were performed as described previously using the anacomp software.11 all recorded variables were tabulated as means sd or sem .
differences in the plasma lipids were evaluated using the mann whitney test , and differences with p < 0.05 were considered statistically significant for all comparisons .
the safety of the radioactive dose intravenously injected into the patients was assured according to the regulations of the international commission on radiological protection.9 the equivalent dose derived from the injected dose for each experiment was 0.1233 msv , well below the 20-msv annual limit for the intake of radionuclides .
table 1 shows the individual physical characteristics and the plasma lipid and apolipoprotein profiles of the obese subjects and the controls .
obese subjects had ldl cholesterol levels similar to those of the controls , but the hdl cholesterol values were lower . on the other hand , fasting triglyceride levels were greater in the obese subjects ( p= 0.019 ) than in the controls .
it is apparent that the curves obtained from the obese subjects did not differ from those of the non - obese controls .
in fact , the calculated cholesteryl ester fcr of the morbid obese subjects was equal to that of the controls .
the triglyceride fcr was lower in the morbid obese subjects than in the non - obese controls . in the non - obese controls ,
, there was a trend ( although not statistically significant ) of a greater triglyceride fcr in comparison to the cholesteryl ester fcr .
in this study , in a group of morbid obese subjects , the removal of cholesteryl esters of an ldl - like nanoemulsion from plasma was similar to that observed in control subjects , but triglyceride removal was slower .
injected into the blood stream , the ldl - like nanoemulsion acquires several circulating apolipoproteins such as apo cii and apo e. apo cii is the co - factor for lipoprotein lipase and can eventually stimulate the enzyme to break down the residual triglycerides present in this nanoemulsion . on the other hand ,
apo e is recognized by the receptors that remove ldl from circulation.12 in several studies from our group , a triglyceride - rich emulsion labeled with radioactive triglycerides and cholesteryl esters has been used to test the chylomicron metabolic pathway in several disorders that affect plasma lipid metabolism.1319 this pathway is also common to very - low - density lipoproteins ( vldl ) that ( similarly to chylomicrons ) undergo degradation of their triglyceride content by lipoprotein lipase using apo cii as a co - factor . in obese subjects ,
the removal of the triglycerides of the chylomicron - like emulsion from plasma was similar to that in the non - obese controls , but the cholesteryl ester removal was slower.20 this finding suggests that , although the lipolysis process occurs normally in the obese , there is difficulty in removing the remnants from circulation , a defect that is associated with atherosclerosis development . in the correlation study on obese subjects that was subsequently performed ,
a negative correlation between cholesteryl ester removal ( i.e. , remnant removal ) and bmi was found ; in contrast , lipolysis correlated positively with bmi.21 one can hypothesize that the excess insulin in the plasma of obese subjects stimulates lipolysis , whereas their obesity inhibits the mechanisms of remnant removal , in which ldl receptors are also involved .
however , when the morbidly obese were compared with obese subjects , the former showed diminished lipolysis , indicating that additional defects appear in individuals with extremely high bmis .
another aspect is alterations in glucagon - like peptide 1 , which modifies the postprandial rise in triglyceride concentration , whose effects are not completely understood in morbid obesity , despite the functions already described.22,23 yet another aspect which may be mentioned is the fact that treatment of obese patients with the cholesterol removing agent sibutramine does not appear to correct the hypertension normally associated to obesity.24 whereas the chylomicron - like emulsion probes the lipolysis and remnant removal of triglyceride - rich lipoproteins , the ldl - like nanoemulsion probes the function of the mechanisms that remove ldl from circulation .
the fact that the removal of the ldl - like nanoemulsion was normal in the morbidly obese indicates that those mechanisms are not altered in these subjects .
this was confirmed by the fact that ldl cholesterol and apolipoprotein b concentrations were both normal , although different removal rates associated with different synthesis rates might have resulted in equal ldl concentrations , a possibility that we explored in this study regarding the removal rates .
the fact that , in both control and obese subjects , the triglycerides of the ldl - like emulsion were removed faster than the cholesteryl ester indicates that lipolysis also occurs in those particles as it does in chylomicron - like emulsions .
the finding that the triglycerides of the ldl - like nanoemulsion were removed more slowly in the morbidly obese than in the controls is noteworthy .
there are significant structural differences between ldl - like particles and chylomicron - like emulsion particles . while in the chylomicron - like emulsion , the triglyceride component is prevalent , constituting up to 70% of the particle composition , in ldl - like particles , the triglyceride content is rather residual . on the other hand ,
the particle diameter of the chylomicron - like emulsion is two to four times greater than that of the ldl - like preparation .
nonetheless , both preparations showed slower triglyceride removal in the morbidly obese subjects ( but not in the obese subjects ) , as compared with the controls . | introduction : obesity increases triglyceride levels and decreases high - density lipoprotein concentrations in plasma . artificial emulsions resembling lipidic plasma lipoprotein structures
have been used to evaluate low - density lipoprotein metabolism . in grade iii obesity , low density lipoprotein metabolism is poorly understood.objective:to evaluate the kinetics with which a cholesterol - rich emulsion ( called a low - density emulsion ) binds to low - density lipoprotein receptors in a group of patients with grade iii obesity by the fractional clearance rate.methods:a low - density emulsion was labeled with [ 14c]-cholesterol ester and [ 3h]-triglycerides and injected intravenously into ten normolipidemic non - diabetic patients with grade iii obesity [ body mass index
higher than 40 kg / m2 ] and into ten non - obese healthy controls .
blood samples were collected over 24 hours to determine the plasma decay curve and to calculate the fractional clearance rate.results:there was no difference regarding plasma levels of total cholesterol or low - density lipoprotein cholesterol between the two groups .
the fractional clearance rate of triglycerides was 0.086 0.044 in the obese group and 0.122 0.026 in the controls ( p = 0.040 ) , and the fractional clearance rate of cholesterol ester ( h1 ) was 0.052 0.021 in the obese subjects and 0.058 0.015 ( p = 0.971 ) in the controls.conclusion:grade iii obese subjects exhibited normal low - density lipoprotein removal from plasma as tested by the nanoemulsion method , but triglyceride removal was slower . |
arginine vasopressin ( avp ) is an antidiuretic hormone produced in the hypothalamus and stored in the posterior pituitary gland 1 .
avp acts principally on renal collecting tubules to increase water reabsorption through stimulating v2 vasopressin receptors and also has direct roles in regulating cardiovascular function , such as increasing peripheral vascular resistance through v1 vasopressin receptors 2 .
central diabetes insipidus ( cdi ) is a rare endocrine disorder that results from a deficiency of avp secretion , and is characterized by a dilute polyuria , plasma hyperosmolality , and relative hypovolemia .
cdi is treated by physiological replacement therapy with the synthetic avp analogue desmopressin ( 1deamino8davp ) , a selective v2 vasopressin receptor agonist , to maintain water volume in the body and normal urine output 3 , 4 .
the maintenance dosage of desmopressin is individually based , with the effective minimum dose for japanese patients being approximately 515 g / day or 120300 g / day via intranasal or orally ( disintegrating tablet ) administration , respectively 5 .
heart failure ( hf ) is a clinical syndrome characterized by the inability of the heart to supply adequate blood flow to peripheral tissues and organs and can be caused by a variety of structural or functional cardiac disorder 6 , 7 .
many symptoms of hf , including ankle swelling and pulmonary edema , can result from sodium and water retention and resolve quickly with diuretic therapy . although hf is not an uncommon condition ,
we report herein a rare case of a patient with left ventricular ( lv ) dysfunction with no history of symptomatic hf who developed cdi .
a 63yearold japanese man was admitted to our hospital in november 2013 because of thirst and polyuria .
the patient had no smoking habit and would only drink a little alcohol several times a month but never heavily .
the patient had been treated with antidepressive agents including paroxetine for depression from 53 to 61 years of age .
the patient presented with asymptomatic atrial fibrillation ( af ) 8 at a routine examination in april 2009 ( at 59 years of age ) and visited a local hospital .
an electrocardiogram ( ecg ) showed af with a heart rate ( hr ) of 43 beats / min .
a chest xray showed an enlarged heart with a cardiothoracic ratio ( ctr ) of 59% without pulmonary edema , and an echocardiogram showed globally reduced lv wall motion with a lv ejection fraction ( lvef ) of 50% ( fig .
his clinical course was uneventful until july 2013 , at which point the patient acutely developed thirst and polyuria , drank 4 l of water , and began to void 20 times throughout the day .
he also experienced a relapse of his depressive symptoms with appetite and body weight ( bw ) loss in october 2013 and visited a psychiatrist .
thereafter , the patient was referred to our hospital because of his persistent thirst and polyuria and was admitted for further detailed examinations . clinical course .
the left ventricular enddiastolic diameter ( lvdd ) , left ventricular endsystolic diameter ( lvds ) , left atrial diameter ( lad ) , inferior vena cava diameter during inspiration and expiration ( iivc and eivc , respectively ) , and fractional shortening ( fs ) were measured by echocardiography . * the left ventricular ejection fraction ( lvef ) was calculated using the modified simpson 's rule , except for the lvef in january 2015 , which was measured using left ventriculography ( simpson 's method).intranasal desmopressin ( 10 g ) was administered once during the desmopressin stimulation test .
the patient experienced a 4kg body weight loss along with resolution of heart failure ( hf ) symptoms including fatigue and lowerextremity edema following 4 days of oral azosemide ( 30 mg / day ) .
bnp , brain natriuretic peptide ; cre , creatinine ; ctr , cardiothoracic ratio ; hr , heart rate ; pra , plasma renin activity ; sbp , systolic blood pressure ; uosm , urinary osmolality at physical examination , the patient was 189 cm tall and weighed 73 kg , and his body temperature , blood pressure ( bp ) , and oxygen saturation by pulse oximeter ( spo2 ) on room air were 36.4c , 104/60 mmhg , and 99% , respectively .
a mild systolic regurgitant murmur was audible , but no jugular vein swelling , chest rale , or peripheral edema was detected .
the volume of urine ( 3600 ml / day ) throughout 1 day was large .
laboratory findings ( table 1 ) showed high levels of serum sodium and chloride , high plasma osmolality ( posm ) and plasma renin activity ( pra ) , low urinary osmolality ( uosm ) , and undetectable plasma avp .
his thyroid function was normal , while levels of plasma brain natriuretic peptide ( bnp ) were high .
a desmopressin stimulation test showed increased uosm along with reduced volume of urine and increased levels of urinary aquaporin2 ( table 2 ) 11 .
dynamic tests for secretion of anterior pituitary hormones showed normal releases of all of prolactin , thyroidstimulating hormone , growth hormone , adrenocorticotropic hormone , cortisol , luteinizing hormone , and folliclestimulating hormone .
magnetic resonance imaging of the brain showed no morphological abnormalities , but the physiological highintensity signal , which would be expected in the posterior pituitary on t1weighted plane images , was undetectable ( fig .
tests for autoantibodies against the anterior and posterior lobes of the pituitary gland were negative .
laboratory findings on admission in november 2013 blood samples were taken in the morning with the patient in the supine position . the reference range for each parameter is shown in parentheses .
the reference range for plasma arginine vasopressin was calculated from the patient 's plasma osmolality , based on the published approximate calculation formula 9 .
desmopressin stimulation test in november 2013 ( day 3 of admission ) the patient refrained from eating and drinking 2 h before and for the duration of the test .
urine samples were taken at 0 ( just before ) , 30 , 60 , 90 , and 120 min after desmopressin ( 10 g ) was administered nasally . as to the patient 's cardiac function ,
an echocardiogram showed globally reduced wall motion ( lvef 46% ) accompanied by an akinetic region at the posterior wall , with normal wall thickness , mild left atrial dilatation , and mild mitral regurgitation .
electrocardiogram records . twelvelead resting electrocardiograms from november 2013 ( time of admission ) ( a ) , june 2014 ( b ) , and october 2015 ( c ) .
posteroanterior chest xrays performed with the patient in a standing position in november 2013 ( time of admission ) ( a ) , june 2014 ( b ) , and october 2015 ( c ) .
the patient started treatment for cdi with intranasal desmopressin at 5 g / day on day 6 of admission and experienced some decreases in thirst , polydipsia , and dilute polyuria ( fig .
the dose of desmopressin was titrated to 7.5 g / day on day 10 , and thereafter , he had almost complete improvements in his cdi symptoms .
his urinary volume and osmolality throughout day 12 of admission were 1100 ml / day and 784 mosm / kg , respectively , and his urinary frequency was 5 times / day .
an echocardiogram performed on day 13 of admission showed cardiac parameters comparable to those at the time of admission . to avoid dehydration or water intoxication and heart failure ,
the patient was instructed to ingest water freely for thirst in the range of 1500 ml / day and to take sodium < 7 g / day and was discharged 14 days after admission .
the patient experienced improvements in his depressive symptoms , regained his appetite and bw , and discontinued antidepressive drugs on the basis of his psychiatrist 's judgment in december 2013 ( fig .
a chest xray showed an enlarged heart with a ctr of 54% , and ecg showed chronic af with a hr of 58 beats / min , both of which were comparable to those performed in november 2013 before therapy with desmopressin .
myocardial scintigraphy with thallium 201 showed decreased uptake in the posterior wall of the left ventricle without redistribution .
left ventriculography showed a relatively preserved global lv wall motion with a lvef of 62% , and an akinetic region in the inferiortoposterior wall of the left ventricle was found .
his endodiastolic volume , endosystolic volume , stroke volume , and cardiac output were 222 , 83 , 138 ml , and 12.7 l / min , respectively . as both the patient 's bp and hr were relatively low , he did not receive medication for lv dysfunction , such as reninangiotensinaldosterone system inhibitors or betablockers 6 , 7 .
the patient came to consume slightly larger amounts of salt in spring 2014 by eating japanese pickles .
in june of the same year , the patient visited our hospital after several days of fatigue , lowerextremity edema , and a 4kg bw gain .
his bw , body temperature , bp , and spo2 on room air were 83 kg , 36.2c , 116/80 mmhg , and 97% , respectively .
no chest rales or heart murmurs , except for preexistent mild systolic regurgitant murmur , were detected , but bilateral pedal edema was observed .
laboratory findings revealed a normal complete blood count , normal serum electrolytes , and normal renal , liver , and thyroid functions , but his plasma bnp level ( 484.0 pg / ml ) was higher than that measured previously ( fig .
xray detected cardiomegaly with a ctr of 62% and bilateral mild pulmonary effusion ( fig .
he was diagnosed with hf and started oral azosemide at 30 mg / day under a continued 7.5 g / day of intranasal desmopressin .
the patient experienced rapid improvements in his fatigue and lowerextremity edema with sufficient diuresis , and his bw returned to 79 kg within 4 days ( fig .
he underwent treatment with strict dietary salt restriction ( 7 g / day ) and adequate water intake ( 1500 ml / day ) without resuming diuretics in combination with desmopressin ( 7.5 g / day ) replacement for cdi .
his subsequent clinical course during desmopressin replacement was uneventful , without recurrence of hf ( figs 1 , 2c and 3c ) .
the patient with a 4year duration of mild lv dysfunction but no history of symptomatic hf developed cdi and started treatment with a minimum fixed dose of desmopressin ( 7.5 g / day ) that was necessary to relieve his thirst and dilute polyuria .
he did not exhibit hyponatremia or low pra , but 7 months after the same desmopressin replacement , he exhibited symptomatic hf without verifiable causes except for excess salt intake , which resolved rapidly during several days of oral diuretics under continued desmopressin therapy . following strict restriction of dietary salt and water
the primary stimulus for avp secretion under normal physiological conditions is an increase in posm .
conversely , an excessive administration of desmopressin can cause decreased posm and hyponatremia in association with excessive water retention 3 , 4 . in the present case , the patient never exhibited hyponatremia during the course of the minimum effective dose of desmopressin therapy for his cdi ( fig .
1 ) . additionally , pra , which would decrease if the extracellular fluid volume increases 12 , was not low during that period .
these findings suggest that excessive retention of water in the body was not clinically detected by measuring electrolytes , posm , or pra during desmopressin treatment in our patient .
few studies have investigated the effects of avp deficiency or desmopressin replacement on cardiac performance in patients with abnormal cardiac function .
however , a study on patients with cdi without a cardiac disorder suggested that avp deficiency induces reversible changes in cardiac function , such as increased hr and lv contractility in association with relative hypovolemia 13 .
that study also suggested that avp deficiency induces subclinically altered lv diastolic function that persists even after treatment with adequate doses of desmopressin . in the present case , the echocardiogram systolic parameters including lvef and fractional shortening were similar before the development of cdi and after desmopressin therapy ( fig .
1 ) . however , other cardiac parameters such as the inferior vena cava diameter 14 and plasma levels of bnp 15 were large or high especially after commencement of desmopressin therapy and before strict dietary sodium restriction , compared with those during the other period
. taken together , these findings suggest that our patient probably became more susceptible to hf after he developed cdi and underwent the desmopressin therapy than had been the case previously .
the patient had chronic af with bradycardia and showed echocardiogram findings of normal lv diameter and wall thickness , mild left atrial dilatation , and mild mitral valve regurgitation , all of which remained almost unchanged during the course of his lv dysfunction > 6 years ( fig .
myocardial scintigraphy with thallium 201 showed decreased uptake in the posterior wall of the left ventricle , and a left ventriculography showed an akinetic region at that area in the absence of coronary artery lesion . based on these results , although his mitral valve regurgitation or af might have contributed in part to his lv dysfunction 2 , the major etiology of the patient 's lv dysfunction remains unclear .
a careful check of cardiac function along with the course of desmopressin therapy for cdi was required in this case .
central diabetes insipidus can be caused by the destruction or degeneration of neurons that originate in the supraoptic and paraventricular nuclei of the hypothalamus .
known causes of these lesions include tumors , local inflammatory , autoimmune or vascular diseases , trauma , and surgery , but many cdi cases are of unknown cause 1 .
the present patient developed acute thirst and polyuria due to avp deficiency without verifiable causal factors ( fig .
t1weighted sagittal magnetic resonance imaging ( a , plane ; b , gadoliniumenhanced images ) showing no morphological abnormalities in the hypothalamus , hypophyseal stalk , or pituitary gland . however , the physiological highintensity signal , which would be expected in the posterior pituitary gland on the plane image ( arrow ) , was not detected . in conclusion
, we reported a patient with cdi and cardiac dysfunction who developed hf during treatment with a minimum effective dose of desmopressin replacement .
this is a single case study and thus does not provide a specific therapeutic proposal , including dosage calculation for desmopressin , on the management of cdi associated with cardiac dysfunction
. however , this case suggests that patients with cardiac dysfunction may become more susceptible to hf after they develop cdi and undergo desmopressin therapy than previously .
therefore , to prevent hf in patients with cdi accompanied by abnormal cardiac function , it is essential to control sodium and water intake using the effective minimum dose of desmopressin .
| key clinical messagecentral diabetes insipidus ( cdi ) results from a deficiency of arginine vasopressin ( avp ) secretion .
it is treated by replacement therapy with the synthetic avp analogue desmopressin . to prevent heart failure in patients with cdi accompanied by cardiac dysfunction ,
controlling sodium and water intake is essential , using the minimum effective dose of desmopressin . |
carotid atherosclerosis is a progressive , multifactorial artery disease , associated with high risks of morbidity and mortality .
several underlying environmental and modifiable factors , such as increased , elevated systolic blood pressure ( sbp ) , dyslipidemia , smoking , and increased body mass index ( bmi ) , play a significant role in the progression of the atherogenic process .
carotid intima - media thickness ( imt ) and plaque assessment by cervical ultrasonography is a noninvasive , feasible , and accurate method for detecting asymptomatic carotid atherosclerosis .
it is also largely used for risk assessment in primary prevention and in clinical trials as a surrogate endpoint for cardiovascular and cerebral events .
both imt and total plaque area ( tpa ) are associated with an increased risk of ischemic stroke and coronary heart disease [ 3 , 4 ] .
it is well established that the presence of carotid plaques represents advanced carotid atherosclerosis , often symptomatic , whereas imt can be assessed in the absence of focal plaques and symptoms [ 4 , 5 ] .
altered calcium homeostasis , including vitamin d deficiency , was recently associated with subclinical atherosclerosis and low bone mineral density , which may predict risk of stroke in women . though the main role of vitamin d is the maintenance of mineral homeostasis and bone health , recent large epidemiological studies have linked vitamin d insufficiency to cardiovascular diseases , infections , and even cancer [ 911 ] .
furthermore , low serum levels of 25-hydroxyvitamin d ( 25(oh)d ) , the metabolite which is used to assess a subject 's vitamin d status , are associated with insulin resistance and metabolic syndrome [ 13 , 14 ] . in the fourth survey of population - based troms study in north norway in 1994 - 1995 , ultrasonography of the right carotid artery and measurements of serum 25(oh)d and total calcium
were performed in a large subgroup , thus allowing us to evaluate the potential relationship between vitamin d status and both imt and plaques .
the troms study is a population - based health survey , which was performed for the first time in 1974 . in the fourth survey of the troms study in 1994 - 1995 , all men and women at age > 24 years and living in the municipality of troms , north norway ,
all subjects aged 5574 years and random 510% samples of the other age groups were invited to a second visit for a more extensive examination which included carotid ultrasound examination .
body mass index ( bmi ) was defined as weight ( kg ) divided by squared height ( m ) .
blood pressure and heart rate were measured with an automatic device ( dinamap vital signs monitor 1846 , critikon inc .
, tampa , fl ) and the mean measurement of the second and the third measurements was used in the statistical analysis .
serum levels of 25(oh)d were analysed by immunometry ( electrochemiluminescence immunoassay ) , using an automated clinical chemistry analyser ( modular e170 ; roche diagnostics ) . according to the manufacturer
, the assay has a total coefficient of variation ( cv ) 7.8% as judged at any of the three different concentrations ( 48.6 , 73.8 and 177.0 nmol / l ) .
nmol / l and a population - based reference range was 27.7107.0 nmol / l for adults as provided by the manufacturer .
, it has recently been described that smokers had 1520% higher serum 25(oh)d than that of nonsmokers .
however , the same pattern was not observed when using other immunological and liquid - chromatography mass spectrometry methods .
this discrepancy is still lacking an explanation , and to avoid this bias , we have included only nonsmokers in our study cohort .
serum total cholesterol was analyzed by enzymatic colorimetric methods ( chod - pap ; boehringer mannheim ) .
the determination of serum calcium was performed on hitachi 917 with reagents from boehringer mannheim , reference range 2.202.60 mmol / l and cv is 2% .
lipid levels were measured twice with an interval of 4 to 12 weeks , and the averages of these values were used in the analysis .
information about smoking habits , angina pectoris , previous myocardial infarction , and stroke was collected from self - administered questionnaires .
the examination of the right carotid artery was performed in the supine position with a high - resolution acuson 128xp/10 art upgraded scanner ( mountain view , ca ) equipped with a linear transducer with 7 mhz in b mode and 5 mhz in pulsed - doppler mode .
imt was measured in 10 mm segments in three locations of the carotid artery : the near and far wall of the common carotid artery ( cca ) and the far wall ( fw ) of the bifurcation .
the loss of parallel configuration of the near and far walls of the cca served as a reference point for the start of the carotid bifurcation .
the ultrasonic images were analyzed offline with a computerized technique for automatic ultrasonic image analysis .
plaques were included in the measurements of imt if they were located in areas predefined for imt registration .
a plaque was defined as a localized protrusion into the vessel lumen with thickening of the vessel wall of > 50% compared to the adjacent imt .
six locations of the carotid artery were examined for plaque presence , the far and near walls of the cca , the bifurcation ( bulb ) , and the ica .
continuous variables are presented as mean standard deviation ( sd ) , unless otherwise stated .
all continuous variables were standardized prior to inclusion in the linear and logistic regression analyses .
the distribution of all variables was determined by measuring skewness and kurtosis and assessments of normal q - q plots of standardized variables .
analyses of the scatter plots and linear regression model were used to assess the relation between serum 25(oh)d and the measurements of carotid atherosclerosis ( mean imt , tpa , and plaques ) .
strata of mean imt ( quartiles ) and tpa ( no plaques or tertiles of tpa ) were created .
a multinomial logistic regression model was applied to assess the effect of serum 25(oh)d and total calcium across strata of mean imt ( using first quartile as a reference ) and tpa ( using no plaques as a reference group ) .
continuous variables : ( age , bmi , sbp , total cholesterol ) and categorical variable season ( summer , fall , winter and spring ) of vitamin d sampling were included in the respective regression models as covariates .
nmol / l , 30.049.9 nmol / l , 50.074.9 nmol / l , 75.0100.0 nmol / l , and > 100.0
nmol / l ) . using the cutoff of the highest mean imt quartile as a clinical endpoint and the 25(oh)d groups as predictors ,
the logistic regression was applied , adjusted for age , bmi , sbp , season for vitamin d sampling , total cholesterol , and smoking status .
all tests were done two sided , and a p value < 0.05 was considered statistically significant .
the study was approved by the regional committee for medical and health research ethics , the data inspectorate , and the norwegian directorate of health .
ultrasound examination of the right carotid artery was performed in 6727 subjects ( 77% of the eligible ) and in 6230 of those serum 25(oh)d measurements were available .
after smokers were excluded , 1994 men and 2200 women were included in the final analysis .
there were no linear associations between serum 25(oh)d and either mean imt or tpa , when adjusted for age , bmi , systolic blood pressure , total cholesterol , and season of blood sampling ( tables 2 and 3 ) . to study the threshold effects , we compared the ors , across quartiles of imt and tertiles of tpa , with the lowest quartile of imt and the no plaque group as reference groups , respectively . in males , a 1 sd increase in serum 25(oh)d was associated with a 30% increased odds for having imt > 1.00 mm , compared to the reference group ( table 4 ) .
however , a 1 sd increase in serum 25(oh)d was associated with a 15% higher odds of having tpa > 18.07 mm versus the absence of plaques ( table 4 ) . in crude analyses ,
the increase in total calcium was associated with the increased odds being in higher levels of mean imt and tpa ( table 5 ) . when adjusted for age , bmi , sbp , and total cholesterol , neither of these associations was significant ( table 5 ) . the logistic regression analysis with 25(oh)d groups as the predictor variable and the sex - specified highest quartile of mean imt as an outcome variable
was performed in the whole population , without the exclusion of smokers as well as in nonsmokers .
neither of the 25(oh)d groups turned out to be a significant predictor of the highest quartile of mean imt in both genders in the whole population , whereas male nonsmokers with serum 25(oh)d > 100 nmol / l were at 77% higher risk to have mean imt > 1.00 mm , compared to those with serum 25(oh)d within the range 75100 nmol / l , but not statistically significant , table 6 .
in our large subgroup of non - smoking general population , men had a significantly higher mean imt compared to women .
impaired calcium homeostasis was not found to have a significant association with mean imt and tpa .
an increase in serum 25(oh)d was independently associated with increased odds of being in the highest quartile of mean imt > 1.00 mm in males and tpa > 18.07 mm in females .
no associations were observed between total calcium and mean imt or tpa . when smokers were included in our cohort , serum 25(oh)d was no longer a significant predictor of higher mean imt in either gender .
male gender , increased bmi , and smoking status are well - established risk factors of carotid stenosis ( 1 , 2 ) , and our finding of higher imt in men supports the results in other studies . recently , a significant contributing impact of deranged calcium homeostasis with decreased serum 25(oh)d and high calcium - phosphorus product has been reported in the observational studies [ 6 , 11 ] .
there is also some evidence that the active vitamin d metabolite , 1,25 dihydroxyvitamin d ( 1,25(oh)2d ) , might exert beneficial physiological effects in both vascular smooth muscle cells and the vascular endothelium . in an observational study by al mheid et al . on 554 healthy volunteers , lower levels of serum 25(oh)d
were associated with vascular stiffness and endothelial function . however , to our knowledge , none of the randomised trials have supported any causal association between vitamin d and arterial disease . in a study by stricker et al .
, 62 patients with peripheral artery disease without vitamin d deficiency received either a single dose of 100,000 iu cholecalciferol or placebo in an observation period of 1 month .
no influence of vitamin d supplementation was demonstrated on either cardiovascular surrogate parameters ( blood pressure , aortic stiffness , and endothelial function ) or parameters of thrombosis ( markers of thrombin generation and markers for fibrinolysis and platelet activation ) .
we have previously reported an association between the thrombogram parameters and serum levels of 25(oh)d . however , similarly to the study by stricker et al .
inconsistent results on vitamin d and peripheral atherosclerosis burden were also demonstrated in the disease state .
thus , in 390 diabetic patients , the hypovitaminosis d ( defined as serum 25(oh)d < 15 nmol / l ) was associated with greater imt . whereas , in a study by yiu et al .
, daily oral supplementation of 5000 iu vitamin d3 was given to 100 patients with type ii diabetes during a 12-week period , and no improvement in vascular function was seen at the end of the study .
our study is , to our knowledge , the largest observational study on the associations between serum 25(oh)d and markers of carotid atherosclerosis .
no convincing associations were observed between serum 25(oh)d and intima - media thickness or plaques in a large subgroup of nonsmokers .
there is accumulating evidence that insufficient vitamin d status has adverse effects on the metabolic profile and may be associated with the severity of arterial disease .
the potential role of vitamin d in atherogenesis , if any , is not completely known .
it has also been speculated that vitamin d may possess some antithrombotic propensities , although no effects of vitamin d supplements were found on the markers of thrombosis [ 21 , 22 ] . in animal models ,
oral supplementation with vitamin d reduced the formation of atherosclerotic plaques by the suppression of t lymphocytes .
moreover , in a study by pilz et al . , low serum 25(oh)d and 1,25(oh)2d were independent predictors of fatal stroke in more than 3000 subjects who were routinely referred to coronary angiography .
one of the possible explanations may be the existence of strong confounders that could either distort the association between vitamin d and atherosclerosis towards or away from the null hypothesis .
alternatively , in studies where the regression analysis is adjusted for smoking status , smoking could still be the nuisance variable that distorts the association between exposure ( serum 25(oh)d levels ) and outcome ( increased imt and/or plaques ) and either magnifies or decreases the statistical associations . in our study , we excluded all smokers and created separate statistical models for vitamin d and total calcium , thus combating powerful confounders .
our findings of possible harmful effects of vitamin d on imt and plaques in general non - smoking population are to our knowledge novel .
even though the findings should be interpreted with caution , a significant 30% higher risk of having mean imt in the fourth quartile versus first quartile per 1 sd increase in 25(oh)d in males is hard to ignore .
moreover , the serum 25(oh)d concentrations > 100.0 nmol / l might be potentially harmful towards the mean thickness of intima media in non - smoking males .
noteworthy , zittermann et al . reported a significant adverse effect of an increased serum 25(oh)d ( > 100
the authors have demonstrated the u - shaped unadjusted association between preoperatively measured 25(oh)d and composite adverse outcome in patients scheduled for cardiac surgery .
this recent finding and our results of a possible harmful impact of increased 25(oh)d definitely require further investigations and confirmative studies .
first of all , smoking status was self - reported ; thus biased self - reporting could have affected the inclusion cohort . secondly , we measured imt and plaques only in the right carotid artery , whereas measurements in both carotid arteries might have been more representative . and finally , we did not have the opportunity to adjust for serum parathyroid hormone ( pth ) , vitamin d binding protein ( dbp ) , and circulating 1,25(oh)2d since the measurements of serum pth were not available for the majority of our study participants and we did not measure dbp in our study .
another major shortcoming is that we were unable to measure the circulating 1,25(oh)2d levels , as in previous studies an inverse association between 1,25(oh)2d and coronary calcification was demonstrated . on the other hand , we did include a substantial amount of subjects , and we included the analysis of total calcium and eliminated the major confounder smoking .
we also adjusted the measurements for age , bmi , and total cholesterol , and the results of our large cross - sectional study are confirmative to previous reports [ 2527 ] .
in conclusion , we demonstrated no consistent association between serum 25(oh)d and mean imt and tpa in a large group of non - smoking men and women .
however , increment in serum 25(oh)d was associated with the elevated risk of being in the highest quartile of mean imt in males and of having the largest tpa in women in our fairly vitamin d sufficient population .
the clinical impact of vitamin d status for carotid artery disease prevention is most likely scanty , and potential clinical trials on subjects without vitamin d deficiency should be performed with caution . | objective .
altered calcium homeostasis has been linked to increased intima - media thickness ( imt ) and plaques .
we aimed to investigate whether serum 25-hydroxyvitamin d ( 25(oh)d ) and serum calcium are associated with imt and plaques in nonsmoking population .
methods .
ultrasound of the right carotid artery with the measurements of imt and plaques was performed in 4194 nonsmoking subjects with available measurements of serum 25(oh)d and total calcium .
linear regression was applied to study the linear relationships between variables .
multinomial logistic regression was used to evaluate predictors of increased imt and total plaque area ( tpa ) , adjusted for age , body mass index , systolic blood pressure , and total cholesterol .
results .
there was no significant linear relationship between mean imt , tpa , and either serum 25(oh)d or total serum calcium .
one sd increase in serum 25(oh)d was independently associated with increased odds of being in the highest quartile of imt in men ( or 1.30 , 95% ci 1.12 , 1.51 ) . in women ,
1 sd increase in serum 25(oh)d was independently associated with increased risk of being in the upper tertile of tpa ( or 1.15 , 95% ci 1.01 , 1.33 ) .
conclusions .
impaired calcium homeostasis has no consistent association with mean imt and tpa ; however , increased serum 25(oh)d may predict subclinical atherosclerosis in nonsmokers . |
enzymatic catalysis , defined as the degree
to which an enzyme is
able to accelerate the rate of a given chemical reaction , remains to be fully understood .
enzymes are able
to achieve rate enhancements in the range of 1010 in comparison to uncatalyzed reactions , exceeding those of any artificial catalysts by many orders
of magnitude .
since the theory of transition
state stabilization was introduced by linus pauling , enzymatic catalysis has been historically defined in terms
of the ability of an enzyme to accommodate the electrostatic and geometric
configuration of a chemical transition state . according to this theory , the rate enhancement of an enzyme - catalyzed
reaction is proportional to the free energy difference between the
enzymatic and unbound transition state , which is of course a truism .
the real question is what is the physical
origin of that free energy difference . over the course of an
enzymatic cycle
, the protein bulk of an enzyme
can sample a myriad of conformations resulting
in a complex hierarchy of dynamic transitions over a wide range of
time scales .
one of the most widely studied
enzymes with regard to the impact of protein dynamics on catalysis
occurring on a time scale of microseconds to milliseconds is dihydrofolate
reductase ( dhfr ) .
this enzyme is essential for
maintenance of the proper intracellular concentration of tetrahydrofolate ,
a coenzyme involved in the biosynthesis of purines , pyrimidines , and
some amino acids .
dhfr catalyzes the
reduction of dihydrofolate to tetrahydrofolate via a protonation step
and a hydride transfer .
protonation of the n5 atom of dihydrofolate
occurs by way of solvent exchange , whereas hydride transfer from the
nicotinamide ring of the cofactor nicotinamide adenine dinucleotide
phosphate ( nadph ) to the c6 position of the substrate is directly
catalyzed by the enzyme . in escherichia coli dhfr ( ecdhfr )
, the hydride
transfer step is mediated by thermally averaged equilibrium fluctuations
of the enzyme that result in a small population
of reactive conformations .
several evolutionarily
conserved residues distal from the active site of ecdhfr have been
implicated as key loci in the motions allowing for hydride transfer .
mutation of these residues leads to a decrease
in the catalytic rate by altering the probabilistic sampling of distinct
enzymatic conformations .
these ecdhfr
motions are slow compared to the time scale of chemical transformation
( femtoseconds ) , and thus can only impact the catalyzed reaction in
a statistical manner .
in contrast , it
is possible that protein dynamics occurring on the femtosecond time
scale , which is the same time scale as chemical bond vibrations , can
dynamically couple to enzymatic barrier crossing . in this manuscript
, we will show that this
is obtained in human dhfr ( hsdhfr ) .
these fast protein motions ,
termed promoting vibrations ( pvs ) ,
are types of nonequilibrium density fluctuations that propagate through
specific structures within a protein .
these motions act to dynamically modulate both the width and height
of the chemical barrier , resulting in an increase in reaction rate .
although pvs have been identified in the reaction coordinates of
several enzymes , fast enzyme dynamics have been shown to have no effect on hydride
transfer catalyzed by ecdhfr .
furthermore ,
the network of coupled protein dynamics of ecdhfr is incapable of
sustaining dynamic correlations between fast vibrational motions at
distances greater than 46 .
this characteristic of the dynamic landscape of ecdhfr obviates
the possibility of the incorporation of fast motions of the protein
bulk in the ecdhfr reaction coordinate , and previous work in our group confirms the absence of a pv in this
enzyme .
recently , interest has
developed regarding the question of how
evolution has altered the catalytic and dynamic properties of dhfr .
a study of hsdhfr conducted by wright and co - workers revealed that
the human enzyme exhibits a dynamic landscape that is highly divergent
from that of ecdhfr .
this effect was
primarily observed in the dynamics of specific mobile loops of the
enzyme , which exhibit smaller scale fluctuations in hsdhfr compared
to those in ecdhfr , which is due to increased flexibility resulting
from the incorporation of additional residues . accompanying
this change
in dynamic landscape in the human enzyme is tighter active site packing .
in hsdhfr ,
ligand flux is mediated by a twisting - hinge motion of the
met20 domain , whereas the opening and closing of the met20 loop modulates
ligand binding and unbinding in ecdhfr .
the human enzyme also lacks
conformational changes that differentiate the michaelis complex and
the product ternary complex in prokaryotic forms of dhfr , including
ecdhfr . because hsdhfr is approximately 10 times more susceptible
to end - product inhibition
, the authors conclude that the evolution
of a new dynamic mechanism in hsdhfr might be an adaptation in response
to the disparate relative concentrations of nadph in relation to nadp that is observed in vertebrate versus prokaryotic cells ( 100:1
ratio in humans and 1:1 in e. coli ) . another relevant study conducted by liu et al . showed
that specific
mutations along the evolutionary path from esdhfr to hsdhfr , also
known as phylogenetically coherent events ( pces ) , affect the binding
properties and catalytic efficiency of the enzyme .
the locations of the pces discussed in this paper are shown
in figure 1 for the hsdhfr system along with
a sequence alignment comparing the human and e. coli enzymes .
the most recent of these pces , a pwpp modification of the
met20 region ( 21-pwnlpadl-27 in ecdhfr and 24-pwpplrne-31 in hsdhfr ) ,
prevents this loop from assuming an open conformation in the human
enzyme and negatively impacts the catalytic rate when expressed individually
in an e. coli chimera enzyme .
mutating two additional
pce regions ( ecdhfr g51 to hsdhfr 62-pekn-65 and a single l28f mutation )
to humanlike sequences in an e. coli variant rectified
the pwpp catalytic deficit , and the end
result is a measurable improvement in catalytic efficiency in hsdhfr
compared to that of ecdhfr . considering the altered dynamic
landscape of hsdhfr in combination
with the effects of specific mutational events on dhfr catalysis ,
our group hypothesized that fast protein dynamics might be dynamically
coupled to hsdhfr - catalyzed hydride transfer . in this work
, we present
the results of a transition path sampling ( tps ) study indicating the presence of a pv in the reaction coordinate
of hsdhfr .
the advantage of tps is that it is completely unbiased ,
unlike other sampling methodologies such as umbrella sampling .
it
is also entirely rigorous , with the only approximations being the
use of quantum mechanical / molecular mechanical ( qm / mm ) simulations
and semiempirical treatment of the quantum region .
we stress that
the trajectories found via tps are classical in nature ; in other words ,
they do not include the effects of hydride tunneling or zero point
energy .
first , the promoting vibration , by compressing the
donor and acceptor , causes the barrier to be both lower and thinner .
second , for other hydride
transfer enzymes , and alcohol dehydrogenase in particular , there is
some indication that tunneling plays a small role .
michaelis complex of hsdhfr showing the locations of the putative
pv residues and a sequence alignment for the human and e.
coli enzymes .
the bound ligands are cyan , the hydride donor is blue ,
and the hydride acceptor is red .
pv residues are depicted as follows :
i17 = green , f35 = orange , 24-pwpplrne-31 of the met20 loop = yellow ,
f32 = violet , and 62-pekn-65 = magenta .
regarding pces , the pwpp modification overlaps
with the yellow residues ; 24-pwpplrne-31 , 62-pekn-65 ( magenta ) , and
l28f ( violet ) mutational events are shown explicitly . to carry out the specific analyses discussed throughout
this paper ,
we first generated a transition path ensemble ( tpe ) for the hsdhfr
hydride transfer reaction consisting of 200 individual reactive trajectories .
we produced these trajectories via a microcanonical tps algorithm
adapted for the study of enzymatic reactions that maintains an acceptance ratio of 25% . to obtain a transition
state ensemble
( tse ) , or a set of atomic coordinates along the stochastic
separatrix , we calculated the commitment
probability of every 10th trajectory in the hsdhfr tpe .
we considered
the time of barrier crossing to correspond to the time necessary for
the committor to rise from 0.1 to 0.9 , and we regarded the transition
state of each trajectory to be the time slice with a committor value
closest to 0.5 . for comparison purposes , we also utilized an ecdhfr
tpe and tse calculated previously .
we performed all steps for system
setup using the program charmm unless otherwise noted .
to obtain initial atomic coordinates
, we utilized the crystal structure
of human dhfr in a ternary complex with the cofactors nadph and folic
acid , which is a substrate mimic .
this structure was solved to a resolution
of 1.20 by wright et al .
( pdb code 4m6k ) . to create
a reactive complex to study hydride transfer using the coordinates
of folic acid
, we changed the protonation states of n1 , n8 , and protonated
c6 and generated atomistic parameters for this new ligand using the
program discoverystudio25 . also using this program , we added c - terminal
residues met1gly3 , which were not resolved in the original
crystal structure .
we employed protonation states of all acidic and
basic amino acids corresponding to said state at physiological ph
as determined by charmm . to ready the complex for qm / mm simulation
,
we isolated the quantum mechanical region ( the dihydronicotinamide
and ribose group of nadph and the majority of dihydrofolate , excluding
the glutamate tail ) as a separate residue , patched the boundary atoms
to the classical regions using the command pres , and constructed a
distinct parameter file for this new residue .
for this new residue ,
we used a nonstandard potential first calculated by garcia - viloca
et al . following this step , we solvated
the entire complex with an 85 - diameter sphere of tip3 water
models , incorporated all crystallographic
waters as tip3 models , and neutralized the charge of the system with
four potassium ions .
we conducted all molecular dynamics ( md )
and qm / mm simulations presented in this work using version 35 of the
program charmm . for classical simulations
, we
implemented the charmm27 all - atom force field . during qm / mm , we simulated
the quantum region using the am1 potential and treated the boundary atoms using the generalized hybrid orbital
( gho ) method .
we conducted all integrations
of forces for md or qm / mm using time steps of 1 fs . the protocol we
utilized to minimize , heat , and equilibrate the hsdhfr system was
selected to mimic that implemented in our previous study on ecdhfr . for minimization , we first constrained all atoms
except tip3p waters and potassium ions using a harmonic potential
of 100 kcal mol and
performed 100 steps of steepest descent ( sd ) followed by 100 steps
of the adopted basis newton raphson method ( abnr ) .
next , we
constrained only the protein and ligand atoms with 100 kcal mol of harmonic force and
conducted 100 steps of abnr .
next , we performed iterative minimization
cycles with the protein and ligand atoms constrained using force constants
of 75 , 50 , and 25 kcal mol , successively .
then , we conducted an unconstrained minimization
using qm / mm for 100 steps of abnr . to heat the system from 0 to 300
k
, we incrementally added kinetic energy for a total of 30 ps while
implementing decreasing constraint levels in the same manner as the
described minimization protocol . using qm / mm , we equilibrated the
system at 300 k for 500 ps with zero applied constraints .
we designated the initial
and final states of the reaction by implementing the following order
parameters : chemical species of the enzymatic reaction with a hydride
acceptor
distance greater than or equal to 1.5 were considered part
of the reactant ( dihydrofolate ) basin , whereas species with a hydride donor
distance greater than or equal to 1.5 and a hydride
acceptor
distance less than or equal to 1.5 were designated as part
of the product ( tetrahydrofolate ) basin . following the next step of
the tps algorithm ,
we connected the reactant and product basins via
an initial , biased qm / mm trajectory of 250 fs for implementation of
the charmm command resd with the parameter that the hydride acceptor
distance was constrained to 1.25 by a harmonic force constant
of 60 kcal mol . using this initial constrained trajectory , we selected a random point
of integration , or time slice , along the trajectory and perturbed
the momentum of each atom in the system by adding a random value as
determined by selection from a zero - mean gaussian distribution multiplied
by a scaling factor of 0.2 .
after rescaling these new momenta to conserve
total energy and eliminate any net angular or linear momenta , we propagated
the system both forward and backward for 250 fs in time to generate
a resultant trajectory of 500 fs in total length .
we utilized this
process of randomly , iteratively perturbed time slices until we achieved
an ensemble of 230 reactive or qm / mm trajectories that connected both
designated configuration basins .
we maintained a ratio of reactive
to nonreactive trajectories ( acceptance ratio ) of approximately 25% .
to ensure complete decorrelation from the original constrained reactive
trajectory
, we disregarded the first 30 generated trajectories in
all of our analyses . to determine
the commitment
probability of a specific set of coordinates
corresponding to a particular time slice along a reactive trajectory ,
we reinitialized the qm / mm dynamics at this point using random velocities
derived from a boltzmann distribution .
all committors for individual
time slices were calculated using 50 trajectories of 250 fs each .
we considered the value of the number of these trajectories landing
in the product basin divided by the total number of trajectories generated
to be representative of the committor value of the time slice . in
this respect , a point with a committor value of 1 would have a 100%
chance of reaching the product basin , whereas a set of atomic coordinates
and velocities with a committor of 0.5 would be equivalent to the
transition state . to determine the level to which a specific
degree of freedom was necessary for the transition state formation
of the enzymatic reactions of hsdhfr and ecdhfr , we conducted committor
distribution analyses . using a transition state as the
starting point , we conducted qm / mm simulations for a trajectory 1
ps in length while constraining the atoms of interest with a harmonic
force constant of 2000 kcal mol .
for all constraints relevant to the atoms of the protein , we constrained
with regard to both the hydride donor and acceptor and the -carbon
of the specific residues ( -carbon in the case of glycine ) .
we calculated the committor value for every 50th slice of this constrained
trajectory , giving 20 committor values for each transition state .
for each committor distribution presented in this study
, we used three
transition states as starter points . in principal component
analysis ( pca ) , one must first determine the eigenvectors of the correlation
matrix of the data ; then , the variance of the data along these eigenvectors
is proportional to the corresponding eigenvalues . if the eigenvalues
of the first few eigenvectors are dominant , then they form a low - dimensional
representation of the data .
the limitation of pca is that it implicitly
linearizes data , which is not a good approximation for the distribution
of transition states on the separatrix . to correct for the linearization
assumption
, we used kernel pca ( kpca ) . in kpca , a nonlinear transformation
of the original data is made to a space where the pca is valid
, pca
is performed there , and then the results are transformed back to the
original space .
we found that quadratic polynomial transformation
( xy ) led to the first
eigenvector dominating the variance .
the direction of the reaction
coordinate is perpendicular to the separatrix ; therefore , we found
the residues that contributed the least to this dominant
eigenvector .
notably , all calculation methods and
parameters reproduced the data from our original ecdhfr study to ensure
that direct comparisons are appropriate .
we utilized
committor distribution analysis to study the reaction
coordinate of hsdhfr in comparison with that
of ecdhfr .
committor distribution for an enzyme - catalyzed reaction
is a rigorous computational method that is utilized to deduce the
complete enzymatic reaction coordinate .
the first step in this
analysis is to use the coordinates of an extant transition state to
initiate a constrained qm / mm trajectory .
if the correct set of reaction
coordinate variables is constrained , the resultant trajectory will
remain on the separatrix .
the next step is to run committor analysis
using points along this new constrained trajectory to obtain a distribution
of committor values .
a correct selection of relevant degrees of freedom
would yield a distribution peak at 0.5 .
because of the complexity
of the enzymatic reactions and the statistical nature of the calculation ,
it is expected that selection of an accurate enzymatic reaction coordinate
will yield a broad distribution centered at 0.5 . incorrect selections
of the reaction
coordinate in an enzymatic system will generally result
in distributions with a peak far from the 0.5 committor value .
using this analysis , we found that a subpicosecond protein
motion is dynamically coupled to the reaction catalyzed by hsdhfr .
committor distribution analysis is used to determine what motions
are on the dividing surface ( the stochastic separatrix ) , and what
motions are orthogonal to it .
the term dynamic coupling refers to
degrees of freedom that are part of the reaction coordinate and occur
on the same time scale as barrier passage .
in other words , the promoting
vibration modulates the barrier and does so on a time scale that is
comparable to passage over the barrier ( i.e. , they are not adiabatically
separable ) .
we observed that the minimal definition of the hsdhfr
reaction
coordinate required the inclusion of the positions of the following
residues : i17 , 24-pwpplrne-31 , f32 , f35 , and 62-pekn-65 ( figure 1 ) .
the residues 24-pwpplrne-31 include the pwpp
modification of the met20 loop ; f32 and 62-pekn-65 also correspond
to previously reported pces in the dhfr protein family .
evolutionarily analogous residues in ecdhfr
( i14 , 21-pwnlpad-27 , f31 , and g51 ) were not part of the ecdhfr reaction
coordinate .
figure 2 shows select committor
distributions generated for both the hsdhfr and ecdhfr systems . to
create each distribution
, we utilized three transition states as starting
points for the production of three constrained qm / mm trajectories
1 ps in length .
we performed committor analysis on every 50th time
slice of each new trajectory to obtain a set of committor values .
we performed calculations while implementing constraints on the following
variables : ( 1 ) the hydride donor and hydride
acceptor
distances ( ecdhfr , figure 2a ; hsdhfr , figure 2d ) , ( 2 ) the positions of the atoms for nadph and
dihydrofolate ligands ( ecdhfr , figure 2b ; hsdhfr ,
figure 2e ) , and ( 3 ) the positions of the ligand
atoms in addition to the distances between the -carbons ( -carbon
in the case of glycine ) of the putative pv residues and the hydride
donor and acceptor ( ecdhfr , figure 2c ; hsdhfr ,
figure 2e ) .
histograms of committor distributions
obtained with increasing
numbers of constraints for the ecdhfr and hsdhfr systems . graphs ( a ) ,
( b ) , and ( c ) correspond to ecdhfr , whereas ( d ) , ( e ) , and ( f ) pertain
to the hsdhfr system .
data shown in ( a ) and ( d ) were obtained while
constraining only the distances between the hydride and the donor
and acceptor , ( b ) and ( e ) illustrate the results of the committor
distribution calculation while applying constraints to all atoms of
the ligands , and ( c ) and ( f ) represent data derived while constraining
the residues of the putative pv ( i14 , 21-pwnlpad-27 , f31 , and g51
in ecdhfr ; i17 , 24-pwpplrne-31 , f32 , f35 , and 62-pekn-65 in hsdhfr ) . in the hsdhfr system , increasing
the number of constraints resulted
in a committor distribution that peaked close to 0.5 .
additionally ,
the incorporation of protein constraints was essential for achieving
an adequate description of the hsdhfr enzymatic reaction coordinate .
all of the distributions calculated for ecdhfr are centered far from
0.5 , demonstrating that the constraints tested were not sufficient
approximations of the enzymatic reaction coordinate . when only the
atomic positions of the ligands were constrained , hsdhfr exhibited
a marked improvement in the centering of the committor distribution ,
whereas ecdhfr did not . because the ligands interact with a large
portion of the protein in both dhfr systems , it is likely that constraining
these atoms resulted in a general constraint on protein conformation .
the gradual transition to a distribution that peaked closer to 0.5
suggests that the reaction coordinate in the hsdhfr system is a wider
tube in configuration space ( i.e. , in protein structure ) .
it must
be reiterated that these differences are qualitative ; however , it
is clear that there are basic physical differences captured by the
variation between panels c and f of figure 2 .
it is also possible that there is a set of protein constraints
that would imply direct coupling of ecdhfr protein motions to hydride
transfer , but given the preponderance of evidence , that seems highly
unlikely .
we observed an increase in structural
variation of the transition states in the tse of hsdhfr when compared
to those sampled by ecdhfr .
first , we calculated the rmsd values for
the position of only the ligand atoms at each transition state versus
those of the average transition state to be 0.0779 and 0.221
in ecdhfr and hsdhfr , respectively .
we then used the atomistic structures
of the transition state conformations of the protein to perform this
calculation and obtained rmsd values of 0.0594 and 0.154 for
the transition state conformations of ecdhfr and hsdhfr , respectively .
acceptor
angle at the transition state in hsdhfr compared to that of ecdhfr .
although the average value for this angle was similar for both enzymatic
systems ( 151.92 and 151.00 in ecdhfr and hsdhfr , respectively ) ,
the standard deviation for this angle was greater in the human enzyme
( 3.2 and 73 in ecdhfr and hsdhfr , respectively ) .
it is
important to note that these rmsd values must be considered qualitative
measures of transition state variation .
we also detected increased
variability in the times of barrier crossing for the hsdhfr tpe in
comparison to those calculated for the ecdhfr tpe .
figure 3a shows box plots representing the distributions
of barrier crossing times exhibited by both the ecdhfr and hsdhfr
systems . in ecdhfr
, the distribution of committors calculated has
a breadth of only 2 fs , whereas the corresponding distribution for
the hsdhfr system spans 17 fs .
these distribution differences are
associated with a p value of less than or equal to
0.05 .
this result may be indicative of an increase in the ruggedness
of the free energy landscape of the hsdhfr reaction compared to that
of the reaction catalyzed by ecdhfr .
the e. coli enzyme
is clearly a much stronger funnel to a single path through the reactive
phase space .
these results demonstrate that the evolutionary changes
allow for greater variation in paths through the reactive phase space
in hsdhfr compared to that of the prokaryotic enzyme . although it
is unclear what exactly this may confer biologically , the physical
observation is definitive .
we can only speculate that this greater
chemical path flexibility may allow for greater responsiveness to
the variable chemical environments found in eukaryotic systems .
( a ) box plots illustrating the differences in the distributions
of barrier crossing times for the ecdhfr tpe ( ectpe ) and hsdhfr tpe
( hstpe ) .
each distribution was calculated using barrier crossing times
from 21 representative trajectories in each tpe . for the ectpe ,
the
shortest and longest times of barrier crossing were 1 and 3 fs , respectively .
the values for the 2nd quartile , median , and 4th quartile were all
2 fs . in the hstpe ,
the corresponding values were as follows : shortest
time = 2 fs , 2nd quartile = 4 fs , median = 6 fs , 4th quartile = 8
fs , and the longest time = 18 fs .
( b ) kpca calculation for the ecdhfr
tse , with putative pv residues at positions 14 , 2127 , 31 ,
and 51 indicated with red circles .
( c ) kpca calculation for the hsdhfr
tse , with putative pv residues at positions 17 , 2431 , 35 ,
and 6265 indicated with red circles .
to determine the contribution of the position of each residue
to
the variance in the hsdhfr and ecdhfr tses , we conducted kernel principal
component analysis .
this technique presents
a way to perform orthogonal transformation of a multidimensional surface ,
such as tse , through nonlinear kernels .
we have previously used this
technique as a way to identify pv residues in the enzymatic reaction
coordinate of human heart lactate dehydrogenase ( ldh ) . in ldh , the residues contributing the least
to the first principal component ( pc ) were able to adequately approximate
the enzymatic reaction coordinate .
the results of this calculation
for the ecdhfr tpe and hsdhfr tpe are displayed in figure 3b and 3c , respectively .
the
residues of the putative pv are highlighted with red circles ( i14 ,
21-pwnlpad-27 , f31 , and g51 in ecdhfr ; i17 , 24-pwpplrne-31 , f32 , f35 ,
and 62-pekn-65 in hsdhfr ) .
in hsdhfr , all but two of the pv residues
corresponded to minima along the first pc , whereas the majority of
these residues in ecdhfr did not .
we found that a compressive
motion between residues i17 and p35
correlates with a dynamic decrease in the hydride donor
acceptor
distance and with the time of barrier crossing in the hsdhfr system .
figure 4 shows the time series representing
specific dynamic distances during an exemplary trajectory along with
the moment of chemical barrier passage ( gray region : 354361
fs ) .
breaking of the hydride donor bond ( red ) and formation
of the hydride acceptor bond ( blue ) are shown in figure 4a along with the donor
figure 4b is a
representation of specific distances between different aspects of
the protein during the same example trajectory .
the compression between
i17 and p35 ( orange ) coincides with a smaller compressive fluctuation
between i17 and the hydride acceptor ( violet ) following an excursion
of p24 toward i17 ( green ) .
this fast motion of p24 toward i17 is the
result of a density fluctuation propagating through the protein bulk ,
which was also the case for the previously identified pv in the reaction
coordinate of human heart lactate dehydrogenase .
distance time series of an exemplary reactive trajectory illustrating
the dynamic contributions of specific residues to chemical barrier
passage in hsdhfr .
( a ) distances corresponding to the chemical reaction
( red = hydride acceptor , blue = hydride donor , and black
= donor acceptor ) .
( b ) dynamic distances within the protein
( orange = i17(atom he1)f35(atom hd1 ) , violet = i17(atom cb)acceptor ,
and green = p24(atom cb)i17(atom cb ) ) .
the gray region represents
the time of barrier passage for this specific trajectory ( 354361
fs ) .
we detected subtle structural
changes between the active sites
of ecdhfr and hsdhfr , which establish a physical link between the
hydride donor acceptor axis and the pv residues in hsdhfr .
figure 5 shows the position of pv residues
near the active site in relation to the hydride donor
the hinges of the met20
loop , p24 and g31 , come into direct contact with i17 and f35 , respectively ,
and these two amino acids are ideally situated on either end of the
hydride donor acceptor axis .
the active site of ecdhfr is too
loosely packed to support these specific contacts .
in particular ,
the close van der waals interaction between i17 and p24 in hsdhfr
is not pronounced in the analogous residues of ecdhfr ( i14 and p21 ) .
tighter packing of the hsdhfr active site may be an important enabling
factor for the incorporation of fast protein dynamics in the hsdhfr
reaction coordinate .
view of the equilibrated structure of the ( a ) ecdhfr and ( b ) hsdhfr
system .
the ligands are cyan , the hydrides are violet , the hydride
donors are blue , and the hydride acceptors are red .
residues of the
met20 loop are yellow , i14/17 is green , and residue f31/35 is orange .
the
committor distribution analysis data presented here represent
our best attempt at isolating the reaction coordinate of hsdhfr .
we
tried many combinations of candidate residues based on the kpca output .
incorporating residues that also corresponded to important mutational
events was key to our elucidation of the reaction
we show
that the subpicosecond protein motion in a specific set
of residues is dynamically coupled to the reaction coordinate of hsdhfr .
contradistinctively , there is no indication
of a pv in the enzymatic reaction coordinate of ecdhfr , suggesting
a change in the form of catalysis from the e. coli to human enzyme .
it could be possible to experimentally validate
this finding through kinetic studies of enzymes that are expressed
utilizing heavy - isotope substitution throughout the protein matrix .
if a pv is in fact dynamically coupled to the
enzymatic reaction coordinate , a
heavy enzyme would
exhibit a decreased probability of barrier crossing , which would not
be the case if there was no such coupling .
experimental and theoretical evidence indicates that the enzymatic rate enhancement
of ecdhfr is primarily governed by electrostatic effects , but electrostatics
do not appear to be the sole factor responsible for catalysis in hsdhfr .
as one of the key mutations differentiating ecdhfr from hsdhfr ,
pwpp
modification of the met20 loop has been implicated in electrostatic
stabilization of the transition state for the ecdhfr - catalyzed hydride
transfer reaction , and the presence of
this modification in a chimera e. coli enzyme results
in a decreased hydride transfer rate .
concurrent with this modification hindering the electrostatic - based
catalysis of ecdhfr , this mutational event also led to a change in
the conformational states of hsdhfr and a more tightly packed active
site .
combined with the other mutational events
that separate the e. coli and human enzymes , this
alteration of the met20 loop allows for a mechanism of catalysis in
the hsdhfr that includes rapid protein dynamics or a pv .
we
also found that the stochastic separatrix , or tpe , of the ecdhfr - catalyzed
reaction is more narrow in terms of the geometry of the transition
state structures and the variability of reactive conformations as
compared to the enzymatic reaction of hsdhfr .
this finding is in agreement
with our committor distribution results demonstrating a smaller degree
of tolerance for changes in the enzymatic conformation of ecdhfr relative
to hsdhfr in order to remain on the separatrix .
our inability to identify
a definitive reaction coordinate for the ecdhfr - catalyzed reaction
also indicates that perturbation of the ecdhfr transition state structure
is more likely to result in deviation from the separatrix compared
to doing so with the human enzyme . when considered within the context
of the conformational landscape model of enzymatic reactions
, these results suggest that fast
distance sampling along the reaction coordinate indicative of a pv
could introduce a degree of finely grained ruggedness to the free
energy landscape of an enzymatic reaction .
thus , it can be argued
that dynamic coupling of protein motion to the reaction coordinate
increases the likelihood of barrier crossing by decreasing the need
for an optimal electrostatic and geometric arrangement of the active
site . as an extension of this conclusion , the breadth of possible
reactive conformations corresponding to a specific conformational
basin appears to be greater for the hsdhfr enzymatic system than for
the ecdhfr system .
an almost philosophical question relates
to the selective pressure
that caused such exquisite design of active sites when the chemistry
is almost never rate limiting .
this is a conundrum for enzymatic active
sites in general , not just promoting vibrations .
the results of this
study indicate that possible selective pressure on the protein matrix ,
which was likely optimized after a static active site , was robustness
to multiple phase space paths rather than optimization of the chemical
rate , although this is purely speculation .
further work is needed
to elucidate possible evolutionary pressures for enzymatic promoting
vibrations . | how
evolution has affected enzyme function is a topic of great
interest in the field of biophysical chemistry .
evolutionary changes
from escherichia coli dihydrofolate reductase ( ecdhfr )
to human dihydrofolate reductase ( hsdhfr ) have resulted in increased
catalytic efficiency and an altered dynamic landscape in the human
enzyme . here
, we show that a subpicosecond protein motion is dynamically
coupled to hydride transfer catalyzed by hsdhfr but not ecdhfr .
this
motion propagates through residues that correspond to mutational events
along the evolutionary path from ecdhfr to hsdhfr .
we observe an increase
in the variability of the transition states , reactive conformations ,
and times of barrier crossing in the human system . in the hsdhfr active
site ,
we detect structural changes that have enabled the coupling
of fast protein dynamics to the reaction coordinate .
these results
indicate a shift in the dhfr family to a form of catalysis that incorporates
rapid protein dynamics and a concomitant shift to a more flexible
path through reactive phase space . |
antigen - presenting cells ( apcs ) activate t cells through a two - signal mechanism : one is initiated by t cell receptor ( tcr ) binding to antigenic peptide presented by major histocompatibility complex ( mhc ) molecules and the second signal involves costimulatory molecules that interact with costimulatory receptors on the t cell surface and leads to t cell cytokine production and their proliferation .
dendritic cells ( dcs ) are believed to be the most potent apcs which have the unique capacity to deliver antigens to t cells and express several costimulatory molecules .
the second signal required for t cell activation which supports cell survival , memory development , proliferation , and cytokines production found on the surface of dcs has been reported such as b7 family members b7 - 1 ( cd80 ) and b7 - 2 ( cd86 ) [ 4 , 5 ] .
binding b7 - 1/b7 - 2 to cd28 is the strongest costimulatory signal delivered by dcs to provide a full activation of t cells , promoting their proliferation and il-2 secretion [ 6 , 7 ] .
cd80 and cd86 have been reported to have particular functions in eliciting t cell activation and inducing differential patterns of cytokine expression supporting type 1 or type 2 t - helper ( th1 or th2 ) response upon binding to cd28 [ 2 , 8 ] .
the primary outcome of cd28-mediated stimulation on molecular level is an increased production of cytokines such as il-2 which is important for t cell proliferation , antiapoptosis .
toll - like receptors ( tlrs ) , as a family of pattern - recognition receptors ( prrs ) , are highly expressed on dc and t cell .
activation of tlr leads to dc maturation and secretion of proinflammatory cytokines , which can induce t cell antitumor immune response .
many polysaccharides as tlr agonists that function as adjuvant and stimulate dcs to prime antigen - specific t and b cell responses have been reported [ 1113 ] . on t cells ,
pretreatment with tlr4 ligand lps enhanced their survival and increased their suppressive activity , whereas tlr4 deficient mice did not respond . both tlr and tcr signaling pathways utilize members of the mapk family .
tlr activation of these pathways influences the subsequent tcr - mediated signaling events [ 15 , 16 ] .
tlr agonists can induce activation of cd4 t lymphocytes , cd8 t lymphocytes , or cytotoxic t lymphocytes ( ctls ) [ 1719 ] .
these findings prompt that tlr agonists may cause the activation of dc and provide signal required for t cell activation .
ycp ( ycp is the acronym of yancheng polysaccharide ) was purified from the mycelium of phoma herbarum ys4108 that inhabits the sediment in the yellow sea area around yancheng , china .
it has a backbone of -1,4-d - glucan with a lower proportion of -1,6-linked glucopyranosyl and glucuronic acid residues as nonreducing terminals , and we have previously found that it possesses a great antitumor potential via enhancement of host immune response [ 20 , 21 ] . however , further studies are still needed to clarify the molecular mechanism of ycp action . in this study
, we mainly focus on the effects and mechanisms of ycp on the specific immunity mediated by dcs and t cells .
all primary antibodies were purchased from ebioscience ( san diego , ca , usa ) and used at concentrations between 1 and 5 g / ml . 1-cyano-4-dimethylaminopyridinium tetrafluoroborate ( cdap ) was purchased from sigma chemical co. ( st . louis , mo , usa ) .
recombinant murine il-4 and recombinant murine gm - csf were purchased from peprotech ( rocky hill , nj , usa ) .
elisa kits for measuring murine ifn- , il-4 , and il-12 were purchased from r&d systems ( minneapolis , mn , usa ) .
anti - mouse tlr2 ( cd282 ) and anti - mouse tlr4 were purchased from ebioscience ( san diego , ca , usa ) .
3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) , ophenylenediamine , and fluoresceinamine ( fla ) were from sigma - aldrich ( st .
rna simple total rna kit was purchased from tiangen biotech ( beijing , china ) .
first - strand cdna synthesis kit was purchased from transgen biotech ( beijing , china ) .
sybr green was purchased from applied biosystems ( foster city , ca , usa ) .
reagents used for cell culture were purchased from gibco ( grand island , ny , usa ) .
male c57bl/6 mice of clean grade ( 68 weeks old ) weighing 1822 g were purchased from the comparative medicine centre of yangzhou university and acclimatized for 1 week prior to use .
b6.129-tlr2/jnju ( tlr2 ko ) and c57bl/10scnjnju ( tlr4 ko ) mice between 6 and 8 weeks old were provided by the model animal research center of nanjing university ( nanjing , jiangsu , china ) . throughout the study ,
the animals were fed by rodent laboratory chow pellets and tap water and were kept with five in each plastic cage in a well - ventilated room , maintained at 21 2c with humidity of 50 10% and a 12/12 h light / dark cycle .
all the procedures were carried out in strict accordance with the china legislation on the use and care of laboratory animals and were approved by the university committee for animal experiments .
mouse splenic t cells were prepared according to the method described previously with slight modification .
briefly , murine spleens were harvested from c57bl/6 inbred mice and minced in rpmi-1640 medium at room temperature .
erythrocytes were removed by hypotonic lysis using nh4cl and adherent cells were separated by plating at 37c for 6 h. the suspended cell populations were collected and used as the lymphocyte populations and applied to a nylon fiber column ( wako , osaka , japan ) .
after the cell suspension sank into the fiber bed thoroughly , the columns were incubated at 37c for 1 h. t cells were gently eluted without adding any additional pressures and b cells were flushed out of the column using a plunger .
the purity of t cells was characterized through fluorescence - activated cell sorting ( facs ) analysis for cd3 expression ; the ratio of cd3 t cells was approximately 90% .
ycp was conjugated with fla by using the cdap - activation method as previously described with slight modification . briefly
, 5 mg cdap was added into an aqueous solution containing 30 mg of ycp with gentle stirring and maintained at ph 9.0 for 2.5 min .
the cdap - activated ycp was then mixed with 2 mg of fla ( ph adjusted to 8.0 ) and incubated at room temperature overnight .
fluoresceinamine - labeled ycp ( fl - ycp ) was separated from the excess - free fla with an amicon ultra-15 centrifugal filter unit ( millipore , billerica , ma , usa ) .
the fla and ycp amounts in fl - ycp were , respectively , quantified by measuring absorbance at 440 nm and phenol - sulfuric acid assay . to perform the competition assay ,
t cells were incubated with medium , pam3csk4 ( 20 nm ) , lps ( 20 nm ) , pam3csk4 ( 20 nm ) + lps ( 20 nm ) , and anti - mouse cd28 ( 5 g / ml ) for 1 h , followed by incubation with fl - ycp ( 1001600 nm ) for 1 h. after washing three times with pbs , cells were examined on a facscalibur flow cytometer ( bd biosciences , san jose , ca , usa ) with a 488 nm laser excitation and a 530 nm emission filter .
data were acquired from a minimum of 100 000 cells and analyzed using the flowjo program ( freestar , ashland , or , usa ) .
t cells were cultured in 96-well microplates at a density of 2 10 cells / ml in rpmi-1640 medium containing 10% fbs , supplemented with 60 mg / l penicillin and 100 mg / l streptomycin .
the cells were pretreated with medium or anti - mouse cd3 ( 5 g / ml ) at 4c for overnight culture and stimulated with ycp ( 100800 nm ) for 48 h in a co2 incubator or with anti - tlr2 ( 20 nm ) , anti - tlr4 ( 20 nm ) , anti - tlr2 ( 20 nm ) + anti - tlr4 ( 20 nm ) , and medium at 37c for 2 h prior to addition of ycp ( 400 nm ) , followed by mtt ( 5 mg / ml ) for another 4 h. the formazan crystals formed from mtt by living cells were fully dissolved in dmso for 10 min . the absorbance was determined at 570 nm in a multiskan spectrum ( thermo fisher scientific , vantaa , finland ) , and induction of cell proliferation was expressed as the proliferation index , calculated by dividing a570 of stimulated cells with a570 of control cells .
bone marrow - derived dendritic cells ( bmdcs ) were generated by culturing monocytes for 7 days in rpmi 1640 medium , 10% fbs , 1% penicillin / streptomycin , 2 mm l - glutamine , 100 ng / ml recombinant murine granulocyte - macrophage colony stimulating factor ( gm - csf ) ( peprotech ) , and 50 ng / ml recombinant murine il-4 ( peprotech ) .
dcs were washed by pbs and stained with anti - mouse cd11c fitc monoclonal antibody ( ebioscience , san diego , ca , usa ) .
an established stable mouse melanoma cell line , b16f10 , was used ( kindly donated by professor heng zheng ) .
cells were cultured in rpmi-1640 medium containing 10% fbs , supplemented with 60 mg / l penicillin and 100 mg / l streptomycin .
b16f10 cells were resuspended at a density of 1 10 cells / ml in a 1.5 ml eppendorf tube with water bath for 30 min at 40c and then putted into 80c refrigerator for 20 min , slow thawing at 37c for 20 min .
after 4 freeze - thaw cycles , cells were centrifuged at 3000 rpm for 20 min .
the supernatant was passed through a 22 m sieve called b16f10 full - cell antigen ( b16ag ) .
adjust the concentration of b16ag to 0.1 mg / ml . dcs cultured for 7 days were stimulated by b16ag for 48 h. dcs ( mdcs or imdcs ) were washed by pbs and stained with anti - mouse mhc class ii pe and anti - mouse cd11c fitc monoclonal antibodies ( ebioscience , san diego , ca , usa ) . for another experiment , dcs cultured for 7 days as described in section 2.7
were stimulated by b16ag and ycp ( 100800 nm ) for 48 h. dcs were washed by pbs at various times ( 6 h , 12 h , 24 h , and 48 h ) and stained with anti - mouse cd80 ( b7 - 1 ) fitc and anti - mouse cd86 ( b7 - 2 ) pe monoclonal antibodies ( ebioscience , san diego , ca , usa ) .
data were acquired from a minimum of 20 000 cells and analyzed using the flowjo program .
t cells were cultured in 96-well microplates at a density of 2 10 cells / ml pretreated with medium or anti - mouse cd3 ( 5 g / ml ) at 4c for overnight culture and stimulated with ycp ( 100800 nm ) for 48 h in a co2 incubator or with anti - tlr2 ( 20 nm ) , anti - tlr4 ( 20 nm ) , anti - tlr2 ( 20 nm ) + anti - tlr4 ( 20 nm ) , and medium at 37c for 2 h prior to addition of ycp ( 400 nm ) .
cell - free supernatants were collected for quantification of ifn- level by commercial elisa kits according to the manufacturer 's protocol described previously .
b16f10 peptide - pulsed dcs were cultured in 96-well microplates at a density of 2 10 cells / ml in rpmi-1640 medium containing 10% fbs , supplemented with 60 mg
b16ag - dcs ( mdcs ) were stimulated with ycp ( 100800 nm ) for 48 h in a co2 incubator or with anti - tlr2 ( 20 nm ) , anti - tlr4 ( 20 nm ) , anti - tlr2 ( 20 nm ) + anti - tlr4 ( 20 nm ) , and medium at 37c for 2 h prior to addition of ycp ( 400 nm ) .
cell - free supernatants were collected for quantification of il-12 level by commercial elisa kits according to the manufacturer 's protocol described previously .
t cells were cultured with mdcs at the ratio of 20 : 1 or without mdcs for 48 h as effector cells ( 2 10 cells / ml ) .
the b16f10 cells were resuspended at a density of 2 10 cells / ml as target cells .
the effector cells and target cells were cocultured and stimulated by medium or ycp ( 100800 nm ) for 48 h. cells were collected for real - time quantity rt - pcr .
the supernatants were obtained and the levels of ifn- , il-12 , and il-4 productions were measured by elisa ( r&d systems ) according to the manufacturer 's protocol as described in section 2.7 .
cells stimulated by medium and cells stimulated by ycp ( 400 nm ) were collected at various times ( 0 h , 6 h , 12 h , 24 h , 48 h , and 96 h ) after washing by pbs and stained with anti - mouse cd3e pe - cyanine5 , anti - mouse cd4 fitc , and anti - mouse cd8a pe monoclonal antibodies ( ebioscience , san diego , ca , usa ) .
the number of target cells was measured by the mrna quantitation of mage - a3 .
total rna was isolated from cell pellet samples using the rna simple total rna kit from tiangen biotech ( beijing , china ) .
rna recovered ( about 2 g ) was reverse transcribed to cdna using random hexamer primers and first - strand cdna synthesis kit from transgen biotech ( beijing , china ) .
b16f10 cells proliferation was assessed by measuring mage - a3 gene expression using the abi prism 7500 sequence detection system to perform qrt - pcr as already described in detail .
briefly , pcr was conducted in 10 l using 20% of the recovered cdna as the template , with 2 m gapdh ( internal standard ) primers ( forward , 5-agaaggctggggctcatttg-3 , 1.5 l ; reverse , 5-aggggccatccacagtcttc-3 , 1.5 l ) or 1 m mage - a3 specific primers ( forward , 5-agctctgcatcgttttgggtt-3 , 1.5 l ; reverse , 5-gttccattatccgctacatctgaa-3 , 1.5 l ) and 5 l 2 sybr green from applied biosystems ( foster city , ca , usa ) .
cycling parameters were 95c 20 s , followed by 40 cycles of 95c 20 s and 60c 30 s. melt curve parameters were 95c 15 s , 60c 60 s , and 95c 15 s. four cell models were prepared to study the signal provided by ycp during in vitro specific immune responses .
the matured dcs ( wt and tlr4 ko ) and t cells ( wt and tlr4 ko ) were cocultured according to the ratio of 1 : 10 while dcs were resuspended at a density of 2 10 cells / ml for 48 h. the mixed cells after being cocultured were used as effector cells ( 2.2 10 cells / ml ) , and the b16f10 cells resuspended at a density of 2.2 10 cells / ml were used as target cells .
the effector cells and target cells were cocultured and stimulated by medium or ycp ( 400 nm ) for 48 h. the number of target cells was measured by quantitative real - time rt - pcr ( qrt - pcr ) as described in section 2.9 . the supernatants were obtained and the levels of ifn- , il-12 , and il-4 productions were measured by elisa ( r&d systems ) according to the manufacturer 's protocol as described in section 2.8 .
the prism 5.0 program ( graphpad software , la jolla , ca , usa ) was used for statistical analysis .
mann - whitney u test or kruskal - wallis test followed by dunn 's post hoc test was performed to determine significant differences where appropriate .
splenic t cells were isolated by nylon fibers to a purity > 90% ( data not shown ) and then cultured with ycp or cona as the positive control in the 96-well plate which had been precoated with acd3 functional antibody or pbs .
the results indicated that ycp could not stimulate t cell alone . with the precoated acd3 ,
ycp could significantly stimulate t cell to proliferate in a dose - dependent manner ( figure 1(a ) ) .
in addition to the proliferative effect , the effect of ycp on cytokine production from t cells was evaluated .
the supernatants collected from nave and ycp - stimulated cells were tested by elisa . with the precoated acd3 ,
ycp could significantly stimulate t cell to produce ifn- in a dose - dependent manner ( figure 1(b ) ) .
the stimulation obtained with ycp on t cell suggests that ycp may be an efficacious second signal in tcr activation just like the contribution of costimulatory molecules on t cell stimulant capable of promoting cell proliferation and producing ifn-. dcs generated with gm - csf and il-4 as immature dcs expressed amounts of cd80 , cd86 , and mhc class ii molecules as fully matured dcs . to determine the purity of monocyte - derived dendritic cells ,
after 7 days generation with gm - csf and il-4 , the expression of cd11c has been significantly increased to 82.6% .
and then followed by medium or b16f10ag for 48 h , the expression of cd11c was 88.2% and 89.3% , respectively ( figure 2(a ) ) . to determine the maturation of dcs , the pattern of expression of various cell - surface makers encoding mhc ii , cd80 ( b7 - 1 ) , and cd86 ( b7 - 2 ) was detected by flow cytometry .
there were more matured dcs that coexpressed both mhc class ii and cd11c stimulated by ag and ycp ( figure 2(b ) ) .
cd80 and cd86 on dcs at different times ( 6 h , 12 h , 24 h , and 48 h ) after the stimulations with medium / ag / ycp+ag / lps+ag were analyzed by using the flowjo program ( figure 2(c)2(f ) ) . with the stimulation of b16f10ag ,
the expressions of cd80 and cd86 on dcs significantly increased to 66.3% and 54.1% at 48 h by contrast with medium group .
ycp assisted the augments of the expressions of cd80 and cd86 on dcs suggesting that ycp could promote the maturation of dcs .
dcs cultured for 7 days were stimulated with medium / ag / ycp+ag / lps+ag for 48 h. the supernatants were obtained and the levels of il-12 productions were measured by elisa .
ycp could significantly stimulate dcs to produce il-12 in a dose - dependent manner ( figure 2(g ) ) .
the stimulation mediated by ycp on dcs suggested that ycp is an activator of cell - surface molecule ( mhc class ii , cd80 , and cd86 ) expression and il-12 production on dcs .
tlrs played a direct role in control of t cell activation including proliferation , cytokine secretion , upregulation of activation markers , and terminal differentiation .
as the receptors on b cells of ycp , tlr2 , and tlr4 had been reported in our previous study , based on these findings , we focused our study on the functional relevance of tlr2 and tlr4 in ycp - mediated tcr activation .
fluoresceinamine - labeled ycp ( fl - ycp ) ( 1001600 nm ) was prepared .
t cells were incubated with pam3csk4 ( a bona fide tlr2 ligand ) , lps ( a bona fide tlr4 ligand ) , and anti - mouse cd28 ( cd28 ligand ) for 1 h , followed by fl - ycp ( 1001600 nm ) for 1 h in competition experiment analyzed by flow cytometry ( figure 3(a ) ) .
the results suggested that ycp as the ligand of tlr2 and tlr4 could compete with pam3csk4 and lps .
cd28 was not the binding target of ycp ; in other words , the ycp t cell activation was achieved by signal provided by tlr without the signal of b7 - 1/b7 - 2:cd28 pathway . to prove this conclusion , we blocked receptors with specific antibodies .
t cells were stimulated with ycp in the presence of anti - tlr2 or anti - tlr4 and then subjected to proliferation assay and total ifn- quantification .
antibodies to tlr2 and tlr4 significantly reduced the proliferation effects of ycp and suppressed the induction of ifn- production with the presence of acd3 ( functional antibody ) which provided first signal for activation of tcr ( figures 3(b ) and 3(c ) ) .
with the presence of anti - tlr2 , t cell proliferation was reduced by 12.1% . with the presence of anti - tlr4 , t cell proliferation
the production of ifn- was decreased by 13.1% . with the presence of anti - tlr4 ,
the reduction was found to be synergetic upon the combined treatment with anti - tlr2 and anti - tlr4 .
therefore , we proposed that tlr2 and tlr4 might be functionally correlated with activation of tcr by ycp as second signal .
as the main receptors of dcs , tlrs have been reported to be important binding targets of polysaccharide vaccine .
anti - tlr4 significantly suppressed the induction of il-12 production ( 51.8% ) . to further confirm this statement ,
the impact that loss of tlr2 or tlr4 has upon ycp activities was evaluated in dcs from either b6.129-tlr2/jnju ( tlr2 ko ) or c57bl/10scnjnju ( tlr4 ko ) mice .
the upregulations of cd80 and cd86 were reduced on tlr2 ko ( 2.5% and 1.4% ) and tlr4 ko mice ( 8.5% and 5.6% ) , especially on tlr4 ko mice ( figure 3(e ) ) .
these findings provided convincing evidence that tlr4 was responsible for activation on dcs by ycp .
dcs cultured for 7 days were stimulated by b16ag for 48 h. t cells were cultured with mdcs at the ratio of 10 : 1 for 48 h as effector cells ( about 2 10 cells / ml ) .
the b16f10 cells were resuspended at a density of 2 10 cells / ml as target cells .
the effector cells and target cells were cocultured and stimulated by medium or ycp ( 100800 nm ) for 48 h. the number of target cells was measured by the mrna quantitation of mage - a3 .
ycp could not kill target cells directly ( without the effector cells ) ( figure 4(a ) ) and could significantly decrease the mrna of mage - a3 on target cells with the incubation of dcs and t cells in a dose - dependent manner ( figure 4(b ) ) .
the results indicate that ycp could strengthen the destruction of target cells in a dose - dependent manner . during the incubation ,
the populations of cd4 t cells and cd8 t cells have been increased significantly in 96 h ( figure 4(c ) ) .
the presence of ycp could strengthen this phenomenon that both cd4 t cells and cd8 t cells have been improved ( figure 4(c ) ) .
the ratio of cd4/cd8 is kept stable at 2.20 0.01 ( medium ) to 2.22 0.02 ( ycp ) which means t cells maintain the biological homeostasis .
the production of ifn- and il-12 reached higher levels and the production of il-4 was suppressed to a lower level ( figure 4(d ) ) .
the results indicate that ycp enhances the cytotoxicity and cytokine production and promotes activation of apcs and the cell - mediated immunity . as tlr4 was the receptor of ycp on dc and t cell , four cell groups were prepared to study the signal provided by ycp during in vitro specific immune responses as follows ( figure 4(e ) ) : ( a ) matured dcs ( wt ) and t cells ( wt ) , ( b ) matured dcs ( tlr4 ko ) and t cells ( wt ) , ( c ) matured dcs ( wt ) and t cells ( tlr4 ko ) , and ( d ) matured dcs ( tlr4 ko ) and t cells ( tlr4 ko ) .
dcs from different groups cultured for 7 days were stimulated by b16ag for 48 h. t cells from different groups were cultured with mdcs at the ratio of 10 : 1 for 48 h as effector cells ( 2.2 10 cells / ml ) .
the b16f10 cells were resuspended at a density of 2 10 cells / ml as target cells .
the effector cells and target cells were cocultured and stimulated by medium or ycp ( 400 nm ) for 48 h. the mrna level of mage - a3 on target cells ( figure 4(f ) ) and the levels of ifn- , il-12 , and il-4 productions were measured ( figure 4(g ) ) .
the results indicated that ycp can suppress the production of il-4 and induce the production of il-12 and ifn- via tlr4 on t cells , while inducing the production of il-4 , il-12 , and ifn- via tlr4 on dcs from these in vitro cell models for studying the ycp - mediated specific immunity .
the target cell killing was enhanced with the activations on t cells and dcs via tlr4 induced by ycp .
many classes of biomacromolecules containing carbohydrate structures have been shown to exert a successful immune response . in our previous study ycp
was demonstrated to stimulate the activation of macrophages and b cells [ 20 , 26 ] , and in recent years some polysaccharides that function as adjuvant and stimulate dc to induce t cell responses have been reported [ 1113 ] .
however , little is known about the molecular mechanisms that how ycp could interfere with the specific immunity mediated by dc and t cell .
thus , we focus on how ycp performs its function on dcs and t cells .
our current study indicates that ycp - induced tlr activation can induce tcr activation at the presence of functional antibody acd3 which is the
first signal of tcr activation on t cells . when the first signal is provided from dc
lps is used in other studies to enhance antigen - driven proliferation on dc [ 28 , 29 ] .
the functional maturation of dendritic cells has been shown previously to be associated with increased levels of certain t cell costimulatory molecules [ 24 , 30 , 31 ] .
bone marrow - derived dendritic cells generated in the presence of gm - csf and il-4 exhibit an intermediate maturation stage with respect to phenotype and in vitro antigen - presenting capacity . in our study , treatment with lps can result in increasing levels of markers on dcs which is in agreement with previous observations .
furthermore , lps will modify b7 ( b7 - 1 and b7 - 2 ) expression of monocytes .
ycp is demonstrated which could increase the expression of cd80 , cd86 , and mhc - ii in dcs and then strongly enhances t cell activation .
then , activated t cells can produce ifn-. furthermore , the secretion of th1-type cytokines il-12 and ifn- was increased while th2-type cytokine il-4 was decreased by ycp in vitro .
this action of ycp can enhance the helper cell response as well as the immune system 's response to foreign antigens .
wang et al . found that sarcodon imbricatus polysaccharide ( sip ) could prominently promote the production of il-2 and ifn- , indicating that sip could positively regulate the immunoreactions ; meanwhile sip significantly decreased the content of il-4 which negatively regulated the immunoreactions .
investigated the effect of lentinan on modulating th1 and th2 responses in patients with digestive cancers .
after lentinan treatment , cd4 ifn- t cell percentages increased significantly , whereas cd4 il-4 t cell percentage decreased significantly , suggesting that lentinan may improve the balance between th1 and th2 . a polysaccharide designated as the d - fraction , isolated from maitake ( grifola frondosa ) ,
can reduce the expression of th2 cytokine il-4 but markedly increase the expression of th1 cytokine ifn- in cd4 t cells and also increase il-12p70 production , indicating that d - fraction promotes the differentiation into th1 cells of cd4 t cells .
cd4 t cells augment the immune response by secreting cytokines that stimulate either a cytotoxic t cell response ( th1 cytokine ) or an antibody response ( th2 cytokine ) .
in addition to the results of the present study , our previous study showed that ycp can also generate a robust antibody response on b cells .
melanoma associated antigens ( mages ) are found in melanoma as tumor - associated antigens ( taas ) and some of these proteins , such as mage-1 , mage - a3 , and mage - a10 , are believed to be the detection and diagnostic target of melanoma .
mage - a3 , which is not expressed in noncancer cells , is highly specific tumor mrna marker , and it can be recognized by t cells which attack the b16f10 cells in a highly specific manner [ 3739 ] .
ycp could not kill melanoma cells directly but could significantly decrease the number of target cells with the incubation of dcs and t cells .
tlr agonists , such as pam3csk4 , lps , are demonstrated to promote dc and t cell activation and the engagement of tlrs in the induction of dc and t cell has been studied in the last few years [ 40 , 41 ] .
competitive inhibition at tlr2 and tlr4 by using pam3csk4 , lps as ligands with fl - ycp , indicates the potential receptors of ycp on t cell .
antibody blocking experiment and gene defect experiment provide convincing evidence that tlr4 is responsible for activation on dc by ycp and tlr2 and tlr4 are responsible for t cell activation by ycp .
second signal of t cell activation to promote t cell proliferation and ifn- secretion via tlr2 and tlr4 .
ycp can effectively promote il-12 secretion and expression of markers ( cd80 , cd86 , and mhc ii ) via tlr4 on dcs .
the data support that ycp is a good candidate for tumor - specific immunotherapy to enhance signals of antigen presentation process , antigen - specific t cell proliferation , th1 cytokine induction , and tumor - specific killing . | ycp , as a kind of natural polysaccharides from the mycelium of marine filamentous fungus phoma herbarum ys4108 , has great antitumor potential via enhancement of host immune response , but little is known about the molecular mechanisms . in the present study , we mainly focused on the effects and mechanisms of ycp on the specific immunity mediated by dendritic cells ( dcs ) and t cells .
t cell /dc activation - related factors including interferon- ( ifn- ) , interleukin-12 ( il-12 ) , and il-4 were examined with elisa .
receptor knock - out mice and fluorescence - activated cell sorting are used to analyze the ycp - binding receptor of t cells and dcs .
rt - pcr is utilized to measure mage - a3 for analyzing the tumor - specific killing effect . in our study , we demonstrated ycp can provide the second signal for t cell activation , proliferation , and ifn- production through binding to toll - like receptor- ( tlr- ) 2 and tlr-4 .
ycp could effectively promote il-12 secretion and expression of markers ( cd80 , cd86 , and mhc ii ) via tlr-4 on dcs . antigen - specific immunity against mouse melanoma cells
was strengthened through the activation of t cells and the enhancement of capacity of dcs by ycp .
the data supported that ycp can exhibit specific immunomodulatory capacity mediated by t cells and dcs . |
metastasis to the bone is a common complication of breast , lung , prostate , gastrointestinal , haematological , melanoma , and other tumours , , . in many patients with primary breast ,
prostate and lung primary tumours , bone metastases ( bom ) are signs associated with the presence of disseminated disease .
these metastases to the bone will usually result in patients experiencing pain which is often palliated using single fraction radiotherapy ( sfrt ) or multiple fraction radiotherapy ( mfrt ) depending on the factors such as cancer type and presence of complication within the bone , , , .
although the distribution of bom have been observed in many skeletal locations , bom that are distal to the elbow and knee joints are considered rare and therefore are infrequently reported .
depending on the primary tumour histopathology , the frequency of skeletal bone metastases ranges from 10% to close to 90% and bone lesions are found more commonly within the axial skeletal versus the appendicular skeleton . although any skeletal bone may be involved with metastatic disease , the vertebral body is most commonly associated with metastatic disease ; long bone involvement or metastases distal to the elbow and knee are rare , , .
this study sought to assess the incidence of distal bone metastases treated with radiotherapy in a provincial programme .
all courses of rt for bone metastases from 2007 to 2011 for patients living in bc were identified in a provincial rt programme .
the bc cancer agency ( bcca ) information system ( cais ) was used to identify 8008 patients who received 16,277 palliative radiotherapy courses for bone metastases from 2007 to 2011 .
information retrospectively collected on these patients included : patient age , gender , primary tumour histology , fractionation schedules , and location of bone metastases .
treated bom were categorized as distal if the bom was located within or distal to the elbow or knee .
patients were grouped by primary tumour site as breast , lung , prostate gastrointestinal , haematological , melanoma , and other .
multivariable linear regression analysis was used to determine associations between primary tumour type and incidence of distal bone metastases .
p values were two - sided , and values less than.05 were considered statistically significant .
we used the spss statistical software package , version 19.0 ( chicago , il ) , for data entry and statistical analysis .
from 2007 to 2011 , 8008 patients in bc were treated with 16 , 277 palliative radiotherapy courses ; 425 ( 3% ) of the courses prescribed were used to treat bones located within or distal to the elbow or knee .
male patients were treated more often than female patients ( 52% ) and sfrt was prescribed 49% of the time ( table 1 ) .
breast and lung malignancies were observed most commonly ( 23% ) , followed by prostate ( 19% ) , other ( 15% ) , lymphoma ( 11% ) , gastrointestinal ( 8% ) and melanoma ( 1% ) ( table 1 ) .
we demonstrated that melanoma malignancies resulted in the highest frequency ( 5% ) of distal bom ; followed by haematological ( 3% ) , lung ( 2% ) prostate ( 2% ) , other ( 2% ) , gastrointestinal ( 1% ) , and breast ( 1% ) .
distal bom where more commonly identified in the lower extremity ( 87% , p<0.001 ) .
distal bom were treated with sfrt significantly more often than non - distal bom ( p<0.001 ) ( fig .
1 ) . table 2 presents the multi - variable analysis performed to determine the associations between distal bom and patient characteristics , which demonstrated that patients with melanoma have a significant higher odds of having distal bone metastases ( p=0.004 ) .
the regression analysis also showed significant correlation between having distal bone metastases treated at abbotsford centre ( odds ratio 2.37 , 95% confidence interval 1.543.64 , p<0.0001 ) ( table 2 ) .
this is the first large scale study to illustrate the incidence of distal bone metastases in patients being treated with palliative radiotherapy ( rt ) . from 2007 to 2011 , 16,277 courses of palliative rt was used to treat bone metastases in 8008 patients ; 425 ( 3% ) of which were rt courses for distal bom .
in addition , distal bone metastases were significantly more likely to be treated with sfrt .
this population - based study demonstrates that patients with melanoma have the highest proportion of distal bom .
this differs from previous reports , which only present that prostate , breast , and lung cancer have the highest absolute number of distal bom , likely because these are the most common cancers with any type of bom , , , , .
here we showed that sfrt was used to treat distal bom more often than non - distal bom ( 66% versus 49% , respectively ) , likely because distal bom are located in regions that do not contain organs at risk such as the spinal cord .
a weakness of this study is that untreated bom are not captured , and therefore the incidence of distal bom may be under - represented .
however , it is unlikely that the proportion of distal bom is markedly different than reported .
in addition , the large population - based nature of this study provides adequate power to identify correlations between distal bom and primary tumour type , while being relatively free of selection bias .
this population based study identified an incidence of distal bom of 3% among patients treated with palliative rt .
mb conceived of the study , participated in the data collection and statistical analysis , and designed and draughted the manuscript .
xxx participated in the acquisition of data , analysis and interpretation of data and helped draughted the manuscript . | purposethis study assesses the incidence of distal bone metastases in palliative radiotherapy ( rt ) patients.material and methodsall courses of rt for bone metastases from 20072011 for patient living in british columbia ( bc ) were identified in a provincial rt programme . treated bone metastases ( bom ) were categorized as distal if the bom was located within or distal to the elbow or knee .
patients were grouped by primary tumour site as breast , lung , prostate gastrointestinal , haematological , melanoma , and other .
the incidence of distal bone metastases and associations with primary tumour types were determined.resultsfrom 2007 to 2011 , 8008 patients were treated with 16,277 courses of rt , of which 425 ( 3% ) were courses of rt for distal bom .
the incidence of distal bom in decreasing order by primary tumour type was melanoma ( 5% ) , haematological ( 3% ) , lung ( 2% ) , other ( 2% ) , prostate ( 2% ) , breast ( 1% ) and gastrointestinal ( 1% ) .
distal bom where more commonly identified in the lower extremity ( 87% , p<0.001 ) .
single fraction rt was used more commonly for distal vs non - distal bom ( 66% vs. 49% ; p<0.001).conclusionthe incidence of distal bom among patients treated with palliative rt was 3% and most commonly identified in patients with melanoma and haematological malignancies . |
skeletal muscle tissues are responsible for the provision of postural control , the coordination of excitation - contraction - relaxation cycles for voluntary movements , the integration of key metabolic and biochemical pathways , and the regulation of heat homeostasis . under normal physiological conditions ,
hence , supramolecular protein complexes with specialized functions , structures , and connections represent a major biochemical feature of muscle fibres .
an excellent example of a large protein assembly present in skeletal muscle is the dystrophin - glycoprotein complex of the sarcolemma [ 15 ] .
the crucial importance of the dystrophin - associated protein complex is exemplified by the pathophysiological fact that primary genetic abnormalities in the dystrophin gene result in progressive muscle wasting diseases , such as duchenne or becker muscular dystrophy [ 68 ] . in normal muscle , the dystrophin - glycoprotein complex provides a trans - sarcolemmal linkage between the actin membrane cytoskeleton and the extracellular matrix component laminin .
the subsarcolemmal dystrophin matrix and the molecular connection between the basal lamina structure and the muscle interior is believed to prevent damage to the muscle surface from potential membrane - distorting forces during contraction - relaxation cycles . in x - linked muscular dystrophy ,
dystrophin deficiency results in a drastic reduction of sarcolemmal glycoproteins that triggers a loss of plasmalemmal integrity .
muscle fibres are more susceptible to contraction - induced injury and their lateral transmission of force is impaired .
cycles of sarcolemmal microrupturing and natural membrane repair mechanisms appear to cause the introduction of ca - leak channels that in turn elevate cytosolic ca - levels and disturb ca - fluxes through the sarcoplasmic reticulum in dystrophic fibres [ 1416 ] .
interestingly , a recent study on the therapeutic effect of upregulating the intramuscular heat shock protein hsp72 to ameliorate the dystrophic phenotype revealed that the serca - type ca - atpase is dysfunctional in severely dystrophic muscle .
these findings strongly indicate that impaired ca - homeostasis plays a key role in x - linked muscular dystrophy .
however , it is not well understood how many molecular and cellular factors are involved in the overall process leading to the highly complex pathology of dystrophinopathy .
thus , in order to determine the hierarchy of secondary pathobiochemical effects that render a dystrophic muscle more susceptible to necrosis , it is crucial to elucidate global alterations due to the disintegration of the dystrophin - glycoprotein complex .
mass - spectrometry - based proteomics suggests itself as a suitable analytical tool for such large - scale and high - throughput approaches to study the effects of dystrophin deficiency .
in contrast to hypothesis - based and targeted bioresearch , proteomics can be considered an unbiased and technology - driven approach for the comprehensive cataloging of entire protein complements [ 1921 ] .
skeletal muscle proteomics in particular is concerned with the global identification and detailed cataloguing of the protein constituents of voluntary contractile fibres in health and disease [ 2224 ] . in the long term , comparative proteomics
promises to be instrumental for the establishment of comprehensive biomarker signatures of myogenesis , muscle repair mechanisms , physiological adaptations and pathological changes , as well as the natural aging process .
most gel electrophoresis - based proteomic studies use high - resolution two - dimensional gel electrophoresis in combination with advanced mass spectrometric analysis for the unequivocal identification of muscle proteins of interest [ 2729 ] . in the case of x - linked muscular dystrophy ,
a variety of mass spectrometric investigations have attempted to determine proteome - wide changes in dystrophin - deficient muscle tissue in order to establish a dystrophy - specific biomarker signature [ 30 , 31 ] .
large - scale proteomic profiling studies have included investigations of serum [ 32 , 33 ] , cardiac muscle [ 34 , 35 ] , and various skeletal muscle tissues [ 16 , 3643 ] from the mdx mouse model of duchenne muscular dystrophy , as well as a proteomic analysis of dystrophic grmd dog skeletal muscle .
although the findings from individual studies do not agree on the exact number and extent of protein alterations within the dystrophic muscle proteome , all investigations concur that dystrophin - deficient fibres exhibit a generally perturbed protein expression pattern .
previous gel electrophoresis - based proteomic profiling studies of dystrophic samples have focused on the cytosolic fraction from 1 , 3 , and 6 months old hind limb muscle covering a pi range of 47 and using coomassie and silver - staining methods [ 36 , 37 ] , crude extracts from gastrocnemius muscle from 9 weeks old hind limb tissue covering a pi range of 310 and using stains - all labeling , preparations from 6 weeks old gastrocnemius muscle covering a pi range of 310 and using fluorescence 2d - dige labeling , crude extracts from 9 weeks old diaphragm tissue covering a pi range of 310 and using hot coomassie staining , crude extracts from 9 weeks old diaphragm muscle covering a pi range of 310 and using fluorescence 2d - dige labeling , crude extracts from 10 weeks old antisense oligomer - treated diaphragm tissue covering a pi range of 310 and using 2d - dige labeling , crude extracts from 9 weeks old extraocular muscle covering a pi range of 310 and using fluorescence 2d - dige labeling , and crude extracts from aged diaphragm muscle covering a pi range of 310 and using fluorescence rubps labeling . in analogy to the above - outlined proteomic studies , this report has focused on aged tibialis anterior muscle from dystrophic mdx mice .
the tibialis anterior is one of the most active lower leg muscles , which exhibits a relatively high degree of resistance to fatigue during periods of intense running , making it an interesting contractile system to study with respect to secondary effects of dystrophinopathy .
in addition , previous experimental gene therapy studies have focused on mdx tibialis anterior muscle .
labeling of proteins with fluorescent dyes has been extensively applied in proteomic investigations and we have used here fluorescent rubps staining for a comparative proteomic survey of dystrophic leg muscle from 8 weeks , 12 months , and 22 months old mdx mice .
the densitometric analysis of two - dimensional gels , covering a pi range of 310 , in combination with mass spectrometry identified significant age - related changes in carbonic anhydrase , aldolase , electron transferring flavoprotein , pyruvate kinase , myosin , tropomyosin , and the small heat shock protein hsp27 in dystrophic mdx tibialis anterior muscle .
materials and electrophoresis - grade chemicals for the proteomic analysis of muscle proteins were purchased from amersham biosciences / ge healthcare , little chalfont , buckinghamshire , uk . for protein digestion , sequencing grade - modified trypsin was obtained from promega ( madison , wi , usa ) . chemiluminescence substrate and
primary antibodies were obtained from vision biosystems novocastra , newcastle upon tyne , uk ( mab ncl - b - dg to the dystrophin - associated glycoprotein -dystroglycan ) and abcam , cambridge , uk ( ab54913 to the ca3 isoform of carbonic anhydrase ; and ab12351 to the small heat shock protein hsp27 ) .
all other chemicals used were of analytical grade and purchased from sigma chemical company , dorset , uk .
the mdx mouse is an established model system of x - linked muscular dystrophy and widely used in basic research and for the evaluation of novel therapeutic options to treat diseases of progressive skeletal muscle wasting .
the mdx mouse is a naturally occurring mutant that is missing the dp427 protein isoform of the membrane cytoskeletal protein dystrophin due to a point mutation in the dmd gene . in analogy to the etiology of patients suffering from duchenne muscular dystrophy , deficiency in full - length dystrophin results in a drastic reduction of all dystrophin - associated glycoproteins in mdx skeletal muscle , making it a suitable animal model for studying secondary pathobiochemical changes due to dystrophin deficiency .
dystrophic tibialis anterior muscle from 8 weeks , 12 months , and 22 months old mdx mice and normal tissues from age - matched c57 mice were obtained from the bioresource unit of the university of bonn .
mice were kept under standard conditions and all procedures were performed in accordance with german guidelines on the use of animals for scientific experiments .
animals were sacrificed by cervical dislocation and muscle tissues quickly removed and quick - frozen in liquid nitrogen . for the mass spectrometry - based proteomic survey of aged mdx skeletal muscle tissue ,
tibialis anterior specimens were shipped to ireland on dry ice and stored at 80c prior to usage . in order to obtain muscle protein extracts ,
4 dystrophic muscle specimens from each age group were pulverized by grinding tissue pieces in liquid nitrogen using a mortar and pestle .
ground muscle powder was solubilized in lysis buffer with the ratio of 100 mg wet weight to 1 ml lysis buffer ( 7 m urea , 2 m thiourea , 4% chaps , 2% ipg buffer ph 310 , 2% ( w / v ) dtt ) . to prevent excess protein degradation ,
following gentle rocking for 30 minutes , suspensions were centrifuged at 4c for 20 min at 20,000 g and the protein concentration determined .
for the separation of muscle proteins , standard two - dimensional gel electrophoresis was carried out by previously optimized methodology using first dimension isoelectric focusing with ph 310 strips , and second dimension slab gel electrophoresis with 500 g protein per gel .
twelve slab gels were run in parallel at 0.5 w / gel for 60 min and then 15 w / gel until the blue dye front had disappeared from the bottom of the gel .
postelectrophoretic staining for the total protein profile was performed with the fluorescent dye ruthenium ii tris bathophenanthroline disulfonate ( rubps ) . as described previously by rabilloud and colleagues ,
a stock solution of rubps dye was prepared . following washing twice for 5 min ,
gels were stained for 6 hours in 20% ( v / v ) ethanol containing 200 nm of ruthenium chelate .
gels were re - equilibrated twice for 10 min in distilled water prior to imaging .
fluorescently labelled proteins from aged mdx tibialis anterior muscle were visualised using a typhoon trio variable mode imager .
gel analysis was performed with progenesis 2d analysis software and protein spots with significantly altered expression levels were identified by mass spectrometry .
protein identification was performed with 2d protein spots from coomassie - stained pick gels , following counter - staining of rubps - labelled analytical gels .
excised protein spots were treated by standardized in - gel tryptic digestion for the generation of representative peptide mixtures .
excision , washing , destaining , and treatment with sequencing - grade trypsin were performed by a previously optimized method .
further recovery was achieved by adding 30% acetonitrile/0.2% trifluoroacetic acid to the gel plugs for 10 min at 37c with gentle agitation .
samples were dried through vacuum centrifugation and concentrated peptide fractions were then suspended in mass spectrometry - grade distilled water and 0.1% formic acid , spun down through spin filters and added to lc - ms vials for identification by ion trap lc - ms analysis .
the mass spectrometric analysis of peptides was carried out with a model 6340 ion trap lc / ms apparatus from agilent technologies ( santa clara , ca , usa ) .
separation of peptides was performed with a nanoflow aglient 1200 series system equipped with a zorbax 300sb c18 analytical reversed phase column using hplc - chip technology .
mobile phases used were a : 0.1% formic acid , b : 50% acetonitrile and 0.1% formic acid .
samples were loaded into the enrichment part of the chip at a capillary flow rate set to 4 l / min with a mix of solvent a and solvent b at a ratio of 19 : 1 .
tryptic digests were eluted with a linear gradient of 5% to 70% solvent b over 6 min , 70% to 100% solvent b over 1 min , 100% to 5% over 1 min .
a 5 min post - time of solvent a was used to remove any potential carry over .
the capillary voltage was set to 2000 v. the flow and temperature of the drying gas were 4l / min and 300c , respectively .
database searches were carried out with mascot ms / ms ion search ( matrix science , london , uk ; ncbi database , release 20100212 ) .
all searches used mus musculus as taxonomic category and the following parameters : ( 1 ) two missed cleavages by trypsin , ( 2 ) mass tolerance of precursor ions 2.5 da and product ions 0.7 da , ( 3 ) carboxymethylated cysteins fixed modification , ( 4 ) oxidation of methionine as variable modifiaction , ( 5 ) percentage coverage was set at over 10% , and ( 6 ) at least 2 matched distinct peptides .
all pi - values and molecular masses of identified proteins were compared to the relative position of their corresponding 2d spots on analytical slab gels .
one - dimensional immunoblotting was employed to verify key findings from the proteomic profiling of aged mdx tibialis anterior muscle .
gel electrophoretic separation and transfer was carried out with a mini - protean ii electrophoresis and transfer system from biorad laboratories ( hemel - hempstead , herts , uk ) .
muscle proteins were transferred to nitrocellulose for 70 minutes at 100 v and at 4c .
blocking of membranes was achieved with a milk protein solution ( 5% ( w / v ) fat - free milk powder in 0.9% ( w / v ) nacl , 50 mm sodium phosphate , ph 7.4 ) for 1 hour .
nitrocellulose sheets were washed and then incubated for 1 hour with secondary peroxidase - conjugated antibodies , diluted in blocking solution .
immunodecorated bands were visualized using chemiluminescence substrate ( roche diagnostics , mannheim , germany ) .
prior to the proteomic analysis of differently aged dystrophic tibialis anterior muscle preparations , the protein complement from extracts of normal muscle samples was gel electrophoretically separated and key proteins identified by mass spectrometry .
this procedure established a select number of reliable landmark protein spots of a typical proteomic muscle map for control purposes .
figure 1 shows a representative fluorescent rubps - labelled gel with the electrophoretically separated protein spot pattern of normal mouse tibialis anterior muscle .
major 2d protein spots were treated by in - gel digestion and the most abundant constituent of this area of the gel identified by mass spectrometry .
table 1 lists the names of identified muscle marker proteins , their international accession number , pi - values , their relative molecular masses , number of matched peptide sequences , percentage sequence coverage , and mascot scores .
identified proteins ranged in molecular mass from 17.1 kda ( spot 22 , myoglobin ) to 70.7 kda ( spot 2 , unknown protein ) , and covered a pi - range from pi 4.6 ( spot 20 , myosin light chain mlc3 ) to pi 8.7 ( spot 15 , fast troponin subunit tni ) .
spots 1 to 22 represent major muscle - associated protein species with apparent molecular mass to isoelectric point ratios of 57 kda / pi 5.2 , 71 kda / pi 5.8 , 59 kda / pi 6.7 , 47 kda / pi 6.7 , 43 kda / pi 6.6 , 40 kda / pi 5.8 , 33 kda / pi 4.7 , 33 kda / pi 4.7 , 37 kda / pi 6.2 , 40 kda / pi 8.3 , 36 kda / pi 8.4 , 30 kda / pi 6.9 , 23 kda / pi 5.6 , 23 kda / pi 5.6 , 22 kda / pi 8.7 , 23 kda / pi 5.7 , 21 kda / pi 5.0 , 19 kda / pi 4.8 , 19 kda / pi 4.8 , 19 kda / pi 4.6 , 12 kda / pi 5.0 , and 17 kda / pi 7.1 , respectively ( figure 1 ) .
electrospray ionization mass spectrometry identified these marker proteins as isoforms of mitochondrial atp synthase , pyruvate kinase , enolase , creatine kinase , actin , tropomyosin , malate dehydrogenase , aldolase , glyceraldehyde-3-phosphate dehydrogenase , carbonic anhydrase , triosephosphate isomerase , troponin , adenylate kinase , parvalbumin , myoglobin , and various myosin light chains ( table 1 ) . following the optimization and initial mass spectrometric identification of muscle marker proteins in normal mouse tibialis anterior muscle , fluorescence
high - resolution two - dimensional gel electrophoresis was employed to detect potential differences in aging - related protein expression patterns in mdx tibialis anterior muscle .
figure 2 summarizes analytical gels with 4 biological repeats of 8 weeks , 12 months , and 22 months old total mdx muscle extracts . panels ta mdx 1 to 4 , ta mdx 5 to 8 and ta mdx 9 to 12 represent 8 weeks , 12 months , and 22 months old muscle preparations , respectively . since the overall 2d spot patterns of normal versus dystrophic tibialis anterior muscle were relatively comparable , a detailed denitometric analysis was carried out in order to evaluate potential differences in individual protein species .
densitometric scanning was performed with a typhoon trio variable imager and progenesis 2d analysis software was used to establish differential expression patterns during muscle aging .
the detailed proteomic survey of dystrophic tibialis anterior revealed distinct age - related changes in 8 muscle protein species between 8 weeks and 22 months old total mdx muscle preparations .
a representative fluorescent 2d master gel of mdx tibialis anterior muscle is shown in figure 3 . as compared to a recent study on senescent mdx diaphragm muscle , which showed drastic age - dependent changes in 11 proteins in this severely necrotic tissue , the more mildly affected mdx tibialis anterior muscle showed less pronounced proteome - wide changes during the aging process .
this finding agrees with the differing pathology of mdx leg muscle versus mdx diaphragm muscle .
skeletal muscle proteins that exhibited significant alterations in expression levels are marked by circles and are numbered 1 to 8 in the 2d gel representing the urea - soluble proteome from mdx tibialis anterior muscle .
besides listing the names of identified proteins , their accession number , pi - values , their relative molecular masses , the number of matched peptide sequences , percentage sequence coverage , and ms / ms scores , this table also shows the fold change of individual proteins affected in dystrophin - deficient mdx tibialis anterior muscle during aging .
proteins species with a changed concentration in mdx tibialis anterior muscle ranged in molecular mass from 23 kda ( heat shock protein hsp27 ) to 224 kda ( myosin 3 ) and covered a pi - range from pi 4.7 ( tropomyosin ) to pi 8.5 ( electron transferring flavoprotein ) . an increased abundance was shown for the ca3 isoform of carbonic anhydrase ( spots 1 and 4 ) , the glycolytic enzyme aldolase ( spot 2 ) , and electron transferring flavoprotein ( spot 3 ) . the key cytosolic enzyme pyruvate kinase ( spot 5 ) , myosin 3 ( spot 6 ) , tropomyosin ( spot 7 ) , and the molecular chaperone hsp27 ( spot 8) were found to be decreased in mdx tissue .
following the mass spectrometric establishment of age - related changes in the urea - soluble mdx tibialis anterior muscle proteome , immunoblotting was used to investigate the concentration of the two most extensively changed new markers ca3 and hsp27 in normal versus dystrophic preparations .
antibodies to the dystrophin - associated glycoprotein -dystroglycan ( -dg ) , which forms the main trans - sarcolemmal linker between the extracellular matrix and the cortical actin cytoskeleton in the fibre periphery , were employed to verify the dystrophic status of mdx tissue samples during aging .
figure 4(a ) illustrates the drastic reduction of -dg in both 8 weeks and 22 months old mdx tibialis anterior muscle , which is characteristic of dystrophinopathy .
equal loading of lanes was ensured by silver staining of gel electrophoretically separated protein preparations ( not shown ) .
immunoblotting of young versus old muscle samples with antibodies to the ca3 isoform of carbonic anhydrase ( figure 4(b ) ) and the molecular chaperone hsp27 ( figure 4(c ) ) showed an increased abundance of the metabolic enzyme and a decreased concentration of the small heat shock protein in dystrophin - deficient muscle .
thus , both the fibre type - specific protein ca3 and the stress protein hsp27 represent suitable candidate biomarkers of the dystrophic phenotype .
duchenne muscular dystrophy is one of the most crippling neuromuscular disorders of childhood , therefore warranting detailed large - scale studies into the establishment of comprehensive biomarker signatures of dystrophinopathy [ 30 , 31 ] . in dystrophinopathy , the almost complete absence of the dp427 isoform of the membrane cytoskeletal protein dystrophin causes a drastic reduction of a large number of surface glycoproteins that in turn triggers a loss of sarcolemmal integrity .
the mdx mouse is a widely used model system for the evaluation of novel treatment options to counter - act the symptoms of x - linked muscular dystrophy and basic biomedical research promises to provide the basis of evidence for the development of novel treatment regimes , such as stem cell therapy , myoblast transfer , or exon skipping therapy .
previous proteomic studies have established a considerable number of novel biomarkers of secondary changes in dystrophin - deficient organisms . besides the cataloging of generally perturbed protein expression patterns , individual proteomic surveys of mdx muscles of differing subtype and age have demonstrated a drastically altered abundance of adenylate kinase isoform ak1 , the luminal ca - binding protein calsequestrin of the terminal cisternae [ 16 , 41 ] , the cytosolic ca - buffering element regucalcin , mitochondrial isocitrate dehydrogenase , and the muscle - specific molecular chaperone cvhsp [ 40 , 41 ] .
a recent aging study of the severely dystrophic mdx diaphragm has demonstrated a drastic increase in the extracellular matrix proteins collagen and dermatopontin , the molecular chaperone b - crystallin , and the intermediate filament protein vimentin , suggesting increased accumulation of connective tissue , an enhanced cellular stress response and compensatory stabilization of the weakened membrane cytoskeleton in severely dystrophic muscle tissue . in the present report
, proteomic profiling showed that during the natural aging of the moderately dystrophic tibialis anterior muscle a number of key skeletal muscle proteins change in abundance .
we have studied aged mdx muscle , because dystrophic mouse muscle tissue progressively deteriorates with age and thus more closely resembles the neuromuscular pathology seen in duchenne patients .
the age - related pathogenesis of mdx muscle is characterized by a drastic loss of myofibres and concomitant replacement by connective tissue [ 6264 ] , progressive motor weakness , the presence of branched fibres that trigger mechanical weakening of the muscle periphery , a reduced life span and increased susceptibility to spontaneous rhabdomyosarcoma , a decline in regenerative potential and alterations in the crucial mtor signaling pathway , and impaired functional and structural recovery after injury .
hence , senescent mdx muscle represents a suitable dystrophic phenotype for determining potential global changes in the protein complement during aging .
this report has summarized the findings of a comparative proteomic analysis of mildly affected mdx tibialis anterior muscle from 8 weeks versus 22 months old mice .
the identification of aldolase , pyruvate kinase , carbonic anhydrase , tropomyosin , myosin , electron transferring flavoprotein and small heat shock protein hsp27 as new indicators of progressive muscular dystrophy might be useful for the establishment of a more comprehensive biomarker signature of dystrophinopathy .
the protein with the highest age - related increase was identified as carbonic anhydrase isoform ca3 . in general ,
carbonic anhydrases catalyze the reversible hydration of co2 and are widely distributed throughout the body .
skeletal muscles express several isoforms of this crucial metabolic enzyme in a fibre - type - specific manner .
the predominant ca3 isoform is mostly present in the cytosolic fraction of type i and iia fibers .
interestingly , metabolic adaptations , altered neuromuscular activity patterns , stretch - induced hypertrophy , and disuse atrophy greatly influence the expression of muscle carbonic anhydrases [ 7173 ] . the higher concentration of the ca3 isoform of carbonic anhydrase in aged mdx muscle , as shown here by mass spectrometry - based proteomics , could be an indication of an increased demand for efficient co2 removal during mdx fibre aging .
on the other hand , since the ca3 isoform is predominantly located in slower - twitching fibre populations , its altered density could also be due to age - related fibre - type shifting in the mdx tibialis anterior muscle .
this would agree with the findings of a recent proteomic survey of middle aged versus aged vastus lateralis muscle , which revealed increased levels of ca3 in senescent human skeletal muscle .
the greatest reduction in a muscle - associated protein during aging of the mdx tibialis anterior was shown to be the small heat shock protein hsp27 .
this indicates a potentially blunted cellular stress response in dystrophic tibialis anterior fibres and demonstrates that marked differences exist with respect to expression levels of small heat shock proteins in moderately affected hind limb muscles versus severely dystrophic diaphragm muscle in the mdx model of dystrophinopathy [ 40 , 41 ] .
while changes in elements of the contractile apparatus suggest downstream effects of dystrophin deficiency on myosin and tropomyosin organization , altered expression levels in electron transferring flavoprotein and glycolytic enzymes indicate perturbed mdx muscle metabolism . the beta - polypeptide chain of the electron transferring flavoprotein is involved in mitochondrial fatty acid and amino acid catabolism and
the enzymes aldolase and pyruvate kinase catalyze the reversible break - down of fructose-1,6-biphosphate into dihydroxyacetone phosphate and glyceraldehyde-3-phosphate and the critical oxidoreduction - phosphorylation step that converts adp and phosphoenolpyruvate to atp and pyruvate , respectively .
alterations in glycolytic enzymes and mitochondrial proteins in mdx tibialis anterior muscle indicate altered flux rates through key metabolic pathway . with respect to the glycolytic pathway
, the activity of four muscle proteins is central to its regulation on the enzymatic level , i.e. the metabolic flux through hexokinase , phosphofructokinase , glycogen phosphorylase , and pyruvate kinase , whereby metabolic silencing of muscle glycolysis is probably mediated by the inactivation of pyruvate kinase .
this central role of pyruvate kinase in muscle metabolism makes its changed abundance in the mdx tibialis anterior muscle a crucial finding .
pyruvate kinase was previously shown to be a suitable biomarker of the general aging process in skeletal muscle tissues [ 77 , 78 ] .
interestingly , the change of metabolic enzymes in aged mdx tibialis anterior muscle , such as pyruvate kinase and aldolase , agrees with the proteomic analysis of golden retriever muscular dystrophy . in the grmd dog model of dystrophinopathy
, targets of the transcriptional control factor of energy metabolism pgc-1 , that is , various glycolytic and oxidative enzymes , were found to be reduced . in conclusion ,
the comparative proteomic survey of dystrophic tibialis anterior muscle from 8 weeks versus 22 months old mdx mice has revealed altered expression levels in a number of critical proteins during skeletal muscle aging .
however , the degree of concentration changes was less pronounced in the moderately dystrophic tibialis anterior muscle as compared to the recently analyzed aged mdx diaphragm .
these differing proteomic findings agree with the pathophysiological concept that the aged mdx diaphragm muscle is more severely affected as compared to moderately necrotic mdx hind limb muscle . in the long - term
, the proteomic identification of new biomarkers of dystrophinopathy might be useful for the establishment of a comprehensive and muscle subtype - specific signature of duchenne muscular dystrophy .
this would be useful for improving diagnostic procedures , monitor disease progression , identify novel therapeutic targets and aid in the evaluation of novel treatments , such as exon - skipping therapy or stem cell therapy . | x - linked muscular dystrophy is a highly progressive disease of childhood and characterized by primary genetic abnormalities in the dystrophin gene .
senescent mdx specimens were used for a large - scale survey of potential age - related alterations in the dystrophic phenotype , because the established mdx animal model of dystrophinopathy exhibits progressive deterioration of muscle tissue with age .
since the mdx tibialis anterior muscle is a frequently used model system in muscular dystrophy research , we employed this particular muscle to determine global changes in the dystrophic skeletal muscle proteome .
the comparison of mdx mice aged 8 weeks versus 22 months by mass - spectrometry - based proteomics revealed altered expression levels in 8 distinct protein species .
increased levels were shown for carbonic anhydrase , aldolase , and electron transferring flavoprotein , while the expressions of pyruvate kinase , myosin , tropomyosin , and the small heat shock protein hsp27 were found to be reduced in aged muscle .
immunoblotting confirmed age - dependent changes in the density of key muscle proteins in mdx muscle .
thus , segmental necrosis in mdx tibialis anterior muscle appears to trigger age - related protein perturbations due to dystrophin deficiency .
the identification of novel indicators of progressive muscular dystrophy might be useful for the establishment of a muscle subtype - specific biomarker signature of dystrophinopathy . |
over the last twenty years , china has achieved unprecedented economic growth , with an accompanying growth of large numbers of wealthy and middle classes , which has led to the requirement of the building of a well - off society in a comprehensive way . to this end , china is currently in the process of reforming its health care system by equipping its hospitals with many modern medicine systems , specifically , medical imaging facilities , as well as building their own . because of the size of its territory and the number of its population as well as the uneven development of economy across the country , the distribution of the modern medical facilities allocates mainly to large cities , such as beijing and shanghai . in order to reach those remote regions , china has begun the development of telemedicine techniques in the late 1980s . in its first attempt taking place in the first decade ( 19902000 ) ,
the main focus resides on the implementation of communication networks with a faster and wider bandwidth , such as isdn ( integrated services digital network ) , in the hope to connect far and wide . as a direct result , built on this digital network service , teleeducation , teleconferencing , and teleconsultation
have flourished albeit mainly as a show case between a number of leading institutes [ 3 , 4 ] . with the advent of world wide web ,
many internet - based services are available and more importantly are free , such as skype , making the services of teleconferencing / consultation not only affordable but also flexible and portable , that is , a network connection being able to set up in an operation room instead of confining to a conference room , bringing visions of practical applications , such as telesurgery into a reality .
subsequently , the first case of teleneurosurgery took place in 2005 between beijing and yan'an with a distance of around 1300 kilometres , performing an operation of tumour removal via a keyhole .
firstly , europe originated imaging field when the nobel laureate , physicist wilhelm roentgen , discovered x - rays that eventually led to the birth of radiology , and thereafter the medical imaging industry . with the application of advanced computer techniques since 1970 , computerised tomography ( ct ) and magnetic resonance imaging ( mri ) were invented , leading to two more awards of nobel prize winners shared between the uk and the usa . with around 80,000 2d images ( e.g. , in geneva hospital ) generated per day
, picture archiving and communications systems ( pacss ) have been developed to manage them in the mid-1980s . by 2005
, most european countries have installed pacs in their hospitals with norway topping the chart with 100% hospitals .
elsewhere more than 70% hospitals in the countries of united kingdom , germany , and italy are equipped with pacs . on the other hand , by 2005
, pacs started to be appreciated in china and to be installed in full version among many leading hospitals . before that , only mini - pacs ( a standalone version ) had been installed .
to a certain extent , europe is also leading the way in ict . in december 1990 , british physicist tim berners - lee created world wide web while working at cern in switzerland , allowing computers to talk to each other through a new language html ( hyper - text markup language ) , which has heralded a new era and changed the way people live forever .
in addition , in 2009 , charles kao , aka the godfather of broadband , was awarded half of nobel prize in physics for his groundbreaking achievements concerning the transmission of light in fibres for optical communication while working at the uk , revolutionised the telecommunications industry , and paved the way to the advent of today 's information age . as of 2010 ,
submarine cables that are laid beneath the sea have linked all the world 's continents , connecting the world into one . in terms of the application of ict to medicine
although the following accounts are neither comprehensive nor conclusive , it is in the intention to be representative . in united kingdom , the national health service ( nhs ) has established a unique national healthcare system providing free health for all since 1948
however , at the advent of the new century , thanks to the accumulated funding constraints and the increased aging populations , the nhs faced long patient and operation waiting lists , shortages in hospital beds and community care , and inadequate medical facilities in intensive care and emergency units as well as nursing staff . this fundamental change has led to the development of the uk 's largest e - health project , nhs direct , which has been in operation since 2000 .
it is a 24-hour nurse - led telephone help line supported by a website , http://www.nhsdirect.nhs.uk/ , covering england and wales . a separate service , nhs24 , http://www.nhs24.com/ , serves scotland
. it currently handles more than half a million telephone calls per day with over 3 million web transactions each month . because of the huge impact it has made to society ,
nhs direct has been acclaimed as the largest and most successful healthcare provider of its kind , anywhere in the world . in the event of collaboration with asia on telemedicine , two european projects , time and width ,
the project time ( teleimaging in medicine a cyber bridge interfaces china with europe on collaborative health care ) ( http://www.mitime.org/time/index.htm ) was funded by ec under asia ict programme between 2005 and 2007 and has borne fruit in a number of publications [ 1215 ] . as a follow - up project , width ( warehousing images in the digital hospital : interpretation , infrastructure , and integration ) is funded by ec fp7 under people marie curie programme to start in may 2011 .
width has 6 european partners and 5 chinese including from the uk , switzerland , germany , greece , norway , italy , and china . on the other hand , in switzerland , in 2001 , the university hospitals of geneva in switzerland and french - speaking countries in africa initiated the raft project ( rseau en afrique fancophone pour la tlmdecine ) by building a multinational telemedicine communication network in order to facilitate distance learning and teleconsultations through the internet - based platform . by 2005
, the network grid had been extended to cameroon , ivory coast , madagascar , and djibouti ( http://raft.hcuge.ch/ ) . as a direct result , through ustilising the internet - based technologies , distance learning and teleconsultation
, this network has connected 18 african countries , extending its activities from french - speaking to english - speaking countries . in 2004 , trieste university at italy hosted an international conference on europacs-2004 in the enlarged europe and attracted more than 400 participants from 47 countries .
trieste university has a long history in the development of open - pacs , in an attempt to initiate an open , scalable , and universal system with accompanying tools to store , exchange , and retrieve all health information .
elsewhere , the project e - learning at university of pisa has developed a niche interactive system for uploading and consulting teaching materials that can be applied to performing self - evaluation tests and exams , targeting at medical students .
furthermore , the university of pisa , division of diagnostic and interventional radiology ( http://www.rad.unipi.it/ ) has cofunded endocas ( centre for computer - assisted surgery ) , with a goal of implementing an imaging - assisted surgery ( ias ) systems to provide information help , action help , and training help , offering assistance on planning surgical intervention , integrating mechanic components of the robots , and simulating complex environment for surgical training , respectively .
norway has a long history of development of telemedicine thanks to its nature of national geography with an elongated shape , forming one of the longest and most rugged coastlines in the world . as early as in the 1990s ,
norway pioneered projects with teleradiology services , which had been in great demand when it comes to consultation in the situations of emergencies , seeking for second opinion and information retrieval between hospitals and the primary health care units . by the end of 2005 ,
nearly all the hospitals in norway had digital x - ray scanners with ris and pacs installed . moreover ,
all the regional health network could communicate with the national health network . in 2006 , in norway , trondheim successfully organised the 24th europacs conference where time project hosted a workshop .
in many ways , the situation in china is quite different from the other countries .
table 1 lists a number of key demographic data in both china and the uk for the purpose of comparison .
being the most populated country in the world , china accommodates 1.33 billion people , representing 20% of the world population .
however , china also has the 3rd largest area in the world , leading to the fact that the density in china is nearly as half as that in the uk with a ratio of 0.55 between the two countries , indicating that people are living far more apart in china than in the uk .
however , in terms of bed numbers in the hospitals , china has half as many beds as in the uk with only 1.4 doctors allocated to per 1000 people . whilst in the uk ,
2.3 doctors are assigned for per 1000 people . with only 6.1% hospitals in china that have been equipped with pacs in comparison with more than 70% in the uk , china is far behind when it comes to the implementation of modern advanced medical equipment with a ratio of 0.087 between the two countries .
hospitals in china are categorized into three four - level grades by the chinese ministry of health who conducted the classification according to the facility , equipment , and staffing that each hospital can offer .
the grades as shown in table 2 include grades 1 , 2 , and 3 with the highest grade being 3a+ that refers to be at an international leading position , whereas grade 3a is expected to be the leaders nationally .
there are only two hospitals that are classified as 3a+ , which are beijing union hospital and beijing hospital 301 of pla . on the other hand , 772 hospitals are classified as grade 3a . in total , as of 2008
, there are 19,246 hospitals and 2 million doctors in service in china . within these hospitals , there are 2.2 million beds with 149 ( 1% ) hospitals having beds over 800 ( grade 3a/3a+ ) and 1930 ( 12% ) offering beds between 300800 ( grade 2a/3b ) . within grade 3 hospitals ,
radiology departments are in place to be responsible for acquiring medical images from modern medical image scanners , including ct ( computerised tomography ) , mr ( magnetic resonance imaging ) , and/or pet ( positron emission tomography ) as well as from film digitisers to digitise x - ray films and many other forms of pictures / images .
figure 1 displays a plot of official statistical data on the number of hospital beds and doctors distributed in both urban and rural regions from 1952 to 2000 in china .
it can be seen from the figure that there is a big gap between rural ( green patterns ) and urban ( in red ) regions . in terms of the number of beds ,
the numbers in the rural areas , in particular from the 1980s , have decreased , whereas in the cities , the number has doubled since the 1980s . on the other hand , the gap between the numbers of doctors is increasing with more and more doctors placed at city hospitals . at the meantime ,
the number of medical doctors and physicians in the counties stagnated or even declined since 1988 .
telemedicine stems from technologies of communications and computer information , within which the clinical care is delivered via a line - telephone , wireless mobile phone , video - conferencing equipment , or internet between medical specialists ( and patients ) in two or more different locations ( e.g. , countries ) for the purpose of conducting consulting , remote medical procedures or examinations .
hence , to provide any kind of teleservice , a telecommunication facility has to be in place first .
china has 1.33 billion people that are distributed over 22 provinces , five autonomous regions , and four metropolitan municipalities .
such a vast population and territory sets a correspondingly vast challenge to keep people in touch with each other .
therefore , starting from late 1990s , china has established more than 2 million kilometres nation - wide optical cable network , built - on asynchronous transfer mode ( atm ) , synchronous digital hierarchy ( sdh ) , and dense wavelength division multiplexing ( dwdm ) technologies , as well as several submarine cables , enabling communications using land - line telephones . in terms of networks that are employed for the purpose of telemedicine , there are three major routes , including the golden health network ( ghn ) , the international medionet of china ( imnc ) network , and the peoples ' liberation army ( pla ) telemedicine network . since the implementation in 1997 , imnc has been widely utilised to telecommunications between medical specialists .
subsequently , there are around 300 hospitals ( 1.5% ) registered on this line across china .
because it is primarily a telephone line employing a low bandwidth , the major activities between these hospitals have been limited to communications with textual data , yielding the network being analogous to an internal telephone line . in order to make the presence of telemedicine felt ,
many hospitals in china have acquired video - based teleconferencing systems in an attempt to deliver telemedical services .
because of the high cost of proprietary video conferencing systems , the hospitals that can afford to install them in china are more likely to be the least that are in need of advice due to their own rich supply of expertise and resources .
furthermore , although china has a small number of telemedicine systems in many of these leading hospitals , they hardly communicate with each other because of different standards of hardware and software that are employed in china , yielding that these systems are essentially standalone . to access rural and regional medical centres ,
connections have to draw from the existing resources that are in considerably less quantity for many hospitals in the rural regions .
because of the wealth that china has grown since the 1980s , china has tapped into a fashion - conscious market , with special interest in those high - tech gadgets . as a result ,
unprecedentedly , china has around 833 million mobile phone users ( 63% ) , thanks to the satellite network systems .
the primary service of a mobile phone is to transfer voice data , though other services have also been deployed , including email reading , web accessing , and messaging .
these services however are provided at a higher cost since internet surfing involves downloading a number of pictures , taking longer to complete , in comparison with landline services . on the other hand , with such large army of mobile users ,
personal health systems can be exploited in the future in china based on the wireless network . as of 2010 , china has 420 million internet users ( 31.6% of its population ) with 277 million accessing web pages via cell phones . since broadband is the most popular way to access the internet with a wired connection , with 98.1% of wired internet users choosing broadband , a total of 364 million people are now online in china , leading china to being the second largest market of internet users after usa with major activities covering online chat , gaming , and internet surfing . because of the very low cost of internet via land lines , web - based practices of telemedicine is much feasible in china , especially when many online meeting applications , such as skype , are at the moment free available . in comparison
the first generation of telemedicine in china started off in 1995 when a case of text - based consultation via emails had been reported , sending from beijing to the us and later the whole world concerning a patient with heavy metal poisoning [ 3 , 4 ] , by which a medicine was later recommended and obtained that eventually cured the patient .
considering the high cost to setup a teleconferencing suite , internet - based applications in medicine are opted for in china in the late 1990s .
it took off from a much publicised event of teleconsultation based on internet in 1998 , by which doctors from both china at xi'an medical university hospital and the usa , stanford university health care , conducted a conferring and reviewing session discussing the cases of two critically ill children , by the application of audio , video , and whiteboard , the emerging facilities at the time . although feasible , much of the event is to prove the variability of webcast architecture , leading to a number of subsequent applications being with the same intention of demonstrations .
the real turning point of telemedical applications in china came in 2005 when telemanipulation / teleneurosurgery was conducted to remove brain tumours using image - guided keyhole surgery technique , after informed consent had been obtained in advance with local ethical committees .
the operating distance is 1300 kilometres away in yan'an , a mountainous region , from beijing , with a home - made frameless stereotactic surgical robotic system cas - bh5 .
the transmission of neuronavigation data , planning , monitoring , and manipulating is done through a digital network with a speed of 2 mbyte / s on the platform of internet . in total
, 10 patients were operated that year with 90% patients improved neurologically with no complications based on the 12-month postoperation followup .
when comparing with local operations where 93.3% improvement is achieved , drawing from over 1000 cases , the accuracy of telesurgery is very much similar to the conventional operation although not conclusive .
with the advances of information and imaging technology , application of robotic systems to the health sector is a burgeoning field in assisting surgeons manipulating , monitoring , and/or guiding operations with the advantages of being higher precision of targeting , persistence of longer duration , and the ability of preoperative planning drawing from patients ' images . as of 2001 , there are around 270,000 cases of image - guided robotic operations conducted per year worldwide . in china , around 6.1% hospitals ( grade 3 ) ( as opposed to 70% in the uk ) are equipped with these modern imaging scanners due to its higher maintenance cost incurred on not only the hardware , but also personnel who will be able to operate the scanners , to perform some repair , and to write some computer code if necessary . therefore sharing
teleneurosurgery is one of the applications in terms of sharing techniques on medical images , telecommunications , and robots . unlike keyhole approaches applied on many other organs ( e.g. , in the abdomen ) , where a microcamera can be inserted to provide an augmented view , for a brain which is a compact organ and extremely high value ,
there is simply no room to accommodate any extra instrument as every tissue in a brain plays an important role to ascertain a person 's normal life .
minimal invasion and sacrifice of healthy tissues are hence the prerequisite for a brain intervention .
therefore , it took much longer in the domain of brain to have a keyhole surgery than in many other domains .
the first case of image - guided neurosurgery took place in 1985 in the usa for the procedure of biopsy using the robotic stereotactic technique , that is , to probe the tumour with a tip through a small burr hole drilled on the skill . in
order to see inside a brain while performing the keyhole surgery , by which a needle / catheter was introduced through the hole to extract tissues , surgeons relied on ct images that performed scanning intraoperatively where the robotic system was mounted on the gantry of the ct .
although this system was applied only for biopsy at the time , it has potentials to progress further .
because it is a robotic system , that is , controlled by a computer , it provides the possibility of being carried out
remotely , giving rise to the name of telerobotic system when coupled with the advances of computer technique .
since a brain resides inside a skull , many approaches have to be developed in order to see and locate the exact position in a brain .
the method employed an external , three - dimensional frame of reference to locate a position in the brain and was utilised on animals as early as in the 20th century .
the frame is a mechanical apparatus attached to an animal 's head and incorporates a cartesian coordinate system . in this way , the movement of a probe to be inserted into the brain can be monitored . however , the variations between the landmarks on a skull and the targets in the brain vary considerably from person to person and from time to time even for the same person , the application of stereotactic device to humans only began in the 1950s .
since then , a number of stereotactic frames have been developed worldwide that can be adjusted manually .
figure 2(a ) illustrates a frame with fiducial markers , which is applied in the navy general hospital in china . to increase the contrast of frame landmarks while being scanned using either ct or mr , contrast agent of copper sulphate solution
is usually filled in the frames of n shapes . in figures 2(b ) and 2(c ) , the positions on those n shape frames are shown on a ct and an mr images ( the dots ) for the same patient and at the same head position , which will be referenced for the procedure of registration in a later stage . to apply such a frame in a surgery ,
two physical spaces have to be registered together to ensure the coordinate systems can be transformed from one to another .
one space is for the frame ( or head space ) , whereas another is for the brain ( brain space ) .
since the brain space is unseen , brain atlases have therefore been developed to help navigating inside a brain . because of the variations in brain size , shape , and status of diseases , a position based on a brain atlas of one size
tends to give considerably deviations from the target , leading to a declined application of such technique to neurosurgery .
the advent of imaging techniques , especially ct and mr , in the early 1980s , has renewed the use of stereotactic apparatus into neurosurgery , where the brain atlases can be entailed by a patient 's brain images , leading to a patient - specific registration of the brain targets . in the event of registration ,
the calculations involve the mapping between a patient 's head ( frame space ) , brain , and images ( image space ) , which are achieved with the help of landmarks as shown in figure 2 , yielding the development of many accurate approaches of registrations , taking forms of linear , affine , and deformable .
on the other hand , wearing a frame is cumbersome in both a scanning suite for acquisition of images and an operation room , while , in the process of a surgery , frameless stereotaxis has gradually replaced the frame . in this way , landmarks ,
that is , fiducial marks , on a skull have to be defined in advance to assist geometric registrations .
significantly , with the benefit of advances of computer hardware and software , frameless stereotactic can achieve the same precision as that with a frame .
in addition , the visualization of 3d volumetric image data preoperatively obtained offers surgeons with surgical planning , tumour boundary delineation , and optimised route to minimise the risk to healthy tissues in the brain .
figure 3 demonstrates the methods using both frame ( a and c ) and frameless stereotactic neurosurgery . in total , about 4,000 operations with frame and 1,500 with frameless stereotactic have been performed at navy general hospital at beijing china , led by professor zengmin tian , the vice president of ngh . with the help of a frame or frameless stereotactic system ,
minimally invasive techniques to remove brain tumour are achievable by locating a precise position on a skull ( and hence the target in the brain ) .
the procedure normally involves the drill of a burr hole ( 110 mm ) , which usually being done manually . because of the lengthy procedure of an operation
, surgical dexterity can be limited to a certain extent by surgeons ' physiological tremor .
therefore , robotic hands or robots have been introduced into the operation theatres in the late 1980s [ 35 , 36 ] , which could replace stereotactic frame and complete precise position location , and precision bone drilling , and are defined as programmable multifunctional manipulators designed to move material , parts , tools or specialized devices through variable program motions for the performance of a variety of tasks [ 37 , 38 ] . in china ,
the application of robotic system for neurosurgery began a decade later than in europe and the usa , which is mainly confined to tumour resection to achieve minimally invasive operations , specifically , the removal of craniopharyngioma , a benign tumour derived from pituitary gland embryonic tissue .
although , at the time , commercially , such a system was available , such as robodoc , the cost was beyond their considerations .
hence , from 1997 to 2007 , in collaborating with beijing university of aeronautics and astronautics , navy general hospital developed their own robotic systems from cas - r-1 and cas - r-2 ( acronym of computer assistant surgery - robot , type 2 ) to cas - bh5 as depicted in figure 3 ( private communications ) .
the robotic system employs a six - jointed plc - controlled ( a programmable logic controller ) and motorised robotic arm ( figure 3(a ) ) as well as a software system that is responsible for location calculation , markers recognition , surgical planning , and intraoperative navigation .
the robotic arm is designed to mimic the joints of a human arm comprising of a shoulder ( joint 3 ) , an elbow ( joint 2 ) , and a wrist ( joint 1 ) , allowing near unlimited range of angular movements and the smoothness of the motion .
in addition , the robot is mounted on an external support ( joint 4 ) incorporating a base ( joint 5 ) that is positioned in such a way as to avoid interference with surgeons ' manoeuvres .
prior to a surgery , four markers attached to a head are applied to position all joints on a trajectory to the intended targets , that is , to allow an accurate registration between a robotic arm , head , and imaging - defined targets . in this way ,
a specially designed probe attached to joint 6 ( the tip ) is able to perform the tasks of biopsy or tumour resection ( red circle in figure 3(c ) ) . on the other hand , to create a skull opening , a power twist drill is passed through the bushing of the probe holder on joint 6 where skull perforation is performed in the exact trajectory to be followed by the probe . in principle , the probe holder can be positioned at any distance from the skull .
as soon as the desired positioning is selected , the robotic software system calculates the distance necessary to reach the intended target within the brain .
the necessary probe length can then be transmitted manually to the probe that is then introduced into the brain manually , or the probe can be fixed to the robot arm whereby the probe introduction can be carried out by the robotic motor system . in terms of the time spending on positing a robot , by using a plc - controlled motor , it takes only a few seconds to move a robotic arm into the intended operative field , to perform probe on the trajectory marked on the skull and to move it out of the area .
such a technique makes it easy to mark the intended site of cranial opening , move the robot out of the field , create the necessary cranial opening , and then rapidly move the robot arm back into the field .
the surgeon thus has unobstructed access to the patient 's head but has the ability to return the robotic arm quickly to the field as desired .
the accuracy of the robotic arm in reaching the target is within the error range of 0.32 mm between planned and actual target [ 39 , 40 ] .
the first case of operation on tumour removal was carried out in china in 1997 with a custom - made robot , cas - r-2 . in the following 10 years , over 2000 cases of operations
the advantages include higher accuracy of targeting , being able to conduct presurgical planning , and synchronised movement between a patient 's head , images , and robotic hand ( robot space ) .
figure 5 illustrates part of tumour that has been dissected as intended using the stereotactic robotic technique . at figures 5(a ) , 5(b ) , and 5(c ) ( t1 mr ) , a tumour occurring in the central part of the brain on different brain locations ( pointed by arrows ) is shown , whereas the bottom row ( t2 mr ) gives detailed information to depict the partial removal of the tumour .
significantly , the advent of robotics in an operating room provides the ability to position the probe - directing system rapidly , to compute the necessary trajectory and target coordinates in an error - free manner , and to remove tumour more completely or as intended .
this has led to the possibility of a robot being controlled remotely , that is , to carry out teleneurosurgery . as a result , in 2005
, the cas - r-2 system was modified into cas - r - bh5 ( figure 4(d ) ) that was employed in performing the first series of teleneurosurgery in china , exclusively for removal of tumours and taking place between beijing where navy general hospital resides and yan'an , a mountainous city , with the distance of 1300 km in between . with five degrees of freedom ( that is , 6 joints ) and dimensions of 280 800 1100 mm , cas - r - bh5 weighs about 40 kg and comprises a robot hand and two sensors that register the robotic space with head space as well as brain mr images .
the system runs three modules that are responsible for conducting surgical planning , target directing , and telemanipulation operations , respectively .
module 1the system of surgical planning offers surgeons with expedient tools to store , retrieve , and analyse relevant data , to study collectively the case history , to visualise reconstructed 3d images in order to define the boundary of a target for more completed removal , and to locate contrally the brain tumours by the safest and least invasive route possible .
the system of surgical planning offers surgeons with expedient tools to store , retrieve , and analyse relevant data , to study collectively the case history , to visualise reconstructed 3d images in order to define the boundary of a target for more completed removal , and to locate contrally the brain tumours by the safest and least invasive route possible .
module 2subsequently , the module of target directing is concerned with robotic arm in arriving at the precise position of the skull to perform the opening and pointing to the direction of the route defined by module 1 .
furthermore , via a rapid and accurate measurement and calculation , the length of the probe ( catheter ) is determined to minimise the trauma to normal tissues .
subsequently , the module of target directing is concerned with robotic arm in arriving at the precise position of the skull to perform the opening and pointing to the direction of the route defined by module 1 . furthermore , via a rapid and accurate measurement and calculation , the length of the probe ( catheter ) is determined to minimise the trauma to normal tissues
.
module 3 in light of communications , module 3 focuses on telemanipulation systems , spanning from network communication , video transmission , and graphics simulation to human - machine interaction , providing technical supports for surgeons who are performing the teleoperation .
in light of communications ,
module 3 focuses on telemanipulation systems , spanning from network communication , video transmission , and graphics simulation to human - machine interaction , providing technical supports for surgeons who are performing the teleoperation .
the teleneurosurgery system is composed of two computer terminals , one being the master to conduct the remote guiding and controlling and another being the operation terminal residing at the same room as the patient , to be applied to conduct the resection of physical tumour .
the platform of telecommunication is the internet with the speed of transmission being 2 mbytes / s .
although it is lower than the current standard of broadband with a typical speed of 20 mbyte / s , the transmission was good enough to allow real - time online visualization and communications .
