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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: gestational diabetes mellitus ( gdm ) , a serious metabolic disorder during pregnancy , may lead to several complications including perinatal morbidity and mortality .
women with gdm have higher risk of cesarean section and are also at greater risk of developing type 2 diabetes in the future . while the risks and complications due to gdm are well established , there is still considerable debate about the best screening and diagnostic methods for gdm . traditionally , an oral glucose tolerance test ( ogtt ) is employed for diagnosis of gdm , and this is considered to be the gold standard . for diagnosis of type 2 diabetes in the nonpregnant state , in 2010 , the american diabetes association included hba1c as a diagnostic test with a cut point of 6.5% ( 48 mmol / mol ) which was later supported by the world health organization .
the international association of the diabetes and pregnancy study groups ( iadpsg ) recommends hba1c in the first trimester of pregnancy to rule out overt diabetes .
an ogtt requires pregnant women to come for the test in the fasting state which can be cumbersome and time - consuming .
in contrast , hba1c has the advantage that it can be measured at any time of the day irrespective of meal timings .
several studies have reported that hba1c levels are lower among pregnant women compared to the general population . however , thus far hba1c has not been considered suitable for diagnosis of gdm during pregnancy due to its lower sensitivity and the lack of reliable cut points .
the utility of hba1c in screening and diagnosis of gdm as well as using it as a prognostic indicator for pregnancy outcomes has not been studied adequately .
the aim of this study was , therefore , to determine the optimal hba1c cut point for diagnosis of gdm and to evaluate its usefulness as a prognostic indicator for pregnancy outcomes among asian indian pregnant women .
this study is part of the women in india with gdm strategy ( wings ) project of the international diabetes federation carried out in tamil nadu , south india .
pregnant women ( n = 1459 ) were screened at their first booking at 15 government primary health centers at kancheepuram and 6 private health centers at chennai in tamil nadu .
booking visit refers to the first antenatal visit of the pregnant women to the health center at which point they were screened for gdm .
written informed consent was obtained in the local language from all participants , and the study was approved by the institutional ethics committee of the madras diabetes research foundation ( mdrf ) .
permission was also obtained from the directorate of public health and the ministry of health , government of tamil nadu , to conduct the study in the primary health centers .
height was measured using a stadiometer ( seca model 213 , seca gmbh co , hamburg , germany ) to the nearest 0.1 cm , and weight was measured with an electronic weighing machine ( seca model 803 , seca gmbh co. , ) to the nearest 0.1 kg . the body mass index ( bmi )
was calculated as weight ( kg ) divided by height ( in meters ) squared .
participants were requested to report in the fasting state ( at least 8 h of overnight fasting ) .
82.5 g oral glucose ( equivalent to 75 g of anhydrous glucose ) was then dissolved in 300 ml of water and was given to the pregnant women who consumed it within 5 min .
further venous samples were drawn at 1 h and 2 h after the ingestion of oral glucose .
pg was estimated by the glucose oxidase peroxidase method using autoanalyzer au2700 ( beckman , fullerton , ca , usa ) .
hba1c was measured using high - performance liquid chromatography using variant ii turbo machine ( biorad , hercules , ca , usa ) .
the intra- and inter - assay coefficients of variation for the glucose and hba1c ranged from 0.78%1.68% to 0.59%1.97% , respectively .
all samples were processed in our laboratory which is certified by the college of american pathologists and by the national accreditation board for testing and calibration laboratories , government of india .
accordingly , in the first trimester , gdm was diagnosed if fasting pg value was between 5.1 and 7.0
mmol / l ( 92126 mg / dl ) and in the 2/3 trimester , if fasting or 1 h or 2 h pg values met or exceeded 5.1 mmol / l ( 92 mg / dl ) , 10.0 mmol / l ( 180 mg / dl ) , and 8.5 mmol / l ( 153 mg / dl ) , respectively .
commonly , infants exceeding the 90 percentile for any specific gestation age are considered macrosomic or large for gestation age . in indians
, 3.45 kg corresponds to the 90 percentile of birth weight and hence the cutoff for macrosomia used is 3.5 kg .
all analyses were done using windows - based spss statistical package ( version 15.0 , chicago , il , usa ) .
receiver operating characteristic ( roc ) curves were plotted using sensitivity and 1-specificity for different hba1c values against gdm diagnosed by iadpsg criteria , and the c - statistic was calculated .
binary logistic regression analysis was used to evaluate the association of hba1c with pregnancy outcomes .
accordingly , in the first trimester , gdm was diagnosed if fasting pg value was between 5.1 and 7.0 mmol / l ( 92126 mg / dl ) and in the 2/3 trimester , if fasting or 1 h or 2 h pg values met or exceeded 5.1 mmol / l ( 92 mg / dl ) , 10.0 mmol / l ( 180 mg / dl ) , and 8.5 mmol / l ( 153 mg / dl ) , respectively .
commonly , infants exceeding the 90 percentile for any specific gestation age are considered macrosomic or large for gestation age . in indians
, 3.45 kg corresponds to the 90 percentile of birth weight and hence the cutoff for macrosomia used is 3.5 kg .
all analyses were done using windows - based spss statistical package ( version 15.0 , chicago , il , usa ) .
receiver operating characteristic ( roc ) curves were plotted using sensitivity and 1-specificity for different hba1c values against gdm diagnosed by iadpsg criteria , and the c - statistic was calculated .
binary logistic regression analysis was used to evaluate the association of hba1c with pregnancy outcomes .
a total of 1459 pregnant women who were screened for gdm under the wings project had booking visit hba1c estimations and pregnancy outcomes data available . mean age of these women was 26.1 3.9 years , bmi was 24.5 4.8 kg / m , mean hba1c was 4.9% 0.5% , and mean week of gestation was 19.5 7.6 weeks .
gdm was identified in 195 women ( n = 33 in the first trimester and n = 162 in the 2/3 trimester ) .
women with gdm were significantly older and had higher bmi at booking , higher hba1c at booking , greater history of previous gdm , and greater family history of type 2 diabetes than women without gdm .
there was no significant difference in the mean week of gestation between women with and without gdm .
comparison of clinical characteristics between women with and without gestational diabetes mellitus to analyze the utility of hba1c in diagnosing gdm , we constructed a roc curve keeping the ogtt as reference diagnostic criteria ( iadpsg ) .
the resulting roc curve showed that a hba1c cutoff of 5.0% ( 31 mmol / mol ) had a sensitivity of 66.2% and specificity of 56.2% with a c - statistic of 0.679 ( confidence interval [ ci ] : 0.6550.703 ) [ figure 1 ] . when the hba1c cut points were increased to 5.5% ( 37 mmol / mol ) and 5.7% ( 39 mmol / mol ) , the specificity improved to 92% and 96.8% , respectively , but the sensitivity drastically came down to 24.6% and 14.9% , respectively .
conversely , when the hba1c cutoff was lowered to 4.7% ( 28 mmol / mol ) , the sensitivity increased to 88.2% , but specificity decreased to 27.8% [ table 2 ] .
receiver operating characteristic curve of glycated hemoglobin versus diagnosis of gestational diabetes mellitus comparison of sensitivity and specificity of different glycated hemoglobin cut points in comparison with the international association of the diabetes and pregnancy study groups criteria to diagnose gestational diabetes mellitus we next compared the baseline clinical characteristics and pregnancy outcomes in women who had a hba1c of 5% ( n = 683 ) and < 5% ( n = 776 ) , irrespective of their gdm status [ table 3 ] .
age , bmi , previous history of gdm , and macrosomia were significantly higher among pregnant women whose hba1c were 5.0% ( 31 mmol / mol ) than those < 5.0% ( < 31 mmol / mol ) .
normal vaginal delivery was significantly higher in women with hba1c < 5.0% ( < 31 mmol / mol ) .
there were no significant differences in any of the maternal and neonatal complications other than macrosomia between the two groups .
comparison of baseline clinical characteristics and pregnancy outcomes of women with glycated hemoglobin < 5.0% ( < 31 mmol / mol ) and 5.0% ( 31mmol / mol ) the unadjusted odds ratio ( or ) for pregnant women ( irrespective of their glycemic status ) with hba1c 5.0% ( 31 mmol / mol ) to have a macrosomic baby was 2.03 ( ci : 1.323.12 , p = 0.001 ) and after adjusting for age , bmi , family history of type 2 diabetes , and previous history of gdm , the or was 1.92 ( ci : 1.242.97 , p = 0.003 ) [ table 4 ] .
binary logistic regression showing risk of adverse outcomes in women with glycated hemoglobin 5.0% ( 31 mmol / mol ) using glycated hemoglobin < 5.0% ( < 31 mmol / mol ) as reference
this study shows the following findings :
hba1c does not have adequate sensitivity and specificity for diagnosis of gdm and hence can not effectively replace an ogtt for diagnosis of gdma hba1c cut point of 5.0% ( 31 mmol / mol ) is associated with an increased risk of macrosomia .
hba1c does not have adequate sensitivity and specificity for diagnosis of gdm and hence can not effectively replace an ogtt for diagnosis of gdm a hba1c cut point of 5.0% ( 31 mmol / mol ) is associated with an increased risk of macrosomia .
hba1c has been a well - established tool for screening and management of type 2 diabetes .
nielson et al . found that in normal pregnancy , hba1c was reduced in the first trimester and it further decreased in the third trimester .
. showed that hba1c was lower at all three trimesters in normal pregnant women compared to their nonpregnant counterparts .
although there is no consensus on the reference range of hba1c in pregnant women , the optimum glycemic goal of < 6.0% ( < 42 mmol / mol ) recommended in pregnant women with preexisting type 1 or type 2 diabetes may be too high for women with gdm .
our study supports earlier studies that due to its low sensitivity , hba1c can not be used as an alternative to ogtt to diagnose gdm . in a study conducted by oshea et al .
, it was shown that 46% of women with gdm could be diagnosed with hba1c cut point of 5.4% ( 36 mmol / mol ) .
similar to these results , our study also showed that hba1c of 5.0% ( 31 mmol / mol ) could identify 46.8% of women with gdm diagnosed by the iadpsg criteria .
the hapo study showed that adverse outcomes were significantly stronger with glucose measures than with hba1c , and they concluded that hba1c could not be used as an alternative to ogtt . a recent study from china suggested that a hba1c cut point of 5.3% ( 34 mmol / mol ) could be used along with fasting pg 79 mg / dl ( 4.4 mmol / l ) as a first step screening test for gdm . another study from brazil which evaluated the performance of hba1c as diagnostic tool for gdm showed that a cutoff of 5.4% ( 36 mmol / mol ) has the optimal sensitivity and specificity , but the sensitivity of 70% is still not satisfactory .
there is growing data to suggest that hba1c can be used for predicting adverse pregnancy outcomes in women with gdm .
have shown that higher hba1c levels are significantly associated with primary and secondary outcomes studied under hapo study .
we found that pregnant women with hba1c of 5.0% ( 31 mmol / mol ) were significantly older and had higher bmi and higher previous history of gdm .
this is confirmed in our study as women with hba1c 5.0% ( 31 mmol / mol ) were at a higher risk of delivering macrosomic babies .
however , other pregnancy outcomes did not statistically differ in women who had hba1c cut points below this level .
found that all gdm women who had hba1c 6.7% ( 50 mmol / mol ) delivered macrosomic babies .
showed that macrosomic babies were born to women with hba1c > 6.3% ( > 45 mmol / mol ) in the last month of pregnancy .
a study from denmark showed that macrosomia was three times higher in women who had hba1c of 5.6% ( 38 mmol / mol ) .
we found that pregnant women who had a hba1c of 5.0% ( 31 mmol / mol ) had higher risk of macrosomia , majority of whom did not have gdm .
this finding is of particular interest because it shows that hba1c may be used as a marker for macrosomia , independent of the diagnosis of gdm .
recently , koyanagi et al . reported a rising prevalence of non - gdm macrosomia worldwide , implying that gdm is not the only reason for macrosomic babies .
reported that majority of macrosomic infants are born to nondiabetic mothers , and women with multiple prior macrosomic infants were at a higher risk of delivering macrosomic babies .
this study has several strengths : first , it is a prospective study with fairly large sample size .
second , our data report on association between hba1c and adverse pregnancy outcomes , especially macrosomia .
one of the limitations of the study was that repeated measures of hba1c were not done during pregnancy , so we could not look at the effect of serial changes in hba1c during pregnancy on outcomes .
earlier studies have reported that changes in hba1c during pregnancy from first to second to third trimester were associated with birth weight .
this study shows that hba1c due to its low sensitivity is not useful for screening or diagnosis of gdm subjects and hence is not an effective replacement for ogtt in diagnosing gdm .
nevertheless , baseline hba1c levels could be used to predict pregnancy outcomes , especially fetal macrosomia .
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### Summary:
: aim : the aim of this study was to determine the optimal glycated hemoglobin ( hba1c ) cut point for diagnosis of gestational diabetes mellitus ( gdm ) and to evaluate the usefulness of hba1c as a prognostic indicator for adverse pregnancy outcomes.methods:hba1c estimations were carried out in 1459 pregnant women attending antenatal care centers in urban and rural tamil nadu in south india .
an oral glucose tolerance test was carried out using 75 g anhydrous glucose , and gdm was diagnosed using the international association of the diabetes and pregnancy study groups criteria.results:gdm was diagnosed in 195 women .
receiver operating curves showed a hba1c cut point of 5.0% ( 31 mmol / mol ) have a sensitivity of 66.2% and specificity of 56.2% for identifying gdm ( area under the curve 0.679 , confidence interval [ ci ] : 0.6550.703 ) .
women with hba1c 5.0% ( 31 mmol / mol ) were significantly older and had higher body mass index , greater history of previous gdm , and a higher prevalence of macrosomia compared to women with hba1c < 5.0% ( < 31 mmol / mol ) .
the adjusted odds ratio for macrosomia in those with hba1c
5.0% ( 31 mmol / mol ) was 1.92 ( ci : 1.242.97 , p = 0.003 ) .
however , other pregnancy outcomes were not significantly different.conclusion:in asian indian pregnant women , a hba1c of 5.0% ( 31 mmol / mol ) or greater is associated with increased risk of macrosomia .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: today , lifestyle is an important and significant strategy in which to prevent non - communicable diseases ( ncds ) ( 1 ) .
worldwide , over one third of mortality rate occurs due to an unhealthy lifestyle , and is the leading cause of prevalence of coronary diseases , hypertension , type ii diabetes , tooth decay , stroke and a number of cancers ( 2 ) . in iran , diseases caused by an unhealthy lifestyle are the most common cause of morbidity and mortality ( 3 ) . the world health organization ( who ) believes that most risk factors that are the major factors in mortality can be managed via changing and modifying lifestyle ( 4 ) . in this regard , one of the objectives that will be determined by the who up to 2020 is to promote a healthy lifestyle among people . to this purpose
, it is suggested that nations consider placing strategies on their agenda that are effective in promoting individual and social life ( 5 ) , and reduce factors like improper physical activities , poor nutrition and drug abuse that direct the lifestyle towards failing health ( 4 ) .
one of these strategies is to increase peoples control on their life choices or their empowerment ( 6 ) . in promoting health
, it is believed that people are empowered such that they take the responsibility of their health and have a healthy lifestyle ( 7 ) . in other words , due to the selective nature of behavior in lifestyle , it is necessary to pay attention to empowerment of each individual ( 8) .
this necessity increases in women , due to the relation between most gender - dependent problems and diseases including breast and uterus cancer , obesity , psychological problems and depression , stress and menopause ( 9 ) . in general
, limited evidence exists in this regard , due to the difficulty of measuring empowerment in society .
recently , several studies in britain were conducted , on the relation between empowerment and health ; however , they were not sufficiently reliable to be referred to , since their focus had not been on health outcomes .
therefore , there is a requirement to conduct studies to specify the relation between various levels of empowerment with individual and social health outcomes ( 10 ) .
moreover , promoting women s empowerment using strategies of ngos is an important factor in their lives , and provides an opportunity for them to choose , and this can bring about expected outcomes ( 11 ) .
the health companions population ( known as hamyaran ) which operates under the supervision of the state welfare organization of iran , is an ngo that operates to create opportunities and resources to promote objectives of social and health development .
empowerment of this population is one of the priorities of the state welfare organization . to this purpose ,
investigating empowerment dimensions of these people , and its relation to healthy lifestyle , can be helpful for future measures .
this paper is part of a larger study on this issue that was conducted to find the relation between individual empowerment and lifestyles of women who are members of the health companions population as a pilot study in northern iran .
the specific objectives of this study are to determine individual empowerment of subjects under study , to determine lifestyle status of subjects under study and to determine factors affecting lifestyle .
sampling of this cross - sectional descriptive - analytic study was conducted from october 2015 to february 2016 among 290 ngos of the health companions population in guilan province .
participants were classified via random sampling and they were selected using health companions population who functioned as volunteers under the supervision of the state welfare organization in guilan province .
inclusion criteria were over 6-month membership at intended population , willingness to participate in the study , and exclusion criteria were suffering from a diagnosed chronic disease and unwillingness to participate in the study . to collect data , a questionnaire with three sections of personal details ( age , duration of membership at population , marital status , number of children , number of family members , job , housing , education , access to computer and the internet , computer capabilities , and socio - economic status of the household ) , and a 37-item questionnaire of individual empowerment
was employed in addition to the questionnaire of health - promoting lifestyle profile ii . in the section of personal details filled by companions , to specify socio - economic status ,
the subjects were asked to rate socio - economic status from very high to very low .
anthropometric items including height , weight and blood pressure in this questionnaire were measured and recorded by administrator using the same measuring tape , scale and weights for all subjects . to measure empowerment , a researcher - made questionnaire with 37 questions
the research group has previously provided this tool and its reliability and validity have been confirmed with cronbach s alpha of 0.81 .
this questionnaire measured individual empowerment of health companions in 8 fields including participation , motivation , cognitive thinking , critical thinking , intention , self - efficacy , perceived control and social support using 37 questions and , where the answers were provided , using 5-item likert scale from strongly agree ( 5 scores ) to strongly disagree ( 1 score ) .
therefore , fields of participation , critical thinking and self - efficacy each with four questions , were scored between 4 and 20 .
scores of motivation , intention and perceived control , with five questions , were between 5 and 25 .
cognitive thinking , with three questions , was scored between 3 and 12 and social support , with seven questions , was scored between 7 and 35 . finally , the total score of individual empowerment was between 37 and 185 while calculating a separate score for each field .
higher scores show higher individual empowerment . in order to facilitate announcement of the results ,
it was decided to consider scores higher than average as favorable . to collect data related to healthy lifestyle in this study ,
this questionnaire measured 6 dimensions of healthy lifestyle including nutrition , taking responsibility of health , interpersonal relations , spiritual growth , physical activity and stress management through 52 questions and they were scored via 4-item likert scale of never ( 1 ) , sometimes ( 2 ) , often ( 3 ) , and always ( 4 ) . four dimensions of nutrition , taking responsibility for health , interpersonal relations and spiritual growth ( each with 9 questions with scoring range of 936 ) and two dimensions of physical activity and stress management ( each with 8 questions with scoring range of 832 ) were scored .
the range of total score for health - promoting lifestyle behavior is between 52 and 208 and for each dimension a separate score is considered . taking higher scores in this questionnaire
the standard questionnaire of health - promoting lifestyle profile ii has been previously translated into persian , and used in numerous studies .
validity and reliability of the persian version of this questionnaire in iran was confirmed by mohammadi zeidi et al . and verified with cronbach s alpha of 0.82 and icc of 0.91 ( 12 ) .
after collecting questionnaires and entering data into the computer , stata 11 software was used to analyze them . to examine the significant difference between mean
score of lifestyle dimensions based on levels of multimode qualitative variables ( including marital status , economic status , and type of housing , job and education ) , one - way anova was employed .
in addition , pearson correlation coefficient was used to examine the relation of various dimensions of health - promoting lifestyle with empowerment . in this study , individual empowerment and healthy lifestyle were considered as independent and dependent variables with significance level of p<0.05 .
multivariate linear regression was used to find the adjusted correlation between empowerment and healthy lifestyle .
with regard to the high design effect of cluster sampling , survey command of stata software was used for this analysis to consider design effect in estimation of variances .
this study has been registered at tehran university of medical sciences with ethical code of 138416 . participating in this study was completely voluntary .
participants were assured that participating in this study would bring about no harm to them .
sampling of this cross - sectional descriptive - analytic study was conducted from october 2015 to february 2016 among 290 ngos of the health companions population in guilan province .
participants were classified via random sampling and they were selected using health companions population who functioned as volunteers under the supervision of the state welfare organization in guilan province .
inclusion criteria were over 6-month membership at intended population , willingness to participate in the study , and exclusion criteria were suffering from a diagnosed chronic disease and unwillingness to participate in the study .
to collect data , a questionnaire with three sections of personal details ( age , duration of membership at population , marital status , number of children , number of family members , job , housing , education , access to computer and the internet , computer capabilities , and socio - economic status of the household ) , and a 37-item questionnaire of individual empowerment was employed in addition to the questionnaire of health - promoting lifestyle profile ii . in the section of personal details filled by companions , to specify socio - economic status ,
the subjects were asked to rate socio - economic status from very high to very low .
anthropometric items including height , weight and blood pressure in this questionnaire were measured and recorded by administrator using the same measuring tape , scale and weights for all subjects . to measure empowerment , a researcher - made questionnaire with 37 questions
the research group has previously provided this tool and its reliability and validity have been confirmed with cronbach s alpha of 0.81 .
this questionnaire measured individual empowerment of health companions in 8 fields including participation , motivation , cognitive thinking , critical thinking , intention , self - efficacy , perceived control and social support using 37 questions and , where the answers were provided , using 5-item likert scale from strongly agree ( 5 scores ) to strongly disagree ( 1 score ) .
therefore , fields of participation , critical thinking and self - efficacy each with four questions , were scored between 4 and 20 .
scores of motivation , intention and perceived control , with five questions , were between 5 and 25 .
cognitive thinking , with three questions , was scored between 3 and 12 and social support , with seven questions , was scored between 7 and 35 . finally , the total score of individual empowerment was between 37 and 185 while calculating a separate score for each field .
higher scores show higher individual empowerment . in order to facilitate announcement of the results ,
it was decided to consider scores higher than average as favorable . to collect data related to healthy lifestyle in this study ,
this questionnaire measured 6 dimensions of healthy lifestyle including nutrition , taking responsibility of health , interpersonal relations , spiritual growth , physical activity and stress management through 52 questions and they were scored via 4-item likert scale of never ( 1 ) , sometimes ( 2 ) , often ( 3 ) , and always ( 4 ) . four dimensions of nutrition , taking responsibility for health , interpersonal relations and spiritual growth ( each with 9 questions with scoring range of 936 ) and two dimensions of physical activity and stress management ( each with 8 questions with scoring range of 832 ) were scored .
the range of total score for health - promoting lifestyle behavior is between 52 and 208 and for each dimension a separate score is considered . taking higher scores in this questionnaire
the standard questionnaire of health - promoting lifestyle profile ii has been previously translated into persian , and used in numerous studies .
validity and reliability of the persian version of this questionnaire in iran was confirmed by mohammadi zeidi et al . and verified with cronbach s alpha of 0.82 and icc of 0.91 ( 12 ) .
after collecting questionnaires and entering data into the computer , stata 11 software was used to analyze them . to examine the significant difference between mean
score of lifestyle dimensions based on levels of multimode qualitative variables ( including marital status , economic status , and type of housing , job and education ) , one - way anova was employed .
in addition , pearson correlation coefficient was used to examine the relation of various dimensions of health - promoting lifestyle with empowerment . in this study ,
individual empowerment and healthy lifestyle were considered as independent and dependent variables with significance level of p<0.05 .
multivariate linear regression was used to find the adjusted correlation between empowerment and healthy lifestyle . with regard to the high design effect of cluster sampling ,
survey command of stata software was used for this analysis to consider design effect in estimation of variances .
this study has been registered at tehran university of medical sciences with ethical code of 138416 . participating in this study was completely voluntary .
participants were assured that participating in this study would bring about no harm to them .
duration of membership at health companions population was less than 5 years for about 71% of respondents .
over half of the respondents were married and living in a 1-to-4-member family . of respondents who had diplomas
of those who mentioned their socio - mental status as average , there was 67.6% .
of eight dimensions of individual empowerment , the highest score belonged to social support with an average of 27.7 and motivation with an average of 21.9 , and the lowest score belonged to self - efficacy with an average of 14 .
of six dimensions of health - promoting lifestyle , spiritual growth and interpersonal relations had the highest scores of 29.6 and 27.5 respectively and physical activity and stress management with average of 17 and 21.1 respectively had the lowest scores .
health - promoting lifestyle of companions with average of 144.3 considering the 0100 scale of hplp ii was at a good level , and individual empowerment with average of 148.3 was high .
as can be seen in table 2 , design effect range of all variables is over one and the highest value is observed in the domain of social support of individual empowerment ( 2.86 ) .
results of correlation coefficient of individual empowerment with its total score , showed that perceived control has the highest correlation with total score of individual empowerment ( r=0.76 ) .
the highest correlation of dimensions of health - promoting lifestyle with its total score , was related to spiritual growth ( r=0.076 ) .
in addition , other dimensions showed a significant correlation with total score of empowerment and health - promoting lifestyle .
table 3 shows the results of correlation test ( pearson ) between dimensions of individual empowerment and health - promoting lifestyle .
the highest correlation is related to empowerment with interpersonal relations ( p<0.01 , r= 0.42 ) and then the relation of perceived control with spiritual growth ( p<0.01 , r=0.39 ) .
the lowest correlation is associated with the relation between perceived control and nutrition ( p<0.05 , r=0.12 ) .
the relation of total score of individual empowerment with lifestyle is also high and significant ( p<0.01 , r=0.36 ) . according to this table , self - efficacy and total empowerment
therefore , with a confidence level of 99% , it can be said that there is a direct relationship between individual empowerment and health - promoting lifestyle in this group .
variables of duration of membership , marital status , having access to the internet , socio - economic status and individual empowerment with p<0.02 in simple regression test , entered multiple regression test .
coefficients of the regression effect of individual empowerment as independent variable on health - promoting lifestyle as dependent variable , showed that empowerment is a good predictor of health - promoting lifestyle .
coefficient of individual empowerment with healthy lifestyle is 21% that shows one unit increase in the individual empowerment score significantly increases health - promoting lifestyle score for 0.21 .
coefficients of the regression effect of other studied variables show that socio - economic status has a significant and reverse relationship with healthy lifestyle behavior .
duration of membership at health companions population was less than 5 years for about 71% of respondents .
over half of the respondents were married and living in a 1-to-4-member family . of respondents who had diplomas
of those who mentioned their socio - mental status as average , there was 67.6% .
of eight dimensions of individual empowerment , the highest score belonged to social support with an average of 27.7 and motivation with an average of 21.9 , and the lowest score belonged to self - efficacy with an average of 14 .
of six dimensions of health - promoting lifestyle , spiritual growth and interpersonal relations had the highest scores of 29.6 and 27.5 respectively and physical activity and stress management with average of 17 and 21.1 respectively had the lowest scores .
health - promoting lifestyle of companions with average of 144.3 considering the 0100 scale of hplp ii was at a good level , and individual empowerment with average of 148.3 was high .
as can be seen in table 2 , design effect range of all variables is over one and the highest value is observed in the domain of social support of individual empowerment ( 2.86 ) .
results of correlation coefficient of individual empowerment with its total score , showed that perceived control has the highest correlation with total score of individual empowerment ( r=0.76 ) .
the highest correlation of dimensions of health - promoting lifestyle with its total score , was related to spiritual growth ( r=0.076 ) .
in addition , other dimensions showed a significant correlation with total score of empowerment and health - promoting lifestyle .
table 3 shows the results of correlation test ( pearson ) between dimensions of individual empowerment and health - promoting lifestyle .
the highest correlation is related to empowerment with interpersonal relations ( p<0.01 , r= 0.42 ) and then the relation of perceived control with spiritual growth ( p<0.01 , r=0.39 ) .
the lowest correlation is associated with the relation between perceived control and nutrition ( p<0.05 , r=0.12 ) .
the relation of total score of individual empowerment with lifestyle is also high and significant ( p<0.01 , r=0.36 ) . according to this table , self - efficacy and total empowerment
therefore , with a confidence level of 99% , it can be said that there is a direct relationship between individual empowerment and health - promoting lifestyle in this group .
variables of duration of membership , marital status , having access to the internet , socio - economic status and individual empowerment with p<0.02 in simple regression test , entered multiple regression test .
coefficients of the regression effect of individual empowerment as independent variable on health - promoting lifestyle as dependent variable , showed that empowerment is a good predictor of health - promoting lifestyle .
coefficient of individual empowerment with healthy lifestyle is 21% that shows one unit increase in the individual empowerment score significantly increases health - promoting lifestyle score for 0.21 .
coefficients of the regression effect of other studied variables show that socio - economic status has a significant and reverse relationship with healthy lifestyle behavior .
various social , economic and environmental factors are associated with health - promoting lifestyle behavior .
although finding the relation between these factors and health is not an arduous task , it is often overlooked by health staff . according to the results of this study
, there is a positive and significant relationship between individual empowerment and health - promoting lifestyle . of dimensions of empowerment , seven dimensions of participation , motivation , critical thinking , intention , self - efficacy , perceived control and social support
are positively and significantly related to health - promoting lifestyle . of demographic details of the subjects , there is a significant relationship between duration of membership at health companions population , socio - economic status and having access to the internet , with health - promoting lifestyle . on the other hand , duration of membership and socio - economic status
, it can be said that , as duration of membership at health companions population increases , people s empowerment also increases and are more inclined to follow health promoting lifestyles . as laverack stated in his study , individual and social empowerment of people can improve health - related outcomes via the effect of factors like participation , management , etc .
socio - economic status can also be a factor to empower individuals by developing freedom of choice and action , and therefore , better and more compliance with health - promoting behavior ( 14 , 15 ) . in examining various dimensions of health - promoting lifestyle , the highest score was related to dimensions of spiritual growth and interpersonal relations , which complies with results of other studies ( 16 , 17 ) .
in addition , the lowest score was related to dimensions of physical activity and stress management .
lower mean score of physical activity and stress management complies with various studies , including the study by malakouti , sousa and cao ( 1820 ) .
, on middle - aged women in yazd , stress management was of a high score ( 16 ) .
this incompliance can be justified by differences in occupational and social features of targeted communities . in terms of individual empowerment ,
this finding complies with results of the study by hall ( 21 ) . in the study by nedjat , social support and dependence
average total score of calculated individual empowerment , shows that participants were able to obtain 80.2% of total score of empowerment inventory and it is a relatively high percent that shows good individual empowerment of these people .
sadi ( 2014 ) reported empowerment of rural women to be 63.3% ( 23 ) . in the study by kiani , women s empowerment to decide for pregnancy ,
higher empowerment in this study can be due to the voluntary nature of ngos and participation of these women in various courses of readiness for cooperation .
on the other hand , it appears that people who are more empowered seek for voluntary activities in society .
total mean score of health - promoting lifestyle shows 69.4% of total score of the inventory , i.e. , higher than average scored by participants .
these results comply with findings of the study by enjezab on women of yazd ( 16 ) .
the similarities in these results are due to selecting middle - aged women as subjects in both studies . however , in most studies where samples were selected from other groups such as teachers , students or nurses , this value was reported to be lower ( 9 , 2527 ) .
results of this study on the relationship between various dimensions of empowerment and health - promoting behavior show that there is a significant and positive relationship between dimensions of participation , motivation , critical thinking , intention , self - efficacy , perceived control and social support with health - promoting lifestyle behavior . in various studies ,
results from investigating the relationship between these dimensions and lifestyle show that health promotion is completely affected by community empowerment ( 28 , 29 ) . in various statements of the who , the effect of empowerment on health promotion and healthier lifestyle are emphasized via individuals participation in health programs and health - oriented decision - making ( 3033 ) . in this regard
, there is evidence on the effect of empowerment on mental health , self - efficacy and self - confidence of people ( 13 , 3437 ) . in two studies
, it is shown that participating in different social groups and programs leads to health promotion and healthy lifestyle behavior ( 13 , 38 ) .
the effect of individual empowerment on health - promoting lifestyle was positive and significant , such that as empowerment increased , health - promoting lifestyle developed for 21% of the study population . in explaining this finding , it can be said that due to the dependency of lifestyle - promoting behavior to decision - making of people ( 13 , 39 ) , all factors affecting decision - making and choices of individuals , comprise following healthier behavior in their lives .
although no study was found that directly investigated these two variables , some studies based on strategies of increasing patients compliance with health - oriented regimes , focus their attention on empowering those people ( 4043 ) .
for instance , in 2011 mccorkle showed that caring patients with the aim of empowerment of cancer patients can result in increased self - efficacy and improve their quality of life and promote a healthier life ( 44 ) .
the relationship between dimensions of individual empowerment and health - promoting lifestyle , shows the significance of most of these correlations .
the highest correlation is found between individual empowerment and interpersonal relations of health - promoting lifestyle and then between perceived control and spiritual growth .
participation as one of the bases of empowerment , requires creating appropriate interpersonal relations . in various groups and populations , due to continuous and permanent interactions ,
interpersonal relations are strengthened ( 45 ) . in his study , hatzidimitriadou reported that membership in a group increases the sense of control and individual empowerment ( 46 ) . in other words , volunteering in ngos , results in increased positive attitudes , development of ethics and accountability in the community ( 47 ) .
since the population of this study were a group of volunteer women who were members of an ngo , deep interactions and interpersonal relations , the spirit of participation in various activities and capability of critical thinking and decision making are considered as part of their routine activities .
this will strengthen individual empowerment and as a result , their community empowerment , and it puts them among empowered people of the society .
there is also evidence for the effect of empowerment on the ability of decision - making ( 48 ) .
in addition , findings of various studies show the relationship between empowerment and health outcomes in individuals and groups ( 49 ) .
therefore , currently one of the effective strategies for comprehensive and sustainable development , is to employ people s empowerment and participation in all dimensions .
the major tool to reach this goal is to establish ngos , based on the needs and abilities of various groups in the essential and assorted issues , to implement development plans ( 50 ) .
therefore , most countries encourage their people to participate in voluntary activities with the aim of promoting environmental or social objectives ( 51 ) .
findings of this study , showed that by increase in empowerment of women under study , their health - promoting lifestyle also improved , and dimensions of social support , motivation and participation had the major role in increasing individual empowerment .
functional importance of these findings , is that some evidence for the effect of empowerment on healthy lifestyle was obtained that was not directly investigated earlier . it is suggested to design interventions to increase the dimensions stated , in order to promote individual empowerment in society .
therefore , while having an empowered community , we will also see a healthier lifestyle .
conducting a complementary study on the relationship between individual empowerment and healthy lifestyle on non - ngo women with a similar methodology , can be a good path for future studies on this issue .
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### Summary:
: introductionaccording to the health - promoting approach , people should be empowered such that they take responsibility for their health and follow a healthy lifestyle .
empowerment is a process in which people confront problems and tasks in their lives in order to better control them .
this study was conducted to specify the relationship between individual empowerment and health - promoting lifestyle among women ngos of northern iran.methodsin this cross - sectional study , 290 women ngos of guilan province were selected randomly using multistage cluster sampling , and were examined using the questionnaire of health - promoting lifestyle profile ii and individual empowerment inventory scale .
data were analyzed using stata 11 software via one - way anova , pearson correlation coefficient and multivariate linear regression.resultsboth variables of individual empowerment and health - promoting lifestyle were of favorable status among the population under study .
the highest score in individual empowerment belonged to the domain of social support and the highest score in lifestyle belonged to spiritual growth .
a significant relationship was found between individual empowerment and health - promoting lifestyle ( p<0.001 ) .
the highest correlation between individual empowerment and dimensions of health - promoting lifestyle was related to interpersonal relationships . in addition
, it was specified that 21% of lifestyle variance could be explained by individual empowerment.conclusionthere is a relation between individual empowerment and health - promoting lifestyle .
individual empowerment is a predictive variable to have a health - promoting lifestyle .
therefore , by increasing individuals empowerment , their healthy lifestyle can be promoted .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: an autopsy determined the cause to be splanchnic hypoperfusion , likely due to the physiologic changes induced by the pneumoperitoneum necessary to conduct the surgery .
we sought to determine whether all reported cases of intestinal ischemia following laparoscopic cholecystectomy ( lc ) shared the same etiology , and if faulty operative technique could be invoked .
a review of all known published cases of intestinal ischemia following lc was conducted to determine the underlying etiology of ischemia , and whether adequate operative technique had been observed .
multiple causes of intestinal ischemia following lc have been described : splanchnic hypoperfusion , thrombosis of the superior mesenteric artery , and thrombosis of the inferior mesenteric vein .
patient factors rather than faulty operative technique would appear to underlie the development of this rare complication .
intestinal ischemia following laparoscopic cholecystectomy ( lc ) is a rarely reported complication , but one that often results in a fatal outcome .
several case reports in the past decade have served to illustrate that ischemia in this context has diverse causes .
, we describe a case of fatal intestinal ischemia following lc of which splanchnic hypoperfusion during pneumoperitoneum was the most likely cause .
a 57-year - old woman was referred for lc following a several month history of biliary colic .
she was moderately obese but otherwise in good health , without any history or known risk factors for cardiovascular disease .
lc was performed without complication according to the standard technique with maintenance of the intraperitoneal pressure between 12 mm hg to 15 mm hg .
the patient was discharged home the same day with a prescription for analgesics to be taken as necessary .
however , at home on the third postoperative day , she suddenly vomited a large volume of blood and died several minutes later in front of her husband .
the coroner was consulted , and an autopsy performed at his request . at autopsy ,
however , the entire small intestine was green and obviously necrotic ; the lumen contained a large amount of blood , as did the stomach .
the mesenteric vessels were meticulously examined : these were normal without evidence of atheromata , arterial thromboembolia , or venous thrombus .
the heart was normal except for mild atheromatosis of the left descending coronary artery ; the other coronary vessels were normal .
the cause of death was recorded as massive small bowel ischemic necrosis due to arterial hypoperfusion , the latter most likely due to elevated intraabdominal pressure during the laparoscopic cholecystectomy procedure .
intestinal ischemia following lc is a rare but devastating complication . a literature review using pubmed and pertinent search terms revealed 7 cases before the present report ( table 1 ) .
the causes of ischemia in the context of lc are diverse , and likely depend on the underlying cardiovascular health of the affected patient .
reported cases of intestinal ischemia after laparoscopic cholecystectomy iap = intraabdominal pressure ; sma = superior mesenteric artery ; imv = inferior mesenteric vein .
lc requires the creation of a pneumoperitoneum via insufflation of carbon dioxide ; resulting increases in pco2 and intraperitoneal pressure lead to changes in pulmonary function and hemodynamic measurements .
pulmonary function is altered as a consequence of increased pco2 that is directly absorbed into the circulation from the peritoneal cavity as well as by increased intraperitoneal volume : vital capacity is diminished with increased dead space and v / q mismatch . increased intraabdominal
pressure leads to decreased blood flow through the splanchnic vessels with relative hypoperfusion of the celiac , superior mesenteric , and renal arteries , despite normal blood pressure .
although these physiologic changes create a theoretical risk of compromised intestinal blood flow in all patients , in the vast majority they are well tolerated with no clinical consequences .
the absence of atheromatosis of the major intraabdominal and mesenteric vessels , and the absence of venous thromboembolism , both excluded by detailed examination at the time of autopsy , eliminates these as causes of ischemia .
likewise , faulty operative technique or intestinal obstruction due to external compression or herniation may also be eliminated .
we conclude that the massive small intestinal ischemia in this patient is therefore most likely splanchnic hypoperfusion occurring at the time of pneumoperitoneum , the physiologic basis of which is described above .
splanchnic hypoperfusion is but one cause of intestinal ischemia following lc . as seen in the accompanying table ,
the most common cause in this small number of cases is thrombosis of the superior mesenteric artery ( sma ) .
patients with underlying atheromatous disease involving the abdominal vessels may be at risk for this complication . however , an alternative explanation in this context is that symptomatic episodes of intestinal ischemia ( mesenteric angina ) before lc have been misinterpreted clinically as symptomatic cholelithiasis .
the resulting laparoscopic intervention may have created a low blood perfusion state resulting in further intestinal ischemia .
one patient died from thrombosis of the inferior mesenteric vein , the cause of which is unexplained , and 1 patient , similarly to our case , developed ischemia secondary to splanchnic hypoperfusion ; he subsequently recovered .
of note , in all cases , there was correct operative technique reported ; where pco2 is specified , it was 15 mm hg or lower .
the question arises whether patients at risk for developing intestinal ischemia during lc can be identified and , if so , can any preventive steps be taken ? given the very small number of cases in which this complication has occurred amid the large number of lcs performed , the negligible incidence of intestinal ischemia likely makes any attempt at screening of the average lc patient even if reliable screening criteria could be identified a futile endeavor .
the patient we describe is illustrative : a previously healthy 57-year - old , moderately obese woman without any other known risk factors for cardiovascular disease would not be considered at increased risk for this complication .
similarly , most of the patients described in the small number of case reports had no identifiable risk factor .
thus , at best , an individualized approach is warranted : measures such as use of lower peritoneal pressure , if technically feasible , and assurance of proper hydration might be considered for those patients with precarious cardiovascular status or a prior history of ischemic enteritis or colitis .
sma thrombosis , inferior mesenteric vein thrombosis , and splanchnic hypoperfusion are all potential causes , all occurring despite correct operative technique .
compromised cardiovascular function may play a role in the development of ischemia in certain patients .
the possibility of survival would appear to correlate with patient age and extent of bowel involved .
preventative measures may be considered on an individualized basis for those patients with severe cardiovascular disease or a prior episode of intestinal ischemia .
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### Summary:
: objectives : intestinal ischemia is a rarely reported complication following laparoscopic cholecystectomy . we describe a case of massive small intestinal necrosis 3 days following this procedure . an autopsy determined the cause to be splanchnic hypoperfusion , likely due to the physiologic changes induced by the pneumoperitoneum necessary to conduct the surgery .
we sought to determine whether all reported cases of intestinal ischemia following laparoscopic cholecystectomy ( lc ) shared the same etiology , and if faulty operative technique could be invoked.methods:a review of all known published cases of intestinal ischemia following lc was conducted to determine the underlying etiology of ischemia , and whether adequate operative technique had been observed.results:multiple causes of intestinal ischemia following lc have been described : splanchnic hypoperfusion , thrombosis of the superior mesenteric artery , and thrombosis of the inferior mesenteric vein .
all occurred despite correct operative technique.conclusions:intestinal ischemia following lc has diverse causes .
patient factors rather than faulty operative technique would appear to underlie the development of this rare complication .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: unilateral obstruction or injury to the vas deferens can result in significant injury to the contralateral testicle ( 1 - 3 ) .
although studies have indicated that the damage can be reversed by repair of the obstructed vas deferens ( 4 ) , other studies have suggested that the effects are irreversible ( 2 ) .
although various pathways have been proposed , the mechanism of contralateral testicular deterioration remains controversial .
the most popular theory , commonly proposed to explain the contralateral testicular damage , has been an autoimmune response ( 3 , 5 ) . currently , the probable mechanism of contralateral injury is accepted as a vasospasm through a sympathetic reflex arc , and hypoxia may be the reason for contralateral injury after unilateral testicular obstruction ( 6 , 7 ) .
succeeding studies have shown decreases in testicular oxygen content and adenylate energy charge , and an increase in biochemical indicators of tissue hypoxia ( 8 , 9 ) .
chemical sympathectomy reduced the increase in biochemical indicators of tissue hypoxia and preserved spermatogenetic function ( 10 - 12 ) .
these findings suggested that contralateral testicular damage resulted from a reflex vasospasm preceded by sympathetic activation .
thus , the present animal study was performed to evaluate the effects of unilateral vasectomy on ipsilateral and contralateral testicular histology and fertility in rats that were chemically sympathectomized neonatally .
the study comprised 40 male albino rats : 20 received a placebo and the other 20 underwent chemical sympathectomy neonatally .
the experimental protocol was approved by the animal ethics committee of akdeniz university , turkey .
the rats were maintained under standard laboratory conditions with a 12 : 12 hr light : dark cycle , with free access to food pellets and tap water .
they were separated equally into the four groups : group 1 : a sham ( control ) group that received a placebo ; group 2 : a group with sham that received 6-hydroxydopamine hydrobromide ; group 3 : a group with unilateral left vasectomy that received a placebo ; group 4 : a group with unilateral left vasectomy that received 6-hydroxydopamine hydrobromide . within 24 hr of birth , 20 rats each received an intraperitoneal injection with placebo ( physiological saline solution 0.2 ml / day , for 7 days ) or 6-hydroxydopamine hydrobromide solution 0.075 mg / g , for 7 days .
dopamine solutions were prepared each day by dilution in physiological saline containing 0.1 mg / ml of ascorbic acid .
when 60 days old , each group of 20 rats was divided into two groups that underwent either a control operation or an operation to create unilateral left vasectomy . to create a unilateral abdominal testis ,
a left inguinoscrotal incision was made in the abdomen , and the left vas deferens was retrieved and exposed . using 4.0 silk suture ,
the incision was closed in 2 layers with 4.0 silk suture , in a running fashion .
briefly , the incision was made , vasa deferentia were exposed , 4.0 silk was passed , but the vasa deferentia were not ligated or transected .
eight weeks after surgery , each male rat was housed with two known fertile female rats for 25 days , and then their testes were harvested .
sections were cut and stained with hematoxylin - eosin , periodic acid - schiff ( pas ) and masson 's trichrome .
the 50 most circular tubules were identified in each testicular biopsy section and their diameters were measured using a 40 objective and an ocular micrometer .
the testicles were evaluated histologically with respect to the following characteristics : 1 ) seminiferous tubular diameter ; 2 ) morphology and progression of maturation of the germinal epithelium ; and
3 ) morphology of the tunica propria and interstitial components , specifically to determine whether fibrosis , hyalinization , or an inflammatory infiltrate could be identified .
germinal epithelial maturity was graded using a modified johnsen testicular biopsy score ( 13 ) ; using a 40 objective , 50 tubules were evaluated and each tubule was given a score from 1 to 10 .
the female rats were observed for an additional 25 days . the number of rats impregnated by each male rat and the number of rats delivered by each female rat were recorded .
the statistical comparisons between mstd and mtbs values of the groups were performed using multi - way analysis of variance .
total fertility rates of the groups were compared using t - test , with p<0.05 taken to indicate significant differences ; all values are presented as the mean ( sd ) .
a total of 6 rats died during study ( 1 from group 1 , 1 from group 2 , 1 from group 3 , and 3 from group 4 ) .
there was no statistically significant difference in histological examination between any of the groups ( p>0.05 ) .
the mtbs of the ipsilateral and contralateral testes were 9.670.50 and 9.780.44 in group 1 , 8.670.5 and 9.780.42 in group 2 , 6.220.44 and 7.110.33 in group 3 , and 7.220.43 and 8.290.48 in group 4 , respectively ( p>0.05 ; fig .
1 ) . while the mstd of the ipsilateral and contralateral testes did not differ significantly between groups , those of the ipsilateral testes of all of the groups were lower than in the corresponding contralateral testes .
the mstd of the ipsilateral and contralateral testes were 242.66689.326 and 249.90675.907 in group 1 , 178.01338.392 and 209.673104.931 in group 2 , 147.1136.667 and 155.89312.954 in group 3 , and 165.44015.449 and 188.14023.360 in group 4 , respectively ( p>0.05 ; fig .
these rates were 88.8% in group 1 , 66.6% in group 2 , 77.7% in group 3 , and 85.7% in group 4 .
total fertility rate of group 4 was significantly higher than that in group 3 ( p<0.05 ) .
unilateral obstruction of the vas deferens can occur for a variety of reasons , including epididymitis , trauma , or iatrogenic damage to the vas during hydrocele repair , orchidopexy , or inguinal herniorrhaphy ( 14 ) .
unilateral injury to the vas deferens has been associated with damage to the contralateral testicle , and this damage is thought to be mediated through antisperm autoantibodies ( 3 , 5 ) .
a current theory of contralateral testicular injury proposes a reflexive decrease in contralateral testicular blood flow and an increase in the biochemical indicators of tissue hypoxia within the contralateral testes ( 6 , 7 ) .
the mechanism by which contralateral testicular blood flow is affected after unilateral vas deferens obstruction has not been thoroughly evaluated .
the vasospasm in the contralateral testis was suggested to arise by a neurovascular pathway triggered by an ipsilateral testicular stimulus , running through a sympathetic reflex arc , resulting in decreased blood flow ( 15 ) .
ipsilateral vas deferens obstruction also resulted in increased levels of lactic acid and hypoxanthine in both testes .
decreases in the blood flow of both testes and oxidative stress have been reported after ipsilateral efferent duct ligation ( 16 ) .
ligation of the vas deferens should increase the pressure within the distal structures and the structure of the tunica albuginea may not allow for expansion to decrease the pressure
. the increased pressure may therefore decrease the blood flow to the ipsilateral testis through increasing the intratesticular pressure .
the increases in the levels of lactic acid and hypoxanthine within the contralateral testis suggest a reflex decrease in contralateral testicular blood flow when the ipsilateral testis is under stress after ligation of the ipsilateral vas deferens .
andiran et al . ( 1 ) reported that unilateral testicular torsion , unilateral vas deferens obstruction , unilateral abdominal testis , and unilateral venous obstruction not only damage the ipsilateral but also the contralateral testes . as the lactic acid and hypoxanthine levels within the contralateral testis were greater than in the controls ,
testicular torsion and vas deferens obstruction seem to share a common pathway , which may be a reflex decrease in contralateral testicular blood flow , for their effects on the contralateral testis ( 1 ) .
an effective circulatory system in the testis is the most critical factor for functional spermatogenesis ( 17 ) .
the increase in biochemical indicators of tissue hypoxia and contralateral testicular damage can be prevented by chemical sympathectomy ( 10 , 11 , 18 ) .
experimentally , the sympathetic nervous system was also activated by partial ligation of the left renal vein in the adult rat , which was blocked by chemical sympathectomy ( 19 ) .
the evaluation of contralateral testis through the model of testicular torsion has revealed a decrease in blood flow and an increase in factors associated with tissue hypoxia , e.g. , lactic acid and hypoxanthine ; chemical sympathectomy reduced the effects of tissue hypoxia and preserved testicular histology , fertility and fecundity ( 9 , 10 ) . additionally , unilateral
undescent has also been reported to result in elevated levels of the products of tissue hypoxia in the contralateral testis ( 20 ) .
( 18 ) evaluated the effects of abdominal testis on ipsilateral and contralateral testicular histology , fertility and fecundity in rats that were chemically sympathectomized neonatally . in their investigation , although fertility and fecundity rates and mtbss were not significantly different , chemical sympathectomy prevented the decrease in mstds that occurred in the contralateral testis .
these findings suggested that the sympathetic system may play a role in contralateral testicular deterioration .
therefore , we evaluated the effects of unilateral vasectomy on ipsilateral and contralateral testicular histology and fertility in rats that were chemically sympathectomized neonatally . although mstd and mtbs were not significantly different between groups , chemical sympathectomy prevented the decrease in total fertility rates of the rats with unilateral left vasectomy in our study .
prevention of this decrease by chemical sympathectomy suggests the sympathetic nervous system may play a role in the testicular degeneration associated with vasectomy .
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### Summary:
: unilateral obstruction or injury to the vas deferens can result in significant injury to the contralateral testicle .
although various pathways have been proposed , the mechanism of contralateral testicular deterioration remains controversial .
the present animal study was performed to evaluate the effects of unilateral vasectomy on ipsilateral and contralateral testicular histology and fertility in rats that were chemically sympathectomized neonatally .
the study comprised 40 male albino rats : 20 received a placebo and the other 20 underwent chemical sympathectomy neonatally . when 60 days old , each group of 20 rats was divided into two groups that underwent either a sham operation or an operation to create unilateral left vasectomy .
eight weeks after surgery , each male rat was housed with two known fertile female rats for 25 days , and then their testes were harvested .
mean seminiferous tubular diameters ( mstd ) and mean testicular biopsy scores ( mtbs ) were determined for each testis . although mstd and mtbs were not significantly different between groups , chemical sympathectomy prevented the decrease in total fertility rates of the rats with unilateral left vasectomy in our study .
prevention of this decrease by chemical sympathectomy suggests that the sympathetic nervous system may play a role in the testicular degeneration associated with vasectomy .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: in this setting , an impoverished developing country ( eg , some of the more impoverished countries in sub - saharan africa , asia , or latin america ) , the patient obtains diagnostic tests at a public hospital with a specific budget for a human immunodeficiency virus ( hiv)screening campaign .
the hiv test returns positive , but the cd4 + count has not dropped to levels that would prompt initiation of antiretroviral therapy ( art ) . after the visit , the patient 's test results remain in the onsite chart , kept in storage and rarely shared with other providers until the patient initiates the next clinical interaction .
additionally , in such resource - limited settings , patients do not typically have healthcare providers ( beyond those assigned in the hospital ) to receive and act upon their test results .
the concept of a dedicated primary care doctor is increasingly a luxury for these patients .
the more common model is either the fee for service urgent care private physician who generally has no incentive or mechanism by which to share information beyond the fee - generating urgent care visit or the public hospital doctor who , if unaided , is too busy to manage the patient 's care at this level of detail .
the loss to follow - up of such patients is notoriously high , and unfortunately , these patients often return to the hospital setting after developing symptoms of advanced aids . in one systematic review ,
rosen et al estimated that fewer than one third of patients testing positive for hiv but not yet eligible for art were retained in pre - art care continuously .
timely follow - up of test results was one of several problems contributing to this statistic .
the problem in this setting is not only the lack of dedicated ambulatory care providers but also how inefficiently clinically important test results are shared outside the setting in which they were ordered . with the responsibility of follow - up resting on impoverished patients , and without the availability of a responsible provider in the ambulatory setting , the likelihood of timely follow - up and initiation of appropriate medical therapy is significantly reduced .
in this second setting ( eg , some countries in latin america , asia , or africa where innovation is encouraged and piloted ) , a middle - income country that has a higher - functioning healthcare system than the first , a patient may receive a diagnosis of hiv during an active case finding campaign . in this setting , because a team of care providers travel to the community to screen and potentially treat at - risk patients , the diagnosis of hiv may be made earlier in a patient 's clinical course .
this healthcare system then may use one of many well - defined processes for maintaining direct contact with patients ( eg , a robust network of community health workers [ chws ] ) or it may finance one or more programs known to assist with patient retention ( eg , food support , travel stipends , the provision of quality primary care in addition to specialized hiv care ) .
these investments increase the likelihood that providers will follow up with a patient and initiate appropriate art therapy when a test result returns positive .
rich et al showed how such a robust system of communication and support contributed to improved adherence and excellent clinical outcomes 2 years after the initiation of art .
nevertheless , because these programs depend largely on the availability , commitment , and initiative of patients , providers , and chws , they are not foolproof .
if information does not flow efficiently through this system ( albeit more robust and patient - centered ) , missed treatment opportunities and losses to follow - up will persist at unacceptable levels .
in this third setting ( eg , the united states , many rapidly developing middle - income countries such as mexico ) , a disenfranchised intravenous drug user presents to an emergency department with an injection - site cellulitis and is admitted to the hospital .
the result does not return until after the patient is discharged , and unfortunately , it returns positive .
there is a strong possibility that the inpatient doctor who ordered the test will not be aware of the result .
in fact , roy et al determined that 72% of physicians were unaware of such potentially actionable test results that were pending at the time of hospital discharge .
interestingly , this often occurs even when test results are available to all providers ( both inpatient and ambulatory ) within the electronic medical record ( emr ) . in these settings ,
lack of awareness of test results is a common cause of delays in diagnosis ( particularly when multiple providers are involved ) and often leads to subsequent patient harm . in the united states , the systems issues that plague test result management are multi - fold , including different providers caring for patients in different settings , poor delineation of responsibility , and lack of systems to flag and alert responsible providers when finalized results become available .
the consequence is a faulty system in which 1 inadvertent error propagates , often resulting in a suboptimal or poor outcome . even if providers are aware of the test result , it is often difficult to track homeless or migratory patients until they present to the acute - care setting with signs of advanced aids
. unfortunately , this may be true for patients who are aware of important pending tests and motivated to follow up . finally ,
even after patients initiate appropriate art , similar problems typically arise in the ambulatory setting with regard to following cd4 + counts and viral loads and assuring adherence to the art regimen . in all of these settings ,
the main challenges are not limited to a lack of resources or caring by the individual providers ; the central issue is the lack of coordination and timely communication among the various providers involved in the care of the patient across different settings , perpetuated by a system ( either paper or electronic ) not set up to reliably ensure timely acknowledgment and follow - up on these test results . from the most impoverished to the richest healthcare delivery systems ,
a central theme is apparent : poor awareness of test results at the time that vital decisions need to be made leads to delays in diagnosis and the subsequent failure to institute appropriate treatments .
what is particularly striking is that developed health - care systems with the most advanced emrs still struggle with this problem . in the united states ,
failure to follow up on test results is an increasingly recognized problem ( estimated at 15%-40% ) , often leading to clinically important treatment delays and unfortunately , malpractice claims . despite the associated morbidity and mortality ,
healthcare delivery systems have not developed effective solutions to address the problem . in general , there is a lack of consensus with regard to what constitutes
best practices in managing test results across the continuum of care . as a result
, individual providers typically have their own systems of managing test results . additionally , despite efficiency gains realized by near instantaneous availability of clinical data from emrs , test result tracking systems are either nonexistent or poorly designed .
the high degree of variability in individual clinician practices and the murky delineation of responsibility for specific test results make it mandatory to establish clear lines of responsibility prior to implementation of any electronic test result management system .
it is not just the availability of information that is necessary to improve the system , but more importantly , the ability to foster improved awareness of that information to the providers responsible for overseeing the care of the patient .
this can be achieved only by seamless integration into clinical workflow . ideally , any test result management system , whether paper or electronic , should include a few basic features : it should flag important tests results , clearly assign responsibility , and facilitate acknowledgment . without these basic features , providers caring for
a patient across a variety of clinical settings will be ineffectual at receiving and acting upon clinically relevant information in a timely manner .
healthcare in boston , massa chusetts , poon et al developed a results management application to help ambulatory clinicians review and act upon test results reliably and efficiently .
dalal et al developed an automated notification system to prompt the responsible inpatient and ambulatory physicians of the results of tests pending at the time of hospital discharge .
the success of these systems is based on not only the fundamental features mentioned above but also thoughtful consideration of the electronic clinical workflow of the patients ' responsible providers .
automated notifications are useful but only when the identities of the responsible providers have been clearly established and notifications are sent via messaging systems in widespread use by these clinicians .
alphanumeric pagers are an excellent option in many settings as the first choice , but clinical messaging systems ( within emrs ) and secure , network email are being used as well . with
the rapid growth and spread of wireless telecommunication networks , advanced mobile devices that access clinical messaging systems and network email using push - notification services are an increasingly attractive option .
the advent of global health as a field focused on , among other objectives , strengthening the systems of healthcare delivery around the world presents an opportunity to determine how best to develop and implement systems of managing test results in a variety of resource - limited settings . the world health organization ( who ) has delineated essential building blocks that contribute to a high - functioning health - care system . according to
who , a well - functioning health information system is one that ensures the production , analysis , dissemination and use of reliable and timely information on health determinants , health system performance and health status . similarly , good health services are those that
deliver effective , safe , quality personal and non - personal health interventions to those that need them , when and where needed , with minimum waste of resources .
therefore , a number of essential components are required in order for medical information to flow efficiently so that health services can be delivered effectively . with regard to test result management ,
developing healthcare systems might benefit from adapting successful strategies and principles from more developed healthcare systems . specifically , these include ( 1 ) identification of responsible providers , ( 2 ) seamless clinical workflow integration , ( 3 ) a mechanism to acknowledge test results , and ( 4 ) a fail - safe escalation hierarchy for unacknowledged test results .
one area in which developing healthcare systems are perhaps ahead of more developed systems is the use of mobile telecommunication networks .
such networks , when used by health workers and their patients , can meaningfully affect the determinants of health outcomes .
for example , the use of mobile technology by health workers has been shown to improve adherence to medication regimens . in kenya ,
text - message reminders ( using short messaging service [ sms ] protocol ) sent to health workers ' mobile phones resulted in a 23% improvement in adherence to malaria treatment guidelines for outpatient pediatric malaria . in tanzania ,
text message reminders sent to chws reduced the average number of days that clients were overdue for community - based hiv case management by 86% . in ghana
, pregnant women can register to receive weekly educational messages and appointment reminders via text message or integrated voice response ( ivr ) systems . in a randomized trial ,
lester et al demonstrated significant improvement in art adherence and rates of viral suppression among hiv patients through weekly interactive sms communication with a clinic nurse .
though there are few published examples of the use of mobile technology for directly notifying providers or clients of test results , qualitative studies in both low- and high - income settings have shown high acceptability among patients .
finally , in places such as zambia , laboratories use text - messaging systems to reduce the time between collection of a blood sample and delivery of results back to the health facility where the sample was obtained . in this way
, the widespread use of mobile technology by health workers , clients , and health facilities offers a unique opportunity to tackle the test result management conundrum in developing healthcare settings . and , if implemented wisely , the use of automated text - messaging services may positively influence the determinants of health outcomes , particularly when used for test results with a high potential to result in meaningful actions taken by providers ( eg , hiv tests , viral loads , cd4 + counts )
. how would such a mobile test - result management system function in developing healthcare settings ?
the overarching goal of any system is to positively influence clinical outcomes by initiating , altering , or discontinuing treatments in a timely manner .
ideally , such a system would draw upon the key principles of test - result management from developed settings : reliable identification of responsible providers , real - time notification of test results , timely acknowledgment and follow - up , and an escalation mechanism such that supervising clinicians or public health authorities can intervene when timely acknowledgment and follow - up do not occur .
for example , when actionable test results ( eg , a positive hiv result associated with a low cd4 + count ) become available , the responsible chw would receive an automated text - message alert prompting a visit to the patient .
patients who register to receive sms or ivr messages ( at the time the tests are drawn ) would receive an alert prompting them that the chw will visit shortly .
( if no chws are identified , the system would provide patients with information about how to seek out a nearby health worker . )
if the chw does not acknowledge the actionable test result after a predefined time interval , the chw 's overseeing clinician then would receive an automated text - message alert .
ultimately , if still left unacknowledged , the automated alert would escalate to more centralized public health authorities .
once acknowledged , mobile healthcare teams ( where they exist ) can coordinate with chws to ensure appropriate follow - up in the patient 's community .
there are many barriers and challenges that need to be addressed for such a mobile test - result notification system to be realized in developing settings .
although an estimated 90% of the world 's population is within coverage of a cellular network , more investment is needed to extend the network 's reach to the most marginalized populations ( these are likely to have a higher burden of treatable diseases and therefore , would serve to gain the most ) . yet
even simple measures such as charging phones remain an obstacle in impoverished areas solar chargers could alleviate this problem , but they are typically cost prohibitive ( although the use of hand crank
also , governments and healthcare providers typically lack experience in and capacity for deploying mobile programs .
there is little guidance available to health systems in low- and middle - income countries to support the scale - up of mobile health programs , including issues around patient privacy , confidentiality , security , interoperability with existing health information systems , and sustainable sources of financing .
finally , contextually appropriate messaging and notification strategies would need to be developed given the sensitivity of certain test results and the likelihood that many households and communities commonly share mobile phones .
mobile health is a nascent industry but one that holds much promise in countries with developing healthcare systems .
mobile test result management systems capable of leveraging automated sms and ivr notifications are promising , but rigorous studies demonstrating the effectiveness of these systems are necessary to advocate for their widespread adoption . key to success
will be integrating strategies and lessons learned from more developed healthcare settings with some of the innovative approaches that have proven effective in developing health systems .
these principles include identification of responsible providers , seamless workflow integration , a mechanism to acknowledge test results , and a fail - safe escalation mechanism for unacknowledged test results .
when such a system is in place , patients and providers alike could feel reassured that important test results will no longer fall through the cracks .
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### Summary:
: overviewacross the globe , the ways in which patients ' test results are managed are as varied as the many different types of healthcare systems that manage these data .
the outcomes , however , are often not too dissimilar : too many clinically significant test results fall through the cracks .
the consequences of not following up test results in a timely manner are serious and often devastating to patients : diagnoses are delayed , treatments are not initiated or altered in time , and diseases progress . in resource - poor settings , test results too commonly get filed away within the paper chart in ways that isolate them and prevent passage to future providers caring for a patient . to make matters worse ,
the onus to act upon these test results often rests on patients who need to return to the clinic within a specified timeframe in order to obtain their results but who may not have the means or are too ill to do so . even in more developed healthcare settings that use electronic records , clinical data residing in the electronic medical record ( emr )
are often stubbornly statickey pieces of clinical information are frequently not recognized , retrieved , or shared easily . in this way , emrs are not unlike paper record systems , and therefore , emrs alone will not solve this problem . to illustrate this problem , consider the case of a patient newly diagnosed with hiv in 3 different healthcare delivery settings .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: it is the most important avoidable cause of premature morbidity and mortality in the world , placing the heaviest burden of morbidity and mortality compared to any other risk factor on people .
the estimated death rate of 4.9 million people in 1999 is expected to rise to 10 million by the 2020s and 2030s , 7 million of which will occur in developing countries .
, it is estimated that about 48% of males and 7% of females are smokers .
most smokers start the habit during adolescence ; less than 2% of them start smoking after their 22nd birthday . in saudi arabia
the prevalence of smoking in adults in 2000 was reported as 11.6% , being higher among males ( 21.1% ) than among females ( 5.2% ) . in the eastern province , saudi arabia ,
a study conducted among female paramedical students in 1999 revealed that the overall smoking prevalence was 5.6% .
smokers are more than 10 times as likely as nonsmokers to develop peripheral vascular disease .
it is estimated that nearly one - fifth of deaths from heart disease is attributable to smoking and the who cancer agency indicated that cigarette smoking has been linked to about 90% of all lung cancer cases .
it also reported that active smoking was linked to cancer of stomach , liver , uterus , kidneys and myeloid leukemia . in women , smoking reduces fertility and increases the rate of spontaneous abortions and complications of pregnancy such as abruptio placenta , placenta previa , bleeding and premature rupture of membranes .
the risk of coronary heart disease is increased among women smokers who use oral contraceptives . among postmenopausal women , current smokers
have an increased risk of osteoporosis than non - smokers and have an increased risk of hip fracture .
smoking poses adverse social , economic and developmental effects on the lives of individuals , their families and communities .
the economic burden includes direct medical care for tobacco - induced illnesses , absence from work , reduced productivity and loss of life from early death .
smoking has been known in saudi arabia for more than 50 years , but of late tobacco imports have increased , with an average annual expenditure of 600 million saudi riyals .
smoking is increasing , particularly among the young in saudi arabia , partly due to aggressive marketing by tobacco companies .
the aim of the present study was to estimate the prevalence and assess the smoking patterns among female non - medical college students in dammam , saudi arabia .
the study population included all female students enrolled in the literature and science colleges in 2005 .
the sample size was calculated by epiinfo based on a prevalence of 12% of smoking among females in previous studies and a worst expected frequency of 3% at a confidence level of 95% .
accordingly , the minimum required sample size was 920 . taking into account the expected response rate based on the pilot study , which was 75% , the sample size was increased to 1200 college students , selected by a multi - stage stratified random sampling technique with proportional allocation as follows .
first , colleges were sub - classified by type into literature and science , then by specialties , and educational levels .
students were selected by systematic random sampling , choosing every fifth student from the list of registered students at each educational level and specialty , till the desired proportionate sample size was obtained .
after data collection , all variables were checked for accuracy and completeness , coded and entered into a personal computer and analyzed using the spss program .
the smoking index was calculated by multiplying the average number of cigarettes smoked per day ( one shisha was considered equal to 18 cigarettes ) by the number of years of smoking .
bivariate analysis was conducted using -test for qualitative data and fisher 's exact p for expected frequency less than 5 in more than 20% of the cells .
of the total sample of 1200 science and literature students , 1020 responded to the self - administered questionnaire , giving a response rate of 85% .
figure 1 demonstrates that 8.6% of the college students were smokers : 2.2% were regular smokers , 3.3% were occasional smokers and 3.1% were ex - smokers .
smoking among literature and science college students , dammam table 1 shows the distribution of female students by college .
smoking was significantly more common among students of the literature college ( 12.1% ) than those of the science college ( 3.4% ) ( p < 0.001 ) .
distribution of female students by smoking and college figure 2 illustrates the type of smoking done by female college students .
more than half of students who smoked were cigarette smokers ( 54.5% ) and 43.2% were shisha smokers .
types of smoking among female literature and science college students table 2 reflects the attributes of tobacco consumption practice among smokers .
the mean age at which smoking was initiated was 16 2.4 years , with a minimum of 11 years .
the mean number of cigarettes smoked per day was 3.4 2.3 , while for shisha it was almost two , and the mean smoking index was 44.4
. characteristics of female smokers table 3 shows the factors related to initiation of smoking .
it was found that parents or friends were the main source of the first cigarette smoked ( 35.2% and 29.5% , respectively ) .
the main motive for smoking was curiosity ( 44.3% ) , followed by relief of tension ( 26.1% ) . about 40% of the students started smoking at home , while about one - fifth started while on trips with friends or in coffee shops .
factors related to initiation of smoking among female college students figure 3 demonstrates what the parents smoked .
most of the fathers smoked cigarettes ( 81.8% ) , while most of the mothers smoked shisha ( 76.9% ) .
types of smoking among parents of female college students , dammam table 4 shows the relationship between students who smoked and parents who smoked .
it reveals that 236 out of 1020 fathers ( about 23% ) and 39 out of 1020 ( 4% ) mothers smoked .
it was found that 15.5% of the fathers who smoked had daughters who smoked , while only 6.6% of non - smoking fathers had daughters who smoked .
the rate was even higher for mothers who smoked , as 33.3% of those mothers had daughters who smoked , compared to only 7.6% daughters of mothers who did not smoke .
the prevalence of smoking among women varies markedly from country to country , ranging from an estimated 7% in developing countries to 24% in developed countries .
stopping further increases in tobacco use among women is one of the greatest challenges in the prevention of disease in the world today .
the present study reveals that the prevalence of smoking among female students of the literature and science colleges in dammam was 8.6% .
recent studies conducted in riyadh ( 2000 ) and in jeddah ( 2006 ) on female college students revealed that the prevalence of smoking was 9% and 14% , respectively .
these figures were higher than those of previous studies conducted in dammam ( 1999 ) and in riyadh ( 1992 ) , which revealed the prevalence of smoking among female college students to be 5.6% and 6.3% , respectively , indicating an increasing prevalence of smoking among female students in saudi arabia .
women / mothers , who are role models for their children and in constant contact with their offspring , should be in good health to function effectively and fulfill their responsibilities to their families and their communities
. the effect of this startling trend among women would be detrimental to health and socioeconomic conditions , if it is allowed to continue .
emro statistics in the late 1990s revealed that the prevalence of smoking among adult females in saudi arabia was higher than in all other gulf countries.[1822 ] in comparison with other emro countries , smoking was more prevalent among adult females in saudi arabia than in sudan , west bank and gaza strip , tunisia , iran , egypt , iraq and jordan.[2329 ] however , it was slightly lower than in pakistan , syria , djibouti and morocco[3033 ] and much lower than in yemen .
smoking was more prevalent among adult males in all emro countries than among females , as female smoking is still socially unacceptable , though the trend is rising .
in developed western countries like the usa , though the prevalence of smoking is decreasing in males , it is still high among females . according to our results , out of the 8.6% female college students who smoked ,
similarly , the study conducted in riyadh in 1993 among female non - medical college students revealed that one - third of students had stopped smoking . according to the present study ,
the mean age at which smoking was initiated was 16 years , the minimum being 11 years .
similar studies had reported comparable findings , where the age at which female college students started smoking ranged between 10 and 22 years .
these figures reaffirm the importance of effective health education as early as possible in schools and through the mass media in order to protect children , especially girls , from adopting this dangerous habit . according to the present study ,
the mean number of cigarettes smoked by non - medical female college students was about three per day .
on paramedical female college students in dammam . however , the two studies had similar findings on the number of shisha smoked per day ( about two ) .
the rate was even higher for mothers who smoked , as one - third of the mothers who smoked had daughters who smoked .
the same results were found in another study conducted in saudi arabia which showed that smokers were more likely to have a parent who smoked .
this shows that parents , particularly mothers , are role models for their daughters . concerning the factors relating to the initiation of smoking
, it was found that parents were the main source of the first cigarette , followed by friends .
other recent studies conducted among female college students in saudi arabia revealed that the main source of first cigarette was friends followed by parents , indicating that among the youth , peer pressure played a role in starting the habit . according to the present study
, most of the students had their first cigarette at home , which is in agreement with the dammam study conducted among paramedical female college students . in our study
similar findings were reported by mandil et al . for the adoption of the habit .
a study conducted in 2002 revealed that the transition from high school to college was a time of personal growth with its attendant developmental stress .
therefore , finding ways to understand psychosocial development and caring for college - age women can effect changes in smoking behavior .
the present study shows that more than half of the students who were smokers smoked cigarettes , while 43% smoked shisha .
moreover , most of the fathers in our study smoked cigarettes , while most of the mothers preferred shisha .
this indicates that shisha smoking among females is common in our society and that the mothers habit affected the habits of their daughters .
some reports reveal an erroneous belief by the public that water pipes held fewer health risks than cigarettes . among the university students in syria , one - third of those sampled thought water pipes were less harmful than cigarettes , while in egypt one - fifth of adult male water pipe users reported that they preferred the use of water pipe to cigarette smoking because it was less harmful .
this may explain the current popularity of shisha . in some societies , gender plays an important role in maintaining the low rate of cigarette smoking among women , though the use of water pipe seems unaffected to the same extent . a recent study in syria examined the perceptions of the use of a water pipe and cigarette smoking by sex of the respondent and sex of the smoker .
the perception of the use of water pipe was generally more positive than cigarette smoking , especially for women .
respondents were particularly enthusiastic about the positive aspects of water pipe , including the fact that it seemed traditional , familiar , and socially attractive .
overall , water pipe smoking may be on its way to becoming the norm in the emr , especially for women and girls.[4244 ] water pipe smoke contains significant amounts of dangerous constituents , including nicotine and heavy metals such as arsenic , cobalt , chromium , and lead.[4547 ] under normal conditions , the smoke produced from a single water pipe use contains approximately the same amount of nicotine - free , dry particulate matter ( tar ) as that present in approximately 20 cigarettes .
however , the tar produced by a water pipe may differ from that produced by a cigarette because in a water pipe , tobacco is heated to about half the temperature of tobacco in a cigarette .
temperature may be positively associated to tar related tumorigenicity and mutagenicity . aside from the direct effect of smoke constituents , since it is customary to share a water pipe in many cultures , the social dimension of the use of water pipe may help the spread of infectious agents .
for example , in aleppo , syria , the majority of water pipe smokers among university students share the same water pipe with their friends . in beirut , lebanon ,
this practice can spread tuberculosis and viruses like herpes and hepatitis , particularly since the temperature of the smoke that comes out of the mouthpiece of the water pipe is similar to that of air .
it may be concluded that the prevalence of smoking among female college students in saudi arabia is increasing .
accordingly , it is recommended that a preventive comprehensive health education program on smoking be initiated for females in middle schools , that stricter tobacco control measures be adopted by the government , and that anti - smoking clinics are established in colleges .
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### Summary:
: background : smoking is the most important avoidable cause of premature morbidity and mortality in the world
. the estimated annual death rate of 4.9 million people in 1999 is expected to rise to 10 million by the 2020s and 2030s , 7 million of which will occur in developing countries.aim:the aim of the present study was to estimate the prevalence of smoking and assess its pattern among non - medical female college students in dammam , saudi arabia.materials and methods : a cross - sectional study was conducted of 1020 female students selected from the literature and science colleges by multi - stage stratified random sampling technique with proportional allocation .
data were collected using a self - administered modified who global youth tobacco survey questionnaire.results:results revealed that occurrence of smoking among female college students was 8.6% .
it was significantly higher among literature college students ( 12.1% ) than among science college students ( 3.4% ) .
the mean age at which smoking started was 16 2.4 years , with a minimum of 11 years .
more than half of the students who smoked were cigarette smokers , while 43.2% were shisha smokers .
there was a strong relationship between parents who smoked and daughters who smoked .
the main motive for smoking was curiosity ( 44.3% ) , followed by relief of tension ( 26.1%).conclusions : it may be concluded that smoking is increasing among female college students in saudi arabia .
accordingly , it is recommended that a preventive comprehensive health education program on smoking be initiated for females in middle schools , that stricter tobacco control measures be adopted by the government , and that anti - smoking clinics be established in colleges .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: eukaryotic nuclear dna , along with a variety of proteins , is organized and packaged into a complex structure called chromatin .
the basic , repeating unit of chromatin is the nucleosome which consists of the dna duplex wound approximately twice around an octamer consisting of two copies each of the four core histones h2a , h2b , h3 and h4 .
nucleosomes undergo further compaction to form the distinct structures seen as chromosomes ( 1 ) .
the function of chromatin is to maintain a restrictive ground state wherein the dna is inaccessible to the transcriptional machinery but is accessible to protein complexes capable of remodeling chromatin locally to allow transcription initiation .
additionally , chromatin has a pivotal role in a number of dna - associated processes , e.g. replication ( 2 ) , repair ( 3,4 ) , kinetochore and centromere formation ( 5 ) .
histones are ideally suited for this role , as both the histone tails and globular domains are subjected to a variety of posttranslational modifications such as acetylation , methylation , phosphorylation , ubiquitination , sumoylation , adp ribosylation , deimination and proline isomerization ( 6 ) .
nucleosomes are remodeled by the action of at least five classes of atp - dependent chromatin remodelers ( 7 ) .
there are many other facets to chromatin biology and chromatin remodeling which involve a variety of proteins associated with nucleosome assembly and disassembly ( 8,9 ) , dna methylation ( 10,11 ) and more , e.g. histone variants ( 12 ) and the involvement of rnai components in heterochromatic gene silencing ( 13 ) .
chromatin biology is complicated and multifaceted and a comprehensive review of this subject is beyond the scope of this article .
in addition to the limited number of reviews cited above , readers are referred to a special issue of cell , volume 128 ( 2007 ) , epigenetics and chromatin organization , which provides a series of excellent reviews on different aspects of chromatin biology and epigenetics .
the chromdb public database ( http://www.chromdb.org ) serves as a repository for chromatin - related proteins .
chromdb was initiated as a national science foundation plant genome project database and focused on arabidopsis thaliana and zea mays ( maize ) .
the database was populated using saccharomyces cerevisiae and animal chromatin - associated proteins as blast queries to search the nearly completed arabidopsis genome and the maize est collection to identify corresponding plant homologs .
chromdb has grown from several hundred plant proteins to over 7000 proteins representing over 30 organisms ( 7474 proteins total : 3328 plants , 1779 animals , 2143 fungi , 167 stramenopiles , 57 protists ) .
currently , the database focuses on chromatin - related proteins that are conserved widely across eukaryotic species .
our main research interest lies in the analysis of the evolution of the plant chromatin proteome . to facilitate this study and to make the database applicable to all researchers and educators interested in chromatin biology ,
we strive to organize chromdb data in a straightforward and comparative manner and provide users with a variety of tools to visualize sequence information and to extract data by way of user - generated customized reports .
our goal is to make information on chromatin - associated proteins readily accessible despite the relative complexity of the processes .
the chromdb database was initiated in 2000 as a project database for a national science foundation plant genome research program grant ( dbi-9975930 ; r. jorgensen , pi ) which focused on the identification and functional analysis of z. mays ( maize ) and a. thaliana genes that contribute to chromatin - based control of gene expression .
one aspect of the aforementioned project was to produce and analyze rnai lines for a set of arabidopsis and maize chromatin - associated genes and display the results at a public database .
chromdb has evolved from a project database to a community database with renewed funding from the plant genome research program ( nsf dbi-0421679 ; pi napoli ) .
the web interface is built in html::mason , taking advantage of mason 's ability to embed perl within html .
perl modules have been developed to access data based on user queries and pass this information back to the web pages for mason to interpret and display the results .
the database is a mysql relational database running in a unix background with the schema developed to account for the type of data used and generated at the website .
genomic - based sequences are limited to plant genomes [ a. thaliana , oryza sativa ( japonica cultivar- and indica cultivar - groups ) , medicago truncatula , populus trichocarpa , physcomitrella patens ( moss ) and z. mays ] and algal and diatom genomes ( chlamydomonas reinhardtii , ostreococcus lucimarinus and phaeodactylum tricornutum ) .
the plant genomes are highlighted on the side toolbar on the chromdb homepage ( shown on the left side in figure 1 ) .
other important plant species are included in the database as transcript - based sequences which are derived from est contigs or singlets . the use of est contigs results in partial sequences especially for larger proteins .
for example , 200 transcript - based sequences are included in the database for hordeum vulgare ( barley ) but only 36% of these transcripts represent the entire , predicted coding sequence .
partial protein sequences , usually protein domains or the c - termini , are used as blast queries when identifying est contigs . the use of a limited span of protein , rather than the entire sequence , limits redundancy that could result from the inclusion of multiple , non - overlapping contigs representing different regions of the same transcript .
transcript - based plant sequences are converted to genome - based as sequencing projects produce sufficient data to make a conversion worthwhile .
for example , maize is being converted from transcript - based to genomic - based due to the rapid accumulation of sequence data from large - scale maize genome sequencing projects .
figure 1.a screen shot of a gene record page along with the side tool bar for accessing database searching , tools , reports and viewers .
a screen shot of a gene record page along with the side tool bar for accessing database searching , tools , reports and viewers .
chromdb does not display whole chromosomes ; thus for genomic - based organisms , the genomic sequence is limited to a span of nucleotides containing the predicted transcript splice model and 5 and 3 untranslated regions .
plant sequences are obtained from a variety of sources , e.g. ncbi databases ( http://www.ncbi.nlm.nih.gov/ ) , the department of energy joint genome institute ( http://www.jgi.doe.gov/ ) , the arabidopsis information resource ( http://www.arabidopsis.org/ ) ; the j. craig venter institute [ http://www.tigr.org/ , formally the institute for genomic research ( tigr ) ] and plantgdb ( http://www.plantgdb.org/ ) .
all plant sequences are curated by chromdb staff members to provide the best transcript models . in many cases ,
we have derived our own transcript models from genomic sequences using fgnesh or fgenesh+ licensed from softberry ( http://www.softberry.com ) .
we make use of multiple sequence alignments to analyze plant proteins and correct models where biological support of the model ( cdna sequences ) is not available .
important animal and fungal model organisms , such as homo sapiens , drosophila melanogaster and s. cerevisiae , are available as transcript - based sequences and are obtained from the ncbi reference sequence ( refseq ) collection ( http://www.ncbi.nlm.nih.gov/refseq/ ) .
we focus on sequenced genomes and do not derive est contigs for non - plant organisms .
these transcripts are rarely curated by chromdb staff , except for predicted transcript models that need substantial improvement as indicated by multiple sequence analysis and only when refseq accessions affect the quality of a phylogenetic tree .
all database sequences are assigned a chromdb i d ( identifier ) which denotes both the transcript and the protein .
these identifiers , as well as formal gene names , loci and aliases are included in the database and can be used to search for gene records .
an explanation of the chromdb identifiers is provided in the help manual under section iv entitled chromdb identifiers .
chromdb identifiers are not gene names and are not intended to take the place of recognized formal names ; these are database identifiers and serve as one level of database organization .
one of the more difficult aspects of the database relates to providing a straightforward hierarchical organization of the range of chromdb protein groups associated with chromatin remodeling .
this difficulty relates to the complexity of the full range of proteins associated with chromatin remodeling .
there are over 90 protein groups displayed at chromdb ; however , they can be grouped into parent categories reflecting different functional aspects of chromatin biology , as shown in supplementary table 1 .
the next , lower level of organization is the individual protein groups , i.e. the three- to five - letter designations .
the more complex groups such as the chr proteins ( swi / snf chromatin remodeling atpase super family ) can be broken down further into distinct phylogenetic groups , e.g. snf2 , chd1 , rad16 ( 14,15 ) .
this protein group classification scheme forms the basis for advanced searching and generating reports , two of our important database tools . a full description of the hierarchy is provided in the supplemental data table 1 .
however , we warn readers that these groups may change slightly by the time this manuscript is published , as we continue to find new ways to organize data in a straightforward manner that allows for ease of navigation through the website .
the major divisions of protein groups are as follows : histones and histone linker proteins , nucleosome organization ( includes assembly and displacement ) , histone modifications , histone modification binding - proteins , modified - histone - binding proteins , dna modifying proteins , non - histone dna - binding proteins , rnai components and chromosome dynamics .
the contents of the database can be searched , compared , and visualized using a variety of search functions , viewers and report tools .
two search options are provided , a limited quick search text box and a menu - driven
the quick search text box is located at the top of every webpage and accepts single entries for gene names , either the chromdb i d , the formal gene name , an alternative alias or a locus . in those cases where the same formal gene name is used for multiple organisms , a list is generated showing each gene name and organism , as well as the chromdb i d .
for example , the argonaute gene designated as ago1 is used for homologous genes in arabidopsis , d. melanogaster , and schizocaccharomyces pombe and a quick search using ago1 will bring up all three entries . additionally , this text box accepts an organism name ( either the scientific or common name ) or an ncbi accession .
advanced search is available from the link on the side menu shown in figure 1 .
this link brings up a menu - driven format that allows the user to customize a search in a variety of ways using three different criteria : organisms , protein groups and the type of report .
for the organisms , the default is an alphabetical list of scientific names , and a link is provided to switch to a taxon classification ( e.g. plants , animals , fungi ) .
for the protein group selection , the default is the functional groups shown in supplementary table 1 , and a link is provided to display an alphabetical list of all protein groups .
alternatively , a link is provided that displays a text box for entering a list of gene names as well as the report selection .
the gene record page is the central navigation portal for accessing information relating to each database gene .
regular users of the database will notice a new look to the chromdb website and the gene record pages .
new additions are ncbi entrez gene link ( if available ) , a chromdb taxon description , a drop - down menu of fasta formatted sequences , the organism / sequence classification ( transcript- or genomic - based ) , and a thumbnail view of the gbrowse display .
figure 1 shows a screen shot of the summary or default page of a gene record page .
the tabular format at the top of the gene record page provides access for additional information such as decorated sequences , ncbi accessions and the gbrowse display .
two more tabs , titled expression and rnai , will appear for some maize and arabidopsis genes , as we have retained the rna gels and rnai information from the previous grant .
more information regarding each tab can be found on the help page at the website ( http://www.chromdb.org/help/genepage.html ) .
chromdb uses the gmod tool , gbrowse ( 16 ) as an individual gene - based visualization tool and not as a genome wide or chromosome visualization tool . for genomic - based organisms ,
the gbrowse view is based on the genomic sequence and the display includes the transcript splice model , protein domains ( aligned against the transcript model ) and ncbi accessions .
the inclusion of the protein domains aligned to exons is useful in discerning the effect of alternate transcript splicing on protein domain structure .
each individual plant can have specialized tracks , for example arabidopsis displays have a track for agrobacterium t - dna insertion events ( 17 ; http://signal.salk.edu/ ) for transcript - based plant organisms , the gbrowse display is based on the transcript and tracks include ncbi accessions and protein domains . for non - plant organisms ,
the gbrowse display is limited to the transcript , protein domains and the refseq accession .
in addition to similarity searching , this tool is useful in determining if a gene is present in the database .
users can select the standard blast programs as well as preset databases such as plants , animals , or fungi . on the results page ,
each match is linked back to that gene 's gene record page where more information can be obtained about that gene .
there is a link on the homepage on the left tool bar and within the web pages .
for example , each plant genome page has a list of appropriate links , e.g. tair ( the arabidopsis information resource ) , among others , for a. thaliana , the craig venter institute ( tigr ) for a number of organisms .
part of our mission is to provide the community with the means to make comparative analyses of chromatin - associated proteins among a diverse group of organisms .
the links for these comparative tools and viewers are provided on the side tool bar on each web page .
most of these features use the same menu - driven interface discussed above for the advanced search feature . examples of these features are : the ability to form fasta files ( entire sequence or a protein domain ) and viewers for pfam and smart protein domains and exon structure ( see supplementary data for examples of the protein and exon viewers ) .
a full description of all chromdb tools will not be listed here , but the contents can be seen in the side tool bar in figure 1 .
information about the selection menus and the tools can be found on the general help page ( http://www.chromdb.org/help/help.html ) .
genomic - based sequences are limited to plant genomes [ a. thaliana , oryza sativa ( japonica cultivar- and indica cultivar - groups ) , medicago truncatula , populus trichocarpa , physcomitrella patens ( moss ) and z. mays ] and algal and diatom genomes ( chlamydomonas reinhardtii , ostreococcus lucimarinus and phaeodactylum tricornutum ) .
the plant genomes are highlighted on the side toolbar on the chromdb homepage ( shown on the left side in figure 1 ) .
other important plant species are included in the database as transcript - based sequences which are derived from est contigs or singlets . the use of est contigs results in partial sequences especially for larger proteins .
for example , 200 transcript - based sequences are included in the database for hordeum vulgare ( barley ) but only 36% of these transcripts represent the entire , predicted coding sequence .
partial protein sequences , usually protein domains or the c - termini , are used as blast queries when identifying est contigs . the use of a limited span of protein , rather than the entire sequence , limits redundancy that could result from the inclusion of multiple , non - overlapping contigs representing different regions of the same transcript .
transcript - based plant sequences are converted to genome - based as sequencing projects produce sufficient data to make a conversion worthwhile .
for example , maize is being converted from transcript - based to genomic - based due to the rapid accumulation of sequence data from large - scale maize genome sequencing projects .
figure 1.a screen shot of a gene record page along with the side tool bar for accessing database searching , tools , reports and viewers .
a screen shot of a gene record page along with the side tool bar for accessing database searching , tools , reports and viewers .
chromdb does not display whole chromosomes ; thus for genomic - based organisms , the genomic sequence is limited to a span of nucleotides containing the predicted transcript splice model and 5 and 3 untranslated regions .
plant sequences are obtained from a variety of sources , e.g. ncbi databases ( http://www.ncbi.nlm.nih.gov/ ) , the department of energy joint genome institute ( http://www.jgi.doe.gov/ ) , the arabidopsis information resource ( http://www.arabidopsis.org/ ) ; the j. craig venter institute [ http://www.tigr.org/ , formally the institute for genomic research ( tigr ) ] and plantgdb ( http://www.plantgdb.org/ ) .
all plant sequences are curated by chromdb staff members to provide the best transcript models . in many cases ,
we have derived our own transcript models from genomic sequences using fgnesh or fgenesh+ licensed from softberry ( http://www.softberry.com ) .
we make use of multiple sequence alignments to analyze plant proteins and correct models where biological support of the model ( cdna sequences ) is not available .
important animal and fungal model organisms , such as homo sapiens , drosophila melanogaster and s. cerevisiae , are available as transcript - based sequences and are obtained from the ncbi reference sequence ( refseq ) collection ( http://www.ncbi.nlm.nih.gov/refseq/ ) .
we focus on sequenced genomes and do not derive est contigs for non - plant organisms .
these transcripts are rarely curated by chromdb staff , except for predicted transcript models that need substantial improvement as indicated by multiple sequence analysis and only when refseq accessions affect the quality of a phylogenetic tree .
all database sequences are assigned a chromdb i d ( identifier ) which denotes both the transcript and the protein .
these identifiers , as well as formal gene names , loci and aliases are included in the database and can be used to search for gene records .
an explanation of the chromdb identifiers is provided in the help manual under section iv entitled chromdb identifiers .
chromdb identifiers are not gene names and are not intended to take the place of recognized formal names ; these are database identifiers and serve as one level of database organization .
one of the more difficult aspects of the database relates to providing a straightforward hierarchical organization of the range of chromdb protein groups associated with chromatin remodeling .
this difficulty relates to the complexity of the full range of proteins associated with chromatin remodeling .
there are over 90 protein groups displayed at chromdb ; however , they can be grouped into parent categories reflecting different functional aspects of chromatin biology , as shown in supplementary table 1 .
the next , lower level of organization is the individual protein groups , i.e. the three- to five - letter designations .
the more complex groups such as the chr proteins ( swi / snf chromatin remodeling atpase super family ) can be broken down further into distinct phylogenetic groups , e.g. snf2 , chd1 , rad16 ( 14,15 ) .
this protein group classification scheme forms the basis for advanced searching and generating reports , two of our important database tools . a full description of the hierarchy is provided in the supplemental data table 1 .
however , we warn readers that these groups may change slightly by the time this manuscript is published , as we continue to find new ways to organize data in a straightforward manner that allows for ease of navigation through the website
. the major divisions of protein groups are as follows : histones and histone linker proteins , nucleosome organization ( includes assembly and displacement ) , histone modifications , histone modification binding - proteins , modified - histone - binding proteins , dna modifying proteins , non - histone dna - binding proteins , rnai components and chromosome dynamics .
the contents of the database can be searched , compared , and visualized using a variety of search functions , viewers and report tools .
two search options are provided , a limited quick search text box and a menu - driven
the quick search text box is located at the top of every webpage and accepts single entries for gene names , either the chromdb i d , the formal gene name , an alternative alias or a locus . in those cases where the same formal gene name is used for multiple organisms , a list is generated showing each gene name and organism , as well as the chromdb i d .
for example , the argonaute gene designated as ago1 is used for homologous genes in arabidopsis , d. melanogaster , and schizocaccharomyces pombe and a quick search using ago1 will bring up all three entries . additionally , this text box accepts an organism name ( either the scientific or common name ) or an ncbi accession
advanced search is available from the link on the side menu shown in figure 1 .
this link brings up a menu - driven format that allows the user to customize a search in a variety of ways using three different criteria : organisms , protein groups and the type of report .
for the organisms , the default is an alphabetical list of scientific names , and a link is provided to switch to a taxon classification ( e.g. plants , animals , fungi ) . for the protein group selection ,
the default is the functional groups shown in supplementary table 1 , and a link is provided to display an alphabetical list of all protein groups .
alternatively , a link is provided that displays a text box for entering a list of gene names as well as the report selection .
the gene record page is the central navigation portal for accessing information relating to each database gene .
regular users of the database will notice a new look to the chromdb website and the gene record pages .
new additions are ncbi entrez gene link ( if available ) , a chromdb taxon description , a drop - down menu of fasta formatted sequences , the organism / sequence classification ( transcript- or genomic - based ) , and a thumbnail view of the gbrowse display .
figure 1 shows a screen shot of the summary or default page of a gene record page .
the tabular format at the top of the gene record page provides access for additional information such as decorated sequences , ncbi accessions and the gbrowse display .
two more tabs , titled expression and rnai , will appear for some maize and arabidopsis genes , as we have retained the rna gels and rnai information from the previous grant .
more information regarding each tab can be found on the help page at the website ( http://www.chromdb.org/help/genepage.html ) .
chromdb uses the gmod tool , gbrowse ( 16 ) as an individual gene - based visualization tool and not as a genome wide or chromosome visualization tool . for genomic - based organisms ,
the gbrowse view is based on the genomic sequence and the display includes the transcript splice model , protein domains ( aligned against the transcript model ) and ncbi accessions .
the inclusion of the protein domains aligned to exons is useful in discerning the effect of alternate transcript splicing on protein domain structure .
each individual plant can have specialized tracks , for example arabidopsis displays have a track for agrobacterium t - dna insertion events ( 17 ; http://signal.salk.edu/ ) for transcript - based plant organisms , the gbrowse display is based on the transcript and tracks include ncbi accessions and protein domains . for non - plant organisms ,
the gbrowse display is limited to the transcript , protein domains and the refseq accession .
in addition to similarity searching , this tool is useful in determining if a gene is present in the database .
users can select the standard blast programs as well as preset databases such as plants , animals , or fungi . on the results page ,
each match is linked back to that gene 's gene record page where more information can be obtained about that gene .
there is a link on the homepage on the left tool bar and within the web pages .
for example , each plant genome page has a list of appropriate links , e.g. tair ( the arabidopsis information resource ) , among others , for a. thaliana , the craig venter institute ( tigr ) for a number of organisms .
part of our mission is to provide the community with the means to make comparative analyses of chromatin - associated proteins among a diverse group of organisms .
the links for these comparative tools and viewers are provided on the side tool bar on each web page .
most of these features use the same menu - driven interface discussed above for the advanced search feature . examples of these features are : the ability to form fasta files ( entire sequence or a protein domain ) and viewers for pfam and smart protein domains and exon structure ( see supplementary data for examples of the protein and exon viewers ) .
a full description of all chromdb tools will not be listed here , but the contents can be seen in the side tool bar in figure 1 .
information about the selection menus and the tools can be found on the general help page ( http://www.chromdb.org/help/help.html ) .
we have not included data on the number of genes for each organism in this article , as those numbers will change by the publication date .
we add new protein groups to reflect new discoveries in the literature , and we continue to devise new tools to enable users to extract information from mysql tables .
new datasets ( organisms and protein groups ) and tools are prepared at our development site , and weekly updates are run to sync production and development to reflect data releases .
a link is provided on the homepage so users can access a list of updated contents .
a phylogenetic classification scheme will be introduced in the future to further subdivide protein groups .
we will post multiple sequence alignments , as well as the phylogenetic trees , in support of the classifications .
the chromdb website changes continually , both in content and appearance , as we strive to present users with a comprehensive database of chromatin - associated proteins for an ever - increasing number of organisms , and as we endeavor to find new ways to display the data in a straightforward and comparative manner .
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### Summary:
: the chromdb website ( http://www.chromdb.org ) displays chromatin - associated proteins , including rnai - associated proteins , for a broad range of organisms .
our primary focus is to display sets of highly curated plant genes predicted to encode proteins associated with chromatin remodeling .
our intent is to make this intensively curated sequence information available to the research and teaching communities in support of comparative analyses toward understanding the chromatin proteome in plants , especially in important crop species such as corn and rice .
model animal and fungal proteins are included in the database to facilitate a complete , comparative analysis of the chromatin proteome and to make the database applicable to all chromatin researchers and educators .
chromatin biology and chromatin remodeling are complex processes involving a multitude of proteins that regulate the dynamic changes in chromatin structure which either repress or activate transcription .
we strive to organize chromdb data in a straightforward and comparative manner to help users understand the complement of proteins involved in packaging dna into chromatin .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: total hip replacement is one of the most successful orthopedic surgeries . annually , about 800 000 total hip replacement is done around the world . unfortunately , this number is increasing , because prevalence osteoarthritis of hip joint is increasing around the world including iran .
quality of life is personal idea and is determined by oneself . to determine quality of life , general and specific instruments exist for each disease .
general instruments like short - form 36 health survey ( sf-36 ) evaluate situation of person broadly . on the other hand , specific instruments for each disease
have created to focus on those aspects of life that are affected by a particular disease ; also , we can utilize these tools to evaluate the effectiveness of various treatments .
in recent years , the outcome of orthopedic surgeries has increasingly been evaluated according to patient - reported questionnaires .
so , self - report questionnaires should be used to achieve more information on patients situations .
self - report questionnaires generally should not include many questions so that the response rate is increased and the risk of data loss is decreased .
the questionnaires should be adapted cross - culturally to maintain the content and construct validity of the original instrument and to prevent population - related and culture - related bias in assessment .
the oxford hip score ( ohs ) , which is a self - administered questionnaire , has been studied extensively and is a reliable , valid , and responsive instrument for evaluating hip pain and disability in patients suffering from osteoarthritis of hip joint , especially those undergoing total hip arthroplasty.[1116 ] it is a 12-item instrument with each item scored by the patient on a 1- to 5-point likert scale .
the global score is given by the sum of the scores for all 12 items resulting in values between 12 and 60 .
permission was obtained from the original questionnaire developer and the technology transfer company of the university of oxford ( oxford , england ) to develop this study .
we followed the recommended process of the mapi research trust as well as the principles ordered by the translation and cultural adaptation working group .
the standard forward - backward translation ( linguistic validation ) was done ; two health professionals ( orthopedic surgeons ) did the forward translation ( english to persian ) independently . after receiving forward translations of the ohs questionnaire from our translators
, we decided to arrange meetings with two forward translators and an orthopedic surgeon to integrate the contents of the translations .
so , we made the opportunity for them to meet each other and we had a discussion on translations .
the session was actually a peaceful settlement about the differences existed between the translations of each of the two translators .
we matched their translations and they tried to convince each other for choosing the most suitable word for each case .
we did the exact things for the backward translations ( persian to english ) as well .
for the next step , in a common meeting in the presence of all four translators and orthopedic surgeon , the original questionnaire of ohs was shown and the backward translated questionnaire was compared with the original one .
we have sent differences we found between our backward translated questionnaire and the original ohs questionnaire to the original questionnaire developer accompanying with the backward translators explanations for each case of difference . at the end , we established the pre final persian version of ohs .
after translation process , the persian version of the ohs was tested in a pilot study by administering to 30 adult patients suffering from osteoarthritis of hip joint ( 30 adult inpatients admitted for hip replacement in alzahra and kashani hospitals ) for cognitive debriefing and to two orthopedic surgeons for clinician 's review . according to the results of the pilot study and feedbacks from the developer , the questionnaire was edited more and the final persian version of the ohs was produced .
the study involved 130 adult persian - speaking patients suffering osteoarthritis of hip joint admitted for primary total hip arthroplasty in two university hospitals ( alzahra and kashani hospitals ) in isfahan in iran from september 2009 until april 2011 .
of these 130 patients , 25 were excluded , five patients did not accept to participate in the study , 15 patients underwent other operative procedures , and five persons were not operated on during the study period . thus , preoperative data were available for 105 patients with hip osteoarthritis .
all of them filled out persian ohs , womac , and sf12 in preoperative examination two weeks before their surgery .
retest was performed on 39 patients two weeks later , when they were admitted for surgery .
access to those hospitals was open to every patient , and our routine patients are a mixture of urban and rural inhabitants from different states of iran .
we used cronbach 's alpha and intra - class correlation coefficient ( icc ) , bland and altman method , respectively , to evaluate the internal consistency and reproducibility of data from the persian ohs .
cronbach 's alpha indicates the average correlation between all items of a scale and the correlation between each item and the whole scale .
we anticipated cronbach 's alpha values greater than 0.9 and 0.8 , which were considered excellent and good , respectively . to analyze the discriminant and convergent validity
, the persian ohs was correlated to the persian version of western ontario and mcmaster universities osteoarthritis index ( womac ) and the persian version of 12-item short form health survey ( sf-12 ) .
the ohs is a 12-item instrument with each item scored by the patient on a 1-to 5-point likert scale .
the global score is generated by summing the scores for all 12 items resulting in values between 12 and 60 .
the distribution of floor and ceiling effects of the persian ohs was determined by calculating the proportion of individuals obtaining the lowest and highest scores , respectively .
the womac is a self - administered , disease - specific instrument that includes subscales for pain , stiffness , and physical function .
scores range from 0 to 20 ( pain ) , 0 to 8 ( stiffness ) , and 0 to 68 ( function ) . the higher the score , the worse is the health state .
the sf-12 is a self - administered generic measure for evaluating the quality of life .
scores are transformed into two weighted summary scores for physical function ( physical component scale [ pcs ] ) and mental health ( mental component scale [ mcs ] ) which can score between 0 and 100 . the higher the score , the better is the health state .
we recoded the scores of ohs and womac into a 0 to 100 point scale with 100 being the best score .
the persian womac and sf12 were recorded in preoperative examination two weeks before their surgery accompanying the ohs . to examine convergent validity
, we assumed that the correlation coefficients describing the relationship between the ohs and the womac and the pcs of the sf-12 would be moderate to high ( r > 0.50 ) . to examine divergent validity , we anticipated the correlation coefficients describing the relationship between the ohs and the mcs of the sf-12 which would be lower than those between the ohs and pcs ( r < 0.50 ) .
pearson correlation was also applied to assess the validity of the persian ohs with respect to the womac and sf12 .
most discrepancies concerned synonyms for specific expressions , for example , the translators have written the pain of your hip instead of the pain from your hip , or have used the words sharp and burning instead of the words shooting and stabbing .
similarly , the phrase walking stick has been used instead of walking aid .
we found no floor or ceiling effects for the persian ohs that were determined by calculating the proportion of respectively .
the persian ohs overall score demonstrated high reproducibility ( icc , 0.93 , p < 0.001 ) and internal consistency ( cronbach 's alpha , 0.94 ) .
mean scores for the first and second administrations were similar ( p = 0.83 ) ( 42.7 12.7 vs 41.4 13.9 , respectively ) .
convergent validity for the persian ohs was observed by the moderate to high correlations between persian ohs scores and the other questionnaire scores [ table 1 ] .
the strongest correlations were between the persian ohs and the womac total score ( r = 0.86 ) , the persian ohs and the womac function score ( r = 0.86 ) , and then between persian ohs and the womac pain ( r = 0.79 ) .
divergent validity for the persian ohs was observed by the low correlation between the persian ohs and the mcs of the sf-12 ( r = 0.40 ) .
our study presents the results of the trans - cultural adaptation and validation of the ohs for iranian patients with persian language , who suffered from hip osteoarthritis . according to the results , items on the persian ohs
were well understood by iranian patients with hip osteoarthritis demonstrating that the translation process was acceptable .
the ohs has been evaluated extensively and is a reliable , valid , and responsive instrument for assessing hip pain and disability in patients undergoing total hip arthroplasty . unlike the studies of wood and mclauchlan and mcmurray et al .
we observed no floor or ceiling effects for the persian ohs , similar to the findings for preoperative patients reported by garbuz et al .
the persian ohs overall score demonstrated high reproducibility ( icc , 0.93 , p < 0.001 ) and internal consistency ( cronbach 's alpha , 0.94 ) .
we found good internal consistency for the persian ohs more than the value reported by dawson et al .
convergent validity for the persian ohs was observed by the high correlations between persian ohs and the womac total score ( r = 0.86 ) and the womac function score ( r = 0.86 ) and then between persian ohs and the womac pain ( r = 0.79 ) .
correlation coefficients between ohs and womac pain and function subscales were 0.76 and 0.88 in a cohort study of 147 patients with a mean age of 68 years .
the correlation coefficients were also ( r = 0.81 - 0.87 ) in a prospective cohort study on 402 patients ( mean age , 61 years ) of ( 29 ) .
we observed that the correlation coefficient describing the relationship between the persian ohs and the womac stiffness subscale was somewhat lower ( r = 0.69 ) , which was consistent with those of ostendorf et al .
divergent validity for the persian ohs was observed by the low correlations between the persian ohs and the mcs of the sf-12 ( r = 0.40 ) , which was slightly lower than the values of ostendorf et al .
( r = 0.49 ) . similar to the values reported by ostendorf et al . and garbuz et al .
( r = 0.53 ; r = 0.60 ) , the correlation coefficient between the persian ohs and the pcs of the sf-12 in our study was moderate ( r = 0.58 ) . in our patient samples ,
when using the original scoring system , mean score for the first administrations was 42.7 , similar to the mean preoperative ohs score reported by dawson et al .
, which was 43.6 ; field et al . reported a mean preoperative value of 41.0 and ostendorf et al . reported a value of 42.5 for dutch patients
our study demonstrated that the trans - cultural adaptation and validation of the persian ohs is a reliable and practicable instrument for self - assessment of function and pain in iranian patients with hip osteoarthritis .
we believe further investigation of the persian ohs in patients after tha is needed to assess the sensitivity to change of this questionnaire .