, that is , within 24 hours , the patient should have the brain images taken , which can be done by any hospital nearby that has the scanning facility .
slave to master : firstly , the patient 's data ( e.g. , mr / ct scan , history record , and the live view of patient in the operation room ) from slave computer are transmitted to the master one .
master to slave : the experts send back the surgical plan and confirmations of surgical procedures as shown in figure 6(b ) , including the precise movement , and the robot arm should comply in order to arrive at the exact location predefined from module 1 on the skull of the patent .
slave to master : the registration data between robotic arm and the fiducial markers on the patient 's skull to ensure the error was below the predefined threshold . otherwise , the following step is repeated .
master to slave : a series commands that the arm should follow in light of six joints instructed from master to slave then to the robot , for example , forward / backward , left / right , up / down , side tilting , anterior / posterior tilting of the supporting device itself , and forward / backward of the probe .
the speed of the movement of robotic arm was at 8 mm / s . before the surgery
, 4 markers are attached to the patient 's skull , which are employed to track the movement of robot arm in relation to the brain images . after the induction of local anesthesia on the skull , the patient 's head is rested stably on an operative frame ( e.g. , a cushion ) .
the robot then automatically registers and verifies the markers via the visual camera and sends back to the master site with registration data to be checked .
tumour removalafter the registration error is below the predefined threshold , the surgeons at the operation room take over and perform subsequent actions : incising the skin , opening a burr hole , equipping robot arm with a catheter , directing into the parenchyma , and extract tumours in the form of liquid .
these surgeons in the operation room have been trained in advance with the knowledge of stereotactic neurosurgery and are capable to handle any intraoperative complications . on the other hand , the experts at the master site are monitoring the process closely online via the camera mounted in the operating room and lending a helping hand both visually and audibly .
the patient undergoes a postoperation ct scan of thin - sliced to ensure the tumour has been resected according to the plan by comparing with the preoperative scans as illustrated in figure 5 .
after the registration error is below the predefined threshold , the surgeons at the operation room take over and perform subsequent actions : incising the skin , opening a burr hole , equipping robot arm with a catheter , directing into the parenchyma , and extract tumours in the form of liquid .
these surgeons in the operation room have been trained in advance with the knowledge of stereotactic neurosurgery and are capable to handle any intraoperative complications . on the other hand
, the experts at the master site are monitoring the process closely online via the camera mounted in the operating room and lending a helping hand both visually and audibly .
the patient undergoes a postoperation ct scan of thin - sliced to ensure the tumour has been resected according to the plan by comparing with the preoperative scans as illustrated in figure 5 .
figure 7 presents four screenshots showing the whole process of the teleoperation , where ( a ) shows the control room where surgical planning takes place ; ( b ) shows the computer screen showing the activities in the remote operation room ; ( c ) shows telementoring and tele - manipulation ; ( d ) shows activities in the operation room . the clinical outcome
is then graded based on glasgow outcome scale ( gos ) , a 5-point score given to victims of traumatic brain injury at some point in their recovery .
these 5 points include vegetative state ( meaning the patient is unresponsive , but alive ; a vegetable in lay language ) , severely disabled ( conscious but the patient requires others for daily support due to disability ) , moderately disabled ( the patient is independent but disabled ) , good recovery ( the patient has resumed most normal activities but may have minor residual problems ) .
gos is a very general assessment of the general functioning of the person who suffers a head injury . between 2005 and 2006 , 32 patients underwent surgery of tumour removal at yan'an with this technique of telesurgery and were all successfully recovered without any complications , the results based on the followups for the patients , which took place between 3 to 14 months after the operations with the average of 12 months .
the mean accuracy of remote fiducial registration is within the range of 1.50 mm , and the standard deviation is 0.32 mm between the planned and actual target .
more specifically , in 2005 , according to the results of 10 patients who were operated on using teleneurosurgery procedure , four patients recovered nearly completely with 5 points on the gos scale , whereas three with 4 gos scores .
in china , navy general hospital is the only one that has the skill to perform keyhole neurosurgery . given the size of the area in china , it would be very difficult for some patients both economically and physically to go to beijing should not for the availability of teleneurosurgery technique , especially that many of patients are young children .
likewise , due to the workload the operation team have in their own hospital , it is very impractical for them to travel around whole china to perform the operations .
therefore , in china , teleneurosurgery is one way forward towards the development of telemedicine , balancing the disparity of medical care distributions . on the other hand , although the techniques of telemedicine / telesurgery are available , the calls for employing them vary from country to country .
for example , in the uk , medical resources are evenly distributed across the whole country and are provided by the nhs , coupled with adequate emergency systems that are equipped with fast means of transportations .
therefore , a patient can be easily located to an intended nearby hospital within required time .
many places , for example , mountainous regions , are difficult to locate even with several means of transportations . significantly , china is still in the developing stage and has a long and bumpy way to go in order to allow everyone access their much needed health care , especially for those 70% peasants who live in the remote areas , far afield from those big cities where modern medical equipment are concentrated . in this case , china should be in favour of the services allowing telemedicine / teleservices to be provided over the internet . by this way , it can save time , personnel , and cost substantially without compromising patients ' safety . considering that over 300 million people are with land telephone line and over 800 million are with mobile phones , teleservices in medicine have huge potentials in china .
one of the promising areas is telesurgery like the one pioneered by the navy general hospital in beijing .
the strength of the approach lies in the knowledge transfer . due to the limited number of experts in the field ,
in particular , it has the ability that one expert team can supervise several telesurgeries that are operated at different locations at the same time , reducing patients ' waiting list , prolonged suffering , and the cost of experts ' time , while maximising the usage of existing resources . in the developing countries
, telemedicine remains to be a way forward to allow health care services being accessed evenly by the people . to this end ,
collaboration with the developed countries is one of the future trends in order to acquire expertise , knowledge , and experience with less cost and in a short - time period .
due to the disparity of economic development in both europe and china , china has to develop telemedicine systems that fit its purpose instead of simply copying the systems that work well in the other countries .
for instance , at the uk , the success of nhs direct owes much to the fact that everyone in the uk ( and many other developed countries ) is connected with 100% of households having land - line telephones and 122% people have mobiles ( many users have more than one handset ) , which proffers advices to anyone who has any doubt on their health . whereas in china , because of the huge number of the population , large number of knowledgeable nurses has to be required to provide the service , which is highly unlikely .
in addition , many peasants who live remotely do not have any means of communications ( more than 30% households without land - lined phones ) nor electricity . centralised local or regional medical centres hence remain to be the trends and can facilitate telemedicine services . in the light of digital divide between rural areas and big cities ,
it is very unlikely in a short period for china to go to paperless ( digital ) hospital .
region - based medical centres or hospitals are still in demand to provide basic health care and advice to the local people .
the nature of relative finance independence of hospitals in china has led hospitals to be competitors as well as collaborators , hampering the development of telemedicine .
a typical example appears to be in the purchase of pacs . on the one hand ,
whereas on the other , some hospitals install pacs with the intention to prevent the data flow going to the other sites , albeit for the benefit of security .
therefore a centralised management system with top - down architecture should be promoted to share and communicate . since over 60% population
own a mobile phone , wireless applications could be considered in providing a reliable , day - to - day modern medical service to any location and in any environment .
this approach should be treated as a strategic means of meeting the priority of providing medical care to everyone .
furthermore , any new gadget can appeal to a huge number of followers in china , and the trend could be exploited towards the application of telemedicine . on the other hand , with respect to telepresence in the operation room
, teleneurosurgery has raised perspective on both necessity and difficulty in china , with a number of issues remaining to be further exploited . at present , due to the low bandwidth of transmission ,
all image data are transferred before the operation to overcome the time lag . since the remote control room is in the position of monitoring rather than teleoperating , near real - time communication of numerical data ( e.g. , the angles of a robot 's position and registration data ) is more important . in this way
, these data can be delivered over either telephone line or mobile phone should the internet network breakdown .
however , real - time overview of the operation room will certainly help to a great extent . additionally , in this study
, all the surgeons in the operations rooms have been very well trained in the control centre in advance and are capable of dealing with any situation of complications that might arise . in the future , however , to take full advantage of telepresence / telemedicine , those trainings can also be concerted via telepresence facilities to reduce the cost especially when local hospitals are underresourced substantially .
although currently all the operations are very well planned and conducted successfully thanks to the experienced surgeons at both control and operation sites , to widen the application of this technique , local level of education should be established , especially in the remit of manual handling of a drilling system in the event of possible failure of the robotic mechanical device .
this again has led to the issue of training of local staff to be able to manipulate the robotic hand manually according to the preplanned path .
although china is getting rich in a number of aspects , accessing adequate medical care is still far afield , especially for those people who live in the countryside .
telesurgery is one way towards bridging this disparity , paving the way for many other similar developments .
it is anticipated that with the advances of computer information and communication technology ( ict ) , telemedicine not only can serve people with regards to health issues , but also can provide additional means of education , closing the gap in both literacy rate and economic development between rural and urban areas . at present , many people from china as well as from many other countries are sceptical about being treated by a robot and still prefer face - to - face consultations , which have hampered the development of telemedicine and its related applications to a certain extent .
sufficient education is therefore in need , in particular , in the field of ict . | with its huge population and vast territory , china faces a great challenge in providing modern advanced health care services to all parts of the country .
the advances of information communication technologies ( icts ) and the advent of internet have revolutionised the means in the delivery of healthcare via telemedicine to remote and underserved populations , which to a certain extent has been very well exploited in china , especially where 70% peasants residing in the rural areas .
this paper reviews the latest development in telemedicine infrastructure in china with the focus on the development of teleneurosurgery , drawing from the results gained from a 3-year networking project between europe and china on telemedicine ( time , 20052007 ) funded by european commission under asia ict programme , with an aim to shape up envisages of future medical care in china .
comparison with its counterparts in europe is also addressed . |
atrichia congenita without ectodermal defects ( isolated form ) is a rare autosomal recessive condition characterized by the shedding of scalp hairs between one and six months of age , after which no growth occurs .
eyebrow , eyelash and body hair may also be sparse or absent ; patients may have a few pubic and axillary hairs .
isolated congenital alopecia has been reported to occur in both sporadic and familial forms . in the isolated familial form ,
inheritance is usually autosomal recessive , although dominant or irregular dominant inheritance has occurred in some families .
the gene locus for familial cases is on chromosome 8p21 - 22 ( alunc - alopecia universalis congenitalis ) , and mutation of the human hairless ( hr ) gene on chromosome 8p21 - 22 produces the clinical picture of atrichia congenita .
human hr gene is a homologue of the murine hairless gene and encodes a zinc - finger transcription factor protein that is expressed in the brain and the skin .
a one - and - a - half - month - old male baby was brought by his maternal grandmother with complaints of frontoparietal scalp hair recession , sparse eyelashes and eyebrows [ figure 1 ] .
the grandmother as well as two of her siblings ( one female and one male ) all had absent scalp , facial , axillary , and pubic hair since infancy .
examination revealed complete absence of scalp , facial , axillary , and pubic hair with no papular skin lesions .
three siblings ( two females and one male ) with absence of scalp , facial , pubic and axillary hair , along with the maternal grandson ( one - and - a - half - months ) having frontoparietal recession , sparse eyelashes and eyebrows there was no history of delayed milestones in the baby , hypohidrosis , bone pain , hearing loss , or seizures .
the condition may be confused with congenital alopecia universalis , vitamin d dependant rickets , and ectodermal dysplasia . in our patients ,
congenital alopecia universalis was ruled out on the basis of lack of any history of sudden , patchy loss of normally appearing hair progressing to the loss of scalp , body , eyelash , andv eyebrow hair .
vitamin d dependent rickets was ruled out on the basis of lack of any history of joint pain , normal serum vitamin d3 and calcium levels , and a normal wrist joint radiograph .
ectodermal dysplasia was excluded as there was no history of delayed milestones or loss of sweating .
teeth , nails , mucosa ( ectodermal components ) , palms , and soles were normal .
the presence of a family history , history of consanguinity , and sparse eyebrows and eyelashes at the time of birth pointed to the diagnosis of atrichia congenita .
our patients met two out of five major criteria supplemented by four minor criteria for the diagnosis of atrichia congenita ( isolated form ) .
diagnostic criteria for atrichia congenita with papular lesions the case is being reported for its rarity . | atrichia congenita is a rare genodermatoses is characterized by a mutation of the human hairless ( hr ) gene on chromosome 8p22 .
there is loss of scalp hair between one to six months of age , after which no growth occurs .
eyebrow , eyelash , and body hair may also be sparse or absent ; patients may have a few pubic and axillary hairs .
the condition may present in isolation or along with other defects . |
coeliac disease ( cd ) , also called gluten - sensitive enteropathy or nontropical sprue , is a unique autoimmune disorder which results from the interaction between gluten and immune , genetic and environmental factors .
originally cd was considered as a malabsorption syndrome of childhood , whereas it is now recognized as a disease which may be diagnosed at any age .
clinical manifestations of cd differ greatly among patients , varying from vague symptoms such as fatigue or malaise to a classical malabsorption syndrome including diarrhoea and steatorrhoea accompanied by abdominal pain or discomfort .
the cornerstone of the treatment of cd is a lifelong gluten - free diet ( gfd ) which leads to noticeable clinical improvement within two weeks in about 70% of patients .
several studies have suggested increased mortality from gastrointestinal malignancies in patients with cd compared to the general population [ 1 , 4,5,6 ] .
particularly the risk of small bowel malignancies , which is generally an uncommon form of cancer , is increased .
in september 2008 , a 70-year - old caucasian male was referred by his general practitioner due to malaise , nausea , vomiting and weight loss of about 8 kg in two months . in his medical history ,
the patient had undergone a hartmann procedure in 1998 because of a perforated diverticulitis . in 1999 a reversal of the hartmann procedure had occurred . in 2001
the patient had been diagnosed with cd by an esophagogastroduodenoscopy with duodenal biopsies for which he was treated with a gfd on which he responded well clinically and which he followed meticulously . at admission
, we performed an esophagogastroduodenoscopy which showed distention of the stomach and duodenum with retention of gastric and enteric fluid ; more than 500 ml was aspirated .
at the point of maximal intubation in the pars horizontalis , a distinct obstruction was suggestive with kinking and fixation but without evident pathology of the mucosa .
biopsies were taken but probably not at the point of maximum obstruction , which could not be reached because of kinking .
histology showed a normal mucosal membrane of the duodenum , without villous atrophy or increased intraepithelial lymphocytes .
additionally , a computed tomography scan of the abdomen was performed which showed an expanded stomach and duodenum up to the ligament of treitz .
an explorative laparotomy was performed , during which a small tumor was palpated near the ligament of treitz .
histological analyses revealed a moderately differentiated , infiltrating adenocarcinoma of 1.5 cm penetrating the muscularis propria and serosa , without invasion of local lymph nodes ( stage t3n0m0 ajcc vi ed 2002 ) ( fig . 1 , fig
malignancies involving the small bowel occur very infrequently in the general population . in the netherlands ,
less than 100 cases a year of small bowel malignancies are revealed while at least 10,000 colorectal carcinomas are diagnosed . despite the fact that the small bowel represents about 75% of the length and 90% of the surface area of the gastrointestinal tract ,
next to the rarity of these lesions , the signs and symptoms patients present are usually vague and poorly defined , often delaying a correct diagnosis .
furthermore , conventional radiographic studies of the upper and lower intestinal tract often appear normal .
the association between carcinoma of the small bowel and cd was first reported in 1958 . since then
, the association between cd and small bowel carcinoma is persistent although only based on a small number of patients of which the greater part is in the form of case reports .
swinson et al . indicated that cd patients have an increased risk of developing small bowel adenocarcinoma ( relative risk 82.6-fold ) .
in addition , results of a national survey in the united states revealed similar results ( relative risk 67-fold ) .
a large population - based study of malignancy in patients which cd performed in sweden confirmed the increased risk for small bowel cancer , although the risk was less compared to previous studies ( 10-fold ) .
possibilities include immunologic disturbance associated with mucosal lymphocyte infiltration , premalignant changes in the damaged surface epithelial cells , increased permeability to oncogenic factors and malabsorption of protective substances such as vitamins a and e [ 10 , 11 ] .
another hypothesis which has been postulated is that small bowel adenocarcinoma in cd arises through an adenoma - carcinoma sequence .
however , currently there are conflicting results regarding this hypothesis . despite the fact that a lifelong gfd leads to noticeable clinical and histological improvement in about 70% of patients , studies about the protective effect of gfd on malignancy are scarce .
most studies suggest that a gfd leads to a decreased incidence of lymphoma , cancer of the mouth , pharynx and oesophagus and a decreased mortality in cd .
however , according to our knowledge only one case report was published about the specific role of gfd concerning small bowel malignancy .
this report did not show a protective effect of gfd , however , further research is needed to confirm this hypothesis . in conclusion ,
the present patient is an illustration that cd is a serious disease with possible serious complications such as duodenal adenocarcinoma . despite the association between cd and small bowel malignancy , currently there are no recommendations for screening for this type of malignancy nor specific protocols for the follow - up of patients with cd . in view of our finding and
based on the present scientific literature , we recommend that clinicians should take into account the possibility of small bowel malignancy in cd patients with vague symptoms such as fatigue or malaise , especially in combination with weight loss , despite a well - maintained gfd . in these patients | coeliac disease ( cd ) is an autoimmune disorder which leads to chronic inflammation of the gut .
furthermore , cd is associated with upper gastrointestinal malignancies , particularly lymphoma of the small intestine .
besides lymphoma , an increased frequency of associated small bowel carcinoma has been described . here
we report the case of a 70-year - old male suffering from cd who was treated with a gluten - free diet presenting with complaints of nausea , vomiting and weight loss of about 8 kg in two months .
he underwent esophagogastroduodenoscopy , which identified distention of the stomach and duodenum and in the pars horizontalis a distinct obstruction was suggestive .
however , histopathological examination showed a normal mucosal membrane .
additionally , a computed tomography scan of the abdomen was performed which showed an expanded stomach and duodenum up to the ligament of treitz . during an explorative laparotomy
a small tumor was palpated near the ligament of treitz .
subsequently , a duodenal segment resection was performed .
after surgery , the patient recovered well and left our hospital in good condition . |
gaucher disease is an autosomal - recessive lysosomal storage disorder caused by mutations in the gene encoding acid beta - glucosidase ( gba1 ) .
the decreased enzymatic activity leads to accumulation of glucocerebroside within characteristic gaucher cells of mononuclear phagocyte origin identified in most tissues .
type 1 ( adult ) is by far the most common type ; it is characterized by accumulation of glucocerebroside in the spleen , liver , and bone marrow and by sparing of the central nervous system .
type 2 ( infantile ) and type 3 ( juvenile ) are the two rare neuropathic forms .
type i disease affects 1 in 50.000 - 100.000 people worldwide and 1 in 400 - 600 people among ashkenazi jewish population ( 1 ) .
few data on the frequency of gaucher disease during reproductive age and pregnancy can be found in the literature and prevalent data have been reported in the netherlands ( 2 per 100,000 live births ) , in australia ( 1 per 57,000 live births ) and portugal ( 3 per 100,000 live births ) ( 2 ) .
pregnancy concurrent with gaucher disease may have several problems , not only by exacerbation of existing signs and symptoms but also by triggering new features ( 3 ) , which may incur additional risk of complications such as changes in haematological parameters ( 4 ) , postpartum infection ( 5 ) , antepartum or postpartum bleeding due to platelet aggregation ( 6 ) , and bone disease ( 3 ) .
specific therapy for the non - neuronopathic manifestations of gaucher disease has been available since 1991 firstly in the form of the macrophage targeted placenta - derived gluco - cerebrosidase ( alglucerase , ceredase , genzyme corporation , ma ) ( 7 ) , and subsequently ( 1994 in usa and 1997 in europe ) by recombinant human enzyme , imiglucerase ( cerezyme , genzyme corporation , ma ) ( 8) .
substrate reduction therapy ( miglustat , zavesca ; actelion pharmaceuticals , allschwill , switzerland ) has more recently been licensed for type 1 of gaucher disease in adult patients with mild to moderate disease for whom enzyme replacement therapy with imiglucerase is not a therapeutic option ( 911 ) .
imiglucerase has a pregnancy category c rating from the food and drug administration ( fda ) with the warning that it is not known whether it is harmful to an unborn baby and with the indication that treatment should be provided to pregnant women only if clearly needed .
in this report , the case of a 27-year - old italian woman ( height , 164 cm ; weight , 55 kg ) was presented during the second month of her first pregnancy .
she had no history of illness until 25 years of age , when she developed general fatigue and bleeding tendency and the symptoms led to diagnosis of gaucher disease .
the diagnosis was based on anemia , thrombocytopenia , elevated levels of serum acid phosphatase and angiotensin - converting enzyme , hepatomegaly , splenomegaly , the presence of gaucher cells in bone marrow aspiration .
dna analysis of the gba gene revealed that she was heterozygous for the c.1448 t > c ( p.l444p ) mutation .
the treatment for the patient started with 19 - 38 units / kg of imiglucerase ( cerezyme ; genzyme , ma , usa ) every two weeks .
menorrhagia improved , hemoglobin concentration and platelet count normalized , biomarkers of disease ( chitotriosidase , acid phosphatase , and angiotensin-1-converting enzyme ) improved , and liver volumes decreased . the patient became pregnant at 27 years of age and , with informed consent , continued imiglucerase treatment throughout all pregnancy , as previously described ( 12 ) at salesi mother and child teaching hospital of ancona ( italy ) in the year 2012 .
genetic counseling was performed at the first obstetric examination and a prenatal testing for gaucher disease and amniotic fluid sampling ( 16th week ) were carried out , thereby , the existence of the disease and other chromosomic anomalies was excluded . by the second month of pregnancy , monthly obstetric ultrasound and blood sampling
were performed and by week 20 of gestation bi - weekly determinations of blood counts revealed that hemoglobin levels were ranging from 9.8 to 10.9 g / dl and platelet counts from 96,000 to 135,000/mm . the prothrombin time and partial thromboplastin time
the biophysical profile studies , as well as doppler analysis , were repeated every two weeks in outpatient clinic of our teaching hospital . in the 39th week of pregnancy ,
an elective cesarean section on maternal choice was carried out and a male baby of 3180 g was delivered .
an uneventful recovery period was observed and the patient was discharged on the 4th postoperative day .
x - ray examination after delivery revealed no bone abnormalities and finally no further increase in the size of the patient 's spleen was observed .
this study was approved by the local departmental ethics committee and written informed consent was obtained from the patient for the case report to be published .
the signs and symptoms of gaucher disease may have an impact on pregnancy and birth , particularly hepatosplenomegaly may be massive and may alter the normal growth in pregnancy ; anemia and thrombocytopenia may be exacerbated by pregnancy and the bleeding tendency may be mild in a nonpregnant patient but may become critical during birth . on the other hand , pregnancy may affect the course of gaucher disease , with regard to signs and symptoms that existed previously as well as the possibility of triggering new features , i.e. bone pains .
the ability of enzyme therapy to rapidly stabilize the disease , especially hematological parameters , has been reported previously ( 13 ) .
there is accumulating evidence that replacement therapy with imiglucerase during pregnancy might stabilize the patient 's condition for physiological changes of pregnancy , and reduce the incidence of complications during delivery and the postpartum period ( 11 , 12 , 14 ) .
the treatment has been related to a reduced risk of spontaneous abortion in women treated with alglucerase and/or imiglucerase , reduced risk of gaucher - related complications during delivery , and a reduced risk of gaucher - related complications during the postpartum period ( 11 ) .
the first antenatal appointment should include a comprehensive assessment of patient , drafting a birth plan , and a multidisciplinary approach to management of pregnancy in such a way that assessment should ideally be performed in a center with experienced experts in pregnancy management .
genetic counseling is recommended and indications for prenatal diagnosis should be explained to the patient .
for example , the facts about whether gaucher disease mutations in one of parents are associated with a risk of neuro - nopathic gaucher disease and the other parent is a carrier of unknown genotype should be explained thoroughly ( 15 ) .
monitoring in pregnancy should be adapted to the needs of the individual patient based on the disease status .
clinicians are advised to concentrate on direct parameters of gaucher disease status , such as platelet count and platelet function that could affect patients during pregnancy and delivery .
ferritin concentrations are often elevated in the serum of gaucher patients as part of the sustained acute inflammatory response ( 16 ) .
this usually does not indicate iron overload but may mask the presence of iron deficiency especially in pregnancy .
iron supplementation is advisable in pregnant gaucher patients with hypochromic microcytic anaemia who do not have evidence of a haemoglobinopathy ( e.g. thalassaemia trait ) , reduced concentrations of serum iron and decreased serum transferrin saturation .
there is accumulating evidence that replacement therapy with imiglucerase during pregnancy might stabilize the patient 's condition for physiological changes of pregnancy , and reduce the incidence of complications during delivery and the postpartum period ( 11 , 12 , 14 ) .
the treatment has been related to a reduced risk of spontaneous abortion in women treated with alglucerase and/or imiglucerase , reduced risk of gaucher - related complications during delivery , and a reduced risk of gaucher - related complications during the postpartum period ( 11 ) .
the first antenatal appointment should include a comprehensive assessment of patient , drafting a birth plan , and a multidisciplinary approach to management of pregnancy in such a way that assessment should ideally be performed in a center with experienced experts in pregnancy management .
genetic counseling is recommended and indications for prenatal diagnosis should be explained to the patient .
for example , the facts about whether gaucher disease mutations in one of parents are associated with a risk of neuro - nopathic gaucher disease and the other parent is a carrier of unknown genotype should be explained thoroughly ( 15 ) .
monitoring in pregnancy should be adapted to the needs of the individual patient based on the disease status .
clinicians are advised to concentrate on direct parameters of gaucher disease status , such as platelet count and platelet function that could affect patients during pregnancy and delivery .
ferritin concentrations are often elevated in the serum of gaucher patients as part of the sustained acute inflammatory response ( 16 ) .
this usually does not indicate iron overload but may mask the presence of iron deficiency especially in pregnancy .
iron supplementation is advisable in pregnant gaucher patients with hypochromic microcytic anaemia who do not have evidence of a haemoglobinopathy ( e.g. thalassaemia trait ) , reduced concentrations of serum iron and decreased serum transferrin saturation .
even when most of the patients with gaucher disease underwent splenectomy before the reproductive period , a reversible massive progressive splenic enlargement during pregnancy occurred ( 17 ) . accumulated data presented here
provide no evidence of any teratogenic effects even when given in the first trimester of pregnancy .
imiglucerase and alglucerase are generally well tolerated in gaucher patients and have an excellent safety record ( 18 ) .
pharmacovigilance data shows that imiglucerase is also well tolerated in pregnant women and there is no evidence of any adverse events specifically related to pregnancy ( 11 ) . even if vaginal delivery is preferred and there is no specific indication for caesarean section , often a surgical delivery is preferred for the risk of splenic rupture during labor .
caesarean sections in gaucher patients are more likely to occur because of disease profile of patients , such as orthopaedic considerations , rather than acute complications during delivery .
caesarean sections can not alone be perceived as an indication of risk of complications , as these are increasingly carried out without medical indication at maternal request or because of caution on the obstetrician 's part , despite associated risks ( 11 ) .
although hemorrhagic tendency may be observed in such patients ( 15 ) , the state could be successfully managed conservatively , for example , by a low transversal yoel - cohen incision or a pfannenstiel curved incision .
surgeons should avoid exploration of the peritoneal cavity unless there is a surgical indication , as palpation of enlarged organs in gaucher disease may precipitate bleeding . because gaucher disease is a disease with multi - organic involvement ,
preoperative assessment should be carried out in order to determine the extent to which the different organs are affected .
general anesthesia should be avoided because of maternal aspiration syndrome and the risk of neurological and respiratory depression in the new - born .
the use of regional anesthesia for patients with gaucher disease undergoing surgery remains controversial ; however , some authors point out that local anesthetics , as for cesarean section , may be safe provided that no other formal contraindications for their use is present , such as clotting parameter alterations , severe spinal deformities or spinal cord abnormalities , that would put the patient at risk of neurological sequelae ( 19 , 20 ) .
perinatal demise of the newborn affected by gaucher disease is very rare and is considered a variant of type 2 gaucher disease that occurs in the neonatal period .
the most frequent features are non - immune hydrops fetalis , in utero - fetal demise , and neonatal distress . in some cases without hydrops ,
neurological signs occur in the first week of life and lead to death within 3 months and some common signs of the disease are hepatosplenomegaly , ichthyosis , arthrogryposis and facial dysmorphy ( 21 ) .
few data to date are available on patients with gaucher disease treated with imiglucerase during the lactation period , on its excretion into human breast milk and its effects on the newborn ( 22 , 23 ) .
even when during breastfeeding , the enzyme was likely to be digested in the child 's gastrointestinal tract suggesting minimal risk to infants ( 24 ) , a continued healthy development of children breast - fed by alglucerase or imiglucerase treated mothers has been reported ( 25 ) .
the european medicines agency and the us food and drug administration indicate that caution should be exercised when imiglucerase is administered in nursing women .
postpartum , bone mineral density assessment using dexa should be considered in cases of prolonged breastfeeding and checked at appropriate intervals after breastfeeding is completed .
the characteristics of the present case have many similarities with the ones stated in the literature since no significant complication occurred during the pregnancy and delivery .
although a mild anemia and thrombocytopenia was observed in the antenatal period , no transfusion was required . knowing the mechanism of autosomal recessive inheritance , especially in most severe forms , a prenatal diagnosis using amniocentesis or chorionic villus sampling should be offered to the patient .
having reviewed the related literature , it seems that if the disease is controlled , with proper therapy and monitoring , mothers are more likely to experience uncomplicated pregnancies and deliveries .
| backgroundgaucher disease is a lysosomal storage disorder due to deficiency of glucocerebrosidase enzyme . in this study ,
a case of enzyme - treated woman during her pregnancy was reported.case presentationa 27-year old woman with type i gaucher disease was managed for pregnancy until delivery .
she underwent elective splenectomy at age 26 years and was treated with 19 - 38 units / kg of imiglucerase . a conservative approach with close monitoring of both mother and baby
was planned.resultsin the 39th week of pregnancy , a healthy male baby of 3180 g was delivered via cesarean section.conclusionapart from mild hematological complications , the pregnancy , the delivery and the puerperium were uneventful . in this case report , the issue of therapy and risk assessment in pregnancy in patients with type i gaucher disease was discussed . |
the immune system has a complex but flexible network of cellular and molecular components to deal with the constraints of limited genetic space in responding to almost limitless types of infectious organisms , i.e. , viral , bacterial , fungal and parasitic variants .
these components are broadly divided into innate and antigen - specific adaptive immunity based on the kinetics of responses following primary exposures to infectious agents , but their specific elements are shaped further to deliver protective subset effects under different conditions of infections .
examples of innate cellular components include the natural killer ( nk ) cells important in early killing of virus - infected cells and for production of early cytokines during a wide range of infections .
cellular constituents of adaptive immunity include cd4 t cells that can be driven into particular lineages to deliver subset cytokines as required for protection under different conditions of infection , and cd8 t cells mediating killing of virus - infected cells and producing cytokines for defense against viral infection .
in addition , innate cytokines induced after infections are key in the endogenous responses because they can promote direct antimicrobial effects and also shape development of the cellular immune responses most beneficial under the conditions of infection being encountered .
the theme of flexibility in responding to infections is carried over to cytokine signaling and function .
although these soluble mediators have well known individual basal effects on particular cells in culture , they can have pleiotropic and overlapping effects , and depending on the conditions of examination , some of these are paradoxical .
examples include members of the janus activated kinase - signal transducer and activator of transcription ( jak - stat ) cytokine family .
these factors stimulate major signaling pathways by inducing receptor oligomerization upon ligand binding to activate receptor associated jaks ( including jak1 , jak2 , jak3 and tyk2 ) , which in turn recruit and activate by phosphorylation the cytoplasmic - resident stats ( including stat1 , 2 , 3 , 4 , 5a , 5b and 6 ) to result in their homo- or heterodimerization , translocation into the nucleus and transcription of unique sets of target genes .
although most of these cytokines have a preference for activating particular stat molecules , the same cytokines often have the ability to conditionally activate more than one stat .
this flexibility suggests that conditions for selecting particular signaling pathways could provide mechanisms for accessing different consequences of jak - stat cytokine exposure as needed .
thorough understanding of how this is controlled to promote important subset responses in a biological context , however , has remained elusive .
new insights are resulting from the characterization of differences in basal and induced total stat levels as well as relative concentrations of stat mixtures .
much of this work has focused on culture studies , but a developing literature examining responses during ongoing infection is also revealing how particular stat concentrations are presented to modify intrinsic immune cellular responsiveness to particular cytokines and how relative concentrations of stat combinations can be dynamically regulated to change the experience of different immune cell subsets to cytokine exposure . to date , the work has primarily focused on nk and cd8 t cell responses to type 1 interferons ( ifns ) but has implications for a broader range of cells and cytokines . taken in its entirety ,
it begins to explain the how and why for the complexity of reported jak - stat pathways as well as the pleiotropic and overlapping cytokine effects .
the picture emerging is one of cytokine receptor preferences for particular stats , but controlled variations in relative stat concentrations to alter the selection of signaling partners .
the original studies investigating cytokine - stat selection used biochemical analyses to identify the major pathways delivering signals from cytokines to cells . in the case of ifns , including type 1 ( ifn/ ) and type 2 ( ifn ) ifns , the pathways promoting their known potent antiviral effects were characterized .
type 1 ifns are a major class of innate cytokines , comprised of products of a single ifn and , depending on the species , 12 ifn genes .
the type 1 ifns activate stat1 and stat2 , inducing their subsequent heterodimerization , and in association with the interferon regulatory factor 9 ( irf9 ) , bind to particular dna sequences in the promoters of genes termed ifn stimulated response elements ( isres ) .
by contrast , the sole type 2 ifn , ifn , activates stat1 homodimers and induces the expression of genes with gamma activating sequences ( gas ) in their promoters .
the recently identified type 3 ifns , ifns , also named interleukin ( il)-28a , -28b and -29 , are another class of cytokines activating stat1 and stat2 for signaling .
further well - characterized cytokine - stat pathways include the activation of stat3 by il-6 and il-10 , the activation of stat4 by il-12 and the activation of stat5 by cytokines that signal through the common gamma chain , including il-2 , il-4 , il-7 , il-9 , il-15 and il-21 . although specific major signaling pathways are associated with exposure to particular cytokines , the factors demonstrate
infidelity such that additional and/or other , non - preferred stats are conditionally activated . in the most extreme case of promiscuity ,
the type 1 ifns have been reported to activate all seven of the different stat molecules ( fig .
1a ) , but there is also a surprisingly long list of cytokines reported to activate both stat1 and stat3 , including ifn , the ifns , il-6 , il-10 and il-21 . because the activated stats themselves are binding to particular promoter sequences in genes to regulate transcription , activation of common stats by different cytokines can elicit common expression of particular target genes ( table 1 ) .
remarkably , the type 1 ifns can differentially regulate the expression of over 100 target genes .
the role for stat1 in induction of many of these , including critical antiviral genes coding for the 2,5-oligoadenylate synthetase ( oas ) , protein kinase r ( pkr ) and the mx protein products , has been definitively established .
in addition , stat1 plays a role in the anti - proliferative and pro - apoptotic effects delivered by type 1 or type 2 ifns , and the pathway contributes to certain immunoregulatory effects mediated by type 1 ifn during the development of innate immune responses to viral infections including the activation of nk cell - mediated cytotoxicity , delivered through perforin- and granzyme - dependent mechanisms , the induction of il-15 expression and interestingly , the inhibition of ifn expression .
certain of the type 1 ifn responses can also be induced by ifn because it activates stat1 . in responses elicited by il-12 ,
the induction of ifn and the cd25 component of the il-2 receptor is stat4 dependent , and under certain conditions , type 1 ifns , but not ifn , can activate stat4 to induce these responses ( see below ) .
pro - survival states and the expression of target genes promoting these , including bcl - xl , survivin , c - myc and cyclin d1 , are linked to stat3 , and activation of stat3 by the variety of cytokines might be predicted to lead to these responses . thus , stimulation of common stats helps explain pleiotropic and overlapping cytokine effects .
( a ) type 1 ifns have been shown to be able to conditionally activate all of the stats .
the key pathways , however , use the preferred stat1/stat2 molecules to stimulate genes through isre and gas promoters for induction of an antiviral state .
( b ) nk cells intrinsically express high levels of stat4 . as a result ,
( c ) cd8 t cells have the potential to respond to type 1 ifn with either stat1 or stat4 activation , but the activation of stat1 is preferred . during the context of responses to infection , the antigen - specific subsets have their stat4 ,
whereas the non - specific cells have stat1 , levels induced . as a result ,
antigen - specific cd8 t cells overcome type 1 ifn stat1-dependent anti - proliferative effects and respond to type 1 ifn with stat4 activation for ifn production .
flexibility in signaling may also help explain the paradoxical biological functions that have been attributed to the cytokines .
studies using cells or mice rendered genetically deficient in particular stats have revealed that there are molecular and biological consequences of experimentally restricting access to stats . in the absence of stat1 ,
the stat1-dependent antiviral and anti - proliferative effects as well as activation of nk cell - mediated cytotoxicity and induction of il-15 expression are blocked during viral infections eliciting high levels of type 1 ifns , but type 1 ifn induction of ifn is revealed . likewise ,
absence of stat1 blocks the stat1-dependent , but allows stimulation of stat1-independent , and novel , ifn gene targets . for il-6 , in the absence of stat3 ,
the cytokine stimulates stat1 phosphorylation and a gene transcription program with induction of an antiviral state that resembles that induced by ifn. thus , if there are biologically relevant conditions modifying availability of particular stat molecules , alternative signaling pathways may provide mechanisms for shaping the unique , pleiotropic , overlapping and paradoxical effects delivered by different members of the jak - stat cytokine family to provide pathways to particular subset responses as needed during infections . as an example , il-10 with its potential to activate both stat3 and stat1 might also be driven to deliver antiviral states in the absence of stat3 .
in addition to direct links between cytokine exposure and stat activation for the induction of gene expression and rapid cellular responses discussed here , the combinations of particular cytokines with particular stats has been shown to play a role in the differentiation of cd4 t cells into specific lineages to shape the availability of cd4 t helper cytokines under different conditions of infections . simply stated , exposure to il-12 uses stat4 to support the differentiation of cd4 th1 type cells producing ifn important during infections with intracellular microorganisms including certain bacteria , whereas exposure to il-4 uses stat6 to support the development of th2 cells producing il-4 important during parasitic infections , and exposure to il-6 uses stat3 to promote th17 cells producing il-17 important during infections with extracellular microorganisms including fungi .
the mechanisms involved in driving cd4 t cell subset development require longer periods and additional genetic modifications to develop .
one way of regulating access to different signaling pathways would be to provide different cell populations , with common expression of cytokine receptors , with differential expression of particular stat levels . because stat1 and stat2 are the preferred type 1 ifn receptor ( ifnar ) signaling molecules , higher level expression of alternative stats
biochemical and flow cytometric studies evaluating stat4 expression within freshly isolated mouse nk cells have demonstrated that these populations are unique in their basal high expression of stat4 and modestly reduced levels of stat1 proteins ( fig .
this can be observed in nk cells prepared from either the spleen or the peritoneal cavity .
ex vivo type 1 ifn exposure results in the preferential activation of phosphorylated ( p)stat4 over pstat1 in these populations .
the mouse system of lymphocytic choriomeningitis virus ( lcmv ) is particularly useful for studying the regulation of type 1 ifn signaling pathways in response to in vivo cytokine exposure because the virus induces a strong innate cytokine response focused on type 1 ifn production , locally detected at hours after infection and broadly observed systemically beginning from around day 1.5 and extending to day 4 of infection . the very earliest type 1 ifn production at the site of infection , the peritoneal cavity , results in an ifnar- and stat4-dependent induction of peritoneal nk cell ifn expression and production of short duration , peaking at about 30 h. this response inhibits viral replication .
thus , nk cells basally experience exposure to type 1 ifn with stat4 rather than stat1 activation , and this leads to ifn production for the benefit of an infected host .
the studies of nk cells provide the first documented differential expression of stats in normal , freshly isolated immune populations .
other reports , however , have also shown differences in stat4 levels in human populations isolated under disease conditions and in culture - derived human dendritic cell ( dc ) subsets . moreover ,
various populations in human peripheral blood leukocytes have different preferences for stat activation after type 1 ifn exposure . in comparison to other cell types ,
exposure to the type 1 ifn , ifn , does not stimulate a strong pstat1 response but does induce pstat3 and pstat5 in b and cd4 t cells subsets .
the observation may help explain a poor induction of stat1-dependent pro - apopotic mrnas in human b and cd4 t cells as well as the apparent pro - survival effects of ifn on these cells as compared with monocytes .
although the stat1 levels are not significantly different in the populations , however , the question of relative stat concentrations in these different cell types has not been addressed because the levels of stat3 and stat5 have not been measured .
thus , there is more work to be done with a broader range of cell subsets , and there may be other mechanisms influencing selection , but taken together , the work to date suggests a model by which differences in stat availability based on relative concentrations influence intrinsic differential cellular responses to type 1 ifns .
another system with work characterizing relative access of a jak - stat cytokine to different stats is the ifn response delivered through its receptor .
stat1 is a predominant transcription factor activated by ifn , but stat3 can also be weakly activated , and in the absence of stat1 , stat3 is highly phosphorylated and drives the expression of genes that are normally induced by ifn. the sophisticated biochemical analysis of the receptor interactions with stat1 as compared with stat3 has demonstrated that a tyrosine residue in the ifn receptor subunit 1 is required for the activation of both stat1 and stat3 , suggesting that the two stats compete for binding to the ifn receptor at the same site .
although the understanding of the stat4/stat1 interactions with the type 1 ifn receptor are less well developed , stat4 is basally associated with ifnar in the context of the high level of stat4 and lower stat1 within nk cells .
thus , a growing literature indicates that the relative concentrations of different stat molecules have consequences for signaling in response to the same cytokine .
the requirements for different concentrations in stat selection are likely to depend on the potential for , and relative affinity of , physical interactions with the respective cytokine receptors .
collectively , these studies highlight the flexibility of intrinsic stat availability in initially selecting cytokine - driven effects and raise the interesting questions of how stat concentrations might be regulated to influence cytokine effects during the dynamic conditions of infections to differentially shape individual cellular responses .
although type 1 ifns can activate stat1 and stat4 , the two stat molecules stimulate opposing gene programs .
as noted above for nk cells , activation of stat4 promotes ifn production , but activation of stat1 inhibits ifn expression while promoting anti - proliferative effects , cytotoxicity and il-15 expression .
the activation of stat1 also results in increased stat1 because the molecule targets promoter sequences in its gene to induce stat1 mrna and protein .
although this pathway has been known for some time , understanding of its biological importance remained largely unappreciated until it was demonstrated that total stat1 levels are dramatically elevated in immune cell populations following infections of mice with lcmv .
biochemical analysis of mixed populations has demonstrated that the ability to use type 1 ifns for stat4 activation inversely correlates with total stat1 levels such that the pathway is accessible prior to infection , blocked at times with increased stat1 levels and apparent again after the stat1 levels have declined .
the original demonstration of this set forth the model of dynamic regulation of stat concentrations .
further characterization of the pathway in nk cells has shown that although these populations basally express and access stat4 in response to type 1 ifns , they have elevated levels of stat1 during the course of lcmv infection . at early times after infection ,
all of the immune cells examined induce stat1 in response to type 1 ifn production , but the kinetics is delayed in nk cells relative to that of the other populations . the condition allows a window of opportunity for type 1 ifn induction of ifn induction though stat4 prior to high level stat1 expression . the delay may be a consequence of reduced immediate activation of stat1 because of the intrinsic lower levels of the molecule in the context of high stat4 and/or because of competition between the high levels of stat4 with low levels of stat1 for access to events at the receptor .
eventually , the stat1 levels do increase in response to type 1 ifn exposure in the nk cells , and once elevated , they are preferentially associated with the type 1 ifn receptor such that although the stat4 levels remain high , they are not activated by the cytokine .
the absence of stat1 results in the ability to continue to use type 1 ifn for stat4 activation . during lcmv infection , however , this is highly detrimental to the host because it allows for continued , dysregulated ifn production and cytokine - mediated disease .
thus , nk cells basally express high stat4 , and type 1 ifn induces ifn through this molecule to help protect against early viral replication , but stat1 is induced to tightly regulate the pathway and protect from cytokine - mediated disease .
the switch might help promote other effector functions because stat1 is important in inducing nk cell cytotoxic function .
thus , passing off from stat4 to stat1 could have many effects including changing effector functions as well as protection from immune dysregulation during infections .
recently , nk cells and dcs isolated from another infection in mice , murine cytomegalovirus ( mcmv ) , have been shown to exhibit different responsiveness to type 1 ifns with alterations in stat1 activation and induction such that the stat1 pathway is preferentially used in dc subsets .
remarkable aspects of the early immune response to this virus as compared with lcmv are that il-12 is elicited along with type 1 ifns , and high systemic levels of nk cell produced ifn are induced through and dependent upon il-12 activation of stat4 .
because the il-12 receptor preferentially activates stat4 , it is tempting to speculate that the biological pressure for il-12 is to provide a pathway to stat4 and ifn in cells that have been conditioned to express high stat1 and as a result , have had their pathway from type 1 ifn to stat4 blocked .
although adaptive t cells , particularly cd8 t cells , can have functions that overlap those of innate nk cells , they have unique challenges to their activation during viral infections .
foremost is that the subsets with t cell receptors ( tcrs ) specific for the antigens of particular infectious organisms are at extremely low frequencies in nave , non - immune hosts and have to be selected and preferentially expanded . in the case of cd8 t cells
, this occurs as a result of antigen presentation by the class 1 major histocompatibility molecules ( mhc ) to the tcr , takes time to develop , and during viral infections , requires proliferation through periods overlapping with the induction of systemic type 1 ifn levels and the opportunity for their delivery of anti - proliferative effects .
in addition , the cells have to be driven into states that allow their needed subset responses , such as ifn production , and type 1 ifns can have both inhibiting and enhancing effects on these .
the picture emerging is one where the concentrations of stat1 and stat4 are being differentially regulated to condition for particular experiences following exposure to type 1 ifn and as a result , facilitate the development of antigen - specific cd8 t cell responses to viral infections .
the stat1 molecules expectedly play an important role in type 1 ifn - mediated inhibition of proliferation and have a novel role in helping to preferentially limit expansion of non - specific cd8 t cells during endogenous immune responses . in the absence of stat1 , a type 1 ifn - mediated inhibition of ex vivo cytokine - driven proliferation of cd8 t cells
is lost , and lcmv infection of stat1-deficient mice results in dysregulated early proliferation of cd8 t cells , through day 4 of infection , without specificity for the known lcmv antigens . how then can antigen - specific cells avoid this inhibition to expand and contribute to defense ?
although stat1 is elevated in all of the splenic leukocytes isolated from mice at different times after lcmv infection , the cd8 t cells responding with dna synthesis and expansion , on days 5 through 8 of infection , are preferentially found within subsets of cells having lower stat1 and higher stat4 protein levels , and the cells expressing receptors specific for lcmv are in this group .
consistent with early reports characterizing t cell lines and cd4 t cell subsets , ex vivo stimulation through the tcr induces elevated stat4 expression in cd8 t cells .
more importantly , the condition of higher stat4 protects against type 1 ifn induction of stat1 and resistance to type 1 ifn - mediated inhibition of proliferation ex vivo , and the presence of stat4 results in enhanced proliferation and reduced stat1 expression in antigen - specific cd8 t cells during lcmv infection .
1c ) such that the non - specific cells have stat1 levels induced and are sensitive to type 1 ifn - mediated inhibition of proliferation , and the antigen - specific cells are induced to express higher levels of stat4 and as a result , develop resistance to type 1 ifn - mediated inhibition of proliferation with selection for preferential expansion in the presence of the cytokines .
although the numbers of antigen - specific cd8 t cells are still difficult to evaluate at days 4 to 6 of lcmv infection , a systemic cd8 t cell - dependent ifn response is detectable at these times . in vivo blocking studies have shown that the response is primarily dependent on antigen - specific cd8 t cells and is greatly enhanced through type 1 ifn- and stat4-dependent pathways .
dissection of the components of stimulation ex vivo shows that there is a high degree of synergism such that although either works alone , small concentrations of type 1 ifn along with small concentrations of antigen enhance ifn production .
type 1 ifn responses of cd8 t cells prepared from uninfected as compared with day 8 lcmv - infected mice , switch from preferentially activating stat1 and a wide range of target genes , including those dependent on this transcription factor , to preferentially activating stat4 and focusing the responses to a narrower target gene range with less expression of the stat1 but continued or elevated expression of the stat4 target genes .
these observations explain the differential effects of type 1 ifn on proliferation and ifn expression by showing that the cd8 t cells are being conditioned to experience the cytokines differentially , and that this is important in shaping the endogenous adaptive immune responses to viral infection .
it is interesting to note that the antigen - specific cd8 t cells now mirror the basal stat expression of nk cells , high for stat4 and lower for stat1 .
how might regulation of stat3 access fit into intrinsic or conditioned cytokine responses during viral infections ?
stat3 can be activated by many of the same cytokines as stat1 , but the functional consequences have been difficult to assign and as noted above , are often cell type and context dependent . the molecule was initially discovered to mediate the acute phase response in hepatocytes in response to il-6 , resulting in the transcription of genes that play a protective role ( e.g. , wound healing ) in acute inflammation .
the il-6-stat3 pathway has also been shown to prevent apoptosis in both pro - b cell lines and primary t cells , in the former case by stat3-mediated induction of bcl-2 , a member of the anti - apoptotic bcl family . in the case of type 1 ifn effects on lymphocyte proliferation or survival ,
the role for stat3 has again been controversial with reports of its positive action being context dependent .
stat3 is the major transducer of il-10 signaling , and in macrophages , this pathway both limits inflammatory cytokine production and prevents proliferation . in stat3-deficient macrophages ,
the cells produce increased levels of inflammatory cytokines in response to the bacterial product , lipopolysaccharide ( lps ) , resulting in chronic enterocolitis in mice . despite these varying functional outcomes in different cell types ,
an interesting feature of stat3 is that , similar to stat1 , the protein levels of stat3 are increased when stat3 is activated by cytokines .
thus , the opportunity to condition cellular responses based on modulation of stat3 levels represents a viable mechanism for controlling cellular functions .
moreover , given the wide range of receptors stimulating stat3 , there is a potential for variations in particular stat levels relative to other stats to have very different consequences for shaping the responses to particular cytokines .
because the molecule is also associated with key cellular processes , including survival , proliferation and apoptosis , understanding the regulation of expression and access could help explain complex events preferentially supporting expansion and maintenance of needed lymphocytes . a stat3 requirement for survival
is underscored by the fact that unlike mice deficient in other stat molecules , mice with targeted deletion of stat3 do not develop past embryonic day 6.5 .
however , conditional deletion of stat3 in various cell types has not always presented a consistent picture of stat3 in promoting survival .
for example , deficiency of stat3 in neurons results in enhanced apoptosis of these cells , whereas stat3 deletion in the mammary epithelium delays the process of involution depending on apoptosis .
interestingly , the most striking evidence for stat3 mediating survival signals is the multitude of studies highlighting the involvement of stat3 in cellular transformation .
the molecule is constitutively activated in many human tumors , and cells with genetically mutated stat3 are resistant to transformation , whereas cells expressing a constitutively active form of stat3 are converted to cancer cells .
consistent with the latter , many of the well - documented target genes of stat3 include those that prevent apoptosis ( bcl - xl and survivin ) and enhance survival and proliferation ( cyclin d1 and c - myc ) .
thus , there is much to be learned about how stat3 is used in the context of evolving immune responses to infection . given that type 1 ifns can mediate different nk and t cell responses based on their ability to signal through stat1 or stat4 , and their preference is influenced by the levels of the stats , infections of mice with lcmv inducing type 1 ifns or mcmv inducing additional cytokines , including ifn and il-6 , provide conditions for changing stat3 levels in responding cells and give the potential for adding this signaling molecule to the mix of relative stat concentrations shaping cellular responses to stat3 activating cytokines .
future studies are needed to better define the modulation of interaction between these stats in the context of a complex cytokine milieu , and studies of viral infections hold promise for evaluating the contributions of regulated stat3 expression in shaping cellular immune functions .
taken together , the studies of nk and cd8 t cells in uninfected and virus - infected mice have demonstrated differential type 1 ifn responsiveness based on intrinsic and induced stat concentrations .
the stat4 levels are basally high in nk cells , allowing them a window of opportunity to immediately respond to type 1 ifns with ifn production , but the pathway is tightly regulated by simultaneous stat1 induction to protect from cytokine - mediated disease .
in contrast , antigen recognition induces stat4 in selected cd8 t cells while stat1 is elevated in non - specific cd8 t cell subsets to allow for preferential expansion of , and type 1 ifn induction of ifn in , the antigen - specific subsets .
the flexible pathways to stat4 allow general access to a highly controlled early nk cell response followed by access to an antigen - specific cd8 t cell response .
because the cd8 t cell induction of ifn is greatly enhanced by tcr stimulation as well as type 1 ifn , the conditions limit this response to periods overlapping viral replication for antigen presentation and type 1 ifn production ( fig . 2 ) .
thus , their stat4-dependent type 1 ifn responses are controlled even though stat1 is not elevated to high levels .
the sophisticated kinetic regulation of these events provides insights into how the immune system uses the resources available to promote optimal defense against infection while protecting against potential immune - mediated disease .
they also suggest approaches for therapeutic intervention by identifying the when and how particular pathways are required .
differential cellular use of type 1 ifns for stat4 activation and ifn production at particular times during lcmv infection .
the lcmv infection in mice provides a powerful system to study the regulation of type 1 ifn effects .
the cytokines are produced locally within a few hours and can be found systemically for several days following infection . prior to infection , nk cell populations are high whereas the cd8 t cells are low for stat4 . as a result , nk cells initially respond to type 1 ifn with stat4 activation and ifn production .
the pathway is tightly regulated , however , because elevated stat1 levels are concurrently induced to block type 1 ifn access to stat4 .
in contrast , the antigen - specific cd8 t cells are being stimulated through their tcr to have elevated stat4 expression whereas the non - specific cd8 t cells are induced to express elevated stat1 .
these conditions promote preferential expansion of the antigen - specific cd8 t cells and their production of ifn in the context of the endogenous type 1 ifns .
thus , there are flexible pathways in different cell types to regulate the consequences of type 1 ifn exposure during viral infection .
although most of the work reviewed here has been performed in the mouse , parallel systems operate in the human . indeed , the first report of type 1 ifn activation of stat4 examined responses in human t cells , and more recent work has shown the response in human nk cells .
in fact , conflicting data suggesting that there were species differences in type 1 ifn activation of stat4 were resolved by the demonstration of differential activation of stat4 based on stat1 concentrations in the mouse , and the correlation between type 1 ifn activation of stat4 with low stat1 levels has been recently reported with cells from individuals chronically infected with hepatitis c virus ( hcv ) as compared with control populations .
thus , the work in the human and mouse is crystallizing to show how partners at the dance can be changed based on availability and concentrations to alter stat pathways of activation and cytokine functions under particular conditions of infection .
why is the block in stat1 induction in antigen - specific cd8 t cells more complete than the delayed induction in nk cells even though both cell types have elevated stat4 ?
the results comparing the lcmv and mcmv infection systems suggest that il-12 access to stat4 is relatively insensitive to high stat1 levels , but almost nothing else is known about how the altered stat4 and stat1 concentrations might affect cellular responses to the range of additional , non - type 1 ifn cytokines having flexibility to stimulate these molecules .
the pathways for stat1 activation to elevated stat1 expression and from tcr stimulation to elevated stat4 have been shown to work during precise periods of viral infections ( fig .
there are also pathways from stat3 activation to elevated stat3 expression , however , and many other yet unidentified mechanisms are likely to be available to regulate relative concentrations of all of the stats in mixtures ( fig . 3 ) .
characterization of these , how they function during immune responses and how they change signaling of the range of cytokines with the potential to activate them will provide novel new insights into how immune responses are regulated .
a variety of cytokines , including all of the ifns , preferentially activate stat1 , and stat1 activation results in the induction of elevated stat1 expression .
other cytokines , including il-6 , il-10 and il-21 , can activate stat1 but have a preference for activating stat3 .
thus , there are a variety of potential known mechanisms for altering stat3 , and the potential for many unknown mechanisms for altering additional stats , to further change intracellular concentrations of stat mixes and the effects of any cytokine with the ability to alternatively activate particular stats .
the results demonstrating the dynamic changes in particular stat concentrations suggest that care must be taken in interpreting long - term studies of the role for particular cytokines or stats after transfer of deficient cell subsets into complete environments .
clearly , both the cytokines and the stats are differentially used during the life of particular cells .
thus , although such long - term studies may provide information on net outcomes , they do not present opportunities for identifying the regulation of access to stats for changing cell or host need , or for revealing key functions depending on particular stats or cytokines that might be delivered downstream of the blocked pathway being tested .
the same caution can be raised about the powerful approach of identifying genetic susceptibilities to classes of infectious organisms to assign unique importance of particular receptors and stats in defense .
the outcome does reveal absolute requirements but does not inform on other effects , flexible pathways and/or on alternative pathways providing other kinds of support .
indeed , in the case of stat3 mutations in humans , the first reports linked the condition to increased susceptibility to fungal infections .
the consequences for long - term maintenance of t cell subsets and control of viral infections were only revealed after more extensive and detailed studies .
thus , a thorough understanding of the mechanisms shaping cytokine and stat functions requires careful dissection of cellular responses in the context of the evolving conditions of different periods of infections . in conclusion
, there is still much to be learned about regulation of cytokine effects and signaling pathways , but existing evidence provides a strong model for continued testing .
clearly , biological outcomes will depend on the integration of multiple rather than additive effects of individual stat signals .
however , the results to date indicate that these are subject to conditioning based on the dynamic regulation of relative stat concentrations
. the consequences of varying stat levels can be predicted to depend on the potential for , and relative affinity of , physical interactions with the respective cytokine receptors . given the range of cytokines activating overlapping stats , the opportunities for differential regulation of their effects with changing stat concentrations are great . with the framework of existing knowledge , the next period of study will be exciting and lead to new understanding about how the immune response is working in its entirety to achieve the best possible response to infections using the available cytokine and signaling resources .
future work holds great promise for not only explaining the how and why endogenous immune responses unfold the way they do , but also for providing insights into new approaches for intervention to promote health over disease in a variety of challenging conditions . | differential use of cellular and molecular components shapes immune responses , but understanding of how these are regulated to promote defense and health during infections is still incomplete .
examples include signaling from members of the janus activated kinase - signal transducer and activator of transcription ( jak - stat ) cytokine family .
following receptor stimulation , individual jak - stat cytokines have preferences for particular key stat molecules to lead to specific cellular responses .
certain of these cytokines , however , can conditionally activate alternative stats as well as elicit pleiotropic and paradoxical effects .
studies examining basal and infection conditions are revealing intrinsic and induced cellular differences in various intracellular stat concentrations to control the biological consequences of cytokine exposure .
the system can be likened to changing partners at a dance based on competition and relative availability , and sets a framework for understanding the particular conditions promoting subset biological functions of cytokines as needed during evolving immune responses to infections . |
a 48-year - old female presented to the emergency department ( ed ) with syncope .
her past medical history was significant for mild mental retardation , former tobacco use , and hernia repair .
the patient stated that while cooking she felt lightheaded , subsequently lost consciousness , and fell to the ground recovering after a few minutes . after recovering , she noticed her exercise tolerance declined , feeling shortness of breath after climbing just one flight of stairs . on review of systems , she reported 2 weeks of menorrhagia .
she denied chest pain , palpitations , diaphoresis , lower extremity edema , orthopnea , and paroxysmal nocturnal dyspnea . on physical examination ,
her heart rate was 88 beats / min , blood pressure 113/48 mmhg , oxygen saturation 98% on room air , and a temperature of 98.4c .
cardiac examination revealed a 3/6 decrescendo diastolic murmur best heard on the right sternal border with radiation to the carotids .
the physical exam was also remarkable for poor dentition with evidence of decay in multiple teeth as well as pallor in mucous membranes , palms , and soles .
laboratory workup was remarkable for profound iron deficiency anemia ( hgb 2.9 g / dl , hct .
10.7% , mean corpuscular volume 54 fl , ferritin 15 ng / ml , iron 261 g / dl , transferrin 218 mg / dl )
her chest x - ray ( cxr ) showed cardiomegaly , computed tomography ( ct ) of the head ruled out hemorrhage , and ct of the abdomen / pelvis revealed multiple myomas which were thought to be the source of bleeding .
due to a new diastolic murmur and cardiomegaly on cxr , a transthoracic echocardiogram ( tte ) was obtained , demonstrating a thickened aortic valve with severe aortic insufficiency .
given high suspicion for ie , a transesophageal echocardiogram ( tee ) was performed revealing an irregularly shaped mobile echodensity ( 1.7 cm1.0 cm ) on the aortic valve consistent with a vegetation ( fig .
1 ) . also , a root abscess was present with fistula formation between the right sinus of valsalva and the right atrium ( fig .
broad - spectrum antibiotics were started with vancomycin and ceftriaxone , and she was transferred to the cardiac care unit .
blood cultures were positive for streptococcus anginosus and antibiotics were narrowed to ceftriaxone for 4 weeks .
cardiac surgery service was consulted for aortic valve repair ( avr ) and repair of the fistula .
tee at the mid - esophageal level showing vegetation ( * ) on the aortic valve and fistula formation ( arrow ) between the aortic root and the right atrium .
the next day the patient underwent avr with a bioprosthetic valve and bovine patch repair of sinus of valsalva fistula .
she remained hemodynamically stable , but required permanent pacemaker implantation due to permanent complete atrioventricular block post - surgery . at a month follow - up ,
the patient has been stable , with normal valvular function , and preserved ejection fraction .
acf is a rare entity occurring in less than 2.2% of native valve endocarditis ( 1 ) .
the most common agents are staphylococcus aureus , streptococcus spp . , enterococcus spp . , and other less common bacterial and fungal infections ( 1 , 2 ) .
it is thought that the process leading to an acf is the resulting inflammation from the bacterial colonization of the valve and surrounding tissue with subsequent abscess formation , creating erosion of the sinus of valsalva ( 13 ) .
the intervalvular fibrosa is particularly prone to infection given its avascularity and infected regurgitation of jet striking subvalvular structures ( 4 ) .
a left - to - right shunt is created from any of the aortic valve sinuses into any of the cardiac chambers without preference , which causes hemodynamic instability ( 2 , 5 ) .
therefore , clinical presentation can range from a non - clinically significant murmur , to a chest pain syndrome , acute coronary syndrome , refractory heart failure , or even aortic dissection ( 6 , 7 ) .
reported cases of aortocavitary fistula sob , shortness of breath ; acs , acute coronary syndrome ; tavr , transcatheter aortic valve replacement ; hocm , hypertrophic obstructive cardiomyopathy . as seen in our index case ,
acf has been documented most commonly in cases of aortic valve ie , more in prosthetic than native aortic valves ( 5 , 8) .
acf rarely presents as a complication of right - sided ie , and when that is the case , it is usually a late presentation ( 9 , 10 ) .
clinical scenarios from primary ie as in our case have also been described with cardiac surgery ( 11 , 12 ) , percutaneous interventions ( 13 ) , chest trauma , and autoimmune process ( 14 ) .
though clinical presentation of acf will be determined mainly by the inciting primary process and the size of the shunt , the variety of the initial clinical presentations can range from acute decompensated heart failure ( 6 ) to asymptomatic patients with only an associated murmur ( 15 , 16 ) .
when symptomatology is poor , a high index of suspicion is required , especially in the background of recent surgery or ie .
the acf murmur has been described as a continuous - thrill murmur ( 17 , 18 ) .
the median time for the echocardiographic diagnosis of the fistula is usually 1 month after onset of symptoms ( 5 ) .
tte is usually the first imaging method used , although tee is a better modality to describe valvular and paravalvular pathology ( 19 , 20 ) , with better delineation of function and morphology ( 19 , 20 ) . in both , tte and tee ,
color doppler can detect a highly turbulent flow between the aorta and the cardiac chambers ( 5 ) .
recently , three - dimensional echocardiography has also emerged as an excellent modality to delineate anatomic and unconventional views of cardiac structures detecting volume data , cropping , and slicing in various planes ( 2123 ) .
ct can be considered as a useful complement in visualizing the cardiac lesions of ie if echocardiography is inconclusive .
it provides assessment of valvular and perivalvular involvement , extra - cardiac lesions , and non - invasive evaluation of the coronary artery anatomy , simultaneously ( 24 ) .
moreover , comprehensive evaluation of anatomic relationships of perivalvular abscess / pseudoaneurysms may be beneficial for presurgical planning ( 25 ) .
this can also be accomplished with cardiac magnetic resonance and aortography ; both of which can provide an excellent anatomical and flow dynamic description of the acf before closure ( 2629 ) .
disadvantages of ct and angiography compared to echocardiography are the contrast - induced nephropathy and radiation exposure .
acf carries severe morbidity and mortality , especially in patients who are elderly , have severe heart or renal failure , require emergent procedures , or are infected with staphylococcus ( 1 , 3 , 5 ) .
acute and subacute ie can present in different forms with subtle clinical signs and symptoms .
physical examination and non - invasive imaging are essential in the diagnosis and management of ie .
paravalvular complications of ie should be treated in a timely manner to avoid further destruction of paravalvular tissue including the conduction system .
the authors have not received any funding or benefits from industry or elsewhere to conduct this study . | infective endocarditis has different presentations depending on the involvement of valvular and perivalvular structures , and it is associated with high morbidity and mortality .
aortocavitary fistula is a rare complication .
we introduce the case of a 48-year - old female with native valve endocarditis , complicated by aortocavitary fistula to the right atrium , and consequently presented with syncope . |
it is a point of convergence of the sutures between the frontal , sphenoid , parietal , and squamous temporal bones .
there are varied patterns of articulation of these bones and sometimes a small epipteric bone may be present .
there are four types of sutural pattern : sphenoparietal , the sphenoid and parietal bones are in direct contact ; frontotemporal , the frontal and temporal bones are in direct contact ; stellate , all the four bones meet at a point ; and epipteric , where there is a small sutural bone uniting all the bones .
the pterion is located superior to the zygomatic arch and posterior to the frontozygomatic suture .
this area is known as the weakest part of the skull , yet it overlies the course of the anterior division of the middle meningeal artery , thus making it vulnerable to rupture , leading to extradural hematoma in the event of a blunt trauma to the side of the head .
in addition , it acts as an important landmark for locating the broca 's motor speech area , anterior pole of the insula , and middle cerebral artery .
pterional or lateral approach is occasionally used in operations involving the broca 's motor speech area and repairing aneurysms of the middle cerebral artery .
differences in the exact location of the pterion have been observed among different races , and this could be due to genetic or environmental influences affecting the craniometric indices of human skull .
sixty - two dry human skulls from the department of human anatomy , university of ibadan , were assembled for this study .
the skulls were from prosected specimens of dissected cadavers used for medical students ' training .
the cadavers were sourced from lagos , ile - ife , ibadan , and other cities within the southwestern region of nigeria .
thirty - seven adult skulls were selected for the study after exclusion of those with deformities and trauma affecting the landmarks for measurement , for example , fractures of the zygomatic arch .
the skulls were divided into 21 males and 16 females using gross dimorphic characteristics for sexual characterization .
inspection of the pterion was carried out and classified into four types : sphenoparietal , frontotemporal , stellate , and epipteric according to previous classifications .
measurements ( figure 1 ) were taken on both sides of the skull from the pterion to the midpoint of zygoma ( mpz ) and to the frontozygomatic suture ( fzs ) using a manual vernier calipers with an accuracy of 0.01 mm .
means and standard deviations were generated and compared using the student 's t - test for the assessment of side and gender differences .
the occurrence of the different types of pterion in the nigerian skulls is represented in figure 2 .
the sphenoparietal type was the most common type ( 86.1% ) , followed by the frontotemporal ( 8.3% ) and stellate ( 5.6% ) types .
the means and standard deviations of the various measurements taken from the pterion are presented in table 1 .
the distance of the pterion posterior to the frontozygomatic suture and superior to the midpoint of the zygomatic arch was compared between the male and female skulls .
the study revealed a statistically significantly higher pterion in the male skulls when compared with the females , but no side differences were observed .
the sphenoparietal type of pterion was observed to be the most common among the nigerian skulls , just as has been reported in different races , for example , indians , turks , and kenyans [ 79 ] . however ,
though the actual determinants of the formation of the pterion are unknown , articulation of the cranial bones is thought to be under genetic influence especially the msx2 gene .
the fact that the development of the calvarium is tightly coordinated with the growth of the brain may explain the prevalence of frontotemporal pattern of sutures among monkey skulls as reported by wang et al . unlike humans with larger brains who have a predominantly sphenoparietal pattern of suture .
the pterion was located 39.74 0.505 mm and 37.95 0.657 mm above the midpoint of the zygomatic archin males and females , respectively .
this is higher than that observed in koreans reported as 36.9 3.8 mm and indians reported as 38.5 mm .
however , it is similar to the 39.31 3.28 mm and 37.35 2.97 mm in males and females , respectively , reported in a kenyan study .
this is likely due to the slightly higher arch of the cranium in africans when compared to the asians .
the pterion has been widely reported to be higher in males than in females [ 7 , 9 ] , and this study reports a similar finding .
reasons proffered for this include the observations from morphometric studies that female skulls are shorter and broader in proportion than male ones and the presence of a more robust zygoma in males .
the pterion was located 31.87 0.642 mm in males and 30.35 0.8358 mm in females posterior to the frontozygomatic suture .
this concurs with description by williams et al . that the pterion lies 30 to 35 mm away from the frontozygomatic suture and similar to the kenyan study which was reported as 30.35 3.61 mm posterior to the frontozygomatic suture
however , there are wide variations with reports of 26.8 4.5 mm and 35 5 mm in koreans and turks , respectively .
there were no significant side variations in the distance from the midpoint of pterion to the frontozygomatic suture and midpoint of zygoma among all the skulls ; hence the landmarks can be used to locate the pterion irrespective of the side .
this information is of great importance for neurosurgical operations in regions where neuronavigation equipments are unavailable and for anthropological identification of nigerian skulls . | the pterion which marks the union of 4 bones of the cranium is located superior to the zygomatic arch and posterior to the frontozygomatic suture .
it is an important neurosurgical landmark for the lateral / pterional approach and has racial differences in both its location and pattern of union of the bones .
this study aims to analyze the location and types of pterion in adult nigerian skulls .
bilateral sides of 37 adult dry skulls were studied .
the pterion types were classified ; linear distances from the centre of the pterion to the midpoint of the zygomatic arch and to the frontozygomatic suture were measured ; these were analyzed for side and gender differences .
sphenoparietal was the most common pterion type ( 86.1% ) followed by frontotemporal ( 8.3% ) , stellate ( 5.6% ) , and epipteric types ( 0% ) .
the mean distances from the pterion to the midpoint of zygomatic arch were 39.74 0.505 mm and 37.95 0.657 mm in males and females , respectively , while the distances to the frontozygomatic suture were 31.87 0.642 mm and 30.35 0.836 mm .
the vertical position of the pterion was significantly higher in males than females .
bilateral occurrence is statistically insignificant .
this information will be of neurosurgical and anthropological importance . |
pregnant women are at increased risk of 2009 influenza a / h1n1 infection and its complications [ 13 ]
. the viral infection does not only cause significant maternal morbidity , but also have a negative impact on pregnancy outcome in women hospitalized because of the infection .
preterm birth rates and stillbirth have been reported to be more prevalent in these women compared to noninfected women [ 47 ] .
as well , it is reported that up to 25% of neonates born from mothers that recovered from severe influenza virus infection during pregnancy were small for gestational age .
it is unclear by which mechanism influenza virus infection causes these negative effects on pregnancy outcome .
we hypothesized that chronic villitis , known to be associated with both intrauterine growth restriction and stillbirth and considered to be of viral origin in a considerable percentage of cases , may be responsible for these effects .
we investigated whether chronic villitis was more prevalent in placentas of neonates whose mother had been affected by influenza infection during pregnancy compared to placentas of a cohort of uncomplicated pregnancies .
we conducted a cohort study to investigate the effects of maternal influenza a / h1n1 infection on the course of pregnancy .
the study protocol was approved by the local medical ethical review board and the clinical investigation was conducted according to the principles expressed in the declaration of helsinki .
, patients filled out a questionnaire on vaccination against influenza virus , influenza like symptoms during the past six months , and , if this was the case , use of anti - influenza antiviral drugs .
influenza like symptoms were defined as fever 38,0c , malaise , and cough eventually combined with myalgia .
patients were prospectively followed up . in case of influenza like symptoms , pcr on influenza on a swab taken from nose and pharynx was done .
clinical cases of influenza virus infection were defined as influenza like symptoms : fever 38,0c , malaise , and cough eventually combined with myalgia during the 2009 influenza pandemic in absence of a confirmatory pcr .
if signs of chronic villitis were present , a pcr on placental tissue with chronic villitis was done .
dna extraction of the paraffin embedded tissue was performed by conventional xylene deparaffinization , two xylene washes 60 minutes rt , two 96% ethanol washes , and one acetone wash followed by protease k digestion ( 18 hours , 56c ) .
nucleic acids were extracted using the total nucleic acid ( for swabs ) or dna ii tissue protocol ( for placental tissue ) with the magna pure lc nucleic acid isolation system ( roche diagnostics , basel , switzerland ) .
each sample was eluted in 200 l buffer , which was sufficient for all real time pcr analyses .
cdna was synthesized by using multiscribe reverse transcriptase ( rt ) and random hexamers ( both from applied biosystems , foster city , ca , usa ) .
each 200 l reaction mixture contained 80 l of eluted rna , 20 l of 10x rt buffer , 5.5 mmol / l mgcl2 , 500 mol / l of each deoxynucleoside triphosphate , 2.5 mol / l random hexamer , and 0.4 u of rnase inhibitor per microlitre ( all from applied biosystems ) . after incubation for 10 minutes at 25c , rt was carried out for 30 minutes at 48c , followed by rt inactivation for 5 minutes at 95c .
the pcr consisted of a final concentration of 1x universal master mix ( applied biosystems ) , 900 nm of each primer , 150 nm of the influenza a probe , and 2 l of target cdna and was made up to a volume of 25 l with nuclease free water ( promega corp .
, madison , usa ) . the real time quantitative pcr amplifications were measured in real time mode using the abi7500 ( applied biosystems ) .
the placentas were weighted without membranes and umbilical cord , and the weight was classified according to gestational age percentiles from pinar et al . .
routine hematoxylin and eosin stained slides from a minimum of 2 umbilical cord sections ( at the fetal and placental side ) , a membrane roll , one sample from the umbilical cord insertion , and two slides of normal placental parenchyma , including decidua and chorionic plate , and additional slides from macroscopical abnormalities were reviewed .
chorioamnionitis was diagnosed based on the presence of polymorphonuclear cells ( neutrophilic granulocytes ) in the chorionic plate or the extraplacental membranes .
diagnosis of funisitis was based on the presence of neutrophilic granulocytes in the wall of the umbilical vein and/or arteries and wharton 's jelly .
chronic villitis was diagnosed as an infiltrate of lymphocytes and macrophages in the placental villi .
in addition to the presence of chorioamnionitis , funisitis , or villitis , the severity of inflammation was graded mild , moderate , or severe , according to the staging and grading system of redline with slight modifications as previously published and presented in table 1 .
placental parenchymal infarction was defined as an area of necrotic villi surrounded by an area of ischaemia with increased hyperchromasia of trophoblast and increased syncytial knotting .
fetal thrombotic lesions were scored as at least 5 avascular fibrotic villi without inflammation or mineralization or if adherent thrombi in stem vessels were present .
an increase of nucleated red blood cells in the fetal circulation is the consequence of either anaemia or hypoxia and was scored when nucleated red blood cells were present in more than two capillaries in a random 10x field .
we compared the results of the histologic examination of the placentas with those of a historic cohort that was published before .
this cohort consisted of placentas derived from uncomplicated pregnancies with spontaneous onset of labor and vaginal delivery .
the placentas in this cohort were examined in the same way and by the same laboratory and pathologist as the placentas collected in this study .
of the participants , 29 ( 20% ) were clinical cases of influenza virus infection , of which 6 were tested by pcr .
all 29 pregnant women gave live birth and both mothers and children were discharged in good health .
of these 15 , 10 were clinical cases and 5 were pcr proven influenza cases .
gestational age at onset of symptoms varied from 2 to 32 weeks with a median of 21 weeks of gestation .
there were 5 patients with infection in the first , 8 in the second , and 2 in the third trimester of pregnancy .
other treatments included amoxicillin ( clavulanic acid ) in 4 patients , paracetamol in 1 patient , and no treatment in 4 patients . in all but one patient , symptoms subsided within 10 days . in 7 out of 15 placentas ( 47% )
when comparing characteristic between those patients whose placentas showed villitis to those whose placentas did not , there were no differences in average duration from infection to delivery ( 153 days ( sd 64 days ) and 140 days ( sd 67 days ) for cases with resp . without chronic villitis ) .
there were also no differences in placental weight , birth weight of the neonates and number of neonates that were small for gestational age . in the placentas with chronic villitis 3 out of 7 placentas were below 10th percentile for placental weight according to gestational age , while all placentas without chronic villitis were of normal weight .
there were no differences in chorioamnionitis , funisitis , infarction , or thrombosis ( results not shown ) .
this appears to be considerably higher than the 24% in our historic cohort and even more different from the 515% reported by redline . comparing the 47% in our cohort with the 24% in our historic cohort yields an absolute difference of 23% with a 95% confidence interval for difference of 3 to 49% .
interestingly , the placentas of all three women treated with oseltamivir demonstrated chronic villitis ( all grade 1 ) .
however , the relatively small number of placentas that we were able to study makes it hard to draw any firm conclusions on these numbers .
a large proportion of cases of chronic villitis however were classified as villitis of unknown etiology ( vue ) and may reflect a noninfectious immune response .
which proportion of chronic villitis has to be attributed to viral infections and which proportion has to be classified as vue is still a matter of debate [ 10 , 13 ] . in some cases
however , it is also reported that 41% of lesions originally classified as vue appear to have a viral origin when examined by electron microscopy . regardless of its etiology ,
recent reports including a meta - analysis have confirmed the association of vue with intrauterine growth restriction and , to a lesser extent , with pregnancy loss [ 15 , 16 ] . in our series
the small size of our study sample limits the ability to detect differences in pregnancy outcomes between cases with and without chronic villitis .
we found no significant difference in birth weight of neonates and there were also no significant differences between placental weight and number of placentas with placental weight < p10 , although the latter almost reached statistical difference ( p = 0 , 07 ) .
we were not able to demonstrate the presence of 2009 influenza a / h1n1 in placental tissue with histological signs of chronic villitis .
second , the pregnant women in our cohort were relatively mildly affected by the virus .
pregnant women who are more severely affected and have viraemia may be more prone to placental infection by influenza . nevertheless , for seasonal influenza a h1n1 placental infection and vertical transmission
third , the virus might also have been cleared from placental tissue since the time between infection and delivery was relatively long in our cohort .
fourth , we may not have selected the appropriate patients and/or placentas to examine , since 10 out of 15 of our cases were clinical cases without a confirmative pcr . however , given that all patients tested by pcr on pharyngeal swab had a proven infection , a large proportion of patients that were not tested but had similar symptoms during the influenza pandemic will probably have had influenza infection as well .
therefore , we expect a large proportion of clinical cases to be actual influenza infections .
this may have decreased the sensitivity of the pcrs , since both natural degradation of rna and the fixation and paraffin - embedding procedure may interfere with the transcription and amplification of rna . on the other hand , we selected those pieces of placental tissue that showed chronic villitis , hypothesizing that we would have optimal chances of finding evidence of placental infection by influenza if present .
our findings are in line with results from turkey . in a series of seven placentas derived from pregnancies complicated by proven pandemic influenza a h1n1 infection , kanmaz et al . performed pcr on random pieces of placental tissue
not related to any histologic abnormalities . like us , they did not find evidence of viral replication in placental tissue .
the increased prevalence of chronic villitis of unknown etiology in women with autoimmune diseases and in ovum donor pregnancies suggests that it results from a disturbance of normal pregnancy tolerance , which is predominantly regulated by several types of t cells .
the immunogenic activity of these cells , however , is influenced by cytokines that are known to be increased in influenza virus infection [ 21 , 22 ] .
we therefore hypothesize that the burst of inflammatory cytokines accompanying influenza virus infection may produce lesions of chronic villitis .
given the limitations of our study and the limited literature on this subject further studies are needed to affirm or deny this hypothesis .
in our small series , we found a high proportion of placentas with chronic villitis in women infected with 2009 influenza a / h1n1 during pregnancy .
we could not demonstrate that chronic villitis was caused by direct infection of the placenta with influenza virus .
our results underline the need for further investigation of whether the immune response triggered by influenza virus infection is responsible for the increased rates of serious adverse events that occur in pregnancies complicated by influenza virus infection . | introduction . pandemic influenza
a / h1n1 infection during pregnancy has a negative impact on several aspects of pregnancy outcome .
as yet , no elucidating mechanism has been revealed for these effects .
we investigated whether placentas of pregnancies complicated by 2009 influenza a / h1n1 infection demonstrated an increased rate of chronic villitis and whether this villitis was caused by influenza virus . methods .
we performed a cohort study on 145 pregnant outpatients during the 2009 - 2010 influenza a h1n1 pandemic .
the placentas of patients with influenza infection were examined for histologic signs of chronic villitis . in case of villitis , polymerase chain reaction ( pcr ) on influenza virus
was performed on placental tissue .
results .
29 patients had influenza infection .
placentas of 15 of these patients were collected and examined . in 7 cases ( 47% )
chronic villitis was detected .
placental weight and birth weight of the neonates did not differ between cases with and without chronic villitis . in all cases pcr
was negative for influenza . conclusion . in our series ,
chronic villitis was present in a high proportion of placentas of pregnancies complicated by 2009 influenza a / h1n1 infection .
we could not demonstrate the presence of influenza virus in placental tissue . |
traf6-flag , traf6-flag ( or traf6 298522 ) , traf2flag ( or traf2 87501 ) , myd88-au1 , myd88-au1 ( or myd88 152296 ) , irak , nik(kk - aa ) , and ha - p46sapk-pcdna3 expression vectors have been described ( 8 , 18 ) .
expression vectors for nh2-terminal flag - tagged htoll and htoll ( amino acids 1666 ) were constructed by inserting pcr - generated cdna fragments lacking the coding sequence for the signal peptide , into the mammalian expression vector pflag - cmv-1 ( eastman kodak co. , rochester , ny ) .
human embryonic 293 or 293 t cells were transiently transfected by the calcium phosphate method with the indicated plasmids .
the total amount of dna was kept constant by supplementation with an empty vector ( pcdna3 ; invitrogen corp . ,
monocytes were separated from fresh blood of healthy donors as described previously ( 19 ) .
2436 h after transfection , cells were lysed in 0.5 ml buffer ( 1% np-40 , 150 mm nacl , 50 mm tris , 1 mm edta , and protease inhibitor cocktail ) .
cell lysates were adjusted to 0.7 m nacl , and the indicated antibodies were added for 14 h at 4c .
after extensive washing ( in lysis buffer with the addition of 0.1% sds ) , the sepharose beads were boiled in sample buffer , and eluted proteins were fractionated by sds - page .
cells were transfected with endothelial leukocyte adhesion molecule luciferase reporter plasmid ( nf-b luc ; 0.1 g ) , pcmv--galactosidase ( gal ; 0.2 g ) , and the indicated expression vectors .
relative nf-b activity was calculated by normalizing relative luciferase activity with gal activity as described previously ( 8) .
data shown are from one out of two to five independent experiments with similar qualitative results .
cells were transfected with nf-b luc ( 0.5 g ) , ha - p46sapk-pcdna3 ( 2 g ) , and the indicated expression vectors .
48 h after transfection , cells were lysed in ripa buffer containing 0.5 mm dithiothreitol , 20 mm -glycerophosphate , 1 mm sodium orthovanadate , 10 mm sodium fluoride , 1 mm pmsf , leupeptin ( 20 g / ml ) , and aprotinin ( 20 g / ml ) .
lysates were cleared by centrifugation , and protein concentration was measured using a commercial bradford protein assay ( promega corp . , madison , wi ) .
equal amounts of each lysate ( usually 500 g ) were incubated on ice with 2 g antiserum to ha ( rabbit polyclonal ; santa cruz biotechnology , inc . , santa cruz , ca ) for 2 h at 4c .
precipitates were boiled in sample buffer and run onto an sds - polyacrylamide gel .
finally , immunoblotting was performed to detect the active phosphorylated form of sapk ( pthr-183/ptyr-185 of hjnk1 ) , or sapk as a control , by using specific antibodies ( anti - pjnk mouse monoclonal , and anti - ha ; santa cruz biotechnology , inc . ) .
an aliquot of the cell lysate was also analyzed for nf-b activation as above .
traf6-flag , traf6-flag ( or traf6 298522 ) , traf2flag ( or traf2 87501 ) , myd88-au1 , myd88-au1 ( or myd88 152296 ) , irak , nik(kk - aa ) , and ha - p46sapk-pcdna3 expression vectors have been described ( 8 , 18 ) .
expression vectors for nh2-terminal flag - tagged htoll and htoll ( amino acids 1666 ) were constructed by inserting pcr - generated cdna fragments lacking the coding sequence for the signal peptide , into the mammalian expression vector pflag - cmv-1 ( eastman kodak co. , rochester , ny ) .
human embryonic 293 or 293 t cells were transiently transfected by the calcium phosphate method with the indicated plasmids .
the total amount of dna was kept constant by supplementation with an empty vector ( pcdna3 ; invitrogen corp . ,
monocytes were separated from fresh blood of healthy donors as described previously ( 19 ) .
2436 h after transfection , cells were lysed in 0.5 ml buffer ( 1% np-40 , 150 mm nacl , 50 mm tris , 1 mm edta , and protease inhibitor cocktail ) .
cell lysates were adjusted to 0.7 m nacl , and the indicated antibodies were added for 14 h at 4c .
after extensive washing ( in lysis buffer with the addition of 0.1% sds ) , the sepharose beads were boiled in sample buffer , and eluted proteins were fractionated by sds - page .
cells were transfected with endothelial leukocyte adhesion molecule luciferase reporter plasmid ( nf-b luc ; 0.1 g ) , pcmv--galactosidase ( gal ; 0.2 g ) , and the indicated expression vectors .
relative nf-b activity was calculated by normalizing relative luciferase activity with gal activity as described previously ( 8) .
data shown are from one out of two to five independent experiments with similar qualitative results .
cells were transfected with nf-b luc ( 0.5 g ) , ha - p46sapk-pcdna3 ( 2 g ) , and the indicated expression vectors .
48 h after transfection , cells were lysed in ripa buffer containing 0.5 mm dithiothreitol , 20 mm -glycerophosphate , 1 mm sodium orthovanadate , 10 mm sodium fluoride , 1 mm pmsf , leupeptin ( 20 g / ml ) , and aprotinin ( 20 g / ml ) .
lysates were cleared by centrifugation , and protein concentration was measured using a commercial bradford protein assay ( promega corp . , madison , wi ) .
equal amounts of each lysate ( usually 500 g ) were incubated on ice with 2 g antiserum to ha ( rabbit polyclonal ; santa cruz biotechnology , inc . , santa cruz , ca ) for 2 h at 4c .
precipitates were boiled in sample buffer and run onto an sds - polyacrylamide gel . finally , immunoblotting was performed to detect the active phosphorylated form of sapk ( pthr-183/ptyr-185 of hjnk1 ) , or sapk as a control , by using specific antibodies ( anti - pjnk mouse monoclonal , and anti - ha ; santa cruz biotechnology , inc . ) .
an aliquot of the cell lysate was also analyzed for nf-b activation as above .
the expression and eventual regulation of specific transcripts encoding for htoll were analyzed in distinct cell types that play a critical role in the natural immune response . in particular ,
human monocytes were separated from healthy donors and treated with the bacterial product lps for different periods of time . as shown in fig .
1 a , specific transcripts for htoll were present in these cells and were induced significantly after treatment with lps .
these observations suggest that modulation of a nonclonal receptor , namely htoll , after exposure to infectious agents may play a regulatory role in the natural immune response . of note , pmn and dendritic cells also transcribed htoll mrna at different levels ( our unpublished observations ) .
a chimeric version of htoll in which the extra cellular portion was substituted with the corresponding region of cd4 ( cd4/htoll ) has been shown previously to induce nf-b activation in jurkat cells ( 2 ) .
we engineered distinct expression constructs encoding for flag epitope tagged htoll ( htoll - flag ) or for a truncated version of htoll that lacks most of the cytoplasmic portion ( htoll - flag ) .
ectopic expression of htoll - flag but not htoll - flag induced nf-b activation in human embryonic 293 t cells at levels similar to the cd4/toll chimeric protein that served as a positive control ( fig . 1 , b and c ) .
from these observations , it is apparent that either cd4-driven or ectopic expression forced aggregation of the cytoplasmic portions of distinct htoll receptors is sufficient to trigger a signaling cascade that leads ultimately to activation of the transcription factor nf-b . given this , one could speculate that an as yet unidentified htoll ligand binds to htoll and induces its oligomerization and subsequent signaling cascade , in a manner similar to il-1 and tnf with their cognate receptors .
htoll shares significant sequence similarity with distinct members of the il-1r family , including il-1ri , il-1racp , and myd88 ; of note , phe 513 and trp 514 in il-1ri , which are conserved in all of these proteins , have been shown to be essential for il-1ri to signal ( fig .
since we have shown recently a homophilic interaction to occur between the il-1racp and myd88 throughout their homologous domains ( 8) , we asked whether htoll and the adapter protein myd88 could interact . upon coexpression , myd88 and
htoll formed an immunoprecipitable complex ; in contrast , a mutant version of htoll , which lacks the region of homology to myd88 and which was unable to induce nf-b activation , failed to bind myd88 ( fig .
2 b ) . a mutant version of myd88 ( myd88 ) , encoding only for the cooh - terminal il-1r like domain , abrogates
il-1ri / il-1racp induced nf-b activation ( 8) ; given this , we analyzed whether myd88 could act as a dominant negative inhibitor of htoll - induced nf-b activation .
as predicted , myd88 specifically inhibited htoll - induced but not tnfr-2induced nf-b activity , lending functional credence to the interaction occurring between htoll and myd88 ( fig .
it is apparent that both il-1r and htoll recruit the adapter protein myd88 to their respective signaling complex .
irak and irak-2 are two additional proximal mediators of the il-1r signaling complex ; irak is recruited to the il-1racp , whereas irak-2 preferentially binds il-1ri ( 8 , 10 ) . given this , we asked whether irak or irak-2 could interact with htoll . upon ectopic expression , irak and
in contrast , irak-2 bound only weakly to htoll compared with il-1ri ( fig .
3 , a and b ) , thus suggesting that it may not represent a relevant htoll signal transducer . nf-b activation induced by a number of cytokine receptors is mediated by members of the traf adapter family .
traf2 , for example , plays a critical role in nf-b activation mediated by tnfr-1 and tnfr-2 ( 21 , 22 ) .
traf6 has been implicated in the il-1 signaling pathway and has been shown to complex with irak and irak-2 downstream to the receptor signaling complex ( 8 , 11 ) .
therefore , we determined whether dominant negative versions of either could act to inhibit htoll - induced nf-b activity .
traf6 but not traf2 significantly impaired htoll - induced nf-b activity , suggesting that traf6 may act as an additional downstream mediator of the htoll - induced nf-b activation cascade ( fig .
the protein kinase nik has been shown to act as a general mediator of traf - induced nf-b activation ( 12 ) ; once activated , nik directly phosphorylates and activates the ikk/ complex , which is responsible for i-b phosphorylation and subsequent nf-b activation ( 1317 ) . dominant negative versions of nik , in which the critical lysine has been mutated to alanine [ nik 429430 ( kk - aa ) ] , act to inhibit nf-b activation induced by fas , tnf , and il-1 ( 12 ) . given this
, we asked whether nik(kk - aa ) could inhibit htoll - induced nf-b activation .
nik(kk - aa ) abrogated nf-b activity triggered by htoll ectopic expression ( fig .
in addition to inducing activation of nf-b , distinct inflammatory cytokines also induce sapk , also known as jnk .
the active phosphorylated form of sapk binds to and phosphorylates the transcription factors c - jun , activating transcription factor 2 ( atf2 ) , and ternary complex factor ( tcf)/elk1 ( 2325 ) . in particular , activation of sapk/ jnk by
the tnfr-1 occurs through a traf2-dependent pathway , as a dominant negative version of traf2 acts to inhibit both nf-b and c - jun activation induced by tnf ( 18 , 26 , 27 ) .
in contrast , a dominant negative version of nik , which abrogates tnf - induced nf-b activation , fails to inhibit c - jun phosphorylation , supporting a model wherein tnf - mediated nf-b and c - jun pathways bifurcate at traf2 ( 28 , 29 ) .
ectopic expression of increasing amounts of htoll - flag but not htoll - flag resulted in activation of sapk as indicated by specific phosphorylation at thr 183 and tyr 185 ( fig .
4 a ) . overexpression of traf6 also activated sapk as described previously ( 28 ) . to identify mediators of htoll - mediated jnk activation
, we cotransfected 293 cells with htoll and dominant negative versions of either myd88 or traf6 .
surprisingly , myd88 but not tra6 acted to inhibit htoll - triggered jnk phosphorylation ( fig . 4 b ) .
importantly , under the same experimental conditions , both myd88 and traf6 abrogated htoll - induced nf-b activation ( 96 and 90% inhibition , respectively ) .
additionally , traf6 alone , as well as myd88 , failed to activate jnk ( data not shown ) .
collectively , these observations indicate that although ectopic expression of traf6 induced sapk , a dominant negative version of traf6 failed to inhibit htoll - induced jnk phosphorylation . given this
, one could speculate that although traf6 overexpression is sufficient to activate jnk/ sapk , endogenous traf6 does not provide a significant contribution to jnk / sapk activation by htoll ( fig .
, myd88 appears to represent the most upstream mediator of the htoll - mediated signaling cascade , which ultimately activates nf-b and c - jun , thus driving transcriptional activation of several cytokines and costimulatory molecules .
therefore , it may represent a potentially useful therapeutic target for controlling the molecular switch from the innate to the adaptive immune response .
( a ) human monocytes were treated with lps ( 100 ng / ml ) for different periods of time and analyzed for their htoll mrna content by northern blotting .
( b ) ectopic expression of htoll - flag and cd4/toll but not the mutant version htoll - flag activate nf-b in 293 t cells in a dose - dependent manner , as measured by nf-b reporter gene activity .
( c ) equal amounts of htoll - flag and htoll - flag are produced upon ectopic expression in 293 t cells ( 3.2 g of each expression construct were used for this experiment ) .
( a ) sequence alignment of human myd88 ( amino acids 152296 ) , htoll ( 667 840 ) , hil-1racp ( 391570 ) , and hil-1ri ( 381569 ) .
dots , conserved amino acids that are essential for il-1ri to signal ( reference 20 ) .
( b ) myd88 associates with htoll but not with a truncated version of htoll ( htoll ) lacking the cytoplasmic region sharing sequence similarity with myd88 .
293 t cells were transfected with htoll - flag , htoll - flag , or il-1racp flag as a positive control together with au1-tagged myd88 .
the presence of myd88 that coprecipitated with the receptors was detected by immunoblotting with a rabbit polyclonal antiserum to myd88 .
( c ) myd88 inhibits htoll - induced but not the unrelated tnfr-2induced nf-b activity
data are expressed as the percentage of relative receptor - induced nf-b activity .
( a ) irak is recruited to htoll but not to the inactive truncated version of htoll ( htoll ) .
cells were transfected and analyzed as in fig . 2 b. ( b ) irak-2 binds very weakly to htoll .
cells were transfected and analyzed as in a. ( c ) traf6 but not the unrelated traf2 attenuates cd4/toll - induced nf-b activity .
a dominant negative mutant version of the downstream molecule nik [ nik(kk - aa ) ] also inhibits cd4/htoll - induced nf-b activity .
( a ) htoll activates sapk / jnk in a dose - dependent manner , as determined by the presence of the active phosphorylated form of jnk ( pjnk ) .
( b ) myd88 ( 152296 ) ( myd88 ) but not traf6 ( 289522 ) ( traf6 ) abolishes htoll - induced sapk / jnk phosphorylation .
the diagram shows that the bifurcation of nf-b and jnk / sapk activation by htoll or tnfr-1 occurs at different levels with respect to the specific trafs . | the human homologue of drosophila toll ( htoll ) is a recently cloned receptor of the interleukin 1 receptor ( il-1r ) superfamily , and has been implicated in the activation of adaptive immunity .
signaling by htoll is shown to occur through sequential recruitment of the adapter molecule myd88 and the il-1r associated kinase .
tumor necrosis factor receptor activated factor 6 ( traf6 ) and the nuclear factor b ( nf-b)inducing kinase ( nik ) are both involved in subsequent steps of nf-b activation .
conversely , a dominant negative version of traf6 failed to block htoll - induced activation of stress - activated protein kinase / c - jun nh2-terminal kinases , thus suggesting an early divergence of the two pathways . |
from march 2003 though november 2004 , admitting physicians in district and provincial hospitals within 9 provinces of thailand in the north , northeast , central , and southern regions were invited to recruit patients of all ages suspected on clinical grounds to have leptospirosis .
clinical features considered were those specifically referred to in the national guidelines ( e.g. , fever , headache , muscle pain , meningism , conjunctival suffusion , and jaundice ) together with hemoptysis , hepatomegaly , diarrhea , hypotension , and reduced urine output . from each patient ,
a 5-ml serum sample was taken to be cultured for leptospira , another 5-ml serum sample was taken for serologic testing , and a third sample was taken 2 weeks later for serologic testing .
. microscopic agglutination test ( mat ) was performed at the world health organization ( who)/united nations food and agriculture organization ( fao)/world animal health organisation ( oie ) collaborating center for reference and research on leptospirosis , brisbane , queensland , australia ( 4 ) .
a positive mat was defined as a single titer of > 1:400 or a 4-fold rise in titer between acute and convalescent phase samples . for leptospira culture ,
100 l of whole blood , 500 l of plasma , and 500 l of serum were each injected into 3 ml of ellinghausen , mccullough , johnson , and harris ( emjh ) medium and supplemented with 3% rabbit serum and 0.1% agarose , then incubated aerobically at room temperature ( 25c30c ) for 6 months and examined every week for 2 months , every 2 weeks during months 3 and 4 , and once a month during months 5 and 6 .
examination was done by placing 1 drop of culture onto a microscopic glass slide and viewing by dark - field microscopy at 200 magnification .
positive cultures were referred to the who / fao / oie collaborating center for reference and research on leptospirosis for identification by using the cross - agglutination absorption test ( 4 ) .
a total of 700 patients with a clinical diagnosis of leptospirosis were recruited during the study period .
all patients had blood samples collected at the hospital for leptospire culture and serologic testing ; convalescent - phase serum samples were obtained during follow - up for 509 ( 73% ) patients . the median age of patients with suspected leptospirosis was 38 years ( range 295 years , interquartile range ( iqr ) 2849 years ) ; 504 ( 72% ) were men .
the number of clinically diagnosed leptospirosis cases by month in the north , northeast , central , and southern regions is shown in figure 1 .
most cases ( 597 , 85% ) were recorded in 4 provinces in the north or northeast ( table ) .
cases were predominantly identified during the rainy season ( june october ) in the north and northeast in 2003 , with a second peak in the northeast , but not the north , during the rainy season of 2004 .
cases of clinical leptospirosis by month for each geographic region , thailand , march 2003november 2004 .
ci , confidence interval . of the 700 patients who received a clinical diagnosis of leptospirosis , 143 ( 20% ) received a confirmed diagnosis of leptospirosis based on leptospira isolation , mat testing , or both ( table ) .
the median age of patients with confirmed leptospirosis was 35 years ( range 1068 years , iqr 2745 years ) ; 121 ( 85% ) were men .
the diagnosis was confirmed after isolation of leptospires from 15 ( 11% ) patients ; the geographic distribution is shown in the table .
the serovars of cultured leptospira were : l. interrogans serovar ( sv . ) autumnalis ( 7 ) , l. interrogans sv .
hebdomadis ( 1 ) , l. interrogans sv grippotyphosa ( 1 ) , and an unidentified serovar ( 1 ) .
an additional 128 patients with culture - negative samples had been exposed to leptospira as determined by mat ; results for 96 ( 75% ) were based on a 4-fold rising titer and for 32 ( 25% ) , on a single raised titer of > 1:400 .
the geographic distribution of the 143 laboratory - confirmed cases is summarized in the table .
most of these patients ( 124 , 87% ) lived in the 4 provinces found in the north and the northeast ( table ) .
the month of diagnosis for confirmed cases is shown in figure 2 ; most were during the rainy season .
cases of laboratory confirmed leptospirosis and positive predictive accuracy of clinical diagnosis by month , thailand , march 2003november 2004 .
the positive predictive accuracy of a clinical diagnosis is defined by the number of laboratory - confirmed cases divided by the number of clinically suspected cases .
results for each of the 9 provinces are shown in the table . when only data from centers that reported at least 10 cases were used , positive predictive accuracy ranged from 3% to 29% .
diagnosing leptospirosis at the point of care is notoriously difficult in the tropical setting , where several common infectious diseases are often hard to differentiate .
positive predictive accuracy for leptospirosis was highest during the rainy season , an observation that is likely related to the higher disease incidence and pretest probability .
variability in positive predictive accuracy was seen among the 3 provinces with the highest number of both suspected and true cases .
the reason for this is unclear but may relate to perceived risk to the community , local policy , or other factors .
the finding that both clinical and confirmed cases of leptospirosis were more common in the north and northeast is consistent with ddc reports . increased incidence in this region may have resulted from > 1 events , such as an increase in the rodent population , a natural reservoir for this pathogen , and a population in which around one third are positive for leptospira in northeast thailand ( 5 ) . alternatively , 1 clone or a small number of bacterial clones may have become adapted for persistence at greater numbers within the natural host or in the environment .
it is also possible that 1 clone or a small number of clones have become adapted for enhanced invasion of the human host .
the most prevalent serovar isolated was l. interrogans serovar autumnalis ( 7/15 [ 47% ] isolates ) , 6 of which were from cases in the north or northeast .
the effect of the low level of accuracy of hospital - based clinical diagnosis of leptospirosis in rural thailand is not known .
a common disease in this setting that is easily confused with leptospirosis is scrub typhus ; both diseases would be predicted to respond to doxycycline , an antimicrobial drug often prescribed for undifferentiated fever .
further studies are required to define the implications of our findings and determine whether routine laboratory testing for leptospirosis should be implemented in thailand . | we defined the positive predictive accuracy of a hospital - based clinical diagnosis of leptospirosis in 9 provinces across thailand .
of 700 suspected cases , 143 ( 20% ) were confirmed by laboratory testing .
accuracy of clinical diagnosis varied from 0% to 50% between the provinces and was highest during the rainy season .
most confirmed cases occurred in the north and northeast regions of the country . |
central giant cell granuloma of the jaws and giant cell tumor of long bones are well - recognized entities revealing benign nature16 .
their clinical behavior8,13,29 , prognostic factors and the histogenesis have been subject of several studies . in spite of that , these questions remain unclear
11,16,23,26
.
morphologic studies performed in order to compare cgcg and gct features have shown that although most of jaw lesions may be distinguished from tumor of long bones on histological appearance , many jaw lesions display the histological profile of the tumor of long bones .
whitaker and waldron29 ( 1993 ) reported that cgcg of the jaws and gct of long bones could represent the development of a single pathologic process that may be influenced by patient 's age , location and other unknown factors .
the true gct of the jaws is rare and local prognosis is considered worse in gct than in cgcg8 .
the biologic behavior of cgcg of the jaws ranges from a quiescent lesion with absence of symptoms , root resorption or cortical perforation , slow growth , and low recurrence rate , to an aggressive pathological process , characterized by pain , rapid growth , root resorption , cortical perforation , and a high recurrence rate8,21 .
the gct of long bones is a rare benign neoplasm , characterized by local aggressiveness , high recurrence rates and metastasis to the lung14,16,21,25 .
an immunohistochemical study to determine the immunoprofile of the mononuclear cells and proliferative compartment of cgcg of the jaws in clinically aggressive and non - aggressive lesions , using antibodies to cd34 , cd68 , factor xiiia , alfa - smooth muscle actin , prolyl 4-hydroxylase , ki-67 , and p53 protein , revealed that these lesions are primarily fibroblastic ( and myofibroblastic ) with macrophages playing a secondary role , and that it is not possible to predict the behavior of the cgcg of the jaws from histologic features , immunophenotypic or proliferation parameters19 .
other studies have demonstrated immunoreactivity of mononuclear cells and multinucleated cells to cd68 in giant cell lesions , suggesting a histiocyte / macrophage origin for a subset of cells of these lesions6,16,19,28 .
cd68 is a transmembrane glycoprotein of unknown function , being strongly expressed by human monocytes and tissue macrophages10,20 .
several groups of investigators have carried out immunohistochemical studies to observe the immunoreactivity , and distribution pattern of the fibronectin and tenascin in dermatologic diseases22 , giant cell granulomas5 , odontogenic cysts18 , and normal oral mucosa , epithelial dysplasia and fibroepithelial hyperplasia3 .
it is found prominently in the matrix of many connective tissues and is more abundant during embryonic development , tissue remodeling4 and within a wide variety of basement membranes1 .
the fibronectin arrangement in focal adhesions stimulates the cellular growth by increasing the cell entry into the s - phase of cell cycle9 . the proliferative activity and newly formed vessels associated to a fibronectin variant support the idea that some types of fibronectin could be important prognosis factors12 .
this protein has demonstrated significant variation in the distribution and immunoreactivity intensity within individual samples of several lesions17 and pathologic processes7 , being strongly expressed in epithelial malignant tumors24 .
the purpose of this study was to investigate the cd68 expression in mononuclear and multinucleated giant cells and the pattern of immunoreactivity and distribution of fibronectin and tenascin between cgcg of the jaws and gct of long bones , in order to analyze if there are differences in the expression of these proteins that could be used to distinguish the studied lesions .
eight cases of cgcg of the jaws were retrieved from the files of the oral pathology discipline of the federal university of rio grande do norte , school of dentistry , and 7 cases of gct of long bones were obtained from the files of the pathology and cytology laboratory of aracaju - se .
microscopic slides on each case were reviewed and histologic features of multinucleated giant cells , mononuclear cells and stroma were assessed . for the immunohistochemical study , 3-m sections were obtained from the formalin - fixed paraffin - embedded surgical specimens of the lesions .
immunohistochemical staining was carried out using the streptavidin - biotin - peroxidase complex ( sabc ) method .
the following antigens were evaluated : cd68 ( macrophage - associated monoclonal antibody , kp-1(2 ) clone , 1:50 ; dako , glostrup , denmark ) , incubated at 4c overnight ; fibronectin ( a-245 clone,1:600 ; dako ) , incubated at room temperature for 120 min and tenascin ( tn2 clone , 1:50 ; dako ) , incubated at 4c overnight . for
the antibodies against fibronectin and tenascin the tissue sections were previously treated with 0.1% and 0.4% pepsin respectively , and for the cd68 the antigen retrieval was processed in citric acid in three cycles for 5 minutes ( 700watts ) .
the immunoreactivity expressed by cd68 , fibronectin and tenascin antigen was evaluated in all the cases by light microscopy , using the following parameters : positive ( + ) and negative ( - ) staining of the mononuclear cells and multinucleated giant cells for the cd68 antigen , and degrees of staining intensity ( intense or weak ) , distribution ( uniform scattered or in focal areas ) and expression pattern ( fibrillar , reticular and fibrillar / reticular ) of the fibronectin and tenascin in the interstitial extracellular matrix and around blood vessels .
cd68 positive cells were detected in many mononuclear cells and in the majority of multinucleated giant cells of the cgcg of the jaws ( figure 1 ) and gct of long bones ( figure 2 ) evaluated .
the immunostaining results for fibronectin and tenascin in cgcg and gct are summarized in tables 14 , and depicted in figures 36 .
+ + = intense immunoreactivity ; + = weak immunoreactivity ; - = negative immunoreactivity .
+ + = intense immunoreactivity ; + = weak immunoreactivity ; - = negative immunoreactivity .
+ + = intense immunoreactivity ; + = weak immunoreactivity ; - = negative immunoreactivity .
+ + = intense immunoreactivity ; + = weak immunoreactivity ; - = negative immunoreactivity .
cd68 positive cells were detected in many mononuclear cells and in the majority of multinucleated giant cells of the cgcg of the jaws ( figure 1 ) and gct of long bones ( figure 2 ) evaluated .
the immunostaining results for fibronectin and tenascin in cgcg and gct are summarized in tables 14 , and depicted in figures 36 .
+ + = intense immunoreactivity ; + = weak immunoreactivity ; - = negative immunoreactivity .
+ + = intense immunoreactivity ; + = weak immunoreactivity ; - = negative immunoreactivity .
+ + = intense immunoreactivity ; + = weak immunoreactivity ; - = negative immunoreactivity .
+ + = intense immunoreactivity ; + = weak immunoreactivity ; - = negative immunoreactivity .
although the origin of the cells present in giant cell lesions has been investigated through histochemistry23 , ultrastructural19,23 and immunohistochemical methods13,23 , the pathogenesis and nature of these lesions are still unclear . whether the cgcg of the jaws and the gct of long bones are really a single pathologic process is also an unanswered question . as stated by whitaker and waldron29 ( 1993 ) , cgcg of the jaws and gct of long bones
could represent the development of a single pathologic process , modified by age of the patient , location , and other unknown factors . in attempt to clarify whether cgcg and gct are separate entities or variants of the same disease , souza , et al.21 ( 1999 ) performed an immunohistochemical study of the cell cycle related proteins p53 , mdm2 , ki-67 and pcna in samples of both lesions .
these authors verified wide expression of mdm2 in cgcg and in gct and no immunoreactivity to p53 in both lesions . comparing the proliferative activity between lesions ,
gct revealed a reduced percentage of ki- 67- and pcna - positive cells . in view of their results
, these authors suggest that p53 inactivation by mdm2 expression may be involved in the pathogenesis of cgcg and gct .
in addition , souza , et al.21 ( 1999 ) state that the differences observed in proliferative activity do not explain the different biological behavior of cgcg and gct , as reactive lesions may show increased proliferative activity .
the authors emphasize that since cgcg and gct occur in different sites , it is difficult to compare accurately their biological evolution
. nevertheless , souza , et al.21 ( 1999 ) suggest that cgcg and gct could represent variants of the same disease . cd68
monocyte - macrophage lineage marker has been often used in the investigation of giant cells .
our results demonstrated positive reactivity of many mononuclear cells and most of multinucleated giant cells to cd68 in both lesions studied , suggesting the existence of a histiocyte / macrophage origin for some of the cellular components of cgcg and gct , as shown by carvalho , et al.6(1995 ) , masui , et al.16 ( 1998 ) , o'malley , et al.19(1997 ) and werner28(2006 ) .
in addition , werner28 ( 2006 ) and wlling , et al.30 ( 2001 ) emphasize that mononucleated histiocytic cells and multinucleated giant cells expressing cd68 antigen are recruited secondarily and do not constitute the actual neoplastic cell population in gct . according to these authors , the proliferatively active neoplastic tumor cells ,
also described as gct stromal cells , constitute varying portions of the tumoral tissue and do not belong to monocytic - histiocytic system .
itonaga , et al.11 ( 2003 ) , in a study performed on cgcg , identified the presence of cell subsets similar to those reported in gct .
these authors verified that mononuclear cells were constituted by two cell subsets , one revealing a macrophagic / osteoclastic - precursor immunoprofile and another subset constituted by mesenchymal cells showing a immunoprofile towards a fibroblast / osteoblast lineage , expressing prolyl-4-hydroxylase and vimentin , being negative for macrophage associated antigens ( cd11a , cd11b ) , leucocyte common antigen ( lca ) and cd68 .
in addition , the conspicuous expression of proliferating cell nuclear antigen ( pcna ) in the latter cell subset led itonaga , et al.11 ( 2003 ) to suggest that the proliferative component of cgcg would be represented by a mesenchymal stromal cell which had the capacity to differentiate along fibroblast / osteoblast lines .
accordingly , liu , et al.15 ( 2003 ) verified the expression of vacuolar h+-atpase ( v - atpase ) , carbonic anhydrase ii
( ca ii ) , cathepsin k , mmp-9 and tartarate - resistant acid phosphatase ( trap ) in multinucleated giant cell of cgcg , thus confirming the characteristics of an osteoclastic phenotype .
moreover , the authors observed strong expression of receptor activator of nf - kb ligand ( rankl ) , an important cytokine necessary and sufficient for osteoclastogenesis , by the spindle stromal cells and the expression ofreceptor activator of nf - kb ( rank ) by round mononuclear and multinucleated giant cells .
therefore , liu , et al.15 ( 2003 ) summarizes that stromal spindle cells of cgcg induce the bone - resorption function by secreting rankl which interacts with its receptor in multinucleated giant cells , similar to the normal situation of mature osteoclasts .
the study performed by wang , et al.27 ( 2006 ) described strong similarities of the osteoclastogenesis process in lesions containing multinucleated giant cells .
these authors , analyzing the mrna and protein levels of c - src , a molecule involved in an important signaling pathway downstream of rank , verified the expression of this component both in cgcg of the jaws and gct of long bones with no significant differences .
these results led wang , et al.27 ( 2006 ) to suggest that c - src may be a common signaling cascade during osteoclastogenesis in cgcg and gct , regardless the location either in the jaws or long bones . only few studies analyzing the constitution of the extracellular matrix of cgcg and gct have been performed . within this subject ,
ueda , et al.25 ( 1996 ) , studying components of vascular basement membranes and matrix metalloproteinases in gct of long bones , reported important findings .
these authors verified weak or absence of expression of collagen iv and laminin in vascular basement membranes close to multinucleated giant cells revealing strong expression ofmmp-9 .
in addition , ueda , et al.25 ( 1996 ) described a reduction of mmp-9 level in multinucleated giant cells present within vessels , suggesting that mmp-9 may be consumed during migration of tumor cells through the blood vessel basement membrane .
therefore , these authors implied that mmp-9 is an important protease for vascular invasion of multinucleated giant cells in gct .
kumta , et al.14 ( 2003 ) , analyzing the expression of vascular endothelial growth factor ( vegf ) and mmp-9 in many osseous lesions , correlating to radiographic staging of osteolytic destruction , observed that lesions in advanced stages and recurrent lesions , including the gct , revealed higher expression of mmp-9 and vegf . according to kumta , et al.14 ( 2003 )
the level of mmp-9 and vegf expression may provide some prognostic indication of biologically aggressive behavior and local disease recurrence in osteolytic lesion affecting bone .
vered , et al.26 ( 2006 ) studied 41 cases of cgcg for the immunoreactivity to vegf and basic fibroblastic growth factor ( bfgf ) in relation to angiogenic activity , assessed by a stereological method for measuring microvascular volume . despite the high levels of immunoreactivity to vegf and bfgf in the lesions , found prominently in mononuclear stromal cells and multinucleated giant cells ,
therefore , vered , et al.26 ( 2006 ) state that vegf and bfgf expression could be related to stimulation of osteoclastogenesis in cgcg , suggesting that high levels of vegf- and bfgf - producing cells in a cgcg would be related to a more aggressive biological behavior .
fibronectin is a glycoprotein of the extracellular matrix and plasma protein that has function in cell adhesion and spreading .
it is found in most body fluids , connective tissues , granulation tissues and basement membranes1,4 .
this protein plays several biological functions and an important role in neoplastic development and other pathological processes , including those that occur in oral cavity
1,2,3,5
fibronectins may be associated with invasion and metastasis .
the variable staining intensity observed between cgcg of the jaws and gct of long bones was not significant to be used as diagnostic differential factor . in the present study ,
fibronectin reticulate / fibrillar was the most common pattern of organization in the evaluated lesions , followed by single fibrillar and reticulate organized patterns .
we have hypothesized that the fibronectin different organization patterns seem non - significant , as the prominence of the mixed pattern revealed that fibronectin can be presented under the reticulate and fibrillar aspects in a single case .
although fibronectin reticular organized pattern had been associated with the presence of inflammatory cells18,22 , our findings do not confirm this fact because this pattern type could be detected in some lesions that did not present inflammatory process .
the fibrillar organized pattern of fibronectin staining , following the collagen fibers , in cgcg of the jaws , described in this study is consistent with previous study carried out by cardoso5 ( 2000 ) .
tenascin is a glycoprotein of the extracellular matrix expressed in epithelial - mesenchymal interactions during embryogenesis and tumorigenesis of several tissues7 that plays an important role as a molecular mediator in proliferation and progression in neoplastic processes24 . in this study
, we observed intense immunoreactivity of tenascin within individual specimens of gct of long bones and cgcg of the jaws and marked variation in its spatial distribution , presenting focal organization in 4 cases of cgcg of the jaws , whereas uniform distribution was detected in all cases of gct of the long bones .
the loose connective tissue between the fascicles presenting inflammatory infiltrate has been associated with increased tenascin expression24 .
like cardoso5 ( 2000 ) and mighell , et al.17(1996 ) , we also observed no association between areas where inflammatory cells were present and tenascin immunoreactivity enhancement .
the findings of this study showed predominant reticulate organized pattern of the tenascin in most of the specimens of both of the analyzed lesions . in some cases , it was observed as a single reticular pattern , but there were cases revealing association with the fibrillar pattern .
the fibrillar pattern isolated was identified following the collagen fibers , similar to that reported by cardoso5 ( 2000 ) and tarqunio22(1999 ) . in this study , tenascin immunoreactivity revealed similarities between both giant cell lesions studied .
despite the low number of tissue specimens of cgcg and gct evaluated , that was a methodological limitation , the findings of the present study revealed immunohistochemical similarities between cgcg of the jaws and gct of long bones , supporting the observation that sometimes these lesions are indistinguishable .
further research is needed to clarify the pathogenesis and nature of these giant cell lesions and other markers have to be investigated in order to answer the question of whether these lesions represent the development of a single pathologic process or not . | objective : this study investigated whether some components of the extracellular matrix and cd68 expression may drive the differences between the central giant cell granuloma ( cgcg ) of the jaws and giant cell tumor ( gct ) of long bones , which present distinct evolution and clinical behavior.material and methods : eight cases of cgcg and 7 cases of gct were selected and immunohistochemically analyzed to verify the pattern of expression of cd68 , tenascin ( tn ) and fibronectin ( fn).results : a large number of the mononuclear cells and multinucleated giant cells cd68 + was observed in both of the studied lesions , indicating histiocyte / macrophage origin .
seven cases of cgcg of the jaws showed intense staining of fn , with uniform distribution predominantly . in all 7 cases of gct of long bones the fn displayed intense expression , with distribution pattern varying from uniform to reticulate / fibrillar .
six cases of cgcg were intensively stained by tn , presenting focal expression in half of specimens , and reticulate / fibrillar pattern of expression in 4 cases .
all cases of gct of the long bones presented intense expression of tn , uniform distribution , and reticulate / fibrillar pattern of expression in four cases.conclusions:the immunoexpression of cd68 in mononuclear cells and multinucleated giant cells and staining patterns of fn and tn were similar in both entities .
these findings indicate that these proteins could not be used to explain the differences between the cgcg of the jaws and gct of the long bones . |
dna double - stranded breaks ( dsbs ) are potentially lethal lesions if unrepaired , and their misrepair can give rise to genome instability , a hallmark of cancer ( jeggo and lavin , 2009 ) . to maintain genome stability in response to such lesions ,
cells employ either homologous recombination ( hr ) or nonhomologous end - joining ( nhej ) pathways to repair dsbs .
hr is initiated by 5 end resection to generate a 3 single - stranded dna ( ssdna ) overhang .
resection is a two - step process initiated by removing a short oligonucleotide through the activities of the mre11-rad50-nbs1 ( mrn ) complex and ctip .
extensive resection is performed by exo1 or dna2-blm in conjunction with replication protein a ( rpa ) , which binds ssdna and removes secondary structures ( sugiyama et al . , 1998 , symington and gautier , 2011 ) .
this , in turn , leads to rad51 nucleofilament formation , which promotes strand invasion of a homologous chromatid , leading to accurate repair ( heyer et al . ,
the broken ends are rapidly bound and protected by the ku70/ku80 heterodimer , which acts as a platform to recruit the dna - pk catalytic subunit ( dna - pkcs ) .
damaged ends are then processed and subsequently joined by the ligase 4 ( lig4 ) , xrcc4 , xlf complex in a template - independent manner , which can lead to inaccurate repair ( lieber , 2010 )
. dsbs may also be repaired through alternative end - joining pathways , such as microhomology - mediated end - joining ( mmej ) , which do not require ku or lig4 . like hr , mmej is initiated by resection , and end - joining is mediated through annealing of short direct repeats of microhomology .
mmej leads to deletions and is frequently associated with chromosomal rearrangements ( mcvey and lee , 2008 ) .
dsb repair is further facilitated through chromatin modification by chromatin remodeling complexes , by incorporation of histone variants , and by histone modification ( smeenk and van attikum , 2013 ) .
histone h3k36 dimethylation has recently been proposed to facilitate nhej , where metnase ( setmar ) directly mediates h3k36 dimethylation near the break site , leading to recruitment and stabilization of nbs1 and ku70 ( fnu et al .
histone h3k36 is also trimethylated , which in mammalian cells is performed uniquely by the setd2/hypb methyltransferase ( edmunds et al . , 2008 ) .
h3k36me3 is associated with transcriptional elongation and is found in gene coding regions , peaking at 3 ends ( edmunds et al . , 2008 ) .
setd2-dependent h3k36 trimethylation facilitates a number of processes within the cell , including splicing , repression of intragenic transcripts , and chromatin accessibility ( li et al .
setd2 is also mutated in a number of cancer types , including breast , lung , acute lymphoblastic leukemia , clear cell renal cell carcinoma , and glioma , supporting its role as a tumor suppressor ( al sarakbi et al .
, 2009 , dalgliesh et al . , 2010 , fontebasso et al . , 2013 , newbold and mokbel , 2010 , zhang et al . , 2012 ) .
setd2-dependent h3k36 trimethylation has recently been shown to regulate dna mismatch repair ( li et al . , 2013 )
. however , setd2-deficient cancers exhibit a wide range of mutations , including insertions , deletions ( indels ) , and chromosomal aberrations ( sato et al . , 2013 , zhu et al . , 2014 ) , suggesting an additional role for setd2 in genome stability .
these contain jmjn - jmjc and tandem - tudor domains that specifically remove the tri- and dimethyl forms of both h3k9 and h3k36 , with preference for the trimethyl form being observed in the case of kdm4a / jmjd2a ( couture et al .
kdm4 family proteins are frequently overexpressed in cancers and are associated with poor patient survival ( berry and janknecht , 2013 , black et al . , 2013 ) . here
in addition , we define a role for setd2 and rad51 in maintaining genome stability through suppressing mmej at break sites .
tumor suppressors can suppress carcinogenesis by preventing genome instability , a hallmark of cancer . to investigate the impact of loss of setd2 , a tumor suppressor , on genome stability following dsb induction , we developed an i - scei - induced loss of function assay ( hprt : i - scei ) .
intron - encoded endonuclease 1 from saccharomyces cerevisiae ( i - scei ) is a rare - cutting endonuclease that has no predicted recognition site in the mammalian genome ( jasin , 1996 ) .
an i - scei recognition sequence was inserted into the endogenous hprt exon 6 in ht1080 ( fibrosarcoma ) cells .
the i - scei site maintains the hprt reading frame and makes only a single amino acid change that does not impair the function of hprt ( figure 1a ) .
following i - scei induction , inaccurate dsb repair generates hprt - negative mutants , which can be selected for using 6-thioguanine ( 6-tg ) , and indels can be detected by pcr amplification using primers flanking the break site ( figure 1a ) . following small interfering rna ( sirna ) knockdown and i - scei induction , the frequency of hprt loss ( mutation frequency ) in setd2-depleted cells was 1.65% , significantly higher ( p = 0.0014 ) than that of nontargeting controls ( 1.1% ) .
these findings resembled those in rad51-depleted cells in which the frequency of hprt loss was also significantly increased to 1.88% ( p = 0.0022 ) compared to nontargeting controls ( figure 1b ) .
setd2 and rad51 codepletion ( sis+sir ) also significantly increased the mutation frequency to 1.52% ( p = 0.037 ) , which was similar to the frequency observed following either setd2 or rad51 depletion ( figure 1b ) .
we confirmed that i - scei protein expression and cleavage efficiency were the same in cells transfected with either control sirna or setd2 sirna ( figures s1a s1c ) .
in addition , hprt gene transcription was not affected by setd2 knockdown , as shown by quantitative rt - pcr ( qrt - pcr ) of spliced and unspliced hprt mrna ( figures s1d and s1e ) .
therefore , the mutation frequency in this system is likely to be directly associated with misrepair of the dsb at the i - scei site .
further , we studied the mutation patterns across the break site of 30 individually isolated hprt - negative clones from each background by pcr amplification and sequencing .
hprt - negative clones from cells treated with nontargeting control sirna indicated the presence of microdeletions of 25 bp , consistent with dsb repair by c - nhej .
larger microdeletions were also observed that were associated with regions of microhomology located either side of the break site , consistent with dsb repair through mmej ( figures 1c and s1f ) .
sequence analysis indicated that setd2-depleted cells exhibited a significant increase in average deletion length ( 11 bp , p < 0.0001 ) compared to control cells ( 5 bp ) ( figures 1c and 1d ) .
rad51-depleted cells exhibited a further significant increase in the average deletion length ( 22 bp , p = 0.014 ) ( figures 1c and 1d ) .
rad51 and setd2 codepletion generated deletion lengths ( an average of 8 bp ) comparable to those observed following setd2 knockdown , which in turn was significantly higher than the nontargeting control ( p = 0.026 ) ( figures 1c and 1d ) .
however , the frequency and size of the insertions was not significantly altered between setd2-depleted cells , setd2/rad51 codepleted and nontargeting controls .
we found that the frequency of microhomology - mediated end - joining ( mmej ) was significantly increased in setd2 and rad51-depleted cells ( p < 0.05 ) ( figures 1e and s1 g ) .
this suggested that hr - deficient cells use mmej as an alternative repair mechanism , which gives rise to a mutation signature of sequence loss between regions of microhomology .
these results support a role for setd2 in tumor suppression by maintaining genome stability through preventing deleterious mutations arising in response to dsbs .
these findings further identify a similar break - induced mutation signature between setd2- and rad51-depleted cells , suggesting an early role for setd2 in hr repair . to test the role for setd2 in dsb repair , we depleted setd2 in u2os and hela cells using two independent sirnas and measured the clonogenic survival following exposure to dna - damaging agents .
setd2 knockdown significantly reduced survival after treatment with mitomycin c ( mmc ) , camptothecin ( cpt ) , or ionizing radiation ( ir ) in u2os cells and hela cells compared to nontargeting controls ( figures 2a and s2a ) .
these results identify a role for setd2 in the cellular response to dna damage and are consistent with a role for setd2 in dsb repair .
western blotting also showed that setd2 knockdown significantly reduced global levels of h3k36me3 , confirming setd2 as the major h3k36me3 methyltransferase in human cells ( figure 2b ) . because rad51 is more likely to be affected among dna repair genes by sirna off - target effects ( adamson et al . , 2012 )
, we also confirmed that setd2 knockdown by sirna did not affect the rad51 protein levels ( figure 2b ) .
we confirmed that setd2 knockdown had no effect on the expression of major hr proteins , including brca2 , rad50 , ctip , mre11 , and rpa and the nhej protein ku80 ( figure s2b ) .
we also observed no changes to the cell cycle after setd2 knockdown ( figure s2c ) .
both mmc and cpt induce s phase - specific dsbs , which are predominantly repaired by hr ( arnaudeau et al . , 2001 ,
, we used a well - characterized gfp - based reporter for hr ( dr - gfp ) .
the hr reporter contains an i - scei recognition sequence , which upon i - scei expression is cleaved to generate a dsb .
dsb repair by hr using a direct repeat within the reporter cassette as a template results in an intact gfp gene ( figure 2c)(pierce et al . , 1999 ) .
setd2 knockdown by two independent sirnas significantly reduced hr repair of an i - scei induced dsb by 67%77% compared to nontargeting control cells ( p < 0.0003 ) ( figure 2d ) . as a control for the hr reporter ,
0.0001 ) , whereas dna - pkcs inhibitor nu7441 had no effect on hr ( figure 2d ) .
in contrast , setd2 knockdown had no effect on repair of an i - scei induced dsb in an nhej reporter ( ires - tk - egfp ) in which joining of two i - scei sites separated by a thymidine kinase gene ( tk ) results in gfp expression ( figures 2e and 2f)(ogiwara et al . , 2011 ) . as a control ,
rad51 had no effect on the same nhej reporter , whereas the dna - pk inhibitor nu7441 reduced nhej activity by 98% ( p
to further investigate the role of setd2 in dsb repair , we examined the rate of repair following ir .
we used h2ax foci as a marker of dna damage and found that following exposure to 5 gy ir , setd2 knockdown significantly delayed removal of h2ax foci at 48 hr ( p < 0.003 ) compared to nontargeting controls , supporting a role for setd2 in efficient dsb repair ( figure 3a ) .
we next investigated the effect of setd2 depletion on the recruitment of hr repair proteins following dna damage .
setd2 knockdown resulted in significantly reduced rad51 foci formation 4 and 8 hr after exposure to ir compared to nontargeting controls ( p <
setd2 knockdown also significantly reduced rad51 foci formation after exposure to cpt ( p <
we also examined the effect of setd2 depletion on rpa recruitment , where we induced dsbs in s phase cells using cpt or mmc .
a significant reduction in rpa foci formation was observed after cpt or mmc treatment following setd2 knockdown compared to nontargeting controls ( p < 0.023 ) ( figures 3c and s2e ) .
setd2 knockdown leads to reduced h3k36me3 in the nuclei as confirmed by costaining the cells with antibodies against h3k36me3 and rpa ( figure s2f ) .
together , these findings support a role for setd2 in promoting hr through facilitating recruitment of rpa and rad51 to dna damage sites .
to further confirm the impact of setd2 loss on the recruitment of hr factors to the site of dsbs , we performed chromatin immunoprecipitation ( chip ) near the i - scei site in the dr - gfp cassette ( figure s3a ) .
the i - scei site is located within the dr - gfp cassette under the control of the highly active cmv promoter , and thus h3k36me3 , which is associated with active transcription , is predicted to be present throughout the cassette .
after induction of dsbs by i - scei , no change in h3k36me3 or setd2 levels was detected proximal or distal to the break site after 8 , 24 , or 48 hr following i - scei transfection ( figure s3b ) .
the results were also quantified by chip - qpcr ( figure s3c ) . although other groups also observed no changes to h3k36me3 level after dsb , globally ( fnu et al . , 2011 ) or locally at the break site ( pei et al . , 2011 ,
2014 ) , we were surprised by the lack of reduction in h3k36me3 or setd2 knowing that regional transcriptional silencing has been observed at sites of dsbs ( shanbhag et al . , 2010 ) .
we speculate that the unexpected maintenance of h3k36me3 at the dsb site by setd2 may serve as an anchor to mediate hr repair .
we monitored recruitment of major hr factors to the dsb site in the chip system .
rpa and rad51 were recruited to the dsb site , resulting in increased binding to the up site 18 hr after i - scei transfection , compared to uncut controls ( figure 3d ) .
setd2 depletion substantially reduced the recruitment of rpa and rad51 after the i - scei cut ( figure 3d ) .
setd2 protein depletion was validated by western blotting ( figure s3d ) , and was further confirmed by the absence of setd2 or h3k36me3 at the break site ( figure 3d ) .
these findings are consistent with the rpa and rad51 foci data , indicating that setd2 is required to recruit rpa and rad51 to dsb sites .
together , these findings confirm a presynaptic role for setd2 in facilitating hr repair of dsbs . to investigate if the methyltransferase activity of setd2 is required for its role in hr , we generated two doxycycline - inducible u2os cell lines , where either a wild - type setd2 cdna or a methyltransferase - dead setd2 cdna was inserted behind a tet operator in the genome of u2os cells .
upon addition of doxycycline to the cell culture medium , the tet operator was derepressed and the wild - type or mutant setd2 was expressed .
the methyltransferase - dead setd2 was mutated at two amino acid residues located within the catalytic site of the set domain , which are conserved with yeast set2 .
the corresponding mutations in yeast were shown to abolish the methyltransferase activity ( rea et al . , 2000 , strahl et al . ,
both arginine ( r ) and cysteine ( c ) residues were mutated in the methyltransferase - dead ( md ) cdna by site - directed mutagenesis ( see supplemental experimental procedures ) .
western blot analysis confirmed that doxycycline - induced expression of wild - type setd2 could restore the level of h3k36me3 after sisetd2 treatment , whereas expression of methyltransferase - dead setd2 could not restore the level of h3k36me3 ( figure s4a ) .
we depleted endogenous setd2 with sirna#3 and examined the rescue of the phenotype by the two constructs .
although the wild - type construct increased foci formation of rpa and rad51 after dna damage in sisetd2 cells , the methyltransferase - dead mutant did not ( figures 4b and 4c ) .
this is consistent with the observation that the wild - type construct partially restored hr efficiency whereas the methyltransferase - dead ( md ) mutant did not ( figure 4d ) .
all wt clones were able to restore hr efficiency , but none of the md clones showed rescue ( figures s4b and s4c ) .
the observation that the methyltransferase - dead setd2 mutant could not rescue hr efficiency suggested that h3k36me3 is required for efficient hr . to test this
first , we overexpressed kdm4a , an oncogene and a demethylase specific for tri- and dimethylated histone h3k36 and h3k9 , predicting that overexpression of kdm4a would reduce the level of h3k36me3 and thus impair hr .
upon addition of doxycycline , kdm4a was overexpressed , which led to a global reduction in h3k36me3 levels ( figure 4e ) .
we then compared the recruitment of the hr factors in this cell line with or without kdm4a overexpression .
we found that kdm4a overexpression inhibited the formation of rpa and rad51 foci after dna damage ( figures 4f and 4 g ) .
accordingly , kdm4a overexpression also reduced hr efficiency as assessed by the hr reporter ( p = 0.0078 ) ( figure 4h ) . using the same cell line without kdm4a cdna integration
, we found that doxycycline itself had no effect on hr efficiency of u2os cells ( figure s4d ) .
these findings indicate that overexpression of the oncogene kdm4a significantly reduces levels of h3k36me3 and hr .
second , we utilized a dominant - negative mutation in the histone h3.3 gene ( h3.3k36 m ) that was recently shown to reduce h3k36me3 specifically without affecting other histone methylations ( lewis et al . , 2013 ) .
accordingly , in cells expressing the mutant h3.3k36 m transgene , global levels of h3k36me3 were depleted compared to the wild - type h3.3 control ( figure 4i ) .
cells expressing h3.3k36 m also exhibited delayed rpa and rad51 foci formation following dna damage ( figures 4j and 4k ) , consistent with similar delays seen in kdm4a overexpressing cells or setd2-depleted cells .
neither kdm4a overexpression nor h3.3k36 m expression affected cell cycle progression as assessed by brdu incorporation ( figures s4e and s4f ) , thus excluding the possibility that the reduction in rpa , rad51 foci formation and hr efficacy was due to cell cycle effects .
the common feature of all three different systems ( setd2 knockdown and rescue , kdm4a overexpression , and h3.3k36 m expression ) was the reduction in the recruitment of hr proteins and hr efficiency associated with reduced h3k36me3 .
lens epithelium - derived growth factor p75 ( ledgf ) binds to h3k36me3 constitutively through its pwwp domain . upon dna damage ledgf recruits ctip , which facilitates the resection step during hr repair ( daugaard et al . , 2012 ) .
accordingly , we were able to coprecipitate ledgf and h3k36me3 , independently of dna damage in u2os cells ( figure 5a ) .
ledgf binding to chromatin was reduced upon setd2 depletion , consistent with preferential binding of ledgf to h3k36me3 ( eidahl et al . , 2013 ) , and
was dsb independent ( figure 5b ) . we predicted that the reduced ledgf binding following setd2 depletion would reduce recruitment of ctip upon damage .
we therefore used micro - irradiation to induce localized dsbs and studied the recruitment of ctip using a u2os cell line expressing gfp - tagged ctip ( sartori et al . , 2007 ) .
we found ctip to be localized to sites of dna damage marked by h2ax ( figure 5c ) .
in contrast , setd2 knockdown resulted in reduced recruitment of ctip to the dna damage site as marked by h2ax ( figure 5c ) .
if setd2 facilitates hr through ledgf binding and ctip recruitment , we would expect setd2 to be epistatic with ctip .
indeed , codepletion of setd2 and ctip ( sic+sis ) showed the same level of reduction in hr as ctip - depletion alone ( figures 5d and s5a ) .
these findings are consistent with the hypothesis that setd2-dependent h3k36me3 promotes recruitment of ctip by ledgf to break sites
. reduced ctip recruitment , together with the reduced rpa foci formation following setd2 depletion , suggested that setd2-dependent h3k36 trimethylation might promote dsb resection . to directly measure resection , we used an in vivo resection assay ( zhou et al . , 2014 ) in which levels of ssdna were assessed at increasing distances from a specific dsb induced by the asisi restriction enzyme , which was found to recruit rad51 ( iacovoni et al .
following dsb induction ( + 4oht ) , ssdna was readily detected 231 bp from the asisi break site ( dsb - ii in aymard et al . , 2014 ) and at reduced levels further from the break site .
following ctip depletion , ssdna levels were significantly reduced at 231 bp ( p = 2.2 10 ) and 918 bp ( p = 0.057 ) from the break site compared to control sirnas ( figure 5e ) .
following setd2 depletion , ssdna levels were also significantly reduced at 231 bp ( p = 0.021 ) and 918 bp ( p = 0.037 ) from the break site compared to control sirnas ( figure 5f ) , and setd2 depletion did not affect dsb induction at this site as assayed by xrcc4 chip ( figure s5b ) , thus confirming a role for setd2 in facilitating dsb resection .
the setd2 gene encoding the h3k36 trimethyltransferase is among the most mutated in human cancers ( lawrence et al . , 2014 ) .
yet , how setd2 functions to suppress the genome instability frequently observed in setd2-deficient cancer cells is unknown ( sato et al . , 2013 , zhu et al . , 2014 ) .
here , we describe a role for setd2-dependent h3k36 trimethylation in facilitating error - free dsb repair and genome stability through efficient hr .
our data support a model in which ledgf is constitutively anchored to h3k36me3-decorated chromatin through its pwwp domain . upon dna damage , chromatin - bound ledgf recruits ctip , which promotes resection an essential step in hr repair ( figure 6a ) . in the absence of setd2-dependent h3k36me3
, the chromatin association of ledgf is compromised , and dna damage - induced ctip recruitment is impaired , leading to reduced resection and hr and an increase in error - prone mmej and subsequent genome instability ( figure 6b ) .
our data do not , however , exclude the possibility that other h3k36me3 binding factors may also contribute to efficient hr .
the involvement of h3k36me3 in hr raises the question as to whether this histone mark is dsb inducible or preset to determine the choice of repair pathways .
we did not detect any appreciable increase in h3k36me3 levels at the break site following i - scei induction .
these findings are consistent with previous observations indicating that h3k36me3 levels do not change following dna damage , either globally ( fnu et al . , 2011 ) or locally ( aymard et al . , 2014 , pei et al . , 2011 )
given that setd2-dependent h3k36 trimethylation strongly correlates with actively transcribed regions of the genome ( edmunds et al .
, 2008 , yoh et al . , 2008 ) , our findings thus support a spatial link between hr and transcriptionally active , h3k36me3 decorated regions of the genome .
indeed , because hr repair is usually error free , this would be expected to protect genome integrity following dsb induction or replication fork collapse at such sites , which would be important in maintaining cell homeostasis .
genome - wide sequencing has suggested certain mutation signatures are associated with specific cancer types ( alexandrov et al . , 2013 ) but lacks the power to identify driver mutations that give rise to that mutation signature .
here , we use a system to determine the impact of individual gene dysfunction on mutation patterns arising during the repair of a single dsb .
we show that , by depleting setd2 or rad51 hr proteins , dsb induction leads to significantly increased mutation frequencies associated with deletions within six cell cycles ( 5 days ) .
these findings identify a role for setd2 , like rad51 , in suppressing break - induced mutations .
further mechanistic analysis indicated that deletions arising from setd2 or rad51 depletion resulted from a significant increase in error - prone mmej .
setd2 loss results in partial resection , short ssdna 3 ends and reduced rpa foci .
these partially resected ends are insufficient to promote efficient hr and are no longer substrates for c - nhej . instead , these partially resected ends become substrates for mmej . similarly , following rad51 depletion , whereas resection proceeds normally , hr is blocked presynaptically , resulting in the accumulation of longer unrepaired resected ends . such ends
are no longer substrates for c - nhej , and , because hr can not proceed , the ends are instead rejoined by mmej . extensive resection following rad51 loss facilitates annealing between regions of microhomology further from the break site , thus leading to larger deletions than those observed for setd2 .
the observations that hr was significantly reduced whereas c - nhej was unchanged following setd2 or rad51 depletion are consistent with this model .
together , these findings suggest a role for hr in maintaining genome stability through suppression of mmej .
mutation study can therefore provide important insights into how loss of these genes contributes to somatic mutation and intratumor heterogeneity , and it highlights the importance of hr repair genes in maintaining genome stability .
setd2 is a tumor suppressor and its loss is an important step in the progression of a number of cancer types ( dalgliesh et al .
, 2010 , fontebasso et al . , 2013 , gerlinger et al . , 2012 ) .
moreover , all three tudor domain - containing kdm4/jmjd2 proteins ( a , b , and c ) are frequently overexpressed in various cancers , including breast , colorectal , lung , and prostate ( berry and janknecht , 2013 ) .
our findings predict that overexpression of these kdm4 family members causes genome instability and tumorigenesis through loss of h3k36me3-mediated hr repair .
the identification of a role for h3k36me3 in facilitating hr repair , in addition to its recently described role in mismatch repair ( li et al . , 2013 ) , establishes the h3k36me3 chromatin mark as an important functional node for maintaining genome stability .
the reporter ht1080 cell line with a functional but i - scei - cleavable hprt gene was generated by inserting an 18 bp i - scei recognition site into exon 6 of the hprt gene using gene targeting .
reporter cells were transfected with the i - scei plasmid 48 hr after sirna knockdowns .
the cells were allowed to repair in a nonselective medium for 5 days before seeding and selecting for hprt - negative cells using 6-tg ( 15 g / ml ) .
the mutation frequency was calculated as the number of hprt - negative colonies divided by number of cells plated , after correcting for plating efficiency . to determine the mutation sequence ,
individual single - cell colonies were isolated , and genomic dna spanning the dsb junction was pcr - amplified using the primers flanking the i - scei site .
sequence alignment of the pcr products was conducted using the dna data bank of japan ( ddbj ) clustalw program .
2014 ] ) were transfected with sirna using the cell line nucleofactor kit v ( amaxa ) according to the manufacturer s instructions .
forty - eight hours after sirna transfection , cells were treated or not with 300 nm of 4-hydroxytamoxifen ( 4oht ) ( sigma ; h7904 ) for 4 hr .
single - stranded dna generated at an asisi - induced dsb was analyzed using the procedure described in zhou et al .
the percentage of ssdna was calculated with the following equation : ssdna% = 1/(2 + 0.5 ) 100 , where ct is calculated by subtracting the ct obtained from mock - digested sample from the bani - digested sample .
u2os - flp - in / t - rex cells express the tet repressor and contain a single flp - in site ( gordon et al . , 2009 ) .
u2os - flp - in / t - rex / drgfp cells were generated by integrating the pdr - gfp expression plasmid ( pierce et al .
, 1999 ) into the u2os - flp - in / t - rex cells .
the kdm4a cdna ( genecopoeia ) was cloned into the pdestfrtto destination vector by a gateway lr reaction ( life technologies ) .
u2os - flp - in / t - rex or u2os - flp - in / t - rex / drgfp cells were cotransfected with the resulting kdm4a destination plasmid and pog44 ( invitrogen ) .
stable clones that express the kdm4a protein under the control of the t - rex system were subjected to the treatments indicated .
p < 0.05 , p < 0.01 , p < 0.001 , and n.s . represents p 0.05 .
l .- p.z . , s.s . , and f.a . designed and performed experiments .
experiments in figure 1 were performed by s.a . , with technology developed by a.c.g.p . ; figure 2 was performed by s.x.p . and s.a .
l .- p.z . ; figure 4 was performed by s.s . and s.x.p . ,
; figure s3 was performed by l .- p.z . ; figure s4 was performed by s.x.p . and s.s . | summarymodulating chromatin through histone methylation orchestrates numerous cellular processes .
setd2-dependent trimethylation of histone h3k36 is associated with active transcription . here
, we define a role for h3k36 trimethylation in homologous recombination ( hr ) repair in human cells .
we find that depleting setd2 generates a mutation signature resembling rad51 depletion at i - scei - induced dna double - strand break ( dsb ) sites , with significantly increased deletions arising through microhomology - mediated end - joining .
we establish a presynaptic role for setd2 methyltransferase in hr , where it facilitates the recruitment of c - terminal binding protein interacting protein ( ctip ) and promotes dsb resection , allowing replication protein a ( rpa ) and rad51 binding to dna damage sites .
furthermore , reducing h3k36me3 levels by overexpressing kdm4a / jmjd2a , an oncogene and h3k36me3/2 demethylase , or an h3.3k36 m transgene also reduces hr repair events .
we propose that error - free hr repair within h3k36me3-decorated transcriptionally active genomic regions promotes cell homeostasis .
moreover , these findings provide insights as to why oncogenic mutations cluster within the h3k36me3 axis . |
acute diarrhea still remains a leading cause of mortality and morbidity in children of the developing countries .
the appropriate management of acute childhood diarrhea consists of fluid and electrolyte therapy , suitable antibiotics ( e.g. in case of shigellosis , severe cholera , etc . ) , and adequate nutritional therapy [ 24 ] .
greater attention to nutritional therapy is particularly important in developing countries , where evidence showed a significant association between the prevalence of diarrhea and children 's growth increments .
if remained un - controlled , prolonged acute diarrhea can reduce growth and increase risk of persistent diarrhea in children .
benefits such as shortened duration of diarrhea , better weight gain , and ameliorating dehydration with a decreased need to intravenous fluid therapy were reported for lactose - free , compared with the lactose - containing formula , in formula - fed children with acute diarrhea [ 7 , 8 ] .
although benefits were reported for lactose - free formula and adverse complications seem to be more likely to occur in children who receive a milk diet containing lactose during acute diarrhea , there is still an argument for the use of a specifically designed lactose - free formula or its routine use for acute childhood diarrhea [ 9 , 10 ] .
therefore , we evaluated the effects of early administration of the lactose - free formula compared with continuing lactose - containing formula in the management of acute diarrhea in formula - fed children under two years old .
this controlled - clinical trial was conducted from january 2009 to august 2009 in the pediatrics clinics at two referral university hospitals in isfahan ( iran ) .
formula - fed children , aged 1 - 24 months , with acute non - bloody diarrhea ( 2 weeks ) were enrolled consecutively in the study .
children who had mucous bloody stools , major systemic illness , severe malnutrition ( weight for age < 60% and/or weight for height < 70% ) , severe dehydration requiring intravenous infusion , severe vomiting , or history of antibiotic therapy were not included .
the required sample size was calculated as 37 subjects in each group considering = 0.05 and study power = 0.8 and expecting a difference of 1 day in duration of diarrhea .
the ethics committee of isfahan university of medical sciences approved the study protocol and written consent was obtained from all parents after full explanation of the study aim and protocol . at admission time
( first visit ) , after obtaining the demographic data and medical history , nude weight was measured with scale with an accuracy of 10 g .
dehydration was assessed and oral rehydration therapy was performed according to world health organization ( who ) guidelines . in children with mild dehydration , 50 ml / kg and in children with moderate dehydration ,
100 ml / kg of oral rehydration solution ( standard who - ors ) was prescribed during the first four hours .
after the initial rehydration phase , for maintenance treatment , children were alternately allocated to receive 100 ml / kg / day of either lactose - free or lactose - containing formula .
children were discharged after confidence of tolerance of oral hydration , and the parents were explained about the red flags and asked to mention to consistency of stools at home and report on the days of follow - up .
the second visit was done seven days after the first visit for measuring weight and evaluating time to diarrhea relief .
the time to diarrhea relief and nude body weight seven days after intervention were considered as outcomes . after gathering data
, we used the independent sample t - test and chi - square test for comparisons . for data without normal distribution appropriate non - parametric tests
this controlled - clinical trial was conducted from january 2009 to august 2009 in the pediatrics clinics at two referral university hospitals in isfahan ( iran ) .
formula - fed children , aged 1 - 24 months , with acute non - bloody diarrhea ( 2 weeks ) were enrolled consecutively in the study .
children who had mucous bloody stools , major systemic illness , severe malnutrition ( weight for age < 60% and/or weight for height < 70% ) , severe dehydration requiring intravenous infusion , severe vomiting , or history of antibiotic therapy were not included .
the required sample size was calculated as 37 subjects in each group considering = 0.05 and study power = 0.8 and expecting a difference of 1 day in duration of diarrhea .
the ethics committee of isfahan university of medical sciences approved the study protocol and written consent was obtained from all parents after full explanation of the study aim and protocol .
at admission time ( first visit ) , after obtaining the demographic data and medical history , nude weight was measured with scale with an accuracy of 10 g .
dehydration was assessed and oral rehydration therapy was performed according to world health organization ( who ) guidelines . in children with mild dehydration , 50 ml / kg and in children with moderate dehydration ,
100 ml / kg of oral rehydration solution ( standard who - ors ) was prescribed during the first four hours .
after the initial rehydration phase , for maintenance treatment , children were alternately allocated to receive 100 ml / kg / day of either lactose - free or lactose - containing formula .
children were discharged after confidence of tolerance of oral hydration , and the parents were explained about the red flags and asked to mention to consistency of stools at home and report on the days of follow - up . the second visit was done seven days after the first visit for measuring weight and evaluating time to diarrhea relief .
the time to diarrhea relief and nude body weight seven days after intervention were considered as outcomes . after gathering data
, we used the independent sample t - test and chi - square test for comparisons . for
remained subjects were 32 male and 39 female children with mean age of 7.13.7 months .
the two groups were similar in demographic and baseline characteristics except that the intervention group had more days with diarrhea at baseline ( p=0.047 ) and dehydration was more severe in the control group ( p=0.015 ) ; table 1 .
comparison of demographic and baseline characteristics between the two groups data are shown as mean ( standard deviation ) or number ( % ) after therapy , those who received lactose - free formula had a significantly more rapid diarrhea relief compared with the controls ( 1.70.7 vs. 2.60.7 days , p<0.001 ) ; fig .
weight significantly increased in both the intervention ( 6.591.94 to 6.631.90 kg , [ ci 95% : 4 to 71 gr ] p=0.03 ) and control ( 6.451.99 to 6.492.00 kg , [ ci 95% : 11 to 65 gr ] p=0.007 ) groups , but there was no difference between the two groups in weight change ( 37100 vs. 3877 gr , p=0.7 ) ; fig .
2 . comparison of diarrhea relief time between the two groups comparison of weight change between the two groups considering differences between the two groups before the intervention , we conducted a multivariate analysis , controlling for baseline values .
multivariate analysis showed that receiving lactose - free formula significantly predicted time to diarrhea relief ( 95% ci : 1.5 to 3.9 , p<0.001 ) while controlling for gender ( p=0.3 ) , age ( p=0.2 ) , dehydration ( p=0.2 ) , and the baseline number of days with diarrhea ( p=0.8 ) .
current issues in dietary management of acute childhood diarrhea include the optimal timing of introduction of foods during illness , the appropriate use of milk - containing treatment regimens , mixed diets containing common staple foods , and the proper use of specific micronutrient supplements .
the aim of our study was to evaluate the effectiveness of early administration of lactose - free milk formula in the management of acute diarrhea in formula - fed children .
we found a significant reduction in the period of diarrhea in children with acute diarrhea who received lactose - free compared with the lactose - containing formula .
however , we did not find any difference with regard to weight changes after therapy .
xu and huang found a significantly shorter duration for diarrhea remission ( 3.171.04 days ) in the lactose - free formula group compared with conventional formula group ( 5.251.58 days ) . in another study , simakachorn and
colleagues found that the median duration of diarrhea was significantly shortened by 20.5 hours in the lactose - free diet group compared to the control group .
they also found significantly higher weight gaining in children with lactose - free diet compared with the control group .
wall and colleagues in a randomized trial compared the efficacy of a low - lactose hydrolyzed milk formula , a lactose - free corn syrup - based milk formula , and a standard lactose - containing formula during re - feeding after rehydration in infants with gastroenteritis .
authors concluded that the routine use of a low - lactose formula is beneficial during re - feeding after rehydration in infants with gastroenteritis .
in contrast to studies indicating beneficial effects of lactose - free formula for the management of acute childhood diarrhea , brown and colleagues in a meta - analysis of 29 randomized controlled trials concluded that most of the children with acute diarrhea can be successfully managed with continued feeding of undiluted non - human milk and routine dilution of milk .
this meta - analysis concluded that routine use of lactose - free formula is therefore , not necessary , especially when ors and early feeding ( in addition to milk ) form the basic approach to the clinical management of diarrhea in infants and children .
this analysis , however , did not address the confounding effects of severity of diarrhea , degree of malnutrition , or young age ( less than 1 y ) in the patients . a recent comparative trial that evaluated the effectiveness of zinc , probiotic bacteria , and lactose - free formula and
their different combinations in the treatment of acute diarrhea in children found no additional value to rehydration therapy for different combination of adjunct therapies except some beneficial effects for those receiving zinc / zinc plus probiotic . although we did not find beneficial effects of lactose - free over lactose - containing formula on weight change during treatment of diarrhea , time to relief from diarrhea
was significantly reduced which in turn may reduce the mortality and morbidity of acute childhood diarrhea . however , there are some limitations to our study which need to be accounted .
there were baseline differences between the two groups probably because allocation was not randomized , though we did a multivariate analysis and controlled baseline differences .
the follow - up was short - term ; we did not determine other factors such as acidosis and plasma electrolyte level , which may indirectly be influenced by lactose - free diet while decreasing the diarrheal duration .
according to the results of the present study , lactose - free , compared with the lactose - containing , formula was shown to be effective in dietary management of acute diarrhea by reducing time to diarrhea relief .
early administration of lactose - free formula for children presenting with acute diarrhea can result in a more rapid remission of diarrhea and thus perhaps less mortality and morbidity .
further trials with longer follow - ups are warranted to evaluate long - term results such as weight change and feeding problems in this regard .
| objectivefew reports are available on some benefits , such as shortened duration of diarrhea and better weight gain , for lactose - free over lactose - containing formula in acute childhood diarrhea .
we evaluated the effects of lactose - free formula in dietary management of acute diarrhea in formula - fed children.methodsthis controlled - clinical trial was conducted on formula - fed children , aged 1 to 24 months , referring with acute non - bloody diarrhea ( 2 weeks ) .
those who had major systemic illness , severe malnutrition , severe dehydration , severe vomiting , or history of antibiotic therapy were not included .
children were allocated to receive lactose - free formula ( intervention , n=37 ) or lactose - containing formula ( control , n=34 ) .
time to diarrhea relief and weight change were compared between the two groups after one week.findingsduring the study , 32 male and 39 female children ( 7.13.7 months ) were included .
those who received lactose - free formula had a significantly shorter time to diarrhea relief compared with the controls ( 1.70.7 vs. 2.60.7 days , p<0.001 ) .
weight significantly increased in both groups , but there was no difference between the two groups in weight change ( 37100 vs. 3877 gr , p=0.673 ) .
multivariate analysis showed that receiving lactose - free formula significantly predicted time to diarrhea relief ( 95% ci : 1.5 to 3.9 , p<0.001 ) controlling for baseline characteristics.conclusionearly administration of lactose - free formula for formula - fed children presenting with acute diarrhea can result in a more rapid relief of acute diarrhea and thus perhaps less mortality and morbidity .
trials with longer follow - ups are warranted to better evaluate long - term results such as weight change and feeding problems in this regard . |
rotavirus ( rv ) is the primary etiological agent causing severe gastroenteritis ( ge ) in children . in 2008 , rv accounted for approximately 453,000 ( 420,000494,000 ) worldwide deaths and 36% of diarrheal hospitalizations among children under 5 years of age.1,2 each year , approximately 65,000 deaths can be attributed to rv in 22 countries of the eastern mediterranean region,3 with rv representing an important cause of severe diarrhea amongst around 102,000 children aged under 5 years in the kingdom of bahrain.46 in comparison with improvements in hygiene and sanitation , vaccination is an effective public health intervention , capable of controlling rv disease.7 since 2006 , the world health organization ( who ) has recommended two live , oral rv vaccines , rotateq ( merck & co , inc , whitehouse station , nj , usa ) and rotarix ( glaxo smithkline vaccines),8 both of which demonstrate good safety , efficacy , and effectiveness profiles in large - scale clinical trials and in reallife settings in many regions globally.917 in 2008 , rotarix was introduced into the kingdom of bahrain s national immunization program as a two - dose schedule at 2 and 4 months of age.4,18 however , since this time , the coverage rate for at least one of the doses has fluctuated from 17%87%.4 in order to assess the public health benefits of introducing the rv vaccine , its impact on rv - associated morbidity and mortality needs to be monitored.19 this study was therefore undertaken to generate baseline information on rvge disease burden , which could be used as a reference for the interpretation of post - vaccine rv disease trends .
these data would allow health care providers and decision makers to design appropriate future plans and assess the benefits of rv vaccination in reducing the burden of severe rvge .
this hospital - based surveillance study assessed the role of rv in causing ge - associated hospitalization in children under 5 years ; evaluated the age and seasonal distribution of rv - associated hospitalization ; identified prevalent rv types ; and assessed outcomes and associated treatment .
this single , referral pediatric hospital - based surveillance study was conducted at the salmaniya medical complex ( smc ) , the kingdom of bahrain , manama over 12 months beginning april 1 , 2006 .
smc is a multispecialty secondary and tertiary government - referral hospital serving approximately 90% of children aged under 5 years in the kingdom of bahrain.20,21 the study design was based on the who 2002 generic protocol for hospital - based surveillance to estimate the burden of rvge in children.22 the study was conducted in accordance with good clinical practice guidelines , the declaration of helsinki , and all necessary local regulatory requirements .
children aged under 5 years hospitalized for ge ( defined as the occurrence of diarrhea [ 3 looser than normal stools / day ] with or without 2 vomiting episodes/24 hours19 ) and whose parents / guardians provided written informed consent were included .
children were excluded if ge had started > 12 hours after hospitalization ( possible nosocomial infections ) . in accordance with the who guidelines , children hospitalized more than once due to new ge episodes were considered as separate cases on each occasion .
parental questionnaires were used to solicit information regarding each child s demographic and ge symptoms .
rv diagnosis was performed using the who - defined ge criteria.19 the severity of rvge was assessed using the vesikari clinical severity scoring manual.23,24 a score of 11 was considered severe rvge .
stool samples were collected on admission and tested on - site for the presence of rv using enzyme immunoassay ( [ eia ] premier rotaclone ; meridian bioscience , river hills drive , oh , usa).25 a random subset of rv - positive samples was further genotyped ( g and p types ) using reverse transcriptase - polymerase chain reaction and reverse hybridization assay at ddl diagnostic laboratory , the netherlands , as previously described.26 the proportion of rv - attributable diarrheal hospitalizations was calculated with exact 95% clopper pearson confidence intervals ( cis)27 using the following formula : demographic characteristics , symptoms of ge , duration of hospitalization , treatment , and outcome at discharge were tabulated by rv status .
seasonality and severity of rvge and distribution of rv g and p types were also described .
data were summarized in frequency tables , with percentages for categorical variables and mean , median , and standard deviation for continuous variables .
lastly , chi - square and fisher s exact tests were used to analyze the association between the clinical characteristics and rv status .
all statistical analyses were descriptive / exploratory and performed using statistical analysis system ( sas ) software ( v 9.2 ; sas institute inc , cary , nc , usa ) .
this single , referral pediatric hospital - based surveillance study was conducted at the salmaniya medical complex ( smc ) , the kingdom of bahrain , manama over 12 months beginning april 1 , 2006 .
smc is a multispecialty secondary and tertiary government - referral hospital serving approximately 90% of children aged under 5 years in the kingdom of bahrain.20,21 the study design was based on the who 2002 generic protocol for hospital - based surveillance to estimate the burden of rvge in children.22 the study was conducted in accordance with good clinical practice guidelines , the declaration of helsinki , and all necessary local regulatory requirements .
children aged under 5 years hospitalized for ge ( defined as the occurrence of diarrhea [ 3 looser than normal stools / day ] with or without 2 vomiting episodes/24 hours19 ) and whose parents / guardians provided written informed consent were included .
children were excluded if ge had started > 12 hours after hospitalization ( possible nosocomial infections ) . in accordance with the who guidelines , children hospitalized more than once due to new ge episodes were considered as separate cases on each occasion .
parental questionnaires were used to solicit information regarding each child s demographic and ge symptoms .
rv diagnosis was performed using the who - defined ge criteria.19 the severity of rvge was assessed using the vesikari clinical severity scoring manual.23,24 a score of 11 was considered severe rvge .
stool samples were collected on admission and tested on - site for the presence of rv using enzyme immunoassay ( [ eia ] premier rotaclone ; meridian bioscience , river hills drive , oh , usa).25 a random subset of rv - positive samples was further genotyped ( g and p types ) using reverse transcriptase - polymerase chain reaction and reverse hybridization assay at ddl diagnostic laboratory , the netherlands , as previously described.26
the proportion of rv - attributable diarrheal hospitalizations was calculated with exact 95% clopper pearson confidence intervals ( cis)27 using the following formula : demographic characteristics , symptoms of ge , duration of hospitalization , treatment , and outcome at discharge were tabulated by rv status . seasonality and severity of rvge and distribution of rv g and p types were also described .
data were summarized in frequency tables , with percentages for categorical variables and mean , median , and standard deviation for continuous variables .
lastly , chi - square and fisher s exact tests were used to analyze the association between the clinical characteristics and rv status .
all statistical analyses were descriptive / exploratory and performed using statistical analysis system ( sas ) software ( v 9.2 ; sas institute inc , cary , nc , usa ) .
overall , 314 children were approached , of whom 75 were excluded as they did not meet the predefined eligibility criteria .
the median age of the 239 children included in the final analysis was 13.0 months ( range 1.056.0 months ) ; 145 ( 60.7% ) children were male , and the majority ( 99.6% ) lived in the area served by the study hospital .
most ge - associated hospitalizations occurred in children aged 623 months ( 169/239 , 70.7% ; figure 1 ) . in the final analysis , 107 children ( 44.8% ) were rv - positive ; 128 ( 53.5% ) were rv - negative ; and four ( 1.7% ) had unknown rv status as their stool samples were not collected .
the proportion of rv - attributable ge hospitalizations was highest in children aged 623 months ( 82/107 ; 76.6% ; figure 1 ) .
the maximum number of rvge cases were recorded during april 2006 ( 26/42 ; 61.6% ) , followed by june and november 2006 ( 8/14 and 4/7 , respectively ; both 57.1% ; figure 2 ) . before hospitalization , 94.4% ( 101/107 ) of rv - positive children and 83.6% ( 107/128 ) of rv - negative children
this association was found to be statistically significant ( p = 0.0204 ; table 1 ) . before hospitalization , vomiting and dehydration
were more commonly observed in rv - positive children , with vomiting being significantly associated with rv - positive status ( p = 0.0008 ; table 1 ) .
the mean ( standard deviation ) duration of hospitalization was 4.1 ( 2.41 ) days , regardless of rv status .
intravenous ( iv ) rehydration therapy was administered to 28.0% ( 30/107 ) of rv - positive and 17.2% ( 22/128 ) of rv - negative children in the emergency room before hospitalization .
almost all children with known rv status ( 234/235 ; 99.6% ) required iv rehydration therapy during hospitalization . by discharge
wild - type g1p[8 [ was the most commonly detected rv strain in ten samples ( 58.8% ) , and g2p[4 [ , g2p[8 [ , and g9p[8 [ were each detected in two samples .
overall , 314 children were approached , of whom 75 were excluded as they did not meet the predefined eligibility criteria .
the median age of the 239 children included in the final analysis was 13.0 months ( range 1.056.0 months ) ; 145 ( 60.7% ) children were male , and the majority ( 99.6% ) lived in the area served by the study hospital .
most ge - associated hospitalizations occurred in children aged 623 months ( 169/239 , 70.7% ; figure 1 ) .
in the final analysis , 107 children ( 44.8% ) were rv - positive ; 128 ( 53.5% ) were rv - negative ; and four ( 1.7% ) had unknown rv status as their stool samples were not collected .
the proportion of rv - attributable ge hospitalizations was highest in children aged 623 months ( 82/107 ; 76.6% ; figure 1 ) .
the maximum number of rvge cases were recorded during april 2006 ( 26/42 ; 61.6% ) , followed by june and november 2006 ( 8/14 and 4/7 , respectively ; both 57.1% ; figure 2 ) .
before hospitalization , 94.4% ( 101/107 ) of rv - positive children and 83.6% ( 107/128 ) of rv - negative children had experienced a severe episode of ge . this association was found to be statistically significant ( p = 0.0204 ; table 1 ) . before hospitalization , vomiting and dehydration
were more commonly observed in rv - positive children , with vomiting being significantly associated with rv - positive status ( p = 0.0008 ; table 1 ) .
the mean ( standard deviation ) duration of hospitalization was 4.1 ( 2.41 ) days , regardless of rv status .
intravenous ( iv ) rehydration therapy was administered to 28.0% ( 30/107 ) of rv - positive and 17.2% ( 22/128 ) of rv - negative children in the emergency room before hospitalization .
almost all children with known rv status ( 234/235 ; 99.6% ) required iv rehydration therapy during hospitalization . by discharge
wild - type g1p[8 [ was the most commonly detected rv strain in ten samples ( 58.8% ) , and g2p[4 [ , g2p[8 [ , and g9p[8 [ were each detected in two samples .
this hospital - based surveillance study indicated rv as a major cause of ge in children under 5 years of age in the kingdom of bahrain and accounts for 44.8% of ge hospitalizations .
this proportion is consistent with previous reports for rvge hospitalizations ( 16%61% ) in middle eastern and north african countries28 and is comparable with more recent estimates of the proportion of rvge from iran ( 40.0%),29 turkey ( 53.0%),30 saudi arabia ( 39.9%),31 and oman ( 49.0%);32 in each of these cases , the same who generic protocol for assessing rvge burden in children aged under 5 years was used.22 these results are also consistent with the worldwide estimates ( 40.0% ) of rvge hospitalizations among children aged under 5 years.33 nevertheless , our findings are higher than previous reports from the kingdom of bahrain in 1990 ( 13.9%)5 and 2002 ( 20.8%),6 and could reflect differences in subject screening and the improved sensitivity of the eia kit we used , which exhibits 100% sensitivity and 97% specificity to rv.25 importantly rvge - related hospitalizations were highest amongst children under 2 years of age , as previously observed in middle eastern countries such as iran , saudi arabia , and jordan.29,31,34 as was also observed in earlier studies conducted in europe35 and saudi arabia,31 we found that the severity of ge - related clinical characteristics depended on the rv status of the child .
the reporting of vomiting in our study was significantly higher in rv - positive than rv - negative children , which is consistent with earlier reports.23,36,37 we found the seasonal distribution of rv to be similar to that previously seen in this region , with rvge occurring throughout the year.2832 while rv exhibits marked seasonal variation in europe and other regions with a temperate climate,36 seasonality is less marked in regions with warmer climates;2832 rvge appears to peak during cooler and drier periods in the middle east.38 in our study , g1p[8 [ was the most commonly detected rv type in the limited number of samples analyzed , followed by g9p[8 [ , g2p[8 [ , and g2p[4[. indeed , g1p[8 [ was also the most commonly detected rv type in recent studies carried out in turkey,30 saudi arabia,31 and other middle eastern countries.28 in iran , g4p[8 [ was the most common rv type,29 and a more diverse strain pattern was noted in oman.39 recent who surveillance data across the eastern mediterranean region revealed considerable strain diversity among 1,290 rv - positive samples characterized from children under 5 years of age who had been hospitalized with rvge.33 g2p[4 [ was the most commonly identified rv type , accounting for 24.0% of all cases , followed by g1p[8 [ ( 17% ) and g9p[8 [ ( 5.0%).33 the main limitation of our study was that , although the study was designed to estimate the incidence of rv - attributable ge - hospitalization , the denominator of births was unavailable to match the numerator data on hospitalization and the incidence could not be estimated .
our study was also limited by the inability to define the catchment area for rvge cases or to obtain accurate birth cohort data for the catchment populations .
the proportion of ge hospitalizations could not be estimated with certainty since data on the number of screened subjects were not available .
the strain diversity observed during the study has to be interpreted with caution since only a limited number of samples were analyzed and this may not warrant any statistically valid conclusions about the rv type distribution in this population .
furthermore , the study considered only hospitalized children with ge and did not include children treated for ge as outpatients or in emergency rooms . however , by considering only the more severe cases leading to hospitalization , our study helps understand the rv - attributable fraction of ge and can be related to the direct cost of hospitalization .
hence , our estimates provide a good representation of severe rvge cases . to our knowledge , this is the first hospital - based surveillance report on rv type distribution to include analyses of the clinical features and associated treatment in the kingdom of bahrain .
the study was conducted according to the who generic protocol22 ( comprising well - established case definitions and standard data collection ) , with typing of both rv g and p types undertaken in an accredited reference laboratory .
although the number of children enrolled was relatively small , this study was conducted in a large hospital serving approximately 90% of the area s pediatric population , suggesting that our findings are likely to be representative of the bahrain population .
rv accounted for 44.8% of ge hospitalizations over a 1-year period from 20062007 in children aged under 5 years in our study .
these data will help in estimating the number of rvge cases in the kingdom of bahrain and serve as a robust baseline for assessing the potential impact of rv vaccination in reducing the burden of rvge following its introduction into the national immunization program in 2008 .
ma was the coordinating investigator , together with hz , for the conduct of the study .
rd managed the study at glaxosmithkline vaccines and contributed to the analysis and interpretation and critically reviewed the study report .
all the authors contributed to the development of the article , revised every draft and approved the final version for submission .
all the authors had full access to the data and the corresponding author had the final responsibility for the submission of the manuscript . | purposerotavirus ( rv ) is the leading cause of morbidity and mortality in children under 5 years of age worldwide .
this study assessed the role of rv as a cause of gastroenteritis ( ge)-associated hospitalization in children , generating baseline information to evaluate the potential impact of the rv vaccine in reducing rvge disease burden in the kingdom of bahrain.methodsthis single , pediatric hospital - based surveillance study was conducted over a period of 12 months beginning april 1 , 2006 .
a total of 314 children aged under 5 years and hospitalized due to ge were enrolled in the study , following collection of written informed consent from parents / guardians .
stool samples were tested for the presence of rv using enzyme immunoassay , and a random subset of rv - positive samples was further genotyped using reverse transcriptase - polymerase chain reaction and reverse hybridization assay.resultsof 314 enrolled children , 239 were included in the final analysis .
rv was detected in 107 children ( 44.8% ) , mostly in the 623 months age group ( 82/107 ; 76.6% ) .
rvge occurred throughout the year , with the highest proportion occurring during april ( 26/42 ; 61.9% ) .
g1p[8 [ was the most commonly detected rv strain ( 10/17 ; 58.8% ) in the limited number of samples analyzed .
vomiting and severe rvge were more commonly observed in rv - positive than rv - negative children before hospitalization ( p = 0.0008 and 0.0204 , respectively).conclusionin our study , rv accounted for over 40% of ge - associated hospitalizations and particularly affected children under 2 years of age .
these data will serve as a baseline for assessing the potential changes in the epidemiology of rv disease and for evaluating the potential impact of the introduction of rv vaccination . |
endometriosis is a common benign gynecological disease characterized by the presence of ectopic endometrial tissue outside the uterus .
it is a well - known fact that endometriosis most commonly affects such organs as the ovaries , utero - sacral ligaments , uterine tubes , pouch of douglas , and rectum .
the bladder , however , is an infrequent site of endometriosis localization , and it is estimated that only 1% of patients suffering from this disease have lesions involving the urinary system .
three leading theories explain the origin of bladder endometriosis :
it develops from mllerian remnants in the vesicouterine / vesicovaginal septum.it is in fact an extension of an adenomyotic nodule of the anterior uterine wall.it results from implantation of regurgitated endometrium .
it is in fact an extension of an adenomyotic nodule of the anterior uterine wall .
from january 2003 to october 2006 , six patients with bladder endometriosis were treated at the department of obstetrics and gynaecology , university hospitals schleswig- holstein , campus kiel .
the symptoms varied from dysmenorrhoea , cystitis , and pain in the abdomen to menorrhagia . in 2 cases , endometriosis of the bladder was an accidental finding in a diagnostic pelviscopy performed for primary sterility .
the eec classification was grade iv in 2 cases and grade ii in 4 cases .
. associated endometriotic lesions involving the ovaries and pouch of douglas were seen in 5 patients .
another patient experienced urethral dilation 3 years prior to laparoscopic treatment of endometriosis , and a third patient underwent laparoscopic bilateral endometrioma enucleation . of 6 patients , 2 with recurrent cystitis , menstrual hematuria , and lower abdominal pain underwent laparoscopic resection of the bladder wall , followed by suturing of the bladder defect with 40 pds in 2 layers .
one of these patients had been treated with gnrh analogues for 3 months for an associated fibroid .
associated endometriotic lesions were treated in 5 patients , treatments including adhesiolysis and myoma enucleation in one patient .
symptomatic relief of hematuria , recurrent cystitis , and abdominal pain was seen in all patients .
after these treatments , all patients were without pain and reported none of the previous symptoms . in the following report
a 38-year - old lady presented with pain in the lower abdomen in a 4-week rhythm although the uterus had been previously resected .
she also suffered from pain while urinating , menstrual hematuria , and hypermenorrhea every 3 weeks to 4 weeks .
symptoms started after the second vaginal birth in 1998 ; a cystoscopy was without pathological findings .
the patient underwent a pelviscopy in 2001 for suspected endometriosis and adhesions and a vaginal cystectomy in 2003 . at the vaginal examination in 2005
, a walnut - sized nodule at the top of the anterior vaginal wall could be touched , which was painful at compression .
we recommended transvaginal excision to remove the nodule and to detach the endometriotic lesions , followed by operative laparoscopy .
the nodule in the anterior vaginal wall was palpated in the septum vesico - vaginale .
the pelviscopy was begun by insufflation of 3.6 liters of co2 and positioning of 3 trocars ; the optic trocar in the caudal umbilicus and two 5-mm trocars in the right and left lower abdomen .
the diagnostic laparoscopy and pelviscopy revealed no adhesions in the upper and mid abdomen , no uterus , an appendix without signs of irritation , a right ovary of normal size , and a left ovary partially adherent to the pelvic wall . on the right twisted tube
, we discovered a 1-cm pedicled hydatid that was displaced by coagulation at the pedicle and sent for histological clarification . in the pouch of douglas on the dorsal
the main manifestation of endometriosis was found in the dome of the bladder , appearing as endometriosis extragenitalis , eec iv. the peritoneum over the bladder was star - like inserted ; the center of the lesion was brown and white colored .
the lesion appeared as a 2 3-cm large nodule infiltrating into the bladder wall ( figure 1 ) .
the gynecologist and urologist cooperated to excise the nodule circularly from the peritoneum with the hook scissors , mobilized on the level of the muscular bladder wall and removed completely with the simultaneous excision of a 2 2.5-cm large adherent lobe of urothelium off the bladder wall .
this resulted in an unobstructed view into the bladder with clean , smooth , and bloodless wound edges ( figure 2 ) . to reduce the tension at the margins of the wound , we laid an extracorporal , knotted pds size 0 suture through all layers of the bladder .
view into the urinary bladder after complete laparoscopic resection of an endometriotic nodule . at the right and left wound slot , the urothelium was closed completely with an intracorporal , knotted pds size 4 suture . for the second layer
, we laid an extracorporal , knotted pds size 0 suture in the middle of the wound to place the peritoneum of the bladder over the wound . on the right and left side , we closed the peritoneum permanently over the wound of the bladder with simple interrupted sutures , size 40 . to check the closure , we filled the bladder with 80 ml to 100 ml of blue solution and no leakage occurred .
after irrigation of the abdominal cavity and placing of a 5-mm robinson drainage for 24 hours , we removed the trocars under vision and closed the skin incisions .
three weeks after the operation , a cystoscopy with contrast medium was performed without pathological findings , and the patient reported no pain . at a control examination
broadly speaking , vesical endometriosis has 2 different causes : a primary and a secondary form .
the secondary form is iatrogenic , occurring after pelvic surgery , such as cesarean delivery or hysterectomy . after iatrogenic dissemination , growth of ectopic endometrium
symptoms , when present , mimic those of recurrent cystitis , or more rarely the condition is characterized by pathognomic menstrual hematuria .
early diagnosis and treatment of urinary tract endometriosis is necessary to avoid loss of kidney function .
transabdominal and transvaginal ultrasound are the initial investigation of choice in evaluation of suspected bladder endometriosis due to immediate availability and easy access . it has proved adequate in determining the site and size of lesion , the degree of infiltration of detrusor and mucosa , and the relationship with concomitant adenomyosis of the uterus .
magnetic resonance imaging can not only delineate the morphologic abnormalities of bladder endometriosis but can also potentially identify other common sites , particularly at the uterosacral ligaments , where ultrasound is less reliable .
cystoscopy is able to visualize the endometriosis foci only when present on bladder mucosal surface , but is unable to define the extent of endometriosis lesion , which can be better detected by tv sonography .
hormonal therapy is a reasonable and effective management for bladder endometriosis reaching into the genital tract .
however , experienced laparoscopists , familiar with endometriosis , can also treat urogenital endometriosis safely and effectively using excision .
segmented resection may be performed , or transurethral resection of the bladder mass , and ureterolysis , if necessary . in cases of submucosal transmural location of the lesion , a minimally invasive combined endoscopic procedure ( laparoscopy and cystoscopy )
represents a good alternative to partial cystectomy for muscle infiltrating bladder endometriosis that does not involve the vesical mucosa .
bladder endometriosis should be considered in women of reproductive age who present with urinary tract symptoms not responding to routine medical management with a view to laparoscopic treatment .
an optimal treatment of bladder and urethral endometriosis should ideally involve a team of experts , ie , gynecological endoscopists , radiologists , and urologists , who are familiar with endometriosis . | background and objectives : genital endometriosis is still an unclear enigma of the female .
it occurs in approximately 20% to 35% of all women of reproductive age .
the urinary tract is seldom affected.methods:we report on a patient with bladder endometriosis with recurrent heavy pain at menstruation within the framework of 5 similar cases.results:a combined vaginal and laparoscopic treatment with excision of the lesion reaching into the dome of the bladder and vaginal wall was performed .
the bladder opening was sutured in 2 layers.conclusion:laparoscopic treatment of bladder endometriosis requires a combined surgical treatment by a gynecologist and urologist or an experienced laparoscopist . |
a 5-month - old feral kitten developed worsening respiratory signs , including tachypnea , coughing and wheezing after standard anthelmintic treatment with fenbendazole at a local shelter .
the kitten was referred to the university of california , davis , william r pritchard veterinary medicine teaching hospital for further evaluation .
thoracic radiographs revealed a severe diffuse bronchointerstitial pattern with bronchial cuffing , ill - defined nodules and lymphadenomegaly .
bronchoalveolar lavage revealed marked neutrophilic and eosinophilic inflammation , and parasitic larvae were observed in a swab of trachea mucus .
parasitic pneumonia should be considered as a cause of respiratory distress in kittens and cats .
lungworm infections have been more commonly reported in free - roaming young and adult cats , but can not be excluded as a differential diagnosis in cats from varied environments and in kittens .
kittens appear to be especially sensitive to lungworm infections , manifested by the development of more severe clinical signs ; thus lungworm infection should always be considered when presented with a kitten in respiratory distress . in the absence of cytologic confirmation of infection via bronchoalveolar lavage or oropharyngeal swab , pcr provides a valuable means for identification of lungworms , such as a abstrusus and troglostrongylus brevior .
a 5-month - old female intact feral kitten presented to the local animal shelter unable to ambulate after being trapped underneath a garage door . a board - certified neurologist ( kmv ) localized the lesion to the c5 to brainstem region .
cervical and thoracic radiographs showed no evidence of cervical fracture , but a mild bronchointerstitial pulmonary pattern was noted . as per routine protocol of the shelter , imidacloprid / moxidectin ( advantage multi for cats ; bayer ) was applied to the kitten and a 5 day course of fenbendazole was initiated at 50 mg / kg po q24h .
the kitten was placed in foster care once able to ambulate 5 days later , and although it continued to improve neurologically , respiratory signs of tachypnea , cough and wheezing suddenly worsened in frequency and severity 9 days later .
on day 10 , the cat presented to the university of california , davis , william r pritchard veterinary medicine teaching hospital for a further diagnostic work - up . on physical examination the kitten weighed 1.7 kg , heart rate was 180 beats per minute , respiratory rate was 70 breaths per minute with wheezing and rectal temperature was 103.0f .
complete blood count results included a mild normocytic normochromic non - regenerative anaemia , likely age related ( haematocrit 27% ; reference interval [ ri ] 3050% ) , and a white blood cell count of 6098/l with neutrophils ( 4025/l ; ri 20009000/l ) , lymphocytes ( 1342/l ; ri 10007000/l ) and eosinophils ( 305/l ; ri 1501100/l ) within ris , and a slight left shift ( band neutrophils 183/l ; ri 0/l ) .
mmol / l ) , hypochloremia ( 115 mmol / l ; ri 117126 mmol / l ) , hyperphosphatemia ( 9.0 mg / dl ; ri 3.26.3 mg / dl ) and increased alanine aminotransferase ( 103
thoracic radiographs revealed a severe diffuse bronchointerstitial pulmonary pattern with peribronchial cuffing and ill - defined pulmonary nodules .
soft - tissue opacities dorsal to the carina and the second and third sternebrae were interpreted as lymphadenomegaly ( figure 1 ) .
parasitic pulmonary disease was considered less likely due to previous treatment with fenbendazole and neoplasia ( lymphoma ) was also lower on the differential list .
( a ) lateral and ( b ) dorsoventral radiographs obtained on day 1 demonstrate a marked diffuse bronchointerstitial pulmonary pattern with peribronchial cuffing and ill - defined pulmonary nodules given the severity of thoracic radiographic findings , the kitten was prepped for anesthesia to undergo a laryngoscopy / bronchoscopy and bronchoalveolar lavage ( bal ) .
premedication was provided with butorphanol 0.6 mg / kg ( 1.03 mg ) subcutaneously , flow - by 100% oxygen was provided and induction was performed with alfaxalone 1.0 mg / kg ( 1.7 mg ) and midazolam 0.3 mg / kg ( 0.5 mg ) intravenously . a jet ventilator was used to provide oxygenation at 180 pulses per minute , and anaesthesia maintained with an alfaxalone constant rate infusion to effect ( 4.12 mg / kg infused ) . a 2.8 mm outer diameter video - endoscope ( flex xc ; karl storz veterinary endoscopy ) was utilized for bronchoscopy .
moderate laryngeal , tracheal and bronchial hyperemia was noted along with excessive mucus accumulation ( figure 2 ) .
bal was performed in the left and right caudal lung lobes and was submitted for cytologic evaluation .
copious amounts of tracheal mucus were expectorated and a swab collected from the oropharynx was also submitted for cytologic evaluation .
( a ) excessive pharyngeal mucus was observed on bronchoscopy and ( b ) the carina was diffusely reddened .
* jet ventilator catheter bal fluid was hypercellular ( 1010 and 1100 cells/l , right and left , respectively ; ri 300400/l ) with 62% and 50% neutrophils ( normal 58% ) and 22% and 25% eosinophils ( normal < 20% ) ( figure 3 ) . increased numbers of multinucleate giant cells , goblet cells and mast cells were also present , along with epithelial hyperplasia .
swabs of oropharyngeal mucus revealed a dense background of mucus with neutrophilic and eosinophilic inflammation , numerous free mucus granules and evidence of mineralization .
five large parasitic ( helminth ) larvae were observed on one slide ( figure 4 ) , including one that appeared to have a dorsal spine on the tail .
the oropharyngeal sample was submitted to the university of teramo for molecular identification of larvae .
dna was extracted using a commercially available kit ( qiagen mini kit ; qiagen ) , and nested pcr was performed targeting the its2 region of aelurostrongylus abstrusus and troglostrongylus brevior , as previously described .
pcr was positive for a abstrusus at the 233 base - pair region and negative for t brevior at the 356 base - pair region ( figure 5 ) .
there are increased numbers of neutrophils and eosinophils , along with scattered macrophages and mast cells ( objective 60 , wright - giemsa stain ; bar = 10 m ) oropharyngeal swab cytology from a kitten with aelurostrongylus abstrusus lungworm infection .
a helminth larva is present in a dense background of mucus with neutrophilic and eosinophilic inflammation ( objective 20 , wright - giemsa stain ; bar = 100 m ) pcr specific for the ribosomal its2 of aelurostrongylus abstrusus .
lane m : gene ruler 100 base - pair marker ; lane 1 : oropharyngeal sample positive for a abstrusus ; lanes 2 and 3 : negative and positive controls for a abstrusus the kitten was discharged with application of a second dose of imidiclopid / moxidectin , and fenbendazole was prescribed at 50 mg / kg po q24h for 10 days .
three weeks later , the kitten was doing clinically well at home but still coughing intermittently .
radiographs revealed marked resolution in the bronchial pattern with partial resolution of peribronchial cuffing and ill - defined nodules ( figure 6 ) .
fenbendazole was prescribed for an additional 5 days at 50 mg / kg / day .
all respiratory signs had resolved at the time of ovariohysterectomy 3 weeks later , and radiographs 2 months after the final treatment were normal .
( a ) lateral and ( b ) dorsoventral radiographs obtained 3 weeks after a second course of fenbendazole ( 10 day course ) .
the soft tissue opacity in the right caudal lung lobe lateral to the cardiac silhouette in ( b ) was considered to represent residual inflammatory infiltrate or a granuloma
lungworm infection in domestic cats is most commonly caused by either a abstrusus or capillaria aerophila , although infection of domestic cats with t brevior is increasingly reported in europe .
t brevior and a abstrusus have a similar indirect biological life cycle with estimated prepatent period of 46 weeks , while c aerophila has a direct life cycle .
it is possible that t brevior , found primarily in the old world , might also be passed vertically in a transmammary fashion from queen to offspring .
adults of a abstrusus reside within the alveolar ducts and alveoli , while t brevior live within the bronchi and bronchioles ; adults of c aerophila live within the submucosa of the trachea and bronchi .
a abstrusus is considered the most common feline lungworm parasite and is found worldwide ; it can infect all cats , regardless of their habitat , lifestyle , breed or sex .
indoor cats are at decreased risk , but possible infection should not be ruled out based upon lifestyle alone .
kittens , owing to their immature immune system , and adult cats that hunt are at an increased risk for lungworm infection with a abstrusus .
while lungworm infection most commonly affects young adult cats , 16% of infected cats were < 6 months of age in one study .
clinical signs of infection vary depending upon worm burden , age , immune response and overall health status of the feline host , and can range from subclinical to severe disease , occasionally including fatal pneumonia .
many cats are subclinical or can have respiratory signs of chronic cough with gradually increasing respiratory difficulty and tachypnea , wheezing , sneezing and nasal discharge , as well as anorexia and fever .
kittens appear to develop more severe disease than adult cats , perhaps because of smaller bronchial diameter , which would be more susceptible to rapid occlusion of the airways by developing worms and concurrent inflammation .
one case report describes a 2-month - old kitten that died from fatal verminous pneumonia and enteritis secondary to infection with a abstrusus , while another discusses reversible pulmonary hypertension in kittens secondary to infection with a abstrusus .
kittens and young cats also develop more severe radiological abnormalities and higher larval worm burdens than adults .
t brevior has been reported as an emerging helminth parasite in europe that can result in clinical signs similar to a abstrusus , although infection with the former lungworm is associated with more severe and life - threatening infection .
kittens and young cats seem to be particularly susceptible to infection with t brevior , and clinical signs include severe cough , respiratory distress and nasal discharge .
a more recent report describes the first known case of irreversible pulmonary hypertension in a kitten infected with t brevior , despite appropriate treatment .
infection with c aerophila can be subclinical or associated with respiratory distress characterized by increased bronchovesicular sounds , wheezing , sneezing and a dry cough .
diagnosis of lungworm infection can be challenging , as clinical signs often imitate other causes of respiratory signs , such as fungal infections , toxoplasmosis and feline asthma .
radiographic abnormalities vary depending upon worm burden and time after onset of infection and range from a mild multifocal bronchointerstitial pulmonary pattern with poorly demarcated nodules ( often in the caudal lung lobes ) to a diffuse alveolar infiltrate .
this test takes 24 h to complete , and three consecutive negative samples are required to rule out parasitic pneumonia .
lungworm larvae can be found in tracheal swabs or washes , as well as on bal cytology , but sensitivity is not as high as with the baermann technique .
infection with a abstrusus or t brevior can be documented by fecal baermann , and specific larval morphology can be used to differentiate the two organisms .
feces , pharyngeal swabs and lower airway samples can be used for detection , but the best sample to submit for pcr appears to be a pharyngeal swab , which is supported by results in this case . while pharyngeal cytology and pcr have not been directly compared , pcr is reported to have a specificity of 100% and sensitivity of 96% .
bal cytology was negative for the parasite in this case , indicating that additional ( pharyngeal ) samples may be needed to obtain a definitive diagnosis .
this case was especially interesting because the kitten s clinical signs initially worsened after anthelmintic treatment .
potential explanations include a severe immune response to a dying worm burden , a mixed infection or potentially a novel case of t brevior infection . a recent report described two littermates with mixed a abstrusus and t brevior infections that were treated appropriately with milbemycin oxime ( 2 mg / kg ) only to have one kitten develop fatal exacerbation of clinical signs .
we consider it likely that worsening clinical signs noted in the kitten of this report were the result of an immune response to dying worms , which incited coughing , wheezing and tachypnea .
given the marked inflammatory response present in bal cytology , treatment with corticosteroids could have been considered .
this case serves as a clinically relevant reminder to include lungworm disease as a differential diagnosis when determining the cause of respiratory signs in cats , especially kittens and free - roaming cats or when the animal s history and origin are unknown .
this case also supports the work currently being performed regarding improvement of diagnostics for feline lungworm , as it can be clinically difficult to diagnose .
diagnosis of lungworm infection in this kitten was obtained via orophargyngeal swab cytology and pcr , with the latter being more sensitive and specific .
diagnosis via cytology has a more rapid turnaround time , but differentiating t brevior from a abstrusus can be morphologically challenging .
pcr allows both diagnosis and speciation ; however , this test is not yet readily available | case summarya 5-month - old feral kitten developed worsening respiratory signs , including tachypnea , coughing and wheezing after standard anthelmintic treatment with fenbendazole at a local shelter .
the kitten was referred to the university of california , davis , william r pritchard veterinary medicine teaching hospital for further evaluation .
thoracic radiographs revealed a severe diffuse bronchointerstitial pattern with bronchial cuffing , ill - defined nodules and lymphadenomegaly .
differentials included infectious etiologies such as toxoplasmosis , feline infectious peritonitis and cryptococcosis .
parasitic infection was considered less likely , owing to previous anthelmintic treatment .
bronchoalveolar lavage revealed marked neutrophilic and eosinophilic inflammation , and parasitic larvae were observed in a swab of trachea mucus .
pcr confirmed the larvae as aelurostrongylus abstrusus .
the kitten recovered with two more rounds of anthelmintic treatment.relevance and novel informationparasitic pneumonia should be considered as a cause of respiratory distress in kittens and cats .
lungworm infections have been more commonly reported in free - roaming young and adult cats , but can not be excluded as a differential diagnosis in cats from varied environments and in kittens .
kittens appear to be especially sensitive to lungworm infections , manifested by the development of more severe clinical signs ; thus lungworm infection should always be considered when presented with a kitten in respiratory distress . in the absence of cytologic confirmation of infection via bronchoalveolar lavage or oropharyngeal swab , pcr provides a valuable means for identification of lungworms , such as a abstrusus and troglostrongylus brevior . |
tooth formation is an advancing process that is regulated by reciprocal interactions between the epithelial and mesenchymal tissues .
these interactions stimulate a subpopulation of mesenchymal cells to differentiate into odontoblasts , which , in turn , form the primary dentin .
but , after dentinal damage these processes may be reactivated , which is subsequently associated with the formation of tertiary dentin including reactive and reparative dentin . in response to minor injuries , the primary odontoblast survive and reactionary tertiary dentin is formed ; meanwhile , reparative dentinogenesis occurs after more intense injuries that lead to odontoblast death . these all demonstrate that postnatal dental pulp contains cells with stem cell properties .
they are highly proliferative , clonogenic , and capable of regenerating a tissue ; the properties that clearly define them as stem cells .
stem cells have several unique properties include the ability of self - renewal , proliferation , differentiation and they can give rise to specialized cell types . stem cells are categorized as embryonic and adult . in comparison to other types of adult stem cells used in tissue engineering , mesenchymal stem cells are more promising for therapeutic purposes .
the most common source of mesenchymal stem cells is bone marrow ; however , it has limitations such as the small number of cells obtained by biopsy and painful process .
currently , several types of adult stem cells have been isolated from teeth , namely dental pulp stem cells ( dpscs ) , stem cells from human exfoliated deciduous teeth ( sheds ) , periodontal ligament stem cells ( pdlscs ) , stem cells from apical papilla ( scaps ) , and dental follicle progenitor stem cells ( dfpcs ) .
sheds are a population of highly proliferative cells which can differentiate into osteogenic and odontogenic cells , adipocytes , and neural cells .
they have several distinct advantages over dental pulp stem cells among which are increased cell population doublings , higher proliferation rate , ease of access , lack of tissue destruction at donor site , reduction or elimination of pain and discomfort , and the possibility of obtaining cells from young patients .
these cells can be regarded as the possible sources of stem cells for future tissue engineering applications .
another field of research following the isolation of stem cells from the desired source is to find the appropriate materials to add to the cell culture medium so that the most efficient material for the proliferation and differentiation of stem cells is introduced .
vitamin d3 metabolites such as 1 , 25-dihydroxyvitamin d3 are the key factors in regulation of bone metabolism .
studies have shown that 1 , 25-dihydroxyvitamin d3 increases the alp activity and osteogenic differentiation of normal permanent dental pulp as well as dental follicle stem cells .
considering the differences between these cells and deciduous stem cells , the current study was designed to investigate the effect of 1,25-dihydroxyvitamin d3 on osteogenic differentiation ( alkaline phosphatase activity and alizarin red staining ) of stem cells of exfoliated deciduous teeth .
fifteen sound exfoliated human deciduous teeth were collected from 10 healthy children aged 6 to 11 years old ( 4 girls and 6 boys ) .
prior to extraction , the patients received oral health education , professional
prophylaxis was done for each patient , and they used 0.2% chlorhexidine mouthwash for one minute .
it was immersed in a digestive solution containing 100 u / ml penicillin , 100 mg / ml streptomycin , and 500 mg / ml claritromycin in 4 ml 0.1 m phosphate - buffered saline ( pbs )
with the addition of 3 mg / ml type i collagenase and 4 mg / ml dispase , for 1 hour at 37c .
the solution was filtered through 70m falcon strainers ( falcon ; fisher , usa ) .
after filtration , the cells were immersed in -mem ( minimum essential medium ) ( gibco ; germany ) , added with 10% fetal bovine serum ( fbs ) ( gibco ; germany ) , 100m 2p - ascorbic acid ,
2 mm l - glutamine , 100 u / ml penicillin , and 100 g / ml streptomycin .
the flasks were incubated at 37c in
a 5% co2 , and the medium was changed twice a week .
after the third passage , the cells were isolated from the culture plate by using 25% trypsin - edta and were cultured for 24 hours .
when the cell density reached 80% , they were used for osteogenic differentiation .
as the control group
, the cells were cultured in osteogenic cell culture medium containing -mem ( gibco ; germany ) , 10% fbs , 50 g / ml l - ascorbic acid 2-phosphate , 50 mm -glycerophosphate , and 0.1 m dexamethasone ( sigma ) . as the case group
, the cells were cultured in osteogenic culture medium supplemented with 100 nm1 and 25(oh)2d3 .
after 21 days of culture , alkaline phosphatase ( alp ) activity was analyzed with a commercially available p - nitrophenylphosphate tablet set ( pars azmoon ; iran ) .
likewise , the mineralization was analyzed by alizarin red staining ( ecm 815 ; millipore , usa ) . for alizarin red staining ,
the cell cultures were rinsed twice with fbs and fixed with 10% formaldehyde for 15 minutes at room temperature .
then , the cultures were stained with alizarin red solution for 20 minutes at room temperature .
the total mineralized tissues were counted according to the manufacturer 's instructions of alizarin red .
quantitative analysis of alizarin red staining was performed by determining the od405 ( optical density ) values of a set of known alizarin red concentrations and comparing these values with the obtained values ( figure 1 ) .
for the analysis of normal distribution of data , non - parametric kolmogorov - smirnov test was used .
after the third passage , the cells were isolated from the culture plate by using 25% trypsin - edta and were cultured for 24 hours .
when the cell density reached 80% , they were used for osteogenic differentiation .
as the control group
, the cells were cultured in osteogenic cell culture medium containing -mem ( gibco ; germany ) , 10% fbs , 50 g / ml l - ascorbic acid 2-phosphate , 50 mm -glycerophosphate , and 0.1 m dexamethasone ( sigma ) . as the case group ,
after 21 days of culture , alkaline phosphatase ( alp ) activity was analyzed with a commercially available p - nitrophenylphosphate tablet set ( pars azmoon ; iran ) .
likewise , the mineralization was analyzed by alizarin red staining ( ecm 815 ; millipore , usa ) . for alizarin red staining , the cell cultures were rinsed twice with fbs and fixed with 10% formaldehyde for 15 minutes at room temperature .
then , the cultures were stained with alizarin red solution for 20 minutes at room temperature .
the total mineralized tissues were counted according to the manufacturer 's instructions of alizarin red .
quantitative analysis of alizarin red staining was performed by determining the od405 ( optical density ) values of a set of known alizarin red concentrations and comparing these values with the obtained values ( figure 1 ) .
for the analysis of normal distribution of data , non - parametric kolmogorov - smirnov test was used .
concentration of alizarin red according to the standard optical density .
the medium was observed by using an inverted light microscope on the days 1 , 3 , 7 , and in the third passage .
the morphology of the cells remained constant during cell passage ( figure 2 ) .
dental pulp stem cells of deciduous teeth at day 3 ( 100 magnification ) .
the results of kolmogorov - smirnov test indicated the normal distribution of data .
compared with the control group
, significant increase was observed in alp activity of sheds after being treated with 1,25(oh)2d3 ( p= 0.002 ) ( table 1 )
. the t - test results of alkaline phosphatase activity quantification of alizarin red staining demonstrated that cells exposed to 1,25(oh)2d3 induced higher mineralized nodules ( p < 0.001 ) ( table 2 , figure 3 ) .
the t - test results of alizarin red staining alizarin red staining of mineralized deposits without ( a , b , c ) and with vitamin d metabolite ( d , e , f ) .
osteogenic differentiation before staining ( a and d ) ( 40 magnification ) ; after staining at 40x ( b and e ) and after staining at 400x magnification ( c and f ) .
the medium was observed by using an inverted light microscope on the days 1 , 3 , 7 , and in the third passage .
the morphology of the cells remained constant during cell passage ( figure 2 ) .
dental pulp stem cells of deciduous teeth at day 3 ( 100 magnification ) .
the results of kolmogorov - smirnov test indicated the normal distribution of data .
compared with the control group
, significant increase was observed in alp activity of sheds after being treated with 1,25(oh)2d3 ( p= 0.002 ) ( table 1 ) .
quantification of alizarin red staining demonstrated that cells exposed to 1,25(oh)2d3 induced higher mineralized nodules ( p < 0.001 ) ( table 2 , figure 3 ) .
the t - test results of alizarin red staining alizarin red staining of mineralized deposits without ( a , b , c ) and with vitamin d metabolite ( d , e , f ) .
osteogenic differentiation before staining ( a and d ) ( 40 magnification ) ; after staining at 40x ( b and e ) and after staining at 400x magnification ( c and f ) .
this study aimed to isolate mesenchymal stem cells from exfoliated deciduous teeth in order to evaluate the effects of 1,25(oh)2d3 on their osteogenic differentiation .
several studies have tried to find the appropriate method for regeneration of damaged bone which is an issue of paramount importance in dentistry . regarding the systemic
advantages of 1,25 ( oh ) 2d3 in bone remodeling and wound healing , in addition to its considerable role in tooth formation
emphasized by in vivo and clinical studies ,
the present study was designed to investigate the effect of 1,25(oh)2d3 on osteogenic differentiation of shed .
deciduous teeth , therefore ,
may be an ideal resource of stem cells to induce bone regeneration .
mesenchymal
stem cells have been extensively characterized in vitro by the expression of markers such as stro-1 , cd146 or cd44 .
the international society for cellular therapy declares that one of the required criteria to define human mesenchymal stem cells is expression of cd105 , cd73 , and cd90 ; in addition to lack of expression of cd45 , cd34 , cd14 , cd11b , cd79a , cd19 , and hla - dr surface molecules . among the limitations of this study was the impossibility of performing flow cytometric analysis of cell surface due to the limited technical and financial resources .
if these cells were end cells , they would not differentiate into other cell lines , so they were certainly considered stem cells .
based on previous studies , differentiation of mesenchymal stem cells such as dpscs and sheds into osteoblast - like cells is
induced by dexamethasone , ascorbic acid , and -glycerophosphate .
these osteogenic
supplements are the most commonly used osteogenic inducer for human mesenchymal stem cells in vitro .
1,25(oh)2d3 was found to
be more effective than dexamethasone in osteogenic differentiation of adipose - derived stem cells .
therefore ,
despite the different categories of stem cells , the effect of 1,25(oh)2d3 was the same on osteogenic differentiation potential .
the in vitro studies on osteoblastic cells revealed that 1,25(oh)2d3 increased the alkaline phosphatase activity .
it was reported that adding 1,25(oh)2d3 ( 10 and 100 nm ) increased the alp activity of dental pulp stem cells and dental follicle stem cells .
likewise , the current study showed that 1,25(oh)2d3 ( 100 nm ) enhanced the alp activity in sheds .
so , the response of stem cells in primary and permanent teeth are the same in this regard .
on the other side , bakopoulou et al .
detected that the alp activity was significantly reduced in presence of hema ( 0.5 mm and 0.1 mm ) and tegdma ( 0.25 mm , 0.1 mm ) in osteogenic culture differentiation , which was unlike the effect of vitamin d3.khanna-jain et al . reported that dental pulp stem cells and dental follicle stem cells formed mineralized matrix when treated with vitamin d3 metabolites in osteogenic differentiation culture .
the alizarin red staining in our study demonstrated that the cells exposed to 1,25(oh)2d3 induced higher mineralized nodules .
application of 1,25 ( oh ) 2d3 in tissue engineering is a suggestion which needs more investigations . | statement of the problem : stem cells from human exfoliated deciduous teeth ( sheds ) are a population of highly proliferative cells , being capable of differentiating into osteogenic , odontogenic , adipocytes , and neural cells .
vitamin d3 metabolites such as 1 , 25-dihydroxyvitamin d3 are key factors in the regulation of bone metabolism .
purpose : the aim of this study was to investigate the effect of 1 , 25-dihydroxyvitamin d3 on osteogenic differentiation ( alkaline phosphatase activity and alizarin red staining ) of stem cells of exfoliated deciduous teeth .
materials and method : dental pulp was removed from freshly extracted primary teeth and immersed in a digestive solution .
then , the dental pulp cells were immersed in -mem ( minimum essential medium ) to which 10% fetal bovine serum was added .
after the third passage , the cells were isolated from the culture plate and were used for osteogenic differentiation . as a control group
, the cells were cultured in osteogenic cell culture medium . as the case group
, the cells were cultured in osteogenic culture medium supplemented with 100 nm 1,25 ( oh)2d3 .
the alkaline phosphatase ( alp ) activity and alizarin red staining were analyzed to evaluate the osteogenic differentiation at day 21 .
the results were analyzed by using t - test .
results : compared with the control group , significant increase was observed in alp activity of sheds after being treated with 1,25(oh)2d3 ( p= 0.002 ) .
alizarin red staining demonstrated that the cells exposed to 1,25(oh)2d3 induced higher mineralized nodules ( p < 0.001 ) .
conclusion : osteoblast differentiation in sheds was stimulated by 1,25(oh ) 2d3 .
it can be concluded that 1,25(oh)2d3 can improve osteoblastic differentiation . |
philosophical , religious , and cultural differences in the concept of death and ways it is defined make discussions of the subject very complex .
advances in medicine and technology that have made it possible to support , repair , or replace failing organs challenge commonly held notions of life and death .
the lack of understanding or even awareness among the public and health professionals and the emotionally charged nature of the subject further complicate discussions .
the growing disparity between the demand for and the availability of transplantable organs is a public health concern globally . in comparison to bridge technologies for end - stage organ failure , such as kidney dialysis and ventricular assist devices , organ transplantation is life - preserving , life - enhancing , and cost - effective . yet ,
when the supply of transplantable organs falls short of demand , unethical and illegal behaviours sometimes occur , including commercial organ trade that victimizes the vulnerable , human killing as a source of transplantable organs , and violations of the allocation system .
while the public supports organ donation after death , its credibility requires clarity in practice and policy . depending on the country and related professional societies , guidelines may or may not exist for the determination of death by neurological and/or circulatory criteria , and when guidelines are in place , they have not always been implemented in a manner that alleviates concerns about the legitimacy of deceased organ donation .
the situation is complicated by variations related to the stage of development of a country s deceased donation program and more generally by the significant diversity in health services and standards of care worldwide .
the world health organization ( who ) and the transplantation society ( tts ) have received requests from a number of countries to address these gaps and to provide guidance to inform clinical practices and health policy for death determination as part of an international effort in support of the istanbul declaration , the who madrid resolution , and the who guiding principles on human cell , tissue and organ transplantation , approved by the world health assembly in 2010 . an international consensus on the determination of death could provide a number of benefits including promoting evidence - based practices , protecting the rights of both patients and health care professionals , improving public and professional confidence in the process of deceased donation , and increasing the number of organs obtained in an ethically legitimate fashion .
on 3031 may 2012 , an invitational forum sponsored by health canada and canadian blood services in collaboration with the who was held in montreal , canada , as part of the planning , scoping , and needs assessment phase in the process of guideline development ( see appendix 1 for forum committees and participants ) .
the 32 invited participants included delegates from a broad range of national and international professional societies involved with death determination in adults and children in acute care settings .
prior to the forum , participants were provided with comprehensive background materials , including a selected bibliography of peer - reviewed articles related to death determination , a literature review on definitions of death , and a draft lexicon of medical terminology relevant to death determination policy and practice .
each section of the meeting began with an expert speaker who provided historical information , reviewed baseline knowledge , and supplied his or her perspective on current issues and controversies .
after each presentation , participants asked questions of the expert and discussed the presentation with the entire group .
reference sheets that offered condensed summaries of existing evidence were distributed , and the participants were then divided into groups where they held extensive discussions about a challenge question relevant to the topic of the session . when the participants reconvened in the plenary , each group s results were discussed , and the collective outputs were reworked until consensus was reached .
consensus was defined as achieving substantial agreement , manifested by all participants indicating that they agreed with or could accept a conclusion , which they would support both within and outside the meeting .
for the purposes of this forum , death was fundamentally considered a biological event . while it was respectfully recognized that legal , ethical , cultural , and religious perspectives on death can impact the utility of recommendations in the field , these perspectives were not topics for debate or discussion at this initial meeting .
table 1 highlights the common terminology that was agreed upon to support clarity and precision in the language used throughout this forum and for subsequent phases of work .
the planning committee worked with participants to identify critical events that comprise the dying sequence , with the understanding that dying is a process whereby biological / physiological functions cease .
death is an event in that dying process , a point when the person can be determined to have died .
the forum identified the tests required for the minimum acceptable clinical standard for determining death in adults and children and recommended additional testing beyond the minimum standard .
a consensus on an operational definition of human death was then derived.table 1participants agreed to the following terminology , in order to improve the clarity of discussions and debate , for use during and subsequent to this forumtermdefinitionactivityphysiologic properties of cells and groups of cells that can be measured by laboratory meansasystole
electricala condition characterized by the absence of electrical , and hence mechanical , activity of the heart , resulting in the absence of contractions of the myocardium and cardiac output / anterograde blood flowasystole
mechanicalthe absence of effective contractions of the myocardium and no cardiac output / anterograde blood flow . may occur in the presence of an organized or disorganized electrocardiac rhythm , e.g. pulseless electrical activityautoresuscitationthe spontaneous resumption of heart contractions causing anterograde circulation that is not induced by cardiopulmonary resuscitation or other external assistance .
examples of heart function such as effective contractions of the myocardium leading to anterograde flow of blood through the aorta and arterial system should be distinguished from examples of heart activity such as atrial natriuretic hormone release or residual pulseless electrical activitybrain deathdiagnosis and confirmation of death based on the irreversible cessation of functioning of the entire brain , including the brainstem ( this forum supports the movement away from this traditional and imprecise terminology in favour of the cessation of neurological function)brainstem deathdiagnosis and confirmation of death based on the irreversible cessation of functioning of the brainstem , predominantly but not exclusively secondary to a supratentorial brain injury ( this forum supports the movement away from this traditional and imprecise terminology in favour of the cessation of neurological function)cardiac arrestthe abrupt cessation of circulation of the blood due to failure of the heart to contract effectively . also known as cardiorespiratory arrest , cardiopulmonary arrest , or circulatory arrestcatastrophic brain injury leading to deathetiologies of high severity that are common causes of brain death , such as , but not limited to , traumatic brain injury , cerebrovascular accidents , and hypoxic - ischaemic encephalopathy
after resuscitated cardiac arrestother forms of catastrophic brain injury that have any degree of residual clinical brain or brainstem function are not under consideration and may include persistent vegetative states , permanent vegetative states , anencephaly , or those conditions related to the historical concept of higher brain ( cortical ) deathcerebral electrical activityelectrical activity of the brain , as measured using an electroencephalogram ( eeg)cessationstoppage , terminationcirculationanterograde flow of blood through the aorta and arterial systemcirculatory death determinationdiagnosis and confirmation of death based on circulatory criteria .
also known as death after cardiac arrest , or death after cardiocirculatory arrest , or death after circulatory - respiratory determination ( this forum supports the movement away from this traditional and imprecise terminology in favour of the cessation of circulatory function)clinicalbased on direct , measurable observation or examination of the patientcomaprolonged absence of wakefulness , awareness , and the capacity for sensory perception or responsiveness to the external environmentconfounding conditionscircumstances during which a diagnostic test may become unreliable and require repetition over time or application of an alternative testconsciousness loss of capacity forlack of current or any future potential for awareness , wakefulness , interaction and capacity for sensory perception of , or responsiveness to the external environmentcriteria
the standard recommended by this forum sets the minimum clinical criteriadead donor rulea principle governing deceased donation practices stating that vital organs should only be taken from dead patients and , correlatively , living patients must not be killed by organ retrieval .
this rule does not apply to , nor preclude , living donation of non - vital organsdeaththe moment in time during the dying process when the individual passes from the state of being alive to that of being deaddeath
concept ofan abstract , unprovable explanation of death , generally based on religious , spiritual , or philosophical beliefsdeath
declaration ofthe point in time at which a health professional , having determined that an individual is dead , formally states this findingdeath operational definition ofbiomedical criteria that describe the state of human deathdeath
determination ofprocesses and tests required to diagnose death in accordance with established criteriadisintegrationloss of intactness , solidness , or cohesion .
such loss can apply to function or to matter ( tissues , etc.)dyingthe process whereby biological / physiological functions cease , thus moving from the state of being alive to that of being deadecmoextracorporeal membrane oxygenation / extracorporeal oxygenation and circulation of blood deployed for life - threatening lung or heart lung failureelectromechanical dissociationa form of mechanical asystole .
a rhythm frequently encountered during cardiac arrest , characterized by organized electrical activity without circulation , traditionally measured by the absence of a palpable pulse or pulsatile arterial blood pressurefixed dilated pupilspupils in mid - position or greater and unreactive to lightfunctionin the context of organs , the primary and fundamental purpose of that organ that can be assessed by observation and examination and is necessary for sustained life .
function should be distinguished from activities , as defined by physiologic properties of cells and groups of cells that can be measured by laboratory means .
examples of brain function such as the capacity for consciousness or ability for unassisted breathing should be distinguished from examples of brain activity such as posterior pituitary antidiuretic hormone release or residual nests of neuronal electrical activityintegrationcombined or coordinated separate elements that provide a harmonious , interrelated whole ; organized or structured so that constituent units function cooperativelyirreversiblepertaining to a situation or condition that will not or can not return or resume . in the context of death determination , there are variable definitions including : 1.loss of function or a condition that can not be restored by anyone under any circumstances at a time now or in the future 2.loss of function or a condition that can not be restored by those present at the time 3.loss of function or a condition that will not resume and will not be restored . also referred to as permanentneuroimagingdiagnostic brain imaging techniques to identify structural brain injury , e.g. ct scan , mrineurological death determinationdiagnosis and confirmation of death based on neurological criteriano effective interventiona therapeutic intervention that is not deployed because it is not effective , not medically indicated under those circumstances , not available or accessiblemechanical ventilationassisted ventilation including bag / mask ventilation , non - invasive support e.g. bipap ( bilevel positive airway pressure ) , conventional mechanical ventilation via artificial airwayrefractory to treatmentdoes not respond to intervention in a clinically meaningful mannerpermanentpertaining to a situation or condition that will not return to its previous state . in the context of death determination , refers to loss of function that will not resume spontaneously and will not be restored through interventionpreconditionspatient - related prerequisites that should be fulfilled prior to application of diagnostic testsrespiratory arrestcessation of breathing . in the context of death discussions
, this may be primary and lead to a subsequent cardiac arrest , or it may be secondary to the loss of brainstem functiontesta procedure performed in diagnosis or detectiontest ancillarya complementary test or an alternative test to one that otherwise , for any reason , can not be conducted or is unreliabletest
clinicala bedside test typically based on physical examination of the patient , but may include the use of a stethoscope and vital signs monitorstest confirmatorya test performed to confirm a previously conducted testtest
laboratorya technical test requiring use of elaborate equipment and medical technologies , e.g. blood testing , diagnostic imagingtest
supplementala test performed in addition to an already conducted testunitythe combination or arrangement of parts into a wholeventricular fibrillationa condition in which there is uncoordinated contraction of the cardiac muscle of the ventricles that causes the cessation of circulation . also referred to as v - fib or vfvital functionnecessary for sustained life participants agreed to the following terminology , in order to improve the clarity of discussions and debate , for use during and subsequent to this forum
forum participants identified several major sequential events in the dying process of patients who have suffered a catastrophic brain injury that will lead to death determined on a neurological basis ( such as , but not limited to , traumatic brain injury , cerebrovascular accidents , and hypoxic - ischaemic encephalopathy after resuscitated cardiac arrest ) ( fig . 1 ) .
this sequence does not apply to patients with anencephaly or with forms of catastrophic brain injury ( such as persistent vegetative states ) where residual clinical brain or brainstem functions are retained ; such patients were not under consideration .
patients entering this sequence are receiving mechanical ventilation ; various other neuroprotective interventions ( such as hyperosmolar therapy , ventricular drainage , decompressive craniectomy ) may have been initiated as well . at n-1 , the patient continues to deteriorate in spite of intervention and the treatment team recognizes that the patient may evolve to brain death . at n-2
however , at this point it is still possible that brain function could return spontaneously or be restored through intervention .
if preconditions are met , confounding factors are absent , and no effective treatment is available or implemented , then by n-3 , the brain has ceased functioning and there is no possibility to resume .
1neurological sequence in the dying process neurological sequence in the dying process preconditions prior to testing brain function at any point in the sequence include an established etiology ; absence of reversible etiologies that would explain the coma ; and the absence of hemodynamic shock associated with inadequate oxygenated circulation to the brain .
confounding conditions that may invalidate testing for cessation of brain function include naturally occurring or therapeutic hypothermia ; the presence of central nervous system ( cns ) depressing drugs that may explain or contribute to coma ; high cervical spine injury ; acquired ( such as severe polyneuropathy ) or therapeutic neuromuscular paralysis ; locked - in syndromes ; and severe acid - base , electrolyte or endocrine abnormalities that may explain or contribute to coma . after agreeing that death is principally established using clinical criteria ( defined as diagnostic testing based on direct , measurable observation or examination of the patient ; see table 1 ) ,
participants came to consensus on the minimum acceptable clinical standards to test for the cessation of brain function ( table 2 ) .
it was also agreed that the validity of a determination of death depends upon the health care professionals performing the clinical determination and those performing and interpreting ancillary laboratory testing possessing the necessary competencies.table 2minimum acceptable clinical standard and additional tests for death after cessation of brain function in adults and childrendescriptionminimum acceptable clinical standardadditional testing ( beyond the minimum clinical standard)n-1catastrophic brain injury : continuing deterioration and progressive loss of brain function despite intervention1 .
evidence for progressing loss of brainstem function1 . neuroimaging that explains the severity of brain injury2 .
absence of brainstem reflexes : pupils mid - position or greater and absent pupillary light reflex ( fixed dilated pupils ) corneal gag / pharyngeal cough / tracheal vestibulo - ocular ( cold caloric ) loss of central drive to breathe nb : performance of apnoea testing should be reserved as the last test of brainstem functionnone : cessation of brain function is a clinical determinationn-3cessation of brain function with no possibility to resume1 .
ancillary laboratory tests e.g. demonstration of brain blood flow or perfusion to be absent refractory intracranial hypertension as measured by icp monitoring transcranial doppler consistent with absent net flow velocity electrodiagnostic testing ( e.g. eeg , absent evoked potentials ) minimum acceptable clinical standard and additional tests for death after cessation of brain function in adults and children forum participants identified several major sequential events in the dying process of patients who suffer a circulatory arrest ( fig . 2 ) .
in situation a , the patient has had a cardiac arrest but no cpr intervention , either because cpr was not medically indicated or the patient ( or surrogate decision maker ) declined it ; this would include terminally ill patients whose end - of - life care involves limiting or withdrawing life - sustaining therapies . at c-1 , the patient s circulation and breathing stop .
after a certain time period ( between 2 and 5 min , based on expert consensus ) , autoresuscitation - the spontaneous , unassisted resumption of circulation - is no longer a possibility under these conditions ( c-2 ) .
since no interventions will be made to attempt to restore circulation , cessation of breathing and circulation is permanent and the patient may be determined to be dead ( c-2 and c-3 occur at the same time ) . in situation b ,
cpr has been used in an attempt to restore circulation and respiration but has been terminated because the patient can not be revived .
once the time interval when autoresuscitation is possible has passed , cessation of breathing and circulation is permanent and the patient may be determined to be dead .
the most common waiting period is 5 min with a range from 2 to 10 min .
the participants came to consensus on the minimum acceptable clinical standards to test for the cessation of circulatory function ( table 3).fig .
2circulatory sequence in the dying processtable 3minimum acceptable clinical standard and additional tests for death after cessation of circulatory function in adults and childrendescriptionminimum acceptable clinical standardadditional testing ( beyond the minimum clinical standard)c-1cessation of circulation and breathing1 .
loss of pulsatile arterial blood pressure by non - invasive measurement6 . coma and fixed dilated pupils7 .
absence of pulse by dopplernb : oxygen saturation pulse oximetry is an unreliable indicator of absence of pulsatile circulationc-2cessation of circulation and breathing with no possibility to resume spontaneously1 . the persistence of c-1 criteria over a period of time as confirmed by continuous observation and intermittent confirmation including repetition of this evaluation at the end of the period .
the time period required is 25 min2 . when breathing and circulation cease following terminated cpr , the time period to reach the point of no possibility to resume spontaneously
use of the same tests for a higher clinical / laboratory standard for c-1 applied after the time interval required to progress from c-1 to c-2 ( 210 min following termination of cpr)c-3cessation of circulation and breathing with no possibility to resume1 .
when cpr will not be provided ( patient fulfils criteria for not providing cpr ) c-3 occurs at the moment of c-22 . following termination of cpr , including a decision not to reinstitute cpr , c-3 and c-2 occur at the same time1 .
nothing in addition to those tests required for c-2 circulatory sequence in the dying process minimum acceptable clinical standard and additional tests for death after cessation of circulatory function in adults and children the inextricable link between circulation and brain function means that the neurological and circulatory sequences integrate at several points in the dying process of patients who have suffered a circulatory arrest ( fig . 3 ) .
once circulation and breathing cease ( c-1 ) , there is a short time period between c1 and n2 ( < 20 s ) during which brain function ceases ( n-2 ) as evidenced by isoelectric eeg [ 1921 ] .
the longer the time period without oxygenated circulation to the brain ( n-2 to n-3 ) the progressively higher likelihood that the cessation of brain function is irreversible , even if oxygenated circulation can be re - established ( either spontaneously or through intervention ) .
the precise time period for the complete cessation of brain function to be non - resuscitable ( through intervention ) is unresolved.fig .
3physiological sequences in the dying process : integrated neurological and circulatory sequence ( applies to patients suffering a circulatory arrest ) physiological sequences in the dying process : integrated neurological and circulatory sequence ( applies to patients suffering a circulatory arrest ) after reviewing the historical taxonomy and definitions of death , and the neurologic and circulatory sequences in the dying process that had been previously discussed and refined , participants came to consensus on an operational definition of human death , that is , a practical and quantifiable description of the state of human death based on measurable and observable biomedical standards .
forum participants agreed on the following operational definition of death : death is the permanent loss of capacity for consciousness and all brainstem functions .
this may result from permanent cessation of circulation or catastrophic brain injury . in the context of death determination ,
permanent refers to loss of function that can not resume spontaneously and will not be restored through intervention .
participants supported avoiding use of anatomically based terms such as brain death or cardiac death that erroneously imply the death of that organ and confuse the general public , health professionals , and policymakers ( organisms die , while organs cease functioning ) .
our operational definition is based on the cessation of function ( the primary and fundamental purpose of an organ that can be assessed by observation and examination and is necessary for sustained life ) rather than activities ( physiologic properties of cells and groups of cells that can be measured by laboratory means ) . while the forum participants understood that the overwhelming majority of deaths in the world occur
after circulation has ceased , and many occur outside health care settings , death determination must focus on the centrality of brain function .
death is a single phenomenon based on permanent cessation of brain function ( loss of capacity for consciousness and brainstem reflexes ) , which occurs along two pathways : ( 1 ) permanent absence of circulation or ( 2 ) subsequent to a catastrophic brain injury , each discerned through a specific set of medical criteria and clinical and laboratory tests two entrances , one end point .
forum participants identified several major sequential events in the dying process of patients who have suffered a catastrophic brain injury that will lead to death determined on a neurological basis ( such as , but not limited to , traumatic brain injury , cerebrovascular accidents , and hypoxic - ischaemic encephalopathy after resuscitated cardiac arrest ) ( fig . 1 ) .
this sequence does not apply to patients with anencephaly or with forms of catastrophic brain injury ( such as persistent vegetative states ) where residual clinical brain or brainstem functions are retained ; such patients were not under consideration .
patients entering this sequence are receiving mechanical ventilation ; various other neuroprotective interventions ( such as hyperosmolar therapy , ventricular drainage , decompressive craniectomy ) may have been initiated as well . at n-1 , the patient continues to deteriorate in spite of intervention and the treatment team recognizes that the patient may evolve to brain death . at n-2
however , at this point it is still possible that brain function could return spontaneously or be restored through intervention .
if preconditions are met , confounding factors are absent , and no effective treatment is available or implemented , then by n-3 , the brain has ceased functioning and there is no possibility to resume .
1neurological sequence in the dying process neurological sequence in the dying process preconditions prior to testing brain function at any point in the sequence include an established etiology ; absence of reversible etiologies that would explain the coma ; and the absence of hemodynamic shock associated with inadequate oxygenated circulation to the brain .
confounding conditions that may invalidate testing for cessation of brain function include naturally occurring or therapeutic hypothermia ; the presence of central nervous system ( cns ) depressing drugs that may explain or contribute to coma ; high cervical spine injury ; acquired ( such as severe polyneuropathy ) or therapeutic neuromuscular paralysis ; locked - in syndromes ; and severe acid - base , electrolyte or endocrine abnormalities that may explain or contribute to coma . after agreeing that death is principally established using clinical criteria ( defined as diagnostic testing based on direct , measurable observation or examination of the patient ; see table 1 ) ,
participants came to consensus on the minimum acceptable clinical standards to test for the cessation of brain function ( table 2 ) .
it was also agreed that the validity of a determination of death depends upon the health care professionals performing the clinical determination and those performing and interpreting ancillary laboratory testing possessing the necessary competencies.table 2minimum acceptable clinical standard and additional tests for death after cessation of brain function in adults and childrendescriptionminimum acceptable clinical standardadditional testing ( beyond the minimum clinical standard)n-1catastrophic brain injury : continuing deterioration and progressive loss of brain function despite intervention1 .
evidence for progressing loss of brainstem function1 . neuroimaging that explains the severity of brain injury2 .
absence of brainstem reflexes : pupils mid - position or greater and absent pupillary light reflex ( fixed dilated pupils ) corneal gag / pharyngeal cough / tracheal vestibulo - ocular ( cold caloric ) loss of central drive to breathe nb : performance of apnoea testing should be reserved as the last test of brainstem functionnone : cessation of brain function is a clinical determinationn-3cessation of brain function with no possibility to resume1 .
ancillary laboratory tests e.g. demonstration of brain blood flow or perfusion to be absent refractory intracranial hypertension as measured by icp monitoring transcranial doppler consistent with absent net flow velocity electrodiagnostic testing ( e.g. eeg , absent evoked potentials ) minimum acceptable clinical standard and additional tests for death after cessation of brain function in adults and children
forum participants identified several major sequential events in the dying process of patients who suffer a circulatory arrest ( fig . 2 ) .
in situation a , the patient has had a cardiac arrest but no cpr intervention , either because cpr was not medically indicated or the patient ( or surrogate decision maker ) declined it ; this would include terminally ill patients whose end - of - life care involves limiting or withdrawing life - sustaining therapies . at c-1 , the patient s circulation and breathing stop .
after a certain time period ( between 2 and 5 min , based on expert consensus ) , autoresuscitation - the spontaneous , unassisted resumption of circulation - is no longer a possibility under these conditions ( c-2 ) .
since no interventions will be made to attempt to restore circulation , cessation of breathing and circulation is permanent and the patient may be determined to be dead ( c-2 and c-3 occur at the same time ) . in situation b ,
cpr has been used in an attempt to restore circulation and respiration but has been terminated because the patient can not be revived . once the time interval when autoresuscitation is possible has passed , cessation of breathing and circulation is permanent and the patient may be determined to be dead .
the most common waiting period is 5 min with a range from 2 to 10 min .
the participants came to consensus on the minimum acceptable clinical standards to test for the cessation of circulatory function ( table 3).fig .
2circulatory sequence in the dying processtable 3minimum acceptable clinical standard and additional tests for death after cessation of circulatory function in adults and childrendescriptionminimum acceptable clinical standardadditional testing ( beyond the minimum clinical standard)c-1cessation of circulation and breathing1 .
absence of pulse by dopplernb : oxygen saturation pulse oximetry is an unreliable indicator of absence of pulsatile circulationc-2cessation of circulation and breathing with no possibility to resume spontaneously1 . the persistence of c-1 criteria over a period of time as confirmed by continuous observation and intermittent confirmation including repetition of this evaluation at the end of the period .
the time period required is 25 min2 . when breathing and circulation cease following terminated cpr , the time period to reach the point of no possibility to resume spontaneously
use of the same tests for a higher clinical / laboratory standard for c-1 applied after the time interval required to progress from c-1 to c-2 ( 210 min following termination of cpr)c-3cessation of circulation and breathing with no possibility to resume1 .
when cpr will not be provided ( patient fulfils criteria for not providing cpr ) c-3 occurs at the moment of c-22 . following termination of cpr , including a decision not to reinstitute cpr , c-3 and c-2 occur at the same time1 .
nothing in addition to those tests required for c-2 circulatory sequence in the dying process minimum acceptable clinical standard and additional tests for death after cessation of circulatory function in adults and children
the inextricable link between circulation and brain function means that the neurological and circulatory sequences integrate at several points in the dying process of patients who have suffered a circulatory arrest ( fig .
once circulation and breathing cease ( c-1 ) , there is a short time period between c1 and n2 ( < 20 s ) during which brain function ceases ( n-2 ) as evidenced by isoelectric eeg [ 1921 ] .
the longer the time period without oxygenated circulation to the brain ( n-2 to n-3 ) the progressively higher likelihood that the cessation of brain function is irreversible , even if oxygenated circulation can be re - established ( either spontaneously or through intervention ) .
the precise time period for the complete cessation of brain function to be non - resuscitable ( through intervention ) is unresolved.fig .
3physiological sequences in the dying process : integrated neurological and circulatory sequence ( applies to patients suffering a circulatory arrest ) physiological sequences in the dying process : integrated neurological and circulatory sequence ( applies to patients suffering a circulatory arrest )
after reviewing the historical taxonomy and definitions of death , and the neurologic and circulatory sequences in the dying process that had been previously discussed and refined , participants came to consensus on an operational definition of human death , that is , a practical and quantifiable description of the state of human death based on measurable and observable biomedical standards .
forum participants agreed on the following operational definition of death : death is the permanent loss of capacity for consciousness and all brainstem functions .
this may result from permanent cessation of circulation or catastrophic brain injury . in the context of death determination ,
permanent refers to loss of function that can not resume spontaneously and will not be restored through intervention .
participants supported avoiding use of anatomically based terms such as brain death or cardiac death that erroneously imply the death of that organ and confuse the general public , health professionals , and policymakers ( organisms die , while organs cease functioning ) .
our operational definition is based on the cessation of function ( the primary and fundamental purpose of an organ that can be assessed by observation and examination and is necessary for sustained life ) rather than activities ( physiologic properties of cells and groups of cells that can be measured by laboratory means ) . while the forum participants understood that the overwhelming majority of deaths in the world occur
after circulation has ceased , and many occur outside health care settings , death determination must focus on the centrality of brain function .
death is a single phenomenon based on permanent cessation of brain function ( loss of capacity for consciousness and brainstem reflexes ) , which occurs along two pathways : ( 1 ) permanent absence of circulation or ( 2 ) subsequent to a catastrophic brain injury , each discerned through a specific set of medical criteria and clinical and laboratory tests two entrances , one end point .
this report describes the initial phase in an international process to develop guidelines and agree upon an operational definition for determining death , based on the cessation of neurological and circulatory functions .
plenary discussions resulted in consensus on seven key areas : death is determined primarily using clinical criteria based on direct observation or examination of the patient , once preconditions have been fulfilled and confounding conditions excluded.the physiological sequences by which circulatory and neurological functions cease , leading up to the moment of death , were set forth to clarify the critical events that occur following a catastrophic injury or illness.clinical tests that constitute the minimum clinical standard for the determination of death were defined for both the neurological and the circulatory sequences . preconditions and confounding conditions that may impede or invalidate death diagnosis were also specified.certain ancillary and/or complementary laboratory tests may be useful in situations in which clinical testing can not be performed or when confounding or special conditions are present ; limitations to using certain of these tests in death determination mean that further research is required to ensure their reliability.a set of precise terminology , which aims to improve clarity in death determination discussions and debate , was agreed upon.an operational definition of human death , based on measurable biomedical standards , was proposed .
participants supported avoiding use of anatomically based terms such as brain death or cardiac death that erroneously imply the death of an organ .
emphasis was placed on the cessation of neurological or circulatory function , and the centrality of brain function for determination of death .
death occurs when there is permanent loss of capacity for consciousness and loss of all brainstem functions .
in the context of death determination , permanent refers to loss of function that can not resume spontaneously and will not be restored through intervention .
this definition is based on the cessation of function ( the primary and fundamental purpose of an organ that can be assessed by observation and examination and is necessary for sustained life ) rather than activities ( physiologic properties of cells and/or groups of cells that can be measured by laboratory means).in order to complete the elaboration of an operational definition of human death that could be used in countries around the world , a broader group of international stakeholders will be needed to develop clinical practice guidelines , based on comprehensive , systematic reviews and grading of existing evidence .
death is determined primarily using clinical criteria based on direct observation or examination of the patient , once preconditions have been fulfilled and confounding conditions excluded
. the physiological sequences by which circulatory and neurological functions cease , leading up to the moment of death , were set forth to clarify the critical events that occur following a catastrophic injury or illness .
clinical tests that constitute the minimum clinical standard for the determination of death were defined for both the neurological and the circulatory sequences .
certain ancillary and/or complementary laboratory tests may be useful in situations in which clinical testing can not be performed or when confounding or special conditions are present ; limitations to using certain of these tests in death determination mean that further research is required to ensure their reliability .
a set of precise terminology , which aims to improve clarity in death determination discussions and debate , was agreed upon .
an operational definition of human death , based on measurable biomedical standards , was proposed .
participants supported avoiding use of anatomically based terms such as brain death or cardiac death that erroneously imply the death of an organ .
emphasis was placed on the cessation of neurological or circulatory function , and the centrality of brain function for determination of death .
death occurs when there is permanent loss of capacity for consciousness and loss of all brainstem functions .
this may result from permanent cessation of circulation or catastrophic brain injury . in the context of death determination ,
permanent refers to loss of function that can not resume spontaneously and will not be restored through intervention .
this definition is based on the cessation of function ( the primary and fundamental purpose of an organ that can be assessed by observation and examination and is necessary for sustained life ) rather than activities ( physiologic properties of cells and/or groups of cells that can be measured by laboratory means ) . in order to complete the elaboration of an operational definition of human death that could be used in countries around the world ,
a broader group of international stakeholders will be needed to develop clinical practice guidelines , based on comprehensive , systematic reviews and grading of existing evidence .
participating organizations : canadian blood services ; australia and new zealand intensive care society ; canadian critical care society ; european society of intensive care medicine ; international federation of emergency medicine ; international pan arab critical care medicine society ; international society for organ donation and procurement ; neurocritical care society ; society of critical care medicine ; united kingdom faculty of intensive care medicine ; university of ottawa loeb chair and research consortium in organ and tissue donation ; world federation of neurology ; world federation of neurosurgical societies ; world federation of pediatric intensive and critical care societies ; world federation of societies of intensive and critical care medicine ; world health organization .
sadek beloucif , france ; dr . mohammed salah ben ammar , tunisia ; dr .
james l. bernat , department of neurology , geisel school of medicine at dartmouth ; dr .
alexander m. capron , professor of law and medicine , university of southern california ; dr .
geoff dobb , australia and new zealand intensive care society ; laura hornby , loeb chair and research consortium in organ and tissue donation ; dr .
edgar jimenez , world federation of societies of intensive and critical care medicine ; dr .
gnter kirste , international society for organ donation and procurement ; dr . niranjan kissoon , world federation of pediatric intensive and critical care societies ; dr . tong kiat kwek , singapore ; dr .
alexander manara , uk faculty of intensive care medicine ; dr . luck noel , world health orgnization ; dr . | introduction and methodsthis report summarizes the results of the first phase in the development of international guidelines for death determination , focusing on the biology of death and the dying process , developed by an invitational forum of international content experts and representatives of a number of professional societies.results and conclusionsprecise terminology was developed in order to improve clarity in death discussion and debate .
critical events in the physiological sequences leading to cessation of neurological and/or circulatory function were constructed .
it was agreed that death determination is primarily clinical and recommendations for preconditions , confounding factors , minimum clinical standards and additional testing were made .
a single operational definition of human death was developed : the permanent loss of capacity for consciousness and all brainstem functions , as a consequence of permanent cessation of circulation or catastrophic brain injury. in order to complete the project , in the next phase , a broader group of international stakeholders will develop clinical practice guidelines , based on comprehensive reviews and grading of the existing evidence . |
single cell suspensions of lymph node cells and thymocytes were prepared from c57bl/6 or cd8 mice thymi .
purified populations of cd4cd8 thymocytes > 96% pure were obtained by anti - cd8 panning of whole thymocyte populations and selecting the adherent cells , as described previously 17 .
cells ( 0.53 10 , as indicated ) were lysed in ice - cold lysis buffer containing either 1% triton x-100 or 60 mm octylglucoside .
after clarification ( 10,000 g for 10 min ) , cell lysates were subjected to immunoprecipitation with the indicated antibodies .
antibodies used for immunoprecipitation were specific for : cd4 ( gk1.5 or rm4.5 ; pharmingen ) , cd8 ( 53 - 67 ; pharmingen ) , or cd8 ( 53 - 5.8 ; pharmingen ) ; and tcr- ( serum 551 ) , lck ( serum 688 ) , or lat ( serum 3023 ) 14 .
antibodies used for immunoblotting were specific for : lat ( serum 3023 ) ; lck ( serum 688 ) ; or phosphotyrosine ( 4g10 ; upstate biotechnology ) . for immunoprecipitations , mabs were directly coupled to cnbr - activated sepharose beads ( amersham pharmacia biotech ) , except when indicated otherwise .
pervanadate treatment ( final concentration of 0.01 mm na3vo4 in the presence of 4.5 mm h2o2 , extemporaneously prepared ) was conducted for 10 min at 37c .
antibodies used for cross - linking were as follows : anti tcr- ( h57 - 597 18 ) , anti - cd4 ( gk 1.5 ; pharmingen ) , and anti - cd8 ( 2.43 19 or 53 - 6.7 [ pharmingen ] ) . thymocytes were cultured for 18 h at 37c , pelleted , and resuspended at 10/ml in ice - cold rpmi containing 1 mm na3vo4 and biotinylated antibodies ( 10 g / ml ) .
after 10 min at 4c , the cells were pelleted and resuspended at 10/ml in rpmi containing 20 g / ml of streptavidin ( southern biotechnology associates ) previously prewarmed at 37c . after a 5-min incubation at 37c
fragments encoding each coreceptor domain , or mutant derivatives thereof , were prepared by restriction enzyme digestion , pcr amplification , or as double stranded oligonucleotides , and ligated to generate the indicated constructs .
the cd8 aam mutation , converting cysteines 227 and 229 to alanines , was introduced using pcr - mediated site - directed mutagenesis 21 .
amino acid sequences ( single letter code ) at the modified junctions were as follows : abb , ldfacd / ittlsl ; aat , licya / rsr ; aba , ldfacd / ittlsl vyfyca / rsrkrvc ; 4aa , gvnqtd / iyiwapl ; 4bb , gvnqtd / ittlsl .
the sequences of all pcr - amplified and oligonucleotide - encoded regions were verified by dideoxy sequencing for the presence of the desired modifications and the absence of additional mutations .
wild - type and mutant cdnas were introduced in pcdna3 ( invitrogen ) for expression in 293 t cells .
total plasmid amount was kept constant among samples by adjusting the amount of empty expression vectors .
expression of the coreceptor derivatives in each sample was verified by cell surface staining with anti - cd4 ( gk 1.5 ) , anti - cd8 ( 53 - 6.7 ) , and anti - cd8 ( 53 - 5.8 ) mabs and cytofluorimetric analysis .
single cell suspensions of lymph node cells and thymocytes were prepared from c57bl/6 or cd8 mice thymi .
purified populations of cd4cd8 thymocytes > 96% pure were obtained by anti - cd8 panning of whole thymocyte populations and selecting the adherent cells , as described previously 17 .
cells ( 0.53 10 , as indicated ) were lysed in ice - cold lysis buffer containing either 1% triton x-100 or 60 mm octylglucoside .
after clarification ( 10,000 g for 10 min ) , cell lysates were subjected to immunoprecipitation with the indicated antibodies .
antibodies used for immunoprecipitation were specific for : cd4 ( gk1.5 or rm4.5 ; pharmingen ) , cd8 ( 53 - 67 ; pharmingen ) , or cd8 ( 53 - 5.8 ; pharmingen ) ; and tcr- ( serum 551 ) , lck ( serum 688 ) , or lat ( serum 3023 ) 14 .
antibodies used for immunoblotting were specific for : lat ( serum 3023 ) ; lck ( serum 688 ) ; or phosphotyrosine ( 4g10 ; upstate biotechnology ) . for immunoprecipitations , mabs were directly coupled to cnbr - activated sepharose beads ( amersham pharmacia biotech ) , except when indicated otherwise .
pervanadate treatment ( final concentration of 0.01 mm na3vo4 in the presence of 4.5 mm h2o2 , extemporaneously prepared ) was conducted for 10 min at 37c .
antibodies used for cross - linking were as follows : anti tcr- ( h57 - 597 18 ) , anti - cd4 ( gk 1.5 ; pharmingen ) , and anti - cd8 ( 2.43 19 or 53 - 6.7 [ pharmingen ] ) .
thymocytes were cultured for 18 h at 37c , pelleted , and resuspended at 10/ml in ice - cold rpmi containing 1 mm na3vo4 and biotinylated antibodies ( 10 g / ml ) .
after 10 min at 4c , the cells were pelleted and resuspended at 10/ml in rpmi containing 20 g / ml of streptavidin ( southern biotechnology associates ) previously prewarmed at 37c .
after a 5-min incubation at 37c , cells were pelleted and lysed in octylglucoside - containing buffer .
fragments encoding each coreceptor domain , or mutant derivatives thereof , were prepared by restriction enzyme digestion , pcr amplification , or as double stranded oligonucleotides , and ligated to generate the indicated constructs .
the cd8 aam mutation , converting cysteines 227 and 229 to alanines , was introduced using pcr - mediated site - directed mutagenesis 21 .
amino acid sequences ( single letter code ) at the modified junctions were as follows : abb , ldfacd / ittlsl ; aat , licya / rsr ; aba , ldfacd / ittlsl vyfyca / rsrkrvc ; 4aa , gvnqtd / iyiwapl ; 4bb , gvnqtd / ittlsl .
the sequences of all pcr - amplified and oligonucleotide - encoded regions were verified by dideoxy sequencing for the presence of the desired modifications and the absence of additional mutations .
wild - type and mutant cdnas were introduced in pcdna3 ( invitrogen ) for expression in 293 t cells .
total plasmid amount was kept constant among samples by adjusting the amount of empty expression vectors .
expression of the coreceptor derivatives in each sample was verified by cell surface staining with anti - cd4 ( gk 1.5 ) , anti - cd8 ( 53 - 6.7 ) , and anti - cd8 ( 53 - 5.8 ) mabs and cytofluorimetric analysis .
lat complexes existed on unstimulated murine t cells and thymocytes , we immunoblotted anti - cd4 and anti - cd8 immunoprecipitates for lat ( fig .
node t cells that express cd4 and cd8 coreceptors on separate cell populations , we found that lat was associated with both cd4 and cd8 ( fig .
1 a , lanes 2 and 3 ) . in immature cd4cd8 thymocytes , which express both cd4 and cd8 on individual cells
, we found that lat associated with cd8 in significantly greater amounts than with cd4 ( fig .
cd4 ) is reciprocal to lck , which binds cd4 > cd8 ( 2425 ; fig .
lat molecules are largely localized to detergent - resistant areas of the plasma membrane referred to as glycolipid - enriched membrane microdomains ( gems [ 22 , 26 ] ) , which also contain subpopulations of both cd4 and cd8 molecules 27 .
thus , it was conceivable that coimmunoprecipitation of lat with cd4/cd8 coreceptors might simply reflect the presence of lat and cd4/cd8 coreceptors in the same gem .
cd8 associations were maintained in anti - cd8 immunoprecipitations of cells solubilized by the detergent octylglucoside , which solubilizes gems ( 2829 ; fig .
1 a , lanes 8 and 9 ) . in fact , detergent solubilization with octylglucoside increased the amount of lat detected in anti - cd8 immunoprecipitates ( fig .
next , we assessed whether lat association with cd8 required both components of cd8 ( and ) . in normal mice ,
cd8 is expressed on thymocytes and thymic - derived t cells as an heterodimer , whereas in cd8 mice , cd8 is expressed as an homodimer 3031 . despite similar amounts of lat in whole cell lysates from normal and cd8 thymocytes ( fig .
1 a , lanes 12 and 13 ) , the amount of cd8-associated lat was markedly greater in cd8 thymocytes than in cd8 thymocytes , although a small amount of lat binding to cd8 homodimers was evident in cd8 thymocytes ( fig .
thus , cd8 contributes to cd8lat association , as it does to cd8lck association 3233 . for lck , cd8 is thought to increase accessibility to a binding site present in the cytosolic tail of cd8 , and this may be the case for lat as well . to determine the molecular basis for lat coreceptor associations , we cotransfected 293 t cells , a human transformed kidney cell line expressing sv40 t antigen , with murine lat ( mlat ) and murine coreceptor molecules ( cd8 and or cd4 ) .
the transfected coreceptor molecules were either wild - type or variants containing altered transmembrane or cytosolic domains ( fig .
surface expression of transfected coreceptor molecules was quantitated by immunofluorescence and flow cytometry and was found to be similar within all experimental groups ( table ) .
lysates from transfected 293 t cells were immunoprecipitated by anti - cd8 mabs ( fig .
both cd8lat and cd4lat complexes were observed in 293 t cells transfected with intact ( aaa , 444 ) coreceptor molecules ( fig . 2 b , lanes 3 and 13 ) , demonstrating that coreceptor
lat complexes could form in nonlymphoid cells and did not require lck , which is not expressed in 293 t cells .
we further documented that lat did not associate with cd4 or cd8 variants expressing either the cytosolic tail of cd8 ( lanes 4 and 12 ) or lacking a cytosolic tail altogether ( lanes 5 and 9 ) .
thus , the cytosolic tails of cd4 and cd8 are the main determinants for lat association , suggesting that a sequence common to both cytosolic tails is involved in lat binding .
sequence homology between the cytosolic domains of cd4 and cd8 is restricted to a short region centered around a cxc cysteine motif that is involved in lck binding 3435 . to examine the possible role of this cytosolic cxc cysteine motif in lat
coreceptor associations , we constructed a cd8 variant ( aam ) in which both cytosolic cysteines c227 and c229 were mutated to alanines ( fig .
we found that substitution with alanine of these two cysteines diminished cd8 associations with murine lat by 70% ( fig .
thus , the cytosolic cxc cysteine motif important for coreceptor associations with lck is also important for coreceptor associations with lat .
because lat molecules contain two molecular species that migrate at either 36 or 38 kd in sds - page 1422 , we wished to determine if coreceptors preferentially associated with one or the other form of lat . to address this issue , we used human lat ( hlat ) because the 36- and 38-kd forms of hlat are easily distinguishable ( fig . 2 c , lysate ) .
we found that cd8 coreceptors were associated almost exclusively with the lower 36-kd form of lat , and that that association was dependent on the cytosolic cysteines in the cd8 tail ( fig .
however , appearance of the lower 36-kd form of hlat is dependent on two juxtamembrane cysteines ( c26 , c29 ) in the cytosolic tail of hlat , which are palmitoylated and responsible for targeting lat molecules to gems 22 .
alanine substitution of both cysteines ( c26/29a ) removes the palmitoylation sites and results in migration of lat as a single 38-kd ( upper ) band ( fig .
interestingly , despite the fact that the c26/29a mutant lat molecule can not be palmitoylated and targeted to gems , we found that the mutant lat can still associate with surface cd8 coreceptor molecules ( fig .
however , we do not yet understand why cd8 normally preferentially associates with the lower band of lat when it is also clearly capable of binding to the upper band .
since the cytosolic cysteine motif in cd8 promotes binding of lat as well as lck , we considered that lat and lck might compete for binding to individual coreceptor molecules . to address this issue , we transiently expressed cd8 , cd8 , and mlat in 293 t cells .
in addition , we also transfected the cells with an excess of either lck - containing vector or empty vector ( fig .
even though lck did not affect the amount of lat present in the cell lysates , lck abrogated the ability of lat to be immunoprecipitated by anti - cd8 ( fig .
lck protein , when present in excess , competes with lat for binding to cd8 .
since lat function in tcr signal transduction depends on its tyrosine phosphorylation , we examined the ability of coreceptor - associated lat molecules to be tyrosine phosphorylated .
treatment of thymocytes with pervanadate to induce activation of intracellular protein tyrosine kinases 36 resulted in tyrosine phosphorylation of cd8-associated lat and recruitment of lat - binding phosphoproteins ( fig .
4 a ) previously identified to include phospholipase c ( plc)-1 and cbl , among others 14 . tyrosine phosphorylation of coreceptor - associated lat molecules was also induced in thymocytes by antibody - mediated coengagement of tcr with either cd4 or cd8 , but was not induced by tcr engagement alone ( fig .
coengagement of tcr with either cd4 or cd8 also resulted in the appearance of phosphoprotein bands indicative of plc-1 and cbl ( fig .
importantly , formation of oligomeric signaling complexes on coreceptor - associated lat molecules occurred with a remarkable degree of specificity , preferentially forming on coreceptor - associated lat molecules that had been coengaged with tcr , compared with lat molecules that had not been coengaged with tcr .
that is , plc-1 and cbl were preferentially recruited to cd4-associated lat molecules by coengagement of tcr with cd4 compared with coengagement of tcr with cd8 ( fig . 4 b , lanes 79 ) ; and , reciprocally , plc-1 and cbl were preferentially recruited to cd8-associated lat molecules by coengagement of tcr with cd8 compared with coengagement of tcr with cd4 ( fig .
the small amount of phosphorylated cbl that was immunoprecipitated by antibodies specific for the nonengaged coreceptor probably reflects the passive and inadvertent capture of some nonengaged coreceptor molecules within the tcr aggregate ( fig
thus , coreceptors can promote lat phosphorylation by tcr - associated zap-70 molecules and the subsequent recruitment of downstream signaling mediators .
this study demonstrates that the lat adaptor molecule associates with cd4 and cd8 surface coreceptors , and that such coreceptor associations are mutually exclusive with lck . indeed ,
the site on the cytosolic tail of cd4 and cd8 coreceptors to which lat binds overlaps the site to which lck binds , resulting in competition between lck and lat for coreceptor binding .
because of their association with coreceptor molecules , lat molecules would be juxtaposed with tcr complexes upon coengagement of mhc peptide complexes . in fact , we found that oligomeric complexes of downstream signaling mediators preferentially formed on those lat molecules that were associated with coreceptors coengaged with the tcr .
thus , this study provides one solution to the problem of how tcr - associated zap-70 molecules can efficiently find their lat substrate .
colocalization of lat with tcr would be a second function performed by cd4 and cd8 coreceptor molecules in tcr signal transduction , with colocalization of lck and tcr being the only previously known function .
in fact , we think that the two coreceptor functions are analogous to one another , in that coengagement of tcr with cd4 or cd8 coreceptor molecules by mhc
peptide complexes serves to physically juxtapose both lck and lat with tcr ( schematized in fig .
, coreceptors promote both the initiation of tcr signaling and the activation of downstream signaling mediators .
it is tempting to consider the implications of our present observations on the distinct but overlapping roles performed by cd4 and cd8 coreceptors in promoting tcr signal transduction in immature cd4cd8 thymocytes during t cell development in the thymus , as cd4 engagement by intrathymic mhc class ii molecules would preferentially promote lck activation 25 , whereas cd8 engagement by intrathymic mhc class i molecules would preferentially promote lat phosphorylation and activation of downstream mediators .
it is conceivable that such coreceptor - induced differences in tcr signal transduction pathways influence lineage choices , but this possibility has not yet been examined . unfortunately , lat knockout mice have not been informative for this issue , as the absence of lat arrests thymocyte development at an early cd4cd8 stage of development that precedes expression of cd4 and cd8 coreceptors as well as the point at which cd4/cd8 lineage determination occurs 37 .
the association of lat with surface cd4 and cd8 coreceptor molecules provides one solution to the problem of how tcr - associated zap-70 molecules manage to contact and phosphorylate lat molecules .
indeed , we found that association of lat with cd4 and cd8 coreceptors was of functional significance for tcr signal transduction , as : ( a ) coreceptor - associated lat molecules were tyrosine phosphorylated upon tcr coreceptor coengagement , and , more importantly , ( b ) the scaffold for oligomerization of downstream signaling mediators preferentially formed on coreceptor - associated lat molecules that were coengaged with tcr .
thus , coreceptor - induced colocalization of lat with tcr promotes downstream tcr signaling events .
lat associations are promoted by the same dicysteine motif in the coreceptor tails that promotes lck binding .
nevertheless , we do not think that lck and lat bind to coreceptor molecules through identical mechanisms .
association of coreceptor molecules with lck involves formation of a zinc - dependent complex between the dicysteine motif in the coreceptor tail and two cysteines in lck 38 , and strictly requires those cysteines to be present in both the coreceptor tail and the lck molecule 3435 .
in contrast , association of coreceptor molecules with lat does not require cysteines in lat , as cd8lat interactions were not abolished by mutation of lat dicysteines to alanines .
in fact , association of coreceptor molecules with lat also does not strictly require the presence of the dicysteine motif in the coreceptor tail , as mutation of these coreceptor cysteines did not abrogate ( but significantly reduced ) association with lat .
thus , we think that the molecular basis for lat coreceptor association is distinct from that of lck coreceptor association despite their overlapping binding sites . that lck and lat actually bind to overlapping sites in the cytosolic tail of cd8 is indicated by cotransfection experiments in which lck was found to disrupt lat binding to cd8 .
such mutual exclusivity of binding may account for the opposite coreceptor binding preferences of lat and lck in cd4cd8 thymocytes , such that lat may be primarily associated with cd8 because lck is primarily associated with cd4 .
however , we favor the possibility that lck and lat have intrinsically different binding preferences for cd4 and cd8 coreceptor molecules such that lck binds cd4 > cd8 and lat binds cd8 > cd4 .
lat molecules are normally palmitoylated and , as a consequence , localized to gems 22 .
coreceptor associations occur even in the absence of lat palmitoylation . indeed , a nonpalmitoylated version of lat ( c26/29a lat ) which does not localize in gems still associated with cd8 .
nevertheless , it should be appreciated that subpopulations of cd4 and cd8 coreceptor molecules are normally present in gems 27 , perhaps as a result of their association with palmitoylated lat molecules .
consistent with such a possibility , we found that detection of cd8lat complexes was significantly increased by solubilization of cells with the detergent octylglucoside , which solubilizes gems far better than the detergent triton x-100 .
surface cd4/cd8 coreceptors are not strictly required for tcr signal transduction , as cd4cd8 t cells , in the absence of either cd4 or cd8 coreceptor molecules , competently transduce tcr signals .
consequently , there must exist coreceptor - independent mechanisms for promoting lat phosphorylation by zap-70 , although they may be less efficient than the physical colocalization resulting from lat association with surface coreceptor molecules .
it is conceivable that trapping of gem - associated lat molecules between aggregated tcr complexes can induce lat phosphorylation . such a possibility is suggested by the finding that tcr stimulation results in the migration of tcr components into gems 3940 .
importantly , however , it is not at all clear that zap-70 actually translocates to gems , a necessary event for zap-70 phosphorylation of gem - associated lat , as conflicting results have been reported 223940 . in any event ,
the inadvertent trapping of lat between tcr aggregates would be expected to be significantly less efficient than the juxtaposition of lat and tcr induced by coengagement of mhc peptide complexes .
it has been suggested that lat phosphorylation by zap-70 may be enhanced by proteins containing src homology 2 ( sh2 ) domains such as 3bp2 , phosphatidylinositol-3 ( pi-3 ) kinase p85 regulatory subunit , or plc-1 .
3bp2 is an sh2-containing adaptor protein that binds to tyrosine - phosphorylated forms of lat and zap-70 41 . because 3bp2 contains only one sh2 domain , it is not evident how it could couple activated zap-70 to lat .
pi-3 kinase p85 subunit is a protein that contains two sh2 domains and has been shown in platelets to couple tyrosine - phosphorylated lat with other tyrosine - phosphorylated molecules such as fcr chain 42 .
similarly , plc- also has two sh2 domains and could conceivably couple zap-70 to lat 43 .
however , because sh2 domain containing linker proteins only bind tyrosine - phosphorylated substrates , they would be expected to promote zap-70 associations with already phosphorylated lat molecules ; they would not be expected to promote lat 's initial phosphorylation by zap-70 .
consequently , the molecular basis for the initial phosphorylation of lat by zap-70 in the absence of cd4 and cd8 coreceptors remains uncertain . in conclusion , this study documents the association of lat with cd4 and cd8 coreceptor molecules in resting t cells and thymocytes , and documents that coengagement of coreceptor molecules with surface tcr results in tyrosine phosphorylation of lat and recruitment of downstream signaling mediators . | linker for activation of t cells ( lat ) is an adaptor protein whose tyrosine phosphorylation is critical for transduction of the t cell receptor ( tcr ) signal .
lat phosphorylation is accomplished by the protein tyrosine kinase zap-70 , but it is not at all clear how lat ( which is not associated with the tcr ) encounters zap-70 ( which is bound to the tcr ) . here we show that lat associates with surface cd4 and cd8 coreceptors and that its association is promoted by the same coreceptor cysteine motif that mediates lck binding .
in fact , lat competes with lck for binding to individual coreceptor molecules but differs from lck in its preferential association with cd8 rather than cd4 in cd4+cd8 + thymocytes .
importantly , as a consequence of lat association with surface coreceptors , coengagement of the tcr with surface coreceptors induces lat phosphorylation and the specific recruitment of downstream signaling mediators to coreceptor - associated lat molecules .
these results point to a new function for cd4 and cd8 coreceptors in tcr signal transduction , namely to promote lat phosphorylation by zap-70 by recruiting lat to major histocompatibility complex
engaged tcr complexes . |
recently , the number of laparoendoscopic single - site surgery ( less ) procedures performed has increased because of advancements in surgical instrumentation , cosmetic considerations and trends toward minimal invasiveness.1 the less procedure was developed due to the unique characteristics of the umbilicus , ie , it is the thinnest portion of the abdominal wall , does not have a muscle layer , and is naturally retracted .
hence , during the less procedure , there may be less tension during wound closure and relatively less scarring in the umbilicus than that in other areas.2 many studies have been performed on less in the gynecologic field , including in gynecologic oncology . the use of less has been reported to be feasible and safe for ectopic pregnancy ; adnexal surgery , including ovarian cyst enucleation ; hysterectomy , including laparoscopic - assisted vaginal hysterectomy and total laparoscopic hysterectomy ; and gynecologic malignancies.311 however , because few gynecologic studies have compared less and conventional laparoscopy , it is unclear whether less is truly better than the conventional surgical approach in reducing objective pain , as reflected by the objective pain scores for both procedures.1215 reproductive - age women diagnosed with a benign adnexal cyst have usually been treated with cyst enucleation to preserve their fertility .
however , in less , it can be technically difficult to achieve the optimal traction - countertraction for enucleation and to control the bleeding from various foci .
hence , we designed the present study to specifically investigate differences in the operative outcomes , postoperative pain , and subsequent convalescence after less and conventional laparoscopic surgery for unilateral cyst enucleation in reproductive - age women with benign adnexal cysts .
our study was a prospective randomized controlled study involving patients who underwent surgical intervention involving less or conventional laparoscopic surgery for cyst enucleation of a unilateral benign adnexal mass .
the study period was december 2009 to september 2010 , and the study involved patients from cheil general hospital and women s healthcare center , seoul , korea . during the study period , 80 patients met the inclusion criteria and agreed to be enrolled in the study .
forty patients were alternately assigned to undergo less , and 40 were assigned to undergo conventional laparoscopic surgery .
the inclusion criteria were as follows : aged between 18 and 45 years ; premenopausal status ; presence of a unilateral adnexal mass , the largest diameter of the unilateral adnexal mass ranging between 4 cm and 10 cm in imaging studies ; and normal cancer antigen ( ca)-125 levels .
the exclusion criteria were as follows : evidence of pelvic or ovarian endometriosis ; previous history of pelvic inflammatory disease ; suspicion of malignancy or pelvic adhesion ; and postmenopausal status . to avoid any possible confounder in the quantification of postoperative pain ,
in addition , a patient who wanted to postoperatively control her pain with patient - controlled analgesia ( pca ) was excluded from the study .
the study protocol was approved by the institutional review board of the cheil general hospital and women s healthcare center .
all the patients were adequately informed of the possible risks and benefits of both less and conventional laparoscopic surgery .
all study participants signed a written consent agreeing to undergo cyst enucleation by less or the conventional method and , if necessary , to allow the use of an additional ancillary port during less .
three experienced surgeons ( yj cho , jm kim and ml kim ) were involved in the protocol , and in the treatment procedure . at the end of the surgery , intraoperative data such as the trocar - introduction time , operation time , intraoperative and postoperative complications , and number of conversions to laparotomy were recorded . during the convalescence period , oral feeding
the visual analog scale ( vas ) , scored from 0 to 10 ( with 0 being no pain and 10 being agonizing pain ) was self - reported at 6 , 24 , and 48 hours after surgery .
follow - up information about the use of analgesics and patient - reported time to recovery end points were obtained from office visits at 4 weeks after the surgery via a modified questionnaire ( appendix a ) that was adapted from those used in a previous study and was administered by the physician members of the research team.16 statistical analysis was performed with spss version 15.0 ( spss , inc , chicago , il ) by using student s t - test , fisher s exact test , and the chi - square test .
the first assistant stood on the right side of the patient to operate the scope . in the case of less , the patients underwent surgery through a single 22.5 cm vertical umbilical incision , performed via the open hasson technique .
we used a homemade wound retractor and a surgical glove as the single - port device .
the distal ring of an alexis retractor ( applied medical , santa rancha margarita , ca ) was used as the intra - abdominal portion of a commercialized wound retractor , and it was rolled up with the wrist portion of a powder - free latex surgical glove ( gammexpf , 70 ; ansell ltd , victoria , australia ) .
the multiple fingers of the glove functioned as a multiport for the laparoscopic instruments and the scope .
we used a rigid 0 or 30 degree , 5 mm or 10 mm laparoscope and standard rigid 5 mm laparoscopic instruments .
the homemade single - port device was inserted into the umbilicus , and two or three 5 mm sheaths and one 10 mm sheath were inserted through the open fingertip portions of the surgical glove and tied with 6 - 0 silk ligatures to prevent gas leakage .
next , carbon dioxide was insufflated to maintain intra - abdominal pressure at 12 mmhg .
once the laparoscope and instruments were in place , the subsequent procedure was similar to the one performed in conventional laparoscopic surgery ( figure 1 ) .
adnexal specimens were extracted with an endopouch ( ethicon endo - surgery , cincinnati , oh ) via the umbilicus . the peritoneum and fascia were approximated and closed layer by layer with 2 - 0 vicryl sutures . for conventional laparoscopy , the 10 mm laparoscope was introduced into the abdominal cavity through the umbilicus , and three 5 mm accessory trocars were suprapubically inserted under direct vision .
all the specimens were extracted using the endopouch via the umbilicus under the guidance of a 5 mm laparoscope .
the first assistant stood on the right side of the patient to operate the scope . in the case of less , the patients underwent surgery through a single 22.5 cm vertical umbilical incision , performed via the open hasson technique .
we used a homemade wound retractor and a surgical glove as the single - port device .
the distal ring of an alexis retractor ( applied medical , santa rancha margarita , ca ) was used as the intra - abdominal portion of a commercialized wound retractor , and it was rolled up with the wrist portion of a powder - free latex surgical glove ( gammexpf , 70 ; ansell ltd , victoria , australia ) .
the multiple fingers of the glove functioned as a multiport for the laparoscopic instruments and the scope .
we used a rigid 0 or 30 degree , 5 mm or 10 mm laparoscope and standard rigid 5 mm laparoscopic instruments .
the homemade single - port device was inserted into the umbilicus , and two or three 5 mm sheaths and one 10 mm sheath were inserted through the open fingertip portions of the surgical glove and tied with 6 - 0 silk ligatures to prevent gas leakage .
next , carbon dioxide was insufflated to maintain intra - abdominal pressure at 12 mmhg .
once the laparoscope and instruments were in place , the subsequent procedure was similar to the one performed in conventional laparoscopic surgery ( figure 1 ) .
adnexal specimens were extracted with an endopouch ( ethicon endo - surgery , cincinnati , oh ) via the umbilicus . the peritoneum and fascia were approximated and closed layer by layer with 2 - 0 vicryl sutures . for conventional laparoscopy
, the 10 mm laparoscope was introduced into the abdominal cavity through the umbilicus , and three 5 mm accessory trocars were suprapubically inserted under direct vision .
all the specimens were extracted using the endopouch via the umbilicus under the guidance of a 5 mm laparoscope .
among the 80 patients selected , 63 were eligible for analysis . in the less group ,
seven patients were excluded because three had deep infiltrating pelvic endometriosis and needed resection of those lesions , one was diagnosed with ovarian endometrioma , one was lost to follow - up , and two needed an additional ancillary port for adhesiolysis and bleeding control . in the conventional laparoscopic surgery group ,
ten patients were excluded because one had severe pelvic adhesion , six had pelvic and/or ovarian endometriosis , and three had incompletely answered the self - reported questionnaires .
these ten excluded patients were successfully treated by the surgical procedure appropriate for their condition .
both the groups were comparable in terms of demographic characteristics and the final pathologic results .
the mean operation times were 42.1 minutes and 36.3 minutes for the less group and conventional laparoscopic surgery group , respectively .
the mean hemoglobin drop was 2.0 g / dl in the less group and was thus higher than that in the conventional laparoscopic surgery group ( p = 0.048 ) .
one patient experienced abdominal distension and pain after oral intake and was diagnosed with paralytic ileus by abdominal radiography .
her initial hemoglobin level was 13.3 g / dl ; at postoperative day 3 , her hemoglobin level was 7.5 g / dl .
the patient had no symptoms or signs of acute bleeding and refused transfusion for anemia correction . on a follow - up visit 2 weeks after the surgery
, the size of the hematoma had decreased to 5 cm and was found to have completely resolved in 6 weeks after surgery .
the data for postoperative pain , including the vas score and the use of additional analgesics after surgery , are shown in table 3 .
there were no statistical differences in the vas scores obtained at 6 , 24 , and 48 hours after surgery .
moreover , the mean number of days of oral analgesic use after discharge was only 1.3 days and 0.9 days in the less and conventional surgery groups , respectively .
most of the patients stated that they would recommend the procedure to a friend or a family member undergoing laparoscopic adnexa - preserving surgery .
ever since laparoscopic surgery was introduced in the gynecologic field , minimally invasive surgery has steadily evolved toward progressively less invasive techniques .
currently , there is an increasing interest in less.17 there are many benefits of less as compared to conventional multiport laparoscopic surgery .
less may result in better cosmesis owing to the relatively hidden umbilical scar , obviation of the use of ancillary ports , and use of the open hasson technique ; therefore , the injury rates by the verres needle and primary trocar are low . additionally , inferior epigastric vessel injury , postoperative wound infection , and hernia formation are potentially reduced .
compared to the conventional 5 mm or 11 mm trocar incisions , a relatively large incision on the umbilicus facilitates easier specimen removal.11 some studies involving retrospective comparsions of hysterectomy have shown that the post - operative pain and narcotic use is less than conventional laparoscopic hysterectomy.13,14 to our knowledge , a prospective study focusing on only cyst enucleation has not yet been published . at present ,
several studies have been published regarding less treatment of adnexal disease and have shown that the results for less are comparable to those for conventional laparoscopic surgery . most of these studies have compared or listed the perioperative outcomes of adnexal surgery procedures such as salpingo - oophorectomy or salpingectomy.3,6,7,1113,1824 however , in our study , we compared the perioperative results including return to work and postoperative satisfaction for less and conventional laparoscopic surgery performed only for cyst enucleation in reproductive - age women with unilateral adnexal cysts .
as reproductive - age women are concerned about the cosmetic problems clinicians have to consider less , but little is known about the safety of adnexa - preserving surgery involving less .
one of the current problems with less is the crowding of instruments at the point of entry into the abdomen .
this problem can be partially offset by using articulating instruments with varying curvatures . in all our procedures
however , if there is a concern about the high cost of flexible laparoscopic instruments , we think that conventional instruments , which are cost effective , could be safely used .
the use of a 30-degree scope or digital endoscope ( olympus endoeye ) would eliminate the cumbersome right - angle light pillar and the camera head , thereby minimizing the possibility of crowding at the entry site .
furthermore , its superior digital image quality owing to the 5 mm telescope would provide excellent visualization.25 less and conventional laparoscopic surgery were comparable in terms of pain .
no statistical differences were observed between vas scores or the requirement for analgesics ( intramuscular or oral ) for both groups .
in addition , given the aforementioned facts , we used a specific convalescence questionnaire , which was previously used , focused on patient - reported time to specific , easily recalled postoperative events .
the patient - reported scar satisfaction scale results were high for both groups , and most of the patients experienced complete recovery and returned to work within 7 days .
although this study was a prospective randomized controlled study , the study population was small .
we think that less is safe and feasible for women who need adnexapreserving surgery , but we could not find major advantages of this method in terms of early convalescence and postoperative pain .
large prospective randomized studies are required to establish the advantages of adnexa - preserving surgery involving less over those of adnexa - preserving surgery involving conventional laparoscopic surgery . however , the postoperative hemoglobin drop was statistically higher in the less group ( 2.0 0.7 g / dl ) than in the conventional surgery group ( 1.7 0.6 g / dl ) .
conversely , none of the patients required blood transfusion , and we think that this difference in postoperative hemoglobin drop can be reduced if we gain more experience in performing less . to our knowledge , this is the first study involving a prospective comparison between less and conventional laparoscopic surgery performed for adnexal preservation . we conclude that performing less is safe and feasible in selected female patients diagnosed with a benign adnexal mass requiring fertility - preserving surgery . | objectiveto compare the operative outcomes , postoperative pain , and subsequent convalescence after laparoendoscopic single - site surgery ( less ) or conventional laparoscopic surgery for adnexal preservation.study designfrom december 2009 to september 2010 , 63 patients underwent less ( n = 33 ) or a conventional laparoscopic surgery ( n = 30 ) for cyst enucleation .
the overall operative outcomes including postoperative pain measurement using the visual analog scale ( vas ) were evaluated ( time points 6 , 24 , and 24 hours ) .
the convalescence data included data obtained from questionnaires on the need for analgesics and on patient - reported time to recovery end points.resultsthe preoperative characteristics did not significantly differ between the two groups .
the postoperative hemoglobin drop was higher in the less group than in the conventional laparoscopic surgery group ( p = 0.048 ) .
postoperative pain at each vas time point , oral analgesic requirement , intramuscular analgesic requirement , and the number of days until return to work were similar in both groups.conclusionin adnexa - preserving surgery performed in reproductive - age women , the operative outcomes , including satisfaction of the patients and convalescence after surgery , are comparable for less and conventional laparoscopy
. less may be a feasible and a promising alternative method for scarless abdominal surgery in the treatment of young women with adnexal cysts |
a significant association between n - ras oncogene activating point mutations and testicular cancer has recently been reported .
we have studied dna samples from the blood and fresh tumor tissues of 17 norwegian testicular cancer patients ( 11 seminomas/6 nonseminomas ) .
point mutations in k - ras-2 and n - ras exons 1 and 2 were studied by denaturing gradient gel electrophoresis ( dgge ) and by oligonucleotide hybridization .
no n - ras mutations were detected in these tumor samples , but two k - ras-2 exon 1 mutations were found in two of the seminoma tumors ( stage i and ii tumors ) using the dgge technique .
the mutations were confirmed by dot blotting and oligonucleotide hybridization and identified as a g-->t and a g-->a point mutation in k - ras-2 codon 12 , leading to a valine and a serine substitution , respectively .
all the white blood cell dnas were negative .
as a positive control for dgge screening , we ran two plasmid constructs carrying human n - ras exon 2 sequences with mutations . to study the role of ras gene activation in testicular cancer , a larger tumor sample population will be investigated.imagesfigure 1 .
afigure 1 .
bfigure 2 . | |
in coronary artery bypass grafting surgery ( cabgs ) , the vessels that must be used are internal mammary arteries ( i m a ) and saphenous veins ( sv ) ; however , svs present a greater risk of occlusion .
nitric oxide ( no ) could be involved in the improvement of sv graft patency rates [ 2 , 3 ] since no plays a pivotal role in vascular homeostasis [ 46 ] .
three isoforms of no synthase ( nos ) exist : neural ( nnos ) , inducible ( inos ) , and endothelial ( enos ) .
although nnos was first described in neurons , it is also present in vascular smooth muscle cell ( vsmc ) .
we have shown in i m a that extraendothelial no is released from nnos present in vsmc [ 9 , 10 ] .
some studies have reported that superoxide ( o2 ) contributes to the development of hypertension [ 12 , 13 ] . in experimental hypertension ,
li et al . have found elevated o2 levels in veins , suggesting that nadph oxidase activity induces hyperreactivity to vasoconstrictors . in agreement with these data ,
we have observed that enhanced nadph oxidase activity drives o2 production in genetically hypertensive rats . in human i
m a , we found that extraendothelial no counter - regulates angiotensin ii ( ang ii ) contractility and that this action is altered in hypertension , probably by an increased oxidative stress and a decreased nnos ability to produce no [ 9 , 10 ] .
in addition , in human umbilical endothelial cells , nadh-/nadph - oxidases play a role in o2 induced by ang ii . in sv ,
alterations of the vascular reactivity , no bioavailability , and oxidative stress could affect sv patency rates .
this notion would be supported by some reports that indicated that functional and structural abnormalities of sv result in graft thrombosis , intimal hyperplasia , and occlusion [ 3 , 18 ] . to the best of our knowledge , despite the fact that hypertension is a major risk factor for coronary disease , the impact of hypertension and the role of oxidative stress in the contractile function of sv grafts have not been studied .
the objective of this work was to evaluate the impact of oxidative stress on ang ii and norepinephrine-(ne- ) mediated vascular reactivity and no bioavailability in sv grafts of hypertensive patients ( ht ) .
discarded sv segments were obtained from cabgs ( centro modelo de cardiologa , tucumn , argentina ) . to establish the impact of hypertension on oxidative stress , no contents and vascular contractility and strict inclusion criteria in relation to the risk factors were taken into account .
patients with diabetes , renal failure , pulmonary disease , peripheral vascular disease in a clinical report , uncontrolled dyslipemia , or active smoking at the time of surgery were not included . to test the influence of hypertension
, patients were divided into two groups , hypertensive ( ht ) and normotensive ( nt ) , according to clinical report .
after surgery , svs were immediately placed in krebs solution ( mm : nacl 118.3 ; kcl 4.7 ; cacl2 2.5 ; mgso4 1.2 ; kh2po4 1.2 ; nahco3 25 ; glucose 11.1 ; na2edta 0.026 ) , maintained at 4c , and transferred to the laboratory .
each vessel was dissected free from connective tissue , and 2 to 5 rings ( 5 mm ) were obtained .
rings were mounted between two stainless - steel wires in organ chambers filled with krebs solution , which had been gassed with 95% o2 and 5% co2 ( ph 7.4 ) .
one wire was anchored , and the other one was connected to an isometric force transducer ( gould uc2 , usa ) and a recorder ( kipp and zonen bd41 , holland ) .
isometric tension was measured under an initial tension of 3 g , which was found to be the optimal tension in which the depolarizing high k solution induced contraction .
all preparations were equilibrated and washed every 15 minutes during 120 minutes . to evaluate endothelial function , a cumulative dose response curve ( cdrc ) to acetylcholine ( ach ) ( 1010 m ) in precontracted ne ( 10 m ) rings was performed .
similar ach responses were observed in all rings from the same patient , which is necessary to point out .
the absence of endothelium - dependent relaxation was observed in rings from 86.4% of the patients ( nt : 12 ; ht : 26 patients ) . according to the objectives of the present work , only these rings ( n = 38 ) were used . in this regard , to rule out the presence of endothelium , immunohistochemical studies were performed ( see below ) . to test endothelium - independent relaxation , sodium nitroprusside ( snp ) 10
snp induced a nearly complete relaxation ( ht : 99 6% and nt : 107 11% of ne pre - contraction ; p : ns ) .
( 1010 m ) or ne cdrc ( 1010 m ) were performed . the maximal contractile response ( rmax ) and negative log of the molar concentration inducing 50% of the rmax ( pec50 )
were measured from the corresponding cdrc . to evaluate the effect of no - mediated inhibition on ang ii and ne reactivity
, rings were pretreated with n-nitro - l - arginine methyl ester ( l - name : 10 m ) for 30 min .
to evaluate the possible role of oxidative stress in vascular reactivity , rings were pretreated with diphenylene iodonium ( dpi : 10 m ) or tempol ( 10 m ) for 30 min . at the end of the experiments ,
nitrites were measured by the griess reaction , which is frequently used to indirectly measure no contents .
we have previously shown that stretching is an ideal condition for in vitro nitrite dosage in isolated human vessels [ 9 , 10 ] . therefore , simultaneous in vitro measurements of vascular reactivity and nitrite release
data with respect to tissue were expressed in pmol / mg of tissue . to corroborate an extraendothelial no presence ,
the possible nos isoforms involved were evaluated by treatment with l - name 10 m , l - name plus l - arginine 10 m , s - methyl - l - thiocitrulline 10 m ( nnos inhibitor ) , or aminoguanidine 10 m ( inos inhibitor ) .
because constitutive nos has been described as a ca / calmodulin - dependent enzyme , some rings were incubated in ca - free media ( krebs solution without cacl2 plus egta 3 mm ) .
the role of oxidative stress on nitrite contents was evaluated with dpi 10 m or tempol 10 m. no release was evaluated in real time , with the membrane - type no - sensitive electrode ( iso - nop ; wpi , usa ) . this electrode records an electrical current that is directly proportional to the no concentration .
the signal was acquired by the apollo 4000 recording system ( wpi , usa ) .
after 120 minutes of equilibration , the electrode was stabilized and the baseline of the current became stable .
the role of oxidative stress in the no content was evaluated with tempol 10 m. at the end of the experiments , the rings were fixed in buffered formol and stained with hematoxylin and eosin ( h&e ) .
immunohistochemistry examination of endothelium was performed with monoclonal cd34 antibodies ( clone : qbend 10 ; bio genex , usa ; dilution : 1/160 ) , anti - enos antibody ( santa cruz , usa , dilution 1/100 ) . in all cases ,
the absence of an endothelial cell layer was observed by h&e , anti - cd-34 , and anti - enos staining .
nnos examination was performed with rabbit anti - nnos antibody ( bd bioscience pharmingen ; dilution : 1 : 100 ) .
the positive control for vsmc presence was performed with anti- actin antibodies ( sigma chemical company , usa ) .
the anti - nnos antibody - stained area was calculated using a light microscope connected to a video camera and an informatics system ( image j 1.43 , md , usa ) calibrated to correspond to an equivalent m value ( 1 m = 18.400 px ) , and the anti - nnos - stained area / total area ratio was calculated . to evaluate the oxidative state protein carbonyl content , conjugated diene ( cd ) ( the primary product of lipid peroxidation ) ,
the reduced / oxidized glutathione ( gsh / gssg ) ratio , and thiobarbituric acid ( tba ) reactive substances ( tbars ) were determined .
rings were homogenized in ice - cold tris - kcl buffer ( 0.15 m , ph 7.4 ) , precipitated by trichloroacetic acid ( tca : 20% ) , and centrifuged .
the pellet was incubated with 2,4-dinitrophenylhydrazine ( dnph : 0.002 g / ml ) , washed with an ethanol - ethylacetate mixture , centrifuged , and redissolved with guanidine hcl / dithiothreitol .
protein carbonyl contents were read spectrophotometrically at 370 nm and expressed as pmol / mg protein .
cd was determined spectrophotometrically at 233 nm using a chloroform - methanol mixture and was correlated with mg of phosphates .