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### Summary:
: introduction : in recent years , outcome assessment related to orthopedic surgeries has increasingly focused on patient - reported questionnaires .
the oxford hip score ( ohs ) , self - administered questionnaire , is a reliable , valid , and responsive instrument for assessing hip in patients undergoing arthroplasty.methods:the study involved 105 adult persian - speaking patients admitted for primary total hip arthroplasty in two hospitals in isfahan in iran from september 2009 until april 2011 .
all of them filled out their scales ( persian ohs , womac , and sf12 ) in preoperative examination.results:mean scores of ohs in first administrations was 42.7 12.7 . the persian ohs overall score demonstrated high reproducibility ( icc,0.93 , p < 0.001 ) and internal consistency ( ca , 0.94 ) .
persianohs had high correlations with womac total score ( r = 0.86 ) , function score ( r = 0.86 ) , and pain score ( r = 0.79 ) , the relationship between the persian ohs and the womac stiffness subscale was somewhat lower ( r = 0.69 ) .
the correlation coefficient between the persian ohs and the pcs of the sf-12 in our study was moderate ( r = 0.58 ) .
persian ohs had low correlation with mcs of the sf-12 ( r = 0.40).discussion : persian ohs had high correlations with womac total score , function score , and pain score .
it had moderate correlation with pcs of the sf-12 and low correlation with mcs of the sf-12.conclusions:our study demonstrated the trans - cultural adaptation and validation of the persian ohs is a reliable and practicable instrument for assessment of function and pain in iranian patients with hip osteoarthritis .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: amyotrophic lateral sclerosis ( als ) is a neurodegenerative disorder characterized by progressive muscular weakness and atrophy .
although the degeneration of upper and lower motor neurons is the pathological hallmark of the disease , several studies indicated that als is a multisystem disorder that also affects cognitive domains ( agosta et al .
, 2010 ; raaphorst et al . , 2010 ; tsermentseli et al . , 2012 ) .
concordantly , neurodegenerative changes beyond the motor - system have been reported ( anderson et al . , 1995 ; grosskreutz et al . , 2006 ; kato et al . , 1997 ; neumann et al . , 2006 ; takeda et al . , 2009 ; wightman et al . ,
1992 ) . besides such structural changes , functional alterations related to als have also been observed across multiple motor and extra - motor regions using a variety of different tasks ( for recent review see tsermentseli et al . , 2012 ) .
most studies observed increased activations of sensorimotor areas and/or recruitment of additional regions , which was interpreted as functional compensation or reorganization within the motor - system ( kew et al .
, 1993 ; kollewe et al . , 2011 ; konrad et al . , 2002 ; mohammadi et al . , 2011 ;
however , some studies also found an activation decrease of sensorimotor and premotor areas ( mohammadi et al .
one major reason for these controversial findings refers to the heterogeneity of the patient populations especially with regard to their particular disease stage during the time of study .
bearing this in mind , it is important to note that neurodegenerative alterations and therewith interrelated functional loss or compensatory processes are certainly subject to change during progression of the disease .
thus , to identify patterns of functional alterations and their putative modifications during als disease - progression , it is indispensable to perform longitudinal within - subject investigations .
the present study was designed to directly address such progression - related functional changes in motor and cognitive functions . to this end ,
brain activity in fourteen als patients and fourteen healthy controls was studied in two fmri sessions separated by a 3-month interval , employing a simple go / nogo - task , in which the additional presentation of task - irrelevant novel stimuli allowed for assessment of novelty - related hippocampal activity .
in addition , to assess the relationship between functional and structural changes emerging during als disease - progression , we performed cross - sectional voxel - based morphometric ( vbm ) analyses on structural volumes acquired with magnetization transfer imaging in an independent , but socio - demographically comparable sample of 26 als patients and 28 controls . the magnetization transfer ratio of tissues depends on the surface chemistry and biophysical dynamics of macromolecules , as well as their tissue concentration ( cosottini et al . , 2011 ; eckert et al . , 2004 ; kato et al . , 1997 ; muller - vahl et al . , 2009 ; wolff and balaban , 1994 ) .
as such it has been shown to be strongly associated with tissue integrity ( grossman et al . , 1994 ) and reduced magnetization transfer ratios have therefore repeatedly been suggested to mirror microstructural alterations like gliosis and changes in axonal density possibly related to early - stage neurodegenerative phenomena ( eckert et al . , 2004 ; kiefer et al . , 2009 ; perez - torres et al .
two patient samples with sporadic als were recruited from the als outpatient clinics of the departments of neurology at the medical school hannover and at the medical school of the otto - von - guericke university magdeburg .
all patients met the criteria for probable or definitive als as defined by the el escorial diagnostic criteria for als ( brooks et al . , 2000 ) and had either a limb or bulbar onset .
exclusion criteria were other neurological conditions that could affect motor performance and cognition ( e.g. stroke , traumatic brain injury ) .
all participants underwent clinical examination on the day of study with active follow - up .
disease severity was assessed using the revised als functional rating scale ( alsfrs - r ; cedarbaum et al . , 1999 ) .
disease duration was defined as time in months between symptom onset and the date of the experiment . from these measures
the disease - progression rate was then calculated as ( 48 alsfrs - r)/disease duration ( ellis et al . , 1999 ) .
in addition , the neuromuscular impairment was quantified by the five - point medical research council ( mrc ) scale .
15 muscles were tested on the right and left for a maximum score of 150 ( sternocleidomastoids , shoulder abductors and adductors , elbow flexors and extensors , wrist flexors and extensors , long finger flexors , thumb opponent , finger abductors and adductors , hip flexors , knee flexors and extensors , and ankle dorsiflexors ) .
good reliability and reproducibility for manual muscle testing in patients with als have previously been shown ( great lakes als study group , 2003 ; andres et al . , 1988 ) .
furthermore , a detailed neuropsychological assessment lasting about 2 h was performed during the baseline visit in the first sample of fourteen patients who took part in the fmri experiment .
forty - two healthy individuals similar to the patients in age and gender were recruited as controls .
twenty - eight of the subjects were included in the vbm study and the remaining fourteen took part in the fmri experiment .
ethical approval for all procedures was obtained prior to study ( vote number 11/06 - 75/11 , ethical committee of the medical faculty of the otto - von - guericke university , magdeburg ) and all participants gave written informed consent before participation .
all experimental procedures have been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments .
subject demographics and all relevant clinical data are shown in table 1 . for neuropsychological assessment a range of standardized neuropsychological tests were employed ( see table 2 ) .
verbal memory performance was tested using the vlmt , a german version of the rey auditory verbal learning task ( lezak et al . , 2004 ) , and non - verbal memory using the rey complex figure test ( shin et al . ,
furthermore , we measured the digit span and the visual memory span ( lezak et al . , 2004 ) . to address executive frontal functions , the ruff figural fluency test ( ruff et al . , 1987 ) , the trail - making test ( soukup et al . , 1998 ) , the regensburger verbal fluency test
( aschenbrenner et al . , 2000 ) , and the copy subtest of the rey complex figure test were employed .
attention was assessed using the d2 attention test ( brickenkamp and zillmer , 1998 ) .
deficient verbal memory performance was defined as abnormal performance ( < 2 standard deviations compared to an age matched reference population ) in 2 different memory tasks ( phukan et al . , 2012 ) .
executive dysfunction was defined as an abnormal performance in 2 different executive tests ( strong et al . , 2009 ) .
adjustments were made for motor disability in tasks that were time dependent ( d2 , ruff figural fluency test , and trail - making test ) .
( 2012 ) , 14% of the patients showed executive dysfunction ( als - ex , single domain ) , 21% had executive paired with memory dysfunction , attention dysfunction or impaired visuo - construction ( als - ex , multi domain ) and 7% showed deficits regarding attentional performance ( non - executive impairment , als - neci ) .
none of the patients fulfilled the neary criteria for frontotemporal dementia ( neary et al . , 1998 ) .
data were acquired on a 3-tesla mr scanner ( siemens magnetom trio , erlangen , germany ) equipped with an 8-channel head coil .
the subjects viewed the stimuli through a mirror attached to the head coil , which reflected the images that were back - projected from an lcd projector onto a screen positioned behind the coil . during each run
187 volumes were acquired with a t2-weighted echo planar imaging sequence ( 32 ac - pc oriented slices , tr 2000 ms , te 30 ms , flip - angle 80 , in - plane resolution 64 64 mm , fov 224 224 mm , no gap , resulting voxel - size 3.5 3.5 3.5 mm ) in an odd even interleaved sequence .
scanning parameters as well as the paradigm were the same for both scanning sessions separated by a 3-month interval .
structural images for the vbm analysis were acquired on a 1.5-t ge , signa horizon lx scanner equipped with a standard quadrature head coil ( general electric , milwaukee , wi , usa ) .
the protocol for magnetization transfer imaging consisted of a proton - density weighted se sequence ( 48 ac pc oriented slices , tr = 2600 ms , te = 20 ms , in - plane resolution 256 256 mm , resulting voxel - size = 3 1 1 mm ) both with ( mt ) and without ( pd ) a preparing saturation pulse ( 1200 hz off - resonance , 1180 flip angle , 16 ms ) . image post - processing included an inter - sequence movement correction by co - registration of the mt and pd images and subsequent calculation of the magnetization transfer ratio maps ( mtr ) by the formula mtr = 100 [ ( pd mt)/pd ] . the fmri experiment consisted of two scanning sessions separated by a 3-month interval , in both of which functional data
were acquired in 4 runs of 6 min each , while subjects performed in a modified go / nogo - paradigm .
all subjects ( 14 patients and their respective controls ) participating in the fmri experiment were scanned twice .
the mean interval between sessions was 95 10 ( standard deviation ; sd ) days ( range : 77112 days ) for the patients and 86 10 ( sd ) days ( range : 71116 days ) for controls .
during the task subjects were repeatedly presented with a target ( church with 3 towers ) and a highly similar non - target stimulus ( church with only 2 towers , see fig . 1 ) ,
subjects were instructed to make a speeded button - press response upon the detection of the target , but not to the highly similar non - target stimulus .
in addition , our design included the presentation of novel stimuli ( indoor and outdoor black - and - white pictures ) , each of which was presented only once throughout the experiment .
the total number of novel pictures matched the number of repetitions of the target and non - target stimuli ( 176 presentations throughout each scanning session ) .
target and non - target stimuli were identical across sessions for all participants , while novel stimuli differed across sessions .
in addition , to avoid memory / habituation - effects , pictures of outdoor - scenes served as novels within one and indoor - scenes in the other fmri session ( counterbalanced across subjects ) . all stimuli
( size of 9.6 5.8 ) were presented 4.4 above a central fixation cross for 1 s and their presentation - sequence was pseudo - randomized with an inter - trial interval varying between 2 and 6 s ( mean : 2.75 s ) to allow for trial separation in an event - related analysis .
fmri data were analyzed using spm8 software ( wellcome department of cognitive neurology , university college london , uk ) and matlab 7.9 ( the mathworks inc . ) .
then the mean image derived from realignment of the 2nd session was co - registered to that of the 1st session and the resultant spatial transformation matrices were applied to all volumes of the 2nd session . finally , all images were spatially normalized to an epi template in mni space and smoothed with an isotropic gaussian kernel of 8-mm full - width at half - maximum .
for statistical analysis , blood - oxygen level - dependent responses were modeled by delta functions at the time of stimulus onsets .
resultant event - regressors ( targets , non - targets , and novels ) were entered into a general linear model and convolved with the standard hemodynamic - response function implemented in spm8 .
the data from both scanning sessions were independently analyzed , resulting in two contrasts of interest per session and subject ( targets > non - targets and novels > non - targets ) .
these individual - subject contrasts then were submitted to second - level random - effects group analyses ( one - sample t - tests ) .
in addition , session - specific contrasts were directly compared against each other by second - level paired t - tests to identify activity changes within 3 months of als disease - progression .
all statistical parametric maps from the group analyses were thresholded at p < 0.001 ( uncorrected ) for voxel - level inference with a minimum cluster - size criterion of 20 contiguous voxels , and subsequent cluster - level correction for multiple testing at p < 0.05 ( fwe - corrected ) . given our a priori hypotheses , the significance of activated clusters in the primary motor cortex , cerebellum , and hippocampus , was assessed using small volume correction ( svc ) .
statistical parametric maps were visualized using mricron ( http://www.mccauslandcenter.sc.edu/mricro/mricron/ ) . to directly compare the magnitude of motor- and novelty - related modulations between sessions , and for correlation of activation differences with clinical measures ,
a region of interest ( roi ) analysis was performed using marsbar ( brett et al . ,
functional rois located in the left primary motor cortex ( m1 ) , bilateral cerebellum , and bilateral hippocampus were defined based on the significant clusters obtained from the second - level paired t - test group analyses on the patients ' data .
targets > non - targets and novels > non - targets were extracted from both fmri measurements .
these values were submitted to repeated - measures anovas ( ranova ) with the factor contrast ( targets > non - targets vs.
novels > non - targets ) and session ( 1st vs. 2nd ) .
if statistical significance ( p < 0.05 ) was obtained , paired t - tests were applied for post hoc comparison .
if significant differences in motor- or novelty - related activity across sessions were observed , correlations between these difference values and the patients ' clinical parameters ( alsfrs - r and mrc - megascore ) were assessed by spearman 's rank correlation .
to relate the patients ' motor- and novelty - evoked activations to those of healthy individuals , mean contrast - estimates from the left m1 and bilateral hippocampus were also extracted from the control subjects ' data .
analysis of the resultant hippocampal data by ranova ( within - subject factor hemisphere and session , between - subject factor group ) revealed no main effects or interactions for the factor hemisphere .
the resultant values then were analyzed by ranova with the within - subject factor session and the between - subject factor group . if significant effects ( p <
0.05 ) were obtained , paired t - tests ( within - subject comparison ) or two - sample t - tests ( comparison between groups ) were applied for post hoc analysis .
in contrast to the fmri experiment , mtr data from our second cohort were acquired only once .
these data thus only allowed for a cross - sectional comparison between patients and controls , but not for assessment of longitudinal structural changes emerging during als disease - progression .
first , pd - weighted images from 52 healthy , aged individuals ( which did not serve as control subjects in the current study ) were normalized to the pd - template included in spm8 .
the resulting normalization parameters were applied to the concurrently acquired mt - images of the 52 subjects . then the normalized mt - volumes were smoothed with a 4-mm fwhm isotropic gaussian kernel and averaged to create a scanner - specific template for normalization of our current vbm data .
mt - images of our als patients and their respective controls were normalized to this template and the resultant normalization parameters were applied to the inherently co - registered mtr - maps .
finally , these normalized mtr - volumes were smoothed with an isotropic gaussian kernel of 4 mm fwhm . for statistical analysis
the normalized and smoothed mtr - images were submitted to an ancova with study group ( als patients vs. controls ) as the main factor , and age , as well as total brain volume as covariates .
controls > als patients revealed significant effects . to assess putative correlations between the disease - related structural changes and the patients ' clinical data we performed regression analyses on the patients ' mtr volumes by adding the their clinical data into the design as covariates ( alsfrs - r scores , mrc - megascores , disease duration and disease progression rate ) , in addition to their age and total brain volumes . beyond that , spherical rois ( 4 mm radius ) were centered at the local maxima showing significant mtr differences between patients and controls in the main ancova ( see table 6 for mni - coordinates of these local maxima where rois were centered ) . from these rois mean mtr - values were extracted and then correlated with the patients ' alsfrs - r scores , mrc - megascores , their disease duration and disease progression rate using spearman 's rank correlation . for the spm analyses , stereotactic coordinates for voxels with maximum t - values within significant activation clusters are reported in mni standard space using an auxiliary voxel - level threshold of p < 0.001 ( uncorrected ) with a minimum cluster - size criterion of 50 contiguous voxels and subsequent cluster - level correction for multiple testing at p < 0.05 ( fwe - corrected ) .
reaction times , correct responses and false alarms ( see table 3 ) were analyzed by ranovas with the within - subject factor session and the between - subject factor group .
these analyses revealed no significant main effects or interactions neither for the reaction times ( session : f(1,26 ) = 0.6 , p > 0.4 ; group : f(1,26 ) = 2.9 , p = 0.1 ; group session : f(1,26 ) = 0.2 , p > 0.6 ) , nor for the correct responses ( session : f(1,26 ) = 1.8 , p > 0.1 ; group : f(1,26 ) = 1.1 , p > 0.2 ; group session : f(1,26 ) = 0.6 , p > 0.4 ) , or for the false alarm rates ( session : f(1,26 ) = 2.9 , p = 0.1 ; group : f(1,26 ) = 0.2 , p > 0.6 ; group session : f(1,26 ) = 0.3 , p >
direct comparison across sessions ( paired t - tests ) also showed no significant differences for any of the behavioral measures of the patients ( reaction times : t(1,13 ) = 0.8 , p > 0.4 ; correct responses : t(1,13 ) = 1.1 , p > 0.2 ; false alarms : t(1,13 ) = 0.8 , p > 0.4 ) , or controls ( reaction times : t(1,13 ) = 0.2 , p > 0.8 ; correct responses : t(1,13 ) = 1.1 , p > 0.2 ; false alarms : t(1,13 ) = 1.6 , p > 0.1 ) . taken together , the behavioral data showed no significant differences between patients and controls and also no significant alterations over scanning sessions within each of the two groups .
for the healthy controls we observed no differences in motor- or novelty - related activity across sessions ( data not shown ) .
the targets > non - targets contrasts from both sessions revealed activity in a variety of regions associated with the motor - system , including the bilateral primary motor cortex ( m1 ) , bilateral cerebellum , supplementary motor area , bilateral inferior frontal gyrus/ anterior insula , striatum , and portions of the thalamus ( see fig . 2a , as well as the upper and middle rows of table 4 ) .
note that maximum t - values and cluster - sizes of these activations are considerably higher during the 1st than during the 2nd session , indicative of a decrease in motor - related activity across sessions .
this assumption was validated by a paired t - test showing that motor - related activity in the left m1 and bilateral cerebellum was in fact significantly higher during the 1st than during the 2nd measurement ( see fig .
2b and bottom rows of table 4 ) . to further corroborate this finding , contrast - estimates from rois located in the left m1 and bilateral hippocampus
these analyses showed a significant main effect for the factor contrast for the left m1 ( f(1,13 ) = 37.8 , p < 0.0001 ) and the left cerebellum ( f(1,13 ) = 5.5 , p < 0.05 ) , as well as a significant contrast session interaction for all 3 rois ( left m1 : f(1,13 ) = 32.9 , p < 0.0001 ; left cerebellum : f(1,13 ) = 8.5 , p < 0.05 ; right cerebellum : f(1,13 ) = 25.7 , p < 0.001 ) .
post hoc comparisons indicated that these effects were due to higher estimates for the contrast
targets > non - targets of the 1st in comparison to the 2nd measurement ( see fig . 2b ; left m1 : t(1,13 ) = 5.2 , p < 0.001 ; left cerebellum : t(1,13 ) = 3.7 , p < 0.005 ; right cerebellum : t(1,13 ) = 4.9 , p < 0.001 ) . to investigate the relationship between the activation decrease within 3 months of disease - progression and the patients ' clinical status
, we correlated the magnitude of the activity reduction with the patients ' alsfrs - r and mrc - megascores ( see fig . 2c ) .
therein , we observed a significant correlation between the motor - related activation decrease and the patients ' alsfrs - r scores for both cerebellar rois ( left cerebellum : = 0.60 , p < 0.05 ; right cerebellum : = 0.54 , p < 0.05 ) , and with the patients ' mrc - megascores for the left m1 ( = 0.61 , p < 0.05 ) . for both sessions , the comparison
novels > non - targets showed activations within multiple ventral occipital and temporal regions , including the middle / superior occipital , fusiform , lingual and parahippocampal gyri ( see fig . 3a and upper / middle rows of table 5 ) . in addition
, stimulus novelty significantly activated the bilateral hippocampus , but due to the chosen threshold of p < 0.001 only during the 2nd measurement .
direct comparison between sessions confirmed that in the bilateral hippocampus the novelty - related activity was higher during the 2nd in comparison to the 1st session ( fig .
direct comparison of these activation changes by ranovas with the factor contrast and session for the bilateral hippocampal roi data showed a significant main effect for the factor contrast ( left hippocampus : f(1,13 ) = 11.5 , p < 0.005 ; right hippocampus : f(1,13 ) = 41.3 , p < 0.05 ) and a significant contrast - by - session interaction ( left hippocampus : f(1,13 ) = 6.7 , p < 0.05 ; right hippocampus : f(1,13 ) = 8.2 , p < 0.05 ) .
post hoc comparisons revealed that these interactions were due to higher novelty - related activity during the 2nd in comparison to the 1st session ( see fig .
3b ; left hippocampus : t(1,13 ) = 5.0 , p < 0.001 ; right hippocampus : t(1,13 ) = 3.9 , p < 0.005 ) .
finally , these novelty - related activation differences showed a negative correlation with the patients ' alsfrs - r scores for both hippocampal rois ( see fig .
3c for graphical illustration ; left hippocampus : = 0.60 , p < 0.05 ; right hippocampus : = 0.63 , p < 0.05 ) .
the within - subject analyses presented so far revealed a decrease of motor - related and an increase of novelty - evoked activity in patients during the 3-month interval .
these data on their own , however , did not allow inferring about the relative course of these changes compared to functionally intact neural activations . to overcome this limitation , beta - estimates from the rois covering the left m1 and bilateral hippocampus
were also extracted from the control subjects ' functional data and compared to those of the patients ' .
for both the hippocampal and motor - related activations analyses by ranovas revealed a significant main effect for the factor session ( left m1 : f(1,26 ) = 8.8 ; p < 0.01 ; hippocampus : f(1,26 ) = 11.6 ; p < 0.005 ) and a group - by - session interaction ( left m1 : f(1,26 ) = 4.4 ; p < 0.05 ; hippocampus : f(1,26 ) = 4.6 ; p < 0.05 ) . for the left m1 , post hoc analyses indicated that the interaction effect was due to a significantly higher motor - related activity in patients compared to controls ( see fig .
4a ) during the first ( t(1,13 ) = 2.3 , p < 0.05 ) , but not during the second session ( t(1,13 ) = 0.7 , p > 0.5 ) .
this pattern was also reflected by a significant decrease of motor - related activity in patients between the 1st and 2nd sessions ( t(1,13 ) = 5.2 , p < 0.001 ; see also fig .
2 ) , which was absent in controls ( t(1,13 ) = 0.5 , p > 0.6 ) . for the hippocampal data ( fig .
4b ) , in contrast , the post hoc analyses did not show significant differences in novelty - evoked activity between groups for any of the two sessions ( 1st session : t(1,13 ) = 0.5 , p > 0.5 ; 2nd session : t(1,13 ) = 1.6 , p > 0.1 ) . within each group
, however , direct comparisons between sessions revealed a significant increase of novelty - evoked activity in the patients ( t(1,13 ) = 5.1 , p < 0.001 ) , but not in controls ( t(1,13 ) = 0.7 , p > 0.4 ) .
the between - group vbm analysis revealed several clusters of significantly reduced magnetization transfer ratio ( mtr ) in als patients compared to healthy controls ( see fig .
the reduced mtrs were located in the bilateral pre-/postcentral gyrus , orbitofrontal cortex , hippocampus , thalamus , striatum , and left inferior frontal gyrus ( see table 6 for mni - coordinates and maximum t - values ) . to assess a putative relationship between these structural changes and the patients ' clinical state , we performed regression analyses using their clinical measures as covariates ( alsfrs - r scores , mrc - megascores , disease duration and disease progression rate ) .
in addition , we assessed putative correlations between the clinical data and the patients ' mean mtr - values extracted from spherical rois centered at the local activation maxima of the between - group analysis .
using these two approaches we did not observe significant correlations between the patients ' mtr - values and their disease state as indexed by any of the clinical measures employed in our analyses ( alsfrs - r scores , mrc - megascores , disease duration and disease progression rate ) .
the fmri part of the current study aimed at investigating how motor- and novelty - related regions are functionally affected within the short interval of 3 months of als - progression .
the volumetric analyses aimed at identifying regions that display alterations of brain tissue in socio - demographically comparable patients at similar disease stages . therefore , we superimposed the statistical maps obtained for the functional differences between measurements with those indicating the structural alterations onto an anatomical template image . by this
means we observed a considerable overlap between the structural alterations identified by the vbm analysis and the functional reduction in motor - related activity in the course of als disease - progression within the left m1 ( illustrated in magenta in fig .
in addition , the motor - related functional alterations within the striatum and thalamus lay in close proximity to the structural alterations affecting these regions ( compare red and blue maps in fig . 6 and activation maxima in table 4 and bottom rows of table 5 ) .
similarly , a comparison between the functional changes in novelty processing across sessions and our vbm results revealed an overlap ( shown in yellow in fig .
6 ) , or at least a very close proximity ( compare red and green maps in fig . 6 ) between structural and functional changes in the bilateral hippocampus ( the corresponding activation maxima are shown in table 4 and bottom rows of table 6 ) .
using a longitudinal within - subject design , the current study revealed motor- and memory - related functional alterations emerging within only 3 months of als disease - progression .
importantly , motor activity decreased during the 3-month interval , while novelty - evoked hippocampal activity increased at the same time .
in addition , these functional changes displayed opposite correlations with clinical measures : while the motor - evoked activity correlated positively with the patients ' clinical status ( alsfrs - r and mrc - megascores ) , the hippocampal activations showed an inverse pattern .
these results indicate that hippocampal and motor - system lesions emerge at different disease stages : the relative decrease in motor activity might reflect a breakdown of compensatory mechanisms due to ongoing progressive neural loss , while the hippocampal activation increase , in contrast , most likely constitutes the initial built - up of compensatory processes to overcome dysfunctions typically observed at the beginning of lesions ( bookheimer et al . , 2000 ; dickerson and sperling , 2008 ; woodard et al . , 2009 ) . finally , our additional cross - sectional voxel - based morphometric ( vbm ) analyses in an independent sample of patients identified structural alterations at the same locations where changes in hemodynamic activity were observed .
thus , our findings indicate a close relationship between functional and structural changes and provide important insights into the temporal dynamics of functional changes during als disease - progression . in line with most previous studies ( kew et al . , 1993 ;
, 2002 ; mohammadi et al . , 2011 ; schoenfeld et al . , 2005 ) , our als patients displayed an increased motor activity compared to controls during the initial measurement .
such increased activity - levels or recruitments of additional regions have previously been interpreted as functional compensation or reorganization within the motor - system .
this network showing increased or exclusive motor - related activity in patients relative to controls comprised regions in the primary sensorimotor and premotor cortices , supplementary motor area , dorsal anterior cingulate cortex , cerebellar motor regions and parts of the striatum and thalamus ( kollewe et al .
, 2011 ; konrad et al . , 2002 ; mohammadi et al . , 2011 ;
these findings were attributed to an als - related cortical hyperexcitability due to the loss of inhibitory interneurons ( maekawa et al . , 2004 ) , accompanied by reduced short - interval intracortical inhibition ( ziemann et al . , 1997 ) and
lower binding rates of the gaba - a receptor ligand flumazenil ( turner et al . , 2005 ) .
importantly , however , at later disease - stages decreased activations in the sensorimotor and premotor areas have also been described ( mohammadi et al . , 2011 ;
the most plausible explanation for this pattern of changes was that after an initial compensatory hyperactivation or recruitment of additional areas , ongoing neurodegeneration during disease progression causes a breakdown of the compensatory mechanisms , finally resulting in decreased activity levels compared to earlier ( within - subject ) measurements or relative to controls .
for one , the pattern of motor - related activations closely resembled that described in previous studies on motor - network functions in als ( kollewe et al .
, 2011 ; konrad et al . , 2002 ; mohammadi et al . , 2011 ;
schoenfeld et al . , 2005 ) and within this network we observed a hyperactivation in patients compared to controls during the initial measurement . beyond that , we observed a relative ( within - subject ) reduction in the patients ' motor - related activity in the left primary sensorimotor cortex and bilateral cerebellum after only 3 months , which is consistent with the idea that degenerative changes in motor regions start long before clinical signs are apparent ( schoenfeld et al . , 2005 ) .
further support is provided by the observation that the magnitude of the activity reductions correlated with the patients ' clinical status : higher alsfrs - r or mrc scores ( less disability/ earlier disease stage ) were accompanied by more pronounced decreases in motor activity during the 3-month interval . taken together , these data corroborate the idea of a continuously changing motor - network function in als , indicating that activity changes in the motor - system caused by progressive neurodegeneration are most pronounced at early stages of the disease , while the breakdown of functional compensatory processes occurs later . traditionally considered as a neurodegenerative disease selectively affecting the motor - system , als is now conceptualized as a multisystem disorder also affecting other cognitive domains . while only a proportion of als patients develop actual clinical signs of frontotemporal dementia ( lomen - hoerth et al . , 2002 )
, milder cognitive and behavioral impairments occur in up to 50% of the patients ( hammer et al . , 2011 ;
lomen - hoerth et al . , 2002 ; phukan et al . , 2012 ; raaphorst et al . , 2010 ) .
in agreement with these data , we observed cognitive deficits in more than 40% of our patients predominantly but not exclusively in the form of executive dysfunctions .
, 1421% also exhibited deficits in the memory domain . however , although memory dysfunctions and concordant hippocampal neurodegeneration ( anderson et al . , 1995 ; grosskreutz et al .
, 2006 ; kato et al . , 1997 ; neumann et al . , 2006 ; takeda et al .
, 2009 ; takeda et al . , 2007 ; wightman et al . , 1992 ) have repeatedly been described , no study so far directly addressed a potential functional affection of the hippocampus in als . particularly , it is not clear how the hippocampal lesions evolve over time and how they translate into hemodynamic activity changes . to bridge this gap ,
the current task - design comprised the presentation of task - irrelevant novel stimuli shown to elicit robust hippocampal activations ( bunzeck and duzel , 2006 ; stoppel et al . , 2009 ) . beyond this robust activation of the hippocampus , the novelty - processing network activated by this task - design comprised occipital vision - related ( fusiform and lingual gyrus ) and temporal memory - related ( parahippocampal gyrus and hippocampus ) regions ( bunzeck and duzel , 2006 ; stoppel et al . , 2009 ) ,
in contrast to the activity in this motor network which was initially higher than in controls and decreased in the 3-month interval the hippocampal activations were higher in the second measurement and not different from that in healthy controls .
such increased hippocampal responses have previously been described in mild cognitive impairment and interpreted as a compensatory recruitment of neural resources to overcome early disease stage dysfunctions ( bookheimer et al . , 2000 ; dickerson and sperling , 2008 ; woodard et al . , 2009 ) .
moreover , similar hyperactivations across different brain areas indicating fresh lesions were observed in other early - stage neurodegenerative disorders such as pre - manifest parkinsonism or huntington 's disease ( buhmann et al . , 2005 ; kloppel et al .
given that most of our patients showed no or only slight memory impairments , the hippocampal hyperactivity in the second measurement might indeed reflect such compensation processes to overcome early lesions ( agosta et al .
, 2010 ; anderson et al . , 1995 ; takeda et al . , 2009 ; tsermentseli et al . , 2012 ) .
consequently , the present results suggest that hippocampal dysfunctions develop at later disease stages than the motor - system lesions .
this is further supported by the inverse correlation between the hippocampal activity increase and the patients ' alsfrs - r scores ( i.e. a more pronounced enhancement was evident at later stages of the disease ) . in this context
it has to be noted that an increase of hippocampal novelty - related activity only was observed in the patient group , while no significant differences were observed in comparison to controls or within the control group across the 3 months .
this is most likely due to the fact that the relative increase of hemodynamic activity in patients is a rather small effect and therefore not significantly different from the stable activity level of controls ( that probably lies in between the activation level of the first and second measurements in als patients ) .
this points out to the importance of longitudinal designs for studying disease - related neurodegenerative changes and provides a convincing explanation why previous studies that compared als patients with controls did not find hemodynamic activity - differences in the hippocampus .
strong decreases of activity in this region are certainly present at later stages of the disease , while early stages seem to be rather indexed by a compensatory activity - increase as observed in the present study . several studies described structural neurodegenerative alterations in als not only within motor areas , but also across multiple frontotemporal , limbic or subcortical structures ( agosta et al . , 2010 ; anderson et al . , 1995 ;
grosskreutz et al . , 2006 ; kato et al . , 1997 ; neumann et al .
1992 ) . as for the functional results , the pattern of changes was quite inconsistent across studies ,
in addition , early - stage neurodegeneration is primarily indexed by microstructural alterations as e.g. intra- or extracellular matrix alterations , gliosis and axonal density - changes ( douaud et al .
, 2013 ; khandelwal et al . , 2011 ; robberecht and philips , 2013 ) , which are not well detected by vbm based on t1-weighted brain volumes ( kabani et al . , 2002 ; van der flier et al . ,
, we employed magnetization transfer imaging , which detects not only volumetric changes ( as e.g. neuronal loss or tissue shrinkage ) but also microstructural alterations dependent of tissue myelination , axonal density , or gliosis ( grossman et al . , 1994 ;
hanyu et al . , 2000 ; van waesberghe et al . , 1999 ; wolff and balaban , 1994 ) .
this analysis is therefore sensitive to lesions present at early stages of neurodegeneration , as it has previously been demonstrated for several other diseases ( eckert et al .
, 2004 ; kiefer et al . , 2009 ; perez - torres et al . , 2014 ;
using a cross - sectional design , we thereby identified mtr - reductions in most previously described regions evident in t1-based volumetric analyses , including the precentral gyrus , frontal / orbitofrontal cortex , hippocampus , thalamus , and striatum .
for most of these regions , however , results were inconsistent across previous investigations on t1-weighted images . beyond that , most previous studies employing mtr to investigate structural differences related to als only focused on changes within the corticospinal tract ( da rocha et al . ,
, 1997 ; tanabe et al . , 1998 ; verstraete et al . , 2014 ) and to date only two studies analyzed mtr on whole - brain data in als using a roi - based approach ( cosottini et al . , 2011 ) or voxel - based volumetric analyses ( cosottini et al . , 2013 ) .
these two studies also revealed changes in mtr across several motor / premotor and extra - motor regions , which is in good agreement with the current results .
importantly , beyond these previously described differences at cortical sites our analyses also revealed mtr reductions across several subcortical areas ( hippocampus , thalamus and striatum ) . while previous imaging analyses ( which mainly relied on t1-weighted brain volumes ) failed to demonstrate als - related structural changes in these regions , histopathological investigations did show their affection during the course of the disease .
thus , our current results suggest that whole - brain vbm analyses based on magnetization transfer imaging are potentially more sensitive to early structural damage in rapid progressive neurodegenerative disorders .
the fmri part of the current study sought to investigate how motor- and novelty related regions are functionally affected within the short interval of 3 months of als progression , while the volumetric analyses aimed at identifying regions showing alterations of brain tissue in another group of socio - demographically comparable patients at similar disease stages using a cross - sectional design .
although the fmri study was performed in a different patient sample than the vbm analysis , we sought to look roughly for commonalities between the functional alterations during disease progression and the structural changes obtained by means of our cross - sectional design .
for this aim , we superimposed the statistical maps obtained for the functional differences between fmri measurements from the first sample with those indexing the als related tissue differences from the larger second sample . despite this rather coarse approach
we observed a high degree of overlap between the disease - related changes in hemodynamic activity and those regions that are subject to structural damage ( see fig .
this was the case not only for functional alterations in motor regions , but also for those in the limbic system .
being aware that structural changes are the result of neural degeneration on a longer time scale , while hemodynamic measures reflect changes on a much shorter one , the high degree of overlap indicates that in als the functional changes occur in the same regions that are subject to structural damage .
given that our structural and functional data were acquired using two separate cohorts on two different scanners , we acknowledge that no hard conclusions should be drawn despite the high degree of overlap between the structural and functional results .
thus , future within - subject comparisons clarifying to which extent the structural and functional changes in fact overlap are mandatory . despite this fact
, we nevertheless believe that our results are of rather high reliability , since the high degree of spatial overlap was achieved not because , but although the data were acquired under conditions of rather high variability ( different scanners/ different cohorts ) .
the current longitudinal study revealed functional alterations in motor- and memory - related regions within only 3 months of als disease - progression .
moreover , we observed als related tissue differences at the same anatomical locations in a second patient sample studied cross - sectionally .
these results indicate that als is a multisystem disorder , in which the hippocampus also is affected . moreover , the dynamics of the observed fmri changes suggest that the functional affection of motor- and memory - related regions emerges at different stages of als : while the motor - system lesions develop rather early , the hippocampal dysfunctions arise later in the course of the disease .
the knowledge of the progression - related dynamics within different functional systems ( motor , memory etc . ) during the course of als is essential for understanding the nature and development of the disease and provides a tremendous potential for fmri to become a diagnostic tool in the future .
the present data highlight the value of longitudinal fmri investigations designed to specifically address particular functional alterations emerging during the progression of als and signify their potential usefulness as a diagnostic tool for neurodegenerative disorders .
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### Summary:
: previous studies have shown that in amyotrophic lateral sclerosis ( als ) multiple motor and extra - motor regions display structural and functional alterations
. however , their temporal dynamics during disease - progression are unknown . to address this question we employed a longitudinal design assessing motor- and novelty - related brain activity in two fmri sessions separated by a 3-month interval . in each session ,
patients and controls executed a go / nogo - task , in which additional presentation of novel stimuli served to elicit hippocampal activity .
we observed a decline in the patients ' movement - related activity during the 3-month interval .
importantly , in comparison to controls , the patients ' motor activations were higher during the initial measurement .
thus , the relative decrease seems to reflect a breakdown of compensatory mechanisms due to progressive neural loss within the motor - system .
in contrast , the patients ' novelty - evoked hippocampal activity increased across 3 months , most likely reflecting the build - up of compensatory processes typically observed at the beginning of lesions .
consistent with a stage - dependent emergence of hippocampal and motor - system lesions , we observed a positive correlation between the alsfrs - r or mrc - megascores and the decline in motor activity , but a negative one with the hippocampal activation - increase . finally , to determine whether the observed functional changes co - occur with structural alterations , we performed voxel - based volumetric analyses on magnetization transfer images in a separate patient cohort studied cross - sectionally at another scanning site . therein , we observed a close overlap between the structural changes in this cohort , and the functional alterations in the other .
thus , our results provide important insights into the temporal dynamics of functional alterations during disease - progression , and provide support for an anatomical relationship between functional and structural cerebral changes in als .
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: a retrospective review was performed for all the patients diagnosed with pc who had undergone surgery at the korea cancer center hospital from january 2007 to december 2011 .
patients who were diagnosed with pc via the surgical specimen , and those who underwent preoperative fna were included in this study .
we excluded patients who had received preoperative chemotherapy or radiotherapy because of possible modifications of the shape and size of the tumor cells . in the end , 7 of the patients ( 5 men , 2 women ) with a mean age of 67 years ( range , 60 to 78 years )
clinicopathological parameters such as tumor size , tumor location , stage , and distant metastases were collected from the patients ' medical records .
the aspirated material was smeared onto 4 glass slides , immediately fixed in 95% alcohol and stained with a papanicolaou stain .
immunocytochemical studies for cytokeratin and vimentin were performed on paraffin sections of the cell blocks in all cases .
all of the surgical resection specimens had been fixed with 10% formalin and embedded in paraffin .
a mean of 6.5 ( range , 5 to 8) hematoxylin and eosin - stained slides per tumor was available .
the cytological features were assessed and compared with the histopathological features of the corresponding surgical specimen .
we defined the epithelial component of pc as malignant tumor cells with glandular or squamous differentiation .
a component of large cell carcinoma was also included in the epithelial component of pc .
we defined the large cell carcinoma component as tumor cells with a tendency to form loosely structured clusters composed of epithelial cells of unequal sizes without glandular or squamous differentiation .
we defined the sarcomatoid component of pc as malignant giant and/or spindle cells.14 the investigated cytological parameters were as follows : component of tumor cells , architectural pattern ( fascicles , single cells , and cluster ) , adhesion ( loose or tight ) , nuclei ( pleomorphism , variability in size , number , and size of nucleoli , nuclear membrane , and chromatin texture ) , nature of the cytoplasm , nuclear - to - cytoplasmic ratio and background .
a retrospective review was performed for all the patients diagnosed with pc who had undergone surgery at the korea cancer center hospital from january 2007 to december 2011 .
patients who were diagnosed with pc via the surgical specimen , and those who underwent preoperative fna were included in this study .
we excluded patients who had received preoperative chemotherapy or radiotherapy because of possible modifications of the shape and size of the tumor cells . in the end , 7 of the patients ( 5 men , 2 women ) with a mean age of 67 years ( range , 60 to 78 years )
clinicopathological parameters such as tumor size , tumor location , stage , and distant metastases were collected from the patients ' medical records .
the aspirated material was smeared onto 4 glass slides , immediately fixed in 95% alcohol and stained with a papanicolaou stain .
immunocytochemical studies for cytokeratin and vimentin were performed on paraffin sections of the cell blocks in all cases .
all of the surgical resection specimens had been fixed with 10% formalin and embedded in paraffin .
a mean of 6.5 ( range , 5 to 8) hematoxylin and eosin - stained slides per tumor was available .
the cytological features were assessed and compared with the histopathological features of the corresponding surgical specimen .
we defined the epithelial component of pc as malignant tumor cells with glandular or squamous differentiation .
a component of large cell carcinoma was also included in the epithelial component of pc .
we defined the large cell carcinoma component as tumor cells with a tendency to form loosely structured clusters composed of epithelial cells of unequal sizes without glandular or squamous differentiation .
we defined the sarcomatoid component of pc as malignant giant and/or spindle cells.14 the investigated cytological parameters were as follows : component of tumor cells , architectural pattern ( fascicles , single cells , and cluster ) , adhesion ( loose or tight ) , nuclei ( pleomorphism , variability in size , number , and size of nucleoli , nuclear membrane , and chromatin texture ) , nature of the cytoplasm , nuclear - to - cytoplasmic ratio and background .
the clinico - pathological data and the original cytological diagnosis are summarized in table 1 .
the mean tumor was 41 mm in diameter ( range , 25 to 70 mm ) .
lobectomy with lymph node dissection was performed in 6 cases , and metastasectomy was performed in only 1 case ( case 5 ) .
case 1 had multiple bronchioloalveolar carcinomas of both lungs and renal cell carcinoma at the time of surgery .
two patients had pleura and chest wall invasion and four had regional lymph node metastasis .
three patients had distant metastases in the brain , adrenal glands , intestine , bone , and lung .
the tumor stages were iia in 1 case , iib in 1 case , iiia in 3 cases , and iv in 2 cases .
adjuvant chemotherapy was performed in 3 patients and adjuvant chemoradiation therapy was performed in 2 patients .
the cell component of the cytological specimens usually reflected the components of the corresponding surgical specimen .
the epithelial component indicated large cell carcinoma in 6 cases , and adenocarcinoma and squamous cell carcinoma in 1 case each .
the spindle cell component was observed in 4 cases , and the gc component was observed in 6 cases ( fig .
the components of tumor cells in the pathological and cytological specimens are listed in table 2 .
the tumor cells were arranged in monolayers , 3-dimensional clusters , or were scattered as single cells .
cohesive clusters were observed in some cases , but loose clusters were observed in most of the cases .
the epithelial tumor cells had an irregular nuclear membrane and a coarsely granular chromatin ( fig .
the nucleus of the giant tumor cells was hyperchromatic to vesicular , bizarre in shape , and more than 5 times the size of the resting lymphocytes , and often much larger .
two different cellular patterns were seen : areas with aggregating pleomorphic sarcomatoid cells and/or entrapped spindle cells , and epithelioid tumor cells .
immunoreactivity to cytokeratin was present in the epithelial cells in all of the cases and in some of the spindle cells in 2 cases ( focal , weakly positive ) .
vimentin was expressed in some of the epithelial components in 4 cases ( focal , strongly positive ) and in the sarcomatoid component of all the cases ( fig .
the clinico - pathological data and the original cytological diagnosis are summarized in table 1 .
the mean tumor was 41 mm in diameter ( range , 25 to 70 mm ) .
lobectomy with lymph node dissection was performed in 6 cases , and metastasectomy was performed in only 1 case ( case 5 ) .
case 1 had multiple bronchioloalveolar carcinomas of both lungs and renal cell carcinoma at the time of surgery .
two patients had pleura and chest wall invasion and four had regional lymph node metastasis .
three patients had distant metastases in the brain , adrenal glands , intestine , bone , and lung .
the tumor stages were iia in 1 case , iib in 1 case , iiia in 3 cases , and iv in 2 cases .
adjuvant chemotherapy was performed in 3 patients and adjuvant chemoradiation therapy was performed in 2 patients .
the cell component of the cytological specimens usually reflected the components of the corresponding surgical specimen .
the epithelial component indicated large cell carcinoma in 6 cases , and adenocarcinoma and squamous cell carcinoma in 1 case each .
the spindle cell component was observed in 4 cases , and the gc component was observed in 6 cases ( fig .
the components of tumor cells in the pathological and cytological specimens are listed in table 2 .
the tumor cells were arranged in monolayers , 3-dimensional clusters , or were scattered as single cells .
cohesive clusters were observed in some cases , but loose clusters were observed in most of the cases .
the epithelial tumor cells had an irregular nuclear membrane and a coarsely granular chromatin ( fig .
the nucleus of the giant tumor cells was hyperchromatic to vesicular , bizarre in shape , and more than 5 times the size of the resting lymphocytes , and often much larger .
two different cellular patterns were seen : areas with aggregating pleomorphic sarcomatoid cells and/or entrapped spindle cells , and epithelioid tumor cells .
immunoreactivity to cytokeratin was present in the epithelial cells in all of the cases and in some of the spindle cells in 2 cases ( focal , weakly positive ) .
vimentin was expressed in some of the epithelial components in 4 cases ( focal , strongly positive ) and in the sarcomatoid component of all the cases ( fig .
there have been only a few reports of cytological analysis in cases of pulmonary pc.14 - 16 most of the previous cytological reports have revealed similar findings .
hummel et al.15 reported that the cytological findings of pc included a conspicuous population of pleomorphic spindle cells arranged singly , in loose clusters , and in fascicles , and as micro - tissue fragments in a necrotic background .
although malignant spindle cells and gcs can be helpful cytological features , pc of the lung is challenging to diagnose because it has very poor cellular differentiation , rare incidence and histological heterogeneity . in this context , histopathological correlation is an important prerequisite of a correct cytological evaluation .
the surgical specimens in case 2 were composed of gc carcinoma , large cell carcinoma , and moderately differentiated adenocarcinoma .
however , the cytologic specimen mainly consisted of clusters of adenocarcinoma and only a few scattered giant tumor cells . because of the morphological continuum and overlapping features that exist between giant tumor cells and poorly differentiated large cell carcinoma , the original cytological diagnosis of this case was " non - small cell carcinoma ( nscc ) , favoring adenocarcinoma . " the cytological specimen in case 4 , which was misdiagnosed as " nscc , favoring squamous cell carcinoma , " mainly revealed tumor diathesis and some malignant squamoid cells , but not spindle cells , although squamous cell carcinoma and spindle tumor cells were marked in the surgical specimens .
however , there is no clear borderline between giant tumor cells and poorly differentiated large cell carcinoma .
fishback et al.2 reported that the single large pleomorphic nucleus of a gc was 4 times larger than that of small resting lymphocytes .
guillan and zelman17 reported that the gcs varied in diameter from 50 m to 120 m , and hellstrom and fisher18 reported that the gcs measured from 80 m to 100 m .
this vague definition of gcs has caused confusion among pathologists . in our study , the size of gcs varied , ranging from 15 m to 65 m .
the nucleus of mononucleated giant tumor cells measured more than 5 times larger than that of small resting lymphocytes .
although the final diagnosis of pc relies in most cases on the close scrutiny of the hematoxylin and eosin stained sections , its immunohistochemical characterization may better highlight the different cell components occurring in these tumors . in several studies
, the epithelial component has been stained to various extents with antibodies to cytokeratin , whereas the sarcomatous component is consistently immunoreactive for vimentin.2,10,19 - 22 in this current study , all tumors displayed dual expression of cytokeratin and vimentin by immunocytochemistry on the cell blocks .
in contrast , the epithelioid tumor cells showed focal or diffuse positivity for cytokeratin and focally expressed vimentin .
the prognosis for pc is generally thought to be worse than that for conventional nsclc.2,9,10,12,23 - 25 a stage - adjusted comparison of 63 cases of psc with an equivalent number of propensity score - matched cases of nsclcs showed the 5-year survival to be significantly different between the 2 groups ( 24.5% for the psc group and 46.3% for the nsclc group),23 favoring the hypothesis that the prognosis of psc may be poorer than that of nsclc.24 the estimated 5-year survival rate in surgical series was up to 33% for pc ( with the median overall survival ranging from 5 to 19 months).2,9,10,23 - 25 pc has been reported to be highly metastatic .
chang et al.10 reported that 7 patients who had undergone surgical resections succumbed to early distant metastases in organs , such as the brain , bone , adrenal gland , and unusual sites including esophagus , jejunum , rectum , and kidney , within a few months . in the present study ,
metastasis was observed in 3 cases : one patient ( case 2 ) had metastasis in both adrenal glands ; one ( case 5 ) had brain , small intestine , and mesentery metastasis at the time of diagnosis ; and the third ( case 7 ) had metastasis to the brain , bone , lung , and both adrenal glands .
two patients died of the disease , 1 patient survived with metastasis , and 4 patients survived without recurrence or metastasis . in conclusion , recognition of pc may be possible when adequate cytological material is present .
the presence of loose clusters of poorly differentiated epithelial cells , malignant spindle cells , mono- , bi- , multi - nucleated gcs , and a necrotic background with lymphocytes and neutrophils is highly suggestive of pc .
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### Summary:
: backgroundpleomorphic carcinoma ( pc ) is a rare pulmonary malignancy . because of its rarity and histological heterogeneity
, cytopathologists might suspect pc only rarely on the basis of its cytological specimen .
in addition , cytological findings from fine needle aspiration ( fna ) specimens have rarely been described .
hence , we investigated the cytological features of fna in the cases of pc.methodswe reviewed 7 fna specimens of pc .
the patients had undergone surgical resection at the korea cancer center hospital between 2007 and 2011 .
the cytological features of pc were assessed and compared with the histopathological features of the corresponding surgical specimen .
immunocytochemical analysis with cytokeratin and vimentin was performed on the cell blocks.resultsthe tumor cells were either dispersed or arranged in loose aggregates , and generally lacked any glandular or squamous differentiation .
pleomorphic or spindle shape tumor cells were observed , and mono- , bi- , or multi - nucleated giant cells were frequently observed . the background showed necrosis and
contained numerous lymphocytes and neutrophils .
immunocytochemically , the tumor cells were positive for cytokeratin and vimentin.conclusionspc displays characteristic cytological features
. it might therefore be possible to make an accurate diagnosis of pc by assessing the degree of nuclear atypia .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: human cytomegalovirus ( hcmv ) prevalence in the general population is about 70% ( 1 ) . the infection is an important cause of morbidity and mortality in infants and immunocompromised individuals such as solid - organ and hematopoietic stem cell transplant recipients , aids patients and patients taking chemotherapy or medicines with suppressed immune system ( 2 - 7 ) .
the hcmv accounts for major viral complications in the post - transplant period ( 6 ) and is also the most common intrauterine infection that may lead to sensorineural hearing loss and mental retardation ( 8 , 9 ) .
these medicines require close monitoring for side effects and although they can help stop the virus from copying itself within the body yet do not eliminate it from the body ( 10 - 12 ) .
immunotherapy is a promising new treatment for infectious diseases involving both therapeutic vaccines and monoclonal antibodies ( 13 ) .
glycoprotein 55 ( gp55 ) is part of glycoprotein b ( gb ) , which is the most abundant constituent of hcmv envelope and the major target for neutralizing antibodies ( 14 , 15 ) .
several monoclonal antibodies have been generated against gb epitopes and showed some degree of neutralization ( 16 ) .
recombinant antibodies ( rab ) are becoming the preferred therapeutic substitutes to full - length monoclonal antibodies due to their characteristics .
single - chain fragment variable antibodies ( scfv ) , which are composed of variable heavy ( vh ) and variable light ( vl ) domains linked together by a flexible polypeptide linker , are one of the most desired rab formats .
the smaller size , human origin , high affinity and specificity and also better penetration to their targets , are beneficial characteristics of human scfvs in medical applications ( 17 - 20 ) .
this study aimed to select specific single chain antibodies against the conserved sequence of gp55 and to evaluate the virus neutralization effects of the antibodies in cell culture using the phage display technology .
a phage antibody display library of scfv was produced , as described previously ( 16 , 17 ) .
escherichia coli bacteria containing phagemid were cultured overnight on 2tyg agar /ampicillin ( tryptone , yeast extract , glucose , agar and ampicillin ) ( merck , germany ) plates at 30c .
the bacterial pellet was transferred to 2ty broth containing ampicillin ( 100 g / ml ) and kanamycin ( 50 l / ml ) , and incubated with constant shaking , at 30c overnight .
the supernatant was passed through 0.3 m filters and stored at 4c ( 22 ) .
peptides ( lvsadgttvtsgstkdtslq ) at a concentration of 10 g / ml were coated on polystyrene immunotubes ( nunc , denmark ) at 4c overnight .
the tubes were washed four times with phosphate buffered saline ( pbs ) , and 4 ml of blocking solution ( 2% skimmed milk ) was added and incubated at 37c for two hours .
the tubes were washed four times with pbs/ tween and four times with pbs . in the next step
, the diluted phage supernatant in the blocking solution ( 1/1 ) was added to the tubes and incubated at room temperature for one hour with occasional inversions .
the tubes were washed and logarithmic phase e. coli was added , followed by incubation at 37c for one hour and centrifugation at 3500 rpm for five minutes . the supernatant was discarded and the bacterial pellet was re - suspended in 2ty broth ( tryptone , yeast extract ) ( merck , germany ) , plated on 2ty agarose / ampicillin plate and incubated at 30c , overnight .
after the panning process , the inserts of selected clones were amplified by pcr ( 94c for one minute , 55c for one minute and 72c for two minutes , performed in 30 cycles ) .
furthermore , 17 l of the pcr product was mixed with 1 l restriction enzyme ( mva - i ) ( roche applied science , germany ) and 2 l of restriction enzyme buffer .
the mixtures were placed in a dry block heater at 37c for two hours and run on a 2% agarose gel .
peptides ( 100 g / ml ) as an epitope were coated in 96 wells of a polystyrene plate and incubated at 4c overnight . after washing with pbs , 150 l of blocking solution (
2% skimmed milk ) was added to each well and incubated at 37c for two hours .
phage rescue supernatant containing the appropriate scfv , diluted with blocking solution ( 1:1 ) , was added to each well and incubated at room temperature for two hours .
nonbinding phages were removed by washing the wells three times with pbs / tween , and three times with pbs .
anti - fd bacteriophage ( 1:100 ) ( sigma , germany ) was added to each well and incubated at room temperature for 1.5 hours .
the wells were washed three times with pbs / tween and three times with pbs .
horseradish peroxidase ( hrp ) conjugated anti - rabbit antibody ( 1:1000 ) ( sigma , germany ) was added to each well and incubated at room temperature for one hour .
the wells were then washed and 150 l of azino - bis-3-ethylbenzothiazoline-6-sulfonic ( abts ) acid ( sigma - aldrich , germany ) solution ( 10 mg abts , 20 ml citrate buffer ( ph4 ) and 6 ml h2o2 ) was added to each well .
the absorbances were read at 405 nm after 30 minutes using an elisa reader . the wells without peptide , with unrelated peptide ( prostate stem cell antigen peptide ) , unrelated scfv ( scfv to her2 ) and with m13ko7 ,
the hcmv ( ad169 ) was diluted in dulbecco s modified eagle s medium ( dmem ) ( sigma - aldrich , germany ) to 50 pfu / ml .
equal amounts of virus and each scfv phage rescue supernatant were mixed and incubated at 37c for one hour .
the mixtures were added to the wells of 24-well plates containing confluent human fibroblast , which were then rocked gently for 45 minutes at 37c .
the mixtures were removed and 1 ml of 1% w / v carboxy methyl cellulose ( cmc ) in 1x dmem , supplemented with 2% fetal calf serum , 0.14% v / v sodium bicarbonate , 100 u penicillin ml , 100 g streptomycin sulfate ml , 0.25 g amphotericin b ml and 0.1 m sodium hydroxide , was added .
the number of micro plaques were counted after ten days and compared with the number of plaques seen in the virus control wells containing virus in dmem .
a phage antibody display library of scfv was produced , as described previously ( 16 , 17 ) .
escherichia coli bacteria containing phagemid were cultured overnight on 2tyg agar /ampicillin ( tryptone , yeast extract , glucose , agar and ampicillin ) ( merck , germany ) plates at 30c .
the bacterial pellet was transferred to 2ty broth containing ampicillin ( 100 g / ml ) and kanamycin ( 50 l / ml ) , and incubated with constant shaking , at 30c overnight .
the supernatant was passed through 0.3 m filters and stored at 4c ( 22 ) .
peptides ( lvsadgttvtsgstkdtslq ) at a concentration of 10 g / ml were coated on polystyrene immunotubes ( nunc , denmark ) at 4c overnight .
the tubes were washed four times with phosphate buffered saline ( pbs ) , and 4 ml of blocking solution ( 2% skimmed milk ) was added and incubated at 37c for two hours .
the tubes were washed four times with pbs/ tween and four times with pbs . in the next step
, the diluted phage supernatant in the blocking solution ( 1/1 ) was added to the tubes and incubated at room temperature for one hour with occasional inversions .
the tubes were washed and logarithmic phase e. coli was added , followed by incubation at 37c for one hour and centrifugation at 3500 rpm for five minutes . the supernatant was discarded and the bacterial pellet was re - suspended in 2ty broth ( tryptone , yeast extract ) ( merck , germany ) , plated on 2ty agarose / ampicillin plate and incubated at 30c , overnight .
after the panning process , the inserts of selected clones were amplified by pcr ( 94c for one minute , 55c for one minute and 72c for two minutes , performed in 30 cycles ) .
furthermore , 17 l of the pcr product was mixed with 1 l restriction enzyme ( mva - i ) ( roche applied science , germany ) and 2 l of restriction enzyme buffer .
the mixtures were placed in a dry block heater at 37c for two hours and run on a 2% agarose gel .
peptides ( 100 g / ml ) as an epitope were coated in 96 wells of a polystyrene plate and incubated at 4c overnight . after washing with pbs ,
150 l of blocking solution ( 2% skimmed milk ) was added to each well and incubated at 37c for two hours .
phage rescue supernatant containing the appropriate scfv , diluted with blocking solution ( 1:1 ) , was added to each well and incubated at room temperature for two hours .
nonbinding phages were removed by washing the wells three times with pbs / tween , and three times with pbs .
anti - fd bacteriophage ( 1:100 ) ( sigma , germany ) was added to each well and incubated at room temperature for 1.5 hours .
the wells were washed three times with pbs / tween and three times with pbs .
horseradish peroxidase ( hrp ) conjugated anti - rabbit antibody ( 1:1000 ) ( sigma , germany ) was added to each well and incubated at room temperature for one hour .
the wells were then washed and 150 l of azino - bis-3-ethylbenzothiazoline-6-sulfonic ( abts ) acid ( sigma - aldrich , germany ) solution ( 10 mg abts , 20 ml citrate buffer ( ph4 ) and 6 ml h2o2 ) was added to each well . the absorbances were read at 405 nm after 30 minutes using an elisa reader . the wells without peptide , with unrelated peptide ( prostate stem cell antigen peptide ) , unrelated scfv ( scfv to her2 ) and with m13ko7 , were also considered as negative controls ( 22 ) .
the hcmv ( ad169 ) was diluted in dulbecco s modified eagle s medium ( dmem ) ( sigma - aldrich , germany ) to 50 pfu / ml .
equal amounts of virus and each scfv phage rescue supernatant were mixed and incubated at 37c for one hour .
the mixtures were added to the wells of 24-well plates containing confluent human fibroblast , which were then rocked gently for 45 minutes at 37c .
the mixtures were removed and 1 ml of 1% w / v carboxy methyl cellulose ( cmc ) in 1x dmem , supplemented with 2% fetal calf serum , 0.14% v / v sodium bicarbonate , 100 u penicillin ml , 100 g streptomycin sulfate ml , 0.25 g amphotericin b ml and 0.1 m sodium hydroxide , was added .
the number of micro plaques were counted after ten days and compared with the number of plaques seen in the virus control wells containing virus in dmem .
dna fingerprinting of selected clones was performed to determine the common patterns after the panning process . as shown in figure 1 , dna fingerprinting of the selected clones demonstrated pattern 1 ( scfv1 ) , ( lanes : 1 , 2 , 5 , 8 , 13 ) with frequency of 25% , pattern 2 ( scfv 2 ) , ( lanes : 4 , 7 , 11 , 14 ) with frequency of 20% and pattern 3 ( scfv3 ) , ( lanes : 3 , 15 , 17 , 20 ) with frequency of 20% .
dominant patterns ( patterns 1 , 2 and 3 ) were selected for further evaluation .
three predominant patterns were differentiated including pattern 1 ( lanes 1 , 2 , 5 , 8 and 13 ) , pattern 2 ( lanes 4 , 7 , 11 and 14 ) and pattern 3 ( lanes 3 , 15 , 17 and 20 ) .
specific binding of the selected scfv antibodies to the corresponding peptide was determined by phage elisa .
the absorbance of wells coated with the corresponding peptide for the three selected scfvs were significantly higher than that of the wells containing no peptide .
table 1 , shows the percentages of plaque reduction when the virus was incubated with scfv .
as the results indicate only scfv2 efficiently neutralized the virus and a neutralizing effect of 68.8% was obtained .
number of plaques are shown in each experiment . following incubation of virus with scfv2 ,
dna fingerprinting of selected clones was performed to determine the common patterns after the panning process . as shown in figure 1 , dna fingerprinting of the selected clones demonstrated pattern 1 ( scfv1 ) , ( lanes : 1 , 2 , 5 , 8 , 13 ) with frequency of 25% , pattern 2 ( scfv 2 ) , ( lanes : 4 , 7 , 11 , 14 ) with frequency of 20% and pattern 3 ( scfv3 ) , ( lanes : 3 , 15 , 17 , 20 ) with frequency of 20% .
dominant patterns ( patterns 1 , 2 and 3 ) were selected for further evaluation .
three predominant patterns were differentiated including pattern 1 ( lanes 1 , 2 , 5 , 8 and 13 ) , pattern 2 ( lanes 4 , 7 , 11 and 14 ) and pattern 3 ( lanes 3 , 15 , 17 and 20 ) .
specific binding of the selected scfv antibodies to the corresponding peptide was determined by phage elisa .
the absorbance of wells coated with the corresponding peptide for the three selected scfvs were significantly higher than that of the wells containing no peptide .
table 1 , shows the percentages of plaque reduction when the virus was incubated with scfv .
as the results indicate only scfv2 efficiently neutralized the virus and a neutralizing effect of 68.8% was obtained .
number of plaques are shown in each experiment . following incubation of virus with scfv2 ,
glycoprotein b is known as the most abundant constituent of the hcmv envelope and the major target for neutralizing antibodies .
it contains three major neutralizing determinants including ad1 , ad2 and ad3 domains ( 23 - 25 ) .
the hcmv peptides derived from the n - terminal part of gb ( netiynttlkygdv ) and c - terminal part of gb ( vtsgstkd ) in the ad2 region and n - terminal part of gh ( paasealdphafhlllntygr ) have been used to develop recombinant anti - hcmv gb and gh .
this resulted in the isolation of eight neutralizing clones with neutralizing effect of 51% - 63% ( 16 ) .
due to recent reports showing the association of cmv diseases with the lack of antibodies against the ad2 region in renal transplant individuals ( 23 ) . in the present study , a well - known peptide in the ad2 region
was considered as the target epitope and a phage display library of scfvs was applied to select specific antibodies against this epitope . the peptide ( lvsadgttvtsgstkdtslq ) , amino acids 789 - 805 derived from the c - terminal end of the part of gb ( gp55 ) was used as an epitope .
it has been shown that the sequence of ad2 domain is conserved among cmv isolates ( 26 ) , which makes it more attractive for cmv targeted therapy .
the vtsgstkd epitope of ad2 has been reported as a major antigenic region on gp55 with the vtsg part , which is essential for recognition .
it was found that 55% of the anti - hcmv seropositive blood samples recognize the tsgstkd epitope , which is part of the applied peptide . in order to select specific scfv antibodies against the epitope ,
after four rounds of panning against the derived peptide , 20 clones were selected and mva- fingerprinting demonstrated the common patterns .
three specific scfvs were selected with frequencies of 25% ( scfv1 ) , 20% ( scfv2 ) and 20% ( scfv3 ) .
lillo et al . , ( 27 ) selected scfv antibodies against a capsular protein ( f1 antigen ) of yersinia pestis by enrichment of a phage library using the panning process .
krishnaswamy et al . , ( 28 ) identified a specific scfv antibody against a. fumigatus membrane fraction ( amf ) as an antigen by panning a phage antibody .
the results of the phage elisa showed that the panning procedure was successfully performed and the selected clones reacted specifically with the peptide .
the three selected scfvs ( scfv1 , scfv2 , scfv3 ) produced positive elisa with the cmv peptide , while no reactivity was detected for the control peptide , helper phage and no peptide wells as negative controls .
furthermore , the unrelated scfv ( scfv to prostate stem cell antigen peptide ) did nt react with the gp55 peptide .
the absorbances of wells coated with peptide of interest for the three selected scfvs were three folds higher than that of the wells containing no peptide .
the specificity of selected scfvs to the corresponding peptides has been shown in different phage elisa assessments .
thathaisong et al . , ( 29 ) showed that the optical density ( od ) of specific scfvs against influenza - a virus h5n1 subtype at 405 nm was two folds higher than negative controls in a positive phage elisa .
specific single chain antibodies to outer membrane protein d ( ompd ) of salmonella typhimurium ( 30 ) , staphylococcal enterotoxin b ( seb ) ( 31 ) and hepatitis b virus ( hbv ) surface antigen ( 32 ) have also been confirmed by phage elisa .
the scfv2 showed a mean plaque reduction of 68.8% while the mean of plaque reduction for scfv1 and scfv3 were 23.7 and 11.6% , respectively .
although monoclonal antibodies have been produced against human cytomegalovirus gb and gh ( 33 , 34 ) , the application of these antibodies in clinical settings is limited due to human anti - mouse antibody ( hama ) response and most of these antibodies have been used for diagnosis purposes .
new treatment options for cmv are urgently needed because currently available therapies have major limitations .
it has also been reported that the management of solid organ transplant recipients for prevention and treatment of cmv disease is a major strategy since a significant correlation between steroid administration for acute cellular rejection ( acr ) and cmv reactivation and disease has been shown in liver transplant recipients ( 35 ) .
the results suggest that the specific scfv2 with neutralizing effect of 68.8% has the potential to be used for prophylaxis and treatment of cmv infections .
as the epitope used for selection of the specific antibodies is a conserved epitope among different cmv strains , the antibody is able to neutralize different cmv strains and would be helpful for clinical applications .
the specific scfvs 1 and 3 with lower neutralizing effects can be used for diagnosis of cmv - related diseases . a combination of the selected neutralizing scfv in this study with the previously selected neutralizing scfvs and scfv against the n - terminal part of gb with 57% and scfv against gh of cmv with 63% neutralizing effects ( 16 ) , could be an effective alternative cocktail for therapy of at risk patients such as transplant recipients and aids patients .
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### Summary:
: background : immunotherapy is a promising prospective new treatment for cytomegalovirus ( cmv ) infections . neutralizing effects have been reported using monoclonal antibodies .
recombinant single chain antibodies ( scfvs ) due to their advantages over monoclonal antibodies are potential alternatives and provide valuable clinical agents.objectives:the aim of this study was to select specific single chain antibodies against gp55 of cmv and to evaluate their neutralizing effects . in the present study , we selected specific single chain antibodies against glycoprotein 55 ( gp55 ) of cmv for their use in treatment and diagnosis.materials and methods : single chain antibodies specific against an epitope located in the c - terminal part of gp55 were selected from a phage antibody display library . after four rounds of panning ,
twenty clones were amplified by the polymerase chain reaction ( pcr ) and fingerprinted by mvai restriction enzyme .
the reactivities of the specific clones were tested by the enzyme - linked immunosorbent assay ( elisa ) and the neutralizing effects were evaluated by the plaque reduction assay.results:fingerprinting of selected clones revealed three specific single chain antibodies ( scfv1 , scfv2 and scfv3 ) with frequencies 25% , 20 and 20% .
the clones produced positive elisa with the corresponding peptide .
the percentages of plaque reduction for scfv1 , scfv2 and scfv3 were 23.7 , 68.8 and 11.6 , respectively.conclusions:gp55 of human cmv is considered as an important candidate for immunotherapy . in this study , we selected three specific clones against gp55 .
the scfvs reacted only with the corresponding peptide in a positive elisa .
the scfv2 with 68.8% neutralizing effect showed the potential to be considered for prophylaxis and treatment of cmv infections , especially in solid organ transplant recipients , for whom treatment of cmv is urgently needed .
the scfv2 with neutralizing effect of 68.8% , has the potential to be considered for treatment of these patients .
the specific scfv1 and scfv3 with lower neutralizing effects can be used for diagnostic purposes .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: the p38 mitogen - activated protein kinase ( mapk ) signaling pathway mediates multiple cellular events , including proliferation , differentiation , migration , adhesion and apoptosis , in response to various extracellular stimuli , such as growth factors , hormones , ligands for g protein - coupled receptors , inflammatory cytokines , and stresses .
it has been shown that p38 mapk signaling is associated with cancers in humans and mice and regulates gene expression through the activation of transcription factors .
long - term exposure of tumor cells to certain types of chemotherapy drugs causes resistance .
the best example is doxorubicin , an anti - cancer drug that often leads to drug resistance .
recent studies on cell resistance to chemotherapy and refractory epilepsy drugs showed that multidrug resistance ( mdr ) transporters , especially p - glycoprotein ( pgp ) encoded by mdr1 , play an important role in multidrug resistance [ 57 ] .
pgp is a membrane - associated protein with 6 transmembrane domains and an adenosine triphosphate ( atp ) binding site .
this energy - dependent structure provides the characteristics of a drug efflux transporter that can pump drugs and other hydrophobic compounds out of cells , reducing the intracellular drug concentration , thus leading to drug resistance [ 810 ] .
pgp expression can be induced by several factors , including cytotoxic drugs , irradiation , heat shock , and other stresses [ 1113 ] .
these factors also activate the p38 mapk signaling pathway [ 1416 ] , suggesting that the p38 mapk signaling pathway may be involved in the regulation of pgp expression . in this study we investigated the effect of a highly selective , potent , cell - permeable inhibitor of p38 mapk ( sb202190 ) on doxorubicin - induced drug resistance associated with pgp in a leukemia cell line .
we demonstrated that p38 mapk is involved in doxorubicin - induced pgp expression , cell resistance to the antiepileptic drug phenytoin sodium , and the chemotherapy drug , doxorubicin , in leukemia cells .
human leukemia cell line k562 was obtained from the blood institute of the chinese academy of sciences ( tianjin , china ) .
doxorubicin hydrochloride ( dox ) , phenytoin sodium ( pht ) , verapamil hydrochloride ( ver ) , rabbit anti - human pgp antibody ) , sb202190 ( 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1h - imidazole ) , u0126 ( 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio ] butadiene ) , cell counting kit-8 ( cck-8 ) , and dab ( 3,3-diaminobenzidine ) were purchased from sigma ( st . louis , mo ) .
anti - p38 antibodies ( total and phosphor t180 - y182 ) were from santa cruz technologies . to generate a resistant cell line k562/dox , k562 cells were cultured in rpmi-1640 medium supplemented with 15% fetal calf serum ( fcs ) , 100 u / ml penicillin , and 100 g / ml streptomycin at 37c overnight , followed by treatment with 10 g / ml doxorubicin at 37c for 2 hours .
the established cell line was then maintained in fresh complete medium supplemented with 0.1 g / ml doxorubicin .
all k562/dox cells were cultured in the absence of dox for 10 days prior to compound treatment .
the cells were then plated and treated with 10 m phenytoin sodium , 10 m doxorubicin , 10 m sb202190 , 10 m u0126 , or 10 m verapamil in dmso for a period of time as indicated below .
k562 and k562/dox cells were seeded on 0.1% poly - lysine coated slides ( sigma ) and fixed in ice - cold acetone for 10 min . after washing 3 times with pbs ,
the cells were permeated with 0.25% triton x-100 plus 5% dmso in pbs for 10 min .
after washing 3 times with pbs , the cells were treated with 1.5% and 3% hydrogen peroxide for 15 min each to block endogenous peroxidase and peroxidase - like activity . following block with 10% goat serum in pbs for 1 hour , the cells were incubated with specific antibody against human pgp ( 1 : 200 dilution ) at 4c overnight .
after incubation of horseradish peroxidase - labeled goat anti - rabbit secondary antibody ( 1:1000 dilution ) at 37c for 1 hour , the cells were washed with 0.1
the cells were then incubated with 0.05% dab substrate in 0.05 mol / l tris - hcl buffer , followed by incubation of 2 drops of 3% hydrogen peroxide for 5~15 min until cells were coloured light brown .
the reaction was stopped by putting the slides into 0.05 mol / l tris - hcl buffer and air - drying .
one microgram of total rna was reversely transcribed using a reverse transcription kit ( mbi fermentas , burlington , canada ) .
the pcr amplification was carried out in a volume of 25 l using the fermentas kit .
the primers of mdr1 and -actin were synthesized by shanghai saibaisheng company ( shanghai , china ) .
the primer sequences were 5-ttttcatgctataatgcgac-3 ( forward ) and 5-tccaagaacaggactgatgg-3 ( reverse ) for mdr1 ( 226 bp ) and 5-cctcgcctttgccgatcc-3 ( forward ) and 5-ggatcttcatgaggtagtcagtc-3 ( reverse ) for -actin ( 620 bp ) .
pcr amplification was performed at 94c for 45 sec , 54c for 45 sec , and 72c for 1 min for 30 cycles .
an initial step to denature rna at 95c for 2 min and a final extension of 5 min at 72c were also performed .
pcr products were then separated on 1.5% agarose gel and analyzed using a gel imaging system ( genegenius , usa ) .
equal amount protein samples were run on sds - page gels and transferred to polyvinylidene difluoride ( pvdf ) membranes . after blocking with 1% skim milk in tbs - t at room temperature for 1 hour
, the membrane was probed with mouse anti - human primary antibody ( 1:500 dilution ) at 4c overnight and subsequently incubated with goat anti - mouse horseradish peroxidase - conjugated secondary antibody ( 1:2000 dilution ) at room temperature for 2 hours .
signals were detected using ecl - plus ( santa clara , ca ) and quantified using the bio - rad2000 gel imaging system with quantity one software ( bio - rad laboratories , hercules , ca ) .
cell viability and drug resistance was determined by cell counting method using cck-8 assay according to the protocol of the manufacturer .
briefly , cells were pre - treated with 10 m sb202190 or 10 m verapamil for 1 hour and treated with phenytoin or doxorubicin for 48 hours . after adding 10 l of the cck-8 solution to each well of the plate
cell viability was calculated using the data obtained from the wells that contain known numbers of viable cells .
the 50% inhibitory concentration ( ic50 ) of each drug was calculated using a weighted regression of the plot .
reversal index ( ri ) was calculated as ri = ic50 without inhibitor / ic50 with inhibitor .
briefly , cells were pretreated with 10 m sb202190 or 10 m verapamil for 1 hour and treated with phenytoin sodium or doxorubicin for 36 hours at a final concentration of 10 m . cells ( 210 in 2 ml culture medium ) were then collected and re - suspended in 0.3 mol / l hcl/50% ethanol .
after centrifugation at 10,000 rpm for 10 min , the supernatant ( 20 l ) was loaded into the column of hplc for the measurement of the intracellular concentration of drug according to the protocol of the manufacturer .
comparisons between groups were performed using either a paired student t - tests or one - way anova , where indicated .
human leukemia cell line k562 was obtained from the blood institute of the chinese academy of sciences ( tianjin , china ) .
doxorubicin hydrochloride ( dox ) , phenytoin sodium ( pht ) , verapamil hydrochloride ( ver ) , rabbit anti - human pgp antibody ) , sb202190 ( 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1h - imidazole ) , u0126 ( 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio ] butadiene ) , cell counting kit-8 ( cck-8 ) , and dab ( 3,3-diaminobenzidine ) were purchased from sigma ( st . louis , mo ) .
anti - p38 antibodies ( total and phosphor t180 - y182 ) were from santa cruz technologies .
to generate a resistant cell line k562/dox , k562 cells were cultured in rpmi-1640 medium supplemented with 15% fetal calf serum ( fcs ) , 100 u / ml penicillin , and 100 g / ml streptomycin at 37c overnight , followed by treatment with 10 g / ml doxorubicin at 37c for 2 hours .
the established cell line was then maintained in fresh complete medium supplemented with 0.1 g / ml doxorubicin .
all k562/dox cells were cultured in the absence of dox for 10 days prior to compound treatment .
the cells were then plated and treated with 10 m phenytoin sodium , 10 m doxorubicin , 10 m sb202190 , 10 m u0126 , or 10 m verapamil in dmso for a period of time as indicated below .
k562 and k562/dox cells were seeded on 0.1% poly - lysine coated slides ( sigma ) and fixed in ice - cold acetone for 10 min . after washing 3 times with pbs ,
the cells were permeated with 0.25% triton x-100 plus 5% dmso in pbs for 10 min .
after washing 3 times with pbs , the cells were treated with 1.5% and 3% hydrogen peroxide for 15 min each to block endogenous peroxidase and peroxidase - like activity . following block with 10% goat serum in pbs for 1 hour
, the cells were incubated with specific antibody against human pgp ( 1 : 200 dilution ) at 4c overnight .
after incubation of horseradish peroxidase - labeled goat anti - rabbit secondary antibody ( 1:1000 dilution ) at 37c for 1 hour , the cells were washed with 0.1
the cells were then incubated with 0.05% dab substrate in 0.05 mol / l tris - hcl buffer , followed by incubation of 2 drops of 3% hydrogen peroxide for 5~15 min until cells were coloured light brown .
the reaction was stopped by putting the slides into 0.05 mol / l tris - hcl buffer and air - drying . after mounting ,
one microgram of total rna was reversely transcribed using a reverse transcription kit ( mbi fermentas , burlington , canada ) .
the pcr amplification was carried out in a volume of 25 l using the fermentas kit .
the primers of mdr1 and -actin were synthesized by shanghai saibaisheng company ( shanghai , china ) .
the primer sequences were 5-ttttcatgctataatgcgac-3 ( forward ) and 5-tccaagaacaggactgatgg-3 ( reverse ) for mdr1 ( 226 bp ) and 5-cctcgcctttgccgatcc-3 ( forward ) and 5-ggatcttcatgaggtagtcagtc-3 ( reverse ) for -actin ( 620 bp ) .
pcr amplification was performed at 94c for 45 sec , 54c for 45 sec , and 72c for 1 min for 30 cycles .
an initial step to denature rna at 95c for 2 min and a final extension of 5 min at 72c were also performed .
pcr products were then separated on 1.5% agarose gel and analyzed using a gel imaging system ( genegenius , usa ) .
equal amount protein samples were run on sds - page gels and transferred to polyvinylidene difluoride ( pvdf ) membranes . after blocking with 1% skim milk in tbs - t at room temperature for 1 hour , the membrane was probed with mouse anti - human primary antibody ( 1:500 dilution ) at 4c overnight and subsequently incubated with goat anti - mouse horseradish peroxidase - conjugated secondary antibody ( 1:2000 dilution ) at room temperature for 2 hours .
signals were detected using ecl - plus ( santa clara , ca ) and quantified using the bio - rad2000 gel imaging system with quantity one software ( bio - rad laboratories , hercules , ca ) .
cell viability and drug resistance was determined by cell counting method using cck-8 assay according to the protocol of the manufacturer .
briefly , cells were pre - treated with 10 m sb202190 or 10 m verapamil for 1 hour and treated with phenytoin or doxorubicin for 48 hours . after adding 10 l of the cck-8 solution to each well of the plate
cell viability was calculated using the data obtained from the wells that contain known numbers of viable cells .
the 50% inhibitory concentration ( ic50 ) of each drug was calculated using a weighted regression of the plot .
reversal index ( ri ) was calculated as ri = ic50 without inhibitor / ic50 with inhibitor .
briefly , cells were pretreated with 10 m sb202190 or 10 m verapamil for 1 hour and treated with phenytoin sodium or doxorubicin for 36 hours at a final concentration of 10 m . cells ( 210 in 2 ml culture medium ) were then collected and re - suspended in 0.3 mol / l hcl/50% ethanol .
after centrifugation at 10,000 rpm for 10 min , the supernatant ( 20 l ) was loaded into the column of hplc for the measurement of the intracellular concentration of drug according to the protocol of the manufacturer .
comparisons between groups were performed using either a paired student t - tests or one - way anova , where indicated .
k562/dox cells were generated by repeating treatments of doxorubicin and confirmed by the induction of pgp expression .
immunocytochemistry showed that wild - type k562 cells had an undetectable level of pgp expression ( figure 1a , left panel ) , whereas most k562/dox cells were pgp - positive and appeared light - brown ( figure 1a , right panel ) , indicating the induction of pgp expression by doxorubicin .
this drug - resistant cell line was further confirmed by the detection of multi - drug resistance 1 gene , mdr1 , in the absence or presence of a p38 inhibitor , sb202190 ( figure 1b ) .
after quantitative analysis of rt - pcr , we found that sb202190 treatment significantly decreased mdr1 expression in k562/dox cells ( figure 1c ; p<0.001 ; n=10 ) .
however , the treatment of u0126 , a highly selective inhibitor of both mek1 and mek2 , had no effect on mdr1 expression .
sb202190 treatment for 48 hours decreased pgp expression , whereas u0126 treatment had no effect ( figure 2a ) .
after quantitative analysis , we found that p38 inhibitor significantly reduced the expression of pgp ( figure 2b ) .
next , we confirmed that sb202190 indeed significantly suppressed phopho - p38 , an active form of p38 , as well as total - p38 in k562/dox cells ( figure 2c , d ) .
first , we examined cell viability by pretreating the cells with p38 inhibitor sb202190 and a positive control , verapamil , followed by treating the cells with phenytoin sodium or doxorubicin .
the inhibition of cell viabilities by phenytoin sodium and doxorubicin was determined by cck-8 assay .
we found that in the presence of sb202190 , phenytoin sodium and doxorubicin significantly decreased the number of living cells ( figure 3a , b ) .
studies confirmed that treatment of 10 m sb202190 or verapamil alone had no effect on cell viability in either k562 or k562/dox cells ( data not shown ) .
the ic50 of phenytoin and doxorubicin in k562/dox cells was significantly higher than that in k562 cells ( 2186.33214.70 vs. 468.8244.67 g / ml and 4.330.50 vs. 0.320.05 g / ml , respectively ) ( table 1 ) , indicating that k562/dox cells were drug - resistant .
after blocking p38 with 10 m sb202190 in k562/dox cells , we observed that the ic50 of phenytoin was significantly decreased , from 2186.33214.70 to 949.83131.31 g / ml , with an ri of 2.30 , similar to that of the verapamil control ( 2.56 ) .
the ic50 of doxorubicin was also lower in cells treated with 10 m sb202190 than in untreated k562/dox cells ( 4.330.50 g / ml and 0.400.09 g / ml , respectively ) , with an ri of 10.83 , similar to that of verapamil ( 12.37 ) .
the intracellular levels of phenytoin and doxorubicin were significantly lower in k562/dox cells than those in k562 cells ( figure 4a , b ) , further confirming the drug resistance of k562/dox cells .
the decrease of the intracellular level of phenytoin and doxorubicin in k562/dox cells was significantly abolished in the presence of sb202190 ( figure 4 ) .
these data clearly demonstrate that p38 is , at least in part , involved in the regulation of drug resistance in k562/dox cells .
k562/dox cells were generated by repeating treatments of doxorubicin and confirmed by the induction of pgp expression .
immunocytochemistry showed that wild - type k562 cells had an undetectable level of pgp expression ( figure 1a , left panel ) , whereas most k562/dox cells were pgp - positive and appeared light - brown ( figure 1a , right panel ) , indicating the induction of pgp expression by doxorubicin .
this drug - resistant cell line was further confirmed by the detection of multi - drug resistance 1 gene , mdr1 , in the absence or presence of a p38 inhibitor , sb202190 ( figure 1b ) .
after quantitative analysis of rt - pcr , we found that sb202190 treatment significantly decreased mdr1 expression in k562/dox cells ( figure 1c ; p<0.001 ; n=10 ) .
however , the treatment of u0126 , a highly selective inhibitor of both mek1 and mek2 , had no effect on mdr1 expression .
sb202190 treatment for 48 hours decreased pgp expression , whereas u0126 treatment had no effect ( figure 2a ) .
after quantitative analysis , we found that p38 inhibitor significantly reduced the expression of pgp ( figure 2b ) .
next , we confirmed that sb202190 indeed significantly suppressed phopho - p38 , an active form of p38 , as well as total - p38 in k562/dox cells ( figure 2c , d ) .
first , we examined cell viability by pretreating the cells with p38 inhibitor sb202190 and a positive control , verapamil , followed by treating the cells with phenytoin sodium or doxorubicin .
the inhibition of cell viabilities by phenytoin sodium and doxorubicin was determined by cck-8 assay .
we found that in the presence of sb202190 , phenytoin sodium and doxorubicin significantly decreased the number of living cells ( figure 3a , b ) .
studies confirmed that treatment of 10 m sb202190 or verapamil alone had no effect on cell viability in either k562 or k562/dox cells ( data not shown ) .
the ic50 of phenytoin and doxorubicin in k562/dox cells was significantly higher than that in k562 cells ( 2186.33214.70 vs. 468.8244.67 g / ml and 4.330.50 vs. 0.320.05 g / ml , respectively ) ( table 1 ) , indicating that k562/dox cells were drug - resistant . after blocking p38 with 10 m sb202190 in k562/dox cells
, we observed that the ic50 of phenytoin was significantly decreased , from 2186.33214.70 to 949.83131.31 g / ml , with an ri of 2.30 , similar to that of the verapamil control ( 2.56 ) .
the ic50 of doxorubicin was also lower in cells treated with 10 m sb202190 than in untreated k562/dox cells ( 4.330.50 g / ml and 0.400.09 g / ml , respectively ) , with an ri of 10.83 , similar to that of verapamil ( 12.37 ) .
the intracellular levels of phenytoin and doxorubicin were significantly lower in k562/dox cells than those in k562 cells ( figure 4a , b ) , further confirming the drug resistance of k562/dox cells .
the decrease of the intracellular level of phenytoin and doxorubicin in k562/dox cells was significantly abolished in the presence of sb202190 ( figure 4 ) .
these data clearly demonstrate that p38 is , at least in part , involved in the regulation of drug resistance in k562/dox cells .
previous studies have shown that the multidrug transporter pgp is involved in cell resistance to chemotherapy and refractory epilepsy .
, we demonstrated that the p38 mapk signaling pathway is involved in doxorubicin - induced drug resistance associated with pgp regulation , and a p38 inhibitor may serve as a pgp antagonist .
pgp is a transmembrane glycoprotein , functioning as a drug transport that actively pumps out a variety of anti - cancer agents and other hydrophobic compounds from the cells , thus reducing intracellular drug concentrations and leading to drug resistance .
it has been shown that long - term exposure of tumor cells to some types of chemotherapy drugs causes resistance .
this is consistent with results of our current study that drug resistance associated with pgp expression can be induced by repeating treatment of doxorubicin .
pgp - overexpressing k562/dox cells allow us to study the effect of p38 inhibitor on drug resistance .
the expression of mdr genes and multidrug transporters , such as pgp , are regulated by many factors , including cytotoxic drugs and stresses [ 1113 ] ; these factors also activate the p38 mapk pathway . both pgp and p38 mapk
are involved in cellular processes ( eg , apoptosis and cell proliferation ) , indicating that there may be a link between p38 mapk and pgp . we and others demonstrated that inhibition of p38 by sb202190 can decrease the expression of pgp and mdr1 , a gene that encodes pgp , in k562/dox ( current study ) and gastric cancer cells , suggesting that p38 mapk signaling is involved in the regulation of pgp .
u0126 , a highly selective inhibitor of mitogen - activated protein kinase / extracellular signal - regulated kinase ( erk ) kinase ( mek ) , can reduce the endogenous expression levels of pgp in the human colorectal cancer cells , hct-15 and sw620 - 14 , and functionally antagonize ap-1 transcriptional activity through noncompetitive inhibition of mek1/2 .
however , in this study we found that the expression of pgp in k562/dox cells was not affected by u0126 , indicating that the mek1/2 signaling pathway is not a major pathway involved in pgp regulation in leukemia cells , and that the effect of u0126 may be cell - type specific . in order to restore the sensitivity of phenytoin and doxorubicin in resistant cells , we applied sb202190 , a p38 mapk - specific antagonist , which leads to the specificity of the p38 mapk pathway on pgp regulation .
k562/dox cells that highly expressed pgp were resistant to doxorubicin and phenytoin sodium , and in the presence of sb202190 these cells reverse their drug resistance to a degree similar to that of the well - known pgp antagonist , verapamil .
this result further confirms that the p38 mapk pathway is involved in multidrug resistance through the regulation of pgp .
our data are consistent with the previous finding that in an acidic environment , the p38 mapk pathway mediated the upregulation of pgp in rat prostate cancer cells .
it has been reported that there are nf-b binding sites in the mdr1 promoter region , suggesting that mdr1 may be activated by nf-b .
p38 mapk can activate nf-b expression , indicating the possibility of that p38 mapk may regulate pgp expression through the activation of this transcription factor .
our study shows that the p38 signaling pathway is involved in doxorubicin - induced drug resistance .
the inhibition of p38 mapk diminishes doxorubicin - induced drug resistance associated with the down - regulation of pgp .
thus , the inhibitors of p38 may provide new chemotherapeutic option to overcome drug resistance in treatment of cancer and epilepsy .
further studies on the mechanisms of p38 inhibitors and the development of effective pgp - specific antagonists with low toxicity will improve the clinical effects of the chemotherapy and anti - epilepsy therapy .
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### Summary:
: summarybackgroundseveral studies have shown that multidrug transporters , such as p - glycoprotein ( pgp ) , are involved in cell resistance to chemotherapy and refractory epilepsy . the p38 mitogen - activated protein kinase ( mapk ) signaling pathway may increase pgp activity .
however , p38-mediated drug resistance associated with pgp is unclear . here ,
we investigated p38-mediated doxorubicin - induced drug resistance in human leukemia k562 cells.material/methodsthe expression of pgp was detected by rt - pcr , western blot , and immunocytochemistry .
cell viability and half - inhibitory concentrations ( ic50 ) were determined by cck-8 assay .
the intracellular concentration of drugs was measured by hplc.resultsa doxorubicin - induced pgp overexpression cell line , k562/dox , was generated .
the p38 inhibitor sb202190 significantly decreased mdr1 mrna expression , as well as pgp , in k562/dox cells .
the ic50 of phenytoin sodium and doxorubicin in k562/dox cells was significantly higher than that in wild - type k562 cells , indicating the drug resistance of k562/dox cells . during the blocking of p38 activity in the presence of sb202190 , cell number
was significantly reduced after the phenytoin sodium and doxorubicin treatment , and the ic50 of phenytoin sodium and doxorubicin was decreased in k562/dox cells .
hplc showed that the intracellular levels of phenytoin sodium and doxorubicin were significantly lower in k562/dox cells than those in k562 cells .
the decrease of the intracellular level of these drugs was significantly abolished in the presence of sb202190.conclusionsour study demonstrated that p38 is , at least in part , involved in doxorubicin - induced drug resistance .
the mechanistic study of mapk - mediated pgp and the action of sb202190 need further investigation .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: the basic principle of radiotherapy is to deliver higher and uniform dose to tumour and to reduce dose to organ at risks ( oars ) as low as possible which reduces the morbidity and in turn improves the quality of life of patients .
although the conventional techniques provide better tumor coverage , there is some limitation in achieving oars tolerance dose . to overcome
this difficulty , modern delivery techniques such as intensity - modulated radiotherapy ( imrt ) and rapid arc have been used widely .
these techniques can deliver a higher dose to tumor and limit the dose to oars .
most of the articles contain conformity index , coverage index , homogeneity index , and dose gradient ( dg ) index for plan evaluation .
an ideal plan is defined as one with full uniform dose coverage ; exact conformed to the target and step - wise fall - off dose outside the target .
introduced one more approach called unified dosimetry index ( udi ) that computes an overall score which integrates contribution from all four dosimetry components mentioned above .
the udi score of each plan can be ranked and selected the least scored plan as a better plan .
this study utilized udi score for the comparison of imrt and rapid arc plans of various sites and also studied the impact of conformity index on udi - based plan evaluation .
dose coverage is defined as 100% of the planning target volume ( ptv ) receiving the prescribed dose .
it gives a measure of how well the ptv is covered by the prescribed dose .
dose conformity , on the other hand , is defined as the ratio of the total volume of all tissues receiving prescribed dose versus the ptv .
dose conformity gives a measure of how well the prescribed dose is confined to the ptv .
dg is defined as the ratio of the volume receiving the prescribed dose and the volume receiving half the prescribed dose .
homogeneity index ( hi ) is defined as the ratio of the maximum dose at any point 2 mm beyond the ptv ( dmax ) to the prescribed dose .
the mathematical logic - based udi formula is : where dik is dosimetry index of each index of the four indices and wk denotes weighting factors that reflect the relative importance of the four components . for ideal plan ,
unified index ( udi ) is equal to 1 : udi = udi ( c ) udi ( cf ) udi ( hi ) udi ( dg ) = 1.0 ( 2 ) where c - coverage index ( di1 ) , cf - conformity index ( di2 ) , hi ( di3 ) and dg - gradient index ( di4 ) .
used the following equations to calculate all indices and explained full detail about udi and method of calculation .
coverage index ( c ) = ptvpi / ptv ( 3 ) conformity index ( cf ) = dvpi / ptv
( 4 ) hi = dmax / dpi ( 5 ) dg index ( dg ) = dvpi / dvhpi ( 6 ) where ptv is the ptv ; ptvpi = ptv receiving the prescribed isodose ( pi ) ; dvpi = dose volume of the pi ; dvhpi = dose volume of the half the prescribed dose ; dmax = maximum dose at any point 2 mm beyond the ptv ; dpi = dose value of the pi . using this approach , we compared two different techniques imrt and rapid arc .
imrt and rapid arc plans were generated by inverse planning method using eclipse treatment planning system ( v-10.0.39 , varian medical systems , usa ) .
all plans use 6mv photon energy and optimized by assigning the target and oar goals .
the udi scoring values of all plans were ranked and lesser value scored plan considered as a better plan for the patient . for determining the impact of conformity index in the comparison of imrt and rapid arc plan ,
a udi score of the plans was calculated by assuming confidence interval = 1 udi at conformity index one of all plan ( udiunit_ci ) , and it was compared against the udi score calculated with actual conformity index . mean and standard deviations of all indices were calculated , and statistical analysis was performed using spss ( version 16.0.0 , spss , chicago , usa ) .
table 1 shows an overview of the mean and standard deviations of dg index , hi , coverage index , conformity index , udi score and udiunit_ci score for imrt and rapid arc techniques planned for each site of the patients .
table 2 shows the mean and standard deviations of the different indices for all 57 patients compared .
mean and standard deviations of dose gradient index , homogeneity index , coverage index conformity index , unified dosimetry index score and unified dosimetry index at conformity index one of all plans score of each treatment site mean and standard deviations of dose gradient index , homogeneity index , coverage index conformity index , unified dosimetry index score , and unified dosimetry index at conformity index one of all plans score of all patients it is observed that the conformity index and udi of rapid arc plan were significantly better than imrt plan . the average dg index , hi , coverage index and conformity index of all sites with rapid arc plans were 0.212 0.05 , 1.123 0.03 , 0.960 0.03 , and 1.056 0.09 ; with imrt plans were 0.190 0.05 , 1.113 0.04 , 0.950 0.04 , and 1.172 0.16 , respectively .
figures 13 show the radar graph of udi scores from rapid arc and imrt plans of various treatment sites . in the figures ,
the lowest score denotes the minimum deviation from an ideal dosimetry plan ( toward to one ) , and highest score denotes the maximum deviation from an ideal dosimetry plan ( outward from one ) .
the radar graph shows that rapid arc plans are better than imrt plans for all treatment sites . the unified dosimetry index score of each patient 's rapid arc and intensity - modulated radiotherapy plans of ca.prostate . the unified dosimetry index score of each patient 's rapid arc and intensity - modulated radiotherapy plans of ca.cervix and endometrium
the unified dosimetry index score of each patient 's rapid arc and intensity - modulated radiotherapy plans of ca.lower third oesophagus .
the method of udi score - based plan evaluation and comparison of different techniques plans can be useful for establishing a benchmark .
although many numbers of indices have been proposed for plan evaluation , it is difficult to understand as to which system is better or useful .
the radiation therapy oncology group has suggested that a quality treatment plan has to evaluate using three separate indices of dose coverage , conformity , and homogeneity .
the udi used here incorporates all four dosimetry indices into a single overall score . in this study , the plans of similar techniques were analyzed and compared to find a better plan for patient treatment using this udi score .
the conformity index can be reduced while selecting lower isodose level as the reference , and therefore increasing the volume of reference isodose .
therefore , in the evaluation of different techniques , this index would help to take clinical decision .
it is observed that the conformity index was significantly higher with rapid arc plan than imrt ( p < 0.001 ) .
while considered the conformity index as unit , the udiunit_ci of both plans did not differ significantly ( p = 0.527 ) .
however , the udi score values of both planning techniques were calculated with actual conformity index was better with rapid arc than imrt significantly ( p < 0.001 ) . subsequent effect of better conformity index , better dg index could be achieved with rapid arc plans . therefore , critical structure dose ( especially prescribed dose region )
was controlled with rapid arc plan significantly than imrt plan ( p < 0.011 ) .
the dose - volume histogram values of different critical structures in the treatment of different sites were controlled significantly with rapid arc plans than imrt . from figures 13 , it is noted that the udi score spikes for few plans . this result was observed in the bulky patients and whose tumor size is relatively large .
the conformity index was relatively lesser due to the higher dose spillage outside the target .
for all plans studied , the coverage index , hi did not differ significantly between both imrt and rapid arc .
therefore , it can be concluded that the higher degree of confined dose delivery of rapid arc technique provides better treatment plans when compared with imrt .
for all compared treatment sites in this study , rapid arc plans scored better udi value as well as better oars sparing . in the comparison of imrt and rapid arc plans using the udi score ,
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### Summary:
: the aim of this study was to evaluate the impact of conformity index in the unified dosimetry index ( udi ) score for two different planning techniques namely intensity - modulated radiotherapy ( imrt ) and rapid arc .
rapid arc and imrt plans of 57 patients were evaluated and compared using udi score which incorporates four indices . to determine the impact of conformity index on the imrt and rapid arc plans , udi at conformity index one of all plan ( udiunit_ci ) score
was calculated by assuming conformity index is equal to one .
mean and standard deviations of all indices were calculated .
rapid arc technique plans of different treatment sites of all patients scored lesser udi than imrt plans , and the conformity index of rapid arc plan was significantly better than imrt plan .
the average dose gradient , homogeneity , coverage , and conformity index of all sites with rapid arc plans were 0.212 0.05 , 1.123 0.03 , 0.959 0.03 , and 1.056 0.09 ; with imrt plans were 0.190 0.05 , 1.113 0.04 , 0.950 0.04 , and 1.172 0.16 , respectively .
udi score value with actual conformity index of rapid arc and imrt plans differed significantly ( p < 0.001 ) .
however , udiunit_ci score values with assumed conformity index equal to one did not differ significantly ( p = 0.528 ) . in the comparison of imrt and rapid arc plans using the udi score ,
the impact of conformity index was significant .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: imaging is widely used in scientific and technological applications because of the interface it provides between vision and intuition . in particular ,
biological imaging is a rapidly growing field , not only in fundamental biology but also in medical science.1 recently , biomedical imaging has received enormous attention in view of its capacity to aid analysis and diagnosis through images at the molecular and cellular levels.2 as a result , a new discipline , known as molecular imaging ( mi ) has emerged , which combines molecular biology and in vivo imaging.3 the aim of mi is to monitor and measure biological processes in living subjects via spectral data .
the measurement and monitoring of biological processes provides information similar to that from a biopsy , but it is noninvasive and performed in real time , thereby offering the possibility of sequential and longitudinal monitoring .
the use of mi techniques permits molecular changes associated with the onset and development of pathologic states to be quantified , and the approach can provide early diagnosis and prognosis of diseases such as cancer .
other applications include the evaluation of the response to therapy , and the study of biological processes in living subjects.46 traditional mi modalities include x - ray computed tomography ( ct ) , optical imaging ( oi ) , positron emission tomography ( pet ) , single - photon emission ct ( spect ) , ultrasound , and magnetic resonance imaging ( mri).7,8 several promising new imaging modalities , such as fluorescence - mediated tomography and photoacoustic tomography , are currently under development.9 each of these diagnostic modalities has its advantages and disadvantages .
for example , mri and ct have high spatial resolution and are able to provide detailed anatomical information , but they lack sensitivity . in contrast ,
pet and spect are highly sensitive , but have limited resolution and can not provide anatomical information . through the development of highly specialized and efficient contrast agents , mri has evolved into a versatile technique with multiple functions and has become one of the most powerful noninvasive imaging tools in the biomedical toolbox .
high resolution and excellent soft tissue contrast are its main advantages over other in vivo imaging techniques .
mri relies on large magnetic fields and radio frequencies ( rfs ) , and makes use of the relaxation times of protons in mobile molecules such as water , lipids , and proteins that are present in organs at different concentrations , to produce high - resolution soft tissue anatomical images with good endogenous contrast.10 in the following sections , we review many of the most innovative approaches that have been adopted in the recent history of mri contrast agents based on nanoparticles ; mainly on superparamagnetic iron oxide nanoparticles .
in this mini - review , we also include polynuclear or particulate contrast agents that are the result of progression from previous ionic agents .
this is the case of the chelates of gadolinium and manganese oxide that were developed from experience with previous gd- and mn - based agents respectively .
in contrast , we have excluded from this review diamagnetic chemical exchange saturation transfer ( cest ) and paramagnetic cest ( paracest ) agents , although they can be included in nanoparticulate systems such as liposomes and polymers.1113 the use of nanoparticles as imaging probes has several advantages over conventional imaging agents .
loadability is one of the advantages where the concentration of the imaging agent can be controlled within each nanoparticle during the synthesis process .
another advantage is the tunability of the surface of the nanoparticles that can potentially extend the circulation time of the agent in the blood or target a specific location within the body .
finally , nanoparticles can act as multifunctional mi agents , since they have two or more properties that can be used simultaneously in multiple imaging techniques , and especially in mri.14
when a strong magnetic field is applied to a sample ( in clinical diagnosis , magnetic fields of 1.5 or 3 t are usually used ) , the magnetic field aligns the magnetic moments of protons in the sample , producing an equilibrium magnetization along the longitudinal axis . a rf pulse , at a resonant frequency ( 5100 mhz ) capable of transferring energy to protons ,
can then rotate their magnetic moments away from the longitudinal axis , in phase , to an angle called the flip angle . upon removal of the radiation , the magnetic moments of the protons
relax to equilibrium.15 in mri , this process is repeated in a quick succession of rf pulses . the time taken by the magnetic moments to return to their original alignment with the magnetic field
this relaxation can be divided into two different , independent processes : 1 ) longitudinal relaxation ( characterized by the parameter t1 ) and 2 ) transverse relaxation ( characterized by the parameter t2 ) .
t1 , called the spin lattice relaxation time , relates to how fast the magnetization parallel to the static magnetic field recovers after a perturbation is applied to the system .
protons that relax rapidly ( short t1 ) recover full magnetization along the longitudinal axis quickly and produce high signal intensities . for protons that relax more slowly ( long t1 ) , full magnetization along the longitudinal axis is not recovered before subsequent rf pulses , and so they inherently produce a lower intensity signal.15 t2 relates to how rapidly the magnetization in the plane perpendicular to the static magnetic field loses coherence . during an rf pulse , proton nuclei spin in phase with each other , whereas after the pulse , the magnetic fields of all the nuclei interact with each other , and energy is exchanged between them . as a consequence , the nuclei lose their phase coherence and tend to spin in a random fashion.16 because t2 decay is the result of the exchange of energy between spinning protons , it is referred to as spin spin relaxation .
longitudinal and transverse relaxation processes are executed independently and simultaneously , although t2 is usually much shorter than t1 , and this difference allows tissues to be differentiated.17 in most cases , the combination of the intrinsic molecular interactions of neighboring molecules and extrinsic magnetic field inhomogeneities means that the observed transverse relaxation time ( t2 * ) is even shorter than the natural t2 that would be caused by pure spin
spin interactions . to eliminate external magnetic field effects and generate the real t2-weighted images based purely on molecular interactions , a spin
this uses 90 pulses to excite the magnetization and one or more 180 pulses to refocus the spins and generate signal echoes named spin echoes.15 the endogenous mri contrast in soft tissue comes from local differences in the proton density ( water concentration ) resulting in different values of t1 and t2 . on this basis , endogenous contrast depends on the chemical and physical nature of the tissues.18 despite the relatively high quality of such images of soft tissues , in some cases there is not enough image contrast to diagnose the pathology of interest .
in these circumstances , the low endogenous sensitivity can be enhanced by increasing the magnetic field ( from 3 to 7 t and beyond ) , acquiring data for longer or designing more sensitive sequences and probes .
they can be small mononuclear or polynuclear paramagnetic chelates ; metalloporphyrins ; polymeric or macromolecular carriers of covalently or noncovalently bonded paramagnetic chelates ; particulate contrast agents ( including fluorinated or nonfluorinated paramagnetic micelles or liposomes ) and paramagnetic or superparamagnetic particles ( eg , iron oxides and gd - labeled zeolites ) ; diamagnetic cest polymers ; diamagnetic hyperpolarization probes ( gases and aerosols ) ; and c - labeled compounds or ions ( eg , 6 li).19 the main role of t1 and t2 contrast agents in mri is to shorten selectively the relaxation times of water protons in the region of interest and thus to provide better contrast for anatomical regions .
contrast is enhanced when one tissue has either higher affinity for the contrast agents or higher vascularity than another .
diseased tissues , such as tumors , are metabolically different from healthy tissues and have taken up the contrast agent in different ways , resulting in a contrast in mri images.19,20
t1-weighted images illustrate anatomy well and are preferred when a clear image of the structures is required .
t2-weighted images produce good pathological information since collections of abnormal fluid appear brighter than the normal tissue background .
although nearly all mri contrast agents affect both t1 and t2 , the effects of contrast agents are usually more pronounced for either t1 or t2 , leading to their categorization as either t1 or t2 contrast agents .
contrast enhancement is measured by the relaxation rate ri = 1/ti ( s ) , where i = 1 or 2 .
the most important parameter for defining the efficiency of a contrast agent is its relaxivity , ri ( ri = ri / cca(mm s ) , where cca is the analytical concentration of ion responsible for the contrast ) .
the r2/r1 ratio is also used to indicate the contrast efficiency ; the higher the ratio , the greater the efficiency of a t2 contrast agent and vice versa for a t1 contrast agent ( figure 1 ) .
the relaxivity for an mri contrast agent is defined as the increase in the relaxation rate of the solvent ( water ) induced by 1 mmoll of the active ion of the contrast agent , and it is calculated according to
ri , obs=[1ti , obs1ti , water]/cca(1 ) the relaxivity is dependent on the magnetic field applied and the temperature , so it should be reported together with both these parameters .