, fractions were homogenized in phosphate buffer plus edta 6.3 mm ( ph 7.4 ) , mixed with tca 10% , and centrifuged .
the supernatant was incubated with nadph 0.3 mm , dtnb 6 mm , and glutathione reductase 0.077 u ( 10 l ) . to calculate gsh , 20 l of the supernatant was incubated for 15 min with dtnb 6 mm ( 0.1 ml ) .
absorbance was read spectrophotometrically at 412 nm and correlated with protein contents , and then the gssg / gsh ratio was calculated .
lipid peroxidation was evaluated in sv fractions homogenized in detergent - free buffer by measurement of malondialdehyde contents .
the results were expressed as mol tbars / mg of protein , as determined by the lowry 's method .
data were expressed as mean standard error ( se ) . to calculate pec50 for each cdrc , the sigmoid equation of the curve fitting program
paired or nonpaired student 's t - test and anova with newman - keuls tests were used when appropriate .
figure 1 shows the vasocontractile response to ang ii ( figure 1(a ) ) and ne ( figure 1(b ) ) on sv rings without endothelium and the effect of dpi in this response .
ang ii ( 1010 m ) produced a dose - dependent contraction in both nt and ht rings ( figure 1(a ) ) ; however , ht showed higher ang ii rmax values ( table 2 ) .
a curve fitness analysis showed no difference in potency ( pec50 ) between nt and ht ( 6.5 0.2 ; n = 7 versus 6.6 0.1 ; n = 6 , resp . ) . in ht , dpi decreased the ang ii dose - dependent contraction ( figure 1(a ) ) and rmax ( table 2 ) , however , this agent did not modify the ang ii response in nt .
tempol did not modify the ang ii rmax in ht or nt ( table 2 ) .
similar to ang ii , ne ( 1010 m ) produced a dose - dependent contraction in both nt and ht ( figure 1(b ) ) .
no difference in the ne pec50 values between nt and ht was found ( 7.6 0.6 ; n = 6 versus 7.1 0.1 ; n = 6 ) . in ht , dpi decreased the ne dose - dependent contraction ( figure 1(a ) ) and rmax ( table 2 ) , however , this agent did not modify the ne response in nt .
tempol did not modify the ne rmax in ht or nt ( table 2 ) . in ht , l - name 10 m
did not modify the ang ii rmax , however , it increased the ang ii reactivity in nt ( table 2 ) .
l - name did not modify the ne rmax in ht , but it did increase the ne reactivity in nt ( table 2 ) .
in contrast to ang ii and ne , the response to kcl 100 mm was similar in ht ( 1115.9 141.7 mg ; n = 22 ) and nt ( 1056.7 193.8 mg ; n = 10 ) .
nitrite contents were present in both nt and ht ( 1066.1 86.3 pmol / mg ; n = 11 versus 487.8 51.6 ; n = 23 ; p < 0.01 ) , despite the endothelial absence . moreover , rubbed maneuvers did not blunt the nitrite contents in either nt ( : 5 13% ,
n = 8 ; p : ns ) or ht ( : 6 12% ,
l - name significantly decreased nitrites in all cases . no differences between nt and ht in the presence of l - name
l - name plus l - arginine did not modify the nitrite contents in ht ( : 11.9 4.2 pmol / mg ; n = 10 ) and nt ( : 15.4 5.2 pmol / mg ; n = 6 ) .
similarly to l - name , s - methyl - l - thiocitrulline ( nnos inhibitor ) inhibited nitrites ( 53.6 6.8% ; n = 8 ; p < 0.01 in ht ) . to test whether nitrite release was mediated by nnos ( a ca / calmodulin dependent enzyme ) ,
ca - free media decreased the nitrite contents in nt ( : 881.6 37.2 pmol / mg ; n = 7 ; p < 0.001 ) and in ht ( : 252.4 43.1 pmol / mg ; n = 6 ; p < 0.01 ) . in any case , aminoguanidine did not modify the nitrite content in nt ( : 54.2 77.4 pmol / mg ;
n = 7 ; p : ns ) and in ht ( : 4.1 53.8 pmol / mg ;
an increase of nitrites was observed with dpi ( 1024.7 45.4 pmol / mg ; n = 7 ; p < 0.001 ) and tempol ( 1071.2 77.8 pmol / mg ;
figure 3 shows typical recorders of direct measurements of no in basal conditions and the effect of tempol in nt ( a ) and ht ( b ) .
nt showed higher no values than ht ( 48.8 4.7 na ; n = 6 versus 17.2 0.8 ; n = 7 ; p < 0.001 ) . in ht ,
tempol 10 m increased no release ( 42.5 2.7 na ; n = 7 ; p < 0.001 ) . in nt ,
tempol did not increase the no contents ( 49.63.6 na ; n = 6 ; p : ns ) . only in ht
, dpi increased no release ( : 24.5 1.8 na ; n = 7 ; p < 0.001 ) . because no was present in sv rings without endothelium and based on previous findings from our laboratory in which we demonstrated the nnos presence in vsmc of i m a rings , immunohistochemistry for nnos was performed in sv . in both nt and ht , the anti - nnos antibody specifically stained vsmc ( figure 4 ) .
the anti - nnos - stained area was higher in nt than ht ( 26 3.2% of the wall area ; n = 7 versus 10 0.6 ; n = 7 , resp . ; p < 0.001 ) .
no immunoreactive product was observed when samples were prepared without the primary antibody . in both nt and ht no staining with the anti - enos antibody
the levels of protein carbonyl groups were higher in ht than in nt ( 21.7 4.4 pmol / mg protein ; n = 5 versus 3.9 0.8 ; n = 4 ; p < 0.05 ) .
determinations of cd showed higher values in ht than in nt ( 0.26 0.012 l / mg phosphates ; n = 6 versus 0.17 0.01 ; n = 6 ; p < 0.05 ) . the levels of gssg were higher in ht than in nt ( 40.3 16.3 mol / mg protein ; n = 7 versus 4.5 1.8 ; n = 7 ; p < 0.05 ) .
in addition , ht had elevated gssg / gsh compared to nt ( 21.9 8.6 ; n = 6 versus 3.1 1.4 ; n = 7 ; p < 0.05 ) .
finally , tbars levels were higher in ht than in nt ( 2.2 0.6 nmol / mg protein ; n = 6 versus 0.1 0.01 ; n = 8 ; p < 0.01 ) .
we studied the contractility response to exogenous agonists in sv rings with endothelial dysfunction and the role of the oxidative stress on this response .
few studies have evaluated the vascular reactivity , or the impact of arterial hypertension , in sv .
lytle et al . have indicated that after 5 years of cabgs , the graft patency of sv exceeded that of i m a .
however , dashwood has indicated that the patency rate is poor , with a high proportion of patients requiring further surgery .
some factors implicated are aging , serum cholesterol , diabetes , harvesting technique , or endothelial integrity .
ca - protein kinase c - pathways are implicated in the reactivity of sv grafts . in the present work
, we found a hyperreactivity to both ang ii and ne in sv from ht patients , which was reverted by antioxidant agents . in this sense , hyperreactivity to vasoconstrictors has previously been observed in doca - salt hypertensive rats .
xu et al . have observed increased reactivity to ne in mesenteric veins , and li et al .
have reported a hyperreactivity to endothelin-1 in the vena cava , which was related to the presence of oxidative stress . in human sv
, hypertension stimulates endothelin-1 release , which not only affects vascular reactivity but also increases oxidative stress . in sv
, sharif et al . have studied the role of oxidative stress , indicating that n - acetylcysteine does not improve endothelial - dependent relaxation and vsmc function .
however , the same authors found that vitamin c improves the endothelial - dependent relaxation . in our vessels ,
antioxidant agents diminish vascular reactivity , suggesting that oxidative stress is implicated in the hyperreactivity of ht .
we also found that the antioxidant agents dpi and tempol increased no release in sv .
this observation is in concordance with previous works from our laboratory on i m a [ 9 , 10 ] , in which we proposed that extraendothelial no release is present through nnos . here , the extraendothelial no presence in sv is supported by the observations that l - name and s - methyl - l - thiocitrulline decrease nitrite levels and that nnos is present in vsmc ( figure 4 ) .
another finding that support that the source of extraendothelial no is through nnos is the lack of staining with anti - enos antibody .
in addition , the nnos presence in sv has previously been demonstrated by webb et al . .
this result is in agreement with previous work in i m a . to the best of our knowledge ,
no comparison of extraendothelial no release in sv between ht and nt has been performed .
dpi increased the no contents in ht , suggesting a higher impact of the oxidative stress in ht patients .
o2 production has been demonstrated to be higher in veins than in arteries [ 36 , 37 ] . in human sv rings ,
have observed that 5-series f2-isoprostanes ( compounds produced by the nonenzymatic peroxidation of arachidonic acid ) do not have vasomotor effects , and antoniades et al .
o2 has been demonstrated not only to be a no - scavenger and a vasoconstrictor but also to produce direct vascular damage . in this sense ,
shi et al . have observed that biological changes in sv grafts are characterized by oxidative stress resulting from higher o2 production and lower superoxide dimutase activity . in our work ,
the increased oxidative damage in hypertension is supported by the observation that the levels of protein carbonyl groups , cd , gssg , gssg / gsh ; and tbars are increased in ht rings .
the present work demonstrates that extraendothelial no counter - regulates contractility in sv used for cabgs .
however , this no action could be altered in hypertensive situations , even if there were no other associated risk factors .
we suggest two mechanisms ( 1 ) increased oxidative stress and ( 2 ) a decreased ability of nnos to produce no .
further studies should be performed to evaluate the implications of these results in sv graft patency rates . | objective . to evaluate the impact of oxidative stress on vascular reactivity to vasoconstrictors and on nitric oxide ( no ) bioavailability in saphenous vein ( sv ) graft with endothelial dysfunction from hypertensive patients ( ht )
. methods .
endothelial function , vascular reactivity , oxidative state , nitrites and no release were studied in isolated sv rings from ht and normotensive patients ( nt ) .
only rings with endothelial dysfunction were used .
results .
ht rings presented a hyperreactivity to vasoconstrictors that was reverted by diphenylene iodonium ( dpi ) .
in nt , no effect of dpi was obtained , but n-nitro - l - arginine methyl ester ( l - name ) increased the contractile response .
no was present in sv rings without endothelial function .
nitrites were higher in nt than in ht ( 1066.1 86.3 pmol / mg ; n = 11 versus 487.8 51.6 ; n = 23 ; p < 0.01 ) and inhibited by nnos inhibitor .
l - arginine reversed this effect .
antioxidant agents increased nitrites and no contents only in ht .
the anti - nnos - stained area by immunohistochemistry was higher in nt than ht .
ht showed an elevation of oxidative state .
conclusions .
extraendothelial no counter - regulates contractility in sv .
however , this action could be altered in hypertensive situations by an increased oxidative stress or a decreased ability of nnos to produce no .
further studies should be performed to evaluate the implication of these results in graft patency rates . |
streptococcus pyogenes is the major causative agent for pharyngitis and tonsillitis , but may also cause , although relatively rarely , invasive infections such as meningitis , sepsis , and necrotizing fasciitis . for such infections , treatment with antimicrobial agents exhibiting superior activity against s. pyogenes must be initiated immediately .
penicillin agents are representative drugs used as the agents of first choice for infections caused by hemolytic streptococci
. however , many reports on the sensitivity of s. pyogenes to antimicrobial agents pertain to strains isolated from patients with pharyngitis or tonsillitis , and there are very few reports focusing on strains isolated from patients with invasive infections .
moreover , most of the drugs evaluated in comparison with the penicillins in these reports are oral antimicrobial agents , and there are no reports on the efficacy of intravenous antimicrobial agents , carbapenems in particular , used for the treatment of invasive infections . in order to select appropriate antimicrobial agents for the treatment of hemolytic streptococcal infections associated with serious symptoms , the minimal inhibitory concentrations ( mics ) and minimal bactericidal concentrations ( mbcs ) of various antimicrobial agents
were examined for s. pyogenes strains isolated from children with invasive infections such as sepsis .
although many reports state that severe infection is associated with specific emm types , no results are available for children in japan .
a total of 12 strains of s. pyogenes isolated from children who were examined for invasive infections at pediatric departments in medical institutions in hokkaido , including our hospital , during the 10-year period between july 2003 and june 2012 were included for this study .
severe infection with s. pyogenes was defined as : ( 1 ) detection of s. pyogenes from originally sterile sites such as blood , cerebrospinal fluid , and puncture fluid and ( 2 ) while s. pyogenes was detected from non - sterile sites such as the skin and pharynx , no other bacteria could be assumed to be the cause and , furthermore , the pathology is not inconsistent with infection due to s. pyogenes .
patients considered to have invasive infections were those with serious symptoms that were almost life - threatening , those requiring a surgical procedure such as drainage and/or debridement , and those requiring hospitalization for 2 weeks or more .
the mic values of seven drugs ( penicillin g , ampicillin , cefotaxime , ceftriaxone , panipenem , meropenem , doripenem ) were measured by the broth microdilution method . for determination of the mbc ,
a culture suspension of 10 l was collected from the plate on which the mic was measured and applied to non - selective culture media in wells containing the drug at a concentration equal to or greater than the mic .
after aerobic cultivation at 35c for 2024 h , the number of colonies were counted , and the concentration that caused a decrease in the volume of the growing bacteria by 99.9 % or more was determined as the mbc .
the laboratory of molecular epidemiology for infectious agents , kitasato institute of life sciences , was requested to perform a determination of the emm types . at the laboratory ,
m protein - coding genes were amplified by pcr and the base sequence at the 5 terminal end of the amplified product was determined ; thereafter , that sequence of 300 bp was transmitted to the cdc reference center and the types were determined by matching with the cdc database .
table 1 shows a list of patients with invasive infections caused by s. pyogenes .
the patients ranged widely in age from 1 day to 15 years , with patients younger than 5 years , including three neonates , accounting for half of all the patients .
the disease was sepsis in four patients , skin and soft tissue infection in three patients , retropharyngeal abscess in two patients , pneumonia plus sepsis in one patient , empyema in one patient , and pyogenic arthritis in one patient .
one patient ( a 15-year - old adolescent girl ) with pneumonia plus sepsis died of shock .
the most prevalent emm type was emm1 ( six strains ; 50.0 % ) , followed by emm12 ( two strains ; 16.7 % ) , while emm3 , emm4 , emm6 , and emm28 were recognized in one strain each.table 1characteristics of children with severe infection due to streptococcus pyogenes
case numberagetype of emm
diagnosisisolatedantibiotic therapyprognosis11 day6pyothoraxpleural fluidabpc 11 dayscure24 days1sepsisbloodabpc 9 dayscure37 days1sepsisbloodabpc 11 dayscure41 year12cellulitisskin swababpc 7 dayscure53 years12sepsisbloodabpc 14 dayscure63 years1sepsisbloodabpc 14 dayscure75 years28arthritissynovial fluidcez 7 dayscure8
5 years4retropharyngeal abscesspharyngeal swabipm / cs 2 dayscure97 years3cellulitisskin exudateipm / cs 7 dayscure1011 years1erysipelaspharyngeal swababpc 7 dayscure1111 years1retropharyngeal abscesspharyngeal swababpc 10 dayscure12
15 years1sepsis + pneumoniabloodpapm / bp one shotdead
abpc ampicillin , cez cefazolin , ipm / cs imipenem / cilastatin sodium , papm / bp panipenem / betamipron
patient was transferred to another hospital for operation
patient was transported in a state of shock in the evening by ambulance and was transferred to an advanced emergency medical care center after receiving basic life support and a single dose of papm / bp , but died the following morning .
the patient had no underlying disease associated with increased susceptibility to infection characteristics of children with severe infection due to streptococcus pyogenes
abpc ampicillin , cez cefazolin , ipm / cs imipenem / cilastatin sodium , papm / bp panipenem / betamipron
patient was transferred to another hospital for operation
patient was transported in a state of shock in the evening by ambulance and was transferred to an advanced emergency medical care center after receiving basic life support and a single dose of papm / bp , but died the following morning .
the patient had no underlying disease associated with increased susceptibility to infection figures 1 and 2 show the distributions of the mics and mbcs for each drug , and table 2 shows the mic90 and mbc90 values .
the drug with the most potent antibacterial activity was doripenem , with mics and mbcs of 0.004 g / ml or less for all the strains .
this drug was followed in antibacterial potency by panipenem , the mic and mbc of which were determined to be 0.004 g / ml or less and 0.008 g / ml for six strains each . for meropenem ,
the drugs showing divergent values of the mic and mbc were : penicillin g for five strains , ampicillin for three strains , cefotaxime for five strains , and ceftriaxone for eight strains .
a comparison of the sensitivities to these four drugs revealed that penicillin g had the highest activity , followed by cefotaxime , ampicillin and ceftriaxone , in that order .
2susceptibilities of s. pyogenes isolates to carbapenemstable 2mic and mbc of s. pyogenes
mic rangemic90
mbc rangembc90
penicillin g0.008 ~ 0.0150.0150.008 ~ 0.0150.015ampicillin0.015 ~ 0.030.030.015 ~ 0.030.03cefotaxime0.015 ~ 0.030.0150.015 ~ 0.030.03ceftriaxone0.015 ~ 0.030.030.03 ~ 0.060.03meropenem0.0080.0080.0080.008panipenem0.004
~ 0.0080.0080.004 ~ 0.0080.008doripenem0.0040.0040.0040.004 susceptibilities of streptococcus pyogenes isolates to penicillins and cephems susceptibilities of s. pyogenes isolates to carbapenems mic and mbc of s. pyogenes
invasive group a streptococcal infections include sepsis , empyema , bone and joint infections , necrotizing fasciitis , and streptococcal toxic shock syndrome . according to a number of reports , the estimated incidence of invasive group a streptococcal infection in children in developed countries is 13 per 100 000 children , and the mortality is 520 % . while among all age groups , the incidence is the highest in elderly people , children younger than 5 years of age are the next most commonly affected age group . among reports on invasive group a streptococcal infections in children ,
mulla from the united states reported the outcomes of 25 children ranging in age from 3 weeks to 17 years with hemolytic streptococcal infections , who were examined between 1996 and 2000 . in regard to the patients age , six children were younger than 1 year old and eight children were 14 years of age , with children younger than 5 years accounting for more than half of the cases . with regard to the presence of invasive disease , 18 patients had bacteremia and three had necrotizing fasciitis .
henriet et al . from france reported that , among 28 children examined between 2000 and 2007 , 15 had joint infections , seven had soft tissue infections , three had pneumonia , and three had toxic shock syndrome , and the median age was 2.9 years .
analyzed the emm types of s. pyogenes isolated from patients with invasive group a streptococcal infections diagnosed at medical institutions in japan between 2003 and 2006 . among the 74 strains obtained ,
many were isolated from patients aged 6080 years , while only 11 strains ( 14.9 % ) were isolated from patients younger than 20 years of age . among the 74 strains , the most prevalent type was emm1 ( 29 strains ; 39.2 % ) , followed by emm49 ( eight strains ; 10.8 % ) and emm12 and emm28 ( five strains each ; 6.7 % ) .
the emm1 type was the most prevalent , which was consistent with the results of the present study . in regard to results reported from europe and the united states , according to one report , out of 247 strains , including those isolated from pediatric patients in spain between 1998 and 2009 , the most prevalent emm type was emm1 ( 60 strains ; 24.3 % ) , followed by emm3 ( 21 strains ; 8.5 % ) and emm4 ( 14 strains ; 5.7 % ) ; these results indicate a high prevalence of the emm1 type .
in germany , evaluation of 586 strains revealed that the most prevalent emm type was emm1 ( 179 strains ; 30.5 % ) , accounting for nearly one - third of all the strains , followed by emm28 ( 107 strains ; 18.3 % ) and emm3 ( 56 strains ; 9.6 % ) .
similarly , emm1 was also the most commonly identified type according to other reports , indicating that invasive infections are strongly associated with the emm1 type [ 7 , 10 ] .
according to recent studies [ 11 , 12 ] , emm12 is the most common among the emm types of s. pyogenes isolated from pharyngitis .
however , the frequencies of emm1 were about half of those in severe infections , showing differences in distributions of emm types between pharyngitis and severe infections .
while there are many reports [ 7 , 9 , 13 , 14 ] examining the associations between emm types and pathogenic factors , such as toxin production and super - antigens , why emm type 1 is commonly detected in severe infections has not been elucidated . among the drugs used in the drug sensitivity testing
, doripenem exhibited the highest activity , with an mic of 0.004 g / ml against all strains .
the mic90 values of panipenem and meropenem were 0.008 g / ml , indicating the superior antibacterial activity of these drugs as compared to that of other agents ( mic90 : 0.0150.03 g / ml ) .
the mic and mbc of carbapenem agents were consistent for all strains , whereas those of the penicillin agents and cephem agents were not quite so consistent .
no reports have focused on s. pyogenes exhibiting resistance to penicillin agents , although there are occasional reports of tolerance [ 1517 ] ; it is considered that , due to the divergence between mic and mbc , the strain requires more time to be killed as compared to other strains , or there is some kind of mechanism against killing in the bacteria .
this phenomenon is considered to be one of the reasons for the recurrence of pharyngitis and tonsillitis caused by s. pyogenes , even after the administration of effective antimicrobial agents [ 18 , 19 ] .
moreover , it is also considered to be one reason why not a few patients treated with penicillin alone have poor outcomes in association with the inoculum effect , a decrease in drug susceptibility when the bacterial load is large in patients with severe infections .
while there are no unified criteria for tolerance , it is said that the mic / mbc ratio is equal to or greater than 16 or 32 [ 19 , 21 ] . in this study , the maximum mic / mbc ratio was twofold , which did not fulfill the definition of tolerance ; however , this phenomenon seems to require attention .
penicillin agents are considered as the drugs of first choice for invasive streptococcal infections , and concurrent use of clindamycin is recommended , with the expectation of its special antimicrobial activities , such as tissue permeability , inhibition of toxin production , and promotion of phagocytic activity .
carbapenem agents have lower mbc than penicillin agents and , moreover , penicillin is highly effective in the log phase while its effect decreases in a steady state .
however , there are results , albeit in vitro , raising the possibility of high efficacy in the early stationary phase .
it is thus considered to be appropriate for the treatment of severe s. pyogenes infection .
as there is no other study comparing the sensitivities of s. pyogenes to penicillin agents , carbapenem agents , and cephem agents , this awaits further basic and clinical investigations . | minimal inhibitory concentrations ( mics ) and minimal bactericidal concentrations ( mbcs ) of various antimicrobial agents were measured against 12 strains of streptococcus pyogenes isolated from children with invasive infections between 2003 and 2012 .
the patients ranged in age from 1 day to 15 years , with patients younger than 5 years , including three neonates , accounting for a half of the patients .
the disease was sepsis in four patients , skin and soft tissue infection in three patients , retropharyngeal abscess in two patients , pneumonia plus sepsis in one patient , empyema in one patient , and pyogenic arthritis in one patient .
one patient with sepsis died , while cure without sequelae was achieved in all the remaining patients .
when classified by type , emm1 ( six strains ) was the most prevalent type , followed by emm12 ( two strains ) .
the mic90/mbc90 values were 0.015/0.015 g / ml for penicillin g , 0.03/0.03 g / ml for ampicillin , 0.015/0.03 g / ml for cefotaxime , 0.03/0.03 g / ml for ceftriaxone , 0.008/0.008 g / ml for panipenem , 0.008/0.008 g / ml for meropenem , and 0.004/0.004 g / ml for doripenem , indicating the superior antimicrobial activities of carbapenem . |
clear cell adenocarcinoma of the urethra ( ccau ) is rare in both sexes but has been more commonly described in the female urethra . even in the female ccau is very rare .
information regarding ccau has been obtained from single case reports and small case series [ 1 , 2 ] .
the ensuing paper contains a review of the literature which has been divided into ( a ) overview which has broadly summarized ccau and ( b ) discussion and narrations from reported cases and case series of ccau .
various internet search databases were used to obtain literature on ccau using the following key words : clear cell adenocarcinoma of urethra ; renal cell carcinoma of urethra ; primary ; metastatic ; secondary .
twenty - six references were identified which were suitable for the review of the literature .
epidemiology . clear cell adenocarcinoma of the urethra most commonly occurs in women with a mean age of 58 years ( range 35 to 80 years ) .
ccacu tends to have similar clinical manifestation to the other urethral carcinomas [ 1 , 3 ] , haematuria
patients tend to present with haematuria and when they are first seen their urine specimens are sent for cytological examination in addition to the urine specimens being sent for microscopy and culture .
the cytological features of ccau include : ( i ) enlarged tumour cells which contain abundant clear cytoplasm with conspicuous vacuoles ; ( ii ) hobnail patterned cells ; ( iii ) and hyaline globules .
urethrocystoscopy
urethrocystoscopy enables the surgeon to visualise the urethral tumour and provides a means by which biopsies are taken for histological examination to establish the diagnosis of ccacu.examination under anaesthesia at urethrocystoscopy enables the surgeon to bimanually examine and assess the urethral tumour for fixity of the tumour and to determine how easy or difficult it might be to completely excise the lesion at operation .
urethrocystoscopy enables the surgeon to visualise the urethral tumour and provides a means by which biopsies are taken for histological examination to establish the diagnosis of ccacu .
examination under anaesthesia at urethrocystoscopy enables the surgeon to bimanually examine and assess the urethral tumour for fixity of the tumour and to determine how easy or difficult it might be to completely excise the lesion at operation .
the following radiological investigations can be used to localize a mass in the urethra as well as show whether there is any urinary bladder wall thickness , pelvic lymph node involvement , or distant metastases .
mri scan may reveal urethral diverticulum containing a nodular enhancing malignancy or a heterogeneous mass in the urethra .
ct scan may reveal urethral diverticulum containing a heterogeneous mass [ 5 , 6 ] .
it could be imagined that if there is no urethral diverticulum the ct scan may demonstrate urethral mass only .
isotope bone scan can also reveal whether or not there is bony metastasis [ 5 , 6 ] .
. for microscopic features , see figures 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , and 13 which show various microscopic and immunohistochemical staining characteristics of the tumour.the microscopic characteristics of ccau are similar to clear cell adenocarcinoma of female genital tract.ccaus tend to exhibit the classic triad of ( a ) tubulocystic , ( b ) papillary , and ( c ) diffuse patterns [ 2 , 3 ] which characterize the tumour.microscopic examination of ccaus shows hobnail and flattened cells with abundant clear cytoplasm , moderate to marked nuclear pleomorphism , and frequent mitotic figures are seen [ 2 , 3].for immunohistochemical staining characteristics , see figures 1to 13 which show various microscopic and immunohistochemical staining characteristics of the tumour .
the microscopic characteristics of ccau are similar to clear cell adenocarcinoma of female genital tract .
ccaus tend to exhibit the classic triad of ( a ) tubulocystic , ( b ) papillary , and ( c ) diffuse patterns [ 2 , 3 ] which characterize the tumour .
microscopic examination of ccaus shows hobnail and flattened cells with abundant clear cytoplasm , moderate to marked nuclear pleomorphism , and frequent mitotic figures are seen [ 2 , 3 ] .
ccaus exhibit positive immunohistochemical staining forpax2 , pax8 , cytokeratin 7 [ 5 , 9],p16 , p53 , ca125 , cam5.2 , ae1/ae3 .
cytokeratin 7 [ 5 , 9 ] , ccaus exhibit negative immunohistochemical staining forpsa [ 2 , 3],pap , thrombomodulin , oestrogen , progesterone , cytokeratin 20 , p63 , cd10 , ceap , wti , afp , s100 .
metastatic involvement from the female genital tract should be excluded .nephrogenic adenoma in which no marked nuclear pleomorphism can be seen on microscopy , in which no mitotic figures can be seen , and in which there exists no infiltrative or solid growth pattern .
nephrogenic adenoma in which no marked nuclear pleomorphism can be seen on microscopy , in which no mitotic figures can be seen , and in which there exists no infiltrative or solid growth pattern .
a small urethral tumour of ccau may be effectively treated by urethrectomy alone.urethrectomy in conjunction with cystoprostatectomy or urethrectomy in combination with anterior exenteration would be considered good options of treatment for ccau.anterior exenteration and pelvic lymph node dissection were the treatment used in most reported cases of ccau ( as in [ 5 , 6 , 9]).consolidation radiotherapy to the pelvis had been given in a case of pelvic lymph node involvement.there is lack of knowledge of the effectiveness of chemotherapy in the treatment of ccau and there has not been any documentation to suggest that chemotherapy is effective in the treatment of ccau ( see in which chemotherapy was given ) .
urethrectomy in conjunction with cystoprostatectomy or urethrectomy in combination with anterior exenteration would be considered good options of treatment for ccau .
anterior exenteration and pelvic lymph node dissection were the treatment used in most reported cases of ccau ( as in [ 5 , 6 , 9 ] ) .
consolidation radiotherapy to the pelvis had been given in a case of pelvic lymph node involvement .
there is lack of knowledge of the effectiveness of chemotherapy in the treatment of ccau and there has not been any documentation to suggest that chemotherapy is effective in the treatment of ccau ( see in which chemotherapy was given ) .
few cases of ccau have been reported and it would appear so far that ccau is an aggressive tumour with low 5-year survival rates .
there is therefore the need to explore for treatment options that would improve the prognosis .
they reported that out of the 19 patients with ccau 18 were from women and 1 from a man .
the ages of the patients ranged from 35 years to 80 years and the average age was 58 years .
they stated that the clinical manifestation and macroscopic findings were similar to those of urethral carcinomas , except for the fact that 12 tumours which were all found in women arose within urethral diverticulum .
they also reported that microscopic examination revealed that the neoplasms exhibited the classic triad of ( a ) tubulocystic , ( b ) papillary , and ( c ) diffuse patterns which characterized the tumour .
furthermore , they reported the following.the tumours exhibited the typical cytological characteristics of clear cell adenocarcinoma which included hobnail cells , flattened cells , and cells with abundant clear cytoplasm.nuclear pleomorphism was typically at least moderate and was marked in almost half of the specimens.they easily found mitotic figures in almost all the specimens.the aforementioned cytological characteristics should be helpful in the distinction of ccau from benign nephrogenic adenoma , even though one of their patients was initially misdiagnosed as having nephrogenic adenoma.they had performed immunohistochemical staining of the tumours for prostate - specific antigen ( psa ) and prostatic acid phosphatase ( pap ) on 13 tumours and all were negative.follow-up was available for 13 patients .
six of the patients did not have any evidence of recurrence up to 10 years postoperatively .
three more patients developed recurrence but they were alive up to 6.5 years following their presentation.scantling et al . reported a 47-year - old woman with a history of chronic recurrent urinary tract infection who was diagnosed with urethral carcinoma during investigation for visible haematuria , hesitancy , straining , and
she had cystoscopy which revealed a papillary urethral mass emanating from urethral diverticulum , and histological examination of the biopsy specimen revealed clear cell adenocarcinoma .
she had a contrast enhanced magnetic resonance imaging ( mri ) scan and computed tomography ( ct ) scan of the pelvis which confirmed the urethral diverticulum containing a nodular enhancing malignancy and enlarged pelvic side wall bilaterally .
she underwent robotic assisted radical anterior exenteration with indiana pouch creation ( radical cystectomy , hysterectomy , urethrectomy , and neobladder construction ) .
the pathology of specimens revealed a 3 cm 1.6 cm 1.2 cm grade 2 ( moderately differentiated ) clear cell urethral adenocarcinoma within urethral diverticulum invading the anterior vagina with negative margins , distal bilateral ureters with negative margins , distal bilateral ureters with negative margins , 21 negative lymph nodes , and a negative hysterectomy specimen .
she was well at her one - year follow - up without any recurrent disease .
the tumours exhibited the typical cytological characteristics of clear cell adenocarcinoma which included hobnail cells , flattened cells , and cells with abundant clear cytoplasm .
nuclear pleomorphism was typically at least moderate and was marked in almost half of the specimens .
the aforementioned cytological characteristics should be helpful in the distinction of ccau from benign nephrogenic adenoma , even though one of their patients was initially misdiagnosed as having nephrogenic adenoma .
they had performed immunohistochemical staining of the tumours for prostate - specific antigen ( psa ) and prostatic acid phosphatase ( pap ) on 13 tumours and all were negative .
six of the patients did not have any evidence of recurrence up to 10 years postoperatively .
three more patients developed recurrence but they were alive up to 6.5 years following their presentation . sheahan and vega vega reported a 54-year - old woman who presented with haematuria and urethral mass on ultrasound scan .
she had biopsies of the mass and histological examination of the specimens revealed clear cell adenocarcinoma with clearing of the cytoplasm , moderate nuclear pleomorphism .
immunohistochemical staining revealed that the tumour cells were positive for cytokeratin 7 and p16 , p53 , ca125 , cam5.2 , and ae1/ae3 .
immunohistochemical staining also showed that the tumour cells were negative for thrombomodulin , oestrogen , progesterone , cytokeratin 20 , p63 , cd10 , ceap , wti , afp , and s100 .
histological examination of the specimen revealed surgically clear margins but 3 out of 6 positive lymph nodes .
she underwent 50.4 gy consolidate radiotherapy due to lack of known benefit of chemotherapy in ccau .
she subsequently had pet scans which showed progression of lymphatic disease but she was alive , one year after her diagnosis .
reported a 42-year - old woman who presented with bloody discharge from her urethra and lower back pain .
papanicolau - stained specimens of her urine showed a small number of papillary or spherical clusters of atypical cells with many benign urothelial cells and squamous cells in the background . a few neutrophils and lymphocytes
the nuclei of the atypical cells showed an increase in the chromatin content with fine granular pattern and irregular contours ; the nucleoli were prominent .
most of the atypical cells had a moderate amount of cytoplasm that was lightly stained green ; however , some atypical cells showed clear , abundant cytoplasm that formed spherical clusters resembling mirror balls .
the cytological findings were reported to be suggestive of a malignant tumour of the urinary tract system and favoured adenocarcinoma .
she had computed tomography ( ct ) and magnetic resonance imaging scans which showed a tumour in the entire urethra . the rest of the intra - abdominal and pelvic organs and lymph nodes were normal .
she had cystourethroscopy which showed two diverticula in the urethral wall in which whitish papillary and villous lesions were found .
the tumours were biopsied and resected transurethrally and histological examination of the specimens was suggestive of clear cell adenocarcinoma ( cca ) but was indeterminate for malignancy .
the final pathological diagnosis of the resected tumour was clear cell adenocarcinoma ( cca ) stage iii pt3n0m0 .
histological examination of the biopsy and transurethral resected specimen showed that the tumour was comprised of papillotubular lesions .
the epithelial cells which covered the tumour were cuboidal and single layered , and some of these showed a
most of the cells had eosinophilic cytoplasm , with the exception of a few which had clear cytoplasm .
the cells that had a clear cytoplasm were positive for periodic acid - schiff reaction .
the epithelial cells exhibited relatively mild cytological atypia and they did not invade the stroma .
immunohistochemical staining showed that the tumour cells were positive forcytokeratin ( ck7),ema , carbohydrate antigen ( ca ) 125.immunohistochemical staining also showed that the tumour cells were focally positive for cd15 ( see figure 5(b ) ) .
immunohistochemical staining showed that the tumour cells were negative forck5/6,ck20,carcinoembryonic antigen , thrombomodulin , uroplakin , prostate - specific antigen , calretinin , oestrogen receptor , progesterone receptor.the aforementioned histological findings were reported to have suggested a diagnosis of ccau , even though definitive diagnosis of malignancy could not be elicited in view of the absence of stromal invasion in both the biopsy and the transurethrally resected specimens .
nevertheless , in the surgically resected specimens , clear atypical cells with papillotubular structure were seen to have invaded all the layers of the urethra and vaginal muscular layer . based upon these findings the final diagnosis was cca .
the authors further reported that the ki-67 labeling index of the tumour cells was about 20% ( see figure 5(c ) ) and about 5% of the tumour cells exhibited strong p53 positivity in the nucleus .
most of the data on ccau have been obtained from case reports and case series [ 1 , 3 , 10 ] .
ccau mostly affects females and up to half of the cases develop in urethral diverticulum [ 3 , 11 , 12 ] .
reported a 44-year - old man with a history of two previous urinary retentions who presented with obstructive urinary symptoms .
he had a rectal examination which revealed an indurated prostate . his serum prostatic - specific antigen ( psa ) was 0.15 ng / ml ( normal ) .
he had cystourethroscopy which revealed a friable solid tumour in the entire urethra and bladder neck .
the tumour was resected transurethrally and histological examination revealed a tumour which was composed of tubular structures lined with cells comprising hyperchromatic nuclei and clear cytoplasm .
tubular and papillary structures were lined with hobnail cells in some areas of the tumour .
he had a ct scan of abdomen and pelvis which showed multiple bilateral internal iliac lymph nodes .
he underwent radical cystoprostatectomy , bilateral pelvic and inguinal lymph node dissection , urethrectomy , and construction of ileal conduit .
histological examination of the cystoprostatectomy specimen confirmed features of clear cell adenocarcinoma of the bladder neck and the tumour in the entire urethra which was similar to the findings of the transurethrally resected specimens .
the perivesical fat , the prostate gland , and seminal vesicles were infiltrated by the tumour .
he received three cycles of methotrexate - vinblastine - epirubicin - cisplatin ( mvec ) chemotherapy but he died of progressive disease 10 months after his cystoprostatectomy .
konnak in 1973 reported the first case of ccau ; since then sporadic cases of ccau have been reported .
konnak used the terminology mesonephric carcinoma for ccau and postulated that the tumour perhaps emanates from the mesonephric duct or intermediate mesodermal vestiges . on the contrary , kawano and associates
some authors [ 15 , 16 ] suggested that there is a clear association between clear cell adenocarcinoma and diverticula of the urethra and that ccau is the most common malignancy arising from diverticula of the urethra .
it has been stated whilst only 10% of carcinoma of the urethra is clear cell adenocarcinoma , one - third of such carcinomas originate in a diverticulum .
pollen and dreilinger in 1984 iterated their support for the homogeneity between the female paraurethral duct and the prostate gland in men based upon the finding of positive immunohistochemical staining for prostate - specific antigen ( psa ) and prostatic specific acid phosphatase ( pap ) .
trabelsi et al . reported a 56-year - old woman who presented with visible haematuria . on examination bleeding
she underwent cystoscopy which revealed a tumour protruding from the posterior urethral wall at the neck of the urinary bladder .
she underwent transurethral biopsy of the tumour and histological examination of the specimen showed an invasive poorly differentiated carcinoma of the urethra .
she underwent total urethrocystectomy including her anterior vaginal wall and pelvic lymph node dissection and ileal conduit construction .
microscopic examination of the specimen showed a tumour which was composed of nests and papillary structures which were lined with cells that had clear cytoplasm with hobnail cells in some areas of the tumour ; the cells exhibited cytological atypia and high mitotic rate ; the tumour cells invaded all the urethral layers but did not involve the urinary bladder .
she did not receive any adjuvant therapy and she was free of disease 3 months after her operation .
. stated the following.pollen and dreilinger were of the opinion that caus arose from the female paraurethral duct and , in their case , the tumour cells were negative for psa.zaviai and associates had reported a neoplasm with similar histological appearance and immunohistochemical features as adenocarcinoma of skene 's paraurethral gland and ducts.their aforementioned findings would support the postulate of abascal junquera and associates that the female clear cell adenocarcinoma arises from the paraurethral duct .
nevertheless , it would seem that female urethral adenocarcinoma has more than one tissue of origin with minority arising from the skene 's glands as suggested by dodson and associates .
morphologically , ccau of the urethra must be differentiated from nephrogenic adenoma of the urethra especially on biopsy .
the predominance of clear cells , severe cytological atypia , high mitotic rate , and necrosis favored the diagnosis of ccau.some authors [ 10 , 20 ] stated that , in view of the rarity ccau , the optimal treatment is not known .
it would appear to be based upon the localization of the primary tumour and the presence of metastasis .
ebisuno and associates stated that radical cystourethrectomy with or without irradiation was performed in most cases .
some authors [ 20 , 21 ] also stated that the response to chemotherapy is also not clear.han et al . reported a 54-year - old woman who presented with painless visible haematuria .
she had vaginal ultrasound scan which revealed a sausage - like elongated mass in the urethra .
the tumour cells were enlarged and had abundant clear or granular cytoplasm with cytoplasmic vacuoles . the nucleus was granular and contained vesicular chromatin with prominent nucleoli .
they stated the following.the histological findings were compatible with clear cell adenocarcinoma.nevertheless , cytologically , it would be necessary to make a differential diagnosis from the other adenocarcinoma or high - grade urothelial carcinoma.oliva and young indicated that ccau accounts for about 1% of male urethral carcinomas and about 15% of female urethral carcinomas.fridman in 2011 reported an 82-year - old woman who underwent transurethral resection of a space occupying lesion which was diagnosed in her urethra and bladder neck ( see figures 1to 11 for various microscopic and immunohistochemical characteristics of the tumour ) .
fridman stated that ccaus are high - grade tumours which are common in women and macroscopically , they are papillary .
fridman summarized the features of ccau as follows.ccaus have various architectural patterns including tubules , cysts , papillae , and diffuse tumour.most tumours have prominent clear cytoplasm due to glycogen and hobnailing.the tumour cells have prominent pleomorphism and marked cytotic activity.there is often muscular invasion and necrosis .the tumour cells are immunoreactive for ck7 , ck903 , ki-67 , and p53 and usually negative for ck20 .
in contrast to majority of urothelial carcinomas , they are also immunoreactive for p504s and negative for p63 .fridman stated that the differential diagnosis of ccau includes nephrogenic adenoma , a metaplastic process which often occurs in young adults with a history of genitourinary trauma , surgery , or stones .
fridman further stated that , in nephrogenic adenoma , reactive changes may exist in the tumour cells as well as mitotic activity , but in such cases there is no marked pleomorphism or invasion .
there are usually no clear cells in nephrogenic adenoma , and if present they are focal , and ki-67 and p53 are usually negative or have minimal staining .
pollen and dreilinger were of the opinion that caus arose from the female paraurethral duct and , in their case , the tumour cells were negative for psa .
zaviai and associates had reported a neoplasm with similar histological appearance and immunohistochemical features as adenocarcinoma of skene 's paraurethral gland and ducts .
their aforementioned findings would support the postulate of abascal junquera and associates that the female clear cell adenocarcinoma arises from the paraurethral duct .
nevertheless , it would seem that female urethral adenocarcinoma has more than one tissue of origin with minority arising from the skene 's glands as suggested by dodson and associates .
morphologically , ccau of the urethra must be differentiated from nephrogenic adenoma of the urethra especially on biopsy .
the predominance of clear cells , severe cytological atypia , high mitotic rate , and necrosis favored the diagnosis of ccau .
some authors [ 10 , 20 ] stated that , in view of the rarity ccau , the optimal treatment is not known .
it would appear to be based upon the localization of the primary tumour and the presence of metastasis .
ebisuno and associates stated that radical cystourethrectomy with or without irradiation was performed in most cases .
some authors [ 20 , 21 ] also stated that the response to chemotherapy is also not clear .
nevertheless , cytologically , it would be necessary to make a differential diagnosis from the other adenocarcinoma or high - grade urothelial carcinoma . oliva and
young indicated that ccau accounts for about 1% of male urethral carcinomas and about 15% of female urethral carcinomas .
ccaus have various architectural patterns including tubules , cysts , papillae , and diffuse tumour .
the tumour cells are immunoreactive for ck7 , ck903 , ki-67 , and p53 and usually negative for ck20 . in contrast to majority of urothelial carcinomas , they are also immunoreactive for p504s and negative for p63 .
fridman additionally stated the following.herawi et al . in 2010 described a clear cell adenocarcinoma which mimicked nephrogenic adenoma due to less prominent nuclear pleomorphism , less prominent nucleoli , and fewer clear cells .
nevertheless , this variant of adenocarcinoma did exhibit extensive muscular invasion and focal hyperchromatic and pleomorphic tumour cells that would not be in nephrogenic adenoma.miller and karnes in 2008 stated that clear cell adenocarcinoma is an aggressive tumour with low rates of 5-year survival . in their case ,
she was well during the subsequent three and half years until 5 months prior to the presentation of the case , when she underwent excision of pelvic lymph node histology of which confirmed clear cell carcinoma similar to the primary tumour . at the time of presentation of the case
the patient was alive without any further recurrence.young and scully described the clinical and pathological characteristics of three previously unreported and 16 previously reported examples of clear cell adenocarcinoma of the urinary bladder and urethra .
they stated that six of the tumours arose in the urinary bladder and 13 in the urethra .
sixteen of the patients were female , and the ages ranged between 35 years and 78 years .
young and scully reported that microscopic examination of the tumours showed various patterns which included tubular glands , cysts , papillae , and diffuse areas .
they had identified cells with abundant glycogen - rich clear cytoplasm and hobnail cells in majority of the tumours .
they stated the following.a young age or a history of genitourinary trauma , operation , or calculus may constitute a clue to the latter diagnosis ; microscopic characteristics such as sheets of clear cells , significant pleomorphism , or mitotic activity would be in favour of the diagnosis of clear cell adenocarcinoma.the follow - up of majority of the patients , most of who underwent a radical operation , was short , but five tumours were known to have metastasized.sun et al
. stated that adequate characterization had been hampered by its rarity ; alpha - methyl - acyl - coa - racemase ( amacr)/p504s had been reported to be positive in prostatic adenocarcinoma , papillary renal cell carcinoma , and gastrointestinal neoplasms ; nevertheless , it had never been previously studied in clear cell carcinomas of lower urinary tract .
they investigated the immunohistochemical staining profile in 4 primary clear cell carcinomas of the urinary tract including p504s .
they retrieved four cases of clear cell adenocarcinoma from their archives : 2 cases from the urinary bladder ( one each from a man and a woman ) and 2 cases from the urethra ( both from women , 1 in a diverticulum ) .
they performed immunohistochemistry for p504s , k903 , cytokeratin ( ck ) 7 , ck20 , ca 125 , and p63 .
sun et al . reported that clear cell carcinomas had distinct immunoreactive profile : strongly positive for p504s , k903 , and ck 7 and negative reactivity for p63 .
two of the cases were also positive for ca 125 and ck 20 ( see figures 12 and 13 as well as table 1 for various microscopic features and immunohistochemical characteristics of some of the tumours ) .
sun et al . concluded the following.the immunohistochemical profile of clear cell carcinoma shares some similarity to conventional urothelial carcinoma ; nevertheless , it deviates from urothelial carcinomas in being positive for p504s and negative for p63.this staining profile may indicate a nonurothelial origin for these tumours and may serve as a useful tool in the differential diagnosis of clear cell adenocarcinoma and may reflect its aetiology.in view of the fact that similar expression of p504s is also seen in nephrogenic adenoma , this marker should not be used to differentiate nephrogenic adenomas from clear cell adenocarcinomas .
herawi et al . in 2010 described a clear cell adenocarcinoma which mimicked nephrogenic adenoma due to less prominent nuclear pleomorphism , less prominent nucleoli , and fewer clear cells .
nevertheless , this variant of adenocarcinoma did exhibit extensive muscular invasion and focal hyperchromatic and pleomorphic tumour cells that would not be in nephrogenic adenoma .
miller and karnes in 2008 stated that clear cell adenocarcinoma is an aggressive tumour with low rates of 5-year survival . in their case ,
she was well during the subsequent three and half years until 5 months prior to the presentation of the case , when she underwent excision of pelvic lymph node histology of which confirmed clear cell carcinoma similar to the primary tumour . at the time of presentation of the case
. a young age or a history of genitourinary trauma , operation , or calculus may constitute a clue to the latter diagnosis ; microscopic characteristics such as sheets of clear cells , significant pleomorphism , or mitotic activity would be in favour of the diagnosis of clear cell adenocarcinoma .
the follow - up of majority of the patients , most of who underwent a radical operation , was short , but five tumours were known to have metastasized .
the immunohistochemical profile of clear cell carcinoma shares some similarity to conventional urothelial carcinoma ; nevertheless , it deviates from urothelial carcinomas in being positive for p504s and negative for p63 .
this staining profile may indicate a nonurothelial origin for these tumours and may serve as a useful tool in the differential diagnosis of clear cell adenocarcinoma and may reflect its aetiology . in view of the fact that similar expression of p504s is also seen in nephrogenic adenoma , this marker should not be used to differentiate nephrogenic adenomas from clear cell adenocarcinomas .
few cases of ccau have been reported and the tumours have been reported to be aggressive with low 5-year survival rates .
ccaus have been treated by anterior exenteration in women and cystoprostatectomy in men and at times by radiotherapy ; chemotherapy has rarely been given . | background .
clear cell adenocarcinoma of the urethra ( ccau ) is extremely rare and a number of clinicians may be unfamiliar with its diagnosis and biological behaviour . aims . to review the literature on ccau
. methods .
various internet databases were used .
results / literature review .
( i ) ccau occurs in adults and in women in the great majority of cases .
( ii ) it has a particular association with urethral diverticulum , which has been present in 56% of the patients ; is indistinguishable from clear cell adenocarcinoma of the female genital tract but is not associated with endometriosis ; and probably does not arise by malignant transformation of nephrogenic adenoma .
( iii ) it is usually , readily distinguished from nephrogenic adenoma because of greater cytological a - typicality and mitotic activity and does not stain for prostate - specific antigen or prostatic acid phosphatase .
( iv ) it has been treated by anterior exenteration in women and cystoprostatectomy in men and at times by radiotherapy ; chemotherapy has rarely been given .
( v ) ccau is aggressive with low 5-year survival rates .
( vi )
there is no consensus opinion of treatment options that would improve the prognosis . conclusions .
few cases of ccau have been reported .
urologists , gynaecologists , pathologists , and oncologists should report cases of ccau they encounter and enter them into a multicentric trial to determine the best treatment options that would improve the prognosis . |
a 39-year - old male with medical history of gastric bypass surgery and chronic alcoholism was admitted for a syncopal episode .
previously , the patient underwent roux - en - y gastric bypass in 2008 after failed lifestyle modifications in effort to reduce body weight . additionally , in the last two and half years , the patient had abused alcohol in the amount of one pint daily , 3 days per week . in the interim
that admission was attributed to alcohol withdrawal seizures after an uneventful hospital course and unremarkable workup .
the patient was at a grocery store on the day of admission when he had one episode of unwitnessed passing out with urinary incontinence .
this presentation was similar to his previous admission 2 weeks earlier with added complaints of four episodes of non - bloody watery diarrhea and dry skin on his lower extremities .
skin examination showed multiple patches of dry , peeling skin on both feet ( image 1 ) .
significant laboratory values included hb 8.7 g / dl ( normal 13.517.5 g / dl ) , and liver functions consistent with alcoholism ( ast 139 u / l [ 820
u / l [ 820 u / l ] , alkaline phosphate 196 u / l [ 2070 u / l ] , and total bilirubin 2.9 mg / dl [ 0.11.0 mg / dl ] ) .
inr was 1.3 with significant electrolyte abnormalities of hypomagnesemia ( 0.9 mg / dl [ 1.62.3 mg / dl ] ) and hypophosphatemia ( 2.2 mg / dl [ 2.54.5 mg / dl ] ) .
multiple patches of dry , peeling skin bilaterally . at this time , the clear history of gastric bypass surgery compounded by alcoholism in conjunction with an extensive and unremarkable initial workup lead to the conclusion that the constellation of signs and symptoms were related to a single etiological process .
he was transfused two units of packed blood cells due to a repeat hb of 6.7 g / dl .
these relatively benign findings , combined with an unremarkable abdominal ct scan just prior to transfusion , prompted the combined decision of the primary team and the gastroenterology consult to defer further esophagogastroduodenoscopy studies . on day 5 ,
the patient had altered mental status for which a head ct and csf analysis were done , and were normal . later that day , the cutaneous lesions worsened to include both hands , extensor surfaces of the arms , and around the mouth .
diarrheal episodes had increased to five times daily . in light of leukocytosis 16 k / ul ( 4.010.8 k / ul ) , infectious disease consult was placed . among important differentials considered were cellulitis , stevens - johnson syndrome , and staphylococcal scaled skin syndrome .
stevens - johnson syndrome , a possible adverse effect of midodrine , was quickly ruled out clinically due to lack of progression of lesions or mucosal involvement .
empirical vancomycin was begun for presumptive cellulitis after an initial set of blood cultures grew methicillin sensitive staphylococcus aureus in one of two bottles .
nutritional indices recommended by a nutritionist included folate 47.2 ng / ml ( 2.814.0 ng / ml ) , b12 1,699 pg / ml ( 211911 pg / ml ) , zinc 0.27 g / ml ( 0.661.10 g / ml ) , and copper 65 g / dl ( 70175 g / dl ) .
the finding of zinc deficiency along with a negative repeat blood culture prompted discontinuation of vancomycin on day 4 of treatment by infectious disease , with the original positive blood culture being attributed to contamination .
the focus of etiology shifted from an infectious process to either a drug reaction or nutritional deficiency with zinc deficiency the working diagnosis .
zinc supplementation ( 220 mg / daily ) was started along with corticosteroids before transfer to a tertiary care center for definite diagnosis of the skin biopsy . peeling skin on left hand , day 17 .
, a rash was noted to have progressed and the patient was still severely orthostatic , requiring continued use of midodrine .
oral steroids were started but discontinued after a dermatology consult attributed the lesions to nutrient deficiency , while also considering stevens - johnsons syndrome , pellagra , and glucagonoma syndrome in the differential diagnoses .
an upper gi small bowel series showed a mild gastro - jejunal stricture measuring 1 cm . with poor oral intake ,
the rash showed continuous improvement with maintained dosages of zinc ( 220 mg / daily ) with gradual clearance of desquamative lesions upon discharge .
the patient was encouraged to continue vigorous oral and nutritional supplementation and to follow up in 2 weeks .
the role of zinc as an essential nutrient in human metabolism has been well known for decades .
zinc has been demonstrated as an integral constituent of the catalytic site of hundreds of metalloproteinases , most notably alkaline phosphatase and carbonic anhydrase .
additionally , zinc finger proteins , a special motif of various steroid hormone receptors , play a special role in structural differentiation of multiple organs including the skin . the decreased enzymatic activity precipitated by zinc deficiency manifests as a broad range of clinical signs and symptoms .
zinc deficiency has been implicated in delayed puberty , impaired cognition , diarrhea , alopecia , dermatitis , immune dysfunction , and delayed wound healing ( 7 ) . the transport protein
this clinical entity was first described as a rash with diarrhea in 1936 by brandt , and first reported by danbolt ( 8) .
later studies revealed low zinc levels correlated with symptoms by demonstrating improvement when zinc was replaced ( 9 ) . a similar clinical entity occurring commonly in later years of life is termed acquired acrodermatitis or acquired zinc deficiency .
the patient in the case report suffered acquired zinc deficiency due to two significant risk factors : alcoholism and gastric bypass surgery .
other conditions linked with zinc deficiency include anorexia nervosa , total parenteral nutrition , pancreatic disorders , malignancies , and renal disorders ( 10 ) .
as mentioned , one important risk factor for zinc deficiency is previous gastric bypass surgery .
gastric bypass surgery is the most common form of bariatric surgery , with roux - en - y bypass the most popular , having doubled in number between 2003 and 2009 .
the procedure entails a portion of the stomach being made into a small 30-ml pouch that is then attached to a distal segment of intestine .
this anastomosis avoids the entirety of the duodenum and the majority of the jejunum that comprise major locations for micronutrient absorption .
perioperative complications are generally quite low while numerous post - operative complications exist ( 11 ) .
one important complication is nutrient depletion secondary to gut manipulation , which alters sites for natural absorption . to that end ,
a useful guideline published in 2008 by aills et al . expertly outlined suggested supplement regimens ( 12 ) .
important causative factors for post - operative nutrient deficiencies are poor follow - up and noncompliance on the part of patients . a study by dalcanale et al .
found the following deficiencies in 75 patients up to 5 years post roux - en - y procedure : magnesium ( 32% ) , zinc ( 40.5% ) , b12 ( 61.8% ) , and vitamin d3 ( 60.5% ) .
most of the deficiencies were attributed to patient non - compliance of post - operative vitamin replacement regimens ( 13 ) an additional study by toh et al .
demonstrated low levels of ferritin ( 15% ) , b12 ( 11% ) , rbc folate , and vitamin d ( 12% ) for similar reasons ( 14 ) .
the reported patient suffered appreciable deficiencies in zinc and copper , with normal b12 and folate indices . aside from the symptoms of hypozincemia , the patient also suffered possible hypocupremia induced peripheral neuropathy in the form of moderate orthostasis requiring the alpha agonist midodrine after failed fluid challenge .
copper , similar to zinc , is an important cofactor in enzymatic reactions , particularly important in processes pertaining to anti - oxidation .
absorption occurs primarily in gastric and duodenal mucosa with recommended daily allowance of 700 g ( 11 ) .
the interaction of copper and zinc at the molecular level of absorption is an important consideration when treating zinc deficiency with zinc supplementation .
zinc upregulates the expression of the important chelator , metallothionein at the level of enterocytes .
copper 's increased affinity for this chelator results in it being bound and trapped in the enterocyte while zinc is free to be absorbed into the bloodstream . for bariatric patients requiring zinc supplementation , co - administration of copper supplementation
the amount of copper in the standard multivitamin was deemed sufficient not to warrant stand - alone dosages .
it is the belief of the authors that zinc deficiency is a common yet significantly underdiagnosed entity .
therefore the diagnosis of zinc deficiency requires a high index of suspicion in bariatric patients due to the large overlap in symptoms amongst nutritional deficiencies .
specifically , the cutaneous manifestations of peeling and later desquamating , ulcerating skin requires considering multiple differential diagnoses .
zinc deficiency falls under the category of necrolytic erythemas that include necrolytic migratory erythema , necrolytic acral erythema , and pellagra .
necrolytic migratory erythema is a blistering , patchy rash that spreads across the abdomen , perineum , and buttocks .
it is associated with glucagonoma , a glucagon - producing tumor of the pancreas , which is definitely diagnosed with ct or ultrasonography .
its histopathology is characterized by parakeratosis and epidermal necrosis in a psoriasiform pattern ( 16 ) .
necrolytic acral erythema shares similar histology but classically the lesions are well circumscribed and dusky with an adherent scale .
pellagra is deficiency of niacin and classically presents as a diarrhea , dermatitis , and dementia in patients .
normal niacin levels , lack of hepatitis c risk factors , and continually normal abdominal imaging were sufficient to rule out these entities .
furthermore , a clear history of post - surgical failure to thrive and hypozincemia with resulting improvement with daily zinc , clinched the diagnosis .
one important feature of the case was the progressive worsening of symptoms despite appropriate multivitamin replacement for chronic alcoholism .
one possibility is that multivitamin replacement causes brief , sustained increases in metabolism demand in highly active tissues such as gastrointestinal mucosa and skin . with such upstrokes in metabolism in the background of decreased zinc level
, relevant zinc - mediated enzymatic reactions would continue to proceed in suboptimal fashion , acutely worsening symptoms .
the basis for this thought is the established thiamine - glucose sequence of replenishment in hypoglycemic patient to guard against iatrogenic induction of wernicke 's encephalopathy and lactic acidosis .
deficiencies in thiamine force pyruvate to convert to lactate , causing lactic acidosis and altered mental status , organ damage , and possibly death ( 18 ) .
it is standard practice to administer thiamine before glucose to ensure such enzymatic pathways are able to handle substrates appropriately .
this question remains unanswered and additional studies are warranted to help revise established treatment protocols for alcoholism and zinc deficiency .
| acquired adult - onset zinc deficiency is occasionally reported in patients with malnutrition states , such as alcoholism , or malabsorptive states , such as post - bariatric surgery .
the defining symptoms of hypozincemia include a classic triad of necrolytic dermatitis , diffuse alopecia , and diarrhea .
we report a case of zinc deficiency in a 39-year - old man with history of gastric bypass surgery and alcoholism . for this patient ,
severe hypozincemia confirmed acrodermatitis , and zinc supplementation was met with gradual improvement . |
-amyloid ( a ) peptide is the main cause of alzheimer 's disease ( ad ) [ 1 , 2 ] .
a has two major types , a40 and a42 , which are derived from a ubiquitous type i transmembrane protein
amyloid precursor protein ( app ) by a two - step secretase ( - and -secretase ) pathway [ 35 ] .
the amyloid cascade hypothesis predicts that the aggregated a peptides in the brain have an early and essential role in the neuronal degeneration that leads to dementia [ 1 , 2 , 6 ] .
the aggregation of a occurs following a conformational conversion from either -helix or random coil to -sheet in a time - dependent manner , indicating that the formation of -sheet structure is the key step for the peptide aggregation [ 7 , 8 ] .
the formation of a aggregates is modulated by several factors , including metal ions [ 9 , 10 ] . in amyloid plaques of ad - affected brain , remarkably high concentration of metals , such as cu ( 400 m ) , zn ( 1 mm ) and fe ( 1 mm ) , has been found [ 11 , 12 ] . in vitro studies , micromolar levels of cu , zn and fe have been shown to sufficiently induce protease - resistant aggregation and precipitation of a .
copper ions are bound to the three his residues of a located at the n - terminus , which forms a 3n1o square - planar motif as verified by epr spectroscopy .
copper - selective chelators can dissolve a deposits extracted from ad postmortem brain specimens and reduce the level of free radical .
curtain and his colleagues previously reported that the addition of cu to oligomeric a40 in a negatively charged lipid membrane can induce a peptides reinserted into lipid membranes , convert a conformation from -strand into -helix , and cause the lipid peroxidation .
their result is the first study to show that instead of -sheet , a in -helical form can also generate free radicals in the presence of cu ions .
recently , our group has characterized the conformation of a in the presence of cu ions into three structural states , stable helix , unstable helix , and random coil , in 40% , 25% , and 5% tfe , respectively , .
therefore , this provides us a possibility to re - examine the relationship between a conformation , and cu - driven free radical generation . in the present study
, we examined the correlation between cu binding affinity , h2o2 formation , and a40 conformation .
it was found that both cu binding affinity and h2o2 formation are well correlated with the conformation of a40 . in general , cu ions can be bound to the different structural forms of a40 .
the copper - binding affinity of a40 in random coiled form is about 3-fold higher than that of a40 in stable helical form . for h2o2 formation
, our result shows in the first time that the generation of h2o2 can also be induced by cu and the stable helical form of a40 .
in contrast , in random coiled form , the formation of h2o2 is inhibited by a40 .
the synthesis of full - length a40 peptide was performed in an abi-433a solid - phase peptide synthesizer using the fmoc protocol .
cleavage and deprotection of the synthesized peptide were performed by treatment with a mixture of trifluoroacetic acid / distilled water / phenol / thioanisole / ethanedithiol .
then the peptide was extracted with 1 : 1 ( v : v ) ether : h2o containing 0.1% 2-mercaptoethanol .
the synthesized a peptides were purified on a reverse - phase c-18 hplc with a linear gradient from 0% to 100% acetonitrile , and the molecular weight of a40 peptides and purity was verified by an maldi - tof mass spectroscopy .
one mg of purified monomeric a40 peptide was dissolved in 1 ml 100% trifluoroethanol ( tfe ) as stock solution . a final a peptide concentration of 30 m in either 5% or 40% tfe was prepared from fresh peptide stock solution diluted in phosphate buffer , ph 7.4 , with or without the same concentration of cu .
a thousand m of cu stock solution was prepared by dissolved cucl2 salt in distilled h2o purged by n2 in a sealed flask and freshly diluted into the designed concentration before it was used .
all measurements were performed in quartz cells with a path length of 0.1 cm .
data were collected at wavelengths from 190 to 260 nm in 0.2 nm increments .
the reported cd spectra were corrected using phosphate buffer , ph 7.4 for the baseline .
an equal molar 300 m of a and cu in 30% glycerol phosphate buffer , ph 7.4 , and 5% and 40% of tfe was employed for spin counting purposes .
epr spectra were obtained at x - band using a bruker emx er073 spectrometer equipped with a bruker te102 cavity and an advanced research system continuous - flow cryostat ( 4.2300 k ) . during epr experiments ,
the sample temperature was maintained at 10 k. the microwave frequency was measured with a hewlett - packard 5246l electronic counter .
tyrosine fluorescence spectroscopy was used to investigate the binding affinity for copper bound to a. a peptide stock solution was diluted in phosphate buffer , ph 7.4 to a final peptide concentration of 10 m with designed concentration of cu .
all measurements were performed in a 96-well plate using a microplate reader ( flexstation 3 , md ) . the excitation and emission wavelengths were selected at 278 and 305 nm , respectively .
reported data were the average obtained from three individual samples and three repeated measurements of each sample .
the binding affinity was calculated using the following equation :
( 1)fxf0=ixii0i=11+ka[cu2+]n ,
where i0 and ix are the fluorescence intensity for free and a/cucomplex , respectively , i is the fluorescence intensity at saturation state , [ cu ] is the copper concentration , and n is the copper binding number .
the related parameters were fitted using nonlinear curve fitting program micro - origin v6.0 ( microcal software , inc . , nothampton , ma ) . in the initial fitting stage , the simplex method , which was set to 100 cycle runs ,
the final fitting parameters were obtained when the value of was less than 0.05 and the parameters and errors for the parameters reached the convergent and steady state .
the production of h2o2 was analyzed using the dichlorofluorescein diacetate ( dcfh - da ) assay .
dichlorofluorescein diacetate was dissolved in 100% dimethyl sulfoxide , deacetylated with 50% v / v 0.05
m naoh for 30 min , and then neutralized ( ph 7.4 ) to a final concentration of 200 m as stock solution .
the reactions were carried out in dulbecco 's pbs , ph 7.4 , in a 96-well plate ( 100 l / well ) containing different concentration of a40 , 30 m cucl2 , tfe ( 5% and 40% ) , deacylated dcf ( 20 m ) , and horseradish peroxidase ( 5 m ) at 37c . for negative control , an extra of 1.0 mm catalase ( aspergillus niger ) was used to quench h2o2 .
fluorescence readings were recorded on a microplate reader ( md , flexstation 3 ) , with the excitation and emission wavelengths selected at 485 and 530 nm , respectively .
uv / vis turbidity assay was used to detect the aggregation process of a. 200 l of 30 m a40 in phosphate buffer , ph 7.4 containing either 5% or 40% tfe was freshly prepared from stock solution .
fresh prepared samples with or without 30 m cu placed in a 96-well plate were incubated at 37c .
turbidity was measured using a microplate reader ( md , flexstation 3 ) at a wavelength of 450 nm .
the aim of this study is to examine the relationship between a40 conformation and other physical and chemical properties such as aggregation , cu - binding affinity , and free radical formation . in the present study ,
we applied 5% and 40% tfe to mimic the conformation of a40 in random coil and stable helix , respectively , .
therefore , the effect of tfe on cu binding affinity and free radical generation has to be evaluated . as depicted in supplemental data
( see supplementary material available online at doi:10.4061/2011/607861 ) , the main effect of tfe on the system is only to quench the dcf fluorescence intensity .
neither was the solubility of cu ions nor the formation of h2o2 affected by tfe .
first of all , we characterized the structural state of a40 with and without cu in either 5% or 40% tfe using circular dichroism ( cd ) spectroscopy .
figures 1(a ) and 1(b ) show the cd spectra for a40 and a40/cu in 5% and 40% tfe , respectively .
it can be seen that , in 5% tfe , the pattern of cd spectrum for a40 in the presence of cu showed a dramatic change compared to that for a40 only , while the cd spectra of a40 with or without cu in 40% tfe did not show any significant change .
the secondary structure of a40 in 5% tfe changes from 63% random coil , 34% -sheet and 3% helix in cu - free state to 45% random coil , 50% -sheet and 5% helix in cu - bound state , representing that there is a structural conversion from random coil into -sheet . on the other hand ,
the conformation of a40 with or without cu in 40% tfe remains stable in which the secondary structure is 74% helix , 14% -sheet , and 12% random coil in cu - free state and 73% helix , 15% -sheet , and 12% random coil in cu - bound state , indicating that there is no conformational conversion of a40 in 40% tfe while adding cu ions .
it has been shown that cu ions are able to accelerate the aggregation of a40 [ 9 , 15 , 18 , 20 ] .
hence , we characterized the aggregation process of a40/cu and a40 in 5% and 40% tfe . the aggregation process of a40/cu and a40 in 5% and 40% tfe was analyzed by turbidity assay as shown in figure 2 . in general , in both 5% and 40% tfe , a40 in the presence of cu was aggregated much faster than that in the absence of cu
furthermore , the aggregation rate in 5% tfe for both a40/cu and a40 was much faster than that in 40% tfe .
the aggregation process in 5% tfe for a40/cu and a40 showed a typical sigmoidal profile . on the other hand ,
the aggregation process in 40% tfe for a40 with and without cu mostly stayed in the nucleation or the early elongating stage within 24 hrs .
the aggregation process for a40 and a40/cu in 5% and 40% tfe echoes with the result obtained in the analyses of secondary structure in which the aggregation ability is correlated with the ability of conformational formation of -sheet . in the present study , we showed that the conformational states for a40/cu and a40 in 5% are dramatically different , while no such conformational change can be observed between a40/cu and a40 in 40% tfe .
although cu ions have been shown to be bound to a in aqueous solution , then , whether cu ions are bound to a40 in 40% tfe is needed to be investigated since the structural state for a40/cu and a40 in 40% tfe shows no difference . to address this issue ,
epr spectroscopy was applied to explore the interaction between cu and a40 in 40% tfe .
as shown in figure 3 , the epr spectra of a/cu both in 5% and 40% tfe showed two major groups of hyperfine peaks , while no hyperfine peak could be observed in the epr spectrum for cu alone , indicating that cu ions were bound to a40 in both 5% and 40% tfe .
the pattern of hyperfine peaks for a40/cu in 5% and 40% tfe is similar , but the position of hyperfine peaks in 40% tfe shifts to low magnetic field .
the related epr parameters of g , a , and g are 2.28 , 156.8 , and 2.06 for a40/cu in 5% tfe , respectively .
the g and g values in 5% tfe are very close to those in the literature measured in aqueous condition , representing that the binding geometry of a40/cu in 5% tfe should adopt a similar 3n1o coordination . on the other hand , the related epr parameters of g , a , and g are 2.40 , 119.6 , and 2.09 for a40/cu in 40% tfe , indicating that the copper - binding geometry is different from the 3n1o mode and
this further indicates that the binding affinity of cu to a40 in random coiled form may be stronger than that to a40 in stable helical form .
as shown in epr studies , cu can be bound to different structural forms of a40 with a different binding affinity . in order to further elucidate the binding property of cu
, we applied the tyrosine fluorescence spectroscopy to determine the cu - binding constant , since tyrosine 10 is the only fluorophore in a amino acid sequence and locates at the binding pocket , in which the binding of cuto a40 may cause the change of tyrosine fluorescence .
the titration curves of cu concentration versus tyrosine fluorescence intensity in 5% and 40% tfe are shown in figures 4(a ) and 4(b ) , respectively .
the binding constants , ka , were estimated using the equation as described in the experimental section with molar ratio of a : cu = 1 : 1 , that is , n = 1 .
the calculated ka values are 0.14 m ( r > 0.97 ) and 0.05 m ( r > 0.96 ) for a/cu in 5% and 40% tfe , respectively .
the ka of a40/cu in 5% tfe is around 3-fold higher than that in 40% tfe , suggesting that cu is bound to random coiled form of a40 more strongly than to stable helical form of a40 which is consistent with the epr studies .
it has been demonstrated that a40 coordinated with cu can cause the reduction of cu and then induce the formation of h2o2 from o2 in a catalytic manner [ 15 , 20 ] . to examine the relationship between free radical formation and a40 conformation ,
figures 5(a ) and 5(b ) show the plots of dcf fluorescence intensity versus a concentration in 5% and 40% tfe as incubated at 37c for 1 hr . in general
, results clearly show that the formation of h2o2 is also correlated with a40 conformation . in 5% tfe ,
the level of h2o2 was reduced to zero when the a40/cu molar ratio 1 , indicating that the formation of h2o2 is inhibited when a40 adopts the random coiled form . unexpectedly but interestingly , unlike the formation of h2o2 which was inhibited in 5% tfe , the level of h2o2 in 40% tfe was increased with an increase of a40 concentration and was much higher than those of either cu or a40 alone , suggesting that the formation of h2o2 was induced when a40 was in the stable helical form .
according to amyloid cascade hypothesis , aggregated a is the main toxic species to cause the alzheimer 's disease .
the various forms of a aggregate have been shown to coordinate with redox active transition metals , such as cu and fe , and induce the generation of reactive oxygen species [ 15 , 20 ] .
however , the correlation of copper - binding affinity , copper - driven aggregation , and free radical formation with a40 conformation still needs to be elucidated . for ion interaction ,
our results demonstrate for the first time that cu ions can interact with both random coiled and stable helical forms of a40 . as a larger a value and binding constant obtained for a40 in 5% tfe ,
the cu - binding affinity for random coiled form of a40 is much stronger than that for stable helical form of a40 .
the possible explanation may be attributed to the different flexibility of a40 conformation in 5% and 40% tfe . as shown in cd spectra ,
the conformation of a40 in 5% tfe is relatively flexible and can easily convert structure from random coil into -sheet while coordinating with cu . on the other hand ,
the overall conformation of a40 with or without cu is relatively rigid and remains in stable helix in 40% tfe , and only the local geometry of cu - binding site is distorted while interacting with cu ions as indicated by the shift of g value .
therefore , the interaction between cu and a40 is strong in 5% tfe and relatively weak in 40% tfe .
the binding affinity for stable helical form of a40 is almost 3-fold lower than that for random coiled form of a40 .
the cu - binding affinity can further be used to account for the differences for cu - driven aggregation between random coiled and stable helical forms of a40 .
it is well known that the formation of -sheet plays a key step for the aggregation of a40 . as shown in the present study ,
the conformation of a40 in 5% tfe is much easier than that in 40% tfe to convert into -strand structure by cu ions .
thus , in the presence of cu , the random coiled form of a40 ( in 5% tfe ) aggregates faster and more severe than the stable helical form of a40 ( in 40% tfe ) . unlike the cascade hypothesis that only -sheet form of aggregated a can produce free radical , we demonstrate that helical form of a40 can also induce the formation of h2o2 . from the aggregation assay , this helical form of a40 may possibly exist as a monomer during the early incubation period .
then , this further implies that monomeric a40 in stable helical form by coordinating with cu can induce the formation of h2o2 .
in contrast , a40 in random coiled form shows to inhibit the formation of h2o2 during the early incubation period .
the inhibition of h2o2 formation becomes more significant with an increase of a40 concentration , and the formation of h2o2 further is completely inhibited when the molar ratio of a/cu molar ratio 1 .
our observation is consistent with a recent study by viles and his colleagues , in which they showed that , at a/cu molar ratio = 1 , the formation of h2o2 is inhibited by both monomeric and fibrillar a peptides .
taken together , it may suggest that helical structure may play a key factor for the generation of h2o2 by a40/cu . in summary ,
our present results demonstrate that both ion interaction and copper - driven free radical formation are well correlated with a40 conformation .
when a40 adopts a stable helical structure , the cu binding affinity is weaker , and the free radical is produced .
on the other hand , when a40 adopts a random coil in 5% tfe , the cu binding affinity is stronger , and the formation of h2o2 is inhibited . | the neurotoxicity of a is associated with the formation of free radical by interacting with redox active metals such as cu2 + .
however , the relationship between ion - interaction , ion - driven free radical formation , and a conformation remains to be further elucidated . in the present study , we investigated the correlation of cu2 + interaction and cu2 + -driven free radical formation with a40 conformation .
the cu2 + -binding affinity for a40 in random coiled form is 3-fold higher than that in stable helical form .
unexpectedly but interestingly , we demonstrate in the first time that the stable helical form of a40 can induce the formation of h2o2 by interacting with cu2 + . on the other hand ,
the h2o2 generation is repressed at a/cu2 + molar ratio 1 when a40 adopts random coiled structure .
taken together , our result demonstrates that a40 adopted a helical structure that may play a key factor for the formation of free radical with cu2 + ions . |
despite differing estimates , about 4 - 8% of the population in india are differently abled .
one in every 10 children is born with or acquires a physical , mental or sensory disability .
these translate into 40 - 90 million children 's , which is a substantial number .
only 35.29% of all people living with disabilities have access to schools . despite improvement in the health care system in the country ,
the situation of differently abled children remains deplorable , particularly in rural areas and among the lower socio - economic population .
differently abled children in india are subject to multiple deprivations and limited opportunities in several dimensions of their lives .
some these include , not being enrolled to schools , lower employment rates , limited awareness of entitlements and services available and lack of social welfare support .
the community - based rehabilitation ( cbr ) is a dynamic program globally for supporting differently abled children 's to lead better quality of life and lead life with dignity , where in their rights are respected and guarded within their own communities and it creates platform for addressing the discriminatory practices in the community .
we are working with nongovernmental organizations ( ngos ) for including people with mental illness in the cbr program .
we have used few case studies from the visit reports to describe the prevailing forms of discrimination and stigma associated with disabilities in the rural communities . due to stigma associated with disabilities ,
families become victims of discrimination and human rights abuse . when poverty , physical neglect and social marginalization intersect , the impact on the disabled can be devastating .
differently abled children 's are kept hidden away at their home , denied basic rights of mobility , education and employment .
such discrimination in some cases starts from the family members and spreads right up to the policy makers and state authorities . as a result of such discrimination the differently abled children 's face chronic ill health , socio - economic burden and destitution .
sometimes it is so difficult to define the marginalization they are outside the margin or within the community meaning , locked in the rooms , institutionalized , families isolating themselves , enrolling in special schools , not admitting that they have children with disabilities ( cwds ) , in the hospitals , etc . social attitudes and stigma play an important role in limiting the opportunities of disabled people for full participation in social and economic life , often even within their own families .
predominantly in the cases of mental or intellectual disability , the family members are reluctant to accept the disability or refer to it as a physical illness and treatable condition .
the pseudo - stigma attached to such disabilities , makes them hide the fact of having a disabled or challenged member at home ultimately leading to social isolation and restrictive behaviors .
there is a fear that they would be victims of disgrace and indignity and thereby family members lose the status or acceptance they enjoy in the community .
such persons would be hidden somewhere and they expect , unrealistically , to overcome the situation without realizing the log term consequences of such self - imposed denial .
i know my son is having less intelligence , may be some degree of intellectual disability , but i do not want to become member of the disability self - help group because i have two more daughters who need to get married , my association with disability self - help group would be self - certifying of my sons problem .
superstitions prevailing in the communities also play a big role in subjecting the people with disabilities to various harmful treatments .
the black - magicians and quacks physically hurt people , subject them to food restrictions etc . claiming to cure the
families often lock or chain their children with intellectual disability having behavioral issues , due to helplessness , ignorance and/or under social pressure .
s , deserted women , during the care givers meeting expressed that she need to tie her child with cerebral palsy under the tree , whenever she goes for her livelihood she started crying as she does not have support from her in laws nor from her parents to take care of her daughter with cerebral palsy .
even the family members of the disabled often tend to avoid such social gatherings in shame or fear that someone would ask about their family member with disability .
differently abled children 's are not exposed to any social gathering , nor does our community recognize the need for children 's participation .
cwds are not been given opportunities in the areas of education , training and employment .
under these circumstances it is natural that the cwds feel rejected or unwanted in the society .
mother of 6 children , witnessed death of two children with muscular dystrophy , and three more children been diagnosed as having same illness said that i do not have any interest in visiting relatives nor attending any social gathering ( functions ) , mainly because people feel pity about my fate and would talk among themselves that i am paying for the sins done in my previous life .
as per the indian laws , all kith and kin in the family are eligible to get their share of inherited property , but in reality , persons with disabilities are denied these rights .
the siblings take responsibility of providing care and they would enjoy the property meant for the person with disability .
families perceive that cwd are incapable of managing their property , they are denied of their property rights and made dependent on the able - bodied siblings .
worst of all would be when family members ensure the chronic condition of the disability by denying treatment or other aids , so that the siblings enjoy the property . during home visit
my elder son need to take care of my younger son with polio , all the property of mine would go to my elder son and his children .
father said that with his clutches he can not do agricultural work - need to depend on others , let that be my elder son. in india the elders arrange majority of the marriages .
if a family has person with disability , eligible boys and girls finding a prospective spouse is almost next to impossible because of the stigma and the disability being seen as a family illness .
i was married at the age of 13 years , i realized that my husband do not talk nor can listen during my first night .
when my son was born , even he did not cry , he also have same problem like my husband . father expressed that
i would settle my son 's wedding with my granddaughter ( daughters daughter ) for two reasons , my younger son with disability would be taken care , secondly i may not be able to get good proposal for my son as we have a disabled person at home .
sexual identity is a critical component of overall personality development and self - esteem , which matures during adolescence .
parents infantilize disabled children and imply that sex is only for the able - bodied and of no relevance to the disabled .
these parental attitudes are transmitted to the child in subtle ways making him / her feel that she / he is inferior and unworthy of love .
parents of cwds encourage dependence and share the general societal perception of disabled persons as essentially child - like , innocent and asexual .
father of 18-year - old daughter in the caregiver meeting , while discussing about the marriage for disabled said that we should not dream about marrying the disabled person , how can they manage their responsibilities and their spouse - it is not irrelevant topic to be discussed in the caregivers meeting .
due to differential gender - based role expectations , education is not considered a priority for disabled girls .
there is an over - representation of disabled boys in education , both in special and mainstream schools .
parents become more protective and restrictive , especially after a disabled girl reaches puberty . travelling to school
is a huge problem , since , besides transport difficulties , the danger of sexual abuse and violation looms large .
there is also the reasoning that there 's little point investing in a disabled girl 's education as they will anyhow never be able to earn .
unfortunately a girl child with disability is seen as a lifelong burden on the natal family because marriage is not a realistic option .
hence , it is concluded to be economically unsound to invest in her education or vocational training .
when we analyzed the annual report of an ngo , we found that men and women with disabilities identified , enrolled in self - help groups was 60 : 40 even though there is no difference in prevalence of disabilities among males and females .
children with disabilities come under the purview of the ministry of social justice and empowerment .
some of the issues like prevention and curative aspects are dealt by the health ministry .
however , no single ministry has taken the responsibility of meeting the holistic needs of cwds .
disability continues to fall in the area of social welfare . although efforts are on to bring it into the rights perspectives , the thinking process is dominated by the charity mode , while providing services for people with disabilities . as disability being state
subject , each state have their own program for persons with disability , but none of the states are able to see in holistic needs of people with disabilities .
some states have been pro - active in increasing awareness among people with disabilities about commitments and entitlements ( tamil nadu , karnataka , and new delhi ) whereas others have lagged in implementing many of the basic entitlements enshrined in the pwd act of 1995 ( bihar , maharashtra , orissa , uttar pradesh ) . in karnataka ,
disability welfare department introduced personnel at the panchayat like village rehabilitation workers , and multipurpose rehabilitation workers to meet the needs of people with disabilities in their respective jurisdictions .
worldwide it is accepted to use international classification of functioning ( icf ) , disability and health , known more commonly as icf , as measurement for quantifying disability , classification of health and health - related domains .
these domains are classified from body , individual and societal perspectives by means of two lists : a list of body functions and structure , and a list of domains of activity and participation . since
an individual 's functioning and disability occurs in a context , the icf also includes a list of environmental factors . despite this common measurement tool for assessing disability
are lacking . in some states individuals with disabilities education act / who disability assessment schedule / icf
there are several unmet challenges , which need to be addressed among disability sector in india .
need for dignified life for children and people with disabilities.need to remove attitudinal barriers among communities and provide rehabilitation of cwds.need to improve infrastructures in mainstream schools to make them disabled friendly and train teachers for optimal support.need to converge between various departments providing services for cwds.need for national harmonization of disability welfare program.need to give executive powers and necessary resources to the commissioner of disabilities for effective implementation and safeguarding rights of pwd.need for promoting and monitoring mechanisms for service outreach below district level.need to improve effective collaborations between government and ngo to avoid duplications.need to adopt to a down to top approach in policy design.need to improve community participation programs .
need for dignified life for children and people with disabilities . need to remove attitudinal barriers among communities and provide rehabilitation of cwds . need to improve infrastructures in mainstream schools to make them disabled friendly and train teachers for optimal support .
need to converge between various departments providing services for cwds . need for national harmonization of disability welfare program . need to give executive powers and necessary resources to the commissioner of disabilities for effective implementation and safeguarding rights of pwd .
need for promoting and monitoring mechanisms for service outreach below district level . need to improve effective collaborations between government and ngo to avoid duplications . need to adopt to a down to top approach in policy design . need to improve community participation programs .
disability sector has recognized the importance of dignity , respect , inclusion , participation , equalization of opportunities and empowerment as key issues of rehabilitation .
the negative attitudes and cultural representations of disability in society are challenged through vigorous awareness - generation and attitudinal change strategies .
the issue of disability must consciously move beyond issues of special education and medical rehabilitation and be mainstreamed into other discourses such as the economy , polity , entertainment , sports , fashion and lifestyle . during the last two decades
, there has been a growing realization that institutional care for the disabled is not entirely suitable for their individual needs , dignity and independence .
there has been relentless advocacy for community care despite the enormous stigma of having a disabled person at home . in india , where family support is the norm and the only form of support available for thousands of years , community care is been thought as a suitable program for meeting the challenges in the disability sector .
community - based rehabilitation is implemented through a joint effort between people with disabilities , their families and communities , and the appropriate health , education , vocational and social services .
cbr depends heavily on the development of positive attitudes and approaches among the people involved .
basic services are provided or facilitated by cbr workers who are minimally qualified , nonprofessionals , but who are highly qualified change agents from their own communities .
cbr recognizes that breaking down barriers to inclusion in society is as important to the mission of the cbr program as is the functional rehabilitation of individuals with disabilities .
thus , the universal mission of cbr is to :
enhance activities of daily life of disabled persons.create awareness in disabled person 's environment to achieve barrier free situations around him and help him in meeting all human rights.create a situation in which the community of the disabled persons , participates fully and assimilate ownership of their integration in to the society .
create awareness in disabled person 's environment to achieve barrier free situations around him and help him in meeting all human rights .
create a situation in which the community of the disabled persons , participates fully and assimilate ownership of their integration in to the society .
community - based rehabilitation is very appropriate in the indian cultural setting , where social and community bonds are strong and deep - rooted .
the challenge is to harness the potential of these bonds for rehabilitation related social action programs .
nevertheless , cbr programs need to draw their resources from existing community development programs and should integrate with them .
cbr builds on and validates existing indigenous knowledge and information systems , while facilitating access to relevant information and ideas from outside the community .
due to stigma associated with disabilities , families become victims of discrimination and human rights abuse .
when poverty , physical neglect and social marginalization intersect , the impact on the disabled can be devastating .
differently abled children 's are kept hidden away at their home , denied basic rights of mobility , education and employment .
such discrimination in some cases starts from the family members and spreads right up to the policy makers and state authorities . as a result of such discrimination the differently abled children 's face chronic ill health , socio - economic burden and destitution .
sometimes it is so difficult to define the marginalization they are outside the margin or within the community meaning , locked in the rooms , institutionalized , families isolating themselves , enrolling in special schools , not admitting that they have children with disabilities ( cwds ) , in the hospitals , etc . social attitudes and stigma play an important role in limiting the opportunities of disabled people for full participation in social and economic life , often even within their own families .
predominantly in the cases of mental or intellectual disability , the family members are reluctant to accept the disability or refer to it as a physical illness and treatable condition .
the pseudo - stigma attached to such disabilities , makes them hide the fact of having a disabled or challenged member at home ultimately leading to social isolation and restrictive behaviors .
there is a fear that they would be victims of disgrace and indignity and thereby family members lose the status or acceptance they enjoy in the community .
such persons would be hidden somewhere and they expect , unrealistically , to overcome the situation without realizing the log term consequences of such self - imposed denial .
i know my son is having less intelligence , may be some degree of intellectual disability , but i do not want to become member of the disability self - help group because i have two more daughters who need to get married , my association with disability self - help group would be self - certifying of my sons problem .
superstitions prevailing in the communities also play a big role in subjecting the people with disabilities to various harmful treatments .
the black - magicians and quacks physically hurt people , subject them to food restrictions etc . claiming to cure the
families often lock or chain their children with intellectual disability having behavioral issues , due to helplessness , ignorance and/or under social pressure .
s , deserted women , during the care givers meeting expressed that she need to tie her child with cerebral palsy under the tree , whenever she goes for her livelihood she started crying as she does not have support from her in laws nor from her parents to take care of her daughter with cerebral palsy .
even the family members of the disabled often tend to avoid such social gatherings in shame or fear that someone would ask about their family member with disability .
differently abled children 's are not exposed to any social gathering , nor does our community recognize the need for children 's participation .
cwds are not been given opportunities in the areas of education , training and employment .
under these circumstances it is natural that the cwds feel rejected or unwanted in the society .
mother of 6 children , witnessed death of two children with muscular dystrophy , and three more children been diagnosed as having same illness said that i do not have any interest in visiting relatives nor attending any social gathering ( functions ) , mainly because people feel pity about my fate and would talk among themselves that i am paying for the sins done in my previous life .
as per the indian laws , all kith and kin in the family are eligible to get their share of inherited property , but in reality , persons with disabilities are denied these rights .
the siblings take responsibility of providing care and they would enjoy the property meant for the person with disability .
families perceive that cwd are incapable of managing their property , they are denied of their property rights and made dependent on the able - bodied siblings .
worst of all would be when family members ensure the chronic condition of the disability by denying treatment or other aids , so that the siblings enjoy the property . during home visit
my elder son need to take care of my younger son with polio , all the property of mine would go to my elder son and his children .
father said that with his clutches he can not do agricultural work - need to depend on others , let that be my elder son.
in india the elders arrange majority of the marriages . if a family has person with disability , eligible boys and girls finding a prospective spouse is almost next to impossible because of the stigma and the disability being seen as a family illness .
i was married at the age of 13 years , i realized that my husband do not talk nor can listen during my first night .
when my son was born , even he did not cry , he also have same problem like my husband . father expressed that
i would settle my son 's wedding with my granddaughter ( daughters daughter ) for two reasons , my younger son with disability would be taken care , secondly i may not be able to get good proposal for my son as we have a disabled person at home .
sexual identity is a critical component of overall personality development and self - esteem , which matures during adolescence .
parents infantilize disabled children and imply that sex is only for the able - bodied and of no relevance to the disabled .
these parental attitudes are transmitted to the child in subtle ways making him / her feel that she / he is inferior and unworthy of love .
parents of cwds encourage dependence and share the general societal perception of disabled persons as essentially child - like , innocent and asexual .
father of 18-year - old daughter in the caregiver meeting , while discussing about the marriage for disabled said that we should not dream about marrying the disabled person , how can they manage their responsibilities and their spouse - it is not irrelevant topic to be discussed in the caregivers meeting .
due to differential gender - based role expectations , education is not considered a priority for disabled girls .
there is an over - representation of disabled boys in education , both in special and mainstream schools .
parents become more protective and restrictive , especially after a disabled girl reaches puberty . travelling to school is a huge problem , since , besides transport difficulties , the danger of sexual abuse and violation looms large .
there is also the reasoning that there 's little point investing in a disabled girl 's education as they will anyhow never be able to earn .
unfortunately a girl child with disability is seen as a lifelong burden on the natal family because marriage is not a realistic option .
hence , it is concluded to be economically unsound to invest in her education or vocational training .
when we analyzed the annual report of an ngo , we found that men and women with disabilities identified , enrolled in self - help groups was 60 : 40 even though there is no difference in prevalence of disabilities among males and females .
children with disabilities come under the purview of the ministry of social justice and empowerment .
some of the issues like prevention and curative aspects are dealt by the health ministry .
however , no single ministry has taken the responsibility of meeting the holistic needs of cwds .
although efforts are on to bring it into the rights perspectives , the thinking process is dominated by the charity mode , while providing services for people with disabilities . as disability being state subject , each state have their own program for persons with disability , but none of the states are able to see in holistic needs of people with disabilities .
some states have been pro - active in increasing awareness among people with disabilities about commitments and entitlements ( tamil nadu , karnataka , and new delhi ) whereas others have lagged in implementing many of the basic entitlements enshrined in the pwd act of 1995 ( bihar , maharashtra , orissa , uttar pradesh ) . in karnataka ,
disability welfare department introduced personnel at the panchayat like village rehabilitation workers , and multipurpose rehabilitation workers to meet the needs of people with disabilities in their respective jurisdictions .
worldwide it is accepted to use international classification of functioning ( icf ) , disability and health , known more commonly as icf , as measurement for quantifying disability , classification of health and health - related domains .
these domains are classified from body , individual and societal perspectives by means of two lists : a list of body functions and structure , and a list of domains of activity and participation .
since an individual 's functioning and disability occurs in a context , the icf also includes a list of environmental factors . despite this common measurement tool for assessing disability
are lacking . in some states individuals with disabilities education act / who disability assessment schedule / icf
there are several unmet challenges , which need to be addressed among disability sector in india .
need for dignified life for children and people with disabilities.need to remove attitudinal barriers among communities and provide rehabilitation of cwds.need to improve infrastructures in mainstream schools to make them disabled friendly and train teachers for optimal support.need to converge between various departments providing services for cwds.need for national harmonization of disability welfare program.need to give executive powers and necessary resources to the commissioner of disabilities for effective implementation and safeguarding rights of pwd.need for promoting and monitoring mechanisms for service outreach below district level.need to improve effective collaborations between government and ngo to avoid duplications.need to adopt to a down to top approach in policy design.need to improve community participation programs .
need for dignified life for children and people with disabilities . need to remove attitudinal barriers among communities and provide rehabilitation of cwds . need to improve infrastructures in mainstream schools to make them disabled friendly and train teachers for optimal support .
need for national harmonization of disability welfare program . need to give executive powers and necessary resources to the commissioner of disabilities for effective implementation and safeguarding rights of pwd .
need for promoting and monitoring mechanisms for service outreach below district level . need to improve effective collaborations between government and ngo to avoid duplications . need to adopt to a down to top approach in policy design . need to improve community participation programs .
disability sector has recognized the importance of dignity , respect , inclusion , participation , equalization of opportunities and empowerment as key issues of rehabilitation .
the negative attitudes and cultural representations of disability in society are challenged through vigorous awareness - generation and attitudinal change strategies .
the issue of disability must consciously move beyond issues of special education and medical rehabilitation and be mainstreamed into other discourses such as the economy , polity , entertainment , sports , fashion and lifestyle . during the last two decades
, there has been a growing realization that institutional care for the disabled is not entirely suitable for their individual needs , dignity and independence .
there has been relentless advocacy for community care despite the enormous stigma of having a disabled person at home . in india , where family support is the norm and the only form of support available for thousands of years , community care is been thought as a suitable program for meeting the challenges in the disability sector .
community - based rehabilitation is implemented through a joint effort between people with disabilities , their families and communities , and the appropriate health , education , vocational and social services .
cbr depends heavily on the development of positive attitudes and approaches among the people involved .
basic services are provided or facilitated by cbr workers who are minimally qualified , nonprofessionals , but who are highly qualified change agents from their own communities .
cbr recognizes that breaking down barriers to inclusion in society is as important to the mission of the cbr program as is the functional rehabilitation of individuals with disabilities .
thus , the universal mission of cbr is to :
enhance activities of daily life of disabled persons.create awareness in disabled person 's environment to achieve barrier free situations around him and help him in meeting all human rights.create a situation in which the community of the disabled persons , participates fully and assimilate ownership of their integration in to the society .
create awareness in disabled person 's environment to achieve barrier free situations around him and help him in meeting all human rights .
create a situation in which the community of the disabled persons , participates fully and assimilate ownership of their integration in to the society .
community - based rehabilitation is very appropriate in the indian cultural setting , where social and community bonds are strong and deep - rooted .
the challenge is to harness the potential of these bonds for rehabilitation related social action programs .
nevertheless , cbr programs need to draw their resources from existing community development programs and should integrate with them .
cbr builds on and validates existing indigenous knowledge and information systems , while facilitating access to relevant information and ideas from outside the community .
community - based rehabilitation as a strategy helps to address the ugly forms of discrimination existing in the community .
the strategy also focuses on enhancing the quality of life for cwds and their families , to meet their basic needs and ensuring inclusion and participation in their own development and also participating in the community development .
the cbr aims not only creates awareness about the rights of people with disabilities among the community members but also guarantees opportunities for their participation in social activities and also excising their rights with in their own communities rather than getting isolated into institutions .
the cbr has become a multi - sectoral approach that empowers persons with disabilities to access and benefit from education , employment , health and social service . | background : persons with disabilities comprise at least 4 to 8 percent of the indian population .
children with disabilities in india are subject to multiple deprivations and limited opportunities in several dimensions of their lives . their families and caregivers also go through lot of stress and challenges in having a person with disability at home which ultimately leads to grave discriminatory practices towards these children.materials and methods : the article attempts to analyze and describe the common discriminatory grounds that children with disabilities commonly face from their immediate families and from the larger community through analyzing the filed visit reports of the basic needs india staff providing on job training ( handholding support ) for the community based rehabilitation workers.results:the case studies describes the various ugly forms of the discriminatory practices seen in the community towards differently abled children , same been categorized as denial of disability , physical restraints , social boycott , denial of property rights , decreased marital life prospects due to disabled member in family , implications on sexuality of people with disability , women with disability , discrepancies in state welfare programs , and problems in measuring disabilities.conclusion:during the last two decades , there has been a growing realization that institutional care for the disabled is not entirely suitable for their individual needs , dignity and independence .
a movement towards community based rehabilitation has picked up pace and contribute toward greater independence and self sustainability of the disabled . |
mixed cortical cell cultures containing both glia and neurons , were prepared from icr mouse brains at 15 - 16 days of gestation . briefly , dissociated neocortical cells ( 3.510 cells / well ) were plated onto primaria - coated 24-well plates ( falcon ) containing a glial bed in plating medium consisting of eagle 's minimal essential medium ( mem ; earle 's salts , supplied glutamine - free ) supplemented with 20 mm glucose , 2 mm l - glutamine , 5% fetal bovine serum , and 5% horse serum .
cytosine arabinoside ( 10 m ) was added 5 days after plating to halt the growth of non - neuronal cells .
cultures were maintained at 37 in a humidified co2 incubator and used for experiments between days 12 and 14 in vitro .
glial cultures were prepared from postnatal mice ( 1 - 3 days ) and plated at 110 cells / well in plating medium supplemented with 10% horse serum , 10% fetal bovine serum , and 10 ng / ml epidermal growth factor .
after 2 weeks in vitro , cytosine arabinoside was added to the cultures , which were fed weekly with the same medium with 10% horse serum used for mixed cultures .
we pre - incubated cultures with esculetin for 2 h before inducing neurotoxicity . for measuring ca uptake ,
cultured cells were washed with hepes - buffered control salt solution ( hcss ) , and then incubated with nmda ( 150 m ) in the presence of mk-801 ( an nmda receptor inhibitor , 10 m ) or esculetin ( 10 , 100 m ) in hcss containing cacl2 ( final activity : 1.0 ci / ml ) .
after 5 min , exposure solution was washed away using four boluses of hcss and cells were lysed by addition of 0.2% sodium dodecyl sulfate solution .
aliquots of lysed cells were added to scintillation counting solution for the counting of beta emissions . for protein preparation ,
cells from three culture plates were pooled in 0.5 ml of 0.1 m phosphate buffer ( ph 7.4 ) and homogenized .
the homogenate was centrifuged for 30 min at 3,000 g at 4 and the supernatant , consisting of the cytosolic+mitochondrial fractions , was used in enzyme assays .
glutathione peroxidase ( gpx ) activity was measured using an nadph reduction assay following the technique of lawrence and burke .
soluble cell proteins were added to a reaction mixture containing reduced glutathione , glutathione reductase , and nadph in phosphate buffer .
the reaction was initiated by adding h2o2 , the absorbance decrease at 340 nm was recorded , and the activity in the absence of cellular protein was subtracted .
one unit of gpx is defined as moles nadph oxidized / min , and results are expressed in moles / min / mg protein .
glutathione reductase ( gr ) activity was measured using an nadph reduction assay following the technique of eklw et al .
gr activity was monitored by decreases in nadph absorbance at 340 nm at 37. results were based on a molar extinction coefficient for nadph of 6.2210 m cm .
one unit of gr is defined as moles nadph oxidized / min , and results are expressed in moles / min / mg protein .
protein content was measured by the method of lowry et al with bovine serum albumin as a standard .
neuronal damage was assessed by measuring the amount of ldh released into the medium by damaged cells 24 h after nmda or bso treatment .
data are expressed as the meanstandard error of the mean ( sem ) and analyzed for statistical significance by one - way anova using a post - hoc student - neuman - keuls test for multiple comparisons .
cultured cortical cells exposed to 150 m nmda or 50 m kainic acid ( ka ) generated moderate ( 50 - 70% ) neuronal damage as indicated by increased ldh release into the bathing medium after 24 h. pre - exposure of esculetin ( 10 , 100 m ) for 2 h attenuated nmda neurotoxicity by 16% and 29% , respectively , but did not show any inhibitory effect against ka neurotoxicity ( table 1 ) . at the highest concentration , esculetin
esculetin dose - dependently and significantly inhibited the neurotoxicity induced by h2o2 or superoxide anions ( o2 ) produced by xanthine / xanthine oxidase co - treatment ( table 2 ) .
morphologically , nmda - induced neurotoxicity manifested as loss of cell bodies ( figure 1a ) ; esculetin rescued many neurons from nmda neurotoxicity ( figure 1b ) . to determine whether esculetin modulates nmda receptors , we tested the effects of nmda alone or nmda+esculetin .
mk-801 , a non - competitive nmda receptor antagonist , blocked ca uptake as well as ldh release . at 100 m ,
esculetin significantly ameliorated the nmda - induced rise in ca uptake ( figure 2 ) . to investigate the interrelationship between lipoxygenase ( lox ) inhibition and glutathione metabolism , we measured esculetin modifiable gsh depletion - induced neurotoxicity .
after 2-h pretreatment with esculetin or vehicle , we exposed the cultured cells to 1 mm bso for 24 h. esculetin , a lox inhibitor ( 1 , 10 or 30 m ) significantly attenuated bso - induced neuronal injury by 46 , 62 , and 73% , respectively ( figure 3a ) .
we morphologically confirmed the protective effect of 30 m esculetin ( figures 3b-3d ) . at 150 m arachidonic acid ,
cycloheximide , an inhibitor of de novo protein synthesis , and vitamin e , an antioxidant , reduced neuronal damage by 25% ( figure 4 ) .
in contrast , esculetin ( 100 m ) significantly inhibited the neuronal injury by 90% ( figure 4 ) .
gpx activity was decreased by bso treatment , but bso did not change gr activity at 24 h compared to the normal group ( figures 5a and 5b ) . here ,
esculetin at 3 - 30 m concentration - dependently elevated gr activity compared to bso alone ( figure 5b ) but caused little change in gpx activity ( figure 5a ) .
these findings suggest that esculetin inhibits oxidized glutathione ( gssg ) accumulation by increasing gr activity , thus sparing the pool of reduced glutathione ( gsh ) .
ischemic injury , especially nmda neurotoxicity , is known to activate calcium - dependent lipoxygenase pathways .
so far , almost all nmda antagonists have failed to protect neuronal cells from cerebral ischemia clinically due to their psychiatric side effects . in this study ,
, we confirmed that the protective effect of esculetin against nmda toxicity can be partly attributed to its ability to modulate nmda receptors directly or indirectly , as demonstrated by the results for calcium uptake .
when nmda receptor overactivation is initiated by excitotoxic insults , release of arachidonic acid and increases in lipoxygenase activity can occur . as in other reports ,
the antioxidant vitamin e and the de novo protein synthesis inhibitor cycloheximide exhibited only weak protective effects .
this means that esculetin may contribute to the diminution of neuronal injury that occurs in ischemic insults by attenuating the nmda receptor - mediated arachidonic acid cascade .
interestingly , esculetin also had a sparing effect on the pool of gsh by halting gsh turnover to gssg .
furthermore , esculetin may contribute to the rescue of neuronal cells from nmda neurotoxicity by scavenging h2o2 or o2 we conclude that esculetin is a potential neuroprotectant in cerebral ischemia that appears to works by modulating nmda receptors and the metabolism of arachidonic acid or glutathione . | recently , loss of endogenous glutathione during n - methyl - d - aspartate ( nmda ) receptor - mediated excitotoxic injury , and the resultant overproduction of reactive oxygen species ( ros ) through an arachidonic acid cascade process in brain , have been implicated in neuronal damage in various neurodegenerative diseases .
glutathione depletion induced by l - buthionine-(s , r)-sulfoximine ( bso ) , an inhibitor of glutathione synthesis , is known to cause arachidonic acid - mediated excitotoxicity in primary mixed cortical cultures .
the aim of this study was to investigate whether esculetin ( 6,7-dihydroxycoumarin ) , an inhibitor of lipoxygenase , protects against neurotoxicity induced by nmda or bso .
we observed that neurotoxicity induced by nmda but not kainic acid was attenuated by esculetin . at the same concentration ( 100 m )
, esculetin attenuated the 45ca2 + uptake elevation induced by nmda .
free radical - mediated neuronal injury induced by h2o2 and xanthine / xanthine oxidase was concentration - dependently blocked by esculetin .
esculetin ( 1 - 30 m ) dose - dependently inhibited bso - induced neuronal injury .
in addition , arachidonate - induced neurotoxicity was completely blocked by esculetin .
bso also reduced glutathione peroxidase ( gpx ) activity , but did not change glutathione reductase ( gr ) activity 24 h after treatment .
esculetin dose - dependently increased gr activity , but did not alter gpx activity .
these findings suggest that esculetin can contribute to the rescue of neuronal cells from nmda neurotoxicity and that this protective effect occurs partly through nmda receptor modulation and the sparing of glutathione depletion . |
maxillofacial trauma represents one of the greatest challenges to public health services worldwide , because of their high incidence and significant financial cost .
they are often associated with morbidity and varying degrees of physical , functional , and esthetic damage .
the road traffic accidents ( rtas ) are now considered a public health hazard of primary magnitude and are likely to increase in the coming years owing to rapid increase in the automobile users .
the country has world 's highest fatality rate in rtas , 20 times that of developed countries . in india , eight people get killed for every 100 vehicles ; whereas , in developed countries like uk , usa , and france ; one person gets killed for every 1,000 vehicles .
maxillofacial injuries occur in significant number of trauma patients and management includes treatment of facial bone fractures , dentoalveolar fractures , and soft tissue injuries , as well as concomitant injuries .
epidemiological assessments of these injuries are essential to reaffirm patterns , identify new trends , plan and evaluate preventive measures and health policies , and develop priority goals for research .
several studies of the incidence and etiology of maxillofacial traumas have been carried out in countries such as austria , germany , new zealand , and united arab emirates .
there is lack of population based data on maxillofacial injuries due to rtas in this part of the country .
this is an important research agenda ; hence , the present study was taken up as an attempt to provide a retrospective analysis of patients treated for maxillofacial injuries and to determine the factors responsible for facial fractures , the age and sex distribution and the type of fracture .
a predesigned questionnaire was used to collect the data for this retrospective analysis . after obtaining permission from the concerned hospital authorities , the hospital records of all the patients treated for maxillofacial injuries in the department during the year 2011 and 2012 ( january 2011december 2012 ) were checked .
the information pertaining to age and sex , distribution , etiology of fracture , type of fracture , and associated injuries was entered in the pro forma .
the data was then computerized and subject to statistical analysis , using statistical package for social sciences ( spss ) windows version 10.0 .
a total of 787 patients were treated for maxillofacial injuries from january 2011 to december 2012 .
men sustained significantly more maxillofacial injuries as compared to females , with an overall ratio of 4.5:1 [ figure 1 ] .
majority of maxillofacial injuries were seen in 2 - 4 decade of life constituting a major proportion ( 76.49% ) of these maxillofacial injuries [ figure 2 ] .
of the eight causes for sustaining maxillofacial injuries , rtas were the most common ( 73.95% ) followed by interpersonal violence ( 13.34% ) [ figure 3 ] . the fracture of the mandible was most common maxillofacial injury ( 44.34% ) followed by mid face fractures ( 18.42 ) [ figure 4 ] .
parasymphysis fracture was the most common ( 38% ) lower third fractures [ figure 5 ] and zygomatic complex fractures were the most common of the middle third fracture ( 55.9% ) [ figure 6 ] .
sex distribution of maxillofacial injuries age distribution of maxillofacial injuries etiology of maxillofacial injuries type of maxillofacial injuries distribution of mandibular fractures distribution of middle third fractures
the higher prevalence of maxillofacial injuries in males as in our study is well - documented in the literature .
males are at greater risk due to their greater participation in activities such as driving vehicles , sports that involve physical contact , an active social life and drug use , including alcohol .
however over the past 3 decades , an increasing prevalence of trauma has been reported among females , mainly in the under-40 age group , probably due to change in women 's social behavior , including their involvement in non - domestic work , a greater active social life , participation in vehicular traffic , and sports as a leisure and health activity .
the fact that majority of the victims were in the 21 - 30 years age group ( 39% ) is also in accordance with other studies .
this is possibly due to behavioral changes and socioeconomic and emotional conflicts to which these young adults are exposed .
this age group is recognized as a phase of great personal independence , social excitement , intense mobility , careless driving on the roads , and exposure to urban violence .
children and individuals over 40 years are less involved in maxillofacial injuries . however , the considerable number of patients in the age group of 010 years ( 14% ) underline the importance of the development and adoption of specific strategies for the prevention of trauma during the childhood , mainly the prevention of falls , traffic accidents , and domestic violence .
rtas were the main cause of maxillofacial injuries ( 74% ) , corroborating other indian and international studies .
the increasing number of rtas in developing countries like india may be attributed to many factors like sharing of roadways by pedestrians and animals with vehicular traffic , low driving standards , large number of old and poorly maintained vehicles on road , large number of two wheelers , defective roads , and widespread disregard for traffic rules . the increasing use of mobile phones and drunken driving is becoming a growing concern for road safety .
the disturbance caused by mobile phone usage can impair driving performance in number of ways like longer reaction time , inability to keep correct lane .
safer roads , effective law enforcement , and public transport policies contributed to a significant decrease in the occurrence of traffic accidents in developed countries over the last 3 decades .
vehicle accident statistics indicate that the best protection against injury includes safety awareness courses , defensive riding skills and a personal commitment to ride safely at all times .
physical violence is another increasingly important etiological factor for maxillofacial injuries . in countries like united states , finland , and switzerland assaults
have been reported as the main cause of maxillofacial injuries . in the present study , assaults were the second most prevalent etiological factor ( 13.34% ) , which reinforces the need for the development of preventive programs , aiming to help individuals , organizations , and communities ; and government agencies plan proactively for the successful mitigation of unexpected violence
. the higher involvement of mandible may be attributed to its prominence and also to its exposed anatomical position on the face .
most of the victims of rtas will try to avoid their head against injury at the time of accidents .
thus , in the process of avoiding their head , may receive maximum impact to the mandible .
the studies conducted by veeresha and shankararadhya , motamedi , ortakoglu et al . , and qudah and bataineh , have also found mandibular fracture to be the most common maxillofacial injury .
the force of the blow is transferred from the chin along the mandible to the condyle causing fractures in the neck , which is one of the weak anatomical locations within the mandible .
the long roots of canines , presence of third molars , and also the abrupt change in the direction between the large , strong body of the mandible , and the thin ascending ramus make the parasymphysis and the angle region , the other two weak anatomical sites susceptible for fractures as found in the studies by veeresha and shankararadhya , motamedi , and orkatoglu et al .
the observed high incidence of nasal and zygomatic complex in most of the middle third fractures is obviously related to the prominent position of these anatomic structures within the facial skeleton , and their greater exposure to external trauma .
however , few cases of nasal fractures are reported in maxillofacial trauma studies as patients are usually referred to ear , nose , and throat ( ent ) and plastic surgeons .
the studies by al khateeb and abdullah have found zygomatic complex as the most common middle third fracture which is coinciding with the results of this study .
the findings of this study indicate the need for development of emergency protocols , effective educational and preventive strategies , and the implementation of policies aimed at preventing and reducing maxillofacial injury and its effects . | introduction : the incidence of maxillofacial injuries is on the rise due to motor vehicle accidents and increased incidence of violence in recent times .
the aim of this retrospective study was to determine the incidence , etiology , and the pattern of fractures in the maxillofacial region.materials and methods : after obtaining permission from the concerned authorities , a predesigned questionnaire was used to collect the necessary data from the department .
a retrospective analysis of 787 patients , who suffered trauma and were managed in the department of oral and maxillofacial surgery , indira gandhi government dental college ( iggdc ) , jammu over a period of 2 years was carried out.results:road traffic accident ( rta ) was the common cause of maxillofacial injuries .
men sustained more injuries as compared to women .
injuries were most commonly sustained in the age group of 11 - 40 years , constituting about 76% of all injuries , mandibular fractures were the most common.conclusion:rtas were the commonest cause for the maxillofacial injuries . |
improvement in the standard of living and changes in diet have led to an increase in the
prevalence of hypertension , arteriosclerosis , and diabetes mellitus . moreover ,
cerebrovascular diseases are
the second - highest cause of death , followed by cancer , in korea1 . among the cerebrovascular diseases ,
the prognosis of stroke after the age of
65 years is usually poor , and more than 80% of patients experience neurological damage2 .
the rate of disability that affects
activities of daily living after a stroke , compared to other chronic diseases , is very high .
long - term rehabilitation is necessary because of the loss of function in the upper and lower
extremities , speech impairment , and cognitive disorders that accompany a stroke3 , 4 . with regard to the recovery of neurological motor functions after a stroke ,
patients show
80% and 95% recovery of the upper and lower extremity function within 3 months ,
respectively5 . despite intensive ,
long - term treatment , most stroke patients fail to show complete recovery of the function of
their upper limbs6 , 7 . among the upper extremity functions ,
various
treatments , including drugs and exercise , are available to enhance motor functions after a
stroke .
other methods include neurological physical therapy focusing on neurodevelopment
therapy and proprioceptive neuromuscular facilitation , occupational therapy , ischemic nerve
block , functional electrical stimulation , constraint - induced movement therapy9 , 10 ,
and robot - assisted therapy for upper limb rehabilitation11 .
these treatment methods may speed up the recovery of patients
upper limb functions but require long - term efforts , and continuous treatment may be
difficult for various reasons .
recent research suggests that transcranial direct current
stimulation ( tdcs ) of the brain can improve physical functions in stroke patients12,13,14,15 .
therefore , we investigated the effects of tdcs on the recovery of upper limb functions in
stroke patients .
the study included 24 patients with stroke diagnosed by a specialist at c hospital located
in d metropolitan city who were receiving rehabilitation treatment for functional recovery .
in all the participants , hemiplegic patients were composed of at least 6 months after the
onset .
participants with a history of stroke , with a personal or family history of epileptic
seizure , who underwent implantation of an artificial cardiac pacemaker , or who showed severe
upper extremity contracture and deformity were excluded , because participation of such
patients was difficult .
patients with hemiplegia who were diagnosed with a stroke were
randomly assigned to either an experimental or a control group .
data were collected from
patients who agreed to participate after they fully understood the research procedures .
all
procedures were approved by the institutional review board committee of c national
university hospital in the korea . fugl - meyer assessment ( fma )
was performed before the experiment for all the patients to
determine the function of their upper limbs16 .
the experimental group received tdcs and physical therapy , whereas
the control group received only physical therapy .
after 4 weeks , the fma was performed
again , and the intervention was performed 5 times per week for 4 weeks .
kolmogorov - smirnov single - sample test was performed to examine the general characteristics
of the participants and the normality of each item .
the data were analyzed with spss 18.0 spss
inc , chicago , il , usa ) for windows , and the significance was set at p < 0.05 .
with regard to shoulder function , the mean standard deviation ( sd ) fma scores were 22.44
5.64 before and 21.00 6.08 after the treatment in the control group , and they were 22.64
6.02 before and 26.82 4.92 after the treatment in the experimental group ; the changes
were statistically significant ( p < 0.01 ) .
the mean sd fma scores for the elbow joint
were 5.00 3.74 before and 5.11 3.72 after the treatment in the control group , whereas
they were 7.09 2.34 before and 8.45 1.92 after the treatment in the experimental group ;
the changes were statistically significant ( p < 0.05 ) . in the control group ,
the mean
sd fma scores for hand function were 8.44 5.22 before and 8.89 5.16 after the treatment .
in the experimental group ,
the mean sd fma scores were 10.45 2.62 before and 12.45
2.16 after the treatment . the changes in both the groups were statistically significant ( p
< 0.05)(table 1table 1.changes score of fma items in a stroke patients of before and after transcranial
direct current stimulationgroupprepost(%)shoulderexperiment ( n=12)22.646.0226.824.92control ( n=12)22.445.6421.006.08elbowexperiment ( n=12)7.092.348.451.92control ( n=12)5.003.745.113.72handexperiment ( n=12)10.452.6212.452.16control ( n=12)8.445.228.895.16values are mean sd , * p<0.05 , * * p<0.01 .
in this study , we applied tdcs , which activates the motor areas in the cerebral cortex , in
combination with physical therapy for patients with a stroke and who experienced hemiplegia
for 6 months .
the changes in the fma scores for the shoulder joint , elbow joint , and hand functions were
greater in the group that received tdcs than in the group that received only physical
therapy .
the fma is considered the best tool for the assessment of a patient s motor
functions .
park and choi17 reported that
fma was more effective than other methods for the assessment of the recovery of upper
extremity motor functions .
in addition , a correlation between the effects of tdcs on upper
extremity functions and fma scores was verified , and fma was found to be a valid method for
the evaluation of upper limb functions .
hummel et al.18 and harvey et al.19 applied tdcs for chronic stroke patients and observed that the
extent of improvement in the motor function performance , including fma scores , was greater
in the tdcs group than in the non - tdcs group .
hummel et al.18 reported that tdcs
applied for chronic stroke patients reduced the reaction time of the paretic hand and
improved the pinch force . in the present study ,
the shoulder joint , elbow joint , and hand
function scores in the experimental group increased significantly , and these findings
support the findings of hummel et al18 .
therefore , the results
can not be generalized to all female patients with stroke .
studies in a larger number of
participants would generate results that are more meaningful .
the validity of the study
would also be improved by clarifying the criteria for the selection of participants .
therefore , additional research needs to focus on
elucidation of the association between changes in the motor areas in the cerebral cortex and
improvement in muscle performance of the upper limbs . | [ purpose ] the purpose of this study was to examine the effects of transcranial direct
current stimulation ( tdcs ) applied to the cerebral cortex motor area on the upper
extremity functions of hemiplegic patients .
[ subjects and methods ] twenty four patients
with hemiplegia resulting from a stroke were divided into two groups : a tdcs group that
received tdcs and physical therapy and a control group that received only physical
therapy .
a functional evaluation of the two groups was performed , and an
electrophysiological evaluation was conducted before and after the experiment .
statistical
analyses were performed to verify differences before and after the experiment .
all
statistical significance levels were set at 0.05 . [ results ] the results showed that
functional evaluation scores for the elbow joint and hand increased after the treatment in
both the experimental group and the control group , and the increases were statistically
significantly different . [ conclusion ] tdcs was effective in improving the upper extremity
motor function of stroke patients .
additional research is warranted on the usefulness of
tdcs in the rehabilitation of stroke patients in the clinical field . |
a body mass index ( bmi ) of 30 or greater is defined as obesity with a bmi less than 35 classified as grade 1 obesity , bmi equal to or greater than 35 but less than 40 classified as grade 2 obesity , whereas a bmi 40 categorized as grade 3 obesity . the risk of coronary heart disease , stroke , diabetes , and cancer increases with increasing bmi / obesity . according to the world health organization ( who ) , more than half a billion adults worldwide were obese in 2014 , with the prevalence of obesity being four times higher in developed / high - income countries as compared to low - income countries . since 1980 ,
a majority of world s population now lives in countries where being overweight and obese kills more people than being underweight .
obesity rates have grown more rapidly in the industrialized countries than other regions of the world and recently the highest prevalence of obesity among the developed countries was seen in the united states ( us ) and australia . in the us , rising obesity rates have become a major public health issue , with more than one - third of adults in the us , i.e. 78.6 million people , being obese and the estimated annual cost of obesity reaching approximately $ 147 billion .
such statistics in a developed nation with high levels of awareness about and efforts to control the obesity epidemic highlight the need for a thorough understanding of the determinants of rising bmi and obesity levels in the country . as in other industrialized countries ,
studies have found differences in obesity prevalence across various socioeconomic , racial / ethnic , gender , and age groups in the us.[5 , 7 , 8 ] for example , overall prevalence of obesity is higher among blue collar workers than white collar workers .
similarly , lower education , lower occupational status , and lower incomes have been associated with higher prevalence of obesity . in terms of racial / ethnic disparities , non - hispanic blacks , american indian / alaska natives , and mexican americans
have the highest obesity prevalence in the us . however , such socio - demographic determinants of obesity have hardly been studied recently taking into account certain important health behaviors associated with obesity .
the behavioral factors known to be associated with obesity include smoking , alcohol consumption , physical activity , and dietary habits.[13 - 16 ] although there is not enough evidence to suggest that smoking leads to loss of weight or a steady state weight , it has been found that those who quit smoking are likely to gain weight and ex - smokers are more likely to be obese .
alcohol consumption , especially heavy drinking , is linked with higher obesity prevalence , so is poor quality diet containing less fruits and vegetables and more fats and sugars .
many americans are not meeting the dietary recommendations as per the dietary guidelines issued by the united states department of agriculture . physical activity
( pa ) has also been established as one of the important predictors of weight gain with those getting enough pa , especially during leisure time , being less likely to be obese . with modern working environments entailing prolonged sedentary work and stresses of modern lifestyle cutting into leisure time ,
not many are able to achieve the recommended levels of pa , at job or during leisure .
previous studies have examined many of the important determinants of obesity . however , except for one study by rohrer et al , we were unable to find studies which have looked at the independent effect of all these possible socio - demographic and behavioral determinants .
rohrer et al primarily focused on determining how the frequency of alcohol use was related to obesity , taking into account age , cigarette smoking , pa , and health status .
however , the study participants were derived only from three community clinics in texas , serving a predominantly lower socioeconomic population rather than a representative national sample .
literature is scarce on research examining variations in obesity or bmi across the possible determinants .
the purpose of this study was to examine the socio - demographic and behavioral determinants of obesity and bmi among adults in a national sample using data from 2010 national health interview survey ( nhis ) .
our aim was to provide a comprehensive assessment of the current obesity and bmi disparities in the united states across racial / ethnic , socioeconomic , and many important behavioral factors .
the nhis uses multistage , cluster sampling and is primarily administered through direct in - person interviews . the household response rate for 2010 nhis was 79.5% .
we limited our analysis to sample adults ( 18 years ) for whom information on bmi and other variables of interest such as smoking was available .
we excluded all observations in which information was missing for any of the variables being studied , except poverty status .
the 2010 nhis included bmi calculated for all persons who self - reported height and weight using the formula : bmi = weight in kilograms / height in meters squared .
obesity was defined as bmi>=30 kg / m . four socioeconomic covariates were considered : education , occupation , poverty status , and home ownership .
poverty status was defined as the ratio of family income to the us federal poverty threshold .
demographic covariates included age , race / ethnicity , gender , and region of residence .
these covariates were measured as categorical variables as shown in table 1 . weighted sample characteristics , percent obese and mean bmi ( n=23,434 ) for continuous variables ( i.e. , frequency of consuming each food item per week ) , the mean and standard deviation of bmi is reported . for continuous variables ,
the % obese for the category below the median and the category at or above the median , separated by a slash , are reported .
p - values for the association between obesity and each covariate . for continuous variables , the mean bmi for the category below the median and the category at or above the median , separated by a slash , are reported .
p - value for the association of bmi and each covariate , the food item variables indicate frequency of consumption per week .
this food category includes fruit , 100% fruit juice , salad , and other vegetables .
this food category includes processed meat and red meat to determine smoking status , respondents were asked have you smoked at least 100 cigarettes in your entire life ? those who replied affirmatively were asked do you now smoke cigarettes every day , some days or not at all ?
current smokers were defined as those who have smoked at least 100 cigarettes in their life and currently smoked every day or some days , former smokers as those who have smoked more than a 100 cigarettes in their life but do not currently smoke , and never smokers as those who have not smoked more than a 100 cigarettes in their life .
alcohol consumption was categorized into six groups : ( 1 ) never drinker who had consumed less than 12 drinks in lifetime ; ( 2 ) former drinker who had consumed 12 drinks in lifetime but none in the past year ; ( 3 ) current infrequent drinker with consumption of 12 drinks in lifetime and between 1 to 11 drinks in the past year ; ( 4 ) current light drinker who had consumed 12 drinks in lifetime and 3 drinks per week in the past year ; ( 5 ) current moderate drinker who had consumed 12 drinks in lifetime , and > 3 drinks and up to 14 drinks per week in the past year among males , or > 3 and up to 7 drinks per week in the past year among women ; ( 6 ) current heavy drinker defined by 12 drinks in lifetime , and > 14 drinks per week in the past year among males , and > 7 drinks per week in past year among women .
the variable for adherence to pa recommendations was computed based on a set of leisure - time physical activity questions which included frequency and duration of vigorous activities , frequency and duration of light or moderate activities , and frequency of strengthening activities .
responses to questions about vigorous and moderate activities were converted into minutes of these activities of 10 minutes or longer per week and the responses to strengthening activity questions were converted into frequency in times per week .
these converted responses were then combined to create a variable to measure adherence to the pa recommendations .
the 2008 us department of health and human services guidelines on pa , recommend that adults should do at least 150 minutes a week of moderate - intensity , or 75 minutes a week of vigorous - intensity aerobic pa , or an equivalent combination of moderate- and vigorous intensity aerobic activity along with muscle - strengthening activities on 2 or more days a week .
respondents were asked to indicate how often during the past month they consumed any of the following food items : fruit , 100% fruit juice , salad , other vegetables , beans , whole grain bread , brown rice , red meat , processed meat , fried potatoes , candy , cookies , donuts , ice cream , non - diet soda , and fruit drinks . for each respondent , we computed frequency of consumption of each food item per week .
food items such as salad , fruits , vegetables and fruit juice were grouped together as fruits and vegetables .
similarly the sugar sweetened beverages group was created by adding soda and fruit flavored drink consumption . finally ,
the food item group meat was created by adding red meat and processed meat consumption .
weighted data were analyzed to report descriptive statistics as well as the distribution of obesity and mean bmi across different categories of the categorical covariates .
bivariate analysis was performed using logistic and linear regression to compute the unadjusted odds ratios and beta coefficients for obesity and bmi , respectively .
multivariate logistic and linear regressions were performed to estimate the association of these covariates with obesity and bmi , respectively . to account for the complex sampling design of nhis survey in all analyses
, we took into account sampling weights , stratification , and primary sampling units in computations .
overall , 28.1% of the adults in the sample were obese and the mean bmi in the sample was 27.6 kg / m .
table 1 shows the weighted sample characteristics including obesity prevalence and mean bmi across different categories of the categorical covariates . for continuous variables of food item consumption ,
table 1 shows obesity prevalence and mean bmi among those who consumed below and those who consumed at or above the median .
significantly higher obesity prevalence and mean bmi were seen among those aged 40 - 54 years than other age groups , and among non - hispanic blacks than other racial / ethnic groups .
males had higher bmi than females ( p<0.001 ) . those with a college degree had lower obesity prevalence and lower bmi than those with lower educational attainment ( p<0.001 ) . those working as laborers had higher obesity prevalence and bmi than those in other occupational categories .
individuals with family incomes at or above 500% of the poverty threshold ( $ 11,137 for 1 person family , $ 14,216 for a family of 2 , and $ 17,373 - $ 45,224 for families with 3 - 9 people for the year 2010 ) had significantly lower obesity prevalence and bmi than those at lower levels of income .
those residing in the south and midwest of us had significantly higher obesity prevalence and bmi than those living in the western us .
higher obesity prevalence and mean bmi were seen among former smokers than current or never smokers , and among former alcohol drinkers than any other alcohol consumption category ( p<0.001 ) . those who did not adhere to the pa recommendations had higher obesity prevalence and bmi than those who did ( p<0.001 ) .
lower consumption of fruits and vegetables ( p<0.001 ) , and consumption of brown rice ( p<0.001 ) were associated with significantly higher obesity prevalence and bmi . on the other hand ,
higher consumption of sugar sweetened beverages , meat , and fried potatoes was associated with significantly higher obesity prevalence and bmi ( p<0.001 for all associations ) .
lastly , lower consumption of whole grain bread ( p=0.026 ) and higher consumption of cheese ( p=0.016 ) were associated with higher obesity prevalence and bmi respectively .
table 2 presents the unadjusted and adjusted odds ratios for the association of obesity with each of the covariates as well as the adjusted regression coefficients ( expected mean difference in bmi ) for the association of bmi with the covariates .
regression of obesity and bmi on covariates ( n=23,434 ) adjusted for all covariates , results of the adjusted analysis were consistent with the unadjusted analyses for most of the covariates .
those aged 40 - 54 had higher odds of being obese and higher bmi ( or=2.38 , =3.29 ) than those aged 18 - 24 years .
non - hispanic blacks had higher odds of being obese and higher bmi ( or=1.62 , =1.66 ) than non - hispanic whites .
individuals in all education categories had higher odds of obesity and higher bmi than those with 16 years of education .
individuals in all occupational groups ( except laborers ) had lower odds of obesity and lower bmi as compared to those in professional occupations once all other socio - demographic and behavioral differences were accounted for .
individuals with incomes below 500% of poverty threshold had higher odds of obesity and higher bmi than those living at 500% of poverty threshold .
those from the midwestern and southern us regions had significantly higher odds of being obese and higher bmi than living in the western us .
the odds of obesity and bmi were higher among former smokers ( or=1.61 , =1.42 ) than current smokers .
former and infrequent alcohol users were more likely to be obese ( or=1.19 and 1.31 , respectively ) , whereas moderate and heavy alcohol users were less likely to be obese as compared to never drinkers .
those who did not adhere to the pa recommendation were more likely to be obese and had higher bmi ( or=1.51 , =0.91 ) than those who did .
higher consumption of fruits and vegetables and brown rice was associated with lower odds of obesity and lower bmi . increased meat , fried potato , and cheese consumption was associated with higher odds of obesity and higher bmi .
consumption of sugar sweetened beverages and whole grain bread were not significantly associated with obesity or bmi .
the purpose of this study was to examine the socio - demographic and behavioral determinants of obesity and bmi in a national sample of adults in us .
we found that old age , non - hispanic black race , 9 - 15 years of education , professional occupations , lower income , and midwestern and southern us region of residence were associated with higher adjusted odds of obesity and higher bmi .
behavioral factors such as former smoking status , infrequent alcohol use , and non - adherence to pa recommendations were also related to higher odds of obesity and higher bmi .
lower consumption of fruits and vegetables and brown rice and higher consumption of meat , fried potato and cheese was associated with higher odds of obesity and higher bmi . in general , our findings regarding age , racial / ethnic , and regional patterns in obesity were consistent with those found in previous studies.[8 , 28,29 ] consistent with previous research , we found higher likelihood of obesity and higher bmi among non - hispanic blacks than any other racial / ethnic group in both unadjusted and adjusted analyses .
however , this finding needs to be interpreted with caution since bmi is used here as a measure of obesity .
previous research has found racial / ethnic differences in the degree of adiposity at a given bmi level,[30 - 33 ] with non - hispanic blacks having higher lean mass and lower fat mass as compared to non - hispanic whites at the same bmi level .
although bmi is known as a standardized measure of obesity , it does not represent adiposity directly .
thus , the racial / ethnic differences found in our study may not necessarily correspond to differences in fat mass or percentage of body fat . in both unadjusted and adjusted analyses ,
lower levels of education and incomes were generally associated with higher likelihood of obesity and higher mean bmi .
these findings are consistent with previous research showing higher obesity and bmi among those at lower levels of socioeconomic position . however , in the adjusted models , those in professional occupations had higher likelihood of obesity and higher bmi than those in non - professional occupations , somewhat inconsistent with the patterns seen in previous studies .
diet , total energy intake , physical activity , sedentary life styles , and other health - risk behaviors might explain much of the observed socioeconomic differences in obesity and bmi and are known to act as proximate , intervening variables in the relationship between socioeconomic status and obesity .
thus , the comparison of unadjusted and adjusted models indicates that dietary factors , pa , and other socio - behavioral factors largely explain education , occupation , and income / poverty influences on obesity and bmi shown here .
consistent with previous findings , we found that former smokers were more likely to be obese and had higher bmi as compared to current smokers .
these previous studies had claimed that quitting smoking was associated with weight gain which is further corroborated by the fact that between 1970 and 2002 the obesity rate for americans has more than doubled , and during the same period , the smoking rate has declined by more than 10% . in our study , even never - smokers had higher odds of being obese and higher bmi as compared to current smokers .
many of the smokers who attempt to quit , claim weight gain as the reason for their adopting smoking again .
although exact mechanism of the association between smoking cessation and weight gain is unknown , many of the smokers do gain weight upon quitting , which is why providing weight management resources along with help in quitting is necessary for the success of cessation as well as weight management programs . in case of alcohol consumption
, we found that current infrequent alcohol users had higher odds of being obese and higher bmi as compared to never users , which was inconsistent with previous findings by rohrer et al who found lower odds of obesity among current infrequent drinkers , and arif et al who had found that the heavy alcohol users were more likely to be obese as compared to never users .
in fact , we found the lowest odds of obesity and lowest bmi among heavy drinkers .
arif et al had used 1988 - 1994 nhanes data , whereas the rohrer study used data collected from patients of clinic serving predominantly low socioeconomic group population .
differences in data collection , time periods , and population along with adjustment for multiple covariates different from the ones in our study , might explain the inconsistency in our findings .
as increased alcohol consumption has been found to be associated with poor diet , we performed additional analysis ( not shown here ) and found that heavy alcohol consumption was indeed significantly associated with higher consumption of unhealthy food items , such as fries and sweets and lower consumption of fruits and vegetables . yet
, our results suggest a possible confounding effect of other factors on the relationship between alcohol consumption , poor diet and obesity .
this interesting finding needs to be explored further , possibly using anthropometric measurements and longitudinal data .
we also found non - adherence to pa recommendation to be associated with higher odds of obesity and higher bmi , which was consistent with previous findings .
lack of pa contributes to the imbalance between energy intake and expenditure and predisposes one to weight gain .
also contributing to the energy imbalance , we found that dietary habits such as lower consumption of fruits , vegetables , and brown rice and higher consumption of cheese , fries and meat , were associated with higher likelihood of obesity and higher bmi .
this finding was consistent with previous research ; however an interesting finding in our study was that of higher consumption of sweets such as candy , cookies , donuts and ice cream , being associated with lower odds of obesity as well as lower bmi .
this finding was consistent in both bivariate and multivariate analyses and should be further explored in future studies .
many of the measures used in our analysis including height and weight used for calculating bmi and obesity , pa , dietary habits , smoking and alcohol consumption status were all self- reported .
although bmi as a measure of obesity correlates with fat distribution , it does not measure percent body fat and it does not account for those with predominantly muscular build .
also , as stated earlier , while self - reporting , there is a general tendency to over - report height and under - report weight and those who are heavier are more likely to under - report their weights . despite this
, bmi based on self - reported height and weight is considered a valid measure of obesity .
similarly , smoking and alcohol consumption are likely to be underreported , whereas leisure time pa might be either under or over - reported .
however , the validity of self - report smoking , self - report alcohol consumption as measures of smoking , alcohol use and pa level has been established .
self - report bias in reporting dietary habits are well known , and thus associations of dietary factors with our outcomes must be interpreted with caution . finally , the cross - sectional nature of the nhis data only allows for reporting the association of the covariates with our outcome of interest and does not permit establishment of causation .
our study is one of the few studies that have tried to shed light on the determinants of obesity and bmi while accounting for a wide range of socioeconomic , demographic and behavioral factors together . using data from a recent and nationally representative survey with a high response rate
, we were able to report the disparities in obesity and bmi across different covariates .
many of our findings were consistent with previous findings reiterating the association of factors such as age , race / ethnicity , education , occupation , region , smoking status , alcohol consumption , pa , and dietary habits with weight gain .
such evaluations of disparities in obesity on a continued basis , not only helps policy makers and public health professionals to monitor the progress of their efforts but also lets them make changes , if required , into the direction and focus of their interventions . persistent racial / ethnic inequalities in the distribution of obesity and bmi call for more targeted and innovative approaches to better understand and reduce such disparities .
given the association of lower socioeconomic status with obesity , public health programs should aim at reaching this subpopulation at an early stage , educating and empowering the children from lower socioeconomic groups so that their disadvantages do not accompany them into adulthood .
healthy habits such as physical activity , healthy diet , and abstinence from tobacco and alcohol , should be instilled in early life in order to achieve long - term health benefits .
re - energizing the physical education program in public schools could be a good start .
additionally , access to healthy food choices could be improved for the socioeconomically disadvantaged population through programs such as community gardens , fresh produce markets , access to physical activity resources and other interventions targeting the environmental factors affecting energy balance .
it is only when public health policies and programs at the population level are able to compliment the medical healthcare services at the individual level that we would be able to achieve a reduction in the huge morbidity and mortality burden of obesity .
our study findings have implications for many low- and middle - income countries facing the rising obesity epidemic .
besides the health and economic impact , obesity has many social and psychological consequences both at the individual and societal level , which makes it immensely important to develop public health programs that could help avoid the adverse social and health consequences of an uncontrolled epidemic .
such consequences are likely to be particularly detrimental to the low - income and middle - income countries . in view of the rising obesity levels in developing countries ,
the development of successful policies and programs that effectively address the obesity epidemic in high - income countries is essential in order to serve as guide for these countries to be better prepared to face the challenges that they are likely to encounter .
similar to the socio - economic disparities in obesity , risk factors such as poor diet and physical inactivity are also no longer restricted to those in high - income countries this suggests that the developed countries , with their abundant resources , could help lead and guide these efforts to achieve the desired global health objectives in terms of reducing obesity as well as the associated social inequalities .
our results could help provide insights into the development of policies and programs that could be expanded and adapted to the needs of low- and middle - income countries struggling with rising obesity rates . until there is a strong policy action to reduce inequalities in socioeconomic conditions and behavioral risk factors ,
overweight and obesity are among the top 5 leading risk factors of mortality in the world .
28.1% us adults were obese in 2010 with a mean bmi of 27.6 kg / m .
these rates keep rising and the obesity prevalence in the us now exceeds 30%.non - hispanic blacks , those residing in the midwestern and southern united states , and those with lower education , income , and occupational status had higher likelihood of obesity and higher bmi than others .
former smokers were more likely to be obese and had higher bmi than current smokers , indicating that providing weight management resources along with help in quitting is necessary for the success of cessation as well as weight management programs.non-adherence to physical activity and lower consumption of fruits , vegetables , and brown rice and higher consumption of cheese , fries and meat were associated with higher likelihood of obesity and higher bmi .
overweight and obesity are among the top 5 leading risk factors of mortality in the world .
non - hispanic blacks , those residing in the midwestern and southern united states , and those with lower education , income , and occupational status had higher likelihood of obesity and higher bmi than others .
former smokers were more likely to be obese and had higher bmi than current smokers , indicating that providing weight management resources along with help in quitting is necessary for the success of cessation as well as weight management programs .
non - adherence to physical activity and lower consumption of fruits , vegetables , and brown rice and higher consumption of cheese , fries and meat were associated with higher likelihood of obesity and higher bmi . | objectives : the aim of this research was to study the socio - demographic and behavioral determinants of obesity and body mass index ( bmi ) in the united states , using a nationally representative sample.methods:we used data from the 2010 us national health interview survey .
analyses were limited to adults 18 years and older ( n=23,434 ) .
multivariate regression analyses were conducted to estimate the associations between covariates and obesity and bmi.results:overall , 28.1% in the sample were obese and the mean bmi was 27.6 kg / m2 . in adjusted models , we found that older age , non - hispanic black race , lower education and income levels , midwestern and southern region of residence , former smoking , infrequent alcohol use , physical inactivity , consumption of less fruits , vegetables , brown rice and more cheese , fried potato and meat , were associated with obesity .
these factors were also associated with higher bmi , along with male gender and higher consumption of meat , fried potatoes and cheese.conclusions and global health implications : the association of many of the socio - demographic and behavioral factors with obesity and higher bmi found in our study was consistent with previous findings .
persistence of such associations suggest a need for better understanding of the underlying mechanism as well as for evaluation of the current programs and policies targeted at reducing the obesity burden in the united states . in view of the rising global obesity epidemic , especially in the low- and middle - income countries , our findings could help guide development of effective health and social policies and programs aimed at reducing the obesity burden in other parts of the world . |
all aspects of the study were conducted in accordance with the declaration of helsinki and received a favorable ethical opinion from the surrey research ethics committee and the university of surrey ethics committee .
they then attended a prescreening session at the university of surrey , typically within 1 month of the laboratory session and during the early afternoon .
bmi and waist circumference were measured , and fasting blood samples were provided for measurement of plasma hba1c , insulin and glucose concentrations , and homeostasis model assessment of insulin resistance ( homa - ir ; calculated using homa calculator version 2.2 software [ diabetes trial unit ; university of oxford , oxford , u.k . ] ) .
twenty - seven male volunteers were recruited ( 8 lean , 11 overweight , and 8 overweight with type 2 diabetes ) . of the patients with type 2 diabetes , average time since diagnosis was 6.9 2.3 years ; three type 2 diabetic patients were controlled by diet and exercise , and the other five patients were treated with combinations of metformin , statins , ramipril , and lisinopril .
one participant within the lean group was a smoker and was required to refrain from smoking for 1 week prior to the study .
none of the participants had undertaken shift work within 5 years of the study or crossed any time zones within 1 month of the study . for 1 week prior to the laboratory study , volunteers were required to maintain prescribed daily feeding times and sleep activity ( sleep 22300630 h ) , which were monitored using wrist actigraphy ( awl ; cambridge neurotechnology , cambridge , u.k . ) , sleep diaries , food diaries , and recorded messages on a laboratory time - stamped answer telephone .
participants also were required to refrain from eating fatty or sugary foods and drinking alcohol or caffeine throughout the week . for the final 3 days of this baseline week
, food was provided by the research team to enable control of timed behavior together with both the quantity and quality of nutrient intake . during these 3 days ,
the daily caloric content of the supplied food was basal metabolic rate times 1.5 with ~35% of energy from fat .
volunteers arrived in the afternoon of day 0 for a night of adaptation . throughout the 2-day study
they were required to remain awake with lights on between 0630 and 2230 h ( 440825 lux in direction of gaze ) and allowed to sleep with lights off between 2230 and 0630 h ( 0
, participants were fed with hourly nutritional drinks ( fortisip ; nutricia , schiphol , the netherlands ) and were allowed to drink water ad libitum .
daily energy intake was basal metabolic rate times 1.1 , divided equally over the waking hours .
four subcutaneous wat biopsies were taken under a local anesthetic ( lidocaine ) from four different sites of the upper buttock region of each participant at 6-hourly intervals for 24 h , beginning at 1030 h on day 1 .
wat biopsies were washed with saline and snap - frozen in liquid nitrogen before storage at 80c .
cdna was synthesized , and expression was measured for genes integral to the circadian clock ( per1 , per2 , per3 , cry2 , bmal1 , and dbp ) and involved in metabolic activity ( reverb , rip140 , and pgc1 ) by quantitative taqman real - time pcr , as described previously ( 21 ) ( table 1 ) .
standard curves ( r > 0.99 ) were generated using human genomic dna ( promega , southampton , u.k . ) , and expression of all genes was normalized to gapdh , as in previous studies of adipose clock gene expression ( 15,16 ) .
taqman pcr primer probe sets prescreen data were analyzed using one - way anova with tukey s post hoc test .
correlation analyses were conducted using linear regression , and the gene expression time course from biopsies was performed using one - way or two - way repeated - measures anova ( factors time and group ) with tukey s post hoc test .
volunteers arrived in the afternoon of day 0 for a night of adaptation . throughout the 2-day study
they were required to remain awake with lights on between 0630 and 2230 h ( 440825 lux in direction of gaze ) and allowed to sleep with lights off between 2230 and 0630 h ( 0 lux ) . during the waking period , participants were fed with hourly nutritional drinks ( fortisip ; nutricia , schiphol , the netherlands ) and were allowed to drink water ad libitum .
daily energy intake was basal metabolic rate times 1.1 , divided equally over the waking hours .
four subcutaneous wat biopsies were taken under a local anesthetic ( lidocaine ) from four different sites of the upper buttock region of each participant at 6-hourly intervals for 24 h , beginning at 1030 h on day 1 .
wat biopsies were washed with saline and snap - frozen in liquid nitrogen before storage at 80c .
cdna was synthesized , and expression was measured for genes integral to the circadian clock ( per1 , per2 , per3 , cry2 , bmal1 , and dbp ) and involved in metabolic activity ( reverb , rip140 , and pgc1 ) by quantitative taqman real - time pcr , as described previously ( 21 ) ( table 1 ) .
standard curves ( r > 0.99 ) were generated using human genomic dna ( promega , southampton , u.k . ) , and expression of all genes was normalized to gapdh , as in previous studies of adipose clock gene expression ( 15,16 ) .
prescreen data were analyzed using one - way anova with tukey s post hoc test .
correlation analyses were conducted using linear regression , and the gene expression time course from biopsies was performed using one - way or two - way repeated - measures anova ( factors time and group ) with tukey s post hoc test .
prescreen data are shown in table 2 . despite efforts to age - match the participants , there was a significant ( p < 0.05 ) difference in age , with post hoc analysis revealing a significant difference only between the overweight and type 2 diabetic groups .
however , subsequent analysis using age as a covariant revealed that age , per se , did not alter gene expression ( data not shown ) .
there were significant ( p < 0.05 ) differences in bmi , waist circumference , fasting plasma glucose and insulin concentrations , hba1c levels , and homa - ir , with highest values occurring in participants with type 2 diabetes .
there was no significant difference in plasma insulin , glucose , hba1c , or homa - ir between the lean and overweight / obese groups , indicating that our overweight / obese participants were insulin - sensitive .
participant prelaboratory data * p < 0.05 vs. overweight / obese group . p < 0.05 vs. lean group . in lean individuals , there was a significant ( p < 0.05 ) effect of time on the expression of multiple genes involved in circadian ( per1 , per2 , per3 , cry2 , bmal1 , and dbp ) and metabolic functions ( reverb , rip140 , and pgc1 ) ( fig .
minimal expression of reverb and dbp occurred around the middle to end of the afternoon . in keeping with circadian gene expression in other tissues ( 10 ) , the phase of per1 , per2 , per3 , and cry2
was slightly delayed compared with reverb and dbp , with minimal expression occurring around the evening light dark transition .
the bmal1 rhythm was in approximate antiphase to the other circadian genes , with maximal expression around the evening light dark transition .
data are presented as means se of n = 811 values , normalized to glyceraldehyde 3-phosphate dehydrogenase .
there was a significant effect of time ( p < 0.05 , one - way repeated - measures anova ) on the expression of each gene .
horizontal bars represent wake ( white ) and sleep ( black ) periods during the laboratory study .
zeitgeber time 0 represents the time of lights on ( equivalent to clock time 0630 h ) .
analysis by two - way repeated - measures anova of the data from all of the experimental groups confirmed a significant ( p < 0.05 ) effect of time on gene expression ( fig .
however , it is surprising that we did not observe a significant effect of experimental group on the expression of any gene . the only gene to exhibit a significant ( p < 0.01 ) time group interaction was bmal1 .
post hoc analysis revealed that bmal1 expression 16.5 h after lights on ( equivalent to clock time 2300 h ) was significantly ( p < 0.05 ) lower in the overweight / obese participants than in the lean or type 2 diabetic groups . comparison of 24-h gene expression profiles in human wat from individuals who are lean ( solid line , ) , overweight / obese ( dashed line , ) , or overweight / obese with type 2 diabetes ( dotted line , ) .
data are presented as means se of n = 811 values , normalized to glyceraldehyde 3-phosphate dehydrogenase .
for all genes , there was a significant ( p < 0.05 ) effect of time but no subject group effect on gene expression ( two - way repeated - measures anova ) . with the exception of bmal1 ( p < 0.01 )
horizontal bars represent wake ( white ) and sleep ( black ) periods during the laboratory study .
zeitgeber time 0 represents the time of lights on ( equivalent to clock time 0630 h ) .
consistent with a previous study ( 22 ) , our data reveal robust diurnal rhythmicity within human wat in vivo . to date , limited data have been published concerning clock gene expression in this tissue .
because of difficulties in acquiring human tissue , some studies have measured clock gene expression in wat collected at a single time point ( 18,19 ) , although it is clearly difficult to infer rhythmical changes from such a temporally limited dataset . an elegant compromise has been to culture explants of tissue taken at a single time point and then measure gene expression in fragments of the biopsies over a 24-h time course ( 17 ) .
although such studies highlight endogenous wat rhythms , they do not reflect gene expression in vivo .
therefore , we measured gene expression in human tissue explants harvested across a whole 24-h cycle to provide in vivo human wat rhythms .
animal models suggest that obesity and type 2 diabetes reduce rhythm amplitude in murine wat because lean c57bl/6j mice exhibit higher amplitude wat rhythms than obese / diabetic kk and kk - a mice ( 15 ) .
other data revealed reduced amplitude rhythms in mice that became obese as a result of a high - fat diet ( 16 )
. however , ando et al . ( 15 ) compared mice with differing genetic backgrounds , which complicate interpretation of their data , and it is possible that dietary intervention could directly regulate gene expression rather than obesity per se .
in contrast to the above studies , we observed minimal differences between clock gene rhythms in human wat .
although murine bmal1 has been implicated in the control of adipogenesis in vitro ( 23 ) , juvenile bmal1 mice develop adipose depots comparably to their wild - type littermates ( 24 ) .
furthermore , the similarity between gene expression profiles in lean individuals and those who were overweight with type 2 diabetes suggests that bmi is not the cause of the time group interaction for bmal1 in the overweight , nondiabetic individuals .
the similarity of gene expression profiles between our experimental groups may partly result from a lack of extreme phenotypic differences .
although there was not a large disparity in bmi between our groups , all participants fell into the current clinical guidelines for lean / healthy , obese , and type 2 diabetic individuals .
therefore , there was no effect of obesity or type 2 diabetes , per se , on human wat rhythmicity .
it remains possible that severely obese individuals may exhibit reduced amplitude wat rhythmicity ; however , interpretation of data from such individuals is complicated by various confounding factors , such as comorbidities associated with type 2 diabetes .
another difference between our study and previous work ( 25 ) is the high level of glycemic control in our participants .
we specifically aimed to investigate the relationship between wat rhythmicity , body weight , and presence of type 2 diabetes .
diurnal adipose gene expression may be modulated by the level of glycemic control and/or drugs taken by diabetic individuals .
alternatively , the association between metabolic state and wat rhythmicity might vary between adipose depots .
however , because of limitations of sampling and experimental group size , we could not directly address these possibilities in the current study .
the persistence of 24-h rhythms in wat from patients with type 2 diabetes now suggests that the link between human circadian and metabolic physiology occurs outside of wat , at least in the earlier stages of the disease . | objectiveprevious animal studies suggest a functional relationship between metabolism , type 2 diabetes , and the amplitude of daily rhythms in white adipose tissue ( wat ) .
however , data interpretation is confounded by differences in genetic background and diet or limited sampling points .
we have taken the novel approach of analyzing serial human wat biopsies across a 24-h cycle in controlled laboratory conditions.research design and methodslean ( n = 8) , overweight / obese ( n = 11 ) , or overweight / obese type 2 diabetic ( n = 8) volunteers followed a strict sleep wake and dietary regimen for 1 week prior to the laboratory study .
they were then maintained in controlled light
dark conditions in a semirecumbent posture and fed hourly during wake periods .
subcutaneous wat biopsies were collected every 6 h over 24 h , and gene expression was measured by quantitative pcr.resultslean individuals exhibited significant ( p < 0.05 ) temporal changes of core clock ( per1 , per2 , per3 , cry2 , bmal1 , and dbp ) and metabolic ( reverb , rip140 , and pgc1 ) genes .
the bmal1 rhythm was in approximate antiphase with the other clock genes .
it is noteworthy that there was no significant effect ( p > 0.05 ) of increased body weight or type 2 diabetes on rhythmic gene expression.conclusionsthe robust nature of these rhythms and their relative phasing indicate that wat now can be considered as a peripheral tissue suitable for the study of in vivo human rhythms .
comparison of data between subject groups clearly indicates that obesity and type 2 diabetes are not related to the amplitude of rhythmic wat gene expression in humans maintained under controlled conditions . |
an aortoenteric fistula ( aef ) is a rare but life - threatening cause of gastrointestinal ( gi ) bleeding .
patients with an aef are commonly encountered as a medical emergency associated with acute gi bleeding .
although the reported incidence of aef is low , immediate recognition and management is essential for the patient 's survival .
aef can be classified into primary and secondary types according to the presence or absence of a prior history of aortic surgery ( 1 ) .
two major concerns for the treatment of aef are bleeding control at the time of the initial presentation and the prevention of late complications associated with bleeding or infection . early diagnosis and
a variety of surgical procedures have been performed to reduce the incidence of postoperative infectious complications .
these include an aortic resection followed by an axillo - bifemoral bypass ( axbfb ) ( 2 ) , in situ aortic reconstruction using a prosthetic graft , antibiotic - impregnated prosthetic graft ( 3 ) , autogenous femoral vein graft ( 4 ) , or cryopreserved aortic allograft ( 5 , 6 ) .
the medical records of five consecutive patients ( all male , mean age 659.2 yr , range 55 - 76 yr ) , who were treated for an aef ( four primary and one secondary aef ; four aortoduodenal and one aortogastric fistula ) between march 1999 and february 2003 were retrospectively reviewed .
preoperatively , intravenous contrast - enhanced abdominal computed tomography ( ct ) scans were performed in all patients .
gastroduodenal fiberscopic examination was performed in one patient , but the bleeding focus could not be located due to a large hematoma in the stomach . for bacterial culture ,
intraoperative tissue culture with periaortic tissue around the aef , and postoperative drain fluid culture were obtained in all patients . for the arterial blood flow reconstruction , axbfb after the aortic aneurysmectomy
was done in two patients and an in situ aortic reconstruction was performed in three patients .
the procedures were selected according to the surgical findings ; axbfb for patients with periaortic purulence and an in situ aortic reconstruction for those with no evidence of gross purulence .
after periaortic debridement and irrigation with 1% povidone iodine solution , the aortic stump or aortic prosthetic graft was covered with the greater omentum .
the greater omentum was dissected from its attachment to the transverse colon and mobilized to the infrarenal aorta through an opening at the transverse mesocolon . before closing
the abdominal wall , closed suction drains were placed around the site of the infrarenal abdominal aorta .
postoperatively , intravenous antibiotics were given for four weeks followed by oral antibiotics for two months or longer .
the selection of antibiotics depended on the results of the bacterial cultures from the blood , retroperitoneal tissue , and peritoneal drainage fluid .
all patients were male with a mean age of 66 yr and presented with acute gi bleeding to the emergency department .
the time interval between the herald bleeding and the episode of large gi hemorrhage was 2.5 hr ( median , range 1 - 48 hr ) .
the underlying causes of the aef were an aortic aneurysm ( three patients with infrarenal abdominal aortic aneurysm [ aaa ] and one patient with type iv thoracoabdominal aneurysm ) and one para - anastomotic pseudoaneurysm in a patient who underwent an aortobifemoral bypass graft eight years earlier due to chronic aortoiliac occlusive disease .
table 2 summarizes the surgical findings and procedures . in a patient with an aortogastric fistula
, gastric fistula opening was located at the posterior wall of the gastric cardia close to the esophagocardial junction . a retrospective review of the preoperative abdominal ct scans revealed periaortic air shadows in four ( 80% ) patients ( fig .
this patient was treated with a sigmoid colectomy and hartmann 's colostomy before axbfb grafting .
table 3 shows the bacteriologic examination results . a postoperative gastropleural fistula ( gpf , fig .
2 ) developed in a patient with taa , and the aortogastric fistula required a second operation to close the gastric fistula and thoracotomy tube for drainage . no mortality or graft infection developed during the follow - up period ( mean 40 months ; range , 24 months to 68 months ) ( table 4 ) .
aef can occur as a rare late complication of aortic reconstructive surgery , or even less frequently , as a complication of an untreated aaa ( 7 ) . the optimal management for aef consists of an early diagnosis before the occurrence of major bleeding , prompt bleeding control by expeditious control of the proximal aorta , closure of the enteric fistula without spilling of the bowel contents , a durable arterial reconstruction , and the prevention of postoperative complications , particularly graft infection .
the role of the primary physician in the emergency department is important for early detection of aef .
although gi hemorrhage , abdominal pain , and a pulsatile abdominal mass are the diagnostic triad of aef , the signs of a pulsatile abdominal mass can be masked by abdominal distension .
capaldo and amin ( 8) reported abdominal pain and a pulsatile mass in 32% and a pulsating abdominal mass in 25% of their aef patients .
herald bleeding occurred in 60% of our patients , and the time interval between the initial bleeding and the massive gi hemorrhage ranged from one hour to two days , which is similar to other reports ( 1 , 8 , 9 ) .
theoretically , this window period provides an opportunity to perform definitive surgical treatment for the aef .
currently , intravenous contrast - enhanced abdominal ct can be easily performed in an emergent situation .
spiral ct provides not only diagnostic clues for an aef but also anatomical information for later aortic surgery .
intravenous contrast- enhanced abdominal ct has been recommended as an initial diagnostic test for aef ( 1 , 3 , 6 , 9 ) .
the ct findings suggestive of aef are air bubbles around the aorta , bowel wall edema around the aorta , the loss of a fatty plane between the aorta and gi tract , and rarely , visualization of the fistula itself . of these findings ,
a periaortic air shadow was reported to be a specific finding for the diagnosis of an aortic graft infection or aef ( 10 , 11 ) .
periaortic air shadows were found in 80% of patients by a retrospective review of the preoperative abdominal ct scans . in order to reduce the risk of infectious complications such as a graft infection , retroperitoneal debridement and irrigation with an antiseptic solution
was performed and pedicled omentum was used to cover up the aortic stump or aortic graft .
there has been some debate regarding the optimal arterial reconstruction procedure after removing the infected aneurysm or aortic graft in patients with an aortic graft infection or infected aortic aneurysm , and either an in situ aortic reconstruction ( 1 , 12 ) or axillobifemoral bypass grafting have been proposed ( 1 , 7 , 9 ) . in previous reports , ( 3 , 5 , 9 ) the great omentum was used to wrap around the prosthetic graft or cover the infected surgical field for the purpose of preventing a graft infection .
however , there have been no randomized clinical studies demonstrating the anti - bacterial effects of omental tissue to date .
regarding the duration of postoperative antibiotic therapy , saers and scheltinga ( 1 ) recommended at least 1 week of antibiotic therapy following negative blood culture and 4 to 6 weeks of systemic antibiotic therapy if blood cultures were positive .
the duration of antibiotic therapy can be determined by the results of blood culture and clinical findings of the patients . in a primary aef
, chronic mechanical irritation between the bowel wall and aneurismal wall appears to provide watertight adhesion between the two tissue planes . to our knowledge
, high - pressure aortic blood can create a one - way fistula into the bowel lumen and massive arterial bleeding can clean the bowel lumen .
these properties of primary aef might be associated with the lower rate of graft infection after an in situ aortic reconstruction . in summary ,
early diagnosis and prompt surgical treatment are essential steps for an improved treatment outcome . for the early diagnosis of an aef
, intravenous contrast - enhanced abdominal ct imaging can provide a rapid and effective diagnosis even in an emergent situation .
while there is no optimal surgical procedure for aef to date , the most appropriate procedure can be selected based on the surgical findings , underlying cause of the aef , and patient status . however
, further studies will be needed to determine the efficacy of the omentum in preventing infections . | in order to establish optimal management for aortoenteric fistula ( aef ) the records of five patients treated for aef ( four aortoduodenal and one aortogastric fistula ) were retrospectively reviewed .
the arterial reconstruction procedures were selected according to the surgical findings , underlying cause , and patient status . in situ
aortic reconstructions with prosthetic grafts were performed on three patients who had no gross findings of periaortic infection , whereas axillo - bifemoral bypass was carried out in the other two patients with periaortic purulence . in all patients , after retroperitoneal irrigation a pedicled omentum was used to cover the aortic graft or aortic stump . in the preoperative abdominal computed tomography ( ct )
scan there was a periaortic air shadow in four out of five patients .
there was no surgical mortality or graft infection observed during a mean follow - up period of 40 months ( range , 24 - 68 months ) .
therefore , the treatment results of an aef can be improved using intravenous contrast - enhanced abdominal ct for rapid diagnosis and selection of an appropriate surgical procedure based on the surgical findings and underlying cause . |
the widely used 450k dna methylation array ( infinium humanmethylation450 beadchip ) includes 485,577 cytosine positions in human dna distributed among all 22 autosomal and 1 sex chromosome pair .
when correlated to rna transcripts , 74.4% of cpgs correspond to coding messenger rna genes , 0.85% are associated to noncoding rnas ( mirnas and long noncoding rnas ) and 24.6% sites do not possess an associated transcript .
analysis of genomic distribution shows 30.9% of the sites are in cpg islands , 23% in cpg shores ( sequences 2 kb upstream and downstream from cpg island ) , 9.7% in cpg shelves ( sequences 2 kb upstream and downstream from shore regions ) and 36.3% are located outside these regions ( open sea ) . following a functional classification ,
41% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp or 1500 bp upstream of transcription start sites , exon 1 and in 5utrs ) , 3.2% correspond to 3utrs , 30.9% to gene bodies and 24.6% to intergenic regions .
the recently developed methylationepic beadchip infinium microarray interrogates the methylation status of 853,307 cpg sites .
there are 439,562 cpgs from the 482,421 cpgs included in the 450k microarray ( 91.1% ) , that are also present in the 850k dna methylation microarray .
the complete list of cpgs shared by both microarrays is shown in supplementary table 1 .
the 42,076 cpg sites ( 8.9% ) that were included in the 450k microarray that are not present in the 850k microarray are listed in supplementary table 2 .
most importantly , the methylationepic beadchip infinium includes 413,745 new cpg sites not included in the 450k microarray that interrogate the dna methylation status of other sequences of the genome , which are shown in supplementary table 3 .
the methylationepic beadchip infinium also interrogates 2880 cng ( c stands for cytosine ; n stands for any nucleotide ; g stands for guanine ) sites that are present in the 450k dna methylation microarray ( supplementary table 4 ) , while 211 cng sites from the 450k microarray are not included in the 850k platform .
the 850k microarray also includes 59 snp sites that are used for quality control processes ( supplementary table 5 ) .
the degree of cpg site overlap between the 450k and the 850k dna methylation microarrays is summarized in figure 1 .
the 413,745 cpg newly added positions in the 850k microarray are localized among all 22 autosomal chromosomes in addition to the x and y sex chromosomes ( figure 2a ) .
chromosome 1 harbors the most positions ( 39,087 , 9.4% ) and chromosome y the fewest ( 179 , 0.04% ) .
according to the manufacturer 's technical notes , most ( 95.1% ) of them used the infinium design ii , while only 4.9% used infinium design i ( figure 2b ) . following a functional classification ,
25.8% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp [ 5.4% ] or 1500 bp [ 10.6% ] upstream of transcription start sites , exon 1 [ 0.4% ] and in 5utrs [ 9.4% ] ) , 0.9% correspond to 3utrs , 40% to gene bodies and 33.3% to intergenic regions ( figure 2c ) .
relative to cpg context , 5.4% of the sites are in cpg islands , 11.4% in cpg shores , 5% in cpg shelves and , most importantly , the vast majority ( 78.2% ) are located outside these regions ( open sea ) ( figure 2d ) .
the observation that the newly included cpgs in the 850k dna methylation microarray are mainly located in sequences with poor cpg content , in addition to its intergenic location ( 33.3% ) , fits with the concept that these cpg sites were incorporated from the human enhancer regions provided by the encode and fantom5 projects .
for example , 4.9% of all the cpg sites are derived from a fantom5 defined enhancer ( figure 2e ) . with respect to encode regulatory elements , 19.3% of the cpgs are located within a known transcription factor binding site identified by chip - seq ( chromatin immunoprecipitation and sequencing ) ( figure 2f ) , 14.6% are in
open chromatin regions defined by faire - seq ( formaldehyde - assisted isolation of regulatory elements sequencing ) ( figure 2 g ) and 53.6% of the cpgs correspond to dnase hypersensitive regions identified by dnase - seq ( figure 2h ) .
the genomic and functional context of all the 853,307 cpgs included in the methylationepic beadchip infinium assay , including those
exported from the 450k microarray ( 439,562 ) with those newly incorporated in the 850k microarray ( 413,745 cpgs ) was analyzed next in whole .
all 22 autosomal chromosomes in addition to the x and y sex chromosomes are present , with chromosome 1 harboring the most positions ( 9.5% ) and chromosome y the fewest ( 0.1% ) ( figure 3a ) . considering infinium design , most cpgs used design ii ( 83.8% ) , whereas 16.2% used design i ( figure 3b ) . to quantify the possible false cpg methylation calling due to the presence of a snp , we analyzed how many 850k cpg probes have an snp within their last ten bases and with a minor allele frequency ( maf ) 5% according to the 1000 human genome project database .
we observed that 8.8% of the cpg sites had an associated snp with the described features ( supplementary table 6 ) . according to associated rna transcripts , 623,483 cpgs ( 73.1% ) correspond to classic coding messenger rna genes , while 21,446 ( 2.5% ) are linked to noncoding rnas ( 9961 for mirnas and 11,485 for long noncoding rnas ) . for 229,824 ( 26.9% ) sites
addressing the issue of functional classification , 35.2% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp [ 9.4% ] or 1500 bp [ 13.9% ] upstream of transcription start sites , exon 1 [ 1.2% ] and in 5utrs [ 10.7% ] ) , 1.9% correspond to 3utrs , 35.9% to gene bodies and 26.9% to intergenic regions ( figure 3c ) . as a function of cpg landmarks , 17.8% of the sites are in cpg islands , 16.9% in cpg shores and 8.8% in cpg shelves , but most of the cpg sites ( 56.5% ) are not in any of these sequences ( open sea ) ( figure 3d ) .
if we combine the two classifications described above , we can determine that for the 300,554 cpg sites located in proximal promoters , 102,892 cpgs ( 34.2% ) are in cpg islands , 84,491 ( 28.1% ) in cpg shores and 18,393 ( 6.1% ) in cpg shelves , while 94,778 ( 31.5% ) are in other regions of the genome without any enrichment of cpg content ( open sea ) ( figure 3 ) .
related to regulatory regions , 3.2% of the cpg sites are within fantom5 described enhancer regions ( figure 3e ) , covering 36.3% of the described fantom5 enhancers with at least one cpg site .
the included fantom5 enhancers are within a cpg island , shelf or shore in 26.6% of cases , whereas the remaining 73.4% are outside these cpg sequence categories .
in addition , 15.6% of the cpgs are located within a known transcription factor binding site ( figure 3f ) , 13.8% are in open chromatin regions ( figure 3 g ) ( covering 38.7% of overlapped open chromatin regions annotated in the encode project ) and 57.4% correspond to cpgs within dnase i hypersensitive regions ( figure 3h ) .
other detailed information about the cpgs included in the 850k array can be found at the manufacturer website . to prove the utility and reliability of the infinium methylationepic beadchip for the analysis of dna methylation
first , we provided a technical validation of the 850k dna methylation microarray data by studying a primary renal tumor ( rcc9 ) , and comparing the obtained results from the well established and fully standardized 450k platform .
we observed that dna methylation data obtained with the new 850k microarray was highly correlated with the methylation levels detected at each cpg site using the 450k microarray ( pearson correlation coefficient r = 0.992 ; p 2.22e-16 ) ( figure 4a ) .
we also performed a replication experiment by hybridizing twice the same sample ( normal colon nc22a ) to the 850k microarray .
we found that the methylation levels detected at cpg sites derived from each experiment were highly correlated and completely interchangeable ( pearson correlation coefficient r = 0.997 ; p 2.22e-16 ) ( figure 4b ) .
given the excellent performance of the 450k microarray for formalin - fixed paraffin - embedded ( ffpe ) samples , we wondered about the reliability of the 850k microarray to determine the dna methylation patterns in this type of archival material . to address this issue , we hybridized to the platform the same dna sample obtained from fresh tissue ( primary renal tumor rcc9 ) or derived after the formalin - fixing paraffin - embedding procedure ( rcc9-ffpe ) .
we observed that the methylation levels detected at each cpg site derived from each sample were highly correlated ( pearson correlation coefficient r = 0.994 ; p 2.22e-16 ) ( figure 4c ) .
cellular heterogeneity is a potential limitation of this part of the study , but as the ffpe section was close to the fresh - frozen section significant cellular heterogeneity is not expected .
we also sought to demonstrate that the newly developed 850k dna methylation microarray was also valuable for the study of 5-hydroxymethylcytosine patterns , as has been shown for the 450k platform . to accomplish this aim
, we carried out oxidation of dna purified from sorted neurons ( n229 ) , a tissue particularly enriched for 5-hydroxymethylcytosine , using the truemethyl array kit ( cambridge epigenetix , cambridge , uk ) where the final eluted fraction followed the methylationepic processing according to the kit instructions for infinium arrays . using this approach
, we were able to determine the 5- hydroxymethylcytosine patterns in the sorted neurons ( figure 4d & e ) .
the genomic distribution of 5-hydroxymethylcytosine , their functional annotation and genomic feature enrichment are shown in supplementary figure 1 . finally , we assessed the ability of the 850k microarray to identify differential methylated cpg sites to allow for the study of various biological and biomedical phenomena . for this purpose , we used unsupervised dna clustering on the 850k methylation data from tissues of varying developmental origin , such as colon mucosa ( nc22a ) and neurons ( n229 ) , and were able to discriminate the two normal tissues based on their methylation profiles ( figure 5 ) .
overall , 1468 cpg sites displayed differential methylation levels with delta beta values higher than 66% between these two cell types ( figure 5 & supplementary table 7 ) .
if we lowered the threshold to a 33% delta value , 73,774 cpg sites are distinct between these normal tissues ( supplementary table 8) . for these differentially methylated cpgs ,
6.2% and 12.6% were located in fantom5 enhancers and open chromatin regions defined by encode , respectively .
most importantly , 40.5% ( at delta beta value of 66% ) ( figure 5 & supplementary table 9 ) and 55.6% ( with delta beta value of 33% ) ( supplementary table 10 ) of these differentially methylated sites corresponded to the newly incorporated cpgs in the 850k , a fraction that due to its enrichment in enhancer sequences could represent a powerful tool to dissect cellular and tissue lineages among different experimental conditions and clinical entities .
the complete 850k and 450k data from all the described samples are available for download from ncbi geo .
the widely used 450k dna methylation array ( infinium humanmethylation450 beadchip ) includes 485,577 cytosine positions in human dna distributed among all 22 autosomal and 1 sex chromosome pair .
when correlated to rna transcripts , 74.4% of cpgs correspond to coding messenger rna genes , 0.85% are associated to noncoding rnas ( mirnas and long noncoding rnas ) and 24.6% sites do not possess an associated transcript .
analysis of genomic distribution shows 30.9% of the sites are in cpg islands , 23% in cpg shores ( sequences 2 kb upstream and downstream from cpg island ) , 9.7% in cpg shelves ( sequences 2 kb upstream and downstream from shore regions ) and 36.3% are located outside these regions ( open sea ) . following a functional classification ,
41% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp or 1500 bp upstream of transcription start sites , exon 1 and in 5utrs ) , 3.2% correspond to 3utrs , 30.9% to gene bodies and 24.6% to intergenic regions .
the recently developed methylationepic beadchip infinium microarray interrogates the methylation status of 853,307 cpg sites .
there are 439,562 cpgs from the 482,421 cpgs included in the 450k microarray ( 91.1% ) , that are also present in the 850k dna methylation microarray .
the complete list of cpgs shared by both microarrays is shown in supplementary table 1 .
the 42,076 cpg sites ( 8.9% ) that were included in the 450k microarray that are not present in the 850k microarray are listed in supplementary table 2 .
most importantly , the methylationepic beadchip infinium includes 413,745 new cpg sites not included in the 450k microarray that interrogate the dna methylation status of other sequences of the genome , which are shown in supplementary table 3 .
the methylationepic beadchip infinium also interrogates 2880 cng ( c stands for cytosine ; n stands for any nucleotide ; g stands for guanine ) sites that are present in the 450k dna methylation microarray ( supplementary table 4 ) , while 211 cng sites from the 450k microarray are not included in the 850k platform .
the 850k microarray also includes 59 snp sites that are used for quality control processes ( supplementary table 5 ) .
the degree of cpg site overlap between the 450k and the 850k dna methylation microarrays is summarized in figure 1 .
the 413,745 cpg newly added positions in the 850k microarray are localized among all 22 autosomal chromosomes in addition to the x and y sex chromosomes ( figure 2a ) .
chromosome 1 harbors the most positions ( 39,087 , 9.4% ) and chromosome y the fewest ( 179 , 0.04% ) .
according to the manufacturer 's technical notes , most ( 95.1% ) of them used the infinium design ii , while only 4.9% used infinium design i ( figure 2b ) . following a functional classification ,
25.8% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp [ 5.4% ] or 1500 bp [ 10.6% ] upstream of transcription start sites , exon 1 [ 0.4% ] and in 5utrs [ 9.4% ] ) , 0.9% correspond to 3utrs , 40% to gene bodies and 33.3% to intergenic regions ( figure 2c ) .
relative to cpg context , 5.4% of the sites are in cpg islands , 11.4% in cpg shores , 5% in cpg shelves and , most importantly , the vast majority ( 78.2% ) are located outside these regions ( open sea ) ( figure 2d ) .
the observation that the newly included cpgs in the 850k dna methylation microarray are mainly located in sequences with poor cpg content , in addition to its intergenic location ( 33.3% ) , fits with the concept that these cpg sites were incorporated from the human enhancer regions provided by the encode and fantom5 projects .
for example , 4.9% of all the cpg sites are derived from a fantom5 defined enhancer ( figure 2e ) . with respect to encode regulatory elements , 19.3% of the cpgs are located within a known transcription factor binding site identified by chip - seq ( chromatin immunoprecipitation and sequencing ) ( figure 2f ) , 14.6% are in
open chromatin regions defined by faire - seq ( formaldehyde - assisted isolation of regulatory elements sequencing ) ( figure 2 g ) and 53.6% of the cpgs correspond to dnase hypersensitive regions identified by dnase - seq ( figure 2h ) .
the genomic and functional context of all the 853,307 cpgs included in the methylationepic beadchip infinium assay , including those
exported from the 450k microarray ( 439,562 ) with those newly incorporated in the 850k microarray ( 413,745 cpgs ) was analyzed next in whole .
all 22 autosomal chromosomes in addition to the x and y sex chromosomes are present , with chromosome 1 harboring the most positions ( 9.5% ) and chromosome y the fewest ( 0.1% ) ( figure 3a ) . considering infinium design , most cpgs used design ii ( 83.8% ) , whereas 16.2% used design i ( figure 3b ) . to quantify the possible false cpg methylation calling due to the presence of a snp , we analyzed how many 850k cpg probes have an snp within their last ten bases and with a minor allele frequency ( maf ) 5% according to the 1000 human genome project database .
we observed that 8.8% of the cpg sites had an associated snp with the described features ( supplementary table 6 ) . according to associated rna transcripts , 623,483 cpgs ( 73.1% ) correspond to classic coding messenger rna genes , while 21,446 ( 2.5% ) are linked to noncoding rnas ( 9961 for mirnas and 11,485 for long noncoding rnas ) . for 229,824 ( 26.9% ) sites
addressing the issue of functional classification , 35.2% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp [ 9.4% ] or 1500 bp [ 13.9% ] upstream of transcription start sites , exon 1 [ 1.2% ] and in 5utrs [ 10.7% ] ) , 1.9% correspond to 3utrs , 35.9% to gene bodies and 26.9% to intergenic regions ( figure 3c ) . as a function of cpg landmarks , 17.8% of the sites are in cpg islands , 16.9% in cpg shores and 8.8% in cpg shelves , but most of the cpg sites ( 56.5% ) are not in any of these sequences ( open sea ) ( figure 3d ) .
if we combine the two classifications described above , we can determine that for the 300,554 cpg sites located in proximal promoters , 102,892 cpgs ( 34.2% ) are in cpg islands , 84,491 ( 28.1% ) in cpg shores and 18,393 ( 6.1% ) in cpg shelves , while 94,778 ( 31.5% ) are in other regions of the genome without any enrichment of cpg content ( open sea ) ( figure 3 ) .
related to regulatory regions , 3.2% of the cpg sites are within fantom5 described enhancer regions ( figure 3e ) , covering 36.3% of the described fantom5 enhancers with at least one cpg site .
the included fantom5 enhancers are within a cpg island , shelf or shore in 26.6% of cases , whereas the remaining 73.4% are outside these cpg sequence categories .
in addition , 15.6% of the cpgs are located within a known transcription factor binding site ( figure 3f ) , 13.8% are in open chromatin regions ( figure 3 g ) ( covering 38.7% of overlapped open chromatin regions annotated in the encode project ) and 57.4% correspond to cpgs within dnase i hypersensitive regions ( figure 3h ) .
other detailed information about the cpgs included in the 850k array can be found at the manufacturer website .
to prove the utility and reliability of the infinium methylationepic beadchip for the analysis of dna methylation , we performed five different tests .
first , we provided a technical validation of the 850k dna methylation microarray data by studying a primary renal tumor ( rcc9 ) , and comparing the obtained results from the well established and fully standardized 450k platform .
we observed that dna methylation data obtained with the new 850k microarray was highly correlated with the methylation levels detected at each cpg site using the 450k microarray ( pearson correlation coefficient r = 0.992 ; p 2.22e-16 ) ( figure 4a ) .
we also performed a replication experiment by hybridizing twice the same sample ( normal colon nc22a ) to the 850k microarray .
we found that the methylation levels detected at cpg sites derived from each experiment were highly correlated and completely interchangeable ( pearson correlation coefficient r = 0.997 ; p 2.22e-16 ) ( figure 4b ) .
given the excellent performance of the 450k microarray for formalin - fixed paraffin - embedded ( ffpe ) samples , we wondered about the reliability of the 850k microarray to determine the dna methylation patterns in this type of archival material . to address this issue , we hybridized to the platform the same dna sample obtained from fresh tissue ( primary renal tumor rcc9 ) or derived after the formalin - fixing paraffin - embedding procedure ( rcc9-ffpe ) .
we observed that the methylation levels detected at each cpg site derived from each sample were highly correlated ( pearson correlation coefficient r = 0.994 ; p 2.22e-16 ) ( figure 4c ) .
cellular heterogeneity is a potential limitation of this part of the study , but as the ffpe section was close to the fresh - frozen section significant cellular heterogeneity is not expected .
we also sought to demonstrate that the newly developed 850k dna methylation microarray was also valuable for the study of 5-hydroxymethylcytosine patterns , as has been shown for the 450k platform . to accomplish this aim
, we carried out oxidation of dna purified from sorted neurons ( n229 ) , a tissue particularly enriched for 5-hydroxymethylcytosine , using the truemethyl array kit ( cambridge epigenetix , cambridge , uk ) where the final eluted fraction followed the methylationepic processing according to the kit instructions for infinium arrays . using this approach
, we were able to determine the 5- hydroxymethylcytosine patterns in the sorted neurons ( figure 4d & e ) .
the genomic distribution of 5-hydroxymethylcytosine , their functional annotation and genomic feature enrichment are shown in supplementary figure 1 . finally , we assessed the ability of the 850k microarray to identify differential methylated cpg sites to allow for the study of various biological and biomedical phenomena .
for this purpose , we used unsupervised dna clustering on the 850k methylation data from tissues of varying developmental origin , such as colon mucosa ( nc22a ) and neurons ( n229 ) , and were able to discriminate the two normal tissues based on their methylation profiles ( figure 5 ) .
overall , 1468 cpg sites displayed differential methylation levels with delta beta values higher than 66% between these two cell types ( figure 5 & supplementary table 7 ) .
if we lowered the threshold to a 33% delta value , 73,774 cpg sites are distinct between these normal tissues ( supplementary table 8) .
for these differentially methylated cpgs , 6.2% and 12.6% were located in fantom5 enhancers and open chromatin regions defined by encode , respectively .
most importantly , 40.5% ( at delta beta value of 66% ) ( figure 5 & supplementary table 9 ) and 55.6% ( with delta beta value of 33% ) ( supplementary table 10 ) of these differentially methylated sites corresponded to the newly incorporated cpgs in the 850k , a fraction that due to its enrichment in enhancer sequences could represent a powerful tool to dissect cellular and tissue lineages among different experimental conditions and clinical entities .
the complete 850k and 450k data from all the described samples are available for download from ncbi geo .
samples included in the study were all from human origin including a primary normal colon ( nc22a ) that was hybridized twice to validate technical reproducibility , primary sorted neurons ( n229 ) , as well as a biopsy of a renal cancer ( rcc9 ) which was divided in two pieces , one was stored as fresh - frozen ( ff ) tissue embedded in optimal cutting temperature ( oct ) compound stored at -80c , while the other piece was formalin - fixed for 8 h followed by paraffin - embedding ( ffpe ) and stored at room temperature ( rcc9-ffpe ) .
all samples were extracted using column - based dna extraction method ( ffpe , e.z.n.a .
, ga , usa ; ff , dneasy blood & tissue kit ; qiagen , hilden , germany ) following manufacturer 's instructions .
all dna samples were treated with rnasea for 1 h at 45c , quantified by the fluorometric method ( quant - it picogreen dsdna assay , life technologies , ca , usa ) , and assessed for purity by nanodrop ( thermo scientific , ma , usa ) 260/280 and 260/230 ratio measurements .
all dnas from ffpe blocks were checked for their suitability for ffpe restoration , as indicated by the infinium hd ffpe qc assay ( illumina inc . ) , by performing a quantitative pcr with 2 ng of ffpe dna .
cq was calculated by subtracting the average value of cq of the interrogated sample from the cq value of a standard provided by the manufacturer .
all ffpe samples had a cq < 5 , which is the recommended threshold for suitability for ffpe restoration .
site - specific oxidation was carried out on 1 g of gdna of the sorted neurons in order to convert 5-hydroxymethyl cytosines ( 5-hmc ) into its formyl derivative 5-formylcytosine ( 5-fc ) using truemethyl array kit ( cambridge epigenetix ) following manufacturer 's instructions .
both aliquots were denatured , and 1 l of oxidant solution was added to one of the aliquots ( ox - bs ) while the other one ( bs ) underwent a mock - up oxidation process by adding 1 l of ultrapure h20 .
after incubating both aliquots at 40c for 30 min , and centrifuging to eliminate precipitates , a bisulfite conversion reaction was performed with the supernatant using supplied reagents .
desulfonation and cleanup process were applied using provided reagents before eluting the dna with 10 l of elution buffer .
the eluate was then transferred directly to methylationepic processing following kit instructions for infinium arrays .
three hundred nanograms of ffpe dna , or 600 ng of ff dna were randomly distributed on a 96-well plate , and processed using the ez-96 dna methylation kit ( zymo research corp . ,
bisulfite - converted dna ( bs - dna ) from ffpe samples was processed as previously described .
an 1-h reaction at 37c was then performed with ppr ( primer pre restore ) reagent and amr ( amplification mix restore reagent ) reagents supplied by the kit manufacturer , in which dna repair is accomplished . dna was cleaned with a zr-96 dna clean & concentrator-5 kit ( zymo research corp . ) and eluted in 13 l of erb . cleaned dna
was then denatured for 2 min at 95c , followed by ligation incubation at 37c for 1 h with rst and cmm reagents .
the resulting material was cleaned with zr-96 dna clean & concentrator-5 kit ( zymo research corp . ) and eluted in 10 l of dih20 .
methylationepic beadarray shares the infinium hd chemistry assay ( illumina inc . ) used to interrogated the cytosine markers with humanmethylation450 beadchip .
thus , the applicable protocol for methylationepic is the same as for humanmethylation450 , which is the infinium hd methylation assay protocol .
eight microliter of restored ffpe bs - dna , 4 l of ff bs - dna or 7 l of samples that underwent oxidation protocol were used to process them following the illumina infinium hd methylation assay protocol , as previously described .
the only difference was that the hybridization volume of processed sample used to load the microarray was 26 l instead of the 15 l used in the case of humanmethylation450 .
the resulting raw data ( idats ) were normalized ( control normalization ) and background corrected using the methylation module ( 1.9.0 ) available on genomestudio ( v2011.1 ) software .
the complete 850k and 450k data from all the described samples are available for download from ncbi geo .
cpg markers present on methylationepic were classified based on its chromosome location , the infinium chemistry used to interrogate the marker ( infinium i , infinium ii ) and the feature category gene region as per ucsc annotation ( tss200 , tss1500 , 5utr , 1st exon , body , 3utr ) .
related to this last classification , categories included tss200 as the region between 0 and 200 bases upstream from the transcriptional start site ( tss ) ; tss1500 category , 2001500 bases upstream tss ; 5utr included the region between the tss and the start site ( atg ) ; cpgs within the first exon of a gene were considered as 1st exon category ; cpgs downstream the first exon including intronic regions until the stop codon , were classified as gene body ; cpgs located downstream the stop codon until the poly a signal were considered as 3utr ; and cpgs that were not classified in any of the previous categories were annotated as intergenic .
when multiple genes or tss were associated with a cpg site , category prioritization was applied following a 5-prime to 3-prime criteria ( tss200 > tss1500 > 5utr > 1st exon > body >
additional criteria included the location of the marker relative to the cpg island ( open sea , island , shore , shelf ) , fantom 5-associated enhancer regions and regulatory regions described on encode project such as transcription binding site sequences , open chromatin regions and digital dnase i hypersensitivity clusters .
samples included in the study were all from human origin including a primary normal colon ( nc22a ) that was hybridized twice to validate technical reproducibility , primary sorted neurons ( n229 ) , as well as a biopsy of a renal cancer ( rcc9 ) which was divided in two pieces , one was stored as fresh - frozen ( ff ) tissue embedded in optimal cutting temperature ( oct ) compound stored at -80c , while the other piece was formalin - fixed for 8 h followed by paraffin - embedding ( ffpe ) and stored at room temperature ( rcc9-ffpe ) .
all samples were extracted using column - based dna extraction method ( ffpe , e.z.n.a .
, ga , usa ; ff , dneasy blood & tissue kit ; qiagen , hilden , germany ) following manufacturer 's instructions .
all dna samples were treated with rnasea for 1 h at 45c , quantified by the fluorometric method ( quant - it picogreen dsdna assay , life technologies , ca , usa ) , and assessed for purity by nanodrop ( thermo scientific , ma , usa ) 260/280 and 260/230 ratio measurements .
all dnas from ffpe blocks were checked for their suitability for ffpe restoration , as indicated by the infinium hd ffpe qc assay ( illumina inc . ) , by performing a quantitative pcr with 2 ng of ffpe dna .
cq was calculated by subtracting the average value of cq of the interrogated sample from the cq value of a standard provided by the manufacturer .
all ffpe samples had a cq < 5 , which is the recommended threshold for suitability for ffpe restoration .
site - specific oxidation was carried out on 1 g of gdna of the sorted neurons in order to convert 5-hydroxymethyl cytosines ( 5-hmc ) into its formyl derivative 5-formylcytosine ( 5-fc ) using truemethyl array kit ( cambridge epigenetix ) following manufacturer 's instructions .
both aliquots were denatured , and 1 l of oxidant solution was added to one of the aliquots ( ox - bs ) while the other one ( bs ) underwent a mock - up oxidation process by adding 1 l of ultrapure h20 .
after incubating both aliquots at 40c for 30 min , and centrifuging to eliminate precipitates , a bisulfite conversion reaction was performed with the supernatant using supplied reagents .
desulfonation and cleanup process were applied using provided reagents before eluting the dna with 10 l of elution buffer .
the eluate was then transferred directly to methylationepic processing following kit instructions for infinium arrays .
three hundred nanograms of ffpe dna , or 600 ng of ff dna were randomly distributed on a 96-well plate , and processed using the ez-96 dna methylation kit ( zymo research corp . , ca , usa ) following the manufacturer 's recommendations for infinium assays .
bisulfite - converted dna ( bs - dna ) from ffpe samples was processed as previously described .
an 1-h reaction at 37c was then performed with ppr ( primer pre restore ) reagent and amr ( amplification mix restore reagent ) reagents supplied by the kit manufacturer , in which dna repair is accomplished . dna was cleaned with a zr-96 dna clean & concentrator-5 kit ( zymo research corp . ) and eluted in 13 l of erb . cleaned dna
was then denatured for 2 min at 95c , followed by ligation incubation at 37c for 1 h with rst and cmm reagents .
the resulting material was cleaned with zr-96 dna clean & concentrator-5 kit ( zymo research corp . ) and eluted in 10 l of dih20 .
methylationepic beadarray shares the infinium hd chemistry assay ( illumina inc . ) used to interrogated the cytosine markers with humanmethylation450 beadchip .
thus , the applicable protocol for methylationepic is the same as for humanmethylation450 , which is the infinium hd methylation assay protocol .
eight microliter of restored ffpe bs - dna , 4 l of ff bs - dna or 7 l of samples that underwent oxidation protocol were used to process them following the illumina infinium hd methylation assay protocol , as previously described .
the only difference was that the hybridization volume of processed sample used to load the microarray was 26 l instead of the 15 l used in the case of humanmethylation450 .
the resulting raw data ( idats ) were normalized ( control normalization ) and background corrected using the methylation module ( 1.9.0 ) available on genomestudio ( v2011.1 ) software .
the complete 850k and 450k data from all the described samples are available for download from ncbi geo .
cpg markers present on methylationepic were classified based on its chromosome location , the infinium chemistry used to interrogate the marker ( infinium i , infinium ii ) and the feature category gene region as per ucsc annotation ( tss200 , tss1500 , 5utr , 1st exon , body , 3utr ) . related to this last classification , categories included tss200 as the region between 0 and 200 bases upstream from the transcriptional start site ( tss ) ; tss1500 category , 2001500 bases upstream tss ; 5utr included the region between the tss and the start site ( atg ) ; cpgs within the first exon of a gene were considered as 1st exon category ; cpgs downstream the first exon including intronic regions until the stop codon , were classified as gene body ; cpgs located downstream the stop codon until the poly a signal were considered as 3utr ; and cpgs that were not classified in any of the previous categories were annotated as intergenic .
when multiple genes or tss were associated with a cpg site , category prioritization was applied following a 5-prime to 3-prime criteria ( tss200 > tss1500 > 5utr > 1st exon > body >
additional criteria included the location of the marker relative to the cpg island ( open sea , island , shore , shelf ) , fantom 5-associated enhancer regions and regulatory regions described on encode project such as transcription binding site sequences , open chromatin regions and digital dnase i hypersensitivity clusters .
our results validate the infinium methylationepic beadchip ( 850k ) as a highly reliable genomic platform for the study of dna methylation patterns and levels in the human genome .
most importantly , in addition to showing great correlation with previous dna methylation platforms , high reproducibility and suitability to study formalin - fixed paraffin - embeded samples and 5-hydroxymethylcytosine , it also represents an extremely valuable tool to decipher how dna methylation changes in unexplored territories , such as enhancer sequences , contribute to cell homeostasis and human diseases .
the obtention of full human dna methylomes requires a large effort in time and budget .
user - friendly dna methylation microarrays , such as the illumina infinium humanmethylation450 beadchip ( 450k ; illumina inc . ,
ca , usa ) , have become widely adopted , but many potentially important regulatory cpg sites are not interrogated .
this study investigates whether the newly developed methylationepic beadchip ( 850k ) can be a reliable tool for more comprehensive dna methylation analyses .
the recently developed epic microarray interrogates the methylation status of 853,307 cpg sites , adding 413,745 cpg positions in comparison to the 450k .
the new cpgs in the 850k microarray are enriched in regulatory regions provided by the encode and fantom5 projects such as enhancers , transcription factor binding sites , open chromatin regions and dnase i hypersensitive regions .
dna methylation data obtained with the new 850k microarray was highly correlated with the methylation levels detected using the 450k microarray .
replication experiments by hybridizing twice at 850k the same sample detected completely interchangeable cpg methylation values .
methylation levels detected from the same dna sample obtained from fresh tissue or formalin - fixing paraffin - embedding were highly correlated .
the samples included in the study were all from human origin including fresh - frozen and formalin - fixed paraffin - embedding .
dna extraction , bisulfite conversion , site - specific oxidation and array hybrydization to the 450k and 850k microarrays was performed according to the manufacturer instructions ( illumina inc . ) .
the recently developed epic microarray interrogates the methylation status of 853,307 cpg sites , adding 413,745 cpg positions in comparison to the 450k .
the new cpgs in the 850k microarray are enriched in regulatory regions provided by the encode and fantom5 projects such as enhancers , transcription factor binding sites , open chromatin regions and dnase i hypersensitive regions .
dna methylation data obtained with the new 850k microarray was highly correlated with the methylation levels detected using the 450k microarray .
replication experiments by hybridizing twice at 850k the same sample detected completely interchangeable cpg methylation values .
methylation levels detected from the same dna sample obtained from fresh tissue or formalin - fixing paraffin - embedding were highly correlated .
the samples included in the study were all from human origin including fresh - frozen and formalin - fixed paraffin - embedding .
dna extraction , bisulfite conversion , site - specific oxidation and array hybrydization to the 450k and 850k microarrays was performed according to the manufacturer instructions ( illumina inc . ) .
| aim : dna methylation is the best known epigenetic mark .
cancer and other pathologies show an altered dna methylome .
however , delivering complete dna methylation maps is compromised by the price and labor - intensive interpretation of single nucleotide methods.material & methods : following the success of the humanmethylation450 beadchip ( infinium ) methylation microarray ( 450k ) , we report the technical and biological validation of the newly developed methylationepic beadchip ( infinium ) microarray that covers over 850,000 cpg methylation sites ( 850k ) . the 850k microarray contains > 90% of the 450k sites , but adds 333,265 cpgs located in enhancer regions identified by the encode and fantom5 projects.results & conclusion : the 850k array demonstrates high reproducibility at the 450k cpg sites , is consistent among technical replicates , is reliable in the matched study of fresh frozen versus formalin - fixed paraffin - embeded samples and is also useful for 5-hydroxymethylcytosine .
these results highlight the value of the methylationepic beadchip as a useful tool for the analysis of the dna methylation profile of the human genome . |
rheumatoid factors ( rfs ) , a class of immunoglobulins ( igs ) that have different isotypes and affinities , were first detected more than 70 years ago , but there is still much to discover about the mechanisms underlying their production , physiological role , and pathological effects .
waaler described an antibody directed against serum gamma - globulins that promoted the agglutination of sheep red blood cells sensitised by subagglutinating doses of rabbit antibodies in 1940 , although it had actually been previously found in patients with liver cirrhosis and chronic bronchitis by kurt meyer in 1922 . in 1948 , rose described these antibodies in patients with rheumatoid arthritis ( ra ) , and in 1952 they were finally christened rfs because of their association with ra .
however , although they owe their name to their first detection in ra patients , rfs are found in patients with other autoimmune and nonautoimmune diseases , as well as - in healthy subjects .
the aim of this review is to describe the clinical applications of testing for rfs .
classic agglutination techniques were initially used because of the ability of igms to induce agglutination .
the first rf detection assay was based on the fact that rf agglutinates sheep red blood cells sensitised with rabbit iggs ( i.e. , the classic waaler - rose test ) [ 2 , 3 ] , and this was followed by the development of other igg carriers such as bentonite [ 5 , 6 ] and latex particles [ 7 , 8 ] .
automated techniques such as nephelometry and enzyme - linked immunosorbent assays gradually replaced the other semiquantitative methods because of their simplicity and greater reproducibility [ 912 ] .
multiplexed immunoassaying is an emerging high - throughput technique for the quantitative detection of multiple analytes from a single biological sample .
although they have yet to be standardised and validated , multiplexed immunoassays can reduce analytical time and enhance accuracy .
however , it is known that rfs can interfere with a number of laboratory immunoassays and lead to false positive results : for example , in patients with high rf levels , the analysis of vancomycin can be compromised if serum rather than plasma samples are used [ 14 , 15 ] .
rfs can also interfere with other laboratory tests , including those designed to detect anticardiolipin antibodies ( especially if igm levels are in the low positive range ) , anti-2gpi antibodies , anti - hcv antibodies , antirubella antibodies , thyroid assays [ 20 , 21 ] , and tests for carbohydrate antigen 199 and various cytokines .
as shown in table 1 , rfs can be detected in patients with many nonrheumatic conditions .
infections and chronic diseases may be characterised by the presence of serum rfs , but unlike those detected in ra patients , the rfs produced during infections are usually transient and not detrimental .
given the ability of rfs to increase the clearance of immune complexes and the fact that rf - producing b cells may behave as antigen - presenting cells ( apcs ) and aid the immune response against the infectious antigens , it is likely that the net impact of rf production during infections is protective for the host [ 24 , 26 ] .
these natural rfs are generally low - affinity , polyreactive igm antibodies produced by cd5-positive b cells [ 27 , 28 ] , and coexistence of rf - positive b cells and nonautoimmune igg antigen in healthy subjects suggests the existence of tolerance mechanisms .
rfs can be found in 4050% of patients with hcv infection , but their frequency can reach 76% .
their production is probably due to chronic stimulation of the immune system by hcv , and , as hcv infection is highly prevalent in various countries ( 1.53% in southern europe ) and represents the first cause of increased serum rfs , hcv antibodies should be sought in all subjects with increased rf levels [ 29 , 30 ] ( figure 1 ) .
rf positivity has also been reported in the healthy population [ 3133 ] , and up to 4% of young caucasians may be rf positive , with a similar distribution between the two genders .
it is thought that genetic and environmental factors are responsible for the worldwide variability in distribution of rfs : for example , their highest prevalence ( up to 30% ) has been observed in north american indians tribes [ 3436 ] .
the rfs found in healthy subjects are different from those present in ra patients as their titres are low / moderate and they are likely to be produced by cd5-expressing b cells as low - affinity , poly - reactive igms without any signs of maturation affinity .
the transient production of low - affinity igm rfs may be induced by polyclonal b cell activators such as bacterial lipopolysaccharides and epstein - barr virus [ 28 , 37 ] , but it has been shown that high rf titres in healthy subjects predict the development of ra . furthermore , igm rfs are sometimes observed in healthy elderly people , which suggests that they may be a consequence of the age - related immune deregulation ( figure 1 ) [ 39 , 40 ] .
rfs are frequently detected in patients with systemic autoimmune diseases , such as systemic lupus erythematosus , mixed connective tissue disease , polymyositis , and dermatomyositis ( table 1 ) [ 24 , 25 ] .
patients with sjogren 's syndrome ( ss ) and those with type ii and iii mixed cryoglobulinemia ( usually hcv related ) have the highest rf titres .
about 60% of the patients with primary ss are rf positive , with males having higher iga rf levels than females .
it is also thought that the disease - related transformation of activated rf - positive b cell clones is involved in the pathogenesis of the lymphoproliferative disorders that develop in about 5% of ss patients .
most ss patients have high titres of polyclonal rfs , whereas monoclonal rfs can be detected in patients with type ii mixed cryoglobulinemia and , to a lesser extent , in ss patients with lymphoproliferative disorders [ 24 , 34 ] .
although rfs can be detected in patients with other connective tissue diseases , rf isotypes are helpful in the management of ra patients from the time of diagnosis until deciding on the choice of therapeutic strategy ( figures 1 and 2 ) [ 44 , 45 ] .
rf testing in ra patients has a sensitivity of 60% to 90% and a specificity of 85% [ 46 , 47 ] .
a number of hypotheses have been postulated in order to explain the possible key role of rfs in ra , including their capacity to increase the elimination of immune complexes by macrophages , the improved cytotoxicity of antiviral antibodies , and the increased elimination of parasites .
it has also been suggested that rfs potentiate the presentation of antigens to t cells by means of the dendritic cell uptake of immune complexes with exogenous antigens and by means of rf b cells , which seem to be more efficient apcs than other b cells ( figure 3 ) .
finally , it is possible that the rapid secretion of large amounts of low - affinity rfs prevents the activation of higher - affinity rf b cells and additional b cells [ 5153 ] . defining rfs as anti - igg or anti - gamma - globulins is inaccurate because it restricts rf reactivity to the igg fc fragment .
igm rfs are the most frequently detected isotype , but igg , iga , ige , and igd rfs can also be observed .
it has been shown that three rf isotypes ( igm , iga , and igg ) are detected in up to 52% of ra patients
moreover , the presence of iga and igg rf isotypes in absence of igm - rf is more prevalent in patients with connective tissue diseases than in ra patients , whereas an increase in both igm and iga rfs is almost exclusively observed in patients with ra [ 55 , 56 ] .
it has long been recognised that rfs play a pivotal role in the differential diagnosis of polyarthritis because they make it possible to identify ra patients . for this reason ,
rf testing has been one of the classification criteria for ra since 1987 and , although many years have passed since their identification , their crucial role in classifying ra has been confirmed by the updated criteria . however , in order to increase the specificity of the latest ra classification criteria , anti - cyclic citrullinated protein / peptide antibody ( acpa ) testing has been added .
a meta - analysis has shown that the pooled sensitivities of acpa and rf are similar , but acpa positivity is more specific for ra than igm rf , igg rf , or iga rf positivity and more specific for early ra than igm rf . on the other hand , sensitivity is reduced because positivity for both acpa and rf is a more stringent criterion than positivity for either alone ; combining acpa and rf positivity is more permissive in terms of sensitivity because the antibodies complement each other , especially for early ra [ 6366 ] .
furthermore , although the cut - off value of each commercial kit is slightly different , it has been suggested that the best acpa cut - off value should be 40 u / ml , which leads to a positive likelihood ratio of 5.49 and a negative likelihood ratio of 0.50 [ 46 , 67 ] .
it has also been shown that rfs are useful in predicting the development of ra , as the detection of igm , iga , and igg rfs may predate its onset by years [ 38 , 68 ] , and it has been reported that their appearance in serum is sequential before diagnosis : first igm rf , then iga rf , and finally igg rf [ 57 , 69 ] .
the detection of igm rfs is also helpful as a prognostic index , and some studies have shown that immunosuppressive treatment can decrease serum rf levels .
however , the clinical usefulness of rfs in monitoring disease activity and treatment response is limited .
it has been shown that a progressive decrease in the rf levels parallels the decrease of clinical activity in patients treated with traditional disease modifying antirheumatic drugs or biologic agents such as infliximab [ 7375 ] , etanercept , adalimumab , rituximab [ 78 , 79 ] , and abatacept or tocilizumab [ 80 , 81 ] .
there are conflicting published data concerning the potential role of rfs in predicting responses to antitumor necrosis factor alpha ( tnf- ) : some studies have found that rf positivity before therapy is insufficient to predict a response [ 8285 ] , whereas others have found that it predicts a negative response [ 86 , 87 ] . in particular , it has been reported that high pretreatment levels of iga rf are associated with a poor clinical response to tnf- inhibitors .
high serum levels of rf are predictors of more severe disease forms and b cell - depleting therapy can have a beneficial effect : rf - positive ra patients have a better response to rituximab than those who are rf negative [ 8992 ] .
it has been demonstrated that low - affinity rfs appear to be key player in immune responses to many infectious organisms , and high - affinity rfs indicate more severe and persistent disease in patients with ra .
rfs are probably the result of the immune response to inflammation ( depending on genetic background ) and may have regulatory effects on ig production by controlling b cell activation . | rheumatoid factors are antibodies directed against the fc region of immunoglobulin g. first detected in patients with rheumatoid arthritis 70 years ago , they can also be found in patients with other autoimmune and nonautoimmune conditions , as well as in healthy subjects .
rheumatoid factors form part of the workup for the differential diagnosis of arthropathies . in clinical practice
, it is recommended to measure anti - cyclic citrullinated peptide antibodies and rheumatoid factors together because anti - cyclic citrullinated peptide antibodies alone are only moderately sensitive , and the combination of the two markers improves diagnostic accuracy , especially in the case of early rheumatoid arthritis .
furthermore , different rheumatoid factor isotypes alone or in combination can be helpful when managing rheumatoid arthritis patients , from the time of diagnosis until deciding on the choice of therapeutic strategy . |
primary biliary cirrhosis ( pbc ) is a chronic cholestatic liver disease recognized at histology as chronic nonsuppurative destructive cholangitis with an autoimmune pathogenesis supported by th1 or th17 cells producing ifn- or il-17 [ 1 , 2 ] .
several inflammatory cell populations , including t and b cells , are found around the affected intrahepatic bile ducts , and chemokines are believed to play a pivotal role for the infiltration of inflammatory cells . a better understanding of the role of specific chemokines in liver injury is ancillary to understanding the molecular mechanisms regulating the autoimmunity process and
the observed patterns of chemokine expression in normal and pbc liver are illustrated in table 1 . in our recent experiments , we cultured epcam - positive cells ( i.e. , biliary epithelial cells and bec ) isolated by immunobeads from explanted liver tissue and examined the production of chemokines by protein array following the stimulation by inflammatory cytokines or toll - like receptor ( tlr ) ligands .
our data illustrated that bec produce proinflammatory chemokines such as cxcl1 , cxcl5 , cxcl6 , and cxcl8 without any specific stimulation as shown in figure 1 .
on the other hand , bec challenged with a tlr3 ligand ( poly i : c ) manifest a th1 shift and the production of ccl3 , ccl4 , ccl5 , and cxcl10 .
such production of th1 chemokines was further prompted by the interaction between cd40 on bec and cd154 on liver infiltrating lymphocytes .
taken altogether , the evidence support the observation that bec induces a proinflammatory environment in the absence of innate immunity stimulation and induces th1-sifted environment when such stimulation is present .
fractalkine is characterized as a type-1 transmembrane molecule with the chemokine domain tethered by a 241-amino acid glycosylated stalk , a 19-amino acid transmembrane region , and 37-amino acid intracellular tail .
the surface - expressed transmembrane fractalkine induces the firm adhesion of leukocytes expressing its receptor cx3cr1 . after shedding by the disintegrins and metalloproteinases ( adam ) 10 and 17
fractalkine is upregulated by inflammation cytokines such as tnf- or ifn- , it has been proposed to contribute to inflammatory diseases by promoting the transmigration of cx3cr1-expressing cells to inflamed tissues in crohn disease , rheumatoid arthritis , atherosclerosis , systemic lupus erythematosus , and most recently pbc .
cx3cr1 is expressed on natural killer cells , monocytes , macrophages , mucosal dendritic cells , cd8 t cells , and a subset of effector - memory cd4 t cells [ 11 , 12 ] .
human th1 cells express high levels of cx3cr1 mrna , different from polarized th2 cells [ 13 , 14 ] .
fractalkine is expressed in limited amounts in the normal human liver , particularly near branches of the hepatic artery and in small bile ducts located at the interface between the portal tract and the hepatic lobule . in the case of acute or chronic viral hepatitis
, fractalkine is detected in the areas of necrosis and inflammatory infiltration and also at the interface between the expanded portal tract and the regenerating nodule .
regenerating epithelial cells of the ductular reaction are also positive for fractalkine . in kidney allograft transplantation
, fractalkine is expressed in renal tubular epithelial cells , and the expression is upregulated by tnf- , the recognized key proinflammatory cytokine in acute rejection .
the cd4 and cd8 t cells expressing cx3cr1 predominantly produce ifn- and tnf- , and these t cells infiltrate the synovium in patients with rheumatoid arthritis . in inflammatory bowel disease ( ibd ) , intestinal microvascular endothelial cells produce high amounts of fractalkine , and ibd mucosa as well as periphery contained significantly more cx3cr1 + cells than control .
fractalkine is a major contributor to t- and monocytic - cell adhesion to endothelial cells . in hcv infection ,
it is unclear whether cx3cr1 positive cells are protective or trigger disease [ 2025 ] .
fractalkine is peripherally expressed dominantly in patients with pbc , and is upregulated in bec of the pbc liver .
cx3cr1 is expressed on infiltrating lymphocytes in the portal tracts and on intraepithelial t cells of injured bile ducts .
bec manifesting senescent features in damaged small bile ducts also overexpress fractalkine . as previously introduced , in our recent work , we separated bec as epcam positive and endothelial cells as cd31 positive by immunobeads and evaluated the production of fractalkine as chemokine by elisa .
figure 2(a ) illustrates the elevated production of fractalkine by endothelial cells challenged with tlr3 ligand ( poly i : c ) or tlr4 ligand ( lps ) .
conversely , bec did not produce fractalkine with any other tlr ligand stimulation ( figure 2(b ) ) , and this was not reversed with the addition of established inflammatory cytokines such as tnf- or ifn-. further , we investigated the production of fractalkine following the interaction between bec or endothelial cells and liver infiltrating lymphocytes .
as shown in figure 3 , mononuclear cells adhered with higher affinity to bec compared to endothelial cells in the tlr4 ligand ( lps ) stimulation , and this adherence was increased more in pbc than in other control diseases .
fractalkine works to modulate inflammation in the bec of pbc , thus suggesting that novel therapies to block fractalkine induced environment may prove beneficial .
based on our data , we propose a working model on the role of fractalkine as chemokine or cell adhesion molecule by vascular endothelial cells and bec , summarized in figure 4 .
first , fractalkine as chemokine from vascular endothelial cells stimulated via tlr3 or tlr4 induce cx3cr1 positive monocytes or nk cells .
second , fractalkine as cell adhesion molecule from tlr4-stimulated bec recruit cx3cr1 positive cells around target cells .
this mechanism may trigger the onset of chronic nonsuppurative destructive cholangitis and autoimmune mechanism perpetuating the cholangitis .
we further submit that th1 chemokines produced by bec stimulated from tlr3 are important contributors to the autoimmune mechanism . | primary biliary cirrhosis ( pbc ) is characterized by the autoimmune injury of small intrahepatic bile duct . on this basis
, it has been suggested that the targeted biliary epithelial cells ( bec ) play an active role in the perpetuation of autoimmunity by attracting immune cells via chemokine secretion . to address this issue , we challenged bec using multiple toll - like receptor ( tlr ) ligands as well as autologous liver infiltrating mononuclear cells ( lmnc ) with subsequent measurement of bec phenotype and chemokine production and lmnc chemotaxis by quantifying specific chemokines , specially cx3cl1 ( fractalkine ) .
we submit the hypothesis that bec are in fact the innocent victims of the autoimmune injury and that the adaptive immune response is critical in pbc . |
periodontitis is a multifactorial disease caused by the presence of periodontopathogenic species in a susceptible host and this susceptibility maybe influenced by environmental and genetic factors .
the most important goal of periodontal therapy is to reduce or eliminate the sub gingival microorganisms , which cause the periodontal disease to maintain periodontal health and if possible to regenerate the lost tissues .
scaling and root planning ( srp ) is considered as a gold standard to attain and maintain periodontal health by elimination of bacterial plaque . as the probing depth increases
, the effectiveness of srp decreases because of limited access to deep pockets , which leads to incomplete removal of periodontopathogens . due to the infective nature of periodontal disease ,
several antimicrobial agents , which are delivered by rinsing , irrigation , systemic administration , and local devices , have been used to overcome the limited efficacy of conventional treatment of periodontitis .
the inherent limitations of systemic and topical chemotherapeutics led to the development of local drug delivery systems .
targeting the antimicrobial agent directly to the infected site and maintaining effective levels for a sufficient time are necessary for successful treatment .
this approach also addresses the critical concerns of unnecessarily exposing the patient to large amounts of systemic antibiotics , which can result in bacterial resistance .
chlorhexidine ( chx ) has long been known as an effective antimicrobial agent and has been used as a topical antiseptic for over 30 years .
its efficacy as a topical mouth rinse to inhibit dental plaque and gingivitis has been well established and demonstrated in study periods for two years without evidence of the development of any bacterial resistance .
it is a bisbiguanide antiseptic and antimicrobial agent , which is used as a locally applied slow release drug delivery system available in the form of gel , varnish , chip , etc . to enhance the efficacy of non - surgical therapy a chemo - mechanical treatment concept
was introduced based on sequential srp and the adjunctive subgingival administration of 35% chx varnish and subgingival placement of a biodegradable chx chip , which biodegrades and releases chx within the pocket over 7 - 10 days maintaining an average concentration in the gcf greater than 12 mg / ml for 8 days .
the objectives of this study were to clinically evaluate the use of chx varnish and biodegradable chx chip when used as an adjunct to srp ( combined therapy ) and also to compare the combined therapy with srp alone .
a total of 15 patients ( 7 males and 8 females ) aged 35 - 55 years reporting to the department of periodontics and implantology , coorg institute of dental sciences , virajpet , coorg were recruited for the study .
subjects diagnosed with chronic periodontitis characterized by at least three sites with a probing depth of 5 - 8 mm , which should bleed on probingpresence of minimum 16 natural teeth ( at least 4 teeth per quadrant)subjects in a good state of general health without any systemic disorder .
subjects diagnosed with chronic periodontitis characterized by at least three sites with a probing depth of 5 - 8 mm , which should bleed on probing presence of minimum 16 natural teeth ( at least 4 teeth per quadrant ) subjects in a good state of general health without any systemic disorder .
subjects with a history of allergy to chxsubjects who are on any medication within the last 6 monthssubjects showing endodontic - periodontic lesionssubjects undergoing orthodontic treatmentsubjects who have undergone surgery in relation to the site within a period of 6 monthssubjects with habits such as smoking , mouth breathing , and tongue thrusting .
subjects with a history of allergy to chx subjects who are on any medication within the last 6 months subjects showing endodontic - periodontic lesions subjects undergoing orthodontic treatment subjects who have undergone surgery in relation to the site within a period of 6 months subjects with habits such as smoking , mouth breathing , and tongue thrusting .
after an initial screening visit for recruitment and signing an informed consent , all patients were subjected for site selection by another examiner who was unaware of the study design .
then , three sites in each patient were randomly assigned for group a , group b , and group c. baseline measurements were recorded on all sites and subjected to different treatment modality accordingly .
the study protocol was approved by the ethical committee of coorg institute of dental sciences , virajpet , karnataka . in each patient , the treatment sites were divided randomly into three groups : group a ( control site ) : periodontal pocket was treated by srp alone [ figure 1 ] .
srp group 5 mm probing depth group b ( chx varnish ) : periodontal pocket was treated with srp plus the application of chx varnish .
the varnish was applied using a blunt needle and the content was slowly released while the needle was moved in a coronal direction from the bottom of the pocket .
then , the excess varnish was gently removed by using gracey 's curette [ figures 2 , 3 and 4 ] .
baseline srp + chx varnish group 5 mm probing depth initial chx varnish application
srp + chx varnish group chx varnish removal after 15 minutes with a curette group c ( chx chip ) : periodontal pocket was treated with srp , and then the biodegradable chx chip was placed inside the pocket .
the chip was inserted apically into the pocket with the rounded end of the chip facing towards the base of the pocket until resistance is met at the base of the pocket and the sites were covered with a periodontal dressing ( coe pack ) [ figures 5 and 6 ] .
baseline srp + chx chip group 5 mm probing depth chx chip insertion srp + chx chip group the following parameters were recorded at baseline , 1 month and 3 months post - operatively :
plaque index ( pi ) ( silness and loe 1964)bleeding on probing ( bop ) evaluated 30 s after pocket probingsulcus bleeding index ( sbi ) ( mhlemann and son 1971)probing pocket depth ( ppd ) linear measurement taken from the gingival margin to base of the pocket using customized acrylic occlusal stent with vertical grooves.clinical attachment level ( cal ) cal was measured using occlusal stent with vertical grooves .
plaque index ( pi ) ( silness and loe 1964 ) bleeding on probing ( bop ) evaluated 30 s after pocket probing sulcus bleeding index ( sbi ) ( mhlemann and son 1971 ) probing pocket depth ( ppd ) linear measurement taken from the gingival margin to base of the pocket using customized acrylic occlusal stent with vertical grooves . clinical attachment level ( cal )
the clinical changes over time within each group and the impact of the treatment modalities on bop ; ppd and cal were statically analyzed with mann whitney u - test .
a total of 15 patients ( 7 males and 8 females ) aged 35 - 55 years reporting to the department of periodontics and implantology , coorg institute of dental sciences , virajpet , coorg were recruited for the study .
subjects diagnosed with chronic periodontitis characterized by at least three sites with a probing depth of 5 - 8 mm , which should bleed on probingpresence of minimum 16 natural teeth ( at least 4 teeth per quadrant)subjects in a good state of general health without any systemic disorder .
subjects diagnosed with chronic periodontitis characterized by at least three sites with a probing depth of 5 - 8 mm , which should bleed on probing presence of minimum 16 natural teeth ( at least 4 teeth per quadrant ) subjects in a good state of general health without any systemic disorder .
subjects with a history of allergy to chxsubjects who are on any medication within the last 6 monthssubjects showing endodontic - periodontic lesionssubjects undergoing orthodontic treatmentsubjects who have undergone surgery in relation to the site within a period of 6 monthssubjects with habits such as smoking , mouth breathing , and tongue thrusting .
subjects with a history of allergy to chx subjects who are on any medication within the last 6 months subjects showing endodontic - periodontic lesions subjects undergoing orthodontic treatment subjects who have undergone surgery in relation to the site within a period of 6 months subjects with habits such as smoking , mouth breathing , and tongue thrusting .
after an initial screening visit for recruitment and signing an informed consent , all patients were subjected for site selection by another examiner who was unaware of the study design .
then , three sites in each patient were randomly assigned for group a , group b , and group c. baseline measurements were recorded on all sites and subjected to different treatment modality accordingly .
the study protocol was approved by the ethical committee of coorg institute of dental sciences , virajpet , karnataka .
subjects diagnosed with chronic periodontitis characterized by at least three sites with a probing depth of 5 - 8 mm , which should bleed on probingpresence of minimum 16 natural teeth ( at least 4 teeth per quadrant)subjects in a good state of general health without any systemic disorder .
subjects diagnosed with chronic periodontitis characterized by at least three sites with a probing depth of 5 - 8 mm , which should bleed on probing presence of minimum 16 natural teeth ( at least 4 teeth per quadrant ) subjects in a good state of general health without any systemic disorder .
subjects with a history of allergy to chxsubjects who are on any medication within the last 6 monthssubjects showing endodontic - periodontic lesionssubjects undergoing orthodontic treatmentsubjects who have undergone surgery in relation to the site within a period of 6 monthssubjects with habits such as smoking , mouth breathing , and tongue thrusting .
subjects with a history of allergy to chx subjects who are on any medication within the last 6 months subjects showing endodontic - periodontic lesions subjects undergoing orthodontic treatment subjects who have undergone surgery in relation to the site within a period of 6 months subjects with habits such as smoking , mouth breathing , and tongue thrusting .
after an initial screening visit for recruitment and signing an informed consent , all patients were subjected for site selection by another examiner who was unaware of the study design .
then , three sites in each patient were randomly assigned for group a , group b , and group c. baseline measurements were recorded on all sites and subjected to different treatment modality accordingly .
the study protocol was approved by the ethical committee of coorg institute of dental sciences , virajpet , karnataka .
in each patient , the treatment sites were divided randomly into three groups : group a ( control site ) : periodontal pocket was treated by srp alone [ figure 1 ] .
srp group 5 mm probing depth group b ( chx varnish ) : periodontal pocket was treated with srp plus the application of chx varnish .
the varnish was applied using a blunt needle and the content was slowly released while the needle was moved in a coronal direction from the bottom of the pocket .
then , the excess varnish was gently removed by using gracey 's curette [ figures 2 , 3 and 4 ] .
baseline srp + chx varnish group 5 mm probing depth initial chx varnish application
srp + chx varnish group chx varnish removal after 15 minutes with a curette group c ( chx chip ) : periodontal pocket was treated with srp , and then the biodegradable chx chip was placed inside the pocket .
the chip was inserted apically into the pocket with the rounded end of the chip facing towards the base of the pocket until resistance is met at the base of the pocket and the sites were covered with a periodontal dressing ( coe pack ) [ figures 5 and 6 ] .
baseline srp + chx chip group 5 mm probing depth chx chip insertion srp + chx chip group
the following parameters were recorded at baseline , 1 month and 3 months post - operatively :
plaque index ( pi ) ( silness and loe 1964)bleeding on probing ( bop ) evaluated 30 s after pocket probingsulcus bleeding index ( sbi ) ( mhlemann and son 1971)probing pocket depth ( ppd ) linear measurement taken from the gingival margin to base of the pocket using customized acrylic occlusal stent with vertical grooves.clinical attachment level ( cal ) cal was measured using occlusal stent with vertical grooves .
plaque index ( pi ) ( silness and loe 1964 ) bleeding on probing ( bop ) evaluated 30 s after pocket probing sulcus bleeding index ( sbi ) ( mhlemann and son 1971 ) probing pocket depth ( ppd ) linear measurement taken from the gingival margin to base of the pocket using customized acrylic occlusal stent with vertical grooves
. clinical attachment level ( cal ) cal was measured using occlusal stent with vertical grooves .
the indices values of pi and sbi were subjected for kruskal wallis test . the clinical changes over time within each group and the impact of the treatment modalities on bop ;
the mean pi of group a ( srp ) at baseline was 1.33 , at 30 days was 0.73 and at 90 days , it was 0.8 and the difference was statistically significant .
the mean pi of group b ( srp + chx varnish ) at baseline was 1.67 , at 30 days was 0.67 and at 90 days was 0.73 and the difference was statistically significant .
the mean pi of group c ( srp + chx chip ) at baseline was 1.67 , at 30 days was 0.67 , and at 90 days it was 0.8 and the difference was statistically significant [ table 1 ] .
kruskal wallis test showing the comparison of plaque index between the 3 groups ( srp , srp+chx varnish and srp+chx chip ) at baseline , 30 day and 90 day the results showed a significant reduction ( p < 0.001 ) in pi scores between baseline and 30 and 90 day . there was a slight increase in the score from 30 to 90 day .
the percentage reduction in bop on 30 day was 66.67% in group a , 86.7% in group b , and 73.3% in group c. the percentage reduction in bop on 90 day was 60% in group a , 86.7% in group b , and 73.3% in group c. the differences were statistically significant [ table 2 , graph 1 ] .
percentages of bleeding on probing between the three groups at baseline , 30 day and 90 day comparing probing pocket depth between the three groups ( scaling and root planning [ srp ] , srp + chlorhexidine [ chx ] varnish and chx chip ) at baseline , 30 day and 90 day the mean sbi of group a ( srp alone ) at baseline was 0.67 , at 30 days was 0.17 and at 90 days was 0.27 .
the mean sbi of group b ( srp + chx varnish ) at baseline was 0.72 , at 30 days was 0.28 and at 90 days was 0.3 .
the mean sbi of group c ( srp + chx chip ) at baseline was 0.72 , at 30 days was 0.22 and at 90 days was 0.3 and the difference was statistically significant .
the results showed a significant reduction ( p < 0.001 ) in index scores between baseline and 30 and 90 day .
there was a slight increase in the score from 30 to 90 day [ table 3 ] .
kruskal wallis test showing the comparison of sulcus bleeding index between the 3 groups ( srp , srp+chx varnish and srp+chx chip ) at baseline , 30 day and 90 day in group a , the average values at baseline were 5.733 0.704 , at 30 day the average values of ppd reduced to 3.333 0.617 and after that until 90 day there was no further reduction in ppd was seen .
however , this difference in reduction of ppd was statistically significance ( p < 0.001 ) . in group b , the average ppd
was reduced from 5.667 0.617 at baseline to 3.133 0.352 at 30 day , which was statically significant .
similar results were seen in group c where ppd reduced from 6.067 0.884 at baseline to 3.267 0.594 at 30 day [ table 4 , graph 2 , figures 7 , 8 and 9 ] .
the ppd between the three groups ( srp , srp+chx varnish and srp+chx chip ) at baseline , 30 day and 90 day mann whitney u test comparison of clinical attachment level between the three groups ( scaling and root planning [ srp ] , srp + chlorhexidine [ chx ] varnish , srp + chx chip ) at baseline 30 day and 90 day 90 day srp + chx varnish group 2 mm probing depth 90 day srp + chx chip group 3 mm probing depth 90 day
srp group 3 mm probing depth in group a , the average value of cal at baseline was 5.733 0.704 , at 30 day the average value reduced to 3.333 0.617 and this gain in cal between 0 and 30 day was statistically significant . in group
b , the baseline value was 5.800 0.775 , at 30 day it was 3.133 0.352 and the difference was statically significance .
similarly , in group c the average value of cal reduced from 6.200 1.082 to 3.400 0.632 at baseline and 30 day respectively , which was statistically significant ; however in all three groups have shown no further improvement in cal from 30 to 90 day [ table 5 , graph 3 ] .
the clinical attachment levels between the three groups ( srp , srp+chx varnish and srp+chx chip ) at baseline , 30 day and 90 day mann whitney u test the differences in percentages of bleeding on probing between three groups ( scaling and root planning [ srp ] , srp + chlorhexidine [ chx ] varnish and srp + chx chip ) at baseline , 30 day and 90 day
the mean pi of group a ( srp ) at baseline was 1.33 , at 30 days was 0.73 and at 90 days , it was 0.8 and the difference was statistically significant .
the mean pi of group b ( srp + chx varnish ) at baseline was 1.67 , at 30 days was 0.67 and at 90 days was 0.73 and the difference was statistically significant .
the mean pi of group c ( srp + chx chip ) at baseline was 1.67 , at 30 days was 0.67 , and at 90 days it was 0.8 and the difference was statistically significant [ table 1 ] .
kruskal wallis test showing the comparison of plaque index between the 3 groups ( srp , srp+chx varnish and srp+chx chip ) at baseline , 30 day and 90 day the results showed a significant reduction ( p < 0.001 ) in pi scores between baseline and 30 and 90 day . there was a slight increase in the score from 30 to 90 day .
the percentage reduction in bop on 30 day was 66.67% in group a , 86.7% in group b , and 73.3% in group c. the percentage reduction in bop on 90 day was 60% in group a , 86.7% in group b , and 73.3% in group c. the differences were statistically significant [ table 2 , graph 1 ] .
percentages of bleeding on probing between the three groups at baseline , 30 day and 90 day comparing probing pocket depth between the three groups ( scaling and root planning [ srp ] , srp + chlorhexidine [ chx ] varnish and chx chip ) at baseline , 30 day and 90 day
the mean sbi of group a ( srp alone ) at baseline was 0.67 , at 30 days was 0.17 and at 90 days was 0.27 .
the mean sbi of group b ( srp + chx varnish ) at baseline was 0.72 , at 30 days was 0.28 and at 90 days was 0.3 .
the mean sbi of group c ( srp + chx chip ) at baseline was 0.72 , at 30 days was 0.22 and at 90 days was 0.3 and the difference was statistically significant .
the results showed a significant reduction ( p < 0.001 ) in index scores between baseline and 30 and 90 day .
there was a slight increase in the score from 30 to 90 day [ table 3 ] .
kruskal wallis test showing the comparison of sulcus bleeding index between the 3 groups ( srp , srp+chx varnish and srp+chx chip ) at baseline , 30 day and 90 day
in group a , the average values at baseline were 5.733 0.704 , at 30 day the average values of ppd reduced to 3.333 0.617 and after that until 90 day there was no further reduction in ppd was seen .
however , this difference in reduction of ppd was statistically significance ( p < 0.001 ) . in group
b , the average ppd was reduced from 5.667 0.617 at baseline to 3.133 0.352 at 30 day , which was statically significant .
similar results were seen in group c where ppd reduced from 6.067 0.884 at baseline to 3.267 0.594 at 30 day [ table 4 , graph 2 , figures 7 , 8 and 9 ] .
the ppd between the three groups ( srp , srp+chx varnish and srp+chx chip ) at baseline , 30 day and 90 day mann whitney u test comparison of clinical attachment level between the three groups ( scaling and root planning [ srp ] , srp + chlorhexidine [ chx ] varnish , srp + chx chip ) at baseline 30 day and 90 day 90 day
srp + chx varnish group 2 mm probing depth 90 day srp + chx chip group 3 mm probing depth 90 day
in group a , the average value of cal at baseline was 5.733 0.704 , at 30 day the average value reduced to 3.333 0.617 and this gain in cal between 0 and 30 day was statistically significant . in group
b , the baseline value was 5.800 0.775 , at 30 day it was 3.133 0.352 and the difference was statically significance . similarly , in group c the average value of cal reduced from 6.200 1.082 to 3.400
0.632 at baseline and 30 day respectively , which was statistically significant ; however in all three groups have shown no further improvement in cal from 30 to 90 day [ table 5 , graph 3 ] . the clinical attachment levels between the three groups ( srp , srp+chx varnish and srp+chx chip ) at baseline , 30 day and 90 day mann whitney u test the differences in percentages of bleeding on probing between three groups ( scaling and root planning [ srp ] , srp + chlorhexidine [ chx ] varnish and srp + chx chip ) at baseline , 30 day and 90 day
the objective of the study was to evaluate the efficacy of two chx containing local drug delivery systems ( ec40 chx varnish and periochip ) over srp for a period of 3 months .
the 3 month time frame was chosen because the effects of locally delivered chx has been shown to be evident for 11 weeks after administration and also 3 months corresponds to the typical recall interval for patients after periodontal treatment . in the present study ,
the full mouth plaque scores from baseline to 30 and 90 day were taken into consideration .
these findings are in accordance with the results obtained in studies conducted by soskolne et al .
the sbi scores showed a significant reduction in both groups from baseline until 90 day .
this can be attributed to the elimination of local factors with srp in all three groups , which is in accordance with studies conducted by carvalho et al .
there was a significant reduction in ppd in the groups a ( srp group ) , b ( srp + chx varnish group ) and c ( srp + chx chip group ) from baseline to 30 days .
however , the reduction in probing depth from 30 days to 3 months was not as significant rather it was consistent .
this is in accordance with the results of the studies conducted by jeffcoat et al .
the intergroup probing depth values when compared to group a ( srp group ) , group b ( srp + chx varnish group ) and group c ( srp + chx chip group ) showed no statistical difference between the groups at 3 months from baseline .
( 2001 ) where significant differences were noted between the groups only at 9 months from baseline .
the results are also in conjunction with grisi ( 2002 ) wherein the probing depth at 3 , 6 , and 9 months did not show any significant differences between the control and test groups .
( 2007 ) also demonstrated that at 6 months all three treatment groups were equally effective .
( 2002 ) in which the differences in probing depth and cals between the control and test groups were not statistically significant at 3 and 6 months . the findings of this study were not in accordance with soskolne et al .
( 1997 ) wherein statistically significant differences were found between test and control groups at 3 and 6 months .
a point to be considered is that in both multicenter studies ( soskolne et al .
1998 ) the period of srp was confined to one hour period without administration of local anesthetic , in a study carried out by badersten et al .
in which srp was carried out with no time limit and under local anesthesia , sites showed improvements in probing depth and clinical attachment gain greater than those obtained in the controlled pockets .
the results are also in conjunction with the study done by carvalho et al . 2007 and
it has reiterated the fact that time limitation may have affected the quality of root planning .
all three groups showed statistically significant reduction in ppd and gain in cal from baseline to 30 day , but there was no significant difference in the scores from 30 to 90 day or rather the scores remained stable and also there was no significant difference in the scores within the groups . in the present study , the cal values followed a trend similar to probing depth reduction , improvement in attachment levels was observed in all the groups when compared to pre - treatment values .
the mean attachment gain was 2.4 mm ( srp group ) , 2.3 mm ( srp + chx varnish group ) and 2.8 mm ( srp + chx chip group ) .
the present study indicated that application of chx varnish and placement of chx chip as an adjunct to srp produced a clinically significant reduction in the ppd , bop and a gain in cal at 30 day and 90 day from baseline when compared to srp alone though the results were not statistically significant .
the limitations of this study could be a smaller sample size over a shorter period and single application / insertion of the varnish / chip .
based on the findings of this study , chx varnish ( ec40 ) and chx chip ( periochip ) did provide clinically significant benefits beyond that achieved with conventional srp after a 3 month period , but the difference was not statistically significant .
long - term studies with a larger sample size and multiple application / insertion are required to determine the potential use of these drugs as an adjunct to conventional periodontal therapy . | background : the purpose of this study was to clinically evaluate the benefits of sub gingival chlorhexidine ( chx ) varnish and biodegradable chx chip application used as an adjunct to scaling and root planning ( srp ) as combined therapy and also to compare the effect of combined therapy with srp alone.materials and methods : fifteen patients with at least three sites with a probing pocket depth ( ppd ) of 5 - 8 mm were considered . following baseline evaluation ,
all three sites were subjected for srp . after completing srp ,
each site was randomly subjected for chx varnish , chx chip application and the 3rd site was left without any medication as a control .
clinical parameters such as sulcus bleeding index , plaque index , bleeding on probing ( bop ) , ppd , and clinical attachment level ( cal ) were recorded at baseline , 1 month and 3 months post-operatively.results:all three groups presented with an improvement in clinical parameters compared to baseline .
the mean reduction in ppd was 2.4 mm in srp sites , 2.5 mm in srp + chx varnish sites and 2.8 mm in srp + chx chip sites .
the mean gain in cal was 2.4 mm in srp sites , 2.3 mm in srp + chx varnish sites and 2.8 mm srp + chx chip sites.interpretation and conclusion : the present study indicated that application of chx varnish and placement of chx chip as an adjunct to srp produced a clinically significant reduction in the ppd , bop and a gain in cal at 30th day and 90th day from baseline when compared to srp alone .
the results though were not statistically significant . |
decays of b mesons to two - body final states containing a charmonium resonance such as a j/ or (2s ) offer a powerful way of studying electroweak transitions .
such decays probe charmonium properties and play a role in the study of cp violation and mixing in the neutral b system .
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\begin{document}$\sqrt{s } = 7~\mathrm{tev}$\end{document } during 2011 and correspond to an integrated luminosity of 0.37 fb .
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data were taken with both magnet polarities to reduce systematic effects due to detector asymmetries .
photon , electron and hadron candidates are identified by a calorimeter system consisting of scintillating - pad and pre - shower detectors , and electromagnetic and hadronic calorimeters .
muons are identified by a muon system composed of alternating layers of iron and multiwire proportional chambers .
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\begin{document}$\sqrt{p_{\mathrm{t}_{1 } } \cdot p_{\mathrm{t}_{2 } } } > 1.3~\mathrm{gev}/c$\end{document}. the di - muon trigger decision in the software trigger requires muon pairs of opposite charge with pt>500 mev / c , forming a common vertex and with an invariant mass in excess of 2.9 gev / c .
in this analysis , the decays \documentclass[12pt]{minimal }
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\begin{document}$b^{+}\rightarrow \psi k^{+}(b^{0}\rightarrow \psi k^{*0},\allowbreak b^{0}_{s}\rightarrow \psi\phi)$\end{document } are reconstructed , where represents (2s ) or j/ , reconstructed in the decay modes .
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\begin{document}$b^{+}(b^{0 } , b^{0}_{s})$\end{document } candidate .
the starting point of the analysis is the reconstruction of either a j/ or (2s ) meson decaying into a di - muon pair .
candidates are formed from pairs of opposite sign tracks that both have a transverse momentum larger than 500 mev / c .
good reconstruction quality is assured by requiring the per degree of freedom of the track fit to satisfy /ndf<5 .
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\begin{document}$\chi^{2}_{\mathrm{vtx}}<20$\end{document } ) .
the resulting di - muon candidate is required to have decay length significance from its associated primary vertex greater than 5 and have an invariant mass between 3020 and 3135 mev / c in the case of a j/ candidate or between 3597 and 3730 mev / c for a (2s ) candidate .
the selected j/ and (2s ) candidates are then combined with a k , k or to create b meson candidates .
pion - kaon separation is provided by the ring - imaging cherenkov detectors . to identify kaons the difference in logarithm of the likelihood of the kaon and pion hypotheses is required to satisfy \documentclass[12pt]{minimal }
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\begin{document}$\delta\log\mathcal{l}^ { k- \pi } > -5$\end{document}. in the case of pions the difference in logarithm of the likelihood of the pion and kaon hypotheses
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\begin{document}$\delta\log\mathcal{l}^ { \pi - k } > -5$\end{document}. as in the case of muons , a cut is applied on the track /ndf provided by the track fit at 5 .
the kaons and pions are required to have a transverse momentum larger than 250 mev / c and to have an impact parameter significance with respect to any primary vertex larger than 2 . in the b channel
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\begin{document}$b^{0}_{s}$\end{document } channel the mass of the kaon pair is required to be \documentclass[12pt]{minimal }
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\begin{document}$1010<\mathrm{m } _ { k^{+ } k^{- } } < 1030~\mathrm{mev}/c^{2}$\end{document}. in addition , we require the decay time of the b candidate ( c ) to be larger than 100 m to reduce the large combinatorial background from particles produced in the primary pp interaction . a global refit of the three - prong ( four - prong ) combination is performed with a primary vertex constraint and with the di - muon pair mass constrained to the nominal value using the decay tree fit ( dtf ) procedure .
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\begin{document}$\chi^{2}_{\mathrm{dtf}}/\mathrm{ndf}$\end{document } ) is required to be less than 5 , where the dtf algorithm takes into account the number of decay products to determine the number of degrees of freedom .
the b candidates , where a muon from the (2s) decay is reconstructed as both muon and kaon , are removed by requiring the angle between the same sign muon and kaon to be greater than 3 mrad .
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\begin{document}$842<\mathrm{m } _ { k^{+ } \pi^{-}}<942~\mathrm{mev}/c^{2}$\end{document } mass window and the number of the \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } \rightarrow \psi k^{+ } k^{-}$\end{document } candidates in a \documentclass[12pt]{minimal }
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\begin{document}$1010<\mathrm{m } _ { k^{+ } k^{-}}<1030~\mathrm{mev}/c^{2}$\end{document } mass window . the number of signal candidates is determined by fitting a double - sided crystal ball function [ 12 , 13 ] for signal together with an exponential function to model the background .
the tail parameters of the crystal ball function are fixed to values determined from simulation .
1mass distributions of ( a ) b
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\begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document}. the total fitted function ( solid ) and the combinatorial background ( dashed ) are shown .
the variation in resolution of the different modes is fully consistent with the energy released in the decays and in agreement with simulation mass distributions of ( a ) b
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\begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document}. the total fitted function ( solid ) and the combinatorial background ( dashed ) are shown .
the variation in resolution of the different modes is fully consistent with the energy released in the decays and in agreement with simulation to estimate the contribution from non - resonant decays bk and \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } \rightarrow \psi k^{+ } k^{-}$\end{document } , the k and kk invariant mass distributions have been studied after relaxing requirements on the k and kk invariant masses , see fig . 2 .
the k and kk invariant mass distributions are then fitted with the sum of a relativistic breit - wigner function convolved with a gaussian , to describe the resonant contribution from the k or , two - body phase space function multiplied by a second order polynomial , to describe the non - resonant k or kk contribution .
the splot technique is used to unfold the k or kk invariant mass of the non - resonant ( in k and kk ) candidates .
this unfolded distribution contains a mixture of combinatorial background and non - resonant bk or \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } \rightarrow \psi k^{+ } k^{-}$\end{document } decays .
is then used to estimate the contribution from the non - resonant b decays , which is subtracted from the total yield to estimate the contribution from resonant bk or \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } \rightarrow \psi\phi$\end{document } decays .
the contribution of the resonant decays can also be extracted by unfolding the contribution of resonant k or kk decays to the k or kk invariant mass distribution .
2mass distributions of k
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\begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } and ( d ) \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document } decays . the total fitted function ( solid ) and the combination of the non - resonant component and the combinatorial background ( dashed )
3mass distributions of a ( a ) b
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\begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } and ( d ) \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document } for resonant component ( full circles ) and non - resonant component ( open circles ) .
the total fitted function ( solid ) and the combinatorial background ( dashed ) are shown .
the fit is described in the texttable 1summary of the signal yields for the six b modes considered and the ratios of the number of j/ and (2s ) decays : n
is the summed signal yield for resonant and non - resonant modes , n
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\begin{document}$n^{\mathrm{res}}_{\psi x}$\end{document } is the signal yield for resonant decays ( through k
or ) . the uncertainties are statistical only
b decay modes
n
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\begin{document}$n^{\mathrm{res}}_{\psi(2s)x}/n^{\mathrm{res}}_{j/\psi x}$\end{document }
b
j/k
141,769410141,7694100.08570.0009
b
(2s)k
12,15413012,154130
b
j/k
35,7702071,2533034,5172090.06120.0018
b
(2s)k
2,22360112122,11161
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7,6549266137,588930.06520.0034
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\begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document }
49525
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49525 mass distributions of k
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\begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } and ( d ) \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document } decays . the total fitted function ( solid ) and the combination of the non - resonant component and the combinatorial background ( dashed )
the fit is described in the text mass distributions of a ( a ) b
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\begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } and ( d ) \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document } for resonant component ( full circles ) and non - resonant component ( open circles ) . the total fitted function ( solid ) and the combinatorial background ( dashed )
the fit is described in the text summary of the signal yields for the six b modes considered and the ratios of the number of j/ and (2s ) decays : n
is the summed signal yield for resonant and non - resonant modes , n
is the signal yield for non - resonant modes only and \documentclass[12pt]{minimal }
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\begin{document}$n^{\mathrm{res}}_{\psi x}$\end{document } is the signal yield for resonant decays ( through k
or ) .
the branching fraction ratio is calculated using
1 where n is the number of signal candidates and is the overall efficiency .
the overall efficiency is the product of the geometrical acceptance of the detector , the combined reconstruction and selection efficiency , and the trigger efficiency .
the simulation samples used are based on the pythia 6.4 generator configured with the parameters detailed in ref . .
the evtgen and geant4 packages are used to generate hadron decays and simulate interactions in the detector , respectively .
the digitized output is passed through a full simulation of both the hardware and software trigger and then reconstructed in the same way as the data .
the overall efficiency ratio is 0.9010.016 , 1.0110.014 and 0.9940.014 for the b , the b and the \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } $ \end{document } channels respectively .
since the selection criteria for bj/x and b(2s)x decays are identical , the ratio of efficiencies is expected to be close to unity .
the deviation of the overall efficiency ratio from unity in the case of the bk decays is due to the difference between the pt spectra of muons for the j/ and (2s ) decays . for the b and \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } $ \end{document } channels this difference is small .
it has been checked that the behaviour of the efficiencies of all selection criteria is consistent in the data and simulation .
since the decay products in each of the pairs of channels considered have similar kinematics , most uncertainties cancel in the ratio .
the different contributions to the systematic uncertainties affecting this analysis are discussed in the following and summarized in table 2 .
table 2systematic uncertainties ( in % ) on the relative branching fractionssource
b
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b
channel
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\begin{document}$b^{0}_{s } $ \end{document } channelnon - resonant decays1.53.4data - simulation agreement1.70.52.0magnet polarity1.40.60.7finite simulation sample size0.30.50.6trigger1.11.11.1background shape0.60.20.2signal shape0.70.80.5angular distribution<0.10.6particle misidentification0.4<0.1<0.1sum in quadrature2.72.24.3 systematic uncertainties ( in % ) on the relative branching fractions the dominant source of systematic uncertainty arises from the subtraction of the non - resonant components in the b and the \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } $ \end{document } decays .
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\begin{document}$b^{0}_{(s)}\rightarrow \psi k^{*0}(\phi)$\end{document } decays directly using the splot technique by unfolding and fitting the \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{(s)}$\end{document } mass distribution of candidates containing genuine k( ) resonances .
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\begin{document}$b^{0}_{(s)}$\end{document } candidates and fitting this distribution to determine the number of non - resonant decays .
the corresponding uncertainties are found to be 1.5 % in the b channel and 3.4 % in the \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } $ \end{document } channel .
the other important source of uncertainty arises from the estimation of the efficiencies due to the potential disagreement between data and simulation .
the corresponding uncertainties are found to be 1.7 % in the b channel , 0.5 % in the b channel and 2.0 % in the \documentclass[12pt]{minimal }
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the trigger is highly efficient in selecting b meson decays with two muons in the final state . for this analysis the di - muon pair is required to trigger the event .
differences in the trigger efficiency between data and simulation are studied in the data using events which were triggered independently on the di - muon pair .
a further uncertainty arises from the imperfect knowledge of the shape of the signal and background in the b meson mass distribution . to estimate this effect ,
a linear and a quadratic function are considered as alternative models for the background mass distribution .
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the maximum observed change in the ratio of yields in the (2s ) and j/ modes is taken as systematic uncertainty .
the central value of the relative efficiency is determined by assuming that the angular distribution of the b(2s)x decay is the same as that of the bj/x .
the systematic uncertainty due to the unknown polarization of the (2s ) in the b meson decays is estimated as follows .
the simulation samples were re - weighted to match the angular distributions found from the data and the relative efficiency was recalculated .
the difference between the baseline analysis and the re - weighted simulation is taken as the systematic uncertainty , as shown in table 2 .
finally , the uncertainty due to potential contribution from the cabibbo - suppressed mode with a misidentified as k is found to be 0.4 % in the b channel and negligible in the b and \documentclass[12pt]{minimal }
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the uncertainty due to the cross - feed between b and \documentclass[12pt]{minimal }
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\begin{document}$b^{0}_{s } $ \end{document } channels with a misidentified as k ( or a k misidentified as ) is negligible .
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\begin{document}$r_{\psi}=\mathcal{b } ( j/\psi \rightarrow \mu ^{+}\mu ^{-})/\mathcal { b } ( \psi(2s)\rightarrow \mu ^{+}\mu ^{- } ) = \mathcal{b } ( j/\psi \rightarrow e^{+}e^{-})/ \mathcal{b } ( \psi(2s)\rightarrow e^{+}e^{- } ) = 7.69\pm0.19$\end{document } . the results are combined using eq .
( 1 ) to give
where the first uncertainty is statistical , the second is systematic and the third is the uncertainty on the r value .
the resulting branching fraction ratios are compatible with , but significantly more precise than , the current world averages of \documentclass[12pt]{minimal }
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\begin{document}$\mathcal{b } ( b^{+}\rightarrow \psi ( 2s ) k^{+})/\mathcal{b } ( b^{+}\rightarrow j/\psi k^{+})=0.60\pm0.07$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$\mathcal{b } ( b^{0}_{s } \rightarrow \psi ( 2s)\phi)/\mathcal{b } ( b^{0}_{s } \rightarrow j/\psi \phi)=0.53\pm 0.10$\end{document } and the cdf result of \documentclass[12pt]{minimal }
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\begin{document}$\mathcal{b } ( b^{0}\rightarrow \psi ( 2s ) k^{*0})/\mathcal{b } ( b^{0}\rightarrow j/\psi k^{*0 } ) = 0.515\pm0.113\pm0.052$\end{document } .
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\begin{document}$b^{0}_{s } \rightarrow \psi(2s)\phi$\end{document } decay is particularly interesting since , with more data , it can be used for the measurement of cp violation in \documentclass[12pt]{minimal }
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physical activity ( pa ) has been identified as a hallmark contributor to individuals ' quality of life , most notably among women .
indeed , several research endeavours have converged on trying to understand the principal determinants of pa , and in particular , those factors that can be associated with individuals ' volitional control of this specific behaviour .
one factor that has been of mounting interest to behavioural scientists is that of affect and/or mood , which is defined as the quality of a subjective feeling state consisting of elements of valence ( i.e. , good / bad ) and activation ( i.e. , high / low ) .
notably , studies have revealed associations between engaging in an acute bout of pa and immediate increases in positive affect . in turn ,
basic affective reactions that are tied to a moderate intensity pa session ( i.e. , increases in positive affect ) have been shown to predict futures bouts of pa six and twelve months later .
indeed , repeated positive affective experiences associated with pa may sustain pa motivation over time and this can facilitate long - term pa participation [ 6 , 7 ] .
it follows then that studying positive affect in the context of specific pa sessions is an outcome worthy of investigation in its own right , and this within broader attempts to understand how to optimally predict sustainable patterns of pa behaviour for successful weight management and greater well - being .
however , experts have highlighted that not all individuals who participate in pa achieve more positive affective states and ensuing increments in general well - being .
indeed , results have not been entirely consistent as some participants have witnessed no change or a worsening of their affect with exercise .
this may have long - term implications in terms of sustaining adequate amounts of pa as well as levels of well - being .
this has led to researchers ' attempts to isolate the conditions under which the specific affective benefits of exercise might be maximized or conversely , thwarted . in particular , one line of research has focused on the intensity of pa and its relationship with exercise - related affect .
meta - analytic findings have revealed that the effect of acute pa on positive - activated affect may be stronger at low - to - moderate intensities [ 4 , 10 ] . in past studies ,
threshold of intensity for the affective benefits of exercise , yet there is insufficient empirical evidence for an inverted u shape dose - response relationship . from a more person - centered view , several researchers now argue that preferred / self - selected intensity might lead to stronger benefits in positive affect than prescribed pa intensities [ 12 , 13 ] .
overall , the evidence is inconclusive as there appears to be significant inter - individual variability regarding the benchmark intensity level that is most conducive to increments in positive affect .
another line of work that has specifically targeted such individual - based differences has also addressed a call for more theoretical research on possible psychological mechanisms underlying the pa - positive affect relationship [ 14 , 15 ] . in particular , it has been suggested and recently emphasized that individuals ' motivational styles toward pa may supply a missing and understudied link . self - determination theory ( sdt ) , one recognized and well - supported motivational theory , distinguishes between two general types of motivation : self - determined motivation and controlled motivation ( or regulation ) .
deci and ryan also classified motivation into several types of behavioural regulations that fall within these two broader categories .
overall , self - determined motivation is rooted in feelings of satisfaction , choice and volition . it is characterized as enjoying an activity for its own sake ( i.e. , intrinsic motivation ) and/or assigning it value and personal importance ( i.e. , identified regulation ) . controlled or non - self - determined motivation can be defined as engaging in an activity , such as exercise , so as to avoid self - inflicted shame or guilt , or to gain a personal reward , namely pride ( introjected regulation ) .
controlled motivation can also be characterized by being motivated according to external demands ( e.g. , to obtain a reward ; external regulation ) .
self - determination theory also postulates another type of motivation , amotivation , which was deemed less relevant in the present study with active participants as it is defined as lacking the intention to act .
self - determination , namely , higher levels of intrinsic and identified styles of motivation , has been associated with healthy intentions to engage in health - promoting behaviours such as pa . in women ,
identified regulation has also stood out as a predictor of the intensity of pa engagement .
higher levels of these self - determined regulations have also been positively associated with desirable psychological variables including pa enjoyment and post - pa positive affect , as well as a battery of well - being indicators such as greater self - esteem and lower depression and state anxiety levels . on the other hand and under the more controlling classifications ,
indeed research has revealed an association between introjected motivation for pa and maladaptive outcomes that include poorer life satisfaction and exercise - dependence symptoms such as strenuous pa .
similarly higher scores on external motivation have been linked to self - esteem issues and higher levels of negative affect .
although motivation toward pa and the perceived intensity of pa have seemingly been construed as independent influences , it could be that they exert an interaction effect on pa - induced affective changes .
such an interplay would be consistent with the expert - noted complexity of underlying mechanisms of the pa - affect ( and well - being ) relationship as well as new evidence regarding the relevance of motivational styles in predicting changes in affect following pa at self - selected intensities [ 15 , 16 ] . especially with introjected regulation , which theory proposes should not be directly linked to well - being ( and affect ) , perceived intensity may exert a particular contributing influence .
while other research , albeit scant , has underscored the possible interplay between self - selected pa intensity and psychological factors such as personality and self - efficacy , to our knowledge no empirical research has properly entertained the interaction with motivation . indeed , an interplay between perceived intensity ( self - selected ) and motivation styles in predicting changes in affect with acute pa could reveal a sustainable mechanism for long - term pa and well - being that lies within individuals ' volitional regulation and that also capitalizes on the physical properties of exercise . therefore , the purpose of this controlled laboratory study was exploratory and was to examine whether there was an interaction between sdt 's motivational regulations to engage in a running activity and ratings of perceived exertion ( rpe ; self - selected intensity ) in predicting pre - to post - pa ( i.e. , running ) changes in positive affect .
the three types of motivation were targeted given evidence cited above as well as recently revealed associations between motivation types and intensity preferences .
lastly , this study was conducted with active women given that the link between pa and affect may be particularly evident in active individuals experienced with pa [ 30 , 31 ] as well as among women [ 32 , 33 ] .
sample to highlight the above underlying theoretical relationships ; in addition , this would contribute to an important need to better understand how to develop successful of pa intervention strategies for women .
fourty - one healthy and active women ( i.e. , > 20 minutes of moderate to vigorous pa three times per week ) with an average age of 40.98 ( sd = 4.93 ) participated in this study . the godin leisure time exercise questionnaire , a common and validated self - report measure of pa levels , was administered as a screening measure and the mean score for this sample was high at 59.71 .
confounding influences in the experimental protocol , it was necessary that participants self - report running as their preferred or most frequent exercise modality . in their leisure time , the women reported running at a mean rpe of 13.37 ( sd = 1.22 ) .
they also gave an average of 8.60 km for a usual run and/or 49.37 minutes per run .
the women in this sample were well - educated ( 82.9% with a university degree or higher ) and all were employed outside of the home .
the positive affect negative affect schedule ( panas ) , comprised of 20 adjective words ( 10 positive , 10 negative ) , was used to assess participants ' affect .
according to the circumplex model , affect can be characterized by the dimensions of valence ( positive , negative ) as well as activation ( low , high ) and experts maintain that the panas focuses on the high activation component of affect . for each adjective , participants rated their response from ( 1 ) not at all to ( 5 ) extremely using the stem indicate to what extent you feel this way right now , that is , at the present moment .
examples of adjectives include : excited ( positive ) and upset ( negative ) . given the focus on positive affect in the present investigation , only these adjectives were summed and analyzed . in the present study ,
the cronbach alpha for the positive subscale pre- and post - running were .88 and .91 , respectively .
the situational motivation scale ( sims ) was employed to measure situational motivation to run .
the sims is comprised of 16 items across four subscales that assess the different behavioural regulations ( intrinsic motivation , identified , external , and amotivation ) . introjected regulation was excluded from the original sims in order to have more succinct instrument for research purposes . in the current study , an enhanced version of the sims with four validated items tapping this type of regulation was used [ 40 , 41 ] .
items measuring introjection in the enhanced situational motivation scale ( sims ) : ( 1 ) because i would feel bad not doing it ; ( 2 ) because i would feel guilty not to do it ; ( 3 ) because i want to avoid feeling guilty ; ( 4 ) because i would regret not doing it . using a 7-point likert scale from ( 1 )
corresponds not at all to ( 7 ) corresponds exactly , participants respond to stem why are you currently [ about to run ] , for items that included because i want to avoid feeling guilty
( introjection ) and because i am doing it for my own good ( identified ) .
average score were calculated for intrinsic motivation , introjected regulation , and identified regulation . as expected from a sample of active participants , scores on external regulation and amotivation were low and variance levels were negligible , which contributed to low internal consistency values .
the cronbach alphas of relevant subscales were acceptable to good with values of .65 , .85 , and .85 for identified regulation , introjected regulation and intrinsic motivation respectively .
the rating of perceived exertion ( rpe ) scale was administered in the minutes immediately following the run to assess the perceived intensity / exertion of the running activity .
more specifically , and similar to previous studies we employed what some experts have coined session rpe
, whereby participants were instructed to rate the overall intensity of the full running session [ 43 , 44 ] .
this was expected to minimize the influence of momentary fluctuations in how participants ' perceived their exertion , which could contaminate the accuracy of the overall evaluation .
numerical values from 6 to 20 make up the [ session ] rpe scale which is also anchored at every odd integer with a brief descriptor ( i.e. , 7 = very , very light ; 13 = somewhat hard ; 19 = very , very hard ) .
the validity and reliability of the rpe are well established given its frequent usage in studies of pa and mood .
this study was approved by the ethics review board of the university of ottawa and was part of a larger project .
, participants provided written informed consent and promptly responded to the sims and the panas prior to the running activity .
participants were taken to a private exercise room equipped with a treadmill , a desk , and a chair and were explained the running protocol . in order to mimic a self - paced run and yet easily log the pace of the run to ensure that at least a moderate - intensity run was being met , the treadmill control panel was physically detached from the treadmill running belt .
this allowed the researcher to easily adjust settings in response to any and all demands from the participant .
the participants completed a 2 - 3 minute warm - up walk at an average speed of 5.17 km / hour .
the researcher remained in proximity in order to speed up or slow down the belt as frequently as necessary , the details and timing of which were duly noted . by weighting any change in pace by the ratio of elapsed time at that pace , an average running pace was computed .
there was minimal conversation and eye contact between participant and researcher and the treadmill was maintained at a grade of zero .
participants ran for a 30-minute duration in order to stay consistent with previous inquiries on acute exercise and affective states ( e.g. , bartholomew et al . , 2005 ) .
afterwards , the belt was slowed to the initial walking speed for a 2-minute cool - down and participants provided the session rpe for the 30-minute running component .
all data were entered into spss version 18.0 ; sums , means , and standard deviations were calculated .
initial data - screening procedures were conducted according to procedures outlined in tabashnick and fidell .
namely , assessments were conducted for data entry errors , missing data , outliers , normality and the basic assumptions of regression analyses .
descriptive statistics and reliability analyses were calculated for affect and motivation variables . a repeated - measures t - test compared pre - run and post - run levels of positive affect as a preliminary examination of whether the run had a significant influence on participants ' affect .
a standardized residual change score for positive affect was calculated for each participant in order to adequately account for participants ' initial affect scores .
namely , a predictor score was computed by regressing post - run affect on pre - run affect and then subtracting this from the observed scores .
the residual change score served as the outcome variable in three separate hierarchical multiple - regression models that were employed to test the interaction between rpe and situational motivation for running .
specifically , separate product terms for rpe and the three motivational regulations were created using standardized scores and each term was added as the last step in their respective regression models .
during the experimental running session , participants ran at an average pace of 9.66 km / hour and provided a mean rpe of 12.79 .
this value reflects a moderate to high intensity that was similar to the usual rpe achieved by the women outside of the laboratory .
all assumptions regarding normality , linearity , and homoscedasticity were met and there was no evidence of collinearity .
positive affect increased significantly from pre- to post - run [ t(40 ) = 4.83 , p < .001 ] . see table 1 for descriptive statistics .
results of the hierarchical regression analyses showed no significant interaction between intrinsic motivation and rpe on residual change scores in positive affect [ fchange ( 1,37 ) = .23 , p = .63 ] nor between identified regulation and rpe [ fchange ( 1,37 ) = 1.82 , p = .19 ] .
however , there was a significant interaction effect of rpe and introjection [ fchange(1,37 ) = 4.20 , = .30p < .05 ] which explained an additional 9% of variance in the change in positive affect from the variables alone . as displayed in figure 1 ,
when participants reported low introjection , the influence of perceived running intensity on the change in positive affect was considerable , with higher rpe being associated with a greater increase in affect from pre- to post - run .
that is , when participants reported high introjected regulation , the change in positive affect was elevated and fluctuated very little with rising rpe values , and even showed a trend toward diminishing slightly .
the results of this experimental study revealed a significant interaction between rpe ( i.e. , intensity ) and introjected regulation but not between rpe and intrinsic or identified styles of motivation in predicting changes in positive affect from pre- to post - running . to our knowledge , this is the first study to have examined this interplay , which builds on previous deliberations by ekkekakis and lind regarding a potentially complex causal chain linking intensity , pleasure from exercise , and adherence , among other factors .
in particular , our investigation offers an important adjunct to a study by duncan and colleagues that revealed associations between sdt 's motivational regulations and pa intensity . specifically , we considered the interaction between these variables in explaining a factor that is being increasingly recognized not only as an important consequence of pa but also as a viable determinant of future participation , namely positive affect .
our results attest to recent suggestion that a person 's motivational style for pa should be considered when attempting to maximize the affective gains of aerobic pa , especially at a self - selected intensity .
the strengths of this investigation include the self - paced nature of the running activity , a situational measure of motivation for running , and the use of a well - controlled laboratory environment .
researchers have observed that up to very high exertion levels , there is a basic positive linear association between exercise intensity and affect .
while the basic linear relationship ( rpe - positive affect ) in this study was in fact positive for low and moderate levels introjection , it deviated markedly for those high in introjection , such that affective changes were fairly stable regardless of perceived intensity .
while introjection has been associated with engaging in vigorous exercise , our results imply that this relationship may have little bearing on the acute mood changes that are experienced through pa , at least among avid female runners .
this interplay may even be worrisome given a downward trend in positive affect as the rpe increased among highly introjected runners .
this could suggest that active females that are high in introjection achieve some form of an immediate feel - good or relief
effect from an activity such as running that serves to prevent or relieve feelings of guilt , thereby materializing irrespective of perceived intensity .
this was evidenced in our study by greater overall gains in affect for those high in introjection , which may be akin to what sabiston and colleagues referred to as the reparative properties of motivation from guilt . on the other hand , among runners
lower in introjected regulation , changes in positive affect were associated with greater variability in rpe which could indicate a greater appreciation of the sensations and physical properties of pa .
lind et al . remarked that individuals usually choose to exercise at a pace that improves or maintains their mood .
our findings show that this may be more applicable for exercisers with lower introjection and who are less driven by internal pressures to exercise , thereby freeing them to experience the pa session more fully .
therefore , in - task affective states and sensation , which were not assessed in our study , could be a source of discrepancy between persons high and low in introjection .
future studies should consider open - ended probes during acute pa sessions in order to ascertain the pertinent sources of affective changes between individuals that differ in motivational style . despite its effect on positive affect in our study , as well its influence on sustaining high intensity pa
, introjection has been associated with several negative psychological consequences that were not assessed in our acute exercise study ( e.g. , lower self - worth and life satisfaction ) . thus ,
cautious interpretation of our results is warranted as immediate improvements in positive affect may not necessarily translate into benefits in general well - being .
this could be disconcerting if we consider that individuals with lower levels of well - being are generally less likely to engage in pa , thus initiating a questionable cycle of regular pa maintenance and compounding issues related to leading a healthy and active lifestyle . on a different note ,
the results showing nonsignificant interactions between more self - determined motivational styles and rpe may be of theoretical significance .
similar to what was discussed above with respect to runners with lower levels of introjection , our results hint that those with higher levels of intrinsic and/or identified regulation could have less contingencies attached to their exercise engagement . in this regard , burton and colleagues found that being intrinsically motivated positively predicted well - being independently of the level of performance
. moreover , they found that fostering intrinsic motivation may diminish certain contingencies between one 's perceived performance and their well - being .
this is consistent with sdt principles and research revealing that despite variability in perceived competence for an activity , self - determined individuals show greater interest , pleasure , and confidence which is exhibited through greater well - being .
thus the relationship between self - determined regulations and well - being is more likely of a direct nature , as indicated in the present study whereby perceived intensity , which could be view as an indicator of their performance , did not significantly shape post - pa changes in positive affect among the women higher on self - determined regulations .
other authors have also noted a direct relationship between identified and intrinsic motivation and post - pa affect as an indicator well - being .
in addition , and from a psychometric perspective , ceiling effects could be partly to blame for nonsignificant findings given the high means and low variances for these variables , especially identified regulation . with respect other possible limitations of this study ,
there are variant opinions in the literature regarding the optimal time point(s ) at which to assess rpe .
we opted to assess session rpe immediately post - pa which is common practice among researchers [ 50 , 56 ] .
however , some authors such as singh et al . argue that post - pa rpe can vary significantly in the few minutes following exercise ( e.g. , between 510 minutes ) and that evaluations of rpe taken 1530 minutes after pa are more stable and valid indicators of participants ' perceived intensity . still , other experts gravitate away from session rpe altogether claiming that repetitive rpe measurements at regular intervals during a pa session provide a more representative measure of intensity [ 58 , 59 ] . in future studies , researchers will need to address concerns over the optimal time point(s ) for rpe measurement(s ) in studying exercise - related affect and they may wish to supplement such inquiries with alternative and objective measures of intensity .
similarly , researchers might wish to consider the use of research designs that capitalize on longitudinal and in - task effects of motivation and intensity on indicators of well - being .
in addition , future studies will need to make use of larger samples and draw from groups of participants that are more diverse in terms of activity levels as this may alleviate some of the psychometric issues regarding the assessment of certain motivational styles .
the use of only active , healthy - weight women could also be considered a drawback of the present study in terms of generalizability and it would be worthwhile to test the given interactions in overweight or obese individuals who may experience pa differently .
researchers have already shown that autonomous regulations are associated with long - term weight management as well as indicators of well - being in obese populations [ 23 , 61 ] and it would be worthwhile to examine the interplay with pa intensity in order to develop optimal interventions strategies for these individuals . yet
it is also interesting to note that some studies have actually shown that affective experiences and pleasure from exercise might not significantly differ between normal weight and overweight women , at least at self - selected intensities [ 29 , 49 ] .
in addition , other experts mention that it is actually fruitful to study an active population as much can be learned regarding the determinants of successful pa engagement and associated consequences and this could then be targeted among the insufficiently active . moreover ,
the purpose of this study was to explore a theory - based interaction mechanism and therefore it was advantageous to select a sample that could maximize the postulated relationships . in sum , while women higher in introjection showed the greatest change in positive affect post - running , further reasoning as to why this increase was not particularly sensitive to the self - selected intensity of the run ( rpe ) , as well as the long - term impact of this relationship on well - being and pa maintenance , is left to future inquiries . | there is evidence that affective experiences surrounding physical activity can contribute to the proper self - regulation of an active lifestyle .
motivation toward physical activity , as portrayed by self - determination theory , has been linked to positive affect , as has the intensity of physical activity , especially of a preferred nature .
the purpose of this experimental study was to examine the interaction between situational motivation and intensity [ i.e. , ratings of perceived exertion ( rpe ) ] in predicting changes in positive affect following an acute bout of preferred physical activity , namely , running .
fourty - one female runners engaged in a 30-minute self - paced treadmill run in a laboratory context .
situational motivation for running , pre- and post - running positive affect , and rpe were assessed via validated self - report questionnaires .
hierarchical regression analyses revealed a significant interaction effect between rpe and introjection ( p < .05 ) but not between rpe and identified regulation or intrinsic motivation . at low levels of introjection , the influence of rpe on the change in positive affect was considerable , with higher rpe ratings being associated with greater increases in positive affect .
the implications of the findings in light of sdt principles as well as the potential contingencies between the regulations and rpe in predicting positive affect among women are discussed . |
ingestion of some amino acids has presumable roles in performance improvement in athletes.[13 ] among them , -alanine supplementation has been suggested to improve performance during high - intensity exercises . on the other hand
, it has been shown that large amounts of h are produced in the muscles during high - intensity exercise and result in ph reduction .
there are many cellular ph buffers defend against exercise - induced acidosis , which include phosphocreatine , inorganic phosphates and histidine - containing dipeptides .
carnosine ( -alanyl - l - histidine ) is the main histidine - containing dipeptide in humans .
additionally , hill et al . and harris et al . , showed that 28 days of -alanine supplementation increased intramuscular levels of carnosine by nearly 60% .
antioxidant function , muscle contractility regulation , and ph buffering , are the possible physiological roles of carnosine in skeletal muscle .
thus , the possible role of carnosine could be prevention of skeletal muscle acidity in improving exercise performance
. prolonged exercise can result in oxidative stress and muscle fatigue , which may be prevented by carnosine due to its antioxidative properties . on the other hand
, -alanine administration could increase carnosine content of skeletal muscles by 4080%.[1719 ] increase of carnosine concentrations in muscle results in altered buffering capacity,[1819 ] and thus affects performance .
furthermore , some studies have shown that carnosine acts as a ca sensitizer for the sarcomeres in muscles[2021 ] and thus could prevent fatigue . however , synthesis of carnosine in muscle is limited by the availability rate of -alanine , which can be overcome by -alanine supplementation . although it has been estimated that carnosine is responsible for nearly 10% of the total buffering capacity in human muscle
there are few studies on -alanine supplementation and its possible effects on endurance exercise ; therefore , the purpose of this study is to assess the effects of -alanine administration on vo2 max , time to exhaustion and lactate concentrations in male physical education students .
these students were fit ( bmi<25 ) and active ( physical activity2 hr / d ) , but not involved in professional sports .
participants age , weight and height were 21.10.7 years , 71.88.8 kg and 1787 cm , respectively , for -alanine ( n=20 ) group and 21.91.5 years , 74.98.3 kg and 1805 cm for placebo group ( n=19 ) , respectively ( ns ) .
before initiating the study , all participants were informed of all procedures of the study and signed an informed consent .
none of the participants had ingested -alanine , or any other nutritional supplements , for a minimum of 3 months before the initiation of the study .
participants were asked to abstain from exercise 24 h before trial initiation and to maintain their current physical activity and dietary patterns .
after pre - testing , the participants were randomly assigned to one of the two groups : a ) -alanine ( 2 g / day ) , b ) placebo ( 2 g dextrose per day).the supplements had the same appearance , and ingested four times per day for 42 consecutive days before post - testing .
all participants completed all experiments , and there were no complaints of side effects of the supplements .
participants were supplemented orally for 6 weeks with either -alanine ( ajinomoto , usa , inc ) or placebo ( dextrose ) .
the study was approved by the ethics committee ( esfahan sport medicine association , iran ) .
supplements were provided in capsules of 400 mg and were administered each day as five divided single doses , with at least 2 h in between ingestions .
venous blood samples were obtained from all participants between 5:00 and 6:00 p.m , after intensive endurance exercising , at the baseline and after intervention .
all measurements were done before the start of the supplementation ( pre ) and after the intervention ( post ) . prior to and following the supplementation protocol , participants performed a continuous graded exercise test ( gxt ) on an electronically braked cycle ergometer ( lode , the netherlands ) to determine vo2 max and time to exhaustion ( tte ) . for each gxt , the primary power output was set at 30 w and elevated 30 w every 2 min until the participant could not maintain the required power output at a pedaling rate of 70 rpm due to fatigue .
plasma samples were obtained for the determination of plasma lactate and glucose concentrations immediately prior to each gxt and 2 min post - exercise .
glucose and lactate were analyzed using ysi auto - analyzer ( yellow springs , oh ) .
statistical analyses were conducted using the statistical program for the social sciences ( spss version 13 , inc , chicago , il ) computer software package .
paired t test was used to analyze before and after test data for each group differences .
table 1 shows the mean sd values of exercise performance indices for the pre - and post - supplementation .
supplementation with -alanine demonstrated a significant increase in vo2 max ( p<0.05 ) . on the other hand , tte and lactate concentrations decreased after 6 weeks of supplementation with -alanine ( p<0.05 ) .
the placebo group showed a significant increase in lactate concentrations ( p<0.05 ) , but a non significant increase in tte .
comparison of exercise performance indices , pre - and post - supplementation ( mean sd ) the post - exercise concentrations of plasma lactate were significantly higher ( p<0.05 ) than baseline in two groups .
however , the post - exercise concentrations of lactate were significantly lower ( p<0.05 ) during the alanine supplementation compared to the placebo group .
dietary intake before each trial was similar for energy and macronutrients [ table 2 ] .
the findings of our study suggest that supplementation with -alanine may improve the endurance exercise performance as measured by the vo2 max , tte and plasma lactate concentrations .
, showed that supplementation with creatine + -alanine resulted in significant increases ( p<0.01 ) in muscle carnosine content .
the increased muscle carnosine content was accompanied with an improvement in vo2 max and tte in response to a maximal graded exercise test performed on a cycle ergometer .
another study demonstrated that supplementation with both -alanine and creatine improved cycling performance ( tte ) .
they concluded that this was due to h buffering by carnosine during this transitional period .
our data demonstrate that the significant improvements in the performance indices with -alanine supplementation were due to ph reduction .
the improvement in tte seen in the placebo group participants might be due to the encouragement provided by our staff and also the participants psychological status .
also , the findings of the present study might have been influenced by the fluctuations in the skeletal muscle response to oral supplementation with -alanine .
our data suggest that supplementation with -alanine may delay the onset of fatigue and thus improve performance during incremental cycle exercise in men .
the glucose concentrations did not change significantly in our study , due to different individual response and insufficient dose or duration .
the participants in this study , however , were male physical education students rather than untrained participants .
however , according to the hypothesis , -alanine supplementation would prevent the drop in intracellular ph during high - intensity contractions and result in less circulating acidosis finally due to elevation of myocellular carnosine content .
furthermore , several studies have shown the importance of ph regulation on performance during endurance exercise , by a pre - exercise alkalosis intervention .
this suggests that the difference between groups is related to the presumable enhancement of muscle carnosine content .
future studies should examine muscle carnosine levels along with vo2 max and plasma lactate concentration during strenuous exercise with variable quantities of -alanine supplementation also , further investigations are necessary to determine the effects of -alanine supplementation during more prolonged and submaximal exercise .
it can be concluded from this study that -alanine administration can reduce acidosis during high - intensity exercise and thus can improve exercise performance in endurance athletes . also it is found that six weeks of supplementation with -alanine at the mentioned prescribed dose did not result in significant changes in glucose concentrations . | objectives : supplementation with -alanine has been proposed to improve performance in some exercises such as cycling and running .
also , it has been demonstrated that great deals of proton ions are produced in the skeletal muscles during exercise that result in acidosis , whereas -alanine may reduce this effect .
therefore , the aim of this study is to assess the effects of alanine supplementation on vo2 max , time to exhaustion and lactate concentrations in physical education male students.methods:thirty-nine male physical education students volunteered for this study .
participants were supplemented orally for 6 week with either -alanine ( 5 * 400 mg / d ) or placebo ( 5 * 400 mg dextrose / d ) , randomly .
vo2 max and time to exhaustion ( tte ) with a continuous graded exercise test ( gxt ) on an electronically braked cycle ergometer ; and serum lactate and glucose concentrations were measured before and after supplementation.results:supplementation with -alanine showed a significant increase in vo2 max ( p<0.05 ) and a significant decrease in tte and lactate concentrations ( p<0.05 ) . a significant elevation in lactate concentrations and a non significant increase in tte
were observed in placebo group .
plasma glucose concentrations did not change significantly in two groups after intervention.conclusion:it can be concluded that -alanine supplementation can reduce lactate concentrations during exercise and thus can improve exercise performance in endurance athletes . |
this study supports the main characteristics of a new genus pygmaiobacter and a new species
pygmaiobacter massiliensis strain marseille - p3336 ( csur p3336 ) ; that was isolated from a stool sample of a healthy 47-year - old pygmy woman.the institut fdratif de recherche ethics committee , gave approval for this study under the number 09 - 022 .
samples were collected for microbial content description as part of the human microbiome description by culturomics . | |
in india among the urban women , the numbers of breast cancer patients were increasing annually , both to aging of the population and increase in age - specific incidences .
case control studies in mumbai and chennai have identified the factors such as null parity , late age at marriage , and late age at first pregnancy are important risk factors . [ 2 , 3 ] .
it has also been suggested that western dietary influences changed the lifestyle of urban women could be one of the major causes of the slowly rising incidence of breast cancer in india
. therefore early detection and search for potential antitumour compounds are important in the control of breast cancer .
we have extracted anthocyanins from red sorghum bran by methanol and acidified methanol and evaluated the antitumour activity in mcf-7 cell lines .
the efficiency of the antitumour compounds seems to be related to the propensity of tumour cells to respond to these sorghum anthocaynins by apoptosis .
recently , considerable attention has been focused on the sequence of events referred to as apoptosis and the role of this process in mediating the lethal effects of antineoplastic agents in breast cancer cells .
apoptosis is a highly regulated process that is characterized by cell shrinkage , membrane blebbing , chromatin condensation and formation of a dna ladder with multiple fragments of 180200 bp caused by internucleosomal dna cleavage .
few recent studies of anthocyanins have demonstrated a significant growth inhibition of some tumour cells including human colon cancer , human cervical carcinoma , human leukaemia , and prostate cancer cells [ 58 ] .
considering the antiproliferative activity of some anthocyanins on certain neoplastic cells , an attempt has been made to evaluate the growth inhibitory activity of a relatively sorghum anthocyanins on mcf-7 cells , and that may provide some new information about therapy of breast cancer .
the bran of sorghum bicolor ( l. ) red sorghum were collected from farmers field in tamil nadu , india and were stored at 20c .
the anthocyanin extraction protocol involved the addition of 10 ml of solvent ( 1% hcl in methanol ) to 0.5 g of sample in 50 ml centrifuge tubes and shaking the samples for 2 hours at low speed ( 75 rpm ) in an orbitory shaker ( neolab ) .
samples were then stored at 20c for overnight in the dark to allow for maximum diffusion of phenolics from the cellular matrix .
samples were then equilibrated to room temperature and centrifuged at 7000 rpm for 10 min and taken for analysis .
residues were rinsed with 10 ml volumes of solvent for two times with shaking for 5 min , then centrifuging at 7000 rpm for 10 min and taken for analysis .
finally , the extracts were mixed well and stored at 20c in the dark until further biochemical analysis . human breast cancer cell line , mcf-7
, cells were maintained in dmem containing 10% fbs , supplemented with additional glutamine ( 0.03% ) and 100 g / ml benzyl penicillin , 100 u / ml streptomycin , and 2.5 g / ml amphotericin .
cells were allowed to grow in tissue culture flasks ( corning , usa ) and were kept in co2 incubator at 37c in a humidified atmosphere of 5% co2 and 95% air . for experimental purpose , cells from exponentially growing culture were used .
briefly , cells were seeded at a density of 3104 cells / well into 24-well plates .
after 24 h , ga extracts were added to the medium at various concentrations and incubated for 24 or 48 h as indicated . at the end of the incubation , 3-(4 - 5 dimethylthiozol-2-yl ) , 25-diphenyl - tetrazolium bromide ( mtt ) ( 2 mg / ml ) per well was added , and the formazan crystals formed were solubilized in acidified isopropanol after aspirating the medium .
the extent of mtt reduction was measured spectrophotometrically at 570 nm , and the cell survival was expressed as percentage over the untreated control .
the methanol and acidified methanol extracts of sorghum bran on cell morphology of mcf-7 cells was investigated .
mcf-7 cells grown in a 6-well plates were treated with different extracts of anthocyanin at 37c for 24 h. morphological changes occurring in the cells were observed under phase - contrast microscope and photographed using ccd camera attached to the te 2000 e microscope ( nikon ) .
nuclear morphology changes characteristic of apoptosis appear within the cell together with a distinctive biochemical event : the endonuclease - mediated cleavage of nuclear dna .
in fact , formation of dna fragments of oligonucleosomal size ( 180200 bp ) is an hallmark of apoptosis in many cell types .
the present protocol provides a method for dna separation of fragmented and intact dna fractions and for their analysis by agarose gel electrophoresis . in apoptotic cells
specific dna cleavage becomes evident in electrophoresis analysis as a typical ladder pattern due to multiple dna fragments .
however , although this protocol is simple and generally able to provide good results , it is only qualitative because of its limitations in dna recovery and solubilization . in order to obtain a cleaner dna , other methods for dna preparation
are required ( in some cases use of proteinase k for deproteinization is recommended ) .
cell suspension at 15 10 cells / ml in complete mem medium tte solution : te buffer ph 7.4 with 0.2% triton x-100 ( store at 4c ) nacl 5 m , ice - cold isopropanol , ice - cold ethanol at 70% , ice cold te buffer ph 7.4 loading buffer 10x tbe buffer for electrophoresis ethidium bromide solution electrophoresis - grade agarose dna molecular weight markers refrigerated cell centrifuge gel electrophoresis apparatus uv transilluminator .
dispense 0.5 ml of cell suspension ( no less than 5 10 , otherwise dna will not be detectable by photography of ethidium bromide stained gel , and no more than 5 10 , to avoid difficult handling of too high amounts of insoluble dna ) in tubes labeled b ( bottom ) . centrifuge cells at 200 g at 4c for 10 min .
add to the pellet in tubes b 0.5 ml of tte solution and vortex vigorously .
this procedure allows the release of fragmented chromatin from nuclei , after cell lysis ( due to the presence of triton x-100 in the tte solution ) and disruption of the nuclear structure ( following mg chelation by edta in the tte solution ) . to separate fragmented dna from intact chromatin , centrifuge tubes b at 20,000 g for 10 min at 4c .
add to the small pellet in tubes b 0.5 ml of tte solution .
add to the 0.5 ml volume present in tubes b , s and t , 0.1 ml of ice - cold 5 m nacl and vortex vigorously .
add to each tube 0.7 ml of ice - cold isopropanol and vortex vigorously .
this step can be shortened by putting samples in a bath of ethanol / dry ice for 1 hr .
after precipitation , recover dna by pelleting for 10 min at 20,000 g at 4c . discard supernatants by aspiration or by rapidly inverting tubes and
carefully remove any drops or fluid remaining adherent to the wall of the tube with a paper towel corner .
this can be a critical step because the pellet could be loosened and transparent , hard to be seen .
rinse the pellets by adding to each tube 0.50.7 ml of ice - cold 70% ethanol .
carefully remove any drops or fluid remaining adherent to the wall of the tube by inverting tubes over an absorbent paper towel for 30 min .
let air dry the tubes in upright position for at least 3 hr before proceeding .
dissolve dna by adding to each tube 2050 l of te solution and place the tubes at 37c for 13 days .
the redissolution of dna may be a critical step , in fact it depends on the dna quantity and size present in the samples .
thus , the nonfragmented dna contained in the b tubes , may need higher volumes of te and longer incubation times in order to be resuspended .
mix the samples of dna with loading buffer by adding 10x loading buffer to a final concentration of 1x .
the addition of loading buffer to samples allows to load gel wells more easily and to monitor the run of samples .
place samples in a heating block at 65c for 10 min and immediately load 1020 l of them to each well of a standard 1% agarose gel containing ethidium bromide 0.5 mg / ml .
run the electrophoresis in standard tbe buffer after setting the voltage to the desired level . during electrophoresis it is possible to monitor the migration of samples by following the migration of bromophenol blue dye contained in the loading buffer .
stop the electrophoresis when the dye reaches about 3 cm from the end of the gel . to visualize dna , place the gel on a uv transilluminator and take photos of the gel . wear eye and skin protection when uv is on .
in the present study , antiproliferative effect of red sorghum bran anthocyanin on breast cancer cell line , mcf-7 was investigated under different concentrations .
the red sorghum bran anthocyanins produced significant morphological alterations on mcf-7 cells in culture . under normal growth condition ( control )
these cells were regular in shape and size , had eccentric nucleus and a relatively small piece of cytoplasm ( figure 1(a ) ) .
after treatment with 1000 g / ml of anthocyanin , cells became irregular in shape and size with altered nuclear : cytoplasm ratio , increased number of nucleoli and multiple cytoplasmic vacuoles ( figure 1(b ) ) .
most of the cells had relatively flattened appearance with long multiple cytoplasmic processes forming cross - bridges with neighboring cells ( figure 1(c ) ) .
this has indicated that anthocyanins may render some changes on the cell surface associated with the adherence to the substratum , as a result of which , treated cells tend to adhere firmly to the growth surface , an opposite behavior of the tumor cells and in vivo systems .
after anthocyanin treatment appearance of multinucleated giant cells were also observed in mcf-7 cells ( figure 1(d ) ) .
the result demonstrate that , with increasing concentrations of sorghum anthocyanins from 15 g / ml to 1000 g / ml the percentage of growth inhibition was 21.31 in methanol extract after 24 hr of anthocyanin exposure .
it was also evident that at all anthocyanin concentrations the percentage of growth inhibition increased with increase in concentrations ( table 1 ) .
thus , the detailed analysis of the results clearly indicated that anthocyanins caused significant growth inhibition of mcf-7 cells in dose - dependent manner ( figure 2 ) .
a similar result was observed when mcf-7 cells were treated with acidified methanol extracts of anthocyanin from sorghum bran .
the result indicated that with increasing concentrations of sorghum anthocyanins from 15 g / ml to 1000 g / ml the percentage of growth inhibition of mcf-7 cells increased progressively from 11.47% to 86.88% in acidified methanol extract after 24 hr of anthocyanin exposure ( table 2 ) , when compared with methanol , acidified methanol extract showed a higher growth inhibition ( figure 3 ) .
finally , we reported that the sorghum anthocyanin caused significant growth inhibition of human breast cancer cell lines even at 1000 g / ml concentration .
pouget et al . and han et al . reported the anticancer activities of flavonoids like flavanones , daidzein , genistein , quercetin , and luteolin in human breast cancer by using mcf-7 cell line .
the significant growth inhibitory activities of red sorghum bran anthocyanins led us to investigate the effect of anthocyanin was also played a role in the induction of apoptosis . as shown in figure 4 ,
the amounts of oligonuceosomal - sized fragments in the mcf-7 cells treated with red sorghum bran anthocyanins were increased when the concentrations of these anthocyanins increased from 62.5 g / ml to 125 g / ml .
reported dna fragmentation in leukemia cells molt 4b by cyanidin 3 glucoside isolated from skin of black glycine max .
on the other hand , we have observed similar results in both methanol and acidified methanol extracts of red sorghum bran anthocyanin .
mukherjee et al . evaluated the antiproliferative activity of enoxacin on a human breast cancer cell line which showed significant result after 5 days of drug exposure .
correct the sentence : the present study demonstrated that these red sorghum bran anthocyanins have significant growth inhibitory effects on human breast cancer .
the above results clearly demonstrated that the anthocyanin has induced significant antiproliferative activity on breast cancer cells .
moreover the compound responsible for antiproliferative activity and the mechanism of action of these compounds are yet to be studied in detail . | breast cancer is a leading cause of death in women worldwide both in the developed and developing countries .
thus effective treatment of breast cancer with potential antitumour drugs is important . in this
paper , human breast cancer cell line mcf-7 has been employed to evaluate the antiproliferative activity of red sorghum bran anthocyanin .
the present investigation showed that red sorghum bran anthocyanin induced growth inhibition of mcf-7 cells at significant level .
the growth inhibition is dose dependent and irreversible in nature . when mcf-7 cells were treated with red sorghum bran anthocyanins due to activity of anthocyanin morphological changes
were observed .
the morphological changes were identified through the formation of apoptopic bodies .
the fragmentation by these anthocyanins on dna to oligonuleosomal - sized fragments , is a characteristic of apoptosis , and it was observed as concentration - dependent .
thus , this paper clearly demonstrates that human breast cancer cell mcf-7 is highly responsive by red sorghum bran anthocyanins result from the induction of apoptosis in mcf-7 cells . |
a 64-year - old male who suffered from osteoarthritis of the right knee underwent a uka ( biomet , swindon , uk ) on the right knee . preexisting
however , 4 months postoperatively , he began to feel a painful snapping sensation on the posteromedial side , particularly when he rose from a chair after prolonged sitting . on examination , moderate swelling and tenderness in the posteromedial corner
were observed and there was a palpable clicking over the posteromedial corner of the knee when the knee was moved from flexion to extension at approximately 60 of flexion .
the passive range of motion ( rom ) was 0 to 135 , and there was no varus - valgus or rotational instability .
the hip - knee - ankle ( hka ) angle changed from 11 varus to 5 varus on the postoperative radiographs .
however , the tibial component was implanted too medially , and the amount of medial overhang was 4 mm on the plain radiograph and posteromedial overhang reached up to 5 mm on the computed tomography ( ct ) images ( figs . 1 and 2 ) .
two years after the operation , the pain caused by the snapping almost disappeared , although he felt a slight snapping sensation . a 71-year - old female with osteonecrosis of the right knee underwent a navigation - assisted uka on the right knee .
however , she began to feel another kind of pain on the medial side 1 month after the operation .
seven months after the operation , she began to feel a snapping sensation over the posteromedial aspect of the right knee .
eight months postoperatively , the pain caused by this snapping got worse , and nonsteroidal antiinflammatory drugs and physiotherapy courses were ineffective . eventually , additional surgery was scheduled . upon clinical examination , moderate swelling and tenderness along
the gracilis tendon were observed , and there was a palpable and painful clicking over the posteromedial corner of the knee when actively extending the knee from flexion at approximately 30 of flexion .
the passive rom was 0 to 130 , and there was no varus - valgus or rotational instability .
neither plain radiographs nor ct images examined in extension position showed an excessive implant overhang .
however , reconstructed ct images revealed an excessive overhang of the mobile bearing up to 4 mm at the posteromedial corner ( fig .
a diagnostic arthroscopy of her left knee was performed first and no intraarticular pathologies that would cause the snapping were found . through an 8-cm medial curved skin incision , the medial patellar retinaculum was incised at the anterior border of the sartorius , and the semitendinosus and gracilis tendons were exposed .
passive extension of the knee reproduced the snapping , which turned out to be a phenomenon of subluxation of the gracilis tendon around the semimembranosus ( fig .
approximately 6 cm of the gracilis tendon around the semimembranosus was resected and the snapping disappeared .
the patient was allowed to walk without any limitation soon after the operation and found that the painful snapping at the posteromedial corner had disappeared . snapping symptom has not recurred for 18 months after the additional operation .
both patients were informed that their data would be submitted for publication , and they gave their consent for the report to be published .
a 64-year - old male who suffered from osteoarthritis of the right knee underwent a uka ( biomet , swindon , uk ) on the right knee . preexisting
however , 4 months postoperatively , he began to feel a painful snapping sensation on the posteromedial side , particularly when he rose from a chair after prolonged sitting . on examination , moderate swelling and tenderness in the posteromedial corner
were observed and there was a palpable clicking over the posteromedial corner of the knee when the knee was moved from flexion to extension at approximately 60 of flexion .
the passive range of motion ( rom ) was 0 to 135 , and there was no varus - valgus or rotational instability .
the hip - knee - ankle ( hka ) angle changed from 11 varus to 5 varus on the postoperative radiographs .
however , the tibial component was implanted too medially , and the amount of medial overhang was 4 mm on the plain radiograph and posteromedial overhang reached up to 5 mm on the computed tomography ( ct ) images ( figs . 1 and 2 ) .
two years after the operation , the pain caused by the snapping almost disappeared , although he felt a slight snapping sensation .
a 71-year - old female with osteonecrosis of the right knee underwent a navigation - assisted uka on the right knee .
however , she began to feel another kind of pain on the medial side 1 month after the operation .
seven months after the operation , she began to feel a snapping sensation over the posteromedial aspect of the right knee .
eight months postoperatively , the pain caused by this snapping got worse , and nonsteroidal antiinflammatory drugs and physiotherapy courses were ineffective . eventually , additional surgery was scheduled . upon clinical examination , moderate swelling and tenderness along the gracilis tendon were observed , and there was a palpable and painful clicking over the posteromedial corner of the knee when actively extending the knee from flexion at approximately 30 of flexion .
the passive rom was 0 to 130 , and there was no varus - valgus or rotational instability .
neither plain radiographs nor ct images examined in extension position showed an excessive implant overhang .
however , reconstructed ct images revealed an excessive overhang of the mobile bearing up to 4 mm at the posteromedial corner ( fig .
a diagnostic arthroscopy of her left knee was performed first and no intraarticular pathologies that would cause the snapping were found . through an 8-cm medial curved skin incision ,
the medial patellar retinaculum was incised at the anterior border of the sartorius , and the semitendinosus and gracilis tendons were exposed .
passive extension of the knee reproduced the snapping , which turned out to be a phenomenon of subluxation of the gracilis tendon around the semimembranosus ( fig .
approximately 6 cm of the gracilis tendon around the semimembranosus was resected and the snapping disappeared .
the patient was allowed to walk without any limitation soon after the operation and found that the painful snapping at the posteromedial corner had disappeared . snapping symptom has not recurred for 18 months after the additional operation .
both patients were informed that their data would be submitted for publication , and they gave their consent for the report to be published .
postoperative complications of uka have often been reported5 ) . however , none have reported the complication of snapping pes syndrome after uka . snapping symptoms around the medial aspect of the knee
have rarely been reported , and in most cases , patients are young or middle - aged .
regarding elderly patients , tensho et al.6 ) presented a case of snapping pes syndrome after total knee arthroplasty and that was the only report after a knee arthroplasty . in their case ,
a bony prominence of residual osteophyte at the posteromedial corner of the tibia and a change in the alignment were presumed to be the two main possible causes of the snapping symptoms .
the most probable cause of snapping in case 1 was an excessive overhang of the tibial tray , which reached up to 5 mm at the posteromedial corner .
gudena et al.7 ) reported that a 2 mm or less overhang of the tibial component is ideal to minimize the pain from excessive medial collateral ligament loading in uka .
excessive medial and posteromedial overhang of the tibial tray should be avoided . the probable cause in case 2 was an excessive posteromedial overhang of the mobile bearing ( fig .
a variety of causes of residual pain after uka , such as aseptic loosening , progression of arthritis on the lateral side , infection , and medial overhang , have been reported5789 ) . however , there are still some uka cases where pain is called " unexplained pain " because of the lack of definite causes .
recent papers have reported that proximal tibial strains following uka and preoperative bone marrow edema could be possible causes of unexplained pain10 ) . snapping pes syndrome
we have experienced a 11 out of 111 uka cases ( 10% ) incidence of tibial component excessive overhang ( 3 mm)9 ) , and three of them felt some pain around the medial side of the knee one year after operation .
however , there were not any other cases that felt snapping sensation even with medial overhang of the tibial component .
the reason was difficult to clarify , but we presumed that this might be because snapping syndrome occurred at the posteromedial corner , not on the medial side .
evaluation of the amount of overhang at the posteromedial corner with ct images will be necessary to elucidate it .
with regard to surgical treatment of snapping syndrome , tendon release at the attachment site has often been reported .
however , there were some failure cases of this harvesting at the attachment site technique .
bollen and arvinte3 ) reported , after the examination of real - time ultrasound , they determined to harvest the semitendinosus tendon .
however , the patient 's painful snapping sensation persisted after the operation . additional operation of harvesting the gracilis tendon resolved the snapping symptom . to prevent conduction of further operation , we wanted to confirm the snapping tendon directly so that we did not utilize the harvesting at the attachment site method
we diagnosed these cases mainly based on clinical examination and did not use dynamic ultrasound in these cases .
though snapping symptoms were easily palpable and diagnosis was confirmed by three expert knee surgeons ( hi , st , and ry ) , we should have examined with real - time ultrasound to reveal the pathology more clearly3 ) .
knee surgeons should be aware of the presence of pes snapping syndrome after uka as a postoperative complication .
meticulous implantation , intraoperative evaluation of the movement of the mobile bearing if mobile bearing uka is performed , and adequate preoperative assessments , including magnetic resonance imaging , would prevent snapping pes syndrome after uka . | snapping pes syndrome is defined as a snapping sensation in the medial knee caused by pes anserinus and rarely occurs .
snapping pes syndrome after unicompartmental knee arthroplasty ( uka ) has not been reported yet .
we experienced two cases with this syndrome after uka .
conservative treatment was effective in one case , while surgical excision of the gracilis tendon was necessary to relieve painful snapping in the other case .
the main cause of the first case might be posteromedial overhang of the tibial tray that reached up to 5 mm .
the probable cause of the second case was posteromedial overhang of the mobile bearing . |
retrograde nailing is an effective and increasingly more popular method of distal femoral fracture fixation1 - 4 ) , and has been shown to produce good clinical outcomes5 - 10 ) .
however , concern remains regarding potential articular damage and angular deformities for comminuted or incompletely reduced fractures11 ) . to avoid intraarticular damage , we have no other choice for the entry point according to the individual anatomy of distal femur , which may cause angular deformities at fracture sites .
periarticular angular deformities are more likely to have a greater effect on the proximate joint than malunions at the diaphyses of the femur . to obtain more accurate reduction using intramedullary nailing , the cerclage technique can be used12 ) . however , extended soft tissue dissection for cerclage with wiring can compromise blood supply to fracture sites .
titanium - nickel memory alloys ( ti - ni smas ) have biomechanical characteristics that are similar to those of bone , e.g. , in terms of thermal shape recovery , superelasticity , biocompatibility , corrosion resistance , and damping properties , which makes alloys of this type interesting for medical applications13,14 ) .
furthermore , ti - ni sma bone fixators present a useful alternative to the materials currently used to treat fractures . in the present study
, we developed a ti - ni sma bone fixator with an optimal transformation temperature ( af=352 ) for human applications15 ) . using this material
, we designed a ti - ni sma ring shaped bone fixator ( sma - rbf , bio - smart , ulsan , korea ) and applied this over the periosteum to replace conventional cerclage with minimal soft tissue dissection .
approval for clinical investigation of this material was obtained from the korean food and drug administration .
the purpose of this study was to identify the effect of the devised sma - rbf on postoperative alignments and union rates after retrograde nailing of distal femoral fractures .
during the period between february 2003 and may 2006 we treated 26 patients with a supracondylar fracture of the femur using the retrograde intramedullary nailing technique , and in the present study , we retrospectively reviewed the clinical documents and radiographs of these patients .
one patient had died before this assessment was performed , and thus , 25 patients were available for evaluation . there were no statistically significant differences in patient demographics or injury characteristics between the groups at inception ( table 1 ) .
two fractures were the result of traffic accidents ; one in a female polytrauma patient with fractures to her ipsilateral humerus , pelvis and thoracic spine , and a subarachnoidal hemorrhage following a motorbike accident , whereas the other patient suffered an accident while walking .
three patients had sustained a prior fracture of the ipsilateral proximal femur and had been treated by internal fixation with a plate , which precluded antegrade intramedullary nailing ; one of these patients had osteogenesis imperfecta .
seven patients had previously undergone total knee arthroplasty to ipsilateral knees and had sustained a periprosthetic supracondylar fracture due to a fall ( fig .
eleven fractures were of class a1 , six were of a2 , and eight were of a3 according to the association for osteosynthesis / association for the study of internal fixation ( ao / asif ) classification .
twenty - two patients were treated using an aim titanium supracondylar nail ( depuy ace , leeds , uk ) and three patients were treated with a t2 supracondylar nail ( stryker trauma , schonkirchen , germany ) .
all operations were performed by one senior surgeon with a tourniquet . with the patient placed in the supine position on a radiolucent table
, a rolled towel was placed under the affected knee to maintain 40-50 of flexion . in all the cases ,
closed reduction was attempted and nailing was done using a limited medial parapatellar approach . the intercondylar entry point in the notch or the slot of the femoral component
the entry point was opened afterwards with an awl and a guide wire was inserted under c - arm radiographic guidance .
the medullary canal was sequentially reamed to 1 mm more than the selected nail diameter and the entry point up was reamed to 12 ( for an aim nail ) or 14 mm ( for a t2 nail ) .
the nail was countersunk approximately 3 mm under the articular surface , and we then checked mediolateral and anteroposterior alignments at the femoral fracture site .
if angular malalignment exceeded five degrees despite several closed reduction trials , we attempted open reduction via a lateral approach using a ca .