most t1 contrast agents currently available are paramagnetic complexes , while those classed as t2 contrast agents are mostly superparamagnetic iron oxides.18
the first generation of exogenous t1 contrast agents ( also called positive contrast agents ) consisted of high - spin paramagnetic metal ions such as manganese ( mn ) , iron ( fe ) , or gadolinium ( gd).21 these contrast agents produce hyperintense signals in t1-weighted images . to obtain significant changes in proton relaxation and therefore a good contrast ,
the paramagnetic ion needs to be in close contact with the protons of the surrounding water molecules .
however , owing to the toxicity associated with these cations ( eg , transient destruction of professional macrophages , exchange with endogenous calcium ions , etc ) , they are used after complexation with low - molecular - weight chelating molecules with no explicit core and surface coating .
it possesses an electron spin of 7/2 and hence seven unpaired electrons promoting spin relaxation due to flipping spins and rotational motion .
free gd ions are cytotoxic and are retained in liver , spleen , and bone.22 to avoid this toxicity , a chelating process is applied to gd , in which large organic molecules form a stable complex around the gd .
examples of chelating compounds are diethylene - triamine - pentaacetic acid ( dtpa ) , 1,4,7,10-tetraazacyclo - dodecane-1,4,7,10-tetraacetic acid ( dota ) , and dipyridoxyl - di - phosphate ( dpdp).19 the chemical structures of such t1 agents are typically characterized by neutral or anionic metal complexes of the type [ m(h2o)(l ) ] or [ m(h2o ) ( l ) ] , where m is the paramagnetic gd or mn ion , and l a macrocyclic or acyclic polyaminopolycarboxylate.23 different types of gd - containing contrast agents have been approved by the european medicines agency ( emea ) and the us food and drug administration ( fda ) ( table 1 ) for use in mri as a contrast agent to provide improved images of organs and tissues . however , although gd is regarded as safe when administered as a chelated compound , the use of some gd chelates in people with renal disease has been linked to a rare but severe complication ; the medical condition referred to as nephrogenic systemic fibrosis .
for this reason , the world health organization ( who ) issued a restriction on the use of gd contrast agents , informing that these compounds were contraindicated in patients with chronic severe renal insufficiency , in those with acute renal insufficiency of any severity due to hepatorenal syndrome , or in the perioperative liver transplantation period , and in newborn babies up to 4 weeks of age.24 because of their low molecular weight , conventional gd - based contrast agents are mostly nonspecific extracellular contrast agents and exhibit rapid extravasation from the vascular space .
in this way , after being intravenously injected , these agents rapidly leak from the blood pool into the interstitium with a distribution half - life ( t1/2 ) of about 5 min .
they are mainly cleared by the kidneys with an elimination t1/2 of about 80 min.19 this limitation , which is inherent to mri , is known as the partial volume dilution effect , and involves a loss of apparent activity in small objects or regions because of the limited resolution of the imaging system .
the partial volume dilution effect has often led to the failure of targeted contrast in vivo.25 extracellular agents are typically gd chelates of linear or macrocyclic polyaminocarboxylate ligands , and constitute the most important class of mri contrast agents available .
initial attempts to target mri focused on coupling gd atoms directly to antibodies or proteins , but these approaches delivered insufficient paramagnetic material to effectively decrease local relaxation times , and provided inadequate mr signal enhancement in t1 images at typical clinical field strengths .
moreover , for certain purposes such as mr angiography ( mra : a special type of mri used to study blood vessels ) the time window for contrast - enhanced images is very narrow , due to rapid extravasation , which limits the acquisition of high - resolution images . for mra
, contrast agents must be blood - pool agents ( also known as intravascular contrast agents ) and are characterized by their high molecular weight ( < 20 kda ) and higher relaxivities .
their large size prevents diffusion through the vascular epithelium and leakage into the interstitial space , and so they reside in the vascular system for an extended period of time .
thus , they are eliminated much more slowly from circulation than their extracellular counterparts , providing a larger imaging time window .
examples of blood - pool contrast agents are gd - based complexes that interact noncovalently with human serum albumin , and gd chelates complexed to polymers ( eg , dextrans , polylysine derivatives , and polyamidoamines [ panam , ge healthcare institute , waukesha , wi , usa]).26 to produce targeting agents , macromolecular constructs , such as liposomes , micelles , fluorinated nanoparticles , dendrimers , and polymers , have been prepared.17,25,27 the resulting nanoparticles have greater paramagnetic metal surface payloads that rotate or tumble more slowly than small - molecule organometallic compounds typically used as blood - pool agents.28 mn , with five unpaired electrons , is another cation used as a contrast agent .
mn - based paramagnetic nanoparticles can be classified into two categories : small - molecule agents and nanoparticulate agents .
small - molecule agents are formed by complexing mn ions with chelates such as dpdp , dtpa , or even porphyrin rings , just as gd chelates are .
the fda approved , in may 1997 , a mn - based contrast agent , the injectable mangafodipir trisodium ( teslacan , st louis mo , usa ) to image the liver .
however , in 2012 , the emea was notified by the marketing authorization holder responsible for telescan the decision to voluntarily withdrawl the marketing authorization in the european union.29 mn chelates can be further modified by their incorporation into lipid bilayers .
such nanoparticulate systems are made of manganese oxides such as mno , mno2 , and mn3o4 .
after dissolution in cells due to proteolytic degradation , these particles convert from t2 contrast agents to t1 contrast agents.30 although mn is a natural cellular constituent that resembles ca , its toxicity is also known from dust containing mn at high doses .
moreover , in view of the capacity of mn to enter cells through calcium channels , mn complexes , dendritic mn chelates , and even mn nanoparticulate systems have potential applications in neuroimaging .
one of the limitations of the majority of the contrast agents used , but that especially affects paramagnetic chelates , is that their efficiency decreases at higher magnetic fields .
for example , gd complexes are optimal at fields below 1 t ; even at the clinical field of 3 t , the t1 relaxivity of gd - based contrast agents is reduced by as much as one - third compared with its maximum , while at higher magnetic fields , r1 falls to zero.31 moreover , for in vitro cell labeling experiments or long - term in vivo cell tracking studies , the clearance of the particles needs to be far slower , which impedes the use of gd - based chelate agents for these purposes .
hence , owing to their short blood circulation times , poor detection sensitivity , and toxicity concerns , mri research has shifted to t2 contrast agents , especially to superparamagnetic iron oxide nanoparticles .
t2 contrast agents ( or negative contrast agents ) decrease the mr signal intensity of the regions they are delivered to .
consequently , they produce hypointense signals in t2- and t2 * -weighted images,32 and thus the affected regions appear darker .
the phenomenon can be said to result from the large heterogeneity of the magnetic field around the nanoparticle through which water molecules diffuse , since diffusion induces dephasing of the proton magnetic moments , resulting in t2 shortening .
t2 contrast agents are also called susceptibility agents because of their effect on the magnetic field .
t2 shortening is a remote effect , whereas the t1 shortening process requires a close interaction between the water molecules and t1 agents , as mentioned.16 another difference with t1 contrast agents is that under high magnetic fields , r2 , the relaxation rate , tends asymptotically to a positive constant.10 iron oxide nanoparticles have been used as t2 contrast agents for more than 25 years .
these iron oxides can be ferromagnetic or superparamagnetic , depending on the size of the core of the nanoparticle .
two iron oxides are generally considered for biomedical applications : magnetite ( fe3o4 ) and its oxidized and more stable form of maghemite ( -fe2o3 ) .
the critical upper size limit for the observation of superparamagnetism is approximately 25 nm for magnetite and 30 nm for maghemite.33 the two compounds fulfill the prerequisites of : 1 ) chemical stability under physiological conditions , 2 ) low toxicity , and 3 ) sufficiently high magnetic moments.34 since these two iron oxides exhibit superparamagnetic behavior , the loss of their net magnetization in the absence of an external magnetic field limits their tendency for self - aggregation , and this helps to obtain a good biological response.10 unfortunately , the ubiquitous van der waals forces induce natural aggregation of the particles , and to circumvent this problem , a large portfolio of chemical approaches exists that stabilize the particles .
polymers are the most widely used stabilizing materials , and can be classified as hydrophilic or amphiphilic , neutral or charged , homopolymers or copolymers .
the polymers can be adsorbed into or anchored onto the iron oxide surface via hydrogen bonds , electrostatic forces , or pseudo - covalent bonding . among the materials used
are poly(ethylene glycol ) ( peg ) , dextran and its derivatives , alginate , chitosan , starch , polyvinyl alcohol , albumin , poly(ethylene imine ) , organic siloxane , sulphonated styrene - divinyl - benzene , and bioactive molecules and structures such as liposomes.35,36 since the biological distribution of the nanoparticles is directly dependent on their size , they have been classified according to the overall size of the particles as follows : 1 ) ultra - small superparamagnetic iron oxide nanoparticles ( uspions ) with diameter ( d ) less than 50 nm , 2 ) superparamagnetic iron oxide nanoparticles ( spions ) with size of hundreds of nanometers , and , ultimately , 3 ) micron - sized particles of iron oxide ( mpio ) with a diameter higher than 1 m.37 while the overall size of the first two classes allows them to be administered intravenously , the larger particles are usually administered orally , limiting their use to the exploration of the gastrointestinal track .
there are also other formulations , such as monocrystalline iron oxide particles ( mion ) and cross - linked iron oxides ( clio).19 several spion formulations for intravenous or oral administration have been approved for clinical use as mri contrast agents by the emea and fda ; however , the majority of the compounds that were approved for intravenous administration have , at present , been taken off the market.38 only the spion for oral administration , gastromark ( amag pharmaceuticals , waltham , ma , usa ; ferumoxsil , silicone - coated spions ) , is currently on the market for gastrointestinal bowel marking .
it is important to note that both the type of coating and its thickness affect the value of r2 , although the influence is unclear as studies report different effects .
for instance , it has been reported that as the coating thickness increases , the ratio r2/r1 decreases.39 this is due to the inner hydrophobic layer excluding water , while the outer hydrophilic peg layer allows water to diffuse within the coating zone . increasing the peg chain length leads to a reduction in r2 values ,
although another study has shown that when water molecules are not excluded from regions close to the spion core , r2 relaxivity increases with increased chain length.40 the overall size of the particle governs its pharmacokinetics and biodistribution .
nanoparticles with a size < 5.5 nm are cleared by the kidneys.41,42 spions whose overall diameter is larger than 200 nm are quickly taken up by phagocytic cells and accumulate in the monocyte phagocyte system ( mps ) , specifically in liver and spleen macrophages . when administered intravenously ,
approximately 80% of the dose is found in liver and 5%10% in spleen , with it having a plasma half - life of less than 10 min.16 therefore , such spions decrease the liver and spleen signal within several minutes of administration .
malignant tumors or metastases have a lack of kupffer cells , and due to the negligible uptake of nanoparticles , they produce a strong contrast between normal and abnormal tissue on t2-weighted images .
uspions evade mps uptake and consequently increase their blood half - life ( > 2 h ) .
spions and uspions are metabolized into a soluble and nonsuperparamagnetic form of iron , which is incorporated into the normal iron pool ( eg , ferritin , hemosiderin , hemoglobin ) within a couple of days.19 as with any nanoparticle , spions can invade small solid tumors and metastatic cells thanks to passive targeting through the enhanced permeation and retention ( epr ) effect .
the epr effect aids nanoparticle uptake by way of leaky vasculature , which permits particles of nanometric size ( more or less , with a hydrodynamic radius of less than 100 nm ) to cross from the vasculature into the interstitium.43 poor lymphatic drainage then aids the entrapment of particles in solid tumors .
spions are used as negative contrast for liver imaging , whereas the typical application of uspions is lymph node imaging .
uspions have been tested as blood - pool agents because they are readily distributed in the intravascular extracellular space . in this way
, uspions are used as contrast for lymphography44,45 and angiography,46,47 as a bone marrow contrast , or as a perfusion agent in brain and kidney .
since biochemical epitopes of interest are often present in nanomolar or picomolar concentrations , particle relaxivities of around 1,000,000 mms are required to achieve acceptable contrast - to - noise ratios at the typical field strength.48 in this way , contrast agents that target specific tissue can increase the sensitivity by increasing the local spion concentration .
to achieve such sensitivity in the nanomolar range , the surface of the spions may be modified by active targeting strategies , such as the addition of ligands that are recognized by molecular signatures of afflicted cells .
polyethyleneimine ( pei ) is one of the more used ligands.49 the same group50,51 has reported that hyaluronic acid targeted iron oxide nanoparticles are efficient probes for targeted mri of cancer cells in vitro and xenografted tumor model in vivo .
other types of ligands have been studied for the targeting of such markers including antibodies , small peptides , lectins , aptamers , engineered proteins , and protein fragments.52 for instance , uspions of less than 10 nm in hydrodynamic diameter were tested for tumor - specific mri targeting . in that study ,
the uspions were stabilized by 4-methylcatechol , and a cyclic arginine glycine aspartic acid ( crgd ) peptide was coupled via the mannich reaction53 ( figure 2 ) .
the peptide rgd binds the v3-integrin , a cell adhesion molecule that is overexpressed in tumor vasculature and invasive tumor cells.54 after the administration of the rgd - nanoparticles , the tumor mri signal intensity decreased by 40%.53 one of the largest drawbacks in using spions is related to the contrast mechanism that they generate .
as mentioned , they are negative imaging agents , which produce a signal - decreasing effect .
the resulting dark signal could be confused with other pathogenic conditions , and renders images of lower contrast than t1 contrasted images . moreover
, the high susceptibility of t2 contrast agents induces distortion of the magnetic field on neighboring normal tissues .
this distortion of the background is called a susceptibility artifact or blooming effect , and generates dark images with no background around the lesions.55 this effect prevents their clinical use in low signal body regions , in organs with intrinsic high magnetic susceptibility ( eg , lung ) , or in the presence of hemorrhagic events.56 in addition to nanoparticles whose metal core is fully based on iron oxides , other nanosystems with different magnetic cores have been introduced to improve the signal sensitivity and enhance mri diagnostics.57 since the transverse relaxivity r2 depends , apart from size , on the saturation magnetization ( ms ) , the optimization of ms is one of the most effective ways to achieve magnetic nanoparticles with high mri sensitivity .
it has been reported that , owing to the higher magnetization , iron nanoparticles have a higher t2 relaxivity than analogous systems containing iron oxides.58 alloy - based nanomaterials are good candidates for developing t2 contrast agents with higher relaxivities .
the substitution of one of the fe ions in an iron oxide for a different magnetic atom ( mn , zn , co , ni , etc ) produces the compounds known as ferrites ( mn - ferrite , zn - ferrite , etc ) characterized by their high ms , and this enhanced ms increases the relaxation rate .
yang et al59 have reported the suitability of mn - ferrites as mri contrast agents .
more pronounced contrast effects are even possible when nonmagnetic ions replace the fe ions.60 interestingly , the substitution of fe with nonmagnetic zn results in an increase in the net magnetization of the nanoparticles .
ms increases with zn doping and becomes maximum with a value of x = 0.4 in ( znm ) fe2o4 ( m = mn , fe).for comparison , the ms of ( znmn ) fe2o4 is 175 emug magnetic atom , whereas the corresponding value for fe3o4 is 96 emug magnetic atom .
the value of r2 reaches 676 mms for the zn - doped magnetic nanoparticles , and 98 mms for fe3o4.61 other nanoparticles with potential applications in mri include gold - iron oxide ( au - fe3o4 ) nanoparticles , metallic ion nanoparticles , porous hollow fe3o4 nanoparticles , and fe - based alloy nanoparticles , such as iron cobalt ( feco ) and iron platinum ( fept ) nanoparticles
. however , metallic nanoparticles are normally very reactive and are subject to fast oxidation in biological solutions .
once they are coated with a layer of polycrystalline fe3o4 or a graphitic shell , these metallic nanoparticles are more stable and provide better contrast in mri .
fept nanoparticles are chemically more stable than fe and feco nanoparticles , and have been shown to have great potential as contrast agents for mri and ct.28 however , it is worth pointing out that such systems could be used for preclinical experiments , but clinical assessment of their acute and long - term toxicity is required .
recently , some paramagnetic ions , such as dysprosium ( dy ) , have been proposed as good alternatives to iron oxide t2 contrast agents in high - field mri , because of their high magnetic moments.62 dy has been used as a chelate ( eg , dy - dtpa ) or as nanoparticles ( eg , dy2o3).63,64 one type of dy - based nanoparticles are -nadyf4 nanoparticles ; their relaxivity has been studied at 3 and 9.4 t in nanoparticles of 5.4 , 9.8 , and 20.3 nm .
it has been reported that their r2 relaxivity is 69 times greater at 9.4 t than at 3 t , and that the larger nanoparticles show higher r2 values than the smaller ones , whereas the r1 relaxivities are almost constant for the three sizes at 3 and 9.4 t. at 9.4 t , the r2/r1 ratio is 306 for nanoparticles of 20.3 nm , 230 for those of 9.8 nm , and 106 for those of 5.4 nm .
one important result of classical outer - sphere relaxation theory65 is that the r2/r1 ratio increases with increasing particle size , and thus , smaller particles are much better t1-shortening agents then larger ones ( figure 3 ) . as a consequence of their larger size and magnetic moments ,
however , a new generation of uspions , with diameters less than 10 nm , has also been reported to have excellent t1-enhancing properties.28,66 compared with paramagnetic ions , spions have higher molar relaxivities , and , when used as blood - pool and tissue - specific agents , may offer advantages at low concentrations.67 as indicated above , spions generate dark or negative contrast at the target site with a marked blooming effect from magnetite susceptibility artifacts . moreover , for mi applications , persistent t2 * effects from circulating spions delay mri by 2472 h after injection . to overcome these limitations , specific off - resonance pulse sequences capable of generating a bright contrast in the presence of spions
have been proposed.68,69 more recently , another approach , called inversion recovery on - resonant water suppression ( iron)-mri , has been developed to obtain a positive contrast.70 unfortunately , these techniques do not eliminate the signal loss or magnetic susceptibility artifacts , but rather exploit them to generate more easily perceived bright images of the contrast effects , often at the expense of the surrounding anatomical detail .
moreover , the undesirable 2472 h delay between injection of the agent and the imaging result remains .
one approach to resolve the prolonged delay between treatment and imaging has been to consider the use of microparticles of iron oxide ( mpio , size range 0.761.63 m ) , which rapidly pass into mps organs.71 however , such large iron oxide particles were prone to aggregation and rapid pulmonary entrapment , and despite the leukocyte mimicking dual homing ligand approach proposed , the potential of the approach will depend on overcoming many challenges.72,73 a more recent approach28 is the preparation of a colloidal iron oxide nanoparticle platform ( cion ) , which is achieved by embedding oleate - coated magnetite particles in a hydrophobic matrix composed of vegetal oil and partially cross - linked phospholipids .
contrary to expectations , this formulation decreased t2 effects , thus allowing positive t1-weighted contrast detection .
a cion may be used to detect biosignatures via voxels at very low nanomolar densities .
conventional mri contrast agents are mostly effective only in a single imaging mode : either t1 or t2 .
they frequently result in ambiguities in diagnostics , especially when the biological targets are small .
the combination of simultaneously strong t1 and t2 contrast effects in a single contrast agent could be a new breakthrough , since it can potentially provide more accurate mri via self - confirmation with better differentiation of normal and diseased areas .
dual contrast agents would eliminate the possible ambiguity of a single - mode contrast agent when some of the in vivo artifacts are present.57 however , the production of such a contrast has proved extremely challenging.74 uspions with a core of less than 10 nm in diameter are capable of producing positive contrast in t1-weighted images when administered in moderate concentrations.75,76 while positive t1 contrast is possible with uspions , this benefit is at the expense of their t2 effects.77,78 for this reason , mixing both types of iron oxides , spions and uspions , to form a single contrast agent could potentially be a good choice .
however , an important problem arises as a consequence of the strong magnetic coupling between the t1 and t2 contrast agents when they are in close proximity : the spin lattice relaxation process of t1 contrast materials is significantly diminished .
one strategy to overcome this phenomenon is the inclusion of a separation layer to modulate the magnetic coupling . to this end
, micellar structures incorporating organic block copolymers , inorganic porous materials , and core shell - type inorganic materials have been considered as possible frameworks.52 for instance , a core shell - type t1t2 dual - mode nanoparticle contrast has been described , where the t1 contrast material , gd2o(co3)2 of 1.5 nm , is located on the shell so as to come into direct contact with water molecules , for high t1 contrast effects ; while the superparamagnetic t2 contrast material , mnfe2o4 of 15 nm , is located at the core , from where it induces a long - range magnetic field for the relaxation of water molecules .
the two materials are separated by sio2 . by adjusting the thickness of the sio2 ,
as the sio2 becomes thicker , t1 quenching reduces and , concurrently , r1 increases ; while the decrease in the t2 effects is relatively weaker .
when the sio2 layer is 16 nm thick , both t1 and t2 contrast effects become larger than the effects of the individual single - mode contrast effects ( figure 4).79 in other studies , colloidal suspensions of fe / fe2o3 nanoparticles capable of providing both t1- and t2-weighted images were synthesized.80 similarly , an iron core ( with its subsequent oxidation giving a ferrite shell ) with added nickel ions to form nickel ferrite shell nanoparticle has been studied ; its surface treated with dopamine - peg to make it dispersible , and it acts as a dual - mode t1 and t2 contrast agent.81 another approach has been the addition of a gd chelate to the polymer coating of spions.82,83 this dual contrast agent efficiently reduces both t1 and t2 relaxation times and achieved a good contrast in mice for both t1- and t2-weighted images .
this unique combination allows for the acquisition of both highly detailed t1- and t2-weighted images with a single imaging nanoprobe .
in addition , such a contrast probe could provide enhanced t1-weighted imaging for brain tumors .
brain barrier ( bbb ) without the use of invasive techniques,84 which limits their application in brain tumor imaging .
a significant advantage of spions is their relatively large surface area , which allows for the efficient addition of biologically active moieties such as bbb - penetrating peptides for noninvasive brain tumor imaging .
for several years , liposomes have been the center of interest with regard to mri probes because of their multiple advantages.85 one is their capability to encapsulate hydrophilic substances in their aqueous inner core .
another advantage is their biocompatibility , understood as the quality of having no toxic or injurious effects on biological systems .
both properties enable liposomes to be utilized as carriers , either for therapeutics or diagnostics in vivo . in this way
, liposomes have been used to carry either gd - based contrast agents ( paramagnetic liposomes ) or fe - based contrast agents ( superparamagnetic liposomes ) . as mentioned above in the
t1 contrast agents section , when administered in vivo , gd chelates rapidly diffuse into tissue and interstitial space and result in decreased lesion / vessel signal intensity and a concomitantly enhanced signal from surrounding tissues . as a result ,
contrast between the lesion / vessel and surrounding tissue is reduced , especially in areas where the vasculature is compromised.85 furthermore , due to the latent toxicity of gd chelates , an efficient renal clearance is highly desirable.19 however , for in vitro cell labeling experiments or long - term in vivo cell tracking studies , the clearance of the particles is required to be far lower , which impedes the use of gd chelates for these purposes , since the rapid reduction in blood concentrations of these agents limits the amount of imaging time available after injection . in this case , active targeting or cell labeling contrast agents must be used . for this , contrast agents must be able to recognize specific molecular sites ( eg , cell - specific receptors ) at the cellular membrane and to accumulate at those sites .
the development of approaches that use probes capable of recognizing and imaging a specific molecular marker of a given pathological process makes diagnosis and therapy much easier .
however , the main problem with approaches that use gd - based contrast agents is the low sensitivity of the resultant mri , so that to reach 50% contrast enhancement it is necessary to have a local concentration of contrast agent of the order of 0.5 mm . to increase the payload of binding groups delivered to the target site , it is possible to use a single carrier that can bear many ligands to bind to molecular markers .
liposomes can be rendered t1 mri active by the incorporation into the bilayer of gd conjugated with lipid moieties ( for instance , gd
dtpa cholesterol ) or by encapsulation of gd within the aqueous space ( for instance , in the form of gd
dtpa).86 as a high payload of gd - containing amphiphilic lipid can be incorporated to the bilayer , the relaxivity per particle increases spectacularly , countering the sensitivity problem.87 nevertheless , the addition of large amounts of gd alone does not guarantee a low detection limit , since negative effects on the sensitivity may result from a disproportionate increase of r2 with respect to r1 , leading to a reduction of the r1/r2 ratio . moreover , relaxivity rates are a function of the exchange rate of the inner and outer sphere water molecules .
as liposome rigidity limits the flux of water between the liposome aqueous cavity and outer bulk water , the physical state of the liposome and its content of cholesterol can modify the overall impact of the contrast agents on local tissue water relaxivity .
moreover , water diffusion at the interface with the phospholipid surface is strongly influenced by the excluded volume provided by the dynamic lipid molecules and head groups.88 these facts mean that the lipid composition of liposomes influences the relaxivity produced by the contrast agents encapsulated or incorporated within them .
the encapsulation of a gd chelate in conventional liposomes results in better circulation properties than those of the free gd chelate , and encapsulated gd stays in the circulating blood for longer than free gd chelate does .
however , recognition by serum proteins may compromise the stability of the encapsulated gd and may thus produce leakage of the gd chelate in circulation .
the leaked gd chelate can diffuse into the extravascular space and diminish the quality of the image .
moreover , conventional liposomes are prone to agglomerate when they are obtained at a high lipid concentration , as is usual in these cases .
if they were prepared with a low lipid concentration , the dose of liposomes required to obtain a significant signal in vivo would involve an excessively high volume of liposomal suspension .
the incorporation of peg into the liposomal membrane by means of a covalent bound creates sterically stabilized liposomes ( stealth liposomes , sequus pharmaceuticals , menlo park , ca , usa ) that have longer blood persistence times .
an example of paramagnetic liposomes are pegylated liposomes encapsulating gd dtpa.89,90 the relaxation characteristics of gd pegylated liposomes differ from those of free gd - dtpa . as expected , the encapsulation significantly lowers the r1 value to 1.04 mms compared with 4.0 mms for free gd - dtpa .
the low relaxivity of liposomal gd is due to the limited access of the gd atoms to the bulk water molecules , because of the liposomal bilayer , which lowers the rate of water exchange between the bulk and the interior of the liposome .
furthermore , if the lipid contains cholesterol , this reduces the permeability coefficient of the water molecules , leading to a greater reduction in the observed relaxivity.91 size is another factor that strongly affects relaxivity .
the difference is due either to the high surface - area - to - volume ratio in small liposomes , which facilitates proton transport across the liposomal bilayer , or to the decreased time of residence of water inside smaller vesicles .
as indicated , the encapsulation of a hydrophilic gd - based contrast agent in liposomes involves a reduction of the relaxivity .
this loss of relaxivity is overcome by an increase in the concentration of gd in the pegylated liposomes , which results in a significant shortening of the blood relaxation time t1 . given the relaxivity problems resulting from the encapsulation of gd inside liposomes , the incorporation of gd chelates into the liposome bilayer is preferable .
this approach is expected to enhance the water contact of the gd chelate , potentially leading to significantly enhanced relaxivity.86 to recognize and image a specific molecular marker of a given pathological process or state ( mi ) , such as inflammation , atherosclerosis , angiogenesis , apoptosis , or the presence of tumors , the paramagnetic liposomes are required to target specific molecular markers . targeted mi of sparsely expressed receptors in tissues , where positive contrast is preferred due to intrinsic t1 and t2 relaxation times , requires amplification strategies because of the relatively low relaxivities of t1-reducing ions , including gd . therefore , it is advantageous to use liposomes in which a large payload of paramagnetic lipids can be incorporated into the bilayers , resulting in efficient t1- and t2-shortening lipidic nanoparticles . as an example , gd - dtpa - bis(steraylamide ) was incorporated into the lipid bilayer of pegylated liposomes .
then multiple human recombinant annexin a5 molecules were covalently coupled to introduce specificity for apoptotic cells .
the resulting contrast agent increased the relaxation rates of apoptotic cell pellets compared with untreated control cells and apoptotic cells that were treated with nonfunctionalized nanoparticles.92 although recent research has progressed in the preparation and formulation of applications of paramagnetic liposomes for nanoparticle imaging systems , the use of liposomes as contrast agents is mainly based on superparamagnetic liposomes .
mlps were the first multifunctional hybrid liposome / nanoparticle assembly , and they have received considerable attention since their introduction in 1988.93 if the superparamagnetic particles are spions , the mlps can be used as t2 contrast agents , whereas if uspions are used , the mlps produce a t1 contrast.75,77,94 the term mlp denotes several types of phospholipid iron oxide constructs with totally different properties .
the original mlps consisted of iron oxide cores , upon which a single phospholipid bilayer was adsorbed .
the size of such mlps is approximately 20 nm ( for a review of the original mlps , see de cuyper95 ) .
another kind of mlps is extruded mlps , which consist of large unilamellar vesicles ( with diameters of the order of a few hundred nanometers ) encapsulating several small nanometer - sized water - dispersible iron oxide cores in the aqueous cavity.96,97 as an alternative to the extrusion method , encapsulation of magnetic particles can also be achieved by sonication , inverse phase evaporation , or a combination of these techniques.98100 finally , a third kind of mlps are formed via the precipitation of iron oxides in the inner space of the vesicles.101 this method has many drawbacks ( for instance , little control over the size and size distribution , and the need for large amounts of starting ferrous and ferric salts , which greatly affects the ph and the peroxidation of unsaturated phospholipids ) .
it has been shown that endosomal localization of different iron oxide particles results in their degradation and reduced mr contrast , the rate of which is governed mainly by the stability of the coating.102 the encapsulation of the iron cores in pegylated liposomes affords , as indicated for paramagnetic liposomes , biological stability that improves the contrast agents . moreover , unlike in the case of spions , liposomes may have some advantages , especially in the field of theranostic agents , eg , nanoscale devices that integrate diagnostic and therapeutic functions .
the versatility of lipid types and lipid conjugation permits the combination of the magnetic cores with drugs , fluorescent lipid conjugates , and ligands to design a single nanosystem that can be detected with multiple imaging techniques.103 an additional advantage of mlps over liposomes or over naked iron oxide nanoparticles is that they can be successfully targeted to body parts of interest , to tumors , for example , and their progression in the body can be followed by mri .
such targeting can be achieved in two ways : 1 ) by attaching antibodies or ligands to the vesicle surface that can be selectively recognized by specific receptors present in the cells ( biological targeting);104 2 ) by applying an external magnet near specific body regions where mlps can then be accumulated ( magnetic targeting).105 both approaches allow a reduction in the total number of targeted nanoparticles used compared with untargeted nanoparticles .
mlps have been used to combine t1 and t2 mri contrast agents in a single system to obtain bilabeled contrast agents.106,107 gd ions were anchored to the surface of mlps , with up to 500 gd ions per magnetic vesicle .
figure 5 is a scheme of a multimodal particle based on a liposomal structure that allows theranostic applications .
this liposome contains several individual cores ( or a cluster of cores ) of spions , and , moreover , it can enclose a drug .
the presence of magnetic nanoparticles makes bioimaging possible or the generation of heat in therapeutic hyperthermia , and also magnetic targeting .
the shell material is responsible for its surface properties , because of the presence of reactive moieties on the surface . in this way
, peg is attached covalently to the surface of phospholipids in order to prevent aggregation and opsonization .
the shell can be tuned to provide binding to molecules ; as an example , the peptide rgd is bound at the distal end of some peg chains for the purpose of targeted drug delivery .
such peptides facilitate the interaction of the liposome with integrins : proteins present on the cellular surface that recognize the peptide rgd .
moreover , the liposomal bilayer can contain a fluorescent probe , which permits its interaction with cells to be visualized by confocal microscopy .
over the last 25 years , various nanoparticles and complexes have been studied as mri contrast agents , and several formulations have been approved for clinical use .
these contrast agents are formed either of transition and lanthanide metals or of iron oxide nanoparticles and , more recently , ferrite nanoparticles .
the transition or lanthanide metals , whose most significant representative is the ion gadolinium ( gd ) , have been extensively used as t1 contrast agents since they increase longitudinal relaxation times . a new generation of t1 contrast agents is formed by gd complexes immobilized in various nanostructured materials ( nanoporous silicas , dendrimers , perfluorocarbon nanoparticles , and nanotubes ) .
iron oxide nanoparticles with overall diameters greater than 50 nm can also be used as mri contrast agents owing to their capacity to shorten t2 * relaxation times in liver , spleen , and bone marrow by selective uptake and accumulation in mps cells .
iron oxide nanoparticles with diameters < 50 nm have been used for lymph node imaging ; moreover , depending on the size of the iron core and their concentration , these small iron oxide nanoparticles can enhance t1 relaxation times .
moreover , iron oxide nanoparticles functionalized with bioactive materials have been used for targeted imaging via the site - specific accumulation .
the presence of dual - mode agents with strong t1t2 contrast effects in a single construct is very challenging , since such dual agents improve the accuracy of biomedical imaging .
moreover , the development of nanomaterials that can filter the mri artifacts allows the discrimination between signals coming from contrast agents or artifacts .
mlps are an example of multifunctional platforms for either multimodal imaging or simultaneous imaging and therapy .
mlps can be carefully manipulated in their composition to incorporate cationic lipids , fluorescent - lipid conjugates , targeting ligands , drugs , and peg , containing all in a single nanosystem .
the most notable limitations associated with the use of such contrast agents are the current detection limits and the lack of tissue specificity .
current detection limits need to be improved for the successful translation of nanoparticles to in vivo applications .
these limitations have been overcome by recent developments in both mri acquisition methods and postsynthesis modification of nanoparticles .
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### Summary:
: magnetic resonance imaging ( mri ) has become one of the most widely used and powerful tools for noninvasive clinical diagnosis owing to its high degree of soft tissue contrast , spatial resolution , and depth of penetration .
mri signal intensity is related to the relaxation times ( t1 , spin lattice relaxation and t2 , spin spin relaxation ) of in vivo water protons .
to increase contrast , various inorganic nanoparticles and complexes ( the so - called contrast agents ) are administered prior to the scanning .
shortening t1 and t2 increases the corresponding relaxation rates , 1/t1 and 1/t2 , producing hyperintense and hypointense signals respectively in shorter times .
moreover , the signal - to - noise ratio can be improved with the acquisition of a large number of measurements .
the contrast agents used are generally based on either iron oxide nanoparticles or ferrites , providing negative contrast in t2-weighted images ; or complexes of lanthanide metals ( mostly containing gadolinium ions ) , providing positive contrast in t1-weighted images .
recently , lanthanide complexes have been immobilized in nanostructured materials in order to develop a new class of contrast agents with functions including blood - pool and organ ( or tumor ) targeting . meanwhile , to overcome the limitations of individual imaging modalities , multimodal imaging techniques have been developed .
an important challenge is to design all - in - one contrast agents that can be detected by multimodal techniques .
magnetoliposomes are efficient multimodal contrast agents
. they can simultaneously bear both kinds of contrast and can , furthermore , incorporate targeting ligands and chains of polyethylene glycol to enhance the accumulation of nanoparticles at the site of interest and the bioavailability , respectively . here , we review the most important characteristics of the nanoparticles or complexes used as mri contrast agents .
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: caloric vestibular stimulation ( cvs ) is a widely used technique developed by robert barany more than a century ago , and is commonly used to diagnose balance disorders or confirm absence of brainstem function .
historically , water or air irrigators have been used to warm or cool the external auditory canal of patients .
both warming and cooling temperature changes lead to density changes in the endolymphatic fluid in the semicircular canals and create convection currents which result in cupular deflection , change in the tonic firing rate of the vestibular nerves and elicit the vestibulo - ocular reflex or horizontal nystagmus .
though there is still debate about the totality of effects that caloric stimulation has on the vestibular organs , the thermoconvection model originally described by barany and wittmaack seems to account for the majority of the induced changes ( at least within earth s gravity ) .
cvs induction has been associated with release of a number of neurotransmitters including serotonin , histamine , acetylcholine and gaba . additionally , the modulation of various networks and nuclei in the brain by cvs including the basal ganglia cerebellum , brainstem , insula , hypothalamus , thalamus , hippocampus and prefrontal cortex suggests significant potential for cvs to modulate both motor and non - motor functions . despite the evidence for widespread activation of neural circuits and a long history to support the clinical safety of cvs , investigation into its potential therapeutic efficacy has been limited .
several groups have demonstrated acute benefits of cvs with hemispatial neglect patients , and a number of studies have explored the therapeutic potential of cvs for pain mitigation . however , all of these studies have suffered from low patient numbers , minimal details about the cvs protocol , no longitudinal treatment , and cursory follow - up , if any . the main impediment to progress for cvs as a mechanism for neuromodulation and/or therapeutic approach has been the lack of a device design suited for prolonged treatments and home use . to address this deficit
we describe the results herein and provide evidence that time - varying thermal cvs waveforms elicit physiological changes in cerebral blood flow velocity , which are consistent with neurostimulation of brainstem centers .
furthermore , we demonstrate the feasibility and safety of longitudinal use of the device for the treatment of episodic migraine .
1 ) is easily operated and consists of a headset , fashioned like music headphones , with aluminum earpieces to conduct heat and a control unit that powers the device and allows for patients to start treatments . the temperature in each ear insert
however , the device has fail - safe and patient - lockout protections , so the patient can only activate the neuromodulation protocols and number of daily treatments prescribed on the sd cards inserted into the control unit .
figure 1.cvs device headset and control unit . cvs device headset and control unit .
the cvs device is based on a peltier unit , an array of diodes comprised of n - type / p - type semiconductor junctions comprised of bismuth telluride ( see fig .
the peltier module used in the cvs device is composed of an array of diodes in a cast epoxy cube to reduce susceptibility to shear stress ( te technology , traverse city , mi ) .
when direct current is passed in one direction through the array , one side heats up with respect to the other . reversing
a pulse - width - modulation algorithm is used to power the peltier devices so that the actual temperature , as measured by a thermistor in the earpiece , matches the waveform target temperature .
the anodized , aluminum earpiece is attached to one side of the peltier array and an aluminum heat sink is attached to the other .
2b shows an enlarged view of the connections to the peltier device as well as the location of a thermistor temperature sensor , which is used in the control circuit that sets the temperature of the earpiece .
the groove in the ear insert is designed to allow for pressure equalization within the ear canal .
the earpiece is covered on its lower base by an ethylene - vinyl copolymer skirt that works to thermally insulate the earpiece from the outer portions of the ear canal and concha .
figure 3.infrared thermographs show the temperatures of the earpiece and heat sink during a square wave thermal waveform with a minimum temperature of 15 c and a maximum temperature of 42 c .
infrared thermographs show the temperatures of the earpiece and heat sink during a square wave thermal waveform with a minimum temperature of 15 c and a maximum temperature of 42 c .
the thermistor in each earpiece samples the temperature at the tip at a rate of 4 hz . the thermistor provides feedback to the control circuit that sets the temperature of the earpiece .
the heating and cooling systems are deactivated if either thermistor detects temperatures 1 c below or above the minimum and maximum temperatures defined by the thermal waveforms , respectively , or if a thermistor fails .
the temperature control circuit accesses a temperature calibration file matched to a specific thermistor whenever the system is powered on .
the device will display an error and will not run a treatment if no calibration file is found .
additionally , because the warming and cooling functions are driven by the system software , any potential failure of the system software would result in inactivation of the device .
the thermistor - recorded temperature is logged with each treatment thus providing a mechanism to confirm treatment compliance . because the thermal load of a subject can lead to a small drop in thermal efficiency later in the run ( thus creating a small divergence between the measured and targeted temperature , see fig .
4 ) , the temperature log can also be assessed to determine whether the subject actually wore the headset during any particular treatment .
figure 4.example of target and actual thermal profiles of the sawtooth time - varying thermal waveform used in this study .
example of target and actual thermal profiles of the sawtooth time - varying thermal waveform used in this study .
the device was manufactured by anuva innovations ( morrisville , nc ) under iso 13485 compliant protocols .
all hardware , software and safety features of the device were tested according to iso 60601 standards by an independent testing laboratory , intertek ( atlanta , ga ) , and the biocompatibility of device materials was independently confirmed under iso 10993 by toxikon ( bedford , ma ) .
proper cvs induction relies on good thermal transfer between the earpieces and the temporal bone .
heat flows around the middle ear space to the inner ear and the vestibular labyrinth .
the horizontal semicircular canal ( hscc ) actually protrudes slightly into the middle ear space and is the structure most immediately and strongly affected during cvs .
the base of the earpiece is insulated with rubber to prevent unwanted heat transfer from other than the shaft and tip .
there is a wide variation in tortuosity of the fleshy part of the ear canal in adults , but the bony canal , which terminates at the tympanic membrane , is similar in all adults . the earpiece tip is sized so that it can not enter the bony canal , but a good fit will ensure that the tip is abutted to the bony canal entrance . though small variations in the fitting of the headset may exist across patients or day - to - day within a given patient ,
cvs is intrinsically well targeted since the thermal stimulus only stimulates the vestibular organs . with conventional irrigation - based cvs
, there is a period of rapid temperature change followed by a constant temperature epoch and then a slower recovery to body temperature as the irrigation stops .
plotted as a time - varying waveform , this would be a very high slew rate change to a plateau followed by a nonlinear ramp back to 37 c .
by contrast , the cvs device allows for defined , time - varying waveform shapes ( for an example , see fig .
4 ) and has a slew rate limited by the thermal capacity of the heat sink , but it can achieve roughly 40 c / minute for heating and 20 c / minute for cooling . notably , the cvs device can repeat a waveform pattern to create cyclic cvs . though the amplitude of the thermal waveform is reduced and the sharp vertices of a sawtooth waveform pattern are smoothed to a shape resembling a sine wave ( due to the thermal conduction properties of the temporal bone ) the frequency is preserved at the inner ear target .
making exact predictions about temperature profiles or tonic firing rates is not possible due to the inter - individual variations in pneumatization of the bone , blood flow , the quality of the fit of the earpiece , etc .
while dizziness and nausea have been associated with conventional irrigation - based cvs , tolerability can be improved and unwelcome side effects can be mitigated by avoiding temperature extremes and by slowing the slew rate .
notably , although primary endpoints will not be discussed in this paper , the waveform shown in fig . 4 has been used in a number of studies to investigate the therapeutic efficacy of the cvs device for a number of neurological and metabolic conditions .
there were only minimal adverse effects in these studies ( and no serious or unexpected adverse effects ) .
additionally , in over 100 patients who have participated in other studies using the time - varying cvs waveform shown in fig .
4 , only 4 patients have reported experiencing dizziness and/or nausea ( transient in all cases ) that could have potentially been related to treatment , and no serious or unexpected adverse events were reported . of the 4 patients who experienced dizziness / nausea during the study , no patient s symptoms were conclusively linked to use of the device ( black and rogers , unpublished observations ) .
in addition to minimizing risk of dizziness and nausea , the precise temporal control of the thermal waveforms allows the device to overcome the therapeutic limitation of scc adaptation which is observed during constant temperature cvs , .
it is important to note that the diminution of nystagmus to constant temperature cvs is due to a physical adaptation of ciliary structures , which respond to the motion of the cupula , rather than the fatigue of afferent nerves .
in fact , bagnall et al .
found that the vesicle structure in the hair cell synapses has evolved to reduce fatigue . by varying the temperature applied to the temporal bone during cvs
, adaptation can effectively be avoided , thus allowing for a therapeutic session limited only by the endurance of the patient .
finally , having independently controlled caloric earpieces allows for unyoking the left and right labyrinths , enabling non - physiological stimulation to be delivered .
the effects of cvs on cerebrovascular dynamics were measured using transcranial doppler sonography ( tcd ) of intracranial blood vessels using a sonoara tcd system ( natus medical , pleasanton , ca ) .
following duke university irb approval , the basilar artery ( ba ) of a single subject was insonated through the suboccipital acoustic window over the course of three cvs treatments . the gosling pulsatility index ( pi ) , a measure of cerebrovascular resistance defined as [ ( peak systolic velocity - end diastolic velocity)/ mean cerebral blood flow velocity ] constituted the primary measure of cerebrovascular dynamics .
successful insonation of the ba was judged by real - time monitoring of the return signal on the tcd unit . a 2 mhz probe
the ultrasound probe was coated with a viscous acoustical coupling gel and held , by hand , at the base of the skull .
the pi time series data was transformed , using a fast fourier transform ( fft ) algorithm ( statplus , analystsoft , walnut , ca ) into a frequency space representation to better highlight the spectral power distribution of the observed oscillations .
three minute long segments of the time series were transformed with no additional digital filtering .
data is presented on a scale from 0.3 to 9.0 cycles per minute ( cpm ) to provide better resolution of the primary observed spectral components . following duke university irb approval ,
a small pilot clinical study with episodic migraine patients was conducted primarily to establish safety and feasibility for use of this device in the home environment .
however , the pilot study was also designed to evaluate different cvs thermal waveforms and gather preliminary evidence of efficacy .
three patients met the following inclusion criteria and were thus enrolled in the study :
a history of at least four , and not more than fourteen total monthly headache days of which between four and nine were migraine headache days .
patients were being treated at the duke university headache clinic.a history of some responsiveness ( incomplete ) to at least one and a maximum of two prophylactic pharmaceutical therapies ( utilized concurrently).subject were at least 18 years of age .
exclusion criteria :
individuals who were pregnant , who had a history of cardiovascular disease , who worked night shifts or who had vestibular migraine , menstrual migraine , post - traumatic migraine , a history of unstable mood disorder or unstable anxiety , moderate or greater hearing loss or a history of traumatic brain injury.a history within the last six months of narcotic or barbiturate use or experience of one or more analgesic rebound headaches .
study parameters :
patients self - reported baseline migraine headache burden.patients used the cvs device for a period of 6 weeks , and treatment with the cvs device consisted of twice daily , roughly 18 minute long sessions ( active only , no placebo).patients maintained a pain diary during the treatment period as well as a 4 week post - treatment observation period where they self - reported headache pain burden using visual analogue scale for pain ( 0 - 10 point subjective pain reporting ) .
the number of headache days was binned over 2-week intervals and was used as a preliminary measure of efficacy.balance was tested using the berg scale prior to treatment and at the end of the 6-week treatment period.patients were required to report any adverse events .
patient # 1 had a device that delivered an in - phase square wave pattern to both ears with a 2-minute period , transitioning from 17 to 42 c .
the ears receiving the warm and cold waveforms were switched on a daily basis to mitigate the hypothetical risk of unintentionally creating a baseline vestibular asymmetry after the treatment period . both the square and the sawtooth thermal waveforms were designed to avoid adaptation of the vestibular response . the square wave program delivered in - phase thermal stimuli to both ears .
this waveform choice was designed to mitigate side effects by negating the asymmetric effect concomitant with unilateral cvs administration .
a history of at least four , and not more than fourteen total monthly headache days of which between four and nine were migraine headache days .
patients were being treated at the duke university headache clinic . a history of some responsiveness ( incomplete ) to at least one and a maximum of two prophylactic pharmaceutical therapies ( utilized concurrently ) .
individuals who were pregnant , who had a history of cardiovascular disease , who worked night shifts or who had vestibular migraine , menstrual migraine , post - traumatic migraine , a history of unstable mood disorder or unstable anxiety , moderate or greater hearing loss or a history of traumatic brain injury . a history within the last six months of narcotic or barbiturate use or experience of one or more analgesic rebound headaches .
patients used the cvs device for a period of 6 weeks , and treatment with the cvs device consisted of twice daily , roughly 18 minute long sessions ( active only , no placebo ) .
patients maintained a pain diary during the treatment period as well as a 4 week post - treatment observation period where they self - reported headache pain burden using visual analogue scale for pain ( 0 - 10 point subjective pain reporting ) .
the number of headache days was binned over 2-week intervals and was used as a preliminary measure of efficacy .
balance was tested using the berg scale prior to treatment and at the end of the 6-week treatment period .
vestibular stimulation , including cvs , has previously been shown to affect cerebrovascular dynamics . to demonstrate that our device provides substantive stimulation of the vestibular system , we measured the effects of treatment on pi using tcd sonography over the course of a cvs treatment . because the thermoconvective effect of cvs is maximized when the horizontal semicircular canal ( hscc ) is in the vertical orientation , we first evaluated the effects of cvs on cerebrovascular dynamics when the subject was supine on a ~22 wedge pillow , a position which puts hscc in the optimal orientation for cvs . in this position , the time - varying sawtooth thermal waveform ( shown in fig .
4 ) induced strong oscillations in pi after ~4 - 5 minutes of cvs treatment ( fig .
these oscillations in pi continued throughout the remainder of the cvs treatment and for approximately 7 additional minutes during the post treatment period .
power spectra analysis of the pi time course show prominent peaks clustered around 1.5 cpm for the mid to late - phase cvs treatment as well as the early post - treatment period as compared to baseline ( fig .
also , peaks ~3.0 - 3.3 cpm ( possible harmonic ) were observed during these periods . in the 21 - 24 minute segment ( early post - cvs ) , the peak power increased , suggesting a possible entrained resonance .
notably , the power spectra for the last 3 minutes of the time series resembles that of the baseline suggesting that pi had returned to pre - cvs values .
figure 5.(a ) pi time course , ( b ) pi power spectrum , ( c ) hr time course and ( d ) hr power spectrum show physiological effects of time - varying cvs treatment when the subject is supine on a 22 wedge pillow .
( e ) time course data for time - varying cvs in the supine position shows an anti - phase relationship between pi ( purple ) and hr ( green ) , with oscillations in pi occurring first .
( f ) pi time course and ( g ) pi power spectrum when the subject is bent forward in a null position .
( h ) pi time course and ( i ) pi power spectrum for constant temperature cvs ( 17 c ) when the subject is supine on a 22 wedge pillow . for panels a - d and
f - i , baseline ( black ) , cvs treatment ( red ) , post - cvs treatment ( blue ) . (
a ) pi time course , ( b ) pi power spectrum , ( c ) hr time course and ( d ) hr power spectrum show physiological effects of time - varying cvs treatment when the subject is supine on a 22 wedge pillow .
( e ) time course data for time - varying cvs in the supine position shows an anti - phase relationship between pi ( purple ) and hr ( green ) , with oscillations in pi occurring first .
( f ) pi time course and ( g ) pi power spectrum when the subject is bent forward in a null position .
( h ) pi time course and ( i ) pi power spectrum for constant temperature cvs ( 17 c ) when the subject is supine on a 22 wedge pillow . for panels a - d and
f - i , baseline ( black ) , cvs treatment ( red ) , post - cvs treatment ( blue ) .
evaluation of heart rate variability also showed that cvs treatment induces significant oscillations in heartbeats per minute ( fig .
5c ) . similar to the case for pi , power spectra analysis for heart rate also revealed significant peaks around 1.5 cpm and ~3.0 cpm , ( fig .
notably , the hr oscillations demonstrated an anti - phase relationship to the pi oscillations ( fig .
the anti - phase relationship was particularly prominent during late - phase cvs . additionally , the onset of the hr oscillations occurred after the first peak in pi and indicating that this entrainment of hr could have been a secondary compensatory response to the fluctuations in pi . to rule out the possibility that the observed cvs - induced changes in cerebrovascular dynamics resulted from a direct thermal effect on vestibular afferent firing ( i.e. that warming or cooling temperatures reaching the nerve itself , affect the tonic firing rate ) , we repeated this experiment ( in the same subject on a separate day ) with the subject sitting up in a slightly forward bent position which leaves the hscc in the horizontal orientation .
we termed this the null position because the horizontal orientation of the hscc should abolish the potential for changes in temperature to generate thermoconvective currents .
5e ) or hr ( data not shown ) and there were no observed peaks in the power spectra that were significantly different from baseline ( fig .
the lack of substantive pi or hr oscillations while the subject was in the null position also rules out the possibility , however unlikely , that the cvs - induced changes in cerebrovascular dynamics observed when the subject was in the supine position were somehow mediated by a placebo - effect due to subjective sensations of warming and cooling temperatures in the ears as this sensory experience remained constant throughout .
finally , to examine the significance of our time - varying thermal waveform , we next evaluated whether pi oscillations could be induced by a constant temperature cvs waveform .
for this , the subject was supine on the ~22 wedge pillow and a constant temperature ( 17 c ) waveform was applied to both ears for 21 minutes .
similar to the time - varying waveform in the null position , constant temperature cvs did not result in oscillations in pi ( fig .
5f ) or hr ( data not shown ) . although further studies will be required to validate these findings , together , these results provide physiological evidence for substantive and prolonged stimulation of the vestibular system by time - varying cvs device treatment .
treatment compliance was above 80% in all cases ( i.e. patients completed at least 67 of the 84 total treatments over the 6 weeks ) .
significantly , no patient reported adverse events that could be attributed to use of the cvs device either during or after the treatment period .
there were no observable changes in balance ( as measured using the berg balance test ) between the baseline and the 6-week post treatment evaluation .
specific comments on the device design generally related to inconvenience of the cabling leading from the headset to the control unit . however , no patient reported significant procedural inconvenience during treatments .
although the primary intent of this pilot study was to gather data on the feasibility and safety of using this portable cvs system in the home setting , evaluation of the of the daily diaries revealed that the number of headaches after 6 weeks of cvs therapy was lower than the self - reported headache frequency prior to entering the study for all 3 patients ( fig .
these results suggest that cvs treatment may provide therapeutic relief for those suffering from episodic migraine headaches and will be validated in a larger scale research clinical trial .
figure 6.the number of headaches reported over the course of the treatment and post - treatment observation periods binned into 2 week intervals . the number of headaches reported over the course of the treatment and post - treatment observation periods binned into 2 week intervals .
irrigation - based cvs has been used for over a century both to diagnose balance disorders and confirm loss of brainstem function . despite a long - standing history of clinically efficacy ,
the primary impediments for evaluating the therapeutic potential of cvs stem from the lack of compatibility of irrigation - based calorics for self - administration in the home and the short duration of vestibular stimulation which can be achieved using constant - temperature cvs .
the solid - state cvs device , described herein , overcomes these limitations of traditional irrigation - based calorics and provides an unprecedented opportunity to evaluate the therapeutic potential of cvs treatment for a number of neurological and metabolic disorders .
one of the most significant advances the device affords over traditional irrigation - based cvs is the ability to provide time - varying thermal waveforms . in this study
, we show that time - varying cvs has a qualitatively different effect on cerebrovascular physiology than does constant temperature cvs . more specifically , time - varying , but not constant - temperature cvs induces significant oscillations in pi ( as well as hr ) after 4 - 5 minutes of treatment ( fig .
analysis of the pi spectral peaks during time - varying cvs indicates that the oscillations occur at 40 seconds ( and 20 second harmonic ) intervals . while these intervals do not match the periods of the warm ( 1.3 minutes ) and cold ( 2.3 minutes ) waveforms , they do fall within the periodicity range of b waves ( sharp rhythmic sawtooth - like oscillations in intracranial pressure ) which are thought to be autoregulatory reactions to changes in arterial blood pressure .
b waves occur at frequencies of 0.5 to 3 oscillations per minute ) , , and due to their slow time constant , are thought to provide a complimentary mode of autoregulation to the rapid autoregulatory responses that are required when going from seated to standing , for example
. this slower mode of autoregulation likely allows for compensation against baseline shifts that could increase intracranial pressure over time .
although much remains unknown about the precise circuit mechanisms that allow for b wave generation , previous studies have provided evidence for a monoaminergic b - wave pacing center in the pons , an area that receives direct innervation from the vestibular nuclei in the brainstem .
what seems clear from the pi data is that the observed oscillations resulted from vestibular stimulation ( since there was an orientation dependence ) and that time variation of the thermal waveforms was necessary ( since the constant temperature cvs run did not generate oscillations ) .
the periods of the applied thermal waveforms did not exactly match the observed oscillation periods , but it is well known that an oscillator can be driven at a non - resonant frequency .
when cvs stimulation stopped , the immediate post - cvs spectra sharpened to a single primary peak at ~1.5 cpm ( plus a harmonic ) . because the pi oscillations fell within the periodicity for b waves ( 0.5 3.0 cpm , ) , it is possible that the pi oscillations resulted from b wave activity .
that is , the time - varying cvs may have entrained the pontine structures responsible for b - wave pacing .
indeed , the finding that there was a significant increase of spectral power at 1.5 cpm when the driving force of cvs stopped ( post - cvs period ) supports the hypothesis that entrainment occurred .
although speculative , the evidence that time - varying cvs seemed to entrain b wave pacing , could provide evidence to suggest a potential mechanism of action for cvs treatment of migraine headaches . more specifically , sliwka et al .
found that migraineurs exhibit a greater coefficient of variation in b wave amplitude compared to healthy controls and patients with tension headaches .
the authors concluded that this was evidence of dysfunction of a brain stem monoaminergic system in migraine .
further investigation into the modulation or entrainment of b wave pacing by time - varying cvs and its potential role in the therapeutic effects for migraine prevention will be an exciting area of future research .
b wave flow abnormalities , if they are truly associated with migraine , are not in and of themselves thought to be causal in the disease . while migraine is associated with vascular flow abnormalities , neuroimaging findings support the hypothesis that migraine is a neurological disorder involving brainstem dysfunction . during acute attack , brain activation is observed in the dorsal pons and also the dorsal midbrain .
this activation persists after pain is controlled through abortive medication ; however , no differences in activation of these regions are observed interictally .
functional imaging studies of irrigation cvs have demonstrated widespread activation patterns in the brain , starting in the vestibular nuclei in the dorsal pons and medulla . for a review ,
although neuroimaging data evaluating patterns of brainstem activation with cvs have been limited due to the difficulty of achieving anatomic resolution with these techniques , a number of tracing studies in animal models have demonstrated that the vestibular nuclei are extensively connected to numerous brainstem regions implicated in migraine headache including the periaqueductal gray , the parabrachial nucleus , the locus coeruleus , the reticular formation , the dorsal spinal and mesencephalic trigeminal nuclei , and the dorsal raphe nuclei , . this extensive anatomic connectivity of the vestibular nuclei with key brainstem regions implicated in migraine headache , together with our evidence for substantive and prolonged stimulation using time - varying thermal cvs waveforms and our preliminary evidence from the episodic migraine pilot ( showing the safety , feasibility and potential efficacy of longitudinal use of the device ) has given us incentive to follow up this work with prospectively powered , randomized , placebo - controlled clinical trial ( nct01899040 ; http://www.clinicaltrials.gov ) .
that trial seeks to gather data in support of an application for regulatory clearance by the u.s .
the temperature ranges and slew rates of the two different thermal waveforms used in the migraine pilot evaluation were selected to minimize adverse events .
importantly , these two waveforms were well tolerated in the migraine pilot study and in a larger scale study .
additionally , in our waveform selection , we sought to balance cvs delivery so that unintentional ( hypothetical ) biasing of baseline vestibular function did not occur . to balance cvs ,
either the waveforms delivered to the two sides were matched or asymmetric waveforms were alternated over time to achieve parity . in future studies , we will seek to establish the physiological sequelae , safety and tolerability of other thermal waveforms .
. whether evaluation of cerebrovascular dynamics in response to cvs waveforms may be beneficial in gauging the potential for therapeutic efficacy will be an interesting area for future studies .
however , it is important to note that given the inherent selectivity of cvs induction ( that is , only the vestibular organs respond to caloric stimulation ) , treatments should be reproducible . by contrast
, two other neuromodulation devices under development for migraine treatment , transcranial magnetic stimulation and cranial electrotherapy stimulation or transcranial alternating current stimulation ( which was recently found to have significantly weaker stimulation that the original models predicted ) , are inherently dependent on patients placing the devices consistently for each treatment session .
inconsistencies with device placement may result in variability in either stimulation intensity or brain regions activated by treatment , and thus are significant limitations of these neuromodulation approaches .
in addition to having potential therapeutic efficacy for migraine headaches , we hypothesize that cvs treatment with our device may be beneficial for a number of clinical disorders .
indeed , a pilot study ( using our device ) demonstrated that with time - varying cvs treatment , two minimally conscious patients , transitioned from involuntary to voluntary behavior and that this effect was time - locked to active treatment .
additionally , a case study in a parkinson s disease patient demonstrated time - locked and persistent improvements in both motor and non - motor symptoms with time - varying cvs treatment .
the findings for durable gains in these two studies , even after cessation of cvs treatment , suggest that cvs therapy may induce mechanisms of neuroplasticity .
additionally , these two studies highlight a range of therapeutic potential for cvs . furthermore , the comorbidity among balance disorders and anxiety disorders ( as well as migraine ) , reviewed by balaban et al . , suggest the vestibular system may play a significant role in regulating psychiatric function and mood .
thus , vestibular neuromodulation may provide a novel therapeutic approach for the treatment of anxiety and mood disorders .
finally , evidence exists which suggest that another form of vestibular stimulation , galvanic vestibular stimulation ( gvs ) , may improve postural stability and gait in parkinson s disease and balance disorders , as well as sensory perception after stroke . thus , vestibular stimulation through our cvs device may also provide therapeutic efficacy in these realms . however , there are a number of qualitative differences between cvs and gvs .
while gvs is believed to primarily affect irregular afferent neurons of the \documentclass[12pt]{minimal }
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\end{document } cranial nerve , cvs affects both regular and irregular neurons .
regular neurons , which outnumber irregular neurons by about 3:1 , seem to play a more prominent role in fine / learned motor skills whereas irregular neurons are thought to be involved in detecting changes in acceleration , . in light of these differences , it is unsurprising that neuroimaging studies have found different patterns of cortical activation in response to cvs and gvs .
finally the kinetics of activation significantly differ between these two modes of vestibular stimulation . in cvs ,
the period of a waveform is limited by the thermal conduction time of the temporal bone , whereas gvs can be implemented up to many 10 s of hz .
however , it is important to note that the actual firing pattern created by time - varying cvs in the vestibular nuclei is complex .
regular neurons have an equilibrium firing rate of ~100 hz and cold cvs lowers the firing rate whereas warm cvs raises it . a temperature ramp
thus , even though the frequency of a cvs thermal waveform is significantly less than 1 hz , the induced firing rate extends for many 10 s of hz above and below the equilibrium firing rate .
furthermore , each ear can be stimulated independently , leading to a highly complex frequency modulation space in the brainstem . from a device usage perspective , the cvs device described herein is a particularly innovative method for vestibular stimulation because it can be administered with little or no technical expertise .
it simply requires an earpiece to be fitted within the external ear canal ( like a headphone or ear plug ) and a pre - set thermal stimulus is then generated from a small stimulation unit .
unlike other rehabilitation techniques , no dedicated space , technical staff or lengthy setup procedures are required . a primary challenge for successful migraine prophylaxis ( or any preventative treatment ) is compliance with therapy over time , and thus , ease - of - use is a crucial element of design . in our pilot study with migraineurs ,
although anecdotal , measures of headache reduction after treatment with the cvs device suggest the importance of performing an adequately powered , randomized controlled trial
. such a trial will be the starting point for actual translational work addressing the needs of migraineurs , and will also lay groundwork for evaluating other therapeutic applications of the cvs device .
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### Summary:
: caloric vestibular stimulation ( cvs ) to elicit the vestibulo - ocular reflex has long been used in clinical settings to aid in the diagnosis of balance disorders and to confirm the absence of brainstem function . while a number of studies have hinted at the potential therapeutic applications of cvs , the limitations of existing devices
have frustrated that potential .
current cvs irrigators use water or air during short - duration applications ; however , this approach is not tenable for longer duration therapeutic protocols or home use . here
, we describe a solid - state cvs device we developed in order to address these limitations .
this device delivers tightly controlled time - varying thermal waveforms , which can be programmed through an external control unit .
it contains several safety features , which limit patients to the prescribed waveform and prevent the potential for temperature extremes . in this paper
, we provide evidence that cvs treatment with time - varying , but not constant temperature waveforms , elicits changes in cerebral blood flow physiology consistent with the neuromodulation of brainstem centers , and we present results from a small pilot study , which demonstrate that the cvs can safely and feasibly be used longitudinally in the home setting to treat episodic migraine .
together , these results indicate that this solid - state cvs device may be a viable tool for non - invasive neuromodulation .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: the most significant recent advancement made in the treatment of facial trauma is the rigid internal fixation and surgical approaches allowing wide exposure of the affected area .
access to the site of affected area may be made either through laceration associated with the injuries or by elective incision .
the main goal in the treatment of facial fractures is healing without postoperative morbidity or long - term deformities .
the coronal incision was initially used in the treatment of nasofrontal injuries for its wide visualization of the operative field .
later , it was being used in cases of fractures involving frontal , nasoorbitoethmoid ( noe ) , severely comminuted or displaced zygomatic arch and its components ; reduction and fixation to reestablish the facial contour is required .
the flap itself permits widespread exposure of the skull and upper and lateral midfacial skeleton , with minimal morbidity . in treating fractures of the zygomatic complex , various approaches , and surgical incisions
have been described ( michelet et al . , 1973 ) , the coronal being one of them .
traditional closed techniques still compete with total exposure of all the fracture lines by multiple incisions ( manson et al . , 1985 ) , or routine coronal incisions ( gruss et al . ,
while there is no doubt due to this wide exposure of the cranial and facial skeleton obtained , a balance has to be maintained between exposure and flap - associated morbidity .
the purpose of this study was aimed to overcome this morbidity using various modifications reviewed in literature .
a prospective study was conducted on ten patients above 10 years of age who required open reduction and internal fixation ( orif ) of the upper and middle third facial fractures with or without other associated fractures of the facial skeleton .
( 1 ) patients with well - defined hairline , ( 2 ) age above 10 years , ( 3 ) patients diagnosed with fracture of frontal bone , nasal , noe , supraorbital , lateral orbital , comminuted zygomatic complex fracture , grossly displaced zygomatic arch fractures and residual defect due to trauma and other associated midface fractures and ( 4 ) clearance from the neurosurgeon .
( 1 ) preoperative facial nerve palsy , ( 2 ) previously operated by the coronal incision , ( 3 ) mentally retarded patients , ( 4 ) patients who have fear of scar , and ( 5 ) medically compromised patients
. mode of injury , age , gender , and social habits is all recorded . in this study group ,
seven patients were operated immediately after facial trauma ( within a period of 10 days from trauma ) , three patients had posttraumatic residual defect due to trauma ( who underwent primary treatment and operated for the correction of residual defect after a period of above 10 days ) . among the study group , seven patients ( four frontal bone and three zygomatic complex fractures )
were planned for the orif and three patients ( two frontal bone and one zygomatic bone fractures ) were planned for reconstruction of the primary defect to maintain the anatomical contour .
parameters evaluated were site of fracture assessed with any of the following computed tomography scan , orthopantomogram , paranasal sinus and submentovertex skull radiographs and intraoperative clinical examination .
time taken to expose the fracture : by the coronal incision from the moment incision placed to complete exposure of the fracture .
. anatomic reduction of fracture : assessed with postoperative radiographs as excellent ( no visible radiographic gap between fracture fragments postoperatively ) and good ( < 7 mm of visible radiographic gap between fracture fragments ) .
infection : assessed by swelling , pain , tenderness , wound dehiscence , or pus discharge at the operated site .
temporal branch of facial nerve weakness : assessed clinically by examination of frontal wrinkling , tight closure of the eyes .
duration of postoperative hospitalization : calculated from the day of operation until the day of discharge ( in days ) .
temporal fossa hollowing : assessed by clinical examination only ( concavity ) has present and not present .
scar at the operated site : assessed by clinical examination as discreet ( thin incision line without fibrosis ) and hypertrophic ( with wide fibrosis ) .
all of the patients were followed for minimum of 12 postoperative weeks , with a regular interval of follow - up at 1 , 2 , 4 , 8 , and 12 to evaluate prognoses and frequency of complication .
( 1 ) patients with well - defined hairline , ( 2 ) age above 10 years , ( 3 ) patients diagnosed with fracture of frontal bone , nasal , noe , supraorbital , lateral orbital , comminuted zygomatic complex fracture , grossly displaced zygomatic arch fractures and residual defect due to trauma and other associated midface fractures and ( 4 ) clearance from the neurosurgeon .
( 1 ) preoperative facial nerve palsy , ( 2 ) previously operated by the coronal incision , ( 3 ) mentally retarded patients , ( 4 ) patients who have fear of scar , and ( 5 ) medically compromised patients . mode of injury , age , gender , and social habits is all recorded . in this study group ,
seven patients were operated immediately after facial trauma ( within a period of 10 days from trauma ) , three patients had posttraumatic residual defect due to trauma ( who underwent primary treatment and operated for the correction of residual defect after a period of above 10 days ) . among the study group , seven patients ( four frontal bone and three zygomatic complex fractures )
were planned for the orif and three patients ( two frontal bone and one zygomatic bone fractures ) were planned for reconstruction of the primary defect to maintain the anatomical contour .
parameters evaluated were site of fracture assessed with any of the following computed tomography scan , orthopantomogram , paranasal sinus and submentovertex skull radiographs and intraoperative clinical examination .
time taken to expose the fracture : by the coronal incision from the moment incision placed to complete exposure of the fracture .
. anatomic reduction of fracture : assessed with postoperative radiographs as excellent ( no visible radiographic gap between fracture fragments postoperatively ) and good ( < 7 mm of visible radiographic gap between fracture fragments ) .
infection : assessed by swelling , pain , tenderness , wound dehiscence , or pus discharge at the operated site .
temporal branch of facial nerve weakness : assessed clinically by examination of frontal wrinkling , tight closure of the eyes .
duration of postoperative hospitalization : calculated from the day of operation until the day of discharge ( in days ) .
temporal fossa hollowing : assessed by clinical examination only ( concavity ) has present and not present .
scar at the operated site : assessed by clinical examination as discreet ( thin incision line without fibrosis ) and hypertrophic ( with wide fibrosis ) .
all of the patients were followed for minimum of 12 postoperative weeks , with a regular interval of follow - up at 1 , 2 , 4 , 8 , and 12 to evaluate prognoses and frequency of complication .
all cases were treated under general anesthesia either with nasotracheal intubation , submental intubation , or oral intubation . initially ,
line of incision is marked with gentian violet ; incision site is infiltrated by 1012 ml of local anesthesia with adrenaline 1:100,000 along the marked incision line .
begins at the upper attachment of the helix extending transversely over the vault of the skull crossing the midline and extending to the helix of the opposite side ( used in upper one - third facial fractures where access is required for frontal bone ) [ figures 1 and 2 ] .
line diagram of bicoronal incision ( note the relationship of the incision to the hair line ) clinical case : ( a ) preoperative profile , ( b ) preoperative computed tomography scan , ( c ) layer by layer incision , ( d ) exposure of the fractured , ( e ) mesh was used to restore contour , ( f ) postoperative x - ray , ( g ) discreet scar , ( h ) postoperative profile begins at the preauricular region in front of the tragus of the ear or little below till the ear lobe attachment and extending transversely over the vault of the skull to the midline ( used in middle facial fractures where access is required for zygoma , zygomatic arch , and lateral orbital rim ) [ figures 3 and 4 ] .
( a ) incision with preauricular extension , ( b ) relation between incision and superficial temporal vessel clinical case : ( a ) preoperative , ( b ) residual defect in lateral orbital rim , ( c ) preoperative computed tomography scan , ( d ) exposure of the fractured site , ( e ) graft fixation done ( f ) postoperative x - rays , ( g ) discreet scar , ( h ) correction of contour defect linear incision with slight forward curvature at the vertex of the skull following the hairline , but 23 cm posterior to it was given in nine cases except in one case were the incision was curvilinear .
continuous locking sutures at the flap margin ( or ) hemostats were used as an aid to achieve hemostasis .
hemostatic clips ( raney 's ) were avoided in our study to minimize hair loss .
the flap is raised to gain exposure of the fractured site . if supraorbital margins had to be exposed neurovascular bundles
were released from the supraorbital notch / foramen and reflected along with the flap . in nine cases ,
lateral dissection was carried out on the outer surface of the temporalis fascia to approximately 2 cm above the zygomatic arch . at that point where the temporalis fascia splits into two layers ,
an incision running anteriosuperiorly at 45 was made through the superficial layer of the temporalis fascia to spare the frontal branches of facial nerve .
in one case , the lateral incision was carried till subperiosteal layer ( dual plane dissection ) by incising the temporalis muscle to expose the lateral orbital rim .
once the plane of dissection was established deep to the superficial layer of the temporal fascia , the dissection was continued inferiorly until the periosteum of the zygomatic arch .
it was then incised and reflected laterally over the arch , the body of the zygoma , and the lateral orbital rim .
it provided excellent exposure of the frontal bone , the upper part of the nose and nasoethmoidal region , the roof , medial and lateral walls of the orbit , the zygomatic bone , and the entire zygomatic arch [ figure 5 ] .
other incisions such as intraoral vestibular and the infraorbital were added for further exposure when necessary . after reduction and fixation ,
double relaxation suturing was done for periosteum and the temporal fascia sutured with 2-3 vicryl , galea , and skin were closed in layers with 23 proline .
pressure dressing was applied for 48 h in all patients and in five cases where the chance of hematoma is expected , there suction drain was used to minimize the postoperative complication of hematoma .
begins at the upper attachment of the helix extending transversely over the vault of the skull crossing the midline and extending to the helix of the opposite side ( used in upper one - third facial fractures where access is required for frontal bone ) [ figures 1 and 2 ] .
line diagram of bicoronal incision ( note the relationship of the incision to the hair line ) clinical case : ( a ) preoperative profile , ( b ) preoperative computed tomography scan , ( c ) layer by layer incision , ( d ) exposure of the fractured , ( e ) mesh was used to restore contour , ( f ) postoperative x - ray , ( g ) discreet scar , ( h ) postoperative profile
begins at the preauricular region in front of the tragus of the ear or little below till the ear lobe attachment and extending transversely over the vault of the skull to the midline ( used in middle facial fractures where access is required for zygoma , zygomatic arch , and lateral orbital rim ) [ figures 3 and 4 ] .
( a ) incision with preauricular extension , ( b ) relation between incision and superficial temporal vessel clinical case : ( a ) preoperative , ( b ) residual defect in lateral orbital rim , ( c ) preoperative computed tomography scan , ( d ) exposure of the fractured site , ( e ) graft fixation done ( f ) postoperative x - rays , ( g ) discreet scar , ( h ) correction of contour defect linear incision with slight forward curvature at the vertex of the skull following the hairline , but 23 cm posterior to it was given in nine cases except in one case were the incision was curvilinear .
continuous locking sutures at the flap margin ( or ) hemostats were used as an aid to achieve hemostasis .
hemostatic clips ( raney 's ) were avoided in our study to minimize hair loss .
the flap is raised to gain exposure of the fractured site . if supraorbital margins had to be exposed neurovascular bundles
were released from the supraorbital notch / foramen and reflected along with the flap . in nine cases ,
lateral dissection was carried out on the outer surface of the temporalis fascia to approximately 2 cm above the zygomatic arch . at that point where the temporalis fascia splits into two layers ,
an incision running anteriosuperiorly at 45 was made through the superficial layer of the temporalis fascia to spare the frontal branches of facial nerve . in one case ,
the lateral incision was carried till subperiosteal layer ( dual plane dissection ) by incising the temporalis muscle to expose the lateral orbital rim .
once the plane of dissection was established deep to the superficial layer of the temporal fascia , the dissection was continued inferiorly until the periosteum of the zygomatic arch .
it was then incised and reflected laterally over the arch , the body of the zygoma , and the lateral orbital rim .
it provided excellent exposure of the frontal bone , the upper part of the nose and nasoethmoidal region , the roof , medial and lateral walls of the orbit , the zygomatic bone , and the entire zygomatic arch [ figure 5 ] .
other incisions such as intraoral vestibular and the infraorbital were added for further exposure when necessary . after reduction and fixation ,
double relaxation suturing was done for periosteum and the temporal fascia sutured with 2-3 vicryl , galea , and skin were closed in layers with 23 proline .
pressure dressing was applied for 48 h in all patients and in five cases where the chance of hematoma is expected , there suction drain was used to minimize the postoperative complication of hematoma .
of 10 patients , 9 patients were male and 1 female , with a male : female ratio of 9:1 .
all the patients ranged from 11 to 45 years with the mean age of 27.1 years .
seventy percent ( 7 cases ) pre- and post - operative radiographs were compared to check for proper reduction and fixation . among them , 20% ( 2 cases ) were assessed as good reduction , 50% ( 5 cases ) as excellent . in the remaining 30% ( 3 cases ) , this surgical incision was performed to maintain the facial contour , which was achieved excellently . the average time taken for exposure of the fracture site by the hemicoronal incision was 27 min with values ranging from 25 min to 30 min .
in case of bicoronal incision , the average time for exposure of fracture is 41 min with values ranging from 35 to 45 min .
surgical access was good in 10% of ( 1 case ) cases and excellent in 90% of cases ( 9 cases ) .
none of the cases in this study encountered any form of infection , except for one patient ( case 3 ) , who had swelling with abscess for almost 8 weeks postoperatively .
it subsided on taking oral medication but recurred once the medication was discontinued and swelling completely subsided after the plate removal on the 12 postoperative week .
however , facial asymmetry in the region of middle one - third of zygomatic arch is persistent till date . in this study , 30% ( 3 cases ) were reported with transient deficit of temporal branch of facial nerve .
out of which , 20% ( 2 cases ) recovered within 2 postoperative week , whereas in 10% ( 1 case ) facial nerve palsy recovered by the end of 12 postoperative week .
the average duration of postoperative hospitalization in 50% ( 5 cases ) with suction drains was 4 days and in 50% ( 5 cases ) without suction drains was 3 days .
the surgeon was comfortable in gaining access to fractured site in all the cases . in 90% ( 9 cases ) , patients were satisfied with the esthetics due to minimal or invisible scar within the hairline . except in 10% ( 1 case ) , where there is persistent facial asymmetry till date even after removal of the plate at 8 postoperative week [ table 2 ] . in this study , no cases of postoperative hemorrhage , hematoma , permanent facial nerve palsy ( temporal branch ) , alopecia , hypertrophic scar or temporal fossa hollowing were reported .
patients with a severe comminution and displacement of the facial skeleton have been treated conventionally through small local incisions with the wiring of bone fragments in position , immobilization of jaws with arch bars , and occasional use of immediate bone grafting for the restoration of facial buttresses . in selected patients ,
the coronal approach provides wide exposure and direct availability of calvarial bone grafts and enables the surgeon to accurately reconstruct the shattered face ( multiple fractures ) with intact bone of similar shape and consistency .
large areas of shattered bone can be easily replaced with intact segments of calvarial bone graft and are subsequently immobilized accurately through the widely exposed operative field .
this technique provides for reconstruction of all fractures or missing bony fragments under direct vision .
it has a great advantage over the conventional techniques of reduction and fixation while treating upper and middle facial fractures .
out of ten patients , there was high male predominance to female with ratio of 9:1 , which correlates with various studies by nysing , lundin et al . and
adam et al . who , had a male to female ratio of 4:1 and study conducted on coronal approach for zygomatic complex fracture by shetty et al .
road traffic accidents are the major cause of fractures of the upper and middle third facial region ( 100% ) .
the age groups of patients ranged from 11 years to 45 years with an average 27.1 years , correlates with results published by zhang et al .
, who had a mean age of 35 years , adam et al . , who had a mean age of 32.33 years and also as reported by ellis et al . , who reported their peak in the second and third decades of life
the classical coronal incision was used as advocated by abubaker et al . in their study on the coronal incision for craniomaxillofacial injuries . in one case ,
curvilinear skin incision was placed till helix and extended to traditional preauricular incision , which was advocated by ramon et al . and munro and fearon .
five cases were operated by hemicoronal incision with preauricular extension , and five cases were treated with bicoronal approach , whereas in four cases , additional local incisions were used to gain access for fractured site . in one case , dual plane dissection ( temporal region )
extended coronal incision into preauricular region with traditional coronal incision will give access to temporomandibular joint ( tmj ) and zygomatic arch as advocated by dunaway and trott .
have all reported excellent access to the upper midfacial skeleton , lefort iii fracture , supraorbital margin , frontonasal suture , nasoethmoidal suture , lateral orbital region , zygomatic complex fracture , zygomatic arch , and tmj by the coronal surgical incision .
even reconstruction of supraorbital , frontal , nasal , and nasoethmoidal region can be done with minimal scar .
6 cases achieved excellent reduction and 1 case achieved good reduction . in the remaining 3 cases , anatomical correction was evaluated esthetically by correcting contour defect which is caused due to trauma , with autografts ( iliac bone ) in one case ( lateral orbital rim ) and alloplast ( titanium mesh ) in two cases ( frontal bone ) .
, who reported that this approach allows for accurate anatomic reduction and fixation of fractures ; marschall et al . reported that reduction of displaced fragments under direct vision and replacement of missing bone with a required material
suction drains were used in 5 cases along with double - layered suturing for incision closure ( galea to galea and skin to skin ) , and pressure dressing for 48 h was applied for all cases in our study .
reported two cases ( repeated episodes of hypertension ) out of 28 patients with hematoma .
shetty et al . reported no case of hematoma in his study of coronal approach for the treatment of the zygomatic complex fractures .
hodges and altman reported that the use of stabilize drains while closing coronal flap will minimize the complication of hematoma . this technique was used in five cases in this study , and findings were correlated .
only one case reported with an abscess which resolved on the removal of mini plate in f z region of lateral orbital rim by giving a local incision and antibiotic therapy .
the findings are similar to various studies of frodel et al . , who reported no cases of flap infection , whereas just four cases of stitch abscess , which resolved on the removal of sutures ; mitchell et al . reported wound infection in six cases who were operated for primary facial trauma due to secondary infection .
of which two cases recovered within 2 postoperative week while in one case , facial nerve palsy recovered by the end of 12 postoperative week .
had 3 cases of facial nerve weakness , out of which two resolved within 9 months and one case persisted till 2 years .
, six out of 69 cases had facial nerve weakness , and one case had a permanent palsy .
had reported nine patients with temporary motor weakness and two patients with permanent facial nerve weakness .
mitchell et al . reported 6 cases of transient deficit of the temporal branch which had resolved within the follow - up period .
dunaway and trott reported three cases with mild temporary facial palsy , which resolved by 6 postoperative week .
none of the cases reported with the problem of alopecia by the end of 12 postoperative week .
reported five cases with telogen phenomenon for 0.51 cm around the scar , which was till 23 months . in one patient , this was permanent . in the patients with hair loss , it was noted that operative time was over 12 h , suggesting prolonged ischemia secondary to the application of raney clips , as a cause for alopecia .
burm and oh reported a surgical procedure to minimize the scar formation and alopecia , by 30 beveling to incision and double relaxation of galeal suturing , as performed on two patients .
surgeon comfort for reduction and fixation of fracture along with correction of contour defect was excellent in all the cases .
findings of this study correlated with the various studies conducted by hartley and kenyon ( 1907 ) , who first reported that these approach will give a good access to anterior cranial vault .
reported that it is the widely accepted approach for the reduction of upper and middle facial fractures along with reconstruction of this area because of its direct accessibility to the fractured site .
all the patients were satisfied with this surgical approach except one patient ( case 3 ) .
it is esthetically also excellent because most of the surgical scar is hidden within the hairline ; even if the incision is extended into the preauricular region , it was not clearly visible .
the duration of postoperative hospitalization , which was to be considered as disadvantage was evaluated . instead of giving multiple incisions for exposing the fracture site in the upper and middle third region of the face which will cause an esthetically unpleasant appearance with minimal hospitalization , a single incision with hidden scar with mild extended postoperative
hospitalization is better . in this study , we have encountered a new complication of facial asymmetry ( case 3 ) at the operated site of temporal region , which might probably be due to fibrosis of temporalis muscle because of dual plane dissection which was advocated by srinivasan et al .
this finding has proved that classical coronal incision advocated by abubaker et al . is better than dual plane dissection .
coronal incisions have advantages such as giving excellent access to the upper and middle third and lateral side till tmj for exact anatomical reduction of fractures along with reconstruction of residual defects . on the other hand
, it has few disadvantages which can be minimized by having a thorough knowledge of the anatomy of related structures involving the surgical site with various modifications in surgical method .
even though the time taken for exposure of the fracture site is more by this approach , multiple fractures ( least number of complications ) can be reduced and fixed with a single incision with a relatively good anatomical reduction .
this incision should not be misused in cases of isolated facial fracture , fractures that can be treated by closed reduction and the fractures that can be corrected with preexisting laceration .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
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### Summary:
: background : coronal incision is a popular and versatile surgical approach to the anterior cranial vault and upper and middle third facial skeleton . the flap itself permits widespread exposure of the fractures in this region . the bicoronal flap was first described by hartley and kenyon ( neurosurgeons ) to gain access to the anterior cranium in 1907 .
it extension as an access flap to the upper and lateral aspect of the face was pioneered by tessier ( 1971 ) .
esthetically , it is pleasing as the surgical scar is hidden within the hair.aims:to evaluate the versatility of coronal incision using various modifications advocated in incision , exposure to fractured site , and closure of flap in treating the upper and middle third facial fractures.materials and methods : a total of ten patients diagnosed with upper and middle third facial fractures requiring open reduction and internal fixation / correction of contour defect were selected after preoperative clinical and radiographic ( computed tomography scan ) evaluation .
all the cases were operated by coronal approach to gain the access to the fracture / defect site for reduction / correction of the defect .
advantages and complication are evaluated.results:excellent access and anatomical reduction by this approach with least number of complications ; if it is performed with healthy knowledge of anatomy of the scalp and temporal region .
certain minimal complications have also been noted using various modifications used in the procedure.conclusion:despite of prolonged surgical time for the exposure , it is very advantages in treating upper and middle third facial fractures due to wide access and discreet scar ( minimal ) .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: the prevalence of childhood obesity has more than tripled over the past three decades .
the latest nhanes data ( 20032006 ) revealed that approximately 35% of 611-year - old children are classified as
[ 1 , 2 ] . this level of obesity in children begets risk factors for cardiovascular disease ( cvd ) and metabolic diseases .
approximately 25% of obese children have 2 cvd risk factors [ 3 , 4 ] and more than 45% of newly diagnosed pediatric diabetes cases are classified as adult - onset type 2 diabetes [ 47 ] .
this alarming evidence has pushed the prevention of childhood obesity to the forefront of today 's scientific research .
this study also reveals that although childhood obesity knows no gender or racial boundary , the rates of obesity are more prevalent in some minority populations . in the 69,000 children ( 517 years ) measured across the united states , a significantly higher prevalence of overweight or obesity
was reported in black children compared to white , hispanic- or other minority children- and boys were reported to have a higher prevalence of obesity compared to girls .
studies have shown that childhood obesity equates over time to as little as a + 2% imbalance between daily energy intake and energy expenditure ( ee ) .
this small positive imbalance can be abated with an increase in regular physical activity ( pa ) .
population - based objective measures of children 's pa levels revealed that only 42% of 611-year - old children acquire the recommended 60 min / day of moderate - to - vigorous pa ( mvpa ) ; for 1215-year - old adolescents , this number drops dramatically to < 10% .
further , the concomitant increase in the amount of time children spend in sedentary pursuits ( e.g. , in the classroom , watching television , or playing video games ) is independently associated with lower levels of pa and daily pa energy expenditure ( paee ) , lower cv fitness , increased risk for obesity , and obesity - related health consequences [ 1015 ] .
these associated risks are exacerbated by the reality that obese children have a preference for sedentary behaviors , spend more time being physically inactive , and may not spend as much energy during pa compared to their nonobese pairs [ 1618 ] .
randomized - controlled intervention studies targeting obese children have successfully increased mvpa participation to enhance body composition and cv fitness [ 19 , 20 ] . however ,
healthy developmental changes in body composition ( e.g. , increased fat - free mass with no change or a decrease in fat mass ) and cv fitness ( e.g. , improved aerobic capacity ) have not been demonstrated in studies of children with a wide range of adiposity .
these results may be attributed to ( 1 ) an insufficient pa dose to correct for energy imbalance in all children , ( 2 ) the imprecise measurements of body composition ( anthropometric measures ) that lack the sensitivity to detect changes in growing children , and ( 3 ) the short duration of the training program , limiting the capacity to detect either small decreases in weight gain or the small excessive increases in weight gain associated with childhood obesity [ 22 , 23 ] .
therefore , we sought to evaluate the efficacy of a 10-month pa intervention on ( 1 ) the prevention of excessive age - related increases in body fatness and ( 2 ) cv fitness .
we hypothesized that black boys who participated in a 10-month after - school pa intervention would prevent excessive age - associated changes in body fatness in addition to a concomitant increase in cv fitness , compared to black boys who did not participate in the 10-month pa program .
black boys ( 812 years of age ) were recruited from five local elementary schools using study fliers .
all 3rd through 5th grade black boys were eligible if they met the following criteria : ( 1 ) weigh < 300 lbs ( equipment limitation ) , ( 2 ) not taking any medications known to affect metabolism , body composition , or fat distribution ( e.g. , ritalin or concerta ) , and ( 3 ) have no known cv , metabolic , or respiratory disease or physical impairment that would limit their participation in regular pa .
twenty - eight percent ( 300 boys ) of the targeted population ( 1050 boys in 3rd5th grade ) were screened by phone to determine their eligibility to participate in the study .
potential participants and their parent or guardian were invited to attend a group information session where they read and signed the informed consent / assent documents in accordance with the medical college of georgia human assurance committee .
participants were given a monetary incentive for the testing portion of the study only ; no additional monetary incentive was offered for attending the intervention .
although recruitment was not based on adiposity , the distribution was representative of the higher weight status in the state of georgia .
of the 157 boys who consented to be in the study , 122 underwent baseline testing .
although siblings were allowed to participate in the study , only one sibling per family was used in the analyses . within each family , the sibling with the most data points
was selected for analysis . in the case of ties , the sibling with the lowest identification number was selected .
the identification numbers were not assigned in any specific order ( e.g. , oldest sibling first ) .
after removing the siblings ( n = 4 ) from the analyses and accounting for those participants who either ( 1 ) were unreachable for baseline or follow - up testing ( n = 26 ) , ( 2 ) declined participation ( n = 9 ) , ( 3 ) became ineligible for participation due to medical reasons ( n = 8) , or ( 4 ) were expelled for disciplinary issues ( n = 4 ) , 106 boys were included in the analyses .
participants reported to the georgia prevention institute of the medical college of georgia for testing prior to the beginning of the study and again after 10 months .
baseline testing began during the summer months before schools started and continued until mid - fall with as many as three boys tested on any given day .
the boys were integrated into the intervention on a rolling basis with each subject given the opportunity to participate in at least 10 months of the intervention .
follow - up testing for the intervention group was conducted within 13 days following the last day of the 10-month intervention .
all testing was conducted by trained study personnel with an exercise physiology or related background .
pubertal status was assessed by study pediatricians based on the criteria established by marshall and tanner [ 24 , 25 ] . utilizing gonad and pubic hair development separately
, the subject was then classified as immature ( tanner 1 - 2 ) , peripubertal ( tanner 3 - 4 ) , or mature ( tanner 5 ) for the analyses . examination of the tanner staging for pubic hair and gonad development was not refused by any of the boys .
height , to the nearest 0.1 cm , and weight , to the nearest 0.1 kg , were measured by standard methods using a wall - mounted stadiometer ( healthometer ) and floor scale ( dectecto ) , respectively . body mass index ( bmi )
was calculated as weight(kg)/height(m ) , and bmi percentile ( bmi% ) was obtained from the centers for disease control age- and gender - specific growth charts .
waist circumference was measured along the narrowest width between the rib cage and the umbilicus .
total body composition was measured in a 3-compartment model as total fat mass ( fm ) , fat - free mass ( ffm ) , and bone mineral content ( bmc ) using dual - energy x - ray absorptiometry ( dxa ; hologic qdr-1000 , waltham , ma ) .
the boys were measured in a supine position while wearing light clothing with no metal and no shoes or jewelry .
bone mineral density and % bf were derived from the three aforementioned components using the dxa scanner software ( hologic , version 2.3.1 ) .
cardiovascular fitness ( vo2 max ) was assessed by the method of indirect calorimetry ( sensormedics vmax 229 cardiopulmonary system , yorba linda , ca ) using a multistage treadmill test .
the treadmill protocol began with a 4-min warm - up at 2 mph and 0% grade .
the speed was then increased 0.5 mph every 2 minutes to 3.0 mph followed by 2%-3% grade increases every 2 minutes until reaching 20% grade ( maximum grade of the treadmill ) or until voluntary exhaustion .
participants were asked to rate their perceived exertion using the 620 point borg scale .
participants were considered to have attained vo2 max if they met two of the following three criteria : ( 1 ) an increase in heart rate ( hr ) < 5 bpm between the final two workloads , ( 2 ) an increase in oxygen consumption ( vo2 ) < 100 ml / min between the final two workloads and ( 3 ) a respiratory exchange ratio > 1.10 . although all participants were encouraged verbally to give a maximal effort , approximately half of them stopped the test voluntarily before reaching vo2 max .
in addition , only about half of the boys who attained vo2 max at baseline also did so at followup . because the achievement of maximal effort is not common in participants of this age , an alternate index of cv fitness
was used : vo2 at an hr of 170 bpm ( vo2 - 170 ) .
vo2 - 170 was determined from individual regression analyses of the vo2 and hr responses to all completed workloads during the treadmill test .
heart rate was monitored throughout the test using a polar hr monitor ( polar usa , lake success , ny ) .
the boys were questioned about their activities , including sleep , over the seven days prior to the interview , starting with the previous day and working backwards .
thus , the seven - day recall included the pa associated with the pa program for the boys in the intervention group at followup .
this interview , typically completed in 2030 minutes , was conducted by trained study personnel .
values of hard pa ( identified as pa that caused increased breathing and moderate movement including activities such as , dancing , aerobics , and soccer ) and very hard pa ( identified as pa that caused hard breathing and quick movements including activities such as , tennis , cycling , and running ) were summed to derive an index of vigorous pa ( vpa ) .
mvpa was calculated as the sum of vpa and moderate pa ( identified as pa that caused normal breathing and some movement including activities such as , walking briskly , volleyball , gymnastics , and gardening ) .
following baseline testing , participants were randomized into either the intervention group ( n = 62 ) or the control group ( n = 44 ) with a ratio of three to two , respectively .
in the instance of siblings , the first to be tested was randomized and the remaining sibling(s ) was / were placed in the same group .
participants in the control group received no intervention and were not allowed to stay for the after - school intervention but rather instructed not to change their daily after - school routine .
participants in the intervention group stayed at their school at the end of each full school day ( 177 8.6 days ) to receive a 2-hour intervention over a 10-month period , excluding school holidays .
briefly , the intervention was conducted by trained study personnel with exercise - related education plus 1 - 2 trained classroom teachers . the program consisted of 30 minutes of homework time during which the boys were provided with a healthy snack followed by 80 minutes of pa .
all the snacks were individually packaged and every day the boys had a choice of something salty ( e.g. , crackers and cheese ) , something sweet ( e.g. , low - fat cookies ) , or a fruit or vegetable .
the pa program included 25 minutes of skills development ( e.g. , how to dribble a basketball ) , 35 minutes of vpa , and 20 minutes of toning and stretching with 5 minutes rest between each component . throughout the intervention
, the boys wore a polar s610 hr monitor ( lake success , ny ) to record their pa intensity and to estimate their ee during the sessions . during the vpa component ,
the boys were asked to maintain an hr of at least 150 bpm .
activities during the vpa component included games such as , basketball , tag , softball , and relay races , all of which were modified to keep all the boys sufficiently active ( 150 bpm ) throughout the 35-minute period . for analyses ,
the intervention group was subdivided into boys who either attended 60% of the intervention ( attenders , att ; n = 31 ) or those who did not attend at least 60% of the intervention ( nonattenders , natt ;
group comparisons of baseline and follow - up values for all outcome variables ( body composition , cv fitness , and pa variables ) were conducted using a 3 2 repeated measures anova using group ( att , natt , and control ) and test session ( baseline and followup ) as main effects .
the relationships at baseline and for the change scores among the outcome variables were determined using pearson correlation analyses . within the intervention group ,
the relationships among the outcome variables , program attendance , and hr during the aerobic portion of the program were all assessed using pearson correlation analysis .
one hundred and six 3rd5th grade boys participated in the study and were randomized into either the intervention ( n = 62 ) or the control ( n = 44 ) group .
all of the boys were classified as prepubertal for gonad and pubic hair development .
there were no significant differences at baseline among the control group , att ( n = 31 ) , and natt ( n = 31 ) for all outcome variables .
all children in the intervention group participated in a minimum of 10 months of pa for the study .
the average attendance for the intervention group was 57.7 3.1% , which varied greatly between the att ( 82.9 1.8% ) and natt ( 32.8 4.5% ) .
heart rate during the vpa component of the daily intervention was not significantly different between att and natt boys and averaged 162.0 1.4 bpm . additionally , no significant differences in total ee per session between the att and natt boys were observed .
the average ee during the 80-minute sessions was 369.8 21.4 kcal / session , of which 192.3 10.8 kcal / session was expended during the vpa component alone .
the baseline and follow - up measures of cv fitness include vo2 - 170 expressed both in absolute terms ( l / min ) and relative to body mass ( ml / kg / min ) ( table 2 ) .
there was a similar improvement in vo2 - 170 across all three groups from baseline to followup when expressed in l / min ( p < .001 ) .
however , the control group demonstrated the only significant increase in vo2 - 170 when expressed in ml / kg / min ( p < .0001 ) . within the att group alone
, there was a positive relationship between the ee during the vpa component of the intervention and changes in vo2 - 170 expressed as l / min ( r = 0.56 ; p = .0018 ) and when expressed as ml / kg / min ( r = 0.47 ; p = .012 ) . additionally , a negative relationship was observed between the vpa component ee and the decrease in % bf ( r = 0.43 ; p = .02 ) .
interestingly , no relationships were observed between either the percent attendance or the average hr maintained during the vpa component of the intervention sessions and the changes in body composition or cv fitness measures in either the att or natt . at baseline , the amount of mvpa reported by the boys was similar among the control , natt , and att groups . additionally , no relationships among mvpa and measures of cv fitness and body composition were observed . at followup ,
the att reported a significant increase ( p = .04 ) in mvpa by 34.8 min / day ; however , no change in mvpa for the control group or the natt group was reported ( figure 1 ) .
there were no significant relationships between the change in reported mvpa and changes in bmi ( r = 0.08 ; p = .44 ) , % bf ( r = 0.12 ; p
= .26 ) , and vo2 - 170 ( r = 0.03 ; p = .73 ) among all three groups .
there was a negative relationship among cv fitness and body composition measures at baseline for the entire population sample and these relationships were stronger at followup ( table 3 ) .
a significant group time interaction was observed for % bf ( figure 2(a ) ) , such that both the att ( p < .0001 ) and natt ( p = .019 ) groups exhibited a decrease in % bf , whereas no significant change was observed in the control group ( p = .18 ) .
additionally , the decrease in % bf among the boys in the att group was significantly greater than the change in the control group ( p = .029 ) .
a group by time interaction for bmi was also observed ( figure 2(b ) ) following the 10-month program , such that the control group exhibited a significant increase in bmi ( p = .0034 ) , whereas no change was observed in the natt group ( p = .06 ) or the att group ( p = .33 ) .
the change in bmi in the att group , a slight decrease , was significantly different than the change in both the natt group ( p = .046 ) and the control group ( p = .009 ) .
further , a similar group by time interaction was observed for changes in fm ( figure 2(c ) ) .
finally , the changes in ffm ( p = .91 ) and bmd ( p = .91 ) and bmc ( p = .85 ) over the 10 months were similar across all groups .
the main finding of this study is that 10 months of after - school pa can prevent further accretion of undesirable levels of adipose tissue in black boys of varying adiposity levels .
the boys who attended the intervention at least 3 days / week had a significant reduction in body fat , bmi , and fat mass compared to significant increases in bmi and fat mass observed in the control group .
although there were similar increases in ffm between the control and att groups , these findings indicated a beneficial effect of physical activity on overall body composition in young black boys .
a unique aspect of the present study included the statistical power to divide the pa intervention group and compare attenders versus nonattenders , those boys who attended at least 60% ( att ) and those who did not ( natt ) , respectively .
this comparison lends support to the beneficial response of pa on body composition , only if consistency is maintained .
attenders exhibited a significant decrease in % bf of 2.25 0.57% compared to a decrease of only 0.63 0.44% in the control group over the 10-month pa intervention .
although the boys who did not attend at least 60% of the intervention gained less fm than the control group , the increase in bmi over the 10 months was similar to the control group .
consequently , this resulted in a similar and nonsignificant decrease in % bf in the natt and control groups .
interestingly , this change in body composition was independent of changes in ffm because all three groups had similar increases in ffm
. moreover , there were similar changes in bmc , bmd , and ffm across all three groups of boys which indicates that although there was an increase in reported mvpa in the att group compared to the control group , this increase in ee associated with participation in the intervention did not hinder increases of lean tissues related to normal growth patterns over the 10 months .
the results of this intervention are comparable , but less dramatic , to another study conducted in our laboratory with young black girls using a similar after - school intervention .
the success of these pa interventions , compared to less successful interventions , is , in part , attributed to the exercise dose ( duration and intensity ) provided by the program .
the average daily energy cost of the program ( 370 21 kcal / session ) is more than twice the magnitude of the proposed energy surplus associated with childhood obesity ( 100165 kcal / day ) [ 23 , 31 ] .
it is speculated that small increases in total daily ee over time will abate the excessive increases in body mass associated with obesity .
this is supported by the fact that only the boys who attended the intervention program on a regular basis reduced their adiposity levels over the 10 months , whereas the boys in the control or natt groups both exhibited an increase in fm over the same time period . although small , there was a positive change in bmi as a result of attending the program .
if these positive changes are compared to the cdc 's age- and gender - specific growth charts , this small reduction or prevention of increasing bmi may have significant physiological and health implications .
for example , at age 8 , a child with a bmi of 21 kg / m is classified as obese according to these standardized charts .
a year later , if that same child increased his bmi by the amount observed in our control group ( 0.5 kg / m ) , he would continue to be classified as obese by age 9 . instead ,
if that same child a year later slightly reduced his bmi as seen in our att group ( 0.2 kg / m ) , he would be classified as overweight rather than obese by age 9 .
if this trend was to continue , by age 12 , that child who participated in regular pa similar to our intervention would have a bmi of 20.2 kg / m and would be classified as a healthy weight , thus reducing his potential risk for obesity - related diseases .
although these findings are promising , with half of the participants in the intervention group not acquiring the desired pa dose ( < 60% attendance ) , it is unclear if this program is generalizable across populations . in the current study ,
the children were asked to play at a vigorous intensity by maintaining an hr of at least 150 bpm during the 35-minute vpa component of the intervention .
using a standardized maximal hr ( hrmax ) of 200 bpm for children , the average hr achieved during the intervention ( 162 bpm ) was 81% of predicted hrmax , which is classified as vigorous intensity pa ( 70%89% hrmax ) .
recent studies along with data from our laboratory suggest that vpa might have a favorable impact on body composition which is to some degree independent of ee [ 14 , 33 ] .
the energy cost of the present intervention was derived from individual linear regressions developed from the hr and vo2 responses during the graded treadmill tests . in support
, the results suggest that the ee was sufficient to abate deleterious changes in body composition and improvements in cv fitness .
additionally , these results are consistent with descriptive studies indicating that accelerometry - measured vpa is strongly related to lower levels of fatness and better fitness than moderate pa ( mpa ) . in the current study
, there were no relationships between the measure of exercise intensity ( hr during the vpa component ) and either body composition or cv fitness ; however , there was a weak relationship between these same factors and the ee during the vpa component .
although the intervention promoted healthy changes in body composition , the intervention had little impact on cv fitness .
the improvements in absolute measures of cv fitness ( l / min ) were similar across all three groups ; however , in contrast to our hypothesis , when expressed relative to body mass , the control group had the greatest increase in cv fitness ( 9.8% ) compared to both the att group ( 5.5% ) and the natt group ( 5.3% ) .
although we can not explain this increase , the effort of the control group during follow - up exercise testing may have been greater than baseline testing . in support of similar postintervention cv fitness changes ,
a recent review of the literature revealed that endurance training at a frequency of 1 to 5 days / week , 20 to 60 min / session , at an intensity of 70% to 90% of maximal hr is necessary for improving cv fitness from 5% to 15% .
however , the most common activities in these studies involved more endurance training , steadystate - type activities ( e.g. , aerobics , running , cycling , etc . ) rather than common children 's games ( e.g. , nonsteady - state games ) employed in the current intervention .
the review further states that boys needed at least 60 min / day of mvpa to promote higher cv fitness .
this suggests that the current intervention , although sufficient to counterbalance the energy surplus of obesity , was not sufficient in intensity and/or duration per session to enhance cv fitness in young boys .
the implementation of this intervention study and its choice of some outcome measures , to our knowledge , have never been implemented and add to the strength of the present findings . the length of the program allowed for revealing the impact of small daily changes in mpa and vpa on body composition as children go through the growth process . the magnitude of the sample size , focusing only on a single gender and race , allowed for the power to emphasize the changes in a population that is at higher risk for obesity and obesity - related diseases .
finally the sensitivity of some of the outcome measures , specifically the dxa , allowed for the detection of small but significant changes in body composition that would not be detected via other measures ( e.g. , skinfolds ) [ 35 , 36 ] .
the levels of pa participation in this study exceed the pa dose recommended in the most recent pa guidelines published by the u.s .
department of health and human services ( 60 min / day of mvpa of which vpa should be included at least 3 days / week ) .
however , using self - report assessments in the form of questionnaires have demonstrated low - to - moderate validity for assessing pa levels in children . in the current study ,
the increase in mvpa in the att group at followup may be attributed to the regularity of the after - school program and the investigators cues about whether or not the subject participated in the program during the administration of the recall at followup .
objective monitoring of mvpa is warranted to assess changes in mvpa in future intervention studies . in conclusion ,
young black boys who attended the 10-month after - school pa intervention at least 3 days / week exhibited healthy weight management compared to both the boys who did not attend the intervention and the control group .
although there were no relationships between the changes in mvpa and body composition , the postintervention decrease in bmi and % bf observed in the att may be indicative of an improved energy balance over the 10 months .
these findings implicate the importance of engaging in physical activity at least 3 days per week to prevent age - associated increases in bmi , fm , and body fat and reduce the risk of childhood obesity .
research should continue in this area , using more objective means of assessing changes in pa participation and assessing program intensity to determine the impact of such a program on total daily energy expenditure .
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### Summary:
: objective . to determine if a 10-month after - school physical activity ( pa ) intervention could prevent deleterious changes in body composition and cardiovascular ( cv ) fitness in young black boys
. methods . following baseline measures , 106 boys ( 812 yrs ) were randomized to either a control group or an intervention group , further divided into attenders ( att ) and nonattenders ( natt ) , participating in 60% or < 60% of the intervention , respectively .
the daily intervention consisted of skills development ( 25 min ) , vigorous pa ( vpa , 35 min ) , and strengthening / stretching ( 20 min ) components .
body composition was measured by dual - energy x - ray absorptiometry . results .
following the intervention , the att exhibited an increase in moderate - to - vigorous pa and a significant reduction in bmi , fat mass , and % bf compared to the control group .
a significant association among the intervention energy expenditure and changes in body composition and cv fitness was observed only in the att group .
conclusion .
an after - school pa program of sufficient length and intensity can promote healthy changes in body composition and fitness levels in black boys who attend at least 3 days / week .
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: the small sample size and lack of control group are the main limitations of the study .
treatment of recurrent or resistant warts is still a very difficult proposition.immunotherapy is an exciting treatment option for such cases.multiple skin test antigens have been used in immunotherapy of warts.there are still no clinical studies from india on candida immunotherapy in the management of warts .
there are still no clinical studies from india on candida immunotherapy in the management of warts .
warts are a common sight in dermatology opds and they constitute the commonest cutaneous manifestation of human papilloma virus ( hpv ) infection . warts ( verrucae ) are primarily treated by destructive therapies such as topical keratolytics , electro fulguration or radiofrequency ablation , liquid nitrogen cryotherapy , and laser vaporization with either pulsed dye laser or co2 laser .
other options include intralesional chemotherapeutic agents such as bleomycin , oral immune modulators like cimetidine , levamisole , or even oral antivirals like cidofovir .
thus , we have patients in whom the warts resolve spontaneously with the simplest treatment or even without any medical treatment , and at the other extreme , there are patients in whom the warts seem to be resistant to every possible mode of treatment .
moreover , even after an individual patient has responded to any of these treatment options , there are chances of relapse and it is quite common to come across patients with multiple troublesome recurrences . in patients with unresponsive or recurrent warts , immunotherapy is an exciting treatment option and over the last few years , various treatment methods have been described that basically stimulate the immunologic response to hpv .
the first and the simplest method is topical application of certain inorganic molecules that are capable of eliciting a contact hypersensitivity reaction with secondary activation of an immunological response , or even topical applications of immune modulators like imiquimod .
another way is to inject the immunotherapeutic agent into the wart tissue itself . in case of multiple warts , it is usually the mother wart or the wart that appeared first that is injected .
the possible mechanism of action of immunotherapy is that the injection or application of the immunotherapeutic agent leads to proliferation of hpv - specific mononuclear cells that mediate an immunologic attack against the wart tissue .
what is interesting is that this immune attack has a potential to resolvethe distant warts as well and not the wart alone that has been primarily injected .
studies have been conducted on the effectiveness of immunotherapy with single as well as multiple test antigens in treatment of warts .
the first such study documented the efficacy of candida skin - test antigen in wart resolution .
subsequent studies were conducted on this antigen and also on other antigens such as mumps , trichophyton , and even a combination of multiple test antigens .
all of these studies have shown that immunotherapy not only causes a resolution of the treated wart but also leads to clearance of distant warts , at least in a subset of the responders .
this study was conducted from november 2010 to january 2012 at our institute on patients suffering from resistant or recurrent veruccae anywhere on the body .
a proper permission was obtained from the local ethics committee before the initiation of the study .
inclusion criteria included patients with multiple veruccae vulgaris that were either resistant to treatment or had relapsed at least once after treatment with any of the tissue - destructive modalities .
the veruccae were termed as resistant only if they failed to show any response to at least one destructive mode of treatment .
patients with any evidence of immunosuppression including hiv infection , patients with any eczematous skin disorder , those with any history of hypersensitivity to candida albicans antigen , pregnant or lactating women , patients on immunosuppressant or immune modulatory drugs , and patients younger than 12 years of age were excluded from the study .
patients were informed about all the modalities of treatment available for warts and the relative merits and demerits of c. albicans immunotherapy including a possible non - response to the treatment regimen .
an informed consent form was then given to the patients and they were made to sign it themselves or by their guardians .
the warts were then photographed and counted and the size of the injected wart was also noted down . before inclusion into the study ,
a test dose of the candida antigen was given intradermally on the flexor aspect of the forearm and the area of injection was marked with a permanent marker pen .
the patient was called after 2 days ( 48 h ) and the reaction at the intradermal injection site was noted down .
erythema and induration at the site were observed , and the response to the test dose was taken as positive if the area of erythema and induration was at least 5 mm in diameter . only those patients who showed a positive response to the candida test antigen were enrolled and taken up for treatment with c. albicans antigen injections .
a cohort of 40 such patients was thus selected for the study and each of these patients received intralesional injection of purified c. albicans antigen solution into the mother wart , i.e. , the earliest or initial wart on the body .
if the patient was not sure about the initial wart , then the largest wart was injected irrespective of its temporal association .
the candida test antigen used was the credisol skin test solution marketed by creative drug industries , mumbai , india .
injections were given into the substance of the wart using a 27-guage needle , and a total of three injections were administered at 3-week intervals .
the volume of the test antigen used was 0.1 ml on every occasion irrespective of the severity of response to the initial intradermal test dose .
thus , a total of 0.3 ml of the test antigen was used in each patient .
the patients were examined at each follow - up injection and any regression in the size of the warts was noted down .
digital photographs were also repeated at every follow - up visit , and adverse events , if any , were also noted down .
each patient was told about the possibility of a febrile reaction after the injection and was advised to take antipyretics in case of such an eventuality .
patients who showed no or a partial response after three injections were called for a final follow - up 1 month after the date of the final injection for eventual evaluation of the therapeutic result . however , all positive responders were further followed up for a period of 6 months after the treatment , and recurrences , if any , were noted down . in those patients where the shaving area of the face was involved ,
shaving blades were prohibited during the treatment as well as for the next 12-weeks of follow - up .
response to treatment was noted down at every follow - up visit with clinical examination of the treated as well as untreated warts along with photographic documentation .
the wart was said to have completely resolved only when there was complete disappearance of the hyperkeratosis and thickening of the skin .
the wart was considered to have shown a partial resolution if there was 50 - 75% reduction in the original diameter of the treated wart .
reduction in the size of the treated wart by < 50% was considered to be a non - response .
patients who did not show any response to the immunotherapeutic regimen were offered alternative treatment options .
of the 40 patients enrolled for the study , six could not complete the course of three injections as they did not come for further injections after their first dose only .
these patients were dropped from the study and thus we had 34 patients available for assessment at the end of the study .
age of these patients ranged from 14 to 36 years , with a mean of 24.3 years .
male patients were more commonly represented in the study group as there were 20 male and only 14 female patients in the study population . the duration of warts ranged from a minimum of 3months to a maximum of 8 years , with a mean of 1.2 years .
sites where the warts were present included the face ( 22 ) , hands ( 18 ) , feet or legs ( 14 ) , and forearms ( 4 ) .
pain at the injection site was the commonest adverse effect reported and this was reported by majority of the patients .
the pain occurred at the time of injection only and was the most severe in intensity when the fingers were injected .
in fact , all patients in whom the fingers , especially the peri - ungual warts were injected , complained of severe pain at the time of injection .
fever or myalgias was reported by three patients only and none of them required any analgesics or antipyretics for this purpose . of the total 34 patients available for analysis of study results , 19 ( 55.9% ) showed a complete resolution of the treated as well as untreated warts [ table 1 ] .
the usual scenario seen in these patients was a progressive reduction in the size of the treated wart that usually started after the first injection only with complete resolution by the third injection [ figures 1 and 2 ] .
simultaneously , with the changes in the surface area of the treated wart , the untreated warts also disappeared gradually without any inflammatory sequalae [ figure 3a and b ] .
response to intralesional candida injection immunotherapy ( a ) recurrent warts on face after multiple destructive procedures .
( b ) same patient after candida immunotherapy ( a ) resistant warts on the face .
( d ) same patient showing resolution of distant warts on the leg ( a ) extensive veruccae on feet since > 8 years .
( c ) almost complete resolution after three doses in two more patients ( 6% ) , the treated wart was seen to respond partially to treatmentregimen , but there were no changes observed in the untreated warts .
in these two patients , warts were seen on the face and hands and it was only the injected wart on the hand that showed a partial resolution after treatment .
both of these patients had multiple verruca plana on the face that failed to show any responseto the treatment regimen .
however , there were five patients who had verucca plana on the face in addition to the presence of common warts on either the hands or the feet . in these patients ,
only the mother wart on hands or feet was injected with the candida test antigen .
three of these five patients showed a positive response to the treatment regimen with resolution of the plane warts on the face along with resolution of common warts on the extra - facial sites . in the follow - up period of 6 months
, none of the non - responders showed any further reduction in size or number of warts , and none of the patients in whom the warts had resolved with the treatment regimen showed any relapse anywhere on the body .
we encountered some technical difficulties while performing the intralesional injections and would like to point them here .
the first and the most important is the possibility of the intralesional drug to ooze out of the wart while injecting . while injecting , the antigen solution tends to come out of the upper surface or the other side of the wart .
this is especially common if one has to inject a wart of smaller size . to overcome this difficulty
, it is better to inject the wart from the upper surface and not from the lateral side .
second , during the second and third injections , it becomes difficult to inject and accommodate the whole volume of the solution in some cases as the injected wart is already resolved to a great extent at that time .
we encountered some technical difficulties while performing the intralesional injections and would like to point them here .
the first and the most important is the possibility of the intralesional drug to ooze out of the wart while injecting . while injecting , the antigen solution tends to come out of the upper surface or the other side of the wart .
this is especially common if one has to inject a wart of smaller size . to overcome this difficulty ,
it is better to inject the wart from the upper surface and not from the lateral side .
second , during the second and third injections , it becomes difficult to inject and accommodate the whole volume of the solution in some cases as the injected wart is already resolved to a great extent at that time .
treatment of warts is not always a simple affairas many patients fail to respond to all conventional modes of treatment .
in addition , there are patients in whom warts do respond but there are multiple troublesome recurrences .
the chances of a relapse are especially high when the warts are present on any part of the face
. the lips and the area around the eyebrows in women and the shaving area in men are the most notorious for these troublesome recurrences to occur ( personal observation ) .
repeating the same destructive procedure again and again in such patients is definitely not a feasible thing to do .
logically , the best possible treatment option in the above - mentioned cases is some form of immunotherapy that will boost the immune response to hpv infection in general .
immunotherapy has been tried with oral immune modulators such as cimetidine and levamisole and also with topical immiquimod and intralesional interferons .
immunotherapy with different skin test antigens like candida , mumps , or trichophyton antigen is a relatively new treatment option for warts that has been studied over the last decade or so .
the first antigen that was tried for immunotherapy of warts was that of candida and the investigators reported success in majority of patients treated with this test antigen .
in a subsequent study , intralesional candida injections were compared with cryotherapy in the treatment of warts .
the study reported complete resolution of the wart treated with candida injections in 74% patients and complete resolution of even the untreated warts in 78% of these responders .
intralesional candida immunotherapy has also been tried in children with recalcitrant warts with a response rate of 47% for the treated wart and 34% for all the body warts .
in addition to candida , some other skin test antigens have been tried with success in treatment of warts .
these include mumps antigen , trichophyton antigen , and even a combination of multiple antigens .
treatment with multiple antigens has , however , shown no advantages over single - antigen injections in terms of efficacy of treatment .
different regimens have been described regarding the candida antigen immunotherapy for warts [ table 2 ] .
in one clinical study , the dose used has been correlated with the amount of induration observed after the test dose .
when the induration observed with the test dose antigen was more than 40 mm , the therapeutic dose administered was 0.1 ml on each occasion , whereas those showing an induration of 20 - 40 mm and 5 - 20 mm were given a dose of 0.2 ml and 0.3 ml , respectively .
we used a similar dose regimen in all patients regardless of the extent of induration achieved after the test dose injection .
this dosage regimen seems to be equally effective as we also achieved a positive response in more than half of our patients .
similarly , some authors have used up to five injections at 3-week intervals , whereas we used only three injections at 3-week intervals for the study .
we found that the person who ultimately shows a resolution of the injected and/or distant warts shows some response after the first or at the most after the second injection only .
persons who fail to show any response after the first or even the second injection are less likely to respond after the third dose as well .
follow - up visit of these patients for the subsequent injections becomes a problem if they fail to see any visible results even after the third dose of candida injection .
the limiting factor in prescribing this treatment option is the absence of any reaction to the test dose of the antigen in a subset of patients .
however , patients showing a negative response to one antigen can be tested with other antigens solutions such as those of trichophytonor mumps .
comparison with select studies on test antigen immunotherapy it is a well - known fact that trauma of any sort including the trauma associated with an intralesional injection can lead to a non - specific immune response against the wart antigens with subsequent resolution of the traumatized wart .
however , going by the high proportion of patients showing a positive response to intralesional immunotherapy , there seems to be a definite role of the test antigens in resolution of warts .
even the eradication of untreated warts after injecting a single wart goes in favor of a systemic immune response against hpv after introduction of the test antigens .
adverse effects that have been reported with candida immunotherapy include febrile reactions , myalgias , erythema , and edema at the injection site and painful purple digit syndrome .
we did not encounter any of these adverse events except mild fever in few patients .
however , majority of our patients did complain of pain at the time of injection , which was severe in some instances .
candida immunotherapy seems to be a promising treatment option in patients in whom the warts are either resistant or not amenable to destructive modes of treatment .
there is no risk of any scarring at the sites of intervention or at the site of warts .
furthermore , treatment of a single wart needs to be done even if there are hundreds of warts on the body .
risk of relapse also seems to be really low , and this is so important in patients suffering from troublesome recurrences of warts .
absence of a control group and small sample size of the study population were important limitations of the study .
therefore , we would definitely recommend more extensive , randomized controlled studies on this subject in larger population groups and with different dosage regimens to make it a routine treatment option to be offered to patients .
multiple intralesional injections with candida test antigen is an effective treatment option in the treatment of recurrent or resistant warts.a single uniform dose can be used for injection irrespective of the extent of induration with the test dose of the injection .
multiple intralesional injections with candida test antigen is an effective treatment option in the treatment of recurrent or resistant warts .
a single uniform dose can be used for injection irrespective of the extent of induration with the test dose of the injection .
absence of a control group and small sample size of the study population were important limitations of the study .
therefore , we would definitely recommend more extensive , randomized controlled studies on this subject in larger population groups and with different dosage regimens to make it a routine treatment option to be offered to patients .
multiple intralesional injections with candida test antigen is an effective treatment option in the treatment of recurrent or resistant warts.a single uniform dose can be used for injection irrespective of the extent of induration with the test dose of the injection .
multiple intralesional injections with candida test antigen
a single uniform dose can be used for injection irrespective of the extent of induration with the test dose of the injection .
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### Summary:
: background : warts are sometimes resistant or they tend to recur after every possible destructive therapy .
immunotherapy with skin - test antigens has been used as a viable therapeutic option in such recalcitrant cases.aim:the aim of the study was to evaluate the response of resistant or recurrent warts to intralesional candida albicans antigen immunotherapy.materials and methods : a total of 40 patients with resistant or recurrent warts who showed a positive test reaction to c. albicans antigen were given intralesional injections of purified c. albicans antigen solution in a single wart at 3-weekly intervals for a total of three doses .
the patients were monitored for resolution of the injected wart as well as other untreated warts .
the patients who responded positively were then followed up for any relapses over the next 6 months .
adverse events , if any , were also documented.results:of the 40 patients enrolled in the study , 34 completed the total treatment protocol of three injections and 6 months of follow - up . in these 34 patients ,
19 ( 56% ) showed a complete resolution of warts at all places on the body .
in addition , two patients ( 6% ) showed a partial or complete resolution of the treated wart , but there was no effect on the untreated warts .
thirteenpatients ( 38% ) failed to show any response to the treatment regimen . in all patients showing resolution of all the warts
, there were no relapses at any site over the next 6 months of follow - up .
the most common adverse effect seen was pain during the intralesional injection.conclusions:intralesional candida immunotherapy seems to be an effective treatment option in more than half of the patients who fail to show a positive response to destructive modes of treatment or in whom there are multiple recurrences.limitations:the small sample size and lack of control group are the main limitations of the study .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: term lifestyle is a relatively common theory that often is used to refer to the way people live and is the full range reflection of social values , attitudes and activities .
lifestyle includes behaviors such as food habits , sleeping and resting , physical activity and exercising , weight controlling , smoking and alcohol consumption , immunization against disease , coping with stress and ability to use family and society supports .
studies have indicated that there is a strong correlation and relationship between health and lifestyle .
american heart association in 2001 introduced lifestyle as an important susceptible cause of mortality in the u.s . and stated that approximately 70 percent of all the physical and mental diseases are related to the lifestyle . moreover ,
currently many health problems such as obesity , cardiovascular disorders , cancer , drug addiction and sexually transmitted diseases are correlated with lifestyle modifications and changes .
inappropriate lifestyle is also one of the effective factors on incidence of chronic diseases such as colon cancer hypertension , chronic obstructive pulmonary disease , renal cirrhosis , stomach ulcers , aids and cardiovascular diseases .
more than one - third of all the mortalities worldwide are due to several threatening behaviors resulting from inappropriateness of individual and social lifestyle most of them are inappropriate diet , low physical activity , smoking and inappropriate sexual behavior .
controlling risk factors in lifestyle and healthy habits such as inappropriate nutrition , lack of physical activity , smoking , alcohol consumption and drug use almost can reduce 50 percent of early deaths .
the study results of charkazi et al . showed that lifestyle of 84 percent of teachers in gorgan city was semi - desirable .
given to the determinant role of lifestyle variable in promoting health and increasing quality of life ( qol ) and due to the sensitive role of teachers as effective human forces in development and evolution of our society and also considering that students follow their patterns and lack of necessary information from teachers lifestyle in esfahan province , the present study aimed to review teachers lifestyle among them in order to determine their lifestyle and providing intervention and strategy , if necessary .
this descriptive - analytical study was conducted on 96 teachers in girl schools of district no . 4 in education department of esfahan province in 2010 with multi - stage sampling method .
4 was randomly selected among 6 districts of education department and 4 schools from each grade ( elementary , secondary and high school ) and 8 teachers from each school were randomly selected .
data collections tools were a two - part questionnaire consisting of demographic variables ( age , number of family members , bmi , marital status , educational level , employment status and financial condition ) and questions related to lifestyle assessment including nutrition , physical activity , coping with stress , smoking and using seat belt completed through self - report data .
it also should be noted that questions related to healthy nutrition assessment had 14 items and those related with stress coping had 6 items and there also were 3 items for smoking assessment , 2 for using seat belt and 7 for physical activity assessment scored by 5-point likert scale ( completely disagree , disagree , no comment , agree , completely agree ) .
eventually , the scores in each part were divided into three categories ( undesirable , semi - desirable and desirable ) .
the validity of the questionnaire conducted through content validity method by health educationalists and its reliability through cronbach 's alpha obtained 80.0 for each part . collecting the data , their analysis conducted through descriptive statistics and kruskal - wallis , and anova .
it should be noted that written consent form was obtained from the teachers participated in the study and they were ensured about confidentiality of information .
the results showed that mean age of the subjects was 40.26 4.82 years , and their mean bmi was 25.08 3.20
mean of household size was 3.75 0.85 and mean working experience was 18.34 6.05 years .
in addition , 93 of them were married ( 96.8% ) . in terms of educational level ,
5 of them were high school graduates ( 5.2% ) , 21 had associate degree ( 21.9% ) , 61 had bsc degree ( 63.5% ) and 9 of them had msc degree ( 9.4% ) .
in addition , 89 of them were formal employment ( 92.7% ) , 3 of them had contract ( 3.1% ) and 4 of them were tuition teachers ( 4.2% ) . in terms of financial conditions ,
2 teachers had a high financial level ( 3.1% ) , 57 teachers had a good financial level ( 59.4% ) and 37 of them had a moderate financial level ( 38.5% ) . in terms of healthy lifestyle , the results showed that totally 82 teachers ( 85.4% ) had a desirable situation , 13 teachers had semi - desirable situation ( 13.5% ) and 1 of them had undesirable lifestyle ( 1% ) .
frequency distribution of various healthy lifestyles ( nutrition , stress management , smoking , physical activity and using seat belt ) are illustrated in [ table 1 ] .
frequency distribution of healthy lifestyles ( nutrition , stress management , smoking , physical activity and using seat belt ) of teachers employed in girl students of education district no .
4 in esfahan in 2010 anova test showed that there was a significant relationship between healthy lifestyle and age ( p = 0.019 ; f = 5.67 ) ; so that in the healthy lifestyle group , mean age of individuals was 39.80 4.63 years and was lower than other groups .
in addition , there was a significant relationship between mbi and healthy lifestyle ( p = 0.045 ; f = 4.27 ) ; so that those with lower bmi had a more healthy lifestyle .
kruskal - wallis test showed that there was no significant correlation between marital status ( p = 0.483 ) , educational level ( p = 0.165 ) and employment status ( p = 0.45 ) .
given to the determinant role of lifestyle in promoting health and increasing quality of life ( qol ) and due to the sensitive role of teachers as effective human forces in development and evolution of our society and also considering that students follow their patterns and lack of necessary information from teachers lifestyle in esfahan province , the present study aimed to review teachers lifestyle among the teachers of girl schools in district no .
the present study was on 96 employed teachers in district no . 4 in isfahan city which showed lifestyle of 1 teacher was undesirable , and
13.5 percent had semi - desirable lifestyle that indicated a lower percentage than the study of bakhtari ( 48 .
however , in the study of rahnavard , lifestyle of 50 percent was undesirable . in the present study ,
24 percent of teachers had a good physical activity which was in accordance with the study of charkazi ( 23.6% ) in the study of dearden , physical activity of women was undesirable and the reasons were individual , familial and social factors such as shortage of facilities and equipments , lack of a secure place for walking and time limitation .
it seems that in the present study also , time limitation for teachers ( due to daily presence in schools ) caused that 76 percent of them had a semi - desirable physical activity . while in the study of farmanbar , only 47.7 percent of study subjects had physical activity and exercise just for risky situations for health . in the study of cahrkazi also 39 percent of they announced they did not have enough time to do the physical activities . in a study sadeghi
has been done in isfahan city , the results showed that 70 percent of the study subjects somehow had physical activity and exercise most common of which were walking and morning exercises .
this might be due to employment of teacher in schools ; however , this issue can be reduced by appropriate planning during a day or some hours can be assigned for teachers physical activity . in the present study ,
78.1 percent of individuals had a desirable stress management which had a higher percentage than the studies of mazouji and farmanbar ; however , it was similar to the study of baroogh ( 79% ) .
the difference was be due to the necessity of stress management in teachers to have an appropriate behavior with students . in the present study ,
24 percent of the teachers had a semi - desirable nutrition and 2.1 percent had an undesirable condition . in the study of charkazi , 4.2 percent of subjects also had an unhealthy diet . in the study of farmanbar ,
58.8 percent of university students also had a risk situation for health in terms of food habits .
the results of a study done in tehran on teacher showed that only 27.7 percent of subjects used safe diet . considering
that lack of appropriate information about nutrition can cause weaken of a healthy nutritional functioning , it seems it was due to lack of adequate information about healthy nutrition .
considering that osteoporosis has a high prevalence among women , regular exercising and physical activity seem necessary for prevention from this disease .
studies have shown that nutrition and physical activity can have an important role in mineral density of bone .
in addition , deviation from proper nutrition in one hand can cause food deficiency and causes diseases resulted from malnutrition and on the other hand is associated with excessive use of fats , meats , sugar and salt which by itself is a factor in incidence of chronic diseases .
it is believed that many chronic diseases such as cardiovascular diseases , myocardial infarction , diabetes , obesity and cancer which cause inabilities and early deaths are associated with diet .
finally , it can be announced that educating healthy lifestyle , particularly about modification of diet , is necessary and effective for teachers .
this also has been proved in other studies.[1922 ] in the present study , lifestyle in terms of using seat belt only was undesirable in 3.1 percent of people and a high percentage of them stated that they use seat belt .
it seems one of the reasons for improvement of using seat belt in these years has been obligatory rule for using safety belt and extensive ads in this regard through mass media . in terms of smoking or exposed to cigarette smoke , 70.8 percent of the study subjects had a desirable lifestyle . in the study of charkizi
, 94 percent of people also stated that they are not smokers ; however , the mentioned study has been done on both sexes .
while in the study of sahebzamani , 3 percent of girls used to smoke cigarette and 26.9 percent used to smoke waterpipe . in the study of samimi , lifestyle of individuals in smoking dimension reported 87.7 percent desirable , 9.8 percent almost desirable and 2.5 percent undesirable .
although smoking increased among university students , it seems cigarette smoking socially is not that accepted among the society of teachers and the fact that they are as a reference for students , a low number of them had undesirable lifestyle and given to low prevalence of smoking among women of iran and conducting this study on employed female teachers , it seems that 29.2 percent of those who had undesirable lifestyle in this regard , have smoker members in their family . in the present study ,
lifestyle had a significant correlation with age and this was also seen in the study of elzeiny ; however , it was not significant in the study of charkizi
. the reason could be due to difference in mean age of both groups of study subjects and also that was because mean age of our study subjects was lower than the charkazi 's .
nevertheless , in this study , lifestyle had no significant correlation with educational level ; it could be due to the reason that they had degree in different majors such as geology , religion and etc , and lack of information about healthy lifestyle thorough other ways such as written and visual media .
considering that a relatively high percent of the study subjects had a semi - desirable lifestyle in healthy nutrition , physical activity and stress management , the necessity of planning for teacher working in schools and also obtaining information in establishing a healthy lifestyle can be very effective .
this article is the result of implementing research project approved by isfahan university of medical sciences no .
hereby , many thanks go to all those who helped us in conducting this study .
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### Summary:
: introduction : the role of individual healthy behaviors like physical activity , nutrition and stress management on reduction of rate of disease mortality and morbidity is well known .
the aim of this study is to determine healthy life style in teachers employed in district no.4 in isfahan , iran , in 2010.materials and methods : the participants of this cross - sectional study were 96 teachers in district no .
4 , selected via random sampling method .
the data collection was performed using a questionnaire including demographic healthy lifestyle questions .
analysis of the data was performed through software spss version 18.results:the mean age of the subjects was 40.26 6.05 years and , bmi mean was 25.08 3.20 .
96.8% of them were married and 3.1% also were single .
1% of the teachers had a weak lifestyle , 13.5%had moderate , 85.4% had a good lifestyle . in terms of nutrition , 2% of the teachers had a weak lifestyle , 23% moderate , 74% good .
76% in terms of physical activity , 29.2% smoking and 21.9% stress had a weak lifestyle.conclusion:according to the results , planning for teachers in school for receiving information about healthy lifestyle is important .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: noncognitive manifestations in dementia have been described in many studies.1 since the 1980s , systematic studies have revealed how important these manifestations are in dementia symptomatology ; they have been shown to constitute early signs of dementia and may even precede the onset of cognitive deficits.2,3 they also have a considerable impact on the daily lives of patients4 and their caregivers.5,6 numerous assessment tools have been developed to assess and analyze these noncognitive manifestations.79 in 1996 , the international psychogeriatric association defined noncognitive manifestations of dementia as signs and symptoms of disturbed perception , thought content , mood , or behavior that frequently occur in patients with dementia.10 furthermore , a distinction was made between behavioral and psychological signs and symptoms .
they include aberrant motor behavior , agitation / aggression , irritability , disinhibition , apathy , and sleeping and eating disorders .
psychological symptoms are mainly noticed during the clinical interview with the patient or by the caregivers .
they consist of mood disorders ( anxiety , depression , and euphoria ) and psychotic signs ( delusion , hallucination , and illusion ) .
prevalence estimates for the presence of psychological or behavioral symptoms among demented patients ( bpsd ) range from 61% to 88%.11,12 this variability is often greater for specific symptoms . for example , the prevalence rates for agitation reportedly vary from 29% to 87% among patients with dementia.13,14 the variability between studies is likely related to methodological differences ( eg , nature and characteristics of the sample , definitions of disorders , and assessment instruments ) as well as to data collection modalities ( eg , evaluator s status ) and life conditions of the subjects ( eg , home , institution , or hospital ) . apathy and agitation constitute the most common behavioral symptoms,1517 while psychological symptoms are particularly represented by depression , anxiety , and delusions.13,17,18 only one study has examined prevalence rates of neuropsychiatric symptoms in belgian patients.19 this study included patients with probable alzheimer s disease , frontotemporal dementia , mixed dementia , and dementia with lewy bodies .
patients were assessed using the behavioral pathology in alzheimer s disease rating scale ( behave - ad),20 with the cohen - mansfield agitation inventory ( cmai)7 and middelheim frontality score ( mfs).21 this study showed that aggressiveness and activity disturbances are the most common symptoms among people with dementia .
some studies compare these symptoms between people with and without dementia;12,15,18 however , at present , no comparative study of belgian people with and without dementia has been carried out .
studies on the nature and prevalence of behavioral and psychological symptoms observed among elderly non - demented subjects ( cognitively impaired non - demented [ cind ] , mild cognitive impairment [ mci ] ) , without characterized cerebral pathology would be interesting .
these studies could be used to distinguish between symptoms appearing mainly in people without dementia and those that are shared by both populations ( ie , people both with and without dementia ) .
an examination of etiological factors is furthermore important for the selection and implementation of treatment .
this study had three aims : ( 1 ) to assess the nature and prevalence of psychological and behavioral symptoms in a belgian sample of people with and without dementia ; ( 2 ) to compare our data with that of previous studies ; and ( 3 ) to compare the prevalence of behavioral and psychological symptoms between people with and without dementia .
a sample of 223 persons with dementia ( d ) and a group of 46 persons without dementia ( nd ) were selected in the context of the qualidem study.22 qualidem was a belgian study examining dementia and had three aims : ( 1 ) to realize an inventory of existing information based on an exhaustive review of scientific and gray literature ; ( 2 ) to develop recommendations for qualitative and humanitarian care ; and ( 3 ) to assess tools and procedures selected during a field study .
qualidem protocol has been validated by belgium s national institute for health and disability insurance ( nihdi ) , the financing body of the study .
we analyzed data from subjects who took part in the qualidem study and for whom we had sociodemographic , diagnostic , state of health , and neuropsychiatric inventory ( npi ) data .
inclusion criteria for the qualidem study were people older than 65 who were institutionalized or had been receiving home care for at least 1 month up to the time of the study and those with specific characteristics ( brief hospitalization in a general or psychiatric hospital ; consultations repeated by a doctor ; demand of a particular financial intervention to the belgian social security system ; demand for home adjustments ; complaint concerning cognitive capacities ; suspicion of dementia formulated by the health care professional ) . a three - step diagnostic procedure was used for clinical diagnosis . in the first stage , as recommended in de lepeleire,23 subjects were assessed with both the activities of daily living24 and the instrumental activities of daily living25 in order to detect those with high sensitivity cognitive losses . in the second and third stages , more specific diagnostic testing
was performed , which included the mini mental state examination ( mmse)26 and camdex - r27 in order to select subjects with dementia ( ie , those with an mmse score < 24 and a camcog score < 70 ) .
the nd group was made up of subjects having some cognitive and/or functional disabilities without meeting the full dsm iv criteria28 for dementia . based on clinical assessment and the results from camdex - r ,
all subjects were assessed with the npi.8 this instrument is specifically used to evaluate neuropsychiatric symptoms among persons with dementia .
npi is an informant - based rating scale ; an experienced professional completes the survey in conjunction with a formal or informal caregiver .
npi evaluates 12 common psychological and behavioral symptoms in dementia , including delusions , hallucinations , agitation/ aggression , depression , anxiety , elation , apathy , disinhibition , irritability , aberrant motor behavior , and sleeping and eating disorders . for each neuropsychiatric symptom
, there is a screening question to determine if the symptom has been present or absent within the past month . for positive responses ,
scripted questions are asked in order to confirm the presence of neuropsychiatric symptoms . if the presence is confirmed , further questions are then asked concerning the frequency ( on a scale from 1 to 4 ) and severity ( on a scale from 1 to 3 ) of the symptom .
the reliability and validity of npi were demonstrated by cummings.29 interrater reliability ranged from 93.6% to 100% , depending on the subdomain .
retest reliability was also shown to be very high ( r = 0.79 ) .
in addition , content validity was shown to be high , as rated by 10 internationally known experts in geriatric psychiatry .
the data were collected in the subject s place of residence ( in their home or in one of 25 different institutions ) by either the family caregiver ( at home ) , or health centers ( in the institution ) in collaboration with the researchers .
a sample of 223 persons with dementia ( d ) and a group of 46 persons without dementia ( nd ) were selected in the context of the qualidem study.22 qualidem was a belgian study examining dementia and had three aims : ( 1 ) to realize an inventory of existing information based on an exhaustive review of scientific and gray literature ; ( 2 ) to develop recommendations for qualitative and humanitarian care ; and ( 3 ) to assess tools and procedures selected during a field study .
qualidem protocol has been validated by belgium s national institute for health and disability insurance ( nihdi ) , the financing body of the study .
we analyzed data from subjects who took part in the qualidem study and for whom we had sociodemographic , diagnostic , state of health , and neuropsychiatric inventory ( npi ) data .
inclusion criteria for the qualidem study were people older than 65 who were institutionalized or had been receiving home care for at least 1 month up to the time of the study and those with specific characteristics ( brief hospitalization in a general or psychiatric hospital ; consultations repeated by a doctor ; demand of a particular financial intervention to the belgian social security system ; demand for home adjustments ; complaint concerning cognitive capacities ; suspicion of dementia formulated by the health care professional ) . a three - step diagnostic procedure was used for clinical diagnosis . in the first stage , as recommended in de lepeleire,23 subjects were assessed with both the activities of daily living24 and the instrumental activities of daily living25 in order to detect those with high sensitivity cognitive losses . in the second and third stages , more specific diagnostic testing
was performed , which included the mini mental state examination ( mmse)26 and camdex - r27 in order to select subjects with dementia ( ie , those with an mmse score < 24 and a camcog score < 70 ) .
the nd group was made up of subjects having some cognitive and/or functional disabilities without meeting the full dsm iv criteria28 for dementia . based on clinical assessment and the results from camdex - r ,
all subjects were assessed with the npi.8 this instrument is specifically used to evaluate neuropsychiatric symptoms among persons with dementia .
npi is an informant - based rating scale ; an experienced professional completes the survey in conjunction with a formal or informal caregiver .
npi evaluates 12 common psychological and behavioral symptoms in dementia , including delusions , hallucinations , agitation/ aggression , depression , anxiety , elation , apathy , disinhibition , irritability , aberrant motor behavior , and sleeping and eating disorders . for each neuropsychiatric symptom
, there is a screening question to determine if the symptom has been present or absent within the past month . for positive responses ,
scripted questions are asked in order to confirm the presence of neuropsychiatric symptoms . if the presence is confirmed , further questions are then asked concerning the frequency ( on a scale from 1 to 4 ) and severity ( on a scale from 1 to 3 ) of the symptom .
the reliability and validity of npi were demonstrated by cummings.29 interrater reliability ranged from 93.6% to 100% , depending on the subdomain .
retest reliability was also shown to be very high ( r = 0.79 ) .
in addition , content validity was shown to be high , as rated by 10 internationally known experts in geriatric psychiatry .
the data were collected in the subject s place of residence ( in their home or in one of 25 different institutions ) by either the family caregiver ( at home ) , or health centers ( in the institution ) in collaboration with the researchers .
data were analyzed using statistica 7.0 ( statsoft , tulsa , ok ) and included the following techniques : student s t - test ( adjusted for unequal variances ) to compare clinical and demographic characteristics between the two groups and to control the impact of health status and age on the presence of bpsd .
whitney u test to compare the frequency and severity criteria between the two groups for each npi domain .
chi - square statistics ( tests ) and contingence coefficient ( cc ) to compare prevalence of bpsd and sex repartition between the two groups .
table 1 shows demographic and clinical characteristics of the study groups . of the total patients , 81% were female
, 83% lived in a long - term - care institution , and 17% lived at home .
significant group differences between the nd and d groups were found for age , health status , mmse total score , and camcog total score .
the nd group was younger and had higher total scores on both the mmse and the camcog . for each npi domain ,
a total of 20.9% of subjects in the d group ( 53/223 subjects ) and 56.5% ( 26/46 subjects ) in the nd group did not present any symptoms .
data from npi domains are presented according to international psychogeriatric association classifications.10 within the d group , the most frequent psychological symptom was depression ( 27.8% ; n = 62 ) , followed by anxiety ( 23.8% ; n = 53 ) and delusions ( 22.9% ; n = 51 ) .
a total of 16.1% ( n = 36 ) of the d group exhibited hallucinations .
prevalence of elation was low ( 11.8% ; n = 26 ) . in the nd group , the most common disorder was depression ( 19.6% ; n = 9 ) .
in contrast , prevalence rates of anxiety ( 8.7% ; n = 4 ) , delusions ( 6.5% ; n = 3 ) , and elation ( 8.7% ; n = 4 ) were distinctly lower .
prevalence of delusions , hallucinations , and anxiety were significantly higher in the d group than in the nd group ( table 2 ) .
frequency and severity of depression were significantly higher in the d group than in the nd group .
there were no differences in frequency and severity of other psychological symptoms . within the d group , the most frequent behavioral symptom was apathy ( 35% ; n = 78 ) , followed by agitation/ aggression ( 34.1% ; n = 76 ) and irritability ( 29.1% ; n = 65 ) .
symptoms in aberrant motor behavior ( 21.5% ; n = 48 ) , disinhibition ( 14.3% ; n = 32 ) , eating disorders ( 22.9% ; n = 51 ) , and sleeping disorders ( 15.2% ; n = 35 ) were also found often . in the nd group , there was a high prevalence of irritability ( 17.4% ; n = 8) and agitation ( 17.4% ; n = 8) .
prevalence of apathy ( 11% ; n = 5 ) and disinhibition ( 8.7% ; n = 4 ) were lower . finally , sleeping disorders ( 6.5% ; n = 3 ) , eating disorders ( 6.5% ; n = 4 ) , and aberrant motor behavior ( 4.3% ; n = 2 )
aberrant motor behavior , and eating disorders were significantly higher in the d group ( table 2 ) .
there were no significant differences between groups regarding disinhibition , sleeping disorders , and irritability .
frequency scores of apathy , irritability , and sleeping disorders were higher in the d group . there were no differences concerning the frequency of other behavioral symptoms
data were analyzed using statistica 7.0 ( statsoft , tulsa , ok ) and included the following techniques : student s t - test ( adjusted for unequal variances ) to compare clinical and demographic characteristics between the two groups and to control the impact of health status and age on the presence of bpsd .
whitney u test to compare the frequency and severity criteria between the two groups for each npi domain .
chi - square statistics ( tests ) and contingence coefficient ( cc ) to compare prevalence of bpsd and sex repartition between the two groups .
table 1 shows demographic and clinical characteristics of the study groups . of the total patients , 81% were female
, 83% lived in a long - term - care institution , and 17% lived at home .
significant group differences between the nd and d groups were found for age , health status , mmse total score , and camcog total score .
the nd group was younger and had higher total scores on both the mmse and the camcog . for each npi domain ,
a total of 20.9% of subjects in the d group ( 53/223 subjects ) and 56.5% ( 26/46 subjects ) in the nd group did not present any symptoms .
data from npi domains are presented according to international psychogeriatric association classifications.10 within the d group , the most frequent psychological symptom was depression ( 27.8% ; n = 62 ) , followed by anxiety ( 23.8% ; n = 53 ) and delusions ( 22.9% ; n = 51 ) .
a total of 16.1% ( n = 36 ) of the d group exhibited hallucinations .
prevalence of elation was low ( 11.8% ; n = 26 ) . in the nd group , the most common disorder was depression ( 19.6% ; n = 9 ) .
in contrast , prevalence rates of anxiety ( 8.7% ; n = 4 ) , delusions ( 6.5% ; n = 3 ) , and elation ( 8.7% ; n = 4 ) were distinctly lower .
prevalence of delusions , hallucinations , and anxiety were significantly higher in the d group than in the nd group ( table 2 ) .
frequency and severity of depression were significantly higher in the d group than in the nd group .
there were no differences in frequency and severity of other psychological symptoms . within the d group , the most frequent behavioral symptom was apathy ( 35% ; n = 78 ) , followed by agitation/ aggression ( 34.1% ; n = 76 ) and irritability ( 29.1% ; n = 65 ) .
symptoms in aberrant motor behavior ( 21.5% ; n = 48 ) , disinhibition ( 14.3% ; n = 32 ) , eating disorders ( 22.9% ; n = 51 ) , and sleeping disorders ( 15.2% ; n = 35 ) were also found often . in the nd group , there was a high prevalence of irritability ( 17.4% ; n = 8) and agitation ( 17.4% ; n = 8) .
prevalence of apathy ( 11% ; n = 5 ) and disinhibition ( 8.7% ; n = 4 ) were lower . finally , sleeping disorders ( 6.5% ; n = 3 ) , eating disorders ( 6.5% ; n = 4 ) , and aberrant motor behavior ( 4.3% ; n = 2 )
prevalence of agitation , apathy , aberrant motor behavior , and eating disorders were significantly higher in the d group ( table 2 ) .
there were no significant differences between groups regarding disinhibition , sleeping disorders , and irritability .
frequency scores of apathy , irritability , and sleeping disorders were higher in the d group . there were no differences concerning the frequency of other behavioral symptoms .
the main goals of this study were to assess the nature and prevalence of psychological and behavioral symptoms in a belgian sample of people with and without dementia and to compare prevalence of behavioral and psychological symptoms between both populations . within
the dementia group , depression , anxiety , and delusions constituted the most frequent psychological symptoms ( from 20% to 30% ) ( table 2 ) .
regarding behavioral symptoms , agitation / aggression ( 34.1% ) , apathy ( 35% ) , and irritability ( 30% ) were reported among a third of people with dementia .
prevalence rates for other npi symptoms varied from 10% to 23% . within the group without dementia ,
regarding behavioral symptoms , irritability and agitation / aggression were the most common symptoms ( 15% ) .
aberrant motor behavior and sleeping and eating disorders were rare ( close to 5% ) .
comparative analysis showed that prevalence rates of depression , elation , irritability , disinhibition , and sleeping disorders did not differ significantly between groups .
delusions , hallucinations , anxiety , apathy , agitation / aggression , aberrant motor behavior , and eating disorders were significantly more frequent in the dementia group . in the dementia group , only the frequencies of apathy , irritability , and sleeping disorders were higher . regarding severity criteria , the scores were higher in the dementia group for apathy and irritability . within the dementia group ,
however , we observed quantitative differences between our data and those from hollingworth et al17 and peters et al,18 whose prevalence rates were much higher than ours ( figure 1 ) .
the sample included in the study by hollingworth et al17 included 1120 individuals diagnosed with late - onset probable ad [ alzheimer s disease ] . their results are higher than those from other npi studies.1518 the group of persons with dementia in peters et al18 included 576 subjects whose average mmse score was clearly higher , and whose average age was lower , than our group of patients with dementia .
indeed , it is possible that some psychological symptoms are more easily detectable among people presenting with minor cognitive disorders than among those presenting with severe cognitive disorders .
using the behave - ad,20 the mfs,21 and the cmai,7 engelborghs et al19 noted the importance of aggressiveness , activity disturbance , and disinhibition in dementia semiology .
the variability between this study and our current investigation is likely related to methodological differences , as well as variation in life conditions , of the subjects ( eg , home , institution , hospital ) . regarding the group without dementia ,
however , peters et al18 reported higher prevalence rates than those shown in previous studies , except for elation and delusions ( figure 2 ) .
their sample contained 193 cind subjects taken from a canadian cohort study of cognitive impairment and related dementias.18 according to these authors , an explanation for these high rates may have emerged within their definition of cind . in another study ,
lyketsos et al12 studied 673 elderly subjects without dementia who were living at home or in an institution .
their observed prevalence rates were much lower than those in our study ( figure 2 ) .
this variability is likely related to various factors , such as demographic and clinical characteristics and size , as well as life conditions of the evaluated populations . moreover ,
the changing nature of the examined symptoms is likely to explain part of this variability .
regarding comparative analysis , lyketsos et al12 showed that all npi symptoms were significantly more frequent in their dementia group than in their non - dementia group , except for sleeping and eating disorders , which were not assessed .
the heterogeneity of reported data may be due to the use of distinct procedures for subject selection and to the nature of the assessed population . despite the fact that our results are roughly similar to those of lyketsos et al,15 this study also found that all npi symptoms are significantly more frequent in their dementia group than in their mci group , except for elation .
this may be explained by the difference between the size of our group without dementia and their mci group .
peters et al18 found similar results to ours except for anxiety ( no significant difference ) and disinhibition .
some disorder prevalence rates did not significantly differ between groups ( ie , depression , elation , irritability , disinhibition , and sleeping disorders ) .
prevalence rates of others npi symptoms ( ie , delusions , hallucinations , anxiety , agitation / aggression , apathy , aberrant motor behavior , and eating disorders ) are significantly higher in the dementia group . among these symptoms ,
some are rarely reported in the group without dementia ( ie , hallucinations , delusions , aberrant motor behavior , and sleeping and eating disorders ) , with prevalence rates close to 5% . as a result
, these symptoms can be considered specific to dementia , with prevalence rates ranging between 15% and 25% of the subjects with dementia .
the other symptoms are more frequent in the dementia group , but have been regularly reported in the group without dementia with prevalence rates close to 10% .
these symptoms are not specific to the dementia group . nevertheless , it is important to note that some studies have reported prevalence rates of hallucinations ( figure 2 ) and aberrant motor behavior ( figure 2 ) as being lower than 5% among persons without dementia .
data concerning eating disorders are more heterogeneous . comparative analysis allows us to suggest a distinction between three categories of symptoms : dementia - specific symptoms , symptoms more frequent in dementia , and symptoms nonspecific to dementia
indeed , it may be hypothesized that dementia - specific symptoms are very likely associated with neurobiological factors .
however , symptoms that are more frequent in dementia and symptoms that are nonspecific to dementia may be related to a variety of environmental , psychological , neurobiological , and somatic factors , the respective weights of which may be variable from one disorder to another and from one subject to another .
the multifactorial etiology of some psychological and behavioral symptoms should have a direct impact on the nature of the interventions on patients with dementia and their families .
thus , based on these hypotheses , a neurobiological approach would be more appropriate when examining the etiological basis of delusions , hallucinations , aberrant motor behavior , and sleeping and eating disorders .
conversely , a biopsychosocial approach would be more advisable to study depression , irritability , elation , sleeping disorders , anxiety , disinhibition , agitation / aggression , and apathy .
the statement that these symptoms are often observed among subjects with other severe somatic pathology3033 is consistent with the multifactor causality hypotheses .
however , since these hypotheses are inconsistent with those of previous studies,3436 they must be verified by further research .
more precise comparison of the symptomatology of disorders between people with and without dementia should give some indication of whether our hypothesis is correct .
the small size of the non - dementia group constitutes a primary limitation of our study .
fourth , neither group was homogeneous regarding age and sex ; additional analyses have been conducted to control for the influence of these factors on the presence / absence of npi symptoms ; the results supported our hypothesis .
fifth , subjects pharmacological treatment was not controlled : some substances could have induced psychological and behavioral disorders ; other substances can reduce , alleviate , or eliminate some symptoms , and the influence of pharmacotherapy on psychological and behavioral symptoms should be the subject of further studies .
the npi assessment tools also had some limitations : information obtained from an informant may have been influenced by his / her expectations and by his / her capacity to deal with critical situations .
proxy assessment is the subject of debate in the literature.37,38 thus , the results must be interpreted according to the definition of symptoms induced by the npi .
indeed , some npi domains include either specific symptoms such as hallucinations or highly heterogeneous syndromes such as aberrant motor behavior .
aberrant motor behavior includes various symptoms , such as intrusive behavior,39 vocally disruptive behavior,40 dyskinesia,41 and ritualized behavior,42 all of which involve different etiological factors .
the small size of the non - dementia group constitutes a primary limitation of our study .
fourth , neither group was homogeneous regarding age and sex ; additional analyses have been conducted to control for the influence of these factors on the presence / absence of npi symptoms ; the results supported our hypothesis .
fifth , subjects pharmacological treatment was not controlled : some substances could have induced psychological and behavioral disorders ; other substances can reduce , alleviate , or eliminate some symptoms , and the influence of pharmacotherapy on psychological and behavioral symptoms should be the subject of further studies .
the npi assessment tools also had some limitations : information obtained from an informant may have been influenced by his / her expectations and by his / her capacity to deal with critical situations . patient
proxy assessment is the subject of debate in the literature.37,38 thus , the results must be interpreted according to the definition of symptoms induced by the npi .
indeed , some npi domains include either specific symptoms such as hallucinations or highly heterogeneous syndromes such as aberrant motor behavior .
aberrant motor behavior includes various symptoms , such as intrusive behavior,39 vocally disruptive behavior,40 dyskinesia,41 and ritualized behavior,42 all of which involve different etiological factors .
we assessed the nature and prevalence of psychological and behavioral symptoms in populations of demented and non - demented people . in both groups , depression was the most common psychological disorder . among behavioral symptoms ,
irritability was most prevalent in the non - dementia group and agitation / aggression was the most common in the dementia group .
comparative analysis revealed a distribution of symptoms in three categories : ( 1 ) those specific to dementia ; ( 2 ) those whose prevalence was higher in dementia ; and ( 3 ) those nonspecific to dementia .
this distinction may prove useful for etiological research and for identifying adequate treatments . in order to better identify etiological factors ,
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### Summary:
: background / aimsthis study assesses and compares prevalence of psychological and behavioral symptoms in a belgian sample of people with and without dementia.methodsa total of 228 persons older than 65 years with dementia and a group of 64 non - demented persons were assessed using the neuropsychiatric inventory ( npi ) in 2004.resultswithin the group without dementia , the most frequent symptoms were depression , agitation , and irritability . within the group with dementia , the most common symptoms were depression , irritability , apathy , and agitation .
prevalence of delusions ( p < 0.05 ) , hallucinations ( p < 0.05 ) , anxiety ( p < 0.05 ) , agitation ( p < 0.05 ) , apathy ( p < 0.01 ) , aberrant motor behavior ( p < 0.01 ) , and eating disorders ( p < 0.05 ) were significantly higher in the group with dementia.conclusiondepression , elation , irritability , disinhibition , and sleeping disorders are not specific to dementia .
agitation , apathy , anxiety , and delusions are more frequent in dementia but were not specific to the dementia group because their prevalence rates were close to 10% in the group without dementia .
hallucinations , aberrant motor behavior , and eating disorders are specific to dementia .
the distinction between specific and nonspecific symptoms may be useful for etiological research on biological , psychological , and environmental factors .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: adrenocortical carcinomas ( accs ) are functional in approximately 60% cases , with a wide range of steroid profile . most commonly , accs secrete cortisol ( 50% ) , followed by androgens , whereas aldosterone secretion is quite rare ( < 2% ) .
combined hormonal secretion is rare and has been described mostly in adenomas . among bi - functional accs ,
co - secretion of cortisol with androgens is more common , while co - secretion of aldosterone and cortisol is extremely rare .
a 37-year - old female patient presented with a history of easy fatigability and extreme weakness of both lower limbs .
, she was found to have hypertension with grade 3 power in both lower limbs along with facial puffiness .
the plasma renin activity ( pra ) was normal , with a very high plasma aldosterone concentration ( pac ) .
the pac / pra ratio , aldosterone to renin ratio ( arr ) was very high ( 39.8 ) .
the 24 h urinary free cortisol was significantly elevated ( 1781.6 g/24 h ) , along with a concomitant high serum cortisol ( 42.26 g/24 h ) .
contrast enhanced computed tomography showed a 6 cm right adrenal mass with a 2 cm right paracaval lymph node [ figure 1 ] .
pre - operatively , she was optimized with potassium supplementation , oral hypoglycemics and required a combination of three antihypertensives for adequate control of hypertension .
, there were multiple lymph nodes in the right paracaval region , with a large node wedged in the retrocaval region and another in the region of the right renal hilum [ figure 2b ] .
adr = adrenal gland ; ln = lymph node ; ivc = inferior vena cava following surgery , she became euglycemic and did not further require any anti - hypertensive medications .
histopathology of the mass revealed right adrenal cortical carcinoma with lympho - vascular invasion - t3n1 with weiss score of 4 [ figure 2d ] .
( a ) operative images - right adrenal mass ; ( b ) large retrocaval lymph node ; ( c ) gross specimen - right adrenal mass with lymph nodes ; ( d ) histopathology shows highly pleomorphic cells with anisonucleosis , macronucleoli and numerous abnormal mitotic figures ( weiss score of 4 ) .
ivc = inferior vena cava follow - up at 6 weeks post - operatively showed a falling arr of 5.5 .
acc is a rare malignancy which often presents at an advanced stage with signs from tumor compression , invasion or metastasis .
most functional acc are cortisol secreting , which may present as overt or subclinical cushing 's syndrome .
aldosterone producing tumors often present with features of primary hyperaldosteronism , notably hypertension and hypokalemia .
edema or facial puffiness is generally absent due to mineralocorticoid escape phenomenon . in a subset of patients with acc
co - secretion has been described in adrenal adenomas and rarely in accs . in patients with co - secretion ,
the level of hybrid steroids like 18-hydroxy - cortisol ( 18-oh - f ) is much higher than in pure aldosterone secreting tumors .
this phenomenon of co - secretion may be explained by the unequal crossover between the genes for 11-hydroxylase ( cyp11b1 ) and aldosterone synthase ( cyp11b2 ) resulting in a chimerical cyp11b1/cyp11b2 gene .
these tumors might have a higher proportion or quantity of cortisol producing cells , which may become clinically significant due to the increased size of the tumor , to secrete relevant amounts of glucocorticoids .
the work - up of patients with primary hyperaldosteronism often involves measurement of renin and aldosterone levels , which help in calculating the arr .
in addition , other adrenal hormones like cortisol and other adrenal steroids also need to be evaluated if symptoms or imaging studies are suggestive of co - secretion .
the dexamethasone suppression test has been shown to be adequate to document and identify the cause of hyper - cortisolism .
the evaluation for co - secretion can include measurement of levels of hybrid steroids like 18-oh - f and 18-hydroxy - corticosterone .
in addition to the weiss scoring used for describing adreno - cortical carcinomas , various immuno - histochemical studies with antibodies against cyp17a , cyp11b1 and cyp11b2 etc .
however , other than an elevated cyp11b2 activity , the other steroidogenic enzymes have shown varied levels of expression in co - secreting accs .
co - secretion has been described in adrenal adenomas and rarely in accs . in patients with co - secretion ,
the level of hybrid steroids like 18-hydroxy - cortisol ( 18-oh - f ) is much higher than in pure aldosterone secreting tumors .
this phenomenon of co - secretion may be explained by the unequal crossover between the genes for 11-hydroxylase ( cyp11b1 ) and aldosterone synthase ( cyp11b2 ) resulting in a chimerical cyp11b1/cyp11b2 gene .
these tumors might have a higher proportion or quantity of cortisol producing cells , which may become clinically significant due to the increased size of the tumor , to secrete relevant amounts of glucocorticoids .
the work - up of patients with primary hyperaldosteronism often involves measurement of renin and aldosterone levels , which help in calculating the arr .
in addition , other adrenal hormones like cortisol and other adrenal steroids also need to be evaluated if symptoms or imaging studies are suggestive of co - secretion .
the dexamethasone suppression test has been shown to be adequate to document and identify the cause of hyper - cortisolism .
the evaluation for co - secretion can include measurement of levels of hybrid steroids like 18-oh - f and 18-hydroxy - corticosterone .
in addition to the weiss scoring used for describing adreno - cortical carcinomas , various immuno - histochemical studies with antibodies against cyp17a , cyp11b1 and cyp11b2 etc . , have been described .
however , other than an elevated cyp11b2 activity , the other steroidogenic enzymes have shown varied levels of expression in co - secreting accs .
in addition to the weiss scoring used for describing adreno - cortical carcinomas , various immuno - histochemical studies with antibodies against cyp17a , cyp11b1 and cyp11b2 etc . , have been described .
however , other than an elevated cyp11b2 activity , the other steroidogenic enzymes have shown varied levels of expression in co - secreting accs .
co - secretion of aldosterone and cortisol , although a rare entity has bearing on management of patients presenting with a functional adrenal tumor .
pre - operative detection of co - secretion helps adequate hormonal manipulation to avoid catastrophic adrenal crisis in the post - operative period .
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### Summary:
: adrenocortical carcinoma ( acc ) co - secreting aldosterone and cortisol is extremely rare .
we report the case of a 37-yearold female who presented with paresis and facial puffiness .
evaluation revealed hypertension , hyperglycemia , severe hypokalemia and hyperaldosteronemia with elevated plasma aldosterone to renin ratio ( arr ) .
urinary free cortisol estimation showed elevated levels . computed tomography scan revealed a right adrenal mass .
radical adrenalectomy specimen revealed acc ( t3n1 ) .
post - operatively , the patient became normotensive and euglycemic with normalization of urinary cortisol and arr .
this case highlights the need for a complete evaluation in patients of hyperaldosteronism if overlapping symptoms of hypercortisolism are encountered , to avoid post - operative adrenal crisis .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: a 55-year - old male presented with a two months history of cough and chest pain .
six months ago , there were no abnormal findings in chest x - ray at that time presented with mild chest pain . at the time of cough with chest pain presentation ,
chest x - ray showed a about 5 cm sized round mass in left hilar area and computed tomography ( ct ) scan of chest showed a about 54 cm lobulated mass arising superior segmental bronchus in lower lobe with interlobar pulmonary artery invasion ( fig .
1a , b ) . positron emission tomography ( pet)-ct was revealed only increasing uptake of hilar mass without anywhere active lesion .
bronchoscopic and ct - guided needle biopsy of the lesion results a histological diagnosis of suggestive sarcoma .
the hilar lesion could be considered as the primary malignant tumor , based on clinical findings including ct scan of chest , pet - ct , and brain mr .
the resected tumor was encapsulated , firm , and areas of focal hemorrhage and necrosis with pulmonary arterial invasion by tumor .
microscopically , the tumor was consisted of a pleomorphic and spindle cell proliferation in a storiform pattern with high grade differentiation ( fig .
by an immunohistochemical staining , the tumor was positive for cd68 , but negative for s-100 protein and desmin .
the histological final diagnosis was typical of malignant fibrous histiocytoma ( mfh ) by the features of microscopic finding and special staining .
there was no evidence of regional lymph node metastasis and bronchial and arterial resection margin were negative .
postoperative recurrence was developed on pleura in pneumonectomy space during first regular follow - up three months after the operation .
the ct scan of chest and pet - ct showed a positive finding of recurrence ( fig .
the patient is still alive without any evidence of cancer recurrence in the chest and another site for 36 months after operation .
a 55-year - old male presented with a two months history of cough and chest pain .
six months ago , there were no abnormal findings in chest x - ray at that time presented with mild chest pain . at the time of cough with chest pain presentation ,
chest x - ray showed a about 5 cm sized round mass in left hilar area and computed tomography ( ct ) scan of chest showed a about 54 cm lobulated mass arising superior segmental bronchus in lower lobe with interlobar pulmonary artery invasion ( fig .
1a , b ) . positron emission tomography ( pet)-ct was revealed only increasing uptake of hilar mass without anywhere active lesion .
bronchoscopic and ct - guided needle biopsy of the lesion results a histological diagnosis of suggestive sarcoma .
the hilar lesion could be considered as the primary malignant tumor , based on clinical findings including ct scan of chest , pet - ct , and brain mr .
the resected tumor was encapsulated , firm , and areas of focal hemorrhage and necrosis with pulmonary arterial invasion by tumor .
microscopically , the tumor was consisted of a pleomorphic and spindle cell proliferation in a storiform pattern with high grade differentiation ( fig .
by an immunohistochemical staining , the tumor was positive for cd68 , but negative for s-100 protein and desmin .
the histological final diagnosis was typical of malignant fibrous histiocytoma ( mfh ) by the features of microscopic finding and special staining .
there was no evidence of regional lymph node metastasis and bronchial and arterial resection margin were negative .
postoperative recurrence was developed on pleura in pneumonectomy space during first regular follow - up three months after the operation .
the ct scan of chest and pet - ct showed a positive finding of recurrence ( fig .
the patient is still alive without any evidence of cancer recurrence in the chest and another site for 36 months after operation .
mfh is the most common soft tissue sarcoma in adult , arising frequently in the extremity , retroperitoneum and trunk .
but primary mfh of the lung is extremely rare , accounting only for less than 0.2% of lung neoplasm . to diagnose primary mfh
, there must be no evidence of tumorous lesion except for primary site by careful physical examination and radiological study . in our case
, there was only pulmonary lesion in ct scan of chest and pet - ct without extrathoracic lesion .
up to date , maeda et al . reviewed the clinical features of only 93 cases of mfh , searching english medical literature in the world .
the median age of these patients was 55 years , range from 10 to 80 years .
but about 32% patients were symptom free . in our case , just six months after first symptom of scanty chest
, the tumor has appeared in ct scan of the chest and pet - ct .
there were relatively fewer symptoms at the early stage and the tumor thought to be having a tendency of rapid growing .
maeda et al . reported that a preoperative diagnosis is difficult and only 4% of their reported cases were histologically diagnosed prior to resection .
maybe , this is because of rarity of mfh and insufficient biopsy specimens by rapid growing .
the differential diagnostic diseases are other sarcoma , malignant schwannoma , spindle - cell and anaplastic carcinoma , and malignant melanoma .
weiss and enzinger have proposed storiform , pleomorphic , myxoid , inflammatory , giant cell , and angiomatoid variants may be occurred .
the cell feature of our case was a pleomorphic spindle - shaped cell arranged in a storiform pattern .
maeda et al . reported , cases without a resection who died of the disease , the median survival was 9 months , range from 2 months to 24 months , and the 5-year survival rate for the patients with resection was significantly better than those without resection .
weiss and enzinger reported that cases with lymph node metastasis account for 12% of all cases .
the 5-year survival rate for the patients without lymph node metastasis was better than those with lymph node metastasis . and
patients with lymph node metastasis at surgery were frequently recurred compared with without lymph node metastasis .
we are thinking of the tumor behavior being rapid growing and relative early recurrence in high grade .
but chemoradiation is likely to be having an effect on recurrent case of mfh the same as our case .
so , we report recurrent case of postoperative primary mfh was treated with chemotherapy and radiotherapy successfully , and we do suggest primary mfh should be considered for careful and closed follow - up to detect recurred case as early as possible and postoperative adjuvant chemotherapy need to a selected patient who has large tumor size and high grade differentiation .
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### Summary:
: we describe herein an extremely rare case of a recurrent primary pulmonary malignant fibrous histiocytoma 3 months after operation that occurred in a 55-year - old man who was treated with chemotherapy and radiotherapy successfully . until now , 36 months later , the patient has shown no evidence of tumor recurrence .
the clinical , radiographic , and pathologic features are reported here together with a brief review of the literature .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: chromosomal analysis ( karyotyping ) of one s own blood is a long - standing classroom exercise .
students and teachers have long needed to be prepared to address the unexpected discovery of an abnormal karyotype ; for example , a female student s rare discovery that she has a 46,xy karyotype indicating that she has complete androgen insensitivity syndrome , is sterile , and may need evaluation for cancer risk .
her discovery would explain her amenorrhea , but is a discovery better made under the care of a physician , not in high - school biology class .
similarly , chromosomal analysis may reveal a balanced translocation ( with relevance for reproductive planning ) , chromosomal inversion ( of potential relevance to fertility ) , or mosaicism of sex chromosomes ( e.g. , 46,xy/47,xxy mosaicism resulting in a rare , mild , and frequently undiagnosed form of klinefelter syndrome ) .
educators asking students to karyotype their own chromosomes should be prepared to refer students to appropriate physicians or genetic counselors and should discuss the privacy of health - related information including karyotypes , as unthinking self - disclosure is a primary risk to the privacy of health - related information .
genetic testing reveals variations in the dna that are associated with a range of traits including eye color , ( in)ability to taste bitterness , continental ancestry , alcohol tolerance , and disease risk . how genetic testing is actually performed for classroom use
sending student samples for analysis by commercial direct - to - consumer companies fails to provide students with hands - on experience in laboratory techniques ( e.g. , dna extraction and amplification ) that they may gain by running polymerase chain reactions on their own dna . in - class
genotyping to identify polymorphisms may focus on dna fingerprinting or identification exercises , or on analysis of genes associated with traits or disease conditions .
the latter borders on and may constitute medical testing , which raises issues about quality control and the ( likely ) failure to conduct the test in a laboratory certified under the clinical laboratory improvement amendments ( clia certification ) , as well as the ethical concerns arising with any medical test : e.g. , informed consent , appropriate use of test results , and privacy protection . attempting to avoid these concerns
, educators may avoid testing exercises focused on disease - associated genes , particularly when students analyze their own genes and/or access their own test results .
educators may instead focus on obvious phenotypic traits ( e.g. , eye color ) or non - deleterious variations like the ptc gene , tas2r38 , that codes for a taste receptor enabling or preventing people from tasting phenylthiocarbamide ( ptc ) , a bitter - tasting compound .
nevertheless , it must be remembered that pleiotropy , whereby one gene can influence more than one phenotypic trait , can result in seemingly benign findings acquiring health - related import as knowledge of genetics grows . particularly if the class is large enough
, another approach is to treat the class as a population with the students individual test results forming an aggregate data set for further study .
the students can test their own dna ; those who prefer not to can be supplied with alternate samples . with this approach ,
focusing on disease - related polymorphisms might be deemed less problematic if individual testing is done anonymously ( i.e. , with no results returnable to individuals ) .
commentators expressed concern about one classroom exercise employing anonymous testing ( 21 ) , both that several participants could potentially identify their results by examining the data , illustrating one disadvantage of working with a relatively small sample size , and that the educators did not indicate whether students could , in fact , opt out of personal testing and work with other samples ( 18 ) . if the total number of participants is small or if there is limited diversity in the sample ( e.g. , only a few male students , or only a few of asian continental ancestry ) , a trait that segregates with genotypes indicating the minority sex or the distinctive continental ancestry may be attributed to those few students in the class , raising question about the protection of their privacy even if only aggregate data are analyzed .
explicit classroom discussion of this concern may be used to introduce a broader discussion of how various types of research can affect community interests , as well as methods of protecting those interests , including community consultation or consent ( 26 ) .
whether or not disease - related polymorphisms are studied , classroom testing may raise some of the issues arising with clinical carrier testing .
knowing patterns of inheritance of particular genes , for example , enables prediction of the likelihood that the offspring of a genetic mother - father pair will inherit a particular gene .
similarly , in reverse , knowing inheritance patterns and an individual s genotype can enable estimation ( with limited accuracy and barring spontaneous mutation ) that persons with particular genotypes are the individual s genetic parents .
thus genetic testing of mother - father - child trios ( unlikely in an educational context)or in the case of some traits , knowledge of the parents phenotypes ( likely)can indicate a student s misattributed parentage ( frequently , misattributed paternity , but also previously undisclosed adoption or use of a gamete donor ) .
inheritance patterns can raise questions about familial relationships even in the case of presumptively neutral traits like bitter taste perception , as the ability to taste ptc is a dominant genetic trait ( and thus at least one parent of a student who can taste ptc should also be able to taste it ) .
when planning for and discussing the potential for such discoveries through classroom testing , instructors may explore the research ethics issue of managing incidental findings that arise in multiple research domains ( 28 ) .
next - generation sequencing technologies determine the order of nucleotides in a single gene , genetic region , or across the whole genome ( in the case of whole genome sequencing , wgs ) .
less costly than wgs is sequencing of the protein - coding genes in the genome ( the exome , approximately 1% of the genome ) in which variations are more likely to have phenotypic relevance .
sequencing of single nucleotide polymorphisms ( snps , a single base pair mutation at a specific locus ) can reveal variations of relevance for both understanding disease risk and pharmacogenomics .
although the cost of both genome and exome sequencing is falling at a rate exceeding moore s law , access to even the $ 1,000 exome is not widely available and is out of reach for most classroom use ( 2 ) . for educational exercises ,
genotyping of students personal genomes typically involves collecting samples ( typically saliva ) and sending them to a commercial company ( e.g. , 23andme ) , whose charges are within the budgets of many undergraduate and graduate students or their schools course budgets . more rarely , samples may be run on university - owned sequencers , potentially affording students hands - on experience of detecting , analyzing and interpreting dna variants to determine , for example , whether or not they are of clinical significance ( 20 ) . currently , personal genomics companies are prohibited from returning health - related genetic information ( 24 ) .
they are thus generally limiting themselves to reporting genetic ancestry , though they had provided disease - risk information in the past and may once again if they satisfy concerns raised by the food and drug administration .
companies like 23andme do return raw data snps to those who submit samples , and individuals can use web - based resources ( e.g. , interpretome or promethease ) to analyze their own results at pharmacogenomic- or disease - relevant loci .
astute students can thus obtain disease - risk and pharmacogenomics information as a result of current classroom sequencing exercises ( 15 ) .
of scientific , and thus ethical concern , is the accuracy of data returned by personal genomics companies . during the time that they were returning disease - risk results , when the government accounting office investigated the accuracy of companies reports , results for identical dna samples sometimes varied between companies and sometimes conflicted with the medical status of the donor ( 16 ) .
in addition to having obvious implications for students reliance on test results , discussion of such conflicting and thus erroneous results can motivate classroom discussion of the ethical importance of reporting accurate information .
reporting individual or aggregate research results that fail to meet rigorous standards of validity risks undermining public confidence in scientific research .
discussion of the gravity of scientific misconduct , as well as the confidence - eroding effects of failing to acknowledge the limitations of one s study or of overstating the importance of research findings , can follow from initial discussion of what students personal genomic information can ( and can not ) tell them and with what degree of confidence .
accurate or not , information presents three sets of ethical issues attending genetic testing / genomic sequencing in any context . the first concerns privacy and the reasons that individuals want to keep their information private .
so - called privacy absolutists simply want to control access to personal information about themselves , either in general or with regard to particular types of information or potential audiences for it .
students who share a lot about their dreams , worries , or weekend activities with friends in person or on facebook , for example , may want to withhold such information from parents or others .
some privacy concerns relate not to control per se , but to the consequences of information s disclosure .
health - related information is regarded as private not only because it may be relevant to personal , even intimate states of affairs and decision - making , but also because its discovery by others presents risks of stigmatization or discrimination . while social responses ( stigmatization ) can not be legislated against , in 2008 the genetic information nondiscrimination act did prohibit discrimination in the united states by employers and health insurers on the basis of genetic information .
using such information in underwriting life , long - term care , automobile , or other insurance , however , is not prohibited .
insurers , who fear the purchase of substantial insurance by those whose genetic testing suggests they are accident prone or at increased risk of late - life dementia , may wish to deny such insurance or charge higher premiums .
whether particular classroom exercises present students with such information about themselves or not , educators should practice preventive ethics ( 7 ) : they should anticipate issues that may arise imminently or , more likely , in the future , and prepare students to address them .
in particular , students who may not anticipate future uses of health information should be cautioned that current self - disclosure of personal genetic information may make that information permanently accessible , for example if it is shared within social media or within social or family circles .
moreover , discussion of the protection of their own health information provides a context to consider the short- and long - term risks to privacy of research participation . the second constellation of ethical issues centers on the right to know personal information about oneself and the concomitant right not to know .
it is hard to imagine students who would prefer to live in blissful ignorance of their ptc gene status ; indeed , they likely already know whether they can taste bitterness in coffee or dark beer .
many health conditions and risks may be well - known to students based on their family or personal medical history . for some , however , learning genetic information would not be welcome .
even pharmacogenomic information seemingly benign or almost unqualifiedly helpful could be unwelcome or could exacerbate underlying problems : while one student , struggling to treat his depression , might find it comforting to learn that genomic variation may account for his difficulty finding an anti - depressant that works , another student s depression may be worsened by learning that his genotype will make it especially difficult to find effective treatment .
finally , information that individuals believe they want may have unexpected and subtle psychosocial sequelae ( 8) . some young women who sought clinical genetic testing for breast cancer risk were nevertheless surprised to find that a positive result altered the way they viewed themselves and their futures , as well as their relationships .
in qualitative studies , they reported feeling like damaged goods ; feeling pressure to speed up dating , marriage , and reproductive plans ; altering career paths ; and feeling out of synch with their peers , like one brca+ woman who experienced ambivalence about her breasts while her friends were focused on more frivolous things like how clothing flattered their bustline ( 9 , 10 , 11 , 27 ) .
the ethical implication of these sequelae is that educators have an obligation to practice preventive ethics to anticipate and mitigate the negative impact of learning personal genomic information .
consideration of the possible impact of learning genetic information should be a part of informed decision making and/or informed consent . moreover , explicit classroom discussion of such potential impact may be used to introduce discussion of researchers obligations to identify , mitigate , and disclose risks of their research , including risks to human participants . even with regard to less weighty genetic information specifically the sort of genetic ancestry information currently available from personal genomics companies upon receiving results ,
samuel richards used genetic testing in his pennsylvania state university sociology class to demonstrate to students how complex race and ethnicity are ( 5 ) .
his colleague in anthropology and genetics , mark shriver , took cheek swab samples from 100 class volunteers for a test shriver developed with partners at dnaprint genomics to measure genetic admixture in populations ( because of the relevance of continental ancestry and admixture for clinical drug trials ) .
richards believed that if dna results indicated continental ancestry differing from students initial beliefs about their race , they might be more open to differences and have deeper discussions about race .
instead the results seemed to reinforce racially constructed categories ( 12 ) , with one student saying , for example ,
and the fact that i m black is more important it s who i m comfortable with .
just because i found out i m white , i m not going to act white .
i m very proud of my black side ( 5 ) . the new york times headline reporting the study ,
dna tells students they are nt who they thought , suggests how dna findings can disrupt individual identity and familial understandings of ancestry , cultural origins , and relationships ( 5 ) . for some , dna evidence of ancestry admixture , for example between those of european and african descent , may raise previously unconfronted questions of sexual relations consensual or not , recent or distant between individuals of different culturally - defined races .
educators using genetic ancestry information need to recognize and prepare to address such intrafamilial or historical questions ( e.g. , the legacy of rape of african - american slaves by european - american slave owners , or miscegenation laws ) .
moreover , some social benefits attach to having a particular ethnic or racial identity , e.g. , eligibility for scholarships or organization membership , or entitlement to settlements from legal disputes or to particular revenue streams ( e.g. , receipt of revenues from businesses run by native american nations or tribes ) .
it remains to be seen how a scholarship committee ( or students themselves ) might interpret the eligibility of a student who has always self - identified as black or african - american , but whose classroom dna test reveals him to be , for example , 62 percent european . when tribal membership usually established on the basis of tribal group relations , a tribal land - base , and cultural continuity is contested , individuals or tribes may employ dna analysis , though thus far its legal status is not established and genetic markers usually can not identify tribal membership ( 6 , 22 ) .
finally , it is instructive to note that the results of dna - testing of richards students were stored in shriver s research database .
this arrangement raises the usual research ethics questions regarding informed consent , consent to subsequent research use of the results , whether and how individuals can withdraw their results , third - party access to results ( with or without court order ) , and whether students will be informed of any findings ( individual or aggregate ) of future research employing their results .
testing / sequencing in the classroom presents a special set of issues because of the opportunity for students to feel pressured by peers or faculty to acquire genetic information about themselves that they do not want . while there is a dearth of empirical data regarding whether students actually feel such pressures ( 20 )
, students have recognized that the opportunity to receive personal genome sequencing at no cost to them may constitute a substantial benefit . whether receiving a ( $ 100 + ) service that most admit they would not get if they were paying for it themselves ( 20 ) constitutes the equivalent of excessive compensation that would be prohibited by an institutional review board ( irb ) in the research context is an open question that likely depends on contextual factors surrounding individual students and courses ( e.g. , whether it is required or elective ) .
classroom discussion of broader ethical questions of pressure to participate in research , informed autonomous decision making , and appropriate levels of compensation of research participants may be pursued when presenting opportunities for personal genome testing .
empirical evidence addressing whether students are making independent , informed decisions about analyzing their own personal genomes and whether efforts to help students make informed decisions are successful would shed valuable light on a major ethical concern surrounding personal genomes in the classroom ( 20 ) . in the absence of such data , given the empirical data that are reported on student uptake of offers of personal genomic testing and their positive responses regarding its educational impact , it is reasonable to assume that while a substantial majority of students at all levels may be interested in personal testing , a minority may not .
care must be taken when incorporating personal genomic testing into course curricula , as some students may not only feel pressured to participate , but also worry that opting out , if permitted , may indicate that they know or suspect something about themselves that they do not want to confirm or enable others to learn .
provision of alternate , non - personal samples for students to use for class exercises particularly if fellow students and faculty members remain uninformed of a student s choice of the alternative may maximally shield students from pressure . in the case of testing that yields raw snp data , students may be invited to submit saliva samples on their own to a designated personal genomics company .
students may then submit their own data for inclusion in the class data set , or may pick up one of the alternate non - personal samples or testing reports ( depending on the nature of the planned exercises ) to use for class exercises .
even if it is not a research activity , educational use of personal testing / sequencing might be helpfully reviewed by an irb or other body experienced in identifying and mitigating risks to privacy and pressure to participate .
when research is conducted on the educational process employing personal genome information , it can be complicated to determine whether the activity constitutes research involving human subjects that should be reviewed by an irb or whether it qualifies for exemption under 45 cfr 46.101(b)(1 ) because it is conducted in established or commonly accepted educational settings , involving normal educational practices , such as ( i ) research on regular and special education instructional strategies , or ( ii ) research on the effectiveness of or the comparison among instructional techniques , curricula , or classroom management methods ( 23 ) .
particularly when the instructional technique under study involves genomic testing / sequencing , something that in a non - educational context would require the informed consent of the person tested , it seems wise to consult an irb about the status of the activity . especially if a commercial company employing its own consent process
is not utilized but testing is done in house or classroom , consideration should be given to obtaining students informed consent , and this process may be used as an opportunity to teach this fundamental doctrine of research ethics . even if informed consent is not considered necessary , enabling students to make an informed decision about participating
the process of facilitating informed decision making has been studied by one group at the icahn school of medicine at mount sinai where instructors designed a 26-hour introductory course discussing ethical and psychosocial implications of genetic testing / sequencing , including familial and reproductive implications , as well as the scientific and technological foundation for analyzing and interpreting wgs results , and the limitations thereof .
students were also told that if they chose to work with their own wgs data later in the course sequence , they could exclude data they did not want to see . following completion of the introductory course
, there was a shift from 47% of students saying that they were able to make an informed choice about analyzing their own genomes to 84% .
those affirming that they knew the risks similarly rose from 47% to 90% , and fewer students were concerned about the consequences of others learning their genetic information and about their privacy .
an increased number expressed belief that using their own genomic information would have educational benefits not afforded by using others results ( 22 ) . despite its employing a small sample ( n = 19 ) of self - selected , motivated , genetics - focused students , this study suggests that students decision making about using their own genomic information can be enhanced .
further study would be required to determine whether a 26-hour course is necessary , or which components of it were most important to their enhanced decision - making ability .
finally , the very idea of using personal genomic testing as a pedagogical tool may be usefully discussed with students as a way of introducing broader questions about the use of resources ( in education and , by analogy , in research ) and of agenda - setting in science . despite substantial interest among educators in employing personal genomic testing to enhance learning , one study found that taking a core genetics course substantially reduced students belief that working with their own genomic information would enhance their learning more than using others ( from 67% pre- to 45% post - course ) and lowered their perceptions of the usefulness of genomic information for themselves , physicians , and patients ( 17 ) .
nevertheless , roughly the same proportion ( 57% pre- and 55% post- ) remained interested in learning their personal genomic information , with general curiosity being the primary reason given before and after the course ( 96% and 94% ) . in light of these findings , instructors and students might have productive discussions of the appropriateness of employing expensive genomic testing / sequencing technologies to satisfy curiosity or to compete for enrollment against other courses using cutting - edge technology .
instructors may draw analogies to broader issues in research ethics such as the multiple influences on determination of which research questions are worth pursuing and investigators responsibilities to help the public interpret and assign value to findings .
careful consideration of how to enable students to make informed decisions about obtaining and using their personal genomic information in educational contexts is incumbent upon educators . in addition
, instructors should anticipate and take measures to mitigate risks to privacy and potential negative psychosocial sequelae .
just as currently unknown pleiotropy can result in seemingly innocuous genetic information having health relevance in the future , what instructors do not know about their students personal circumstances and preferences may render seemingly innocuous exercises or information problematic for some students .
these risks should be neither overblown nor ignored . with thoughtful planning , many can be mitigated .
moreover , explicit discussion of these ethical considerations provides an opportunity to enhance students research ethics skills by encouraging consideration of underlying ethical values and issues pertinent to multiple research contexts .
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### Summary:
: rapidly decreasing costs of genetic technologies especially next - generation sequencing and intensifying need for a clinical workforce trained in genomic medicine have increased interest in having students use personal genomic information to motivate and enhance genomics education .
numerous ethical issues attend classroom / pedagogical use of students personal genomic information , including their informed decision to participate , pressures to participate , privacy concerns , and psychosocial sequelae of learning genomic information .
this paper addresses these issues , advocates explicit discussion of these issues to cultivate students ethical reasoning skills , suggests ways to mitigate potential harms , and recommends collection of ethically relevant data regarding pedagogical use of personal genomic information .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: pantothenate kinases ( pank ) catalyze the
rate - limiting step in
the biosynthesis of coa and regulate the concentration of this essential
cofactor .
coa is found in all living organisms , where
it acts as an acyl group carrier in various synthetic and oxidative
metabolic pathways such as the tricarboxylic acid cycle and fatty
acid metabolism .
four closely related isoforms of panks have been
identified in mammals : pank1 , pank1 , pank2 , and pank3 ,
which are encoded by three genes .
recently , the scientific
community has shown interest in the pank2 and pank1 isoforms because
of their role in pank - associated neurodegeneration ( pkan ) and diabetes ,
respectively .
pkan is
a rare and neurological disorder caused by mutations in
the human pank2 gene .
pkan is inherited in an autosomal recessive pattern
and leads to progressive dystonia , dysarthria , parkinsonism , and pigmentary
retinopathy .
classic pkan develops around age 3 , and most patients
are at risk of early death because there are no fda approved treatments
for the disease .
the pank2 isoform is highly expressed in human neuronal
tissues and the pank2 mutations are predicted to
result in significantly lower coa levels , thereby reducing neuronal
metabolism and function in pkan patients .
pank2 knockout
mice were generated to investigate the complex pathogenesis of pkan
but unfortunately did not reproduce the human disease .
the single pank1 and pank2 knockout
mice did not show a neurodegenerative phenotype probably due to compensation
by the other pank enzymes .
double knockout
mice were either embryonic lethal or died in the first few weeks after
birth , making potential treatments difficult to test .
therefore , the lack of tools to investigate the relationship
between coa levels and neurodegeneration limits our understanding
of the mechanisms by which pank2 mutations result
in neurodegeneration .
limitation of the coa supply by genetic
deletion of pank1 activity
blunts the hepatic coa increase in response to fasting and leads to
a deficit in fatty acid oxidation and impaired gluconeogenesis .
the key role of coa in metabolic control is
highlighted by the phenotype of the pank1lep double knockout
mouse .
the abnormally high level of coa
in the lep liver is reduced
by the deletion of the pank1 gene , resulting in normalization
of the hyperglycemia and hyperinsulinemia characteristic of the lep mouse .
the pank1pank2 double knockout mice have a severe metabolic phenotype related to
decreased fatty acid and ketone oxidation and do not survive to weaning . taken together
, these data demonstrate the impact
of reduced intracellular coa on oxidative metabolism and , in particular ,
the fuel switching during the transition from the fed to the fasted
state . these data and
the fact that a genome - wide study showed an association between pank1 variants and insulin levels in humans suggest that pank inhibitors
may be useful therapeutics for type ii diabetes .
the above background
and our interest in understanding coa physiologic
functions led us to hypothesize that it is possible to discover compounds
acting as pank modulators that can be used in animals to regulate
coa synthesis .
one approach to pkan treatment would be to identify
pank1 or pank3 activators that would stimulate coa synthesis in tissues
lacking pank2 .
the pank inhibitors would serve as
tools to investigate the role of coa in adult tissues and accelerate
the identification of bypass drugs to treat pkan disease .
additionally ,
pank inhibitors may have direct therapeutic application for type ii
diabetes .
recently , we reported a high throughput screening
( hts ) study of
3200 unique molecules with known biological activity that identified
small molecule inhibitors and activators of pank .
however , the identified compounds could not be used as
ideal probes for studying the role of pank in a disease due to their
higher affinity toward other proteins .
therefore , we decided to expand
our high throughput screen to the larger , more chemically diverse
compound libraries available at st .
we screened a total of 521133
compounds to discover novel small - molecule
modulators of pank activity using a luciferase - based method we previously
reported .
the screened compound library
contained 504956 small molecules and 16177 natural products acquired
from various commercial sources and collaborators or synthesized at
st .
the pank3 isoform was selected to conduct the hts because
it has a wide tissue distribution in mammals and can be purified in
large amounts .
all pank isoforms have homologous catalytic domains ,
and so we expected that the pank3 modulators would act against other
pank isoforms in a similar fashion .
all compounds were screened
at 12 m concentration in the
pank3 hts assay , and the data was analyzed in both activator and inhibitor
modes ( figure 1 ) .
the assay provided averaged z value of > 0.7 for activators and > 0.5 for
inhibitors ,
demonstrating the robustness and quality of our assay ( figure 1a , d ) .
the discriminatory power of the primary screen
was assessed using receiver operating characteristic ( roc ) analysis using 448 representative compounds .
true positives
were defined as any compound yielding a well - behaved , saturating sigmoidal
curve in the dose
roc
analyses indicated excellent discriminatory power ( auc 0.9
for both assays ) and suggested that a cutoff of 40% for activators
and 50% for inhibitors would retain 80% of true positives
( figure 1c , f ) .
on the basis of this roc analysis ,
we classified 9687 compounds ( 2415 activators and 7272 inhibitors )
as actives for further analysis .
these active compounds
were then subjected to dose response analysis for pank1 , pank3 ,
luciferase interference , and cytotoxicity assays ( see supporting information ) .
the dose response analysis
showed that the compounds identified as pank3 modulators similarly
modulated pank1 activity .
( b ) scatter plot of percentage activity
of each well from 1642 384-well plates analyzed in activator mode
[ green , the positive control for the activator screen consisted of
a reaction mixture lacking atp ; red , negative control ( dmso vehicle
with complete assay components ) ; blue , compound with activity above
cutoff ; black , compounds with activity below cutoff . note : y - axis is normalized % activity , not raw count . ] .
( c ) roc
analysis of activators showing true ( y axis ) versus
false ( x axis ) positive rates of percentage compound
activity .
( e ) scatter
plot of percentage activity of each well analyzed in inhibitor mode
[ green , the positive control for the inhibitor screen contained 60
m acetyl - coa ; red , negative control ( dmso vehicle with complete
assay components ) ; blue , compound with activity above cutoff ; black ,
compounds with activity below cutoff . note : y - axis
is normalized % activity , not raw count . ] .
the most promising 100 activators
and 100 inhibitors were selected
based on their potency , curve filter , hill number , absence of cytotoxicity ,
and luciferase interference activity .
these compounds were then clustered
together based on their structural similarities . to ensure the synthetic
tractability of the compounds , a similarity search on each of the
scaffolds
was performed against the initial actives
to generate preliminary structure activity relationships ( sar )
and deprioritize singleton hits .
representative compounds of each
cluster are shown in figure 2 , and the details
of their dose response analysis are provided in supporting information , tables s1 and s2 .
structures of representative
compounds with different chemical
scaffolds characterized as ( a ) activators ( 14 ) and ( b ) inhibitors ( 58 ) as identified from the hts . ec50 and ic50 values ( m ) represent the activity of the compounds for pank3
( see supporting information , tables s1 and s2 , for detail dose response analysis ) . reagents and conditions : ( a )
etoh , hydrazine ( 5 equiv ) , 30 min , 160 c , mw , 74% ; ( b ) etoh ,
methyl 4-acetyl-5-oxohexanoate ( 1.5 equiv ) , 15 min , 80 c , mw ,
79% ; ( c ) thf , naoh , 2 h , rt , 99% ; ( d ) dmf , 3-(methylthio)aniline ( 1.2
equiv ) , hbtu ( 1.3 equiv ) , et3n ( 1.5 equiv ) , 4 h , rt , 41% .
several compounds with a core tricyclic scaffold
( represented by
compound 7 ) were in the curated actives list of inhibitors .
thus , we focused our efforts on the synthesis of compounds with the
tricyclic scaffold to characterize an active compound from the hts
inhibitor list and to generate preliminary structure
our synthetic efforts began with a microwave assisted
reaction between 2-chloronicotinonitrile ( 9 ) with hydrazine .
the reaction yielded 10 , which
was then reacted with methyl 4-acetyl-5-oxohexanoate to obtain tricyclic 12 .
the hydrolysis of methyl
ester 12 followed by its coupling with 3-(methylthio)aniline
provided the required compound 7 .
the activity
of 7 was determined for each of the principle
pank isoforms using a radiochemical enzyme assay ( figure 3 ) .
the ic50 calculated for compound 7 was 25 nm for pank3 , whereas
the ic50s for pank1 and pank2 were 70 and 92 nm ,
respectively .
response analysis
using the hts assay showing that compound 7 inhibited
each of the pank isoforms at about the same level .
although the radiochemical
and hts pank assays were robust and reproducible , the hts assay ( table 1 ) consistently yielded higher ic50 values
than the radiochemical assay ( figure 3 ) .
the
key difference between the two assays was the atp concentration . the
hts assay employed 100 m atp , whereas the radiochemical assay
used 2.5 mm atp .
compound 7 appeared more potent in the
radiochemical assay because the pank was saturated with atp , and the inhibitor binds to the atp
ic50 ( pank1 = 70
1.1 nm , pank2 = 92 2.0 nm , and pank3 = 25 1.8 nm ) .
ten points dose
response curve in
triplicate ( sd are provided in supporting information ) . calculated using chembiodraw
ultra
. using
the same approach as described for the synthesis of 7 , compounds 1433 , having diversification
on the right - hand side of the molecule ( r1 ) , were synthesized
through intermediate 13 .
removal of the substituent on the aromatic ring ( compound 15 ) resulted in decreased activity , indicating its importance .
interestingly , when we moved the sme substituent from meta- to para - position , the activity of
the compound ( 14 ) was reduced by 30-fold . replacing the
aromatic ring with a cyclohexane ring in compound 17 decreased
the activity against pank1 and abrogated the activity against
pank3 .
insertion of an additional methylene group between the amino
moiety and the phenyl group gives rise to benzylamino derivatives ,
which exhibited either a complete or a significant loss of potency
( compare compounds 22 and 25 ; 27 and 30 ; 26 and 29 ) .
compound 16 , where the amino group of the anilino moiety is methylated ,
was completely inactive against pank1 and pank3 ( compared to 15 ) , demonstrating the necessity of having a hydrogen bond
donor at that position . by comparing the relative activities of compounds 21 , 22 , and 23 ;
26 , 27 , and 28 ; 7 and 14 ; and 19 and 20 suggested that the substitution
at the meta - position on the aniline ring was most
important for the activity observed .
a small set of synthesized compounds
( 3442 ) with the modification on
the tricyclic core ( r2 ) did not show any improved activity
( < 30% inhibition at 10 m ) ( figure 4 ) . to evaluate the effect of structural modifications on overall
intrinsic potential of the molecule
, we used a well - accepted metric :
lipophilic ligand efficiency ( lipe ) , which combines both potency and
lipophilicity . compared to 7 ,
modification
on the left side of the molecule ( r2 , tricyclic
core ) .
all these modifications resulted in inactive compounds ( < 30%
inhibition at 10 m ) .
overall , this sar analysis shows that the side chain of tricyclic
compound 7 is tolerant to multiple modifications , providing
an ample opportunity to expand the series in search of more potent ,
druglike molecules .
additionally , the higher potency of compound 15 and 23 toward pank1 compared to pank3
suggested that further medicinal chemistry efforts might ultimately
lead to selective inhibitors of different pank isoforms .
as shown in figure 5a , compound 7 lowered both the vmax and km for atp .
the pattern
of compound 7 inhibition with respect to pantothenate
was mixed ( figure 5b ) .
a thermal shift analysis was performed to confirm that compound 7 bound to the atp
compound 7 did not stabilize the protein
to thermal denaturation when atp was absent but increased the protein s
thermal stability when added in the presence of 2 mm atp / mg .
the apparent binding constant for compound 7 in these
experiments was 0.3 0.08 m . these results clearly demonstrate
that compound 7 binds to the atp pank3 complex .
( a ) analysis
of atp kinetics in the presence of 0 ( ) , 0.038 ( ) ,
and 0.128 ( ) m of compound 7 showing mixed - type
inhibition with respect to atp .
( b ) analysis of pantothenate kinetics
in the presence of 0 ( ) , 0.038 ( ) , and 0.128 ( )
m of compound 7 showing mixed inhibition with
respect to pantothenate .
( c ) thermal stabilization of pank3 by compound 7 in the presence of 2 mm atp / mg .
the ability of compound 7 to inhibit
coa biosynthesis
in cultured c3a cells was evaluated in a metabolic labeling experiment .
compound 7 caused a dose - dependent decrease in [ h]pantothenate incorporation into coa ( figure 6 ) .
there was no effect on cell viability at the concentrations
of compound 7 used in this experiment .
these data confirm
that compound 7 acts as a pank inhibitor in cultured
cells by blocking de novo coa biosynthesis .
c3a cells were labeled
with [ h]pantothenate for 24 h in the absence or presence
of the indicated concentrations of compound 7 .
the cells
were extracted , and the amount of radioactive coa was determined by
binding the extract to de-81 filters as described under methods .
in
summary , we conducted a hts of 521133 compounds and identified
novel pank activators and inhibitors capable of modulating pank activities
and cellular coa levels .
the hts correctly identifies inhibitors based
on a preliminary sar of tricyclic compound 7 , and the
kinetic experiments show that the inhibitor acts by binding to the
atp enzyme complex .
improvement of the selectivity , efficacy ,
and other druglike properties of these pank modulators will require
extensive medicinal chemistry efforts which are beyond the scope of
this work .
our future endeavors will include the further optimization
of identified scaffolds which may lead to a druggable agent capable
of selectively modulating pank activity in vivo .
indeed , the development
of modulators of pank activity could represent a promising approach
to the treatment of pkan and diabetes .
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### Summary:
: pantothenate
kinase ( pank ) is a regulatory enzyme that controls
coenzyme a ( coa ) biosynthesis .
the association of pank with neurodegeneration
and diabetes suggests that chemical modifiers of pank activity may
be useful therapeutics .
we performed a high throughput screen of > 520000
compounds from the st .
jude compound library and identified new potent
pank inhibitors and activators with chemically tractable scaffolds .
the hts identified pank inhibitors exemplified by the detailed characterization
of a tricyclic compound ( 7 ) and a preliminary sar .
biophysical
studies reveal that the pank inhibitor acts by binding to the atp
enzyme
complex .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: we report on a patient who was born with a birth weight of 2,320 g at 38 weeks ' postgestation ( < 3rd centile ) . because of the low birth weight , she required hospitalization at the newborn care unit .
she was diagnosed with diabetes without ketoacidosis on the fourth day of postnatal life ( blood glucose 19.5 mmol / l ) .
the initial treatment was intravenous insulin ( 0.04 units kg h ) , followed by subcutaneous injections of nph insulin ( 0.9 units kg day ) .
at the age of 2 weeks and following a clinical diagnosis of neonatal diabetes , analysis of the kcnj11 gene was undertaken ( 3 ) .
we found a novel , heterozygous missense mutation , r365h ( c.1094g > a ; p.arg365his ) , in the proband .
1 ) . standard oral glucose tolerance tests revealed a normal glucose tolerance with normal insulin and c - peptide values in the parents and paternal grandmother , but the paternal grandfather had impaired fasting glycemia combined with impaired glucose tolerance ( 0-h glycemia 5.8 mmol / l ; 2-h glycemia 9.4
katp channel mutations causing tnd in the proband and adult - onset diabetes in a parent and/or grandparent have been previously reported ( 3 ) . the r365h mutation identified in our family
was thought likely to be pathogenic because the arginine residue at codon 365 is conserved in dog , rat , and mouse , was not present in 298 control chromosomes , and has not previously been reported in patients with hyperinsulinism .
channels containing the r365h mutation were expressed in xenopus oocytes and their surface density , activation by the metabolic inhibitor azide , and blocking by the sulfonylurea tolbutamide were measured ( 4 ) .
unexpectedly , there was no difference between the wild - type and mutant channels , which calls into question the pathogenicity of this mutation .
in the meantime , the patient developed hypoglycemia on insulin treatment , and at the age of 7 months , the insulin therapy was discontinued .
the nine - value 24-h glycemic profile 7 months after stopping insulin administration was normal ( 4.74.15.57.87.67.13.95.35.2
mmol / l ) , and a1c was 4.3% ( within the diabetes control and complications trial range ) , ( high - performance liquid chromatography , variant ii ; biorad , hercules , ca , u.s . ) .
currently , with the subject at the age of 2 years , a1c is 5.1% .
thus , we had to revise our initial diagnosis of pnd due to a kcnj11 mutation ( 3 ) . genetic analysis of the methylation status within the tnd critical region on chromosome 6 was performed using the methylation - specific pcr method , as previously described ( 5 ) .
a 6q24 duplication was identified , and family member testing demonstrated that the duplication was also present in the unaffected father and paternal grandmother ( fig .
we report a novel kcnj11 variant ( r365h ) and a 6q24 duplication in a proband with tnd and her unaffected father .
the paternally inherited 6q24 duplication is likely to be the etiological mutation as a consequence of overexpression of paternal genes within the duplicated chromosome 6q24 region .
the maternal allele is methylated and therefore inactive : only genes on the paternally derived chromosome are transcribed .
the probands father is unaffected because his 6q24 duplication is maternally inherited and , therefore , inactive ( 2 ) .
the impaired fasting glycemia and impaired glucose tolerance in the paternal grandfather may be influenced by his age ( 53 years ) , weight ( bmi 28.5 kg / m ) , and maternal history of type 2 diabetes .
we conclude that the r365h is likely to be a rare variant of no clinical significance . in pnd patients ,
screening of the kcnj11 mutations is recommended , and if it is negative , mutations in other genes should be investigated ( e.g. , insulin gene , abcc8 , etc . )
our case report highlights the difficulty in testing neonates before the clinical course ( transient versus permanent ) is known and when no supportive signs ( e.g. , macroglossia and umbilical hernia in 6q24 abnormalities ) are present .
thus , in these patients , we recommend screening for kcnj11 gene mutations and 6q24 abnormalities simultaneously , as making the genetic diagnosis earlier may influence treatment ( patients with katp channel mutations are often sensitive to sulfonylureas , while patients with 6q24 abnormalities are usually treated with insulin ) . finally ,
when a novel mutation in a known gene is found , functional studies and investigations of other neonatal diabetes genes can play an important role .
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### Summary:
: objective neonatal diabetes is a heterogeneous group of disorders with diabetes manifestation in the first 6 months of life . the most common etiology in permanent neonatal diabetes is mutations of the atp - sensitive k+ channel subunits ; in transient neonatal diabetes , chromosome 6q24 abnormalities are the most common cause.research design and methods we report a sporadic case of diabetes without ketoacidosis diagnosed on the fourth day of life.resultsanalysis of the kcnj11 gene found a novel r365h mutation in the proband and her unaffected father .
the functional analysis did not support pathogenicity of this variant .
when the patient 's diabetes remitted in the seventh month of life , the 6q24 region was analyzed and a paternally inherited duplication was identified.conclusionsour case reports a coincidental novel kcnj11 variant in a patient with transient neonatal diabetes due to a 6q24 duplication , illustrating the difficulty in testing neonates before the clinical course of neonatal diabetes is known .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: it is believed that many surgeons use a drain following thyroid surgery to obliterate the dead space and evacuate collected blood and serum .
this fear prompts surgeons to use a routine drain after any type of thyroid surgery .
although the rate of bleeding might increase in a subtotal thyroidectomy due to vascularized remnant tissue , postoperative bleeding is actually quite rare and occurs in only 0.31% of patients after a thyroidectomy [ 1 , 2 ] .
many studies have suggested that drains may be blocked with clotted blood ; therefore , the surgeon is not alerted even if major bleeding occurs [ 14 ] .
in addition , numerous studies have also failed to show any benefits of drainage in thyroid surgery [ 35 ] .
hence , the goal of this prospective study was to evaluate the necessity of drainage after thyroid surgery .
this interventional , randomized , double - blind , prospective study was approved by the local ethics committee at the turkish ministry of health , and informed consent was obtained from all the patients .
the subjects were 400 patients who underwent thyroid surgery between january 2010 and january 2012 .
these patients were randomized via a computer - generated random number table into two groups according to whether or not drains were inserted at the time of surgery .
group 1 consisted of 200 patients without drains and group 2 consisted of 200 patients with drains .
the indications for surgery , procedures performed , local complications ( infection , seroma , bleeding , hematoma , laryngeal nerve palsy , and hypoparathyroidism ) , necessity for reoperation , and length of hospital stay were recorded for all of the participants .
those with substernal goiter or malignant disease requiring lymphatic dissection were excluded from the study .
in addition , nondifferentiated cancer patients and those undergoing anticoagulant therapy were also not included .
depending on the thyroid disorder , either a total thyroidectomy or a lobectomy plus an isthmectomy was performed .
the duration of the operation was defined as the time from the first incision to the last suture placement .
additionally a closed suction drain with negative pressure ( hemovac evacuator , zimmer inc . ,
warsaw , in , usa ) was inserted through a separate wound in the patients in group 2 .
an ultrasound of the neck using the b mode with a linear frequency of 7.5 mhz was performed on both groups at the postoperative 24th hour of surgery by the same radiologist .
the volume of fluid collection in the operative bed was calculated by measuring the maximum diameter in three dimensions , and the fluid which collected in the drain was measured separately .
the drains were removed from all of the patients after 24 hours since less than 100 cc had been collected .
postoperative pain was assessed according to a visual analogue scale ( vas ) with scores ranging from 0 ( no pain ) to 10 ( worst pain imaginable ) at the postoperative sixth hour and postoperative first day . a standard analgesic protocol comprised of diclofenac sodium ( 75 mg intramuscularly ) and oral paracetamol ( 500 mg twice a day )
thyroid gland volumes were calculated according to the ultrasound measurements of all the patients , and the correlation between thyroid volume and volume of fluid was evaluated in the thyroid bed .
the differences between the two groups were analyzed with student 's t - test , and a value of p < 0.05 was accepted as being significant .
between january 2010 and january 2012 , we performed 400 thyroidectomies in our clinic and analyzed 403 surgical interventions in these patients ( mean age 46.8 12.9 years ; range 1782 ) .
the male to female ratio was 1 : 7.51 , and there was equal distribution in both groups based on the type of surgery and size of the nodule .
the patients ' characteristics are presented in table 1 , and there were no significant differences with regard to gender , age , hormonal status , or histopathological results between the two groups ( p = 0.39 , p = 0.45 , p = 0.24 , and p = 0.32 , resp . ) .
similar average operating times occurred in group 1 ( 86.45 ( 50120 ) 18.93 min ) and in group 2 ( 88.80 ( 45120 ) 21.33 min ) ( p = 0.19 ) .
however , the mean vas score was significantly lower in group 1 than in group 2 at the postoperative sixth hour ( 3.64 ( 27 ) 1.06 and 4.95 ( 28 ) 1.05 , resp . )
( p = 0.002 ) and at the postoperative first day ( 2.08 ( 15 ) 0.74 and 3.09 ( 15 ) 0.77 , resp . )
an intramuscular analgesic was requried for all of the patients in group 2 , whereas 162 ( 81% ) of the patients in group 1 needed this medication .
the amount of fluid collection in the thyroid bed was assessed by usg for both groups at the postoperative 24th hour , and the results are shown in table 3 .
student 's t - test was applied to detect any difference in the means of fluid collection between the groups , but there was no statistically significant difference in the volume of fluid collection ( p = 0.117 ) . in group 2 , the amount of fluid collected in the suction drain was noted over a 24-hour period , with an average finding of 53.32 ml ( range 3090 ml / day ) .
the mean volume of the thyroid gland was 54.31 ( 17.3116.4 ) 22.48 ml and 53.72 ( 16.8120.4 ) 21.61 ml in groups 1 and 2 , respectively .
a linear regression analysis was performed , and no significant statistical difference was seen in the amount of collection according to the volume of the thyroid gland in groups 1 and 2 ( p = 0.73 and p = 0.16 , resp . ) .
the groups were also divided into toxic and nontoxic subgroups , and there was no significant difference in the volume of fluid collection ( p = 0.192 ) .
the complications that we observed are shown in table 4 , and the complication rates were similar between the groups ( p = 0.43 ) .
two cases of hematoma ( 1% ) , four cases of seroma ( 2% ) , one case of transient recurrent laryngeal nerve palsy ( 0.5% ) , one case of reaction to the silk sutures ( 0.5% ) , and eight cases of transient hypoparathyroidism ( 4% ) occurred in group 1 , whereas three cases of hematoma ( 1.5% ) , three cases of seroma ( 2% ) , two cases of wound infections ( 1% ) , one case of persistent single - side recurrent laryngeal nerve injury ( 0.5% ) , two cases of reaction to the silk sutures ( 1% ) , and six cases of transient hypoparathyroidism ( 3% ) were present in group 2 ( p = 0.56 ) .
two of patients in group 1 and three in group 2 required single aspiration due to seroma . in addition , one patient in group 1 and two more in group 2 needed surgical intervention for postoperative bleeding and hematoma .
the alerting symptom was the sudden increase in neck volume with dyspnea . in the second intervention ,
the average length of hospital stay was 1.10 ( 13 ) 0.33 days for group 1 and 1.53 ( 16 ) 0.80 days for group 2 , and there was a statistically significant difference noted after conducting an analysis with a two - sample t - test ( p = 0.004 ) .
the mean follow - up period was 23.60 ( 1236 ) 7.51 months . in the end
, we concluded that the presence or absence of the drain did not contribute significantly to the postoperative complications .
drains have been traditionally used in most of the surgical procedures involving the thyroid , and there is limited evidence to suggest that they provide any benefit [ 47 ] .
our study failed to show any advantage in the routine use of the drain after thyroid surgery .
arterial bleeding near the trachea leads to decreased space , which subsequently compresses the airway and produces significant edema in the soft tissues of the larynx and pharynx .
all this causes in the syndrome known as suffocating hematoma , and immediate treatment and surgical revision in the operating theater are then required .
this complication appears very infrequently , with figures ranging from 0.3 to 2.5% ; however , when it does exist , it presents a big challenge for both the surgeon and the anesthesiologist [ 810 ] .
the risk is greater in patients with intrathoracic goiter and those with graves ' disease .
suffocating hematoma tends to appear between two and six hours after surgery , and most patients report coughing , vomiting , or nausea prior to the hemorrhage .
possible causes for this complication include displacement of an improperly applied suture , the opening of a vessel in which diathermia was used for coagulation , or drooling of an area that has been improperly cauterized .
in our series , postoperative bleeding occurred in three patients ( two in group 1 and one in group 2 ) two hours after surgery .
surgical drains neither prevent this complication from occurring nor contribute to early detection [ 36 , 10 ] .
in fact , hemorrhage can appear , and the container may be empty because the blood has clotted inside the drain .
they keep blood from collecting in the subcutaneous plane , but the blood may dissect the deep plane to the prethyroid musculature in the paratracheal region , leading to compression of the airway at that level .
two large nonrandomized studies of 250 and 400 patients have also documented that the use of drains after thyroid surgery produces no benefits [ 8 , 11 ] . in our study , there was an absence of fluid in the thyroid bed on usg , but it was present in the suction drain .
this could be caused by the drain itself , since by virtue of the inflammation caused by their presence , they may actually increase the drainage .
in addition , the vacuum created by the negative suction of the drain may prevent the lymphatics from sealing off , thus causing an increase in seroma formation and drainage [ 4 , 12 ] .
furthermore , a possible relationship between drain insertion and infective complications has been observed in some studies [ 1 , 11 , 13 ] .
however , we found no relationship between wound infection and drain usage in our study ( table 4 ) .
two studies also investigated the relationship between drain insertion and postoperative pain [ 1 , 14 ] , and the authors noted an approximate 50% reduction in the vas score in the group in which no drains were used .
these results indicate that drain insertion might be directly associated with higher levels of postoperative discomfort due to increased pain .
demonstrated that thyroid surgery without the use of a drain decreases the length of hospital stay while producing no increase in patient morbidity .
we also observed that performing a thyroidectomy without the use of drain decreased the length of hospital stay .
meticulous hemostasis and an adequate surgical technique are the keys for avoiding hemorrhage and hematoma formation .
herranz and latorre suggested using a drain in cases in which there is extensive dead space or retrosternal goiters and also noted that routine drainage of the thyroidectomy bed is not effective in decreasing the rate of postoperative complications after thyroid surgery . in a meta - analysis ,
corsten et al . concluded that the use of suction drains in thyroid surgery to prevent postoperative hematoma is not evidence based .
furthermore , khanna et al . reported no significant differences between patients with drains and those without in the collection of the thyroid bed when this was assessed by postoperative usg . in this study
, we also demonstrated that the use of a drain for thyroid surgery was not effective for decreasing the rate of complications associated with toxic thyroid disorders .
additionally , a total thyroidectomy performed without a drain is safe and effective for patients with graves ' disease or toxic goiter .
most studies have revealed that drainage is unnecessary after routine thyroid surgery [ 1 , 35 , 13 , 14 ] . however , many of these studies had a small number of patients and a retrospective design .
this prospective study contains the largest number of patients on this topic in the literature .
we only recommend that they should be used in thyroidectomies for patients with substernal goiter and nondifferentiated cancer along with those who are undergoing anticoagulant therapy or require lymphatic dissection .
we believe that thyroidectomies without drains are safe for differentiated thyroid cancer , graves ' disease , and other toxic goiters .
our randomized prospective study verified that routine drain placement after thyroid surgery is not necessary nor is it effective in decreasing the rate of postoperative complications .
meticulous hemostasis and attention to finer details during surgery are more important for achieving this goal .
thyroid surgery without the drain decreases the length of hospital stay without increasing patient morbidity .
hence , we found no positive evidence that the use of drains improves patient outcomes .