5-cm skin incision . to maintain reduction , one or two sma - rbfs were used instead of cerclage wiring .
a fixator was first cooled in ice - cold saline ( when cooled the fixator is malleable and easily uncoiled ) .
fixator sizes were determined either preoperatively from radiological images or intraoperatively based on direct measurements .
an appropriately sized bone fixator was then applied to the reduced fracture site , and then irrigated with warm saline , which caused the fixator to contract to its original ring shape . after confirming that alignments are acceptable
, the nail is locked with interlocking screws distally and proximally using an aiming device .
mobilization was started on the first day postoperatively with continuous passive motion and active / active assisted quadriceps exercises .
partial weight bearing was allowed after early signs of callus formation ( usually from the 6th postoperative week ) .
thirteen patients were treated by closed reduction and retrograde nailing ( group n ) , and 12 patients were treated by open reduction and additional internal fixation using a sma - rbf ( group s ) .
patients in groups n and s were followed regularly , and examined radiologically at 4 , 8 and 12 weeks , and at 6 months postoperatively and then annually when necessary .
fracture union was assessed using plain radiographs and was confirmed by the presence of bridging callus formation on anteroposterior and lateral radiographs and by the absence of pain at fracture sites during clinical examinations and weight bearing .
functional evaluations were performed using the sanders ' grading system16 ) , and angular deformities were measured in anteroposterior and lateral radiographs .
the statistical package for the social sciences ( spss ver . 12.0 , spss inc . ,
mean follow - up for patients in group n was 26.8 months ( range , 12 - 96 months ) , whereas mean follow - up in group s was 25.9 months ( range , 12 - 57 months ) .
the type of fractures according to the ao / asif classification in group n were a1 : 7 ( 54% ) , a2 : 3 ( 23% ) , a3 : 3 ( 23% ) ; and in group s were a1 : 4 ( 33% ) , a2 : 3 ( 25% ) , a3 : 5 ( 42% ) . the mean operative time in group n was 63 min ( range , 55 - 80 min ) , which was shorter than that of group s ( 97 min ; range , 82 - 120 min ) ( p=0.001 ) .
one distal interlocking screw was used due to severe comminution in 2 cases of a3 type fractures in group s , and two screws used in the other cases .
fractures healed after an average of 12.2 weeks ( range , 9 - 15 weeks ) in group n and 11.6 weeks ( range , 10 - 13 weeks ) in group s ( p=0.351 ) .
nail dynamization was performed in one patient in group n. functional grading was similar in the two groups . according to sanders ' criteria , results were graded as excellent in 2 ( 15% ) patients in group n and in 2 ( 17% ) patients in group s ; good in 7 ( 54% ) and 6 ( 50% ) ; fair in 4 ( 31% ) and 3 ( 25% ) ; and as poor in 0 ( 0% ) and 1 ( 8% ) , respectively .
two patients in each group achieved a flexion of < 100 and the others 100. one case of heterotopic ossification was noted in group s in a patient with a subarachnoid hemorrhage ( fig .
2 ) . there were no cases of implant failure , knee sepsis , or deep or superficial infections .
the mean angle of coronal angular deformity was valgus 0.8 ( range , varus 2.3-valgus 4.5 ) in group n and valgus 0.7 ( range , varus 1.0-valgus 2.4 ) in group s ( p=0.892 ) . however , mean angles of sagittal angular deformity were extension 1.0 ( range , flexion 3.2-extension 3.1 ) in group n and 0 ( range , flexion 2.1-extension 1.2 ) in group s , which were significantly different ( p=0.022 ) .
there was no case of shortening or further angulation until complete union in both groups .
although the intramedullary nailing reduces the frequency of angular deformity for femur diaphyseal fractures , angular malalignment has been reported to occur at a frequency of up to 42% in studies on distal femoral metaphyseal fractures11,17,18 ) .
these were mostly varus angulations caused by incomplete facture reduction , which is well known in distal femoral fractures because of adductor muscle pull , a laterally applied injury force , and medial comminution , which is commonly associated .
when we perform total knee arthroplasty , we often use an intramedullary guide for the femur side . if there is femoral bowing , or even if not , we sometimes encounter cases requiring a femoral intramedullary guide entry point on the cartilage medial to the intercondylar notch to locate the guide at the center of the medullary canal . however , there is no compromise when nailing for a distal femoral fracture because of cartilage damage or the fixed tka implant slot . in such cases ,
angulation of the distal fragment seems to be more common when the fracture is treated with a nail .
moreover , rotational malunion remains a problem after intramedullary nailing , which is not encountered during plating procedures19 ) .
furthermore , the intramedullary nailing method is less stable to varus - valgus loading than the plate method , although it provides adequate internal splinting for rapid healing and functional outcomes that are comparable to those of lateral fixation devices20,21 ) .
when distal femoral fractures can not be accurately reduced by intramedullary nailing , several alternatives are available , i.e. , acceptance of the malalignment , alteration of the fixation method from closed intramedullary nailing to open plate fixation , or additional mini - open reduction and cerclage wiring .
cases of diaphyseal femur fracture with more than 5 of angulation are poorly corrected by remodeling , and limb mechanics are affected .
however , for distal femoral metaphyseal fractures , alignment assessment is difficult , and few studies have been conducted on the outcome of malalignment .
the authors have performed the mini - open method when cases of malalignment and poor fracture fragment contact were encountered .
the cerclage technique is sometimes required in unstable fractures or femoral fractures during total hip arthroplasty , especially during revisions22,23 ) .
however , concerns have been expressed about additional periosteal dissection because of the possibility of disturbing the blood supply and causing non - union or delayed union . to minimize soft tissue dissection and injury to blood supply ,
we found no case of non - union or delayed union in the s group , which needed additional open reduction and sma fixation .
one case of heterotopic ossification was encountered , but this female patient was bed - ridden as a result of head trauma .
it was retrospective and not randomized , and patient numbers were insufficient to perform statistical analysis , which precluded accurate comparisons .
in addition , the patients were heterogeneous and associated degenerative joint changes were not considered an outcome measurement . yet , the described retrograde nailing method using an sma has not been previously studied , and our study showed that when reduction of a distal femoral fracture with retrograde nailing was difficult additional mini - open reduction and fixation with a ring shaped sma did not increase the risk of union delay or non - union and resulted in good postoperative alignment . however , no differences in functional outcomes were noted after an average follow - up of 26 months . a larger patient cohort and longer follow - ups
when reduction of a distal femoral fracture with retrograde nailing was difficult additional mini - open reduction and fixation with a ring shaped sma did not delay or prevent bony union and resulted in good postoperative alignment . | purposeto identify the effects of using a ti - ni shape memory alloy ring shaped bone fixator ( sma - rbf ) during the retrograde nailing of supracondylar femoral fractures.materials and methodsthe authors reviewed 25 patients with a supracondylar femoral fracture treated by retrograde intramedullary nailing with or without sma - rbf ( group s , 12/25 ; group n , 13/25 ) .
radiological measurements of angular deformity were performed and functional assessments were made using the sanders grading system.resultsall fractures healed after an average of 12.2 weeks ( range , 9 - 15 weeks ) in group n and after 11.6 weeks ( range , 10 - 13 weeks ) in group s ( p=0.351 ) .
the mean angle of coronal angular deformity was valgus 0.8 ( range , varus 2.3-valgus 4.5 ) in group n and valgus 0.7 ( range , varus 1.0-valgus 2.4 ) in group s ( p=0.892 ) .
the mean angle of sagittal angular deformity was 1.0 in extension ( range , flexion 3.2-extension 3.1 ) in group n and 0 ( range , flexion 2.1-extension 1.2 ) in group s ( p=0.022 ) .
however , functional grading evaluations revealed no differences between the two groups.conclusionswhen reduction of a distal femoral fracture with retrograde nailing was difficult additional mini - open reduction and fixation with a ring shaped sma did not delay or prevent bony union and resulted in good postoperative alignment . |
tumor samples were collected from 505 consecutive lung adenocarcinoma patients who underwent curative surgical resection at seoul national university bundang hospital between may 2003 and december 2012 .
clinicopathological information and follow - up data were obtained by reviewing the medical and the pathological records of the enrolled patients .
smoking history was defined as smokers who have smoked 100 cigarettes , and never - smokers who have never smoked or smoked less than 100 cigarettes in their life time .
h.c ) independently reviewed the hematoxylin and eosin stained slides and classified the diagnosis according to the international association for the study of lung cancer / american thoracic society / european respiratory society ( iaslc / ats / ers ) criteria and the 2015 world health organization ( who ) classification system .
histological grade was based on the predominant histological subtypes as follows : grade 1 , lepidic ; grade 2 , acinar and papillary ; and grade 3 , solid and micropapillary .
tumors were staged according to the american joint committee on cancer , seventh staging system .
progression - free survival ( pfs ) was measured from the date of surgery until disease progression or death .
overall survival ( os ) was measured from the date of surgery to the time of death or last follow - up visit .
the most representative areas were obtained for each tumor sample and arranged for tissue microarray ( tma ) .
tissue cores with a diameter of 2 mm were embedded within tma blocks , which were sectioned into series of 4-m - thick slices and then stained with hematoxylin and eosin ; immunohistochemical labeling was then performed .
immunohistochemistry for aqp1 , e - cadherin , and vimentin were performed according to the antibody manufacturer s instructions .
the following antibodies were used : aqp1 ( b-11 , 1:100 , santa cruz biotechnology , santa cruz , ca , usa ) , e - cadherin ( spm471 , 1:150 , thermo fisher scientific , carlsbad , ca , usa ) , and vimentin ( 1:100 , 4a4 , zeta corporation , arcadia , ca , usa ) .
aqp1 overexpression was defined when 25% showed membranous staining with loss of polarization , as previously reported .
immunohistochemical stains of e - cadherin and vimentin were scored using a semi - quantitative evaluation for each case .
the intensity of staining was scored on a four - point scale as follows : 0 , no staining ; 1 , weak staining ; 2 , moderate staining ; and 3 , strong staining .
the final score was calculated by multiplying the intensity score to the fraction score , generating a total range of 0300 .
scores of 0100 and 101300 were considered as negative and positive , respectively , as previously reported .
translocation of anaplastic large cell lymphoma kinase ( alk ) was evaluated in 440 cases by fluorescence in situ hybridization analysis using a probe to alk ( vysis lsi alk dual color , break - apart rearrangement probe , abbott molecular , des plaines , il , usa ) ; translocation was observed in 28 out of 440 cases ( 6.4% ) .
epidermal growth factor receptor ( egfr ) mutations of exon 18 to 21 and kras mutations of codon 12 and 13 were evaluated in 484 and 413 cases using polymerase chain reaction and direct dna sequencing , as previously described .
egfr and kras mutations were identified in 49.0% ( 237/484 ) and 6.1% ( 25/413 ) of cases , respectively .
the association between immunohistochemistry results and clinicopathological variables was assessed by chi - square test , fisher exact test , or spearman s rank correlation test .
kaplan - meier analysis with log - rank test and multivariate cox regression analysis were performed for survival analysis .
tumor samples were collected from 505 consecutive lung adenocarcinoma patients who underwent curative surgical resection at seoul national university bundang hospital between may 2003 and december 2012 .
clinicopathological information and follow - up data were obtained by reviewing the medical and the pathological records of the enrolled patients .
smoking history was defined as smokers who have smoked 100 cigarettes , and never - smokers who have never smoked or smoked less than 100 cigarettes in their life time .
h.c ) independently reviewed the hematoxylin and eosin stained slides and classified the diagnosis according to the international association for the study of lung cancer / american thoracic society / european respiratory society ( iaslc / ats / ers ) criteria and the 2015 world health organization ( who ) classification system .
histological grade was based on the predominant histological subtypes as follows : grade 1 , lepidic ; grade 2 , acinar and papillary ; and grade 3 , solid and micropapillary .
tumors were staged according to the american joint committee on cancer , seventh staging system .
progression - free survival ( pfs ) was measured from the date of surgery until disease progression or death .
overall survival ( os ) was measured from the date of surgery to the time of death or last follow - up visit .
the most representative areas were obtained for each tumor sample and arranged for tissue microarray ( tma ) . tissue cores with a diameter of 2 mm were embedded within tma blocks , which were sectioned into series of 4-m - thick slices and then stained with hematoxylin and eosin ; immunohistochemical labeling was then performed .
immunohistochemistry for aqp1 , e - cadherin , and vimentin were performed according to the antibody manufacturer s instructions .
the following antibodies were used : aqp1 ( b-11 , 1:100 , santa cruz biotechnology , santa cruz , ca , usa ) , e - cadherin ( spm471 , 1:150 , thermo fisher scientific , carlsbad , ca , usa ) , and vimentin ( 1:100 , 4a4 , zeta corporation , arcadia , ca , usa ) .
aqp1 overexpression was defined when 25% showed membranous staining with loss of polarization , as previously reported .
immunohistochemical stains of e - cadherin and vimentin were scored using a semi - quantitative evaluation for each case .
the intensity of staining was scored on a four - point scale as follows : 0 , no staining ; 1 , weak staining ; 2 , moderate staining ; and 3 , strong staining .
the final score was calculated by multiplying the intensity score to the fraction score , generating a total range of 0300 .
scores of 0100 and 101300 were considered as negative and positive , respectively , as previously reported .
translocation of anaplastic large cell lymphoma kinase ( alk ) was evaluated in 440 cases by fluorescence in situ hybridization analysis using a probe to alk ( vysis lsi alk dual color , break - apart rearrangement probe , abbott molecular , des plaines , il , usa ) ; translocation was observed in 28 out of 440 cases ( 6.4% ) .
epidermal growth factor receptor ( egfr ) mutations of exon 18 to 21 and kras mutations of codon 12 and 13 were evaluated in 484 and 413 cases using polymerase chain reaction and direct dna sequencing , as previously described .
egfr and kras mutations were identified in 49.0% ( 237/484 ) and 6.1% ( 25/413 ) of cases , respectively .
all statistical analyses were performed using spss ver . 18.0 ( spss inc . , chicago , il , usa ) .
the association between immunohistochemistry results and clinicopathological variables was assessed by chi - square test , fisher exact test , or spearman s rank correlation test .
kaplan - meier analysis with log - rank test and multivariate cox regression analysis were performed for survival analysis .
the tumor specimens in this study were from 505 lung adenocarcinoma patients , consisting of 247 male ( 48.9% ) and 258 female ( 51.1% ) patients .
the mean age of patients was 62.9 years ( range , 21 to 83 years ) , with 302 non - smokers ( 59.8% ) and 203 smokers ( 40.2% ) .
with respect to tumor pathology , 274 samples ( 54.3% ) were pathologic stage i , 93 ( 18.4% ) were stage ii , 109 ( 21.6% ) were stage iii , and 29 ( 5.7% ) were stage iv . according to the new iaslc / ats / ers adenocarcinoma classification and the 2015 who classification , 298 ( 59.0% ) were acinar , 83 ( 16.4% ) were papillary , 73 ( 14.5% ) were solid , 33 ( 6.5% ) were lepidic , 8 ( 1.6% ) were micropapillary , and 10 ( 2.0% ) were invasive mucinous subtypes .
venous invasion , lymphatic invasion , and perineural invasion were observed in 25.7% ( 130/505 ) , 50.1% ( 253/505 ) , and 7.1% ( 36/505 ) of samples , respectively .
aqp1 expression was observed in the vascular endothelial cells and the apicolateral surfaces of hyperplastic type ii pneumocytes around tumors .
aqp1 was also detected in myoepithelial cells of bronchial glands and red blood cells ( data not shown ) .
there was a significant association of aqp1 overexpression with venous invasion ( p=.035 ) and lymphatic invasion ( p=.039 ) .
the recurrence rate of patients with aqp1 overexpression was significantly higher than that of patients without aqp1 overexpression ( p=.029 ) .
aqp1 overexpression was not associated with higher histological grade ( p=.097 ) , pleural invasion ( p=.131 ) , and other clinicopathological variables or molecular characteristics , such as egfr and kras mutation and alk rearrangement . in total ,
immunohistochemical analyses of e - cadherin and vimentin were performed for 479 and 471 cases , respectively .
loss of e - cadherin expression was observed in 201 of 479 cases ( 42.0% ) , and expression of vimentin was observed in 192 of 471 cases ( 40.8% ) .
we compared the association of aqp1 overexpression to expression of e - cadherin or vimentin ( table 2 ) and found that aqp1 overexpression was correlated with loss of e - cadherin expression ( p=.011 ) and expression of vimentin ( p<.001 ) . at the time of analysis , the median pfs was 31.0 months ( range , 1 to 84 months ) and the median os was 39 months ( range , 1 to 84 months ) . during this time ,
194 patients ( 38.4% ) suffered tumor recurrence and 90 patients ( 17.8% ) died from cancer .
survival analysis using kaplan - meier and cox proportional hazards analyses were performed to evaluate the prognostic impact of aqp1 overexpression . as shown in fig .
2 , kaplan - meier revealed that pfs of patients with aqp1 overexpression was significantly shorter than that of the patients without aqp1 overexpression group ( p=.018 ) .
univariate analysis indicated that larger tumor size ( p<.001 ) , higher histological grade ( p=.032 ) , pleural invasion ( p<.001 ) , venous invasion ( p<.001 ) , lymphatic invasion ( p<.001 ) , perineural invasion ( p=.043 ) , ptnm stage ( p<.001 ) , and aqp1 overexpression ( 46.1 months vs. 56.2 months , p=.020 ) were associated with shorter pfs ( fig .
multivariate cox regression analysis demonstrated aqp1 overexpression to be an independent factor indicating poor prognosis with regard to pfs in patients with lung adenocarcinoma ( hazard ratio [ hr ] , 1.429 ; 95% confidence interval [ ci ] , 1.033 to 1.977 ; p=.031 ) .
larger tumor size ( hr , 1.797 ; 95% ci , 1.336 to 2.418 ; p<.001 ) , pleural invasion ( hr , 1.372 ; 95% ci , 1.007 to 1.871 ; p=.045 ) , lymphatic invasion ( hr , 1.547 ; 95% ci , 1.113 to 2.151 ; p=.009 ) , and ptnm stage ( hr , 2.179 ; 95% ci , 1.586 to 2.995 ; p<.001 ) were also independent prognostic factors associated with shorter pfs in lung adenocarcinoma .
for os , larger tumor size ( p<.001 ) , higher histological grade ( p=.021 ) , pleural invasion ( p<.001 ) , venous invasion ( p<.001 ) , lymphatic invasion ( p<.001 ) , perineural invasion ( p=.001 ) , vimentin expression ( p=.045 ) , and ptnm stage ( p<.001 ) reached statistical significance by univariate analysis .
aqp1 overexpression was not associated with os ( 63.2 months vs. 70.1 months , p=.237 ) . in multivariate analysis ,
larger tumor size ( hr , 1.775 ; 95% ci , 1.137 to 2.771 ; p=.012 ) , venous invasion ( hr , 2.129 ; 95% ci , 1.352 to 3.354 ; p=.001 ) , and ptnm stage ( hr , 4.789 ; 95% ci , 3.026 to 7.578 ; p<.001 ) were statistically significant .
the tumor specimens in this study were from 505 lung adenocarcinoma patients , consisting of 247 male ( 48.9% ) and 258 female ( 51.1% ) patients .
the mean age of patients was 62.9 years ( range , 21 to 83 years ) , with 302 non - smokers ( 59.8% ) and 203 smokers ( 40.2% ) .
with respect to tumor pathology , 274 samples ( 54.3% ) were pathologic stage i , 93 ( 18.4% ) were stage ii , 109 ( 21.6% ) were stage iii , and 29 ( 5.7% ) were stage iv . according to the new iaslc / ats / ers adenocarcinoma classification and the 2015 who classification , 298 ( 59.0% ) were acinar , 83 ( 16.4% ) were papillary , 73 ( 14.5% ) were solid , 33 ( 6.5% ) were lepidic , 8 ( 1.6% ) were micropapillary , and 10 ( 2.0% ) were invasive mucinous subtypes .
venous invasion , lymphatic invasion , and perineural invasion were observed in 25.7% ( 130/505 ) , 50.1% ( 253/505 ) , and 7.1% ( 36/505 ) of samples , respectively .
aqp1 expression was observed in the vascular endothelial cells and the apicolateral surfaces of hyperplastic type ii pneumocytes around tumors .
aqp1 was also detected in myoepithelial cells of bronchial glands and red blood cells ( data not shown ) .
there was a significant association of aqp1 overexpression with venous invasion ( p=.035 ) and lymphatic invasion ( p=.039 ) .
the recurrence rate of patients with aqp1 overexpression was significantly higher than that of patients without aqp1 overexpression ( p=.029 ) .
aqp1 overexpression was not associated with higher histological grade ( p=.097 ) , pleural invasion ( p=.131 ) , and other clinicopathological variables or molecular characteristics , such as egfr and kras mutation and alk rearrangement .
in total , immunohistochemical analyses of e - cadherin and vimentin were performed for 479 and 471 cases , respectively .
loss of e - cadherin expression was observed in 201 of 479 cases ( 42.0% ) , and expression of vimentin was observed in 192 of 471 cases ( 40.8% ) .
we compared the association of aqp1 overexpression to expression of e - cadherin or vimentin ( table 2 ) and found that aqp1 overexpression was correlated with loss of e - cadherin expression ( p=.011 ) and expression of vimentin ( p<.001 ) .
at the time of analysis , the median pfs was 31.0 months ( range , 1 to 84 months ) and the median os was 39 months ( range , 1 to 84 months ) . during this time ,
194 patients ( 38.4% ) suffered tumor recurrence and 90 patients ( 17.8% ) died from cancer .
survival analysis using kaplan - meier and cox proportional hazards analyses were performed to evaluate the prognostic impact of aqp1 overexpression . as shown in fig .
2 , kaplan - meier revealed that pfs of patients with aqp1 overexpression was significantly shorter than that of the patients without aqp1 overexpression group ( p=.018 ) .
univariate analysis indicated that larger tumor size ( p<.001 ) , higher histological grade ( p=.032 ) , pleural invasion ( p<.001 ) , venous invasion ( p<.001 ) , lymphatic invasion ( p<.001 ) , perineural invasion ( p=.043 ) , ptnm stage ( p<.001 ) , and aqp1 overexpression ( 46.1 months vs. 56.2 months , p=.020 ) were associated with shorter pfs ( fig .
multivariate cox regression analysis demonstrated aqp1 overexpression to be an independent factor indicating poor prognosis with regard to pfs in patients with lung adenocarcinoma ( hazard ratio [ hr ] , 1.429 ; 95% confidence interval [ ci ] , 1.033 to 1.977 ; p=.031 ) .
larger tumor size ( hr , 1.797 ; 95% ci , 1.336 to 2.418 ; p<.001 ) , pleural invasion ( hr , 1.372 ; 95% ci , 1.007 to 1.871 ; p=.045 ) , lymphatic invasion ( hr , 1.547 ; 95% ci , 1.113 to 2.151 ; p=.009 ) , and ptnm stage ( hr , 2.179 ; 95% ci , 1.586 to 2.995 ; p<.001 ) were also independent prognostic factors associated with shorter pfs in lung adenocarcinoma . for os , larger tumor size ( p<.001 ) ,
higher histological grade ( p=.021 ) , pleural invasion ( p<.001 ) , venous invasion ( p<.001 ) , lymphatic invasion ( p<.001 ) , perineural invasion ( p=.001 ) , vimentin expression ( p=.045 ) , and ptnm stage ( p<.001 ) reached statistical significance by univariate analysis .
aqp1 overexpression was not associated with os ( 63.2 months vs. 70.1 months , p=.237 ) . in multivariate analysis ,
larger tumor size ( hr , 1.775 ; 95% ci , 1.137 to 2.771 ; p=.012 ) , venous invasion ( hr , 2.129 ; 95% ci , 1.352 to 3.354 ; p=.001 ) , and ptnm stage ( hr , 4.789 ; 95% ci , 3.026 to 7.578 ; p<.001 ) were statistically significant .
in the present study , we assessed the expression of aqp1 in 505 lung adenocarcinomas and evaluated the relationship between aqp1 overexpression and various clinicopathological factors and molecular characteristics , as well as the expression of emt - related markers .
our study showed that aqp1 overexpression significantly correlated with several aggressive pathological factors and can be used as an independent prognostic factor for pfs in lung adenocarcinoma .
recently , the functional roles of aqp1 protein expression have been studied in various cancers .
previous studies have demonstrated that aqp1 is upregulated in several cancer tissues in vitro and in vivo , and aqp1 overexpression is associated with poor prognosis .
hoque et al . reported that upregulated aqp1 in lung cancer may play a role in cancer cell proliferation by resisting apoptosis .
machida et al . showed that aqp1 overexpression with a loss of polarization is associated with invasive growth and poor prognosis in lung adenocarcinomas .
consistent with previous observations , our study showed that aqp1 overexpression tended to be more frequently observed in the high grade histological subtypes of adenocarcinomas although it was not statistically significant .
we also analyzed the association of aqp1 overexpression with emt - related markers ( e - cadherin and vimentin ) .
the loss of e - cadherin and increased expression of vimentin , both hallmarks of a mesenchymal phenotype , were frequently observed in tumors with aqp1 overexpression .
we hypothesized that aqp1 may participate in tumor progression through emt ( loss of e - cadherin and vimentin expression ) in lung adenocarcinoma .
emt is implicated in tumor progression , invasion , metastasis , and poor prognosis in lung cancer . recently
aqp1 has been suggested to function as a linker molecule to promote emt , or to stabilize the cytoskeletal complex .
similar results were reported in various cancers including colorectal cancer , breast cancer , and brain tumors , which is in line with our results .
the exact biological and functional mechanism of aqp1 to promote emt needed to be clarifiied by further studies . of note
, some studies demonstrated that tumor tissue showed intratumoral heterogeneity of aqp1 expression in brain tumor .
thus , it is necessary to clarify the intratumoral heterogenous distribution of aqp1 overexpression in cancer and its clinical significance . in conclusion
, we demonstrated that overexpression of aqp1 was significantly associated with venous invasion , lymphatic invasion , higher pathological stage , and cancer recurrence in lung adenocarcinomas .
aqp1 was also deemed an independent marker of poor prognosis with regard to pfs . in particular ,
increased expression of aqp1 protein was associated with the expression of vimentin and loss of e - cadherin expression , suggesting that aqp1 overexpression may be involved in tumor cell invasion through facilitating emt . | background : aquaporin 1 ( aqp1 ) overexpression has been shown to be associated with uncontrolled cell replication , invasion , migration , and tumor metastasis . we aimed to evaluate aqp1 expression in lung adenocarcinomas and to examine its association with clinicopathological features and prognostic significance .
we also investigated the association between aqp1 overexpression and epithelial - mesenchymal transition ( emt ) markers.methods:we examined aqp1 expression in 505 cases of surgically resected lung adenocarcinomas acquired at the seoul national university bundang hospital from 2003 to 2012 .
expression of aqp1 and emt - related markers , including ecadherin and vimentin , were analyzed by immunohistochemistry and tissue microarray.results:aqp1 overexpression was associated with several aggressive pathological parameters , including venous invasion , lymphatic invasion , and tumor recurrence .
aqp1 overexpression tended to be associated with higher histological grade , advanced pathological stage , and anaplastic lymphoma kinase ( alk ) translocation ; however , these differences were not statistically significant .
in addition , aqp1 overexpression positively correlated with loss of e - cadherin expression and acquired expression of vimentin .
lung adenocarcinoma patients with aqp1 overexpression showed shorter progression - free survival ( pfs , 46.1 months vs. 56.2 months ) compared to patients without aqp1 overexpression .
multivariate analysis confirmed that aqp1 overexpression was significantly associated with shorter pfs ( hazard ratio , 1.429 ; 95% confidence interval , 1.033 to 1.977 ; p=.031).conclusions : aqp1 overexpression was thereby concluded to be an independent factor of poor prognosis associated with shorter pfs in lung adenocarcinoma .
these results suggested that aqp1 overexpression might be considered as a prognostic biomarker of lung adenocarcinoma . |
since 1980 , when the first implantable cardiac defibrillator ( icd ) was implanted , the use of cardiovascular implantable electronic devices ( cied ) including permanent pacemaker and icd has become more popular . with growing indications ,
infectious endocarditis is a rare complication following implantation of these devices . in suspected cases , transthoracic echocardiography ( tte )
but , the incidence of constrictive pericarditis due to icd lead infection in dual active fixation method is rare .
a 62-year - old man with history of diabetes and opium addiction for 20 years was admitted in 2007 at sina hospital , isfahan , iran , for evaluation of arrhythmia .
however , left ventricular ( lv ) function was severely reduced with ejection fraction of 20%
the patient was placed on anti - arrhythmia drugs ( amiodarone , 400 mg , orally , three times in day ) and discharged .
one year later , the patient was admitted with repeated vt with severe lv dysfunction arrhythmia which did not respond to medical therapy .
tte was unremarkable , tee showed large vegetations on the icd lead in ra and also vegetations on the anterior leaflet of tricuspid valve .
the result of the blood culture was positive for staphylococcus aureus which was methicillin sensitive .
septal motion abnormality ( septal bounce ) the decision was made to remove the device surgically throw median sternotomy in february 2011 .
the cultures of excised vegetations were positive for staphylococcus aureus , e.g. , video 1 , figure 2 .
leaflet of tricuspid valve , infected implantable cardiac defibrillator leads one month after the surgery , the patient returned to hospital with chest pain , malaise , and no fever .
pericardial effusion with air - fluid level in a patient with previous implantable cardiac defibrillator ( non - contrast chest ct .
mediastinal window ) one month later , the patient returned with severe lower extremity edema .
echocardiography showed echo findings were as follows : moderate pulmonary arterial hypertension ( pap = 45 - 50 mmhg ) , moderate circumferential pericardial effusion with high - density material ( 18 mm ) at pericardium [ figures 4 - 8 ] .
mitral valve inflow deceleration time constrictive pericarditis mitral valve velocity propagation significant respiratory variation in tricuspid inflow significant respiratory variation in mitral inflow sever diastolic dysfunction in mitral valve ( doppler ) surgical removal of vegetation of ant .
generator and lead removal during ten months follow - up after pericardiotomy , dyspnea and edema improved significantly with no evidence of life - threatening arrhythmias .
constrictive pericarditis following icd implantation is rare . with less frequent use of patch electrodes ,
koich keno et al . reported a case of delayed pericarditis 23 days after icd implantation with active fixation atrial lead method , but no endocarditis .
they believed that constant contact of the atrial screw with pericardium was the cause of pericarditis .
they believe that mechanical injury was the main reason for pericarditis . in all cases of reported icd infection with large vegetations ,
icd - related endocarditis is an uncommon but a serious complication , the incidence ranging from 0.5 to2% with high mortality rate close to 35% .
the most common bacteria causing icd infection is staphylococcus aureus as it was in our case . | the usage of implantable cardiac defibrillator ( icd ) since 1980s is becoming more popular these days .
the rate of both , endocarditis and constrictive pericarditis are low but it still needs attention .
we are reporting a rare case of icd endocarditis as a result of toe infection in a diabetic patient .
this was followed by infectious pericarditis after device removal by open heart surgery and then delayed constrictive pericarditis . |
in the past , traditional dental luting agents such as zinc phosphate were commonly used
for cementation of crowns . in spite
of disadvantages , such as solubility , lack of retention and low ph ,
this luting agent has been successfully
used and was the most
researched cement for over a century . nowadays , some new adhesive luting agents have
been tested in order to reduce microleakage , increase retention , and improve physical
properties .
currently , vegetable polyurethanes , which combine the versatility of polymer formulation
with the global concern in producing new biomaterials through resources that preserve
the environment , became one of the main studied categories of materials . in dentistry , membrane
material , sealers and irrigating
solution
additionally , since this polyurethane
has been described as being biocompatible , osteoconductor , osteoinductor , antimicrobial ,
osseointegrable and absorbable , it has been used in bone prosthesis ,
alveolar healing and plastic surgery . in a study conducted by camargo , et al .
( 2010 ) , the castor oil bean showed less inflammatory
response in subcutaneous tissue of rats when compared with calcium hydroxide cement .
this polyurethane has elasticity , good compatibility , versatility , composition and
structure that can be modified in accordance with specific requirements , characteristics that enable great
applicability in the biomedical area and open a new field to the development of dental
luting agents .
in addition to the innumerable uses of the castor oil , the plant from
which it is extracted can be found in many parts of the world .
it is greatly exploited
in brazil and india , and can be
produced on a large scale , which makes it interesting from an economic and ecological
point of view . in the oral environment , dental luting agents must withstand masticatory and
parafunctional stresses in different clinical situation .
they should
maintain their integrity while transferring stresses from crowns or fixed partial
dentures to tooth structure .
once the retention of crowns is intimately related to
mechanical properties of the dental luting agents , the strength of these materials
associated to the ability of resisting crack propagation , which causes ruptures , can predict the clinical success .
resistant dental luting agents provide better stress distribution , less probability of
compressive or tensile failures and great probability of clinical success .
diametral tensile strength ( dts ) is a mechanical property that must be
assessed because several
cements are extremely friable and have a susceptibility to mechanical failure .
this test is widely used due to its
relative simplicity and reproducible results . additionally , it is the most common method
for assessing the tensile strength of friable materials because it avoids the
difficulties inherent to the flexural tensile strength test .
in addition to the mechanical properties , physical properties , such as film thickness
( ft ) of luting agents , can directly affect long - term clinical success .
current iso standards require a ft at the time of
seating inferior to 25 m for water - based luting cements , and no greater than 50 m for resin - based
cements .
low ft can improve
seating and decrease marginal gaps ; whereas improved marginal adaptation can also reduce
plaque accumulation , periodontal disease and cement dissolution .
so , the purpose of this study was to
compare the dts and ft of an experimental polyurethane dental luting agent derived from
castor oil with traditional zinc phosphate cement .
experimental groups , manufacturers and compositions of materials used in this
study cop= castor oil poliurethane ; mdi= methylene diphenyl disocyanato the castor oil polyurethane ( cop ) was supplied in sachet .
pure cop contained the
prepolymer and polyol separately , while cop 10% and cop 50% included another separation
containing 10% or 50% in weight of filler ( calcium carbonate - caco3 ) ,
respectively .
the quantity of filler , in % weight , was determined by the manufacturer in
relation to the sum of the polyol and the prepolymer weights .
the mixing method of cop
groups includes 2 min of manual mixing into the sachet and other 2 min of mixing with a
spatula on a teflon plate to obtain the final material ( figure 2 ) .
homogeneous paste of castor oil polyurethane ( cop ) obtained after manual mixing
and mixing with a spatula on a teflon plate zinc phosphate was mixed within 90 s with a spatula following the incremental technique .
the proportion of 1.4 g/0.4 ml recommended by the manufacturer was converted to 1.4 g of
powder/1.54 g of liquid .
dts test : it was conducted with 40 cylindrical specimens ( 6.0 mm in
diameter x 3.0 mm in height ) divided into the 4 experimental groups ( n=10 ) . immediately
after the mixing
, the materials were inserted in a teflon mold , which was put in a
mechanical press under constant load in an atmosphere of 100% relative humidity at 37c
for 1 h. subsequently , all specimens
were finished and polished with 400-grit abrasive sic papers ( buehlermet abrasive
papers , buehler , lake bluff , il , usa ) and were stored in distilled water at 37c for 24
h before the mechanical test .
the specimens were subjected to a compressive load ( 10 kn )
in a universal testing machine ( mts-810 material test system , eden praire , mn , usa ) at a
crosshead speed of 0.5 mm / min until fracture .
the results were recorded and transformed
in tensile values ( mpa ) by the computer software ( test star ii , international business
machines corporation - ibm , armonk , ny , usa ) , connected to the system . the results were
submitted to one - way anova and tukey 's test ( =0.05 ) in order to compare the values of
dts among the 4 experimental groups .
ft test : it was conducted with 40 specimens divided into the 4
experimental groups ( n=10 ) .
the method for determining ft was described in iso
9917 . for pure cop ,
cop 10%
and cop 50% , immediately after the mixing , the dental luting agent was sandwiched
between two uniform thickness glass plates ( 2 cm ) with faces precisely
parallels .
a load of 15 kg was applied vertically on the top of the glass plate .
after
10 min , the thickness of the two plates with cement was determined using a micrometer
( absolute digimatic micrometer 227 , mitutoyo sul america ltda , suzano , sp , brazil ) .
the
ft of the cement was calculated by subtracting the thickness of the glasses without the
mixed material from the overall thickness . for zinc phosphate group , after 3 min of the
beginning of the mixture , the cement was inserted between the two glass plates and
submitted to the ft test as cited above .
dts and ft values of each material are given in tables
1 and 2 .
diametral tensile strength ( mpa ) of dental luting agents and standard
deviations different uppercase letters indicate significant differences by one - way anova
followed by hsd tukey test one - way anova ( p=1.01e-4 ) cop = castor oil polyurethane dts= diametral tensile strenght film thicknesses ( m ) of dental luting agents and standard deviations different uppercase letters indicate significant differences by one - way anova
followed by hsd tukey test one - way anova ( p=2.4e-10 ) cop = castor oil polyurethane according to the statistical analysis , all cops ( pure cop , cop 10% and cop 50% )
demonstrated significantly higher ( p<0.05 ) dts than zinc phosphate .
the addition of
the filler ( caco3- 10% and 50% w / w ) increased dts .
no significant differences
( p>0.05 ) were found between the dts values of cop 10% and cop 50% . as shown in table 2 , ft values of cop 10% ,
cop
50% and zinc phosphate were inferior to 25 m ; only the pure cop exceeded this value .
there was a positive influence of filler addition in the ft , since the comparisons pure
cop vs. cop 10% , pure cop vs. cop 50% , cop 10%
vs. cop 50% were minor than 0.05 i.e. , the mean values of ft
diminished as the quantity of filler was incorporated .
this research assessed dts and ft of an experimental polyurethane dental luting agent
derived from castor oil , pure or with different quantities of caco3 ,
comparing then with zinc phosphate cement .
the zinc phosphate cement and cop differ each other because their viscous and elastic
components ; zinc phosphate is water - based cement which is more friable after the final
setting reaction , while cop is a polymer similar to resinous cements .
when submitted to
tensile forces , cop shows a curve characteristic of polymers that undergo flow after the
linear region of elasticity , with plastic deformation occurring until rupture .
the results showed that the addition of caco3 improves the dts of cop
independently of its percentage .
calcium carbonate diminishes the plastic deformation of
cop causing an increase in the final resistance since this filler can fill any pores in
the matrix , allowing the cement resists to higher loads .
the presence of
caco3 was also proven interesting because it provides radiopacity without
affecting biocompatibility .
zinc phosphate cement had the lower dts ( 4.88 mpa ) when compared with cop .
dts of cops
( cop 10% and cop 50% ) was higher than values of glass ionomer cement ( 18 mpa ) and seems to be more close to the
literature results of resin cements which ( 40 - 45 mpa ) .
with respect to ft , there is no agreement on its minimum value , but values between
50 - 100 m seem convenient .
the
american dental association specification n. 8 restricts the zinc
phosphate ft ranging from 25 m to 40 m , but literature values of ft of a number of luting materials show
that it can range from 10 m to 152 m , depending on the nature of the
material .
the results of this study showed that all evaluated dental luting agents , except pure
cop , had ft means inferior to 25 m , meeting the relevant iso standard .
the differences found among the ft of
the evaluated materials may be explained by the nature and composition of them .
while
the setting of zinc phosphate cement occurs by means of a water - based reaction with the
growth of the crystalline network of the zinc phosphate , the polymerization of cop includes a moisture curing
kinetics which is determined by a reaction between the isocyanate and hydroxyl groups ,
showing a volumetric expansion and consequently higher ft .
the obtained results of zinc
phosphate ( 20.04 m ) are in agreement previous studies .
ft of cop diminished with the addition of caco3 making it lesser than that of
some resin cements . considering that
the volumetric expansion is linked to the quantity of organic matrix and that the
experimental cement may be composed by only organic matrix ( pure cop ) or by an organic
matrix with mineral filler ( cop+caco3 )
, the incorporation of filler
diminishes the expansion and consequently provides better ft values . despite the clinical importance of the two properties evaluated in this study ,
the
assessment of single properties is not sufficient to identify the best dental luting
agent .
however , if an experimental dental luting agent does not have appropriate ft and
dts it will not allow the prosthesis serves the functions adequately over a long period .
among the various materials used for cementing indirect restorations and fixed dental
prostheses ,
although one of the experimental materials tested in this study provided better
results compared with zinc phosphate cement , further research is required .
comparison
with other cements and evaluation of other properties , such as adhesion , hydrolytic
degradation and microleakage , can be the focus of future researches .
finally , the
introduction of new materials , particularly those not derived from petroleum will enrich
the available arsenal of luting materials .
within the limitations of this study , it was conclude that : 1 ) the polyurethane dental luting agent derived from castor oil ( cop ) with the addition
of caco3 filler showed better values of dts and ft when compared with zinc
phosphate cement .
2 ) for both dts and ft , the best composition of the experimental polyurethane dental
luting agent was the one with the addition of 50% w / w of caco3 .
the authors of this paper thank to fapesp for financial support ( grants 06/02821 - 8 ;
06/00082 - 3 ; 07/02441 - 3 ) | the need to develop new dental luting agents in order to improve the success of
treatments has greatly motivated research.objectivethe aim of this study was to evaluate the diametral tensile strength ( dts )
and film thickness ( ft ) of an experimental dental luting agent derived from castor
oil ( cop ) with or without addition of different quantities of filler ( calcium
carbonate - caco3).material and methodseighty specimens were manufactured ( dts n=40 ; ft n=40 ) and
divided into 4 groups : pure cop ; cop 10% ; cop 50% and zinc phosphate ( control ) .
the
cements were mixed according to the manufacturers ' recommendations and submitted to
the tests .
the dts test was performed in the mts 810 testing machine ( 10 kn , 0.5
mm / min ) . for ft test
, the cements were sandwiched between two glass plates ( 2
cm2 ) and a load of 15 kg was applied vertically on the top of the
specimen for 10 min .
the data were analyzed by means of one - way anova and tukey 's
test ( =0.05).resultsthe values of dts ( mpa ) were : pure cop- 10.941.30 ; cop 10%- 30.060.64 ; cop
50%- 29.870.27 ; zinc phosphate- 4.880.96 .
the values of ft ( m ) were : pure cop-
31.093.16 ; cop 10%- 17.054.83 ; cop 50%- 13.034.83 ; zinc phosphate- 20.000.12 .
one - way anova showed statistically significant differences among the groups ( dts -
p=1.01e-40 ; ft - p=2.4e-10).conclusionthe experimental dental luting agent with 50% of filler showed the best
diametral tensile strength and film thickness . |
many epidemiological studies have shown that regular flavonoid rich fruit intake is associated with delayed parkinson 's disease ( pd ) , alzheimer 's disease ( ad ) , ischemic diseases and aging effects ( ono et al . , 2003 ; savaskan et al . , 2003 ; marambaud et al . , 2005 ;
data from in vitro and animal studies suggest that among the sources of antioxidants , phytochemicals in berry fruits ( e.g. , anthocyanin and caffeic acid ) have a beneficial role in brain aging and neurodegenerative disorders because of their anti - oxidative , anti - inflammatory , anti - viral and anti - proliferative properties ( youdim et al . , 2001 ) .
since oxidative stress and inflammation appear to be involved in brain aging and in neurodegenerative diseases ( casadesus et al . , 2002 ) , it is theorized that increased consumption of antioxidants may be effective in preventing or ameliorating these changes .
the neuroprotective effects of strawberry , bilberry , black currant , blackberry , blueberry and mulberry , were demonstrated by many scholars ( basu et al . , 2010 ; rendeiro et al . ,
neuroinflammatory processes in the brain are believed to play a crucial role in the development of neurodegenerative diseases , especially due to increased production of reactive oxygen species ( ros ) ( zheng et al .
, 2003 ; shaffer et al . , 2006 ) . because of low activity of antioxidant defense systems , the brain is susceptible to oxidative stress more than other organs ( rahman , 2007 ; uttara et al . , 2009 ) .
moreover , many neurotransmitters are autoxidized to generate ros ( lau et al . , 2003 ) .
in agreement with these observations , there is evidence that increased oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases such as ad , pd , ischemic diseases and aging ( esposito et al . , 2012 ) .
the neuroprotective effects of many polyphenols rely on their ability to cross the blood - brain barrier and directly scavenge pathological concentrations of reactive oxygen and nitrogen species and chelate transition metal ions ( aquilano et al . , 2008 ) .
different polyphenolic compounds were shown to have scavenging activity and the ability to activate key antioxidant enzymes in the brain , thus breaking the vicious cycle of oxidative stress and tissue damage ( lau et al . , 2003 ; esposito et al . , 2012 ) .
there is a growing interest in the potential of natural polyphenols in berries ( chen et al . , 2013 ; rios de souza et al .
preclinical evidence has indicated that flavonoids may exert powerful actions on mammalian cognitive function and may reverse age - related declines in memory and learning .
these beneficial effects are mainly in demand in preventing against brain damage , such as ischemic and neurodegenerative diseases , reducing neuronal apoptosis , and improving memory , learning and cognitive functions ( kovasova et al . , 2010 ; angeloni et al . ,
, we made an attempt to clearly describe the beneficial effects of various types of berries as promising neuroprotective agents .
strawberry tree ( arbutus unedo l. ; ericaceae family ) is an evergreen shrub , a native mediterranean species that are also cultivated in other regions of eastern europe .
the most abundant antioxidants are caffeic acid , ellagic acid , and certain flavonoids including anthocyanins , tannins , catechin , quercetin , kaempferol , gallic acid derivatives , vitamins c , e and carotenoids ( table 1 ; hakkinen et al . , 2009 ; simirgiotis et al . , 2010 ; karlund et al . ,
( 2001 ) studied the inhibitory effects of strawberries on cyclooxygenase ( cox ) in vitro , which is a key enzyme that plays an important role in the conversion of arachidonic acid to various eicosanoids involved in inflammation .
extracts from strawberries are moderately effective in inhibiting cox-1 , and are more potent inhibitors of cox-2 as well .
cox-2 is the main promoter of inflammatory prostaglandins , while cox-1 is known to produce some gastroprotective prostaglandins .
selective inhibition of cox-2 could be important because the inflammatory process is involved in the etiology of a wide range of neurodegenerative diseases , including ad and pd ( ferencik et al . , 2001 ) .
previous studies have shown that strawberry extracts offer protection to age - induced deficits by enhancing gtpase activity , calcium content , oxotremorine - enhanced k - evoked striatal dopamine ( da ) release , and alterations in membrane rigidity and are effective in preventing the loss of sensitivity in purkinje cells ( joseph et al . , 1998 ;
in addition , strawberry extracts can improve cognitive function as shown by morris water maze performance .
another study has demonstrated that strawberry extracts can improve motor behavioral performance on the rod walking ( joseph et al . , 1998 ) .
these findings suggest that phytochemicals present in strawberry benefit age - related deficits in addition to the known beneficial effects on cancer and other cardiovascular diseases .
young rats exposed to fe particle radiation showed neurochemical and behavioral changes which are similar to those seen in aged organisms ( joseph et al . , 2000 ) . some scholars ( joseph et al . , 1998 , 1999 ; bickord et al . , 2000
2001 ) have reported that maintaining rats for 2 months in antioxidant diets containing strawberry extracts can prevent the occurrence of neurochemical and behavioral changes that are characteristic of ageing .
precisely , maintaining rats for 2 months in diets containing strawberry extracts increased oxotremorine - enhanced dopamine release from striatal slices when compared to control diet - fed animals .
in addition to the improvement in dopaminergic function , there were improvements in motor behavior , spatial learning and memory ( joseph et al .
( 2002 ) showed that diet ( 2% strawberry extracts ) reduced the effects of oxidative stress following exposure to fe particles .
these results suggest that antioxidant - rich diet may serve as effective countermeasures to prevent neurochemical and behavioral changes following exposure to heavy particles .
strawberry and vitamin e are shown to have equal protective effects on age - related deficits ( joseph et al . , 1998 ) .
bilberries provide significant health benefits because of their high levels of anthocyanins , flavonols , vitamins c , e , and manganese and contain carotenoid , lutein , and zeaxanthin ( murray et al . , 2009 ;
the biological function ( including benefits for eyes , mouth , gum health ) , powerful anti - inflammatory ( luo et al . , 2014 ) , anti - hyperglycemic ( stefanut et al . , 2013 ) and antioxidative effects ( davarmanesh et al .
, 2013 ; baum et al . , 2014 ; calo and marabini , 2014 ) can protect blood vessels and improve blood circulation ( pantelidis et al .
a number of studies have shown that aging and particularly brain aging are associated with free radicals action ( grady and craik , 2000 ; liu et al . , 2003 ) .
glutathione and its related enzymes participate in the maintenance of oxidant homeostasis and in the aging process and are associated with a gradual pro - oxidizing shift in the glutathione redox state . there is a close link between glutathione metabolism and oxidant homeostasis that can be manifested as learning and synaptic plasticity deficits under the condition of low glutathione content ( sayre et al . , 2008 ; johnson , 2012 ) .
there are few suitable animal models to study the supplemental antioxidant functions in age - related deficits in learning and memory .
oxys rats with inherited features of accelerated aging and high sensitivity to oxidative stress are potential genetic murine models . these rats have significantly shortened lifespan ( 28% shorter than wistar rats ) . therefore , oxys rats have become a murine animal model to elucidate the basic mechanisms of age - related changes in brain functions , such as learning and cognitive deficiencies in age - related diseases ( obukhova et al . , 2009 ) .
( 2006 ) reported that the level of glutathione in the brain of young oxys rats is 1.3 times lower as compared to wistar rats .
at the same time , superoxide dismutase activity was higher in 3-month - old oxys rats than in age - matched wistar rats .
it is known that in many cells the expression of genes whose products exhibit antioxidant activity might be induced by reactive oxygen species generation .
therefore , a simultaneous increase in superoxide dismutase activity and a decrease in glutathione level might indicate the increased level of ros generation in the brain of young oxys rats .
the above data support the theory that the reduction of cellular expression and activity of antioxidant proteins is a fundamental cause of the aging process and neurodegenerative diseases .
memory loss is accompanied but not necessarily caused by accumulation of oxidative damage to lipids , proteins , and nucleic acids , all of which can disrupt neuronal function .
they also demonstrated that the bilberry extract is effective in decreasing lipid peroxides and increasing superoxide dismutase activity in the brain .
furthermore , long term supplementation of bilberry extract prevents learning and memory deficits in oxys rats .
it is known that high efficiency of bilberry extract might be provided by its flavonoids , which have high free radical scavenging activity and disease - fighting properties ( rahman , 2007 ; uttara et al .
anthocyanins are the major group of polyphenols in blackcurrant , accounting for about 80% of the total amount of quantified compounds ( ghosh and konishi , 2007 ) .
-amyloid ( a)-induced formation of ros is also inhibited by flavonols from blackcurrant ( li et al . , 2004 ) .
polyphenolic substances present in blackcurrant fruits have been reported for antioxidant , antimicrobial , antiviral , and antibacterial properties ( krisch et al . , 2009 ; molan et al . , 2010
; bragoulo and molan , 2011 ; szachowicz - peteleska et al . , 2012 ; tabart et al . , 2012 ; vepsalainen et al . , 2013 )
( 2013 ) investigated the effects of anthocyanin - rich blackcurrant extracts on neuroprotection and amyloid precursor protein ( app ) expression in human sh - sy5y neuroblastoma cells overexpressing app751 isoform under ad - related stress conditions .
they also found that the cells which were treated with anthocyanin - rich blackcurrant extracts experienced significantly reduced ros production .
these findings indicate that anthocyanin - rich blackcurrant extracts exhibit a beneficial effect through their promising antioxidant activity .
polyphenols , which are abundant in bilberry and blackcurrant , have been shown to inhibit the formation and extension of a fibrils and to destabilize the preformed a fibrils in vitro ( vepsalainen et al . , 2013 ) .
they also investigated the effects of both bilberry and blackcurrant - fed apde9 mice ; and found both berry extract - fed apde9 mice showed similar reductions in total app - normalized app c - terminal fragments levels , while the dietary effects on soluble a40 and a42 levels and the ratio of a42/40 in the dorsal cortex were different .
interestingly , bilberry supplementation reduced both soluble a40 and a42 levels as compared to blackcurrant - fed mice , whereas a reduced ratio of insoluble a42/40 and moderately increased soluble app levels were observed in blackcurrant - fed mice , but not in bilberry - fed mice .
these important findings clearly suggest that the increased ratio of a42/40 is a key pathogenic feature and that soluble app is known to exert neuroprotective effects .
berry supplements may have an inhibitory effect on -secretase expression , preventing cognitive decline and mitigating ad - like pathology in a mouse model of ad . on the other hand
, the decreased ratio of insoluble a42/40 in blackcurrant - fed mice may be attributed to the modulation of -secretase function than -secretase inhibition ( vepsalainen et al . , 2013 ) .
bilberry and blackcurrant supplemented diets also attenuated behavioral abnormalities in apde9 mice . under a stressful swimming condition ,
a black currant diet increased swimming speed , ruling out the possibility that this is derived from some kind of motor impairment .
the most striking effect of berry extracts was observed in the food - motivated spatial working memory task , in which both bilberry and blackcurrant attenuated the apde9 genotype - linked impairment .
a moderate beneficial effect of the berry extracts was also observed in the strategy of solving the morris swim task : both the time spent near the pool wall and search rotations while swimming were decreased in the bilberry and blackcurrant fed mice ( vepslinen et al . ,
interestingly , hyperactivity was alleviated to some extent by both bilberry and blackcurrant diets , but significance was found only in the blackcurrant - fed mice .
these finding suggests that the flavonols and anthocyanin - rich blackcurrant extracts exert protective effects under stress conditions .
however , the fact that moderate alterations in long - lasting supplementation of apde9 mice with bilberry or blackcurrant revealed beneficial effects on app and a metabolism .
in addition , these supplementations alleviated behavioral abnormalities in a well - characterized ad mouse model .
based on these results , it is anticipated that bilberry- and blackcurrant - derived phytochemicals could display beneficial neuroprotective effects on behavioral outcome and app processing and a accumulation ( vepsalainen et al . , 2013 ) .
blackberry fruits are well known to be a rich source of antioxidants , rich polyphenols ( kaume et al . , 2012 ) manganese , folate , fibers , cyaniding-3-o - glucoside , vitamin c , salicylate and high tannin .
the biological functions of blackberries include anti - hyperglycemic ( stefanut et al . , 2013 ) , antioxidative , antiseptic , antibacterial / antiviral , anticancer properties . in addition , they can normalize cholesterol , delay the process of aging , relieve pains , and strengthen blood circulation ( jiao and wang , 2000 ; siriwoharn et al . , 2006 ) .
( 2013 ) reported that wild blackberries , brigantinus and vagabundus collected from braganc ( northeast region of portugal ) demonstrated attainable neuroprotective effects by reducing intracellular ros levels , modulating glutathione levels and inhibiting the occurrence of caspases during treatments .
these effects protected neuronal cells against oxidative injury , one of the most important features of neurodegeneration . in vitro studies have also reported that blackberries have potent anti - inflammatory and antiproliferative properties ( wang and jiao , 2000 ; dai et al . , 2007 ) .
in addition , the antioxidants present in these fruits improved behavioral performance in motor neuron tests in aged rats .
the balance and fine motor coordination in cognitive test were also improved in the morris water maze , demonstrating the measures of spatial working memory and learning ( shukitt - hale et al . , 2009 ) .
blueberries are a rich source of flavonoids , notably anthocyanins , caffeic acid , flavanols and hydroxycinnamates ( cao et al . , 1999 ;
the consumption of blueberries has been reported to prevent oxidative stress , inhibit inflammation ( sweeney et al . , 2002 ) and kidney injury ( nair et al . , 2014 ) , and improve vascular health ( erlund et al . , 2008 ) .
these beneficial effects have been attributed to their relatively high flavonoid content , in particular , anthocyanins .
a recent study has demonstrated that blueberry supplementation can alleviate age - related behavioral deficits and high - fat diet - related behavioral declines ( carey et al . , 2014 ) .
the protein kinases , such as map kinase ( mapk ) , extracellular signal - regulated kinase ( erk ) and transcriptional activator cyclic - amp response element binding protein ( creb ) , are involved to mediate the beneficial effects of learning and memory produced by various phytochemicals present in blueberries .
the imbalance in calcium homeostasis and the accumulation of ab protein promote oxidative stress , aging and neurodegeneration .
a alone or together with glutamate , inhibits pka and its downstream signaling target creb , in embryonic neurons .
creb is closely associated with learning and memory at the synaptic sites that are affected in ad .
it is further complicated by age - related differences in memory , signal processing , and susceptibility to ros ( impey et al . , 1999 ;
( 2010 ) reported that blueberry extract treatment offers protection from a42 in middle - aged ( neurons isolated in 1012 months ) and old neurons ( neurons isolated around 24 months ) in a defined culture medium .
importantly , blueberry extract induces a cellular upregulation of glutathione synthesis , a major antioxidant that lowers production of cellular ros , indicating that the blueberry extract is a potent antioxidant .
this is confirmed by the findings that a treatment lowered the major redox buffer , glutathione , consistent with oxidative depletion , but blueberry extract treatment reversed this loss by increasing the level of glutathione .
the increase in glutathione level in old neurons after a treatment is escorted by enhanced perk signaling in an age - dependent manner , showing the greatest increase in the mapk activity ( brewer , 1998 ) . however , blueberry extract treatment can reduce perk activity ( brewer et al . , 2013 ) .
erk1/2 is essential for protection against neurodegeneration because of inflammation / oxidative stress and is required for memory formation .
studies have suggested that prolonged activation of perk signaling possibly has an adverse effect and moderate perk activity is protective and has beneficial effects .
thus , at least in part , blueberry extract may decrease endogenous perk expression level and its activity , because the overall oxidative stress load and a levels can be reduced with blueberry extract . as mentioned above
( 1998 ) reported that blueberry extract pretreatment prevents calcium dysregulation and inhibit creb and erk activities through ros stress response , suggesting that blueberry extract can improve the cognitive function in aging rats by regulating transient stress signaling and ros generation .
a preclinical study has demonstrated that blueberry supplementation enhances motor and memory performance in aged animals ( youdim et al . , 2000 ; casadesus et al . , 2004 ) .
changes in brain - derived neurotrophic factor - mediated protein synthesis , such as arc / arg3.1 , are directly related to blueberry consumption .
inhibition of creb / brain - derived neurotrophic factor pathway effectively blocks the changes in spatial memory in the blueberry - supplemented animals ( williams et al .
anthocyanins have been identified in the specific cerebral regions responsible for cognitive function , including the hippocampus and neocortex ( andres - lacueva et al . , 2005 ) .
furthermore , anthocyanins distribution in the hippocampus might be related to increased neuronal signaling in this region ( casadesus et al . , 2000 ) .
( 2006 ) conducted a study involving psychopharmacological screening to evaluate potential effects of a lyophilized extract of different cultivars from vaccinium ashei , reade ( ericaceae ) berries , which are commonly known as rabbit eye blueberries and are shown to have memory - enhancing , anxiolytic and locomotion increasing properties in mice , as well as the protective effects against free radical - induced dna damage in the brain .
these results are reliable with the hypothesis that flavonoids ( including anthocyanins ) can show beneficial effects on cell signaling and decrease oxidative damage .
these results also suggest that flavonoids might directly act on cognitive function , which may help prevent age - related and pathological degenerative processes in the brain .
( 1999 ) found that 8 week dietary supplementation of blueberry extracts was effective in reversing age - related deficits in the brain and behavioral dysfunctiond in aged ( 19 months ) f344 rats .
in addition , blueberry supplemented animals showed positive effects on cognitive behavior , motor performance ( e.g. , rod walking and the accelerating rotarod ) , carbachol - stimulated gtpase activity , and oxotremorine enhanced da release .
a study showed that after 6 weeks of blueberry - supplemented diets , neuronal loss in the hippocampus was reduced in rats with cerebral ischemia ( sweeney et al .
there is evidence that in addition to morris water maze performance , the cognitive declines in object recognition were effectively reversed by blueberry supplementation ( goyarzu et al . , 2004 ) .
chronic treatment with blueberry reduces ischemia / reperfusion - induced apoptosis and cerebral infarction ( wang et al . , 2005 ) .
( 2005 ) show that blueberry causes a rapid but transient increase of ox-6-positive microglia in the striatum and the globus pallidus of normal f344 male rats .
additionally , the number of striatal th - positive nerve fibers was increased in animals fed with blueberry supplemented diet .
supplementation of blueberries in adult mice ( aged 3 months ) improved performance in memory tasks and had a protective effect on dna damage in the hippocampus and cerebral cortex ( barros et al . , 2006 ) .
short - term dietary supplementation of antioxidant rich blueberries can decrease the level of oxidative stress in brain regions and can ameliorate age - related deficits in neuronal and behavioral functions to generate a heat shock protein 70 mediated neuroprotective response to stress in rats .
therefore , supplementeation of blueberries shows beneficial effects by increasing antioxidant level , enhancing anti - inflammatory activities and regulating various signaling pathways at different time points ( galli et al . , 2006 ) .
a 2-month dietary supplementation of blueberries alleviated deficits in learning performance induced by bilateral hippocampal injections of kainic acid , reduced the loss of ca1 pyramidal neurons ( duffy et al . , 2008 ) , and reversed the deficits in cognitive performance ( shukitt - hale et al . , 2007 ) .
short - term blueberry - enriched diet prevents and reverses object recognition memory declines in aged fischer-344 rats ( malin et al . , 2011 ) .
( 2003 ) showed that amyloid precursor protein / presenilin-1 transgenic mice that were given a diet containing blueberry extract from 4 to 12 months of age showed no behavioral deficits in y - maze performance .
2010 ) indicated that wild blueberry juice supplementation for 12 weeks improved memory function in old adults with mild memory decline .
the central cholinergic system is essential for the regulation of cognitive functions ( sarter and bruno , 1997 ; silman and sussman , 2005 ; zimmerman and soreq , 2006 ) .
agonists of cholinergic receptors and inhibitors of acetylcholinesterase have been extensively used to increase endogenous acetylcholine levels and thus overcome cognitive deficits .
acetylcholinesterase metabolizes acetylcholine to choline and acetyl - coenzyme a. papandreou et al . ( 2009 ) reported that administration of polyphenol - rich wild blueberry extract to healthy adult mice attenuated brain oxidative stress ( mda levels ) , increased brain ascorbate and glutathione levels , and decreased acetylcholinesterase activity .
thus , supplementation of polyphenols - concentrated blueberry extract significantly enhances the cognitive function of adult mice by increasing cerebral antioxidant level and inhibiting acetylcholinesterase activity .
these findings stress the critical impact of wild blueberry bioactive components on brain function . shukitt - hale et al .
( 2008 ) reported that blueberry polyphenols attenuated kainic acid - induced learning impairments in rats , which were similar to those observed in aged animals .
the reason for the similarity in behavioral deficits between aged and kainic acid - injected rats , as mentioned above , might be the increase in inflammation , which is a factor of inducing cognitive deficits .
young rats give a diet supplemented with a 2% blueberry extract for 2 months , prior to the injection of an inflammatory stimulus into the hippocampus , exhibit significantly less impairments in their spatial learning and memory abilities .
furthermore , rats fed with the blueberry diet prior to kainic acid injection exhibited less activation of the inflammatory marker mhc class ii marker ( ox-6 ) , increased expression in the neurotrophic factor insulin - like growth factor-1 along with decreased levels of inflammatory cytokines interleukin-1 , tumor necrosis factor- , and transcription factor nuclear factor kappab .
thus , the mechanism by which blueberry polyphenols protects the brain is to decrease the deleterious effects of an inflammatory stimulus by altering the expression of inflammation - related genes .
experimental autoimmune encephalomyelitis presents with pathological and clinical features similar to those of multiple sclerosis , including inflammation and neurodegeneration . a study by xin et al .
( 2012 ) has demonstrated that in relapsing - remitting experimental autoimmune encephalomyelitis models , blueberry - supplemented mice showed lower motor disability scores and improved cumulative and final motor scores compared to control diet - fed mice .
these findings demonstrated that blueberry supplementation is beneficial in multiple experimental autoimmune encephalomyelitis models , suggesting that blueberries , which are easily administered orally and well - tolerated , may provide benefits to multiple sclerosis patients .
mulberries ( morus alba l. , moraceae ) are used in oriental traditional medicine for anti - inflammatory , diuretic , antitussive , antipyretic ( asano et al . , 2001 ) and anti - hyperglycemic purposes ( stefanut et al . , 2013 ) .
high amounts of anthocyanins from berries are consumed in the common diet and used in some therapeutic applications ( mitcheva et al . , 1993 ; dugo et al . , 2001 ) .
cyanidin-3-o--d - glucopyranoside ( c3 g ) , which is an aglycon of anthocyanin , has free radical scavenging and inflammation suppressing activities and offers protection to an endothelial dysfunction ( seeram et al . ,
2003 ) . in an effort to reduce the level of ros - induced damage ,
the mulberry fruit extract and c3 g were evaluated to determine whether they can prevent ros generation and reduce the degree of neuronal damage .
the data show that the neuroprotective effect of the mulberry fruit extract is the result of c3 g in the h2o2-induced oxidative damage in pc12 cells ( kang et al . , 2006 ) .
in addition , c3 g offered more effective neuroprotection in oxygen - glucose deprivation - induced cerebral ischemia than the mulberry fruit extract at the same concentration ( kang et al . , 2006 ) .
the result suggests that c3 g is a major neuroprotective compound in the mulberry fruit extract in oxygen - glucose deprivation - induced cerebral ischemic cytotoxicity in pc12 cells . in in vivo experiments ,
mulberry fruit extract and c3 g reduce infarct volume in middle cerebral artery - occluded animal models .
additional studies have demonstrated that the mulberry fruit extract has neuroprotective effects in both in vitro and in vivo ischemic oxidative stress models , suggesting that c3 g is a major neuroprotective constituent of the mulberry fruit extract ( kang et al . , 2006 ) .
oxidative stress and inflammation are major factors contributing to aging and the development of age - related neurodegenerative diseases .
numerous natural antioxidant / anti - inflammatory compounds found in plant food matrices , like fruits , especially berries ( such as strawberry , bilberry , blackcurrant , blackberry , blueberry and mulberry ) can offer neuroprotective effects ( table 2 ) ( essa et al . , 2012 ; subash et al .
furthermore , the berry fruit may exert their effects directly through alterations in cell signaling to improve / increase neuronal communication , calcium buffering , neuroprotective stress shock proteins , plasticity , antioxidant / anti - inflammatory action , stress signaling pathways and inhibition of acetylcholinesterase .
these modifications , and others that are being studied , may mediate the enhancements in cognitive and motor behavioral performance by berries .
thus , nutritional interventions rich in phytochemicals ( for example anthocyanins and caffeic acid ) such as berry fruits may be a valuable asset in preventing against aging by reducing or delaying the development of age - related neurodegenerative diseases ( figure 1 ) .
extensive clinical trials need to be done to further validate the effects of berry fruits and bring novel therapeutic agents for brain - related diseases .
neuroprotective effects of berry fruits graphic representation showing the possible mechanism of berry fruits against neurodegenerative diseases ( ndd ) . | recent clinical research has demonstrated that berry fruits can prevent age - related neurodegenerative diseases and improve motor and cognitive functions .
the berry fruits are also capable of modulating signaling pathways involved in inflammation , cell survival , neurotransmission and enhancing neuroplasticity .
the neuroprotective effects of berry fruits on neurodegenerative diseases are related to phytochemicals such as anthocyanin , caffeic acid , catechin , quercetin , kaempferol and tannin . in this review
, we made an attempt to clearly describe the beneficial effects of various types of berries as promising neuroprotective agents . |
ovarian cancer is a highly fatal gynecologic cancer and the fifth leading cause of cancer mortality in women , with 14,030 deaths occurring in the united states per year ( 1 ) . in korea ,
ovarian cancer is the ninth leading cause of death with an age - standardized cancer mortality rate of 2.3 per 100,000 women during 2010 ( 2 ) .
most cases of ovarian cancer are discovered at an advanced stage and quickly progress , resulting in a poor outcome .
the national cancer institute 's surveillance , epidemiology , end results program ( seer ) reported that 68% of ovarian cancer cases diagnosed during 2002 - 2008 presented at the late distant stage with very low 5-yr survival rates ( < 26% ) ( 3 ) . due to the rapid progression and low survival rate ,
there is usually only a short window of time to evaluate the environmental factors affecting the course of the disease .
obesity is a health problem with increasing prevalence and is directly related to various morbidities . among gynecologic cancers ,
hormone - related cancers such as endometrial cancer and breast cancer are associated with obesity ( 4 ) .
recently , there have been some epidemiologic and experimental studies indicating that abdominal adiposity and weight gain increase ovarian cancer risk .
they may also increase tumor invasiveness and tumor cell migration in various cancer types ( 5 , 6 ) .
however , there have been only a few clinical studies determining the role of obesity in ovarian cancer outcomes .
advanced ovarian cancer patients are frequently found in a cachectic state with ascites which may contribute significantly to body weight , but do not counted as real body mass .
most patients with ovarian cancer are operative candidates who then receive adjuvant chemotherapy , which can jeopardize their health status . in asian populations ,
the categorizing standard of obesity is different from that applied to people of western populations , and the incidence of comorbidities is different ( 7 , 8) . therefore , there is still some question regarding whether obesity truly has an adverse effect on the outcome of ovarian cancer among asian patients .
the aim of this study was to analyze the relationship between obesity and the survival of patients with advanced epithelial ovarian cancer in korean population .
a total of 236 patients who underwent staging laparotomy for advanced ( stage iii and iv ) eoc between january 2000 and february 2009 were included .
medical records of 320 patients were retrieved from korea university anam hospital ( 86 patients ) , korea university guro hospital ( 44 patients ) , gachon university gil medical center ( 120 patients ) , kyung hee medical center at gangdong ( 22 patients ) and cha university hospital ( 48 patients ) , korea .
all cases were reviewed for height , weight , age , comorbidities and treatment - specific details .
patients with a non - epithelial tumor histology , synchronous or metachronous tumors , borderline tumors and early stage ( stage i and ii ) ovarian cancer were excluded .
the patients ' primary surgery was performed at the gynecological department of one of the five different hospitals , and the staging procedure was done at the time of surgery .
cytoreductive surgery was considered to have achieved optimal debulking when the residual disease was < 1 cm .
postoperatively , all patients received at least six cycles of taxane - platin based chemotherapy .
four patients had three or fewer cycles of neoadjuvant chemotherapy before staging operation and no patient had intraperitoneal chemotherapy .
recurrence was defined as the earliest date when abnormal findings were detected on a follow - up ct scan .
time of survival was defined as the time from diagnosis to recurrence for progression free survival ( pfs ) , and from diagnosis to death irrespective of cause for overall survival ( os ) . for censored cases survival
patients were evaluated for complications , rates of optimal debulking ( residual disease<1 cm ) , pfs , os and platinum sensitivity , i.e. recurrence more than 6 months from completion of platinum - based chemotherapy .
body weight and height were checked at diagnosis which means within three months before operation .
body mass index ( bmi ) was calculated using the quetelet 's index expressed in kg / m and categorized into five groups : underweight ( bmi<18.5 ) , normal ( 18.5bmi<23 ) , overweight ( 23bmi<25 ) , obese i ( 25bmi<30 ) and obese ii ( 30.0bmi ) .
the bmi cut - off points followed those proposed by the regional office for western pacific region of world health organization ( who ) ( wpro criteria , 2000 ) ( 9 ) .
survival was analyzed using the kaplan - meier method and compared using a log - rank test .
statistical analysis was performed using the chi - square test , fisher 's exact test and anova , where appropriate .
this study was approved by institutional review board of korea university anam hospital ( ed10313 ) and 4 other hospitals .
the subjects ' informed consent was waived by the boards due to the observation design of this study .
this study was approved by institutional review board of korea university anam hospital ( ed10313 ) and 4 other hospitals .
the subjects ' informed consent was waived by the boards due to the observation design of this study .
five ( 2.1% ) , 85 ( 36.0% ) , 69 ( 29.2% ) , 64 ( 27.2% ) , and 13 ( 5.5% ) patients were underweight , normal weight , overweight , obese i and obese ii , respectively
. age at diagnosis significantly differed across the five bmi groups ( p = 0.003 ) .
the initial hb of underweight patients ( 10.41.8 g / dl ) was significantly lower compared to the other bmi groups ( p = 0.049 ) .
no significant differences were seen in parity , medical history , surgery optimality , stage , ca 125 , ca 19 - 9 and follow - up time among the five bmi categories .
histologic characteristics showed an even distribution across the five bmi groups , and the serous type comprised majority for each bmi group ( table 1 ) .
the median pfs was 9.5 months ( 1 - 132 months ) and the 5-yr os was 47% , considering all stages for the whole series ( data not shown ) . during the median follow - up period of 21.4 months ( range , 1 - 141 months ) ,
a total of 56 deaths were recorded ( 23.7% ) with an even distribution across the bmi strata ( table 1 ) .
table 2 shows the univariate and multivariate hazard ratios ( hrs ) and 95% confidence intervals ( cis ) for ovarian cancer mortality associated with bmi and surgical optimality .
underweight patients ( bmi<18.5 ) had a significantly higher mortality compared to patients in the reference group ( 18.5bmi<23 ) ( hr , 8.622 ; 95% ci , 1.871 - 39.737 ; p=0.006 ) .
the hr and 95% ci comparing women who had non - optimal surgery versus patients with optimal surgery was 2.302 ( 95% ci , 1.326 - 3.995 ; p=0.003 ) .
our analysis did not find any other factors affecting the survival of stage iii and iv ovarian cancer ( data not shown ) .
in the subcohort analysis , within 97 patients with serous ovarian cancers after optimal surgery , underweight patients had significantly poorer survival than other bmi groups ( p=0.031 ) and obese ii patients showed no significant difference in survival compared to normal weight , overweight and obese i patients ( p=0.097 ) ( fig .
however , among the three mid - range bmi groups overweight and obese i patients had a higher rate of survival than normal weight patients ( p=0.012 ) ( fig .
ovarian cancer is one of the most lethal cancers in women ; however , there is little data available on the precise nature of the relationship between obesity and ovarian cancer mortality .
furthermore , there is lack of study about the effect of obesity to the ovarian cancer survival in asian population .
this study examined whether bmi group following asian criteria has an association with the ovarian cancer survival .
we found that an underweight bmi and surgical optimality are independent risk factors for the survival of patients with advanced ovarian cancer .
however , there was no significant relationship between ovarian cancer survival and the other four bmi groups . only in the subgroup analysis with a serous histology and with optimal surgery , patients in the overweight and obese i groups had better survival than normal weight patients .
the median pfs and 5-yr os of our data ( 15 months and 47% , respectively ) are pretty better than the pfs and os expected in advanced ovarian cancer patients .
it seems because only patients who completed over six cycles of chemotherapy were included in this study .
these results are notable because to date obesity has been shown to have only two effects on ovarian cancer survival , either a worse prognosis or no effect .
( 10 ) reported that obesity was independently associated with both shorter time to recurrence and shorter overall survival for patients with advanced ovarian cancer .
our study is both of a different ethnic population and a large sample size . according to study by yang et al .
however , bmi was measured one year before the diagnosis of ovarian cancer , and the bmi definition used were different from those in our study .
the pathophysiology of both obesity and cancer biology is complicated , and therefore , the approach to reveal the association between them should be carefully considered . despite the technical challenge that may be present during surgery , our patients had consistent optimal cytoreduction rates across the bmi groups .
this important factor in the rate of survival was emphasized in the study of matthews et al . who suggested that maximal effort should be directed towards debulking tumor mass in obese patients with eoc ( 12 ) .
they reported that survival rates are similar between obese and non - obese patients when optimal debulking status is the same .
underweight patients in our study showed a significantly poor survival rate than other bmi groups .
although elevated bmi is associated with an increased prevalence of comorbidities , the survival rate of obese ii patients was not different from normal weight patients .
these findings are similar to a study done by skrnisdttir and sorbe ( 13 ) .
however , the study population in underweight and obese ii groups was very small ( five and thirteen , respectively ) so , we obtained the reliability of our results through subgroup analysis with normal , overweight and obese i patients .
serous carcinoma is the most common type of ovarian cancer with a frequency of over 90% ( 14 ) .
nearly all comprehensively staged patients present in advanced stage with tumor disseminated throughout the abdominal and pelvic cavities .
some characteristics of serous carcinoma , including early metastasis and dissemination , can be key targets for improving the prognosis of overweight and obese i patients .
it is possible that overweight status in asians with advanced ovarian cancer is a protective factor since asian obesity appears to be associated with a better prognosis than western obesity .
overweight and obese i patients may be resistant to the toxicity of chemotherapy due to better initial nutritional status .
one potential confounder in advanced serous ovarian cancer is that there is a discrepancy between real body mass and bmi due to the burden of ascites and multi - organ metastasis . the international obesity task force of the who proposed a system of classification based on bmi , and selected a bmi of 30.0 as the cutoff point for obesity based on mortality and morbidity outcomes ( 15 ) .
in recent years , accumulating evidence has suggested that the who criteria for classifying obesity in adult caucasians may not be appropriate for asian populations .
asian populations have a high level of abdominal fat at a lower bmi relative to caucasians , and show an increasing trend toward obesity .
therefore , the regional office for the western pacific region of the who , the international association for the study of obesity and the international obesity task force proposed a separate classification of obesity for asian population in 2000 .
this led to the proposal that overweight adults be designed as such in asia with a bmi23.0 , and that obesity be defined by a bmi25.0 ( wpro criteria ) ( 9 ) .
to the best of our knowledge , this is the first study analyzing ovarian cancer survival using the asian bmi criteria .
however , there are still many unanswered questions about the association between asian bmi criteria and ovarian cancer survival .
some strengths and limitations of the present study should be considered when interpreting these findings .
strengths include appropriate bmi criteria for asian patients , subgroup analysis excluding the effects of important variables , directly measured anthropometric variables and a study population consisting of advanced epithelial ovarian cancer . to the best of our knowledge ,
this is the first epidemiologic evidence showing that overweight and obese status is associated with a better prognosis . with regard to limitations ,
a history of medical disease was not detailed for patients enrolled in the study . nevertheless , overweight and obese i patients had better outcomes despite being older and potentially being at higher risk for cardiovascular disease .
further large scale studies including other asian countries will be needed to confirm the observed associations in this study . in conclusion ,
underweight and surgical optimality are independent risk factors for advanced ovarian cancer survival in korean patients .
bmi does not influence the survival of advanced eoc patients except in underweight patients . in the subgroup of serous histology and optimal debulking surgery , patients of the overweight and obese i groups have higher rates of survival than normal - weight patients .
these results provide a useful reference for planning further studies and for predicting the prognosis of asian patients with ovarian cancer . | controversy remains regarding the effect of obesity on the survival of patients with ovarian cancer in asia .
this study examined the impact of obesity on the survival outcomes in advanced epithelial ovarian cancer ( eoc ) using asian body mass index ( bmi ) criteria .
the medical records of patients undergoing surgery for advanced ( stage iii and iv ) eoc were reviewed .
statistical analyses included anova , chi - square test , kaplan - meier survival and cox regression analysis . among all 236 patients ,
there were no differences in overall survival according to bmi except in underweight patients . in a multivariate cox analysis ,
surgical optimality and underweight status were independent and significant prognostic factors for survival ( hr , 2.302 ; 95% ci , 1.326 - 3.995 ; p=0.003 and hr , 8.622 ; 95% ci , 1.871 - 39.737 ; p = 0.006 , respectively ) . in the subgroup of serous histology and optimal surgery
, overweight and obese i patients showed better survival than normal weight patients ( p = 0.012 ) .
we found that underweight bmi and surgical optimality are independent risk factors for the survival of patients with advanced ovarian cancer .
high bmi groups ( overweight , obese i and ii ) are not associated with the survival of advanced eoc patient . however , in the subgroup of eoc patients with serous histology and after optimal operation , overweight and obese i group patients show better survival than the normal weight group patients.graphical abstract |
subfoveal choroidal neovascularization ( cnv ) is a leading cause of visual loss in the geriatric population .
peripapillary cnv membranes ( ppcnvm ) have been associated with multiple etiologies [ 2 , 3 ] .
while ppcnvm , associated with optic nerve head drusen ( onhd ) , is a well - described entity in the pediatric population , it is not reported in the adult population ( pubmed search november 29 , 2014 ) .
herein , we report a case of ppcnvm associated with onhd impacting the macula , which required treatment with an anti - vascular endothelial growth factor ( anti - vegf ) agent .
a 75-year - old male presented with a chief complaint of blurry vision in the right eye ( od ) of 3 months duration .
the patient 's past medical and ocular history were significant only for herpes zoster ophthalmicus on the left side years prior . on examination ,
his treatment was initiated with 3 ranibizumab injections , administered every 5 weeks , without any impact on the amount of subfoveal fluid .
subsequent treatment with a single aflibercept injection resulted in complete resolution of subfoveal fluid ( fig .
the patient 's visual acuity has been stable for 15 months with no recurrence of subfoveal fluid .
the association of onhd with macular threatening ppcnvm and its favorable response to anti - vegf agents is already known in the pediatric population [ 2 , 3 , 4 ] .
the natural course of ppcnvm is variable and unpredictable ; however , macular threatening ppcnvm requires treatment .
while the role of anti - vegf therapy as a treatment for this specific subset of choroidal neovascular membranes was not studied as part of the major neovascular age - related macular degeneration ( amd ) studies , its use for the treatment of other forms of cnv is well established .
bevacizumab , ranibizumab and aflibercept are 3 anti - vegf agents used for the treatment of neovascular amd .
ranibizumab and bevacizumab are monoclonal antibodies that selectively bind to vegfa [ 7 , 8 ] .
aflibercept , however , is a fusion protein composed of the binding domains of vegf receptor 1 , vegf receptor 2 , and the fc portion of igg that selectively binds to vegfa , vegfb , and platelet growth factor ( pigf ) .
aflibercept has been shown to have a greater affinity for vegf and a longer half - life than both ranibizumab and bevacizumab .
the comparison of age - related macular degeneration treatment trials ( catt ) demonstrated equivalence in efficacy between ranibizumab and bevacizumab in the treatment of wet amd , as did the inhibition of vegf in the age - related choroidal neovascularization ( ivan ) trial . the vegf trap - eye : investigation of efficacy and safety in wet amd ( view 1 and view 2 ) trials similarly demonstrated an equivalence between ranibizumab and aflibercept in treating wet amd .
inadequate response to 1 anti - vegf agent is not uncommon when treating cnv , and , on occasion , changing to an alternate anti - vegf agent has demonstrated efficacy . currently , there are no studies that have investigated the response of aflibercept in treatment - resistant peripapillary cnv , likely due to the rarity of this presentation .
previous studies have concluded that aflibercept may provide an efficacious therapeutic response in cases of cnv due to amd that initially failed to respond to bevacizumab and ranibizumab .
further studies are required to investigate whether aflibercept is an equally efficacious therapeutic alternative to treatment resistant peripapillary cnv . based upon our experience , an individualized treatment plan for adult patients with ppcnvm , associated with onhd ,
| optic nerve head drusen can be associated with peripapillary choroidal neovascularization , in both the pediatric and adult population .
these membranes can involve the macula , causing significant visual loss . herein , we present a case that required treatment with an anti - vegf agent .
the patient failed to respond to the initial agent , but subsequently responded to a change of agent .
adult patients with macular degeneration involving peripapillary choroidal neovascularization associated with optic nerve head drusen may require individualized treatment plans . |
yttria tetragonal zirconia polycrystal ( y - tzp ) ceramics serve as an upcoming material for engineering applications and are also considered important for restoration medicine .
the excellent mechanical characteristics such as high strength and fracture resistance are attributed to the ceramic 's unique ability known as transformation toughening , a stress - induced phase transformation from tetragonal ( t ) to monoclinic ( m ) [ 1 , 2 ] .
the stabilizing effect of yttria ( y2o3 ) makes it possible for y - tzp ceramic to be processed in the metastable tetragonal ( t ) structure .
this is essential since the retention of the ( t ) phase at ambient temperature allows it to transform to the monoclinic ( m ) structure under external applied stress [ 3 , 4 ] .
despite its outstanding properties , kobayashi et al . discovered that the y - tzp ceramics suffer a serious limitation for applications near 250c in moist environment .
the findings revealed that the ceramic can suffer a slow , tetragonal to monoclinic phase transformation at the samples surface in a humid atmosphere , followed by microcracking and a major loss in strength known as low temperature degradation ( ltd ) [ 610 ] .
since then , experiments have been conducted with y - tzp in an attempt to understand the basic micromechanisms of the ageing - induced ( t)-(m ) phase transformation and to suppress the ltd phenomenon [ 1119 ] .
based on research findings , the addition of ceramic oxides ( mgo , al2o3 , zno , cao , and ceo2 ) helps overcome if not prevent the low temperature degradation occurrence in y - tzp ceramics [ 9 , 2024 ] .
ceo2 is generally used to stabilize the tetragonal phase of zirconia and is also known to increase the sintering of glass ceramics and strength and thermal stability .
ce - tzp in contrast to y - tzp has complete resistance to ltd and higher toughness . enhancing the moderate ce - tzp properties would result in the material becoming a primary candidate for biomedical applications which has the requirements in terms of phase stability and long - lasting characteristics in the human body .
fischer and stawarczyk found that the ce - tzp / al2o3 nanocomposite offers superior mechanical properties compared to conventional y - tzp . also , when compared to conventional y - tzp the flexural strength is in the same range whereas the fracture toughness increases with the addition of ceria .
investigating the effect of ceria addition both supported the statement that ceria increases the fracture toughness of y - tzp [ 28 , 29 ] .
kuroda et al . have shown that several reactions occur during sintering with the addition of cuo which can be beneficial or detrimental for densification of y - tzp .
more recently , tribological studies on 3 mol% y - tzp ceramics showed that the addition of 1.8 mol% of cuo led to a reduction of friction coefficient from 0.2 to 0.6 under dry sliding conditions , while another study revealed that the ferromagnetic behaviour of y - tzp with enhanced coercivity could be achieved by doping 0.3 mol% nio in the zirconia matrix .
the susceptibility of y - tzp to low temperature degradation ( ltd ) is a shortcoming for a material that has excellent mechanical properties and biocompatibility .
the addition of cerium oxide , ceo2 , makes the material inert to ltd but is counterbalanced by modest strength and hardness , making its usage in biomedical applications limited and difficult . in order to overcome this limitation ,
the amount of ceo2 addition to y - tzp is altered along with the sample preparing factors such as sintering methods and temperatures , mixing methods , and the purity of powders used .
two commercial type powders were prepared for this experiment : the 3 mol% of yttria - stabilized zirconia powder manufactured by kyoritsu ltd . , japan , under the code name of kz-3yf as the base powder and cerium oxide , ceo2 , manufactured by sigma aldrich , as the dopant .
three different compositions of cerium oxide were mixed with y - tzp , that is , undoped 0.3 wt% , 0.5 wt% , and 1.0 wt% .
the powders were mixed using the wet milling method , using zirconia balls as mixing media and ethanol as the mixing medium .
the slurry was oven dried at 60c sieved , and the powder was uniaxially pressed at 0.3 mpa into discs and rectangular bars .
these samples were then sintered under atmospheric condition at a rate of 10c / min , with temperatures ranging from 1250c to 1450c , and a holding time of 2 hours before cooling down to room temperature .
the sintering temperatures 1250c to 1450c are used for this research as it is sufficient to retain the tetragonal phase of y - tzp . besides that , this temperature range is the optimum temperature range to obtain the high mechanical properties of y - tzp .
disc samples are polished on a polishing machine with grinding papers of different roughness ( 120 , 240 , 600 , and 800 cc - cw ) and finally are polished using diamond paste of 6 microns and a polishing cloth .
the bulk density of the sintered samples was measured based on archimedes ' principle , using the water immersion method with a standard mettler toledo balance ag204 densimeter .
polished samples were used to determine vickers 's hardness and fracture toughness ( kic ) using vickers 's indentation method . an indentation with a force of 9800
the fracture toughness was calculated for the measured value obtained using niihara 's formula as stated below :
( 1)kic=0.016(e / h)1/2c3/2,kic being the fracture toughness ( mpam ) ; e is young 's modulus ( gpa ) , h the vickers hardness ( gpa ) , and c the crack length ( m ) measured from the center of indentation .
the microstructure of the material was evaluated using sem ( scanning electron microscope ) whereby the grain structures and grain sizes were determined using the average grain intercept method .
the average grain intercept method is used to measure the grain size of a material by drawing randomly positioned line segments on the micrograph , counting the number of times each line segment intersects a grain boundary , and finding the ratio of intercepts to line length .
figure 1 shows the influence of various ceo2 additions to the bulk density of y - tzp .
the sintering temperature acts as a controlled variable ; ceo2-y - tzp samples are sintered over a temperature range of 12001450c .
the addition of ceo2 was beneficial in aiding densification as the increase in density was proportional to the increasing sintering temperature .
the figure shows that samples with ceo2 content > 0.3 wt% achieved much higher values of densities as compared to samples of lower amount and undoped samples .
the highest value of density was recorded by the samples with 0.5 wt% ceo2 , which is approximately 97% of the theoretical value ( 6.09 g / cm ) .
a significant reduction in density values was observed at higher temperatures , a phenomenon probably attributed to the grain growth .
besides that , the decrease could also be associated with agglomeration of the ceo2-y - tzp particles during the preparation and sintering processes .
the effect of sintering temperatures and ceo2 doped samples on the vickers hardness of y - tzp is shown in figure 2 .
the undoped sample recorded a hardness value of 9.6 gpa ( 978.9 hv ) . when sintered at 1250c , the doped samples showed higher hardness values at 1250c but was still below the theoretical value .
the highest value recorded was 13.2 gpa ( 1346 hv ) at a sintering temperature of 1400c , before facing a drop in value with further sintering at 1450c .
overall , from observation it was found that samples with 0.5wt % and 1.0 wt% dopant addition sintered at temperatures > 1300c achieved hardness values higher than the theoretical vickers hardness of y - tzp .
the decrease in hardness at high temperatures ( > 1400c ) can probably be associated with the increased proportion of transformable tetragonal phase and associated pseudoplasticity .
the influence of the addition of ceo2 on young 's modulus of the sintered sample is depicted in figure 3 . at 1250c , all doped samples reached almost the theoretical value of young 's modulus ( 210 gpa ) whereas the undoped y - tzp was low ( ~177 gpa ) .
the 0.3 wt% ceo2-y - tzp doped sample shows a decline in e value from 1350c onward , and in contrast 0.5 wt% ceo2-y - tzp kept increasing up till 1450c and attained the highest value of ~211 gpa .
an inversly proportional relationship exists between the bulk density and young 's modulus as a lower density results in a higher elasticity in the material .
it can be inferred that the bulk density is an important parameter governing the matrix stiffness of y - tzp .
similar results were seen in work from other researchers [ 37 , 38 ] . the influence of the varying amounts of ceo2 and various sintering temperatures on the fracture toughness was seen in figure 4 .
it was clearly seen that the addition of ceo2 was beneficial in enhancing the fracture toughness of y - tzp . at 1250c ,
the undoped sample only reached the fracture toughness value of 4.5 mpam while doped samples achieved the higher toughness ranging from 4.8 mpam to 5.5 mpam .
all samples showed improvement in toughness when sintered 1300c , except for the 0.3 wt% doped samples , which in contrast displayed a decreasing trend up till 1450c .
sintering at higher temperature ( 1300c ) seems to have an outstanding effect on the fracture toughness of y - tzp , especially with the addition of 0.5 wt% ceo2 .
the highest value of fracture toughness recorded was 6.4 mpam for 0.5 wt % ceo2 addiition at 1400c .
the high fracture toughness of ceo2-y - tzp sample could be attributed to the resistance of the material to crack propagation and also the grain growth .
figures 5 and 6 illustrate the effect of the addition of 0.3 wt% and 0.5 wt% ceo2 on the microstructure of the ceramic .
the 0.5 wt% doped sample revealed a microstructure that is more homogenous than the microstructure of the 0.3 wt% ceo2 doped specimens .
the average grain intercept is determined from two of the best samples ( polished ) with different dopant content sintered at 1400c .
it is seen that the samples containing 0.3 wt% ceo2 have a bigger grain size with an average value of 2.13 m whereas the samples with 0.5 wt% ceo2 have a smaller grain size value of 1.586 m .
the current work shows that adding ceo2 to y - tzp has proved to be beneficial .
the bulk density was found to be higher with a value of ~5.9 g / cm ( ~97% of the theoretical density ) , with the addition of up to 0.5 wt% ceo2 when sintered at 1400c .
all samples with 0.5 wt% ceo2 produced the highest values for mechanical properties ; the vickers hardness was increased to ~13.2 gpa ; young 's modulus was enhanced to ~211 gpa ; fracture toughness was increased to ~6.4 mpam .
also , sintering temperature had an important role in determining the mechanical properties and microstructure of the material as most increment in values occurred at temperatures 1300c . | the effects of ceo2 addition on the sintering behavior and mechanical properties of y - tzp have been investigated over a wide sintering regime by pressureless sintering .
it has been revealed that small additions of ceo2 ( 0.31.0 wt% ) to y - tzp were beneficial in enhancing the mechanical properties and hydrothermal ageing resistance of y - tzp .
sintered samples were used to evaluate the bulk density , vickers 's hardness , young 's modulus , and fracture toughness of the material .
ceo2 doped y - tzps were sintered at relatively low temperatures ( 1250c and 1350c ) retaining high bulk density ( > 97% of theoretical density ) and high young 's modulus ( > 200 gpa ) without sacrificing tetragonal phase stability .
the optimum level of dopant was found to be at 0.5 wt% for sintering between 1250c and 1450c using the standard 2 h holding time cycle , with sintered body exhibiting excellent combination of properties when compared to the undoped ceramics . in this experiment , the addition of 0.5 wt% recorded a bulk density reading of 5.9 g / cm3 , vickers hardness value of 13.2 gpa , young 's modulus value of 211 gpa , and fracture toughness of 6.4 mpam1/2 , respectively , in a temperature range of 14001450c . |
transcranial magnetic stimulation ( tms ) and transcranial direct current stimulation ( tdcs ) are non - invasive brain stimulation techniques that , by means of magnetic fields and low intensity electrical current , respectively , aim to interefere with and modulate cortical excitability , at the level of dorsolateral prefrontal cortex , in patients with major depression and poor response to standard antidepressants . while the clinical efficacy of tms in major depression has been extensively investigated over the last 10 years , tdcs has attracted research interest only in the last years , with fewer randomized clinical trials ( rcts ) in the field .
nevertheless , in spite of the different rationale and mechanism of action of the two techniques , tdcs recent acquisitions , in relation to the treatment of major depression , seem to parallel those previously obtained with tms , in terms of treatment duration to achieve optimal benefit and patient 's history of drug - resistance .
after briefly introducing the two techniques , the article examines possible common pathways of clinical use for tms and tdcs , emerging from recent rcts and likely orienting future investigation with non invasive brain stimulation for the treatment of major depression . | |
metabolism lies in the heart of cell biology . understanding how cancer cells cope with metabolic needs for their unique biology
it began with the landmark observation reported more than 80 years ago by otto warburg that cancer cells consumed more glucose and produced a large amount of lactate even in a well - oxygenized environment , a process known as aerobic glycolysis or the warburg effect [ 1 , 2 ] . while normal differentiated cells maximize atp production by mitochondrial oxidative phosphorylation of glucose under normoxic conditions , cancer cells generate much less atp from glucose by aerobic glycolysis . despite being less efficient in atp production , glycolysis is a much more rapid process [ 3 , 4 ] .
cancers commonly deregulate pathways that enhance glycolysis , including activation of the pi3 k - atk - mtor pathway and upregulation of hif-1 and c - myc [ 5 , 6 ] .
the increase in aerobic glycolysis together with its dynamic process in cancer cells enables glycolytic intermediates to be redirected for the biosynthesis of cellular building blocks ( nucleotides , amino acids , and lipids ) while also producing atp .
therefore , the warburg effect / aerobic glycolysis meets the demands of cancer and proliferating cells for macromolecular synthesis and energy production [ 7 , 8 ] . as a result
, cancer cells display enhanced glucose uptake and produce higher levels of lactate [ 1 , 2 ] .
the warburg effect was explored for the common clinical detection of tumors by fluorodeoxyglucose ( 2-deoxy-2-(f)fluoro - d - glucose ) positron emission tomography ( fdg - pet ) . in the glycolytic process
, pyruvate kinase ( pk ) catalyzes the last reaction , transfer of a high - energy phosphate group from phosphoenolpyruvate ( pep ) to adp , producing atp and pyruvate .
pyruvate is then either reduced to lactate by lactate dehydrogenase ( ldh ) in the cytosol or enters the mitochondria to produce atp through the tricarboxylic acid ( tca ) cycle ( figure 1 ) .
along the glycolysis pathway , intermediate metabolites can be channeled to synthesize amino acids , nucleotides , and lipids ( figure 1 ) if the rate of flux through the pathway is controlled .
pk is an ideal candidate for this control because ( 1 ) pk catalyzes the last reaction of the pathway ( figure 1 ) and ( 2 ) the reaction is essentially irreversible ( figure 1 ) [ 9 , 11 ] .
therefore , lowering pk activity is expected to produce less pyruvate ( figure 1 ) or prevent complete conversion of glucose to pyruvate ( 1 molecule of glucose to 2 molecules of pyruvate ) .
this enables the upstream glycolytic intermediates to accumulate and thus contribute to the shift of metabolism towards the anabolic phase for amino acids , nucleotides , and lipid production ( figure 1 ) .
cancer cells explore this logic by predominantly using pkm2 , an isoform of pk , as its activity can be dynamically regulated between the less active pkm2 dimer and the highly active pkm2 tetramer .
pk consists of four isoforms : the l ( pkl ) and r ( pkr ) isoforms encoded by the pklr ( 1q22 ) gene and the m1 ( pkm1 ) and m2 ( pkm2 ) isoforms encoded by the pkm2 ( 15q23 ) gene .
the full - length pkr isoform is expressed in red blood cells while the pkl isoform missing exon 1 is detected in liver and kidney [ 5 , 13 , 14 ] .
the highly active pkm1 is expressed in tissues that consistently need high levels of energy , like skeletal muscle , heart , and brain [ 5 , 10 ] .
pkm2 is expressed in most cells except adult muscle , brain , and liver [ 12 , 16 , 17 ] and is the predominant pk in proliferating and cancer cells . while pkl , pkr , and pkm1 form stable tetramers ( the active form of pk ) , pkm2 exists as both dimers and tetramers [ 18 , 19 ] .
the pkm2 dimer has a higher km towards pep than the tetramer and thus is less active in converting pep to atp and pyruvate [ 19 , 20 ] .
while tetrameric pkm2 favors atp production through the tca cycle , dimeric pkm2 plays a critical role in aerobic glycolysis ( figure 1 ) .
therefore , the dynamic equilibrium between dimer and tetramer pkm2 allows proliferating cells to regulate their needs for anabolic and catabolic metabolism .
this not only explains why cancer cells predominantly express pkm2 but also reveals the existence of mechanisms that regulate this dynamic equilibrium . to ensure pkm2 expression ,
immunohistochemical analysis revealed that pkm2 is commonly expressed in colon cancer , renal cell carcinoma ( rcc ) , and lung cancer .
pkm2 has been suggested to be a marker for rcc [ 29 , 30 ] and testicular cancer .
elevation of serum pkm2 levels was reported in patients with colon cancer , breast cancer , urological tumors , lung carcinoma , cervical cancer , and gastrointestinal tumor .
recently , mass spectrometry has demonstrated increases in pkm2 , and the predominant presence of pkm2 was confirmed in rcc , bladder carcinoma , hepatocellular carcinoma , colorectal cancer , lung carcinoma , and follicular thyroid adenoma .
high levels of pkm2 associate with poor prognosis for patients with signet ring cell gastric cancer . a unique pattern of four expressed genes , including pkm2 , was reported to predict outcomes for mesothelioma patients undergoing surgery .
butyrate displays anticolon cancer effects along with the inhibition of pkm2 expression in neoplastic but not nontumor colon tissues .
shikonin , a derivative of a chinese herb with antitumor activities , induces necrosis and inhibits pkm2 expression in cancer cell lines .
finally , the spry2 tumor suppressor was reported to inhibit hepatocarcinogenesis via the mapk and pkm2 pathways .
overexpression of pkm2 upregulates bcl - xl in gastric cancer and promotes the proliferation and migration of colon cancer cells [ 43 , 44 ] .
knockdown of pkm2 using specific sirna inhibited cancer cell 's proliferation and invasion in vitro and the formation of xenograft tumors in vivo [ 41 , 45 ] .
the needs of energy production ( atp ) and synthesis of cellular building blocks for proliferating cancer cells dictate the shift from oxidative to glycolytic metabolism even under normoxic conditions , the warburg effect or aerobic glycolysis [ 2 , 7 , 8 ] . under hypoxic conditions , cells metabolize glucose by anaerobic glycolysis , a process that is regulated by two master transcription factors , hypoxia - inducible factor ( hifs ) , and c - myc .
consistent with pkm2 being essential for aerobic glycolysis , a relationship exists among hif-1 , c - myc , and pkm2 .
it was first demonstrated by christofk and colleagues in 2008 that knockdown of pkm2 in a panel of cancer cell lines decreased the rate of glycolysis and proliferation . introducing pkm2 but
not pkm1 to the knockdown cells not only enhanced glycolysis but also increased the ability to form xenograft tumors .
this research elegantly revealed that pkm2 is important and that the level of pk activity is essential , as the defects in pkm2 knockdown cells in supporting tumorigenesis could not be corrected by overexpression of the more active isoform pkm1 .
furthermore , in comparison to pkm1 rescued cells , reintroducing pkm2 into knockdown cells rescued the deficiency of cell proliferation under hypoxic conditions .
this investigation also suggests that pkm2 may contribute to the adaptive response ( hypoxia response ) of cells to hypoxia , which is specifically relevant to tumorigenesis as solid cancers consistently face hypoxia intratumorally .
it is thus a typical characteristic that cancers consistently execute hypoxia response . in the heart of this response
hif-1 is a heterodimeric transcription factor , consisting of hif-1 and hif-1. the subunit is constitutively expressed , while the subunit is directly regulated by oxygen ( o2 ) levels [ 48 , 49 ] . under normoxic conditions , hif-1 is hydroxylated at prolines ( p ) 402 and 564 by three prolyl hydroxylase domain proteins ( phd1 - 3 ) in the presence of oxygen , -ketoglutarate , iron , and ascorbate .
this results in the ubiquitination of prolyl - hydroxylated hif-1 by the von hippel - lindau ( vhl ) tumor suppressor and the subsequent degradation of hif-1 [ 51 , 52 ] . under hypoxic conditions ,
hif-1 is stabilized as a result of inhibiting prolyl hydroxylation , allowing hif-1 to dimerize with hif-1 in the nucleus .
this leads to transcription of a set of genes to cope with reduced o2 availability [ 5355 ] .
hif-1 transactivates the glucose transporters glut1 and glut3 , hexokinase ( the first kinase in the glycolysis pathway ) , lactate dehydrogenase a ( ldha ) , and pyruvate dehydrogenase kinase 1 which phosphorylates and inhibits pyruvate dehydrogenase ( pdh ) ( figure 1 ) .
consistent with the warburg effect 's association with synthesis of cellular building blocks , hif-1 also transactivates glucose-6-phosphate dehydrogenase ( g6pd ) to channel glucose-6-p into the pentose phosphate shunt for nucleotide and amino acid synthesis ( figure 1 ) .
therefore , the collective actions of hif-1 transcription activity seem to shift cells from oxidative metabolism to glycolysis ( figure 1 ) . in line with these observations , pkm2 shares an intimate connection with hif-1 .
the first intron of the pkm2 gene contains the functional hypoxia - response element ( hre ) , thus also making it a target of hif-1 .
pkm2 interacts with hif-1 , a process that requires the prolyl hydroxylase 3 ( phd3 )
this association and hydroxylation induces pkm2 to interact with hif-1 , which plays a role in hif-1-mediated transactivation of target genes including the ldha , pdk1 , and vegfa ( encoding the vascular endothelial growth factor ) genes .
additionally , pkm2 binds to p300 and enhances its recruitment to the hre sites of hif-1 target genes . taken together ,
pkm2 functions as a hif-1 coactivator by enhancing the warburg effect in cancers [ 21 , 22 ] .
the regulation between hif-1 and pkm2 also occurs under normoxic conditions , by changes in other signalling events which act to stabilize hif-1 in cancer cells .
activation of mtor is inhibited by tumor suppressors tsc1/tsc2 and facilitated by the pi3 k - akt pathway .
consistent with this knowledge , abnormal activation of the pi3 k - akt - mtor pathway and loss of function of tumor suppressors vhl , tsc1/2 , and pten have been demonstrated to stabilize hif-1 [ 57 , 58 ] .
activation of mtor by downregulation of tsc1/2 and pten induced pkm2 expression via stabilization of hif-1 .
pkm2 makes essential contributions to mtor - mediated aerobic glycolysis , as knockdown of pkm2 reduced glucose consumption and lactate production in cells with elevated mtor activation .
the difference between these is the inclusion of exon 9 and exclusion of exon 10 for pkm1 and vice versa for pkm2 ( figure 2 ) [ 5 , 15 ] .
this mutually exclusive pattern of splicing is mediated by members of the heterogeneous nuclear ribonucleoprotein ( hnrnp ) family , hnrnpa1 , hnrnpa2 , and hnrnp1/ptb ( polypyrimidine track binding protein ) [ 23 , 60 ] .
binding of these proteins to the dna sequence flanking exon 9 prevents its inclusion , resulting in the inclusion of exon 10 [ 23 , 60 , 61 ] . in order to achieve predominant expression of the m2 isoform ,
cancer cells have a strategy to preferentially splice the m2 isoform over m1 through c - myc - mediated upregulation of hnrnpa1 , hnrnpa2 , and ptb ( figure 2 ) [ 23 , 61 ] .
this finding is supported by the discovery that cells with high levels of c - myc activity also demonstrated high pkm2/pkm1 ratios [ 23 , 62 ] .
these observations are well in line with a large body of evidence indicating that c - myc stimulates glycolysis and is required to coordinate with hif-1 to regulate the cellular response to hypoxia [ 24 , 46 ] .
thus , evidence suggests that pkm2 plays a role in c - myc - mediated cancer metabolism and in c - myc 's communication with hif-1 .
adding to this attractive possibility is a recent demonstration that pkm2 also upregulates c - myc transcription [ 63 , 64 ] , suggesting another positive feedback loop involving pkm2 in regulating the warburg effect . taken together , pkm2 is an integrated piece in the network of glycolysis regulation together with hif-1 and c - myc .
the importance of hnrnpa1 , hnrnpa2 , and ptb in splicing pkm2 has also been explored by tumour suppression activity .
the micrornas mir-124 , mir-137 , and mir-340 inhibit colorectal cancer growth by repressing the expression of these hnrnas favouring pkm1 splicing , thereby inhibiting aerobic glycolysis or the warburg effect .
cancers have developed a complex regulation of pkm2 to meet the needs for energy and synthesis of nucleotides , amino acids , and lipids .
the latter two mechanisms directly or indirectly affect pkm2 activity through physical interaction and by regulating the pkm2 dimer - tetramer dynamic .
in addition to the above discussion of hif-1 and c - myc - mediated transcription and splicing of pkm2 , transcription of the pkm2 gene is also regulated by the sp1 and sp3 transcription factors [ 5 , 22 , 66 ] . the network of pi3 k - akt - mtor ( mammalian target of rapamycin ) plays a critical role in cell metabolism , proliferation , and survival and is one of the most frequently activated pathways in cancer owing to the activation of kinases and the inactivation of tumor suppressors , tsc1/2 ( tuberous sclerosis 1/2 ) and pten .
mtor activity induces pkm2 expression through the combination of hif-1 and c - myc [ 59 , 69 ] .
elevation in pten function reduces glucose uptake and the warburg effect and inhibits pkm2 expression . in a feedback manner , pkm2 is able to sustain mtor activation in serine - depleted medium by enhancing endogenous serine synthesis
. taken together , evidence supports that the upregulation of pkm2 plays an important role in the mtor - mediated warburg effect in tumors .
tumor cells express high levels of dimer pkm2 [ 14 , 32 ] . among the four pk isoforms , pkm2 is the only one to be allosterically regulated between a less active dimer and an active tetramer [ 18 , 19 ] .
these different forms of pkm2 regulate glucose metabolism through either the tca cycle or glycolysis .
accumulating evidence supports the concept that the less active pkm2 dimer drives aerobic glycolysis , while the active pkm2 tetramer produces pyruvate for oxidative phosphorylation ( figure 1 ) [ 12 , 7274 ] .
pkm2 is regulated by fructose-1,6-biphosphate ( fbp ) , an upstream intermediate of glycolysis which when bound to pkm2 activates tetramerization through high affinity association [ 7577 ] .
binding of tyrosine - phosphorylated peptides dissociates fbp from the pkm2 tetramer , resulting in conversion to the pkm2 dimer .
the less active pkm2 dimer is critical in mediating aerobic glycolysis in tumor cells based on high levels of lactate production and lower oxygen consumption . disrupting the binding of the phosphotyrosine peptide in a pkm2 mutant ( m2ke )
increased pkm2 kinase activity , which was associated with reduction in lactate production and elevation of oxygen consumption . in supporting the low levels of cellular pyruvate kinase activity being critical for aerobic glycolysis , replacing pkm2 with pkm1 led to an increase in cellular pyruvate kinase activity , decreasing lactate production and elevating oxygen consumption [ 12 , 74 ]
in addition to the above mechanism regulating pkm2 activity , pkm2 was also controlled by tyrosine phosphorylation . it was observed in 1988 that pkm2 was tyrosine - phosphorylated in v - src - transformed chicken embryo cells .
this phosphorylation reduced the affinity of pkm2 towards its substrate phosphoenolpyruvate ( pep ) . in vitro ,
although this investigation suggested that v - src phosphorylated pkm2 , the sites of phosphorylation remain unknown .
recent development demonstrated that pkm2 was phosphorylated at several tyrosine residues , including y105 , by fibroblast growth factor receptor type 1 ( fgfr1 ) .
phosphorylation at y105 causes fbp to dissociate from the pkm2 tetramer , which results in pkm2 dimers and promotes the warburg effect based on the production of lactate .
conversely , abolishing y105 phosphorylation by substitution with phenylalanine ( y105f ) elevated the kinase activity , resulting in decreased lactate production and increased oxygen consumption . taken together , evidence demonstrates that phosphorylation at y105 plays a role in the conversion of pkm2 tetramers to dimers .
more importantly , regulation of pkm2 dimer and tetramer conversion is critical for tumorigenesis . while the less active pkm2 dimer enhances xenograft tumor formation , enforced formation of active pkm2 ( ke and y105f mutations ) and replacing pkm2 with pkm1 inhibited the formation of xenograft tumors [ 72 , 73 , 79 ] . in line with this concept ,
the conversion between dimer and tetramer pkm2 is also used in tumour suppression to inhibit tumorigenesis .
the death - associated protein kinase ( dapk ) tumor suppressor activates pkm2 by stabilizing the pkm2 tetramer via a direct association .
this reduces cancer metabolism or the warburg effect , which may be one aspect of dapk - mediated tumor suppression [ 80 , 81 ] . in line with these observations , several small molecule
pkm2 activators have been identified . among them , dasa-58 ( the substituted n , n-diarylsulfonamide ncgc00185916 ) and tepp-46 ( the thieno-[3,2-b]pyrrole [ 3,2-d]pyridazinone ncgc00186528 ) activate pkm2 by inducing pkm2 tetramerization . unlike fbp - induced activation , the tetramer induced by these compounds is resistant to tyrosine - phosphorylated peptide - mediated conversion to the pkm2 dimer .
this suggests that fbp and these small molecule activators bind pkm2 at distinct sites , but , importantly , all inhibit tumorigenesis [ 74 , 82 , 83 ] . additionally , a new set of chemical platform bases , the quinolone sulfonamide - based pkm2 activators ,
similar to dasa-58 and tepp-46 , these activators also stabilize the pkm2 tetramer via binding to a pocket distinct from fbp binding and thus prevent the pkm2 tetramer from tyrosine - phosphorylated peptide - mediated disruption .
quinolone sulfonamide - based pkm2 activators reduce carbon flow towards the serine biosynthetic pathway , rendering cells to serine auxotrophy .
in addition to the above two small molecule pkm2 activators , a third activator was recently reported by the same research group based on modifications to one of their previous compounds .
in contrast to these , potent small molecule pkm2 inhibitors which may in part induce cell death by inhibiting pkm2 activity have also been developed .
furthermore , the pyruvate kinase activity of pkm2 can be inhibited by association with several distinct proteins . while the nuclear promyelocytic leukemia ( pml ) protein functions as a tumor suppressor , cytosolic pml was reported to specifically inhibit tetrameric but not dimeric pkm2 activity , thereby contributing to the warburg effect .
prolactin signal promotes cell proliferation by inducing its receptor to associate with pkm2 , leading to pkm2 activity reduction .
the muc1-c oncoprotein was reported to promote breast cancer tumorigenesis in part via inhibiting pkm2 activity .
although interaction of muc1-c cys3 with pkm2 c - domain cys474 results in activation of pkm2 , oncogenic signals from egfr ( epidermal growth factor receptor ) can alter the association of muc1-c and pkm2 , thereby leading to inhibition of pkm2 activity .
egfr phosphorylates muc1-c at tyrosine 46 , causing muc1-c to interact with pkm2 at lys433 .
this association inhibits tetrameric pkm2 activity and thereby increases aerobic glycolysis along with glucose uptake .
pkm2 was also found to interact with human papillomavirus 16 ( hpv16 ) protein e7 , which may contribute to hpv16-induced cervical cancer .
a potential therapeutic protein tem8-fc , consisting of a portion of the tumor endothelial marker 8 ( tem8 ) and the fc domain of human igg1 , was found to associate with pkm2 . whether this interaction contributed to tem8-fc - associated tumor suppression was not clear .
consistent with the knowledge that pkm2 plays a critical role in regulating aerobic glycolysis and biosynthesis for cellular building blocks , pkm2 is activated by serine but inhibited by alanine and phenylalanine when bound to these amino acids . a reduction in activity
was reported by acetylation of pkm2 at lysine ( k ) 305 in response to high levels of glucose .
this modification reduces pkm2 activity and its affinity towards the pep substrate , resulting in pkm2 degradation via chaperone - mediated autophagy . as a result
, acetylation enhances cell proliferation by increasing the availability of glycolytic intermediates for anabolic synthesis [ 94 , 95 ] .
acute increases in intracellular levels of ros ( reactive oxygen species ) induce oxidation of pkm2 at cys358 .
this reduces pkm2 activity , which allows the accumulation of glucose-6-phosphate and thus shifts glucose flux through the pentose phosphate pathway ( ppp ) to generate reduced nadph ( figure 1 ) .
as ppp is the major pathway of generating reduced nadph , oxidation - mediated inhibition of pkm2 is therefore a mechanism of detoxification during oxidative stress .
consistent with this notion , substitution of c358 with s358 to produce oxidation - resistant mutants sensitized cells to oxidative stress and inhibited xenograft tumor formation .
a similar antioxidative stress function of pkm2 is also mediated through binding to cd44 , a major cell adhesion molecule .
cancer stem cells are known to be cd44 positive , so this interaction is consistent with cd44 promoting cancer progression , metastasis , and chemoresistance [ 97 , 98 ] .
consistent with these observations , pkm2 was reported to bind cd44 , resulting in receptor tyrosine kinase - mediated phosphorylation of pkm2 and inhibition of pkm2 activity .
this enhanced glucose flux through the ppp pathway to generate reduced nadph and counteract oxidative stresses through detoxification [ 61 , 100 ] .
in addition to its cytoplasmic presence to regulate aerobic glycolysis , pkm2 was also detected in the nucleus in response to interleukin-3 and apoptotic signals [ 101 , 102 ] .
nuclear pkm2 binds oct 4 through its c - terminal region ( residues 307531 ) , enhancing oct-4-mediated transcription ( figure 3 ) .
nuclear pkm2 was also reported to be a coactivator of hif-1 ( figure 3 ) .
egfr signaling was reported to activate src tyrosine kinase , which in turn phosphorylates -catenin at y333 .
pkm2 binds to tyrosine - phosphorylated -catenin in the nucleus and contributes to -catenin - mediated transactivation of cyclin d and c - myc , thereby promoting both cell proliferation and tumor progression ( figure 3 ) .
since the binding of tyrosine - phosphorylated peptides maintains pkm2 in its dimer status , these observations suggest that dimerized pkm2 binds and enhances -catenin function , in which a new kinase activity rather than pyruvate kinase activity might be involved .
indeed , it was very recently reported that the pkm2 dimer contributes to its nuclear function and possesses protein tyrosine kinase activity .
surprisingly , instead of using high - energy atp , pkm2 uses the high - energy phosphate from pep as a phosphate donor to phosphorylate its protein substrates .
the pkm2 dimer phosphorylates stat 3 at y705 in the nucleus and thus enhances stat 3 transcription activity [ 26 , 105 ] ( figure 3 ) .
taken together , while tetramer pkm2 is a pyruvate kinase , dimer pkm2 can also act as a protein tyrosine kinase .
the last decade has seen a high reemergence of interest in the warburg effect , the typical cancer cell metabolism that was reported almost 90 years ago . the detailed molecular and genetic knowledge accumulated in the last few decades of extensive cancer research
mutations in several enzymes of the tca - cycle were discovered , including isocitrate dehydrogenases 1 and 2 ( idh1 and idh2 ) , succinate dehydrogenase ( sdh ) , and fumarate hydratase ( fh ) [ 106109 ] .
these mutations collectively reduce tca - cycle - mediated oxidative phosphorylation , resulting in an accumulation of metabolites for the biosynthesis of amino acids , nucleotides , and lipids as well as increases in glucose uptake . the increases in glucose uptake together with aerobic glycolysis yield a robust elevation of lactate production .
although recent development suggests that the by - product of aerobic glycolysis ( lactate ) contributes to overall tumorigenesis [ 111 , 112 ] , it is also critical for cancer cells to efficiently export lactate to maintain the flux of glycolysis and to prevent cellular acidification .
cancer cells accomplish this task in part by upregulation of the monocarboxylate transporters ( mcts ) .
another strategy to reduce the cellular burden of lactate accumulation during aerobic glycolysis may be the prevention of a complete conversion of glucose to lactate ( 1 glucose for every 2 lactate molecules ) by reducing the conversion of pep to pyruvate .
accumulating evidence obtained in the last 10 years demonstrates that pkm2 's glycolytic enzyme activity is regulated by oncogenes and tumor suppressors [ 2125 ] .
a shift of the dimer - tetramer dynamic towards dimerization is critical for pkm2 to promote the warburg effect , leading to cell proliferation and tumorigenesis .
surprisingly , in addition to its glycolytic pyruvate kinase activity in the cytosol , the pkm2 dimer also displays protein tyrosine kinase activity in the nucleus and nuclear pkm2 promotes the transcriptional activities of hif , -catenin , stat 3 , and oct 4 [ 21 , 26 , 63 , 103105 ] .
this all indirectly contributes to cancer metabolism and other aspects of tumorigenesis . in light of this new development
, future research should determine the contributions of the cytosolic versus nuclear pkm2 dimer to aerobic glycolysis .
effort is currently underway to target pkm2 for cancer therapy , which is part of the current attempt in targeting cancer metabolism .
as nearly complete knockdown of pkm2 does not completely inhibit cancer cell proliferation , the utility of pkm2 inhibition in targeting cancer should be cautious .
( 1 ) pkm2 is also expressed in normal tissue [ 16 , 17 ] and the function of pkm2 in normal tissues has not yet been determined ; ( 2 ) genetic changes in pkm2 have not been reported in primary cancers ; ( 3 ) despite modulation of pkm2 which affects formation of xenograft tumors , whether tissue - specific manipulation of pkm2 impacts tumorigenesis is still on the waiting list ; ( 4 ) as pkm2 was detected in cancer stroma [ 113 , 114 ] , whether it plays a role in tumorigenesis by affecting cancer - associated fibroblasts is not clear ; ( 5 ) while aerobic glycolysis has been a hot topic in the last decade , its impact on cancer stem cells ( cscs ) has not been addressed .
as it is becoming increasingly clear that cscs play a critical role in tumorigenesis , especially in tumor progression and metastasis , it would appear critical to understand whether targeting cancer metabolism in general and pkm2 in particular will have an inhibitory effect on cscs .
this knowledge became important as it was suggested that glioma cscs ( gscs ) may not use aerobic glycolysis to the same degree as differentiated cancer cells . | aerobic glycolysis is the dominant metabolic pathway utilized by cancer cells , owing to its ability to divert glucose metabolites from atp production towards the synthesis of cellular building blocks ( nucleotides , amino acids , and lipids ) to meet the demands of proliferation .
the m2 isoform of pyruvate kinase ( pkm2 ) catalyzes the final and also a rate - limiting reaction in the glycolytic pathway . in the pk family
, pkm2 is subjected to a complex regulation by both oncogenes and tumour suppressors , which allows for a fine - tone regulation of pkm2 activity .
the less active form of pkm2 drives glucose through the route of aerobic glycolysis , while active pkm2 directs glucose towards oxidative metabolism . additionally , pkm2 possesses protein tyrosine kinase activity and plays a role in modulating gene expression and thereby contributing to tumorigenesis .
we will discuss our current understanding of pkm2 's regulation and its many contributions to tumorigenesis . |
relapsing polychondritis ( rp ) is an uncommon disorder of unknown etiology that is characterized by recurrent and progressive inflammation of cartilaginous structures .
a minority of patients with rp develop central nervous system ( cns ) manifestations , and limbic encephalitis has also been reported .
glucocorticoid has been used as the first - line therapeutic agent , but a standardized second - line therapeutic protocol for rp with cns manifestations has not been established .
the effects of anti- tumor necrosis factor ( tnf ) - agents have been reported recently .
we report a patient with limbic encephalitis associated with rp who was refractory to initial high - dose glucocorticoid therapy , but subsequently responded to infliximab and discontinued therapy without recurrence .
we also reviewed cases of rp with cns manifestations using pubmed with regard to clinical manifestations and treatment .
a 58-year - old japanese male architect was brought to our institution by his wife , presenting with amnesia , disorientation , emotional liability and urinary incontinence .
one year prior to admission , he had bilateral ear pain with swelling and erythema which improved without any treatment over a 4-week period .
nine months prior to admission , he experienced iritis and scleritis in addition to recurrent pain in bilateral auricles .
subsequent biopsy of the left auricle revealed infiltration of inflammatory cells in the perichondrium ( fig .
1 ) . diagnosis of rp was made based on mcadam s criteria , modified by damiani and levine .
inflammation of bilateral auricles disappeared without any treatment , while iritis and scleritis were controlled by topical glucocorticoid therapy .
one month prior to admission , he developed difficulty with drawing architectural drafts and finding his way home , together with emotional liability and urinary incontinence .
past medical history revealed well - controlled diabetes mellitus by diet and dipeptidyl peptidase-4 inhibitor ( hba1c was 6.4 to 6.7% ) . on admission ,
his body temperature was 36.7c , blood pressure was 112/66 mmhg and heart rate was 68 beats per minute .
head , eye , ear , nose , chest and abdominal examinations were unremarkable . neurological examination revealed poor tandem gait and poor finger - nose - finger test , but other examinations such as the cranial nerve , sensory and motor systems were unremarkable .
his mini - mental states examination ( mmse ) result was 16 out of 30 .
antinuclear antibodies , anti - neutrophil cytoplasmic antibodies , rheumatoid factor , anti - thyroid peroxydase antibody , anti - thyroglobulin antibody , urinalysis and serological tests for human immunodeficiency virus ( hiv ) and treponema pallidum were all normal or negative .
cerebrospinal fluid ( csf ) analysis showed 33 cells/l with 32 polymorphonuclear leukocytes , glucose 81 mg / dl and protein 92 mg / dl .
csf cultures for bacteria and mycobacterium tuberculosis and polymerase chain reaction of herpes simplex virus and cytology were also negative .
both anti - n - methyl - d - aspartate type glutamate receptor ( glur ) n2b antibody and anti - glur 2 antibody were positive in csf , but neither were positive in serum .
whole body fluorine-18 fludeoxyglucose positron emission tomography ( [ 18f]fdg - pet ) with ct to detect tumor revealed no abnormal uptake . comparing current brain magnetic resonance imaging ( mri )
result with the previous ones indicated limbic system atrophy resulting in ventricular enlargement ( fig .
2-a , b ) . diffusion weighted image , fluid - attenuated inversion recovery image ( flair ) and gadolinium enhancement showed no abnormality .
electroencephalogram showed diffuse dominant theta waves with no spike . considering his clinical symptoms like emotional lability and amnesia , limbic system atrophy in mri and increased number of csf cells
limbic encephalitis was diagnosed . because other causes such as hiv encephalitis , herpes simplex encephalitis , tumor - associated limbic encephalitis or hashimoto encephalopathy were ruled out , limbic encephalitis associated with rp was diagnosed , clinically .
a course of intravenous 1 g methylprednisolone for 3 days was administered , followed by oral prednisolone 1 mg / kg per day .
his head mri had no change but mmse score was improved gradually , ataxia disappeared through 4 doses of infliximab over a 3-month period , and problematic behavior disappeared . because of his stable condition as well as the high cost of infliximab , he and his wife refused further infliximab therapy .
the patient was followed up for an additional 9 months after stopping prednisolone without recurrence ( fig .
, he could continue active daily living independently , but could not resume his work .
rp , a rare episodic and progressive inflammatory disease presumed to have autoimmune etiology , was first described in 1923 .
rp affects cartilage in multiple organs , such as the ear , nose , larynx , trachea , bronchi , and joints .
in addition , it can affect proteoglycan - rich tissues such as the eyes , aorta , heart and skin .
mcadam criteria modified by damiani and levine , which is commonly used as a criterion to confirm the diagnosis of rp , consists of : a ) at least 3 of 6 clinical criteria ( bilateral auricular chondritis , nonerosive seronegative inflammatory polyarthritis , nasal chondritis , ocular inflammation , respiratory chondritis and audiovestibular damage ) ; b ) 1 or more of the previously - mentioned clinical criteria and biopsy confirmation of cartilage inflammation ; or c ) chondritis at 2 or more separate anatomic locations with response to steroids and/or dapsone .
we searched medline in march 2014 using ( " polychondritis , relapsing " [ mesh ] or " relapsing polychondritis " ) and ( " encephalitis " [ mesh ] or " limbic encephalitis " [ mesh ] or encephalitis or encephalopathy or " limbic encephalitis " or " meningoencephalitis " [ mesh ] or meningoencephalitis or " nervous system " ) as keywords .
we retrieved a total of 54 articles , 26 of them including 31 cases that met inclusion criteria ( case report or case series written in english or japanese ) ( table 1 ) .
as shown in table 1 , 28 out of 31 patients have been treated with a high dose of glucocorticoid .
twenty - two out of those 28 patients had symptoms which were well - controlled by initial therapy , but only one could discontinue glucocorticoid therapy .
additional therapy ( cyclophosphamide , intravenous immune globulin , tacrolimus , plasmapheresis , methotrexate and cyclosporin ) showed no remarkable effect and 3 patients died .
only one who was treated with inflixmab had a good outcome , so we chose infliximab as a second - line agent .
this is the first case report of rp with cns manifestations treated with an anti - tnf- agent who did not show deterioration of signs and symptoms after stopping therapy .
infliximab may be a good choice for rp with cns manifestation refractory to initial glucocorticoid therapy .
infliximab has a large molecular weight , so it is impossible for it to permeate the blood - brain barrier .
one potential explanation is that breakdown of the blood - brain barrier by inflammation may permit infliximab to access cerebral parenchyma , resulting in the suppression of tnf- mediated inflammatory processes .
although theoretically it may be reasonable to stop infliximab when neurologic symptoms are stable , if breakdown of the blood - brain barrier by inflammation is important for the effect of infliximab , it would be wise to closely observe the clinical course when discontinuing infliximab .
anti - tnf- agents may be a treatment of choice for rp with cns manifestations refractory to initial glucocorticoid therapy .
in addition , anti - tnf- agents may be discontinued , but it would be prudent to closely observe the clinical course when stopping infliximab .
histopathological examination of ear biopsy ( hematoxylin - eosin stain ) showed infiltration of inflammatory cells ( histiocytes , lymphocytes , neutrophils and eosinophils ) in perichondrium and chondrium .
( a ) t2wi one year before admission ; ( b ) t2wi of day 1 showed ventricular enlargement compared to one year before admission ; ( c ) there was no change after 6 months .
other patients received no treatment or the results were unknown . * the clinical course after the second pulse is not shown . | abstractcentral nervous system ( cns ) manifestations are rare complications of relapsing polychondritis ( rp ) .
the majority of patients respond well to glucocorticoid therapy , but need to maintain it .
some patients are refractory to initial glucocorticoid therapy and to additional immunosuppressants , and end up with an outcome worse than at therapy initiation .
the standardized therapeutic protocol for this condition has not been established .
the effects of anti - tumor necrosis factor ( tnf ) - agents have been reported recently .
we experienced a patient with rp and limbic encephalitis who was refractory to initial high - dose glucocorticoid , but subsequently responded to infliximab and did not show deterioration of signs and symptoms after stopping therapy .
we report this case together with a systematic literature review .
this is the first case report of rp with cns manifestations successfully treated by an anti - tnf- agent without recurrence after discontinuation . |
during the late 1970s and early 1980s , the lack of commercial development for drugs that treat rare diseases became an important political issue in the us . these so - called
orphan drugs were largely neglected by the pharmaceutical industry because they represented small markets that were unlikely to be profitable .
the lobbying and public awareness efforts of a number of rare disease patient organizations in the us eventually culminated in the passage of the orphan drug act of 1983 ( oda ) , which provides industry with support and incentives to develop orphan drugs , defined as drugs intended for use in treating a condition that affects less than 200,000 persons in the us . the substantial increase in the development of drugs for rare diseases over the past three decades is often directly attributed to the passage of the oda , and the act is widely considered to be a success . since the oda was passed in 1983 , more than 400 orphan drugs have been developed and marketed in the us , which suggests that the incentives are having an effect .
moreover , the last 1015 years have been the most successful period of development for orphan drugs . according to the fda ,
nearly 200 orphan drugs enter development each year and approximately one third of new drugs approved by the fda are for the treatment of rare diseases .
a concurrent trend that is contributing to the shift toward niche market development is recent advances in new genomic technologies that are now making
the completion of the human genome project the international effort to map the entire human genomelaid the foundation for the development of new health care technologies and therapies , including genetic tests to assist in the diagnosis and prevention of disease and drug therapies that are tailored to the genetic characteristics of individual patients. pharmacogenomics , the study of the influence that genetic factors have on drug response , has emerged from genomics - related research and the development of new diagnostic approaches based on biomarkers .
there is increasing interest in the pharmaceutical sector toward pairing pharmaceutical products with diagnostic tests that can stratify broader disease categories into rarer disease genotypes .
significantly , a growing number of products in clinical development now rely on a clinical biomarker , which suggests the mounting importance of pharmacogenomic - based drug development .
advances in pharmacogenomic research and increasing industry interest in personalized medicine have important implications for the way that orphan drug policies are interpreted and applied . in the us ,
the office of orphan products development ( oopd ) has indicated that pharmacogenomic products are treated the same as any other orphan drug submissions .
however , concerns have been raised regarding orphan drug legislation generally and the impact of pharmacogenomics in this context in particular . some express concern that orphan drug incentives are not adequately targeted to the diseases with greatest unmet medical needs and that the oda has favored the development of treatments for diseases that can , through omics data and technologies , be recast as rare , or belong to the larger , more lucrative therapeutic class of oncology products. moreover , there are concerns that the oda does not adequately distinguish between true orphan drugs and trojan applicants that seek to co - opt the benefits for drugs that should not qualify as orphans. however , in 2013 , the fda made a number of important amendments to the oda regulations that purport to address many of the challenges raised by the evolving drug development environment , including advances in pharmacogenomics .
the oda has served as a model for legislation in a number of other jurisdictions , including europe , japan , and australia .
although the legal framework implemented in these countries is similar , the definition of an orphan disease , the criteria which must be satisfied to obtain a designation of orphan status , the incentives provided to encourage the development of orphan drugs and the authorization process for orphan drugs , varies from jurisdiction to jurisdiction .
although for years the canadian government denied the need for the country to develop its own orphan drug policy , the government has recently reversed its policy and in december 2012 , released a draft orphan drug policy for discussion .
the release of the 2013 amendments to the oda regulations provides an opportunity to examine what lessons canada can learn from the fda 's experience in drafting its own orphan drug policy , or indeed , whether a canadian orphan drug policy is even necessary given the increasingly lucrative nature of niche markets .
this article considers whether orphan drug legislation can be drafted in a way that will maximize benefits and minimize concerns relating to the impact of pharmacogenomics on orphan drug research and development . after reviewing the issues that may arise at the intersection of orphan drug policies and pharmacogenomics
, this article will discuss the potential impact of pharmacogenomics at two critical points : orphan designation and approval of the drug product . at each of these points ,
the relevant aspects of current us orphan drug legislation are examined , focusing on the extent to which recent amendments may address concerns that have been raised previously . this analysis will then provide the foundation for a critical review and recommendations regarding the proposed new canadian orphan drug framework . in recent years , many concerns have been raised about the potential impact of pharmacogenomics and new genomic technologies on our understanding of how disease categories are delineated , and subsequently , how the concept of rare disease should be defined for the purposes of orphan drug policies .
it is estimated that over 80% of rare diseases are genetically - based , so it makes sense that pharmacogenomics could play an important role in the discovery and development of new treatments for rare disease .
a 2013 report by thomson reuters suggests that the tremendous growth in orphan drug development over the past decade coincides with the increasing focus on personalized medicine , and that orphan disease markets will propel the evolution of [ personalized ] medicine. as argued by haffner and colleagues , [ i]n an environment in which medicine is increasingly adapted to the needs of patients , the incentives of the orphan drug act could arguably take on even greater importance. the obama administration 's announcement in january 2015 of a \documentclass[12pt]{minimal }
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personalized approaches to medicine that take into account individual differences in people 's genes , environments , and lifestyleis yet another indication of the burgeoning importance of this area of research and development .
many pharmacogenomic drugs have already qualified for orphan drug status under the oda , although at present these drugs represent only a small fraction of the total number of products that have received orphan drug designation . in certain cases
, there may be a legitimate need to incentivize research into pharmacogenomic treatments that may only be effective in a particular subset of patients with a particular genetic biomarker .
the nuffield council on bioethics , for example , highlights that stratifying more common diseases into rarer disease genotypes may result in some patient subsets being so small that developing specific medicines targeting these groups may not be financially viable for drug developers . in such cases
, orphan drug policies may provide the necessary incentives to encourage pharmaceutical companies to develop medicines for these narrow populations , perhaps even allowing drugs that would have otherwise failed to be targeted to a smaller subpopulation in which the drug is more likely to be safe and/or effective . on the other hand ,
[ t]he nature of pharmacogenomics drugs may allow some pharmaceutical companies to game the system and abuse the oda 's built - in incentives for drug development. the oda provides incentives to manufacturers at two stages .
first , in the development phase , sponsors can apply for an orphan drug designation , which gives them access to a variety of support and incentive measures , including a tax credit of 50% for the costs of clinical research , access to the oodp 's clinical research grants program , a waiver of fda user fees , and development and regulatory assistance .
second , if the drug is then approved , the manufacturer is granted a seven - year period of market exclusivity for the orphan indication(s ) for which the drug is approved .
this means that the fda will not approve another drug for the same indication(s ) during the exclusivity period , unless the holder of the exclusive license consents or can not supply sufficient quantities of the drug , or the other product is shown to be clinically superior ( and therefore not the
the exclusivity applies only to the approved orphan indication(s ) and does not prevent the same drug from being designated or approved for a different use .
pharmacogenomics can impact the way that the oda operates at a number of points in the product lifecycle .
first , the use of pharmacogenomics to identify patient subsets can impact the way that disease categories are defined , and thus the size of the target population for the purposes of orphan designation .
as the science advances , the nomenclature and classification of disease is becoming increasingly complex and consequently , the number of potential orphan diseases appears to be on the rise .
herder argues that new insights from genomics and epigenomics are rendering the boundary between common and rare disease increasingly mutable , potentially exploding the scope of legislated definitions of orphan disease. however , orphan designation is only the first step toward market approval for an orphan drug product , and many orphan designated drugs never reach the market or are not ultimately approved for the orphan indication .
second , pharmacogenomics can have an impact on the way that orphan exclusive approval is granted within the rare disease or condition , or orphan subset , for which the designation was given .
pharmacogenomics can contribute to the narrowness of approved indications because stratification of the disease may increase the specificity with which the approved indication is defined , and may lead to multiple , narrow approvals within a single orphan designated disease or subset .
repurposing efforts that is , the discovery of new useful activity in an older clinically used drugallowing some drugs to achieve multiple orphan drug designations and approved indications . in these cases
, orphan designation and exclusive approval may add to the profitability of a drug that has already been approved and widely marketed for other uses .
the oda thus provides an incentive for sponsors to invest in studying potential new uses of a drug for rare diseases .
one concern that has long plagued the oda is the potential for drug developers to exploit the benefits of the act by artificially subdividing diseases to create subgroups of patients that fall under the orphan drug prevalence threshold a practice referred to as
salami slicing. a classic example of salami slicing is the drug epogen ( epoetin alpha ) , which received an orphan designation from the fda in 1986 for the treatment of anemia associated with end - stage renal disease .
after the drug was approved by the fda in 1989 , the drug became widely prescribed for a wide variety of patients with anemia , not only anemia caused by end - stage renal failure .
consequently , through off - label use prescribing a drug for indications not formally approved by drug regulators in patients without end - stage renal disease , epogen became a blockbuster drug and generated billions of dollars in revenue for its manufacturer .
loughnot argues that pharmacogenomics could potentially take salami slicing to a new level by allowing drug developers to
genetically subdivide diseases that affect a large portion of the population into groups small enough to qualify for orphan drug status. the potential to stratify broader disease categories based on biomarker status significantly increases the potential number of orphan subsets that may be defined for the purpose of orphan designation .
the fda acknowledges that what is considered a distinct disease or condition may change over time as scientific understanding evolves , which would affect prevalence determinations. maher and haffner point out that
[ w]hether such increasingly precise disease descriptions constitute separate diseases would normally appear to be an academic exercise , unless , as is now more often the case , a specific therapy targeting a specific mutation is developed. that is , the very development of a new treatment can play an integral role in shaping the classification of a new rare disease or condition , or orphan subset . the potential for
salami slicing is not a new concern , having been previously considered in earlier revisions of the us orphan drug legislation . in discussing the criteria for orphan drug designation ,
the 1991 notice of proposed rulemaking stated that a subset of a common disease or condition would qualify for designation only if the subset is medically plausible and that arbitrary
however , the 1992 regulations offered little guidance on the meaning of this rather ambiguous phrase , providing only that the concept of medically plausible is interpreted flexibly depending on the specific facts of each case .
moreover , a request to further define the term arbitrary in this context was rejected on the basis that every fda decision on arbitrariness would necessarily be highly fact dependent. according to herder , until recently , the addition in 1992 of the requirement that the disease in question be considered
medically plausible was the only relevant constraint that has been adopted by the fda to distinguish between rare diseases that have long been identified as such and those which have been reclassified as rare by virtue of new scientific insights. the question of orphan subsets was addressed again , in more detail , in the most recent regulatory amendments . in the 2011 notice of proposed rulemaking
[ b]ecause the term medically plausible has not been further clarified through regulations or guidance , it has been misinterpreted to mean any medically recognizable or any clinically distinguishable subset of persons with a particular disease or condition , and that inappropriate application of the medically plausible concept could result in artificially narrow subsets .
although it can certainly be beneficial for manufacturers to develop more effective treatments for some subsets of common diseases and conditions , it could be said that this departs from the original intent of the oda to target very rare diseases that currently lack any effective treatments .
indeed , the fda believes such an interpretation would frustrate the intent of the oda and divert resources away from research and development of true orphan drugs by allowing
a non - rare disease or condition to be artificially subdivided into smaller groups for establishing subsets that are under the prevalence limit for designation. the fda therefore proposed to amend the regulation to remove the term medically plausible and instead provide a description of how an appropriate subset may be identified for the purpose of orphan drug designation. the final rule adopted in 2013 added a definition of orphan subset to clarify that an appropriate subset may exist where use of the drug in a subset of persons with a non - rare disease or condition may be appropriate but use of the drug outside of that subset ( in the remaining persons with the non - rare disease or condition ) would be inappropriate owing to some property(ies ) of the drug. the fda insists that the 2013 amendments are consistent with the agency 's longstanding approach to identifying
some may continue to question how effectively the fda can distinguish between true and
artificial subsets of disease , particularly since evaluation of orphan subsets is complex and is often based on uncertain evidence .
may be difficult given the potential information asymmetries between the fda and the companies it regulates. if there were significant concerns about the fda 's previous approach , the fact that it maintains that the new language is consistent with this approach may undermine confidence in the potential for the new language to better distinguish between real and
however , an analysis of the revised wording , along with some examples of subsets that have been accepted or rejected by the fda , suggests that these concerns may have been mitigated somewhat . in the 2013 final rule , although the fda accepted that biomarker - based and other targeted treatments could be used to define subsets , it expressly rejected the proposition that an orphan subset can exist whenever there is a basis for using the drug in the subset of interest , regardless of whether the drug can also be used in the remaining persons with the disease or condition. at the core of the analysis is consideration of the property or properties of the drug that preclude its use in the remaining persons with the non - rare disease or condition , outside of the orphan subset [ emphasis added]. according to the stated approach of the fda , sponsors have to not only demonstrate why this subset should be targeted for an orphan drug treatment , but also why the drug can not also be used outside the subset .
the 2013 amendments provide specific guidance as to what factors may or may not inform whether an appropriate orphan subset exists .
this is likely due to the fact that that much of the new wave of concern around
salami slicing has arisen from the impact of new genomic technologies on the classification of rare diseases and conditions .
first , the 2013 final rule provides that where a drug 's mechanism of action suggests that the drug would not have significant activity outside of a subset of patients with a particular type of tumor or biomarker , this may establish an orphan subset .
second , the final rule indicates that where previous clinical experience with the drug indicates that the drug does not demonstrate significant activity in a particular subset of patients , this may inform whether an acceptable orphan subset exists .
pharmacogenomic research can assist in the identification of patient subsets that are more likely to respond to drug therapy .
third , the final rule lists the drug 's toxicity profile as a relevant factor in defining orphan subsets .
for example , patients with a particular non - rare disease or condition who are refractory to , or intolerant of , other less toxic drugs could be a subset for the purposes of a more toxic drug , whereas other patients with the same disease or condition would not be appropriate candidates for that drug .
pharmacogenomic research is often used to identify those patients who may be at a higher risk of adverse drug reactions due to their genetic profile .
the fda may grant multiple orphan designations for a particular disease or orphan subset indeed , this has become common practice .
however , it is worth noting that the fda 's acceptance of an orphan subset for one drug does not mean that the same subset will be accepted for subsequent applicants .
the fda states that the prevalence estimate may be narrowed owing to one or more properties of the drug that allow for the existence of an orphan subset [ emphasis added]. that is , although a particular orphan subset may be designated for a given drug product , this same orphan subset may be rejected for another drug because the appropriateness of the subset is evaluated separately based on the specifics of each drug . similarly , the factors that are not sufficient to define an orphan subset are informative in the pharmacogenomic context .
the 2013 final rule also notes that clinical trial eligibility , a sponsor 's plans to study the drug only for a
select indication , the particular grade or stage of a disease , or a low likelihood of use in a broader population are not sufficient in themselves to establish an orphan subset . with pharmacogenomics , biomarkers can be used to prospectively select patient subpopulations a strategy known as enrichment of the study population that are more likely to respond to a given drug therapy so that the treatment effect is more likely to be detected . in such cases , later - stage studies
may only be conducted in patient groups with a particular biomarker status . according to the fda , restricting clinical trial eligibility to biomarker - positive patients , or only choosing to study the drug in a particular patient subset , is not sufficient to establish an orphan subset .
again , the sponsor is required to show not just that the drug is more effective or less likely to cause adverse effects within the subset population , but also that the difference between the subset and the larger group is sufficient to prevent the drug from being a viable treatment option for patients in the larger group .
as such , this approach does show some potential for limiting what could be seen as abuse , particularly since the sponsor bears the burden of showing that drug is inappropriate for use outside of the orphan subset .
loughnot suggested in 2005 that the increased precision that pharmacogenomics brings to pharmacology might eventually help provide the fda with the ability to define
medically plausible. ultimately , as the science advances , pharmacogenomics will likely increase not only the number of potential rare diseases and orphan subsets , but also the precision with which these diseases and subsets may be delineated . as noted by haffner and colleagues , advances in genomics and proteomics
have led to increasingly precise disease definitions. as advances in pharmacogenomics increase the ability of researchers to measure non - response , this may raise the bar for sponsors who are trying to establish an orphan subset .
maher and haffner note that a clearer understanding of drug non - response is often revealed when response is stratified. consequently , as the lines between response and non - response are more clearly defined , orphan subsets will hopefully become less prone to manipulation .
salami slicing would primarily arise in circumstances where the prevalence of the broader disease category is over 200,000 cases in the us , but the prevalence of the medically plausible subtype is less than 200,000 cases .
there are a few cases where a pharmacogenomic - based biomarker was likely a determinative factor in bringing an orphan subset below the prevalence threshold .
for example , although non - small cell lung cancer accounts for 85% of the approximately 400,000 cases of lung cancer in the us thus placing the disease well above the orphan designation threshold in 2010 , xalkori ( crizotinib ) received an orphan designation for the treatment of alk - positive , met - positive , or ros - positive non - small cell lung cancer. in this case , the biomarker status of the non - small cell lung cancer ( ie alk - positive , met - positive , or ros - positive ) appears to have been the factor that brought the indication under the prevalence threshold to qualify for orphan drug status . as another example , in 2011 , there were some 960,000 americans living with melanoma , but in 2010 , zelboraf ( vemurafenib ) received an orphan designation for the treatment of patients with iib to stage iv melanoma positive for the braf ( v600 ) mutation. if , as the 2013 amendments suggest , the stage of disease alone is usually not sufficient to define an appropriate orphan subset ( see below ) , then the biomarker selection for the braf(v600 ) mutation appears to be important to bringing the target population below the 200,000 patient prevalence threshold . in clarifying their longstanding approach to eligibility for orphan subsets ,
the guidance provided by the fda in the 2013 amendments may help to decipher the reasons behind their decisions to deny orphan drug designations to some pharmacogenomic drugs in the past .
for example , before receiving approval for the breast cancer ( specifically , her2-positive metastatic breast cancer ) drug herceptin ( trastuzumab ) in september 1998 , its manufacturer , genentech , applied for orphan drug status with the fda , but the designation was denied . at the time
, there was an estimated 165,000 metastatic breast cancer patient in the us , of whom approximately 30% , or 49,500 people , had her2 overexpressing tumors
well below the 200,000 cut - off for orphan drug designation . however , the fda denied herceptin orphan drug status . while the exact reason for the denial was unclear , the oopd indicated that the most common reasons for refusal is disagreement between drug sponsors and regulatory authorities over how the target population is defined . in 2002 , shah suggested that herceptin was most likely not approved for orphan designation because the size of the population of her2 overexpressers was underestimated , as it is overexpressed in cancers other than that of the breast. in particular , it was clear from the success of clinical trials that herceptin could potentially also be used in the treatment of a range of other possible cancers including bladder , pancreatic , ovarian , colorectal , and prostate .
however , it seems unlikely that the possibility of treating multiple types of cancer with herceptin was in fact the reason for the denial since in the 2013 final rule , the fda explicitly states that [ a ] drug that shows promise in multiple , different rare diseases or conditions may be eligible for multiple designations , one for each
disease or condition , because fda considers the prevalence within each disease or condition. rather , it is more likely that the reason for the denial was that the stage of the disease ( metastatic , or stage iv breast cancer ) was not an acceptable subset to limit the target population since fda currently considers stage i breast cancer to be the same disease or condition as stage iv breast cancer when evaluating orphan drug designation requests for products that treat breast cancer. since breast cancer was estimated to affect nearly three million women in the us in 2011 , the sponsor presumably failed to demonstrate that the drug would not be effective in a broader subset of patients ( ie in her2-positive breast cancer in other stages ) . in most cases ,
stratification based on genetic biomarkers does not appear to be necessary to bring the target population below the 200,000 patient threshold ; pharmacogenomic data is often used to stratify diseases that are already rare enough to be eligible for orphan designation .
indeed , greenbaum notes that the vast majority of pharmacogenomics drugs fall within the literal definition of an orphan drug. that is , most orphan designations that have been granted for pharmacogenomic drug products are for diseases where the broader disease category already falls below the 200,000 person threshold , such as chronic myelogenous leukemia , pancreatic cancer , or acute lymphoblastic leukemia .
moreover , biomarker status is only one of many different factors that can be used to subdivide disease categories .
most rare diseases and orphan subsets granted orphan designation are already subdivided based on a wide range of factors such as chronic or acute state , age of the target population ( eg adult vs. pediatric ) , or the underlying cause of disease , just to name a few .
only once a larger body of examples is available to examine will it be possible to fully assess to what extent the oda remains open to abuse through salami slicing. the clarification provided in the 2013 final rule does seem to reduce the potential for abuse , and experience to date seem to suggest that instances in which subsets are artificially created to bring products below the orphan drug prevalence threshold will be fairly rare ; in most cases , subdivision based on biomarkers may be entirely legitimate .
while this is an issue that should continue to be monitored , other jurisdictions , like canada , can learn from the fda 's recent efforts to define the orphan subset concept in a way that minimizes the potential for abuse .
concerns about the potential misappropriation of orphan designation through salami slicing may be tempered by the fact that obtaining an orphan designation is only the first step toward market approval . while many of the benefits under the oda accrue
as soon as an orphan designation is received ( namely assistance in clinical trials , the 50% tax credit for clinical trial costs , and access to federal grants ) , these benefits will ultimately be of little value if they do not lead to market authorization for the indication ( or a subset thereof ) for which the orphan designation was granted .
the seven - year market exclusivity , also known as orphan exclusive approval , is arguably the most important incentive for drug developers seeking orphan designation .
the bar for obtaining orphan designation is significantly lower than that for obtaining market approval and only a small fraction of orphan designated drugs ever reach the us market . as noted by maher and haffner ,
[ e]valuation of [ a request to consider a subset of a prevalent disease for orphan designation ] frequently rests on both incomplete knowledge of disease etiology and uncertain therapeutic mechanism of action , and is both difficult and complex. for example , as of 2012 , there had been 2661 successful orphan product designations granted by the fda , which had led to 408 approved orphan products ( representing about 15% of orphan drug designations ) . as with other orphan drugs , only a portion of pharmacogenomic drugs that receive an orphan designation are approved for the us market , though the proportion of designated pharmacogenomic - based drugs ultimately approved appears to be somewhat higher than for other classes of drugs .
once a drug that has been granted orphan designation is approved for a rare disease or condition , the market exclusivity provisions in the oda prevent the fda from approving such drug for such disease or condition for a period of seven years from the date of approval , unless the holder of the first approval consents or can not supply sufficient quantities of the drug .
the regulations specify that once a designated drug receives exclusive approval , no approval will be given to a subsequent sponsor of the same drug for the same use or indication for 7 years unless one of the exceptions applies .
same drug is defined by regulation to mean a drug that contains the same active moiety or principal molecular features as a previously approved drug and is intended for the same use .
same drug if it can be shown to be clinically superior to the
first drug. the regulations define a
clinically superior drug as one that is shown to provide a significant therapeutic advantage over and above that provided by an approved drug , through greater safety , efficacy , or other
major contribution to patient care. a sponsor can obtain an orphan designation for a previously approved drug for the same rare disease or condition if it can present a plausible hypothesis that its drug may be clinically superior to the first drug , and then can receive its own exclusive orphan drug approval if it can demonstrate this clinical superiority .
the plausible hypothesis of clinical superiority standard for orphan designation is easier to establish than the
is achieved through liberally granting designation based on a plausible hypothesis of clinical superiority , allowing drugs to benefit from development incentives that flow from designation. maher and haffner note that unlike the fda market approval review , the orphan designation review takes place at an earlier stage of product development , sometimes even prior to any clinical studies having been performed. it is also worth noting that demonstration of clinical superiority at the approval stage need not be on the same basis as the hypothesis presented at the designation stage . the current approach to requiring proof of clinical superiority at the approval stage
was called into question by the september 2014 decision of the us district court for the district of columbia in the case of depomed inc .
the case arose from a 2012 complaint launched by the pharmaceutical firm depomed challenging the fda 's decision to deny orphan drug exclusivity for the drug gralise .
other drugs with the same active ingredient ( gabapentin ) had previously been approved and marketed for the same indication ( post - herpetic neuralgia ) .
having provided a plausible hypothesis of clinical superiority over these earlier products , gralise was granted orphan drug designation .
however , fda refused to grant exclusive approval because the sponsor had not proved the clinical superiority of its product .
none of the previously approved products had received orphan drug designation , however , so depomed argued that the clinical superiority requirement should not apply .
looking at the relevant statutory provisions , the district court found that a plain - language reading of the oda mandates the fda to recognize exclusivity for any drug that the fda has designated [ as an orphan drug ] and granted marketing approval. it was
therefore not open to the fda to impose the additional requirement of proving clinical superiority as a condition of granting exclusivity . as a result ,
the fda was ordered by the district court to grant orphan drug exclusivity for gralise without requiring proof of clinical superiority or imposing any additional conditions on depomed. it is important to note that the district court 's decision expressly acknowledges that fda can still impose conditions for orphan drug designation because it has been granted the authority in the oda to make regulations on this issue .
therefore , the fda can still use clinical superiority to determine whether a drug for which orphan drug designation is sought is the same drug as one previously designated and approved . in the court 's view , this should allay fda 's concerns that removing the clinical superiority requirement for exclusive approvals could lead to sponsors evergreening or obtaining
serial exclusivity for their products ( an issue discussed in more detail in the next section ) , contrary to the policy goals of the oda .
a sponsor can only obtain orphan drug exclusivity for a product that has been designated as an orphan drug , and the fda can deny this designation to the sponsor of a drug that is the same as ( ie not clinically superior to ) a previously approved drug . following this decision , in december 2014 , the fda issued a clarification of policy in which the agency stated that the district court decision was limited to the specific case of gralise and that as such , the agency will continue to apply its existing regulations which require the sponsor of a designated drug that is the
same as a previously approved drug to demonstrate that its drug is clinically superior to that drug upon approval in order for the subsequently approved drug to be eligible for orphan drug exclusivity. this seems to be a fairly aggressive position , considering that the district court 's decision questioned the fda 's authority to impose these conditions on exclusivity , and has led to considerable speculation about its implications .
the issue is unlikely to be definitively resolved unless and until these issues are relitigated in further court challenges or appeals .
while considerable uncertainty remains , the specific implications for the issues discussed in this article may be limited , given that fda 's authority to set conditions for orphan drug designation remains undisturbed , and the specific factual context of the depomed case where the same drug had been previously approved but not designated as an orphan drug are quite unusual .
the pharmaceutical sector is fiercely competitive and brand name drug companies are always seeking ways to squeeze more profits out of drug products , particularly those that are approaching patent expiry or that are no longer under patent protection . a common tactic is to seek new periods of patent protection or market exclusivity by discovering new uses or new target populations for existing drug products a strategy often referred to as
repurposing. the national institutes of health in the us have described repurposing as a key initiative to fight against stagnation in drug development. in particular , repurposing is an important strategy in the development of therapies for rare diseases : the sheer number of unmet medical needs to be found among orphan and rare disease suggests that drug repurposing among existing clinically used drugs may be a major solution to this societal medical need. in addition , drug developers may seek to reformulate or improve upon existing drug products in order to qualify for a new period of market exclusivity .
it is worth noting that once the patent and market exclusivity periods have expired on a pharmaceutical product , anyone may seek an orphan designation and orphan exclusive approval for that product .
a common means of extending the lifecycle of a patented drug product is through drug reformulation where a drug company modifies the characteristics of an existing drug product enough to qualify for a new patent or period of data exclusivity .
pharmaceutical companies are often accused of evergreening their products by making trivial and needless modifications to patented medicines in order to extend the term of patent protection or exclusivity .
the fda has acknowledged that one of the potential concerns with allowing new periods of orphan exclusivity for an already approved drug is that it could permit inappropriate evergreening of exclusive approval periods by allowing a sponsor to apply for a new designation ( and then exclusive approval ) near the end of a previous exclusivity period . as noted by the fda
evergreening would allow orphan exclusivity to be extended indefinitely for the same drug for the same use without any meaningful benefit to patients , a result at odds with the seven - year exclusivity period provided by the statute. however , reformulation is not considered to be inappropriate if the sponsor can demonstrate clinical superiority ; a sponsor that improves its own previously approved drug can be eligible for a new exclusivity period if clinical superiority is shown .
as the fda notes , the requirement of clinical superiority is intended to encourage the
development of potentially safer and more effective orphan drugs rather than encouraging minor modifications to already approved drugs that confer no meaningful benefit to patients. indeed , if the intent is to create incentives to improve treatment options , then arguably it should n't matter who develops the clinically superior alternative , whether the original sponsor or a competitor . in this way , the oda may incentivize research into improved versions or new applications of existing drug products for a rare disease or condition .
it is important to note that the scope of the market exclusivity is determined by the approved indications , not by the orphan designation .
the fda generally grants orphan - drug designation for use of a drug in all patients with a rare disease or condition and expects sponsors to seek approval on this basis , but sometimes the approval will be narrower if the data submitted only supports use in a subset of patients or indications . in the 2013 amendments ,
the fda set out to clarify the scope of market exclusivity by replacing the term subset [ of uses] with
select indication(s ) or use(s). the regulations now provide that if orphan exclusive approval is limited to only particular indication(s ) or uses(s ) within the rare disease or condition for which the drug was designated , fda may later approve the drug for additional indication(s ) or uses(s ) within the rare disease or condition not protected by the exclusive approval. that is , the sponsor may obtain multiple periods of seven - year market exclusivity for each approved indication or use that falls within the orphan designation .
each new period of market exclusivity will begin to run from the date of approval for the new ( ie not previously approved ) indication or use thus staggering the market exclusivity based on the approval date of each new orphan indication .
loughnot expresses concern that pharmacogenomics could contribute to evergreening tactics by help[ing ] identify patients who are susceptible to adverse drug reactions , allowing a sponsor to create significantly better clinical trial results without altering a drug at all by including only patients who are less likely to have adverse reactions .
enriching clinical trial populations with patients who are most likely to benefit from the drug under study has become common practice in the development of pharmacogenomic products , as well as in other areas of drug development . as noted above ,
the 2013 amendments make clear that for the purposes of orphan designation , clinical trial eligibility and the decision to only study the drug in a particular patient population are insufficient to establish an acceptable orphan subset .
subsequently , at the approval stage , if the issue is the sponsor attempting to evergreen an existing orphan exclusive approval on its own product , simply retargeting a drug at a narrower patient population within an already approved orphan indication would likely not be sufficient to obtain a new period of market exclusivity .
an already approved drug could be targeted toward an unapproved indication within the orphan designation , since the oda clearly permits multiple approvals within a designation . however
it is common for an orphan designated drug to be ultimately approved for a narrower indication than was set out in the designation .
for example , the pharmacogenomic - based drug xalkori ( crizotinib ) initially received orphan designation for the treatment of alk - positive , met - positive , or ros - positive ( three different types of biomarkers ) non - small cell lung cancer but has so far only received fda approval for treatment of alk - positive non - small cell lung cancer .
a broader orphan designation widens the potential scope of orphan exclusive approvals that may be obtained under a single designation ; if the scope of the orphan designation is too narrow , additional designation will likely need to be obtained . as noted above in the context of orphan designation , pharmacogenomics
may both multiply the number of potential rare diseases and orphan subsets that may be designated and increase the precision with which these diseases and subsets may be defined ;
[ w]ith therapies becoming increasingly guided by a more complete understanding of both genomics and proteomics , the number of potential orphan diseases should be expected to increase. similarly , at the approval stage , pharmacogenomics may narrow the scope of approved indications within a designated rare disease or subset since the approval may specify increasingly precise conditions of use or target populations .
accordingly , pharmacogenomics may increase the trend toward having multiple orphan approvals within a single orphan designation since pharmacogenomics increases the stratification of disease and the specificity with which disease subtypes and/or subpopulations may be defined .
nonetheless , as discussed in the next section , the potential for off - label prescribing may erode the distinction between approved and unapproved indications within an orphan designation . through subgroup analyses , pharmacogenomics can assist in drug repurposing efforts by identifying new targets for treatment or pinpointing patient subpopulations in which an existing drug may be more effective . as greenbaum notes , [ n]ew technological tools are now being used to determine
if there are additional targets of current drugs on the market , or so called off - targets. such repurposing is an example of
retrospective pharmacogenomic drug development , where sponsors can use data generated in previous clinical trials to identify potential new indications .
in addition , pharmacogenomic research may be able to rescue drugs that may have failed all comer clinical trials by defining a more appropriate patient population and conducting enriched clinical trials ; traditional randomized clinical trials may mask treatment efficacy by including participants for whom the drug has poor efficacy .
further , pharmacogenomics enables genetic profiling of subpopulations at an increased risk of adverse drug events .
the fda , for example , has acknowledged that if new science enables us to determine that the adverse events are restricted to a small , identifiable segment of the population , public health could be improved by making the drug available to others who could benefit without undue risk. consequently , pharmacogenomics may even allow drugs that have been withdrawn from the market due to rare but serious adverse events to be reintroduced for a specific subpopulation under more restricted terms of authorization .
a previously approved drug may receive an orphan designation for an unapproved use , regardless of whether the previous approval was for a rare or non - rare disease or condition . even where a drug was previously approved for a common indication
, it can receive orphan designation for a different indication that qualifies as an orphan disease , and manufacturers can be granted market exclusivity for an off - patent drug for orphan indications . the support and incentives provided under the oda may result in previously discarded treatments being revived or in new orphan applications for already successful drug products .
haffner and colleagues note that orphan exclusive approval under the oda is important for the development of older drugs namely , drugs that are no longer covered by patent protection .
sponsors may be able to maximize the sales potential for existing drugs by obtaining new orphan designations .
the fda has explicitly stated that [ a ] drug that shows promise in multiple , different rare diseases or conditions may be eligible for multiple designations , one for each disease or condition , because fda considers the prevalence within each disease or condition. gleevec ( imatinib ) , for example , has received seven separate orphan designations and orphan exclusive approvals . according to a report by thomson reuters ,
about 15% of orphan drugs analyzed in one study had subsequent launches for additional rare diseases .
the fda openly encourages drug developers to pursue orphan indications for drugs that have already been approved for more common conditions .
the oopd has created a database of products that have received both orphan status designation for a rare disease and a market authorization for the treatment of more common diseases .
this database offers sponsors a useful tool for finding special opportunities to develop niche therapies that are already well - advanced through development and thus represent a far easier lift to drug developers than beginning with an untested new therapy compound. even drugs that have achieved blockbuster sales in broad patient markets may be eligible for orphan designation where that same drug can also be used to treat an orphan condition . indeed , the fda has granted orphan designation to over 100 drugs with existing approvals for more common diseases , including to some highly successful blockbuster drugs such as prozac ( fluoxetine ) , viagra ( sildenafil citrate ) , and neurontin ( gabapentin ) . however , of these only gabapentin has received market authorization for an orphan indication .
it is problematic when incentives are used in situations where they are not intended or needed which could be the case where orphan drug policies intended to encourage sponsors to develop products that would not otherwise be financially viable
are used simply to enhance the profitability of products that would already viable without any incentives . in these situations , concerns of abuse or exploitation may be raised .
the oda does not consider the previous profitability of the drug in determining whether a new orphan application should receive an orphan designation , and
subsequently , orphan exclusive approval .
rather , the oda is aimed at encouraging the development of orphan treatments that would otherwise not be developed .
even if a drug had already achieved blockbuster sales for another indication , this does not necessarily translate into making it financially viable to pursue new orphan indications for the drug product since this additional research and development can entail significant cost .
thus , the incentive to investigate and test the drug 's potential for a particular indication could be necessary and useful , even if the drug is already profitable .
therefore , on balance , it is legitimate to allow orphan designations for drugs previously approved for other indications , including common diseases .
it is quite fair to say that [ f]rom the perspective of the patient with a rare disease , whether a drug is also effective in treating a more prevalent disorder is irrelevant. from this perspective , anything that could encourage a sponsor to identify and test the drug as a potential therapy for the patient 's condition might be beneficial . finally ,
as noted above , the potential for off - label prescribing may erode the value of the orphan drug exclusivity : although manufacturers are prohibited from marketing drugs for off - label uses , physicians are free to prescribe drugs off - label , potentially allowing generic drugs to be prescribed for indications that are protected by orphan exclusivity .
typically , generic drugs must have the same labeling as the innovative drug product to which they are compared in their application for market approval .
however , where the innovator has patent or exclusivity protection for a particular use or condition , if the generic copies these protected elements in the innovator 's labeling , they risk an infringement action
. however , the generic may seek permission from the fda to carve out the protected language , which would theoretically limit the generic 's market to conditions or uses that are not covered by patent or exclusivity rights . while a detailed discussion of the carve - out policy is beyond the scope of this article
, it is worth noting that these provisions could potentially undermine an innovator 's incentive to pursue repurposed orphan applications for existing
drug products .
as noted by mahn , innovative drug companies are concerned that the carve out rule , coupled with the practice of prescribing and substituting generic drugs off label , threatens their ability to recover the large investments needed to discover new uses or to improve the safety or efficacy profiles for old drugs.
in the past , suggestions that canada consider a us - style orphan drug framework had been rejected by health canada on the basis that sufficient flexibility and incentives already existed in the canadian legislation . in a 1997 policy statement ,
health canada cited several mechanisms which could be applied to orphan drugs , including tax incentives , fee reductions for drugs with small market potential , and access to unapproved drugs through the special access program .
health canada concluded that these mechanisms were sufficient because approximately 60% of us - approved orphan drugs were available in canada .
the number of persons with rare diseases in canada may not be sufficient to support substantial clinical trial research and development in the area of orphan drugs. currently , canadians may access orphan drugs through the health canada 's special access program , by participating in clinical trials , or where the drug has been approved through the regular drug approval process .
[ t]here has not been significant pressure from industry or special interest groups in canada to develop an orphan drug policy. recently , however , there have been renewed calls for an orphan drug policy in canada . the canadian organization for rare disorders , for example ,
has argued that without an orphan drug policy , manufacturers have no motivation to seek market approval for orphan drugs in canada and consequently , canadians with rare disorders run the risk of being among the last in the developed countries to gain access to new medicines , if at all. biotecanada , the national association representing the biotechnology industry , also strongly supports the development of a canadian orphan drug framework , stating that the initiative will help canada to compete in attracting investment to nurture [ orphan drug ] products into the marketplace , and see new canadian solutions for unmet medical needs developed in canada. orphan drug policies have also been framed as a means to promote personalized medicine : a decade ago , in a report to the canadian government , the external advisory committee on smart regulation noted that
[ w]ith the recent developments in pharmacogenomics , and the resulting ability to target treatments for sub - groups
of the population , it may be timely to consider implementing a legislative framework to facilitate access to these drugs. according to the canadian organization for rare disorders , health canada 's proposed orphan drug framework would be the first step towards canada taking a leadership position in personalized medicine. despite its earlier position that there was no need for an orphan drug policy in canada , in october 2012 , the federal government announced its plans to develop an orphan drug framework for the designation , authorization and monitoring of orphan drugs . soon after in december 2012 , health canada released an initial draft discussion document for a canadian orphan drug framework , which proposed a regulatory framework for orphan drugs , including provisions relating to orphan drug designation , scientific and clinical protocol advice , special market authorization for orphan drugs , and post - market assessment and management .
the criteria for orphan designation outlined in the proposed canadian framework mirror those of the european legislation , with the exception that they do not consider the economic viability of the drug .
in particular , the canadian framework adopts the same prevalence criteria as the european union legislation in the definition of an
orphan drug : a drug that is intended for the diagnosis , treatment , mitigation or prevention of a life - threatening , seriously debilitating , or serious and chronic disease or condition affecting not more than five in 10 thousand persons in canada. as is the case in other jurisdictions , health canada 's proposed legislation contains a series of incentives including priority review for marketing authorization , fee reductions for small to medium enterprises , and scientific and clinical protocol advice .
currently , the canadian orphan drug framework is still in the draft discussion phase , and health canada has provided only a high - level description of the elements of the proposed framework . as such
, it is too early to assess how pharmacogenomics products will be handled under the canadian orphan drug policy .
as the proposed framework is developed and implemented , canada can learn from the us experience with the oda and the debate surrounding how rare diseases are categorized and understood in order to maximize the benefits and minimize the potential for abuse of an orphan drug regime .
in particular , health canada will have to consider how orphan subsets will be defined for the purposes of orphan designation and the terms and conditions for the approval and market exclusivity for orphan drugs . under the draft orphan drug framework , to qualify for orphan designation
, the drug must either not currently be authorized for the canadian market , or if already approved , must provide a potentially substantial benefit for the patient distinguishable from the existing therapy. the proposed framework also permits a sponsor to submit an application for orphan designation on the basis of a designation from a recognized country if the proposed orphan drug and indication in canada is the same to that under which the foreign designation was issued . as in the us ,
a single drug may be eligible for multiple orphan designations for different rare diseases . while the draft canadian framework contains many of the same types of incentives that are currently offered under the us system ,
there are some important incentives that are not included in the draft framework or that are offered under more restricted terms .
perhaps most significantly , the oda offers a tax credit for 50% of the cost of clinical trials , whereas the draft canadian orphan drug framework does not currently propose any type of specific tax credit for orphan designated drugs .
in addition , under the us system , drugs that have been granted an orphan designation are exempt from the application fee that sponsors must normally pay to the fda when making a regulatory submission ; these application fees can be over two million dollars per product depending on the type of application .
in contrast , although few details are provided at this stage , the canadian draft framework proposes only a fee reduction rather than a fee exemption , and further this reduction is aimed at small and medium enterprises , whereas the us exemption is available to any applicant . as a result , the financial incentives available upon orphan designation under the proposed canadian orphan drug framework are arguably less enticing than those offered under the oda and consequently , may be less likely to encourage the creation of artificial subsets . finally ,
as noted above , concerns about the potential misappropriation of orphan designation through salami slicing should be tempered by the fact that designation is only the first step toward market approval , and only a fraction of orphan drugs will ultimately be approved . under the canadian draft framework
, the government will be required to maintain and make available to the public a list of orphan drug designations. this is in line with the orphan drug product designation database maintained by the fda and the register of designated orphan medicinal products maintained by the european medicines agency ( ema ) .
however , it is worth noting that while the fda does not make public any information about the drug products that have been denied orphan designation , in europe , the ema also maintains a register of drug products that have been refused orphan drug designation , which includes links to various documents giving the basis for the decision . making public information on which sponsors have applied for and been refused orphan designation could potentially add a level of public scrutiny to the activities of pharmaceutical companies that apply for orphan drug designation , hopefully discouraging companies from making questionable applications for designation
this type of transparency measure is important for ensuring the accountability and responsiveness of the orphan drug regime , and health canada should aim to make public information about both successful and unsuccessful applications for orphan designation .
as the orphan drug framework develops , health canada must establish criteria for defining acceptable orphan subsets , particularly given the controversy that had arisen over this subject south of the border .
although designation will ultimately be assessed on a case - by - case basis , the long running uncertainty in the us around the definition of a
medically plausible subset , and the subsequent need for clarifications under the 2013 amendments demonstrate the need for regulatory authorities to provide more detailed guidelines around what factors may and may not inform the existence of an acceptable orphan subset particularly in light of advances in new genomic technologies and evolution in our understanding of disease categories .
the us approach of requiring the sponsor to demonstrate why patients outside of the orphan subset are not good candidates for the drug appears to be a good approach particularly as it puts the onus on the sponsor to justify their framing of the target population .
further , given that the current canadian draft framework proposes to allow a sponsor to submit an application on the basis of designation of a recognized country , the policies adopted by the fda in respect of orphan subsets may very well lead to a corresponding orphan designation in canada if the us orphan designation is used as the basis for the application . at the approval stage
, the canadian orphan drug framework does not propose any new category of exclusivity for orphan drug products .
these provide eight years of exclusivity for an innovative drug , defined as a drug that contains a medicinal ingredient not previously approved in a drug and that is not a variation of a previously approved medicinal ingredient. the exclusivity operates by preventing a competitor from obtaining market approval for another drug based on a direct or indirect comparison with the innovative drug .
an additional six months of exclusivity is provided for innovative drugs that have been the subject of studies relating to use of the drug in pediatric populations .
similar provisions for new chemical entity and pediatric exclusivity exist in the us but are distinct from orphan drug exclusivity . by
proposing linkage to the innovative drug and pediatric exclusivity periods , health canada 's draft orphan drug framework appears to indicate that up to an eight - and - a - half year exclusivity period would be available for orphan drugs .
this period is longer than the seven - year period of market exclusivity granted for orphan drugs in the us , though shorter than the ten years provided by the european orphan drug legislation .
however , there is an important distinction between how the existing data exclusivity offered by health canada operates in comparison with the orphan exclusivity offered under the us orphan drug regime : in the us , drugs that have been previously approved are still eligible for the seven - year orphan exclusivity for each new approved orphan indication , whereas under the proposed canadian system , a previously approved drug would not be eligible for a new period of exclusivity if the sponsor subsequently sought approval for an orphan indication .
that is , the sponsor would not be granted a fresh period of eight - year exclusivity for a new orphan indication if the drug has already been approved for another indication because the exclusivity is only available for innovative drugs . at most ,
perhaps , an extension ( similar to the six - month pediatric extension ) might lengthen a previously granted period of exclusivity . unless there are plans to alter the existing exclusivity to allow an orphan drug to receive multiple periods of exclusivity , the draft canadian orphan drug framework is unlikely to provide much incentive for pharmaceutical companies to pursue research into new orphan applications for existing drugs that have already been approved , or that are no longer under patent protection . while only granting a single period of data exclusivity for each drug product has the advantage of preventing abuse of the orphan drug incentives through evergreening strategies , the disadvantage is that this approach will do little to encourage the repurposing of existing drug products for orphan indications , or the development of clinically superior reformulations of existing treatments for orphan diseases .
health canada will need to do more than simply apply the existing eight- or eight - and - a - half - year exclusivity to the orphan drug context if it wants to provide these incentives .
rather , as demonstrated by the controversy around the assessment of clinical superiority under the us orphan drug policy , health canada will have to clearly articulate the circumstances under which orphan drug products will be eligible for market exclusivity . as the canadian framework takes shape , the orphan exclusivity provisions should encourage the development of alternative treatments that offer genuine and significant benefits for patients by setting strict standards for what constitutes a clinically superior product that entitles a sponsor to a new exclusive approval for what would otherwise be the
same drug. the difficulty is that it is currently unclear how the canadian exclusive approval would work , and therefore , how the issue would play out under the new framework .
greater uncertainties may exist for orphan drugs given the complexities of the diseases , the small and vulnerable populations and the treatment environment itself , and that consequently ,
greater abilities to plan for and resolve those uncertainties are needed once the drug is on the market. accordingly , the draft canadian orphan drug framework includes expanded transparency measures , including the publication of the key information on when an application for market authorization for an orphan drug has denied .
health canada notes that [ t]he publication of negative decisions and the basis for these decisions is important for orphan drugs in circumstances when an existing marketed re - purposed drug could continue to be used off - label. unfortunately , the fda does not currently make publicly accessible , in their online orphan drug database or otherwise , any specific information about when an application for market authorization for an orphan - designated drug has been unsuccessful .
given the rapid evolution of the field of pharmacogenomics , its impact on orphan drug policies will need to be continually reassessed to ensure that policies remain responsive to the current drug development paradigm .
the recent revision of the oda regulations in 2013 is encouraging because the fda has at least demonstrated that it is alive to the concerns raised by the increasing detail with which potential orphan subsets can be defined .
although questions remain about whether the amendments will be sufficient to prevent abuse of the provisions , it appears likely that some of the concerns have been mitigated .
the us experience can provide lessons for other jurisdictions , including canada as it develops its new orphan drug policy . a central dilemma in addressing the issue of salami slicing in orphan designation
artificial subdivision of disease categories an assessment that must be made with reference to the purposes of the orphan legislation .
however , it is important to note that this does not necessarily imply that developing treatments for artificial groups that do n't qualify as orphan subsets is not valuable . as noted by herder , [ i]n the abstract , it is not obvious that orphan disease policy should prioritize diseases that have long been understood as rare over diseases like breast cancer that may soon be subdivided into multiple rare diseases since either may be in need of new and improved treatment options .
rather , the more important question is whether the objectives of the oda are fulfilled by incentivizing development of such treatments . in pharmaceutical development
, there is a general desire to encourage the development of more and better treatment options for patients with rare diseases . while this is a worthy goal , we should bear in mind that the more specific objective of orphan drug legislation is to target incentives toward the development of drugs that would not otherwise be developed due to a lack of financial viability .
some argue that orphan drug legislation is overinclusive because it provides incentives where they are not really needed .
the marked increase in applications for orphan designations in recent years has led to suggestions that the oda risks becoming a victim of its own prodigious success , and tends to confirm suspicions that the orphan drug market is one that the pharmaceutical industry now views as potentially lucrative .
there are orphan drugs that are actually very profitable due to the very high prices that can be charged for rare disease therapies or to extensive off - label use for more common conditions both of which are in themselves sources of significant controversy .
the outstanding question is whether it is better to err on the side of offering orphan incentives more liberally , even if this leaves the policy open to abuses such as salami slicing , or to be more restrictive in granting incentives but risk of dissuading the development of some orphan drugs , such as those that target biomarker - based orphan subsets .
another area that should be of interest to canada as it develops its orphan drug framework is the potential to encourage the use of pharmacogenomics research to repurpose drugs for use in treating rare disorders . in the us ,
the possibility of a new period of orphan drug exclusivity provides an incentive for manufacturers to invest in repurposing , and there is even talk of adding a new type of extension to exclusivity or patent terms to further stimulate this activity . as discussed above , the exclusivity proposed in the canadian draft framework for orphan drugs would need to be modified to provide an effective incentive for repurposing approved drugs for new orphan indications .
finally , the impact of pharmacogenomics on orphan drug development makes it all the more important to ensure that orphan drug policies be implemented with a high level of transparency and coordination . in introducing the draft orphan drug framework ,
the canadian government stated that a key focus of this new approach will be on international information - sharing and collaboration for the development and regulation of orphan drugs. specifically , the proposed canadian orphan drug regulations would allow health canada to operationally align and participate in well - established activities of the us and the european union including designation , scientific / protocol advice and pre- and post - market information sharing. transparency is also addressed through plans to publish the basis for decisions on marketing authorization .
as the science of drug development becomes more and more complex , measures to minimize abuse of incentives and to enhance the safety of off - label use by sharing information among regulatory agencies , manufacturers , and the public are essential .
this work was funded by the government of canada through genome canada and the ontario genomics institute ( ogi-064 : enhanced care for rare genetic diseases in canada ) , and through a second genome canada grant ( ethical and legal issues of cancer initiating stem cell research ) .
| advances in pharmacogenomic research and increasing industry interest in personalized medicine have important implications for the way that orphan drug policies are interpreted and applied .
concerns have been raised about the potential impact of pharmacogenomics and new genomic technologies on our understanding of how disease categories are delineated , and subsequently , how the concept of rare disease should be defined for the purposes of orphan drug policies .
this article considers whether orphan drug legislation can be drafted in a way that will maximize benefits and minimize concerns relating to the impact of pharmacogenomics on orphan drug research and development . after reviewing the issues that may arise at the intersection of orphan drug policies and pharmacogenomics ,
this article will discuss the potential impact of pharmacogenomics at two critical points : orphan designation and approval of the drug product . at each of these points ,
the relevant aspects of current us orphan drug legislation are examined , focusing on the extent to which recent amendments may address concerns that have been raised previously .
this analysis will then provide the foundation for a critical review and recommendations regarding the proposed new canadian orphan drug framework . |
nod / ltj female mice were purchased from charles river ( calco , italy ) .
animal care procedures were performed according to protocols approved by the hospital san raffaele institutional animal care and use committee ( iacuc no .
blood glucose was measured in the morning three times a week using a glucometer ascensia breeze 2 glucose meter ( bayer , leverkusen , germany ) .
a diagnosis of diabetes was made after a glucose measurement of 300 mg / dl to ensure no spontaneous disease reversal .
a relapse of disease in treated animals was considered following two consecutive glucose measurements of 200 mg / dl , levels at which spontaneous diabetes reversal never occurred in any of the tested mice ( data not shown ) . after diabetes onset , female nod mice ( aged 22 7 weeks ) were treated with varying doses of the non fc - binding anti - cd3 f(ab)2 clone 145 - 2c11 ( bio express , west lebanon , nh ) or mab isotype control ( golden syrian hamster igg ; ebioscience , san diego , ca ) , according to the glycemia levels , the same day they were found to have diabetes .
rapamycin ( rapamune ; wyeth europe , taplow , u.k . ) was diluted in water and administered by gavage once a day at 1 mg / kg , a dose that we and others previously demonstrated not to be toxic to pancreatic islets ( 14,16 ) .
recombinant human il-10 ( bd biosciences , mountain view , ca ) was diluted in pbs and administered twice a day at a dose of 0.05 mg / kg i.p .
glucose tolerance was monitored via tail - vein sampling at time 0 ( just before glucose solution injection ) and 5 , 10 , 15 , 20 , 25 , 30 , 45 , 60 , 75 , 90 , and 120 min after glucose solution injection .
all statistical analyses were performed using a two - tailed student 's t test . a p value of < 0.05 was deemed significant .
nod / ltj female mice were purchased from charles river ( calco , italy ) .
animal care procedures were performed according to protocols approved by the hospital san raffaele institutional animal care and use committee ( iacuc no .
blood glucose was measured in the morning three times a week using a glucometer ascensia breeze 2 glucose meter ( bayer , leverkusen , germany ) .
a diagnosis of diabetes was made after a glucose measurement of 300 mg / dl to ensure no spontaneous disease reversal .
a relapse of disease in treated animals was considered following two consecutive glucose measurements of 200 mg / dl , levels at which spontaneous diabetes reversal never occurred in any of the tested mice ( data not shown ) .
after diabetes onset , female nod mice ( aged 22 7 weeks ) were treated with varying doses of the non fc - binding anti - cd3 f(ab)2 clone 145 - 2c11 ( bio express , west lebanon , nh ) or mab isotype control ( golden syrian hamster igg ; ebioscience , san diego , ca ) , according to the glycemia levels , the same day they were found to have diabetes .
rapamycin ( rapamune ; wyeth europe , taplow , u.k . ) was diluted in water and administered by gavage once a day at 1 mg / kg , a dose that we and others previously demonstrated not to be toxic to pancreatic islets ( 14,16 ) .
recombinant human il-10 ( bd biosciences , mountain view , ca ) was diluted in pbs and administered twice a day at a dose of 0.05 mg / kg i.p .
glucose tolerance was monitored via tail - vein sampling at time 0 ( just before glucose solution injection ) and 5 , 10 , 15 , 20 , 25 , 30 , 45 , 60 , 75 , 90 , and 120 min after glucose solution injection .
all statistical analyses were performed using a two - tailed student 's t test . a p value of < 0.05 was deemed significant .
to define a suboptimal dose of anti - cd3 amenable to combinational therapy studies ( i.e. , having a second agent that improves the action of the first ) and to identify the influence of starting glycemia on the ability to reverse disease , we first grouped nod mice based on degree of hyperglycemia and treated with various dosages of anti - cd3 ( fig .
using this strategy , several suboptimal anti - cd3 dosages leading to a wide range of diabetes reversal rates ( i.e. , from 35 to 75% ) were identified .
the efficacy of the anti - cd3 treatment was strictly dependent on the dosage , as previously shown ( 17,18 ) , but was also influenced by glucose levels at time of treatment .
figure 1b shows the glucose levels of each of the animals treated with the best effective anti - cd3 dosage ( i.e. , 50 g 3 doses in mice with 300349 mg / dl glycemia levels ) , demonstrating a rapid and uniform diabetes reversal in six of eight animals treated ( fig
female diabetic nod mice were grouped based on glycemia levels ( 300349 [ gray bar ] and 350400 mg / dl [ white bar ] ) and treated with various anti - cd3 dosages .
percentages of diabetes reversal and number of mice in each group are shown ( a ) .
glucose levels of the eight nod mice with 300349 mg / dl glycemia and treated with three 50-g doses of anti - cd3 ( 75% diabetes reversal ) are shown ( b ) .
female diabetic nod mice with 300400 mg / dl glycemia were treated with rapamycin alone ( 1 mg / kg daily during the entire time of observation [ ] ) or anti - cd3 alone ( 50 g 3 doses at days 0 , 1 , and 2 [ ] ) .
although rapamycin has a strong immunomodulatory capacity in pre - diabetic nod mice ( 14,16 ) , rapamycin monotherapy did not lead to disease reversal in any of the diabetic mice ( fig .
1c ) . to test whether rapamycin cooperates with anti - cd3 to reverse diabetes and reinforce the development of long - term tolerance , diabetic nod mice were treated with rapamycin and the anti - cd3 dosage that showed the weaker ability to reverse disease ( i.e. , 35% diabetes reversal ) .
the concomitant administration of rapamycin and anti - cd3 did not lead to enhanced diabetes reversal in any of the animals tested ( fig .
2a ) ; rather , the inclusion of rapamycin blocked the ability of anti - cd3 to impart its beneficial effect .
the deleterious influence of rapamycin on the anti - cd3 reversal capacity was even observed when rapamycin was coadministered with dosages of anti - cd3 able to cure 65% of diabetic nod mice ( fig .
of 10 diabetic nod mice , 9 remained diabetic when treated with rapamycin and anti - cd3 at the highest effective dose ( i.e. , 70% diabetes reversal ) ( fig . 2c ) .
female diabetic nod mice were treated with anti - cd3 alone or in conjunction with rapamycin ( 1 mg / kg ) or il-10 ( 0.05 mg / kg ) during the entire time of observation .
mice with 350400 mg / dl glycemia were treated with 25 g anti - cd3 ( one dose ) alone ( ) or in conjunction with rapamycin ( ) ( a ) .
mice with 300349 mg / dl glycemia were treated with 18 g anti - cd3 ( one dose ) alone ( ) or in conjunction with rapamycin ( ) ( b ) .
mice with 300400 mg / dl glycemia were treated with 50 g anti - cd3 ( three doses ) alone ( ) or in conjunction with rapamycin ( ) ( c ) .
mice with 300400 mg / dl glycemia were treated with 50 g anti - cd3 ( three doses ) alone ( ) or in conjunction with il-10 ( ) ( d ) .
percentages of diabetes reversal and number of mice are shown . to evaluate whether other pro - tolerogenic compounds acted similarly to rapamycin when combined with anti - cd3 , we tested il-10 , an immunomodulatory cytokine with known tolerogenic potential in vivo ( 14,19 ) .
when coadministered with anti - cd3 in diabetic nod mice , the addition of il-10 did not interfere with the anti - cd3 therapeutic activity ( fig .
these data demonstrate that the tolerogenic capacity of anti - cd3 during the first induction phase is completely halted by rapamycin , but not by il-10 therapy , in diabetic nod mice . to define whether the detrimental effect of rapamycin on anti - cd3 therapy was related to changes in the t - cell compartment , we tested the percentages of circulating cd4 and cd8 t - cells and the frequency of cd4cd25foxp3 t - cells in the pancreatic lymph nodes of nod mice treated with anti - cd3 alone or in combination with rapamycin . circulating cd4 t - cells
were depleted upon anti - cd3 treatment irrespective of the presence of rapamycin , whereas cd8 t - cells were only partially affected ( fig .
similarly , rapamycin did not alter the frequency of cd4cd25foxp3 t - cells in the pancreatic lymph nodes 2 weeks after anti - cd3 treatment ( fig .
contrary to what was previously demonstrated ( 6 ) , anti - cd3treated nod mice did not show a selective increase of cd4cd25 t - cells in the pancreatic lymph nodes compared with control nod mice .
this might be due to the reduced anti - cd3 dosage used in our study ( i.e. , 50 g 3 doses ) compared with that used by belghith et al .
( i.e. , 50 g 5 doses ) ( 6 ) , which might lead to different kinetics in the expansion of cd4cd25 t - cells .
t - cell frequency and phenotype in nod mice treated with anti - cd3 or anti - cd3 and rapamycin .
diabetic nod mice were treated with anti - cd3 alone ( 50 g 3 doses , n = 6 , [ ] ) or in combination with rapamycin ( 1 mg / kg per day , n = 5 , [ ] ) .
peripheral blood was collected at different time points after treatment , and circulating cd4 ( left ) and cd8 ( right ) t - cells were analyzed by a fluorescence - activated cell sorter .
( ) , mean sd of peripheral cd4 and cd8 t - cells in nondiabetic nod mice within cd45 cells ( n = 10 ) ( a ) .
pancreatic lymph nodes from normoglycemic untreated ( n = 4 ) , diabetic untreated ( n = 5 ) , anti - cd3treated ( 50 g 3 doses ) ( n = 2 ) , and anti - cd3 ( 50 g 3 doses ) plus rapamycin treated ( n = 5 ) nod mice were analyzed by a fluorescence - activated cell sorter 3 weeks after treatment .
one representative plot for each group , after cd4 t - cell gating , is shown .
the big gate includes all cd25 t - cells , whereas the small gate includes only cd25foxp3 t - cells .
percentages of means sd of cd25 t - cells ( left panel ) and cd25foxp3 t - cells ( right panel ) within cd4 t - cells in the pancreatic lymph nodes are shown ( c ) .
the anti - cd3 maintenance phase in nod mice is a stable condition of tolerance that is no longer dependent on the presence of the antibody .
given our previous results , we tested whether rapamycin negatively affects this stable condition of reversed type 1 diabetes .
five weeks after anti - cd3mediated diabetes reversal , normoglycemic nod mice were treated with rapamycin .
quite remarkably , all previously cured mice returned to a state of hyperglycemia within 7 weeks of rapamycin administration , whereas rapamycin - untreated animals showed no signs of diabetes recurrence ( fig .
4a ) . to further delay the interval between anti - cd3 intervention and rapamycin administration ,
normoglycemic nod mice were treated with rapamycin 30 weeks after anti - cd3mediated diabetes reversal .
consistent with the aforementioned observations , two of three anti - cd3cured nod mice returned to a diabetic state within 10 weeks of rapamycin administration , whereas all rapamycin - untreated animals remained normoglycemic ( fig .
this phenomenon was reversible and strictly dependent on the presence of rapamycin , as all mice treated for 230 days with rapamycin promptly returned to a normoglycemic state upon drug removal .
delayed rapamycin administration in anti - cd3treated nod mice . according to their glycemia levels ,
thirty - five days after anti - cd3 mab mediated diabetes reversal , four mice were treated with rapamycin ( ) and 24 were left untreated ( ) .
percentages of diabetes reversal and number of mice are shown ( a ) . according to their glycemia levels ,
one hundred and eighty days after anti - cd3mediated diabetes reversal , three mice were treated with rapamycin ( ) and 21 were left untreated ( ) .
intraperitoneal glucose tolerance test was performed in three normoglycemic untreated nod mice , three diabetic nod mice , six normoglycemic anti - cd3cured mice , and three mice previously cured with anti - cd3 but again diabetic upon rapamycin treatment .
intraperitoneal glucose tolerance test was performed in the latter group during rapamycin treatment ( i.e. , 320 days after anti - cd3 mab treatment ) .
the average glucose levels per each time point and the best - fitting curve for each group are shown ( c ) .
the area under the curve ( auc ) from time 0 to 120 min after glucose injection for all the animals included in panel b is shown ( d ) . to evaluate the metabolic parameters underlying this finding ,
a glucose tolerance test was performed in nod mice previously treated with anti - cd3 and under rapamycin treatment .
although two of three rapamycin - treated mice were hyperglycemic at the time of analysis ( as shown in fig .
4b ) , all three demonstrated a glucose response similar to that observed in diabetic untreated nod mice , while control anti - cd3cured nod mice showed a glucose response superimposable to that of normoglycemic untreated animals ( fig .
overall , rapamycin reverts the stable tolerance condition established in anti - cd3cured nod mice while it is administered .
with prior data demonstrating that rapamycin is a protolerogenic compound both in vitro and in vivo ( 12,13,15 ) as well as information from the first clinical trials with anti - cd3 in recent - onset type 1 diabetic patients suggesting that this form of therapy can be improved ( 8,9 ) , we tested the specific hypothesis that rapamycin would augment the therapeutic effectiveness of anti - cd3mediated type 1 diabetes reversal in nod mice . against all expectations , we observed that rapamycin not only blocks the ability of anti - cd3 treatment to cure overt hyperglycemia in nod mice but also breaks its curative effect while it is administered .
ctla-4 neutralizing antibodies ( 6 ) block the reversal capacity of anti - cd3 in the induction phase while cyclophosphamide breaks anti - cd3mediated tolerance during the maintenance phase ( 20 )
. the mechanisms by which these compounds counteract the anti - cd3 effect have been elucidated .
cyclosporine a blocks t - cell activation and t - cell depletion mediated by activation - induced cell death , and anti
ctla-4 monoclonal antibodies impede the generation and/or function of inducible tgf-dependent tregs , whereas cyclophosphamide depletes tregs .
in addition , rapamycin allows t - cell activation and activation - induced cell death ( 11 ) , permits generation of inducible tregs ( 2123 ) , and does not selectively deplete tregs ( 12,24,25 ) .
in fact , t - cell frequency and phenotype in anti - cd3rapamycin treated mice were identical to those of anti - cd3treated nod mice .
it is therefore unlikely that rapamycin alters the anti - cd3 activity through the same inhibitory mechanisms demonstrated for the abovementioned compounds .
it has been recently proposed that the reduced il-2 production by t effector cells in nod mice is the root cause of the progressive loss of treg
t effector cell balance in the islets , leading to -cell destruction ( 26 ) .
rapamycin , by inhibiting signal transduction delivered by il-2 , might directly interfere with this pathway , nowadays considered so crucial for maintaining immunological tolerance in nod mice .
. alternative mechanisms that can explain the unique effect of rapamycin in anti - cd3treated nod mice may be related to -islet physiology .
it has recently been demonstrated that rapamycin induces fulminant diabetes in the psammomys obesus mouse model of nutrition - dependent type 2 diabetes by increasing insulin resistance and reducing -cell function and mass through increased apoptosis ( 27 ) .
the fundamental function of mammalian target of rapamycin signaling in -cells , which is blocked by rapamycin , has been confirmed by others ( 28,29 ) .
rapamycin might therefore have a negative effect directly on the islets rather than blocking the activity of anti - cd3 in nod mice .
however , this hypothesis is in contrast to previous observations by our group ( 14 ) and others ( 16 ) in pre - diabetic nod mice wherein rapamycin monotherapy significantly protected animals from disease development . in addition , diabetic nod mice treated with rapamycin did not develop a more aggressive disease , in terms of glycemia , than untreated mice ( a.v . , unpublished data ) .
an alternative hypothesis is that rapamycin interferes with -cell proliferation , as demonstrated in specific experimental settings such as pregnancy ( 30 ) and transgenic mice ( 31 ) .
however , at this time , there are no data indicating that anti - cd3 leads to -cell proliferation .
indeed , currently available data suggest the opposite : recovery of metabolic control following anti - cd3 therapy may be due to mending of -cells that had been already present but not functional in the pancreas at the moment of hyperglycemia rather than -cell proliferation ( 18,32 ) .
future experiments will investigate the pancreata of nod mice treated with rapamycin , with or without anti - cd3 , in order to further understand the mechanisms underlying its deleterious action .
rapamycin monotherapy in long - lasting type 1 diabetic patients does not aggravate the autoimmune disease but , rather , improves the suppressive function of ntregs ( 15 ) .
one should therefore expect that rapamycin behaves similarly in the case of new - onset type 1 diabetes .
however , the disease in early - onset type 1 diabetic subjects appears both metabolically and immunologically different from that observed in long - lasting patients , and rapamycin might have a different outcome in diverse patient populations .
a clinical trial combining rapamycin and il-2 recently began with new - onset type 1 diabetic patients ( http://www.clinicaltrials.gov/ct2/show/nct00525889?term=rapamune+and+il-2&rank;=1 ) .
our data suggest caution in designing rapamycin - based combinational treatments because the addition of rapamycin to anti - cd3 therapy in early - onset diabetic nod mice completely abolished the anti - cd3 therapeutic effect . | objectivenon fc - binding anti - cd3specific antibodies represent a promising therapy for preserving c - peptide production in subjects with recent - onset type 1 diabetes
. however , the mechanisms by which anti - cd3 exerts its beneficial effect are still poorly understood , and it is questionable whether this therapeutic approach will prove durable with regard to its ability to impart metabolic preservation without additional actions designed to maintain immunological tolerance .
we used the nod mouse model to test whether rapamycin , a compound well - known for its immunomodulatory activity in mice and humans , could increase the therapeutic effectiveness of anti - cd3 treatment in type 1 diabetes.research design and methodsrapamycin was administered to diabetic nod mice simultaneously with anti - cd3 or to nod mice cured by anti - cd3 therapy .
the ability of this combined therapy to revert type 1 diabetes and maintain a state of long - term tolerance was monitored and compared with that of anti - cd3 therapy alone.resultsrapamycin inhibited the ability of anti - cd3 to revert disease without affecting the frequency / phenotype of t - cells .
rapamycin also reinstated diabetes in mice whose disease was previously reversed by anti - cd3 .
withdrawal of rapamycin in these latter animals promptly restored a normoglycemic state.conclusionsour findings indicate that , when combined with anti - cd3 , rapamycin exerts a detrimental effect on the disease outcome in nod mice for as long as it is administered .
these results suggest strong caution with regard to combining these treatments in type 1 diabetic patients . |
zinc is an essential element in the
nutrition of animals , human beings , and plants [ 1 , 2 ] .
zinc plays an important role in replications , gene expressions , and the metabolism of
nucleic acids and different proteins
there are many methods for
zinc determination such as spectrophotometry
[ 35 ] , atomic absorption spectrometry [ 6 , 7 ] , flame atomic absorption spectroscopy , graphite furnace atomic absorption
spectrometry [ 9 , 10 ] ,
inductively coupled plasma atomic emission spectroscopy ,
and inductively coupled plasma mass
spectrometry .
but some are expensive ,
some are time - consuming , some are troublesome , and others are toxic .
preconcentration
technique [ 1316 ] can improve the detection limit as well as the selectivity of
the method .
flow injection analysis ( fia ) [ 1719 ] can improve analytical
procedures such as high analytical throughput , precision , accuracy , low
reagents , and sample consumption .
therefore , a new method was proposed for online preconcentration and
spectrophotometric determination of low - concentration zn ( ii ) in water .
the
proposed method is based on the fact that zn reacts with pan to produce zn ( ii)-pan complex
formation in the ph 9.3 buffer solution . to avoid the extraction procedure
, it
has been found that the pan reacts
with zinc to form a water - soluble complex at the presence of tween-80 .
a model zj - la automatic metallic element
analyzer developed and produced successfully by professor xinshen zhang of our own laboratory was used .
the analyzer has the functions of flow injection analysis ( fia ) , ion
chromatography , and automatic sample injection .
the function of automatic
reference fia was applied to determine zn ( ii ) .
two analytical pumps ( shanghai
huxi analytical instrument plant , shanghai ,
china ) of fia
were used to deliver solutions .
one was used to deliver elution solution and
reagent solution while the other delivered sample solution .
polytetrafluoroethylene ( ptfe ) tubing of 0.5 mm in internal diameter was used as the channels
for all solutions to circulate .
ultraviolet and visible spectra were
obtained using a spectrumlab s54 ( lengguang technology co. , ltd , shanghai ,
china ) .
data
acquisition and processing were performed
with hw-2000 chromatography software ( qianpu software co. , ltd , shanghai , china ) running under windows xp .
all reagents used including 1-(2-pyridineazo)-2-naphthol
( pan ) ( tianjin ruijinte chemical reagent plant , tianjin , china ) , tween-80 , sodium tetraborate ,
o - phenanthroline , ddtc , and sodium hexametaphosphate ( chengdu fang zhou chemical reagent plant , chengdu , china ) , oxalic acid dihydrate , lithium
hydroxide monohydrate , ammonium thiocyanate , and citric acid ( beijing donghua chemical reagent plant , beijing , china ) were of analytical grade and all
solutions were prepared with deionized water .
zinc
stock solution : zinc stock standard solutions at a concentration of
1000 mg l were obtained from china environmental monitoring terminus , beijing , china .
working solutions were prepared by suitable
dilution of the stock solution . mixed
chromogenic reagent solution ( r ) : 10 ml of 4 10 mol l pan
ethanol solution , 20 ml of 8% tween-80 , 20 ml of ph 9.3 of sodium tetraborate
suffer solution , 1.5 ml of 10% sodium hexametaphosphate , 1 ml
of 0.1% ddtc , and 2 ml of o - phenanthroline
were mixed and the mixture solution was
diluted to 100 ml .
elution
solution : a total of 1.26 g of oxalic acid dihydrate , 1.576 g of citric acid monohydrate , and 1.15 g of lithium hydroxide
monohydrate were dissolved in 1000 ml
of water .
the preconcentration column developed by our laboratory was filled with
macroporous spherical resin with 4-(2-pyridylazo ) resorcinol ( par ) functional
group .
the par functional group can form chelate complex with determined metal
ion in the preconcentration column and the chelate complex can be eluted by the
eluent solution of oxalic acid - citric acid - lithium hydroxide .
the filling
granularity of resin was 80100 m . the
preconcentration column length was 40 mm and its internal diameter was 5 mm . by filling the preconcentration column , mix resin and deionized water into
pasty matter and drop slowly into column with burette .
after sedimentating for
a moment , resin was dropped slowly until the preconcentration column became
full .
then , cover strainer , cover on the column , and wash a few minutes with
deionized water for further work .
the sample
solution was injected into preconcentration column to preconcentrate with a
six - way injection valve . when the instrument was put into analyzing position , the concentrated zn
( ii ) was eluted by eluent solution , pushed to the three - way cock , and mixed
with chromogenic reagent solution in the
reaction coil . the zn ( ii)-pan complex was
then formed and carried into the flow cell .
the
absorbance intensity of zn ( ii)-pan complex was
determined at 560 nm by detector and transformed to a signal of the peak which
was recorded by hw-2000 software on a pc .
in the flow
injection analysis , the absorbance of complex is affected by ph of the reaction
medium .
the effect of ph on
the zn ( ii)-pan complex
was tested in the ph range 411 .
the test results showed that the maximum peak
height could be obtained when ph range was between 8 and 10 .
the effect of pan concentration on the peak height were tested from 5 10
mol l to 1.2 10
the results showed
that the peak height rose when concentration of pan changed from 5 10 mol l to 4 10 mol l , and the peak height declined
when concentration of pan changed from 4 10 mol l to 1.2 10 mol l. therefore , 4 10 mol l pan was selected for further
work . according to
the reaction kinetics , the reaction rate will inevitably increase when
increasing concentrations of the reagent .
however , the reaction rate will not
increase when concentrations of the reagent reaches a certain value .
therefore ,
when pan consumption reaches a certain value , the concentration of pan which
reacts with zinc should be certain because zinc concentration is fixed in the
sample .
thereby , the absorbency will not increase when increasing pan
concentration . on the contrary , the background color ( baseline noises ) will
increase because the free state pan in the system will increase .
thus , tween-80 was selected as reagent which could increase
solubility of the system .
the test results in figure 3 showed that the peak
height increased when concentration of tween-80 changed from 1.0% to 1.6% and
that the peak height did not increase even when concentration of tween-80
changed from 1.6% to 2.2% . therefore , 1.6% tween-80 was selected as the optimal
concentration for the further work . in the fia , the
flow rate could affect the sensitivity of the proposed method .
in the study ,
the flow rate was adjusted by the interior diameter of pump pipe . therefore
,
the effect of the flow rate of reagent solution on the peak height was studied
from 0.10 ml min to 0.72 ml min .
the test results
showed that the best flow rate could be obtained when the flow rate of reagent
( r ) was
set at 0.23 ml min . in the
flow injection analysis , the reaction time is adjusted by the length of
reaction coil .
the
study results in figure 4 showed that 3 m of the
reaction coil length was the
optimum and further increase of reaction coil length did not increase peak
height .
length of preconcentration column and
filling granularity could affect the effect of preconcentration . in the study ,
filling granularity of 80 100 m was selected and length of concentration
column from 20 mm to 100 mm was tested .
the results showed that the shorter the
length of concentration column , the higher the sensitivity .
however , if the
length of concentration column was too short , the concentration of
preconcentration zn ( ii ) in samples was not adequate , which lowered the
sensitivity .
the highest sensitivity was obtained when the length of
concentration column was 40 mm .
the results in figure 5 showed
that the preconcentration time was longer , the zinc concentration of the
preconcentration column would be higher which causes the analysis sensitivity
to be higher but the sampling frequency to be
lower . taken a comprehensive consideration of analysis sensitivity and
sampling frequency , preconcentration time of 5 minutes was decided for further
work .
too high - preconcentration rate would
increase analysis sensitivity but augment pressure of preconcentration column
which would cause leakage or pump pipes rupture .
too low - preconcentration rate
would reduce analysis sensitivity and sampling frequency but augment the
preconcentration time .
taken a comprehensive consideration of analysis
sensitivity and sampling frequency , preconcentration rate of 1.5 min l
was chosen for further work . if eluent concentration was too low , zn ( ii ) in
preconcentration column would elute incompletely .
if eluent concentration was
too high , it would waste reagent . in the study ,
nitric acid and oxalic
acid - citric acid - lithium hydroxide were tested as eluents .
the experimental
results showed the sensitivity of the eluent of oxalic acid - citric acid - lithium hydroxide was higher than that of nitric acid .
thus , oxalic
acid - citric acid - lithium hydroxide was selected as eluent solution and its
concentrations were 0.01 mol l ,
the sensitivity was greatly affected by flow rate of
eluent . if flow rate was too high , the sensitivity augmented and baseline
noises increased .
if flow rate was too slow , zn ( ii ) in concentration column
would elute incompletely , which caused sensitivity decline .
the result showed that flow rate of
0.5 ml min of eluent was optimal for further work .
the interference of some
possibly coexisting foreign metallic ions was examined to make sure the proposed
method can be applied to determine zn ( ii ) in water .
the tolerance limit was
defined as the concentration of added solutions causing less than 5% relative
error during the determination of 10 g l of zn ( ii ) .
no
interference was observed from a great deal of k , na ,
nh , mg , ca , cr , cr ,
al , co , hg , as , and so
forth .
however , the metallic ions of ni , cd , mn ,
pb , cu , and fe interfered . the
interference was eliminated ( 100 g l of each ion of ni ,
cd , mn , pb , cu , and
fe ) by adding 1.5 ml of
10% sodium hexametaphosphate , 1 ml of 0.1% ddtc , and 2 ml of o - phenanthroline as appropriate
masking reagents into mixed
chromogenic reagent solution .
thus , the results showed that the proposed
method had good selectivity . to assess the reproducibility and accuracy of the proposed method , under
the selected conditions above
the linearity was tested by a series of working standard
solutions of zn ( ii ) ranging from 0.5 g l to 400 g l.
test showed that the peak height versus zn ( ii ) concentration was linear within
the range of 2.0 g l to 360 g l. the curve was h = 0.427c + 3.638 ( h : peak height ; c :
concentration of zn ( ii ) ( g l ) ) and the correlation coefficient
obtained was 0.9995 and the detection limit ( 3 multiples of baseline noises )
was 0.42 g l. the test of precision was conducted by eight
injections of 5.0 g l and 50 g l of zn ( ii )
standard solution , respectively .
the results
showed that the relative standard deviation ( rsd )
was calculated as 3.55% and 2.14% , respectively .
the proposed method was applied to determine zn ( ii ) in water samples
using the zj - la automatic metallic element analyzer .
the analysis results of
samples were shown in table 1 . to prove the
accuracy of the method , a test of recovery would be conducted by accurately
adding known concentration of zn ( ii ) into sample whose original zn ( ii )
had been determined .
the
test results in table 1 showed that the recovery was between 97.1% and 104.8%
and analytical results of the proposed method were satisfactory . therefore ,
the
proposed method was suitable for determination of trace amounts of zn ( ii ) in
water .
the proposed method was significant with regard to the development of a
simple , reliable , and sensitive flow injection method for online
preconcentration and determination of trace amounts of zinc ( ii ) .
high sensitivity ,
selectivity , broad determination range ( 2.0 g l to 360 g l )
of zn ( ii ) , low - detection limit ( 0.42 g l ) , and fully automated analysis were just some of the
advantages of the proposed method .
especially , compared with other existing
methods of zinc preconcentration , eluent concentration is even low to mmol l.
the detection limit of the proposed method can be further improved by
increasing preconcentration time . the model zj - la automatic metallic
elements analyzer employed in this study was cheap , small , and easy to move .
the proposed method has been successfully applied to determine zinc ( ii ) in
water .
the method is suitable for determination of zn ( ii ) in both water samples and other samples for its good
selectivity and the above mentioned advantages . | a simple , sensitive , reliable and flexible flow injection spectrophotometric method is proposed for on - line preconcentration and determination of trace amounts of zinc in water . at the presence of tween-80 in ph 9.3
buffer solutions , the shade of color of zn ( ii)-pan complex is in a linear relation to the zinc amount at the point of the maximum absorption peak of 560 nm .
the optimal experimental conditions , including reaction conditions and preconcentration conditions , had been obtained .
the linear range of the proposed method
was between 2.0 and 360 g l1 and the detection limit was 0.42 g l1 .
the relative standard deviation was 3.55% and 2.14% for
5.0 g l1
and 50 g l1
of zinc standard solution ( n = 8) .
the method had been successfully applied to zinc determination in water samples and the analytical results were satisfactory . |
noise is one of the physical factors in industries , and , today , more attention is being paid to its harmful effects . after smoking and air pollution , noise pollution
world health organization ( who ) also considers noise as the third dangerous pollutant of megacities .
moreover , damage to the hearing system , interference in conversation , effects on the organs of vision , effects on balance system , social disorders , psychological as well as nervous effects , impacts on electrolytes , physiological effects , and mental effects are among the noise health effects on human body .
almost two thousand years ago , pliney stated that the individuals living near noisy waterfalls tend to suffer from hearing loss sooner than other people .
the results of a study which was conducted in 2000 showed hearing loss as the main harmful effect of long exposure to occupational noise .
furthermore , neghab et al . performed a study on the workers of petrochemical complex and revealed that noise had led to an increase in blood pressure and hearing loss in the exposed workers .
in the same line , smith et al . investigated the body 's physiological responses when being exposed to high - noise levels and showed that exposure to repeated , continuous noise caused the incidence of physiological as well as psychological responses and resulted in changes in heart rate as well as blood pressure [ 6 , 7 ] .
moreover , according to the study by motamedzade and ghazaiee , exposure to higher than 85 db noise levels increased both systolic and diastolic blood pressure , affected the working efficiency , and led to interference in conversation .
ising and michalak compared the effects of noise in both field and laboratory conditions and showed that exposure to 97 db noise level had resulted in physiological as well as psychological changes in half of the study subjects .
overall , according to the report by who , noise causes 4 million dollars health damage every day [ 10 , 11 ] , and occupational noise can put the industrial workers ' health at a high risk .
based on what was mentioned above and since noise can have both physiological and psychological effects on humans , the present study was conducted in order to investigate the effects of noise exposure on blood pressure and heart rate in steel industry .
the present cross - sectional study aimed to investigate the effect of occupational noise exposure on heart rate and systolic as well as diastolic blood pressure . considering ci = 95% and power of 90% ,
the sample size of the study was determined as 50 subjects who were selected through simple random sampling .
the inclusion criteria of the study were being physically and psychologically healthy , not smoking , not using alcohol , not taking hypnotic drugs , and not working in shifts .
the participants ' demographic information was gathered through a questionnaire , and their systolic as well as diastolic blood pressure and heart rate were measured using beurer bc16 pulse meter .
in addition to assessing the workplace noise , a sample of the devices ' noise was also obtained . in doing so
, a microphone was attached to a worker 's collar near his ear and the device 's noise was recorded for 10 minutes . considering the features of steel industry , this study was conducted with exposure to 85 , 95 , and 105 db noise levels in controlled experimental conditions for 3 consecutive days . at first
, the subjects were placed in a quiet room with the background noise level of 40 db and their systolic as well as diastolic blood pressure and heart rate was measured .
then , they were exposed to the noise recorded from the workplace at 85 , 95 , and 105 db levels for 5 minutes and systolic as well as diastolic blood pressure and heart rate was measured again . in order to eliminate the sequence effect , the experiments were performed with 24 h intervals .
finally , the data were entered into the spss statistical software , and paired t - test was used in order to compare the means of the variables before and after noise exposure .
the demographic characteristics of the study subjects are presented in table 1 . in this study , age , weight , height , systolic and diastolic blood pressure , and
moreover , the results of workplace noise analysis showed that , among the 60 areas under study , 13 ( 23% ) had over 85 db noise level and 23 ( 39% ) had caution range noise level , that is , 6585 db .
table 2 presents the subjects ' systolic as well as diastolic blood pressure and heart rate before and after noise exposure in the controlled experimental conditions .
as the table depicts , the mean of systolic blood pressure has increased at all noise levels except for 85 db ; however , the changes were not statistically significant ( p > 0.05 ) .
on the other hand , diastolic blood pressure increased a little at all the three noise levels . besides
, a decrease was observed in the mean of heart rate at all the noise levels ; nevertheless , the changes were not statistically significant ( p > 0.05 ) .
up to now , the mechanism noise that affects blood pressure has not been identified .
nevertheless , several studies have shown high secretion of vasoconstrictors in urine as a result of being exposed to higher than 90 db noise levels , which might represent the biological effects of noise exposure on blood pressure . in the study by ising and michalak no significant difference was observed in the subjects ' heart rate after acute noise exposure . in general ,
acute exposure to 90100 db noise levels increases the catecholamines [ 5 , 13 ] which might have led to an increase in systolic as well as diastolic blood pressure .
however , the changes in blood pressure were not statistically significant , which is consistent with the findings of the present study . on the other hand , motamedzade and
ghazaiee showed that exposure to higher than 85 db noise levels resulted in an increase in both systolic and diastolic blood pressure , affected the working efficiency , and led to interference in conversation , which is in line with the results of the current study . in the study by neghab et al .
, a significant difference was found between the exposed and the reference groups ' mean blood pressure , which might be due to the industry under investigation as well as the study design .
moreover , the exposed and the reference groups were similar in the present study , while neghab et al . selected two separate groups ( control and exposed ) in order to investigate the effects of noise .
overall , since blood pressure and heart rate might be affected by various factors , the results obtained regarding the effects of noise exposure on these parameters must be interpreted with caution .
the findings of the present study were in agreement with those of the previous studies conducted on the issue . according to the results ,
no significant difference was observed in blood pressure and heart rate before and after acute exposure to 85 , 95 , and 105 db noise levels . | background and objectives .
this study aimed to investigate the effect of noise exposure on blood pressure and heart rate of steel industry workers . materials and methods . in the present cross - sectional study ,
50 workers were selected from a steel company in fars province , iran , and exposed to 85 , 95 , and 105 db noise levels for 5 minutes .
the participants ' blood pressure and heart rate were measured using beurer bc16 pulse meter both before and after the exposure .
results .
the study results showed no significant difference in blood pressure and heart rate before and after the exposure .
however , the workers ' systolic blood pressure had increased compared to before the exposure ; of course , the difference was not statistically significant ( p > 0.05 ) . besides , although the subjects ' heart rate had reduced in comparison to before the exposure , the difference was not statistically significant ( p > 0.05 ) . conclusion .
no significant change was observed in blood pressure and heart rate after acute exposure to 85 , 95 , and 105 db noise levels . |
for many years , the identity of components of microtubule - organizing centers ( mtocs ) that nucleate microtubule assembly and establish microtubule polarity was a central unanswered question in the field of mitosis and the cytoskeleton .
the discovery of -tubulin ( oakley and oakley , 1989 ) , the key finding that allowed this question to be answered , came from a genetic screen in the fungus aspergillus nidulans designed to identify genes important for microtubule function ( weil et al . ,
the existence of members of the tubulin superfamily beyond the microtubule proteins - and -tubulin was completely unexpected at the time , and the discovery of -tubulin is a powerful validation of the value of forward genetics , in which well - designed screens let the cell tell us what is important rather than us imposing our preconceived notions on the cell .
-tubulin is ubiquitous in eukaryotes , and genome - sequencing projects have revealed that there are one to three -tubulin genes in eukaryotic genomes ( findeisen et al . , 2014 ) .
it localizes to structurally diverse mtocs and , with few exceptions , is required for microtubule nucleation ( reviewed by job et al . , 2003 ) .
ring - shaped structures called turcs ( -tubulin ring complexes ) that contain -tubulin and associated proteins nucleate microtubule assembly in vitro ( zheng et al . , 1995 ) and in vivo ( figure 1 , a
d ) ( reviewed by teixido - travesa et al . , 2010 ; kollman et al . , 2011 ; lin et al . , 2014 ) .
proteins that associate with -tubulin have been identified and designated grips ( -tubulin ring proteins ) or gcps ( -tubulin complex proteins ) ( figure 1d ) .
five of these proteins , gcp26 , are structurally related and contain conserved regions called grip motifs ( gunawardane et al . , 2000 ;
the turc consists of gcps that bind to -tubulin and to each other , forming a complex in which a ring of -tubulin molecules effectively mimics the plus end of a microtubule .
it forms a pre - existing nucleus for microtubule assembly . at physiological tubulin concentrations , spontaneous assembly of tubulin into microtubules
instead , almost all microtubule growth occurs from preformed nucleating structures , principally the turc .
( c ) the finding that gcp26 all bind to -tubulin raises the possibility that gcps and -tubulin may assemble into alternative tusc - like structures ( kollman et al . , 2011 ) that assemble along with tuscs to form the turc .
( d ) designations for gcps in various organisms in which they have been studied extensively .
-tubulin complexes nucleate microtubules from the centrosome and other mtocs in both interphase and mitosis or from microtubule surfaces in mitosis . in interphase and mitosis
, -tubulin has been shown to regulate microtubule plus - end dynamics . in mitosis , it plays a role in the spindle assembly checkpoint ( sac ) and the control of mitotic exit . in interphase , -tubulin plays an important role in inhibiting apc / c , thereby promoting the transition from the g1 to the s phase .
it also appears to regulate e2f1-mediated gene expression and complexes with rad51 , suggesting a role in the rad51-mediated dna damage response . beyond gcp26 ,
turcs contain three additional , more recently discovered , core subunit proteins , mozart1 ( mitotic spindle - organizing protein associated with a ring of -tubulin , or gcp9 ) and mozart2a and mozart2b ( gcp8a and gcp8b ) , which are closely related to each other ( hutchins et al . , 2010 ) .
mozart1 is highly conserved in eukaryotes , except in saccharomyces cerevisiae and close relatives ( hutchins et al . , 2010 ;
lin et al . , 2014 ) , and it is essential for mitotic spindle assembly and function in humans and schizosaccharomyces pombe ( hutchins et al .
arabidopsis thaliana has two mozart1 homologues , gip1 and gip2 , that interact with gcp3 .
they are not essential individually , but gip1/2 double mutants are embryonic lethal ( nakamura et al . , 2012 ) .
mozart2a and mozart2b are apparently restricted to vertebrates , in which they play a role in recruitment of the turc to the centrosome ( teixido - travesa et al .
in addition to the core turc components , various phyla have proteins , unrelated in sequence to gcp29 , that are involved in targeting -tubulin complexes to mtocs ( reviewed extensively by lin et al . , 2014 ) .
high - resolution structures of human -tubulin ( aldaz et al . , 2005 ; rice et al . , 2008 ) and gcp4 ( guillet et al . , 2011 ) and lower - resolution structures of small -tubulin complexes ( tuscs , containing two molecules of -tubulin and one molecule each of gcp2 and 3 ) from s. cerevisiae ( kollman et al . , 2008 , 2010 ) have been determined , and these and other findings have led to a persuasive model for the structure of the turc ( kollman et al . , 2011 ) . in this model , gcp26 each binds directly to -tubulin and assemble into a structure that contains 13 -tubulin molecules ( perhaps containing five tuscs and one molecule each of gcp4 - 6 bound to -tubulin ; figure 1a ) .
gcp3 contains a hinge region , and in the current model , movement about this hinge would alter the positioning of -tubulin molecules . in one position
, the -tubulin molecules would be too far apart to nucleate microtubule assembly , but in the other position , the straight position , they would have exactly the same spacing as microtubule protofilaments and would form a perfect template for microtubule assembly .
control of the conformation of gcp3 could therefore regulate the ability of the turc to nucleate microtubule assembly .
important unanswered questions in this area are the exact arrangement of gcp26 in the turc and the positioning of mozart1 , 2a , and 2b in the complex . the hinge model ,
although persuasive , needs to be tested rigorously , and if it is correct , it raises the question of what controls the rotation of gcp3 around its hinge .
although microtubule nucleation from mtocs is extremely important , in many phyla mtocs are not apparent . even in vertebrate mitosis
, centrosomes are not always required for the formation of a functional spindle ( khodjakov et al . , 2000 ) , and when centrosomes are present , many microtubules may be nucleated independent of the centrosome .
( 2005 ) showed convincingly that cortical microtubules in higher plant cells are nucleated from the sides of existing microtubules at a characteristic angle of 42 with respect to existing microtubules , that -tubulin is at the branch points , and that lateral microtubule nucleation is -tubulin dependent .
( 2005 ) demonstrated that microtubules are nucleated from -tubulin complexes at the sides of cytoplasmic microtubules in s. pombe .
these data established the principle that -tubulin complexes function in nucleation of microtubules from the sides of existing microtubules .
data obtained over the intervening decade have identified the augmin complex as a key player in lateral microtubule nucleation .
augmin is an eight - subunit complex that is important for localizing -tubulin to mitotic spindles and for centrosome - independent microtubule generation in mitotic and meiotic spindles ( goshima et al . , 2008 ; ho et al . , 2011 ; petry et al . , 2011 ; hsia et al . ,
2014 ) . in higher plants , augmin colocalizes with gcps , and they are corecruited to cortical microtubules .
knockdown of augmin with a microrna reduces branching lateral nucleation of microtubules ( liu et al . ,
2014 ) . in xenopus extracts , augmin and turcs along with the microtubule assembly factor tpx2 and the gtp - bound rangtpase nucleate microtubule assembly , forming fan - like structures consistent with the possibility that the microtubules are in branched arrays ( petry et al . ,
microtubule nucleation persists if augmin or tpx2 are depleted , but fan - like arrays do not form .
monitoring the plus - end microtubule - tracking protein eb-1 revealed that new microtubules are nucleated from the sides of existing microtubules and that the newly nucleated microtubules have the same polarity as the microtubules from which they are nucleated .
such lateral nucleation of microtubule assembly in a mitotic apparatus would result in the proliferation of spindle microtubules but , importantly , would not disrupt the polarity of microtubules in the spindle .
these and related findings support a persuasive although not completely proven model that the augmin complex binds to microtubule walls and recruits the turc through nedd1 or other proteins to initiate new microtubule nucleation ( uehara et al . , 2009 ; zhu et al . , 2009 ; johmura et al . ,
the fact that augmin apparently plays a role in lateral microtubule nucleation in both plants and animal suggests that augmin - mediated lateral microtubule nucleation is widespread in eukaryotes . however , many organisms do not have homologues of all augmin subunits ( lin et al . , 2014 ) , and the fact that the polarity of microtubules nucleated from the sides of microtubules in s. pombe is opposite to that of augmin - mediated nucleation ( janson et al . , 2005 ) suggests there are at least two lateral nucleation mechanisms .
the theme that has emerged from two decades of research is that -tubulin complexes are almost universally involved in microtubule nucleation .
mechanisms have evolved , however , to control and target these complexes , such that microtubule nucleation occurs when and where it is needed .
the traditional view has been that each protein has a single function , but increasingly , proteins have been shown to be multifunctional . in this regard , increasing evidence indicates that -tubulin has functions beyond microtubule nucleation ( cuschieri et al . , 2007 ) .
in many ways , this makes sense , because -tubulin is at the hub of the microtubule cytoskeleton and , along with other mtoc components , is in a unique position to receive and send signal molecules transported by microtubule motors .
the problem with interpreting some of these data , however , is that , because -tubulin has a critical role in mitosis and disruption of mitosis has many consequences , it is difficult to distinguish putative non microtubule - nucleation functions of -tubulin from consequences of mitotic failure or alteration of microtubule nucleation . nevertheless , we believe that there is ample evidence that -tubulin has important functions beyond microtubule nucleation ( summarized in figure 1e ) .
there have been repeated reports from multiple phyla that mutations or deficiencies in -tubulin or gcps alter plus - end microtubule dynamics ( paluh et al . , 2000 ;
, 2001 ; zimmerman and chang , 2005 ; bouissou et al . , 2009 ) and that -tubulin alters the distribution of the plus end
tracking protein bim1 in s. cerevisiae ( cuschieri et al . , 2006 ) and its homologue , eb1 , in drosophila ( bouissou et al . , 2014 ) .
considering that there is constant bidirectional transport along microtubules , one idea is that -tubulin complexes at mtocs could bind catastrophe or rescue factors or the motor molecules that transport them .
this would facilitate loading of molecules that affect microtubule dynamics onto motor molecules , thereby affecting their transport to microtubule plus ends and , consequently , affecting microtubule dynamics at plus ends .
consistent with this idea , -tubulin mutations alter binding of motor proteins and/or microtubule stability factors ( vogel et al .
, 2001 ; zimmerman and chang , 2005 ; cuschieri et al . , 2006 ) .
for example , some -tubulin mutations alter the binding of kinesin-14 and kinesin-5 to -tubulin complexes , while others alter the interaction of cytoplasmic dynein with spindle pole bodies ( spbs ; li et al .
, 2005 ; rodriguez et al . , 2008 ; olmsted et al . , 2014 ) , although the binding of type 5 and 14 kinesins may be involved in regulating microtubule nucleation rather than plus - end microtubule dynamics ( olmsted et al . , 2014 ) .
the second proposed explanation comes from work in drosophila , in which turc proteins localize along astral microtubules and are proposed to act as rescue factors that arrest microtubule disassembly ( bouissou et al . ,
it is worth noting that the proposed rescue activity of turcs distal to mtocs might not be intrinsic to the turc but a function of its binding to rescue factors , as discussed above .
another possibility that is unfortunately difficult to test is that -tubulin mutations alter the microtubule lattice and thereby alter microtubule dynamics .
there have also been persuasive reports of perturbation of the regulation of mitotic exit by alterations of -tubulin or gcps .
alanine - scanning mutants of human -tubulin expressed as the sole -tubulin in s. pombe allowed cells to go through anaphase and cytokinesis when spindle formation was disrupted ( hendrickson et al . , 2001 ) .
mutations in the gcp4 and gcp5 homologues of s. pombe caused a failure of mitotic arrest in the presence of the antimicrotubule agent thiabendazole ( vardy and toda , 2000 ) .
similarly , a -tubulin mutation in a. nidulans caused mitotic exit before successful completion of mitosis in a strain in which the establishment of spindle bipolarity was delayed by a type 14 kinesin deletion ( prigozhina et al .
another allele caused a premature mitotic exit even when the microtubule cytoskeleton was completely disassembled with benomyl ( prigozhina et al .
likewise , rna interference ( rnai ) of dgrip84 ( gcp2 ) in drosophila caused an untimely mitotic exit in the presence of colchicine ( colombie et al .
thus genetic or rnai perturbations of -tubulin or gcps in three organisms and four laboratories all point to a microtubule nucleation - independent role for -tubulin complexes in the control of mitotic exit . a possibly related area
in which results from several labs have indicated a non microtubule nucleation function for -tubulin is coordination of mitotic events and/or the spindle assembly checkpoint ( sac ) . in a systematic alanine mutagenesis screen
, a number of -tubulin mutants were shown to cause premature anaphase entry and defective cytokinesis , suggesting that -tubulin regulates the sac and orderly mitotic exit ( hendrickson et al . , 2001 ) .
an a. nidulans -tubulin allele caused late mitotic events ( chromosomal disjunction , spindle elongation , and mitotic exit ) to become disordered ( prigozhina et al . , 2004 ) .
the failure of chromosomal disjunction in this instance was not due to lack of microtubule assembly or force generation , because chromosomes were often stretched dramatically in anaphase , even though they did not disjoin .
with respect to mitotic regulation and the sac , it is worth noting that there are reports that the sac / mitotic regulatory proteins cdc20 and bubr1 are in a complex with -tubulin in mammalian cells ( muller et al . , 2006 ) and that mad2 binds to alp4p ( gcp2 ) in s. pombe ( mayer et al . , 2006 ) .
finally , there is incontrovertible evidence that -tubulin plays a role in the regulation of cell cycle progression in interphase . in a. nidulans ,
a -tubulin allele , mipad159 , causes failure of inactivation of the anaphase - promoting complex / cyclosome ( apc / c ) at the g1/s boundary ( nayak et al . , 2010 ) .
this failure correlates with failure of the apc / c activator cdh1 to dissociate from the spb , and it can be overridden by deletion of cdh1 ( edgerton - morgan and oakley , 2012 ) .
importantly , this failure occurs if microtubules are present or if they are completely depolymerized , so it is not a microtubule nucleation phenomenon ( nayak et .
( a. nidulans has a single gene with functional domains of bub1 and bubr1 ) and mps1 , thereby abrogating the sac ( edgerton et al . , 2015 ) .
note also that analyses of an alp4 ( gcp2 ) mutation indicate alp4 has an essential function in g1 in s. pombe ( vardy and toda , 2000 ) .
this mutation , moreover , can cause septation , even when mitosis is arrested , by allowing inappropriate recruitment of the sid1 kinase to the spb ( vardy et al . , 2002 ) .
there are also reports that -tubulin modulates the activity of e2f transcription factors , which control the expression of a number of genes that are necessary for dna replication and centrosome duplication ( hoog et al .
the key challenge in understanding the various nonnucleation functions of -tubulin will be to move from phenomenology to molecular mechanism .
although the functional significance is not yet clear , there is strong and intriguing evidence of the association of -tubulin with tumor suppressor proteins , particularly those involved in dna repair .
it binds to rad51 in nuclei in response to dna damage ( lesca et al . , 2005 ) and coimmunoprecipitates with atr , brca1 , and c53 from nuclei ( zhang et al .
, 2007 ; hubert et al . , 2011 ; horejsi et al . ,
brca1/bard1 binds to and monoubiquitinates -tubulin , and there is evidence that this ubiquitination is important in the regulation of centrosome number ( hsu et al . , 2001 ;
starita et al . , 2004 ) , a particularly interesting finding , since centrosome amplification is important to cancer progression .
-tubulin expression or localization pattern is altered in a variety of cancers , including some multiple myelomas , non small cell lung cancers ( maounis et al . , 2012 ; dementyeva et al . , 2013
) , ductal hyperplasia and breast cancer ( niu et al . , 2009 ; cho et al . ,
2010 ) , gliomas and glioblastoma cell lines ( katsetos et al . , 2006 ) , and medulloblastomas and medulloblastoma cell lines ( caracciolo et al . , 2010 ) .
spontaneous mutations in one of the two human -tubulin genes , tubg1 , are associated with lissencephaly and microcephaly ( poirier et al .
2014 ) . further effort devoted to studies of both the microtubule nucleation - dependent and nucleation - independent functions of -tubulin in different cell and tissue contexts should shed light on the relevance of this cytoskeleton protein in health and disease . | tremendous progress has been made in understanding the functions of -tubulin and , in particular , its role in microtubule nucleation since the publication of its discovery in 1989 .
the structure of -tubulin has been determined , and the components of -tubulin complexes have been identified .
significant progress in understanding the structure of the -tubulin ring complex and its components has led to a persuasive model for how these complexes nucleate microtubule assembly . at the same time
, data have accumulated that -tubulin has important but less well understood functions that are not simply a consequence of its function in microtubule nucleation .
these include roles in the regulation of plus - end microtubule dynamics , gene regulation , and mitotic and cell cycle regulation .
finally , evidence is emerging that -tubulin mutations or alterations of -tubulin expression play an important role in certain types of cancer and in other diseases . |
although uncommon in pregnancy , liver disease may cause a significant risk for the mother and fetus .
liver diseases presenting during pregnancy are usually specific to pregnancy including preeclampsia , hellp syndrome ( haemolysis , elevated liver enzymes , low platelets ) , obstetric cholestasis , hyperemesis gravidarum , and acute fatty liver disease .
the diagnosis of liver disease during pregnancy poses a challenge because of overlapping features of these disorders . in a study of 4377 obstetric patients , ch'ng et al . reported liver dysfunction in 3% but with no liver - associated maternal mortality .
although most of these patients had a pregnancy - related liver disease , liver disease can occur coincidentally in pregnancy .
it is characterized by ongoing inflammation , destruction and fibrosis of intrahepatic and extrahepatic bile ducts . because of incapability of bile ducts to regenerate effectively or to regenerate at all , the disease often results in cirrhosis , portal hypertension and liver failure .
the disease normally starts from age 30 to 60 , though may begin in childhood .
symptoms of itching , fatigue , jaundice and weight loss usually indicate progression of the disease , however initially histologic progression can be clinically asymptomatic .
diagnosis is made by magnetic resonance cholangiopancreatography ( mrcp ) or endoscopic retrograde cholangiopancreatography and sometimes an additional liver biopsy is performed .
although not proven effective on the natural history of the disease , ursodeoxycholic acid ( udca ) seems to reduce symptoms and improve laboratory findings . in end - stage
we report a woman with primary sclerosing cholangitis diagnosed prior to pregnancy who developed an exacerbation of sclerosing cholangitis during pregnancy with successful outcome .
a 34-year - old woman , gravida 1 , para 0 , was diagnosed with primary sclerosing cholangitis 12 years before her pregnancy by endoscopic retrograde cholangiopancreatography .
characteristic abnormalities of both the intra and extrahepatic bile ducts , with a long extrahepatic stenosis were evident . this was treated with a 12 cm 10-french endoprothesis for 4 weeks . on repeated colonoscopy
mrcp 4-years before pregnancy showed stenosis of the mid - part of the common hepatic duct without prestenotic dilatation . based on these findings stenting of the extrahepatic stenosis was repeated for a period of 4 weeks .
she had no history of alcohol abuse or viral hepatitis , neither was there any evidence of associated autoimmune hepatitis .
she was treated with ursodeoxycholic acid ( 12 mg / kg / day ) . in the period from initial diagnoses
untill pregnancy , laboratory findings remain stable . during her pregnancy in the first trimester transaminases were slightly elevated , which normalized in the second trimester with maintenance of ursodeoxycholic acid use ( figure 1 ) .
she was admitted in the hospital at 21 weeks of gestational age because of an imminent premature delivery complicated by an urinary tract infection .
after successful treatment with antibiotics and tocolysis she was dismissed . at 26 weeks of gestational age symptoms of itching started and 2 weeks later she developed an exacerbation of her sclerosing cholangitis : abnormal liver function tests ( figure 1 ) and by ultrasound thickening of the common hepatic duct were noted , but no dilatation .
magnetic resonance cholangiopancreatography showed a stenosis of the hepatical duct with prestenotic dilation ( figure 2 ) .
u / l ) , bilirubin ( umol / l ) and bile acid ( umol / l ) throughout the pregnancy and postpartum ( pp ) .
u / l ) , bilirubin ( umol / l ) and bile acid ( umol / l ) throughout the pregnancy and postpartum ( pp ) .
the biliary ducts are alternatingly dilated and stenosed . the major stenosis is found at the level of the junction of the right and left hepatic duct .
this mrcp image in a 35-year old pregnant woman is very suspect for primary sclerosing cholangitis .
the biliary ducts are alternatingly dilated and stenosed . the major stenosis is found at the level of the junction of the right and left hepatic duct .
this mrcp image in a 35-year old pregnant woman is very suspect for primary sclerosing cholangitis .
she was admitted again at 32 3/7 weeks of gestation because of jaundice : progression of pruritus and dark urine .
liver function test including serum bile acids were elevated ( figure 1 ) and serology was negative for hepatitis a , b and c viruses and hiv .
other laboratory tests included hemoglobin 12.9 g / dl ( 1216 g / dl ) , platelet count 61010/l ( 15040010/l ) , uric acid 0.17 mmol / l ( 0.120.34 mmol / l ) , glucose 6.3 mmol / l , no proteinuria . in comparison with previous ultrasound no changes were noted . in conclusion : the preexisting liver disease combined with laboratory results and clinical presentation could account for a diagnosis of exacerbation of primary sclerosing cholangitis with obstetric cholestasis .
she was treated with increased dosage of ursodeoxycholic acid ( 35 mg / kg / day ) .
frequent controls of the activated partial thromboplastin time were normal ; therefore vitamin k was not added . at 36 weeks
she spontaneously delivered a healthy boy of 2660 gram ( p60 ) with meconium - stained amniotic fluid .
one month after delivery her liver tests were still elevated with elevated bilirubin but pruritis had disappeared .
further follow up showed that bilirubin levels and liver tests returned to her pre - pregnancy values and remained stable with a follow up of two years .
our patient suffered from multiple peripartal exacerbations and had fetal complications including preterm labor and meconium - stained amniotic fluid . besides idiopathic cholestasis of pregnancy other causes of liver disease due to pregnancy had been ruled out .
the maternal and fetal effects of primary sclerosing cholangitis are similar to the effects of idiopathic cholestasis of pregnancy : pruritis , elevated bile acids , placental insufficiency , preterm labor and sudden fetal death .
primary sclerosing cholangitis may represent an extreme alteration of maternal - fetal bile salt metabolism as in obstetric cholestasis .
the mechanism of obstetric cholestasis has been suggested to be related to hormonal influences of increased estrogens on the bile salt export pump and the basolateral membrane of hepatocytes resulting in impaired transfer of bile acids from mother to fetus across the placenta , leading to potentially toxic levels of bile acids in the fetus .
the elevation of the bile acid levels possibly also affects myometrial contractility and may cause vasoconstriction of chorionic veins in the placenta , possibly resulting in preterm deliveries and fetal distress .
transporter gene mutations in the hepatocyte may play an important role as disease modifiers in primary sclerosing cholangitis .
it is conceivable that interaction of these mechanisms may result in severe exacerbations of primary sclerosing cholestasis .
management of idiopathic cholestasis of pregnancy is 2-fold : symptomatic treatment ( ursodeoxycholic acid ) and delivery of the fetus .
the course of primary sclerosing cholangitis is highly variable ; a flare of activity may occur throughout pregnancy or post partum . in all cases
the main presenting symptom was severe pruritus and in two cases the pruritus was the reason to induce labor ( 30 and 38 weeks gestation ) .
one women had a successful stenting soon after delivery and another had a liver transplantation six years after her last pregnancy .
in three cases signs of fetal asphyxia were noted ( either meconium staining or low apgar scores ) .
one woman had a dysmature child with birth weight 2140 gram at 38 weeks gestational age . in only two women treatment with ursodeoxycholic acid
it is proposed that ursodeoxycholic acid can displace more hydrophobic endogenous bile salts from the bile acid pool and thereby protect the hepatocyte membrane from their damaging toxicity , enhance bile acid clearance across the placenta from the fetus and protect in vitro rat cardiomyocytes from damage by endogenous bile salts .
low dose udca has shown relief of pruritus and improved liver tests along with less prematurity .
furthermore it is more effective in reducing pruritus , aminotransferase activity and bile acid levels than cholestyramine or dexamethasone with fewer preterm deliveries or side effects .
more recently lindor et al . suggested that long term higher dosage of udca may improve serum liver tests , but does not improve survival and may have severe adverse effects .
data are insufficient to support the widespread use of high dose ursodeoxycholic acid outside of clinical trials .
women should be aware of the lack of robust data concerning improvement in pruritus , protection against stillbirth and safety to the fetus or neonate .
since primary sclerosing cholangitis may represent an extreme alteration of maternal - fetal bile acid metabolism as in obstetric cholestasis , we treated our case with ursodeoxycholic acid with success .
although , long term follow up of women with primary sclerosing cholangitis is lacking and few cases are described , these cases suggest that primary sclerosing cholangitis has both maternal and fetal effects on pregnancy : exacerbation of primary sclerosing cholangitis , increased risk of fetal asphyxia and preterm birth ( 15% ) .
the association of exacerbation of primary sclerosing cholangitis and obstetric cholestasis implies a high recurrence rate in a subsequent pregnancy . in conclusion ,
although , primary sclerosing cholangitis has both maternal and fetal effects on pregnancy , the overall outcome is favorable .
it often improves pruritus but data on protection against stillbirth and safety to the fetus or neonate are scarce . | primary sclerosing cholangitis is a progressive disease , and coincidentally in pregnancy it is rare .
it is characterized by progressive inflammation and destruction of bile ducts finally resulting in liver failure .
a rare case of primary sclerosing cholangitis in pregnancy is presented .
the course of the pregnancy was marked by threatened preterm delivery and exacerbation of cholestasis .
she was successfully treated with ursodeoxycholic acid ( udca ) .
although , primary sclerosing cholangitis has both maternal and fetal effects on pregnancy , the overall outcome is favorable .
only few cases have been reported using high dose ursodeoxycholic acid for primary sclerosing cholangitis in pregnancy , it often improves pruritus but has no protection against stillbirth .
data on the safety to the fetus or neonate and long - term outcome are scarce . |
genitourinary involvement causes untoward symptoms of atrophic vaginitis including dryness , burning , dyspareunia , vulvar pruritus , and discharge .
these symptoms are localized , affect up to 50% of postmenopausal women , and significantly reduce their quality of life .
for this reason , many investigations have been performed to find out effective , safe , and acceptable therapeutic methods for atrophic vaginitis .
although systemic administration of estrogen can improve the localized symptoms of atrophic vaginitis , women are often reluctant to use systemic hormone replacement therapy and prefer local administration of estrogen .
different forms of estrogen have been widely prescribed for the local treatment of atrophic vaginitis ; however , vaginal estrogen creams has been the most common form of local estrogen therapy .
patients often consider these preparations to be unhygienic due to vaginal leakage and need for the use of some form of sanitary protection .
these factors cause poor compliance , reduced medication acceptability , and undertreatment , which lead to ineffective symptom control .
vagifem is a low dose slow - release small vaginal tablet , which contains 25 g of 17--estradiol .
it is placed deep into the vagina by an applicator , adheres to the vaginal mucosa , and has minimal discharge .
vagifem has been reported to be an easy and hygienic method of treatment for atrophic vaginitis .
this study was designed to compare the efficacy , acceptability , and safety of vagifem in a larger sample size with vaginal estrogen cream .
after approval of the study by the ethics committee of isfahan university of medical sciences ( research project number 390225 ) and obtaining informed consent , this randomized clinical trial was performed in a private clinic in isfahan , isfahan province , iran between 2010 and 2012 .
one hundred and sixty postmenopausal women aged 50 - 70 years with clinical diagnosis of vaginal atrophy who did not need systemic estrogen therapy for the treatment of vasomotor symptoms or prophylaxis of osteoporosis , and had no history of vaginal bleeding over the previous year entered the study .
women were excluded if they had any history of carcinoma of the breast or endometrium , abnormal genital bleeding , acute thrombophlebitis , or thromboembolic disorders associated with previous estrogen use , or current urinary or vaginal infection .
in addition , women who underwent hormone replacement therapy , treated with systemic or vaginal estrogen within 6 months prior to the study , or had any contraindication for estrogen therapy were excluded from the study . using simple randomization ,
women were randomly divided into vagifem ( from novo nordisk ) or vaginal estrogen cream ( equin from actoverco ) treatment groups ( 80 women in each group )
. patients of the first group were commenced on vaginal estrogen cream of actoverco company , one tube every night for 14 nights ; then , one tube 2 nights in 1 week ( two tubes every week ) for 10 weeks . in the second group , vaginal estrogen cream from novo nordisk was replaced with vagifem 25 mcg ; however , the treatment plan was similar to the first group .
in addition to recording demographic data , four main symptoms of atrophic vaginitis including dysuria , dyspareunia , vaginal itching , and dryness were evaluated by patients self - report , and vaginal atrophy was assessed by the gynecologist . at the beginning of the study ,
patients were asked to report whether they had no symptom or had mild , moderate , or severe symptoms .
then , the women were examined by a single gynecologist , and the degree of vaginal atrophy was determined and recorded .
follow - up sessions were arranged at 2 weeks , 4 weeks , 8 weeks , and 12 weeks after the beginning of the treatment .
the gynecologist who examined the women was unaware of the treatment group . at the end of the study
this is just confined to the response of the patients to the questions included in the checklist .
medication acceptability was checked after 2 weeks of the treatment and at the end of the study .
patients were asked about the presence and degree of pain [ using visual analog scale ( vas ) ] during application of the medication , vaginal leakage of the medication , need to use sanitary towels to clean leakage , and user - friendliness . a 3-point scale ( very easy , easy , or difficult )
women were also asked about whether they had experienced vaginal bleeding , recurrent vaginal discharge , stress , or urge incontinence .
data were analyzed by statistical package for the social sciences ( spss ) 16 software ( equine cream manufactured by aldo - union , s.a , barcelona , spain ) .
for demographic data , no significant difference was found between the two groups in baseline data [ table 1 ] .
demographic characteristics of the two groups the rate of pre- and posttreatment symptoms showed that there was no significant difference between the two groups and they had the same rate of response .
comparison of pre- and posttreatment within the groups showed a significant improvement of all the symptoms ; however , there was no significant difference between the two groups in posttreatment frequency of patients with a different intensity of symptoms .
comparison of patients with different intensities of vaginal atrophy between the two groups before and after treatment comparison of patients with different intensities of vaginal dryness between the two groups before and after treatment comparison patients with different intensities of itching between the two groups before and after treatment comparison of patients with different intensities of dyspareunia between the two groups before and after treatment comparison of patients with different intensities of dysuria between the two groups before and after treatment in contrast to the vagifem treatment group , most women receiving vaginal estrogen cream experienced discharge of medication following application . throughout the 12-week treatment period [ figure 1 ] .
number of women reporting leakage of medication from the vagina after 2 weeks and 12 weeks of treatment with vagifem and vaginal estrogen cream after 2 weeks of treatment , only 13% of the vagifem - treated women reported leakage of medication from the vagina compared with 53 ( 66% ) of those who had received vaginal estrogen cream .
the difference between the treatment groups was highly significant ( p < 0.0001 ) . at the end of the study , only 6%women in the vagifem treatment group reported leakage compared with 68% in the vaginal estrogen cream group ( p < 0.0001 ) .
the need for using sanitary towels was also significantly higher in the vaginal estrogen cream treatment group throughout the trial .
after 2 weeks of treatment , vaginal leakage of the medication necessitated the use of sanitary towels by only 7 ( 9% ) of the women receiving vagifem compared with 35 ( 44% ) of those in the vaginal estrogen cream treatment group ( p < 0.0001 ) .
following reduction of drug application frequency to twice - weekly , none of the women treated with vagifem reported the need for sanitary towels while 23% women receiving vaginal estrogen cream still needed to use them ( p < 0.0001 ) .
regarding overall user - friendliness , women reported vagifem to be a significantly more user - friendly medication [ tables 6 and 7 ] .
comparison of user - friendliness of medications between the two groups according to patients reported scores
it is well - understood that use of local estrogen in the form of vaginal cream has some limitations for patients .
these limitations mainly include difficulties in application such as the obligation for usage at bedtime and somehow leakages from the vagina marking the underwear .
patient acceptability and preference is a very important aspect of local estrogen therapy , which significantly affects patients compliance .
since long - term therapy is likely to be necessary for women suffering from atrophic vaginitis , treatment should be easy and convenient . despite the well - documented efficacy of treatment with vaginal estrogen creams , compliance with therapy
the food and drug administration ( fda ) approved the 10 mcg estradiol vaginal tablet formulation ( vagifem ) for the treatment of vva in menopausal women .
estradiol vaginal tablets previously were approved in a 25 mcg formulation in the united states and the european union .
the company discontinued the sale of vagifem 25 mcg in the united states on july 30 , 2010 .
this poor compliance is mainly caused by vaginal leakage of medication , which usually necessitates the use of sanitary protection .
this study showed that both vagifem and vaginal estrogen cream significantly improve the signs and symptoms of atrophic vaginitis .
although there was no significant difference between these two medications in treating the symptoms , patients were more satisfied with vagifem regarding user - friendliness and hygienic issues .
two other previous studies also confirmed our findings , and showed that women treated with vagifem were more likely to remain on therapy than those who were treated with a vaginal cream .
studied 96 women randomly classified them into two equal groups of vaginal estrogen cream and estrogen tablet .
their results showed that vaginal tablets were as effective as estrogen cream ; however , patients acceptance was significantly higher in the tablet - treated group .
similarly , rioux et al . performed a multicenter , open - label , randomized , parallel group study on 159 menopausal women to compare treatment with vagifem and vaginal estrogen cream .
they concluded that treatment with vagifem and with vaginal estrogen cream was equivalent in relieving symptoms of atrophic vaginitis .
in addition , they demonstrated that vaginal tablets have a localized effect with no significant increase in the systemic estradiol level or estrogenic side effects .
they also reported greater patient acceptance and lower withdrawal rates in vaginal tablet therapy compared with the vaginal cream group . based on findings of several studies ,
estradiol vaginal tablets such as vagifem were found to be an effective alternative to traditional therapeutic methods for atrophic vaginitis in postmenopausal women .
this is consistent with the results of previous clinical trials , which treated and followed up patients for up to 2 years .
it is not a difficult decision to select one treatment from a group of choices when the efficacy is similar . in the presence of similar effectiveness
vagifem is the tablet form of local estrogen preparation and therefore , it has an easier method of application with fewer adverse hygienic effects such as vaginal leakage .
this is a very important factor , which affects patient adherence to the treatment in long term .
in summary , we conclude that vagifem is an appropriate medication for the treatment of atrophic vaginitis , which is as effective as vaginal estrogen creams and is more userfriendly .
all authors contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . | background : atrophic vaginitis is a disease , which affects up to 50% of postmenopausal women .
this study compared the effectiveness and user - friendliness of vagifem ( an estradiol vaginal tablet ) and vaginal estrogen cream in the treatment of atrophic vaginitis.materials and methods : one hundred and sixty postmenopausal women with symptoms of atrophic vaginitis were randomly divided into two groups of treatment with vagifem or with vaginal estrogen cream for 12 weeks .
patients used the medication daily for the first 2 weeks of the study , and twice weekly .
severity of vaginal atrophy and four main symptoms of atrophic vaginitis including dysuria , dyspareunia , vaginal itching , and dryness were evaluated and compared before and after treatment .
in addition , patients were asked regarding user - friendliness and hygienic issues of medications.results:both vaginal estrogen cream and vagifem significantly improved symptoms of atrophic vaginitis but in terms of effectiveness for the treatment symptoms of atrophic vaginitis , there was no significant difference between the two medications .
vagifem compared to estrogen cream resulted in significantly lower rate of hygienic problems ( 0% versus 23% , p
< 0.001 ) , and was reported by the patients as a significantly easier method of treatment ( 90% versus 55% , p
< 0.0001).conclusion : this investigation showed that vagifem is an appropriate medication for the treatment of atrophic vaginitis , which is as effective as vaginal estrogen creams and is more user - friendly . |
there is a plethora of tools that focus in proteomics sequence analysis , alignment and visualization but there are few examples for web servers and other software tools dedicated to peptide analysis .
there are three major classes where the tools for peptide analysis can be classified : ( i ) the peptide / protein identification tools like raid_dbs ( 1 ) , peptizer ( 2 ) , denovoid ( 3 ) and peptidefinder ( 4 ) ; ( ii ) tools for the mapping of post - translational peptide modifications like modi ( 5 ) , for peptide phosphorylation like phoscalc ( 6 ) and proteolysis ( 7 ) ; ( iii ) tools for the investigation of specific characteristics of the peptide fragments like remus ( 8) and unimap ( 9 ) for the property of uniqueness , like signalp ( 10 ) for the signaling property , of the peptide mhc binding affinity like biodmhc ( 11 ) and mhcpred ( 12 ) .
however , to our knowledge , there is not any web server dedicated to the broad analysis of peptide characteristics performing peptide clustering and visualization in the peptide feature space .
here , we present a web application which can provide information regarding human peptide sequence analysis and clustering based on their related characteristics ( annotated as
features in the uniprot database ) along with information regarding peptide uniqueness and peptide similarity .
pepserve is an application for analyzing and clustering peptide sequences according to a set of selected peptide features .
the clusters of peptide sequences can be visualized as a graph where all the peptide sequences that share the same characteristics can be viewed .
the user may already have or may produce a peptide sequence list . in the second case , a peptide list can be retrieved in the following ways : ( i ) from a selected enzymatic digestion of a group of proteins ; ( ii ) from the exhaustive peptide combinations around a specified position or within a position range of a protein sequence ; and ( iii ) from the mapping on a given list of molecular masses , filtered or not with regular expression constraints as shown in figure 1a .
figure 1.(a ) the information flow of the system the input can be protein(s ) which is ( i ) digested or ( ii ) filtered by position and peptides , either sequences or molecular masses .
the output is a list of peptide sequences which is subjected to feature retrieval and visualization .
( b ) a characteristic example of the feature - driven peptide visualization : the distribution of the peptides produced after tryptic digestion of three proteins from three different families ( p14921 , p22676 and p62166 ) according to the selected features ( calcium binding and dna binding ) . (
a ) the information flow of the system the input can be protein(s ) which is ( i ) digested or ( ii ) filtered by position and peptides , either sequences or molecular masses .
the output is a list of peptide sequences which is subjected to feature retrieval and visualization .
( b ) a characteristic example of the feature - driven peptide visualization : the distribution of the peptides produced after tryptic digestion of three proteins from three different families ( p14921 , p22676 and p62166 ) according to the selected features ( calcium binding and dna binding ) . in the first searching mode , the user can enter one or more protein uniprot accession numbers ( ac ) or protein names and select a set of enzymes that will computationally digest the specified proteins . usually , tryptic digestion is preferred because it has high specificity and also 90% of the peptide fragments have less than 20 residues . generally , the more enzymes are selected , the more peptides are produced with short length .
this mode is useful when known protein sets are available and the researcher expects to find peptide characteristics and subsequently protein characteristics . in the second searching mode , the protein accession number or the protein name
is specified , along with the amino acid position of interest in the protein sequence .
this position will define the part of a protein sequence which can be either around a selected position or within selected position boundaries in the protein sequence .
the system searches for sequence characteristics within the specified positions and therefore this mode is preferred when a known protein is prone to mutagenesis or other protein states where the location of the sequence under investigation is important .
the third searching mode performs directly the analysis if a list of peptide sequences is at hand , whereas the final searching mode expects a list of peptide molecular masses in order to translate them to possible peptide sequences and then analyze them .
the final search mode facilitates researchers using tandem mass spectrometry ( ms / ms ) analysis and for this reason there are more input parameters such as measured mass tolerance , protein molecular mass , isoelectric point range and pattern matching to refine the results .
the system uses the file repositories from our previous works ( 4,9 ) which contain peptide fragments classified according to their molecular mass , derived from extensive digestion of human proteins in the uniprot database .
the current repositories contain uniprot s release 6/2010 and it has been scheduled for an update once a year .
in addition , the system exploits the information from the uniprot flat files regarding the sequence positional features ( feature table
ft lines ) according to the position of the peptide fragment inside the corresponding protein .
complementary to the annotated features , peptide sequence uniqueness as well as sequence similarity are calculated and included in the collected peptide characteristics .
we define a peptide feature space with n dimensions defined from the collected features mentioned before .
the number of dimensions is related with the number of peptide features which is selected by the user .
the total number of features is 41 , where the 38 features are those included in the sequence annotation tag of uniprot and the three more features are described below .
each sequence has three possible states for each feature corresponding to ( yes , partially ,
when a sequence falls within the uniprot position boundaries for the given feature , the sequence state is characterized as
partially state is when a part of the sequence is lying within the feature boundaries .
for each two sequences si and sj , the system calculates their euclidean distance as :
where cm , i and cm , j are the arithmetic state values ( + 1 , 0 , 1 ) of ( yes ,
no ) for the m - th characteristic of sequences si and sj , respectively .
the peptides are clustered according to this euclidian distance in the peptide feature space , and subsequently they are visualized within a graph .
the graph contains nodes and edges where the nodes represent the peptides and the edges have a weight which corresponds to the euclidian distance between the two nodes .
the performed clustering can be considered as a supervised one , since the classes are predefined from the combinations of the sequence features ( i.e. all features included in the uniprot annotation scheme plus some extra features regarding uniqueness , sequence similarity and protein commonality ) .
the clustering metric is the euclidean distance in this feature space that controls the length of the edges in the graph topology .
each peptide from the list is annotated according to the uniprot sequence positional features ( regions : topological domain , transmembrane , domain , calcium binding , zinc finger , dna binding , nucleotide binding , coiled coil , motif , compositional bias ; molecule processing : signal , transit peptide , propeptide , peptide ; amino acid modifications : lipidation , disulfide bond , non - standard residue , modified residue , glycosylation , cross - link ; experimental information : mutagenesis , sequence uncertainty , sequence conflict , non - adjacent residue , non - terminal residue ; sites : active site , metal binding , binding site , site ; secondary structure : helix , turn , beta strand ; natural variations : alternative sequence , natural variant ) enriched with the feature of sequence uniqueness . when a peptide is characterized as unique
, it means that it exists only in one protein in the set of human proteins registered in the uniprot .
this feature is provided for all other species in uniprot database in order to compare with human peptide sequence uniqueness .
the system produces a peptide sequence list accompanied by the corresponding list of peptide features ( table 1 ) .
all possible features are available to be selected for the clustering procedure . in the feature list , there are also two new characteristics related to the peptide pair - wise similarity : the sequence similarity and the number of common proteins the two peptide sequences can be found in .
the sequence similarity is an index which shows the smallest number of edits to change one sequence into the other .
table 1.an example of pepserve outputmolecular masspeptideinformationunique peptideprotein and features632.34stellapeptide atlasyesq4kmx7no features found check across speciescheck enymatic digestion.1016.46tyacfvsnlpeptide atlasyesp06731domainig - like 7check across speciescheck enymatic digestion.587.34avatarpeptide atlasnoq04609topological domaincytoplasmic ( probable)check across speciesq8iwk6topological domainextracellular ( potential)check for other unique sequencesq8n122no features found when peptide sequence
are served as an input , the system responds with the corresponding monoisotopic molecular mass and the peptide characteristics . as shown ,
avatar is not unique in human proteome because it can be found in three proteins , whereas stella and tyacfvsnl are unique sequences and their corresponding features are displayed .
stella , avatar and tyacfvsnl are served as an input , the system responds with the corresponding monoisotopic molecular mass and the peptide characteristics . as shown ,
avatar is not unique in human proteome because it can be found in three proteins , whereas
tyacfvsnl are unique sequences and their corresponding features are displayed . when the desired features are selected , the system calculates the euclidian distance between the peptides .
this way , the peptide relations in the feature space can be dynamically visualized in a graph by using properly created graphml files handled by prefuse , a java - based visualization toolkit ( 13 ) .
the nodes of the graph are displayed as red circles representing the peptides and when they are double clicked they display the peptide sequences that belong to the same cluster along with the cluster s features .
the edge color varies according to the edge weight where a red edge represents the smallest weight .
the user may already have or may produce a peptide sequence list . in the second case , a peptide list can be retrieved in the following ways : ( i ) from a selected enzymatic digestion of a group of proteins ; ( ii ) from the exhaustive peptide combinations around a specified position or within a position range of a protein sequence ; and ( iii ) from the mapping on a given list of molecular masses , filtered or not with regular expression constraints as shown in figure 1a .
figure 1.(a ) the information flow of the system the input can be protein(s ) which is ( i ) digested or ( ii ) filtered by position and peptides , either sequences or molecular masses .
the output is a list of peptide sequences which is subjected to feature retrieval and visualization .
( b ) a characteristic example of the feature - driven peptide visualization : the distribution of the peptides produced after tryptic digestion of three proteins from three different families ( p14921 , p22676 and p62166 ) according to the selected features ( calcium binding and dna binding ) . (
a ) the information flow of the system the input can be protein(s ) which is ( i ) digested or ( ii ) filtered by position and peptides , either sequences or molecular masses .
the output is a list of peptide sequences which is subjected to feature retrieval and visualization .
( b ) a characteristic example of the feature - driven peptide visualization : the distribution of the peptides produced after tryptic digestion of three proteins from three different families ( p14921 , p22676 and p62166 ) according to the selected features ( calcium binding and dna binding ) . in the first searching mode , the user can enter one or more protein uniprot accession numbers ( ac ) or protein names and select a set of enzymes that will computationally digest the specified proteins . usually , tryptic digestion is preferred because it has high specificity and also 90% of the peptide fragments have less than 20 residues . generally , the more enzymes are selected , the more peptides are produced with short length .
this mode is useful when known protein sets are available and the researcher expects to find peptide characteristics and subsequently protein characteristics . in the second searching mode , the protein accession number or the protein name
is specified , along with the amino acid position of interest in the protein sequence .
this position will define the part of a protein sequence which can be either around a selected position or within selected position boundaries in the protein sequence .
the system searches for sequence characteristics within the specified positions and therefore this mode is preferred when a known protein is prone to mutagenesis or other protein states where the location of the sequence under investigation is important .
the third searching mode performs directly the analysis if a list of peptide sequences is at hand , whereas the final searching mode expects a list of peptide molecular masses in order to translate them to possible peptide sequences and then analyze them .
the final search mode facilitates researchers using tandem mass spectrometry ( ms / ms ) analysis and for this reason there are more input parameters such as measured mass tolerance , protein molecular mass , isoelectric point range and pattern matching to refine the results .
the system uses the file repositories from our previous works ( 4,9 ) which contain peptide fragments classified according to their molecular mass , derived from extensive digestion of human proteins in the uniprot database .
the current repositories contain uniprot s release 6/2010 and it has been scheduled for an update once a year .
in addition , the system exploits the information from the uniprot flat files regarding the sequence positional features ( feature table
ft lines ) according to the position of the peptide fragment inside the corresponding protein .
complementary to the annotated features , peptide sequence uniqueness as well as sequence similarity are calculated and included in the collected peptide characteristics .
we define a peptide feature space with n dimensions defined from the collected features mentioned before .
the number of dimensions is related with the number of peptide features which is selected by the user .
the total number of features is 41 , where the 38 features are those included in the sequence annotation tag of uniprot and the three more features are described below .
each sequence has three possible states for each feature corresponding to ( yes , partially ,
when a sequence falls within the uniprot position boundaries for the given feature , the sequence state is characterized as
partially state is when a part of the sequence is lying within the feature boundaries .
for each two sequences si and sj , the system calculates their euclidean distance as :
where cm , i and cm , j are the arithmetic state values ( + 1 , 0 , 1 ) of ( yes ,
no ) for the m - th characteristic of sequences si and sj , respectively .
the peptides are clustered according to this euclidian distance in the peptide feature space , and subsequently they are visualized within a graph .
the graph contains nodes and edges where the nodes represent the peptides and the edges have a weight which corresponds to the euclidian distance between the two nodes .
the performed clustering can be considered as a supervised one , since the classes are predefined from the combinations of the sequence features ( i.e. all features included in the uniprot annotation scheme plus some extra features regarding uniqueness , sequence similarity and protein commonality ) .
the clustering metric is the euclidean distance in this feature space that controls the length of the edges in the graph topology .
each peptide from the list is annotated according to the uniprot sequence positional features ( regions : topological domain , transmembrane , domain , calcium binding , zinc finger , dna binding , nucleotide binding , coiled coil , motif , compositional bias ; molecule processing : signal , transit peptide , propeptide , peptide ; amino acid modifications : lipidation , disulfide bond , non - standard residue , modified residue , glycosylation , cross - link ; experimental information : mutagenesis , sequence uncertainty , sequence conflict , non - adjacent residue , non - terminal residue ; sites : active site , metal binding , binding site , site ; secondary structure : helix , turn , beta strand ; natural variations : alternative sequence , natural variant ) enriched with the feature of sequence uniqueness . when a peptide is characterized as unique
, it means that it exists only in one protein in the set of human proteins registered in the uniprot .
this feature is provided for all other species in uniprot database in order to compare with human peptide sequence uniqueness .
the system produces a peptide sequence list accompanied by the corresponding list of peptide features ( table 1 ) .
all possible features are available to be selected for the clustering procedure . in the feature list , there are also two new characteristics related to the peptide pair - wise similarity : the sequence similarity and the number of common proteins the two peptide sequences can be found in .
the sequence similarity is an index which shows the smallest number of edits to change one sequence into the other .
table 1.an example of pepserve outputmolecular masspeptideinformationunique peptideprotein and features632.34stellapeptide atlasyesq4kmx7no features found check across speciescheck enymatic digestion.1016.46tyacfvsnlpeptide atlasyesp06731domainig - like 7check across speciescheck enymatic digestion.587.34avatarpeptide atlasnoq04609topological domaincytoplasmic ( probable)check across speciesq8iwk6topological domainextracellular ( potential)check for other unique sequencesq8n122no features found when peptide sequence
are served as an input , the system responds with the corresponding monoisotopic molecular mass and the peptide characteristics . as shown ,
avatar is not unique in human proteome because it can be found in three proteins , whereas stella and tyacfvsnl are unique sequences and their corresponding features are displayed .
stella , avatar and tyacfvsnl are served as an input , the system responds with the corresponding monoisotopic molecular mass and the peptide characteristics . as shown ,
avatar is not unique in human proteome because it can be found in three proteins , whereas
tyacfvsnl are unique sequences and their corresponding features are displayed . when the desired features are selected , the system calculates the euclidian distance between the peptides .
this way , the peptide relations in the feature space can be dynamically visualized in a graph by using properly created graphml files handled by prefuse , a java - based visualization toolkit ( 13 ) .
the nodes of the graph are displayed as red circles representing the peptides and when they are double clicked they display the peptide sequences that belong to the same cluster along with the cluster s features .
the edge color varies according to the edge weight where a red edge represents the smallest weight .
the user may already have or may produce a peptide sequence list . in the second case , a peptide list can be retrieved in the following ways : ( i ) from a selected enzymatic digestion of a group of proteins ; ( ii ) from the exhaustive peptide combinations around a specified position or within a position range of a protein sequence ; and ( iii ) from the mapping on a given list of molecular masses , filtered or not with regular expression constraints as shown in figure 1a .
figure 1.(a ) the information flow of the system the input can be protein(s ) which is ( i ) digested or ( ii ) filtered by position and peptides , either sequences or molecular masses .
the output is a list of peptide sequences which is subjected to feature retrieval and visualization .
( b ) a characteristic example of the feature - driven peptide visualization : the distribution of the peptides produced after tryptic digestion of three proteins from three different families ( p14921 , p22676 and p62166 ) according to the selected features ( calcium binding and dna binding ) . (
a ) the information flow of the system the input can be protein(s ) which is ( i ) digested or ( ii ) filtered by position and peptides , either sequences or molecular masses .
the output is a list of peptide sequences which is subjected to feature retrieval and visualization .