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### Summary:
: background .
the use of a suction drain in thyroid surgery is common practice in order to avoid hematomas or seromas . the aim of this study was to determine the efficacy of routine drainage after thyroid surgery
. methods . in this prospective randomized trial ,
400 patients who underwent either a total thyroidectomy or lobectomy for thyroid disorders were randomly allocated to either the nondrainage ( group 1 ) or the drainage ( group 2 ) group .
the volume of fluid collection in the operative bed , postoperative pain , complications , and length of hospital stay were then recorded . results .
both groups were homogeneous according to age , gender , thyroid volume , type of procedure performed , and histopathological diagnosis .
after assessment by usg , no significant difference was found between the groups in the fluid collection of the thyroid bed ( p = 0.117 ) , but the length of hospital stay was significantly reduced in group 1 ( p = 0.004 ) . conclusions . in our experience ,
the use of drain for thyroid surgery is not a routine procedure .
however , it should be used in the presence of extensive dead space , particularly when there is retrosternal or intrathoracic extension , or when the patient is on anticoagulant treatment .
this trial was registered with clinical trials.gov nct01771523 .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: study participants were recruited ( response rate 87% ) through the computerized registration system at a large general medicine clinic in new york city during 2007 . all english- or spanish - speaking adults with type 2 diabetes for > 6 months treated with medication were eligible .
sociodemographic factors , diabetes history , and comorbidities were self - reported , whereas a1c was ascertained using electronic medical record review . in a face - to - face interview , beliefs about the chronicity , cause , consequences , and controllability of diabetes were measured based on self - regulation theory and the brief illness perception questionnaire ( 2,4 ) .
medication beliefs were assessed using five items adapted from the beliefs about medicines questionnaire ( 5 ) .
additional questions assessed difficulty in taking diabetes medications , perceptions of low and high glucose , and familiarity with the a1c test .
the tests were used to identify demographic and medical characteristics associated with beliefs regarding disease and medication .
logistic regression was used to determine predictors of such beliefs after multivariable adjustment for characteristics with at least one statistically significant association in univariate analysis .
the tests were used to identify demographic and medical characteristics associated with beliefs regarding disease and medication .
logistic regression was used to determine predictors of such beliefs after multivariable adjustment for characteristics with at least one statistically significant association in univariate analysis .
the 151 study subjects were predominantly latino ( 58% ) and african american ( 34% ) , with low income ( 89% < $ 30,000 per year ) .
participants had long - standing diabetes ( mean 13 years ) , and 55% were using insulin .
. twelve percent of the sample believed that they have diabetes only when their glucose levels are high , and 36% said that they will not always have diabetes .
more than half ( 56% ) thought that the glucose levels are not high until they reach > 200 mg / dl , and 42% thought that a glucose level of 110 mg / dl is too low .
over half of the participants ( 54% ) thought that they can feel when the glucose levels are high , and 29% expected a doctor to cure them of diabetes .
half ( 49% ) thought that the consequences of diabetes are minimal , that they have little control over diabetes , and that diabetes has few symptoms .
only one - third ( 37% ) of subjects had heard of the a1c test . among these patients ,
twenty - three percent thought that they do not need to take medication if blood glucose levels are normal .
thirty - nine percent were very worried about side effects of their diabetes medications , 16% were worried about becoming addicted to them , and 18% said that they were hard to take .
insulin use and higher mean a1c were associated with several suboptimal disease and medication beliefs ( table 1 ) .
the univariate testing demonstrated that insulin use , mean a1c , oral diabetes medication use , sex , and education had at least one significant relationship to a poor disease or medication belief . in a multivariable - adjusted model including these covariates ,
participants with less than a high school education were more likely ( odds ratio 2.3 [ 95% ci 1.05.2 ] ) to believe that they have diabetes only when the glucose levels are high .
insulin users were more likely ( 2.9 [ 1.27.9 ] ) to report being able to feel high glucose levels and to report that diabetes significantly disrupted their social life ( 4.2 [ 1.214.0 ] ) but less likely to report believing that their doctor can cure diabetes ( 0.4 [ 0.20.9 ] ) .
patients taking oral medications were more likely to believe that the consequences of diabetes are limited ( 4.7 [ 1.613.4 ] ) and to report that diabetes significantly disrupted their social life ( 7.8 [ 2.227.7 ] ) but less likely to believe that it is not important to take medication when glucose levels are normal ( 0.3 [ 0.10.8 ] ) .
patients with higher a1c ( per unit increase ) were more likely to report feeling little control over their diabetes ( 1.3 [ 1.01.6 ] ) , to report believing that their doctor can cure diabetes ( 1.3 [ 1.11.7 ] ) , to consider a glucose level up to 200 mg / dl within normal limits ( 1.3 [ 1.01.7 ] ) , to believe that it is not important to take medication when the glucose levels are normal ( 1.3 [ 1.01.6 ] ) , and to believe that their diabetes medications were hard to take ( 1.4 [ 1.11.8 ] ) .
women were more likely to report that diabetes significantly disrupted their social life ( 2.3 [ 1.05.3 ] ) .
misconceptions about diabetes were common . twelve percent of the sample believed that they have diabetes only when their glucose levels are high , and 36% said that they will not always have diabetes .
more than half ( 56% ) thought that the glucose levels are not high until they reach > 200 mg / dl , and 42% thought that a glucose level of 110 mg / dl is too low .
over half of the participants ( 54% ) thought that they can feel when the glucose levels are high , and 29% expected a doctor to cure them of diabetes .
half ( 49% ) thought that the consequences of diabetes are minimal , that they have little control over diabetes , and that diabetes has few symptoms .
only one - third ( 37% ) of subjects had heard of the a1c test . among these patients ,
twenty - three percent thought that they do not need to take medication if blood glucose levels are normal .
thirty - nine percent were very worried about side effects of their diabetes medications , 16% were worried about becoming addicted to them , and 18% said that they were hard to take .
insulin use and higher mean a1c were associated with several suboptimal disease and medication beliefs ( table 1 ) .
the univariate testing demonstrated that insulin use , mean a1c , oral diabetes medication use , sex , and education had at least one significant relationship to a poor disease or medication belief . in a multivariable - adjusted model including these covariates , participants with less than a high school education were more likely ( odds ratio 2.3 [ 95% ci 1.05.2 ] ) to believe that they have diabetes only when the glucose levels are high .
insulin users were more likely ( 2.9 [ 1.27.9 ] ) to report being able to feel high glucose levels and to report that diabetes significantly disrupted their social life ( 4.2 [ 1.214.0 ] ) but less likely to report believing that their doctor can cure diabetes ( 0.4 [ 0.20.9 ] ) .
patients taking oral medications were more likely to believe that the consequences of diabetes are limited ( 4.7 [ 1.613.4 ] ) and to report that diabetes significantly disrupted their social life ( 7.8 [ 2.227.7 ] ) but less likely to believe that it is not important to take medication when glucose levels are normal ( 0.3 [ 0.10.8 ] ) .
patients with higher a1c ( per unit increase ) were more likely to report feeling little control over their diabetes ( 1.3 [ 1.01.6 ] ) , to report believing that their doctor can cure diabetes ( 1.3 [ 1.11.7 ] ) , to consider a glucose level up to 200 mg / dl within normal limits ( 1.3 [ 1.01.7 ] ) , to believe that it is not important to take medication when the glucose levels are normal ( 1.3 [ 1.01.6 ] ) , and to believe that their diabetes medications were hard to take ( 1.4 [ 1.11.8 ] ) .
women were more likely to report that diabetes significantly disrupted their social life ( 2.3 [ 1.05.3 ] ) .
our findings demonstrate that patients with diabetes , despite having long - standing disease and regular outpatient diabetes care , frequently hold beliefs regarding disease and medication that are inconsistent with a chronic disease model of diabetes .
insulin use and worse a1c levels were associated with higher rates of several of these beliefs , as were female sex , lower education levels , and oral medication use , suggesting a potential need for additional attention when treating diabetic patients with these characteristics .
the newly observed misconceptions and related predictors may represent important opportunities for targeting barriers to successful diabetes management .
furthermore , patients displayed unrealistic expectations of treatment , as exemplified by the finding that one - third expected their doctor to cure them of diabetes .
equally troubling is the fact that half of the patients thought that a glucose level up to 200 mg / dl is normal , and 42% stated that the glucose level up to 110 mg / dl is too low .
these frequent misconceptions may be even more prevalent and have a greater effect in populations with more limited access to care and more poorly controlled a1c levels than those observed in this population .
diabetes is a complex disease that involves monitoring of multiple indexes , assessment of risk factors , and , frequently , multiple medications . as noted in other studies , misconceptions and inadequate knowledge
however , there has been limited research examining how behavioral theory driven assessments of patients ' knowledge and beliefs about the disease and
its treatment relate to successful diabetes management , particularly among inner - city adults with long - standing diabetes . in summary
, we found that disease and medication beliefs inconsistent with a chronic disease model of diabetes were common among urban minorities with diabetes despite long - standing disease and regular medical care .
these misconceptions may be logical targets for interventions to improve diabetes self - management in lower - income , minority populations .
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### Summary:
: objectiveto determine diabetic patients ' knowledge and beliefs about the disease and medications that could hinder optimal disease management.research design and methodsa cross - sectional survey of 151 type 2 diabetic patients characterizing diabetes knowledge and beliefs about the disease and medications was conducted.resultsmean diabetes duration was 13 years .
over half of the patients ( 56% ) believed that normal glucose is 200 mg / dl , 54% reported being able to feel when blood glucose levels are high , 36% thought that they will not always have diabetes , 29% thought that their doctor will cure them of diabetes , one in four ( 23% ) said there is no need to take diabetes medications when glucose levels are normal , and 12% believed they have diabetes only when glucose levels are high.conclusionsdiabetes knowledge and beliefs inconsistent with a chronic disease model of diabetes were prevalent in this sample .
suboptimal knowledge and beliefs are potentially modifiable and are logical targets for educational interventions to improve diabetes self - management .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: cholelithiasis is uncommon in childhood even though recent series show increased detection of this disease .
in contrast to adults , the epidemiology of cholelithiasis in children is unknown , as most of the studies deal with a limited number of patients .
the frequency of pediatric gallstones documented in studies showed a prevalence of 0.2 - 1.9% .
the increased use of ultrasonography in diagnosis and monitoring of gastrointestinal and genitourinary pathologies , has led to increased detection of asymptomatic gallbladder calculi .
the aim of this study was to review the clinical presentation , predisposing factor and outcome of gallstones in children through a cross - sectional study between 2000 to 2011 in a referral children 's hospital in babol , a northern city in iran .
this cohort study was performed on children with cholelithiasis in amirkola children 's hospital from 2000 to 2011 .
data on age at diagnosis , gender , clinical presentation , predisposing factors , ultrasonographic findings , gallstone density in abdominal x - ray , possible accompanying renal stone , were collected and management , follow up duration , and outcome recorded .
we defined gallstones as echogenic foci in the gallbladder or bile ducts that produced posterior acoustic shadowing in ultrasonography . in some cases
the data was analyzed by spss version 18 and a p - value < 0.05 considered being significant .
of all 66 patients 27 ( 40.9% ) were females with the mean age at diagnosis 6.64.5 years ( range 2 months to 17 years ) .
the most common predisposing factor for cholelithiasis was ceftriaxonee therapy ( 18 cases , 27.3% ) .
predisposing factors in children with gallstones abdominal pain was the most common initial symptom ( 44 patients , 67% ) that led to abdominal ultrasonography and detection of gallstones . other symptoms or signs included vomiting in 23 ( 35% ) , fever in 11 ( 17% ) , diarrhea in 9 ( 14% ) , agitation in 4 ( 6% ) , hepatomegaly or spleno - megaly in 4 ( 6% ) , jaundice in 3 ( 4.5% ) ; 5 ( 7.5% ) patients were asymptomatic and their gallstone was incidentally found in ultrasonography . in 18 cases of ceftriaxone induced cholelithiasis , the stones dissolved within 1 month after cessation of therapy .
urolithiasis accompanying cholelithiasis was recorded in 3 cases ( 4.5% ) 2 of whom had cystic fibrosis and 1 was obese .
in all cases , ultrasonographic stone location was in gallbladder , simultaneous occurrence of stone in common bile duct ( cbd ) and cystic duct was seen in 2 and 1 patient respectively . in 19 ( 29% )
patients the gallstones were solitary and in 47 ( 71% ) multiple . among 59 performed abdominal x - rays , 47(80% ) had radiolucent gallstones .
three patients who were asymptomatic and had radiolucent stones received ursodeoxycholic acid ( udca ) .
cholecystectomy was done in 6 patients ( two cases of chronic abdominal pain , 1 case of cystic fibrosis , 1 case of major thalassemia and two cases of cholecystitis ) .
the duration of follow up ranged from 1 month 6 years with a mean of 12.517.8 months in all 42 patients with ultrasonographic follow up .
resolution of stones was documented in 21 ( 50% ) patients ( 18 patients after discontinuation of ceftriaxone therapy , 3 patients after receiving udca ) , in the remaining 21 ( 50% ) patients there was no significant changes in the ultrasonographic appearance of stones in the gallbladder .
three patients with cholelithiasis died , one because of end stage renal disease due to polycystic kidney disease ( pkd ) , one because of renal failure and hemorrhage complicating thrombocytopenia associated with fanconi anemia , and the last one because of metabolic disease ( mitochondrial type ) .
this study reviews the experience with 66 children with gallstones during a 12 year period .
it is not possible to determine the incidence of gallstones in childhood because the total number of patients who had abdominal ultrasonography in this period is not available . in most studies , the mean age of diagnosis ranges from 5 to 10 years . in our study
some previous studies have shown a female predominance , while some others found a gender ratio of equal to 1 particularly in children < 12 years of age . in the present series 59.1% of patients were males and 40.9% females , this difference is not statistically significant . in our study ,
the most common ( 27.3% ) predisposing factor was ceftriaxone whereas in a multicenter study in italy , ceftriaxone was responsible for only 6% of cholelithiasis cases .
this can be related to the widespread use of ceftriaxone in treatment of pediatric infectious diseases in our area .
hemolytic diseases were the second predisposing factor ( 13.6% ) , however , in most studies the hemolytic diseases were the main risk factor for inducing cholelithiasis .
cystic fibrosis ( cf ) was responsible for 7.5% of patients with cholelithiasis in our research .
in some previous studies , same as in ours , cf was the significant risk factor for gallstones in children while in more recent studies , cf was not a common risk factor .
in some studies , and in our study , idiopathic gallstones were more common than other causes of cholelithiasis . in a multicenter study in italy , hemolytic diseases , obesity and family history were common predisposing factors for inducing cholelithiasis whereas in our study obesity was responsible for only 3% of patients and family history has not been evaluated . in our experience ,
abdominal pain was the most common ( 67% ) complaint same as in other studies .
7.5% of our patients were asymptomatic whereas in a multicenter study 35.3% of patients were asymptomatic compared to 80% of asymptomatic adult patients .
we found stones located mostly in gallbladder as seen in ultrasonography , whereas 3% of the patients had stones simultaneously in the common bile duct ( cbd ) and 1.5% in cystic duct .
in other studies , gallstones were the most common finding in ultrasonography whereas 15.8% of patients had stones in the cbd . in children , more radiopaque gallstones
abdominal x - ray does not appear to be an initial investigation method for diagnosing children 's cholelithiasis .
guidelines for management of cholelithiasis are available for adults , but little is known about management of childhood cholelithiasis .
conservative management with periodical clinical and ultrasound follow up is recommended . in our study , 6 ( 9% ) patients underwent cholecystectomy and 3 ( 4.5% ) patients received udca that dissolved gallstones during follow up without relapse . in a study , 7.2% of patients responded to udca initially but in 50% of them stone recurred and cholecystectomy was performed on 35% of patients . in another study , most of the children underwent cholecystectomy ( 13 of 19 patients ) . in a study 3.1% of asymptomatic patients and 59% of symptomatic
patients necessitated cholecystectomy , but in our study , most of the patients , received conservative management because the most predisposing factor for inducing cholelithiasis was ceftriaxone therapy where resolving stones occurred after discontinuation of the drug . in this study ,
two of them had cystic fibrosis ( cf ) ; so , in our area , cf must be considered when urolithiasis and cholelithiasis are accompanying .
cf is not only a predisposing factor for gallstones , but also predisposes patients to urolithiasis .
in conclusion , ceftriaxone therapy , hemolytic diseases , hepatobiliary disease and cf were the most common predisposing factors of cholelithiasis , and in 30% of patients , no predisposing factor was detected . in our series , cholecystectomy was required in a few patients .
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### Summary:
: objectivecholelithiasis rarely occurs in children but the increased use of ultrasonography has led to increased detection of gallstones in patients .
the epidemiology and predisposing factors of cholelithiasis vary in different populations .
the aim of this study was to describe the clinical presentation , predisposing factors and to evaluate management and outcome of patients referred to amirkola children 's hospital jn babol.methodsthis cohort study was performed on children with cholelithiasis referred during 2000 to 2011 .
cholelithiasis was diagnosed with ultrasonography .
the data was obtained based on history , physical exam , clinical and paraclinical investigations and analyzed by spss version 18 .
p - value < 0.05 was considered being significant.findingsfrom the 66 patients with cholelithiasis , 39 ( 59.1% ) were males .
the mean age at diagnosis was 6.64.5 years .
the most common predisposing factor included ceftriaxone therapy ( 27.3% ) , hemolytic diseases ( 13.6% ) , hepatobiliary diseases ( 7.5% ) and cystic fibrosis ( 7.5% ) . in 30.3% of patients ,
no predisposing factor was detected .
the most common complaint was abdominal pain ( 67% ) . among the patients in whom abdominal x - ray
was performed , only 20% had radiopaque gallstones ; 6 ( 9% ) patients underwent cholecystectomy.conclusionaccording to this study , ceftriaxone therapy and hemolytic diseases were the most common predisposing factors in children with cholelithiasis in our area and cholecystectomy had not been needed in most patients .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: ocular flutter is an eye movement disorder characterized by purely horizontal rapid saccadic oscillations lasting for few beats and stops spontaneously .
opsoclonus and nystagmus are multidirectional and are influenced by fixation , thus can be differentiated from ocular flutter .
postinfectious ocular flutter and truncal ataxia are a rare entity which has been described after infections with enterovirus , mumps , cytomegalovirus , and human immunodeficiency virus .
however , there are no reported cases of parainfectious ocular flutter and truncal ataxia in association with dengue virus infection .
hereby , we report a patient with dengue fever who had ocular flutter and truncal ataxia and responded to intravenous steroids .
a 14-year - old male child presented with fever associated with chills of 3 days duration .
on the 3 day of fever , he noticed giddiness associated with difficulty in sitting and standing ; needing support of family members ; and swaying while walking .
there was blurring of vision but no dysphagia , dysarthria , weakness or incoordination of limbs , sensory symptoms , or myoclonic jerks . on examination , his vitals were stable .
oculomotor abnormality in the form of sudden conjugate horizontal saccadic oscillations without intersaccadic interval ( ocular flutter ) occurring spontaneously , with eyes open and closed , during pursuit and saccades , and mainly in horizontal than vertical direction of gaze was seen [ video 1 ] .
liver function test showed transaminitis ( aspartate aminotransferase : 110 iu / l ; alanine aminotransferase : 156
the patient had symptomatic improvement in the form that his ataxia improved and ocular flutter decreased [ video 2 ] .
he was able to walk without support and was discharged with tapering dose of oral steroids .
a syndrome of benign encephalitis with ocular oscillation and truncal ataxia was described in the 1960 's by smith and walsh and later cogan .
patients developed ocular flutter or opsoclonus and shivering movements of the head and body with preserved consciousness following a prodrome of malaise and mild fever .
ocular flutter is the saccadic intrusions characterized by pathologic fast saccade , followed by corrective saccade without an intersaccadic interval .
the role of omnipause neurons in the brainstem is to inhibit the excitatory burst neurons which are required to initiate a saccade .
opsoclonus and ocular flutter are due to the loss of the inhibitory control of saccadic burst neurons by pontine omnipause cells .
wiest et al . reported three patients with similar syndrome as reported by smith , walsh , and cogan secondary to enterovirus infection .
patients had ocular flutter and truncal ataxia with the symptoms following an upper respiratory infection and resolved without sequelae within a few weeks .
idris and sokrab reported two patients with falciparum malaria infection who had severe ocular flutter and cerebellar ataxia .
the most common etiologies of ocular flutter are paraneoplastic syndrome , brainstem encephalitis , metabolic - toxic disturbance , and idiopathic . in children ,
paraneoplastic causes include malignancies , such as small cell lung cancer , testicular germ cell tumor , and breast cancer .
the parainfectious occurrence of ocular flutter and cerebellar ataxia in association with dengue fever has not been reported previously .
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### Summary:
: ocular flutter is an eye movement disorder characterized by purely horizontal rapid saccadic oscillations lasting for a few minutes which stops spontaneously .
postinfectious ocular flutter and truncal ataxia are a rare entity .
there are reported cases of opsoclonus myoclonus ataxia in association with dengue virus infection .
however , there are no reported cases of parainfectious ocular flutter and truncal ataxia in association with dengue virus infection .
hereby , we report a child with dengue fever who had ocular flutter and truncal ataxia .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: transfusion - related acute lung injury ( trali ) is a clinical syndrome associated with blood transfusions and is typically characterized by the sudden onset of dyspnea due to bilateral noncardiogenic pulmonary edema.1 although classic trali is defined as acute lung injury ( ali ) that occurs within 6 hours following transfusion , late - onset ali associated with blood transfusion has also been observed clinically .
it is proposed that this condition is an independent disease entity termed delayed trali syndrome.2 a recent laboratory investigation using the two - event animal model proposed that a trali event could be halted by the depletion of platelets from the recipient animal,3 and in addition , there are a few reports on the participation of platelets in trali.4,5 however , the clinical evidence that supports this rationale is poorly understood . in this report , a patient who experienced a recurrence of delayed trali syndrome is presented .
the patient was a 61-year - old woman who was diagnosed with advanced nonsmall - cell lung cancer with multiple brain metastases .
however , febrile neutropenia ( 38.5c , neutrophil 753/l , c - reactive protein 41.50 mg / dl ) and rapidly progressing anemia ( hb 5.2 g / dl ) due to intestinal tract bleeding developed after chemotherapy .
she was immediately administered two bags ( 600 ml ) of plasma- and leukoreduced red blood cells ( rbcs ) on hospital day 1 .
no apparent complications were observed within 6 hours after transfusion ; however , 48 hours after the transfusion , sudden development of dyspnea and rapid progression of hypoxemia were observed .
there was no evidence of microbial , pneumocystis , tubercular , or fungal infection in the lungs .
methylprednisolone sodium succinate was administered for pulmonary edema , but pulmonary infiltrates and hypoxemia continued to worsen ( figure 1b ) . on day 23 , chest radiography showed a significant improvement in pulmonary shadows ( figure 1c ) .
however , bleeding from the intestinal tract continued , and two additional transfusions , each comprising two bags of rbcs , were required .
no adverse events were associated with the two additional transfusions . on day 32 , an additional transfusion of two bags of rbcs was performed and on day 34 , there was a rapid deterioration of the respiratory condition again .
an autopsy was performed , and the pathological findings of the lungs indicated diffuse alveolar damage with hyaline membrane formation ( figure 2a ) . to elucidate the contribution of platelets to ali , we tried an immunohistochemical staining for the platelet using anti- cd41 antibody against the pulmonary specimen obtained from the autopsy .
the results clearly demonstrated the sequestration of platelets in the patient s lung ( figure 2b ) .
these findings strongly suggested the contribution of platelets to the pathogenesis of ali in this case .
antileukocyte antibodies were not detected in the serum of donors from whom the blood products that led to both of the ali episodes were obtained .
however , antiplatelet immunoglobulin m antibodies , in addition to the various antileukocyte antibodies ( anti - human leukocyte antigen ( hla)-i , anti - hla - ii , antimonocyte , and unclassified antineutrophil antibody ) , were detected in the serum of the recipient .
classic trali is defined as an ali that occurs within 6 hours after blood transfusion.6 however , this diagnostic criterion for classic trali was not met in our case because the two ali episodes did not occur within 6 hours after the corresponding transfusions . a recent report on the two - event animal model revealed that the environmental status of the recipient can affect the procedure in trali.3 looney et al described that platelets in the circulation of the recipient are essential for developing ali and act as terminal effector cells.3 the pathological findings also showed marked sequestration of platelets in the lung in which ali developed .
however , some antiplatelet antibodies have been shown to directly activate platelets.7,8 platelets have been shown to contain members of the chemokine family of the c - c class , such as regulated on activation , normal t - cell expressed , and secreted and monocyte chemotactic peptide-3 , as well as the cxc class , such as interleukin-8 and epithelial neutrophil - activating peptide-78.9 moreover , recent studies have shown that human platelets express toll - like receptor 410 and can sequestrate in the lung in response to lipopolysaccharide ( lps ) in a neutrophil - dependent manner.11,12 taken together , these findings suggest that circulating platelets contribute to the development of trali throughout the whole process . in the case documented here ,
antileukocyte antibodies were not found in the donors , but antiplatelet antibodies were observed in serum from the recipient . it is hypothesized that severe bacterial infection associated with systemic chemotherapy plays a role in lps - priming , and the transfusion of stored rbcs , which can contain any biological response modifier ( eg , lysophosphatidylcholine , arachidonic acid , or hydroxyeicosatetraenoic acid ) , acts as a second hit initiating trali.13 however , antiplatelet antibodies in the recipient might inhibit intra - alveolar platelet recruitment , resulting in a delay of platelet sequestration .
consequently , it is considered that this delay of platelet sequestration due to antiplatelet antibodies could cause a 48-hour eclipse time from the corresponding blood transfusion to the development of ali .
the present case report suggests an important role for the recipient s platelets and antiplatelet autoantibodies in the development of trali ( figure 3 ) .
these clinical findings could represent modulation of a trali event by environmental factors in the recipient , rather than in the donors .
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### Summary:
: a 61-year - old woman with lung cancer developed delayed transfusion - related acute lung injury ( trali ) syndrome after transfusion of plasma- and leukoreduced red blood cells ( rbcs ) for gastrointestinal bleeding due to intestinal metastasis .
acute lung injury ( ali ) recurred 31 days after the first ali episode .
both ali episodes occurred 48 hours after transfusion .
laboratory examinations revealed the presence of various antileukocyte antibodies including antiplatelet antibody in the recipient s serum but not in the donors serum .
the authors speculate that antiplatelet antibodies can have an inhibitory effect in the recipient , which can modulate the bona fide procedure of ali and lead to a delay in the onset of ali .
this case illustrates the crucial role of a recipient s platelets in the development of trali .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: hepatitis e is an enterically transmitted disease that spreads through faecal contamination of drinking water .
it occurs both in the form of epidemics as well as sporadic infection in developing countries [ 1 , 2 ] .
it is endemic to the indian subcontinent , where the seroprevalence rate ranges between 4 and 20% .
hepatitis e virus affects young to middle aged adults and causes high mortality in pregnant women , 2030% as compared to 0.21% in general population .
it has been implicated as an important aetiological agent for sporadic fulminant hepatic failure ( fhf ) in developing countries .
hev belongs to the genus hepevirus in the family hepeviridae and has a 7.2 kb positive - sense single - stranded rna genome .
the orf1 , orf2 , and orf3 encode nonstructural proteins including an rna - dependent rna polymerase ( rdrp ) , a capsid protein , and a small protein that possibly induces immune suppression in hev - infected patients , respectively [ 6 , 7 ] . presently ,
genotypes 1 and 2 have been identified exclusively in humans , and genotypes 3 and 4 have been found in humans and several animal species .
genotypes 1 and 2 have been isolated in asia , africa , north america ; genotype 4 has been identified only in asia ; and genotype 3 has been found in almost every country .
all parts of india have been experiencing repeated outbreaks and sporadic cases of hev since 1955 [ 1114 ] with genotype 1 being prevalent in the human population . with the development of knowledge about the circulation of subtypes of hev ,
this study is aimed at the molecular characterization of hev isolates to determine the most prevalent genotype in northwest india ( rajasthan ) .
the present study was carried out on 585 acute hepatitis patients attending the opd or admitted in wards of gastroenterology department of the sms medical college and hospital jaipur , rajasthan a tertiary care centre , from september 2006 to december 2009 .
the study was approved by the institutional ethics committee and informed written consent was taken from the patients . on the basis of disease severity the study comprised three hev - induced groups : acute viral hepatitis ( avh ) , acute liver failure ( alf ) , and acute - on - chronic liver disease ( aclf ) , respectively .
avh is marked by appearance of jaundice with or without prodrome and raised alt and ast levels .
alf is defined as the rapid development of hepatocellular dysfunction , specifically coagulopathy and mental status changes ( encephalopathy ) in a patient without known prior liver disease and with an illness of < 24 weeks duration .
aclf is defined by the asian pacific association for the study of liver ( apasl , 2008 ) as an acute hepatic insult manifesting as jaundice and coagulopathy , complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease ( cld ) .
patients with stable compensated chronic liver disease , significant comorbid illnesses like coronary artery disease , renal failure , and cerebrovascular disease were excluded from the study .
10 ml blood samples were collected from all the cases . the serum was separated and stored at 80c taking precaution avoiding repeated freezing and thawing of the samples by aliquating them separately for serology and pcr .
the clinical symptoms of the patients were recorded simultaneously and all the biochemical tests were performed .
serum samples were screened using commercially available micro - elisa for markers of hepatitis e ( eiagen hev igm , adaltis , spain ) .
the kit was coated with recombinant proteins for open - reading frames ( orfs ) 1 and 2 with 98% sensitivity and specificity .
the following biochemical parameters were done for all the patients : ( i ) serum alanine aminotransferase ( alt ) , ( ii ) serum aspartate aminotransferase ( ast ) , ( iii ) alkaline phosphatase ( alp ) , and ( iv ) total bilirubin ( tb ) .
rna extraction from serum of acute hepatitis e cases was done by gitc chloroform phenol method with minor modification .
cdna synthesis was carried out using mulv rt enzyme , reverse primer ( 20 pmol / ml ) ( external antisense : 5-ccg aat tca aag gca tcc atg gtg ttt gag aat gac- 3 ) ( promega ) , rnase out ( 20 u/l , invitrogen ) , 0.1 m dtt , and 5 l templates at 42c for one hour .
after cdna synthesis pcr amplification was carried out using the specific previously validated primers selected from nonstructural orf1 region ( gene bank accession no .
the primers used were external sense : 5- ccg gat cca cac aca tct gag cta cat tcg tga gct- 3 , external anti - sense : 5- ccg aat tca aag gca tcc atg gtg ttt gag aat gac- 3 , internal sense : 5- gga att cga ctc cac cca gaa tta ctt- 3 , and internal anti - sense 5- gga att cac agc cgg cga tca gga cag- 3. these two sets of primers were designed to produce 343 bp segment of orf1 region .
the thermal cycling conditions were as follows : initial denaturation 94c for 5 minutes followed by 30 cycles of denaturation for 30 seconds at 94c , annealing for 30 seconds at 59c , and extension for 30 seconds at 72c , as well as final extension for 7 minutes at 72c .
positive and negative control was included in every reaction to rule out false positive and negative . amplified final pcr products ( 343 bp of rdrp region )
the expected bands were excised from the gel and purified with a qia quick gel extraction kit ( qiagen , hilden , germany ) .
the purified dna were subjected to sequencing pcr using big dye terminator ready reaction mixture , reverse primer ( 3.2 pmoles/l ) at thermal cycling conditions : 25 cycles of denaturation 94c for 10 seconds , annealing 55c for 5 seconds and extension 60c for 4 minutes .
cycle sequencing pcr product was purified by adding 0.1 volume of 3 m sodium acetate ( ph = 4.5 ) and 2.5 volume of absolute alcohol and dissolved in 25 l of template suspension reagent ( tsr ) and heated at 95c for 5 minutes and snap - chilled to denature the dna .
the sample was run through performance - optimized polymer ( pop6 ) and electrophoreses at 12.1 kv for 3 hours in 1 xgenetic analyser buffer on abi prism 310 sequencer .
amplicon sequences of hev rdrp region were compared to an online database for the best possible match using the blast ( basic local alignment search tool ) program of national center for biotechnology information ( http://www.ncbi.nlm.nih.gov/ ) and clustal - x version 2.0 .
the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( 1000 replicates ) is shown next to the branches .
the tree is drawn to scale , with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree .
the evolutionary distances were computed using the kimura 2-parameter method and are in the units of the number of base substitutions per site .
all positions with less than 95% site coverage were eliminated , that is , fewer than 5% alignment gaps ; missing data and ambiguous bases were allowed at any position .
only those sequences , which had our target region of hev rdrp , were taken for phylogenetic analysis .
for data management and statistical analysis , spss-10 software ( spss inc . , chicago , il , usa ) was used .
difference between avh , alf , and aclf with respect to various liver function tests was calculated using the anova ( analysis of variance ) .
in the study , a total of 585 acute hepatitis cases were included . amongst these 205 ( 35.04% )
these were grouped into 100 patients with acute viral hepatitis ( avh ) , 75 with acute liver failure ( alf ) , and 30 with acute on chronic liver failure ( aclf ) .
age presentation , sex distribution , and characteristic features of different groups were as shown in table 1 .
the mortality rate in pregnant females was 6/27 ( 22.22% ) and in nonpregnant females was 1/40 ( 2.5% ) .
there was no mortality in 100 patients of avh , while 9/75 ( 12% ) alf patients and 2/30 ( 6.67% ) sahf patients died .
variation was observed in the different groups with respect to clinical symptoms like fever , pruritus , anorexia , and pedal edema as shown in table 2 . the age and lft profile ( ast , alt , and alp ) was significantly higher in alf cases than sahf and avh but no significant difference
was obtained in total serum bilirubin as shown in table 3 . due to the transient viremic nature of hev infection ,
80/205 samples were selected ( within 1 week of the illness ) for hev rna detection by nested reverse transcriptase polymerase chain reaction ( nrt - pcr ) .
the amplification products of rdrp region of orf1 ( 343 nt ) from 6 avh patients , 7 alf patients , and 4 acute on chronic patients were subjected for sequence analysis and submitted to genbank with accession nos .
fj231471 , fj231472 , fj231473 , fj231474 , fj231475 , fj231476 , fj231477 , fj231478 , fj231479 , fj231480 , fj231481 , fj231482 , fj231483 , fj231484 , fj231485 , fj231486 , fj231487 .
gene sequences of the present isolates along with standard strains from different geographical areas were used for the construction of the phylogenetic tree .
figure 1 shows the phylogenetic tree constructed for the 17 new sequences and their closest matching sequences in genbank . to further classify the subtype
fj231474 , fj231475 , fj231477 , fj231480 , fj231481 , fj231482 ; 7 alf with accession nos .
fj231472 , fj231473 , fj231476 , fj231483 , fj231484 , fj231485 , fj231486 ; remaining 4 the accession nos .
belong to aclf ) hev isolates 12 belonged to genotype 1a and 5 belonged to genotype 1c .
all avh and aclf isolates belonged to genotype 1a and two alf isolates belonged to genotype 1a and the remaining five were clustered with genotype 1c .
hev isolates which belonged to genotype 1c had 90.1%93.5% sequence homology with subtype 1c and hev isolates which belonged to genotype 1a had 91.5%94.2% sequence homology with subtype 1a .
there was a high degree of variability found between genotypes 3 and 4 , with a divergence of 31.2% and 30.1% , respectively .
iu / l , 9452317 ; p = 0.01 ) than genotype 1a and the prothrombin time was lower in the genotype 1c patients ( 61% , 4277 versus 84% , 7096 ; p = 0.05 ) .
up to now , divergent hev sequences have been reported from many countries and fall into 4 major genotypes , namely , genotypes 1 , 2 , 3 , and 4 . based upon nucleotide differences
, it is proposed that five , two , ten , and seven subtypes for hev genotypes 1 , 2 , 3 , and 4 designated respectively . however , inspite of the genomic diversity of hev , studies conducted so far [ 20 , 21 ] indicate that all strains identified till date comprise a single serotype [ 9 , 22 ] . it is reported that genotypes 1 and 4 continue to circulate in humans and pigs , respectively , from india [ 23 , 24 ] . however ,
identification of two different isolates from the same region of japan indicated that there could be significant diversity between strains from the same region .
subsequently diversity was found between various isolates from argentina , austria [ 26 , 27 ] , spain , china , and taiwan [ 29 , 30 ] .
growing reports suggest that different hev genotypes and subtypes play important role in disease severity .
genotypes 1 and 2 have similar epidemiological and sporadic features and can result in acute hepatitis , acute liver failure , and acute - on - chronic liver failure while genotypes 3 and 4 were normally considered to cause acute , self - limiting illness followed by a complete recovery .
thus in human genotypes 3 and 4 seem to be less virulent than genotypes 1 and 2 and do not cause severe liver diseases .
hev genotypes may affect the effectiveness of viral transmission and , in turn , the severity of hev , associated hepatitis ; therefore we classified patients into three groups on the bases of disease severity .
nevertheless , several genotypes circulate in the neighbouring countries , such as china , thailand , or vietnam , where genotypes 3 and 4 have also been identified from human and swine infections .
studies demonstrated that human and swine hevs from western and southern india belong to different genotypes ; genotype 1 circulates in humans whereas genotype 4 in pigs [ 3335 ] and the majority of the indian isolates belong to subtypes 1a , 1b , 1c , and 1d , though hev genotypes appear to be segregated geographically .
single ind - fhf isolates were studied to form genotype i d . although all other indian isolates were from either western or southern parts of india , the exact location of isolation of isolate ind - fhf is not known . in a recent report from north
india subtypes 1a and 1c are prevalent with the subtype 1c showing a trend towards fulminancy .
this diversity of genotypes and subtypes is useful in understanding epidemiological phenomena such as geographical spread of the virus or the transmission in the community . in the present study ,
the strains phylogenetically clustered into genotype 1 and formed two subgroups a and c , sharing 91.5%94.2% and 90.1%93.5% homology with intersubgroup and 79.585.3% , 80.184.8% , and 78.380% intrasubgroup identity homology with subgroup b , d , and e , respectively .
interestingly , we also observed that the patients with genotype 1c tend to have more clinical manifestation than those with genotype 1a infection .
the present study indicates that genotype 1 is the main prevalent type of the genotypes in humans in north west india with subgroup a and c. avh and alf patients have different subtypes a and c , respectively , which raises our insight on the molecular basis of hev disease severity .
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### Summary:
: genotyping and subtyping are important to understand epidemiology of the hepatitis e virus so as to improve control measures to prevent transmission of virus in the community . hence , the aim of the current study was to identify the prevalent hev genotypes in rajasthan in acute sporadic hepatitis e cases with varying degree of liver failure .
we studied hepatitis e virus ( hev ) isolates from hospitalized patients in rajasthan , western india . in a total of seventeen hev sequences , six acute viral hepatitis ,
seven acute liver failure , and 4 acute- on - chronic cases were analyzed .
subtypes 1a and 1c of hev are prevalent in northwest india .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: vitamin d deficiency has emerged as a major health problem in saudi arabia in both genders and all age groups .
the reported prevalence in patients visiting primary health care centers ( phcs ) is between 28% in healthy adult males , 80% in healthy school children , and 100% in young adult females .
this reported wide variability in the prevalence could be related to the use of different cut - off points for the definition of vitamin d deficiency , or the differences in the methods used in analyzing the blood samples for vitamin d levels .
related literature since 1983 indicates that the problem of vitamin d deficiency in saudi arabia is not recent . in articles published in the last few years to highlight the size of the problem ,
several factors have been identified as contributors to the low level of vitamin d in the saudi population .
these include the religious practice whereby women wear hijab , limited exposure to the sun in spite of abundant sunlight all year round , and poor dietary intake .
the relationship between vitamin d , rickets , osteomalacia and osteoporosis is well established , but vitamin d has recently been identified as an immunomodulator that could be linked to such conditions as bronchial asthma , atopic dermatitis and food allergy , type 1 diabetes mellitus , and an increased susceptibility to infections . in 2003 ,
the american academy of pediatrics recommended giving supplements of 200 iu / day of vitamin d to all infants starting in the first 2 months after birth as a primary means of preventing rickets . in 2008 ,
the policy was revised and the dose increased to 400 iu / day starting in the first few days of life to continue through childhood to adolescence .
despite these widely advocated recommendations , few studies have addressed the practice of health care workers with regard to vitamin d supplementation for infants .
a study of military pediatricians and family physicians found that pediatricians were 40% more likely than family physicians to prescribe vitamin d to exclusively breast - fed infants , and the most common reason for not prescribing vitamin d was the belief that infants were adequately exposed to sunlight . in this study ,
63% of the family physicians prescribed vitamin d to exclusively breast - fed infants compared to 89% of the pediatricians . in addition , only 29% of the family physicians gave supplements to breast- and bottle - fed infants compared to 66% of the pediatricians . a similar finding
was reported in a study of pediatricians and family physicians in connecticut , usa , where 88.6% of the pediatricians reported counseling parents about vitamin d for infants compared to 52.3% of the family physicians .
another american survey of 155 pediatric primary health care providers published in 2004 , showed that 47.7% of the surveyed physicians prescribed supplementation for exclusively breast - fed infants .
of those physicians who prescribed supplementation , 27.1% recommended it for all infants ( both breast- and bottle - fed ) . in this study ,
females , pediatricians , and those who had 21 or more years of experience were more likely to recommend vitamin d. in saudi arabia , there are no recommendations on vitamin d supplementation by the ministry of health ( moh ) .
consequently , the practice of giving infants supplements of vitamin d is left to the health institutions , and therefore varies from one institution to another .
furthermore , no studies were found that address the practice of giving infants in saudi arabia supplements of vitamin d. the objective of this study was to measure knowledge , attitude and practice ( kap ) of general practitioners ( gps ) working in phc in the dammam area , towards vitamin d supplementation for infants .
this was a cross - sectional study of gps working in the moh phcs in dammam , saudi arabia .
the total number of gps working in dammam phcs during the period of data collection ( march 2014 ) was 110 , and all were invited to participate in the study . a 10-min self - administered 2-part questionnaire developed by the researchers after discussions with experts and a literature review was used to achieve the objective of the study .
the first part covered demographic data that included : age , gender , marital status , number of children , nationality , place of graduation , number of years since graduation , number of years of practice as a gp , and previous work experience in institutions other than phc .
the participants were asked to state their level of agreement with 20 neutral sentences by choosing from 3 responses ( agree , neutral , disagree ) . for the questions on practice ,
the questionnaire was pilot tested in 20 gps working in the neighboring al khobar area .
some questions were modified after the analysis of the pilot testing . before the final form was prepared , the questionnaire was revised after an assessment for content validity by two experts in the field
. the main outcome of the study was to determine gps kap about vitamin d supplementation to infants .
a set of questions was used to explore gps information on the importance of vitamin d , their perception of the role of vitamin d in preventing disease , their practice of prescribing vitamin d for infants and whether they asked parents about their compliance with giving their infants the vitamin d supplements .
the collected data was managed , coded and entered to spss 20.0 for macintosh ( spss inc . ,
chicago , il , usa ; 2010 ) and was checked for any abnormality before analysis . statistical analysis included descriptive analysis of all the variables , expressed as the mean ( standard deviation [ sd ] ) or the median if not normally distributed .
a knowledge score was calculated by combining the results of the 11 knowledge questions ( 3 points were given for each correct answer giving an optimum score of 33 to denote that all knowledge questions had been answered correctly ) .
the responses were re - coded as the following : score 3033 were regarded as excellent knowledge ( mean + 2sd ) , scores 2729 were regarded as good knowledge ( mean + 1sd ) , scores 2426 ( mean 1sd ) were regarded as fair knowledge , and scores < 24 ( mean 2sd ) were regarded as poor knowledge .
practice score was calculated by combining the results of the questions on practice . to check for correlation between different study variables and the outcomes of the study
, bivariate correlation was used for categorical variables , and independent t - test was used for continuous variables .
in addition , multivariate regression analysis of knowledge and practice scores ( as dependent variables ) and all demographic variables ( as independent variables ) was done .
of the 110 gps who were working in dammam phc during the study period , 99 answered the questionnaire .
six questionnaires were excluded from the analysis for being incomplete ( missing data more than 30% ) .
the mean age of participants was 33.4 ( sd 5.5 ) years , and the majority were females ( 74% ) .
a large proportion was married ( 81% ) with a median number of children of 2 .
seventy - three percent of doctors were saudi and 55.8% had graduated from saudi arabian universities . the mean duration of practice as gp was 6.64 years ( sd 1.6 ) . around 30% of the participants
demographic characteristics of gps participating in the survey the mean and sd for the knowledge score was : mean = 27 , sd = 3 .
the analysis showed that 30% of the gps had excellent knowledge , 32% had good knowledge , 23% had fair knowledge , and 14% had poor knowledge [ table 2 ] .
frequency distribution of knowledge score of gps regarding vitamin d supplementation the attitudes of gps were generally positive ; 92.2% believed that vitamin d supplementation was important for infants health .
of the 93 physicians surveyed , 56 physicians ( 60.2% ) regularly prescribed vitamin d supplementation for all infants , 8 physicians ( 8.6% ) recommended supplementation for exclusively breast - fed infants only , and 7 physicians ( 7.5% ) did not recommend vitamin d to infants in their practice .
twenty - two gps ( 23.6% ) did not respond to the questions on the practice of supplementation . on the question of advice given to parents
, 63% of physicians stated that they regularly advised parents to expose their infants to the sun in order for them to get adequate vitamin d. in addition , 53% stated that they spent time explaining to all parents the importance of vitamin d supplementation for their infants health . on the question of follow - up of infants
, 33.3% of the physicians stated that they asked parents about their compliance with giving supplements of vitamin d at each encounter regardless of the reason for the visit to the clinic , 28% asked about compliance during
well baby visits only , and 3.2% stated that they did not ask about compliance with supplementation at all [ table 3 ] .
practices of gps regarding vitamin d supplementation there was no significant relationship between knowledge and practice scores , with age , gender , marital status , nationality , place of graduation , years of practice as gp or previous work experience in places other than phc ( p > 0.05 ) .
in addition , the knowledge and practice scores were regressed as dependent variables , while all other variables were entered as independent variables .
this high rate is important for the reduction of nonresponse bias since the objective of the survey was to measure kap of physicians .
more than half of the participants in this study had either excellent or good knowledge scores .
this could help in the future design of programs to change kap of gps by providing a general idea of the current situation . of the physicians surveyed , more than half prescribed vitamin d supplementation for all infants . in addition , just a minority of gps prescribed vitamin d for exclusively breast - fed infants only .
this result was not comparable to the reported prevalence in other studies . in the american study on the prescribing pattern in las vegas , only about one - third ( 27.1% ) of the physicians recommended supplementation for all infants ( both breast- and bottle - fed ) .
the study of military physicians showed a similar finding in which 29% of family physicians advised supplements for both breast- and bottle - fed infants .
these figures show that gps in saudi arabia were more likely to prescribe vitamin d supplementation for both breast- and bottle - fed infants
. this could be due to the high prevalence of vitamin d deficiency in saudi arabia and the higher emphasis put on using vitamin d supplementation to ameliorate the effect of low exposure to sunlight .
in addition , the positive attitude of almost all of the surveyed physicians regarding vitamin d supplementation could be a factor in the higher rates of prescribing vitamin d. there are some limitations to the study .
these include small sample size , which might be a reason for the absence of a significant association between demographic variable and knowledge and practice scores .
another limitation is that large percentage of gps did not respond to the questions on their practice .
the reason for this high nonresponse to a specific section of the questionnaire is not clear .
it is unlikely to be related to questionnaire design , as we did not have this problem in the pilot study . in addition , the survey was anonymous so participants could not have been worried about any adverse consequences for admitting their true practice .
whatever the reason for their nonresponse , this could have a negative impact on the accuracy of determining prescribing patterns . finally , one of the problems that gps caring for infants in saudi arabia may face is that there is no specific recommendation from the saudi moh on vitamin d supplementation .
besides , different pediatric societies have different practice guidelines for vitamin d supplementation , and none of these recommendations has been endorsed by the moh . the consequence of this is the variation in gps practice as observed in this study .
the saudi moh should establish its own clinical practice guidelines or endorse one of the existing guidelines for use , to ensure that gps follow the latest , evidence - based practice and that all health institutions maintain the same standards of care .
this study is the first to provide data on gps kap of supplementing infants with vitamin d in saudi arabia .
around one - third of gps had excellent knowledge of vitamin d supplementation and almost all of them had a positive attitude toward supplementation .
the practice of giving infants supplements of vitamin d was suboptimal , given the high prevalence rate of vitamin d deficiency .
consequently , it is important for the moh to develop clinical practice guidelines to improve the practice of supplementation and to maintain proper standards of care .
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### Summary:
: background : with the report of high prevalence rate of vitamin d deficiency in the literature since the 1980s , the condition can be considered an epidemic in saudi arabia .
however , no specific steps have been taken by the ministry of health to tackle the problem . moreover ,
little is known about general practitioners ( gps ) knowledge , attitude , and practice ( kap ) of vitamin d supplementation for infants.objectives:to measure the kap of gps , of vitamin d supplementation for infants in dammam , saudi arabia.materials and methods : this was a cross - sectional study of gps practicing in primary healthcare centers in the dammam area of saudi arabia .
all the 110 gps working in primary health centers ( phcs ) in dammam were invited to participate in the study .
data was collected with the help of a self - administered questionnaire .
data was analyzed using spss 20.0 .
statistical analysis included descriptive analysis of all the variables , expressed as the mean ( standard deviation [ sd ] ) or the median if not normally distributed.results:ninety-three gps working in the dammam area participated in the study .
thirty percent had excellent knowledge of vitamin d. ninety two percent had a positive attitude toward vitamin d supplementation and sixty percent regularly prescribed vitamin d supplementation for infants.conclusion:the majority of gps had a positive attitude toward vitamin d supplementation for infants .
however , their knowledge and practice need improvement .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: gorlin goltz syndrome , also known as nevoid basal cell carcinoma syndrome ( nbccs ) , is a rare inherited autosomal dominant disorder .
although the prevalence of this syndrome varies with different ethnic groups , it is estimated to be 1 in 57,000 to 1 in 164,000 . however , in indian scenario , nbccs has been rarely reported .
it is characterized by multiple keratocystic odontogenic tumors ( kcots ) , multiple basal cell carcinomas ( bcc ) , skeletal , ophthalmic , neurological abnormalities , and facial dysmorphism .
recently , genetic studies suggest markers such as ptch1 , ptch2 , and sufu to be responsible for the syndrome . among them , mutation in patched1 ( ptch1 ) gene which acts as a negative regulator of hedgehog signaling pathway has been identified as the main cause of nbccs . as the syndrome is a hereditary condition with a 50% chance of inheritance in offsprings of affected patients , genetic screening and counseling of patients and family members become important to screen for familial predisposition of this syndrome .
the genetic mapping of individuals would help in early diagnosis and management of suspected disease , thus decreasing the severity of abnormalities .
antenatal diagnosis for pregnancies in increased risk patients is also possible by ultrasound scan and extraction of dna from fetal cells by amniocentesis .
also , this provides an opportunity for the development of future drugs for treatment or prevention of syndrome in subsequent generations .
the purpose of this present article is to describe , in detail , the clinical , radiological , and histopathological findings in five cases of gorlin goltz syndrome from north india diagnosed in our department , to discuss the role of gene mutation analysis , and to predict the recurrence of kcots depending on its histopathologic nature .
also , this article aims to correlate the incidence of the present study 's findings of gorlin goltz syndrome with cases published in literature in indian patients and compare the prevalence and frequency of major and minor criteria noted in indian population with other studies in different parts of the world .
this study was conducted in 5 patients of nbccs diagnosed in our department from september 2013 to october 2014 .
all 5 patients were females with chief complaint of swelling , pain , or foul fluid discharge on at least one side of maxilla or mandible and a detailed extraoral and intraoral examination was done raising a suspicion of cyst or tumor involvement .
it was followed by panoramic radiographs which confirmed the presence of multiple maxillary and mandibular cyst - like lesions [ figure 1 ] .
orthopantomogram of patient ( case 1 ) showing multiple mandibular and maxillary cyst - like lesions a detailed clinical examination of head and neck region was done in all patients according to the diagnostic protocol suggested by lo muzio which revealed the presence of macroencephaly with increased occipitofrontal circumference ( > 55 cm ) , frontal bossing , ocular hypertelorism with increased intercanthal distance ( > 36 mm ) , or palmar and plantar pits on systemic examination [ figure 2 ] .
due to characteristic clinical findings and radiographic presentation of multiple cyst - like lesions , gorlin goltz syndrome was suspected .
aspiration of suspected cystic cavities revealed white keratin - like material in all patients raising suspicion of kcots .
further investigations and radiological evaluation of the chest , skull bones , hands , feet , long bones , pelvis , and spine were carried out for all patients . computed tomography examination of head and face
multisystem evaluation of patients with dermatologic , ophthalmic , cardiac , ear , nose , and throat and neurologic examination was advised for all patients .
extraoral photograph of patient showing macroencephaly with increased occipitofrontal circumference and ocular hypertelorism ( case 3 ) incisional biopsy or marsupialization of cystic cavities also revealed keratin - like cheesy material .
histopathological examination showed 56 layers of thick stratified squamous epithelium with cuboidal to columnar palisaded basal cell layer with dark - staining nuclei and a corrugated surface with parakeratinization .
histopathological examination depicting features of parakeratinized keratocystic odontogenic tumor the diagnosis of nbccs was made based on the presence of major and minor criteria as suggested by kimonis et al .
all the patients were enquired about any family history ( fh ) of cysts and tumors or other features of syndrome and first - degree relatives examined for any positive findings . the detailed clinical and radiographic presentation and positive diagnostic findings of syndrome
a search was done of the available literature ( pubmed , google scholar , mozilla , and internet explorer ) on cases of gorlin goltz syndrome reported in india from 1977 to present , which revealed 48 patients in 38 case reports being published .
a summary of the diagnostic features of syndrome in these patients is presented in table 4 .
the frequency of findings in these patients in indian literature combined with 5 patients in our case series was then compared with studies in different parts of the world and is presented in table 5 .
diagnostic findings of nbccs in five cases being reported clinical features associated with kcot 's in five nbccs patients being reported clinical features associated with kcot 's in five nbccs patients being reported total number of nbccs cases reported in indian literature along with their diagnostic findings comparison of diagnostic criteria with other studies in various parts of world
preoperative paresthesia or anesthesia was not noted in any of the patients on clinical examination .
all the patients had unilocular or multilocular radiolucent cavities of maxilla or mandible with number of cysts ranging from 2 to 5 .
the most common site of cystic involvement was mandibular body region followed by mandibular angle - ramus region and maxillary posterior region .
inferior alveolar nerve canal displacement or antrum involvement was seen in all the patients due to large size of cystic lesions
. retained deciduous teeth associated with cystic cavities were found in 80% of the patients though the association of retained deciduous teeth with nbccs has not been reported in literature so far .
comparing the findings of the present case series with other reports in indian literature , none of our patients had bcc , syndactyly or polydactyly , pectus deformity , bridging of sella turcica , pigmented nevi , or fh of syndrome as opposed to these findings in some earlier reports in indian population .
as all the patients reported in this series were from north india , this suggests wide variation in syndrome manifestations within the same ethnic and racial population .
cleft lip and palate had been reported in only one indian patient with nbccs previously .
the second such finding has been found in one of our patients . on comparison of diagnostic findings of this syndrome in indian patients with other parts of the world , the incidence of bifid or fused ribs
medulloblastoma ( med ) has not been found in any indian patient in contrast to other studies conducted worldwide .
small unilocular cysts were enucleated and in larger cysts , marsupialization followed by enucleation was done as treatment protocol .
in all the patients , enucleation was followed by aggressive curettage and two cycles of liquid nitrogen spray cryotherapy of 1 min each separated by a thaw interval of 5 min .
the patients were kept on a strict , regular postoperative follow - up regimen of 1 , 3 , 6 , and 12 months and biannually thereafter to detect early recurrence or for the appearance of any other manifestations of the disease .
gorlin goltz syndrome ( nbccs ) was first recognized in 1894 by jarisch and white .
dr . robert gorlin and dr . robert goltz ( 1960 ) delineated the different clinical features in their study on multiple naevoid basal cell epithelioma , jaw cysts and bifid rib syndrome .
the main clinical manifestations of this syndrome include multiple bcc , kcots of jaws , palmer - plantar pits , rib and skeletal anomalies and facial dysmorphism , cleft lip / palate , eye anomalies like cataract , bitot 's spots , etc . ) .
various low frequency neoplasms such as meds , meningiomas , ovarian and cardiac fibromas , epidermoid cysts , and defects of stomatologic system , including mandibular prognathism , high arched palate , malocclusion , impacted teeth , ameloblastoma , squamous cell carcinoma , and odontogenic myxoma , have also been reported . in certain occasions
very few cases of nbccs had been reported previously in indian literature probably representing underrecognition . in order to establish a diagnosis of the gorlin
the presence of two major and one minor or one major and three minor criteria are necessary to establish diagnosis .
bcc ( multiple or one occurring under the age of 20 years)histologically proven kcots of the jawspalmar or plantar pits ( three or more)bilamellar calcifications of the falx cerebribifid , fused , or markedly splayed ribsfirst - degree relative with nbccs .
bcc ( multiple or one occurring under the age of 20 years ) histologically proven kcots of the jaws palmar or plantar pits ( three or more ) bilamellar calcifications of the falx cerebri bifid , fused , or markedly splayed ribs first - degree relative with nbccs .
macrocephaly ( adjusted for height)congenital malformation : cleft lip or cleft palate , frontal bossing , coarse face , and moderate or severe hypertelorismother skeletal abnormalities : sprengel deformity , marked pectus deformity , and marked syndactyly of the digitsradiological abnormalities : bulging of sella turcica , vertebral anomalies such as hemi vertebrae , fusion , or elongation of vertebral bodies , defects of the hands and feet , or flame - shaped hands or feetovarian fibromamed .
macrocephaly ( adjusted for height ) congenital malformation : cleft lip or cleft palate , frontal bossing , coarse face , and moderate or severe hypertelorism other skeletal abnormalities : sprengel deformity , marked pectus deformity , and marked syndactyly of the digits radiological abnormalities : bulging of sella turcica , vertebral anomalies such as hemi vertebrae , fusion , or elongation of vertebral bodies , defects of the hands and feet , or flame - shaped hands or feet in all of our five cases , clinically at least two major and one minor criteria were present .
histopathologically , all the lesions were associated with parakeratinization , intramural epithelial remnants , and satellite cysts suggesting it to be kcot .
dominguez and keszler ( 1988 ) suggested that all these features were more frequent among nbccs keratocysts than among solitary keratocysts .
hence , the clinical and histopathological features confirmed the diagnosis of gorlin goltz syndrome for all the five cases .
donatsky and hjrting - hansen reported that the significantly higher occurrence of proliferative epithelial remnants in the connective tissue wall of the cyst might be an explanation for the high recurrence rate of kcots seen in patients with nbccs .
as the biological behavior of kcots associated with nbccs is more aggressive and these cysts have higher recurrence rates ( 82% ) compared with solitary keratocysts ( 61% ) , treatment by marsupialization or enucleation with adjuvant chemical cautery using carnoy 's solution or cryosurgery should be undertaken in these patients .
cryotherapy produces cellular necrosis and destroys the residual cysts and epithelial remnants while maintaining the inorganic osseous framework intact . in our patients ,
enucleation with or without marsupialization was followed by liquid nitrogen spray cryotherapy ( 196c ) for the treatment of cystic cavities . the literature reports wide variation in the incidence of kcots in nbccs patients ranging from 62% to 100% .
usually , multiple keratocysts are found in nbccs ranging from 1 to 30 in number , average being 5 . in our case series ,
the most common site which is usually found is the mandibular molar or ramus region ( 44% ) followed by incisor - canine region ( 18% ) . in our case series , mandibular body region
was affected in 4 patients ( 80% ) , crossing midline , and extending to contralateral side in two ( 40% ) of them .
in contrast , the majority of the maxillary cysts occur commonly in incisor - canine region ( 15% ) and molar - tuberosity region ( 13% ) .
the bilateral maxillary cysts in one of our patients were found in premolar - molar region .
palmar - plantar pits usually occur in 3587% of the syndromic patients and in 80% develop by the age of 15 years and in 85% after the age of 20 years .
pits were found to be present in 80% of our cases ( 4 patients ) .
they are usually more commonly found on palms ( 77% ) than on soles ( 80% ) , but plantar pits were seen in three of our patients ( 60% ) as compared to 2 patients with palmar pits ( 40% ) .
ectopic calcifications of falx cerebri have been found in as low as 21.2% to as high as 92% patients in different reports worldwide whereas in our series , they were found in 60% of the cases [ figure 4 ] . computed tomography scan showing calcification of falx cerebri and tentorium cerebella ( case 1 ) relative macroencephaly with an increased occipitofrontal circumference ( > 55 cm )
ovarian cysts and fibroma are usually a finding in 2550% of affected women with nbccs and often present bilaterally ( 75% ) .
this is consistent with our finding in only one affected patient ( 20% ) but being present unilaterally .
bifid , fused , wide , partially missing , or underdeveloped ribs are usually found in 1658% of nbccs patients .
epidermoid cysts have been found to occur in 50% of the cases in literature . in our case series ,
cleft lip and palate and sprengel deformity are rare in this syndrome with 09% and 422% occurrence .
scoliosis of spine rarely being reported in indian literature was also found in one of our patients [ figure 5 ] .
multiple impacted teeth were seen in all of these patients with a novel feature of retained deciduous teeth seen in 4 patients ( 80% ) which may be another associated finding with this syndrome not being reported previously .
right 5 rib anteriorly and left 5 rib posteriorly nbccs is a hereditary condition mostly caused by mutations in the ptch1 gene which was isolated in 1996 as the human homolog of the drosophila segment polarity gene ptch .
more than 225 mutations in the ptch1 gene in gorlin goltz syndrome have been found so far .
the ptch1 gene has been mapped to the long arm of chromosome 9q22.3 located from base pairs 95,442,981 to base pairs 95,516,964 .
the ptch1 gene provides instructions for producing the patched-1 protein , which functions as a receptor . a protein
together , they trigger signals that play a role in cell growth , cell specialization , and determining the patterning of different parts of developing body .
mutation in ptch1 gene prevents the production of patched-1 or leads to the production of an abnormal version of the receptor .
an altered or missing patched-1 receptor can not effectively suppress cell growth and division . as a result
, cells proliferate uncontrollably to form the tumors that are characteristic of gorlin goltz syndrome .
its pathogenesis supports the knudson two - hit hypothesis which states that normal cells require two mutagenic hits ( two distinct episodes of dna damage ) to produce cancer .
patients with retinoblastoma , nbccs , and similar syndromes have a constitutional ( germline ) defect in the dna sequence in one of the two copies of a tumor suppressor gene .
if a second dna injury or if loss of the normal remaining allele occurs at the same locus ( the second hit ) , the cell may become malignant .
in nbccs , various tumors and hamartomas ( bcc , odontogenic keratocysts , meningiomas , and ovarian fibromas ) exhibit loss of heterozygosity but other lesions , for example , palmar pits do not .
various physical anomalies ( bifid rib , macroencephaly ) apparently need only one - hit .
although bcc has a low metastatic potential of 0.00280.55% , recently an nbccs patient had been diagnosed with recurrent bcc with lung metastasis . as cumulative ultraviolet ( uv )
light is the main risk factor , these patients are warned against prolonged sun exposure .
about 7080% of the individuals with nbccs have an affected parent and about 2030% of the probands have a de novo mutation . the risk to a sib of a proband
depends on the genetic status of the parents : if a parent of the proband is affected , the risk to the sibs is 50% ; when the parents are clinically unaffected probably because the effects being so mild that is , has never been diagnosed , the risk to the sibs of a proband appears to be low .
in the present case series , the syndrome did not affect the patient 's parents or siblings and there were no familial antecedents in any of our 5 patients .
location of gene for this syndrome by gene mutation analysis offers the possibility that dna markers can be used in risk estimation and presymptomatic identification of the patients .
( 2008 ) identified a heterozygous germline mutation in ptch2 gene in affected members of a chinese han family with nbccs .
however , no ptch2 mutations were found in 11 cases of nbccs or in individuals with familial cases of nbccs who did not have identifiable ptch1 mutations .
an isolated case of gorlin goltz syndrome has been found to have mutation in sufu gene located on chromosome 10q24.32 in the absence of mutation in ptch1 gene . as all of these three genes belong to sonic hedgehog pathway ,
whether mutations in a single gene or a combination of these genes is probably responsible for the various manifestations of nbccs needs to be further studied .
furthermore , a new treatment strategy based on the understanding and inhibition of the hedgehog pathway can provide for specific drug treatment of disease in future to suppress tumor growth .
thus , antenatal diagnosis for pregnancies at increased risk for syndrome is possible by analysis of dna extracted from fetal cells ( obtained by amniocentesis or by chorionic villous sampling ) and ultrasound scans .
it can thus be helpful in detecting serious fatal developmental malformations such as fibromas of heart .
some fetuses with syndrome may have difficult deliveries due to large heads requiring assistance in delivery by either forceps or caesarean section .
all the 5 patients have been kept on regular periodic follow - ups , and no recurrence of kcot 's or new syndromic manifestations have been found in any patient until date .
bcc ( multiple or one occurring under the age of 20 years)histologically proven kcots of the jawspalmar or plantar pits ( three or more)bilamellar calcifications of the falx cerebribifid , fused , or markedly splayed ribsfirst - degree relative with nbccs .
bcc ( multiple or one occurring under the age of 20 years ) histologically proven kcots of the jaws palmar or plantar pits ( three or more ) bilamellar calcifications of the falx cerebri bifid , fused , or markedly splayed ribs first - degree relative with nbccs .
macrocephaly ( adjusted for height)congenital malformation : cleft lip or cleft palate , frontal bossing , coarse face , and moderate or severe hypertelorismother skeletal abnormalities : sprengel deformity , marked pectus deformity , and marked syndactyly of the digitsradiological abnormalities : bulging of sella turcica , vertebral anomalies such as hemi vertebrae , fusion , or elongation of vertebral bodies , defects of the hands and feet , or flame - shaped hands or feetovarian fibromamed .
macrocephaly ( adjusted for height ) congenital malformation : cleft lip or cleft palate , frontal bossing , coarse face , and moderate or severe hypertelorism other skeletal abnormalities : sprengel deformity , marked pectus deformity , and marked syndactyly of the digits radiological abnormalities : bulging of sella turcica , vertebral anomalies such as hemi vertebrae , fusion , or elongation of vertebral bodies , defects of the hands and feet , or flame - shaped hands or feet in all of our five cases , clinically at least two major and one minor criteria were present .
histopathologically , all the lesions were associated with parakeratinization , intramural epithelial remnants , and satellite cysts suggesting it to be kcot .
dominguez and keszler ( 1988 ) suggested that all these features were more frequent among nbccs keratocysts than among solitary keratocysts .
hence , the clinical and histopathological features confirmed the diagnosis of gorlin goltz syndrome for all the five cases .
donatsky and hjrting - hansen reported that the significantly higher occurrence of proliferative epithelial remnants in the connective tissue wall of the cyst might be an explanation for the high recurrence rate of kcots seen in patients with nbccs .
as the biological behavior of kcots associated with nbccs is more aggressive and these cysts have higher recurrence rates ( 82% ) compared with solitary keratocysts ( 61% ) , treatment by marsupialization or enucleation with adjuvant chemical cautery using carnoy 's solution or cryosurgery should be undertaken in these patients .
cryotherapy produces cellular necrosis and destroys the residual cysts and epithelial remnants while maintaining the inorganic osseous framework intact . in our patients ,
enucleation with or without marsupialization was followed by liquid nitrogen spray cryotherapy ( 196c ) for the treatment of cystic cavities . the literature reports wide variation in the incidence of kcots in nbccs patients ranging from 62% to 100% .
usually , multiple keratocysts are found in nbccs ranging from 1 to 30 in number , average being 5 . in our case series ,
the most common site which is usually found is the mandibular molar or ramus region ( 44% ) followed by incisor - canine region ( 18% ) . in our case
series , mandibular body region was affected in 4 patients ( 80% ) , crossing midline , and extending to contralateral side in two ( 40% ) of them .
in contrast , the majority of the maxillary cysts occur commonly in incisor - canine region ( 15% ) and molar - tuberosity region ( 13% ) .
the bilateral maxillary cysts in one of our patients were found in premolar - molar region .
palmar - plantar pits usually occur in 3587% of the syndromic patients and in 80% develop by the age of 15 years and in 85% after the age of 20 years .
pits were found to be present in 80% of our cases ( 4 patients ) .
they are usually more commonly found on palms ( 77% ) than on soles ( 80% ) , but plantar pits were seen in three of our patients ( 60% ) as compared to 2 patients with palmar pits ( 40% ) .
ectopic calcifications of falx cerebri have been found in as low as 21.2% to as high as 92% patients in different reports worldwide whereas in our series , they were found in 60% of the cases [ figure 4 ] . computed tomography scan showing calcification of falx cerebri and tentorium cerebella ( case 1 ) relative macroencephaly with an increased occipitofrontal circumference ( > 55 cm )
ovarian cysts and fibroma are usually a finding in 2550% of affected women with nbccs and often present bilaterally ( 75% ) .
this is consistent with our finding in only one affected patient ( 20% ) but being present unilaterally .
bifid , fused , wide , partially missing , or underdeveloped ribs are usually found in 1658% of nbccs patients .
epidermoid cysts have been found to occur in 50% of the cases in literature . in our case series ,
cleft lip and palate and sprengel deformity are rare in this syndrome with 09% and 422% occurrence .
scoliosis of spine rarely being reported in indian literature was also found in one of our patients [ figure 5 ] .
multiple impacted teeth were seen in all of these patients with a novel feature of retained deciduous teeth seen in 4 patients ( 80% ) which may be another associated finding with this syndrome not being reported previously .
chest x - ray showing scoliosis of spine , bifid right 4 rib posteriorly , right 5 rib anteriorly and left 5 rib posteriorly
nbccs is a hereditary condition mostly caused by mutations in the ptch1 gene which was isolated in 1996 as the human homolog of the drosophila segment polarity gene ptch .
the ptch1 gene has been mapped to the long arm of chromosome 9q22.3 located from base pairs 95,442,981 to base pairs 95,516,964 .
the ptch1 gene provides instructions for producing the patched-1 protein , which functions as a receptor .
together , they trigger signals that play a role in cell growth , cell specialization , and determining the patterning of different parts of developing body .
mutation in ptch1 gene prevents the production of patched-1 or leads to the production of an abnormal version of the receptor .
an altered or missing patched-1 receptor can not effectively suppress cell growth and division . as a result , cells proliferate uncontrollably to form the tumors that are characteristic of gorlin goltz syndrome .
its pathogenesis supports the knudson two - hit hypothesis which states that normal cells require two mutagenic hits ( two distinct episodes of dna damage ) to produce cancer .
patients with retinoblastoma , nbccs , and similar syndromes have a constitutional ( germline ) defect in the dna sequence in one of the two copies of a tumor suppressor gene .
if a second dna injury or if loss of the normal remaining allele occurs at the same locus ( the second hit ) , the cell may become malignant . in nbccs , various tumors and hamartomas ( bcc , odontogenic keratocysts , meningiomas , and ovarian fibromas ) exhibit loss of heterozygosity but other lesions , for example , palmar pits do not .
various physical anomalies ( bifid rib , macroencephaly ) apparently need only one - hit .
although bcc has a low metastatic potential of 0.00280.55% , recently an nbccs patient had been diagnosed with recurrent bcc with lung metastasis . as cumulative ultraviolet ( uv )
light is the main risk factor , these patients are warned against prolonged sun exposure .
about 7080% of the individuals with nbccs have an affected parent and about 2030% of the probands have a de novo mutation . the risk to a sib of a proband
depends on the genetic status of the parents : if a parent of the proband is affected , the risk to the sibs is 50% ; when the parents are clinically unaffected probably because the effects being so mild that is , has never been diagnosed , the risk to the sibs of a proband appears to be low .
so , genetic counseling involving family members in regular screening appears to be mandatory . in the present case
series , the syndrome did not affect the patient 's parents or siblings and there were no familial antecedents in any of our 5 patients .
location of gene for this syndrome by gene mutation analysis offers the possibility that dna markers can be used in risk estimation and presymptomatic identification of the patients .
( 2008 ) identified a heterozygous germline mutation in ptch2 gene in affected members of a chinese han family with nbccs .
however , no ptch2 mutations were found in 11 cases of nbccs or in individuals with familial cases of nbccs who did not have identifiable ptch1 mutations .
an isolated case of gorlin goltz syndrome has been found to have mutation in sufu gene located on chromosome 10q24.32 in the absence of mutation in ptch1 gene . as all of these three genes belong to sonic hedgehog pathway ,
whether mutations in a single gene or a combination of these genes is probably responsible for the various manifestations of nbccs needs to be further studied .
furthermore , a new treatment strategy based on the understanding and inhibition of the hedgehog pathway can provide for specific drug treatment of disease in future to suppress tumor growth .
thus , antenatal diagnosis for pregnancies at increased risk for syndrome is possible by analysis of dna extracted from fetal cells ( obtained by amniocentesis or by chorionic villous sampling ) and ultrasound scans .
it can thus be helpful in detecting serious fatal developmental malformations such as fibromas of heart .
some fetuses with syndrome may have difficult deliveries due to large heads requiring assistance in delivery by either forceps or caesarean section .
all the 5 patients have been kept on regular periodic follow - ups , and no recurrence of kcot 's or new syndromic manifestations have been found in any patient until date .
early diagnosis of this syndrome is important for counseling of patients to prevent harmful exposure to uv and ionizing radiations that increase the risk of developing bcc .
regular follow - ups by multispecialists can be offered to prevent substantial morbidity because of complications .
thus , early diagnosis of patients can be used in a preventive multidisciplinary approach to provide a better prognosis . on comparison of our cases of nbccs with those in indian literature and with other studies in various parts of the world , wide variation in manifestations in different ethnic groups and within the same population
further , research on mutation of different genes related to syndrome can provide for gene replacement therapy which can be a boon for these patients .
this study was approved by the ethical committee of punjab government dental college and hospital , amritsar .
the authors have obtained the necessary patient consent forms where the patients have given their approval for participation in the investigation , followed by representation in the concerned article .
the patients do understand that the authors will ensure that their identities wo n't be revealed , however anonymity can not be guaranteed .
this study was approved by the ethical committee of punjab government dental college and hospital , amritsar .
the authors have obtained the necessary patient consent forms where the patients have given their approval for participation in the investigation , followed by representation in the concerned article .
the patients do understand that the authors will ensure that their identities wo n't be revealed , however anonymity can not be guaranteed .
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### Summary:
: objective : in indian scenario , gorlin goltz syndrome ( nevoid basal cell carcinoma syndrome [ nbccs ] ) has been rarely reported .
the clinical , radiological , and histopathological findings and major and minor criteria in five cases of nbccs in north indian population have been presented along with a discussion of the role of gene mutation analysis in early diagnosis of syndrome.materials and methods : the diagnostic findings of gorlin goltz syndrome in 5 patients were compared with other reports in indian population and with reports of this syndrome in other parts of the world.results:the most common features seen were keratocystic odontogenic tumors ( 100% ) , calcifications of falx cerebri ( 60% ) , palmar - plantar pits ( 80% ) , rib anomalies ( 80% ) , macroencephaly ( 60% ) , ocular hypertelorism ( 80% ) , and frontal bossing ( 60% ) in our series
. retained deciduous teeth seen in 80% patients whose association has not been previously reported has been presented .
none of our patients had basal cell carcinoma , syndactyly or polydactyly , pectus deformity , bridging of sella turcica , pigmented nevi , or family history of this syndrome in contrast to such findings in other indian patients .
medulloblastoma has not been reported in any indian patient so far compared to this finding in other studies conducted worldwide.conclusions:combining the features of 48 patients in 38 cases of nbccs being published in indian literature with five cases of our series and on comparison with other studies in the world , a wide disparity in different ethnic groups and a wide variation in presentation of syndrome within the same population is suggested .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: although many theories have been proposed , the mechanism of ovarian carcinogenesis is still unclarified .
several risk factors for epithelial ovarian cancer ( eoc ) are identified . however , by far the most significant risk factor for eoc is a family history of this disease ; a woman with one first - degree relative with ovarian cancer has a three - fold increased risk of developing eoc herself .
at least two hereditary syndromes predispose to familial ovarian cancer : the hereditary breast - ovarian cancer ( hboc ) syndrome and the lynch syndrome .
mutations in the brca1 and brca2 tumor suppressor genes account for 6585% of all hereditary ovarian cancers . a recent population - based study showed a combined brca1 and brca2 mutation frequency of 13.3% among 1,342 women with ovarian cancer .
the lifetime risk of ovarian cancer in brca1 and brca2 mutation carriers is approximately 4060 and 1025% , respectively [ 7 , 8 ] . mutations in mismatch repair genes mlh1 , msh2 , msh6 , pms1 , and pms2 in the lynch syndrome account for 1015% of hereditary ovarian cancers , with a lifetime risk of 810% of developing ovarian cancer [ 2 , 8 , 9 ] . to date , reliable screening methods for ovarian cancer are not available and screening for ovarian cancer in the general population does not reduce mortality . even in high risk populations screening
the only proven method to dramatically reduce the incidence of ovarian cancer in high - risk patients is a prophylactic bilateral salpingo - oophorectomy ( bso ) [ 12 , 13 ] .
it not only reduces the risk of ovarian cancer by up to 96% but also halves the risk of breast cancer ( bc ) in pre - menopausal women [ 1215 ] .
it has several advantages over genetic tests including lower costs , greater acceptability and a reflection of shared genetic and environmental factors .
moreover , criteria for genetic testing rely almost exclusively on family history information . in various studies
however , the question is raised how accurate family history is to predict mutation status [ 3 , 4 , 6 ] .
several reasons are given for a low validity of family history taking as diagnostic test for hereditary cancer , i.e. small families or families with few women , changeable penetrance , sporadic cancers , new mutations or inadequate family history taking .
there is no literature available on the adequacy of family history taking in eoc patients .
the purpose of this population - based study was to describe adequacy of family history taking in eoc patients and to identify factors that determine this adequacy .
secondly , we aimed to acquire insight in the reliability of presence or absence of written notifications of family history in medical records by comparing data in medical records with data collected thru self - administered questionnaires .
to evaluate adequacy of family history taking in eoc patients and factors that determine this adequacy , we used population - based data from a retrospective study in 11 hospitals in the eastern part of the netherlands : one university clinic and ten community hospitals . the population - based netherlands cancer registry ( ncr ) registered 1,178 patients with primary eoc in these hospitals between 1996 and 2006
hospital records of the patients were studied by trained registrars using a standard case record form .
data on patient characteristics , tumor characteristics , therapy and recurrence were collected . after excluding 66 patients without histological confirmed eoc , data of a total number of 1,112 patients were available .
data on family history taking included degree and age at diagnosis of all relatives diagnosed with eoc , bc , or colorectal cancer ( crc ) .
for this study adequate family history taking was defined as a written notification of the presence or absence of relatives with breast or ovarian cancer .
factors that may be correlated with family history taking were tested using univariable logistic regression analysis .
factors tested were ; age of the patient at diagnosis ( < 40 , 4060 , > 60 year ) , hospital type ( general , teaching , university hospital ) , year of diagnosis ( 19961999 , 20002003 , 20042006 ) , menstrual state ( pre- or postmenopausal ) , cancer in patient s history ( yes or no ) , bc in patient
s history ( yes or no ) , cervical cancer in patient s history ( yes or no ) , endometrial cancer in patient s history ( yes or no ) , colon cancer in patient s history ( yes or no ) , ca125 at diagnosis ( 35 or > 35 ) , risk of malignancy index ( 200 or > 200 ) , karnofsky score ( 70 or > 70 ) , stage of disease ( early = figo < iib or advanced = figo iib ) , surgery ( yes or no ) , chemotherapy ( yes or no ) , histology ( serous , endometrioid , mucinous , adenocarcinoma not otherwise specified or other ) , grade of differentiation ( grade i or ii or iii ) , number of recurrences ( 0 , 1 , 2 , 3 or 4 ) and inclusion in a trial ( yes or no ) .
all p values presented are two - sided , and associations were considered significant if the p value < 0.05 . since correlation between certain factors
was expected all significant indicators ( p < 0.05 ) were entered in a multivariable model using a stepwise forward approach .
statistical analyses were performed using statistical package for social sciences 16.0 for microsoft windows ( spss inc . ) . to measure the reliability of presence or absence of written notifications on family history in medical records , we compared data in the medical files with data collected by self - administered questionnaires . in 2008
, these questionnaires were sent to all living patients diagnosed with ovarian cancer between 1989 and 2008 in seven of the 11 hospitals .
the questionnaire database included 308 patients of whom 150 were also included in the above described eoc database .
the other 158 cases were diagnosed before 1996 or after 2006 , or were non - eoc cases ( fig . 1 ) .
another three cases were excluded since data on family history were missing in both the eoc database and the questionnaires .
data on family history taking included type of malignancy and age at diagnosis of first - degree relatives .
a comparison was made based on the number of relatives with a malignancy , the type of malignancy ( being bc , eoc or crc ) , and age at diagnosis . cancer cases among family members mentioned in the self - questionnaire but diagnosed after the last follow - up date of the patient were excluded .
agreement between the databases on number of relatives with a malignancy , type of malignancy , and age at diagnosis with an acceptable margin of error of 5 years , was defined as total agreement .
partial agreement was defined as agreement on number of relatives with a malignancy and type of malignancy.fig .
eoc epithelial ovarian cancer , oc ovarian cancer , bc breast cancer overview of databases used in this study .
figure 1 shows an overview of this database and the other databases used in this study . for 41% ( 456/1,112 ) of the cases ,
documentation on family history of breast and ovarian cancer was found in the medical records .
table 1 shows characteristics of patients with or without an adequate documentation on family history .
univariable logistic regression analysis showed age , hospital type , year of diagnosis , menstrual state , bc in the past , karnofsky score , histology , having surgery or chemotherapy , recurrence , and inclusion in a trial , to be significantly correlated with adequacy of family history taking .
multivariable logistic regression analysis identified age , hospital type , and having surgery or chemotherapy , as significant independent prognostic factors ( table 2 ) .
patients who were younger , diagnosed in a university hospital , or who underwent chemotherapy and/or surgery , were more likely to have an adequate documentation on family history.table 1associations between patient , tumor and treatment characteristics and adequacy of family history taking in univariable logistic regression analysis ( n = 1,112)characteristicsadequate n(%)inadequate n(%)p valueage ( years)<0.01 > 60200 ( 32%)429 ( 68% ) 4060216 ( 52%)202 ( 48% ) < 4039 ( 66%)20 ( 34%)hospital type<0.01 general hospital182 ( 33%)371 ( 67% ) teaching hospital188 ( 42%)264 ( 58% ) university hospital86 ( 81%)20 ( 19%)year of diagnosis0.019 19961999166 ( 38%)274 ( 62% ) 20002003181 ( 41%)265 ( 59% ) 20042006109 ( 49%)113 ( 51%)menstrual state<0.01 premenopausal113 ( 57%)86 ( 43% ) postmenopausal311 ( 37%)527 ( 63%)cancer in patients history0.230 no372 ( 41%)536 ( 59% ) yes82 ( 46%)97 ( 54%)breast cancer in patients history<0.01 no410 ( 41%)599 ( 59% ) yes44 ( 56%)34 ( 44%)colon cancer in patients history0.464 no448 ( 42%)621 ( 58% ) yes6 ( 33%)12 ( 67%)endometrial cancer in history0.131 no443 ( 42%)607 ( 58% ) yes11 ( 30%)26 ( 70%)cervical cancer in patients history0.593 no448 ( 42%)628 ( 58% ) yes5 ( 50%)5 ( 50%)ca-125 at diagnosis0.174 3560 ( 49%)63 ( 51% ) > 35360 ( 42%)491 ( 58%)risk of malignancy index ( rmi)0.984 20056 ( 44%)71 ( 56% ) > 200286 ( 44%)364 ( 56%)karnofsky score<0.01 < 7051 ( 24%)159 ( 76% ) 70395 ( 46%)459 ( 54%)stage0.602 early ( < iib)113 ( 43%)152 ( 57% ) advanced ( iib)316 ( 44%)394 ( 56%)chemotherapy<0.01 no98 ( 25%)297 ( 75% ) yes357 ( 50%)359 ( 50%)surgery<0.01 no40 ( 19%)166 ( 81% ) yes416 ( 46%)488 ( 54%)histology<0.01 serous205 ( 48%)221 ( 52% ) mucinous37 ( 42%)52 ( 58% ) endometrioid78 ( 47%)89 ( 53% ) adenocarcinoma nos84 ( 33%)169 ( 67% ) other45 ( 40%)67 ( 60%)grade of differentiation0.297 156 ( 41%)81 ( 59% ) 2100 ( 42%)140 ( 58% ) 3225 ( 47%)257 ( 53%)number of recurrence<0.01 0229 ( 35%)426 ( 65% ) 1145 ( 47%)163 ( 53% ) 258 ( 53%)51 ( 47% ) 314 ( 61%)9 ( 39% ) 410 ( 59%)7 ( 41%)inclusion in trial0.034 no421 ( 40%)623 ( 60% ) yes33 ( 54%)28 ( 46%)adequate family history taking was defined as a written notification of the presence or absence of relatives with breast or ovarian canceradenocarcinoma not otherwise specifiedclear cell , brenner , mixed , undifferentiated and other rare epithelial typestable 2associations between patient , tumor and treatment characteristics and adequacy of family history taking in multivariable logistic regression analysis ( n = 1,112)characteristicsadequateinadequateodds ratio95% cilowerupperage > 60200 ( 32%)429 ( 68%)1.00 4060216 ( 52%)202 ( 48%)1.651.232.22 < 4039 ( 66%)20 ( 34%)3.271.706.26hospital type general hospital182 ( 33%)371 ( 67%)1.00 teaching hospital188 ( 42%)264 ( 58%)1.631.222.19 university hospital86 ( 81%)20 ( 19%)8.744.8415.79surgery no40 ( 19%)166 ( 81%)1.00 yes416 ( 46%)488 ( 54%)1.681.022.76chemotherapy no98 ( 25%)297 ( 75%)1.00 yes357 ( 50%)359 ( 50%)2.251.633.12adequate family history taking was defined as a written notification of the presence or absence of relatives with breast or ovarian cancer associations between patient , tumor and treatment characteristics and adequacy of family history taking in univariable logistic regression analysis ( n = 1,112 ) adequate family history taking was defined as a written notification of the presence or absence of relatives with breast or ovarian cancer adenocarcinoma not otherwise specified clear cell , brenner , mixed , undifferentiated and other rare epithelial types associations between patient , tumor and treatment characteristics and adequacy of family history taking in multivariable logistic regression analysis ( n = 1,112 ) adequate family history taking was defined as a written notification of the presence or absence of relatives with breast or ovarian cancer 147 cases in the eoc database also completed a questionnaire . in 36% ( 53/147 ) of the cases , full agreement was found regarding the number and types of malignancy , as well as ages at diagnoses within 5 years . another five per cent ( 8/147 ) agreement was found for the number and type of malignancy in relatives . in 59% ( 86/147 ) of the cases , the information on family cancer history was discordant . in those 86 cases , medical records reported more malignancies than self - administered questionnaires in 11% ( 9/86 ) , self - administered questionnaires reported more malignancies in 23% ( 20/86 ) , and data were missing in the medical records where the self - administered questionnaires reported a negative family history in 66% ( 57/86 ) .
our population - based study shows that , adequate documentation on family history was present in only 41% of all medical records of eoc patients .
this percentage is in agreement with ranges found in literature regarding patients with crc and data required in primary care settings [ 1720 ] . in univariable analysis a slight improvement over time was seen which is encouraging however family history is still poorly recorded .
this is especially true when taking into account our limited definition of an adequate history covering the majority of hereditary ovarian cancers , namely the brca mutation carriers , but leaving others unattended .
previous studies on crc defined adequate family history taking by presence of a notification of relatives with crc only [ 18 , 20 ] . therefore , we feel that we used an appropriate definition for adequate family history taking in this study , but encourage clinicians to apply a much broader definition .
we believe a comprehensive adequate family history should include information on first and second degree relatives , the type of tumor they developed ( especially colorectal or endometrial carcinoma ) and the age at onset .
since families are getting smaller one should also note the total number of first and second - degree relatives to put it in perspective .
an additional problem is that with the decreasing number of relatives family history taking is expected to be less accurate in the future .
it is important to ask about second degree relatives since 50% of the mutations are paternally derived and the fathers are likely to be unaffected . though it is known that information on second degree relatives is less reliable than information on first degree relatives .
also the absence of malignancies should be noted . in case of suspicion of hereditary cancer
having chemotherapy or surgery often requires involvement of more specialists , longer therapeutic relationships and hospitalization , leading to more opportunities to ask about family history .
regarding age , in younger patients family history taking is probably more accurate because physicians are aware of an earlier age at onset in the majority of mutation carriers .
we urge specialists to keep in mind the possibility of a hereditary malignancy , even in the elderly patients .
with respect to hospital type , specialized gynecologists in academic hospitals more often recorded family history .
since it is shown that treatment by a gynecologic oncologist improves outcome , more and more eoc patients will be treated by specialized gynecologists and discussed in multidisciplinary tumor boards , which eventually may improve adequacy of history taking . with the result of the multivariable analysis
the question raised if gynecologists perform worse in family history taking compared to other physicians . to answer this question
family history data of all 4,858 bc and all 342 eoc patients diagnosed between 2005 and 2008 were compared .
data , extracted from medical records , included number of first - degree relatives with the same malignancy ( being bc or eoc ) , age of the youngest relative with the same malignancy and number of first - degree relatives with another malignancy . in 30% ( 104/342 ) of all eoc patients , compared with 64% ( 3,103/4,858 ) of all bc patients , documentation on presence or absence of first - degree relatives with the same malignancy was found .
24% ( 81/342 ) of eoc patients and 20% ( 989/4,858 ) of bc patients had any documentation in their medical record about first - degree relatives with other malignancies .
so family history regarding first - degree relatives with the same malignancy was taken twice more frequently in bc patients .
, patients can be more aware of bc in their family and both patient and physician can be more forthcoming towards this subject
. the clinician and patient can also be more aware of bc as a hereditary cancer , since there has been a lot of attention for this subject .
family history taking on first - degree relatives with another malignancy is as likely to be forgotten by surgeons in bc patients as by gynecologists in eoc patients .
although the large sample size is an apparent strength of this study , we are aware of some limitations .
it is possible that specialists did not register anything in the absence of relatives with a malignancy . in that case
the family history was considered to be absent although it in fact was examined correctly . in order to determine this possible bias , we compared the data with self - administered questionnaires .
. showed that reliability of self - reported family history taking varies by cancer site and by degree of relative . for first - degree relatives ,
supported the first statement but also states that negative family history reports for ovarian cancer are less useful .
especially abdominal malignancies are reported inaccurately probably because many organs are within the abdominal cavity and it is often referred to as abdominal cancer . in the current study
, we were unable to assess the reliability of family history taking by verifying it with a population - based registration and therefore chose a comparison with self - administered questionnaires .
data of 147 eoc cases of mainly long - term ovarian cancer survivors were studied . in nearly 60% of the cases
no agreement was found which was largely due to missing data in the eoc database in absence of any relatives with a malignancy in the questionnaires .
data from medical records were gathered in 2007 and the questionnaires were filled out by patients in 2008 .
it is likely that if gynecologists asked about family history , they did so in the early stages of treatment and never pursued it over time
. patients may also have developed more awareness on cancer in their family during the years since their diagnoses . as mentioned previously ,
these 147 cases were mainly long - term survivors . while analyzing data of these cases , 58% of these patients showed an adequate documentation in their medical records , compared to 41% in the whole eoc group .
an unselected patient group with respect to survival may worsen the results in our study even more .
it is of upmost importance to have an accurate tool for the identification of hereditary cancer .
the risk of eoc can currently not be reduced by screening but a prophylactic bso offered to high - risk patients can reduce the risk significantly .
brca mutation carriers also have an improved sensitivity to platinum chemotherapy and novel therapeutic agents such as poly ( adp - ribose ) polymerase inhibitors have increased activity in these patients .
our study shows that family history taking over the last 10 years was inadequate in the majority of eoc patients .
moreover , a recent study shows that family histories change significantly over time and updates on family history every 510 years are recommended .
data on sensitivity of family history as a predictor of mutation carrier status are conflicting . in various studies
the proportion of brca gene mutation carriers having a first or second degree relative with ovarian or bc varied from 92% to 6269% [ 4 , 5 ] .
it appears reasonable to offer genetic testing to all non - mucinous eoc patients in order to fully benefit from preventive measures like a bso .
but since genetic testing is not routinely offered in many countries and even when it is offered it is not performed in all cases , there is still a place for family history taking .
family history taking also plays an important role in the counseling of patients with a possible hereditary tumor .
moreover , patients with familial ovarian cancer but without mutation can be offered a bso .
the upcoming of electronic health record systems can be helpful by turning family history taking into a fixed item in consultations with cancer patients .
further education is needed for the physician to increase awareness of hereditary cancer since taking an adequate family history is still essential to provide high - standard care to patients with cancer and their families even in the era of genetic testing .
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### Summary:
: the aim of this study was to evaluate the adequacy of family history taking in epithelial ovarian cancer ( eoc ) patients and to identify factors that determine adequacy .
furthermore , the validity of family history taking was assessed by comparison with self - administered questionnaires .
medical records of all 1,112 eoc patients registered by the nation - wide cancer registry and diagnosed in eleven dutch hospitals between 1996 and 2006 were reviewed .
adequate family history taking was defined as a written notification of the presence or absence of relatives with breast or ovarian cancer .
factors that were correlated with family history taking were identified using univariable and multivariable logistic regression .
147 patients filled in a postal questionnaire .
an adequate family history was taken in 41% of all cases .
younger age , an academic hospital and having undergone surgery and/or chemotherapy were associated with adequate family history taking .
the comparison with self - administered questionnaires showed a disagreement in 64% mainly due to missing data in medical records .
documentation on family history is either absent or inadequate in the medical records in the majority of eoc patients .
these data urge for better uptake of hereditary cancer risk assessment .
different strategies for this assessment like improved family history taking and genetic testing in eoc patients should be explored .
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: nodal involvement and tumor size are the most important prognostic factors for breast cancer patients at different risk of disease recurrence and/or death ( 1 ) .
many clinicopathologic variables other than tnm stage have been identified to predict the clinical course of the disease . however , identifying all high - risk patients is still difficult . beyond the conventional clinicopathologic variables , therefore , novel prognostic factors are needed to be explored to predict the outcome more accurately ( 2 ) .
molecular markers such as p53 and bcl-2 have been extensively evaluated for their prognostic value since the 1990s ( 2 - 5 ) .
although the prognostic value of p53 and bcl-2 expression in breast cancer has been evaluated in a number of studies , the results were controversial .
treatments were also various with mastectomy ( 6 , 7 ) breast conservative therapy ( 8 - 11 ) or the combination of the two ( 12 - 14 ) . given these observations , we evaluated the prognostic value of p53 and bcl-2 expression on the treatment outcome , in relatively homogeneous population consisted of early stage breast cancer patients who underwent breast conservative treatment .
in addition , the correlation between these markers and triple - negative phenotype was also investigated .
between march 2000 and february 2002 , 125 patients underwent radiotherapy after breast conserving surgery for breast cancer . of these ,
100 patients whose immunohistochemical staining for p53 and bcl-2 was performed at the time of diagnosis were the cohorts for this retrospective study .
performance status of the patients was good with ecog grade of 0 for 27 patients and grade of 1 for 73 patients .
sixty patients received chemotherapy : cyclophosphamide , methotrexate and 5-fluorouracil in 30 patients ; doxorubicin - containing regimen in 27 patients ; others in 3 patients .
sixty - two patients received hormonal therapy and 35 of them also received chemotherapy . as regards to radiotherapy ,
whole breast irradiation was given , up to 50.4 gy at 1.8 gy / fraction by using two opposing tangential photon beams .
the clinicopathologic findings according to the expression of p53 and bcl-2 are summarized in table 1 .
median duration of follow - up was 69 months ( range ; 21 - 88 ) .
the specimens were immunostained to determine the expression of p53 , bcl-2 and c - erbb2 as well as the status of estrogen receptor ( er ) and progesterone receptor ( pr ) .
immunostaining was performed in the usual manner , using antibodies to p53 ( dako , glostrup , denmark ; 1:1,200 ) , bcl-2 ( dako , 1:50 ) , c - erbb2 ( novacastra laboratories ltd .
, new castle upon tyne , uk , 1:200 ) , er ( dako , 1:50 ) and pr ( dako , 1:50 ) .
the percentage of tumor cells with nuclear staining for p53 was graded semi - quantitatively : 0% , 1 - 25% , 26 - 50% , 51 - 75% or > 75% . for statistical analysis ,
p53 expression was considered as negative if 25% of tumor cells were stained and as positive if > 25% of tumor cells were stained .
er / pr and bcl-2 expression were judged as positive when 10% of tumor cells were stained in nucleus and membrane , respectively .
c - erbb2 expression was considered as positive if the score was 3 , and negative if the score was 0 , 1 or 2 .
differences in categorical variables between the parameters were compared by using standard chi - square test or fisher 's exact test .
the actuarial survival rates were calculated by using the kaplan - meier method and statistical significance of differences between the actuarial survival rates was evaluated by the log - rank test .
cox proportional hazard model was used for multivariate analysis . for all statistical tests , p values
patients with p53 expression were more likely to have node - positive and er - negative tumors .
expression of p53 was correlated with triple - negative phenotype ( p=0.011 ) , while bcl-2 negativity was correlated with triple - negative phenotype ( p<0.001 ) .
inverse relationship was demonstrated between p53 and bcl-2 expression ( p=0.004 , table 1 ) .
the 5-yr loco - regional relapse - free and distant metastasis - free survival rates were 91.7% and 90.9% , respectively .
on univariate analysis , age less than 40 yr and t2 stage were significant prognosticators predicting poor loco - regional relapse - free survival ( table 2 ) . when incorporating these variables into cox proportional hazard model , however , both lost the statistical significance .
given the association between bcl-2 expression and er status , we tested interaction of the two variables . compared with bcl-2(-)/er(- ) phenotype ( n=14 ) , other phenotypes ( bcl-2[-]/er[+ ] , bcl-2[+]/er[- ] , and bcl-2[+]/er[+ ] ) achieved higher loco - regional relapse - free survival rate ( 77.9% vs. 93.9% , p=0.033 , fig .
1 ) . on multivariate analysis incorporating age , t stage and the presence of bcl-2(-)/er(- ) phenotype , the bcl-2(-)/er(- )
phenotype was the only adverse prognostic factor for loco - regional relapse - free survival ( p=0.018 ) . when the use of chemotherapy and hormonal therapy were added in the model , the prognostic significance of the bcl-2(-)/er(- )
for the distant metastasis - free survival , age less than 40 yr , t2 stage and p53 positivity ( fig .
patients with bcl-2(-)/er(- ) phenotype had an inferior distant metastasis - free survival rate than those with other phenotypes , but the difference was not statistically significant ( 77.9% vs. 93.0% , p=0.153 ) .
on multivariate analysis incorporating aforementioned variables as well as nodal and er status , which were correlated with p53 expression , only p53 expression was significant ( p=0.009 ) .
the statistical significance of p53 expression remained even after the use of chemotherapy and hormonal therapy were added in the model ( p=0.011 ) . in subgroup analysis with triple - negative phenotype ( n=19 ) ,
bcl-2(-)/er(- ) phenotype was not associated with loco - regional relapse ( p=0.204 ) , and p53 expression was not associated with distant metastasis ( p=0.331 ) .
there have been several reports that investigated the prognostic value of p53 or bcl-2 expression on loco - regional failure after breast conservative therapy .
compared 66 patients who experienced local recurrence with 139 matched controls in terms of the expression of several oncogenes along with histological factors .
they found that bcl-2 negativity carried a 17 times higher risk of local recurrence in patients > 50 yr ( 10 ) .
noguchi et al . ( 9 ) and turner et al . ( 15 ) conducted similar case - control studies and noted the association between local recurrence and p53 positivity .
however , a case - control approach has the limitation of inevitable biases in selecting control cases although the process is random .
in contrast , other studies which analyzed consecutive patients failed to show any prognostic significance of these markers on local control ( 8 , 11 , 16 )
. this might be partly due to the small number of events , that is , local recurrences in early stage breast cancer patients receiving breast conservative therapy .
we also failed to show that any of the two molecular markers was associated with loco - regional relapse - free survival .
instead , the combined variable of bcl-2(-)/er(- ) phenotype was proven to be a significant prognostic factor in univariate and multivariate analyses . given the association between bcl-2 expression and er status in a number of studies including ours ( 11 , 17 - 19 ) ,
showed that the risk of death in bcl-2(-)/er(- ) phenotype is higher than those in other three phenotypes ( 20 ) .
demonstrated that the bcl-2(-)/er(- ) phenotype had a 5.7 times higher risk of overall recurrence compared with bcl-2(+)/er(- ) phenotype on multivariate analysis ( 12 ) .
likewise , an analysis of several molecular markers in combination provides more accurate prognostic information than that of the single variable , even in the low - risk group like our population . for the distant metastasis
, there are some limited data on the prognostic value of these markers in the breast conservative treatment setting .
aforementioned case - control studies noted a higher risk of distant metastases in patients with p53 expression ( 9 , 15 ) .
again , however , consecutive population - based studies failed to verify the relationship , despite the larger number of patients and events ( 11 , 17 ) . in the current study ,
p53 positivity was an independent prognostic factor predicting distant metastasis - free survival after adjusting conventional clinicopathologic variables including nodal and er status and treatment - related factors such as the use of chemotherapy and/or hormonal therapy .
recently , a number of investigators are seeking for new therapeutic strategy for triple - negative breast cancer , which is associated with inferior distant metastasis - free survival ( 21 - 23 ) .
unlike other reports , however , triple negativity was not a significant risk factor for distant metastasis in the present study . however , the correlation between p53 expression and triple - negative phenotype was demonstrated .
although the prognostic impact of p53 expression in triple - negative tumors was not established in our study as well as others ( 25 ) , p53 expression can give therapeutic implication for those patients with triple - negative tumors .
. showed that the rate of pathologic complete remission was higher in patients with p53-positive tumors than those with p53-negative tumors after neoadjuvant chemotherapy for triple - negative breast cancer ( 26 ) . despite the small number of patients and events , our study demonstrated the prognostic significance of bcl-2 and p53 expression on loco - regional control and distant metastasis , respectively .
this is partly because our population is relatively homogeneous , consisted of early stage breast cancer patients treated with breast conserving surgery and radiotherapy .
moreover , accrual period of the patient was relatively short , and therefore our patients were treated in a relatively uniform fashion regarding loco - regional therapy .
but , the delivery of systemic therapy was somewhat various as decision to undergo systemic therapy was based on the risk of the individual patient and the subsequent gain with systemic therapy .
unlike hormonal receptor expression or c - erbb2 expression , there is currently no specific treatment available to target tumors with either p53 expression or bcl-2 negativity .
but , breast cancer patients may be stratified into more detailed subtypes by these markers as well as other more relevant molecular markers , which are in the process of development . although more diversified patient and tumor specific treatment strategies need to be elucidated from future trials , this approach will eventually benefit patients undergoing treatment for breast cancer . in conclusion , results from this study show that a bcl-2(-)/er(- ) phenotype and p53 positivity are useful molecular markers predicting loco - regional relapse - free and distant metastasis - free survival , respectively , in patients treated with breast conserving surgery and radiotherapy .
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### Summary:
: prognostic value of p53 and bcl-2 expression on treatment outcome in breast cancer patients has been extensively evaluated , but the results were inconclusive .
we evaluated the prognostic significance of these molecular markers in patients treated with breast conserving surgery and radiotherapy .
one hundred patients whose immunostaining of p53 and bcl-2 expression was available among 125 patients who underwent radiotherapy after breast conserving surgery and axillary lymph node dissection were enrolled into this study .
eighty - seven patients also received adjuvant chemotherapy and/or hormonal therapy .
conventional clinicopathologic variables and treatment - related factors were also considered .
the 5-yr loco - regional relapse - free and distant metastasis - free survival rates were 91.7% and 90.9% , respectively .
on univariate analysis , age , t stage and the absence of bcl-2 & estrogen receptor ( er ) expression were associated with loco - regional relapse - free survival .
when incorporating these variables into cox proportional hazard model , only bcl-2(-)/er(- ) phenotype was an adverse prognostic factor ( p=0.018 ) .
as for the distant metastasis - free survival , age , t stage , and p53 expression were significant on univariate analysis . however , p53 expression was the only prognosticator on multivariate analysis ( p=0.009 ) .
a bcl-2(-)/er(- ) phenotype and p53 expression are useful molecular markers predicting loco - regional relapse - free and distant metastasis - free survival , respectively , in patients treated with breast conserving surgery and radiotherapy .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: a 158 cm , 40 kg , 26-year - old female patient visited the hospital complaining of hoarseness and intermittent respiratory distress .
it was found in a bronchial endoscope examination through the nasal cavity that an elliptic tumor mass about 4 cm in diameter was blocking almost all of the top part of the glottis .
the computed tomography ( ct ) showed that the tumor mass may have originated from the left aryepiglottic fold .
the boundary between the tumor mass and other surrounding parts was relatively well distinguished , and the round tumor mass caused the laryngeal cartilaginous structure to deviate to the right ( fig .
1 ) . no abnormality was found in a preoperative blood test , electrocardiography ( ekg ) and chest pa image .
there was no specific finding in the family and personal history . in tests related to the airway , the mouth opening and neck extension were normal , and the mallampati score was 3 .
initially , we decided to carry out awake orotracheal intubation using the gvl and a flexible bronchoscope .
considerations were given to the patient 's demand to avoid a tracheostomy if possible , taking her young age into account , as well as her discomfort with the intubation through her nasal cavity during the bronchoscopy test .
when arriving at the operation room , her blood pressure was 110/70 mmhg , her heart rate was 80 beats / min and her peripheral arterial oxygen saturation ( spo2 ) was 97% .
the ent department prepared for an emergency tracheostomy because there was a high probability of airway obstruction during the induction process .
4% lidocaine was sufficiently sprayed onto the backside of the tongue , the soft palate , the pharynx and the larynx in the oral cavity using the gvl .
subsequently , 0.75 g / kg of remifentanil was injected intravenously , and the injection was continued at a rate of 0.075 g / kg / min .
oxygen was supplied through an oxygen mask at a rate of 5 l / min .
an assistant exposed the tumor mass at the top of the glottis when the gvl blade was introduced into the oral cavity .
another operator inserted an endotracheal tube with an inner diameter of 7.0 mm at the right of the gvl blade and introduced the tip of a flexible bronchoscope , of which the outer diameter was 5.2 mm .
however , only a part of the glottis was shown due to the solid tumor mass , and the deviated laryngeal cartilaginous structure and the bronchoscope tip could not proceed .
although attempts were made to move the bronchoscope tip forward several times while varying the moving direction , the introduction of the bronchoscope was halted for fear of the risk of hemorrhage and damage to the vocal cords .
subsequently , the solid tumor mass was pushed to the left using the gvl tip at the entrance of the vocal cord , and the patient 's cricoid cartilage was pushed to the left posterosuperior direction by an assistant . a part of the vocal cord
endotracheal intubation was successfully performed using an endotracheal tube with an inner diameter of 7.0 mm into which a stylet was inserted ( fig .
2 ) . propofol 80 mg and rocuronium 30 mg were injected intravenously as preparation for the induction . while securing the airway , the patient grimaced in pain once , but she followed the instructions well and did not execute any defensive motion .
anesthesia was maintained with sevoflurane 1.2 vol% and remifentanil 0.2 - 0.3 g / kg / min , and methylprednisolone 125 mg was administered to prevent postoperative laryngeal edema .
the intraoperative vital signs of the patient were stable and there were no specific findings during the operation , which continued for 2.5 hours .
extubation was carried out after confirming the recovery of consciousness and spontaneous breathing after the operation had finished .
the patient was transferred to the postanesthetic care unit , and there were no specific findings in that ward as well .
the tumor mass was diagnosed as a schwannoma in a histological examination and the patient was discharged 7 days after the operation .
an operation method through the oral cavity was determined considering the patient 's repulsion upon the suggestion of a method through the nasal cavity , which she had experienced before .
we decided to use the gvl , as it was expected that the bronchoscope tip would not easily reach the entrance of the vocal cord due to the change in the anatomical structure caused by the supraglottic mass when the flexible bronchoscope was introduced through the oral cavity .
there have been many reports of cases in which difficult airway management was successfully performed using the gvl and a bronchoscope together .
moore and wong reported that they could perform intubation relatively easily in an obese patient , despite the fact that a challenging airway management situation had been expected , as they took advantage of the flexibility of the bronchoscope as they watched the gvl monitor .
introduced the protocol of using the gvl in conjunction with a flexible bronchoscope for patients in whom a challenging airway management scenario is expected . for patients of cormack - lehane grade 1 or 2 based on an oral cavity observation with the gvl
, they suggested the method of moving the tip of a flexible bronchoscope directly to the vocal cord while watching its progress on the gvl monitor . for patients of grade 3 or 4
, they suggested a method in which the operator attempts endotracheal intubation , checking the vocal cord through the images displayed by the bronchoscope , while an assistant observes the moving direction of the flexible bronchoscope tip through the gvl monitor and gives directions to the operator .
xue et al . reported that they performed endotracheal intubation successfully in a relatively easy manner by applying the method described above with 13 patients for challenging endotracheal intubation scenarios were expected . in the present case ,
a method involving the use of a flexible bronchoscope in conjunction with the gvl was chosen to secure the airway , but the endotracheal intubation process failed .
the first possible reason for the failure was that the bronchoscope tip was not moved forward owing to the tumor mass and the anatomical change of the aryepiglottic fold .
asai and shingu stated that the small space due to the anatomical structure of the posterior pharynx and the larynx is a typical reason for a failure of endotracheal intubation using a flexible bronchoscope .
it was also reported that the tip of the endotracheal tube could be resisted by an anatomical structure such as the aryepiglottic fold when it was inserted through the top of the bronchoscope .
second , the bronchoscope was not strong enough to push the tumor mass and the surrounding structures away due to its thinness .
hakala and randell reported that the success ratio was high when a thick endoscope was employed . in our case , the thin bronchoscope was not strong enough to move the tumor mass , which was a large , solid and fixed schwannoma that extended from the left aryepiglottic fold to the epiglottis . in most case reports in which endotracheal intubation succeeded with a flexible bronchoscope ,
it was reported that a bronchoscope could be inserted by pushing the tumor mass aside with the tip , although careful operation of the bronchoscope was required in the case of a pyriform cyst due to the risk of hemorrhaging and dropout [ 10 - 12 ] .
the final reason may be that the tumor mass was not moved aside and thus impeded the tip of the gvl blade , which was simply used as a camera to watch the moving direction of the bronchoscope tip .
additionally , the assistant was not given an active assignment to expose the vocal cord , such as by pressing the part near the cricoid cartilage .
moorthy et al . studied airway management with 801 patients who had a supraglottic mass and classified them into four categories ( moorthy 's airway category ) based on their clinical symptoms and the findings using a laryngoscope and a flexible bronchoscope .
they recommended the use of airway management algorithms during induction for the patients in each category .
the patients for whom airway management is difficult are grades as grades 2b , 3 or 4 .
the patient in the present case report was classified into moorthy 's airway category grade 3 or 4 because only a part of the vocal cord was observed after making considerable effort to do so .
thus , the recommended method of endotracheal intubation was through a flexible bronchoscope or directly through a laryngoscope , and tracheostomy had to be considered if a challenging situation arose .
the advantages of using the gvl are as follows : first , the visible part of the airway can be enlarged on the monitor and the operator can observe the details , even when the blade is not accurately located at the centerline of the vocal cord .
second , the tip of the gvl blade is bent by 60 degrees , allowing structures that are not readily observed by general laryngoscopes to be observed ; moreover , the blade can be easily bent .
therefore , the success ratio of intubation can be increased using the gvl and a flexible bronchoscope together in an organized manner . as a result of the findings of this article
, we recommend an airway management method that includes the operation of a gvl blade and cooperative pressing of the cricoid cartilage by an assistant for the awake orotracheal intubation of a patient with a fixed supraglottic mass .
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### Summary:
: intubating patients with a huge , fixed supraglottic mass causing an obstruction of the glottis is difficult to most anesthesiologists .
we attempted awake fiberoptic orotracheal intubation assisted by glidescope videolaryngoscope ( gvl ) following topical anesthesia with 4% lidocaine spray and remifentanil infusion .
the glottis could not be identified by the gvl view . however , by entering toward the right side of the mass with bronchoscope , the glottis was found . due to stiffness of the mass
, we were unable to further enter the area using the bronchoscope .
alternatively , we attempted to expose the glottis by gvl blade and then successfully intubated the patient by manually pressing the cricoids cartilage
. gvl is nonetheless an excellent instrument in airway management compared to fiberoptic bronchoscope for patients with a huge and fixed supraglottic mass .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: there are an estimated 123,000 new cases of cervical cancer in india every year with 67,000 deaths in women in india alone , .
this cancer has a long precancerous phase , of about a decade , in most women and screening methods like the papanicolaou ( pap ) smear , colposcopy and hpv dna test are available for population screening yet , the disease continues to claim the lives of women in their forties and fifties , particularly from low socioeconomic groups .
human papilloma virus ( hpv ) is the principal causative factor in most cervical cancers though chemical carcinogenesis is also known .
hpv vaccine has been developed against major high risk hpv ( hrhpv ) types , yet it is not available universally and chemical carcinogenesis has been reported in murine models .
a large number of cases of cancers are still detected in women in an advanced stage .
what is worse is that a large number of precancerous lesions , low - grade squamous intra - epithelial neoplasia ( lsil ) and high - grade squamous intra - epithelial neoplasia ( hsil ) which are also detected by pap smear remain untreated due to lack of treatment facilities or recurrences .
these may regress spontaneously , after medical or surgical treatment , or progress to invasive cancer over some years , , .
ayurveda has described cancer like lesions ( arbuda , granthi , dushtavrana , sannipatikvyadhi ) in various texts and described different therapies for the same .
based on the concepts of rasayana therapy and arbudachikitsa in ayurveda , kuttan et al . and hastak et al .
, demonstrated the preclinical and clinical potential of oral turmeric oil ( to ) for oral cancer , , , .
reverse pharmacology principles were applied and safety studies have been conducted with turmeric oil in animals and in healthy volunteers , .
baseline data on cancer screening , standard antimicrobial therapy and persistent lsil was reported from women who came for follow up .
subsequently a pilot study on integrative therapy of lsil persistent after antimicrobial treatment and treated with oral administration of a standardized extract of to [ nbfr-03 ] was carried out and gave encouraging results .
out of 19 women with persistent lsil who were treated for 12 weeks , none progressed and 16 regressed to atypical or negative smears .
this communication describes the follow up data of women in the above study who could be followed up till 36 months ( n = 18 ) , and compares them with other lsil cases treated with antimicrobials alone but who did not take turmeric oil treatment and came for follow up .
the interim data with documentation of colposcopy and cytology was presented at the national colposcopy and cytology conference at tata memorial hospital in 2012 .
it is important to note that it is after screening a very large number of women ( > 1500 ) the precancer cases were identified and out of these , few came for follow up as the hospital caters to a population from far and near and also to cases outside mumbai .
all women with a diagnosis of lsil in pap smears were advised treatment with antimicrobials including their husbands ( when applicable ) and all were advised to come for a six monthly follow up for repeat pap smears , whether they participated in the turmeric oil study or not , .
those with persistent or recurrent lsil or hsil were referred to other hospitals for colposcopy , hr hpv dna test and cervical biopsy when indicated .
a total of 14 cases who had undergone integrative treatment with antimicrobials ( azithromycin , forcanazole , secnidazole , and if endocervicitis persisted , cephadroxyl ) followed by standardized oral extract of to as described earlier came for follow up in our clinic ( group 1 ) .
another four cases who had completed the study , but could not come to our clinic regularly for follow up , had their pap smears collected in their nearby clinics or hospitals and communicated their report telephonically . some study cases ( group 1 ) have come for a longer follow up upto seven years .
ten cases who did not participate in the to study but only took the antimicrobials also came for follow up smears up to 36 months ( group 2 ) .
the mean age of group 1 ( antimicrobials followed by turmeric oil treatment group ) and group 2 ( only antimicrobial therapy ) was 41 2.6 and 46 3.5 years respectively ; the range was 2767 vs 3665 years .
the mean parity of group 1 was 1.7 0.2 vs 4 3.5 in group 2 ; the range was from 0 to 3 vs 2 to 8 .
the findings in repeat pap smears at 36 months are reported in table 1 .
the microphotographs of representative pap smears classified as per the bethesda classification from 1 case each of study group 1 and 2 are given in fig . 1 .
other diagnostic tests were employed as required : hr hpv dna in 2/18 in group 1 , 4/10 in group 2 ; colposcopy at follow up 5/18 cases in group 1 and 7/10 cases in group 2 ; and cervical biopsy in 3/18 in group 1 , and 3/10 in group 2 .
none of the cases has developed hsil or invasive cancer as confirmed by these investigations .
17 had an uneventful treatment period with integrative therapy but reported transient hematuria for 2 h under conditions of domestic shock ( suicide in the family and unplanned stay in hospital for several hours in hospital ) .
she was treated with ayurvedic therapy for the single episode of hematuria for three months .
her smear had regressed from lsil to negative during study period but there was mild abnormality and slight increase in nucleocytoplasmic ratio after one year and ten months .
there was no hematuria or other urinary abnormality during or after treatment and smear regressed to negative status second time again .
case no 12 , had an extensive erosion covering 80% of cervix and history of recurrent post - coital bleeding prior to the study .
she had undergone cauterization of cervix earlier and her previous cervical biopsy report was chronic cervicitis , negative for cervical cancer or neoplasia .
however following a report of lsil in our screening program she underwent standard antimicrobial therapy followed by oral turmeric oil extract therapy for 12 weeks and had regression in the pap smear to mild atypia at 12 weeks .
she came for six monthly follow ups and had recurrence of cervical erosion , lsil in pap smears after two years but not on colposcopy .
she underwent douching with panchavalkal kwath in another ayurvedic hospital and took oral ayurvedic formulations and responded to treatment as her erosion healed and postcoital bleeding stopped .
her repeat smear in our laboratory after three years was inflammation ( negative ) and she has decided to undergo six monthly pap smears and colposcopy .
cervical cancer is preventable because the carcinogenesis is usually slow and there are opportunities to detect and treat precancerous lesions by pap smears , colposcopy , or by hpv dna tests .
secondary chemoprevention has been attempted with compounds like folic acid , retinoic acid analogues , grape extract , fluorouracil , and other plant extracts which showed variable success and side effects , .
we have reported on the chemopreventive potential of integrated treatment of antimicrobials followed by oral turmeric oil ( to ) extract , 0.2 ml twice daily , for 12 weeks in a group of women who had persistent low - grade squamous intra - epithelial lesion ( lsil ) in their pap smears after antimicrobial therapy . in this study we report their post - therapy long - term follow up with pap smears and colposcopy when needed . although the numbers are small , integrated treatment appears to have not only arrested or reversed pap smear abnormalities but also appears to have persistent post - therapeutic benefit .
similarly one case who had mild deterioration in pap smear improved again when she was administered to for two months .
this indicates the possibility of repeating the to therapy without side effects , if needed .
have reported a recurrence rate from 5 to 15% in cervical neoplasias after surgical treatment in long term follow up , .
in conclusion integrative therapy with antimicrobials and oral to extract appears to be a viable non - invasive option to surgical or cold knife therapy for reversal or arrest of low grade squamous abnormality detected by pap smear and confirmed by colposcopy .
the limitation of this report is the small sample size and hence we recommend a larger long term study .
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### Summary:
: cervical cancer is preventable because the carcinogenesis is slow and there are opportunities to detect precancerous lesions by papanicolaou ( pap ) smears , colposcopy , or hpv dna tests and to treat them by antimicrobials , surgery or cold co2 vapourization .
we have earlier reported on the chemopreventive potential of integrated treatment with antimicrobials therapy followed by a standardized oral turmeric oil ( to ) extract upto 12 weeks in women who had persistent low - grade squamous intra - epithelial lesion ( lsil ) in their pap smears . in this communication
we report their post - therapy follow up for 36 months ( n = 18 ) with pap smears .
we were also able to follow up for 36 months control cases ( n = 10 ) who had only standard therapy with antimicrobials.during 36 months of follow up none of the cases with integrated treatment , progressed to hsil or cancer .
out of 15/18 cases which had a regression of pap smear , all 15 remained free of lsil from 6 to 36 months post - therapy showing persistent therapeutic effect of integrated therapy . in one case
there was recurrence of lsil in pap smear , ten months post - therapy , which regressed to mild atypia after a second course of oral to for 8 weeks.in the control group , persistence of lsil after antimicrobials was observed in pap smears in 5/10 cases when followed up by pap smears up to 36 months .
this preliminary report indicates some post - therapeutic benefit with integrative treatment as compared to the use of antimicrobials alone . a large scale
controlled study is warranted .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: the nurses ' health study ii ( nhsii ) is an ongoing prospective study of u.s .
a total of 109,172 participants remained after exclusion for diagnosis of any type of diabetes , cancer , or cardiovascular disease at baseline ( 2% ) or for missing information on childhood body shape ( 2.5% ) , bmi at age 18 years ( 0.7% ) , or baseline bmi ( 0.3% ) .
this study was approved by the institutional review board of the partners health care system ( boston , ma ) . at baseline ,
body fatness at ages 5 , 10 , and 20 years of age was assessed by recall of somatotypes or 9-level pictorial diagrams developed by stunkard et al .
( 9 ) representing sizes ranging from extreme thinness ( category 1 ) to obesity ( category 9 ) .
the use of recalled somatotypes has been validated in both older ( 10 ) and younger ( 11 ) women by comparison with childhood records .
somatotypes at ages 5 , 10 , and 20 years correlated moderately with recorded bmi ( r = 0.60 , 0.65 , and 0.66 , respectively ) ( 10 ) .
the validity did not differ by adult bmi at the time of report ( 10 ) .
weight change was the difference between weight at age 18 years and weight at baseline in 1989 .
the correlation between recalled weight at age 18 and documented weight from college or nursing school records was 0.84 , and the correlation between self - reported and technician - measured adult weight was 0.96 ( 13 ) .
a supplementary questionnaire was mailed to confirm the self - report of diabetes diagnosis and to distinguish different types of diabetes ( 14 ) .
the national diabetes data group diagnostic criteria were used for cases reported through 1997 and required confirmation of at least one of the following : 1 ) one or more symptoms ( weight loss , hunger , thirst , or polyuria ) and elevated glucose ( fasting 7.8 mmol / l [ 140 mg / dl ] or random plasma or 2-h glucose 11.1
mmol / l [ 200 mg / dl ] ) , 2 ) no symptoms but occurrence of elevated plasma glucose as described above on at least two different occasions , or 3 ) use of insulin or oral hypoglycemic medication ( 15 ) . for cases occurring after 1998 ,
the cutoff of fasting plasma glucose was changed to 7.0 mmol / l ( 126 mg / dl ) in accordance with revised criteria ( 16 ) .
we excluded women classified as having only gestational diabetes mellitus or type 1 diabetes . in the nurses ' health study , the supplemental questionnaire was highly reliable regarding diabetes diagnosis ( 14 ) . in a random sample of 84 women classified as being a case subject , medical records were available for 62 of these women and an endocrinologist confirmed the diagnosis in 61 women ( 98% ) ( 14 ) .
age was calculated as months from the reported birth date to date of questionnaire return .
race , smoking status , birth weight , prematurity , multiple gestation birth , age of menarche , being breast - fed , alcohol consumption , and family history of diabetes were self - reported at baseline or in 1991 .
parity and age at first birth were measured biennially . physical activity , in met units ,
was derived from the average time spent in certain activities ( e.g. , jogging or running ) in 1989 , 1991 , 1997 , and 2001 .
differences in participant characteristics by childhood somatotype at age 10 years were compared using or linear regression .
person - years were calculated based on date of return to date of diagnosis , death , or 1 july 2005 , whichever came first .
multivariate cox proportional hazard models were used to estimate the relative risk ( rr ) for the associations of somatotypes at ages 5 , 10 , and 20 years and bmi at age 18 years with type 2 diabetes risk .
we used category 2 as the reference because most women had reported this category at both ages 5 and 10 years .
in one multivariate model , estimates were adjusted for age ( continuous ) , race ( african american , hispanic , asian , or white ) , parity ( 0 , 12 , or 3 ) in combination with age at first birth ( > 24 years ) , family history of diabetes ( maternal , paternal , or both ) , smoking ( current , past , or never ) , and physical activity ( quintiles ) . in a second model among participants not missing birth weight information ( n = 87,349 , case subjects = 2,771 ) , estimates were adjusted for characteristics influencing childhood size including age , race , family history of diabetes , birth weight ( < 5.5 , 5.56.9 , 7.08.4 , 8.59.9 , or 10 pounds ) , prematurity or multiple gestation birth status of the woman ( yes or no ) , and age of menarche ( < 12 , 12 , 13 , 14 , or > 14 years ) . to test for significant trends ,
linear models were fitted using the median values of each category of exposure ( e.g. , 17.0 , 19.0 , 21.0 , 23.5 , 28.0 , and 35.0 for bmi at age 18 years ) .
the differences in somatotype categories ( between ages 5 and 10 and between ages 10 and 20 ) or in weight ( between age 18 years and baseline ) were used to assess whether change in size was associated with diabetes development .
models were additionally adjusted for starting size ( e.g. , adjustment for age 5 somatotype in evaluating the difference between ages 5 and 10 ) , because where a woman began in size may reflect weight gain or loss ( i.e. , heavier women have the possibility to lose more weight ) .
the cumulative effect of overweight across the life course before reported diabetes was evaluated using the combination of somatotype 5 at age 10 , bmi 25 kg / m at age 18 , and bmi 25 kg / m at baseline ( i.e. , to represent adulthood ) .
the reference group comprised women who reported no overweight at any of those time points .
analyses were performed using sas ( version 8.2 ; sas institute , cary , nc ) .
at baseline , body fatness at ages 5 , 10 , and 20 years of age was assessed by recall of somatotypes or 9-level pictorial diagrams developed by stunkard et al .
( 9 ) representing sizes ranging from extreme thinness ( category 1 ) to obesity ( category 9 ) .
the use of recalled somatotypes has been validated in both older ( 10 ) and younger ( 11 ) women by comparison with childhood records .
somatotypes at ages 5 , 10 , and 20 years correlated moderately with recorded bmi ( r = 0.60 , 0.65 , and 0.66 , respectively ) ( 10 ) .
the validity did not differ by adult bmi at the time of report ( 10 ) .
weight change was the difference between weight at age 18 years and weight at baseline in 1989 .
the correlation between recalled weight at age 18 and documented weight from college or nursing school records was 0.84 , and the correlation between self - reported and technician - measured adult weight was 0.96 ( 13 ) .
a supplementary questionnaire was mailed to confirm the self - report of diabetes diagnosis and to distinguish different types of diabetes ( 14 ) .
the national diabetes data group diagnostic criteria were used for cases reported through 1997 and required confirmation of at least one of the following : 1 ) one or more symptoms ( weight loss , hunger , thirst , or polyuria ) and elevated glucose ( fasting 7.8 mmol / l [ 140 mg / dl ] or random plasma or 2-h glucose 11.1
mmol / l [ 200 mg / dl ] ) , 2 ) no symptoms but occurrence of elevated plasma glucose as described above on at least two different occasions , or 3 ) use of insulin or oral hypoglycemic medication ( 15 ) . for cases occurring after 1998 ,
the cutoff of fasting plasma glucose was changed to 7.0 mmol / l ( 126 mg / dl ) in accordance with revised criteria ( 16 ) .
we excluded women classified as having only gestational diabetes mellitus or type 1 diabetes . in the nurses ' health study ,
the supplemental questionnaire was highly reliable regarding diabetes diagnosis ( 14 ) . in a random sample of 84 women classified as being a case subject , medical records were available for 62 of these women and an endocrinologist confirmed the diagnosis in 61 women ( 98% ) ( 14 ) .
age was calculated as months from the reported birth date to date of questionnaire return .
race , smoking status , birth weight , prematurity , multiple gestation birth , age of menarche , being breast - fed , alcohol consumption , and family history of diabetes were self - reported at baseline or in 1991 .
physical activity , in met units , was derived from the average time spent in certain activities ( e.g. , jogging or running ) in 1989 , 1991 , 1997 , and 2001 .
differences in participant characteristics by childhood somatotype at age 10 years were compared using or linear regression .
person - years were calculated based on date of return to date of diagnosis , death , or 1 july 2005 , whichever came first .
multivariate cox proportional hazard models were used to estimate the relative risk ( rr ) for the associations of somatotypes at ages 5 , 10 , and 20 years and bmi at age 18 years with type 2 diabetes risk .
we used category 2 as the reference because most women had reported this category at both ages 5 and 10 years .
in one multivariate model , estimates were adjusted for age ( continuous ) , race ( african american , hispanic , asian , or white ) , parity ( 0 , 12 , or 3 ) in combination with age at first birth ( > 24 years ) , family history of diabetes ( maternal , paternal , or both ) , smoking ( current , past , or never ) , and physical activity ( quintiles ) . in a second model among participants not missing birth weight information ( n = 87,349
, case subjects = 2,771 ) , estimates were adjusted for characteristics influencing childhood size including age , race , family history of diabetes , birth weight ( < 5.5 , 5.56.9 , 7.08.4 , 8.59.9 , or 10 pounds ) , prematurity or multiple gestation birth status of the woman ( yes or no ) , and age of menarche ( < 12 , 12 , 13 , 14 , or > 14 years ) . to test for significant trends ,
linear models were fitted using the median values of each category of exposure ( e.g. , 17.0 , 19.0 , 21.0 , 23.5 , 28.0 , and 35.0 for bmi at age 18 years ) .
the differences in somatotype categories ( between ages 5 and 10 and between ages 10 and 20 ) or in weight ( between age 18 years and baseline ) were used to assess whether change in size was associated with diabetes development .
models were additionally adjusted for starting size ( e.g. , adjustment for age 5 somatotype in evaluating the difference between ages 5 and 10 ) , because where a woman began in size may reflect weight gain or loss ( i.e. , heavier women have the possibility to lose more weight ) .
the cumulative effect of overweight across the life course before reported diabetes was evaluated using the combination of somatotype 5 at age 10 , bmi 25 kg / m at age 18 , and bmi 25 kg / m at baseline ( i.e. , to represent adulthood ) .
the reference group comprised women who reported no overweight at any of those time points .
analyses were performed using sas ( version 8.2 ; sas institute , cary , nc ) .
there were 3,307 incident cases of type 2 diabetes over 16 years of follow - up , for an incidence rate of 197 cases per 100,000 person - years .
table 1 shows the characteristics for all women and by reported childhood somatotype at age 10 years categorized from 1 to 6 .
women with somatotype 1 at age 10 years were more likely to be of minority race , to have birth weight < 5.5 pounds , to have been born prematurely , and to have had a later age at menarche ; whereas women with somatotype 6 were more likely to have birth weight > 10 pounds , to actively smoke , to be nulliparous , and to have had a positive parental history of diabetes .
somatotypes at ages 5 , 10 , and 20 years were associated with each other . of the women reporting somatotype 6 at age 10 years , 41% reported somatotype 6 at age 5 years , and 28% at age 20 years .
weight and bmi were also positively associated with somatotype ; a 12 kg / m increase in adolescent or adult bmi was observed per increase in somatotype category .
baseline characteristics of nhsii participants by somatotype at age 10 years means sd presented unless indicated .
n = 109,172 . * reported in 1991 questionnaire ( rather than at baseline in 1989 for all other covariates ) .
somatotypes at ages 5 , 10 , and 20 years and bmi at age 18 years ( table 2 ) were all significantly and positively associated with diabetes risk ( all ptrend < 0.0001 ) .
compared with having a somatotype of 2 , the rr of diabetes associated with a somatotype 6 at age 5 was 2.19 ( 95% ci 1.792.67 ) , at age 10 years was 2.57 ( 2.203.01 ) , and at age 20 years was 5.67 ( 4.926.54 ) .
the rr among women who reported a bmi at age 18 > 30 kg / m was 8.72 ( 7.5810.02 ) compared with women with a bmi of 1819 kg / m . among a subgroup not missing birth weight information ( n = 87,175 ) , adjustment for childhood factors including birth weight did not substantially change these estimates .
childhood and adolescent size and rr ( 95% ci ) of type 2 diabetes in adulthood in nhsii data are rr ( 95% ci ) unless otherwise indicated .
* model 2 adjusted for age , race , smoking status , parental history of diabetes , parity , age at first birth , and adult physical activity .
model 3 adjusted for age , race , parental history of diabetes , birth weight , multiple birth , prematurity , age of menarche ( n = 87,175 , case subjects = 2,681 in subgroup not missing birth weight information ) .
type 2 diabetes risk increased among women who reported increases in size whether by somatotype between ages 5 and 10 and between ages 10 and 20 or by weight gain since age 18 ( table 3 ) . compared with women reporting no change in somatotype , one or more unit increases in somatotype at these ages
were associated with approximately twice the risk , whereas decreases in somatotype at these ages were associated with reduced risk .
weight gain since age 18 , which is the difference between baseline weight and weight at age 18 , was also significantly associated with risk of diabetes . compared with women who had little change in weight ( i.e. , 4.9 pounds ) ,
even a weight gain of 58 pounds doubled risks , whereas weight gain of > 25 pounds increased risk by > 20 times .
weight loss was associated with increased risk until after adjustment for bmi at age 18 to account for greater weight loss being associated with larger adolescent body size . in analyses stratified by bmi at age 18 years , weight loss of 10 pounds was significantly associated with a reduced risk of diabetes among women who were overweight ( rr 0.45 [ 95% ci 0.220.91 ] ) or obese ( 0.45 [ 0.280.72 ] ) in adolescence but not among those who were lean ( 1.72 [ 0.763.90 ] ) .
changes in size and rr ( 95% ci ) of type 2 diabetes in nhsii data are rr ( 95% ci ) unless otherwise indicated .
n = 109,172 . * model 2 adjusted for age , race , smoking status , parental history of diabetes , parity , age at first birth , and adult physical activity .
model 2 variables with additional adjustment for earlier size , i.e. , somatotype at age 5 and 10 years and bmi at age 18 years .
the prevalences of overweight by somatotype 5 at ages 5 , 10 , and 20 years were 7 , 12 , and 11% , respectively .
most of the cases ( 83% ) in the cohort occurred among women who were overweight ( bmi 25 kg / m ) at baseline with an incidence rate of 562 per 100,000 person - years . among these women ,
the prevalences of overweight by somatotype 5 were 12 , 21 , and 23% , respectively . to evaluate the cumulative effect of being overweight across the life course ,
women were jointly categorized as being overweight using somatotype at age 10 , bmi at age 18 years , and bmi at baseline ( with mean age of 34 years ) ( fig .
women who were overweight only as an adult ( bmi > 25 kg / m ) had an adjusted rr of 8.23 ( 95% ci 7.419.15 ) compared with women who were never overweight at any age .
the adjusted rr increased to 15.10 ( 13.2117.26 ) for women who were also overweight at age 10 ( somatotype 5 ) and age 18 ( bmi > 25 kg / m ) .
however , women who were overweight at age 10 but came off the trajectory and became lean in adulthood ( i.e. , not being overweight in adulthood ) did not have a significantly increased risk of diabetes ( 1.02 , 0.741.40 )
. adjusted rrs ( 95% ci ) of type 2 diabetes by somatotype 5 at age 10 , bmi 25 kg / m at age 18 years , and bmi 25 kg / m at baseline ( average age of 34 years ) , after adjustment for age , race , smoking status , parental history of diabetes , parity , age at first birth , and physical activity .
number of cases of type 2 diabetes for each category : 503 for none ; 50 for 10 years ; 21 for 18 years ; 10 for 10 and 18 years ; 1,469 for 34 years ; 260 for 10 and 34 years ; 664 for 18 and 34 years ; and 441 for all ages .
among 109,172 women followed for > 16 years , somatotypes at ages 5 and 10 years were positively associated with the risk of incident type 2 diabetes after adjustment for major risk factors .
however , women who were overweight at age 10 but were lean in adulthood did not have an increased risk of diabetes associated with childhood overweight , underscoring the importance of continued efforts to control adiposity among overweight children .
limited studies have investigated the long - term consequences of childhood overweight and the risk of type 2 diabetes in adulthood , with inferences hindered by small numbers of case subjects and/or lack of differentiation of diabetes type ( 28 ) .
in addition , some studies included case subjects with young age at onset ( < 30 years old ) ( 3,5,7 ) .
one study using data based on records ( 185 cases ) showed that the cumulative incidence of diabetes was doubled ( from 4.2 to 8.4% ) among women in the largest bmi group at age 11 years ( > 17.4 kg / m ) compared with those in the smallest bmi group ( 15.3 kg / m ) ( 4 ) .
in contrast , a study of older french women using recalled body shape , found an association between childhood thinness and diabetes risk ( 8) .
however , this birth cohort vastly differed from the nhsii as demonstrated by > 60% of their case subjects having reported extreme leanness ( somatotype 1 ) at age 8 years , possibly due to their nutritional status having been affected by world war ii ( 8) .
in addition to absolute size , longitudinal changes through different ages are also relevant to the risk of diabetes ( 17,18 ) . under normal development , infants lose weight after 6 months of age and continue doing so until 5 years of age when their adiposity rebounds ( 18 ) .
although we were unable to assess age at adiposity rebound , earlier rebound has been associated with increased risk of type 2 diabetes ( 17 ) , and there is some suggestion that age at rebound matters more than the absolute size of the child at any point in time because it corresponds to weight gain .
our findings of increased risk associated with increases in somatotype and weight gain support the importance of change in size in addition to absolute size . these findings offer support for continued weight reduction efforts across the life span .
moreover , in analyses of the cumulative effect of overweight through childhood , adolescence , and adulthood , the risk for type 2 diabetes was greatest among women reporting overweight by all three measures , even though the difference was marginal when compared with adolescent overweight .
however , women who became lean in adulthood did not have an increased risk of diabetes associated with childhood overweight .
similar observations have been made from other studies of youth overweight and type 2 diabetes or related traits , with findings becoming nonsignificant after accounting for adult bmi ( 6,19,20 ) .
tracking of metabolic risk factors ( e.g. , hdl and triglycerides ) has also been observed after 21 years of follow - up from childhood ( 21 ) , supporting the fact that cumulative overweight is linked with prolonged exposure to metabolic irregularities , putting children on the path to -cell dysfunction earlier .
it should be noted that although there is tracking of overweight to obesity from childhood to adulthood , the trajectory is not fixed from youth ( 18,22 ) .
it has been shown that 1030% of overweight children do not go on to be overweight as adults with conflicting evidence as to whether there is greater tracking in girls than in boys ( 22 ) .
conversely , the majority of adults who are overweight were not in childhood ( 22 ) as confirmed here with only 1220% of the overweight women reporting childhood overweight .
our study had some limitations . although experts have recommended bmi for the measurement of childhood size ( 18,23 ) , we used recalled somatotype .
however , two previous studies have shown that the accuracy of recall between childhood somatotypes and recorded childhood bmi did not differ by adult bmi ( 10,11 ) .
the misclassification of childhood size is thus more likely to be nondifferential , which can not explain the observed positive association between childhood size and type 2 diabetes risk .
in addition , our findings were in agreement with those of other studies that used childhood measures of weight and height ( 4,20 ) .
the generalizability of our findings may be limited to white women who comprised > 95% of our study population .
more research is needed in minority populations who have higher rates of overweight and diabetes ( 1,24 ) .
last , we can not rule out the possibility of residual confounding by unmeasured confounders because of the observational nature of the study . as for strengths ,
the nhsii included a large number of women ( > 3,000 case subjects ) and adjusted for many risk factors .
follow - up for previous studies required recontacting participants in adulthood with low to moderate success rates ( 2060% ) ( 5,7,19,20 ) , whereas we evaluated somatotypes at baseline and > 90% of the women remained for follow - up . in summary ,
women who do not continue to be overweight in adulthood do not have increased risks .
it remains important then to promote lifestyle changes from youth so that the adverse trajectory could be avoided .
multiple interventions to address childhood overweight have been suggested ( 23 ) , but these remain to be fully tested .
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### Summary:
: objectiveto determine how childhood overweight , in conjunction with other life course weight characteristics , relates to the development of type 2 diabetes in adulthood.research design and methodsamong 109,172 women in the nurses ' health study ii , body fatness at ages 5 , 10 , and 20 years was assessed by recall using 9-level pictorial diagrams ( somatotypes ) representing extreme thinness ( category 1 ) to obesity ( category 9 ) .
recalled weights at age 18 years and adulthood were used to derive bmi .
self - reported cases of type 2 diabetes were confirmed by supplementary questionnaire.resultssomatotypes at ages 5 and 10 years were positively associated with diabetes risk ( ptrend < 0.0001 ) .
the adjusted relative risk ( rr ) of women with somatotype 6 ( vs. 2 ) at age 5 years was 2.19 ( 95% ci 1.792.67 ) and at age 10 years was 2.57 ( 2.203.01 ) .
increases in size by somatotype or by weight gain since age 18 were associated with increased risk . compared with women who were never overweight at any age , women who were overweight as an adult ( bmi > 25 kg / m2 ) but
not previously had an adjusted rr of 8.23 ( 7.419.15 ) .
the adjusted rr was 15.10 ( 13.2117.26 ) for women who were also overweight at age 10 ( somatotype 5 ) and 18 ( bmi > 25 kg / m2 ) . increased childhood size
was not associated with risk among women who did not continue to be overweight in adulthood.conclusionsincreased body size starting from childhood is associated with a greater risk of diabetes in adulthood .
however , women who become lean in adulthood do not have an increased risk .
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: with the improvement of perinatal care , the frequency of infant death has reduced considerably , but the incidence of neurological disabilities related to perinatal brain damage has not decreased in western countries over the last decades [ 13 ] .
perinatal brain injury due to asphyxia , cerebral ischemia , cerebral hemorrhage , or intrauterine infection is the major contributor to perinatal morbidity and mortality as the immature brain is highly susceptible to damage .
injury to the newborn during the perinatal stage is the underlying etiology for a host of developmental disabilities that includes spastic motor deficits such as cerebral palsy [ 4 , 5 ] and cognitive , behavioral , attentional , socialization and learning difficulties [ 69 ] .
as brain development substantially influences the progression and hallmarks of brain injury [ 10 , 11 ] , it is not possible to apply therapeutic procedures used for adult ischemia to newborns . in term newborn infants , hypoxic / ischemic ( h / i )
presently , optimal management of h / i brain injury involves prompt resuscitation , careful supportive care , and treatment of seizures .
although hypothermia is a promising new therapy , and recent studies suggested that head or whole - body cooling administered within 6 hours of birth reduces the incidence of death or moderate / severe disability at 12 to 22 months , there is undeniable need for the identification of new therapeutic targets for the implementation of clinical trials to address treatment of h / i encephalopathy .
accordingly , epidemiological and experimental data have allowed researchers to identify a number of potential targets for neuroprotective strategies .
animal models have led to the elucidation of biochemical events involved in neurodegeneration and neuroprotection [ 1418 ] ; however , important differences among species have been described [ 19 , 20 ] .
the initiation and development of injury to the neonatal brain is complex , with multiple contributing mechanisms and pathways resulting in both early and delayed injury . as in other types of acute central nervous system ( cns ) injuries ,
tissue damage and neurodegeneration initiate a cascade of inflammatory response depending on the nature and extent of damage , which is characterized by the involvement of damaged neurons , microglial , astrocytes , endothelial cells , and recruited blood leukocytes [ 2225 ] .
microglial cells are the main nervous component of the innate immune system , playing a key role in the phagocytosis of cell debris to repair damage and maintain tissue homeostasis , but active producers of inflammatory mediators .
astrocytes rapidly respond to extracellular changes and are the main cell type responsible for the restoration of blood - brain barrier , new glia limitans formation , and the establishment of a long - term glial scar .
in addition , vascular damage induces massive influx of blood leukocytes , particularly monocytes and neutrophils , which are also actively involved in inflammatory processes .
it is important to note that the glial and inflammatory response after perinatal brain damage differs from the mature brain due to key ongoing postnatal developmental processes .
importantly , neuronal dendritic arborization , establishment of synaptic contacts , axonal growth , myelination , and glial differentiation take place during the first two - three postnatal weeks in rodents . at the molecular level , several studies have described a distinctive expression of growth factors , adhesion molecules , inhibitors of axonal growth , and cytokines [ 3335 ] , determining the neonatal brain 's particular response to injury , showing increased susceptibility to excitotoxicity [ 11 , 36 , 37 ] and to proinflammatory molecules [ 38 , 39 ] . in this regard ,
it becomes evident that descriptions of the glial and inflammatory cell changes in adult injury models can not be extrapolated to animal models of perinatal brain damage . in the present study
we have used the experimental model of h / i - induced neonatal injury initially described by vannucci and coworkers [ 17 , 40 ] for the rat , and adapted to the mouse in several laboratories [ 14 , 18 , 41 ] with the advent and increased usage of transgenic and knock - out mice . as most studies describing detailed neuropathological and glial and inflammatory cellular changes after neonatal h / i have used the rat model , the goal of our study was to provide a neuropathological followup of tissue damage and detailed morphological and quantitative analysis of astroglial , microglial and leukocytic response following h / i to the postnatal day 7 mice at nine different survival times ranging from 3 hours after hypoxia to 100 days , focusing both on the ipsilateral and the contralateral hemispheric changes .
this short- and long - term temporal description aims to help in the future design of novel experimental approaches towards the development of neuroprotective strategies .
ninety - nine c57bl6 mice ( from twenty litters bred in harlan labs , france ) of different postnatal ages were used in this study .
animals were housed under controlled temperature ( 22c 2c ) , with a 12 hour light cycle period and pelleted food ( global diet 2014 ) and water ad libitum .
experimental procedures were approved by the ethical commission of autonomous university of barcelona ( ceeah protocol no . 811 ) .
all efforts were made to minimize the number of animals and animal suffering in every step .
hypoxic / ischemic ( h / i ) brain damage was induced in postnatal day 7 ( p7 ) c57/bl6 mice by permanent left carotid occlusion and exposure to hypoxia as previously described . briefly , a midline ventral skin incision was made under isoflurane anesthesia ( 4.5% v / v for induction and 2.5% v / v for maintenance , and 0.6 l / min of o2 ) ; the left carotid artery was exposed and sutured with a 8/0 silk surgical suture .
after surgery , pups were returned to their dam for at least 1.5 hours to recover .
later , litters were placed for 55 minutes in a hypoxic chamber containing 8% of oxygen balanced with nitrogen , with controlled humidity and temperature maintained at 37c .
the mean index of postnatal mouse mortality due to surgery or hypoxia was 19.31% , with 18.46% for males and 20.00% for females , showing no statistical differences between genders .
as 18 animals died during surgical procedure or hypoxia , only 81 animals were analyzed in this study .
intact control mice were sacrificed at p7 , p10 , p14 , p21 , and adult .
lesioned pups were sacrificed at 3 , 12 , 24 , 48 , and 72 hours , and at 7 , 14 , 30 , and 100 days after hypoxia .
p7 , 3 hrs , 12 hrs , 24 hrs ; group ii
p21 and/or adult , 14 days , 30 days , and 100 days . for histological and immunohisto - chemical analysis , mice were i.p .
anaesthetized ( ketamine and xylazine 80/10 mg / kg ) and perfused intracardially using 4% paraformaldehyde in phosphate buffer ( pb , ph 7.4 ) .
subsequently , brains were removed , postfixed for 4 hours in the same fixative , cryoprotected in 30% sucrose , frozen with dry co2 , and finally stored at 80c until use .
brains were serially cut in a cryostat ( leica cm3050 s ) in 30 m thick sections and stored in 20c mounted on flex ihc slides ( dako ) . to determine the injury score ,
one series of parallel sections from each animal ( 610 mice / survival time ) was air dried at room temperature for an hour , rinsed and incubated with nissl solution ( 0.1% toluidine blue in walpole buffer 0,2 m and ph 4,5 ) at room temperature for 3 minutes and washed with distilled water .
the degree of tissue damage was calculated following the injury score detailed on table 1 ( for 3 to 72 hrs ) and table 2 ( for 7 to 100 days ) .
three animals from each control age group and four representative animals from each postlesion survival time ( injury scores = mean 2 s.d . ) were processed for the immunohistochemical demonstration of astrocytes ( by glial fibrillary acidic protein , gfap labeling ) , microglia / macrophages ( by iba-1 labeling ) , neutrophils ( by ly-6b.2 labeling ) , and t - cells ( by cd3 labeling ) .
single immunohistochemistry was initiated by blocking the endogenous peroxidase ( 2% h2o2 in 70% methanol for 10 min ) and incubation of sections mounted on slides for 1 h in blocking buffer ( bb ) containing 10% fetal calf serum and 3% bovine serum albumin in tris - buffered saline ( tbs , ph 7.4 ) with 1% triton x-100 ( tbst ) at room temperature ( rt ) .
slides were then incubated overnight at 4c and 1 h at rt with one of the following primary antibodies diluted in bb : hamster monoclonal anti - cd3 ( abd serotec no .
mca2690 , dilution 1 : 250 ) , rabbit polyclonal anti - gfap ( dako no .
z0334 , dilution 1 : 1500 ) , rabbit polyclonal anti - iba-1 ( wako no .
019 - 19741 , dilution 1 : 3000 ) , and rat monoclonal ly-6b.2 ( abd serotec no .
later , sections were washed with tbst and incubated at rt for 1 h with respective biotinylated secondary antibodies : anti hamster ( vector labs no .
ba9100 , dilution 1 : 500 ) , anti - rabbit ( vector labs no .
ba1000 , dilution 1 : 500 ) , and anti - rat ( vector labs no .
ba4001 , dilution 1 : 500 ) , followed by washes with tbst and incubation for 1 h with streptavidin peroxidase ( vector laboratories no .
the peroxidase reaction was visualized by incubating the sections in 3,3-diaminobenzidine and hydrogen peroxide using the dab kit ( sk-4100 ; vector laboratories , usa ) for gfap , iba-1 and ly-6b.2 . for cd3
, slides were treated by the glucose oxidase - dab - nickel method , and the reaction was terminated by washing with 0.1 m acetate buffer ( ph 6.0 ) . finally , sections were dehydrated and coverslipped in dpx .
sections were analyzed and photographed with a dxm 1200f nikon digital camera joined to a nikon eclipse 80i microscope , and plates were arranged using adobe photoshop cs .
imagej software ( national institute of health ) was used for quantitative analysis of immunoreacted sections .
images from 5 sections / animal were taken , representing the following regions : corpus callosum ( cc ) , caudate putamen ( cp ) , hippocampus ( h ) , neocortex ( n ) , and thalamus ( t ) ( figure 1 ) .
micrographs were captured using the 40x objective ( for the cc and the hippocampus at 72 hours after hypoxia ) or the 20x objective ( rest of areas and survival times ) . in group
i , ii and iii sections were 240 m apart , and bregma levels ( bls ) analyzed included ( approx . ) : anterior bl1 , 0.26 mm & bl2 , 0.02 mm ; posterior
bl3 , 1.82 mm ; bl4 , 2.06 mm ; bl5 , 2.30 mm . in group iv , sections were 300 m apart , and bl analyzed included : anterior bl1 , 0.32 mm & bl2 , 0.02 mm ; posterior
bl3 , 1.82 mm ; bl4 , 2.12 mm ; bl5 , 2.42 mm .
image analysis was used to obtain the area occupied by glial cells , using a modification from a previously described method .
initially , in each section , the mean intensity of grey ( immunoreactive labeling ) in the contralateral region was measured .
subsequently , by using the mean intensity of grey as the threshold value , we measured in both hemispheres the percentage of the total area occupied by immunoreactive staining showing an intensity of grey above the threshold ( i.e. , representing reactive cells ) .
all samples for demonstration of atrocytes and microglia were done simultaneously in order to reduce variability on dab intensity .
the regions analysed are shown in figure 1(b ) and included the hippocampus ( h1 , h2 , h3 ) , neocortex ( n ) , caudate - putamen ( cp ) , medial third ventricle ( m3v ) , lateral third ventricle ( l3v ) median fissure ( mf ) , and thalamus ( t ) in at least 4 representative animals of each lesioned group and 3 animals / control group , with 3 sections / animal , was analysed . in groups
i , ii and iii sections were 240 m apart , and counted bregma levels ( bls ) included : bl1 , 1.82 mm ; bl2 , 2.06 mm ; bl3 , 2.30 mm . in group iv , sections were 300 m apart , and counted bl included : bl1 , 1.82 mm ; bl2 , 2.12 mm ; bl3 , 2.42 mm . all data was corrected by abercrombie correction method , with an average of length ( t ) = 0,848 .
all experiments were performed so as to reduce variations , and data are presented as mean s.e.m .
two - way anova followed by bonferroni posthoc analysis , along with t - test , was used to determine statistical significance as required ( graphpad , prism 3 ) .
analysis of toluidine blue - stained sections ( figures 2 and 4 ) was used to evaluate the extent of brain damage in both hemispheres at 3 , 12 , 24 , 48 , and 72 hours and at 7 , 14 , 30 , and 100 days after hypoxia . in general ,
microscopic evaluation showed mild changes in the contralateral hemisphere [ mainly in hippocampus ( hp ) and corpus callosum ( cc ) ] , and extensive tissue damage and neuronal loss in the ipsilateral hp and cc at all survival times analyzed , although the caudate putamen ( cp ) was also usually affected .
damage in the cortex ( cx ) and the thalamus ( tl ) was not always seen and showed the highest variability . in order to better characterize lesion progression , a semiquantitative injury score was calculated for each region and animal ( tables 1 and 2 , figures 3 and 5 ) . from 3 to 72 hours after hypoxia , damage was characterized by neurodegeneration and increased cellularity due to gliosis , and the description of the injury score rating is depicted in table 1 . at 7 days after hypoxia , damage was mainly characterized by atrophy of gray and white matter areas , and therefore a different injury score rating was defined , which is depicted in table 2 . from 3 hours to 7 days after hypoxia , no apparent tissue damage or ventricle swelling in the contralateral hemisphere was observed using the nissl staining ( figures 2 and 4 ; right side of the panel ) .
interestingly , at 14 days after hypoxia , scattered patches of neurodegeneration with a mild reduction in cellular density when compared to intact age - matched control brains were observed in the ca field of the hp ( figure 4 ) , showing a mean injury score in the contralateral hp of 0.92 0.2 ( table 2 , figure 5 ) .
in addition , the contralateral cc was also damaged in the 30- and 100-day survival groups , showing approximate 40% of atrophy ( mean cc atrophy scores of 1.31 0.59 and 0.86 0.38 , resp . ) ( table 2 , figure 5 ) , accompanied by evident ventricle swelling ( figure 4 ) .
no apparent changes in the contralateral dentate gyrus ( dg ) , caudate - putamen , neocortical layers and thalamus were seen .
h / i injury in hippocampusas early as 3 hours after hypoxia , hippocampal tissue disruption with disorganization of ca cytoarchitecture and the presence of patches of neurodegeneration ca pyramidal neurons was observed in the ipsilateral hemisphere ( figures 2(b ) , 2(e ) and 2(f ) ) , but showing a high degree of variability between animals ( figure 3 ) . from 12 to 72 hours after hypoxia , the hippocampal ca field was visibly damaged in all animals , displaying a degenerating pyramidal cell layer with massive neuronal cell loss in ca1 and ca3 ( figures 2(i)2(af ) , left panel ) , showing a mean injury score of ca field of 3.27 0.74 between 12 and 72 hours after hypoxia ( table 1 , figures 2 and 3 ) .
in addition , at 12 hours , the dentate gyrus ( dg ) also showed neuronal injury and layer disruption , which was most evident at the 12- and 24-hours survival times ( figure 2(i ) ) . at 7 days after hypoxia ,
massive atrophy of the hippocampus was observed , showing mean total hippocampal injury scores ranging from 5 2.2 to 10.42 1.46 ( out of 12 , table 2 ) , where the 30-day survival group showed the lowest score ( figures 4 and 5 ) .
ca pyramidal neurons , but less than 50% reduction in dg neuronal density ( figures 4 and 5 ) .
interestingly , only in the 33% of the animals , the ipsilateral hippocampus was observed 100 days after hypoxia .
as early as 3 hours after hypoxia , hippocampal tissue disruption with disorganization of ca cytoarchitecture and the presence of patches of neurodegeneration ca pyramidal neurons was observed in the ipsilateral hemisphere ( figures 2(b ) , 2(e ) and 2(f ) ) , but showing a high degree of variability between animals ( figure 3 ) . from 12 to 72 hours after hypoxia , the hippocampal ca field was visibly damaged in all animals , displaying a degenerating pyramidal cell layer with massive neuronal cell loss in ca1 and ca3 ( figures 2(i)2(af ) , left panel ) , showing a mean injury score of ca field of 3.27 0.74 between 12 and 72 hours after hypoxia ( table 1 , figures 2 and 3 ) .
in addition , at 12 hours , the dentate gyrus ( dg ) also showed neuronal injury and layer disruption , which was most evident at the 12- and 24-hours survival times ( figure 2(i ) ) . at 7 days after hypoxia ,
massive atrophy of the hippocampus was observed , showing mean total hippocampal injury scores ranging from 5 2.2 to 10.42 1.46 ( out of 12 , table 2 ) , where the 30-day survival group showed the lowest score ( figures 4 and 5 ) .
hippocampal damage induced approximately a 1040% of remaining ca pyramidal neurons , but less than 50% reduction in dg neuronal density ( figures 4 and 5 ) .
interestingly , only in the 33% of the animals , the ipsilateral hippocampus was observed 100 days after hypoxia .
h / i injury in corpus callosumfrom 3 hours post - hypoxia , the ipsilateral corpus callosum showed increased cellularity ( figure 2(f ) ) and the presence of scattered apoptotic cells ( data not shown ) .
the density of cells in the ipsilateral corpus callosum was notably increased at 48 and 72 hours post - hypoxia ( figures 2(v ) , 2(ad ) and 3 ) , when ventricle swelling started to become evident ( figures 2(r ) and 2(z ) ) . at 7 and 14 days post - hypoxia , increased cellularity was still observed ( figures 4(f ) and 4(n ) ) , but this was minimum from 30 days ( figure 4(v ) ) .
important atrophy of the white matter accompanied by ventricle swelling was seen in all animals at 7 days after hypoxia , but it was more remarkable at 14 days after hypoxia , showing mean corpus callosum atrophy score ( 14100 days ) of 2.33 0.84 , which represented an approximate 50% tissue loss ( table 2 , figure 5 ) .
from 3 hours post - hypoxia , the ipsilateral corpus callosum showed increased cellularity ( figure 2(f ) ) and the presence of scattered apoptotic cells ( data not shown ) .
the density of cells in the ipsilateral corpus callosum was notably increased at 48 and 72 hours post - hypoxia ( figures 2(v ) , 2(ad ) and 3 ) , when ventricle swelling started to become evident ( figures 2(r ) and 2(z ) ) . at 7 and 14
days post - hypoxia , increased cellularity was still observed ( figures 4(f ) and 4(n ) ) , but this was minimum from 30 days ( figure 4(v ) ) .
important atrophy of the white matter accompanied by ventricle swelling was seen in all animals at 7 days after hypoxia , but it was more remarkable at 14 days after hypoxia , showing mean corpus callosum atrophy score ( 14100 days ) of 2.33 0.84 , which represented an approximate 50% tissue loss ( table 2 , figure 5 ) .
h / i injury in caudate - putamen , neocortex , and thalamusat 3 hours after hypoxia , we observed increased cellularity and disorganization of white and gray matter areas , mainly in the dorsal part of caudate - putamen ( figure 3 ) , showing a mean injury score ( 372 hours ) of 1.02 0.81 corresponding to less than 40% of striatal area damaged ( table 1 , figure 3 ) , but showing important variability between animals ( figure 3 ) . at 7 to 100 days after hypoxia , there was apparent caudate - putamen atrophy ( figures 3 and 5).the neocortex and the thalamus showed mild changes , that were only apparent in a minority of animals at all times analyzed , giving very variable results ( figures 3 and 5 ) .
neocortical damage , when present , was characterized by scattered radial columns of neurodegeneration and tissue damage , mainly until 12 hours after hypoxia . at 7 days after hypoxia mild
cellular damage in the thalamus was even less frequent but could be observed in some animals , affecting the rostral thalamic nuclei ( figure 3 ) .
however , probably as a consequence of ventricle swelling , different grades of thalamic atrophy were seen in most animals at 7 days ( table 2 , figure 5 ) .
at 3 hours after hypoxia , we observed increased cellularity and disorganization of white and gray matter areas , mainly in the dorsal part of caudate - putamen ( figure 3 ) , showing a mean injury score ( 372 hours ) of 1.02 0.81 corresponding to less than 40% of striatal area damaged ( table 1 , figure 3 ) , but showing important variability between animals ( figure 3 ) . at 7 to 100 days after hypoxia , there was apparent caudate - putamen atrophy ( figures 3 and 5 ) .
the neocortex and the thalamus showed mild changes , that were only apparent in a minority of animals at all times analyzed , giving very variable results ( figures 3 and 5 ) .
neocortical damage , when present , was characterized by scattered radial columns of neurodegeneration and tissue damage , mainly until 12 hours after hypoxia . at 7 days after hypoxia
cellular damage in the thalamus was even less frequent but could be observed in some animals , affecting the rostral thalamic nuclei ( figure 3 ) .
however , probably as a consequence of ventricle swelling , different grades of thalamic atrophy were seen in most animals at 7 days ( table 2 , figure 5 ) .
astrocytes were analyzed by gfap immunostaining and studied in control intact brains from p7 , p10 , p14 , p21 and adult mice , and in the contralateral and ipsilateral hemisphere of hypoxic / ischemic brains from 3 hours to 100 days after hypoxia .
the distribution and immunostaining intensity of gfap+ cells changed during postnatal development ( figures 6(a)6(c ) ) , showing increased gfap levels at earlier ages , as has been previously reported [ 4649 ] .
briefly , in addition to the gfap+ radial glial processes still observed at p7 ( figure 7(g ) ) , at the p7p10 age range , the most intense gfap+ astroglial cells were found in cortical layer i , the hippocampal fissure ( figure 6(a ) ) and white matter areas including the corpus callosum ( figure 6(a ) ) , and the fimbria . at p14 ,
gfap immunoreactivity was generally decreased but it was maintained in cortical layer i , the hippocampal fissure and white matter tracts ( figure 6(b ) ) . by p21 in the adult pattern of gfap+ cell distribution
was established , showing the strongest immunoreactivity in the astroglial endfeet surrounding blood vessels ( as in the hippocampal fissure , figure 6(c ) ) and in the white matter .
an astroglial response in the contralateral hemisphere was generally observed , mainly from 3 to 72 hours after hypoxia , and being importantly decreased by 7 days and longer survival times .
increase in gfap immunoreactivity due to astrogliosis was mainly seen in the hippocampal region ( mainly in the hippocampal fissure and the fimbria ) and in the cingulum region of the corpus callosum ( figures 6(d)6(f ) compared to age - matched controls in 6(a)6(c ) ) .
astroglial changes in the contralateral hippocampus were maximal at 2448 hours after hypoxia ( figure 6(e ) ) .
in addition , mild changes were also noted in the neocortex ( figures 7(h ) and 7(i ) ) , but no apparent changes were observed in the contralateral caudate - putamen ( figures 7(b ) and 7(c ) ) and thalamus . at 7 days after hypoxia , contralateral hemispheres showed no changes in gfap+ cell distribution when compared to age - matched controls . in this sense , it is important to note that the contralateral hippocampal and corpus callosum atrophy observed from 14 days post - hypoxia ( figure 5 ) was not accompanied by noticeable astroglial changes in these areas at late survival times . increased gfap immunostaining and changes in astroglial distribution and astrogliosis were seen in the ipsilaterally damaged hemisphere from 3 hours to the last survival time analyzed ( figures 68 ) .
the most intense astroglial response was found in the damaged hippocampus although the corpus callosum , the caudate - putamen , the neocortex and the thalamus also showed noticeable astroglial reactivity . at 3 hours after hypoxia , the ipsilateral hemisphere already showed an increase in astroglial gfap labeling as well as astrogliosis when compared to the contralateral side ( figures 6(d ) and 6(g ) ) . at this survival time , and at 12 hours after hypoxia , reactive astrocytes mainly covered the hippocampal fissure , and the molecular and polymorphic layers of the ca field , but no reactive astrocytes were seen within ca pyramidal cell layer or in the dg . at these early survival times , the area occupied by reactive astrocytes was significantly increased in the il side ( figure 8) . at 24 hours , but mainly at 4872 hours after hypoxia , astroglial processes started to cover the degenerating ca1 and ca3 pyramidal layers and reactive astrocytes concentrated in the hippocampal fissure , the molecular layer and the polymorphic layer of ca1 , adjacent to the white matter ( figures 6(h)6(m ) ) .
astroglial cell response was at this time also evident , to a lower extent , in the dg , mainly in the hilus ( figure 6(j ) ) .
as depicted in figure 8 , the percentage of gfap+ area in the hippocampus was high and significant in il hippocampus at all survival times . at 7 days after hypoxia , an intense glial scar formed in the degenerated pyramidal layer , around the blood vessels in the hippocampal fissure and in the hippocampal limits ( figure 6(n ) ) . at 14 days after hypoxia ,
astroglial response in the dg was noticeably decreased although increased gfap+ cells were often seen in the hilus ( figures 6(o ) , 6(s)6(t ) ) .
the glial scar was maintained until 100 days after hypoxia ( figures 6(p ) and 6(q ) ) .
an increase in gfap immunostaining and cell density when compared to the contralateral side was already seen at 3 hours after hypoxia ( figure 6(d ) and 6(g ) ) , however maximum response was observed at 2472 hours after hypoxia ( figures 6(h)6(k ) ) , when reactive astrocytes presented a marked increase in gfap intensity , showing hypertrophy and increased process thickness . by 7 days , astrogliois clearly diminished ( figures 6(n ) and 6(q ) ) , and at 14 days after hypoxia , gfap immunostaining was strongly decreased and was indistinguishable from controls ( figures 6(o)6(r ) ) . it should be noted that no striking changes were observed in the quantification of the astroglial response when compared to the contralateral side ( figure 8) .
an increase in astroglial gfap immunoreactivity was noted in the caudate - putamen at 3 hours after hypoxia ( figure 7(d ) ) although no changes in astroglial distribution were seen until later . from 24 hours
, astroglial response was mildly increased until 72 hours , when maximum gfap labeling was reported ( figure 7(e ) ) .
astroglial gfap expression was close to control values by 14 days after hypoxia ( figure 7(f ) ) , although glial scarring in the caudate - putamen remained in some animals at longer survival times , showing variability ( figure 8) .
notably , the area occupied by reactive astroglial cells in the ipsilateral caudate - putamen was above contralateral values at all survival times analyzed even though variability was found in some time points ( figure 8)
. in the neocortex , increased gfap expression and mild astrogliosis were first observed in layers v - vi at 312 hours after hypoxia ( figure 7(j ) ) , and it spread to upper layers from 24 to 72 hours ( figure 7(k ) ) , showing significant increases in astroglial response area ( figure 8) . at longer survival times ,
astrocytic response was clearly diminished ( figures 7(l ) and 8) and was practically absent by 14 days after hypoxia . in the thalamus ,
changes in astrocytes were not observed until 24 hours after hypoxia , showing strong variability between animals ( figure 8) .
astroglial response was characterized by patches of reactive astrocytes mainly in the rostral thalamus and only until 7 days after lesion , when glial scarring was noticed . at longer survival times , it was clearly diminished .
intense microglial iba-1 staining was observed at p7 and gradually decreased until adulthood . in postnatal animals ,
primitive ramified microglial cells were mainly found in the gray and white matter ( figures 9(a ) , 10(a ) and 10(g ) ) although some amoeboid microglial cells were seen in the cingulum of the corpus callosum , as previously reported . in the hippocampus ,
in addition , round - shaped iba-1 + macrophages were observed in the pia , very prominently in the medial fissure and in the ventricle linings , as has already been reported . at p10 ,
microglial cells were slightly more ramified , and an increase in cell density was noted , specifically in the corpus callosum , where microglial cells showed a parallel orientation to axon fibers . by p14 , microglial cells showed decreased iba-1 immunostaining ( figure 9(b ) ) and ramified resting morphology as described for the adult brain . at this age , iba-1 + macrophages
were strongly diminished in the meninges and ventricles . by 21 days after birth , only highly ramified resting microglial cells
were observed in the brain parenchyma , showing very low iba-1 staining ( figure 9(c ) ) .
microglial activation was generally observed in several areas of the contralateral hemisphere from 3 to 48 hours after hypoxia ( figures 9(d ) and 9(e ) ) . increased expression of iba-1 and changes in microglial cell morphology towards reactive ramified cells mainly , but also
amoeboid cells to a lower extent , were seen in most areas analyzed , but mainly in the hippocampus ( very prominently in the hippocampal fissure , figures 9(d ) and 9(e ) ) and the corpus callosum ( figures 9(d)9(f ) ) and other white matter tracts like the anterior commissural and external capsule , where microglial response was seen until 4872 hours after hypoxia .
after 14 days fter hypoxia , only in the hippocampal fissure and corpus callosum of some animals , mild - activated microglia was observed . in the caudate - putamen ( figures 10(b ) and 10(c ) ) , neocortex ( figures 10(h ) and 10(i ) ) and
thalamus ( data not shown ) , activated ramified microglial cells were seen mainly until 48 hours after hypoxia .
hippocampusat 3 hours after hypoxia , microglial response in the ipsilateral hippocampus closely resembled that seen in the contralateral side ; however , reactive microglial cells tended to accumulate surrounding the blood vessels in the hippocampal fissure only in the ipsilateral hippocampus ( figure 9 , compare 9(d ) and 9(g ) ) . by 12 hours
, reactive microglial cells changed to pseudopodic / ameboid morphologies and persisted in the fissure , significant differences between il and cl hippocampus were observed ( figure 11 ) . at 24 hours , increased iba-1 + macrophages were observed in the third ventricle , and the microglial response was maintained in the hippocampal fissure ( figure 9(h ) ) , but iba-1 + round - shaped microglia / macrophages started to cover the degenerating ca fields ( figure 9(h ) ) .
notably , at this time , although morphological and distribution changes in the microglial response versus the contralateral hippocampus were evident ( figure 9 , compare 9(e ) and 9(h ) ) , the area occupied by reactive microglial cells did not differ significantly from the contralateral side ( figure 11 ) , probably as a consequence of the reduced total cell area of pseudopodic / ameboid cells versus ramified cells . from 48 hours
to 7 days after hypoxia , a massive increase in microglia / macrophage cell intensity was evident in the fissure and ca field ( figures 9(i ) , 9(j ) , 9(l)9(n ) ) , showing a 57-fold increase in the area occupied by reactive microglia / macrophages when compared to the contralateral hippocampus ( figure 11 ) . at longer survival times ,
microglial response was strongly decreased , showing scattered reactive ramified and macrophages in the fissure and ca only until 14 days ( figures 9(o ) , 9(s ) , and 9(t ) ) , but no presence of reactive microglia / macrophages at 30 and 100 days ( figures 9(p ) , 9(q ) ) .
it should be noted that only scattered activated microglial cells were present in the dg , and always located in the hilus , correlating with the above described astroglial response in this area which is mostly spared in this neonatal injury model as a consequence of its late development [ 53 , 54 ] .
at 3 hours after hypoxia , microglial response in the ipsilateral hippocampus closely resembled that seen in the contralateral side ; however , reactive microglial cells tended to accumulate surrounding the blood vessels in the hippocampal fissure only in the ipsilateral hippocampus ( figure 9 , compare 9(d ) and 9(g ) ) . by 12 hours
, reactive microglial cells changed to pseudopodic / ameboid morphologies and persisted in the fissure , significant differences between il and cl hippocampus were observed ( figure 11 ) .
at 24 hours , increased iba-1 + macrophages were observed in the third ventricle , and the microglial response was maintained in the hippocampal fissure ( figure 9(h ) ) , but iba-1 + round - shaped microglia / macrophages started to cover the degenerating ca fields ( figure 9(h ) ) .
notably , at this time , although morphological and distribution changes in the microglial response versus the contralateral hippocampus were evident ( figure 9 , compare 9(e ) and 9(h ) ) , the area occupied by reactive microglial cells did not differ significantly from the contralateral side ( figure 11 ) , probably as a consequence of the reduced total cell area of pseudopodic / ameboid cells versus ramified cells . from 48 hours
to 7 days after hypoxia , a massive increase in microglia / macrophage cell intensity was evident in the fissure and ca field ( figures 9(i ) , 9(j ) , 9(l)9(n ) ) , showing a 57-fold increase in the area occupied by reactive microglia / macrophages when compared to the contralateral hippocampus ( figure 11 ) . at longer survival times , microglial response
was strongly decreased , showing scattered reactive ramified and macrophages in the fissure and ca only until 14 days ( figures 9(o ) , 9(s ) , and 9(t ) ) , but no presence of reactive microglia / macrophages at 30 and 100 days ( figures 9(p ) , 9(q ) ) .
it should be noted that only scattered activated microglial cells were present in the dg , and always located in the hilus , correlating with the above described astroglial response in this area which is mostly spared in this neonatal injury model as a consequence of its late development [ 53 , 54 ] .
corpus callosumthe corpus callosum , like other white tracts including the internal and external capsules , showed microglial response characterized by the presence of reactive ramified cells elongated in parallel to axonal tracts , from a few hours after the insult ( figure 9(g ) ) , and some ameboid microglia / macrophages observed at 2472 hours after hypoxia ( figures 9(h)9(k ) ) and until 7 days ( figure 9(n ) ) , when response diminished ( figures 9(o ) and 9(r ) ) , almost returning to basal level at 14 days after hypoxia .
however , it should be noted that in this region only mild differences in relation to the contralateral side were seen , with no statistically significant differences shown in the iba-1 + area at any timepoint ( figure 11 ) , this pattern of microglial response in the ipsilateral versus contralateral white matter correlated with the mild response of astroglial cells described above although the changes in glial cells of the contralateral corpus callosum , which also results mildly atrophied , may be masking the increases in glial response in the ipsilateral side .
the corpus callosum , like other white tracts including the internal and external capsules , showed microglial response characterized by the presence of reactive ramified cells elongated in parallel to axonal tracts , from a few hours after the insult ( figure 9(g ) ) , and some ameboid microglia / macrophages observed at 2472 hours after hypoxia ( figures 9(h)9(k ) ) and until 7 days ( figure 9(n ) ) , when response diminished ( figures 9(o ) and 9(r ) ) , almost returning to basal level at 14 days after hypoxia .
however , it should be noted that in this region only mild differences in relation to the contralateral side were seen , with no statistically significant differences shown in the iba-1 + area at any timepoint ( figure 11 ) , this pattern of microglial response in the ipsilateral versus contralateral white matter correlated with the mild response of astroglial cells described above although the changes in glial cells of the contralateral corpus callosum , which also results mildly atrophied , may be masking the increases in glial response in the ipsilateral side .
caudate - putamen , neocortex , and thalamusin general , in these areas , microglial response was also seen as early as 3 hours after hypoxia and lasted until 7 days although it showed a high degree of variability and very few significant differences in compared to the contralateral hemisphere ( figure 11 ) .
reactive microglial cells mainly showed an activated ramified morphology and increased iba-1 labeling ( figures 10(d)10(f ) and 10(j)10(l ) ) although some pseudopodic / amoeboid microglial cells were seen from 12 to 72 hours after lesion , when maximum responses were seen ( figures 10(e ) and 10(k ) ) . in the caudate - putamen , iba-1 +
cells have shown the higher activation in the ventral - lateral region . at 714 days after hypoxia , in all three regions , microglial response remained as patches of reactive ramified microglial cells ( figures 10(f ) and 10(l ) ) .
in general , in these areas ,
microglial response was also seen as early as 3 hours after hypoxia and lasted until 7 days although it showed a high degree of variability and very few significant differences in compared to the contralateral hemisphere ( figure 11 ) .
reactive microglial cells mainly showed an activated ramified morphology and increased iba-1 labeling ( figures 10(d)10(f ) and 10(j)10(l ) ) although some pseudopodic / amoeboid microglial cells were seen from 12 to 72 hours after lesion , when maximum responses were seen ( figures 10(e ) and 10(k ) ) . in the caudate - putamen , iba-1 +
cells have shown the higher activation in the ventral - lateral region . at 714 days after hypoxia , in all three regions , microglial response remained as patches of reactive ramified microglial cells ( figures 10(f ) and 10(l ) ) .
only scattered neutrophils were seen in the medial or lateral third ventricle at p7p21 , in decreasing numbers with hardly countable cells at p21 . at these ages
we also observed a few cells in blood vessels located in hippocampus and neocortex of both hemispheres ( figures 12(a)12(c ) ) .
scattered neutrophils were also seen in the meninges / median fissure . in comparison to adults
, neonates are known to have weakened neutrophil response and reduced tendency to extravasate from blood vessels [ 5557 ] . at 3 and 12 hours after hypoxia , some neutrophils were observed inside the blood vessels in the neocortex , caudate - putamen , and in the hippocampus , but also in the lateral side of third ventricle . by 2472 hours
, neutrophil cell numbers decreased in the blood vessels of neocortex and in the third ventricle ( figure 13 ) . at 714 days after hypoxia
, some neutrophils were observed in the medial third ventricle ( figure 13 ) , the neocortex , and the thalamus . at 30 and 100 days after injury
, there was hardly any cell found in the brain blood vessels or the parenchyma ( figure 13 ) .
hippocampusneutrophils were observed in the ipsilateral hippocampus as early as 3 hours after hypoxia ( figure 13 ) .
cells were usually found distributed in the hippocampal fissure , the dentate gyrus , or the fimbria . at 12 hours after hypoxia , the number of cells increased and was localised in the ca3 region , in the parenchyma as well as inside the blood vessels . in the hippocampal fissure , the dentate gyrus and in the fimbria , most of the neutrophils were inside the blood vessels ( figure 13 ) . at 24 hours after hypoxia , neutrophils were observed throughout the hippocampus but mainly localised in ca1 region and the fimbria ( figure 13 ) . by 48 hours after hypoxia , neutrophils were not observed in the dentate gyrus though a few cells were present near ca3 and the fimbria ( figure 13 ) .
neutrophils appeared to be evenly distributed throughout the hippocampus after 72 hours after hypoxia , but significantly higher density of cells were observed at 7 days after hypoxia ( figures 12(d)12(f ) and 13 ) . at this time of maximum neutrophil numbers , the cells were mostly observed near the hippocampal fissure , ca1 and ca3 region , with the majority of cells in the parenchyma , but usually concentrated near the blood vessels ( figures 12(e ) and 12(f ) ) . at 14 days after hypoxia , the amount of cells rapidly decreased although a few cells were still found , in close opposition to blood vessels in the hippocampal fissure and around the ca3 region . at 30 days after hypoxia , very few neutrophils inside the blood vessels could be identified , and at 100 days after hypoxia no neutrophils were seen inside the hippocampus .
neutrophils were observed in the ipsilateral hippocampus as early as 3 hours after hypoxia ( figure 13 ) .
cells were usually found distributed in the hippocampal fissure , the dentate gyrus , or the fimbria . at 12 hours after hypoxia , the number of cells increased and was localised in the ca3 region , in the parenchyma as well as inside the blood vessels . in the hippocampal fissure , the dentate gyrus and in the fimbria , most of the neutrophils were inside the blood vessels ( figure 13 ) . at 24 hours after hypoxia , neutrophils were observed throughout the hippocampus but mainly localised in ca1 region and the fimbria ( figure 13 ) . by 48 hours after hypoxia , neutrophils were not observed in the dentate gyrus though a few cells were present near ca3 and the fimbria ( figure 13 ) .
neutrophils appeared to be evenly distributed throughout the hippocampus after 72 hours after hypoxia , but significantly higher density of cells were observed at 7 days after hypoxia ( figures 12(d)12(f ) and 13 ) . at this time of maximum neutrophil numbers , the cells were mostly observed near the hippocampal fissure , ca1 and ca3 region , with the majority of cells in the parenchyma , but usually concentrated near the blood vessels ( figures 12(e ) and 12(f ) ) . at 14 days after hypoxia , the amount of cells rapidly decreased although a few cells were still found , in close opposition to blood vessels in the hippocampal fissure and around the ca3 region . at 30 days after hypoxia , very few neutrophils inside the blood vessels could be identified , and at 100 days after hypoxia no neutrophils were seen inside the hippocampus .
ventriclesan elevated number of cells were also present in the third ventricle , both medially and in the ipsilateral side of the third ventricle as early as 3 hours after hypoxia ( figures 12(g ) and 13 ) . at 1248 hours ,
the quantity of cells progressively decreased , but they were mostly distributed in the medial part ( figure 13 ) . by 7 days after hypoxia , correlating with increased numbers also in hippocampus , an increase in neutrophils both in the medial and ipsilateral side of the ventricle could be seen ( figure 13 ) .
finally , by 14 to 100 days , no neutrophils were seen in the lateral side of the third ventricle although scattered cells were located in the medial part .
an elevated number of cells were also present in the third ventricle , both medially and in the ipsilateral side of the third ventricle as early as 3 hours after hypoxia ( figures 12(g ) and 13 ) . at 1248 hours ,
the quantity of cells progressively decreased , but they were mostly distributed in the medial part ( figure 13 ) . by 7 days after hypoxia , correlating with increased numbers also in hippocampus , an increase in neutrophils both in the medial and ipsilateral side of the ventricle could be seen ( figure 13 ) .
finally , by 14 to 100 days , no neutrophils were seen in the lateral side of the third ventricle although scattered cells were located in the medial part .
caudate - putamen , neocortex , and thalamusfrom 3 hours to 72 hours , only a few neutrophils were located in the caudate - putamen region ( figure 13 ) .
an increase in the number of cells was seen at 7 days ( figures 12(h ) and 13 ) , correlating with previously described areas . at longer survival times , no neutrophils were seen in this region.at 3 hours after hypoxia , some neutrophils were distributed in the blood vessels of different layers of the neocortex ( figure 12(i ) ) , being the time showing the highest density ( figure 13 ) . from 12 to 72 hours a reduction in neutrophil cell counts was generally observed although by 72 hours a few cells remained in the upper layers of neocortex . at 7 days after hypoxia , there was a mild increase in neutrophils located inside the cortical blood vessels in both hemispheres . at 30 and 100 days ,
almost no neutrophils were present in the neocortex , and if so , they were located inside the blood vessels ( figure 13).in the thalamus , very few cells were observed as compared to the other regions analysed .
no neutrophils were observed from 3 to 48 hours after hypoxia , and only a few cells were seen at 72 hours , 7 and 14 days ( figures 12(j ) and 13 ) . from 30 days , neutrophils were no longer present in the thalamus ( figure 13 ) .
from 3 hours to 72 hours , only a few neutrophils were located in the caudate - putamen region ( figure 13 ) .
an increase in the number of cells was seen at 7 days ( figures 12(h ) and 13 ) , correlating with previously described areas . at longer survival times , no neutrophils were seen in this region . at 3 hours after hypoxia , some neutrophils were distributed in the blood vessels of different layers of the neocortex ( figure 12(i ) ) , being the time showing the highest density ( figure 13 ) . from 12 to 72 hours a reduction in neutrophil cell counts was generally observed although by 72 hours a few cells remained in the upper layers of neocortex . at 7 days after hypoxia , there was a mild increase in neutrophils located inside the cortical blood vessels in both hemispheres . at 30 and 100 days , almost no neutrophils were present in the neocortex , and if so , they were located inside the blood vessels ( figure 13 ) . in the thalamus ,
no neutrophils were observed from 3 to 48 hours after hypoxia , and only a few cells were seen at 72 hours , 7 and 14 days ( figures 12(j ) and 13 ) . from 30 days , neutrophils were no longer present in the thalamus ( figure 13 ) . in the control brain and at all ages analysed , scattered lymphocytes were only located in the ventricles and meninges , although scattered single cells were sometimes seen in the hippocampus , neocortex , always inside the blood vessels ( figures 12(k ) , 12(l ) and 12(m ) ) . at all time points analysed after hypoxia
, no changes were seen in the contralateral or the ipsilateral hemisphere when compared to control .
in this study we have performed a detailed short and long - term analysis of neuropathological changes , astroglial , microglial response , and leukocyte recruitment following h / i to the neonatal mouse brain , describing massive damage and cellular changes in the ipsilateral hemisphere , but also not negligible changes in the contralateral side .
our description of neuropathological changes in the ipsilateral hemisphere is in agreement with previous reports [ 18 , 42 , 58 , 59 ] , showing hippocampal damage as the most striking feature of hypoxic / ischemic damage in the neonatal mouse , whereas damage to caudate - putamen , neocortex , and thalamus is highly dependent on the postnatal age and the duration of the hypoxia .
hippocampal damage with tissue disruption , neuronal damage , and disorganization of the ca cytoarchitecture was observed as early as 3 hours after hypoxia followed by milder damage to dg at later survival times , which is maintained relatively spared due to its postnatal development . at 7 days after hypoxia , significant atrophy of hippocampal area
this temporal pattern of neurodegeneration is consistent with the observation from other studies showing that h / i damage in an immature brain evolves more rapidly than its adult counterpart [ 40 , 60 ] .
we observe subcortical white matter damage and long - term atrophy , which has been described as a hallmark of neonatal h / i in preterm infants , where the oligodendrocytes in the periventricular white matter are considered one of the most vulnerable cell types to h / i damage [ 61 , 62 ] . in rodent models , neonatal h / i injury has been shown to cause axonal degeneration and disturbances in myelination [ 64 , 65 ] . following h / i in the p9 mouse ,
several authors have reported decreased expression levels of myelin basic protein ( mbp ) and proteolipid protein ( plp ) , decreased neurofilament expression , and the presence of apoptotic cells in the corpus callosum within 24 to 72 hours after injury [ 66 , 67 ] .
white matter damage has been related to the loss of immature oligodendrocytes in the tracts as well as the loss of subventricular zone ( svz ) progenitors after h / i , inducing a depletion of oligodendrocyte precursors [ 68 , 69 ] .
another area showing consistent damage and atrophy in the mouse model of h / i is the caudate - putamen , and the neocortex to a lesser extent and showing higher variability . in this regard , a recent study by selip and coworkers using the neonatal rat model have shown that rats with moderate or severe loss of mbp having significantly increased axonal degeneration in the temporal - parietal cortex , caudate - putamen , thalamus , and internal capsule .
moreover , pups without evidence of severe white matter loss exhibited mild selective grey matter injury , as evidenced by mild axonal injury and neuronal degeneration , in the cortex , internal capsule , and caudate - putamen ; structures central to language processing and understanding , and motor and sensory function .
injury in these regions , even if mild , may be implicated in the neurocognitive disturbances noted in preterm survivors who do not demonstrate other clinical or radiological evidence of overt periventricular white matter injury .
it is interesting to note that we here describe in the mouse that caudate - putamen and cortical atrophy are mainly noted as a long - term effect but show very disperse injury scores at early survival times .
moreover , the effect of h / i in the contralateral hemisphere has been studied extensively to suggest that it can not be used as an efficient control for histological assessment of brain damage in mice , in contrast to what has been described previously in the neonatal rat [ 60 , 72 , 73 ] , providing an significant difference in these species response to h / i .
previous studies using the rat model of h / i have demonstrated that the blood flow to the contralateral cerebral hemisphere structures is relatively unchanged during hypoxia , and that the contralateral hemisphere , when evaluated several weeks after the injury , shows no tissue alterations or atrophy , suggesting that the contralateral hemisphere can be used as a control reference for the evaluation of the extent of damage in the ipsilateral hemisphere in the rat .
some molecular changes in kinases and proinflammatory molecules have been described in both hemispheres in h / i neonatal rats [ 75 , 76 ] .
also jansen and low histologically assessed a hypertrophy of the contralateral hemisphere in adult rats that had undergone perinatal h / i . in the mouse brain , several laboratories have shown that it does not suffer apparent changes during the first week following h / i as is also commonly used as a reference to evaluate the ipsilateral hemisphere .
interestingly , inflammatory gene profiling in p9 mouse brain after h / i shows more than 140 genes involved in the tissue response during the first 72 hours ; however , only microglial expression of osteopontin showed an increase in contralateral subcortical white matter .
while mice and rats show distinguished regional features in tissue damages , it would be interesting to analyze the molecular changes in hi - neonatal mice .
nevertheless , it should be noted that we here report that analysis of the h / i mouse brain up to 3 months after the injury shows some degree of atrophy in the contralateral hippocampus and the corpus callosum , accompanied by ventricle swelling , an event that has been reported earlier .
there are reports stating compensatory reorganizational changes occurring in the contralateral hemisphere in some animals following neonatal h / i brain injury and that this plasticity may be functionally advantageous .
moreover , the presence of significant cognitive deficit in apparent unilateral focal brain injury also indicates towards the involvement of contralateral hemisphere . in this sense
, it should be noted that h / i animals undergo systemic hypoxia , which has been shown to induce changes in gene expression and cell activity by itself .
as an example , change in the expression of certain cytokines , like hypoxia inducing factor alpha ( hif ) , and p - akt to the same extent in both the ipsi as well as contralateral hemisphere showed that hypoxia is sufficient to regulate multiple mediators that may contribute , but may not be sufficient to induce long - term neuronal damage .
as reviewed by sofroniew and vinters , many gray matter astrocytes in healthy cns do not express gfap at immunohistochemically detectable levels or express low levels as in neonates . in our immunohistochemically processed sections ,
although gfap expression was seen in control neonatal brains , an increase in gfap immunostaining was observed after h / i from early time points ( 312 hours ) in comparison to p7 age - matched control , implying an onset of astrogliosis .
notably , changes in astroglial morphology by gfap immunostaining were first seen at 3 hours post - hypoxia both in the ipsilateral as well as contralateral hemisphere . in the contralateral hemisphere , increased gfap and astroglial hypertrophy showed a maximum response at 2448 hours after hypoxia but decreased at longer survival times . in the contralateral side , astroglial response was very restricted to the corpus callosum and the area of the hippocampal fissure , but never covering the ca - neuronal layer .
however , in the ipsilateral h / i - damaged hemisphere , the increase in gfap expression and cell hypertrophy peaked at 14 days after hypoxia and was evident in the corpus callosum , the caudate - putamen , the neocortex , and the hippocampus , where the long - term glial scar persisted till 100 days after hypoxia .
as the radial glia mature , they show gfap expression and some give rise to gfap - expressing radial neural stem cells ( nscs ) that persist in juvenile and adult forebrain , while others become astrocytes [ 8183 ] .
some of these radial nscs remain constitutively active throughout life in the subventricular zone of the lateral ventricles and in the subgranular zone of the hippocampal dentate gyrus , where they are the predominant source of adult neurogenesis
. this might be the reason of concentration and persistence of glial scar or gfap+ cells at 14100 days after hypoxia in hilus and hippocampal fissure in our study .
notably , gfap expression after h / i was also found most highly concentrated in layers showing high content of synaptic contacts including the hippocampal fissure in the neonatal brain as seen in our study , which is in concordance with reports where astrocytes appear to influence developmental synaptic pruning by releasing signals and thereby tag them for elimination by microglia [ 84 , 85 ] .
the role of reactive astrogliosis in the evolution of ischemic brain lesions especially in neonates is at present not clear , but recent studies have suggested that reactive astrocytes provide essential metabolic support to neurons during transient ischemia and that failure of astrocyte functions may contribute to neuronal degeneration [ 86 , 87 ] .
additionally , in adult transgenic mice , experimental disruption of astroglial scar formation following stroke is associated with loss of barrier functions along the margins of infarcts , resulting in increased spread of inflammation and increased lesion volume .
moreover , adult mice lacking gfap [ gfap(/ ) ] show attenuated reactive gliosis , reduced glial scar formation after focal brain ischemia as compared to injured developing brain where there is only an increase in the survival of newborn neurons .
astrocytes also play a vital role in white matter , regulating molecules such as glutamate in the extracellular space and preventing excitotoxic damage to neighbouring oligodendrocytes and axons .
consistent with previous reports , we noted an increase in gfap expression in white matter astrocytes accompanied by hypertrophy and process thickening in ipsilateral hemispheres . in control postnatal mice ,
we observed amoeboid and ramified microglia throughout gray and white matter from p7 to p14 mice , as has been described previously .
as the brain development continues after birth , microglial cells need to adapt to the changes in the microenvironment . until p14 , we observed groups of amoeboid microglial cells which are present in the developing corpus callosum , cingulum , and fimbria .
these cells are proposed to be involved in the phagocytosis of cellular debris and contribute to the axonal nerve fiber remodeling and synapsis during normal development [ 9395 ] . in the present study , we have observed morphologically activated microglia from 3 to 72 hours after hypoxia in the contralateral corpus callosum , with a peak of response at 24 hours . this microglial response to hypoxic conditions in the subcortical white matter
has been extensively studied by the group of ling and coworkers , who have demonstrated that hypoxia - activated microglial cells in the developing white matter produce several inflammatory mediators including cytokines , chemokines , and reactive oxygen species which are detrimental for white matter development and oligodendrocyte survival ( reviewed in ) , which may account for the long - term contralateral corpus callosum atrophy we observe , although the microglial response in the contralateral corpus callosum is transient , in agreement with the findings of zaidi and coworkers in the p7 rat model , that did not observe activated microglia after 14 days of hypoxia in the contralateral hemisphere .
interestingly , in agreement with our observations , cowell and coworkers have shown a transient contralateral microglia activation in the cortex , white matter and hippocampus after an unilateral transection of mca in neonatal rat brain .
obviously , microglial response in the ipsilaterally damaged corpus callosum is very striking , showing reactive ramified and ameboid / macrophagic forms from 3 hours to 14 days after hypoxia , with a peak of response at 4872 hours .
it is now evident that the developing brain is highly susceptible to hypoxic damage because of its high oxygen and energy requirements [ 99 , 100 ] , and that white matter at this developmental stage is vulnerable .
moreover it have been described that the myelin from the degenerating axons is phagocytosed by microglia . in this sense ,
as long - term atrophied white matter is observed after microglia returns to a resting state , we may suggest that activated microglial cells may not be sufficient to complete phagocytosis and avoid the inhibition of oligodendrocyte precursors differentiation . as most of this knowledge
is mainly obtained from results in rat models and several differences has been described between rodents , a more detailed description on the late effects on oligodendrocytes , their precursors , myelination and axonal degeneration in neonatal mice brain hypoxic ischemic injury is needed .
interestingly , microglial response in the contralateral gray matter areas was more evident that the astroglial response , and activated microglial cells were seen as early as 3 hours after hypoxia in the hippocampus , but also in the caudate - putamen and cortex ( see figures 9 and 10 ) ; however , contralateral microgliosis was very transient and only persisted until 4872 hours depending on the regions . in the contralateral hippocampus ,
microglial response was mostly evident in the hippocampal fissure , and not so widespread as in the ipsilaterally damaged side , where we describe a layer - specific activation of microglia as early as 3 hours after hypoxia , with a maximum response from 48 hours to 7 days , followed by a patchy pattern at later time points .
this has also been demonstrated at early time points in rat model as mentioned previously by cowell and coworkers .
remarkably , hippocampal microglial response was first observed surrounding the blood vessels in the hippocampal fissure , which have been suggested to be more vulnerable to ischemic episodes than those from other hippocampal areas .
interestingly , this is known to be one of the sources of microglia progenitors during late embryonic life in the rat , showing , during early postnatal development an outside - to - inside microglia distribution pattern towards the pyramidal or granular cell layers . from 24 hours onwards evident neuronal damage when evident neuronal damage takes place in the ipsilateral hippocampus and then ameboid / macrophagic phagocytic microglia populate the neurodegenerating ca areas .
the association between microglia activation and injury development raises the question whether this reaction is detrimental or beneficial [ 103105 ] .
however , it is now established that , as macrophages do in the periphery , microglia has two different patterns of activation and function in response to cns injury ( revised by [ 26 , 103 , 106 ] ) .
opposite effects have been described in neonatal h / i mice and rats using minocycline , a tetracycline derivative that nonspecifically blocks all microglia activation .
in rat brain , this treatment protects the brain tissue in some reports [ 19 , 20 ] but only have a transient protective effect in others .
in contrast , tissue damaged increases in minocycline - treated h / i mice , especially in cortex , caudate - putamen and thalamus without significant effects on hippocampus .
additionally , selective depletion of microglia before a transient mcao in a p7 rats does not change the volume of injury but enhances cytokines production compared to not depleted animals , suggesting a beneficial role of microglial cells .
these evidences made a complete characterization of neonatal mice microglial response essential , in order to define the better window and target for protective therapies .
new insides in the physiological activity of microglia ( called surveillance instead of resting ) , joined to adult mri and behaviour assessment [ 78 , 110 ] , would be beneficial to promote phagocytic and anti - inflammatory response of microglia than a complete blocking of their activation in order to obtain better outcomes of therapies applied to injured developing brain .
the neonates are known to have weakened neutrophil response and reduced tendency to leukocyte extravasation from blood vessels [ 5557 ] .
previous studies have demonstrated that neutrophils contribute to the long - term hypoxic / ischemic brain injury in the neonatal rat brain [ 111 , 112 ] .
we here report that neutrophils appeared as early as 3 hours after hypoxia in blood vessels of most of the regions studied , especially in the neocortex and third ventricle , in agreement with previous reports showing that neutrophils are seen in brain blood vessels rather early [ 111 , 113 , 114 ] . however , there are limited studies reporting neutrophils in the neonatal parenchyma after hypoxia , and the results are variable ; we observed neutrophil recruitment to the injured mouse parenchyma ( mainly hippocampus and caudate - putamen ) after 72 hours to 7 days after hypoxia , whereas other studies have shown neutrophils accumulated in the injured rat parenchyma at 1224 hours after hypoxia , peaking at 7296 hours [ 113 , 115 ] .
notably , neutrophils accumulate in the same areas of microglia / macrophage accumulation , contributing in the removal of cellular debris and the release of cytokines to further attract more immune cells to the injury site [ 114 , 116 ] .
the negligible lymphocytic infiltration reported here is in accordance with previous reports where no cd3 + cells were detected in the neonatal p1 rat brain at 48 hours after hypoxia and lps induction .
furthermore , there are reports of very low expression of cd3 chain of the t - cell receptor in p3 , p7 , and p14 mice brain in contrast to adult .
since many investigators are using transgenic and knockout mice to determine the importance of specific molecules in the evolution of damage after brain injury , there is an urgent need to perform comparative studies on the relative vulnerability of the mouse brain in comparison to other species . a mouse model of hypoxic - ischemic encephalopathy
has paved a way for the description of the specific molecular mechanisms associated with this destructive disease , by the use of genetically modified animals .
our major finding describing the short- and long - term effects as well as the involvement of the contralateral hemisphere may serve as a valuable resource for functional definition of neuroprotection or damage as well as will aid in selecting the time and mode of intervention in the broad therapeutic window .
to summarize , this study describes qualitatively and quantitatively the tissue damage , glial response , and inflammatory cell recruitment after brain injury induced by carotid occlusion and systemic hypoxia ( 8% o2 , 55 minutes ) to the postnatal day 7 mouse brain , analyzing changes from 3 hours to 100 days after hypoxia . in general , massive tissue injury and atrophy in the ipsilateral hippocampus , corpus callosum and caudate - putamen are consistently shown , with neutrophil recruitment and earlier microgliosis , but persistent long - term glial scarring until 100 days after hypoxia .
remarkably , in the contralateral hippocampus and corpus callosum , milder atrophy is delayed in areas that show the activation of astrocytes and microglial during the first 72 hours .
this study highlights that care should be taken when using the contralateral hemisphere as control while studying ipsilateral h / i injury in postnatal mouse brain .
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### Summary:
: understanding the evolution of neonatal hypoxic / ischemic is essential for novel neuroprotective approaches .
we describe the neuropathology and glial / inflammatory response , from 3 hours to 100 days , after carotid occlusion and hypoxia ( 8% o2 , 55 minutes ) to the c57/bl6 p7 mouse . massive tissue injury and atrophy in the ipsilateral ( il ) hippocampus , corpus callosum , and caudate - putamen are consistently shown .
astrogliosis peaks at 14 days , but glial scar is still evident at day 100 . microgliosis peaks at 37 days and decreases by day 14 .
both glial responses start at 3 hours in the corpus callosum and hippocampal fissure , to progressively cover the degenerating ca field .
neutrophils increase in the ventricles and hippocampal vasculature , showing also parenchymal extravasation at 7 days .
remarkably , delayed milder atrophy is also seen in the contralateral ( cl ) hippocampus and corpus callosum , areas showing astrogliosis and microgliosis during the first 72 hours .
this detailed and long - term cellular response characterization of the ipsilateral and contralateral hemisphere after h / i may help in the design of better therapeutic strategies .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: ascende - rt ( androgen suppression combined with elective nodal and dose escalated radiation therapy ) is a randomized trial testing the role of brachytherapy ( bt ) boost compared to external beam radiation therapy ( ebrt ) boost after whole pelvis ebrt for intermediate- to high - risk prostate cancer .
however , given limited follow - up to date , it is unclear whether this improvement in biochemical recurrence - free survival ( brfs ) may translate to a mortality benefit for patients treated with a brachytherapy boost .
similarly , two smaller single - center studies found a brfs benefit with the addition a bt boost in a similar patient population ; in both cases , the secondary endpoint of overall survival was not improved [ 2 , 3 ] .
while national comprehensive cancer network ( nccn ) guidelines began including ebrt + bt as a treatment option for intermediate - risk disease starting in 2002 , the first randomized study to support this practice was published in 2005 , and may have supported the use of a bt boost before the preliminary ascende - rt results were recently reported .
recent retrospective data suggests that the benefit of bt boost may be limited to patients with high - risk disease , whereas intermediate - risk patients appear to have similar cancer - specific survival whether they receive ebrt + bt or ebrt alone .
other work has suggested that high - risk prostate cancer represents a heterogeneous disease with variable outcomes dependent on the number of risk factors [ 6 , 7 , 8 ] . among patients managed with radiation
, we have recently shown that patients with favorable high - risk disease ( stage t1c , gleason 4 + 4 = 8 , and prostate - specific antigen [ psa ] < 10 ng / ml or stage t1c , gleason 6 , and psa > 20 ng / ml ) have much better cancer - specific outcomes than others with high - risk disease and similar outcomes as patients with unfavorable intermediate - risk disease .
in particular , estimated 8 year prostate cancer - specific mortality ( pcsm ) for favorable high - risk disease was 2.1% , compared with 7.1% for other high - risk disease ( p = 0.049 ) and 2.5% for unfavorable intermediate - risk disease ( p = 0.710 ) .
this finding raises the possibility that patients with favorable high - risk disease might be candidates for less aggressive forms of therapy than is typically recommended for high - risk disease .
based on the possible benefit of bt boost for patients with high - risk disease seen so far in the ascende - rt trial and in retrospective data , we used a large , national cancer database to investigate whether patients with favorable high - risk prostate cancer benefit from ebrt + bt compared to ebrt alone .
given the lack of a survival benefit seen in retrospective data for intermediate - risk disease , and the similarities in outcome between intermediate - risk disease and favorable high - risk disease , we hypothesized that patients with favorable high - risk prostate cancer would not have a survival benefit from ebrt + bt versus ebrt alone .
the surveillance , epidemiology , and end results ( seer ) database is a population - based cancer registry that covers 28% of the us population and 97% of incident cancers .
the registry collects cancer diagnostic , treatment , and survival data along with patient demographic characteristics . to match the ascende - rt enrollment criteria
, we studied 45,078 patients in the seer database with ct1c - t3an0m0 prostate cancer diagnosed between 2004 - 2011 who had intermediate- or high - risk prostate cancer as defined by nccn , and were managed with ebrt alone or ebrt + bt .
patients were excluded if they had a psa over 40 ng / ml ( as in ascende - rt ) or received surgical treatment of their cancer .
patients were considered to have intermediate - risk disease if they had at least one intermediate - risk factor ( t2b - c , gleason 7 , or psa 10 - 20 ng / ml ) with no high - risk features , and patients were considered to have high - risk disease if they had at least one high - risk factor ( t3a , gleason 8 - 10 , or psa > 20 ng / ml ) . in addition , we considered patients to fall into the subset of favorable high - risk disease if they had stage t1c disease with gleason 4 + 4 = 8 and psa < 10 ng / ml , or stage t1c disease with gleason 6 and psa > 20 ng / ml . because of recently reported possible inaccuracies in the recording of a minority of psa values in the seer database [ 12 , 13 ] , we excluded 2,178 out of 47,256 initial patients ( 4.6% ) who had values for psa and psa interpretation recorded in seer that were discordant ( e.g. psa < 4.0 ng / ml recorded as positive / elevated or psa > 4.0 ng / ml recorded as negative / normal ) .
otherwise , we only included patients who had known values in seer for clinical and demographic factors , including t stage , gleason score , psa , reason for not undergoing surgery , marital status , age , income level , education level , year of diagnosis , and race .
statistical analyses were conducted using stata / mp 14.0 ( statacorp , college station , tx , usa ) .
fine and gray competing risks regression was used to determine differences in the risk of pcsm between groups of patients after controlling for t stage ( t1c vs. t2 vs. t3a ) , gleason score ( 6 vs. 7 vs. 8 - 10 ) , the logarithm of the psa , race ( white vs. black vs. other ) , reason for not receiving surgery ( not recommended vs. other ) , marital status ( married vs. other ) , age at diagnosis , year of diagnosis , income quartile , and educational quartile .
median values were compared using the kruskall - wallis test , and proportions were compared using the test or t - test , as appropriate .
the surveillance , epidemiology , and end results ( seer ) database is a population - based cancer registry that covers 28% of the us population and 97% of incident cancers .
the registry collects cancer diagnostic , treatment , and survival data along with patient demographic characteristics . to match the ascende - rt enrollment criteria
, we studied 45,078 patients in the seer database with ct1c - t3an0m0 prostate cancer diagnosed between 2004 - 2011 who had intermediate- or high - risk prostate cancer as defined by nccn , and were managed with ebrt alone or ebrt + bt .
patients were excluded if they had a psa over 40 ng / ml ( as in ascende - rt ) or received surgical treatment of their cancer .
patients were considered to have intermediate - risk disease if they had at least one intermediate - risk factor ( t2b - c , gleason 7 , or psa 10 - 20 ng / ml ) with no high - risk features , and patients were considered to have high - risk disease if they had at least one high - risk factor ( t3a , gleason 8 - 10 , or psa > 20 ng / ml ) . in addition , we considered patients to fall into the subset of favorable high - risk disease if they had stage t1c disease with gleason 4 + 4 = 8 and psa < 10 ng / ml , or stage t1c disease with gleason 6 and psa > 20 ng / ml . because of recently reported possible inaccuracies in the recording of a minority of psa values in the seer database [ 12 , 13 ] , we excluded 2,178 out of 47,256 initial patients ( 4.6% ) who had values for psa and psa interpretation recorded in seer that were discordant ( e.g. psa < 4.0 ng / ml recorded as positive / elevated or psa > 4.0 ng / ml recorded as negative / normal ) .
otherwise , we only included patients who had known values in seer for clinical and demographic factors , including t stage , gleason score , psa , reason for not undergoing surgery , marital status , age , income level , education level , year of diagnosis , and race .
statistical analyses were conducted using stata / mp 14.0 ( statacorp , college station , tx , usa ) .
fine and gray competing risks regression was used to determine differences in the risk of pcsm between groups of patients after controlling for t stage ( t1c vs. t2 vs. t3a ) , gleason score ( 6 vs. 7 vs. 8 - 10 ) , the logarithm of the psa , race ( white vs. black vs. other ) , reason for not receiving surgery ( not recommended vs. other ) , marital status ( married vs. other ) , age at diagnosis , year of diagnosis , income quartile , and educational quartile .
median values were compared using the kruskall - wallis test , and proportions were compared using the test or t - test , as appropriate .
patient characteristics for the cohort are shown in table 1 . within the entire cohort , median follow - up was 3.6 years , with 28% of patients
men with favorable high - risk disease tended to be diagnosed slightly earlier than those with unfavorable intermediate - risk or other high - risk disease . the majority ( 76% ) of patients with favorable high - risk disease had t1c , gleason 8 , and psa < 10 ng / ml versus a minority ( 24% ) with t1c , gleason 6 , and psa 20 - 40 ng / ml .
approximately 20% of patients received ebrt + bt , and patients with favorable high - risk disease were slightly more likely to receive ebrt + bt compared to intermediate - risk or other high - risk patients . among patients with favorable high - risk disease , 25.4% of those with t1c , gleason 4 + 4 = 8 and psa < 10 ng / ml and 15.3% of those with t1c , gleason 6 , and psa > 20 ng / ml received brachytherapy .
brachytherapy , iqr interquartile range patients with favorable high - risk disease had similar 5 year pcsm whether they received ebrt + bt or ebrt alone ( 1.6% vs. 1.8% , adjusted hazard ratio [ ahr ] : 0.56 , 95% confidence interval [ ci ] : 0.21 - 1.52 , p = 0.258 ) .
in contrast , others with high - risk disease had a significantly reduced rate of pcsm at 5 years if treated with ebrt + bt compared to ebrt alone ( 3.9% vs. 5.3% , ahr : 0.73 , 95% ci : 0.55 - 0.95 , p = 0.022 ) . like those with favorable high - risk disease , patients with intermediate - risk disease did
not have significantly improved 5 year pcsm if they received ebrt + bt compared to ebrt alone ( 0.8% vs. 1.0% , ahr : 0.83 , 95% ci : 0.59 - 1.16 , p = 0.270 ) ( figure 1 ) .
prostate cancer - specific mortality among patients with intermediate - risk ( a ) , favorable high - risk ( b ) , or other high - risk ( c ) prostate cancer treated with external beam radiation therapy ( ebrt ) or ebrt + brachytherapy ( bt )
patient characteristics for the cohort are shown in table 1 . within the entire cohort , median follow - up was 3.6 years , with 28% of patients
men with favorable high - risk disease tended to be diagnosed slightly earlier than those with unfavorable intermediate - risk or other high - risk disease . the majority ( 76% ) of patients with favorable high - risk disease had t1c , gleason 8 , and psa < 10 ng / ml versus a minority ( 24% ) with t1c , gleason 6 , and psa 20 - 40 ng / ml .
approximately 20% of patients received ebrt + bt , and patients with favorable high - risk disease were slightly more likely to receive ebrt + bt compared to intermediate - risk or other high - risk patients . among patients with favorable high - risk disease , 25.4% of those with t1c , gleason 4 + 4 = 8 and psa < 10 ng / ml and 15.3% of those with t1c , gleason 6 , and psa > 20 ng / ml received brachytherapy .
patients with favorable high - risk disease had similar 5 year pcsm whether they received ebrt + bt or ebrt alone ( 1.6% vs. 1.8% , adjusted hazard ratio [ ahr ] : 0.56 , 95% confidence interval [ ci ] : 0.21 - 1.52 , p = 0.258 ) .
in contrast , others with high - risk disease had a significantly reduced rate of pcsm at 5 years if treated with ebrt + bt compared to ebrt alone ( 3.9% vs. 5.3% , ahr : 0.73 , 95% ci : 0.55 - 0.95 , p = 0.022 ) . like those with favorable high - risk disease , patients with intermediate - risk disease did not have significantly improved 5 year pcsm if they received ebrt + bt compared to ebrt alone ( 0.8% vs. 1.0% , ahr : 0.83 , 95% ci : 0.59 - 1.16 , p = 0.270 ) ( figure 1 ) .
prostate cancer - specific mortality among patients with intermediate - risk ( a ) , favorable high - risk ( b ) , or other high - risk ( c ) prostate cancer treated with external beam radiation therapy ( ebrt ) or ebrt + brachytherapy ( bt )
in this study , we found that treatment with ebrt + bt boost vs. ebrt alone was not associated with an improvement in pcsm among those with favorable high - risk disease ( defined as t1c , gleason 4 + 4 = 8 , psa < 10 ng / ml or t1c , gleason 6 , psa > 20 ng / ml ) , while others with high - risk disease experienced 27% reduction in the adjusted hazard rate of pcsm and an absolute risk reduction of 1.4% ( 5.3% to 3.9% ) at 5 years .
this study adds support to the notion that patients with radiation - managed high - risk disease represent a heterogeneous group .
previous work has suggested that the survival outcomes of favorable high - risk patients are much better than the outcomes of other high - risk patients and similar to the outcomes of intermediate - risk patients .
here , we show that while patients with other high - risk disease might generally benefit from intensification of radiation therapy via brachytherapy boost , the survival benefit may not extend to the subset of patients with favorable high - risk disease .
the recent report of a brfs benefit with brachytherapy boost from the ascende - rt randomized trial is likely to increase interest in the use of brachytherapy for patients with intermediate- to high - risk disease . while the trial has not matured far enough to show a cancer - specific or overall survival benefit , it is possible that the striking 53% improvement in brfs will translate to a statistically significant mortality benefit with longer follow - up .
until longer follow - up is obtained in the ascende - rt trial , retrospective data might be used to guide application of these preliminary results .
similar to the preliminary results of ascende - rt , two smaller randomized trials , with 218 and 51 patients , have previously shown 31% and 58% improvements in brfs , respectively .
neither study showed an improvement in overall survival , although follow - up was relatively short ( 7.1 and 8.2 years , respectively ) , and the studies may have been underpowered for overall survival compared to ascende - rt , which enrolled nearly 400 patients [ 2 , 3 ] .
retrospective data also suggests that the combination of ebrt + bt is superior to ebrt alone , even when very high ebrt doses ( 86.4 gy ) are used .
while only a prospective randomized non - inferiority trial can definitively prove that brachytherapy may be safely omitted in patients with favorable high - risk prostate cancer , these retrospective data may be used until future data from ascende - rt or other studies are available .
our results are important because it is desirable to identify patients who might not benefit from a bt boost .
brachytherapy for prostate cancer is associated with inherent risks , including an increase in the rate of urethral stricture , persistent urinary retention requiring further procedures [ 16 , 17 ] , or urinary incontinence .
in addition , more severe complications such as recto - urethral fistulization or bladder neck contracture may rarely occur .
finally , although not directly related to the procedure , there are inherent risks associated with the use of anesthesia .
although the seer dataset does not contain information regarding complications from bt , it is possible that the patients in our cohort who received bt are likely to experienced more side effects or toxicity than patients treated with ebrt alone . in the absence of a survival benefit for intermediate - risk or favorable high - risk disease ,
these patients might be better served by omitting bt from their radiation treatment ; although prospective randomized trial data , either as a subgroup analysis of ascende - rt or a separate trial focusing on favorable high - risk disease , will be needed before definitive conclusions can be made regarding the appropriate level of therapy for these patients .
the present study supports the notion that favorable high - risk disease may represent a distinct disease entity within the high - risk group , and that patients with favorable high - risk disease may generally be good candidates for less aggressive therapy than is typically recommended for high - risk disease .
recent data have suggested that patients with favorable high - risk disease , who have clinical t1c ( non - palpable ) disease and only one other high - risk feature ( either gleason 4 + 4 = 8 and psa < 10 ng / ml or gleason 6 and psa > 20 ng / ml ) , have significantly better outcomes than others with high - risk disease and similar outcomes as those with unfavorable intermediate - risk disease .
our results showing a benefit to bt boost for patients with other high - risk but not favorable high - risk or intermediate - risk disease , are consistent with the view that favorable high - risk and intermediate - risk prostate cancer likely represent similar levels of risk .
therefore , it is reasonable to consider similar treatment paradigms for these two risk categories . on the other hand ,
patients with other high - risk disease are likely to represent a subpopulation of men who would benefit most from a higher intensity of therapy , including long - term adt and bt boost .
therefore , our results are susceptible to the general limitations of this study design , including selection bias . while we attempted to adjust for potential differences between the ebrt + bt and ebrt groups , some factors such as the use of high - dose - rate ( hdr ) versus low - dose - rate ( ldr ) bt , seed type , dose of radiotherapy , quality of radiation therapy ,
the specifics regarding ebrt or bt technique , receipt of hormone therapy , or bulk of disease ( e.g. number of cores positive and percentage of core involvement ) , are not available in the seer database . only a large randomized trial can fully account for all possible sources of selection bias .
in addition , the heterogeneity of the practice of bt , which we were not able to measure or adjust for , limits the general applicability of our results .
second , our risk classification depended on the recorded values of psa in the seer database , which may contain a number of errors , possibly due to misplacement of a decimal point .
however , preliminary investigations by the seer program have determined that only approximately 5% of psa values led to incorrect classification of psa category among < 10 ng / ml , 10 - 20 ng / ml , and > 20 ng / ml .
based on the results of schymura et al . , we attempted to account for these erroneous values by removing patients who were listed as having a positive psa despite psa < 4 ng / ml or who were listed as having a negative psa despite psa > 4 ng / ml ; this approach identified 4.6% of patients with possibly incorrect psa values .
although this approach may not have accounted for all possible errors in psa values , the relatively low rate of incorrect psa categorization combined with the likely random nature of these coding errors leads us to believe that these errors are not likely to systematically bias our findings or impact our conclusions .
third , it is possible that we did not find a benefit to bt boost among favorable high - risk patients due to a lack of power or due to the relatively short median follow - up of 3.6 years .
however , the favorable high - risk subgroup had a large number ( n = 2,785 ) of patients .
while a low event rate may still lead to underpowering of the comparison , this low rate of pcsm underscores our conclusions that patients with favorable high - risk disease may not benefit from intensification of therapy in the form of bt boost .
studies with longer follow - up , including ascende - rt , will be required before definitive recommendations for patients with favorable high - risk disease can be made .
while previous work in other cohorts has suggested an increased rate of toxicity associated with bt for prostate cancer [ 1 , 16 , 17 ] , the seer database does not contain information about toxicity or quality of life , so we were unable to study differences in these important outcomes between the ebrt and ebrt + bt groups .
in addition , it is possible that patients who did not receive bt were more likely to receive hormonal therapy , which is also associated with increased toxicity and side effects [ 21 , 22 , 23 , 24 , 25 , 26 ]
. physicians should continue to weigh the patient - specific risks of toxicity from bt , hormonal therapy , and ebrt when personalizing treatment for patients with unfavorable - risk prostate cancer .
patients with favorable high - risk prostate cancer ( t1c , gleason 4 + 4 = 8 , and psa < 10 ng / ml or t1c , gleason 6 , and psa > 20 ng / ml ) do not appear to derive a pcsm benefit from ebrt + bt compared to ebrt alone , while others with high - risk disease were 27% less likely to die from prostate cancer if they were treated with ebrt + bt .
pending long - term survival data from the ascende - rt trial , these results suggest that personalization of therapy is possible within high - risk prostate cancer , and raise the possibility that adding bt boost may not provide a survival benefit for the favorable high - risk subgroup .