( b ) a characteristic example of the feature - driven peptide visualization : the distribution of the peptides produced after tryptic digestion of three proteins from three different families ( p14921 , p22676 and p62166 ) according to the selected features ( calcium binding and dna binding ) . in the first searching mode , the user can enter one or more protein uniprot accession numbers ( ac ) or protein names and select a set of enzymes that will computationally digest the specified proteins . usually , tryptic digestion is preferred because it has high specificity and also 90% of the peptide fragments have less than 20 residues . generally , the more enzymes are selected , the more peptides are produced with short length .
this mode is useful when known protein sets are available and the researcher expects to find peptide characteristics and subsequently protein characteristics . in the second searching mode , the protein accession number or the protein name
is specified , along with the amino acid position of interest in the protein sequence .
this position will define the part of a protein sequence which can be either around a selected position or within selected position boundaries in the protein sequence .
the system searches for sequence characteristics within the specified positions and therefore this mode is preferred when a known protein is prone to mutagenesis or other protein states where the location of the sequence under investigation is important .
the third searching mode performs directly the analysis if a list of peptide sequences is at hand , whereas the final searching mode expects a list of peptide molecular masses in order to translate them to possible peptide sequences and then analyze them .
the final search mode facilitates researchers using tandem mass spectrometry ( ms / ms ) analysis and for this reason there are more input parameters such as measured mass tolerance , protein molecular mass , isoelectric point range and pattern matching to refine the results .
the system uses the file repositories from our previous works ( 4,9 ) which contain peptide fragments classified according to their molecular mass , derived from extensive digestion of human proteins in the uniprot database .
the current repositories contain uniprot s release 6/2010 and it has been scheduled for an update once a year .
in addition , the system exploits the information from the uniprot flat files regarding the sequence positional features ( feature table
ft lines ) according to the position of the peptide fragment inside the corresponding protein .
complementary to the annotated features , peptide sequence uniqueness as well as sequence similarity are calculated and included in the collected peptide characteristics .
we define a peptide feature space with n dimensions defined from the collected features mentioned before .
the number of dimensions is related with the number of peptide features which is selected by the user .
the total number of features is 41 , where the 38 features are those included in the sequence annotation tag of uniprot and the three more features are described below .
each sequence has three possible states for each feature corresponding to ( yes , partially ,
when a sequence falls within the uniprot position boundaries for the given feature , the sequence state is characterized as
partially state is when a part of the sequence is lying within the feature boundaries .
for each two sequences si and sj , the system calculates their euclidean distance as :
where cm , i and cm , j are the arithmetic state values ( + 1 , 0 , 1 ) of ( yes ,
no ) for the m - th characteristic of sequences si and sj , respectively .
the peptides are clustered according to this euclidian distance in the peptide feature space , and subsequently they are visualized within a graph .
the graph contains nodes and edges where the nodes represent the peptides and the edges have a weight which corresponds to the euclidian distance between the two nodes .
the performed clustering can be considered as a supervised one , since the classes are predefined from the combinations of the sequence features ( i.e. all features included in the uniprot annotation scheme plus some extra features regarding uniqueness , sequence similarity and protein commonality ) .
the clustering metric is the euclidean distance in this feature space that controls the length of the edges in the graph topology .
each peptide from the list is annotated according to the uniprot sequence positional features ( regions : topological domain , transmembrane , domain , calcium binding , zinc finger , dna binding , nucleotide binding , coiled coil , motif , compositional bias ; molecule processing : signal , transit peptide , propeptide , peptide ; amino acid modifications : lipidation , disulfide bond , non - standard residue , modified residue , glycosylation , cross - link ; experimental information : mutagenesis , sequence uncertainty , sequence conflict , non - adjacent residue , non - terminal residue ; sites : active site , metal binding , binding site , site ; secondary structure : helix , turn , beta strand ; natural variations : alternative sequence , natural variant ) enriched with the feature of sequence uniqueness . when a peptide is characterized as unique , it means that it exists only in one protein in the set of human proteins registered in the uniprot .
this feature is provided for all other species in uniprot database in order to compare with human peptide sequence uniqueness .
the system produces a peptide sequence list accompanied by the corresponding list of peptide features ( table 1 ) .
all possible features are available to be selected for the clustering procedure . in the feature list , there are also two new characteristics related to the peptide pair - wise similarity : the sequence similarity and the number of common proteins the two peptide sequences can be found in .
the sequence similarity is an index which shows the smallest number of edits to change one sequence into the other .
table 1.an example of pepserve outputmolecular masspeptideinformationunique peptideprotein and features632.34stellapeptide atlasyesq4kmx7no features found check across speciescheck enymatic digestion.1016.46tyacfvsnlpeptide atlasyesp06731domainig - like 7check across speciescheck enymatic digestion.587.34avatarpeptide atlasnoq04609topological domaincytoplasmic ( probable)check across speciesq8iwk6topological domainextracellular ( potential)check for other unique sequencesq8n122no features found when peptide sequence
stella , avatar and tyacfvsnl are served as an input , the system responds with the corresponding monoisotopic molecular mass and the peptide characteristics . as shown ,
avatar is not unique in human proteome because it can be found in three proteins , whereas stella and tyacfvsnl are unique sequences and their corresponding features are displayed .
stella , avatar and tyacfvsnl are served as an input , the system responds with the corresponding monoisotopic molecular mass and the peptide characteristics . as shown ,
avatar is not unique in human proteome because it can be found in three proteins , whereas stella and
tyacfvsnl are unique sequences and their corresponding features are displayed . when the desired features are selected , the system calculates the euclidian distance between the peptides .
this way , the peptide relations in the feature space can be dynamically visualized in a graph by using properly created graphml files handled by prefuse , a java - based visualization toolkit ( 13 ) .
the nodes of the graph are displayed as red circles representing the peptides and when they are double clicked they display the peptide sequences that belong to the same cluster along with the cluster s features .
the edge color varies according to the edge weight where a red edge represents the smallest weight . at last
feature - driven peptide visualization and clustering demonstrates the common peptide features of the various protein fragments .
for example , in figure 1b we show the distribution of the peptides produced after tryptic digestion of three proteins from three different families ( p14921 : ets family ; p22676 : calbindin family ; and p62166 : recoverin family ) according to the selected features ( calcium binding and dna binding ) .
figure 2.immune peptide sequences as differentiation antigens from prostate cancer disease and melanoma case are clustered according to the two selected features , disulfide bond and beta strand .
immune peptide sequences as differentiation antigens from prostate cancer disease and melanoma case are clustered according to the two selected features , disulfide bond and beta strand .
also , protein p27487-dipeptidyl peptidase 4 , which is a protein with 766 amino acids , has 12 positions in its sequence where eight of them can affect tryptic peptides .
there are 39 peptides out of 59 which are unique in the specified protein and they are not affected by experimental mutation of amino acid(s ) on the biological properties of the protein .
table 2.peptide clustering using the features of sequence uniqueness and mutagenesisfeature properties for peptide clusteringnumber of peptidesunique and mutagenesis4unique and not mutagenesis39not unique and mutagenesis4not unique and not mutagenesis12tryptic digestion of p27487 dipeptidyl peptidase 4 produces 59 peptides which are grouped into 4 clusters .
peptide clustering using the features of sequence uniqueness and mutagenesis tryptic digestion of p27487 dipeptidyl peptidase 4 produces 59 peptides which are grouped into 4 clusters . in a third example , a group of immune peptide sequences were served as an input to pepserve ( 14 ) .
there were 13 out of 14 peptides from gut carcinoma that were found to be unique in the human proteome . in the melanoma case ,
54 out of 59 peptide sequences were unique , in prostate cancer all 5 peptides were unique and finally in breast cancer both peptides were also unique .
the majority of the peptides from each tumor state share the same features , mainly the peptides from the melanoma are placed in the lumenal , melanosome subcellular compartment where there is a non - membrane region of a membrane - spanning protein .
these 59 peptides are located in 7 proteins . respectively , the prostate peptides are found in two proteins with peptidase s1 protein domain , disulfide bonds and beta strand regions as peptide features .
peptide clustering using the two features , disulfide bond and beta strand shows us the distinction between the two sets of peptides ( figure 2 ) .
the peptides related to gut carcinoma were found in one protein and those related to breast cancer in two proteins .
the immune peptides and especially the differentiation antigens are considered very useful in the design of monoclonal antibodies since targeted monoclonal antibody therapy is employed to treat diseases .
the finding of unique differentiation antigens is considered very crucial and promising in this direction of targeted therapy .
when using the property of uniqueness , a biologist will be able to find the functions of specific peptides with high selectivity .
thus , one may perform studies on the opposite direction from the studies based on sequence and function similarity .
that is , the property of sequence uniqueness may imply peptide function uniqueness and perhaps may give insights in the de novo design of differentiation antigens or antimicrobial peptides .
peptide clustering and visualization offers useful information for the research community , since common peptide characteristics for a set of sequence annotations can emerge from a list of peptide fragments
. the biologists can view groups of peptides that share the same characteristics , from a protein enzymatic digestion or mapped on a list of molecular masses from mass spectrometry .
furthermore , peptide feature clustering can be useful in the analysis of cell lines , and specifically cancer cell lines , and also to contribute in providing supplementary information concerning normal and disease states .
pepserve is hosted by an apache server on a linux platform and incorporates a script - based curation protocol of the repository files and the uniprot database .
the system responds using reporting procedures based on dynamically generated html files ( using cgi perl scripts ) .
the updating procedure has been scheduled to run twice a year using uniprot s swiss - prot database .
pepserve is part of a group of tools , databases and web services developed in the biomedical research foundation , academy of athens , called
funding for open access charge : biomedical research foundation , academy of athens , 4 soranou ephessiou , 115 27 athens . | peptides , either as protein fragments or as naturally occurring entities are characterized by their sequence and function features .
many times the researchers need to massively manage peptide lists concerning protein identification , biomarker discovery , bioactivity , immune response or other functionalities .
we present a web server that manages peptide lists in terms of feature analysis as well as interactive clustering and visualization of the given peptides .
pepserve is a useful tool in the understanding of the peptide feature distribution among a group of peptides .
the pepserve web application is freely available at http://bioserver-1.bioacademy.gr / bioserver / pepserve/. |
although not the most prevalent of human malignancies , a unique combination of physiology , anatomy and biology leads to an extremely poor prognosis for all but a few patients diagnosed with pancreatic ductal adenocarcinoma ( pdac ) .
pancreatic cancer is known for its early metastasis and aggressive invasion of surrounding tissues . due to the lack of early detection methods and a paucity of recognizable symptoms ,
patients are usually diagnosed at late tumor stages without surgical therapy options.1 approximately 80% of pancreatic cancer patients present with locally advanced , non - resectable or distant metastatic disease . even for patients diagnosed with surgically resectable local disease ,
when augmented with adjuvant chemoradiotherapy , typically only 3% - 4% can achieve long - term cure.2 in addition , pancreatic cancer is highly resistant to conventional systemic chemotherapies such as gemcitabine , paclitaxel , 5-fluorouracil , leucovorin and platinum - based drugs.3,4 this chemoresistance is a function of the desmoplastic architecture of pancreatic tumor stroma which contributes to exaggerated hypovasculature.4 this weak blood perfusion applies equally to nutrients and leads to an aggressive biology for pdac whereby these tumor cells can survive under extreme nutrient deprivation . despite recent advances in genotyping and development of matched targeted therapeutics that demonstrate impressive efficacy in vitro , these have not to - date provided worthwhile advancement in clinical strategies.5 unfortunately
, currently available therapies provide at best only marginal improvement in median survival , let alone a guarantee of the complete eradication of pancreatic cancer . in 2000 ,
izuishi and coworkers reported a study comparing the survival of pancreatic cancer cells and normal human fibroblasts in media lacking glucose , amino acids and serum , to mimic the nutrient deprivation conditions encountered in vivo.6 four pancreatic cancer cell lines , including panc-1 , aspc-1 , bxpc-3 , and kp-3 , were shown to survive for 48 h under nutrient deficiency , whereas normal fibroblasts died within 24 h under the same conditions.6 since normal tissues seldom encounter nutrient deprivation , the austerity of pancreatic cancer cells has become a potential novel biochemical target for cancer therapy .
significant effort has been invested in searching for anti - austerity agents that selectively target pancreatic cancer cells under nutrient - deprived conditions . in 2006 , a coumarin - based natural product , angelmarin ( 1 , figure 1 ) , was isolated from the root of the japanese medicinal plant , angelica pubescens .
the 23-carbon structure contains a columbianetin ( 2 , figure 1 ) core and a p - hydroxycinnamoyl group ( blue , figure 1 ) connected via an ester linkage .
angelmarin was found to exhibit cytotoxicity against panc-1 cells preferentially under nutrient - deprived conditions , inducing 100% cell death at a concentration of 0.01 g / ml within 24 h.7 the core coumarin ( 3 ) structure of angelmarin ( 1 ) ( 1 , figure 1 and 3 , figure 2 ) is found in a number of pharmacologically active compounds and hydroxycoumarin derivatives in particular have been shown to exhibit promising antitumor , anti - flammatory and anti - viral effects.8 intrigued by the selective cytotoxicity of angelmarin against pancreatic cancer cells under nutrient - deprived conditions and the frequent occurrence of coumarins in natural compounds , our laboratory launched structure - activity relationship ( sar ) studies based on the core coumarin structure of angelmarin . in 2011 ,
our group reported the discovery of a novel geranylgeranylated ether coumarin derivative ( 4 , figure 2 ) that was found to induce panc-1 cell death at a concentration of 6.25 m within 24 h , with preferential cytotoxicity under nutrient - deprived conditions.8 since this discovery , a library of other isoprenylated coumarin derivatives with systematic variations in the tail length ( 5 , 10 or 15 carbons ) and substitution position on the coumarin scaffold ( at 3- , 6- or 7-position ) has been completed.9 the 6-substituted farnesylated ether coumarin derivative ( 5 , figure 2 ) displayed the highest cytotoxic activity with an lc50 value of 4 m and induced apoptosis - like morphological changes in panc-1 cells after a 24-hour incubation , making this isoprenylated ether coumarin our most promising lead compound.9 in order to better understand the preferential cytotoxicity of our anti - proliferative coumarin derivatives , we have begun studies to probe the cellular mechanism of action of these compounds . for these studies , we selected compound 5 because of its potent and selective cytotoxicity against panc-1 cells under nutrient - deprived conditions . the studies presented herein explore the effect of several cell culture medium components on the cytotoxic activity of 5 .
the cell growth inhibition activity of 5 against two additional pancreatic cancer cell lines , bxpc-3 and capan-2 , is also reported .
human pancreatic cancer cell lines , panc-1 , bxpc-3 , and capan-2 were cultured in dulbecco modified eagle 's medium ( dmem ) . to prepare dmem ,
one bottle of dmem powder ( sigma - aldrich ) was dissolved in 800 ml of hplc grade water .
sodium bicarbonate solution ( 49.1 ml ) and antibiotic - antifungal solution ( 10 ml , 100x ) were added to the mixture .
the volume was brought to 900 ml and the solution was adjusted to a ph between 7 - 7.4 .
heat - inactivated fetal bovine serum ( fbs , 100 ml ) was added to the neutralized solution and filtered via a 0.2-m corning filter .
the following electrolytes and vitamin solution were added in concentrations as follows : cacl2(2h2o ) , 265 mg / l ; fe(no3)(9h2o ) , 0.1 mg / l ; kcl , 400 mg / l ; mgso4(7 h2o ) , 200 mg / l ; nacl , 6400 mg / l ; nahco3 , 700 mg / l ; nah2po4 , 125 mg / l ; phenol red , 15 mg / l ; hepes buffer ( 25 mm , ph 7.4 ) ; and mem vitamin solution ( 1x / l ) ( life technologies , inc . , rockville , md ) , which completed the preparation of the ndm . for nrm ,
additional nutrients were supplement at concentrations as follows : d - glucose , 1000mg / l ; l - glutamine , 2 mm ; mem amino acids solution and mem nonessential amino acids solution ( life technologies , inc . ) , 20 ml and 10 ml respectively for 1 l medium ; fbs , 100 ml for 1 l medium . for all special media combinations , corresponding combinations of nutrients were added at concentrations identical to those in nrm . for media with dialyzed serum , the same concentration of dialyzed serum ( life technologies , inc . )
was added as a replacement of fbs ( 100 ml for 1 l medium ) . to a cell - containing t-75 culture flask
, a 2 - 5 ml aliquot of filtered trypsin - edta was added to detach the cells after the aspiration of dmem and a pbs wash .
the trypsin - containing flask was placed in the incubator ( 37c and 5% co2 ) for 2 - 3 minutes and then monitored under a microscope with 10x amplification .
detached cells were transferred to a 15 ml centrifuge tube and pelleted for 3 minutes at 1000 rpm .
the cell pellet was re - suspended thoroughly in the complete dmem and transferred to an appropriately - labeled t-75 flask with additional dmem to reach a final volume of 25 ml . in vitro cytotoxicity assay : pancreatic cancer cells ( panc-1 , bxpc-3 or capan-2 ) , were seeded in 96-well plates at a density of 23,000 cells per well and incubated in a fresh dmem , ( sigma - aldrich ) at 37 c , 5% co2 for 24 h. after rinsing with pbs , cells were subjected to the addition of nrm , ndm , or special media conditions .
serially diluted solutions of synthesized compounds ( 5.5% v / v dmso in ndm ) were added to the cells up to a series of concentrations of 100 m , 50 m , 25 m , 12.5 m and 6.25 m , followed by a 24 h incubation at 37 c , 5% co2 .
cytotoxicity was assessed on pbs washed cells by the addition of fresh dmem containing 10% wst-8 cell counting reagent ( dojindo ) . following a 3 h incubation at 37 c ,
5% co2 , absorbance values were measured with a plate reader at 450 nm , and cell viability was calculated using the equation : at least two replicate experiments were conducted for each medium condition reported , and similar results were obtained .
compound 5 was synthesized as previously described.9 briefly , to an oven - dried 100 ml round bottom flask prepared with a magnetic stirring bar , a rubber septum cover , and a nitrogen inlet , 6-hydroxycoumarin ( 486.4 mg , 3.0 mmol ) and 4 ml of anhydrous n , n - dimethylformamide ( dmf ) were added .
the solution was cooled to 0 c in a salt - ice bath and sodium hydride ( 120 mg of 60% mineral oil suspension , 3.0 mmol ) was added to the flask .
farnesyl bromide ( 1.0 ml , 3.69 mmol ) dissolved in 1 ml of anhydrous dmf was cooled to 0c and added dropwise to the reaction flask through the rubber septum using a syringe .
the reaction mixture was left stirring under nitrogen with warming to room temperature overnight and concentrated in vacuo .
the desired product was obtained by column chromatography using a solvent system of 100% hexanes , 20:1 hexanes : ethyl acetate , 15:1 hexanes : ethyl acetate , 10:1 hexanes : ethyl acetate , 7:3 hexanes : ethyl acetate , and 2:1 hexanes : ethyl acetate .
the identity of the purified product was confirmed by nmr spectroscopy ( h - nmr , c - nmr ) and high - resolution mass spectrometry ( hrms ) .
h - nmr ( 300 mhz , cdcl3 ) : 1.60 - 1.76 ( 12h ) , 1.96 - 2.12 ( 8h ) , 4.56 ( d , 2h , j=6 hz ) , 5.09 ( m , 2h ) , 5.48 ( t , 1h ) , 6.43 ( d , 1h , j=9 hz ) , 6.92 - 7.66 ( 4h ) ; c - nmr ( 75 mhz , cdcl3 ) : 15.93 , 16.29 , 17.70 , 25.71 , 26.29 , 27.04 , 39.53 , 39.69 , 109.75 , 117.00 , 117.83 , 119.14 , 123.31 , 123.77 , 128.33 , 131.38 , 135.56 , 141.96 , 143.27 , 148.40 , 155.33 , 161.05 .
human pancreatic cancer cell lines , panc-1 , bxpc-3 , and capan-2 were cultured in dulbecco modified eagle 's medium ( dmem ) . to prepare dmem ,
one bottle of dmem powder ( sigma - aldrich ) was dissolved in 800 ml of hplc grade water .
sodium bicarbonate solution ( 49.1 ml ) and antibiotic - antifungal solution ( 10 ml , 100x ) were added to the mixture .
the volume was brought to 900 ml and the solution was adjusted to a ph between 7 - 7.4 .
heat - inactivated fetal bovine serum ( fbs , 100 ml ) was added to the neutralized solution and filtered via a 0.2-m corning filter .
for all medium conditions , the following electrolytes and vitamin solution were added in concentrations as follows : cacl2(2h2o ) , 265 mg / l ; fe(no3)(9h2o ) , 0.1 mg / l ; kcl , 400 mg / l ; mgso4(7 h2o ) , 200 mg / l ; nacl , 6400 mg / l ; nahco3 , 700 mg / l ; nah2po4 , 125 mg / l ; phenol red , 15 mg / l ; hepes buffer ( 25 mm , ph 7.4 ) ; and mem vitamin solution ( 1x / l ) ( life technologies , inc . , rockville , md ) , which completed the preparation of the ndm . for nrm , additional nutrients were supplement at concentrations as follows : d - glucose , 1000mg / l ; l - glutamine , 2 mm ; mem amino acids solution and mem nonessential amino acids solution ( life technologies , inc . ) , 20 ml and 10 ml respectively for 1 l medium ; fbs , 100 ml for 1 l medium .
for all special media combinations , corresponding combinations of nutrients were added at concentrations identical to those in nrm . for media with dialyzed serum , the same concentration of dialyzed serum ( life technologies , inc . )
was added as a replacement of fbs ( 100 ml for 1 l medium ) .
to a cell - containing t-75 culture flask , a 2 - 5 ml aliquot of filtered trypsin - edta was added to detach the cells after the aspiration of dmem and a pbs wash .
the trypsin - containing flask was placed in the incubator ( 37c and 5% co2 ) for 2 - 3 minutes and then monitored under a microscope with 10x amplification .
detached cells were transferred to a 15 ml centrifuge tube and pelleted for 3 minutes at 1000 rpm .
the cell pellet was re - suspended thoroughly in the complete dmem and transferred to an appropriately - labeled t-75 flask with additional dmem to reach a final volume of 25 ml .
in vitro cytotoxicity assay : pancreatic cancer cells ( panc-1 , bxpc-3 or capan-2 ) , were seeded in 96-well plates at a density of 23,000 cells per well and incubated in a fresh dmem , ( sigma - aldrich ) at 37 c , 5% co2 for 24 h. after rinsing with pbs , cells were subjected to the addition of nrm , ndm , or special media conditions .
serially diluted solutions of synthesized compounds ( 5.5% v / v dmso in ndm ) were added to the cells up to a series of concentrations of 100 m , 50 m , 25 m , 12.5 m and 6.25 m , followed by a 24 h incubation at 37 c , 5% co2 .
cytotoxicity was assessed on pbs washed cells by the addition of fresh dmem containing 10% wst-8 cell counting reagent ( dojindo ) .
following a 3 h incubation at 37 c , 5% co2 , absorbance values were measured with a plate reader at 450 nm , and cell viability was calculated using the equation : at least two replicate experiments were conducted for each medium condition reported , and similar results were obtained .
compound 5 was synthesized as previously described.9 briefly , to an oven - dried 100 ml round bottom flask prepared with a magnetic stirring bar , a rubber septum cover , and a nitrogen inlet , 6-hydroxycoumarin ( 486.4 mg , 3.0 mmol ) and 4 ml of anhydrous n , n - dimethylformamide ( dmf ) were added .
the solution was cooled to 0 c in a salt - ice bath and sodium hydride ( 120 mg of 60% mineral oil suspension , 3.0 mmol ) was added to the flask .
farnesyl bromide ( 1.0 ml , 3.69 mmol ) dissolved in 1 ml of anhydrous dmf was cooled to 0c and added dropwise to the reaction flask through the rubber septum using a syringe .
the reaction mixture was left stirring under nitrogen with warming to room temperature overnight and concentrated in vacuo .
the desired product was obtained by column chromatography using a solvent system of 100% hexanes , 20:1 hexanes : ethyl acetate , 15:1 hexanes : ethyl acetate , 10:1 hexanes : ethyl acetate , 7:3 hexanes : ethyl acetate , and 2:1 hexanes : ethyl acetate .
the identity of the purified product was confirmed by nmr spectroscopy ( h - nmr , c - nmr ) and high - resolution mass spectrometry ( hrms ) .
h - nmr ( 300 mhz , cdcl3 ) : 1.60 - 1.76 ( 12h ) , 1.96 - 2.12 ( 8h ) , 4.56 ( d , 2h , j=6 hz ) , 5.09 ( m , 2h ) , 5.48 ( t , 1h ) , 6.43 ( d , 1h , j=9 hz ) , 6.92 - 7.66 ( 4h ) ; c - nmr ( 75 mhz , cdcl3 ) : 15.93 , 16.29 , 17.70 , 25.71 , 26.29 , 27.04 , 39.53 , 39.69 , 109.75 , 117.00 , 117.83 , 119.14 , 123.31 , 123.77 , 128.33 , 131.38 , 135.56 , 141.96 , 143.27 , 148.40 , 155.33 , 161.05 . hrms ( ci ) calcd . for c24h31o3 : 367.22733 ; found : 367.22801 .
compound 5 was prepared via a williamson ether synthesis using farnesyl bromide 6 and 6-hydroxycoumarin sodium alkoxide 7 pre - generated by treating the hydroxycoumarin with sodium hydride ( figure 3 ) .
the desired compound was purified by flash column chromatography and was thoroughly characterized by 1h- and 13c - nmr spectroscopy and high resolution mass spectrometry .
compound 5 was tested in vitro for its cytotoxic activity against panc-1 cells under different medium conditions ( figure 4 ) .
our previous studies have shown that 5 exhibits selective cytotoxicity against panc-1 cells under nutrient - deprived conditions with no activity observed under nutrient - rich conditions.9 the role of three essential medium components , amino acid ( aa ) , glucose ( glu ) and serum ( ser ) were systematically evaluated to account for the difference in the cytotoxic activity of 5 observed under nutrient - rich vs nutrient - deprived conditions . to better focus on the contribution of glucose to the viability of panc-1 cells in the presence of 5
, we have also run assays in the presence of dialyzed serum ( dia ser ) .
cell culture medium lacking all three nutrients ( nutrient - deprived medium , ndm ) and medium containing all three nutrients ( nutrient - rich medium , nrm ) were selected as controls .
the survival of panc-1 cells was examined within 24 h after the exposure to 5 by using the wst-8 reagent to assay for cell viability .
media compositions that demonstrate < 25% cell death in the presence of 5 are designated inactive and media compositions that demonstrate > 25% cell death in the presence of 5 are designated active . in this study , exposure of panc-1 cells to 100 m of compound 5 for 24 hours led to complete cell death in ndm and the cell culture medium combination of ndm and aa , whereas less than 40% cell death was observed under all other medium conditions examined ( figure 5 ) .
for example , when panc-1 cells are deprived of glucose or very low levels of glucose are present , complete or almost complete cell death is observed upon 24-hour exposure to 100 m of compound 5
. the relatively unimportant role of the presence of aa in the observed cytotoxic activity is highlighted by the medium combination of ndm and aa ( i.e. , lacking both serum and glucose ) , which displayed a similar effect on the cytotoxicity of 5 as pure ndm .
the cytotoxicity of 5 was also investigated in vitro against two other pancreatic cancer cell lines , bxpc-3 and capan-2 ( figure 6 ) .
similar to panc-1 , both cell lines exhibited a preferential sensitivity to compound 5 only under nutrient - deprived conditions ( ndm ) , with lc50 values of 5 m for both cell lines ( table 1 ) .
numerous anti - austerity agents have been reported to possess preferential cytotoxicity against panc-1 under nutrient - deprived conditions .
key nutrients were examined to obtain information about the sensitivity of panc-1 cells to other potential anticancer agents including troglitazone6 , ly2940026 , kigamicin d10 , pyrvinium pamoate11 , arctigenin12 , and damnacanthal.13 among all compounds , troglitazone induced necrotic panc-1 cell death under the depletion of glucose and serum .
kigamicin d , pyrvinium pamoate and arctigenin induced necrotic panc-1 cell death under glucose deprivation . however ,
when panc-1 cells were exposed to damnacanthal , it was observed that the lack of serum was key to obtain cytotoxicity .
our data suggest that glucose may be the key component linked to the activity of compound 5 .
it has been established for some time that cancer cells activate an altered metabolic regime that emphasizes the use of glycolysis over oxidative phosphorylation , classically referred to as the warburg effect.14 our observations would suggest that while glucose is plentiful even in supplemented ndm or ndm containing serum , compound 5 is less cytotoxic , presumably because of metabolic reorganization .
however , in the absence of serum or when the supply of glucose is limited by the addition of dialyzed serum to ndm , compound 5 becomes increasingly effective .
this would imply that the efficacy of 5 targets a salvage pathway when glycolysis is less critical for cancer cell survival .
the regulated cytological degradation known as autophagy is characteristically a part the biology of cancer cells and provides a mechanism by which cells can obtain essential nutrients from the dissolution of endogenous reservoirs ( e.g. , mitochondria ) .
interestingly , autophagy has been demonstrated to be integral to the survival of pdac cells such as panc-1 in the presence of chemotherapeutic drugs such as gemcitabine and 5'-fluorouracil.15 with respect to the correlation between glycolytic dependency and autophagy , it is of note that ( see figure 4 ) under limited glucose availability ( ndm + dia ser and ndm + dia ser + aa ) , compound 5 only becomes fully effective at higher concentrations .
it has not escaped our attention that the structure of 5 is in some regards similar to known inhibitors of autophagy ( e.g. , chloroquine ) .
autophagy as a therapeutic target has been explored extensively of late16 , both from the standpoint of inhibition15 , and hyper - stimulation17 at least in panc-1 cells .
the anti - proliferative activity displayed by compound 5 against the two other pdac cell lines , bxpc-3 and capan-2 , in this study hint at a broad utility for this coumarin derivative . since autophagy is of such prime importance to the progression of pancreatic tumors , the hallmark of which is extreme physiological stress , we are presently investigating our hypothesis that the mode of action of 5 is via an inhibition of autophagy . | pancreatic cancer is one of the most devastating forms of human cancer .
the lack of effective clinical treatments for pancreatic cancer has led to one of the lowest five - year survival rates among all cancers .
recently , our laboratory has developed a novel series of isoprenylated coumarin derivatives that have exhibited anti - pancreatic cancer activity exclusively under nutrient - deprived conditions . in this study , we report the effect of the various cell culture medium components on the preferential cytotoxicity of our lead isoprenylated coumarin compound against the pancreatic adenocarcinoma cell line panc-1 . in particular , our findings show a clear link between observed cytotoxicity and glucose deprivation , suggesting that our compound targets a salvage pathway when glycolysis is no longer an option for cancer cell survival .
the cytotoxicity of our lead compound was also examined in vitro against two other pancreatic cancer cell lines , bxpc-3 and capan-2 under both nutrient - rich and nutrient - deprived conditions . |
helicases are enzymes that separate in an energy - dependent manner stretches of duplexed dna and/or rna into single - stranded components . currently , based on characteristic motifs and the sequence comparisons , three superfamilies ( sf1 through 3 ) and two smaller families ( f4 , f5 ) of helicases have been identified ( 1 ) .
superfamily 1 and 2 contain helicases which share seven or more recognized signature amino acid motifs while sf3 and f4 and f5 helicases are characterized generally by three conserved motifs ( 2 ) ; the f4 and f5 proteins are largely bacterial and bacteriophage proteins .
currently , it should be cautioned that many helicases are not bona fide helicases , but may only function as rna translocases , perhaps to fulfill functions in the remodeling of ribonucleoprotein complexes ( rnp ) .
dead - box and the related deah , dexh and dexd ( 3 ) helicases are the most numerous members of sf2 and are ubiquitously present in eukaryotic genomes .
these helicases share eight conserved motifs and are commonly refered to as the dexh / d family of helicases .
humans , arabidopsis and saccharomyces have 38 , 55 and 25 such entities , respectively ( 4 ) .
differing from dna helicases and dexh proteins , dead helicases are poor in unwinding long nucleic acid duplexes and are best suited for separating short rna hybrids .
this suggests that the specificity determinants for dead helicases may be through the recognition of protein factors . in this regard ,
a better understanding of the roles for dead proteins depends on the clear characterization of their respective interacting proteins .
although the precise substrate for most helicases awaits definition , dead helicases are generally thought to participate pleiotropically in many aspects of rna metabolism including transcription , mrna splicing , mrna export , translation , rna stability and mitochondrial gene expression ( 58 ) .
uap56 , brr2 , prp16 , prp22 and prp43 play roles in rna - splicing ( 4,9 ) , while dbp5 ( 10,11 ) and ddx3 ( 12 ) chaperone rnas from the nucleus into the cytoplasm .
eif4a and ded1 serve for translation of mrnas while rh1b , ski2 , dob1 , dhh1 helicases contribute to mrna stability ( 4 ) .
other dead helicases act in ribosome biogenesis through regulation of small nucleolar rnas and ribosomal rnas ( rrnas ) interactions ( 13,14 ) .
finally , neurospora and trypanosoma dead proteins contribute to mitochondrial gene expression ( 15,16 ) ; a cryptococcus dead helicase is required for cryptococcosis pathogenesis ( 17 ) , and the dipteran chironomus tentans uses a hrp84 dead helicase to regulate mrna transport from the nucleus into the cytoplasm onto polyribosomes ( 18 ) .
given that helicases significantly contribute to normal cellular metabolism , are they similarly essential to viruses ?
the operational answer appears to be a qualified yes. indeed , when dead / deah - box helicase motif ( interpro ipr001410 ) was used to search the embl - ebi database , 1561 matches to individual viral sequence entries were found ( ) , suggesting that many viruses have evolved to encode directly helicase or helicase - like proteins .
the strongest biological evidence which supports the importance of a helicase in the virus life cycle comes from those viruses with an rna genome .
hence , all positive - strand rna viruses encode one or more helicase / helicase - like open reading frame ( orf ) which , aside from the rna - dependant rna polymerase , is the most highly conserved viral sequence .
although less ubiquitous , helicases are also found in other types of viruses ( see some examples listed in table 1 ) .
direct mutagenesis studies have confirmed that a helicase function is biologically required for the replication of many viruses including vaccinia virus ( 19 ) , poliovirus ( 20 ) , alphaviruses ( 21 ) , brome mosaic virus ( 22 ) , nidoviruses ( 23,24 ) and flaviviruses ( 2527 ) .
in 1981 , the first cases of acquired immunodeficiency syndrome ( aids ) were described in american homosexual men .
thereafter , within three short years , french and american scientists confirmed that the human immunodeficiency virus ( hiv ) is the causative agent for aids . in the ensuing 20 years ,
> 20 million individuals have died from aids ; and currently , in 2006 , 50 million people worldwide are infected by hiv-1 with 3 million incremental aids deaths and 45 million new infections occurring annually .
hiv-1 is a retrovirus of the lentivirus genus with an rna genome of 9 kilobases which encodes nine polypeptides .
the major hiv-1 structural proteins are encoded by three genes , gag ( group - specific antigen ) , pol ( polymerase ) and env ( envelope ) , while the accessory proteins , vif , vpu , vpr and nef , and the regulatory proteins , tat and rev , are the primary translation products of multiply - spliced mrna .
hiv-1 infects cd4 + human t - cells and macrophages and integrates as a provirus into the host cell 's dna .
gene expression of hiv-1 is governed transcriptionally by a viral protein , tat ( 28,29 ) , via its binding to a nascent viral tar rna ( 30 ) , and post - transcriptionally by a second viral protein rev ( 31,32 ) through its association with the viral rre rna . both tat and rev interact with several host cell proteins in their transcriptional and post - transcriptional functions ( 33 )
hiv-1 does not encode for any rna helicase ; however , findings suggest that host cell rna helicases may be involved in the reverse transcription of hiv-1 rna , in hiv-1 mrna transcription and in the nucleus - to - cytoplasm transport of viral mrna .
a recent unexpected finding revealed the possibility that an rna helicase may potentially contribute roles in hiv-1 particle assembly and reverse transcription ( 34 ) . using proteomic analyses ,
roy et al . ( 34 ) reported that the deah protein rna helicase a ( rha ) was found associated with hiv-1 gag and packaged into hiv-1 virions in an rna - dependent manner . when rha was knocked down in cells , hiv-1 particles which were produced from these cells were significantly less infectious .
first , it is conceivable that rha participates in the formation of infectious virus particles either by shaping gag
second , roy et al . ( 34 ) reported evidence that hiv-1 particles that do not contain rha showed reduced virion - endogenous reverse transcriptase activity . in this respect
, it may be that rha assists hiv-1 reverse transcriptase to more efficiently copy rna by unwinding rna secondary structure or by promoting the interaction of viral rna with the nucleocapsid protein in order to assemble a better reverse transcription complex .
separate from reverse transcription , the unwinding of highly structured rnas might also be reasoned to be important for transcription ( 35 ) . however , direct evidence for an rna helicase role has been somewhat elusive .
first , in vaccinia virus , it has been postulated that the nph - ii helicase assists transcription by strand - separating duplexed rna structures to prevent r - loop formation behind the elongating rna polymerase ( 36 ) .
second , rha has been invoked to provide a factor - recruitment role , bridging at the promoter the creb - binding protein ( 37 ) and rna polymerase ii ( 37 ) .
third , the p68 dead - box helicase was shown recently to be a novel transcriptional co - activator for p53 's transcriptional function ( 38 ) .
interestingly , in the latter two instances , neither the atpase nor the helicase activity of rha and p68 is apparently required for their attributed transcriptional roles . for hiv-1 ,
two recent studies provide clues that rna helicases may also serve co - factor function for transcription from the viral long terminal repeat ( ltr ) .
fujii et al . ( 39 ) observed that rha conserves in its n - terminus two double - stranded rna - binding ( dsrbd ) domains characterized previously for the tar rna - binding protein , trbp ( 40,41 ) .
these investigators found in both reporter and virus replication assays that rha activated , in a tar rna - binding dependant manner , hiv-1 ltr - directed transcription ( 39 ) .
next , cocude et al . ( 42 ) separately described that the expression of a dexh rna helicase , rh116 , was significantly induced after infection of hela - cd4 cells by hiv-1 .
rh116 was found localized in the nucleus of hiv-1 infected cells and was observed to augment the transcription of unspliced hiv-1 transcripts . while both studies offer tantalizing evidence for roles by two different rna helicases in hiv-1 transcription , critical details on how the helicases function by binding to tar rna and how this binding might cooperate with the activities of the viral transcriptional activator , tat ,
pending more direct experimental tests , it remains unclear whether rha and rh116 provide direct roles in transcription or indirectly influence the milieu of polymerase ii initiation / elongation at the ltr .
downstream from transcription , the fate of hiv-1 encoded rna is regulated at the step of export of unspliced / partially spliced moieties from the nucleus into the cytoplasm .
unspliced and partially spliced viral rnas code for genomic rnas that are packaged into progeny virions and structural proteins .
hence , the egress of these rnas from the nucleus into the cytoplasm is critical to the life cycle of the virus .
exit of hiv rnas from the nucleus is a significant issue because unspliced / partially spliced cellular mrnas are routinely retained in and not permitted export from the nucleus ( 4346 ) .
thus , it was established that the hiv-1 encoded rev protein binds a highly secondary structured element ( rev responsive element ; rre ) present in all unspliced and partially spliced hiv transcripts ( 4757 ) ; and this binding specifically distinguishes , for purposes of nuclear export , viral transcripts from cellular rnas .
new evidence now suggests that rna helicases are also co - factors for rev - directed export of hiv-1 mrnas ( 58 ) . in its role of transporting unspliced and incompletely spliced viral rnas from the nucleus , rev directly interacts with nuclear export receptor crm1 ( 59,60 ) , and crm1 is required for rev - mediated export of hiv rnas ( 59,61,62 ) .
a recent report provides data that an rna helicase , ddx3 , is an additional player in the rev
thus , it was shown that ddx3 over - expression enhanced rev - dependent , but not other export , pathway ; and that ddx3 is a nucleo - cytoplasmic shuttling protein which binds crm1 and rev .
moreover , ddx3 's necessity for rev / rre / crm1 function was demonstrated by knock - down of cell endogenous ddx3 . finally , because ddx3 locates to nuclear pore complexes ( npc ) , yedavalli et al .
( 12 ) further proposed that this human helicase , like the analogous yeast dbp5p ( 11 ) , may function with rev / crm1 to remodel and thread large unspliced hiv-1 rnas through the nuclear pore , facilitating their final release to the cytoplasmic side of the npc .
the above ddx3 results are consistent with two additional papers which described similar findings for a related rna helicase , ddx1 .
thus , pomerantz and co - workers ( 63 ) showed that ddx1 binds directly to the n - terminus of rev and to the rre - rna motif and participates in the export of unspliced hiv-1 rna from the nucleus to the cytoplasm .
additionally , they illustrated that reduced expression of ddx1 in astrocytes explains the previously observed tissue restricted function of hiv-1 rev ( 64 ) . fully spliced viral mrnas encoding for viral tat , rev and nef , proteins have been shown previously to exit the nucleus using the cellular mrna export pathway .
as yet , the involvement of dbp5 in export of spliced hiv-1 viral rna has not been fully clarified .
the story of hiv-1 and rna helicases is , however , likely to be more complex than and unlikely to conclude simply with rha , rh116 , ddx1 and ddx3 ( figure 1 ) .
splicing is a multiple - step process requiring the recognition of splice sites by spliceosomes .
it is generally believed that remodeling of rna rna and rna protein interactions within the spliceosome is catalyzed by a family of dead / dexh box rna helicases . to date , seven mammalian proteins that are rna helicases have been implicated in mrna splicing ( 67,68 ) . whether there is specific preference by subclasses of rna helicases for viral mrna splicing remains to be clarified .
moreover , how cellular rna helicases might contribute to the translation of viral mrnas also require further investigation .
( 69 ) and krishnan and zeichner ( 70 ) have provided evidence that the expression of several cellular rna helicases including ddx24 , ddx21 , ddx18 , ddx11 and ddx9 is modulated during hiv-1 infection ; however , the precise cellular role and significance of these helicases for hiv-1 pathogenesis have not be characterized .
interestingly , krishnan and zeichner reported microarray data which examined the transition of hiv-1 infection from latency to productive replication , and found that several cellular rna helicases were upregulated ( 71 ) . for future understanding of functions
, it will be important to design experiments which can segregate helicases which serve direct , although perhaps overlapping and redundant , roles on hiv-1 from those that might participate indirectly in the viral life cycle .
nevertheless , the convergence of evidence would support that several discrete cellular rna helicases contribute importantly to the efficient execution of several steps in the hiv-1 replicative cycle .
given that the hiv-1/aids disease burden has reached pandemic global proportions , new antiviral strategies that target molecularly delineated mechanisms used by this virus are urgently needed ( 72 ) .
is there a possibility that host cell helicases can be therapeutic targets for anti - hiv-1 chemotherapy ?
implicit within this question is the concept that one could attack a host cell protein in order to treat an infecting pathogen .
although targeting a cellular protein involved in a viral pathway risks obvious cytotoxicity , this approach avoids the inherent problem posed by rapid hiv-1 mutation to all currently utilized chemotherapeutics targeted to virus - encoded proteins .
we note that inhibition of cell - encoded enzymes in medical therapy is not an unprecedented strategy .
suppression of angiotensin - converting enzyme ( ace ) is widely used to treat hypertension , congestive heart failure , myocardial infarction , endothelial dysfunction and renal disease ( 73 ) .
elsewhere , aromatase inhibitors have been used to treat hormone - dependent breast cancer ( 74 ) , and inhibitors of cellular secretory proteases are contemplated for alzheimer 's disease ( 75 ) .
we recently inhibited the cellular polyprotein convertase , furin , at minimal toxicity to the cell in order to block hiv-1 replication ( 76 ) .
thus , a priori exclusion of cellular helicase as an antiviral target is not warranted .
guarded optimism that small molecule helicase inhibitors can be developed against viruses arises from encouraging progress in non - retroviral systems . unlike hiv-1 , human herpesviruses physically encode helicases .
the herpes simplex virus ul5 and ul9 genes are helicases in superfamily 1 and 2 , respectively ( 77 ) .
two recent studies provide proof - of - concept that the hsv helicase primase can be targeted at low host cell toxicity by two new classes of drugs , amino - thiazolyphenyl - molecules ( 78 ) and thiazole amide derivatives ( 79 ) .
in addition , other studies suggest that the ns3 protein , a rna helicase encoded by hepatitis c virus and related west nile virus and japanese encephalitis virus can be targeted to inhibit viral replication ( 8082 ) . this conceptual break through in drug development is important because it indicates that target discrimination between different helicases by small molecule inhibitors is possible . of relevance to hiv-1 ,
a synthetic immunomodulator murabutide was shown recently to suppress hiv-1 replication in macrophages and t cells .
murabutide was shown to inhibit the activity of rna helicase rh116 , blocking its positive transcriptional activity for hiv-1 gene expression ( 42 ) .
if one looks beyond the signature motifs conserved amongst helicases , then it becomes clear that the different proteins are widely divergent in their coding sequences . in principle
, this suggests that individual helicases can be abrogated with specificity in a knowledge - directed manner . in theory ,
a helicase can be attacked by ( i ) inhibition of ntpase activity through direct competition for ntp binding , ( ii ) inhibition of substrate binding through direct competition at active site , ( iii ) allosteric mechanisms to affect ntp - binding / ntp hydrolysis and/or polynucleotide binding , and ( iv ) inhibition of unwinding activity by steric hinderance of helicase translocation along the polynucleotide substrate ( 83,84 ) . because the ntp - binding and substrate - binding pockets may be sufficiently similar between various helicases , specificity of inhibition through these sites
on the other hand , the tremendous variations in sequence and sizes of helicases , in their oligomerization states , in their discrete domains responsible for protein protein interactions and/or for targeting to specific nucleic acids ( 85 ) , and in their differential localizations within cells ( 86 ) offer interventional possibilities outside of the ntp- or polynucleotide - binding sites .
we are in the preliminary stages of screening ring - expanded nucleoside analogs found previously to be successful ntpase / helicase inhibitors of west nile virus , hepatitis c virus and japanese encephalitis virus ( 81,82 ) .
we have observed that a few of these candidate inhibitors have substantial anti - hiv-1 activity at doses that do not incur cytotoxicity to cells treated in tissue culture for 1 week .
further studies are needed before concluding that these compounds exert specific inhibition of ddx3 , one of the other cellular helicases , or some other target altogether .
there is another area where a cellular helicase activity and hiv-1 are likely to intersect .
an emerging research focus is the role of small interfering rnas ( 87 ) and micrornas ( mirna ) as innate cell defenses against viruses including hiv-1 ( 8790 ) . in human cells ,
the precursor for mirna ( pre - mirna ) is processed by dicer ( figure 2a ) which is a ribonuclease with a bona fide rna helicase domain ( 91,92 ) . a surprising recent finding revealed that the human trbp , which has been shown to be a potent binder of the hiv-1 tar rna rna ( 40,41 ) , is an indispensable dsrna - binding partner of dicer which allows the latter to associate with pre - mirna ( 91,92 ) . without trbp , dicer 's mirna processing activity is lost .
helicase protein dicer requires trbp to process duplex - structured mirnas in order that the cell can use such matured mirnas for antiviral defense , it could be speculated that hiv-1 has evolved to restrict this defense by the ability to transcribe viral tar rna to squelch trbp away from dicer ( y. bennasser , m. l. yeung and k. t. jeang , manuscript submitted ) ( figure 2b ) .
if this thinking is correct , then hiv-1 has developed mechanisms not only to co - opt the active functions of a virus - propitious cellular helicase ( i.e. ddx3 ) but also to inactivate the role of a second virus - pernicious helicase ( i.e. dicer ) for purposes of selfish gain . in a separate perspective
, virus infection can trigger through double - stranded viral rnas an innate antiviral immune response .
thus viral dsrnas can be recognized by cellular proteins [ pattern - recognition receptors ( prrs ) ] which initiate antiviral responses by inducing the production of a variety of cytokines including type i interferons ( ifn- and ifn- ) and initiating additional inflammatory and adaptive immune responses .
recently , dexd / h rna helicases such as rig-1 ( retinoic acid inducible gene-1 ) ( 93 ) and mda5 ( melanoma differentiation - associated gene 5 ) ( 94 ) have been identified as suppressors of viral replication by binding to virus associated dsrna and activating type i interferon - dependent antiviral immunity . over - expression of rig-1 and mda5
currently , it remains speculative whether helicases like rig-1 and mda5 may recognize hiv-1 dsrna and trigger an innate immune response .
intriguingly , several reports exist in the literature that hiv-1 infection does induce activation of type 1 interferons ( 95,96 ) . in conclusion , by studying helicase proteins one can gain insights into normal cellular metabolic processes , abnormal inherited human diseases ( e.g. bloom syndrome , werner syndrome , cockayne 's syndrome and xeroderma pigmentosum ; all diseases with mutations in cellular helicases ) , and remarkably also the biology of viruses .
schematic representation of the involvement of cellular rna helicases in reverse transcription , transcription and post - transcriptional regulation of hiv-1 rnas .
rha has been found to be a part of the hiv-1 virion and may participate in hiv-1 reverse transcription as well as particle assembly .
rha and rh116 have been reported to participate in hiv-1 transcription through interaction with the viral leader rna , tar .
ddx1 and ddx3 act with rev and rre - containing rnas for the transport of unspliced and partially spliced hiv-1 rna from the nucleus to the cytoplasm .
involvement of dicer rna helicase , trbp and hiv-1 tar rna in the biology of the cell 's micro rna pathway .
( b ) a schematic representation of a potential mechanism for tar rna - dependent suppression of dicer activity .
tar rna is suggested to bind to trbp and decoy it from dicer preventing dicer - dependent mirna processing activity . | viruses are replication competent genomes which are relatively gene - poor .
even the largest viruses ( i.e. herpesviruses ) encode only slightly > 200 open reading frames ( orfs ) .
however , because viruses replicate obligatorily inside cells , and considering that evolution may be driven by a principle of economy of scale , it is reasonable to surmise that many viruses have evolved the ability to co - opt cell - encoded proteins to provide needed surrogate functions .
an in silico survey of viral sequence databases reveals that most positive - strand and double - stranded rna viruses have orfs for rna helicases . on the other hand ,
the genomes of retroviruses are devoid of virally - encoded helicase . here
, we review in brief the notion that the human immunodeficiency virus ( hiv-1 ) has adopted the ability to use one or more cellular rna helicases for its replicative life cycle . |
one of the major risk factors for the development of coronary heart disease is high low - density lipoprotein ( ldl ) cholesterol .
ldl is a lipoprotein made up of outer phospholipids , apolipoproteins , free cholesterol and inner triglycerides ( tgs ) and cholesterol ester .
-quantification is the standard method for estimating ldl , which includes ultracentrifugation and chemical precipitation .
-quantification requires ultracentrifuges and takes time , delaying the turnaround time and hence can not be employed in day to day practice .
automated methods are available for direct ldl estimation which is expensive and requires significant time for analysis .
came up with a formula for estimating ldl using total cholesterol , high - density lipoprotein ( hdl ) cholesterol and tgs . according to friedewalds formula , tgs divided by five gives the value of very - ldl ( vldl ) .
tgs > 400 mg / dl , disorders related to lipoproteins ( type iii hyperlipoproteinemia ) and secondary hyperlipoproteinemias are conditions wherein friedewalds equation for calculating ldl can not be employed since these conditions decrease the accuracy of friedewalds equation in estimating ldl .
the accuracy and targets to be achieved regarding the analytical performance of ldl cholesterol were issued by national cholesterol education program ( ncep ) panel . as per ncep guidelines , precision should be < 4% , bias < 4% and total analytical error should be < 12% . falsely , low values of ldl cholesterol has been reported in conditions such as diabetes mellitus , advanced renal disease and liver failure due to elevated tgs in these conditions . in spite of these well - established limitations of friedewalds equation in estimating ldl cholesterol , it remains to be widely employed .
many studies have been published questioning the accuracy of friedewalds equation in measuring ldl cholesterol especially at levels of tgs above the normal range .
this study was taken up to study the accuracy of friedewalds calculated ldl in comparison to direct ldl method .
the current study was a validation study . assuming a paired mean difference of 12.39 67.0 between the ldl measured by the direct and friedewald method , the sample size required for the study was estimated to be 234 to achieve 80% power of the study and 5% significance level .
lipid profile was estimated in 248 samples which were estimated in venous blood drawn after 12 h of fasting between the age group of 2070 years .
patients with tgs more than 400 mg / dl , diabetes mellitus , advanced renal disease and patients with liver failure , patients receiving lipid - lowering drugs were excluded from the study .
the principle of the methods were total cholesterol cholesterol oxidase - peroxidase method ; tgs -enzymatic , colorimetric method ( gpo / pap ) with glycerol phosphate oxidase and 4-aminophenazone ; hdl - cholesterol homogeneous enzymatic colorimetric assay ; and direct ldl cholesterol the homogeneous enzymatic colorimetric assay : this automated method for direct ldl - cholesterol estimation is based on micellar solubilization of ldl - cholesterol by a nonionic detergent and the interaction of a sugar compound and lipoproteins ( vldl and chylomicrons ) .
biorad internal and external controls were run for total cholesterol , tgs , hdl and ldl cholesterol .
friedewalds calculated ldl was calculated by the formula : ldl cholesterol = total cholesterol hdl cholesterol tgs/5 ( vldl cholesterol ) .
categorical data were reported as frequency and continuous data were reported as mean and standard deviation .
paired t - test was used to test the difference in ldl concentration obtained by the direct method and friedewalds calculated method .
data were categorized into three groups based on the tg levels [ table 1 ] .
lipid profile was estimated in 248 samples which were estimated in venous blood drawn after 12 h of fasting between the age group of 2070 years .
patients with tgs more than 400 mg / dl , diabetes mellitus , advanced renal disease and patients with liver failure , patients receiving lipid - lowering drugs were excluded from the study .
the principle of the methods were total cholesterol cholesterol oxidase - peroxidase method ; tgs -enzymatic , colorimetric method ( gpo / pap ) with glycerol phosphate oxidase and 4-aminophenazone ; hdl - cholesterol homogeneous enzymatic colorimetric assay ; and direct ldl cholesterol the homogeneous enzymatic colorimetric assay : this automated method for direct ldl - cholesterol estimation is based on micellar solubilization of ldl - cholesterol by a nonionic detergent and the interaction of a sugar compound and lipoproteins ( vldl and chylomicrons ) . biorad internal and external controls were run for total cholesterol , tgs , hdl and ldl cholesterol .
friedewalds calculated ldl was calculated by the formula : ldl cholesterol = total cholesterol hdl cholesterol tgs/5 ( vldl cholesterol ) .
categorical data were reported as frequency and continuous data were reported as mean and standard deviation .
paired t - test was used to test the difference in ldl concentration obtained by the direct method and friedewalds calculated method .
data were categorized into three groups based on the tg levels [ table 1 ] .
the study group consisted of 248 patients , 151 were males and 97 were females .
table 2 indicates there was no statistical difference between direct ldl values and friedewalds calculated ldl values ( p = 0.140 ) .
table 3 shows there is no statistical difference between direct ldl values and friedewalds calculated ldl at different ranges of tgs , 200 mg / dl ( p = 0.47 ) , 201300 mg / dl ( p = 0.32 ) and 301400 mg / dl ( p = 0.22 ) . table 4 shows pearson correlation which indicates good correlation between direct ldl and friedewalds calculated ldl ( correlation coefficient 0.98 ) . figure 1 indicates scatter plot representing good correlation between direct ldl and friedewalds calculated ldl with correlation coefficient of 0.98 .
figure 2 indicates scatter plot which indicates good correlation between direct ldl and friedewalds calculated ldl at tgs < 200 mg / dl with a correlation coefficient of 0.982 .
figure 3 indicates scatter plot representing good correlation between direct ldl and friedewalds calculated ldl at tgs 201300 mg / dl with correlation coefficient of 0.964 .
figure 4 indicates scatter plot representing good correlation between direct ldl and friedewalds calculated ldl at tgs 301400 mg / dl with correlation coefficient of 0.968 .
comparison between direct ldl method and friedewalds calculated ldl ( n=248 ) comparison between direct ldl method and friedewalds calculated ldl at different serum level of tg ( mg / dl ) pearson correlation between direct ldl and friedewalds calculated ldl scatter plot representing the correlation between direct ldl and friedewalds calculated ldl at triglycerides less than 400 mg / dl ( r = 0.981 , r = 0.963 ) scatter plot representing the correlation between direct ldl and friedewalds calculated ldl at triglycerides < 200 mg / dl ( r = 0.982 , r = 0.966 ) scatter plot representing the correlation between direct ldl and friedewalds calculated ldl at triglycerides 201 to 300 mg / dl ( r = 0.981 , r = 0.964 ) scatter plot representing the correlation between direct ldl and friedewalds calculated ldl at triglycerides 301 to 400 mg / dl ( r = 0.968 , r = 0.938 ) person correlation showed that there exists good correlation between direct ldl versus friedewalds formula ( correlation coefficient = 0.98 ) [ table 4 ] .
the primary target for diagnosis and management of hypercholesterolemia is ldl cholesterol as per ncep adult treatment panel report .
, there was no significant difference between the overall mean of direct ldl cholesterol with that of friedewalds calculated ldl cholesterol [ table 2 ] .
there was no significant difference between direct ldl and friedewalds ldl at different tg levels ranging below 200 mg / dl , 201300 mg / dl , and 301400 mg / dl [ table 3 ] .
observed that friedewalds calculated ldl were lower when compared to direct ldl with tg more than 180 mg / dl .
observed in their study that friedewalds method overestimated ldl cholesterol by 7 mg / dl when compared to direct ldl method .
observed that friedewalds ldl method underestimated ldl cholesterol by 10.39% in comparison with direct ldl method .
observed that friedewalds formula underestimated ldl cholesterol especially when tg value is > 150 mg / dl .
knopfholz et al . observed no significant difference between direct ldl and friedewalds ldl at tgs below 150 mg / dl and above 150 mg / dl which was comparable to findings of this study .
kannan et al . observed friedewalds calculated ldl underestimated ldl cholesterol in comparison to direct ldl method .
they exhibit a negative bias as observed in studies done by rifai et al . and this may result in placing a patient into low risk who actually belongs to high - risk hypercholesterolemia .
nauck et al . in their study observed , direct ldl method has no advantage when compared to calculated ldl method and recommended further validation for direct homogeneous methods .
miller et al . in their study observed no advantage of direct ldl method in comparison to calculated ldl method at tg value below 400 mg / dl .
mora et al . observed in their study the nonassociation of direct ldl with friedewalds ldl in nonfasting samples and they could not demonstrate any advantage of direct ldl in comparison to friedewalds calculated ldl .
they also stated using direct ldl may misclassify the patients into low - risk ncep category because the results of direct ldl were 510 mg / dl lower when compared to friedewalds calculated ldl .
observed friedewalds calculated ldl was accurate for any value of tg below 400 mg / dl . in this study , we observed a similar finding since the ldl cholesterol calculated by friedewalds formula correlated well with direct ldl at tgs below 400 mg / dl [ figures 14 ] .
choi et al . observed that direct ldl values were 5% higher than calculated ldl and in diabetics , the difference was much higher .
observed friedewalds calculated ldl method underestimated ldl levels in comparison to direct method and they concluded that direct ldl method is better than friedewalds calculated ldl in diabetics .
chatterjee and mendez observed there was a good correlation between friedewalds calculated ldl and direct ldl method which is in agreement with the findings of the present study .
lindsey et al . observed friedewalds calculated ldl underestimated ldl levels by 20 mg / dl when compared to direct ldl method .
kaur observed there was no significant difference between the ldl values measured by direct ldl method and friedewalds calculated method in patients with metabolic syndrome .
friedewalds calculated ldl can not be employed in individuals with tg value more than 400 mg / dl .
derived a new formula which correlated well with direct ldl values at tgs higher than 400 mg / dl .
chaudhari et al . observed 38.2% of the study group were classified as high - risk group when ldl was estimated by direct ldl method and 24.9% were classified as high risk when ldl was calculated by friedewalds calculated ldl .
observed friedewalds calculated ldl showed a positive bias at tg level < 150 mg / dl and at tg level between 301 and 400 mg / dl , friedewalds calculated ldl showed a negative bias in comparison to direct ldl .
krishnaveni and gowda observed friedewalds calculated ldl correlated well with direct ldl except at tg level below 100 mg / dl and they observed at tg below 100 mg / dl , anandaraja 's calculated ldl performed better than friedewalds calculated ldl .
charuruks and milintagas observed in their study , direct ldl to be more accurate and precise than friedewalds calculated ldl and they suggested direct ldl method to be used in individuals with tg level > 200 mg / dl .
observed in their study , there was no significant difference between friedewalds calculated ldl and direct ldl at tg concentrations below 100 mg / dl but the ldl values obtained by friedewalds equation was significantly lower when compared to direct ldl method at tg concentrations between 101200 mg / dl and 201300 mg / dl which is not in agreement with the findings of this study .
mohan et al . observed in their study underestimation of ldl of 2025 mg / dl by friedewalds calculated ldl when compared to direct ldl method at tg between the range of 300400 mg / dl .
observed ldl cholesterol measured by direct ldl method was significantly lower than friedewalds calculated ldl and differences in the ldl cholesterol concentrations had no relation with tg concentrations .
gasko observed anandaraja 's calculated ldl correlated better than friedewalds calculated ldl with direct ldl in a brazilian population .
observed the original friedewalds calculated ldl correlated best with direct ldl levels in comparison to modified friedewalds formula and they suggested the chances of error in calculated ldl increases with increase in tgs .
teerakanchana et al . observed a high bias of 20.9 mg / dl at tgs between 301 and 400 mg / dl and a lower bias at tgs below 300 mg / dl with friedewalds calculated ldl in comparison to direct ldl .
sahu et al . observed a significant difference between direct ldl and friedewalds calculated ldl at tg values below 300 mg / dl and there was no significant difference at tg values above 300 mg / dl . according to sahu et al .
, the possible explanation for such a result could be the masking or removal of ldl cholesterol due to the detergent used in direct method .
the sample size of this study was only 248 which is the limitation of the present study .
friedewalds formula can be used to estimate ldl cholesterol , and direct ldl should be employed only in those cases wherein friedewalds formula can not be used like nonfasting samples , patients with tgs more than 400 mg / dl , disorders related to lipoproteins ( type iii hyperlipoproteinemia ) and secondary hyperlipoproteinemias .
| background : one of the risk factors for the development of coronary heart disease is high low - density lipoprotein ( ldl ) cholesterol levels .
national cholesterol education program atp iii guidelines suggest drug therapy to be considered at ldl - cholesterol levels > 130 mg / dl .
this makes accurate reporting of ldl cholesterol crucial in the management of coronary heart disease .
estimation of ldl cholesterol by direct ldl method is accurate , but it is expensive .
hence , we compared friedewald 's calculated ldl values with direct ldl values.aim:to evaluate the correlation of friedewalds calculated ldl with direct ldl method.materials and methods : we compared ldl cholesterol measured by friedewald 's formula with direct ldl method in 248 samples between the age group of 2070 years . paired t - test was used to test the difference in ldl concentration obtained by a direct method and friedewald 's formula .
the level of significance was taken as p < 0.05 .
pearsons correlation formula was used to test the correlation between direct ldl values with friedewald 's formula.results:there was no significant difference between the direct ldl values when compared to calculated ldl by friedewalds formula ( p = 0.140 ) .
pearson correlation showed there exists good correlation between direct ldl versus friedewalds formula ( correlation coefficient = 0.98 ) .
the correlation between direct ldl versus friedewalds calculated ldl was best at triglycerides values between 101 and 200 mg / dl.conclusion : this study indicates calculated ldl by friedewalds equation can be used instead of direct ldl in patients who can not afford direct ldl method . |
heavy metal toxicity due to occupational and domestic exposures raises apprehension over their potential effects on human health and the environment .
chronic arsenic toxicity , also known as arsenicosis , is a global health problem affecting millions of people and mainly caused by drinking of arsenic - contaminated ground water .
the worst affected countries include india , bangladesh , pakistan , japan , and the united states .
arsenic is a naturally occurring metalloid having four primary oxidative states : + 5 , + 3 , 0 , and -3 , among which the trivalent arsenite form ( as2o3 ; as iii ) and the pentavalent arsenate form ( as2o5 ; as v ) are of utmost biological relevance .
the inorganic trivalent species of arsenic are of great concern to the toxicologists compared to the organic one .
arsenic exposure shows various systemic manifestations , such as pigmentation and keratosis of skin , bronchitis , obstructive and/or restrictive pulmonary diseases , liver disorders allied with non - cirrhotic portal fibrosis , gastrointestinal disorder , polyneuropathy , cancer of various organs like skin , lung , liver , kidney and urinary bladder , anemia etc .
arsenic toxicity - acute , subchronic or chronic is known to interfere with hematopoietic and immune systems .
the first tissue that encounters arsenic in the body after its systemic absorption is blood .
arsenic exposure may lead to its accumulation in erythrocytes and cause anemia associated with leucopenia with characteristic neutrophil depletion and thrombocytopenia .
arsenic exerts its toxicity by inactivating numerous enzymes , including the enzymes involved in cellular energy pathways , dna replication , and repair .
apart from this , arsenic attacks cellular redox systems resulting in the generation of excess reactive oxygen species ( ros ) in the form of superoxide anion ( o2- ) and hydroxyl radical ( oh ) leading to substantial oxidative damages of proteins , lipids , and dna . as erythrocytes lack any replenishing machinery and have a considerably higher average life of about 120 days , it therefore can carry the impression of long - term toxic and/or pathological insult .
moreover , in vitro sodium arsenite even at a very low concentration is reported to induce apoptosis in rat bone marrow mesenchymal stem cells . however , a detailed haematological picture in chronic arsenic intoxication has hardly been reported in toxicological studies .
alfa lipoic acid ( ala ) , is an endogenously produced coenzyme and is being used by researchers as a dietary supplement with good bioavailability , both in humans and experimental animals .
the role of ala as anti - oxidant and in the prevention of hematological abnormalities caused due to heavy metal toxicity ( such as lead , cadmium and copper ) has been reported earlier and it has been found that the co - administration of ala with heavy metals maintains normal antioxidant activity and normalize hematological parameters , especially anemia , in rats .
ala is also reported to prevent arsenic toxicity via protecting against oxidative damages in acute and chronic animal model as well as in vitro studies .
a few studies have been undertaken to find the corrective approach of ala on hematological alterations by heavy metals , but protective nature of ala on blood or its components due to subchronic exposure of arsenic in rats are currently lacking .
therefore , the aim of the present study is to find out the protective role(s ) of ala , if any , on arsenic - induced hematological alterations and oxidative stress using rats as experimental animal .
subchronic arsenicosis was developed in wister rats according to standardized protocol of our laboratory . in short , a dose of as2o3 ( 3 mg / kg body weight / rat / day ) was selected and administered per orally over a period of 28 days .
ala was dissolved in olive oil and was administered per orally once daily ( 25 mg / kg body weight / rat ) after about 2 hours of the meal . the dose of ala is calculated keeping the dose for human ( per kg body weight ) essentially as described previously .
twenty - four adult male wister rats weighing 13010 g were selected for this experiment .
the animals were maintained under standard laboratory conditions ( 14 h light : 10 h dark , 252c ) with free access to food and water .
all animal experiments were performed according to the ethical guidelines suggested by the institutional animal ethics committee ( number : 796/ac/03/cpcsea/18 - 01 dated 18.01.2013 ) .
rats were randomly grouped into three , consisting eight rats in each group as control ( group i ) , arsenic - treated ( group ii ) , and arsenic - treated and supplemented with ala ( group iii ) .
all the animals of groups i , ii and iii were provided with a normal diet composed of 71% carbohydrate , 18% protein , 7% fat , and 4% salt mixture and vitamins .
both the group ii and iii rats were gavaged arsenic trioxide solution where only the rats of group iii were given ala per orally after the meal . to overcome the impact of any altered food intake , group i rats were pair - fed with other experimental groups ii and iii .
food and water intake and body weight of the rats were monitored throughout the experimental period .
blood was collected under sterile condition from anesthetized rats by cardiac puncture and kept in both ethylenediaminetetraacetic acid ( edta ) and heparinized vials for hematological and biochemical analyses , respectively .
some portion of edta - treated blood was also used for scanning electron microscopy ( sem ) of erythrocytes .
complete blood counts ( total and differential ) and estimation of hematological indices , including total haemoglobin ( hb ) , packed cell volume ( pcv ) ,
mean corpuscular haemoglobin ( mch ) , mean corpuscular volume ( mcv ) , mean corpuscular haemoglobin concentration ( mchc ) were performed using an automated cell counter ( beckman counter , france ) .
neutrophil to lymphocyte ratio ( nlr ) and platelet to lymphocyte ratio ( plr ) were calculated with the help of absolute count of neutrophil , lymphocyte , and platelet .
plasma concentration of tas was estimated based on the inhibition of radical cation abts [ 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid ) radical cation ] , which has characteristic long wavelength absorbance maxima at 734 nm and calculated from trolox standard curve as described earlier .
the absorbance of the stock solution containing 7 mm abts ( produced from 12 - 16 hour incubation of 7 mm abts and 2.45 mm potassium persulphate in 0.01 m phosphate buffer saline , ph-7.4 ) was adjusted to about 0.700 at 734 nm with 0.01 m phosphate buffer saline , ph-7.4 .
change in absorbance ( a ) was calculated from absorbance reading just before adding sample and after 6 minutes of sample addition was recorded and converted to mm trolox equivalent .
tos was estimated according to the method of erel , which is based on the generation of colored complex of ferric ion in the presence of oxidative components and xylenol orange in acidic medium and calculated from h2o2 standard curve . in short ,
140 l of sample was added to 900 l of reagent 1 ( containing 150 m xylenol orange , 140 mm nacl and 1.35 m glycerol in 25 mm h2so4 solution , ph 1.75 ) and mixed .
the initial reading ( a1 ) was obtained from subtracting absorbance at 800 nm ( secondary wavelength ) from that of 560 nm ( primary wavelength ) .
44 l of reagent 2 ( containing 5 mm ferrous ion and 10 mm o - dianisidine in 25 mm h2so4 solution ) was then added to the above mixture and incubated for 4 minutes .
the value of tos was then obtained using a ( = a2-a1 ) from the h2o2 standard curve .
oxidative stress index ( osi ) was calculated from the ratio of tos and tas { osi=[(tos , m h2o2 equivalent)100/(tas , m trolox equivalent ) ] } according to the standard method and expressed as arbitrary units . for this purpose , the result unit of tas
peripheral blood smear was prepared on grease free glass slides and photographed using zeiss light microscope ( zeiss , thornwood , ny ) and progress capture pro 2.5 software ( genotypic optical systems , gmbh , jena , germany ) using 1000 magnification after staining with the leishmann stain .
briefly , erythrocytes were directly fixed overnight with 2.5% glutaraldehyde solution in pbs , ph 7.2 , and post - fixed by keeping overnight in 1% osmium tetraoxide in the same buffer .
the suspensions were dehydrated in an ethanol series . after drying with carbon dioxide by the critical point method and
sputter coating with gold , samples were examined on a sem ( vegaii lsu , tescan , czech republic ) .
one - way anova followed by tukey s hsd post hoc analysis was performed to check any statistical difference between the parameters of the studied groups .
statistical analysis was carried out using the statistical program packages spss version 16.0 for windows ( spss inc . ,
subchronic arsenicosis was developed in wister rats according to standardized protocol of our laboratory . in short , a dose of as2o3 ( 3 mg / kg body weight / rat / day ) was selected and administered per orally over a period of 28 days .
ala was dissolved in olive oil and was administered per orally once daily ( 25 mg / kg body weight / rat ) after about 2 hours of the meal . the dose of ala is calculated keeping the dose for human ( per kg body weight ) essentially as described previously .
twenty - four adult male wister rats weighing 13010 g were selected for this experiment .
the animals were maintained under standard laboratory conditions ( 14 h light : 10 h dark , 252c ) with free access to food and water .
all animal experiments were performed according to the ethical guidelines suggested by the institutional animal ethics committee ( number : 796/ac/03/cpcsea/18 - 01 dated 18.01.2013 ) .
rats were randomly grouped into three , consisting eight rats in each group as control ( group i ) , arsenic - treated ( group ii ) , and arsenic - treated and supplemented with ala ( group iii ) .
all the animals of groups i , ii and iii were provided with a normal diet composed of 71% carbohydrate , 18% protein , 7% fat , and 4% salt mixture and vitamins .
both the group ii and iii rats were gavaged arsenic trioxide solution where only the rats of group iii were given ala per orally after the meal . to overcome the impact of any altered food intake , group i rats were pair - fed with other experimental groups ii and iii .
food and water intake and body weight of the rats were monitored throughout the experimental period .
blood was collected under sterile condition from anesthetized rats by cardiac puncture and kept in both ethylenediaminetetraacetic acid ( edta ) and heparinized vials for hematological and biochemical analyses , respectively .
some portion of edta - treated blood was also used for scanning electron microscopy ( sem ) of erythrocytes .
complete blood counts ( total and differential ) and estimation of hematological indices , including total haemoglobin ( hb ) , packed cell volume ( pcv ) ,
mean corpuscular haemoglobin ( mch ) , mean corpuscular volume ( mcv ) , mean corpuscular haemoglobin concentration ( mchc ) were performed using an automated cell counter ( beckman counter , france ) .
neutrophil to lymphocyte ratio ( nlr ) and platelet to lymphocyte ratio ( plr ) were calculated with the help of absolute count of neutrophil , lymphocyte , and platelet .
plasma concentration of tas was estimated based on the inhibition of radical cation abts [ 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid ) radical cation ] , which has characteristic long wavelength absorbance maxima at 734 nm and calculated from trolox standard curve as described earlier .
the absorbance of the stock solution containing 7 mm abts ( produced from 12 - 16 hour incubation of 7 mm abts and 2.45 mm potassium persulphate in 0.01 m phosphate buffer saline , ph-7.4 ) was adjusted to about 0.700 at 734 nm with 0.01 m phosphate buffer saline , ph-7.4 .
change in absorbance ( a ) was calculated from absorbance reading just before adding sample and after 6 minutes of sample addition was recorded and converted to mm trolox equivalent .
tos was estimated according to the method of erel , which is based on the generation of colored complex of ferric ion in the presence of oxidative components and xylenol orange in acidic medium and calculated from h2o2 standard curve . in short
, 140 l of sample was added to 900 l of reagent 1 ( containing 150 m xylenol orange , 140 mm nacl and 1.35 m glycerol in 25 mm h2so4 solution , ph 1.75 ) and mixed .
the initial reading ( a1 ) was obtained from subtracting absorbance at 800 nm ( secondary wavelength ) from that of 560 nm ( primary wavelength ) .
44 l of reagent 2 ( containing 5 mm ferrous ion and 10 mm o - dianisidine in 25 mm h2so4 solution ) was then added to the above mixture and incubated for 4 minutes .
the value of tos was then obtained using a ( = a2-a1 ) from the h2o2 standard curve .
oxidative stress index ( osi ) was calculated from the ratio of tos and tas { osi=[(tos , m h2o2 equivalent)100/(tas , m trolox equivalent ) ] } according to the standard method and expressed as arbitrary units . for this purpose , the result unit of tas
peripheral blood smear was prepared on grease free glass slides and photographed using zeiss light microscope ( zeiss , thornwood , ny ) and progress capture pro 2.5 software ( genotypic optical systems , gmbh , jena , germany ) using 1000 magnification after staining with the leishmann stain .
briefly , erythrocytes were directly fixed overnight with 2.5% glutaraldehyde solution in pbs , ph 7.2 , and post - fixed by keeping overnight in 1% osmium tetraoxide in the same buffer .
the suspensions were dehydrated in an ethanol series . after drying with carbon dioxide by the critical point method and
sputter coating with gold , samples were examined on a sem ( vegaii lsu , tescan , czech republic ) .
one - way anova followed by tukey s hsd post hoc analysis was performed to check any statistical difference between the parameters of the studied groups .
statistical analysis was carried out using the statistical program packages spss version 16.0 for windows ( spss inc . ,
assessment of hematological profile indicated that hemolytic and inflammatory responses are the primary events associated with arsenic toxicity in rats after arsenic treatment ( table 1 ) .
a significant reduction of erythrocyte count ( p=0.021 ) associated with decreased total hb content ( p=0.031 ) was noticed in arsenic - treated rats compared to that of control .
mcv was found to be significantly increased ( p=0.041 ) in arsenic - treated rats compared to that of control , suggesting macrocytosis .
in addition , the toxic effect of arsenic also caused significant neutropenia ( p=0.022 ) , lymphocytopenia ( p<0.001 ) , and thrombocytopenia ( p=0.023 ) indicating compromised hemostatic and immune system .
eosinophil count shows insignificant increase ( p=0.123 ) whereas monocyte ( p=0.993 ) and basophils ( p=0.684 ) shows insignificant variations after arsenic treatment .
neutrophil to lymphocyte ratio ( nlr ) and platelet to lymphocyte ratio ( plr ) , the derived hematological indices representing inflammation , were also altered after arsenic treatment .
though nlr showed insignificant elevation ( p=0.330 ) , plr was found to be significantly increased ( p=0.005 ) in arsenic - treated rats .
ala supplementation resulted in partial restoration of erythrocyte ( p=0.503 ) , hb ( p=0.180 ) , and mcv ( p=0.807 ) , though insignificant , but leads to significant restoration of lymphocyte count ( p=0.006 ) and thereby partial restoration of total leucocyte count ( p=0.093 ) compared to the arsenic - treated group , but failed to achieve normal status .
neutrophil ( p=0.748 ) , eosinophil ( p=0.086 ) , basophil ( p=0.986 ) and monocyte ( p=0.999 ) count does not show any significant improvement .
it also significantly reduces the nlr value ( p=0.029 ) associated with a non - significant reduction of plr ( p=0.195 ) compared to the arsenic - treated group .
effect of ala supplementation on hematological profile of as2o3 treated male rats data represented as meanstandard error of mean .
tukey hsd post hoc analysis , p<0.05 ; control versus treated ; control versus supplemented ; treated versus supplemented hematological alterations were found to be accompanied by poikilocytosis , i.e. morphological alteration of erythrocytes , characterized by the formation of echinocytes and spherocytes and such changes were not seen in the control group ( figure 1a and b ) .
the peripheral smear of arsenic - treated rats contains about 19.77% echinocytes and 6.78% spherocytes compared to 1.98% echinocytes and 0.99% spherocytes in control .
this morphological response was found to be partially prevented by ala supplementation ( figure 1c ) .
ala co - administration reduces the echinocytic content to about 4.69% and that of spherocytes to 1.56% .
light microscopic images ( 100 ) of erythrocytes obtained from control ( a ) , arsenic - treated ( b ) , and ala - supplemented ( c ) rats .
arsenic - induced echinocytic transformation of erythrocyte was confirmed by sem analysis , which showed distinct membrane blebbing , increased number of spherocytes and microcytosis , but these were almost absent in the control group ( figure 2a and b ) .
interestingly , ala supplementation gave substantial protection from arsenic - induced echinocytic transformation ( figure 2c ) .
sem images of erythrocytes obtained from control ( a ) , arsenic - treated ( b ) , and ala - supplemented ( c ) rats .
echinocytic and microcytic spherocyte transformations of erythrocytes are shown by thick and thin white arrow , respectively .
arsenic treatment caused a significant oxidative stress in rats as evident from the elevated plasma tos ( p<0.001 ) , osi ( p<0.001 ) and reduced plasma antioxidant status ( p<0.001 ) compared to the control group .
ala , known for its antioxidant potential , was seen to significantly prevent oxidative stress by lowering plasma tos ( p<0.001 ) and osi ( p<0.001 ) status in spite of arsenic supplementation .
ala also helped in restoring the antioxidant capacity as shown by a significant increase in plasma tas ( p=0.005 ) in la supplemented group compared to the arsenic group ( figure 3 ) .
plasma redox status of control ( a ) , arsenic - treated ( b ) , and ala - supplemented ( c ) rats .
assessment of hematological profile indicated that hemolytic and inflammatory responses are the primary events associated with arsenic toxicity in rats after arsenic treatment ( table 1 ) .
a significant reduction of erythrocyte count ( p=0.021 ) associated with decreased total hb content ( p=0.031 ) was noticed in arsenic - treated rats compared to that of control .
mcv was found to be significantly increased ( p=0.041 ) in arsenic - treated rats compared to that of control , suggesting macrocytosis .
in addition , the toxic effect of arsenic also caused significant neutropenia ( p=0.022 ) , lymphocytopenia ( p<0.001 ) , and thrombocytopenia ( p=0.023 ) indicating compromised hemostatic and immune system .
eosinophil count shows insignificant increase ( p=0.123 ) whereas monocyte ( p=0.993 ) and basophils ( p=0.684 ) shows insignificant variations after arsenic treatment .
neutrophil to lymphocyte ratio ( nlr ) and platelet to lymphocyte ratio ( plr ) , the derived hematological indices representing inflammation , were also altered after arsenic treatment .
though nlr showed insignificant elevation ( p=0.330 ) , plr was found to be significantly increased ( p=0.005 ) in arsenic - treated rats .
ala supplementation resulted in partial restoration of erythrocyte ( p=0.503 ) , hb ( p=0.180 ) , and mcv ( p=0.807 ) , though insignificant , but leads to significant restoration of lymphocyte count ( p=0.006 ) and thereby partial restoration of total leucocyte count ( p=0.093 ) compared to the arsenic - treated group , but failed to achieve normal status .
neutrophil ( p=0.748 ) , eosinophil ( p=0.086 ) , basophil ( p=0.986 ) and monocyte ( p=0.999 ) count does not show any significant improvement .
it also significantly reduces the nlr value ( p=0.029 ) associated with a non - significant reduction of plr ( p=0.195 ) compared to the arsenic - treated group .
effect of ala supplementation on hematological profile of as2o3 treated male rats data represented as meanstandard error of mean .
tukey hsd post hoc analysis , p<0.05 ; control versus treated ; control versus supplemented ; treated versus supplemented
hematological alterations were found to be accompanied by poikilocytosis , i.e. morphological alteration of erythrocytes , characterized by the formation of echinocytes and spherocytes and such changes were not seen in the control group ( figure 1a and b ) .
the peripheral smear of arsenic - treated rats contains about 19.77% echinocytes and 6.78% spherocytes compared to 1.98% echinocytes and 0.99% spherocytes in control .
this morphological response was found to be partially prevented by ala supplementation ( figure 1c ) .
ala co - administration reduces the echinocytic content to about 4.69% and that of spherocytes to 1.56% .
light microscopic images ( 100 ) of erythrocytes obtained from control ( a ) , arsenic - treated ( b ) , and ala - supplemented ( c ) rats .
arsenic - induced echinocytic transformation of erythrocyte was confirmed by sem analysis , which showed distinct membrane blebbing , increased number of spherocytes and microcytosis , but these were almost absent in the control group ( figure 2a and b ) .
interestingly , ala supplementation gave substantial protection from arsenic - induced echinocytic transformation ( figure 2c ) .
sem images of erythrocytes obtained from control ( a ) , arsenic - treated ( b ) , and ala - supplemented ( c ) rats .
echinocytic and microcytic spherocyte transformations of erythrocytes are shown by thick and thin white arrow , respectively .
arsenic treatment caused a significant oxidative stress in rats as evident from the elevated plasma tos ( p<0.001 ) , osi ( p<0.001 ) and reduced plasma antioxidant status ( p<0.001 ) compared to the control group .
ala , known for its antioxidant potential , was seen to significantly prevent oxidative stress by lowering plasma tos ( p<0.001 ) and osi ( p<0.001 ) status in spite of arsenic supplementation .
ala also helped in restoring the antioxidant capacity as shown by a significant increase in plasma tas ( p=0.005 ) in la supplemented group compared to the arsenic group ( figure 3 ) .
plasma redox status of control ( a ) , arsenic - treated ( b ) , and ala - supplemented ( c ) rats .
in the present study , hematological alterations and associated redox imbalance were seen due to arsenic treatment , which were found to be maintained near normal status after ala supplementation ; also in favour of the contemporary report on the protective potential of ala against arsenic toxicity .
the counts of erythrocyte , leucocyte , and platelet along with total hemoglobin content , which were depleted due to arsenic insult showed to be prevented on ala supplementation .
although no significant prevention was noted in other hematological indices , but an order of control was seen in this regard due to ala supplementation in arsenic - treated rats .
this finding is in partial agreement with related recent reports of hematoprotective effect of ala against heavy metal toxicity , but again , it is to be noted that in these studies ala is used in combination with other drugs .
the partial restoration of hematological parameters after ala supplementation is in agreement with previous reports , which show that despite its antioxidant potential , ala fails to significantly improve hematological parameter .
arsenic - neutrophil interaction and its deleterious effect on the neutrophils , which potentiates its anticancer activity is well documented in the literature .
neutropenia is so profound in arsenic - treated rats that even the ala supplementation fails to restore its count even to near normal .
nlr and plr are two derived measures obtained from the neutrophil , lymphocyte , and platelet absolute counts are recently reported as the marker of systemic inflammation and related with disease outcome .
recent reports also suggest that nlr is elevated in chronic stress in rats and it can serve the purpose of a physiological marker in experimental animals .
both nlr and plr were found to be elevated after arsenic exposure indicating systemic inflammation caused by arsenic toxicity .
ala administration significantly reduces these two scores in spite of arsenic exposure even below the status in the sham control group .
the differential responses of nlr and plr in the case of arsenic treatment and ala supplementation suggests that in the case of prognosis of arsenic toxicity , a combination of these two should be taken into account as also suggested by other authors .
the anti - inflammatory activity of ala in this model of arsenicosis also supports the anti - inflammatory potential as reported in both acute and chronic model in rats .
arsenic imposes its damaging effects via impairment of enzyme by forming strong complexes with thiols .
it also generates ros during its metabolism in cells leading to redox imbalance and enhanced lipid peroxidation .
arsenic - induced oxidative stress and toxicity is bring about by generated free radicals and potentiated by arsenic metabolites especially the organic derivatives of arsenic .
dimethylarsine ( a trivalent form of arsenic ) can react with molecular oxygen to form a ( ch3)2as .
all these free radicals along with others , like oh ultimately damage various cellular macromolecules , including proteins and dna .
generation of ros , beyond the body s endogenous antioxidant balance was known to cause redox imbalance favoring an oxidative milieu .
noticeable increase in plasma tos associated with a decrease in plasma tas in comparison to that of the control group indicates a state of oxidative stress due to arsenic toxicity , which further supports the notion that redox imbalance is one of the significant events leading to damages due to arsenicosis .
these alterations lead to marked elevation of osi , suggesting a fair reduction of the antioxidant system parallel to increased ros production after arsenic exposure .
ala , a naturally occurring antioxidant and coenzyme of many enzymes , includes key enzymes of energy metabolism pathway .
ala and its derivative dihydrolipoic acid ( dhla ) quench a number of reactive oxygen species in both lipid and aqueous phases , chelate transition metals , prevent membrane lipid peroxidation along with protein damage via interactions with vitamin c and glutathione and thus they are considered as potent antioxidants .
ala shows a significant prevention of redox alteration induced by arsenic treatment , as confirmed by the tos , tas , and osi status in the supplemented group and this is in accordance with other previous reports in this field .
these findings / observations suggest that the beneficial effect of ala supplementation in preventing the hematological alteration and inflammatory changes in a rat model of arsenicosis may be attributed to its antioxidant potentials as also suggested by others .
microscopic study has shown that arsenic exposure leads to poikilocytic response and reduced discocyte content in rats .
the observed major shape transformations being echinocytic and spherocytic , which are in agreement with the previous reports on arsenic affected human populations .
sem image analyses confirm these findings and also indicate microcytosis in the arsenic - treated groups .
these changes in erythrocyte morphology may be attributed to increased oxidative stress and inflammatory status due to arsenic exposure , which is suggestive of eryptotic changes .
oxidative stress within erythrocytes that occurs due to arsenic exposure and accumulation is most likely responsible for membrane injury , methemoglobin formation , osmotic fragility , and destruction of cells .
ala treatment ameliorates poikilocytic effect of arsenic and increases discocyte content , reduces microcytosis and membrane blebbing in the peripheral blood .
this may be explained by the unique ability of the ala which could neutralize free radicals within aqueous and lipid regions of the cell .
this uniqueness of the ala , allows it to be transported easily across the cellular membrane and thereafter alleviate the ros induced damages by its antioxidant capacity and/or by the gsh enhancing ability . in the present study , the use of one selected concentration of ala may not reflect the optimum doses in favour of its ameliorating capacity . moreover , the study could not provide any clue related to its action at the molecular level .
this study was also unable to highlight the status of arsenic accumulation in plasma and erythrocytes , though a clear indication exists regarding the hemato - protective ability of ala under arsenic insult .
based on the experimental data obtained from the rat arsenicosis model , it may be concluded that ala as a food supplementation has a beneficial effect against arsenic toxicity .
it brings about this protection by restoring near normal hematological , oxidative , and inflammatory status .
further in - depth studies in this direction may help generating knowledge about the defensive role(s ) of ala against arsenic toxicity . | background : arsenic toxicity is a major global health problem and exposure via contaminated drinking water has been associated with hematological and other systemic disorders .
the present investigation has been conducted in adult male rats to evaluate the protective ability of -lipoic acid ( ala ) against such hematological disorders.methods:twenty-four adult male wister rats ( b.wt.13010g ) were grouped and accordingly group i ( control ) received the normal diet , group ii ( treated ) was given arsenic orally for 28 consecutive days as arsenic trioxide ( 3 mg / kgbw / rat / day ) whereas group iii ( supplemented ) received the same dose of arsenic along with ala ( 25 mg / kgbw / rat / day ) as oral supplement .
hematological profile , plasma oxidant / antioxidant status , and erythrocyte morphology were assessed .
statistical analysis was done by one - way anova using spss software ( version 16.0).results : arsenic exposure caused reduction of erythrocyte ( p=0.021 ) , leucocyte ( p<0.001 ) , and hemoglobin ( p=0.031 ) associated with echinocytic transformation as evidenced by light and scanning electron microscopic studies .
the other significantly altered parameters include increased mean corpuscular volume ( p=0.041 ) and lymphocytopenia ( p<0.001 ) with insignificant neutropenia and eosinophilia .
altered serum oxidative balance as evidenced by decreased tas ( p<0.001 ) and increased tos ( p<0.001 ) with osi ( p<0.001 ) was also noted .
the dietary supplementation of ala has a beneficial effect against the observed ( p<0.05 ) arsenic toxicities .
it brings about the protection by restoring the hematological redox and inflammatory status near normal in treated rats .
arsenic - induced morphological alteration of erythrocytes was also partially attenuated by ala supplementation.conclusion:it is concluded that arsenicosis is associated with hematological alterations and ala co - supplementation can partially alleviate these changes in an experimental male rat model . |
cultures , susceptibility testing and dna extraction - one hundred and
eight cultures had previously been tested for drug susceptibility using the ogawa - kudoh
standard proportion method ( canetti et al .
1963 )
at the central laboratory of rio grande do sul ( ipb / lacen - rs ) , the regional reference
laboratory for mycobacterium - related disease in the city of porto
alegre , south region of brazil .
this study was approved by the ethical committee of the
school of public health and the state foundation of health research and production of
brazil under protocols 465/09 and 03/2010 , respectively .
nucleic acids were extracted from the m. tuberculosis culture using the
cetyl trimethylammonium bromide method , as described previously ( van soolingen et al .
1994 ) at the centre of scientific and
technological development of the state foundation in production and health research ,
porto alegre .
the genes encoding rpob , katg and the
inha promoter , which are related to rmp and inh , respectively , had
been sequenced as part of an earlier study ( verza et al .
2009 , maschmann et al .
the h37rv
( atcc27294 ) reference strain was used as a control for both dst and genotyping .
amplification of part of rpob , katg , inha and is6110 - the multiplex
pcr was performed using the primers described in supplementary data .
the reverse primers
were biotin labelled and the pcr - multiplex was standardised in a final volume of 50 l
containing 200 m of each dntp , 10 mm tris - hcl ( ph 8.3 ) , 50 mm kcl , 2 mm mgcl2 ,
10 pmoles of primers rif1 and rif2 each , 25 pmoles of primers
katg1 and katg2 , inha1 and
inha2 and is1 and is2 each , 2.5 u of taq dna polymerase
( invitrogen / usa ) and 1 l ( 100 ng/l ) of purified bacterial dna .
the pcr reactions were
carried out as follows : 3 min at 95c , 30 cycles of 1 min at 95c , 1 min at 60c , 1.5
min at 72c and 4 min at 72c .
each amplification experiment included a positive
( m. tuberculosis h37rv dna , 100 ng ) and a negative control ( water ) .
for standardisation of the amplification reactions
, the pcr products were analysed using
polyacrylamide gel electrophoresis , followed by detection in ultraviolet light after
staining with ethidium bromide and comparison of the amplicons to a molecular weight
marker .
reverse - line blot hybridisation and colorimetric detection - reverse
hybridisation and colorimetric detection were performed based on the previously
described protocol ( maschmann et al .
the
membranes were washed in 2x ssc/0.1% sodium dodecyl sulfate ( sds ) for 5 min at 50c and
were incubated in 2x ssc with 5% bovine serum albumin ( bsa ) at 50c for 15 min .
twenty - five microlitres of each pcr reaction was added to 150 l of 2x ssc/0.1% sds and
the mixture was denatured at 100c for 10 min and then transferred to an ice bath .
the
denatured pcr product was then added to a polyethylene tube containing a membrane in 1.4
ml of the hybridisation solution ( 2x ssc/0.1% sds ) .
after hybridisation , the membranes were washed in 2x
ssc/0.5% sds at 57c for 10 min each .
the membranes were then treated with tris - buffered
saline ( tbs ) ( 100 mm tris - hcl , 150 mm nacl , ph 7.5 ) containing 6% bsa for 30 min at
50c .
the membranes were incubated in tbs containing 0.33 g / ml streptavidin - alkaline
phosphatase ( ap ) conjugate for 15 min at room temperature ( rt ) .
the unbound conjugate
was removed by washing in tbs for 10 min followed by the addition of ap buffer ( 100 mm
tris - hcl , 150 mm nacl and 5 mm mgcl26h2o , ph 9.5 ) for 10 min at
rt .
the hybridisation was visualised by adding 40 g / ml 5-bromo-4-chloro-3-indoyl
phosphatase and 82.5 g / ml nitro blue tetrazolium ( sigma ) in the ap buffer and
incubation for 10 min at rt . a purple precipitate was observed when there was a perfect
match between the probe and the biotinylated pcr product .
the colorimetric reaction was
blocked with distilled water and the membranes were dried at rt .
sensitivity and specificity of the assay - the analytical sensitivity
of the test was determined by performing the assay with a serial 10-fold dilution of
m. tuberculosis h37rv dna ( 100 ng to 1 pg ) . to determine the
specificity of line - tb / mdr , 100 ng of genomic dna from each of the following bacteria
were submitted to the entire procedure : neisseria meningitidis , streptococcus
pneumoniae , salmonella enterica , haemophilus influenzae , escherichia coli
( obtained from ipb / lacen - rs ) , mycobacterium marinum , mycobacterium
intracellulare , mycobacterium scrofulaceum , mycobacterium gordonae , mycobacterium
avium , mycobacterium smegmatis , mycobacterium kansasii , mycobacterium xenopi ,
mycobacterium fortuitum - peregrinum and mycobacterium phlei
( obtained from oswaldo cruz foundation , brazil ) ( verza et al .
probe design and binding to membrane - the oligonucleotide probes were
designed using the software primer express v.2.0 ( applied biosystems ) .
our final assay
setup was composed of ( i ) five probes for the wild type ( wt ) rpob
allele and five probes for each of the different rpob mutant alleles ,
( ii ) one probe each for the wt and mutant katg and ( iii ) one probe each
for the wt and mutant inha promoter alleles . additionally , we designed
a probe specific for hybridisation with the insertion sequence is6110
( supplementary data ) . for the immobilisation of the dna probes ,
a negatively - charged nylon membrane ( biodyne
c , pall corporation ) was activated in a solution of 16% ( w / v )
( 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ) ( acros organics ) for 20
min at rt and was washed in distilled water for 2 min at rt .
then , 10 l of each of the
oligonucleotide probes , containing a 5-terminal amino group , were diluted to the
appropriate concentration ( supplementary data ) in 0.5 m nahco3 ( ph 8.4 ) and
plotted into 15 circles ( 5 mm ) previously designed on the membrane , followed by
incubation for 1 min at rt .
the membranes were then incubated in 0.1 m naoh for 10 min ,
washed in distilled water for 2 min at rt , washed in 2x ssc/0.1% sds for 10 min at 50c
and finally incubated in 20 mm edta ( ph 8.0 ) for 15 min at rt .
the membranes were
finally transferred into a polyethylene tube and stored at 4c for later use .
assay interpretation - the panel f in figure demonstrates the positions of the probes for rpob ,
katg , inha and is6110 .
a
hybridisation result was considered positive when a purple colour was observed in the
circle where a particular probe had been plotted .
the probes in rpob
were located within the 81 bp resistance - determining region ( rdr ) and hybridisation with
all five wt probes was considered to indicate an rmp - susceptible strain .
when one of
these signals was lacking , independent of any hybridisation to probes 6 - 10 , the strain
was considered to be an rpob - mutated strain .
a strain was considered
mutated for the inha gene when there was a lack of hybridisation to
probe 12 and/or hybridisation to probe 11 occurred ; the wt katg gene
was represented by hybridisation to probe 13 and a positive signal for probe 14 was
observed when the gene was mutated .
the hybridisation to probe 15
( is6110 ) was an additional confirmation of the m.
tuberculosis complex ( mtbc ) .
a : example of the wild type ( wt ) result to the three genes
rpob , katg and inha ; b :
example of a strain mutated in the 531 region of the rpob gene
with no hybridisation with the rif5wt probe and hybridisation with the rif4 m ;
c : hybridisation with the 14 katgm probe ; d : hybridisation
with the probe number 11 ; e : hybridisation was not observed with the rif4
probe , what suggested that there are mutation in the rpob gene
codon 526 , although the method have not allowed to identified the mutation ; f :
schematic presentation of line - tb / mdr .
probes position : rpob
wt probes , circles 1 - 5 ( rif 1 , rif 2 , rif 3 , rif 4 , rif 5 ) ;
rpob mutated probes , 6 - 10 ( rif1 m , rif2 m , rif3 m , rif4 m ,
rif5 m ) ; 11 , inham ; 12 ,
inhawt ; 13 , katgwt ; 14 ,
katgm ; 15 , is6110
.
genotype
mtbdrplus assay - the genotype mtbdrplus assay was
performed according to the manufacturer s instructions ( crudu et al .
65 were tested
using the genotype mtbdrplus assay . in order to guarantee the highest
fidelity possible between the line - tb / mdr assay and the genotype
mtbdrplus assay , the same dna aliquot was used in both
experiments .
a positive hybridisation signal was observed with the probe specific for
is6110 for all 108 isolates ; no non - mycobacterial dnas or
mycobacterium species other than those of the mtbc presented a
signal at this membrane region .
the test was therefore 100% sensitive and specific for
strains belonging to the mtbc ( a in figure ) . among
the 108 cultures ,
55 were considered sensitive to both of the drugs and presented the wt
alleles for rpob , katg and inha in
the dst .
sequence analysis demonstrated the presence of at least one mutation in the
rpob gene in 53 of the mdr - tb samples and among these , 31 presented
an additional mutation in katg .
there were also three samples that
presented mutations in the inha promoter region and 10 presented
mutations in both katg and inha .
nine isolates ( 17% )
had no mutation in either of the inh - r - associated genes ( table i ) .
table icorrelation between line - tb / mdr and sequencingline - tb / mdr pattern line - tb / mdr result - location of mutationsequencing data positive probesgene
( probe ) nucleotide / amino acid changesstrains n ( % ) 1,2,3,4 *
rpob ( rif4 m )
s531ltcg - ttg / ser - leu37 ( 69.8)1,2,3,4 *
rpob ( rif5m)s531wtcg - tgg / ser - trp1 ( 1.88)1,2,4,5 *
rpob ( rif3m)h526dcac - gac / his - asp4 ( 7.5)1,3,4,5 *
rpob ( rif2m)h526ycac - tac / his - tyr4 ( 7.5)1,2,3,5*lack
hybridisationh526lcac - gtc / his - leu1 ( 1.88)1,2,3,5*lack
hybridisation at ( rif2/3m)h526rcac - cgc / his - arg1 ( 1.88)1,2,3,5*lack
hybridisation at ( rif2/3m)h526pcac - ccc / his - pro1 ( 1.88)1,2,3,5*lack
hybridisation at ( rif2/3m)h526lcac - ctc / his - leu1 ( 1.88)1,3,4,5*lack
hybridisation at ( rif2/3m)d516ygac - tac / asp - tyr2 ( 3.77)1,3,4,5 *
rpob ( rif1m)d516vgac - gtc / asp - val1 ( 1.88)11 *
inha ( inham )
-15c - t13 ( 24.6)12 *
inha ( inhawt)no mutationwt40 ( 75.4)14 *
katg ( katgm )
s315tagc - acc / ser - thr41 ( 77.3)13 *
katg ( katgwt)no mutationwt12 ( 22.7)1,2,3,4,5,12,14*wtno mutationwt55 ( 100)wt : wild type .
the line - tb / mdr method achieved 100% sensitivity ( 55/55 ) among the wt samples and , as
such , identified the inh - s and rmp - s isolates ; this was demonstrated by hybridisation to
the five wt rpob probes ( probes 1 - 5 ) and the wt inha
and katg probes ( probes 12 and 13 ) ( a in figure ) . among the 53 mdr strains , 38 had a mutation in codon 531 .
the line - tb / mdr assay was able
to correctly identify all of the mutations occurring at codon 531 when the snp involved
resulted in an amino acid ( aa ) change of serleu ; these strains exhibited hybridisation
signals with probes 1 through 4 and 9 , but had a lack of hybridisation to probe five ( b
in figure ) . among the 12 additional isolates with
rpob mutations , the line - tb / mdr method correctly identified eight
codon 526 mutants , of which four presented a hisasp
codon 516 in the line - tb / mdr assay correctly identified a single
strain that had the aspval aa change .
four mutants at codon 526 and two mutants at
codon 516 did not have a complementary mutant probe in our assay , but they lacked
hybridisation signals with the wt 526 probe 4 , as expected , and probe 2 , which
represented the wt codon 516 ( e in figure ) . the
comparison of the results obtained for all of the isolates using the line - tb / mdr assay
( n = 108 ) and the rpob rdr sequencing as a reference is summarised in
table ii .
this assay reported a kappa value
( k ) [ 95% confidence interval ( ci ) ] and agreement of 1 ( 99.5 - 100.0 ) .
table iicomparison of the results obtained for the identification of mutation with
the sequencing and with drug susceptibility testing ( dst ) results compared results ( n =
108)(n =
108 ) line - tb / mdrline - tb / mdrline - tb / mdrline - tb / mdrkappa sesp line - tb / mdrseqwt / seq wtmut / seq wtwt / seq mutmut / seq mut(95% ci)p(%)(% )
inh wt586458334488.9
( 82.5 - 95.2 ) < 0.00177.690.6inh res5044rmp wt5555550053100
( 99.5 - 100.0 ) < 0.001100100rmp res5353
line - tb / mdr line - tb / mdrline - tb / mdrline - tb / mdrline - tb / mdrkappa sesp dstwt / dst - smut / dst - swt / dst - rmut / dst - r(95% ci)p(%)(% )
inh wt5555 55 0124188.7
( 82.2 - 95.1 ) < 0.00177100inh res4153 rmp wt5555550053100
( 99.5 - 100.0 )
< 0.001100100rmp res5353 ci : confidence interval ; inh : isoniazid ; rmp : rifampicin ; se : sensitivities ;
sp : specificities .
ci : confidence interval ; inh : isoniazid ; rmp : rifampicin ; se : sensitivities ;
sp : specificities .
we also evaluated the performance of the line - tb / mdr method in 53 inh - resistant
isolates . according to the sequence data
, 44 of these isolates carried mutations in
katg and/or inha and 41 ( 77.3% ) were
katg 315 ( agc - acc ) ( serthr ) . among these samples ,
the line - tb / mdr
method was able to correctly identify 36 ( 88% ) , with a positive signal at probe 14
( katgm ) and a lack of hybridisation at probe 13
( katgwt ) ( c in figure ) .
three
isolates with a mutation in codon 315 gave a positive signal for probe 13
( katgwt ) , while two of the isolates did not hybridise to any of the
katg probes ( e in figure ) .
among the 12 isolates , 11 ( 91.6% )
hybridised to probe 13 ( katgwt ) , as
expected , but one isolate again failed to hybridise to any of the katg
probes .
in addition , 40 of the 53 mdr strains ( 75.4% ) presented a
wtinha genotype ; among these , 37 ( 92.5% ) showed hybridisation to
probe 12 ( inhawt ) and a lack of hybridisation to probe 11
( inham ) ( d in figure ) .
three
isolates presented discordant results when compared to the sequencing data ; one sample
hybridised to probe 11 , corresponding to a mutated region of the gene
( inham ) , while two isolates did not hybridise to any of the inha
probes ( inham ) .
the other strains ( n = 13 ; 25% ) contained the mutation
c-15 t in the promoter region of inha and 10 ( 77% ) strains correctly
demonstrated hybridisation to probe 11 ( inham ) , while three failed to
hybridise to either probe 11 or 12 . in conclusion , the overall results of the
line - tb / mdr assay ( n = 108 ) and the combined sequences of katg
and inha demonstrated a k ( 95% ci ) of 0.889 ( 82.5 - 95.2 ) for
katg and inha and an negative predictive value and
positive predictive value of 94.8% and 100% , respectively ( table ii ) .
sixty - five of the 108 isolates were also analysed using the genotype
mtbdrplus assay and 25 susceptible strains with wt , inh and rmp
sequences were correctly identified . among the 40 rif - resistant strains that also had
rmp - r genotypes , 37 ( 92.5% )
the concordance rates between the line - tb / mdr
and mtbdrplus assays for drug resistance ( dr ) detection in these
samples were 93.6% ( 63/65 ) for the inh sequences and 87.4% ( 62/65 ) for the rmp sequences
( table iii ) .
the mtbdrplus
did not identify three rmp - r strains that were correctly identified by line - tb / mdr and
sequencing and three sequencing - confirmed inh - r isolates were not identified by
line - tb / mdr .
table iiicomparison of the results obtained for the identification of genotype
mtbdrplus with the sequencing and with line - tb / mdr resultscompared results ( n =
65)(n =
65 ) hain
strip hain
striphain
striphain
striphain
stripkappa sesp seqwt / seq wtmut / seq wtwt / seq mutmut / seq mut(95% ci)p(%)(% )
inh wt272525324096.9 < 0.00187.0100.0inh res3840(89.9 - 98.6)rmp wt282525033790.5
< 0.00192.5100.0rmp res3740(84.3 - 86.4 ) hain striphain striphain striphain stripkappa hain stripline - tb / mdrwt / line - tb / mdr wtmut / line - tb / mdr wtwt / line - tb / mdr mutmut / line - tb / mdr mut(95% ci)p
inh wt252525023893.6 < 0.001 inh res4040(86.7 - 97.5 ) rmp wt282525033787.4 < 0.001 rmp res3740(84.3 - 96.2 ) ci : confidence interval ; inh : isoniazid ; rmp : rifampicin ; se : sensitivities ;
sp : specificities .
ci : confidence interval ; inh : isoniazid ; rmp : rifampicin ; se : sensitivities ;
sp : specificities .
mdr - tb is defined by simultaneous resistance to inh and rmp , the main drugs used for tb
treatment ( laurenzo & mousa 2011 ) . because
m. tuberculosis is a clonal organism , most of the acquisition of dr
in m. tuberculosis is due to point mutations in the chromosome ( sreevatsan et al .
1997 , ramaswamy & musser 1998 ) that accumulate and are preserved in
the different lineages ( brosch et al .
these traits of
mycobacterium render it suitable for the development of assays for
the detection and eventual characterisation of the presence of well - described inh - r and
rmp - r snps ( cardoso et al .
2011 ) . in brazil , as recommended by the ministry of health ,
tb diagnosis and dst can be performed using solid media ( lj or ogawa - kudoh ) and liquid
media ( middlebrook 7h10 ) , but these methods have several limitations , mainly related to
the delay of diagnosis , which can be as long as 12 weeks ( ms 2010 ) . for dr detection , tests based on reverse blot
hybridisation probes , such as the line probe assay ( inno - lipa assay rif tb ,
innogenetics ) and the genotype mtbdrplus assay , are currently widely
used in many countries .
these tests have sensitivities ranging from 71 - 100% and
specificities of 96 - 100% ( bwanga et al .
while inno - lipa is
restricted to the identification of rmp resistance , genotype is able to identify both
rmp and inh resistance .
however , both of these methods are expensive and not widely
available in developing countries ( mkinen et al .
2006 , maschmann et al . 2013 ) .
recently ,
a molecular test for the detection of m. tuberculosis and mutations
conferring rmp resistance directly from sputum ( genexpert , cepheid sunnyvale , ca , usa )
was developed , but the performance characteristics of this procedure might be different
when implemented in environments with low or high tb burdens ( dorman et al .
2013 ) . for the evaluation of our reverse hybridisation - based assay for the
detection of rmp - r and inh - r , we chose a panel of strains with mutations similar to
those reported in other publications .
the mutations were predominantly localised in
codons 531 , 526 and 516 of the rpob gene ( sekiguchi et al .
1996 , arago et al . 2007 ) and at position -15 of the inha
regulatory region ( palomino 2009 ) .
all of the 108
isolates analysed by line - tb / mdr showed specific hybridisation to the is6110
probe , which was used for m. tuberculosis identification
( amongst other members of the mtbc ) , indicating that this method can be a reliable tool
for tb diagnosis .
this result is similar to that found previously by our research group
when testing a method to detect tb ( detec - tb ) ( michelon
et al .
in addition , the 55 dna samples that did not contain mutations
in the genes associated with resistance and were drug susceptible according to the dst ,
were correctly identified by the line - tb / mdr method . of the 53 samples with mutations in
the rpob gene , 100% ( 53/53 ) hybridised to at least one
rpob - mutated probe or showed no hybridisation to the wt probe ,
confirming that the isolate was not susceptible to rmp ; there was complete agreement ( k
= 1 ) between the current test and the sequencing results ( table ii ) .
these data suggest that the line - tb / mdr method can be
used to reliably detect resistance to this drug , as observed by aslan et al .
( 2008 ) , when using a commercial kit and samples , both
mutated or not , in the rpob gene . when snps for which a mutant probe
was developed were present , we observed a corresponding signal for that mutant and a
lack of hybridisation to the wt probe
. four isolates carried snps that had no mutant
probe represented and , as expected , demonstrated a lack of signal in the wt probe only
( e in figure ) , which indicated the presence of an
snp associated with rmp - r .
the sensitivity for detecting rmp resistance was 100% , with
sequencing used as the reference method .
this is promising for the rapid detection of
mdr / tb isolates because rmp mono - resistant isolates are rarely observed ( malhotra et al .
previous studies reported
that approximately 95 - 100% of rmp - resistant clinical isolates carry mutations in an 81
bp region of the rpob gene ( palomino
2009 ) .
therefore , in cases with a lack of snps in this region , we do not
expect our assay to be useful for detecting rmp - r .
although most of the methods used to
detect mutations related to inh resistance use only the katg gene as a
target , maschmann et al .
( 2011 ) demonstrated that
the concomitant evaluation of katg and the inha
regulatory region improves sensitivity for identifying inh resistance ( aslan et al .
2008 ) . in this study , only three
inh - resistant isolates had mutations in the inha gene , but the
frequency of inh - resistant isolates containing the snp ( cg ) at position 315 of
katg ( serthr ) ranged from 50 - 100% in the literature , including
literature from brazil ( hillemann et al . 2005 ,
costa et al .
this aa change seems to
lead to a decrease in catalase peroxidase enzyme activity , which has been described as
essential for the activation of the drug ( zhang et al .
when the
line - tb / mdr method was compared to dst , which is a reference for mdr / tb identification ,
the overall agreement was 88.7% ( 82.2 - 95.1 ) . among the 12 dna samples
that were not
correctly identified as inh - resistant , nine were found to have an inha
and katg wt genotype by sequencing even though they were
identified as mdr by dst ; only three showed no hybridisation to the probes used in our
assay .
some studies report that approximately 10 - 30% of inh - r m.
tuberculosis strains have no mutations in dr - associated genes ( hillemann et al .
2005 ) and may have other resistance
mechanisms , such as efflux pumps or cell envelope permeability ( mkinen et al .
the prevalence of katg and
inha mutations , as well the frequency and type of gene mutations ,
varies greatly among different geographic regions , with ranges of 35 - 91% and 20 - 35% ,
respectively ( piersimoni & scarparo 2003 ,
silva et al .
the inclusion of other genes , such as the
oxyr - ahpc intergenic region , may increase the sensitivity of the
test , although the relationship between inh resistance and mutations in this region are
not yet fully understood . in previous work ,
mutations in this gene have frequently been
observed to be associated with mutations in other genes and have been found alone in
very few isolates ( costa et al . 2009 ) .
the line - tb / mdr method achieved satisfactory results for the identification of snps
involved in inh resistance , with a sensitivity of 91.6% ( 99/108 ) , which is similar to
the performance of genotype mtbdrplus and other commercial kits ( bonington et al .
our aim was to evaluate the potential
contribution of a new in - house
test for accelerating the laboratory detection of rif
and inh resistant strains , particularly when mdr - tb is suspected .
the discrepancies between the line - tb / mdr method and dna sequencing might be due to ( i )
the specificity of the probes , ( ii ) the analysis of hetero - resistant strains or ( iii )
the lack of sensitivity of the hybridisation .
it should be noted that the line - tb / mdr
test identified three isolates as inh - resistant using inham probe
hybridisation , while the sequencing data identified them as wtinha .
this could be due to the higher sensitivity of the hybridisation - based assays compared
to sequencing - based assays for detecting mixed bacterial populations ( van deun et al .
the line - tb / mdr method described in this study offers advantages over
alternative techniques and provides results 6 h after amplification of the target ,
considerably decreasing the time needed for dst .
we are now in a second - phase trial for
the evaluation of the reproducibility of this test in different laboratories .
the hope
is that this test will be an additional tool for the diagnosis of mdr - tb and will be
routinely applied in laboratories in regions with high tb and tb / hiv burdens and limited
financial resources .
probe set for identification of the wild type ( wt ) and mutant
genotypes sequencelength probes concentrationprobes(5'-3')(bp ) ( pmoll)rif1wtcagccagctgagccaattcat21wt5.0rif2wtttcatggaccagaacaaccc20wt10.0rif3wtcgctgtcggggttgacc17wt10.0rif4wtttgacccacaagcgccgact20wt5.0rif5wtctgtcggcgctggggc16wt10.0rif1mccaattcatggtccagaa21mutant5.0rif2mgttgacctacaagcgccg18mutant10.0rif3mggttgaccgacaagcgcc18mutant10.0rif4mctgttggcgctggggc16mutant10.0rif5mcgactgtgggcgctgg16mutant10.0
katg wttcaccagcggcatcgag17wt0.4katgmtcaccaccggcatcgag17mutant0.4inhawtcggcgagacgataggttgtc20wt0.6is6110tgcccgtcccgccgatctc18wt1.0
primers used for amplification and standardisation of multiplex pcr for
genes rpob , katg , inha and is6110 sizegeneprimer sequence ( 5'-3')(bp)is6110is1
cgtgagggcatcgaggtggc245 is2
bio - gcgtaggcgtcggtgacaaa rpobrif1
ggtcgccgcgatcaaggagt157 rif2
bio - tgcacgtcgcggacctcca katgkatg1
catgaacgacgtcgaaacag232 katg2
bio - cgaggaaactgttgtcccat inhainha1
cctcgctgcccagaaaggga248 inha2
bio - atcccccggtttcctccggt | drug - resistant tuberculosis ( tb ) threatens global tb control and is a major public
health concern in several countries .
we therefore developed a multiplex assay
( line - tb / mdr ) that is able to identify the most frequent mutations related to
rifampicin ( rmp ) and isoniazid ( inh ) resistance .
the assay is based on multiplex
polymerase chain reaction , membrane hybridisation and colorimetric detection
targeting of rpob and katg genes , as well as the
inha promoter , which are all known to carry specific mutations
associated with multidrug - resistant tb ( mdr - tb ) .
the assay was validated on a
reference panel of 108 m. tuberculosis isolates that were characterised by the
proportion method and by dna sequencing of the targets . when comparing the
performance of line - tb / mdr with dna sequencing , the sensitivity , specificity and
agreement were 100% , 100% and 100% , respectively , for rmp and 77.6% , 90.6% and 88.9% ,
respectively , for inh . using drug sensibility testing as a reference standard , the
performance of line - tb
/ mdr regarding sensitivity , specificity and agreement was 100% ,
100% and 100% ( 95% ) , respectively , for rmp and 77% , 100% and 88.7% ( 82.2 - 95.1 ) ,
respectively , for inh .
line - tb / mdr was compared with genotype mtbdrplus for 65
isolates , resulting in an agreement of 93.6% ( 86.7 - 97.5 ) for rif and 87.4%
( 84.3 - 96.2 ) for inh .
line - tb / mdr warrants further clinical validation and may be an
affordable alternative for mdr - tb diagnosis . |
radiographers understand the importance of getting the most diagnostic information from as few radiographs as possible . in the world of pediatric scoliosis imaging , pediatric orthopedic surgeons , radiologists and orthotists must obtain more than just spine data from each and every radiograph .
scoliosis is a lateral curvature of the vertebral column in the coronal plane . until the end of the 19th century
after x - rays were discovered , their medical uses soon followed and the technique of radiography evolved ( 2 ) .
radiology is a standard modality for the evaluation of pre - screened individuals and for following the progress of curves in individuals with scoliosis ( 3 ) .
whole spine scanography ( wss ) is a radiological examination that exposes the whole body to x - ray radiation .
wss is often repeated during the treatment period , which results in a much greater radiation exposure than that in routine x - ray examinations ( 4 ) .
scoliosis patients routinely undergo sequential studies , and it is estimated that the typical patient with scoliosis will have approximately 22 radiological examinations over a 3-year treatment period ( 5 ) .
whole spine image is taken using 14 36 inch grid cassettes with film in the film - screen processing system , but digital radiographic system uses auto image paste methods . the auto image paste method ( aipm )
establishes the top - to - bottom height before scanning , acquires images by moving the detector and x - ray tube , and then pastes together the acquired images . on analog devices , the film - screen method projects the whole body on a sheet of film .
conversely , the digital method involves the use of a detector , and applies aipm , which assembles multiple images of each body part in order to overcome the size limitations of the detector .
the aims of the current study were to evaluate the effective dose of wss using aipm , and to confirm the effectiveness of the method by comparing the effective dose ( ed ) in aipm with the effective dose resulting from the film - screen method .
in addition , this study will extract the dap - ed conversion factor which is easily available for patient dosimetry by analyzing the correlation of dap with ed .
wss is a radiological examination that observes the postero - inferior and antero - superior displacement of the pelvis , the internal and external rotation of pelvic bones , supero - inferior rotation of scapular bones and analyzes the asymmetric shape of the whole body .
this study was carried out with 50 patients who underwent examinations for diagnosis of scoliosis at the hospital from november 2012 to january 2013 .
this study evaluated all the patients who visited the clinic to diagnose scoliosis during the study period .
the average age of the patients was 55.5 years , the average patient weight was 64.1 kg , and the average bmi was 22.89 kg / m ( table 1 ) .
a definium 6000 digital radiography system ( ge medical systems , milwaukee , us ) was used to scan the entire backbone of each patient from the cervical spine to the coccyx .
the radiography system was composed of a non - tiled 41 41 cm amorphous silicon photodiode with a cesium iodide scintillator and a thin film transistor array ( 2048 2048 pixels and 0.2 mm pixel pitch ) ( 6 ) .
auto exposure control ( aec ) was used for ap projections , with a tube voltage of 80 kvp , while lateral projections used both the aec mode at 85 kvp and the manual exposure control mode at 92 , 93 , and 95 kvp .
all of the antero - posterior projections were acquired in aec mode , while 60% of lateral projections were acquired in aec mode . in aec mode , the exposure dose is determined by automatic process of the diagnostic medical equipment . however , in manual mode , the radiographer set up the exposure parameter of c - spine , t - spine and l - spine lateral projection regarding the thickness of the patients . in lat projection of wss , for obese patients , it may not generate enough exposure to make adequate diagnostic information .
this is because the considerable difference between c - spine thickness and l - spine thickness in obese patients makes an uneven density image in digital image processing .
this error of system is the reason why some obese patients have to retake the x - ray image in examinations .
since obese patients have bigger thickness difference between c - spine and l - spine in lateral projection than ap projection , manual mode was used to prevent the retakes for obese patients especially in lateral projection based on the radiographer s judgment . before the wss examination , the radiographer checked the patient s spine length from the cervical spine to the coccyx .
once they set up the range from starting point to end point , including the patient s c - spine and pelvic bone prior to exposure , the equipment determines the number of shot image by dividing the total length of scan range .
the starting point should be at the level of about 10 cm superior to the external auditory meatus covering all seven cervical spines and the end point should be at the level of pubic symphysis covering the coccyx .
scanning parameters were as follows : x - ray target angle , 12.5 ; anode rotation speed , 10,800 rpm ; and focal spot size , 0.6 mm/1.25 mm ; original filter , tube insert 0.8 mm al equivalent and tube housing 0.3 mm al equivalent .
prior to applying aipm , digital imaging and communications in medicine ( dicom ) tags for the dose area product ( dap ) of each image were collected by investigating the dicom tag data of each partial image , and additional information was identified including x - ray field size , tube voltage , the use of aec mode , and distance between x - ray tube and detector ( 7 ) .
dicom tag data of each image was found on a centricity ca1000 workstation ( ge medical systems , milwaukee , us ) .
tag 0018,115e revealed the information for dap as well as tube voltage , tube current , radiation time , and distance from the source to the detector ( figure 1 ) ( 8) . the aipm established the top - to - bottom height before scanning , acquired images by moving the detector and x - ray tube , and then pasted together the acquired images .
the device used in this study produced a 90 cm - long whole spine scanography within 12 seconds by acquiring and pasting together three partial images ( figure 2 ) . to evaluate ed of ap and lateral projections for each patient
, ed was calculated using pcxmc v2.0 ( stuk , finland ) , a pc - based monte carlo program for calculating patient radiation doses in medical x - ray examinations .
the pcxmc 2.0 hermaphrodite mathematical phantoms were based on the work of cristy and eckerman ( 9 ) .
the tissue weighting factors and effective dose calculations were based on icrp 103 ( 10 ) .
factors for evaluating effective doses were derived from information in the dap of dicom tag data .
the pcxmc computer simulation used patient height , weight , gender , x - ray target angle , filter information , detector size , distance between detector and x - ray tube , tube voltage , and dap information .
these data were used in analysis of frequency , group average analyses , tendency analysis , and correlation analysis by spss v20.0 ( ibm corporation , new york , usa ) .
for the whole spine scanography of the 50 patients , an average of 6.1 images were taken for reconstruction of ap projections , while an average of 6.5 images were acquired for reconstruction of lateral projections , which equated to the acquisition of 7.37% more images in later versus ap projections .
for female patients , the average dap value was 2559 dgy cm for ap projections , and 9507 dgy cm for lateral projections . for male patients ,
the dap was 5591 dgy cm for ap projections , and 12975 dgy cm for lateral projections . for female patients ,
the ed was 0.43 msv for ap projections , and 0.53 msv for lateral projections . for male patients ,
for all patients , the average ap projection dap was 4257 dgy cm , while the average lateral projection dap was 11449 dgy cm , which implied that the lateral projection dap was 168% larger than the ap projection .
furthermore , the average ed of ap projections was 7% higher than the average ed of lateral projections ( table 2 ) .
abbreviations : ap , antero - posterior ; dap , dose area product ; ed , effective dose ; lat , lateral ; max , maximum ; min , minimum ; sd , standard deviation ; var , variance
. evaluation of the impact of aec mode on lateral projections revealed that using aec mode , the average dap value was 10,439 dgy cm , and the corresponding ed was 0.60 msv .
however , using the manual mode , the average dap value was 12964 dgy cm and ed was 0.77 msv .
thus , using the manual mode resulted in 24% higher dap values and 27% higher ed values .
analysis of the correlation of all patients revealed that r values for the correlation of dap and ed were 0.94213 and 0.76803 for ap and lateral projections , respectively , which indicated a significant correlation ( figure 3 ) .
dap is converted to ed by the following equation , where the conversion factor b values are 0.119334e-4 in ap projection and 6.27011e-5 in lat projection and the constants are 0.11954 , and 0.0463 , respectively .
y = ed ( mgy ) , x = dap ( dgy cm ) , a = constant , b = conversion factor .
pearson correlation coefficients resulting from analysis of the correlation of effective dose and bmi were 0.771 and 0.546 for ap and lateral projections , respectively , which indicated a higher significant correlation for ap projections ( p < 0.01 ) ( table 3 ) .
abbreviations : aec , auto exposure control ; dap , dose area product ; ed , effective dose ; lat , lateral ; max , maximum ; min , minimum ; sd , standard deviation ; var , variance . for normal bmi patients ( bmi : 18.5
kg / m ~ 23 kg / m ) , the pearson correlation coefficients of total dap for antero - posterior and lateral projections were respectively 0.739 , and 0.638 ( p < 0.05 ) and the pearson correlation coefficients of total effective dose for ap and lateral projections were 0.638 ( p < 0.01 ) , and 0.522 ( p < 0.05 ) , respectively .
for obese patients ( bmi > 23 kg / m ) , the pearson correlation coefficients of total dap for ap and lateral projections were 0.091 , and 0.017 ( p < 0.01 ) , respectively and the pearson correlation coefficients of total ed for ap and lateral projections were 0.591 ( p < 0.01 ) , and 0.078 ( p < 0.05 ) , respectively .
the correlation coefficients of aec with bmi also indicated the significance level of 0.586 ( p < 0.01 ) .
the correlation of dap with ed in each statistical group is mentioned in table 4 .
abbreviations : aec , auto exposure control ; ap , anterior - posterior ; bmi , body mass index ; dap , dose area product ; ed , effective dose , bmi , body mass index , aec , auto exposure contro .
the calculated ed following use of aipm for all patients was 0.6276 msv and 0.6716 msv for the ap and lateral projections , respectively , which were lower than the 0.798 msv ed for the ap projection , and higher than the 0.597 msv ed for the lateral projection reported by mogaadi et al . (
however , using aec , ed of lateral projections was 0.605 msv , which was slightly different .
accordingly , the results indicated that use of aec mode with aipm contributed significantly to a reduction in the exposure dose . in wss examinations , the conversion factors to ed from dap in ap projections and lateral projections
these conversion factors could be simply used to convert dap value into ed in a clinical field . by using the dap - ed conversion factor
, one can easily estimate the patient - specific ed value and use it as the reference value for reducing patient s dose . on analysis of bmi
, it can be detected that the pearson correlation coefficients in ap projections are higher than those in lateral projections .
so , it is considered that ed for patients in ap projections is higher than that in lateral projections .
the reason why the correlation of dap with bmi in ap projections are higher than that in lateral projections is considered due to the increase of exposure area in ap projections . in obese patients , since the difference of exposure area between ap projections and lateral projections is not so big , the correlation of bmi with dap value is low . in normal weight patients ,
since there is a considerable difference of exposure area between ap projections and lateral projections , the correlation of bmi with dap value is considered as high .
furthermore , the use of automatic exposure control is considerably related to bmi . in the analysis of correlation of dap with ed regarding to sex and bmi
, there is a high correlation between dap and ed , especially higher in lat projection than ap projection .
therefore , in wss examination , an effort to reduce dap will help reduce the patient s effective dose .
moreover , since this correlation of dap with ed is higher in aec mode than the manual mode , using aec can be applied to reduce the patient s effective dose .
compared to the single exposure method used with the film - screen method , use of aipm exhibited only a slight difference in ed .
however , the significant reduction in exam time and convenience to both the examiner and patient contribute to the value of aipm .
( 11 ) was carried out among patients between 16 years and 22 years and hansen et al .
compared to these studies , the subjects of this study were adults aged between 26 years and 74 years .
so , given the age of the patients , most of the patients were large - bodied adults .
considering this situation , it is assessed that in the wss examinations , using aipm is more useful to reduce the patient exposure dose than using the single exposure film - screen method .
it is because these two studies used wedge - shaped aluminium filter resulting in a reduced exposure dose by weakening the strength of x - ray beam exposed to the chest region .
this method is only available in single exposure method , and not in aipm ( table 5 ) .
this study was performed only among adult patients aged from 26 to 74 , and did not include pediatric patients .
so , this study has an important limitation , and further studies for pediatric patients should be performed in the future . in conclusion , to reduce patient exposure dose , it is essential to consider the detector response characteristics and the final image quality of the whole spine scanogram . in particular , understanding the exact characteristics of the device , and applying aec mode are useful for reducing patient exposure dose . | background : whole spine scanography ( wss ) is a radiologic examination that requires whole body x - ray exposure .
consequently , the amount of patient radiation exposure is higher than the radiation dose following routine x - ray examination.objectives:several studies have evaluated the patient effective dose ( ed ) following single exposure film - screen wss .
the objective of this study was to evaluate patient ed during wss , based on the automatic image pasting method for multiple exposure digital radiography ( apmdr ) .
further , the calculated eds were compared with the results of previous studies involving single exposure film - screen wss.patients and methods : we evaluated the ed of 50 consecutive patients ( m : f = 28:22 ) who underwent wss using apmdr . the anterior - posterior ( ap ) and lateral ( lat ) projection eds were evaluated based on the monte carlo simulation.results:using apmdr , the mean number of exposures was 6.1 for ap and 6.5 for lat projections .
lat projections required more exposures ( 6.55% ) than ap projections .
the mean ed was 0.6276 msv ( ap ) and 0.6716 msv ( lat ) .
the mean ed for lat projections was 0.6061 msv in automatic exposure control ( aec ) and 0.7694 msv in manual mode .
the relationship between dose - area - product ( dap ) and ed revealed a proportional correlation ( ap , r2 = 0.943 ; lat , r2 = 0.773 ) .
compared to prior research involving single exposure screen - film wss , the patient ed following wss using apmdr was lower on ap than on lat projections.conclusion:despite multiple exposures , ed control is more effective if wss is performed using apmdr in the aec mode . |
in the last few decades , computer simulation has become an important tool to investigate various phenomena in cardiac biology , including studies of single ion channel properties , action potentials of the myocyte [ 3 , 5 ] , dynamics of action potential propagation in tissue , subcellular calcium dynamics , etc . in spite of the advancement of computational technology , the simulation of action potential waves in three - dimensional ( 3d ) cardiac tissue with a realistic geometry is still considered as a large - scale simulation . general - purpose computing on gpus ( gpgpu ) is a recently emerging technology [ 1 , 4 , 8 ] , which uses gpus , instead of cpus , to compute large simulations in parallel .
. each gpu may contain 128240 stream processors whereas today s cpus contain 2 , 4 , or 8 cores . in this paper
, we demonstrate that the gpu is about 30~40 times faster than the cpu , enabling it to perform whole heart electrophysiology simulations within practical time . in this study , we chose the simulation of the propagation of the action potential in cardiac tissue , which is modeled as the propagation of a wave in an excitable medium . therefore , this technique can be applied to a number of phenomena in physics , chemistry , and biology .
the first was a 2d homogeneous sheet , and the second was an anatomic rabbit ventricular model with
fiber rotation , that is , an anisotropy that varies from point to point in the heart .
the gpu simulation was performed with a single nvidia geforce 8800 gt 1 gb graphic random - access memory ( ram ) and an nvidia geforce 9800 gx2 1 gb graphic ram .
these graphic cards were installed into a system with a dual - core 2.0 ghz amd opteron processor and 4 gb error correction code ( ecc ) ram .
the cpu simulation was performed with an 8-node high performance - computing ( hpc ) cluster .
each node has two dual - core 2.0 ghz amd opteron processors ( i.e. , 4 cores in each node ) and 4 gb ecc ram .
all 2d simulations , and all 3d simulations with one gpu , were performed with the nvidia geforce 8800 gt .
3d simulations with two or four gpus were performed with the nvidia geforce 9800 gx2 . because these gpus support only single precision , all floating - point calculations were done using single precision across both gpu and cpu simulations .
when the gpu kernel code is executed , it is similar to a cpu based parallel implementation accomplished through a series of threads , with each thread running independently in parallel .
similar to a cpu implementation , it was necessary to synchronize all threads after each ordinary differential equation ( ode ) or partial differential equation ( pde ) kernel execution .
we can then thread these intra - gpu as they control the processing within a single gpu .
in addition to having to manage threads intra - gpu , it was also necessary to have inter - gpu threads to control each gpu .
for instance , the nvidia geforce 9800 gx2 graphics card has two gpus on one card . in order to utilize each gpu
as with a cpu cluster with distributed memory , it is also necessary to manage the distributed gpu memory . however , unlike a cpu where data can be moved from one cpu to another , gpus can and must communicate with the cpu memory , that is , data is transferred from one gpu to the other gpu via the main ram ; gpu1ramgpu2 .
the cardiac tissue was modeled using the following partial differential equation:\documentclass[12pt]{minimal }
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\begin{document}$$ { \frac{\partial v}{\partial t } } = - { \frac{i}{{c_{m } } } } + \nabla \cdot d\nabla v , $ $ \end{document}where v is the transmembrane voltage , i is the total ionic current , cm is the transmembrane capacitance , and d is the diffusion tensor .
we solved this reaction - diffusion equation with the forward euler method , using the technique of operator splitting .
the time step was adaptively varied between 0.01 and 0.1 ms and the space step was 0.015 cm .
details of the modeling of cardiac tissue are described in our previous study . for each time step ,
the ode part was solved once and the pde part was solved four times for the 2d simulation and six times for the 3d simulation ( fig .
1sample gpu code to solve the reaction - diffusion equation in 2d tissue . at each time step , the ode part is called once and the pde part is called four times .
the ode kernel code and the pde kernel code are in the appendix sample gpu code to solve the reaction - diffusion equation in 2d tissue . at each time step , the ode part is called once and the pde part is called four times .
the ode kernel code and the pde kernel code are in the appendix to test the gpu code , we induced spiral waves in 2d and 3d tissue using cross - field stimulation , that is , two successive perpendicular rectilinear wave fronts . in each case
2).fig . 2action potential propagation in 2d tissue and in the anatomic heart model .
the spiral wave was induced by cross - field stimulation . c spiral wave breakup in 2d tissue .
g electrocardiogram from the anatomic heart simulation f action potential propagation in 2d tissue and in the anatomic heart model .
the spiral wave was induced by cross - field stimulation . c spiral wave breakup in 2d tissue .
g electrocardiogram from the anatomic heart simulation f for the 2d tissue simulations , the benchmark protocol involved pacing the tissue from the corner for 3 s of simulated time at a pacing cycle length of 150 ms .
tissue size was varied from 100 100 ( 1.5 cm 1.5 cm ) to 800 800 ( 12 cm 12 cm ) .
for the 3d tissue simulations , the benchmark protocol consisted of pacing the whole heart from the apex for 3 s of simulated time , at a pacing cycle length of 150 ms . finally , we investigated where the computational
we split the program into three parts , the ode calculation , the pde calculation , and the data transfer . in order to measure the data transfer time ,
the ode calculation and the pde calculation were skipped , and the total time elapsed was then assigned to data transfer .
then , skipping the ode calculation , we could measure the time for the pde calculation plus the data transfer .
the time for the pde calculation was then estimated by subtracting the data transfer time from the ( pde + data transfer ) time .
the time for the ode calculation was obtained by subtracting the ( pde + data transfer ) time from the time for the whole simulation .
when tissue is homogeneous , parallel computation is very efficient . to compute 1 s of simulated time in 100 100 tissue , a single gpu took 8.2 s , whereas the cpu took 201 s. for larger tissue ( 800 800 ) the gpu took 283 s , while the cpu took 13,113 s. this is
because as the tissue size becomes larger , the boundary / non - boundary ratio becomes smaller and the parallel computation becomes more efficient . in these cases ,
3comparison between gpu and cpu . a time to compute 3 s of simulation time in 2d tissue .
righty - axis is acceleration ( i.e. , computation time with cpu / computation time with gpu ) .
b time to simulate the whole heart for 1 s of the simulation time .
c computation ratio of the ode , the pde , and the data transfer for each simulation comparison between gpu and cpu . a time to compute 3 s of simulation time in 2d tissue .
righty - axis is acceleration ( i.e. , computation time with cpu / computation time with gpu ) .
b time to simulate the whole heart for 1 s of the simulation time .
c computation ratio of the ode , the pde , and the data transfer for each simulation to simulate the anatomic rabbit ventricular model for 1 s , the hpc cluster with 32 cpus ( 8 nodes ) took 45 min . on the other hand ,
one gpu took about 72 min and two and four gpus took about 43 and 27 min respectively for the same simulation ( fig .
on the other hand , the bottleneck of the computation with gpus is mainly in the pde part ( fig .
we demonstrate that gpus are substantially faster than cpus in the simulation of action potential propagation in cardiac tissue .
a single gpu simulation of the whole heart is only 1.6 times slower than the simulation in an hpc cluster , and two or four gpus are even faster than the hpc cluster , making the gpu a new tool for cardiac simulations .
however , like parallel cpu implementations , management of threads and memory must be well thought out if maximum performance is to be achieved . | in this technical note we show the promise of using graphic processing units ( gpus ) to accelerate simulations of electrical wave propagation in cardiac tissue , one of the more demanding computational problems in cardiology .
we have found that the computational speed of two - dimensional ( 2d ) tissue simulations with a single commercially available gpu is about 30 times faster than with a single 2.0 ghz advanced micro devices ( amd ) opteron processor .
we have also simulated wave conduction in the three - dimensional ( 3d ) anatomic heart with gpus where we found the computational speed with a single gpu is 1.6 times slower than with a 32-central processing unit ( cpu ) opteron cluster .
however , a cluster with two or four gpus is faster than the cpu - based cluster .
these results demonstrate that a commodity personal computer is able to perform a whole heart simulation of electrical wave conduction within times that enable the investigators to interact more easily with their simulations . |
ebus echobronchoscopy is a relatively recent examination method , introduced in clinical practice in 1992 .
it is a combination of echography and bronchoscopy , allowing the physician to directly examine the tumors and adenopathies situated next to the airways , both extrabronchial and extratracheal ones , inaccessible to traditional bronchoscopy . a real time ,
ultrasound guided sampling of cytology and histopathology specimens for diagnostic purposes is also possible with fine needle aspiration .
there are 2 types of ebus : - radial probe ebus ( rp - ebus ) for evaluation and bioptic sampling of peripheral tumors - convex probe ebus ( cp - ebus ) for evaluation and sampling of tumors and lymph node stations 2,3,4,7,10,11 outside of the bronchial tree but right next to the bronchial wall .
a doctor who wishes to perform ebus should be familiar with mediastinal anatomy , the characteristics of ultrasound , and must have a good bronchoscopy and echography knowledge .
main indications of ebus - tbna : - diagnostic and staging of non - small cell lung cancer ( nsclc ) - evaluation of mediastinal tumors ( ex : lymphomas ) - evaluation of mediastinal adenopathies of unknown etiology ( sarcoidosis , lymph node metastasis of a known tumor ) .
the contraindications of ebus are similar to the contraindications of bronchoscopy and they include : - myocardial infarction - life threatening arrhythmias - poorly controlled heart insufficiency - uncooperative patient - high risk for hemorrhage : current anticoagulation therapy , coagulopathies , thrombocytopenia , high urea and creatinine levels .
the examination can be performed in local anesthesia with 2% xylocaine , under conscious sedation or under general anesthesia with laryngeal mask or endotracheal tube sized at least 8 .
the introduction of ebus tbna has brought important changes to the staging and diagnostic strategy of lung cancer .
the introduction of ebus has changed this paradigm , ebus being a minimally invasive method with a cost / efficiency value slightly superior to mediastinoscopy .
we present the experience of the bronchoscopy department of the pulmonology clinic of cluj - napoca with ebus - tbna as a tool for the diagnosis and staging of tumors in contact with the bronchial wall and mediastinal and hilar adenopathies of unknown etiology . during the period august 2014
january 2016 we examined 152 patients with no direct or indirect signs of lung tumor at traditional bronchoscopy .
the examinations were carried out with a fuji echoendoscope , under general anesthesia with laryngeal mask .
all patients underwent fine needle aspiration ( tbna ) for diagnosis after rigorous evaluation , measuring and description of the visualized lymph node stations .
ebus preoperative staging for non - small tumors has been performed in 4 patients , starting with n3 contralateral to the tumor , followed by n2 , n1 .
an experienced cytopathologist was part of the team and she was present in the operation room where she performed the quick - diff rapid coloration and the cytological examination .
the real - time examination provided us a direct feedback on the quality of the sampled tissue , it confirmed that the tissue was indeed taken from a lymph node ( > 30% lymphocytes from nucleated cells ) or from the tumor , and identified within 13 minutes the existence of tumoral cells or infirmed the supposition of a malignant disease .
these results were confronted with the results of the histopathological examination with immunohistochemical staining , performed by a laboratory of reference .
the distribution of the patients according to their presumed diagnostic was as follows : adenopathies of unknown etiology : 25 patients ; suspect of lung tumor : 81 patients ; sarcoidosis : 28 patients ; suspect of lymphoma : 2 patients ; lung or bone metastasis of unknown origin : 6 patients ; unknown adenopathies with a documented tumor ( breast , prostate , cervical , renal ) in personal history : 10 patients .
the average age of our patients was 54.43 years and 64% came from urban and 36% from rural background .
the results of the onsite examination rose showed a positive diagnostic rate of 59.2% from the total number of the patients from our study , for 62 patients the results were negative , and for 90 patients we could make up a diagnosis on the spot . regarding the diagnostic yield of tumors : in 97 patients suspected of having primary lung tumor or other tumors we could confirm the existence of tumoral cells on site with microscopic examination rose in the case of 79 patients , representing 81% of the patients .
ebus - tbna brought the final histological confirmation ( tumors , sarcoidosis , limfoma ) in 82.8% of the cases .
a tumor confirmation was obtained in 61.8% of the total number of patients and in 95% in the case of the patients who were suspected of having tumor .
the histological profile of the cases was the following : adenocarcinoma : 29 patients ; small cell lung cancer : 19 patients ; squamous cell carcinoma 8 patients ; non - small cell carcinoma : 18 patients ; metastatic carcinoma : 5 patients ( 1 metastases from prostate carcinoma , 2 metastases from renal carcinoma,1 metastasis of melanoma and 1 metastasis of colonic carcinoma ) .
we present one of our clinical cases to illustrate a clinical and imaging pattern of ebus indication and to emphasize the importance of this method for the diagnosis in pulmonology .
a 38 year old female , non - smoker , with no personal or familiar medical history , with professional exposure to toxic substances ( worker in a factory of car cables ) is admitted for intense precordial pain of a sudden onset , without radiation .
electrocardiogram showed sinus rhythm , a heart rate of 90 b / min , intermediate qrs , inferior - lateral t waves .
myocardial infarction was excluded by a normal coronarograpy while angio - computed tomography ( angio - ct ) excluded lung embolism ( no evidence of pulmonary emboli ) .
however , the angio - ct showed a 2 cm suspicious nodule in the right lower lobe ( rll ) , lower from the bronchial bifurcation towards the basal segments , small bilateral pleural effusions , mainly in the right side , and multiple adenopathies : right interbronchial , subcarinal , barety space , right paratracheal , with dimensions varying up to 2.5 cm . at this moment
we performed a traditional bronchoscopy which evidenced the following : no modifications of the trachea , slightly enlarged tracheal bifurcation , hypervascularization of the main bronchus .
, we can choose from : mediastinoscopy , open lung biopsy , ebus - tbna .
we pick the less invasive procedure available that can bring a rapid result : ebus with on - site cytodiagnostic .
the ebus examination showed enlarged mediastinal and hilar lymph nodes , their size varying between 13 cm .
the on - site cytological examination brought the following : lymph node stations 7 , 11r : moderate cellularity , mainly made up of tumor cells , frequent red blood cells , rare eosinophils and polymorphonuclear cells .
the tumor cells are medium sized , mononucleated , with excentric nucleus , one macronucleoli / nucleus , cytoplasmatic vacuoles .
the conclusions of the cytological examination was of non - small cell carcinoma ( adenocarcinoma ) .
the final histopathological examination with immunohistochemical staining concluded that the final diagnosis was ttf-1 positive adenocarcinoma of the lung .
the next steps in order to offer our patient a proper staging and pre - therapeutic evaluation would have been a head and abdominal computed tomography ( for evaluation of possible brain / liver / abdominal metastasis ) , followed by referral to medical oncologist for chemotherapy , but an unfortunate complication occurred : bilateral infrapopliteal deep venous thrombosis diagnosed clinically and by echo - doppler .
a proper diagnosis and staging of lung cancer is very important , as it is the key to treatment .
computer tomography and pet - ct continue to play a key role in the diagnosis and staging of lung cancer . in order to achieve histological confirmation , which allows a modern cancer therapy ,
twenty years ago tissue samples from tumors localized in the mediastinum was obtained by mediastinoscopy or open lung biopsy with thoracotomy .
the introduction of the minimally invasive method ebus has changed the diagnostic and staging methodology of lung cancer . the cost / efficiency rate is high , when compared with surgical procedures .
the sensitivity of ct , pet - ct and ebus - tbna for lung cancer diagnosis and staging is 76.9% , 80% and 92.3% respectively according to a study by yasufuku et al .
the specificity of the methods was 55.3% , 70.1% and 100% , and the diagnostic accuracy 60.8% , 72.5% and 98% . in our study
the sensitivity of the method and diagnostic accuracy in lung cancer was 95 % confirmation from the total number of cancer suspicions .
the accp s acquire registry has begun collecting and analyzing data on 1317 patients undergoing ebus at 6 hospitals ( all or nearly all were academic centers training fellows ) .
ebus proved to be quite safe , indeed , with the following reported complications occurring within 24 hours : risk of pneumothorax with ebus alone : 0.2% , or 1 in 500 . in comparison the risk of pneumothorax after transbronchial biopsy was of : 2.7% , or 1 in 37 .
the study reported seven pneumothorax complications of which 4 required tube thoracostomy and the others resolved without intervention .
the risk of bleeding requiring intervention was reported at 0.2% , or 1 in 439 and one patient succumbed from bleeding ( 1 in 1317 or 0.08% ) .
the overall 24-hour complication rate was 1.4% , but it was < 1% if transbronchial biopsy was not performed .
about 1015% of lung cancers occur in non - smokers of which 2/3 are women . in case of young patients ,
lung cancer has a few particular characteristics : it appears in a large percent in women , the predominant histological type being adenocarcinoma , it is usually diagnosed in advanced stages and survival rate is similar to the one in case of older patients .
the particular features of our case were the young age , lack of smoking history , atypical clinical presentation and the presence of a lung nodule accessible only by ebus , mediastinoscopy or thoracotomy , but not by traditional bronchoscopy .
for example : was it a lung embolism , although it was refuted by angio - ct ?
the ulterior onset of a deep venous thrombosis , most probably in the context of a paraneoplastic syndrome suggests a possible lung embolism not seen on ct , which can explain the initial symptoms ( sudden onset of severe chest pain ) .
the m ( metastasis ) status is also not perfectly clear : the ct - based stage showed mx but the bilateral pleural effusion of possible malignant origin could have classified it as m1 , stage iv .
the influence of environmental factors : the patient was a factory worker , possibly exposed to : chrome , rubber , nickel , cadmium , carbon electrodes . according to the journal of thoracic disease , the exposure to chrome , nickel , cadmium and rubber derivatives has been proved to have carcinogenic effects in humans , and the carbon electrodes are possible to be carcinogens .
a review of the literature shows that patients with lung adenocarcinoma have a 20x increased risk to have thrombophlebitis / deep venous thrombosis .
young patients with adenocarcinoma of the lung are frequently diagnosed in advanced stages , stadium iv . according to journal of thoracic disease , regarding adenocarcinoma patients ,
the average time passed from first contact with a doctor until consultation by a respiratory specialist is of 27 days , and 23 more days are generally required to complete the investigations and have a final diagnosis .
the unusual presentation form , the quick investigations and the availability of ebus - tbna allowed a very quick diagnosis .
the total time from the first manifestation of the tumor ( with severe chest pain ) lead to emergency admission to hospital and start of the diagnostic pathways , leading to a definitive diagnosis of lung adenocarcinoma in just 12 days .
the 3rd edition of the accp ( american college of chest physicians ) guidelines regarding lung cancer recommends the minimally invasive technique ebus as a gold standard for the diagnosis and staging of lung cancer with involvement of the n2 , n3 lymph node stations , before mediastinoscopy , thoracotomy or other more invasive techniques . in our case ,
regarding the cost - effectiveness of ebus compared with mediastinoscopy , gargoum et al . concluded that an ebus based diagnostic pathway results in cost effective patient management compared with traditional mediastinoscopy even in the first year of service . in a 2009 study from the uk , medford et al . also found that ebus can save health community costs , pointing out the need for new , more precise coding procedures for ebus to reflect the true value and difficulty of the procedure .
our team has experienced the same problem : lack of proper procedure code for ebus - tbna , the available codes were those of standard bronchoscopy with biopsy or lymph node puncture .
endobronchial ultrasound examination combined with fine needle aspiration ebus - tbna is a minimally invasive diagnostic and staging examination tool of the mediastinum .
it is also well tolerated by patients , which is a major advantage , and grants ebus a top position among the diagnostic and staging methods of lung cancer . by combining the ebus technique with rose ( rapid on site cytological examination ) we can obtain a cytological confirmation on the examination site , during the examination .
it is a valuable and cost efficient tool for the mediastinum and exo - bronchial tumors in contact with the bronchial wall . by examination of the mediastinal and hilar lymph node stations ,
ebus tbna is a quick staging tool for lung cancer , avoiding thoracotomies and mediastinoscopies , which are more invasive and with more risk for the patient . by the introduction of this method in our country
one year ago , we can diagnose patients with lung and mediastinal tumors , which can not be diagnosed by traditional bronchoscopy . | background and aimendobronchial ultrasound ( ebus ) is a recent minimally invasive , safe examination method for the mediastinum , with a good diagnostic precision.this method makes possible real time examination with transbronchial fine needle aspiration , diagnostic transbronchial needle aspiration ( tbna ) and staging of non - small pulmonary tumors , as well as diagnosis of mediastinal and hilar adenopathies of various causes.methodswe present the experience of the bronchoscopy department of the pulmonology clinic of cluj - napoca with ebus - tbna as a tool for the diagnosis and staging of tumors in contact with the bronchial wall and mediastinal and hilar adenopathies of unknown etiology . during the period august 2014
january 2016 we examined 152 patients with no direct or indirect signs of lung tumor in traditional bronchoscopy .
rapid on site evaluation ( rose ) was available for all patients.resultsour study is a retrospective study of 152 ebus - tbna examinations .
the average age of our patients was 54.43 years and 64% came from urban and 36% from rural background .
ebus - tbna brought the final histological confirmation ( tumors , sarcoidosis , limphoma ) in 82.8% of the cases .
a tumor confirmation was obtained in 95% of the patients who were suspected of having tumor.for a better understanding of the importance of this method in the daily clinical practice we present a case of peripheral pulmonary neoplasm with mediastinal and hilar adenopathies , where the contribution of ebus - tbna to a rapid diagnosis was essential.conclusionby the introduction of this method in our country one year ago , we can diagnose patients with lung and mediastinal tumors , which can not be diagnosed by traditional bronchoscopy .
this brings a valuable contribution to the improvement of lung cancer staging and diagnostic . |
there is some evidence that proliferation of pleural mesothelial cells ( mc ) occurs soon after deposition of asbestos fibers . to study this effect
, we instilled a single dose of 0.1 mg crocidolite into the lungs of mice for 1 and 6 weeks and counted labeled nuclei after 3h - thymidine ( 3ht ) injection .
short fibers ( < 1 micron ) induced little change in the lung ; they were mostly phagocytized and had a minimal effect on mc labeling .
long fibers up to 20 microns damaged the bronchiolar epithelium and were incorporated into connective tissue . increased 3ht uptake was seen in fibroblasts and epithelial cells and also in mc , which peaked at 2% labeled at 1 week compared to near 0% labeling in controls .
no fibers were found in or near labeled mc , which suggested that a cytokine generated in the lung during the early response phase might induce mc proliferation . to look for a cytokine effect in vitro ,
we instilled asbestos into rat lungs and , after 1 and 6 weeks , bronchoalveolar and pleural lavage fluids as well as macrophages were collected .
alveolar macrophages contained fibers , but pleural macrophages ( pm ) did not .
after short - term culture , macrophage supernatants and the lavage fluids were tested on rat lung mc in culture . at 1 week
, pm secreted growth factor(s ) for mc , and the mitogenic effect was more pronounced with lavage fluids .
no effects on mc were found using material prepared 6 weeks after asbestos .
the early mc growth increase was not blocked by antibodies to cytokines , such as platelet - derived growth factor , fibroblast growth factors , or tumor necrosis factor , but was inhibited by anti - keratinocyte growth factor ( anti - kgf ) .
the results show that an early growth phase of mc after asbestos exposure appears unrelated to particle translocation to the pleura but is associated with cytokine release , most likely kgf , by lung cells.imagesfigure 3 . | |
snake bite was added to the world health organization ( who ) neglected list of tropical diseases in 2009 .
an estimated 5.5 million people are bitten by snakes per year resulting in 400,000 amputations and between 20,000 and 125,000 deaths annually .
a relatively recent report highlighted that one of the highest burdens exist in sub - saharan africa .
the incidence of snake bite envenomation in central sub - saharan africa was 3.33/100,000 population with a mortality rate of 0.53/100,000 population .
these areas should have access to antivenom and protocols to guide the management of these cases .
hospitals where snake bites are uncommon should also have protocols on standby in the event of such cases presenting .
the burden of human immunodeficiency virus ( hiv ) in developing countries is substantial . according to who statistics from 2011 ,
there are over 5.6 million people living with hiv in south africa and over 16 million acquired immune deficiency syndrome ( aids ) orphans living in sub - saharan africa .
the effect of hiv on snake bites in south african children has not been described in the literature .
hiv has been associated with multiorgan dysfunction but never directly associated with snake bites . the case discussed below
is that of a 1-year - old child from a poor community , 300 km west of johannesburg . the burden of mortality for snake bite victims has been shown to occur in poor communities .
a 1-year - old child was referred to our tertiary center from a peripheral hospital .
he presented 2 days after the snake bite with severe left upper limb swelling extending from the hand up into the axilla and involving the shoulder joint .
features of cytotoxic envenomation with a wide area of necrotic skin on the volar aspect of the arm and forearm required extensive debridement postinitial emergency fasciotomy ( fig .
polyvalent antivenom ( south african institute of medical research polyvalent snakebite antiserum ) administration was delayed and once obtained was given 4 days after the snake bite .
this antivenom is made from the venom of 10 different species including adders , mambas , and cobras . during admission to the pediatric intensive care unit ( picu ) ,
a persistent thrombocytopenia occurred , caused by either the hiv infection or the snake 's venom ( cytotoxic envenomation presentation in keeping with bitis arietans [ puff adder ] envenomation ) .
repeated lower respiratory tract infections and overwhelming sepsis ultimately resulted in the child 's death .
left : initial presentation with swelling and demarcation of superficial tissue necrosis extending from the left hand up to the axilla .
maggots were found in the axillary region . despite extensive slough that had to be removed ,
there is substantial morbidity and mortality related to admission of an hiv positive child to a picu .
argent showed that these children are more commonly affected by severe respiratory tract infections as in the case described above . before the advent of antiretroviral medication , the mortality rate was 100% in children with aids and 20% in cases of severe pneumonia .
hiv may in fact cause a pancytopenia due to either bone marrow hypoplasia or myelofibrosis .
symptomatic hiv associated thrombocytopenia occurs most commonly in undiagnosed children with hiv under 2 years of age .
initiation of antiretroviral therapy has been shown to be effective in treating hiv - related thrombocytopenia but the response is variable .
hiv - related thrombocytopenia was found to be a poor prognostic factor with no specific correlation to the patient 's immune status .
this case shows similar findings to presentations from a study done in eshowe , south africa
although this patient had a fasciotomy of both his arm and forearm , the tissues were not found to be typical of a compartment syndrome , putting the diagnosis of compartment syndrome in to question .
the skin and subcutaneous tissues of the arm and forearm in fact became necrotic and subsequently required extensive debridement .
children in this series were also at greatest risk of developing bite site complications and severe and gross swelling .
the snake species that bit this child had a combination of hemorrhagic and cytotoxic venom .
this was evidenced by massive tissue necrosis of the arm as well as pancytopenia that required transfusion with both packed red cells and platelets .
the snake that commonly causes these symptoms of envenomation and is also commonly found in the area in which the patient lived is the african puff adder ( b arietans ) .
although the species was not positively identified by a specialist , the clinical features were typical of b arietans .
also this is the main cytotoxic species found in this area of south africa , which explains why myotoxic and cytolytic components within the venom contributed to local tissue necrosis .
other digestive hydrolases , secondary effects of inflammation , ischemia from thrombosis and compartment syndrome also contribute to local necrosis .
currier et al showed that the african puff adder has a high degree of inter - specimen variability in terms of protein expression , immunogenicity , and activity of snake venom metalloproteinases .
this variation may have implications for efficacy of antivenom given to patients and may have future consequences in selecting specimens for antivenom production .
the effect of hiv on envenomation has not been discussed before in the literature it is possible that the combination of the hiv - related immunodeficiency was exacerbated by the envenomation in this patient that led to his demise .
this case highlights the challenge of treating a cytotoxic snake bite in a young child with hiv and the detrimental outcome of delay in treatment . hiv infection and envenomation were co - contributory in leading to the thrombocytopenia and overwhelming sepsis in this case . | abstractsnake bites occur commonly in the rural areas of south africa .
hospitals where snake bites are uncommon should always have protocols on standby in the event of such cases presenting .
this is the first reported case documenting the effect of human immunodeficiency virus ( hiv ) on snake bite in south african children.a case report and review of relevant information about the case was undertaken.we present a case of a 1-year - old child referred from a peripheral hospital following a snake bite to the left upper limb with a compartment syndrome and features of cytotoxic envenomation .
the patient presented late with a wide area of necrotic skin on the arm requiring extensive debridement .
the underlying muscle was not necrotic .
polyvalent antivenom ( south african institute of medical research polyvalent snakebite antiserum ) administration was delayed by 4 days after the snake bite .
the patient was also diagnosed with hiv and a persistent thrombocytopenia possibly due to both hiv infection and the snake bite venom .
lower respiratory tract infections with subsequent overwhelming sepsis ultimately resulted in the child 's death.the case highlights the challenge of treating a snake bite in a young child with hiv and the detrimental outcome of delayed treatment .
a protocol is essential in the management of snake bites in all hospitals.level iv , case report.this case highlights the interaction of snake bite envenomation and hiv infection on thrombocytopenia . |
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