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### Summary:
: purposerecent retrospective data suggest that brachytherapy ( bt ) boost may confer a cancer - specific survival benefit in radiation - managed high - risk prostate cancer .
we sought to determine whether this survival benefit would extend to the recently defined favorable high - risk subgroup of prostate cancer patients ( t1c , gleason 4 + 4 = 8 , psa < 10 ng / ml or t1c , gleason 6 , psa > 20 ng / ml).material and methodswe identified 45,078 patients in the surveillance , epidemiology , and end results database with ct1c - t3an0m0 intermediate- to high - risk prostate cancer diagnosed 2004 - 2011 treated with external beam radiation therapy ( ebrt ) only or ebrt plus bt .
we used multivariable competing risks regression to determine differences in the rate of prostate cancer - specific mortality ( pcsm ) after ebrt + bt or ebrt alone in patients with intermediate - risk , favorable high - risk , or other high - risk disease after adjusting for demographic and clinical factors.resultsebrt + bt was not associated with an improvement in 5-year pcsm compared to ebrt alone among patients with favorable high - risk disease ( 1.6% vs. 1.8% ; adjusted hazard ratio [ ahr ] : 0.56 ; 95% confidence interval [ ci ] : 0.21 - 1.52 , p = 0.258 ) , and intermediate - risk disease ( 0.8% vs. 1.0% , ahr : 0.83 , 95% ci : 0.59 - 1.16 , p = 0.270 ) .
others with high - risk disease had significantly lower 5-year pcsm when treated with ebrt + bt compared with ebrt alone ( 3.9% vs. 5.3% ; ahr : 0.73 ; 95% ci : 0.55 - 0.95 ; p = 0.022).conclusionsbrachytherapy boost is associated with a decreased rate of pcsm in some men with high - risk prostate cancer but not among patients with favorable high - risk disease .
our results suggest that the recently - defined favorable high - risk category may be used to personalize therapy for men with high - risk disease .
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: healing is the process of cure and restoring of integrity of injured tissues . during wound healing ,
cells communicate with each other through the use of specific molecules that are usually proteins .
" growth factors are a subclass of cytokines that specifically stimulates the proliferation of cells .
a platelet present in blood of mammals is important for its role in blood coagulation .
platelets activated by the coagulation cascade particularly thrombin and sub endothelial collagen ; release a number of growth factors from their alpha granules into the wound site .
these factors can activate the proliferation and differentiation of the local osteoprogenitor cells into bone forming cells leading to the formation of new bone matrix and mineralization .
platelet - rich plasma ( prp ) is an autologous concentrate of platelets suspended in plasma .
it is a proven source of growth factors such as platelet - derived growth factors ( pdgfs ) and transforming growth factor - beta 1 and 2 ; vascular endothelial growth factors positively influence repair and regeneration of tissues . by combining with calcium chloride and thrombin , prp releases these growth factors .
prp gel also contains a native concentration of fibrinogen . as a result of its fibrin content
, prp gel permits stabilized coagulation of blood thereby favoring regeneration of the osseous defects particularly in the early stage .
soft tissue healing is also substantially improved through the application of prp , by increasing collagen content and regulating key cellular processes , such as mitosis , cell differentiation , and metabolism .
preparation of prp includes two stages of preparation and biochemical handling of blood . whereas , platelet rich fibrin ( prf ) - is a second generation platelet concentrate having several advantages over prp ; such as , ease of preparation and lack of biochemical handling of blood , which makes this preparation strictly autologous .
prf is a leukocyte and prf biomaterial with a specific composition and three - dimensional architecture .
prf has a dense fibrin network with leukocytes , cytokines , structural glycoproteins and also growth factors .
leukocytes that are concentrated in prf scaffold play an important role in growth factor release , immune regulation , anti - infectious activities , and matrix remodeling during wound healing .
the slow polymerization mode of prf and cicatricial capacity creates a physiologic architecture favorable for wound healing .
the use of prf dressings may be a simple and effective method of reducing the morbidity associated with donor sites of autogenous free gingival grafts .
yelamali and saikrishna found better and faster wound healing and bone formation with prf , and also he stated preparation of prf is simpler than prp .
this study was undertaken at the department of oral and maxillofacial surgery , after obtaining ethical clearance .
thirty patients having bilateral mandibular third molar impaction ( fully / partially erupted with all type of angulations ) in both male and female aged between 18 and 30 years were included in the present study with an informed consent and were divided into two groups .
group i - control groupgroup ii - this group was further divided into two subgroups group iia and group iib ( split moth method was used on one side of each patient as a control group and another side as intervention group)group iia - was the one in which left extraction socket was filled with prp gelgroup iib - in which right extraction socket was filled with prf .
group i - control group group ii - this group was further divided into two subgroups group iia and group iib ( split moth method was used on one side of each patient as a control group and another side as intervention group ) group iia - was the one in which left extraction socket was filled with prp gel group iib - in which right extraction socket was filled with prf .
preoperative evaluation with intraoral periapical radiograph [ figure 1 ] and complete blood investigation was done for all patients .
intraoral periapical radiographs radiographic image showing method to evaluate bone density patient venous blood was collected and subjected for centrifugation then 2 ml of conventional prp ( cprp ) was treated with 60 l of citrate inhibitor sterile 10% calcium chloride solution and allowed to coagulate .
then , squeezed to release the platelet - derived thrombin and collected in serum rich plasma .
then , autologous thrombin rich plasma was mixed with cprp in the 1:4 ratios and allowed to coagulate .
armamentarium for platelet rich plasma and platelet rich fibrin preparation concentrated platelet rich plasma platelet - rich plasma gel after addition of cacl2 and autologus thrombin armamentarium required for prf is same as that of prp except citrate phosphate dextrose adenine ( cpda ) anticoagulant solution and 10% cacl2 .
around 5 ml of whole venous blood was collected in each of two sterile vacutainer tube of 6 ml capacity without anticoagulant .
the vacutainers were placed in the centrifuge at 3000 rpm for 10 min after which we obtained red lower fraction containing red blood cells , upper straw colored cellular plasma , and the fibrin clot was obtained in the middle . the straw colored upper layer was removed , and middle layer with fraction 2 mm below dividing line was collected which will be the prf . in group
i , the extraction socket was closed primarily without prp and prf in the socket . in group
iia , prp in the mandibular left extraction socket and in group iib mandibular right extraction socket 2 - 3 ml of prf was placed .
the extraction socket evaluated on 1 , 2 , and 7 postoperative day for any wound dehiscence and recorded as present / absent .
probing depth ( pd ) measured using michigan o probe with william 's markings from " free gingival margin ( fgm ) " to the bottom of the pocket in nearest mm .
the average of pd was recorded at the three different positions , i.e. distobuccal ( db ) , distal ( d ) , distolingual ( dl ) on the distal surface of second molar taken preoperatively , 1 , 2 , 4 , and 6 month postoperatively .
alveolar bone height ( abh ) measured distal to second molar was measured with a caliper from cementoenamel junction ( cej ) to the alveolar crest on intraoral periapical radiographs ( iopa ) at 1 , 2 , 4 , and 6 month postoperatively .
bone density was measured on direct iopa using densitometric analysis device [ figures 1 and 5 ] pre- and post - operatively at 1 , 2 , 4 , and 6 month .
radiographic schematic image showing method to evaluate bone density . a = free gingival margin , b = cementoenamel junction , c = depth of the pocket , d = alveolar crest the bone density calculated using below formula : where , point a = point at alveolar crest just mesial to third molar .
point a = point just mesial to third molar at the level of apical third .
point b and b = points in similar fashion but distal to mandibular third molar .
where , point c = mesial point in the third molar socket at the alveolar crest level .
point c - mesial point in the third molar socket at the level of apical third .
mean gray level histogram values of the digitalized iopa images of third molar extraction socket of both control and study groups were calculated at preoperative , 2 , 4 , and at 6 month postoperative period in adobe photoshop ( 7.0 ) software ( adobe systems for windows and os x ) .
the digitalization of the iopa radiographs was carried out using scanner ( lexmark international corporation manufacturer for laser scanner and printers ) and similarly densitometric analysis was done using densitometer ( the little genius densitometer # 07 - 444 , cardinal health ) with standard protocol .
point d and d - points is in similar fashion but in the distal aspect of the third molar extraction site as shown in figure 1 .
, armonk , ny , usa . all results were calculated using the mean value and standard deviation for each of the parameters considered and checked for statistical significance using student unpaired t - test .
the differences in the results of pd , abh , and bone density at 2 , 4 , and 6 months were compared between each group using student unpaired t - test .
patient venous blood was collected and subjected for centrifugation then 2 ml of conventional prp ( cprp ) was treated with 60 l of citrate inhibitor sterile 10% calcium chloride solution and allowed to coagulate . then , squeezed to release the platelet - derived thrombin and collected in serum rich plasma
. then , autologous thrombin rich plasma was mixed with cprp in the 1:4 ratios and allowed to coagulate .
armamentarium for platelet rich plasma and platelet rich fibrin preparation concentrated platelet rich plasma platelet - rich plasma gel after addition of cacl2 and autologus thrombin
armamentarium required for prf is same as that of prp except citrate phosphate dextrose adenine ( cpda ) anticoagulant solution and 10% cacl2 . around 5 ml of whole venous blood
was collected in each of two sterile vacutainer tube of 6 ml capacity without anticoagulant .
the vacutainers were placed in the centrifuge at 3000 rpm for 10 min after which we obtained red lower fraction containing red blood cells , upper straw colored cellular plasma , and the fibrin clot was obtained in the middle .
the straw colored upper layer was removed , and middle layer with fraction 2 mm below dividing line was collected which will be the prf . in group
i , the extraction socket was closed primarily without prp and prf in the socket . in group
iia , prp in the mandibular left extraction socket and in group iib mandibular right extraction socket 2 - 3 ml of prf was placed .
the extraction socket evaluated on 1 , 2 , and 7 postoperative day for any wound dehiscence and recorded as present / absent . probing depth ( pd ) measured using michigan o probe with william 's markings from " free gingival margin ( fgm ) " to the bottom of the pocket in nearest mm .
the average of pd was recorded at the three different positions , i.e. distobuccal ( db ) , distal ( d ) , distolingual ( dl ) on the distal surface of second molar taken preoperatively , 1 , 2 , 4 , and 6 month postoperatively .
alveolar bone height ( abh ) measured distal to second molar was measured with a caliper from cementoenamel junction ( cej ) to the alveolar crest on intraoral periapical radiographs ( iopa ) at 1 , 2 , 4 , and 6 month postoperatively .
bone density was measured on direct iopa using densitometric analysis device [ figures 1 and 5 ] pre- and post - operatively at 1 , 2 , 4 , and 6 month .
a = free gingival margin , b = cementoenamel junction , c = depth of the pocket , d = alveolar crest the bone density calculated using below formula : where , point a = point at alveolar crest just mesial to third molar .
point a = point just mesial to third molar at the level of apical third .
point b and b = points in similar fashion but distal to mandibular third molar .
where , point c = mesial point in the third molar socket at the alveolar crest level .
point c - mesial point in the third molar socket at the level of apical third .
mean gray level histogram values of the digitalized iopa images of third molar extraction socket of both control and study groups were calculated at preoperative , 2 , 4 , and at 6 month postoperative period in adobe photoshop ( 7.0 ) software ( adobe systems for windows and os x ) .
the digitalization of the iopa radiographs was carried out using scanner ( lexmark international corporation manufacturer for laser scanner and printers ) and similarly densitometric analysis was done using densitometer ( the little genius densitometer # 07 - 444 , cardinal health ) with standard protocol .
point d and d - points is in similar fashion but in the distal aspect of the third molar extraction site as shown in figure 1 .
ibm corp . , armonk , ny , usa . all results were calculated using the mean value and standard deviation for each of the parameters considered and checked for statistical significance using student unpaired t - test .
the differences in the results of pd , abh , and bone density at 2 , 4 , and 6 months were compared between each group using student unpaired t - test .
the extraction socket evaluated on 1 , 2 , and 7 postoperative day for any wound dehiscence and recorded as present / absent .
probing depth ( pd ) measured using michigan o probe with william 's markings from " free gingival margin ( fgm ) " to the bottom of the pocket in nearest mm .
the average of pd was recorded at the three different positions , i.e. distobuccal ( db ) , distal ( d ) , distolingual ( dl ) on the distal surface of second molar taken preoperatively , 1 , 2 , 4 , and 6 month postoperatively .
alveolar bone height ( abh ) measured distal to second molar was measured with a caliper from cementoenamel junction ( cej ) to the alveolar crest on intraoral periapical radiographs ( iopa ) at 1 , 2 , 4 , and 6 month postoperatively .
bone density was measured on direct iopa using densitometric analysis device [ figures 1 and 5 ] pre- and post - operatively at 1 , 2 , 4 , and 6 month .
a = free gingival margin , b = cementoenamel junction , c = depth of the pocket , d = alveolar crest the bone density calculated using below formula : where , point a = point at alveolar crest just mesial to third molar .
point a = point just mesial to third molar at the level of apical third .
point b and b = points in similar fashion but distal to mandibular third molar .
where , point c = mesial point in the third molar socket at the alveolar crest level .
point c - mesial point in the third molar socket at the level of apical third .
mean gray level histogram values of the digitalized iopa images of third molar extraction socket of both control and study groups were calculated at preoperative , 2 , 4 , and at 6 month postoperative period in adobe photoshop ( 7.0 ) software ( adobe systems for windows and os x ) .
the digitalization of the iopa radiographs was carried out using scanner ( lexmark international corporation manufacturer for laser scanner and printers ) and similarly densitometric analysis was done using densitometer ( the little genius densitometer # 07 - 444 , cardinal health ) with standard protocol .
point d and d - points is in similar fashion but in the distal aspect of the third molar extraction site as shown in figure 1 .
ibm corp . , armonk , ny , usa . all results were calculated using the mean value and standard deviation for each of the parameters considered and checked for statistical significance using student unpaired t - test .
the differences in the results of pd , abh , and bone density at 2 , 4 , and 6 months were compared between each group using student unpaired t - test .
the results were evaluated based on clinical observation , radiographic analysis , and densitometric analysis of the recorded data .
table 1 , shows dehiscence in 5 ( 33.3% ) out of 15 cases , group iia showed dehiscence in 3 ( 20% ) cases , whereas group iib did not show any sign of wound dehiscence . the decrease in pd , corresponding to the distal surface of the mandibular second molar is seen in all the three groups [ table 1 ] .
we observed that the difference in the decrease in abh at 2 , 4 , and 6 months postoperatively in the group iib was statistically significant [ table 2 ] .
mean and sd values of probing depth ( pd ) ( fgm to bottom of the pocket , in mm ) in group i , ii ( a ) and ii ( b ) mean and sd values of alveolar bone height ( abh ) distal to 2 molar ( cej to alveolar crest , in mm ) in group i , ii ( a ) and ii ( b ) in group iia and group iib , there was a highly significant difference in bone density of the bone adjacent to the extraction socket and the bone formed in the extraction socket at 2 , 4 , and 6 months postoperatively when compared with group i. but group iia and groupiib showed no significant difference in bone density of the bone adjacent to the extraction socket and the bone formed in the extraction socket at 2 and 4 months and at 6 month postoperatively [ table 3 ] . mean and sd values of bone density on iopa ( in pixels ) in group i , ii ( a ) and ii ( b ) :
growth factors are a realistic way to improve and expedite both soft tissue and bony wound healing .
all these growth factors increase the population of wound healing cells and recruits other angiogenic growth factors to the wound site .
it is , therefore , a reasonable hypothesis that increasing the concentration of platelets in bone defects may lead to improved , faster healing .
extraction of impacted mandibular third molars causes multiple periodontal defects at the distal root of the second molar .
there are various augmentation procedures performed at the time of tooth extraction to maintain or enhance ridge form for prosthetic reconstruction , periodontal health or implant placement .
one of the most recent and innovative technique which has come up is the use of prp .
the use of prp has shown to be a valid technique for promoting bone regeneration at the distal surface of the mandibular second molar following extraction of impacted third molars .
kazemi and fakhrjou has referred that prp could have a positive influence also on cartilage repair .
kim et al . concluded that addition of prp , prf and concentrated growth factor had significantly increased bone formation at the 6 week .
the prp is activated to form prp gel thus causing degranulation of -granules present in the platelets and releasing the growth factors .
have reported the possibility of hypersensitivity to bovine thrombin which may cause antigenic reactions in many individuals . in our technique , autologous thrombin was prepared and was mixed with prp to form an autologous platelet gel .
this platelet gel was free of eliciting any antigen - antibody reaction as it was prepared from patients own blood .
the anticoagulant used in our study was cpda since it has been observed that ethylenediaminetetraacetic acid is potentially more harmful than cpda . in our study ,
the abh distal to second molar was 2.77 2.430 at the end of 6 months [ table 1 ] .
these results did not show a significant periodontal breakdown in terms of pd and abh distal to second molar . in the present study , a significant decrease in the pd
was observed in all the three groups at 2 , 4 , and 6 months postoperatively , but the greatest decrease was present in group iib [ table 2 ] where the extraction sockets were treated with prf .
observed a notable reduction in pd and improvement in the probing attachment level distal to second molars at 12 and 18 weeks in those extraction cases treated with prp compared with controls where prp was not used .
measurement of the abh was performed periodically at 2 , 4 and 6 month postoperatively , and it revealed no significant difference between mean values of abh distal to second molar ( cej to alveolar crest , in mm ) at preoperative and 2 months , but it is significant at 4 months and 6 months when compared with group i and group iia , with group i and group iib and with group iia and group iib which concludes that group iia and group iib significant abh at 4 and 6 months when compared to group i. overall group iib shows significant abh at 4 and 6 months when compared to group i and group iia [ table 2 ] .
mean values of bone density on iopa ( in pixels ) was highly significant at 2 months , 4 months and 6 months when compared with group i and group iia and with group i and group iib .
however , there was no significant difference between mean values of bone density on iopa ( in pixels ) at preoperative , 2 months , 4 months , and 6 months when compared with group iia and group iib .
similarly , it was highly significant at and 2 months , 4 months , and 6 months when compared with group i and group iia and with group i and group iib
. however , there was no significant difference between mean values of bone density on iopa ( with densitometer ) at preoperative , 2 months , 4 months , and 6 months when compared with group iia and group iib .
it shows new bone formation by prp and prf and encouraging results when compared to control group [ table 3 ] .
yelamali and saikrishna found better bony density with prf compared prp after 4 months follow up .
similary martnez et al . stated that various pdgfs ( ppp , prr and prf ) helps in promotion of wound healing and angiogenesis both in vitro and in vivo .
the limitation of this study was that the 6 months postoperative follow - up is a short duration to comment on the significance of periodontal breakdown distal to second molar .
however , this duration of time was adequate enough to evaluate the effects of prp and prf in initiating and enhancing both hard and soft tissue healing
. in future extra time , extra resources , and long term studies need for good results .
thisstudy indicates a definite improvement in the periodontal health distal to second molar after third molar surgery in cases treated with prf as compared to the prp group and control group .
the procedure of prf preparation is simple , quick , and cost effective as compared to prp , but prp and prf have demonstrated good results and both can be incorporated as an adjunct to promote wound healing and osseous regeneration in human mandibular third molar extraction sites .
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### Summary:
: aim and objectives : this study attempted the evaluation of the efficacy of platelet - rich plasma ( prp ) and platelet - rich fibrin ( prf ) in alveolar defects after removal of bilateral mandibular third molars.materials and methods : a total of 30 patients reporting to department of oral and maxillofacial surgery and having bilateral mandibular third molar impaction in both male and female aged between 18 and 30 years were included in this study .
prf and prp were placed in extraction site and recalled at 2nd , 4th , and 6th month postoperatively .
data were statistically analyzed using ibm spss software for windows , version 19.0 .
ibm corp .
, armonk , ny , usa.results:this study showed decreased probing depth in prf group compared to prp and control one .
this signifies a better soft tissue healing of extraction sockets with prf as compared to the prp and the control group and increase in the bone density highlights the use of prp and prf certainly as a valid method in inducing hard tissue regeneration.conclusion:this study indicates a definite improvement in the periodontal health distal to second molar after third molar surgery in cases treated with prf as compared to the prp group and control group .
hence , prp and prf can be incorporated as an adjunct to promote wound healing and osseous regeneration in mandibular third molar extraction sites .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: sebaceous glands generally arise in close association with hair follicles to form the pilosebaceous apparatus , but they are also found independently .
sebaceous glands are unevenly distributed over the body surface , being most numerous on the face and scalp , less numerous over the thorax and abdomen , and sparest on the extremities .
ectopic sebaceous glands , consisting of small yellow nodules of the lips and oral cavity ( fordyce 's spots ) , were first described in 1896 .
many otherwise healthy persons were found to have fordyce 's spots , so that they are no longer considered a rarity or a disease entity .
then the lesions have been recognized in many tissues of ectodermal origin including salivary glands , prepuce and vulva . in the esophagus , which is derived from endoderm ,
ectopic sebaceous glands in the esophagus were first reported in autopsy cases microscopically , and the lesions were observed grossly in one autopsy case .
recently , this condition diagnosed by esophageal endoscopy and biopsy has been reported in several living individuals [ 5 , 6 ] .
we report a case with ectopic sebaceous glands diagnosed by endoscopy and biopsy , and with yearly follow - up endoscopy for 3 years .
there are few reports with detailed follow - up endoscopy over several years [ 5 , 6 , 7 ] .
an asymptomatic 56-year - old japanese man visited our hospital in may 2008 for a routine medical examination , including upper gastrointestinal endoscopy .
blood tests showed a slightly elevated total cholesterol level of 263 mg / dl , although other tests were within normal limits .
endoscopic examination revealed more than 100 yellowish plaques 120 mm in diameter scattered over the mucosal surface of the middle and lower thoracic esophagus ( fig .
the smaller lesions had a fine , granular appearance and the larger lesions had lobulated margins , giving them a flower - like appearance .
biopsy specimens stained with hematoxylin - eosin ( h&e ) showed sebaceous glands associated with lymphocytic infiltration and an excretory duct , and overlying squamous epithelium ( fig .
repeat endoscopic examination at 1 and 2 years revealed that the lesions had decrease slightly in size or number compared with the first examination ( fig .
endoscopic examination at 3 years revealed that the lesions had increased slightly in size or number compared with the preceding two examinations , to resemble the first endoscopy ( fig .
several cases diagnosed by endoscopy and biopsy have been reported [ 5 , 6 ] .
the apparently low incidence of this condition may be due to the lack of obvious clinical signs and symptoms .
most cases were discovered incidentally by endoscopy during a routine medical examination or at an annual follow - up examination .
the patient was asymptomatic , and he was a smoker and a social drinker with mild hyperlipidemia .
there is very limited evidence in the literature to support a possible pathogenic role of smoking , drinking or hyperlipidemia in this condition [ 5 , 7 ] .
. showed that patients with this condition typically have < 10 lesions and that lesions are typically > 1 mm in size .
wei et al . indicated that the number of lesions varied from 1 to more than 100 in one patient , with a diameter of 120 mm .
several studies have indicated that the lesions may have several different shapes , such as elevated form and lobulated plaque form [ 9 , 10 ] . the lesions were found predominantly in the upper thoracic esophagus , or mostly in the middle and lower thoracic esophagus [ 8 , 9 , 10 ] . in our case , the patient had more than 100 lesions of lobulated plaque form measuring 120 mm in diameter in the middle and lower thoracic esophagus .
further reported cases with a longitudinal distribution of lesions and postulated that the distribution of sebaceous glands in the esophagus may be significant .
however , there are little data to support this theory to date . in our patient ,
the factors affecting the pattern and distribution of sebaceous glands in the esophagus require further study .
there are few reports of these lesions that include detailed follow - up endoscopic findings over several years [ 5 , 6 , 7 ] .
follow - up examinations are recommended at intervals of 612 months . in most cases reported to date
, follow - up did not show any change in the size or number of lesions .
no malignant change arising from such ectopic lesions has been reported , suggesting a favorable prognosis .
the lesions fluctuated over the 3 years , but there were no obvious overall changes in size or number and no treatment was needed .
whether sebaceous glands in the esophagus are truly ectopic or metaplastic is a point of greatest interest , as the esophagus originates mostly from the endodermal layer .
however , this condition has not been described in children , even in large necropsy studies .
sebaceous glands have been found in salivary glands and have been attributed to metaplasia ( acquired differentiation ) .
it would therefore not be surprising for some of the numerous esophageal gland lobules to undergo sebaceous metaplasia . in the present case
, there were no obvious changes but some fluctuations in appearance on endoscopic examination over 3 years .
it is unknown whether these endoscopic findings participate with the origin of sebaceous glands in the esophagus .
however , further research is needed to clarify the mechanisms underlying the development of ectopic sebaceous glands in the esophagus .
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### Summary:
: sebaceous glands in the esophagus are rare and are of particular interest because of their as yet unknown origin .
we report a case with ectopic sebaceous glands diagnosed by esophageal endoscopy and biopsy , with follow - up endoscopic examinations for 3 years .
few cases with follow - up endoscopic findings have been reported . in our case
, there were no significant overall changes during 3 years of follow - up , but the lesions fluctuated over time .
while taking the endoscopic findings of the present or past cases into account , we discuss the possible pathogenic mechanisms of this condition .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: to study about the correlation between the glycemic status and increase in fetal abdominal circumference in gestational diabetes patients and its relationship with fetal birth weight .
seventy - five gestational diabetes mellitus ( gdm ) patients were taken up for study with duly informed consent and suggested for anthropometry profile and glycemic profile with hba1c .
seventy - five gestational diabetic mothers who have attended a secondary level diabetic clinic and on regular follow - up were included in the study .
pre - gdm mothers , patients with co - morbid disease were excluded from the study .
indian women have an 11-fold increased risk of developing glucose tolerance during pregnancy and thereby increasing incidence of fetal macrosomia .
the placenta is a highly potent endocrine organ producing steroid and protein hormones , therefore strongly influences maternal carbohydrate metabolism .
permanent glucose oversupply to the fetus stimulates the fetal pancreatic islet cells to increase insulin production and it gradually induces their hypertrophy and hyperplasia .
the mean blood glucose in normal pregnancy is 5.0 - 5.6 mmol / l ( 90 - 100 mg / dl ) . the post - prandial blood glucose level in pregnancy elevated from 7.2 to 7.8 mmol / l ( 130 - 140 mg / dl ) due to the result of placental anti - insulin hormones .
glucose tolerance improves in normal early pregnancy due to the effect of human chorionic gonadotropin ; however , there is a progressive decrease in glucose tolerance after the 20 gestational week associated with placental anti - insulin hormones .
insulin secretion is increased during normal pregnancy with gestational age . however , the effect of insulin is enhanced by insulinotropic hormones before the 20 gestational week , but is decreased thereafter by the effect of anti - insulin hormones .
thus , the glucose homeostasis is changed in the direction of diabetes mellitus in normal pregnancy , therefore glucose tolerance gradually deteriorates for which pregnancy is often called as diabetogenic .
the definitions include birth weight greater than 4000 - 4500 g or greater than 90% for the newborn adjusted for race , sex , and gestational age .
identification of these at - risk pregnancies may allow intervention to reduce the risk , to provide appropriate counseling , and to implement appropriate plans for monitoring and follow - up care during pregnancy and after delivery .
maternal diabetes is one of the strongest risk factors associated with giving birth to an infant that is considered large for gestational age .
pre - gestational and gestational diabetes result in fetal macrosomia in as many as 50% of pregnancies complicated by gestational diabetes and in 40% of those complicated by type 1 diabetes mellitus .
studies of macrosomic infants of diabetic mothers reveal a greater amount of total body fat , thicker upper - extremity skin fold measurements , and smaller ratios of head to abdominal circumference than macrosomic infants of non - diabetic mothers.maternal weight prior to pregnancy can affect the weight of the fetus .
women who are obese are more likely to have larger infants.excessive weight gain in pregnancy is a risk factor for macrosomia .
the risk is greater for women with obesity than for women without obesity.gestational age is associated with macrosomia .
birth weight increases as gestational age increases . prolonged pregnancies ( > 41 weeks ) are associated with an increased incidence of macrosomia .
macrosomic infants account for about 1% of term deliveries and 3 - 10% of post - term deliveries.multiparity and grand multiparity increase the risk of macrosomia .
parity has been reported to be associated with 100 - 150 g of weight gain at birth.a history of macrosomia can influence future pregnancies .
women who previously delivered a macrosomic fetus are 5 - 10 times more likely than women without such a history to deliver a baby considered large for gestational age , the next time they become pregnant.fetal sex influences macrosomic potential .
recent studies have confirmed this association.excessive amniotic fluid defined as greater than or equal to 60 percentile for gestational age has recently been associated with macrosomia.despite these so - called risk factors for macrosomia , much of the variation in birth weights remains unexplained .
maternal diabetes is one of the strongest risk factors associated with giving birth to an infant that is considered large for gestational age .
pre - gestational and gestational diabetes result in fetal macrosomia in as many as 50% of pregnancies complicated by gestational diabetes and in 40% of those complicated by type 1 diabetes mellitus .
studies of macrosomic infants of diabetic mothers reveal a greater amount of total body fat , thicker upper - extremity skin fold measurements , and smaller ratios of head to abdominal circumference than macrosomic infants of non - diabetic mothers
prolonged pregnancies ( > 41 weeks ) are associated with an increased incidence of macrosomia .
macrosomic infants account for about 1% of term deliveries and 3 - 10% of post - term deliveries .
parity has been reported to be associated with 100 - 150 g of weight gain at birth .
women who previously delivered a macrosomic fetus are 5 - 10 times more likely than women without such a history to deliver a baby considered large for gestational age , the next time they become pregnant .
excessive amniotic fluid defined as greater than or equal to 60 percentile for gestational age has recently been associated with macrosomia . despite these so - called risk factors for macrosomia ,
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### Summary:
: aim : to study about the correlation between the glycemic status and increase in fetal abdominal circumference in gestational diabetes patients and its relationship with fetal birth weight.materials and methods : seventy - five gestational diabetes mellitus ( gdm ) patients were taken up for study with duly informed consent and suggested for anthropometry profile and glycemic profile with hba1c .
fetal abdominal circumference was measured during routine scans .
the patients were followed up till delivery and the fetal birth was noted.inclusion criteria : seventy - five gestational diabetic mothers who have attended a secondary level diabetic clinic and on regular follow - up were included in the study.exclusion criteria : pre - gdm mothers , patients with co - morbid disease were excluded from the study.expected results : fetal abdominal circumference correlated well with fluctuating glycemic control and fetal birth weight .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: it is not unusual to find unexpected hypermetabolic foci as a result of scanning using whole - body positron emission / computed tomography ( pet / ct ) . known as incidentalomas ,
parotid gland incidentalomas ( pgis ) are defined as new focal intraglandular deposits of radiotracer in patients without prior history of parotid disease.1 these deposits are most commonly due to benign lesions such as warthin tumor ( wt),1
2
3
4
5 but they can also be caused by metastatic lymph nodes or malignant tumors.6
the parotid gland is the salivary gland that most commonly develops tumors , most of which are benign .
it presents as a slow - growing mass in the tail of the gland , and is usually asymptomatic .
it generally occurs between the sixth and seventh decades of life , predominantly in men .
it has a strong association with smoking , which is therefore considered a risk factor for its development.7
8
9
10 wt can be multicentric or bilateral , and it rarely becomes malignant . to our knowledge ,
only two previous studies have reported the prevalence of pgi , and both were performed in asia.2
11 furthermore , clinical features such as the role of smoking have not been considered in their etiology . the present study aimed to determine the prevalence of parotid incidentalomas in f - fluorodeoxyglucose ( f - fdg ) pet / ct and evaluate the presence of smoking in this group of patients .
we also reviewed the database of parotidectomies performed in our hospital to assess the relationship between wt and smoking .
we conducted a retrospective review of the reports of pet / ct performed in 4,250 patients in our hospital between june 2009 and february 2013 .
we identified 16 cases of parotid focal uptake in patients without known disease of the parotid glands .
the group consisted of 10 men and 6 women , 48 to 88 years of age .
in most cases , pet - ct was indicated as an extension study of diagnosed neoplasms or to confirm locoregional relapse after treatment . in two cases ,
we reviewed the history of smoking in the 16 cases : 13 had smoked or were current smokers .
we identified additional studies performed on the parotid masses , finding that only 5 patients underwent cytologic analysis by fine needle aspiration cytology ( fnac ) . in 8 of the 16 cases , the lesion was located in the right parotid gland , in 7 cases it was located on the left , and in 1 case it was bilateral .
we analyzed the database of parotidectomies performed in our department in the previous 10 years and focused on the pathologic diagnosis and the presence or absence of smoking in each case .
combined f - fdg whole - body pet / ct ( gemini tf , philips , amesterdam ) with 64-slice ct was performed .
standard patient preparation included : 6-hour fasting , hydration , and serum glucose level of less than 150 mg / dl before tracer injection .
patients were asked to rest quietly in supine position , and vesical evacuation was done before the acquisition of images .
low - dose ct and pet from the base of the skull to the proximal thighs were performed , with an additional acquisition of head and neck images .
an abnormal pet - ct finding was defined as a significant increased uptake , higher than that of the surrounding normal tissue .
we conducted a retrospective review of the reports of pet / ct performed in 4,250 patients in our hospital between june 2009 and february 2013 .
we identified 16 cases of parotid focal uptake in patients without known disease of the parotid glands .
the group consisted of 10 men and 6 women , 48 to 88 years of age .
in most cases , pet - ct was indicated as an extension study of diagnosed neoplasms or to confirm locoregional relapse after treatment . in two cases ,
we reviewed the history of smoking in the 16 cases : 13 had smoked or were current smokers .
we identified additional studies performed on the parotid masses , finding that only 5 patients underwent cytologic analysis by fine needle aspiration cytology ( fnac ) . in 8 of the 16 cases , the lesion was located in the right parotid gland , in 7 cases it was located on the left , and in 1 case it was bilateral .
we analyzed the database of parotidectomies performed in our department in the previous 10 years and focused on the pathologic diagnosis and the presence or absence of smoking in each case .
combined f - fdg whole - body pet / ct ( gemini tf , philips , amesterdam ) with 64-slice ct was performed .
standard patient preparation included : 6-hour fasting , hydration , and serum glucose level of less than 150 mg / dl before tracer injection .
patients were asked to rest quietly in supine position , and vesical evacuation was done before the acquisition of images .
low - dose ct and pet from the base of the skull to the proximal thighs were performed , with an additional acquisition of head and neck images .
an abnormal pet - ct finding was defined as a significant increased uptake , higher than that of the surrounding normal tissue .
we found pgis in 16 patients who underwent pet / ct between june 2009 and february 2013 , corresponding to 0.4% of patients undergoing pet / ct .
the incidentalomas were more frequent in men ( 62.5% , 10 cases ) , and the average age at diagnosis was 68 years .
the most common indication for the test was lung cancer ( 7 cases , corresponding to 43.75% ) , followed by breast cancer ( 3 cases ) , giant cell vasculitis ( 1 case each of horton disease and takayasu arteritis ) , and 1 case each of locally advanced oropharyngeal cancer , gallbladder carcinoma with liver metastasis , peritoneal carcinomatosis of gynecologic origin , and retroperitoneal lymphoma .
abbreviations : fnac , fine needle aspiration cytology ; pet / ct ; positron emission / computed tomography ; suvmax , standardized uptake value .
three of five masses had an inflammatory component without a malignant component , one was considered a mixed tumor suggestive of pleomorphic adenoma , and the other was inconclusive .
of the 16 patients , only the patient with the pleomorphic adenoma underwent surgery ; a superficial parotidectomy was performed and the histology was confirmed . in the remaining 11 cases ,
fnac was not performed but the mass was monitored by periodic clinical follow - up .
the average standardized uptake value of parotid tumors was 6.51 , ranging from 2.87 to 16.1 .
a review of the database of parotidectomies performed in our department revealed that 311 patients were surgically treated for parotid gland tumors in the previous 10 years and 80 cases ( 25.7% ) were identified as wt .
a history of smoking was identified in 93.8% of patients with wt ( 75 cases ) .
this percentage was high compared with the prevalence of smoking in pleomorphic adenomas , which was 38.8% .
our findings show that the prevalence of pgis on pet / ct in our institution is similar to that reported in the two previous studies in asia .
when analyzing the presence of cigarette smoking in our patients with pgis , we found it was a common factor in 81.25% of the patients . in our study , fnac was performed in five cases .
the remaining patients did not undergo additional testing on the pgi due to the advanced stage of the primary tumor , the absence of symptoms , or the unlikelihood of metastasis based on clinical and tomographic features . in the largest series published to date ,
wang et al found 58 pgis in 19,333 patients , representing a prevalence of 0.3%.11 they obtained histologic confirmation in 51 patients , 41 with benign lesions ( mainly represented by pleomorphic adenomas and wt ) and 10 with malignant tumors . in a smaller sample ,
horiuchi et al detected four cases of abnormal parotid fdg uptake in 1,872 pet scans , with an estimated prevalence of 0.2% ; all four cases were diagnosed with wt.2 other series of cases also found wt as the main cause of pgi.1
3
as pgis are likely benign lesions , metastasic disease should not be suspected initially without histopathologic confirmation , even in patients with known malignancy elsewhere.1
4
5 nevertheless , these lesions warrant further radiologic and histopathologic correlations .
ultrasonography and fnac are useful tools to characterize parotid masses , avoiding erroneous staging of primary malignancies .
the objective of cytologic diagnosis of parotid masses is the differential diagnosis between benign and malignant lesions.12 complex histopathology and heterogeneity of cellular patterns of salivary gland tumors explain why typing of primary salivary malignancies by fnac is a challenge .
the accuracy of fnac depends on the experience of the clinician who performs the procedure and the pathologist who evaluates cytologic material.12
we found the prevalence of smoking in parotid tumors surgically treated in our hospital was significantly higher in patients with wt than in patients with other tumors ( p < 0.001 ) . more than 90% of patients with wt were smokers , and cigarette use in patients with other benign and malignant neoplasms was lower , 38 and 55.4% , respectively .
several other authors have studied the relation between wt and smoking . according to their publications , between 80 and 94% of patients with wt smoke,7
8
9
13 and the risk for development of wt correlates with the level and duration of smoking.7
9 sadetzki et al found a remarkably high odds ratio ( 15.3 ) for developing wt in the presence of smoking.9
based on the high prevalence of wt as pgi on pet / ct and the strong relationship with smoking , we suggest diagnosis other than warthin tumor should be considered for pgi in nonsmokers .
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### Summary:
: introduction parotid gland incidentalomas ( pgis ) are unexpected hypermetabolic foci in the parotid region that can be found when scanning with whole - body positron emission / computed tomography ( pet / ct )
. these deposits are most commonly due to benign lesions such as warthin tumor .
objective the aim of this study was to determine the prevalence of pgis identified in pet / ct scans and to assess the role of smoking in their etiology .
methods we retrospectively reviewed all pet / ct scans performed at our center in search of pgis and identified smoking status and standardized uptake value ( suvmax ) in each case .
we also analyzed the database of parotidectomies performed in our department in the previous 10 years and focused on the pathologic diagnosis and the presence or absence of smoking in each case .
results sixteen cases of pgis were found in 4,250 pet / ct scans , accounting for 0.4% .
the average suvmax was 6.5 ( range 2.8 to 16 ) .
cytology was performed in five patients ; it was benign in four cases and inconclusive in one case .
thirteen patients had a history of smoking .
of the parotidectomies performed in our center with a diagnosis of warthin tumor , we identified a history of smoking in 93.8% of those patients .
conclusions the prevalence of pgis on pet / ct was similar to that reported by other authors . warthin tumor is frequently diagnosed among pgis on pet / ct , and it has a strong relationship with smoking .
we suggest that a diagnosis other than warthin tumor should be considered for pgis in nonsmokers .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: in genetic epidemiological research , both case - control and case - parent trio designs have been used widely to evaluate genetic susceptibilities to human complex diseases and markers , such as single - nucleotide polymorphisms ( snps ) , to localize disease gene variants [ 1 - 5 ] . the sample size for detecting associations between disease and snp markers
is known to be highly affected by disease prevalence , disease allele frequency , linkage disequilibrium ( ld ) , inheritance models ( e.g. , additive , dominant , and multiplicative models ) , and effect size of the genetic variants ( e.g. , odds ratio , relative risk , etc . ) [ 4 , 6 , 7 ] .
previous studies have shown that a population based case - control design can be more powerful than a family - based study design in identifying genes predisposing human complex traits , both for qualitative traits and for quantitative traits [ 8 - 11 ] . however , some studies reported that the case - parent design is much more powerful than the case - control design in evaluating genetic risk for common complex diseases , because case - control studies are susceptible to bias due to phenotype misclassification or population stratification [ 12 , 13 ] .
recently , genome - wide association studies ( gwass ) using thousands of cases and controls reported many susceptibility snps for 237 human traits by the end of june , 2011 ( www.genome.gov/gwastudies ) .
since a gwas evaluates hundreds of thousands of snp markers , it requires a much larger sample size to achieve an adequate statistical power [ 14 - 18 ] . in genetic association studies , the observed signal for association
is referred to be statistically significant if the p - value is less than a preset threshold value ( ) of 0.05 to reject a null hypothesis of genetic association .
testing a large number of snp markers leads to a large number of multiple comparisons and thus increases false positive rates .
either the bonferroni correction or the false discovery rate is generally applied to avoid false positive ( type i error ) rates . however , the bonferroni - corrected p - value , the significance threshold set to 0.05 divided by the total number of snps analyzed in a gwas , is too strict to consider the correlations among snp markers ( e.g. , p = 1 10 for 500,000 ( 500 k ) snps , p = 5 10 for 1 million ( m ) snps ) [ 16 , 19 ] . therefore , estimating a sufficient sample size to achieve adequate statistical power is critical in the design stage of genetic association [ 20 - 24 ] .
statistical power is the probability to reject a null hypothesis ( h0 ) while the alternative hypothesis ( ha ) is true .
although a researcher collects a large number of samples , all samples may not be necessary to be analyzed to detect evidence for association .
a large sample size improves the ability of disease prediction ; however , it is not cost - effective that a researcher genotypes more than the effective sample size .
unless researchers estimate sample size and statistical power at the research design stage , it leads to wasted time and resources to collect samples .
an effective sample size can be defined as the minimum number of samples that achieves adequate statistical power ( e.g. , 80% power ) . on the other hand ,
too small a sample size to detect true evidence for an association increases false negative rates and reduces the reliability of a study .
false negative rates are increased by multiple factors that cause systematic biases , and such biases reduce statistical power .
the statistical power of 80% is used widely to avoid false negative associations and to determine a cost - effective sample size in large - scale association studies [ 7 , 22 , 23 ] . however
, many researchers tend to overlook the importance of statistical power and sample size calculations . in this study
, we evaluated statistical power with increasing numbers of markers analyzed under various assumptions and compared the sample sizes required in case - control studies and case - parent studies .
we computed the effective sample size and statistical power using a web browser program , genetic power calculator developed by purcell et al .
we conducted power and sample size calculations under various assumptions about genetic models ( i.e. , allelic , additive , dominant , recessive , and co - dominant models ) , minor allele frequencies ( mafs ) , pair - wise ld , disease prevalence , case - to - control ratio , and number of snp markers ( i.e. , single snp , 500 k snps , and 1 m snps ) . the values tested for heterozygous odds ratio ( orhet ) were 1.3 , 1.5 , 2 , and 2.5 .
the power and sample sizes were calculated under different ranges of factors , such as maf of 5% , 10% , 20% , and 30% ; ld of 0.4 , 0.6 , 0.8 , and 1 ; disease prevalence of 0.01% , 0.1% , 5% , and 10% ; and case - to - control ratio of 1:1 , 1:2 , 1:3 , and 1:4 .
the bonferroni p - value that was specific to the number of snp makers tested was applied to cover 3 billion base pairs of the human genome ( i.e. , p = 0.05 for a single snp marker , p = 1 10 for 500 k snp markers , and p = 5 10 for 1 m snp markers ) .
we fixed the proper range of sample sizes from 100 to 2,000 cases , because the power is too low when the sample size is below 100 cases ( or trios ) , and the cost is too high to realistically collect samples when the sample size is above 2,000 [ 7 , 22 ] .
we calculated the sample size to achieve 80% statistical power according to the genetic models and the heterozygous ors using a single snp marker in a case - control study under the assumptions of 5% disease prevalence , 5% maf , complete ld , 1:1 case - to - control ratio , and 5% type i error rate ( ) ( table 1 ) .
the dominant model required the smallest sample size to achieve 80% power compared to other genetic models ( e.g. , 90 cases ) .
in contrast , the effective sample size to test a single snp under the recessive model was too large to collect with a limited budget , even if the homozygous or is greater than 4 ( e.g. , 1,536 cases ) .
it reveals difficulty in detecting a disease allele that follows a recessive mode of inheritance with a moderate sample size .
the sample size and statistical power for the allelic test in a case - control study under the different assumptions of orhet , maf , disease prevalence , ld , and case - to - control ratio by allowing a 5% type i error rate are shown in fig .
1a , a lower sample size was required to test allelic association for a single snp with a larger maf at the same risk of disease ( or ) under the assumptions of 5% disease prevalence , 5% , complete ld , and 1:1 case - to - control ratio .
the minimum number of cases decreased from 1,974 cases for a snp with a maf of 5% to 545 cases for a snp with a maf of 30% under the same assumption .
a high - risk allele showing a high or requires a smaller sample size to be detected under the same assumption .
while an allele with an or of 1.3 requires 1,974 cases and 1,974 controls to be significantly detected in a case - control study , a snp with an or of 2.5 can be detected in a study of 134 cases and 134 controls under the assumption of a maf of 5% , disease prevalence of 5% , type i error rate of 5% , and d ' of 1 ( fig .
the higher prevalence and the higher ld were associated with increased statistical power : for instance , as the ld increased from 0.4 to 0.6 , 0.8 , and 1 , the statistical power obtained from a study of 1,000 cases and 1,000 controls was obviously increased from 26.5% to 49.2% , 72.8% , and 88.4% , respectively , under the assumption of or 1.3 , 5% maf , 5% prevalence , and 5% level ( fig .
in addition , a 1:4 case - to - control ratio , which is the golden standard ratio for the numbers of cases and controls to be collected in a case - control study , showed the most effective sample size to achieve 80% statistical power . in many clinical settings ,
researchers are able to obtain more data from affected individuals than healthy individuals . on the other hand , there are more healthy participants than participants with a disease in a population - based study .
therefore , the minimum numbers of cases and controls required to achieve 80% statistical power depend on the study design . for a snp with an allelic or of 2 and 5% maf ,
127 cases and 508 controls are required in the case of a 1:4 case - control ratio , whereas 248 cases and 248 controls are required in the case of a 1:1 ratio to achieve 80% statistical power under the assumption of 5% prevalence , complete ld , and 5% level . in table 2
, we compared the number of cases to the number of case - parent trios to perform a case - control study and a study using case - parent trios by increasing the number of snps being analyzed .
genetic association studies with larger numbers of snp markers require a larger sample size to reduce false positive association due to testing multiple hypotheses .
the sample size required in a case - parent study is generally larger than that of a case - control study design .
for instance , 248 cases and 248 controls ( 496 individuals ) were required to detect a snp with an orhet of 2 and 5% maf in a case - control study , whereas 282 case - parent trios ( 846 individuals ) were required under the assumption of 5% disease prevalence and complete ld by allowing a 5% level . however , the sample sizes required in both study designs increase tremendously in a gwas . under the same assumptions as shown above , the number of samples increased from 248 cases for a single snp analysis to 1,206 cases and 1,255 cases for analyses of 500 k snps and 1 m snps , respectively , based on the threshold of p - value , calculated using a strict bonferroni correction for multiple hypotheses comparisons . the statistical power to test the same number of subjects was higher for the case - control design than for the case - parent trio design ( fig .
both designs of the case - control study and case - parent study are used widely in the field of genetic epidemiology for studying associations between genetic factors and the risk of disease . over the past 2 decades , there has been a steep increase in the number of genetic association studies , and these studies have successfully reported a number of gene variants associated with human complex diseases [ 1 , 4 , 5 , 28 ] .
recently , gwass , a new frontier in genetic epidemiology , have identified thousands of new gene variants related to human diseases .
the population - based studies with a large sample size have increased statistical power , which leads to smaller variance .
however , it requires too much money and takes too long to collect a sufficient number of samples , and these large - scale studies are more likely to be affected by systematic bias and noise [ 25 , 30 ] . in the current study , we demonstrated the effective sample sizes that are required to achieve 80% statistical power for a case - control study and case - parent study separately under various assumptions regarding effect size , maf , disease prevalence , ld , case - to - control ratio , and number of snps .
a lower sample size is required under the dominant model in any assumption , while the recessive model requires too many samples under the same assumptions to achieve adequate statistical power .
further , we confirmed that a lower sample size is required for testing more common snps with stronger effect sizes and increased ld between marker allele and disease allele .
a lower sample size is required to study a common disease than a rare disease .
the statistical power increases by increasing the number of controls per case ; however , a case - to - control ratio exceeding 1:4 does not yield a significant increase in statistical power . among the parameters tested , under the assumption of a high level of ld between a marker and disease variant ,
common variants are more informative than rare variants in ld - based indirect association studies on complex diseases .
it means that researchers can reduce the cost by choosing common variants to be genotyped at the design stage of an ld - based association study . in general
, the case - control study design is more powerful than the case - parent study design [ 8 , 10 ] .
since patients with family histories of the disease are more likely to inherit disease - predisposing alleles than patients without family histories of the disease , researchers can improve the statistical power by sampling patients with affected relatives and by comparing to controls without any family history in case - control association studies .
genome - wide case - control studies have been used to identify genetic variants that predispose to human disease with model assumptions for parameters , such as the inheritance model .
such studies are powerful in detecting common variants with moderate effect in the occurrence of a disease ; however , a study with a large number of snp markers by using 500 k or 1 million chips requires a large number of samples ( e.g. , thousands of cases and controls ) to achieve adequate statistical power
. a researcher can rarely successfully conduct a large - scale association study without collaboration using a high - throughput microarray chip , in which most embedded snps reveal a small effect size between 1.3 and 1.6 .
therefore , researchers who are planning a genetic association study must calculate the effective sample size and the statistical power in the design phase to perform a cost - effective study that reduces false negative and false positive test results .
although we could not cover all plausible conditions in study design , the estimates of sample size and statistical power that were computed under various assumptions in this study may be useful to determine the sample size in designing a population - based association study .
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### Summary:
: a sample size with sufficient statistical power is critical to the success of genetic association studies to detect causal genes of human complex diseases .
genome - wide association studies require much larger sample sizes to achieve an adequate statistical power .
we estimated the statistical power with increasing numbers of markers analyzed and compared the sample sizes that were required in case - control studies and case - parent studies .
we computed the effective sample size and statistical power using genetic power calculator .
an analysis using a larger number of markers requires a larger sample size . testing a single - nucleotide polymorphism ( snp )
marker requires 248 cases , while testing 500,000 snps and 1 million markers requires 1,206 cases and 1,255 cases , respectively , under the assumption of an odds ratio of 2 , 5% disease prevalence , 5% minor allele frequency , complete linkage disequilibrium ( ld ) , 1:1 case / control ratio , and a 5% error rate in an allelic test . under a dominant model , a smaller sample size
is required to achieve 80% power than other genetic models .
we found that a much lower sample size was required with a strong effect size , common snp , and increased ld .
in addition , studying a common disease in a case - control study of a 1:4 case - control ratio is one way to achieve higher statistical power .
we also found that case - parent studies require more samples than case - control studies .
although we have not covered all plausible cases in study design , the estimates of sample size and statistical power computed under various assumptions in this study may be useful to determine the sample size in designing a population - based genetic association study .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: a stroke is the rapidly developing loss of brain function due to a disturbance in the blood
supply to the brain .
this can be due to ischemia caused by blockage or due to a
hemorrhage1 .
after a stroke , motor ,
sensory , perceptual , or cognitive deficits may occur , and these impairments can have various
impacts on individual functioning through generation of disabilities and affect
rehabilitation potential2 .
stroke survivors have difficulty in balance and postural control for standing upright
because they are impaired by asymmetric posture , abnormal body imbalance , and deficit of
weight transfer3 .
asymmetric movement also
decreases ability to stand upright , disorients the body midline and space , and hinders
appropriate alignment between vertebrae , trunk rotation , selective movement between trunk
and extremities , anterior - posterior tilt of the pelvis during weight transfer , protective
reaction , and equilibrium reaction4 .
previous studies have demonstrated the particular importance of trunk control in stable
walking and decreasing falling risk in stroke patients5 , 6 .
in essence , it describes the
muscular control required around the lumbar spine for maintenance of functional stability .
the core has been described as a box , with the abdominals in the front , paraspinal and
gluteal muscles in the back , the diaphragm as the roof , and the pelvic floor and hip girdle
musculature as the bottom7 .
particular
attention has been paid to the core because it serves as a muscular corset that works as a
unit to stabilize the body and spine , with and without limb movement . in short ,
the core
serves as the center of the functional kinetic chain . in the world of alternative medicine ,
the core has been referred to as the powerhouse , the foundation or engine of all limb
movement8 . with regard to impaired trunk
control and poor balance ,
previous studies have advocated efficient neuromuscular control
for trunk stability and accurate trunk muscle recruitment patterns for control of spinal
load in relation to a given task and posture9 , 10 .
many recent studies have reported on core stability and its affect on athletes and patients
with low back pain8 , however , few studies
on the relationship between core stability and balance ability and gait in patients with
hemiplegia have been reported .
therefore , the purpose of this study was to examine the
effect of core stabilization exercise on dynamic balance and gait functions in stroke
patients .
a total of 16 stroke patients who voluntarily agreed to active participation were included
in this study .
the selection criteria were : 1 ) independent gait ability with or without
walking aid for a minimum of 15 m ; 2 ) a mini - mental state examination score greater than
24/3011 ; 3 ) adequate vision and hearing
for completion of the study protocol , as indicated by the ability to follow written and oral
instructions during screening ; and 4 ) the capacity to understand and follow instructions .
exclusion criteria were 1 ) a history of previous stroke or other neurologic diseases or
disorders ; 2 ) patients with pusher syndrome ( defined as leaning to the hemiparetic side and
giving resistance to any attempt at passive correction ) ; 3 ) terminal illness ; and 4 ) pain ,
limited motion , or weakness in the non - paretic lower extremity that affected performance of
daily activities ( by self - report ) .
the subjects were randomly divided into the core stabilization exercise group
( eight subjects ) and the control group ( eight subjects ) .
subjects in both groups
participated in a general training program for five sessions , 60 minutes per week , during a
period of four weeks .
subjects in the core stabilization exercise group practiced additional
core stabilization exercises for three sessions of 30 minutes per week , for a period of four
weeks .
general characteristics of the core stabilization exercise group and control group
are shown in table 1 . the core stabilization exercise consisted of three subparts , bed exercises , wedge
exercises , and ball exercises using a swiss ball .
first , the bed exercises without devices
consisted of bridge exercise , bridge exercise with legs crossed , bridge exercise with one
leg , curl - ups with straight reaching , curl - ups with diagonal reaching , bird dog exercise ,
and side bridge exercise .
second , the wedge exercises consisted of curl - ups with straight
reaching , curl - ups with diagonal reaching , and curl - ups with arms crossed . finally , the ball
exercises consisted of bridge exercise , bridge exercise to the side , bridge- ups , abdominal
curl - ups , bird dog exercise , and push - ups .
dynamic balance ability was measured using the timed up and go test ( tug ) .
subjects were
seated in a chair with armrests and then instructed to stand ( using the armrests , if
desired ) and walk as quickly and as safely as possible for a distance of 3 m. subjects then
turned around , returned to the chair , and sat down .
the time from the point at which their
spine left the back of the chair until they returned to that same position was recorded
using a stopwatch .
high intrarater ( icc=0.99 ) and interrater ( icc=0.99 )
reliability have been demonstrated using this measure12 .
the standard gaitrite walkway contained six sensor pads encapsulated in
a rolled - up carpet with an active area of 3.66 m in length and 0.61 m in width .
as the
subject walked along the walkway , the sensors captured each footfall as a function of time
and transferred the gathered information to a personal computer for processing of the raw
data into footfall patterns .
the gaitrite system was used to measure the spatiotemporal
parameters , including gait velocity , cadence , step length , and stride length13 .
the spss statistical package , version 18.0 , was used in performance of all statistical
analyses .
the paired
t - test was used to determine whether there were changes in balance and gait function between
before and after the training . the independent t - test
tug , timed up and go test . * p<0.05 differences in balance and gait function after exercise
the before and after tug scores for subjects in the core
stabilization exercise group showed a significant decrease , from 33.0618.39 sec to
27.6413.73 sec ( p=0.029 ) ; the control group showed no significant difference , ( from
30.3312.58 sec to 24.858.76 sec , p=0.057 ) .
gait parameters in the core stabilization exercise group showed significantly increased
gait velocity ( from 44.83 18.83 cm / s to 58.9118.21 cm / s , p=0.024 ) and cadence ( from
74.5513.85 steps / min to 84.0714.00 steps / min , p=0.041 ) , however , no significant increase
was observed in affected side step length ( from 35.9812.95 cm to 41.5410.58 cm , p=0.160 )
and stride length ( from 69.5121.99 cm to 87.7118.89 cm , p=0.075 ) . the only significant
difference observed between the core group and control group was in velocity ( p=0.039 ) .
the
control group did not show a significant increase in gait velocity ( from 37.69 11.03 cm / s
to 37.3910.11 cm / s ) and cadence ( from 75.9011.73 steps / min to 77.5110.68 steps / min ) ,
affected side step length ( from 29.495.25 cm to 30.928.05 cm ) , and stride length ( from
53.7716.59 cm to 58.4014.54 cm ) .
this study examined the effect of core stabilization exercise on dynamic balance and gait
functions of stroke patients . in this study ,
ranges of tug scores have been reported for various samples of elderly people . in a previous
study ,
men and women without known pathology , aged 70 to 84 years ( mean=75 years ) , had a
mean tug score of 8.50 seconds ( range=710)12 .
al.14
reported that conduction biofeedback and conventional physical therapy programs resulted in
a decrease in tug from 23.08 before participation in an exercise program to 14.62 after
participation in an exercise program . in our study , the before and after tug score for
subjects in the core stabilization exercise group showed a significant decrease , from
33.0618.39 sec to 27.6413.73 sec ( p=0.029 ) ; no significant difference ( from 30.3312.58
sec to 24.858.76 sec ) was observed in the control group ( p=0.057 ) .
core training presumably
improved the balance of the lumbo - pelvic - hip complex , corrected postural alignments , and
increased balance of the whole body . as a result ,
dynamic balance ability for transfer of
center of gravity ( cog ) showed gradual improvement15 .
the common features of walking after
stroke include decreased gait velocity and asymmetrical gait pattern17 , 18 .
achievement of
normal gait patterns and speed is usually the ultimate goal of gait training .
bohannon et
al.19 reported that mean comfortable
gait speed ranged from 127.2 cm / sec for women in their 70s to 146.2 cm / sec for men in their
40s .
mean maximum gait speed ranged from 174.9 cm / sec for women in their 70s to 253.3 cm / sec
for men in their 20s .
both gait speed measures were reliable ( coefficients0.903 ) and showed
significant correlation with age ( r0.210 ) , height ( r0.220 ) , and the strengths of lower
extremity muscle actions ( r=0.1900.500 ) .
holden et al.20 reported that the velocity of gait in hemiparetic subjects ( n=10 )
was 41% of normal .
duncan et al.21
investigated the effect of a home program aimed at improvement of endurance , balance , and
strength for stroke subjects whose mean duration after onset was 66 days .
after eight weeks ,
mean gait speed increased by 25 cm / sec among patients .
yang et al.22 studied dual task programs in stroke subjects and measured
the speed of 5 m of walking .
they found that gait speed showed a significant increase after
participation in the dual task program , from 86.52 cm / sec to 115.35 cm / sec in chronic
patients after stroke ( p<0.05 ) . in our study , the core stabilization exercise group
showed a significantly increased gait velocity ( from 44.8318.83 cm / s to 58.9118.21 cm / s ,
p=0.024 ) and cadence ( from 74.5513.85 steps / min to 84.0714.00 steps / min , p=0.041 ) , and the
only significant difference observed between the core group and control group was in
velocity ( p=0.039 ) .
these findings are consistent with those of previous studies and suggest
that core stabilization exercise increased posterior tilt of the pelvis and cog transfer
during the swing phase through core training .
lamoth et al.23 studied that trunk coordination has an effect on gait parameters
and that flexible adaptations in trunk coordination to changes in walking velocity are
considered a hallmark of unaffected gait . and as found for cadence , the core stabilization
exercise group showed a larger increased than the control group but there was no significant
difference between groups .
we suggest that core training might improve the stability of the
lower trunk and pelvis and result in increased ability with regard to static balance ,
dynamic balance , and weight support of the more affected side and ultimately may contribute
to a more stable gait . through this research ,
the core stabilization exercise was found to be effective in balance
and gait functions of stroke patients .
we expect that this core stabilization exercise will
be used at stroke patient care centers in physical therapy as an effective form of training
for balance and gait functions .
further research is needed in order to confirm the
generalization of these findings and to identify which stroke patients might benefit from
treadmill gait training .
|
### Summary:
: [ purpose ] the purpose of this study was to determine the effects of core stabilization
exercise on dynamic balance and gait function in stroke patients .
[ subjects ] the subjects
were 16 stroke patients , who were randomly divided into two groups : a core stabilization
exercise group of eight subjects and control group of eight subjects . [ methods ]
subjects
in both groups received general training five times per week .
subjects in the core
stabilization exercise group practiced an additional core stabilization exercise program ,
which was performed for 30 minutes , three times per week , during a period of four weeks .
all subjects were evaluated for dynamic balance ( timed up and go test , tug ) and gait
parameters ( velocity , cadence , step length , and stride length ) . [ results ] following
intervention , the core exercise group showed a significant change in tug , velocity , and
cadence . the only significant difference observed between the core group and control group
was in velocity .
[ conclusion ] the results of this study suggest the feasibility and
suitability of core stabilization exercise for stroke patients .
|
<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: esophageal adenocarcinoma ( eac ) is one of the rapidly rising cancers in the western world . despite improvements in multimodality treatment ,
the major risk factor for eac is the premalignant lesion barrett 's esophagus ( be ) , in addition to age , male gender , and caucasian ethnicity . as a consequence of gastro - esophageal reflux disease ( gerd )
, the normal squamous epithelium of the lower esophagus can be replaced by columnar intestinal cells , including goblet cells , representing be . per year 0.120.5% of the patients diagnosed with be will develop eac , following a multimorphological sequence , in which intestinal metaplasia evolves to low - grade dysplasia ( lgd ) , high - grade dysplasia ( hgd ) and ultimately to invasive adenocarcinoma .
the prevalence of be in the general population is estimated at 2% , and among patients with gerd even at 10% . since the prognosis of advanced eac is relatively poor , patients with be are subjected to intensive endoscopic surveillance with biopsy sampling to identify those patients with neoplastic progression at an early stage . however , because the annual risk of developing eac from be is relatively low , most be patients will not progress to cancer and do not benefit from this surveillance . it can be anticipated that genetic susceptibility could play a role in the initiation of eac in be patients . from this perspective the identification of single nucleotide polymorphisms ( snps ) , which identifies high - risk patients , could make the surveillance of be patients more cost - effective and could be helpful by diagnosing patients with eac in an early and curable stage and thereby increase the prognosis remarkably .
recently , the first genome - wide association study ( gwas ) on eac and the premalignant lesion be was published .
this study revealed three snps associated with eac : rs10419226 ( crtc1 ) , rs11789015 ( barx1 ) , and rs2687201 ( foxp1 ) .
in addition , evidence was found that rs9936833 ( foxf1 ) , previously associated with be , was also associated with eac and that the snps : rs2178146 and rs3111601 near rs9936833 had even a stronger association with eac .
rs9936833 was first identified in a gwas on be performed by su et al . in 2012 , simultaneously rs9257809 ( mhc ) was found to be associated with be .
the aim of the present study was to validate the association between these six previously identified snps and the risk of eac in an independent and large case
patients diagnosed with an adenocarcinoma of the distal esophagus or esophagogastric junction ( egj ) and treated at the department of surgery , erasmus mc cancer institute , university medical centre , rotterdam , between january 1996 and december 2013 ( n = 761 ) were selected for the study . in addition , patients treated in the academic medical centre at the university of amsterdam , between 1994 and 2004 were included as well ( n = 310 ) .
the control group was derived from the rotterdam study , a population - based prospective cohort study . in brief , this is an ongoing large population - based cohort study , which started in january 1990 .
all inhabitants , who were aged 55 years and older , living in ommoord , a district in rotterdam , the netherlands , were invited to participate . this population - based control group provided reference groups of allele frequencies , which reflect the local general european population .
the tissue samples were obtained from the resection specimens and used according to the code of proper secondary use of human tissue in the netherlands established by the dutch federation of medical scientific societies ( http://www.federa.org ) .
nonmalignant tissue from the resection specimen ; tumor negative lymph nodes or tumor negative resection margins , confirmed by an experienced gi - pathologist , were macro dissected from microscopic sections of fresh frozen - or formalin fixed paraffin embedded tissues .
a multiplex snapshot assay was designed ; multiplex polymerase chain reaction was used to amplify the regions of the snps : rs2178146 ( foxf1 ) , rs10419226 ( crtc1 ) , rs9936833 ( foxf1 ) , rs2687201 ( foxp1 ) , rs11789015 ( barx1 ) , and rs3111601 ( foxf1 ) ( hg19 ) .
the amplified fragments of the normal dnas were analyzed by snapshot using the abi prism snapshot multiplex kit ( life technologies , carlsbad , ca , usa ) .
data analysis was performed using genemarker analysis software version 2.4.0 ( softgenetics , state college , pa , usa ) [ figure 1 ] .
result of snapshot analysis design of primers and probes for controls , genome - wide snp genotyping was performed using infinium ii assay on the humanhap550 and human 660-quad genotyping beadchips ( illuminainc , san diego , ca , usa ) .
approximately 30 million snps were imputed using 1000 g phase 1 v3 populations as reference .
all variants tested here had an imputation quality of 0.9 or higher , suggesting near perfect imputation .
departures from hardy weinberg equilibrium were tested for using the goodness - of - fit test .
logistic regression analyses were used to calculate odds ratios ( or ) with 95% confidence intervals ( 95% ci ) .
the major allele homozygous was set as a reference and was compared with the minor allele .
p values were corrected for multiple testing using bonferroni correction ( p = 0.05/6 = 0.008 ) before considered significant .
logistic regression was performed with spss version 20.0 ( spss , chicago , il , usa ) and meta - analysis of the original gwas , and the current study was performed using r library rmeta .
patients diagnosed with an adenocarcinoma of the distal esophagus or esophagogastric junction ( egj ) and treated at the department of surgery , erasmus mc cancer institute , university medical centre , rotterdam , between january 1996 and december 2013 ( n = 761 ) were selected for the study . in addition , patients treated in the academic medical centre at the university of amsterdam , between 1994 and 2004 were included as well ( n = 310 ) .
the control group was derived from the rotterdam study , a population - based prospective cohort study . in brief , this is an ongoing large population - based cohort study , which started in january 1990 .
all inhabitants , who were aged 55 years and older , living in ommoord , a district in rotterdam , the netherlands , were invited to participate . this population - based control group provided reference groups of allele frequencies , which reflect the local general european population .
for cases , the tissue samples were obtained from the resection specimens and used according to the code of proper secondary use of human tissue in the netherlands established by the dutch federation of medical scientific societies ( http://www.federa.org ) .
nonmalignant tissue from the resection specimen ; tumor negative lymph nodes or tumor negative resection margins , confirmed by an experienced gi - pathologist , were macro dissected from microscopic sections of fresh frozen - or formalin fixed paraffin embedded tissues .
for cases , a multiplex snapshot assay was designed ; multiplex polymerase chain reaction was used to amplify the regions of the snps : rs2178146 ( foxf1 ) , rs10419226 ( crtc1 ) , rs9936833 ( foxf1 ) , rs2687201 ( foxp1 ) , rs11789015 ( barx1 ) , and rs3111601 ( foxf1 ) ( hg19 ) .
the amplified fragments of the normal dnas were analyzed by snapshot using the abi prism snapshot multiplex kit ( life technologies , carlsbad , ca , usa ) .
data analysis was performed using genemarker analysis software version 2.4.0 ( softgenetics , state college , pa , usa ) [ figure 1 ] .
result of snapshot analysis design of primers and probes for controls , genome - wide snp genotyping was performed using infinium ii assay on the humanhap550 and human 660-quad genotyping beadchips ( illuminainc , san diego , ca , usa ) .
approximately 30 million snps were imputed using 1000 g phase 1 v3 populations as reference .
all variants tested here had an imputation quality of 0.9 or higher , suggesting near perfect imputation .
departures from hardy weinberg equilibrium were tested for using the goodness - of - fit test .
logistic regression analyses were used to calculate odds ratios ( or ) with 95% confidence intervals ( 95% ci ) .
the major allele homozygous was set as a reference and was compared with the minor allele .
p values were corrected for multiple testing using bonferroni correction ( p = 0.05/6 = 0.008 ) before considered significant .
logistic regression was performed with spss version 20.0 ( spss , chicago , il , usa ) and meta - analysis of the original gwas , and the current study was performed using r library rmeta .
a total of 1071 cases were initially analyzed , due to technical failure of 972 cases reliable data were obtained that was compared with 6206 controls regarding the six previously mentioned snps .
clinical data were available of 550 cases from rotterdam . the median age of these patients was 63 years ( range : 1984 years ) and 80% was male .
almost half of the patients received some form of neoadjuvant therapy and all patients underwent esophagectomy .
ninety percent was diagnosed with an invasive adenocarcinoma , of which 35% arose clearly from be .
most tumors were located in the distal esophagus ( 41.3% ) or at the egj ( 40.5% ) .
the majority of the tumors were moderately or poorly differentiated ( 36.5% and 45.1% , respectively ) .
the most common pathological tumor stage ( pt ) was pt3 ( 53.8% ) and half of the patients appeared to have positive lymph nodes ( pn1 - 3 ) , whereas four patients had distant metastasis ( pm ) according to the tnm - classification of the american joint committee on cancer staging manual 7 edition .
after surgery , 40.4% of the cases developed recurrence of disease ( locoregional disease or distant metastasis ) .
the mean overall survival of the 550 cases was 53.8 months ( 95%ci : 49.558.2 ) and the 5-year overall survival was estimated at 38.2% .
the distribution of genotype frequencies for the investigated snps was consistent with hardy weinberg equilibrium ( p > 0.05 ) , except for foxf1 rs3111601 ( p = 0.013 ) .
the allelic association of the six snps with eac showed significantly increased risk for the minor allele of rs10419226 ( crtc1 ) ( or = 1.17 , p = 0.001 ) and significantly decreased risk for the minor allele of rs11789015 ( barx1 ) ( or = 0.85 , p = 0.004 ) .
none of the other four snps were significant in the currently studied population , although direction and effect size were consistent with previous gwas results .
the meta - analysis of the original gwas and the current study revealed more accurate effect estimate and improved level of significance for rs10419226 ( crtc1 ) ( or = 1.18 , p = 6.66 10 ) and rs11789015 ( barx1 ) ( or = 0.83 , p = 1.13 10 ) , and in addition for rs2178146 ( foxf1 ) ( or = 0.87 , p = 9.37 10 ) , while there was no significant allelic association with eac in the currently studied population [ table 1 ] . logistic regression and
meta - analysis genotypic association analysis showed a dose effect for the significantly associated snps .
the gt genotype for rs10419226 ( crtc1 ) increased the risk of eac in comparison with the gg genotype ( or = 1.07 , p = 0.428 ) , which became significant for the tt genotype ( or = 1.39 , p = 0.001 ) .
the rs11789015 ( barx1 ) ag genotype decreased the risk of eac in comparison with the aa genotype ( or = 0.95 , p = 0.456 ) , this decrease in risk became significant for the genotype gg ( or = 0.57 , p = 3.68 10 ) [ supplementary table 2 ] .
a total of 1071 cases were initially analyzed , due to technical failure of 972 cases reliable data were obtained that was compared with 6206 controls regarding the six previously mentioned snps .
clinical data were available of 550 cases from rotterdam . the median age of these patients was 63 years ( range : 1984 years ) and 80% was male .
almost half of the patients received some form of neoadjuvant therapy and all patients underwent esophagectomy .
ninety percent was diagnosed with an invasive adenocarcinoma , of which 35% arose clearly from be .
most tumors were located in the distal esophagus ( 41.3% ) or at the egj ( 40.5% ) .
the majority of the tumors were moderately or poorly differentiated ( 36.5% and 45.1% , respectively ) .
the most common pathological tumor stage ( pt ) was pt3 ( 53.8% ) and half of the patients appeared to have positive lymph nodes ( pn1 - 3 ) , whereas four patients had distant metastasis ( pm ) according to the tnm - classification of the american joint committee on cancer staging manual 7 edition .
after surgery , 40.4% of the cases developed recurrence of disease ( locoregional disease or distant metastasis ) .
the mean overall survival of the 550 cases was 53.8 months ( 95%ci : 49.558.2 ) and the 5-year overall survival was estimated at 38.2% .
the distribution of genotype frequencies for the investigated snps was consistent with hardy weinberg equilibrium ( p > 0.05 ) , except for foxf1 rs3111601 ( p = 0.013 ) .
the allelic association of the six snps with eac showed significantly increased risk for the minor allele of rs10419226 ( crtc1 ) ( or = 1.17 , p = 0.001 ) and significantly decreased risk for the minor allele of rs11789015 ( barx1 ) ( or = 0.85 , p = 0.004 ) .
none of the other four snps were significant in the currently studied population , although direction and effect size were consistent with previous gwas results .
the meta - analysis of the original gwas and the current study revealed more accurate effect estimate and improved level of significance for rs10419226 ( crtc1 ) ( or = 1.18 , p = 6.66 10 ) and rs11789015 ( barx1 ) ( or = 0.83 , p = 1.13 10 ) , and in addition for rs2178146 ( foxf1 ) ( or = 0.87 , p = 9.37 10 ) , while there was no significant allelic association with eac in the currently studied population [ table 1 ] . logistic regression and meta - analysis
the gt genotype for rs10419226 ( crtc1 ) increased the risk of eac in comparison with the gg genotype ( or = 1.07 , p = 0.428 ) , which became significant for the tt genotype ( or = 1.39 , p = 0.001 ) .
the rs11789015 ( barx1 ) ag genotype decreased the risk of eac in comparison with the aa genotype ( or = 0.95 , p = 0.456 ) , this decrease in risk became significant for the genotype gg ( or = 0.57 , p = 3.68 10 ) [ supplementary table 2 ] .
in this study two snps , rs10419226 ( crtc1 ) and rs11789015 ( barx1 ) , were replicated to be associated with eac . in the performed meta - analysis including the data of the eac cohort from the original gwas ,
the level of significance was improved compared with the original findings of the gwas , confirming the association of crtc1 and barx1 with eac .
control study revealed a significantly increased risk of eac for the minor allele t of rs10419226 ( crtc1 ) and a significantly decreased risk of eac for the minor allele g of rs11789015 ( barx1 ) .
both snps showed a dose - effect in the genotypic analysis ; two minor alleles gave a stronger effect than one minor allele .
in addition , rs2178146 ( foxf1 ) showed an improved level of significance in the meta - analysis while it did not reach significance in the allelic analysis of the present study cohort .
this could be explained by a smaller sample size compared to the population used for the original gwas resulting in a decreased power to detect the association .
however , because all cases were retrieved clinically , and controls with eac were excluded , no attenuation had taken place .
only two of the six previously identified snps , appeared to be significantly associated with eac in the current study , however , a consistency of the direction of effect and effect size was seen for all variants , suggesting that all these variants may play a role in eac .
rs10419226 is an intronic variant in the crtc1 gene , which is encoding for creb - regulated transcription co - activator that has been found previously to be associated with the oncogenic activity .
the down - regulation or loss of lkb1 , a tumor suppressor kinase , activates crtc1 signaling and the transcriptional activity of the downstream targets of crtc1 .
in addition , altered lkb1/crtc1 signaling has been demonstrated to induce a migratory and invasive phenotype in esophageal cancer cell lines .
the homolog of barx1 has been found to be associated with the differentiation of the esophagus and trachea in developing mouse embryos and in addition to be associated with the down - regulation of the wnt pathway in stomach morphogenesis and differentiation . identifying these snps
associated with eac suggests that genetic susceptibility might play a role in the initiation of eac and could be of importance for the surveillance of be patients . since the prevalence of be patients
is valued at 2% in the general population , and the annual risk of developing eac from be is estimated at 0.120.5% , most patients with be will not benefit from endoscopy surveillance .
however because of the relatively poor prognosis of eac , it is of utmost importance to diagnose eac patients in an early and curable stage of the disease .
therefore , it could be of additional value to incorporate snps associated with eac in the surveillance program of be , in order to only select the high - risk patients for developing eac .
control study replicated the association of rs10419226 ( crtc1 ) and rs11789015 ( barx1 ) with the risk of eac .
these findings indicate a possible genetic contribution to the development of eac and might contribute to gain more insight in the etiology of this cancer .
in addition , snps associated with eac could be helpful by identifying patients at increased risk for malignant progression during surveillance and/or screening programs aimed to improving the survival of these patients by diagnosing eac in an early and curable stage .
anna m.j . van nistelrooij : departments of pathology and surgery , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
van der korput : department of pathology , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
mark i. van berge henegouwen : department of surgery , academic medical centre , amsterdam , the netherlands .
carel j. van noesel : department of pathology , academic medical centre , amsterdam , the netherlands .
katharina biermann : department of pathology , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
spaander : department of gastroenterology and hepatology , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
hugo w. tilanus : department of surgery , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
j. jan b. van lanschot : department of surgery , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
albert hofman : department of epidemiology , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
andr g. uitterlinden : departments of internal medicine and epidemiology , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
wijnhoven : department of surgery , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
dinjens : department of pathology , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
ronald van marion : department of pathology , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
linda broer : department of internal medicine , erasmus mc cancer institute , university medical centre rotterdam , the netherlands .
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### Summary:
: objective : recently , single nucleotide polymorphisms ( snps ) associated with esophageal adenocarcinoma ( eac ) and barrett 's esophagus ( be ) were identified ; rs10419226 ( crtc1 ) , rs11789015 ( barx1 ) , rs2687201 ( foxp1 ) , rs2178146 ( foxf1 ) , rs3111601 ( foxf1 ) , and rs9936833 ( foxf1 ) .
these findings indicate that genetic susceptibility could play a role in the initiation of eac in be patients .
the aim of this study was to validate the association between these previously identified snps and the risk of eac in an independent and large case
control study.design:six snps found to be associated with eac and be were genotyped by a multiplex snapshot analysis in 1071 eac patients diagnosed and treated in the netherlands .
allele frequencies were compared to a control group derived from the rotterdam study , a population - based prospective cohort study ( n = 6206 ) . logistic regression analysis and meta - analysis
were performed to calculate odds ratios ( or).results : rs10419226 ( crtc1 ) showed a significantly increased eac risk for the minor allele ( or = 1.17 , p = 0.001 ) , and rs11789015 ( barx1 ) showed a significantly decreased risk for the minor allele ( or = 0.85 , p = 0.004 ) in the logistic regression analysis .
the meta - analysis of the original gwas and the current study revealed an improved level of significance for rs10419226 ( crtc1 ) ( or = 1.18 , p = 6.66 1010 ) and rs11789015 ( barx1 ) ( or = 0.83 , p = 1.13 108).conclusions : this independent and large dutch case
control study confirms the association of rs10419226 ( crtc1 ) and rs11789015 ( barx1 ) with the risk of eac .
these findings suggest a contribution of the patient genetic make - up to the development of eac and might contribute to gain more insight in the etiology of this cancer .
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: cross - sectional surveys over more than 20 years have demonstrated hyperactive sympathetic nervous system ( sns ) responses in approximately 9000 blacks ( africans ) from the north - west province in south africa .
sns responses and prevalence of lifestyle - related diseases were apparent in urban - dwelling africans , which underscored the urban environment as a potent chronic psychosocial stressor .
other studies support the role of sns hyperactivity or early stages of neurogenic hypertension associated with cardiometabolic risk markers such as disturbed vascular blood pressure responses , stress hormones , inflammation , fibrinolysis , structural wall changes in the left ventricle and sympathovagal disturbance .
thus the process of chronic exposure to psychosocial stress will result in high allostatic load or overload , which may be detrimental to cardiometabolic health .
it is possible that prolonged , uncontrollable psychosocial stress impairs stress appraisal or coping ability at both physiological and psychological levels , inducing neural fatigue . despite improved medical treatment regimens , the emerging burden of cardiometabolic disease among urban black africans urgently needed an innovative approach . to date ,
experimental trials of hypertension treatment or prospective population studies have not attempted to explain the role of hyperactive sns or neural response pathways ( brain ) contributing to the cardiometabolic ( heart ) disease burden in africans .
therefore , an innovative brain - heart link approach motivated the design of the international metabolic syndrome institute award - winning sabpa project .
our objectives were to conduct the first psychophysiological prospective cohort study in sub - saharan africa , addressing ( i ) the brain - heart link and ( ii ) neural response pathways to describe plausible mechanisms for cardiometabolic morbidity and mortality in a bi - ethnic cohort including both sexes .
all teachers ( n = 2170 ) , enrolled in the 43 schools of the dr kenneth kaunda education district ( klerksdorp and potchefstroom ) , north - west province south africa , were invited to participate ( figures 12 ) . to control for seasonal variation of the pro - coagulation markers ,
ambulatory autonomic dysfunction and cortisol data were used to obtain relevant effect sizes based on differences in biological profiles and genotyping hypothalamic - pituitary - adrenal ( hpa ) axis variation .
resulting sample sizes of 50416 would enable explanation of biological differences and detection of single nucleotide polymorphisms ( snps ) with a statistical power of 0.8 , and level of significance of 0.05 .
figure 1.geographical location of klerksdorp and potchefstroom in the north - west province , south africa .
figure 2.the sympathetic activity and ambulatory blood pressure in africans ( sabpa ) prospective cohort study population .
geographical location of klerksdorp and potchefstroom in the north - west province , south africa .
the sympathetic activity and ambulatory blood pressure in africans ( sabpa ) prospective cohort study population .
the target population included urban - dwelling well - educated black ( african ) and white african ( caucasian ) male and female teachers , ensuring participants with similar socioeconomic standing ( figure 2 ) .
all volunteering teachers had medical aid benefits and were screened to meet study eligibility criteria during the recruitment phase ( figure 2 ) .
those complying formed the respondent group of 409 ( figure 2 ) , and those not complying formed the non - respondent group ( n = 62 ) ( table 1 ) .
the black teachers preferred to be informed and recruited in separate sex groups and the protocol , especially the amount of blood drawn and hair sampling , was not well received .
time constraints were the main obstacle for participation in the caucasian teachers cohort and mixed - sex informed recruitment sessions were not a problem .
data are currently available for 409 teachers of phase i from which 359 were followed up in phase ii .
table 1.comparing respondents and non - respondents in terms of basic variablesvariablesrespondents ( n = 409)non - respondents ( n = 62)difference ( 95% ci)p - value ( t-/x tests)socio - demographic profile at baseline age , years44.70 9.646.39 9.21.69 ( 4.250.87)0.195 sex , men , n ( % ) 202 ( 49.4)10 ( 16.1)33.3 ( 20.246.3)<0.0001 africans , n ( % ) 200 ( 48.9)15 ( 24.2)24.7 ( 11.537.9)0.0003 teachers , grades 17 , n ( % ) 181 ( 44.3)39 ( 62.9)18.6 ( 5.331.9)0.006self - reported anthropometric measurements body mass index ( kg / m)28.85 6.630.26 5.01.41 ( 3.130.31)0.108self - reported comorbidities at baseline cardiovascular disease , n ( % ) 44 ( 10.8)21 ( 33.9)23.1 ( 14.132.1)<0.0001 hypertension drugs , n ( % ) 96 ( 23.5)27 ( 43.6)20.1 ( 8.431.7)0.0008 diagnosed diabetics , n ( % ) 12 ( 2.9)8 ( 12.9)10.0 ( 4.615.3)0.0003 epilepsy , n ( % ) 3 ( 0.73)6 ( 9.68)9.0 ( 5.412.5)<0.0001 antidepressant drugs , n ( % ) 4 ( 1.0)33 ( 53.2)52.2 ( 46.857.7)<0.0001 blood donor , n ( % ) 0 ( 0.0)6 ( 9.7)9.7 ( 6.812.6)<0.0001values are expressed as arithmetic mean or number of participants ( % ) .
differences are expressed as absolute values ( 95% ci ) . comparing respondents and non - respondents in terms of basic variables values
the sabpa study abode by the institutional guidelines and terms of the declaration of helsinki ( revised 2004 ) and was approved by the ethics review board of the north - west university , potchefstroom campus ( nwu-0003607s6 ) .
consent and cooperation agreement were obtained from the department of education north - west , south african democratic teacher unions and headmasters of schools .
the nature , benefits and risks of the study were explained in detail in english and native language to the involved parties and teachers , and their written informed consent was obtained before participation .
recruitment was systematically done over a 3-month period prior to clinical assessments during phases 1 and 2 . a registered nurse and a trained african fieldworker provided volunteers with a detailed description of planned measurements , the protocol and expected outcomes with an opportunity to ask questions .
participants medical history and treatment information were screened for eligibility ( 21.71% of 2170 invited teachers ) and , on approval , for inclusion in the main study .
late autumn 2008 and 2009 , and 409 participants completed phase i ( figure 2 ) .
n = 2170 ) , including 44% of all primary school teachers ( grades 1 to 7 ) participated in the sabpa study ( figure 2 , table 1 ) .
the differences almost throughout could be attributed to the tightly controlled lower comorbidities required for the respondent group .
follow - up ( phase 2 ) data collection was completed after 3 years in 201112 .
contact between baseline and 3-year follow - up was sustained via mobile cellular messaging system and feedback workshops to explain results and referrals .
the participants were encouraged to follow medical prescribed treatment regimens resulting from the study s clinical assessments .
three months prior to 3 years after phase 1 , all respondents who completed phase i were invited to take part in the follow - up ( figure 1 ) .
the non - respondents ( n = 62 ) were encouraged to enter the service delivery programme of the hypertension research and training clinic ( on campus ) for ambulatory blood pressure screening .
respondents are registered teachers , allowing a trained employee and the principal investigator to keep track of the mortality rates of the respondent group only , in collaboration with the north - west departments of education and internal affairs .
the data were collected in a well - controlled overnight facility at the north - west university in order to habituate participants to their environment , to avoid the white - coat effect , while maintaining high data quality .
well - controlled conditions were maintained in assessing optimal sns responses during exposure to acute mental laboratory stressors .
individualized health profile reports and referrals were received by each respondent within 1 week after data collection . despite the intense clinical assessment programme
, feedback reports indicated that participants were comfortable with the experimental setting and 97.8% expressed their interest for future participation .
seasonal variations were avoided and gold - standard methods were applied at both phases , i.e. validated questionnaires , measurements and calibrated apparatus ( figure 3 ; table 2 ) .
repeat measurements were collected at both phases 1 and 2 of the brain - heart link measures [ hpa and sympathy - adrenal - medullary axis ( sam ) , renin - angiotensin - aldosterone system ( raas ) , ambulatory blood pressure and electrocardiogram ( ecg ) , and objectively assessed lifestyle ( figure 4 ) , neurotrophins , renal function , metabolism , pro - inflammatory , pro - thrombotic , functional and structural endothelium ] .
psychophysiological stress tasks , namely the cold pressor and the stroop word - colour conflict ( stroop ) tests were administered at phase i and were applied for 1 min in a counterbalanced design . for blood sampling ,
a sterile winged infusion set was left in situ with a heparin block to prevent clotting ( 0.5 ml of heparin sodium - fresenius 5000 iu / ml in 50 ml normal saline solution ; fresenius kabi , port elizabeth , south africa ) .
the infusion set was thoroughly flushed with 23 ml saline and the first 2 ml of blood discarded , before sampling was done for coagulation measures . for each task ,
beat - to - beat blood pressure and 12-lead ecg were assessed throughout stress testing followed by a 10-min recovery period .
blood ( total 150 ml ) and saliva sampling ( total 6 ml ) was done at rest and at 10 and 30 min post - task , respectively .
participants received a monetary incentive according to performance during completion of the stroop task as motivation to excel , and perception of stressors was scored .
additional measurements at phase 2 included 24-h diet , 24-h urine , 7-day objective physical activity status , differentiated blood cell count , genetic damage , microvascular ( retinal ) endothelial function under mydriatic conditions , and chronic stress ( hair samples ) ( table 2 ) .
figure 3.mean temperature over the 4-month data collection periods of the sabpa prospective cohort study during late summer - late autumn .
figure 4.obtaining anthropometric ( 4a ) and 24h ambulatory blood pressure and 24h ecg ( including heart rate variability ) measures ( 4b ) during the sabpa prospective cohort study data collection phases .
table 2.summary of brain - heart clinical assessments and apparatusassessments200809201112medical history & related questionnaires: duration of stay , education , marital status , alive family members , health ( cardiometabolic , inflammation , depression , renal , arthritis , cancer , reproduction ) , sleep apnoea , ambulatory & dietary diary , mental stress perceptionfeedback of results ( incl .
referrals and incident findings): individual & groupchronic medications nervous system , kidney , hypertension , arrhythmia , diabetes , coagulants , hormones , inflammation , analgesics , depression / anxiety , proton pump , epilepsy , supplementslifestyle objective & self - reported smoking & alcohol habits diet , standardized dinner & breakfast 24-h anthropometry ( height , body mass , waist and neck circumferences) number of times measured ( invicta stadiometer ip 1465 , precision health scale , a & d company holtain 7-mm wide metal tape)33 objective total energy expenditure ( actical mini mitter , bend or ; 7-days actiheart , camntech ltd) 7-day changes in lifestyle habits over timepsychosocial battery measures with known heritability : life orientation , personalityx predictors of development / worsening of hypertension : coping , depression , cognitive distress measures which could moderate the effects of environment : fortitude , mental health , self - regulation , job stresscardiovascular assessment resting bp & 12-lead ecg ( riester ce 0124 & 1.3 m littman ii s.e . stethoscope 2205 ; finometer , finapres medical systems ; norav pc - ecg 1200) 24-h ambulatory bp & -ecg ( cardiotens & cardiovisions 1.19 , meditech)biochemical assessments ( trained technicians ; accredited laboratories ) bodily fluids sampling times ( urine , blood , saliva) serum brain - derived neurotrophic factor serum cotinine ( smoking ) and liver enzymes ( alcohol misuse) differentiated blood count serum electrolytes serum oxidative stress citrate haemostasis plasma inflammatory profile, plasma essential amino acids plasma renin - angiotensin - aldosterone system , endothelial function ( l - arginine - no synthase) urine / blood / saliva hpa & sam axis hormones , serotonin hair whole blood hiv status ( pmc medical , daman , india ) , pareekshak test ( bhat bio - tech , bangalore , india)retinal vessel analysisx static & dynamic measures ( incl .
dynamic saliva stress hormone responses ) ( dva plus 12100003 , imedos)intraocular eye pressurex ( tono - pen - avia applanation tonometer ce 0120 , reichert)ultrasound carotid intima - media thickness ( cimt) ( sonosite micromaxx , sonosite inc . , bothell , wa)dna genome ( pcr based ) nuclear & mitochondrial genome sequencing snp analyses , epigenetics telomere lengthmetabolism serum lipogram & insulin , naf glucose , urine metabonomicsrenal function 24-h serum creatinine & 8-h urinex , no measures.including sympathetic nervous system ( sns ) responses ( 1-min exposure to mental stress ( cold pressor & stroop colour - word - conflict tests).cimt , plaque score and stenosis at optimal angles obtained from baseline .
mean temperature over the 4-month data collection periods of the sabpa prospective cohort study during late summer - late autumn .
obtaining anthropometric ( 4a ) and 24h ambulatory blood pressure and 24h ecg ( including heart rate variability ) measures ( 4b ) during the sabpa prospective cohort study data collection phases .
summary of brain - heart clinical assessments and apparatus including sympathetic nervous system ( sns ) responses ( 1-min exposure to mental stress ( cold pressor & stroop colour - word - conflict tests ) .
a complete list of collaborators and publications from the sabpa study can be found at http://stressed - project.co.za / data - dissemination/. the overarching aim of the study was to assess the interaction between brain and cardiometabolic responses ( brain - heart link ) and the neural response pathways in a bi - ethnic cohort including both sexes in order to provide estimates of the proportion of individuals at high risk of future vascular events .
we have demonstrated that objective lifestyle risk factors , such as alcohol misuse , mostly explained differences in blood pressure , the metabolic syndrome and sub - clinical vascular disease between black and white south africans . among black africans we observed disturbed sympathovagal balance suggestive of sympathetic activity modulation , augmented -adrenergic vascular , pro - coagulant , attenuated nitric oxide metabolite , stress hormone and emotional distress responses .
diminished - adrenergic responsiveness was previously suspected in urban africans and is again demonstrated by the increased 24-h bp , augmented -adrenergic responses and associated vascular hypertrophy in the sabpa cohort . moreover ,
our findings complement a notion of autonomic dysfunction via depressed heart rate variability and early structural vascular changes in african men , mostly facilitated by hypervigilant defensive coping .
these structural vascular changes could counteract sympathetic vasoconstriction and contributed to their stress - induced ischaemia .
this will thus impair neuronal re - uptake of norepinephrine , thereby potentiating sympathetic signalling .
indeed , reduced perfusion of the heart was observed with a higher prevalence of silent ischaemic events in hypervigilant defensive coping african men , especially if low testosterone levels prevail , i.e. 9.5 events [ 95% confidence interval ( ci ) : 5.1 - 14.0 ] vs 1.5 ( 0.0 - 5.3 ) when compared with caucasian counterparts .
overall , cardiometabolic vulnerability was more evident in african men compared with african women and caucasian men and women , particularly when utilizing defensive coping .
fight , in - control , positive affect and eliciting - adrenergic responses , whereas emotional avoidance coping relates to defeat ,
flight or loss - of - control responses , which have been associated with poorer well - being and an -adrenergic response profile .
however , our findings contradict this notion as african men reported behavioural defensive coping in - control responses coupled with physiological emotional avoidance loss - of - control responses . indeed
, they revealed more metabolic syndrome markers , attenuated stress hormone levels and autonomic dysfunction .
an apparent dissociation evidently occurs between behavioural and physiological defensive coping responses suggesting loss - of - physiological control. it could further imply that an overly taxing situation or challenging environment , where chronic stress is experienced , may mask control .
another urgent matter , at the heart of the rising metabolic syndrome epidemic , was the lack of ethnic - specific cut - points for central obesity in prospective studies .
we addressed this crucial matter and an ethnic - specific waist circumference ( wc ) cut - point model for africans was proposed .
furthermore , supporting the brain - heart link , we revealed that the validated ethnic - specific wc cut - point model ( african men , 90 cm ; -women , 98 cm ) was associated with cognitive emotional distress and sub - clinical atherosclerosis .
preliminary causal analyses are progressing and thus far receiver operating characteristic ( roc ) wc cut - point models strongly support the use of only wc and blood pressure as predictors of the metabolic syndrome .
mcnemar 's case - control test was used to calculate change in risk ( unpublished , figure 5 ) .
we demonstrated no change in risk for behavioural defensive coping responses { 4.21% change , p = 0.147 [ odds ratio ( or ) 1.39 , 95% ci : 0.90 - 2.15]}. pertaining to physiological responses , we observed a decreased trend for autonomic dysfunction [ 5.38% , p = 0.051 ( or 0.64 , 95% ci : 0.40 - 1.00 ) ] , but no change in risk for a hypertensive state [ 3.62% , p = 0.313 ( or 0.71 , 95% ci : 0.43 - 1.14 ) ] or usage of hypertension drugs [ 1.95% , p = 0.313 ( or 1.22 , 95% ci : 0.74 - 2.01 ) ] . increased risk was however revealed for waist circumference [ 8.64% , p < 0.0001 ( or 3.58 , 95% ci : 1.86 - 7.47 ) ] and type 2 diabetes ( 6.98% , p <
findings underscore chronic behavioural defensive in - control responses , which were accompanied by physiological pathological changes , possibly indicating loss - of - control responses .
the ongoing challenge will be to describe neural response pathway mechanisms for cardiometabolic morbidity and mortality from a dissociative stress perspective .
figure 5.cardiometabolic risk markers presenting change in risk over 3 years in the sabpa prospective cohort .
heart rate variability ( hrv ) suggests increased autonomic dysfunction ( standard deviation r - r interval , 50 - 100 ms ) ; waist circumference , joint interim statement .
cardiometabolic risk markers presenting change in risk over 3 years in the sabpa prospective cohort .
heart rate variability ( hrv ) suggests increased autonomic dysfunction ( standard deviation r - r interval , 50 - 100 ms ) ; waist circumference , joint interim statement .
main weaknesses include the relatively small sample size , which was restricted in order to control for seasonal variation .
additionally , the requirement for an overnight stay and 2 days of testing likely may have had a considerable restrictive effect on the initial response rate .
key strengths are the generation of a unique highly phenotyped cohort in a well - controlled research setting . a complex set of gold - standard measures were obtained in the first psychophysiological prospective cohort study in sub - saharan africa .
the focus in this study is on sns activity contributing to cardiometabolic disease and emotional distress .
current findings address the original sabpa design and problem statement and prospective findings may confirm the american heart association 2014 statement that depression needs to be considered as a risk factor in preventive cardiology .
the cohort data and samples are embedded in a single large regional research centre at the study research institute of the hypertension in africa research team , north - west university ( potchefstroom campus ) , south africa .
data are password protected and electronic participant records are stored for a maximum of 20 years in the secured facility .
the electronic records have been flagged to facilitate ease of contact and follow - up .
therefore , in addition to the measures that were undertaken , it will be possible to access measurements undertaken at both phases 1 and 2 . to ensure maximum data dissemination , all national and international expert collaborators signed a contract ( memorandum of understanding ) for sharing of data and author input .
potential collaborators are invited to contact the corresponding author and principal investigator at [ leone.malan@nwu.ac.za ] .
this work was financially supported by : the metabolic syndrome institute france ; and the north - west university , medical research council , national research foundation , pa & alize malan trust , north - west department of education and roche diagnostics , south africa .
the funding organizations played no role in the design or conduct of the study ; collection , management , analysis and interpretation of the data ; or preparation , review or approval of the manuscript .
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### Summary:
: adapting to an over - demanding stressful urban environment may exhaust the psychophysiological resources to cope with these demands , and lead to sympathetic nervous system dysfunction .
the evidence that an urban - dwelling lifestyle may be detrimental to the cardiometabolic health of africans motivated the design of the sympathetic activity and ambulatory blood pressure in african prospective cohort study .
we aimed to determine neural mechanistic pathways involved in emotional distress and vascular remodelling .
the baseline sample included 409 teachers representing a bi - ethnic sex cohort from south africa .
the study was conducted in 200809 and repeated after 3-year follow - up in 201112 , with an 87.8% successful follow - up rate .
seasonal changes were avoided and extensive clinical assessments were performed in a well - controlled setting .
data collection included sociodemographics , lifestyle habits , psychosocial battery and genetic analysis , mental stress responses mimicking daily life stress ( blood pressure and haemostatic , cardiometabolic , endothelial and stress hormones ) .
target organ damage was assessed in the brain , heart , kidney , blood vessels and retina .
a unique highly phenotyped cohort is presented that can address the role of a hyperactive sympathetic nervous system and neural response pathways contributing to the burden of cardiometabolic diseases in africans .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: among the rare group of natural killer - cell ( nk - cell ) malignancies , there are two which primarily involve the peripheral blood and bone marrow : aggressive nk - cell leukemia ( ankl ) and chronic lymphoproliferative disorder of nk - cells ( clpdnk ) .
ankl is an ebv - associated tumor most prevalent among asian young adults ( median age of 42 years ) 1 .
it has a fulminant clinical course , presenting with fever and hepatosplenomegaly , complicated by multiple organ failure , coagulopathy and hemophagocytic syndrome , and frequently resulting in death within 2 months .
in contrast , clpdnk has no demonstrable association with ebv or racial predilection and tends to occur in older adults ( median age of 60 years ) 2 .
clinically , clpdnk is similar to t - cell large granular lymphocytic leukemia with most patients incidentally found to have cytopenia(s ) and/or a persistent increase in circulating mature nk - cells 3 .
given the stark contrast in the clinical course and management of ankl and clpdnk , accurately discriminating between these two entities is very important .
morphologically , the tumor cells from both diseases can be similar and indistinguishable from their normal counterpart large granular lymphocytes .
immunophenotypically , although they are cell surface cd3 ( scd3)-negative and cd2-positive , the published data regarding these rare entities suggest that they can be very different in other surface antigen expression .
ankl is thought to usually exhibit a cd56+/cd16/cd57 phenotype 1,46 , whereas clpdnk shows a cd56/cd16+/cd57 + phenotype 7,8 . here
, we report an unusual case of nk - cell neoplasm having an immunophenotype similar to that associated with ankl but with clinical and laboratory features typical of the much more indolent clpdnk .
the patient is a 65-year - old caucasian male with a 16-year history of leukopenia and neutropenia with his white blood count ( wbc ) varying from 1.8 10/l to 3.5 10/l and his absolute neutrophil count ( anc ) ranging 0.5 10/l to 0.7 10/l .
he was completely asymptomatic , denying fevers , night sweats , or unexplained weight loss and had no documented history of neutropenic infection . at the time of his follow - up ,
the wbc was 2.7 10/l with neutrophils 0.6 10/l and lymphocytes 2.1 10/l , hemoglobin 9.7 g / dl and platelets 127 10/l .
ultrasound revealed an enlarged spleen ( 16.2 15.8 9.0 cm ) without focal masses .
the peripheral blood smear showed an increase in granular lymphocytes with bland nuclei and abundant pale - staining cytoplasm containing fine to coarse azurophilic granules ( fig.1b ) .
no nuclear atypia , such as enlargement , folding , or open chromatin , was noted .
peripheral blood flow cytometric immunophenotyping revealed significantly increased nk - cells ( 84% of gated lymphoid events and 66% of total analyzed events ) that were cd2-positive , scd3-negative , and cd5-negative , with abnormally diminished cd7 expression ( fig.1a ) .
the neoplastic cells were cd56-positive , displayed aberrantly diminished cd16 , and lacked cd57 expression ( fig.1a ) .
further , they exhibited aberrantly uniform expression of cd94 and nkg2a , and a complete absence of expression of the killer cell immunoglobulin - like receptors ( kirs ) , cd158a , cd158b , and cd158e ( fig.1a ) .
hematoxylin and eosin - stained bone marrow biopsy showed a slight increase in interstitial lymphocytes ( 1015% of cellularity ) which by immunohistochemical studies were positive for cd3 ( fig.1c ) , granzyme b ( fig.1d ) and tia-1 ( fig.1e ) and distributed in an interstitial as well as an intrasinusoidal pattern . in situ hybridization for ebv - encoded rna ( eber ) was negative and no clonal t - cell receptor gene rearrangements were detected by peripheral blood pcr studies .
cytogenetic studies revealed a normal male karyotype . in conjunction with clinical and pathological features , a diagnosis of chronic lymphoproliferative disorder of nk cells
( a ) immunophenotying by flow cytometric analysis on peripheral blood showed a distinct population of surface cd3-negative nk - cells ( in red ) with partial loss of cd7 .
they were positive for cd56 , and negative for cd8 , cd16 , and cd57 .
they had uniform strong expression of cd94 and nkg2a and they completely lost the expression of kirs , such as cd158a , cd158b , and cd158e .
( b ) wright - giemsa - stained peripheral blood smear showed a population of lymphocytes with intermediate - sized reticulated chromatin and azurophilic granules in abundant amounts of pale cytoplasm ( original magnification 1000 ) .
( c e ) immunohistochemical studies on the bone marrow biopsy revealed a linear ( or intrasinusoidal / intravascular ) distributed neoplasm cells that were positive for cd3 ( c ) , granzyme b ( d ) and tia-1 ( e ) .
the diagnosis of nk - cell leukemia is challenging for practicing pathologists because nk - cells lack a singular lineage defining antigen and are phenotypically similar to normal cytotoxic t - cells .
for this reason , flow cytometric immunophenotyping is a primary laboratory tool for this purpose as it allows for the accurate identification of nk - cells through combined assessment of antigens which are expressed by these cells such as cd2 , cd7 , cd16 , and cd56 , and antigens which are not expressed such as scd3 and cd5 .
furthermore , flow cytometry is also critically important for determining if the nk - cells are leukemic as making this determination rests solely on the detection of phenotypic aberrancy , as nk - cells , by definition , do not harbor uniquely rearranged antigen receptor genes to serve as a marker of clonal cellular expansion .
the power of flow cytometry to detect and define nk - cell phenotypic aberrancy has been greatly enhanced by the analysis of a group of nk - cell receptors including the kir antigens cd158a , cd158b , and cd158e and also cd94/nkg2a heterodimers .
these utility of these assays in identifying nk - cell leukemia has been most extensively studied in clpdnk , where distinction of the leukemic nk - cells from normal nk - cell expansion is problematic due to the indolent nature of the condition .
the published literature indicates that clpdnk is typically brightly cd16-positive and cd57-positive ( majority of cases ) , a subset having diminished or absent expression of cd56 710 .
abnormal uniform expression of a single kir antigen , or abnormal absence of all kir antigens , is seen in essentially all clpdnk cases , and over one - half have abnormally uniform expression of cd94/nkg2a heterodimers 8,10,11 .
although these features are included in the who description of this provisional entity 12 , there are few large studies with complete clinical phenotyping and therefore these observations are based on collectively , fewer than 200 published cases .
aggressive nk - cell leukemia is believed to have a phenotype distinct from that of clpdnk .
much of the previous data regarding the phenotype of ankl are generated from immunohistochemical studies , which limits their utility in regards to comparison to flow cytometry 6 .
recently , comprehensive flow cytometric immunophenotyping studies for ankl from a japanese group of 22 cases 1 , european group of 12 cases 4 , and a chinese group of 29 cases 5 revealed that all cases have bright cd56 expression and lack cd57 expression .
the vast majority of cases do not express cd16 or cd8 46 , although some cases are cd16 positive 1 .
however , our understanding of the disease phenotype is only based on a very small number of published cases .
although cldpnk and ankl have dramatically different clinical manifestations , the paucity of data regarding the specificity of the disease phenotypes can be problematic from the laboratory standpoint , particularly in the reference laboratory setting where there may be little or no clinical information provided . because of this , it can be difficult to render a definitive pathologic diagnosis which guides the clinical management of the case .
this is illustrated by this case presented here as the leukemic nk - cells had a cd56+/cd16/cd57 , immunophenotype putatively of ankl .
nevertheless , the clinical , morphologic and pathologenetic ( ebv status ) were those of clpdnk ( table1 ) , treatment of which depends on the severity of clinical symptoms and cytopenias .
ankl is highly associated with ebv infection , and thus absence of ebv should make one question the diagnosis . given the indolent behavior and negative ebv infection ,
the diagnosis of chronic lymphoproliferative disorder of nk - cells was favored , despite the aggressive immunophenotype .
whether this will eventually evolve into an aggressive form is uncertain and long clinical follow - up is warranted .
immunophenotyping plays a key role in distinguishing ankl from cldpnk , however , it could not be used as sole criteria and clinical / laboratory findings are equally important .
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### Summary:
: key clinical messagedistinguishing chronic lymphoproliferative disorder of nk - cells from aggressive nk - cell leukemia is critical because they have distinct clinical course and management .
immunophenotyping plays a key role in distinguishing these two entities , however , it could not be used as sole criteria and clinical / laboratory findings are equally important .
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: c57bl/6j ( b6 ) male mice ( jackson laboratory ) were maintained on a high - fat diet ( hfd ) ( 60% fat , labdiet , 5001 ; richmond , in ) or a standard rat diet ( 10% fat ) for 3 months starting at 2 months of age .
male b6 ob / ob mice , b6.cg-nfkb1/j , and control littermates were purchased from jackson laboratories .
tlr2 , tlr4 , myd88 , and toll - interleukin-1 receptor ( tir ) domain containing adaptor protein inducing interferon- ( trif ) knockout male mice on a b6 background were provided by dr .
all of the animal experiments were performed under protocols approved by the university of alabama at birmingham . to isolate the elvs , visceral adipose tissue of 5-month - old mice
was washed with pbs and cut into small pieces < 4 mm , transferred to six - well plates containing 3 ml / well of dulbecco 's modified eagles medium ( invitrogen ) supplemented with 50 g / ml gentamicin and 10% fbs with bovine sera exosomes predepleted using a method as described previously ( 23 ) , and cultured in a 37c incubator in an atmosphere of 5% co2/95% air .
the cultured supernatants were used for elv purification , which was accomplished by differential centrifugation using a previously described method ( 24 ) .
concentrated elvs were analyzed using a hitachi h7000 electron microscope ( electronic instruments ) as previously described ( 24 ) .
thymus exosomes were isolated from male b6 mice using a method as described previously ( 25 ) .
pkh67 and pkh26 kits were used for labeling bone marrow derived macrophages ( bmdm ) according to the manufacturer 's instructions ( sigma ) .
bmdms from 7-day primary cultures of femoral bone marrow from 6- to 8-week - old female wild - type mice were generated as described previously ( 26 ) and detailed in the supplemental methods in the online appendix available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0216/dc1 .
the trafficking of injected macrophages was monitored by fluorescence - activated cell sorter ( facs ) analysis of labeled macrophages in adipose tissue , liver , spleen , and bone marrow . as a means to monitor the proliferation potential of injected macrophages , 24 h before mice were killed , 1 mg of bromodeoxyuridine ( brdu ; sigma ) , dissolved in 300 l pbs , was injected intraperitoneally with a second dose 3 h before the mice were killed .
the leukocytes were then isolated from each tissue including adipose , liver , spleen , and bone marrow using protocols as described previously ( 24 ) and stained with anti - brdu antibody .
once stained , the leukocytes were analyzed by facs using a protocol described previously ( 27 ) .
mice were injected intravenously with elvs ( 30 g / mouse ) every 3 days for 21 days .
after the last injection , glucose tolerance and insulin tolerance tests were performed using a method as described ( 7 ) and detailed in the supplemental methods .
c2c12 myocytes were purchased from american type culture collection and cultured in -minimal essential medium supplemented with 10% fbs .
once 80% confluency was attained , the c2c12 myocyte culture medium was replaced with conditioned media from bone marrow precursor cells that had been pretreated with wild - type ( wt)elvs or obelvs or thymus exosomes ( 10 g / ml ) for 14 days in the presence of granulocyte monocyte colony stimulating factor ( gm - csf ; 20 ng / ml ) . the effects of combined medium with or without addition of anti tnf- and anti il-6 antibodies on the [ h]glucose uptake of c2c12 myocytes was assessed as described ( 28 ) and detailed in the supplemental methods .
a recombinant mouse rbp4 ( r&d systems , catalog number 3476-lc ) was purchased and used for stimulating bmdms ( 1 10/100 l in rpmi160 medium ) .
twenty - four hours after the stimulation , the cell culture supernatants were harvested and assayed for tnf- and il-6 using an elisa . to determine the in vivo effects of a mouse rbp4 on the induction of il-6 and tnf- , wild - type b6 mice or tlr4 knockout b6 mice were injected intravenously with mouse rbp4 ( 250 g / mouse in 200 l of pbs ) .
six hours after the injection , serum levels of il-6 and tnf- were measured using a standard elisa . to determine whether the cells pretreated with recombinant mouse rbp4 respond to subsequent elv rbp4 stimulation ,
the bmdms treated with mouse rbp4 were washed with pbs 3 times and then cultured in the presence of wtelvs or obelvs ( 10 g / ml ) for an additional 24 h. tnf- and il-6 in the supernatants was quantified using an elisa .
the details of other methods used for this study have been published by this laboratory and are described in the supplemental methods .
statistical differences between groups were determined by anova with multiple comparisons using fischer 's post hoc analysis .
the student 's t test was used for comparisons when only two parameters were evaluated .
to isolate the elvs , visceral adipose tissue of 5-month - old mice was washed with pbs and cut into small pieces < 4 mm , transferred to six - well plates containing 3 ml / well of dulbecco 's modified eagles medium ( invitrogen ) supplemented with 50 g / ml gentamicin and 10% fbs with bovine sera exosomes predepleted using a method as described previously ( 23 ) , and cultured in a 37c incubator in an atmosphere of 5% co2/95% air .
the cultured supernatants were used for elv purification , which was accomplished by differential centrifugation using a previously described method ( 24 ) .
concentrated elvs were analyzed using a hitachi h7000 electron microscope ( electronic instruments ) as previously described ( 24 ) .
thymus exosomes were isolated from male b6 mice using a method as described previously ( 25 ) .
pkh67 and pkh26 kits were used for labeling bone marrow derived macrophages ( bmdm ) according to the manufacturer 's instructions ( sigma ) .
bmdms from 7-day primary cultures of femoral bone marrow from 6- to 8-week - old female wild - type mice were generated as described previously ( 26 ) and detailed in the supplemental methods in the online appendix available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0216/dc1 .
the trafficking of injected macrophages was monitored by fluorescence - activated cell sorter ( facs ) analysis of labeled macrophages in adipose tissue , liver , spleen , and bone marrow . as a means to monitor the proliferation potential of injected macrophages , 24 h before mice were killed , 1 mg of bromodeoxyuridine ( brdu ; sigma ) , dissolved in 300 l pbs , was injected intraperitoneally with a second dose 3 h before the mice were killed .
the leukocytes were then isolated from each tissue including adipose , liver , spleen , and bone marrow using protocols as described previously ( 24 ) and stained with anti - brdu antibody .
once stained , the leukocytes were analyzed by facs using a protocol described previously ( 27 ) .
mice were injected intravenously with elvs ( 30 g / mouse ) every 3 days for 21 days . after the last injection , glucose tolerance and insulin tolerance tests were performed using a method as described ( 7 ) and detailed in the supplemental methods .
c2c12 myocytes were purchased from american type culture collection and cultured in -minimal essential medium supplemented with 10% fbs .
once 80% confluency was attained , the c2c12 myocyte culture medium was replaced with conditioned media from bone marrow precursor cells that had been pretreated with wild - type ( wt)elvs or obelvs or thymus exosomes ( 10 g / ml ) for 14 days in the presence of granulocyte monocyte colony stimulating factor ( gm - csf ; 20 ng / ml ) . the effects of combined medium with or without addition of anti tnf- and anti il-6 antibodies on the [ h]glucose uptake of c2c12 myocytes was assessed as described ( 28 ) and detailed in the supplemental methods .
a recombinant mouse rbp4 ( r&d systems , catalog number 3476-lc ) was purchased and used for stimulating bmdms ( 1 10/100 l in rpmi160 medium ) .
twenty - four hours after the stimulation , the cell culture supernatants were harvested and assayed for tnf- and il-6 using an elisa . to determine the in vivo effects of a mouse rbp4 on the induction of il-6 and tnf- , wild - type b6 mice or tlr4 knockout b6 mice were injected intravenously with mouse rbp4 ( 250 g / mouse in 200 l of pbs ) .
six hours after the injection , serum levels of il-6 and tnf- were measured using a standard elisa . to determine whether the cells pretreated with recombinant mouse rbp4 respond to subsequent elv rbp4 stimulation ,
the bmdms treated with mouse rbp4 were washed with pbs 3 times and then cultured in the presence of wtelvs or obelvs ( 10 g / ml ) for an additional 24 h. tnf- and il-6 in the supernatants was quantified using an elisa .
the details of other methods used for this study have been published by this laboratory and are described in the supplemental methods .
statistical differences between groups were determined by anova with multiple comparisons using fischer 's post hoc analysis . the student 's t test was used for comparisons when only two parameters were evaluated .
others have demonstrated previously that exosomes are released by 3t3-l1 , a precursor adipocyte cell line ( 29,30 ) .
we found that more elvs are released in 30-min ex vivo adipose tissue cultures of age - matched wild - type ( b6 ) mice fed a hfd over 3 months ( hfdelvs , 16.5 1.2 g / g of adipose weight ) and of leptin - deficient ( ob / ob ) b6 mice ( obelvs , 14.4 1.1 g / g of adipose weight ) when compared to adipose tissue cultures of wild - type lean b6 mice ( wtelvs , 4.1 1.0 g / g of adipose weight ) .
the quantity of elvs released from adipose tissue increased over a period from 30 min to 6 h of ex vivo adipose tissue culture ( supplemental fig .
s1a ) , that is , 16.522.2 g / g hfdelvs ; 14.4 23.2 g / g obelvs ; and 4.16.7 g / g wtelvs .
electron microscopy examination revealed vesicles that measured 60100 nm in diameter and had a cup - shaped morphology ( supplemental fig .
s1b ) . neither calnexin nor lamp-1 were detectable when the elvs were analyzed by immunoblotting ( supplemental fig .
s1c ) , indicating that our elv preparations were free of contaminating nonexosome membrane proteins ( 31 ) .
further evidence that the vesicles were exosomes was obtained through analysis of the protein composition using linear ion trap mass spectrometery ( ltq lc / ms ) ( supplemental table s1 ) .
these analyses indicated a protein composition typical of exosomes derived from other cell types ( 3235 ) ( supplemental table s2 ) .
specific proteins were at undetectable levels in wt - elvs when compared with protein detected in obelvs and hfdelvs ( supplemental table s1 ) , but in both cases the elvs contained proteins known to be involved in cell metabolism , membrane trafficking , multiple small gtp - binding proteins , integral membrane proteins , and several class e vacuolar protein sorting proteins .
the fatty acid composition of the obelvs was almost exclusively palmitic acid ( n = 8 , 40.22 3.82% ) and stearic acid ( 52.71 4.42% ) ( supplemental table s3 ) . because there are minor differences in the protein composition of elvs released from b6 mice fed an hfd in comparison with obelvs ( supplemental table s1 ) , we focused on the b6 obelvs released from adipose tissues at 30-min ex vivo culture for the remainder of study because a larger amount of adipose tissue for isolation of elvs was available from ob / ob mice of the same age .
we determine whether obelvs released from adipose tissue are taken up by cells of the immune system .
we labeled obelvs with pkh67 dye before injecting them intravenously into b6 mice fed an hfd or standard diet for 3 months starting at 2 months of age .
facs analysis of single - cell suspensions of several tissues that had been harvested 24 h after the injection of obelvs indicated that more than 1.1% of total blood cells or liver leukocytes had taken up obelvs .
approximately 80% of the cells having taken up obelvs were cd11bf4/80 monocytes ( supplemental fig .
s2a and b ) , but no b cells , t cells , nor natural killer cells had taken up obelvs ( data not shown ) .
this result is unlikely because of pkh67 dye leakage from obelvs to the monocytes as no pkh67- positive macrophages were detected in the tissues of mice having been injected intravenously with free pkh67 dye ( data not shown ) .
facs analysis of the gated pkh67 cells isolated from peripheral blood further indicated that the monocytes ( cd11bf4/80pkh67 ) that had taken up the obelvs expressed higher levels of the monocyte receptors icam-1 ( intracellular adhesion molecule-1 ) , cd204 , and mhcii ( major histocompatibility complex ii ) than did monocytes that had taken up elvs isolated from lean , wild - type b6 mice ( wtelvs ) ( fig .
1a ) , although both wtelvs and obelvs were taken up by the monocytes with equal efficiency ( data not shown ) .
in addition , at days 1 and 7 after injection of the elvs there were higher levels of the proinflammatory cytokines il-6 and tnf- in the sera of the mice that had been injected with obelvs than in the sera of b6 mice that had been injected with wtelvs ( fig .
analysis of elisa results indicated that the injection of elvs does not induce the host to generate antibodies against the injected elvs ( data not shown ) , implying that induction of inflammatory cytokines may not lead to further activation of adaptive immune responses .
the experiments as described above were also repeated in b6 mice fed a standard rat diet , and similar results were obtained ( data not shown ) , suggesting that hfd preconditioning is not required for obelv - mediated activation of monocytes .
a : wild - type b6 mice fed an hfd for 3 months starting at 2 months of age were injected intravenously with the pkh67-labeled obelvs or wtelvs ( 30 g / mouse ) .
twenty - four hours after the injection , cd11bf4/80pkh67 cells from peripheral blood were analyzed for the presence of cd204 , icam-1 , and mhcii markers .
results were pooled from five independent experiments ( n = 5 mice / experiment ) and are presented as the means se , * p < 0.05 , * * p < 0.01 .
b : mice treated as described above at day 1 and 7 , peripheral blood was collected at 4 h after the elv injections , and the serum concentration of il-6 and tnf- were determined using a standard elisa , * p < 0.05 , * * p < 0.01 ( n = 5 mice per group ) .
i.v . , intravenous . to determine whether the obelv - activated macrophages produce factors that affect insulin sensitivity , we determined the effect of conditioned medium harvested from bone marrow precursor cells that had been pretreated with obelvs for 14 days on glucose uptake and the insulin response of myocytes .
the conditioned medium was harvested from 14-day cultures of bone marrow cells that had been pretreated with obelvs or wtelvs and cultured in the presence or absence of gm - csf .
in the course of these experiments , we noted that on day 4 after the addition of the elvs to the day 0 cultured bone marrow precursor cells that the bone marrow precursors exhibited differentiation into macrophages instead of dendritic cells and that the differentiation into macrophages occurred when the dendritic cell differentiation factor gm - csf had been added to the day 0 cultures ( supplemental fig .
these activated macrophages secreted higher quantities of macrophage colony - stimulating factor , il-6 , and tnf- into the culture supernatants than did the bone marrow precursors that had been stimulated with wtelvs ( supplemental fig .
furthermore , elevated obelv concentrations were associated with the promotion of bone marrow precursor differentiation ( supplemental fig .
upon addition of the conditioned medium to the myocyte cultures , the levels of phosphorylation of akt were lower in the myocytes cultured with the conditioned medium harvested from macrophages pretreated with obelvs than in the myocytes cultured with conditioned medium from wtelvs ( fig .
furthermore , basal and insulin - stimulated transport of glucose was inhibited in myocytes treated with obelv - conditioned medium , indicating that insulin function is also impaired ( fig .
induced impairment of insulin responses was not observed when bone marrow precursors were stimulated with thymus exosomes ( fig .
2b ) , suggesting that obelv - mediated impairment of insulin responses in myocytes is adipose tissue exosome specific .
adipose obelvs promote the differentiation and proliferation of bmdms and impair activation of the insulin signaling pathway in vitro and macrophage infiltration into adipose tissues in vivo .
c2c12 cells at 80% confluency were cultured for 24 h in the presence of conditioned medium harvested from 14-day cultures of bone marrow cells that had been pretreated with obelvs , wtelvs , or thymus exosomes ( 10 g / ml ) at day 0 of the culture and cultured in the presence or absence of gm - csf . after a 3-h starvation ,
the c2c12 cells were stimulated with insulin ( 100 nmol / l ) for 20 min and either lysed for western blot analysis of phosphorylated akt ( a ) or used for glucose uptake testing ( b ) .
results presented are representative of a minimum of three experiments ( a ) or data are the means se of three experiments with two replicates of each ( b ) .
c : b6 mice fed an hfd were intravenously injected with a mixture ( 1:1 ) of 4 10 of bmdms that had been preincubated with obelvs with bmdms that had been preincubated with wtelvs .
the percentage of injected macrophages infiltrating adipose tissue , liver , spleen , and bone marrow were determined by facs analysis of pkh67 and pkh26 14 days after the injection .
the proliferation of injected fluorescent dye labeled cd11bf4/80 macrophages was then determined by facs analysis of brdu cells in the adipose tissue , liver , spleen , and bone marrow .
a representative graph of facs analysis of macrophage infiltration in adipose tissue is shown and the data represent the means se from five mice from each group . * * p < 0.01 .
in addition , in agreement with the data published by other groups ( 3638 ) , addition of anti
il-6 neutralizing antibodies to the conditioned medium harvested from the obelv - treated wild - type bone marrow precursor cells led to a partial reversal of the impaired responses ( supplemental fig .
s5 ) . to assess the homing of the macrophages that had taken up the elvs , we injected b6 mice fed an hfd over 3 months with a mixture of pkh26-labeled bmdms that had been preincubated with obelvs ( bmdms ) with pkh67- labeled bmdms that had been preincubated with wtelvs ( bmdms ) .
facs analysis of the tissues harvested 14 days after the bmdms or bmdms injections revealed that the number of pkh26 macrophages were remarkably higher in adipose tissue and liver but not in the spleen and bone marrow ( fig .
analysis of the proliferation of the infiltrating macrophages ( cd11bf4/80pkh26 ) using a brdu incorporation assay suggested that the macrophages that had been pretreated with obelvs proliferated faster than those pretreated with wtelvs ( fig .
preferential homing to and faster proliferation of macrophages prepulsed with obelvs , but not wtelvs , in adipose and liver tissue was also observed in the same - aged b6 ob / ob mice ( data not shown ) .
the tlr pathway has been shown to play a role in the development of obesity ( 14 ) ; therefore , we repeated the above studies using bmdms from tlr2 knockout , tlr4 knockout , and b6 wild - type mice .
elisa analysis of supernatants obtained from the obelv - stimulated bmdms show there were much higher levels of il-6 and tnf- in the culture medium of the obelv - treated bmdms from tlr2 knockout and wild - type b6 mice than the obelv - treated bmdms from tlr4 knockout mice ( fig .
facs analysis of the cells indicated that the obelv - induced expression of cd204 was also lower in the obelv - treated bmdms from the tlr4 knockout mice than the obelv - treated bmdms from the wild - type b6 mice and the tlr2 knockout mice ( supplemental fig .
similar results were obtained when icam and mhcii expression were analyzed by facs ( data not shown ) .
together , these data suggest that obelvs are capable of utilizing tlr4 signaling to induce a macrophage inflammatory response . to further substantiate the involvement of the tlr4 signaling pathway
, we sought to determine if knockout of either myd88 or trif affected the response , as both these molecules can be used as adaptors for the tlr4 signaling pathway ( 3942 ) .
knockout of trif , but not myd88 , substantially inhibited the obelv - mediated induction of il-6 and tnf- ( fig .
the tlr4/trif pathway plays a role in adipose obelv - mediated macrophage activation and the impairment of the insulin response .
a : bmdms from wild - type b6 , tlr2 knockout , or tlr4 knockout mice were treated with obelvs ( 10 g / ml ) , and the quantity of il-6 and tnf- in 24 h culture supernatants was measured using a standard elisa .
b : bmdms from wild - type b6 , myd88 , or trif mice were treated with obelvs ( 10 g / ml ) at 0 h of the culture , and the quantity of il-6 and tnf- in the 24 h culture supernatants was determined as done in fig .
c : the c2c12 cells were cultured for 24 h in the presence of conditioned medium harvested at 24 h after addition of obelv ( 10 g / ml ) to bmdms from tlr4 knockout b6 mice or wild - type b6 mice .
data are the means se of three experiments with two replicates ( a c ) .
in addition , the combined medium harvested at 24 h after addition of obelv ( 10 g / ml ) to bmdms from tlr4 knockout b6 mice led to better glucose uptake or insulin response of myocytes than from wild - type b6 mice ( fig .
we found that rbp4 is present in elvs isolated from the adipose tissue of b6 ob / ob mice and in significantly higher amounts than wtelvs ( fig .
the addition of recombinant rbp4 ( rrbp4 ) alone to bmdms from wild - type b6 mice induced the production of il-6 and tnf- in a concentration - dependent manner ( supplemental fig .
this rbp4-induced production of il-6 and tnf- was dependent on tlr4 because there was less induction of either cytokine when tlr4 knockout macrophages were used in experiments ( fig .
this result was also supported by in vivo data indicating that 6 h after intravenous injection of recombinant rbp4 ( 250 g / mouse ) in tlr4 knockout b6 mice , a reduced induction of serum tnf- and il-6 had occurred ( supplemental fig .
s7b ) . nuclear factor ( nf)-b activation has been shown to play a role in tlr4-driven inflammatory responses . this rbp4-induced production of il-6 and tnf- was also attenuated when bmdms from nf-b p50 knockout mice were used ( supplemental fig .
s7c ) . collectively , these data suggest that rbp4-mediated induction of tnf- and il-6 is regulated through tlr4/nf-b pathway .
obelv rbp4 induces the production of macrophage proinflammatory cytokines via activation of the tlr4/trif pathway .
a : fifty micrograms of obelvs and wtelvs were lysed in protein lysis buffer , and each lysate was resolved by page in a 10% sds gel for western blot analysis .
the signal intensity of each protein was quantified using an odyssey infrared imaging system ( li - cor ) .
the ratios of signal intensity of each protein in obelvs : wtelvs were calculated and plotted ( bar graphs ) .
b : bmdms from wild - type b6 mice or b6 mice with different gene knockouts were treated with 5 g / ml of the rbp4 . the quantity of il-6 and tnf- produced
c : bmdms from wild - type b6 mice were treated with a mouse rbp4 ( 5 g / ml ) or dialyzed solution as a control for 24 h. cells were washed with pbs three times and cultured in the presence of obelvs ( 10 g / ml ) for an additional 24 h. the supernatants were harvested and il-6 and tnf- quantified by elisa .
data represent the means se of five replicate wells , * p < 0.05 , * * p < 0.01 . to address the possibility that elv rbp4 utilizes a different pathway to stimulate tnf- and il-6 production by bmdms than does free rbp4
the bmdms were pretreated 24 h before the addition of obelvs with mouse rbp4 or its dialyzed buffer as a control .
elisa analysis of the supernatants confirmed that obelvs or rbp4 alone could induce the production of tnf- and il-6 ; however , pretreatment of the bmdms with rbp4 attenuated the ability of the adipose obelvs to induce the production of tnf- and il-6 ( fig .
4c ) , suggesting that rbp4 may compete with the same pathway as obelvs to induce tnf- and il-6 . to test the effects of elvs in vivo , b6 and tlr4
knockout mice were injected intravenously ( 30 g / mouse ) every 3 days for 3 weeks with elvs released from the adipose tissue of lean wild - type mice or ob / ob mice .
glucose uptake , insulin response , and serum levels of tnf- and il-6 were determined after the last injection . remarkably , the injection of elvs released from the adipose tissue of ob / ob mice , but not wild - type mice , led to the development of glucose intolerance ( fig .
5c ) were much lower in the tlr4 knockout mice that were treated with the obelvs than the wtelvs .
thus , elvs released from the adipose tissue of ob / ob mice substantially enhance the development of insulin resistance and impair glucose tolerance and induction of inflammatory cytokines in a tlr4-dependent manner .
injection of obelvs leads to the intolerance of glucose uptake , insulin resistance , and induction of inflammatory cytokines of mice .
a and b : wild - type b6 mice or tlr4 knockout of b6 mice ( n = 10 ) were injected intravenously with obelvs or wtelvs ( 30 g / mouse in 200 l of pbs ) every 3 days for 21 days .
one day after the last injection ( day 22 ) , mice were fasted either overnight before receiving an intraperitoneal injection of 2 mg of dextrose / g body wt for glucose tolerance testing ( a ) or fasted for 4 h before receiving recombinant human insulin ( 1 unit / kg i.p . ) for insulin responsiveness testing ( b ) .
data are means se , * p < 0.05 , * * p < 0.01 .
in addition , serum tnf- and il-6 was also quantified using a standard elisa at 4 h after the last injection ( c ) .
others have demonstrated previously that exosomes are released by 3t3-l1 , a precursor adipocyte cell line ( 29,30 ) .
we found that more elvs are released in 30-min ex vivo adipose tissue cultures of age - matched wild - type ( b6 ) mice fed a hfd over 3 months ( hfdelvs , 16.5 1.2 g / g of adipose weight ) and of leptin - deficient ( ob / ob ) b6 mice ( obelvs , 14.4 1.1 g / g of adipose weight ) when compared to adipose tissue cultures of wild - type lean b6 mice ( wtelvs , 4.1 1.0 g / g of adipose weight ) .
the quantity of elvs released from adipose tissue increased over a period from 30 min to 6 h of ex vivo adipose tissue culture ( supplemental fig .
s1a ) , that is , 16.522.2 g / g hfdelvs ; 14.4 23.2 g / g obelvs ; and 4.16.7 g / g wtelvs .
electron microscopy examination revealed vesicles that measured 60100 nm in diameter and had a cup - shaped morphology ( supplemental fig .
s1b ) . neither calnexin nor lamp-1 were detectable when the elvs were analyzed by immunoblotting ( supplemental fig .
s1c ) , indicating that our elv preparations were free of contaminating nonexosome membrane proteins ( 31 ) .
further evidence that the vesicles were exosomes was obtained through analysis of the protein composition using linear ion trap mass spectrometery ( ltq lc / ms ) ( supplemental table s1 ) .
these analyses indicated a protein composition typical of exosomes derived from other cell types ( 3235 ) ( supplemental table s2 ) .
specific proteins were at undetectable levels in wt - elvs when compared with protein detected in obelvs and hfdelvs ( supplemental table s1 ) , but in both cases the elvs contained proteins known to be involved in cell metabolism , membrane trafficking , multiple small gtp - binding proteins , integral membrane proteins , and several class e vacuolar protein sorting proteins .
the fatty acid composition of the obelvs was almost exclusively palmitic acid ( n = 8 , 40.22 3.82% ) and stearic acid ( 52.71 4.42% ) ( supplemental table s3 ) .
because there are minor differences in the protein composition of elvs released from b6 mice fed an hfd in comparison with obelvs ( supplemental table s1 ) , we focused on the b6 obelvs released from adipose tissues at 30-min ex vivo culture for the remainder of study because a larger amount of adipose tissue for isolation of elvs was available from ob / ob mice of the same age .
we determine whether obelvs released from adipose tissue are taken up by cells of the immune system .
we labeled obelvs with pkh67 dye before injecting them intravenously into b6 mice fed an hfd or standard diet for 3 months starting at 2 months of age .
facs analysis of single - cell suspensions of several tissues that had been harvested 24 h after the injection of obelvs indicated that more than 1.1% of total blood cells or liver leukocytes had taken up obelvs .
approximately 80% of the cells having taken up obelvs were cd11bf4/80 monocytes ( supplemental fig .
s2a and b ) , but no b cells , t cells , nor natural killer cells had taken up obelvs ( data not shown ) .
this result is unlikely because of pkh67 dye leakage from obelvs to the monocytes as no pkh67- positive macrophages were detected in the tissues of mice having been injected intravenously with free pkh67 dye ( data not shown ) .
facs analysis of the gated pkh67 cells isolated from peripheral blood further indicated that the monocytes ( cd11bf4/80pkh67 ) that had taken up the obelvs expressed higher levels of the monocyte receptors icam-1 ( intracellular adhesion molecule-1 ) , cd204 , and mhcii ( major histocompatibility complex ii ) than did monocytes that had taken up elvs isolated from lean , wild - type b6 mice ( wtelvs ) ( fig .
1a ) , although both wtelvs and obelvs were taken up by the monocytes with equal efficiency ( data not shown ) . in addition , at days 1 and 7 after injection of the elvs there were higher levels of the proinflammatory cytokines il-6 and tnf- in the sera of the mice that had been injected with obelvs than in the sera of b6 mice that had been injected with wtelvs ( fig .
analysis of elisa results indicated that the injection of elvs does not induce the host to generate antibodies against the injected elvs ( data not shown ) , implying that induction of inflammatory cytokines may not lead to further activation of adaptive immune responses .
the experiments as described above were also repeated in b6 mice fed a standard rat diet , and similar results were obtained ( data not shown ) , suggesting that hfd preconditioning is not required for obelv - mediated activation of monocytes .
a : wild - type b6 mice fed an hfd for 3 months starting at 2 months of age were injected intravenously with the pkh67-labeled obelvs or wtelvs ( 30 g / mouse ) .
twenty - four hours after the injection , cd11bf4/80pkh67 cells from peripheral blood were analyzed for the presence of cd204 , icam-1 , and mhcii markers .
results were pooled from five independent experiments ( n = 5 mice / experiment ) and are presented as the means se , * p < 0.05 , * * p < 0.01 .
b : mice treated as described above at day 1 and 7 , peripheral blood was collected at 4 h after the elv injections , and the serum concentration of il-6 and tnf- were determined using a standard elisa , * p < 0.05 , * * p < 0.01 ( n = 5 mice per group ) .
to determine whether the obelv - activated macrophages produce factors that affect insulin sensitivity , we determined the effect of conditioned medium harvested from bone marrow precursor cells that had been pretreated with obelvs for 14 days on glucose uptake and the insulin response of myocytes .
the conditioned medium was harvested from 14-day cultures of bone marrow cells that had been pretreated with obelvs or wtelvs and cultured in the presence or absence of gm - csf . in the course of these experiments
, we noted that on day 4 after the addition of the elvs to the day 0 cultured bone marrow precursor cells that the bone marrow precursors exhibited differentiation into macrophages instead of dendritic cells and that the differentiation into macrophages occurred when the dendritic cell differentiation factor gm - csf had been added to the day 0 cultures ( supplemental fig .
these activated macrophages secreted higher quantities of macrophage colony - stimulating factor , il-6 , and tnf- into the culture supernatants than did the bone marrow precursors that had been stimulated with wtelvs ( supplemental fig .
furthermore , elevated obelv concentrations were associated with the promotion of bone marrow precursor differentiation ( supplemental fig .
upon addition of the conditioned medium to the myocyte cultures , the levels of phosphorylation of akt were lower in the myocytes cultured with the conditioned medium harvested from macrophages pretreated with obelvs than in the myocytes cultured with conditioned medium from wtelvs ( fig .
furthermore , basal and insulin - stimulated transport of glucose was inhibited in myocytes treated with obelv - conditioned medium , indicating that insulin function is also impaired ( fig .
induced impairment of insulin responses was not observed when bone marrow precursors were stimulated with thymus exosomes ( fig .
2b ) , suggesting that obelv - mediated impairment of insulin responses in myocytes is adipose tissue exosome specific .
adipose obelvs promote the differentiation and proliferation of bmdms and impair activation of the insulin signaling pathway in vitro and macrophage infiltration into adipose tissues in vivo .
c2c12 cells at 80% confluency were cultured for 24 h in the presence of conditioned medium harvested from 14-day cultures of bone marrow cells that had been pretreated with obelvs , wtelvs , or thymus exosomes ( 10 g / ml ) at day 0 of the culture and cultured in the presence or absence of gm - csf . after a 3-h starvation ,
the c2c12 cells were stimulated with insulin ( 100 nmol / l ) for 20 min and either lysed for western blot analysis of phosphorylated akt ( a ) or used for glucose uptake testing ( b ) .
results presented are representative of a minimum of three experiments ( a ) or data are the means se of three experiments with two replicates of each ( b ) . * p < 0.05 ; * * p < 0.01 .
c : b6 mice fed an hfd were intravenously injected with a mixture ( 1:1 ) of 4 10 of bmdms that had been preincubated with obelvs with bmdms that had been preincubated with wtelvs .
the percentage of injected macrophages infiltrating adipose tissue , liver , spleen , and bone marrow were determined by facs analysis of pkh67 and pkh26 14 days after the injection .
was then determined by facs analysis of brdu cells in the adipose tissue , liver , spleen , and bone marrow .
a representative graph of facs analysis of macrophage infiltration in adipose tissue is shown and the data represent the means se from five mice from each group . * * p < 0.01 .
in addition , in agreement with the data published by other groups ( 3638 ) , addition of anti tnf- and anti
il-6 neutralizing antibodies to the conditioned medium harvested from the obelv - treated wild - type bone marrow precursor cells led to a partial reversal of the impaired responses ( supplemental fig .
s5 ) . to assess the homing of the macrophages that had taken up the elvs , we injected b6 mice fed an hfd over 3 months with a mixture of pkh26-labeled bmdms that had been preincubated with obelvs ( bmdms ) with pkh67- labeled bmdms that had been preincubated with wtelvs ( bmdms ) .
facs analysis of the tissues harvested 14 days after the bmdms or bmdms injections revealed that the number of pkh26 macrophages were remarkably higher in adipose tissue and liver but not in the spleen and bone marrow ( fig .
analysis of the proliferation of the infiltrating macrophages ( cd11bf4/80pkh26 ) using a brdu incorporation assay suggested that the macrophages that had been pretreated with obelvs proliferated faster than those pretreated with wtelvs ( fig .
preferential homing to and faster proliferation of macrophages prepulsed with obelvs , but not wtelvs , in adipose and liver tissue was also observed in the same - aged b6 ob / ob mice ( data not shown ) .
the tlr pathway has been shown to play a role in the development of obesity ( 14 ) ; therefore , we repeated the above studies using bmdms from tlr2 knockout , tlr4 knockout , and b6 wild - type mice .
elisa analysis of supernatants obtained from the obelv - stimulated bmdms show there were much higher levels of il-6 and tnf- in the culture medium of the obelv - treated bmdms from tlr2 knockout and wild - type b6 mice than the obelv - treated bmdms from tlr4 knockout mice ( fig .
facs analysis of the cells indicated that the obelv - induced expression of cd204 was also lower in the obelv - treated bmdms from the tlr4 knockout mice than the obelv - treated bmdms from the wild - type b6 mice and the tlr2 knockout mice ( supplemental fig .
similar results were obtained when icam and mhcii expression were analyzed by facs ( data not shown ) .
together , these data suggest that obelvs are capable of utilizing tlr4 signaling to induce a macrophage inflammatory response . to further substantiate the involvement of the tlr4 signaling pathway
, we sought to determine if knockout of either myd88 or trif affected the response , as both these molecules can be used as adaptors for the tlr4 signaling pathway ( 3942 ) .
knockout of trif , but not myd88 , substantially inhibited the obelv - mediated induction of il-6 and tnf- ( fig .
the tlr4/trif pathway plays a role in adipose obelv - mediated macrophage activation and the impairment of the insulin response .
a : bmdms from wild - type b6 , tlr2 knockout , or tlr4 knockout mice were treated with obelvs ( 10 g / ml ) , and the quantity of il-6 and tnf- in 24 h culture supernatants was measured using a standard elisa .
b : bmdms from wild - type b6 , myd88 , or trif mice were treated with obelvs ( 10 g / ml ) at 0 h of the culture , and the quantity of il-6 and tnf- in the 24 h culture supernatants was determined as done in fig .
c : the c2c12 cells were cultured for 24 h in the presence of conditioned medium harvested at 24 h after addition of obelv ( 10 g / ml ) to bmdms from tlr4 knockout b6 mice or wild - type b6 mice .
data are the means se of three experiments with two replicates ( a c ) .
in addition , the combined medium harvested at 24 h after addition of obelv ( 10 g / ml ) to bmdms from tlr4 knockout b6 mice led to better glucose uptake or insulin response of myocytes than from wild - type b6 mice ( fig .
we found that rbp4 is present in elvs isolated from the adipose tissue of b6 ob / ob mice and in significantly higher amounts than wtelvs ( fig .
the addition of recombinant rbp4 ( rrbp4 ) alone to bmdms from wild - type b6 mice induced the production of il-6 and tnf- in a concentration - dependent manner ( supplemental fig .
this rbp4-induced production of il-6 and tnf- was dependent on tlr4 because there was less induction of either cytokine when tlr4 knockout macrophages were used in experiments ( fig .
this result was also supported by in vivo data indicating that 6 h after intravenous injection of recombinant rbp4 ( 250 g / mouse ) in tlr4 knockout b6 mice , a reduced induction of serum tnf- and il-6 had occurred ( supplemental fig .
s7b ) . nuclear factor ( nf)-b activation has been shown to play a role in tlr4-driven inflammatory responses . this rbp4-induced production of il-6 and tnf- was also attenuated when bmdms from nf-b p50 knockout mice were used ( supplemental fig .
s7c ) . collectively , these data suggest that rbp4-mediated induction of tnf- and il-6 is regulated through tlr4/nf-b pathway .
obelv rbp4 induces the production of macrophage proinflammatory cytokines via activation of the tlr4/trif pathway .
a : fifty micrograms of obelvs and wtelvs were lysed in protein lysis buffer , and each lysate was resolved by page in a 10% sds gel for western blot analysis .
the signal intensity of each protein was quantified using an odyssey infrared imaging system ( li - cor ) .
the ratios of signal intensity of each protein in obelvs : wtelvs were calculated and plotted ( bar graphs ) .
b : bmdms from wild - type b6 mice or b6 mice with different gene knockouts were treated with 5 g / ml of the rbp4 . the quantity of il-6 and tnf- produced
c : bmdms from wild - type b6 mice were treated with a mouse rbp4 ( 5 g / ml ) or dialyzed solution as a control for 24 h. cells were washed with pbs three times and cultured in the presence of obelvs ( 10 g / ml ) for an additional 24 h. the supernatants were harvested and il-6 and tnf- quantified by elisa .
data represent the means se of five replicate wells , * p < 0.05 , * * p < 0.01 .
to address the possibility that elv rbp4 utilizes a different pathway to stimulate tnf- and il-6 production by bmdms than does free rbp4 , we used a competitive assay .
the bmdms were pretreated 24 h before the addition of obelvs with mouse rbp4 or its dialyzed buffer as a control .
elisa analysis of the supernatants confirmed that obelvs or rbp4 alone could induce the production of tnf- and il-6 ; however , pretreatment of the bmdms with rbp4 attenuated the ability of the adipose obelvs to induce the production of tnf- and il-6 ( fig .
4c ) , suggesting that rbp4 may compete with the same pathway as obelvs to induce tnf- and il-6 .
to test the effects of elvs in vivo , b6 and tlr4 knockout mice were injected intravenously ( 30 g / mouse ) every 3 days for 3 weeks with elvs released from the adipose tissue of lean wild - type mice or ob / ob mice .
glucose uptake , insulin response , and serum levels of tnf- and il-6 were determined after the last injection . remarkably , the injection of elvs released from the adipose tissue of ob / ob mice , but not wild - type mice , led to the development of glucose intolerance ( fig .
5c ) were much lower in the tlr4 knockout mice that were treated with the obelvs than the wtelvs .
thus , elvs released from the adipose tissue of ob / ob mice substantially enhance the development of insulin resistance and impair glucose tolerance and induction of inflammatory cytokines in a tlr4-dependent manner .
injection of obelvs leads to the intolerance of glucose uptake , insulin resistance , and induction of inflammatory cytokines of mice .
a and b : wild - type b6 mice or tlr4 knockout of b6 mice ( n = 10 ) were injected intravenously with obelvs or wtelvs ( 30 g / mouse in 200 l of pbs ) every 3 days for 21 days .
one day after the last injection ( day 22 ) , mice were fasted either overnight before receiving an intraperitoneal injection of 2 mg of dextrose / g body wt for glucose tolerance testing ( a ) or fasted for 4 h before receiving recombinant human insulin ( 1 unit / kg i.p . ) for insulin responsiveness testing ( b ) .
data are means se , * p < 0.05 , * * p < 0.01 . in addition , serum tnf- and il-6 was also quantified using a standard elisa at 4 h after the last injection ( c ) .
in this study , we found that elvs released from adipose tissue of ob / ob mice induce macrophage activation in a tlr4-dependent manner and that the rbp4 that is incorporated in these elvs plays a role in the induction of macrophage activation .
the exposure of wild - type macrophages to obelvs resulted in an increased production of the proinflammatory cytokines il-6 and tnf- , enhanced the migration of macrophages into adipose tissue and the liver , and promoted the development of insulin resistance .
in contrast , the intravenous injection of obelvs into tlr4 knockout mice did not result in the development of insulin resistance , and treatment of tlr4 knockout macrophages with obelvs did not enhance the production of il-6 or tnf-. adipose obelv rbp4 protein can induce the production of macrophage il-6 and tnf- in a tlr4-dependent manner . using an ex vivo adipose tissue culture approach , we provide evidence that elvs are secreted from adipose tissue . unlike the data for the exosomes released from in vitro cultured cells , the data generated using the exosomes released from adipose tissue as demonstrated in this study are more relevant to what may take place in an in vivo obesity mouse model .
the exosomes could be taken up by resident macrophages leading to their activation in adipose tissue .
because of resident macrophage activation , more monocytes / macrophages may be recruited into adipose tissue to further augmented inflammatory responses with more elvs released from adipose tissue as the size of adipocytes increased .
this increase in adipose cell size is one of the major features of obesity and may contribute to an increased amount of elvs circulating in the peripheral blood .
therefore , activated monocytes circulating in the peripheral blood could be further expanded by these elvs circulated in the peripheral blood .
although the factors driving monocytes / macrophages to preferentially infiltrate adipose tissue and liver are not clear , this study provides strong evidence supporting the idea that the macrophage tlr4-mediated pathway plays a role in obelv - mediated activation of macrophages .
a physiological role of tlr4 in insulin resistance was demonstrated previously in a model in which tlr4 knockout mice were fed an hfd ( 13,14,17 ) .
we found that the obelv - mediated induction of il-6 and tnf- occurs through activation of the tlr4 pathway of macrophages .
our data further suggest that the exosome - mediated induction of il-6 and tnf- of macrophages is dependent on the tlr4/trif pathway .
this is consistent with recent data suggesting that signaling through the trif - mediated pathway can activate nf-b leading to the production of il-6 and tnf- ( 40,4244 ) .
the preferential signaling through the tlr4/trif pathway rather than the tlr4/myd88 pathway is of particular interest with respect to the potential mechanisms by which the obelvs may act to stimulate the macrophages .
it has been reported that the tlr4-trif pathway is activated in endosomes , whereas the tlr4-myd88 pathway is activated at the plasma membrane level ( 41,45 ) .
the biogenesis of exosomes is considered to be initiated in endosomes , and it is conceivable that the proteins contained in exosomes may preferentially home to the endosome compartment of the cells where they are taken up and that this may influence the effects of the exosomes on the cells in terms of the type of response they elicit and the magnitude of the response .
currently , it is unknown whether the activation of the tlr4/trif pathway and the induction of il-6 and tnf- by obelvs is dependent on obelv rbp4 .
a neutralizing anti - rbp4 antibody to block obelv rbp4mediated induction of il-6 and tnf- would further confirm the results presented in fig .
we have been unable to develop a satisfactory neutralizing anti - rbp4 antibody , and there is no commercially available anti - rbp4 antibody for use in neutralization experiments at the present time .
finally , we propose a hypothetical model in which obesity may cause dysregulation of the adipose exosome protein and/or fatty acid sorting machinery , resulting in the unregulated sorting of certain proteins / lipids into exosomes .
factors , such as inflammatory cytokines that are high - risk indicators for developing obesity and insulin resistance , may play a direct role in dysregulation of the cell sorting machinery .
because of the dysregulation , the exosomes released by the adipose tissue in obese mice contain proinflammatory favorable proteins .
these exosomes are capable of a potent stimulatory effect locally and at some distance from the adipose tissue , which could mediate both the obesity - associated inflammatory responses and the development of insulin resistance .
we propose that the contribution of adipose elvs to obesity - associated insulin resistance is likely multifactorial .
it is conceivable that if a single protein could be responsible for certain biological effects without regulating other effects , the formation of exosomes as a complex to execute their immune regulatory function would seem to waste host energy to assemble .
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### Summary:
: objectivewe sought to determine whether exosome - like vesicles ( elvs ) released from adipose tissue play a role in activation of macrophages and subsequent development of insulin resistance in a mouse model.research design and methodselvs released from adipose tissue were purified by sucrose gradient centrifugation and labeled with green fluorescent dye and then intravenously injected into b6 ob / ob mice ( obese model ) or b6 mice fed a high - fat diet .
the effects of injected elvs on the activation of macrophages were determined through analysis of activation markers by fluorescence - activated cell sorter and induction of inflammatory cytokines using an elisa .
glucose tolerance and insulin tolerance were also evaluated .
similarly , b6 mice with different gene knockouts including tlr2 , tlr4 , myd88 , and toll - interleukin-1 receptor ( tir ) domain containing adaptor protein inducing interferon- ( trif ) were also used for testing their responses to the injected elvs.resultselvs are taken up by peripheral blood monocytes , which then differentiate into activated macrophages with increased secretion of tumor necrosis factor- ( tnf- ) and interleukin-6 ( il-6 ) .
injection of obelvs into wild - type c57bl/6 mice results in the development of insulin resistance .
when the obelvs were intravenously injected into tlr4 knockout b6 mice , the levels of glucose intolerance and insulin resistance were much lower .
rbp4 is enriched in the obelvs .
bone marrow derived macrophages preincubated with recombinant rbp4 led to attenuation of obelv - mediated induction of il-6 and tnf-.conclusionselvs released by adipose tissue can act as a mode of communication between adipose tissues and macrophages .
the obelv - mediated induction of tnf- and il-6 in macrophages and insulin resistance requires the tlr4/trif pathway .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: first , alumni were interviewed in order to evaluate what they felt were the most important skills needed for the work they had just started .
the aim of this first step was not to constitute a conclusive qualitative study , but rather to inform the creation of survey items , a method successfully used in other studies ( 13 ) .
second , a survey - based investigation was performed , using categories established from the interviews , to provide a statistical breakdown of the views of the alumni .
nine alumni who had already worked as medical practitioners from one to three years were interviewed by phone .
these alumni were selected with the objective of maximising the ranges of age , ethnicity , gender and geographical location .
the initial enquiry sought to establish what they felt to be important for a ( new ) physician to know .
semi - structured interviews addressed the informants ' views on their medical training programme and their current work situation .
the questions presented were used as headlines for conversation and follow - up questions were asked where appropriate .
it was felt that the issues dealt with during the interview might in fact be easier to discuss with a distance from the interviewer .
following these interviews , key competences were extracted and reformulated as skills , knowledge or attitudes relevant to working as a physician .
the following quote from one of the interviews illustrates how these extractions were made : it [ being a competent physician ] is first and foremost about a good doctor
it 's also about listening to the patient 's needs , being a good listener that you have a nice manner .
( quotation translated from swedish and adjusted for clarity)competences identified in this quotation include being able to ( a ) establish a strong doctor
patient relationship , ( b ) show the patient respect , ( c ) listen to the patient 's needs and ( d ) present information to the patient intelligibly .
it [ being a competent physician ] is first and foremost about a good doctor
it 's also about listening to the patient 's needs , being a good listener that you have a nice manner .
( quotation translated from swedish and adjusted for clarity ) the second step in this analysis classified these 52 competences into a more manageable number .
the number of competences was reduced by merging interview statements expressing similar meaning . since the author
( not being a physician himself ) was not fully acquainted with medical terminology , two senior staff members from the medical school were consulted during this process .
formulation of the competences to be included in the survey aimed to preserve as far as possible the original wording as used by the informants at interview because changing the expressions employed might have altered the specific meaning attributed to them .
these categories are as follows : medical knowledge and skills eight items.interpersonal skills ( patient relationships ) six items.interpersonal skills ( staff and workplace issues ) eight items.life-long learning skills five items.intrapersonal skills five items .
interpersonal skills ( patient relationships ) six items . interpersonal skills ( staff and workplace issues )
the alumni had then been working for at least one year and a maximum of two - and - a - half years .
letters with a url pointing to the electronic survey were sent to respondents ' home addresses .
two reminders were sent to non - respondents , in approximately two - week intervals . of the non - respondents , 10 were contacted briefly by telephone and asked if they could comment on why they had not answered the survey .
the 32 competences derived from the preliminary interviews each headlined a set of questions . under this headline ,
three subsidiary questions were asked in which the respondents were asked to rate how important they thought the competence in question was , how they rated their own competence in the specific area identified and how much attention they felt this competence should have been given during their undergraduate training .
an important principle , originating from the design of this study , is that interpretation of data has been done relatively .
that is , any mark on the scale in the questionnaire should not be viewed as an absolute , but rather interpreted in relation to other answers .
for instance , a noted five for importance is only considered in relation to how other competencies have been rated , giving the result that one competence was felt to be more important than another .
this more careful interpretation allows for the reasonable assumption that newly graduated doctors are not the best judges of , for instance , their own competence , as have been shown in other studies ( 14 ) .
in addition , each questionnaire contained several free - text sections in which respondents were encouraged to add , in their own words , any further information or comments they felt to be relevant or appropriate .
these comments were coded according to topic and used for clarification and explanation of results in the discussion .
this survey was not piloted , as a pilot was seen as conflicting with the ambition to create survey questions from interview statements .
a pilot with the purpose of changing survey questions was seen as incompatible with an inductive approach and the ambition to preserve the wording from interview respondents .
nine alumni who had already worked as medical practitioners from one to three years were interviewed by phone .
these alumni were selected with the objective of maximising the ranges of age , ethnicity , gender and geographical location .
the initial enquiry sought to establish what they felt to be important for a ( new ) physician to know .
semi - structured interviews addressed the informants ' views on their medical training programme and their current work situation .
the questions presented were used as headlines for conversation and follow - up questions were asked where appropriate .
a more honest rather than politically correct view of physicians work was desired . therefore , the use of phone interviews was not chosen entirely for practical reasons .
it was felt that the issues dealt with during the interview might in fact be easier to discuss with a distance from the interviewer .
following these interviews , key competences were extracted and reformulated as skills , knowledge or attitudes relevant to working as a physician .
the following quote from one of the interviews illustrates how these extractions were made : it [ being a competent physician ] is first and foremost about a good doctor
it 's also about listening to the patient 's needs , being a good listener that you have a nice manner .
( quotation translated from swedish and adjusted for clarity)competences identified in this quotation include being able to ( a ) establish a strong doctor
patient relationship , ( b ) show the patient respect , ( c ) listen to the patient 's needs and ( d ) present information to the patient intelligibly .
it [ being a competent physician ] is first and foremost about a good doctor
it 's also about listening to the patient 's needs , being a good listener that you have a nice manner .
( quotation translated from swedish and adjusted for clarity ) the second step in this analysis classified these 52 competences into a more manageable number .
the number of competences was reduced by merging interview statements expressing similar meaning . since the author
( not being a physician himself ) was not fully acquainted with medical terminology , two senior staff members from the medical school were consulted during this process .
formulation of the competences to be included in the survey aimed to preserve as far as possible the original wording as used by the informants at interview because changing the expressions employed might have altered the specific meaning attributed to them .
these categories are as follows : medical knowledge and skills eight items.interpersonal skills ( patient relationships ) six items.interpersonal skills ( staff and workplace issues ) eight items.life-long learning skills five items.intrapersonal skills five items .
interpersonal skills ( patient relationships ) six items . interpersonal skills ( staff and workplace issues )
the alumni had then been working for at least one year and a maximum of two - and - a - half years .
letters with a url pointing to the electronic survey were sent to respondents ' home addresses .
two reminders were sent to non - respondents , in approximately two - week intervals . of the non - respondents , 10 were contacted briefly by telephone and asked if they could comment on why they had not answered the survey .
the 32 competences derived from the preliminary interviews each headlined a set of questions . under this headline , three subsidiary questions were asked in which the respondents were asked to rate how important they thought the competence in question was , how they rated their own competence in the specific area identified and how much attention they felt this competence should have been given during their undergraduate training .
an important principle , originating from the design of this study , is that interpretation of data has been done relatively .
that is , any mark on the scale in the questionnaire should not be viewed as an absolute , but rather interpreted in relation to other answers .
for instance , a noted five for importance is only considered in relation to how other competencies have been rated , giving the result that one competence was felt to be more important than another .
this more careful interpretation allows for the reasonable assumption that newly graduated doctors are not the best judges of , for instance , their own competence , as have been shown in other studies ( 14 ) .
in addition , each questionnaire contained several free - text sections in which respondents were encouraged to add , in their own words , any further information or comments they felt to be relevant or appropriate .
these comments were coded according to topic and used for clarification and explanation of results in the discussion .
this survey was not piloted , as a pilot was seen as conflicting with the ambition to create survey questions from interview statements .
a pilot with the purpose of changing survey questions was seen as incompatible with an inductive approach and the ambition to preserve the wording from interview respondents .
the findings are structured according to the category of competence identified in the first interview study .
competences under each category are presented as a table giving the mean respondent score and standard deviation for each question .
the results in the category of medical knowledge and skills are shown in table 1 .
as we shall see , the other categories deal more with generic or transferable skills ( 15 ) . competence regarding medical knowledge and skills two areas stand out in respondents ' views on important competences .
specifically , knowledge of basic biomedicine and perform medical research ( mean scores 3.6 and 3.2 , respectively ) were seen to be significantly less important than other competences .
this view was also reflected by responses regarding coverage where a score of 3 indicated
knowledge of terms and conceptions , while considered more important , was also rated close to 3 regarding coverage . in contrast , the mean coverage scores for all the other competences were closer to 4 , indicating that the alumni felt that more attention should have been devoted to these areas .
practical skills and patient examination skills appeared to be of most concern , both regarding coverage and self - assessed competence .
patient in general , the alumni felt that competences in this category were very important .
however , they also found themselves ( with the exception of breaking bad news ) fairly competent with respect to their interpersonal skills .
this category deals with relations at work and ancillary duties ( such as administration and night duty ) that the alumni at interview felt to be of importance . the questions and responses are listed in table 3 .
competence regarding interpersonal skills staff and workplace issues overall the alumni felt less competent in many areas concerning decision - making , conflict management and handling night duty .
they felt ill - prepared for the possible eventuality that they might be reported to the board .
several of the informants in the interviews stressed the importance of continuing education and related competences ( table 4 ) .
competence regarding life - long learning skills in general , these competences were judged to be very important . however , there was marked variation in self - assessed abilities in this area .
learning from the books and colleagues appeared less problematic than finding and understanding the scientific articles .
there was also a desire for more training in finding scientific articles and , notably , in critical thinking skills .
the final category , intrapersonal skills , addresses how the respondent personally handles workplace issues as interns or as newly registered physicians ( table 5 ) .
competence regarding intrapersonal skills the largest differences between importance and self - assessed ability were found in this category .
this may not be surprising because this category deals with situations that are stressful or psychologically demanding .
the alumni clearly wished to be more prepared in dealing with stressful situations . in the free - text sections many comments concerned ability to work hard. a substantial number of alumni appeared to be unhappy that they were expected to work hard and for long hours , even though they often acknowledged that the job demands this commitment .
this study has highlighted a number of areas where the alumni feel either well - prepared or unprepared for the next step and their first positions .
most competences listed in the survey were seen to be important in the work of physicians .
this suggests , even though the interview sample was small , that the interviews covered the majority of areas that the alumni felt to be important .
the following conclusions can be drawn from the results of the survey : the alumni considered that training in knowledge of symptoms , diseases and treatments was significantly more important than basic biomedical knowledge.the alumni reported a lack of training and preparedness regarding practical competences including patient examination skills.many of the skills listed as important are in fact generic skills.the alumni were generally confident about their skills regarding doctor
patient relationships and cooperation with other staff.there were many concerns concerning their abilities to handle stressful situations at work .
issues identified included leadership , conflicts , being reported to the board and handling night duty .
respondent scores on intrapersonal skills , including ability to prioritise , stress management and balancing work with free - time , reflected similar concerns.the alumni generally considered that life - long learning skills are important . while they felt fairly confident in this respect , browsing for and understanding scientific articles were areas where more training would have been helpful .
the alumni considered that training in knowledge of symptoms , diseases and treatments was significantly more important than basic biomedical knowledge .
the alumni reported a lack of training and preparedness regarding practical competences including patient examination skills .
issues identified included leadership , conflicts , being reported to the board and handling night duty . respondent scores on intrapersonal skills , including ability to prioritise , stress management and balancing work with free - time , reflected similar concerns .
the alumni generally considered that life - long learning skills are important . while they felt fairly confident in this respect , browsing for and understanding scientific articles were areas where more training would have been helpful .
the results in the category of medical knowledge and skills are shown in table 1 .
as we shall see , the other categories deal more with generic or transferable skills ( 15 ) . competence regarding medical knowledge and skills two areas stand out in respondents ' views on important competences .
specifically , knowledge of basic biomedicine and perform medical research ( mean scores 3.6 and 3.2 , respectively ) were seen to be significantly less important than other competences .
this view was also reflected by responses regarding coverage where a score of 3 indicated
knowledge of terms and conceptions , while considered more important , was also rated close to 3 regarding coverage .
in contrast , the mean coverage scores for all the other competences were closer to 4 , indicating that the alumni felt that more attention should have been devoted to these areas .
practical skills and patient examination skills appeared to be of most concern , both regarding coverage and self - assessed competence .
patient in general , the alumni felt that competences in this category were very important .
however , they also found themselves ( with the exception of breaking bad news ) fairly competent with respect to their interpersonal skills .
this category deals with relations at work and ancillary duties ( such as administration and night duty ) that the alumni at interview felt to be of importance . the questions and responses are listed in table 3 .
competence regarding interpersonal skills staff and workplace issues overall the alumni felt less competent in many areas concerning decision - making , conflict management and handling night duty .
they felt ill - prepared for the possible eventuality that they might be reported to the board .
several of the informants in the interviews stressed the importance of continuing education and related competences ( table 4 ) .
competence regarding life - long learning skills in general , these competences were judged to be very important . however , there was marked variation in self - assessed abilities in this area .
learning from the books and colleagues appeared less problematic than finding and understanding the scientific articles .
there was also a desire for more training in finding scientific articles and , notably , in critical thinking skills .
the final category , intrapersonal skills , addresses how the respondent personally handles workplace issues as interns or as newly registered physicians ( table 5 ) .
competence regarding intrapersonal skills the largest differences between importance and self - assessed ability were found in this category .
this may not be surprising because this category deals with situations that are stressful or psychologically demanding .
the alumni clearly wished to be more prepared in dealing with stressful situations . in the free - text sections many comments concerned ability to work hard. a substantial number of alumni appeared to be unhappy that they were expected to work hard and for long hours , even though they often acknowledged that the job demands this commitment .
this study has highlighted a number of areas where the alumni feel either well - prepared or unprepared for the next step and their first positions .
most competences listed in the survey were seen to be important in the work of physicians .
this suggests , even though the interview sample was small , that the interviews covered the majority of areas that the alumni felt to be important .
the following conclusions can be drawn from the results of the survey : the alumni considered that training in knowledge of symptoms , diseases and treatments was significantly more important than basic biomedical knowledge.the alumni reported a lack of training and preparedness regarding practical competences including patient examination skills.many of the skills listed as important are in fact generic skills.the alumni were generally confident about their skills regarding doctor
patient relationships and cooperation with other staff.there were many concerns concerning their abilities to handle stressful situations at work .
issues identified included leadership , conflicts , being reported to the board and handling night duty .
respondent scores on intrapersonal skills , including ability to prioritise , stress management and balancing work with free - time , reflected similar concerns.the alumni generally considered that life - long learning skills are important . while they felt fairly confident in this respect , browsing for and understanding scientific articles were areas where more training would have been helpful .
the alumni considered that training in knowledge of symptoms , diseases and treatments was significantly more important than basic biomedical knowledge .
the alumni reported a lack of training and preparedness regarding practical competences including patient examination skills .
issues identified included leadership , conflicts , being reported to the board and handling night duty .
respondent scores on intrapersonal skills , including ability to prioritise , stress management and balancing work with free - time , reflected similar concerns .
the alumni generally considered that life - long learning skills are important . while they felt fairly confident in this respect , browsing for and understanding scientific articles were areas where more training would have been helpful .
the inductive approach used in this study has produced a list of important competences that differ somewhat from those found in other lists .
for example , life - long learning was considered to be very important while other lists seldom include these items .
most of the lists have not discriminated between different forms of medical knowledge ( basic and applied ) , an important finding in the present study .
one interpretation of table 1 is that the alumni feel themselves to be adequately prepared regarding basic biomedical theory as required for their current positions and duties , and even in some cases overly prepared .
however , the respondents felt they lacked practical skills and clinical knowledge of symptoms , diseases and treatments competences that have more direct practical applicability in the workplace than knowledge of the underlying anatomical or biomedical structures .
there are , however , many similarities with studies that have employed predefined lists of competences . regarding the doctor
patient relationship , alumni generally felt themselves to be fairly competent in the area and did not advise more training . in another study , student
graduates similarly provided high self - estimates of interpersonal competence compared to other general competences ( 10 ) .
one explanation of these results is that whereas the alumni consider these competences to be very important , they do not consider these to be teachable or
learnable in a medical programme . a quote from one of the interviews might illustrate this opinion : my competence in the doctor
patient relationship is much more because of my personality traits and my personal experience than anything we learned during the programme .
it should not be taught in an academic setting.however , there is evidence to suggest that students change their attitude toward training in communication skills after completing medical school .
( 16 ) reported a similar attitude to learning interpersonal skills . in their study , students realised the value of specific courses in communication skills only after they had completed them . my competence in the doctor
patient relationship is much more because of my personality traits and my personal experience than anything we learned during the programme .
no evident problems were identified regarding relations with other staff in the areas of cooperation and professional respect .
in contrast , several comments in the free - text sections of the surveys indicated that the alumni felt themselves to be unprepared for the loneliness of leadership and that they had to be much firmer than expected.we are very well trained for working in teams by the methods from the programme , but when i am the only doctor on the er a dark night , i alone must decide what to do.due to the number of similar comments , competence regarding firmness and standing your ground should possibly be considered for inclusion if this study is repeated .
we are very well trained for working in teams by the methods from the programme , but when i am the only doctor on the er a dark night , i alone must decide what to do .
the present study is consistent with earlier studies reporting that alumni generally feel themselves unprepared for dealing with critical and stressful situations .
they feel uncomfortable with their competence regarding prioritisation , stress management and issues regarding clinical leadership . however , the overall results suggest a rather consistent view that these skills are
one of the interviewed alumni who had been working for 2 years remarked : this is not possible to learn or train for prior to working , and then things sort themselves out after a while .
you are less stressed out , can prioritize better , and when your confidence increases , the night duty feels ok and even exciting at times.goldacre et al .
( 4 ) asks whether mismatches between education and work are short - term hindrances rather than critical flaws of undergraduate education .
some comments in the free - text sections as well as interviews with more experienced alumni suggest that these problems sort themselves out over a relatively short period of time . however , it may be possible to prepare students better for stressful situations in the first year of employment .
there is evidence that shadowing a physician whose post a student is to take over can provide more security and familiarity ( 5 ) .
this is not possible to learn or train for prior to working , and then things sort themselves out after a while .
you are less stressed out , can prioritize better , and when your confidence increases , the night duty feels ok and even exciting at times . concerning life - long learning skills ,
the alumni highlighted a need for more training in finding and understanding scientific articles and in developing their critical thinking skills .
nevertheless , an important finding was that the alumni generally did not consider that an ability to actually do medical research was an important competence as a physician .
it seems clear that a majority of the alumni picture their future careers in clinical practice rather than in research .
a 30 ects - credit student research project is currently scheduled for inclusion in the revised curriculum , with the expectation that this will raise students ' interest in medical research .
the author would like to stress that interpretation of means in tables 15 should be done relatively rather than absolute . viewing the mean ratings as absolute would give rise to unreasonable conclusions .
more of everything ( since almost all competencies have been given positive numbers on preferred coverage ) .
instead , interpretation of data has consistently been done relatively , so that a high rating of one competence only means something in so far as it is rated higher than other competencies .
the following limitations of the study are noted : all interviews and subsequent analysis were carried out by the author alone .
ideally , more researchers would have been involved in the process of selecting items for the survey .
additionally , this study has explored the views of graduates of a single swedish medical school .
even if the results are perhaps most relevant to consider for the educators at that particular institution , there are features of this study that make results more general .
the study does not ask questions about particular features of local curriculum but rather of general competences related to the first years of medical practice .
in addition , as all swedish physicians are accredited to work at the hospital of choice in sweden as well as in other eu countries , the curricula have to be similar enough to make the results presented in this study possible to apply to other institutions .
graduates are seldom of the view that their education has provided them with a key set of skills and competences for their chosen career . nevertheless
, medical training programmes may hope to be better attuned to the needs of the trainee .
medical students , in view of long periods of internship combined with a tradition of clinical supervision , are likely to be better prepared for their future work than are students in other fields . however , despite these advantages , the alumni respondents in this study expressed several concerns about their preparedness for work as a physician . as noted by some alumni , perhaps the perfect preparation for work as a physician is not attainable .
real - life situations such as encounters with sick patients , decision - making under pressure and conflicts with colleagues can not be easily or realistically simulated .
in addition , it may not be enough to discuss these situations in class or to read about them in books .
it is therefore likely that the perfect medical curriculum that fully prepares graduates for working life may be impossible to realise .
the views of the alumni are likely to be helpful when revising the content of the medical curriculum .
even so , it is important to bear in mind that there may be other equally important views regarding what medical education should focus upon .
what do senior physicians consider to be the most important areas for inclusion ; what in retrospect did they find most useful in their education ?
finally , in addition to preparation for work , are there other aims or agendas that must shape the medical curriculum ? further studies will be required to address these issues .
the author has not received any funding or benefits from industry to conduct this study .
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### Summary:
: backgroundalthough medical programmes are often thoroughly evaluated , these evaluations more seldom include workplace points of view .
the present study focuses on how well a swedish medical programme was judged to prepare students for work as a physician.methodsthirty-two competences in physicians ' work were identified through interviews .
a subsequent questionnaire was completed by 123 programme alumni who had worked for 12 years in different parts of the country .
alumni were asked to rate the importance of each competence , their self - assessed competence as well as how these competences were addressed during their medical training.resultsthe subsequent analysis identified areas where their training programme , according to the alumni , failed to prepare them satisfactorily .
problem areas included competences in clinical skills , handling stressful situations and in applied rather than foundational knowledge about common symptoms and diseases.conclusiondespite extensive practical training , medical education still faces some problems in the transition from education to work .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: this procedure is performed to improve nasal function or for cosmetic reason . during the past ten years
the patients usual complaint is expressed as a sense of disharmony or inappropriateness of the shape of the nose relative to the rest of the face .
the motivation for seeking rhinoplasty arises from a culturally inspired desire for beauty and attractiveness for their own sake or as a recognized preliminary necessity to make friends and attract the opposite sex .
mayer found that 16 out of 30 patients ( 53 percent ) who seeking rhinoplasty had psychological problems .
one patient was diagnosed as psychotic and 2 as severely neurotic , the one having a depressive reaction and the other an obsessive and phobic reaction.1 recognition of patients with psychological problems which may result in an unfavorable postoperative outcome is essential.2 problems encountered by patients can lead to requests for repeated procedures , depression and adjustment problems , social isolation , familial dysfunction , self - destructive behaviors and anger towards the surgeon and his or her staff.3 problems encountered by the surgeon can include distress to themselves and their colleagues , harassment by patients for further surgical procedures , complaints and legal action.4 the surgical success rate may be increased if we can detect these patients preoperatively and if the patients have enhanced self - knowledge in the postoperative period.5 there is no consensus about the psychological aspects of rhinoplasty in the literature .
disagreement persists as to whether these patients are psychologically stable or have psychiatric disorders.6 some studies found that patients seeking cosmetic surgery do not express greater psychopathological abnormalities than controls,7,8 while the other studies found that this group of patients expresses greater psychopathological abnormalities.911 to our knowledge , there are no reports on the psychological aspects of rhinoplasty in patients of asian ethnicity .
the objective of this study was to evaluate the psychological status of patients seeking rhinoplasty , compared with normal population .
we prospectively collected data from patients presenting themselves for elective rhinoplasty in srinagarind hospital , khon kaen university , thailand and our private clinics from august to december 2010 .
all patients who fell into this category were asked to complete a self - administered psychological test questionnaire ( ghq-28 ) preoperatively .
we assumed that the age range of those seeking rhinoplasty was between adolescence and middle age and that the individuals were physically active .
the control group was randomly selected from students and university staff who were comparable in terms of age and physical status .
the thai general health questionnaire is a validated tool for detecting psychiatric disorders in non - psychiatric clinical settings and communities for thais.12 we selected the 28 items version of the general health questionnaire ( ghq-28 ) , which is the most popular version and has sub - scales to detect the main psychological components ( somatic symptoms , anxiety / insomnia , social dysfunction , severe depression ) . a cut - off point of 6
statistical analysis were performed using stata ( version 10 , statacorp lp , texas , usa ) .
the mental health status was compared between the study and control group by using relative risk with 95% confidence interval .
the student s t - test and chi - square test were used as appropriate .
we recruited 196 participants . of these , 98 were patients seeking rhinoplasty and 98 were controls .
the mean ages were 23.69 and 29.28 years for the rhinoplasty group and control group respectively .
more than a half of the individuals in both groups had earned a bachelor degree ( table 1 ) .
the study group had a 5.5 times ( 95% ci 1.25 to 24.17 , p = 0.01 ) higher risk of poor mental status when compared with controls ( table 2 ) .
we also divided the poor mental status participants into four groups , according to the ghq-28 scores , and found that participants seeking rhinoplasty were prone to have anxiety , insomnia and social dysfunction 2 times more than controls ; this difference is statistically significant ( p = 0.03 and 0.04 respectively ) ( table 3 ) .
it is the most conspicuous structure in the human body and never covered by clothes . at puberty there
begins a period of increased nasal growth13 which continues through adolescence and results in a structure which is capable of being feminine or masculine in its contours and proportions.10 augmentation rhinoplasty is the common procedure for non - caucasian noses.14,15 asians generally have a shorter , wider and less projecting nose .
reduction rhinoplasty , with dorsal hump reduction and some form of lower lateral cartilage reduction , is more popular in caucasians.1618 the dorsum and tip projection are the main area of concern for most patients in our area.19 the patients usually feel lack of self - esteem when faced with other people.20,21 we conducted this prospective study to evaluate the psychological status in patients seeking a rhinoplasty operation .
it is a general phenomenon that people seeking plastic surgeries are in the higher possibility to have psychological problems . among patients seeking a consultation for a cosmetic procedure , up to 47.7 percent meet criteria for a mental disorder.22 however , there is no consensus about the psychological aspects of rhinoplasty in the literature . to our knowledge
we found that patients seeking rhinoplasty were statistically more prone to have psychological problems than controls .
our results disagree with those of a previous report that found no statistically significant higher psychological problem in rhinoplasty patients.8 this may be because our sample size was large enough to detect a clinically important effect . this study may have limited application in western countries , due to differences in anatomical and cultural motivations .
patients with psychological abnormalities are more likely to report unfavourable results.2 realistic expectations and motivational factors should be sought and thoroughly evaluated by surgeons ( eg , checklists , interview ) during their initial encounter with the patient .
if any doubt exists , it would be propitious to allow a period of reflection and contemplation on the part of the patient , schedule a second interview , or request a psychiatric consultation before any firm decision for surgery is made.23
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### Summary:
: background : rhinoplasty is the most common operation performed by our facial plastic surgery unit .
recognition of patients with psychological problems which may result in an unfavourable post - operative outcome is essential .
the objective of this study was to evaluate the psychological status of patients seeking rhinoplasty , compared with the general population.methods:we prospectively collected the data from patients who requested rhinoplasty in srinagarind hospital , thailand and controls .
we used the general health questionnaire-28 ( ghq-28 ) for psychological problems detection.results:we included a total of 196 participants , 98 in study group and 98 in control group .
the study group has a 5.5 times ( 95% ci 1.25 to 24.17 , p = 0.01 ) higher risk for poor mental status more than control group.conclusions:the patients seeking rhinoplasty group were more likely to have psychological problems when compared with the control group .
surgeons should be aware of the patient s psychological status before performing the operation .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: historically , mitochondrial ros ( mtros ) production and oxidative damage have been associated with aging and age - related diseases such as parkinson s disease ( morais et al . , 2014 ) .
in fact , the age - related increase in ros has been viewed as a cause of the aging process ( forster et al . , 1996 ) while mitochondrial dysfunction is considered a hallmark of aging ( lpez - otn et al . , 2013 ) , as a consequence of ros accumulation . however , pioneering work in caenorhabidits elegans has shown that mutations in genes encoding subunits of the electron transport chain ( etc ) ( dillin et al . , 2002 ) or genes required for biosynthesis of ubiquinone ( asencio et al . , 2003 , wong et al . , 1995 )
the lifespan extension conferred by many of these alterations is ros dependent , as reduction of ros abolishes this effect ( lee et al .
moreover , chemical inhibition of glycolysis or exposure to metabolic poisons that block respiratory complex i ( ci ) ( rotenone , paraquat , or piericidin a ) or complex iii ( ciii ) ( e.g. , antimycin a ) also prolong lifespan in c. elegans in a ros - dependent manner ( dillin et al .
, 2002 , schmeisser et al . , 2013 , schulz et al . , 2007 , yang and hekimi , 2010a ) .
various studies have shown that ros act as secondary messengers in many cellular pathways , including those which protect against or repair damage ( ristow and schmeisser , 2011 , yee et al . , 2014 ) .
ros - dependent activation of these protective pathways may explain their positive effect on lifespan .
the confusion over the apparent dual nature of ros may , in part , be due to a lack of resolution as without focused genetic or biochemical models it is impossible to determine the site from which ros originate .
a promising path to resolving ros production in vivo is the use of alternative respiratory enzymes , absent from mammals and flies , to modulate ros generation at specific sites of the etc ( rustin and jacobs , 2009 ) .
the alternative oxidase ( aox ) of ciona intestinalis is a cyanide - resistant terminal oxidase able to reduce oxygen to water with electrons from reduced ubiquinone ( coq ) , thus bypassing ciii and complex iv ( civ ) ( fernandez - ayala et al . , 2009 ) .
ndi1 is a rotenone - insensitive alternative nadh dehydrogenase found in plants and fungi , which is present on the matrix - face of the mitochondrial inner membrane where it is able to oxidize nadh and reduce ubiquinone , effectively bypassing ci .
2010 ) have demonstrated that allotopic expression of ndi1 in drosophila melanogaster can extend lifespan under a variety of conditions and rescue developmental lethality in flies with an rnai - mediated decrease in ci levels . to determine the role of increased ros production in regulating longevity , we utilized allotopic expression of ndi1 and aox , along with drosophila genetic tools to regulate ros production from specific sites in the etc . we show that ndi1 over - reduces the coq pool and increases ros via reverse electron transport ( ret ) through ci .
importantly , restoration of coq redox state via ndi1 expression rescued mitochondrial function and longevity in two distinct models of mitochondrial dysfunction .
initially , we asked whether increased mtros production is a general feature of aging in flies by measuring ros production in fly brains using two fluorescent probes , mitosox ( for mitochondrial matrix ros ) and h2dcf ( for total cellular ros levels ) , and a redox - sensitive gfp based reporter for in vivo mitochondrial h2o2 ( mth2o2 ) ( mtorp1-rogfp ) ( albrecht et al . , 2011 ) .
we observed a consistent increase in ros in old flies in two wild - type strains ( dahomey and oregon r ) ( figures 1a , 1b , s1a , and s1b ) . in dahomey flies
, we observed that with age , dorsal flight muscle mitochondrial ultrastructure became increasingly rounded and swollen with the appearance of perturbed cristae structure at 75 days ( d ) ( figures 1c , s1c , and s1d ) .
further , in both strains , high - resolution respirometry and enzymatic assays showed a decrease in ci - linked respiration ( ci - respiration from here on ) and in the enzymatic activity of ci and ciii ( figures 1d and 1e ) .
aconitase activity initially decreased from 5 to 25 d but remained constant as the flies continued to age ( figure s1e ) . at this age
( 25 d ) , no decrease in locomotive activity ( figure s1f ) or increase in ros ( figures 1a , 1b , s1a , and s1b ) was observed .
western blot analysis showed that only the levels of ci and aconitase were significantly affected with age ( figures 1f , 1 g , and s1g s1j ) .
however , ci concentration was decreased at very late ( 75 and 85 d ) ages , suggesting a shift in mitochondrial metabolism supported by an increase in impl3 ( lactate dehydrogenase a homologue ) expression ( figure 1h ) . based on our previous results
, we hypothesized that decreasing ros and compensating for a loss in ci respiration would extend lifespan .
we and others have previously reported that allotopic expression of ndi1 decreases ros in mitochondria isolated from old individuals ( sanz et al . , 2010 ) .
surprisingly , measurement of ros levels using mitosox , h2dcf , and mtorp1-rogpf revealed in fly brains a ndi1-mediated increase in ros that was partially abolished by aox expression ( figures 2a , 2b , and s2b ) .
neither ndi1 nor aox had any detectable effect on respiration alone ( figure s2c ) .
strikingly , the increase in ros elicited by ndi1 was similar to that caused by feeding flies with a dose of rotenone able to significantly inhibit ci ( figures s2d s2f ) .
aox expression completely abolished lifespan extension conferred by ndi1 ( figure 2c ) , suggesting that ndi1 lifespan extension is dependent on over - reduction of the etc and consequently increased ros levels .
expression of aox alone had a mild effect on wild - type ros production ( figures 2a , 2b , and s2b ) and did not shorten lifespan ( figure 2c ) .
yang and hekimi , 2010a ) , we hypothesized that the observed ndi1-mediated increase in ros production was perhaps a signal that led to an extension in lifespan . to further test this possibility
, we generated another model in which we expressed ndi1 but alleviated the over - reduction of the etc and prevented additional ros formation by decreasing ci levels ( via knockdown of nd-39 ( cg6020 ) , the fly homolog of ndufa9 , figures 2d and s2 g ) .
these flies , hereafter referred to as ci > 2;ndi1 > dagal4 flies , had reduced ci activity ( figure s2h ) and , like aox > 2;ndi1 > dagal4 flies , had decreased ros levels compared to ndi1 expressing flies ( figures 2e , 2f , and s2i ) . as with aox expression ,
ci knockdown suppressed lifespan extension conferred by ndi1 ( figure 2 g ) , further supporting the hypothesis that over - reduction of the etc downstream of ci can increase ros production and extend lifespan . to verify a change in the redox state of coq , we measured the percentage of reduced and oxidized coq in ndi1-expressing and controls flies , observing a remarkable increase in the proportion of reduced coq in ndi1 flies ( figure s2j ) .
as expected , co - expression of ndi1 with aox or decreasing ci levels restored the redox state of coq to normal ( figure s2j ) . to exclude the possibility that the ros signal originated downstream of coq
, we knocked down the civ subunit , levy ( cg17280 , fly homolog for human cox6a ) , to over - reduce cytochrome c and ciii in aox - expressing flies ( hereafter referred to as civ > 2;aox > dagal4 flies ) .
aox was required in this model to maintain the coq pool oxidized ( figure s2k ) and prevent ret through ci .
civ > 2;aox > dagal4 flies displayed significantly decreased civ - linked respiration ( figure s2l ) but normal food intake and body weight ( figures s2 m and s2n ) .
however , stabilization of reduced cytochrome c did not produce a ros signal ( figure s2o ) or extend lifespan ( figure s2p ) , supporting a site specificity for this longevity - extending ros signal . finally , to confirm that ros and not changes in the redox state of coq were responsible for ndi1 lifespan extension , we co - expressed ndi1 with a mitochondrially targeted catalase ( mtcat ) ( mockett et al . , 2003 )
expression of mtcat restored h2o2 levels to normal ( figures 2h and 2i ) without , as expected , affecting superoxide ( figure 2j ) or the redox state of coq ( figure s2r ) . since lifespan of mtcat > 2;ndi1 > dagal4 flies was similar to control flies
, we can conclude that mtros are responsible for ndi1 lifespan extension ( figure 2k ) .
significantly , expression of mtcat alone reduced both mth2o2 levels ( e.g. , more than aox ) and lifespan ( figure 2k ) , indicating that excessive reduction of ros can be detrimental for lifespan . to investigate how ndi1 increases ros , we dissected mtros production in vivo by adding specific etc inhibitors to the food of ndi1 flies ( figure 2l ) .
rotenone is a specific ci q - site inhibitor that prevents ci binding to coq .
rotenone feeding in wild - type flies caused a significant decrease in ci nadh : decylubiquinone ( nadh : dq ) activity , while non - physiological nadh : hexaammineruthenium ( nadh : har ) activity , which is not dependent on coq binding , was unaffected ( figure s2f ) , indicating that levels of ci were not reduced .
rotenone feeding significantly decreased ros production in ndi1 expressing flies ( figures 2 m and s2 t ) , while increasing it in wild - type flies ( figures s2d and s2e ) , suggesting that ret is the source of ndi1-mediated ros production .
ret in vitro is strongly membrane potential - dependent requiring a high enough membrane potential so as to make forward electron transport through ci unfavorable . collapsing membrane potential by feeding fccp ( a h ionophore ) like rotenone depleted ndi1-mediated ros production , whereas myxothiazol ( a ciii qo site inhibitor that blocks entry of electrons into ciii ) did not ( figure 2 m ) , strongly supporting the existence of ret in vivo in ndi1-expressing flies .
interestingly , we have recently shown that during ischemia reperfusion ret is the main source of superoxide in vivo ( chouchani et al . , 2014 ) .
if ros signaling is site specific and works to maintain mitochondrial function , we hypothesized that it should be possible for beneficial ros signaling to rescue deleterious mitochondrial phenotypes induced by oxidative stress . to test this
, we knocked down sod2 ( the only mitochondrial matrix superoxide dismutase in drosophila ; figure s3a ) and observed a significant increase in levels of superoxide in fly brains ( figures 3a and 3b ) while levels of mth2o2 were significantly decreased ( figure s3b , right ) , which is in agreement with the reported positive correlation between sod2 activity and mth2o2 levels ( rodrguez et al . , 2000 ) .
sod2 knockdown dramatically shortened longevity ( figure 3c ) and , in addition , severely affected ci - respiration ( figure 3d ) , with a significant decrease in the enzymatic activity of ci , cii , and aconitase ( figure 3e ) .
western blot analysis showed that levels of ci and aconitase were unchanged ( figure s3d ) , suggesting that , as has been shown for aconitase , high levels of superoxide inhibit the activity of these enzymes ( gardner and fridovich , 1992 ) .
mitochondrial ultrastructure from dissected dorsal flight muscle was unaffected by sod2 knockdown ( figures s3e and s3f ) , showing that high levels of superoxide recapitulate some ( e.g. , increased mortality and , decreased ci activity ) , but not all features of aging ( e.g. , alteration in mitochondrial morphology ) .
although high levels of superoxide in the absence of sod2 are detrimental , this situation is unlikely to occur in vivo in aged flies since sod2 levels ( figures s1 g and s1h ) and superoxide dismutase activity ( sohal et al . ,
1990 ) are reported to increase with age . to assess the role of coq - mediated ros production , we expressed either aox or ndi1 , which have opposite effects on mtros and the redox state of the coq pool , in sod2 knockdown flies .
aox expression rescued the increase in superoxide elicited by sod2 knockdown , but further decreased the concentration of mth2o2 ( figures 3a , 3b , and s3b ) , and did not extend lifespan or rescue decreased etc function ( figures 3c3e ) .
ndi1 expression partially rescued lifespan ( figure 3c ) , along with ci respiration and activity ( figures 3d and 3e ) , despite further increasing ros levels ( figures 3a , 3b , and s3b ) . to confirm that ndi1 rescue of lifespan was mediated via ret ,
we reduced ci levels in sod2 knockdown flies expressing ndi1 ( hereafter sod2 > ci ; ndi1 > actgs ) using the inducible geneswitch system ( nicholson et al . , 2008 ) .
strikingly , ndi1 rescue of lifespan disappeared when ret was prevented by depletion of ci levels ( figure s3
these results indicate that sod2 knockdown perturbs coq reduction due to decreased activity of ci and cii , which results in increased superoxide production . in this model ,
re - establishing ret - derived ros with ndi1 rescued ci activity , suggesting that ret may be necessary for the maintenance of mitochondrial function and lifespan extension . in figure 1 , we demonstrated that mitochondrial quality was decreased in aged flies .
interestingly , we also found that levels of pink1 and parkin decreased in parallel over time ( figures 4a and 4b ) .
experimental manipulation of pink1 and parkin levels has previously been shown to alter drosophila lifespan ( rana et al .
decreases in pink1 activity have been related to a loss of ci activity ( morais et al . , 2014 ) , while parkin loss of function mutations cause the accumulation of aberrant mitochondria ( greene et al . ,
2003 ) . strikingly , decreases in pink1 and parkin levels ( figures 4a and 4b ) preceded the decline in respiration observed in old flies ( figure 1d ) .
it has been recently shown that pink1 is required for reduction of coq by ci via phosphorylation of ndufa10 ( morais et al . , 2014 ) , although this was not confirmed by a later report ( pogson et al . , 2014 ) .
since pink1 protein levels decrease with age , we hypothesized that this decrease could be responsible for the decline in ci - respiration . to test the relevance of coq - mediated ros signaling in a more physiological context , we used the inducible geneswitch system to drive expression of an rnai construct against pink1 specifically in adult flies ( pink1 > dags flies , figure s4a ) .
pink1 knockdown did not increase ros levels ( figures 4c and s4b ) ; however , we did observe a significant decrease in ci - linked respiration ( figure 4d ) and nadh : dq ci activity , but not nadh : har activity ( figure 4e ) or ci protein levels ( figure s4c ) , showing that reduction of coq by ci was impaired , as was observed in sod2 knockdown flies ( figures 3d and 3e ) and 50-d - old wild - type flies ( figures 1d and 1e ) .
furthermore , pink1 knockdown flies displayed decreased mobility , time spent flying , and lifespan compared to controls ( figures 4f and 4 g ) . as in sod2 knockdown flies , ndi1-mediated ros production ( figure 4c ) was able to rescue ci - linked respiration and ci activity in pink1 knockdown flies ( figures 4d and 4e ) .
however , unlike in sod2 knockdown flies , cii and aconitase activities were also rescued ( figure 4e ) , suggesting that sod2 may be necessary for ndi1-mediated rescue of cii and aconitase .
locomotive activity , time spent flying , and lifespan were also partially or totally rescued by ndi1 expression in pink1 knockdown flies ( figures 4f and 4 g ) .
interestingly , reduction of ci levels from adulthood decreased ci respiration to levels comparable with 50-d - old wild - type flies and pink1 knockdown flies ( figure 4h ) , whereas locomotion and lifespan were similar to that observed in flies with induction of the driver alone ( compare figures 4i and 4j with figures s4d
moreover , ndi1 did not restore respiration in this background ( data not shown ) , just as it failed to when ci knockdown and ndi1 expression were driven with a stronger driver ( figure s2c ) .
this indicates that the rescue of mitochondrial function conferred by ndi1 in sod2 and pink1 knockdown flies is not due to direct compensation by ndi1 of decreased ci activity , rather ndi1 rescues ci function through over - reduction of the coq pool .
therefore , we propose that over - reduction of coq pool generates , via ret , a ros signal necessary for homeostasis .
interruption in electron flow through ci would prevent coq reduction and thus cause the deleterious phenotypes associated with aging and age - related pathologies . in summary , we show that mtros production increases with age and that un - detoxified ros can be detrimental to drosophila lifespan , while increasing ros production specifically from reduced coq , possibly via ret , acts as a signal to maintain mitochondrial function ( notably ci ) and extend lifespan ( figure s4j ) .
these results are similar to those reported in worms where small doses of rotenone ( schmeisser et al . , 2013 ) , paraquat , and mutations in ci that increase ros extend lifespan ( yang and hekimi , 2010a ) .
however , we did not observe lifespan extension in flies fed with varying doses of rotenone or paraquat ( figures s4h and s4i ) .
it is possible that an intact ci is required for lifespan extension in fruit flies , as metformin , which increases lifespan by blocking ci and increasing ros in worms ( de haes et al . , 2014 ) , fails to do so in fruit flies ( slack et al . , 2012 ) .
if the mechanism we describe here is conserved in mammals , manipulation of the redox state of coq may be a strategy for the extension of both mean and maximum lifespan and the road to new therapeutic interventions for aging and age - related diseases .
female flies were used in all experiments . see table s2 for details of the different fly strains used in this study .
flies were maintained at 25c on standard media in a controlled 12:12 hr light : dark cycle at a density of 20 flies / vial and transferred to new food every 2 to 3 d. for all lifespan experiments , at least 80 flies were used and each experiment was repeated at least two to four times . statistical analysis for all lifespan experiments
mitosox and dichlorofluorescein ( h2dcf ) were used to measure either mitochondrial matrix superoxide or total cellular ros levels , respectively . following dissection ,
fly brains were incubated in mitosox or h2dcf for 10 min and imaged immediately .
for in vivo ros imaging brains dissected from lines carrying the mtorp1-rogfp reporter construct were imaged under ex .
nm / em . 510 nm . the total ( average ) fluorescence intensity of each individual brain imaged was quantified using imagej .
dorsal flight muscles were dissected and suspended in 0.16 m sym - collidine buffer ph 7.4 ( electron microscopy sciences ) supplemented with 5% glutaraldehyde ( fixing solution ) .
muscle sections were imaged using a jeol jem-1400 plus transmission electron microscope fitted with an 11 mpx olympus quemesa digital camera .
the images were manually segmented using gimp ( http://www.gimp.org/ ) , and the resulting segmented image was further analyzed using the imagej particle analysis tool .
state 4 respiration was initiated by addition of 5 mm pyruvate and 5 mm proline .
ci - linked respiration ( ci+ciii+civ in figures ) was inhibited by adding 0.5 m rotenone , and ciii - linked respiration was stimulated by addition of 20 mm glycerol 3-phosphate ( ciii+civ in figures ) .
ciii - linked respiration was inhibited with the addition of 2.5 m antimycin a. civ respiration ( civ in figures ) was initiated by the addition of 4 mm ascorbate and 2 mm tmpd .
all assays were adapted to a 96-well plate with a final volume of 300 l .
ci ( nadh dehydrogenase or nadh : hexaammineruthenium ( har ) oxidoreductase activity ) , cii ( malonate - sensitive succinate : dichlorophenolindophenol ( dcpip ) oxidoreductase activity ) , ciii ( antimycin - sensitive decylubiquinol : cytochrome c oxidoreductase activity ) , civ ( potassium cyanide - sensitive cytochrome c oxidase activity ) , cv ( oligomycin - sensitive nadh - coupled atpase activity ) , and mitochondrial aconitase activities were measured according to standard procedures .
all etc activities were normalized to mitochondrial density calculated by citrate synthase ( cs ) .
live tracking of the flies was performed using a high - sensitivity the imaging source usb camera ( equipped with a computar 1/3 varifocal lens [ 2.812 mm ] ) , for 60 min per experiment .
the recorded video files were used to calculate distance moved per hour for each fly using the ethovision xt video tracking software ( noldus information technology ) .
data were analyzed with prism 6 ( graphpad ) using either one - way anova with newman - keul s post hoc test or using the unpaired student s t test .
p < 0.05 was taken as statistically significant . in all figures p < 0.05 , p < 0.01 , p < 0.005 , and p < 0.001 denotes significant difference from all other groups unless otherwise indicated by line art .
a. sanz designed and performed experiments , supervised the project , and co - wrote the manuscript .
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### Summary:
: summaryincreased production of reactive oxygen species ( ros ) has long been considered a cause of aging .
however , recent studies have implicated ros as essential secondary messengers . here
we show that the site of ros production significantly contributes to their apparent dual nature .
we report that ros increase with age as mitochondrial function deteriorates .
however , we also demonstrate that increasing ros production specifically through respiratory complex i reverse electron transport extends drosophila lifespan .
reverse electron transport rescued pathogenesis induced by severe oxidative stress , highlighting the importance of the site of ros production in signaling .
furthermore , preventing ubiquinone reduction , through knockdown of pink1 , shortens lifespan and accelerates aging ; phenotypes that are rescued by increasing reverse electron transport .
these results illustrate that the source of a ros signal is vital in determining its effects on cellular physiology and establish that manipulation of ubiquinone redox state is a valid strategy to delay aging .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: cardiac performance is determined by the coordinated and dynamic interaction between several cell types and various components of the extracellular matrix ( ecm ) . in response to stress conditions such as acute ischemia , pressure and volume overload and congenital and acquired valvular cardiac diseases
, heart evokes a diverse and complex array of cellular responses involving both cardiomyocytes ( cms ) and nonmuscle cells that initiate and sustain a process of structural remodeling of the myocardium .
cardiac remodeling ( cr ) is clinically manifested by changes in the size , shape , and function of the heart . from a histopathological point of view
, it is characterized by structural derangements and rearrangements of the tissue components of the myocardial chamber wall and it involves cm death , cardiofibroblast ( cf ) proliferation , fibrotic processes , rarefaction of coronary vasculature , and cm hypertrophy . ultimately cardiac remodeling leads to heart failure ( hf ) , a final threatening condition with poor prognosis .
prolonged treatment with angiotensin converting enzyme inhibitors and beta - adrenergic receptor antagonists ( -blockers ) has partially attenuated the onset and the progression of the adverse process ; nonetheless the associated morbidity and mortality remain unacceptably high .
undoubtedly , new and more efficacious interventions are highly desired to counteract the progression towards hf . moreover
, an inexpensive treatment targeting the fundamental causes of myocardial remodeling would be more appealing since this would combine an improved outcome with reduced health care costs . as in the case of
aspirin and -blockers , the application of existing drugs to new target of diseases and disorders may be a valid alternative to the development of new drugs and pharmaceutical formulas . over the past few years , a virtual explosion of new knowledge took place in many biomedical fields . among these , a primary position is occupied by the physiological and pathophysiological relationship that exists between the thyroid gland and the cardiovascular ( cv ) system .
the influence of thyroid hormones ( ths ) on the cv system involves the regulation of key processes related to maintenance of cardiac contractility , electrophysiological functions and cardiac structure [ 24 ] . to exert its effects
, the principal product of thyroid gland t4 must be converted into its biologically active form , t3 , by type 1 and type 2 deiodinase enzymes in peripheral tissues .
current evidence shows that t3 levels significantly decline after myocardial infarction ( mi ) both in animal models and in patients due to the reduced conversion of t4 into t3 and the concomitant increased conversion of t4 into the inactive rt3 by the upregulation of type 3 deiodinase .
although low t3 state has been long time considered a beneficial adaptive mechanism activated under stress conditions , several studies have shown that the low t3 syndrome may have an adverse prognostic impact on various acute and chronic cardiac disorders [ 711 ] .
importantly , many of the cardiac alterations observed in subclinical hypothyroidism are reversed once thyroid function has been normalized [ 12 , 13 ] . on the basis of the evidence obtained from cells , animals , and even humans finding , it seems likely that timely treatments targeting the th signaling may promote endogenous regeneration of the damaged myocardium [ 1416 ] .
th plasma levels increase promoting the switch from the fetal into the adult isoforms of key myocardial proteins such as myosin heavy chain ( mhc ) isoforms and . in the early phases of cardiac disease progression , the activation of a fetal gene reprogramming occurs as compensatory response that prompts cell de - differentiation . in this context
, low t3 level may favour cardiac repair but , in the long term , it can lead to end - organ damage .
we will briefly review the major reported evidence supporting the hypothesis that a low thyroid signaling , namely , the one resulting in a postischemic myocardium is maladaptive ( i.e. , hypothyroid heart ) and th replacement , may counteract progression toward cardiac pathological remodeling and failure by inducing beneficial effects on myocyte survival , reduction of interstitial fibrosis in the non - infarcted myocardium , prevention of microvascular loss , and blunting pathological hypertrophy .
cm death is now recognized as a critical factor in the development of heart diseases [ 1822 ] .
myocyte apoptosis is an important event after acute myocardial infarction and may be responsible for a significant portion of myocyte death during the acute ischemic stage , as well as progressive loss of surviving myocytes during the subacute and chronic stages [ 23 , 24 ] .
in addition to the infarcted area , profound ongoing myocyte apoptosis has been demonstrated in the border area after mi [ 23 , 24 ] . moreover ,
inhibition of myocyte apoptosis improves left ventricular ( lv ) remodeling and cardiac function .
one of the major cardioprotective mechanisms involves the activation of phosphatidylinositol 3 kinase / protein kinase b ( pi3k / akt ) and extracellular regulated kinase 1 and 2 ( erk1/2 ) signaling .
t3 replacement in cm cultures prevented serum starvation - induced apoptosis through an akt - mediated mechanism .
accordingly , t3 can inhibit cm apoptosis and activate akt signaling in the border area after acute myocardial infarction . in a langendorff rat heart model of ischemia reperfusion injury ,
t3 treatment during the reperfusion period significantly enhanced the recovery of function and reduced myocyte apoptosis through the activation of the pi3k / akt and erk1/2 axis .
an emerging mechanism of cardioprotection implies regulation of mitochondria function and biogenesis . actually , besides their contribution to bioenergetic function ,
mitochondria are integrally involved in regulating myocardial calcium flux , myocyte cell death and remodeling events , reactive oxygen species ( ros ) generation and antioxidant response , and in furnishing cardioprotective responses to physiological insults .
not surprisingly , mitochondrial dysfunction plays a critical role in the occurrence and progression of hf [ 28 , 29 ] underlining the importance of new therapeutic strategies aiming to preserve mitochondrial integrity .
ths are well - known regulators of mitochondrial biogenesis and function [ 30 , 31 ] .
changes in thyroid status are associated with bioenergetic remodeling of cardiac mitochondria and profound alterations in the biochemistry of cardiac muscle , with repercussions on its structure and contractility .
a recent study reported that early t3 administration prevents cardiac remodeling and decreases death of stressed cms by rescuing mitochondria function and biogenesis in a model of postischemic hf .
the proposed mechanisms underlying the t3 beneficial effects are the upregulation of mitochondrial transcription factor a ( mttfa ) and peroxisome proliferator activated receptor gamma coactivator 1 alpha ( pgc-1 ) and the opening of the protective mitochondrial atp - dependent potassium channel ( mitok - atp ) .
recently , it has been demonstrated that hf can also be aggravated through myocyte necrosis which is largely initiated at the level of the mitochondria following ca overload .
aberrant ca homeostasis is a universal feature of human and experimental hf and is widely attributed to decreased sarcoplasmic reticulum calcium atpase ( serca ) expression or chronic serca inhibition by phospholamban ( plb ) .
reduction of serca level in cardiac disease enhances cytosolic ca overload which besides compromising cm contractility , initiates a pathway cascade leading to mitochondrial dysfunction and necrotic cell death
. ths are well - established potent regulators of serca2a pump expression and cardiac muscle contractility .
interestingly , there is a very close correlation between the rate of sarcoplasmic reticulum ca uptake and the plb to serca2a ratio in hypothyroid , euthyroid , and hyperthyroid hearts , and this determines the contractile effectiveness of the heart .
evidences has provided that the diminished expression of the serca associated to hypothyroidism can be restored by correcting the hormonal disorder through th therapy [ 36 , 37 ] . moreover , in an ex vivo study on human myocardial biopsies , t3 supplementation at physiological doses restored ca handling which was altered after long - term t3 deprivation .
thus , it is likely that the low - t3 state , observed during the evolution of cardiac disease , may favor adverse remodeling by inducing a reduction of mitochondrial biogenesis and function and by enhancing the activation of necrotic pathways driven by mitochondrial ca overload .
recently , some mirnas have been identified with critical importance in inhibiting mitochondrial - mediated cell death pathways [ 39 , 40 ] . in particular mirna-30 family members
are abundantly expressed in the heart , but their levels decreased in response to cm oxidative stress or myocardial ischemia / reperfusion [ 4143 ] .
preliminary , unpublished data from our laboratory strongly indicate that in a rat model of myocardial ischemia / reperfusion , early three - day infusion of physiological doses of t3 is able to maintain myocardial contractility and mitochondrial function in the border area of the ischemic injury and that this effect is linked to the maintenance of mirna-30a expression levels . in summary ,
overall available data support the hypothesis that an antiapoptotic and antinecrotic effect of th may be important in preventing adverse cellular responses in cardiac disease and that this effect seems to be mediated , at least in part , by a th action on cardiac mitochondrion .
non - cm cell types , such as fibroblasts , endothelial cells , smooth muscle cells , and immune cells , which play a fundamental role in both physiological and pathological condition .
as reported by many investigators , in the clinical context of myocardial damage , cf undergoes a transformation from a quiescent cell , primarily involved in the extracellular matrix homeostasis , to myocf , an activated cell type which plays a central and dynamic role in wound healing .
the process of cellular metamorphosis is associated with several different phenotypic endpoints including cellular proliferation , chemotactic migration , ecm remodeling and the coordinated production of specific cytokines , interleukin ( e.g. , il-1 and il-6 ) , chemokine , and growth factors ( e.g. , tnf- and tgf- ) [ 4450 ] . among the main effects of ecm remodeling , the continuous synthesis and deposition of interstitial collagen , along with deregulation of matrix metalloproteinases ( mmps ) and tissue inhibitors of mmps ( timps ) , impair the cardiac diastolic function and compromises the systolic mechanics leading to hf progression [ 47 , 5154 ] .
ths seem to play a pivotal role in ecm homeostasis . however , the only available data deal with hyperthyroidism or hypothyroidism experimental models .
it has been reported that mrna for collagen type i , the major fibrillar collagen in the heart , is downregulated by th treatment both in the myocardium and in cf culture . in similar experiments
th - induced hypertrophy is accompanied by an increase of mmp1 , mmp2 , and timp2 without signs of cardiac fibrosis or inflammatory response [ 56 , 57 ] .
t3 can also repress the activation of some transcription factors such as activator protein 1 ( ap-1 ) [ 58 , 59 ] that are involved in activation of mmps and collagen gene expression .
hypothyroidism condition increased expression of collagen precursor in rat cf culture , while th treatment reversed this effect only in the presence of thyroid hormone receptor beta and alpha ( tr and tr ) . altogether
, the above findings suggest that th may selectively blunt the onset of cardiac fibrosis and play an important role in regression of cardiac fibrosis via endocrine pathways .
however , the effect of physiological th treatment on ecm remodeling in oxidative stressed myocardium has never been reported .
we believe that unravelling this important issue with further translational studies may be crucial in the perspective of a new therapeutic approach with th in cardiac disease .
recent studies have shown that the expression of several mirnas which play direct and indirect roles in the regulation of cardiac fibrosis is altered following mi or other fibrotic pathologies [ 41 , 61 ] . among them
mirna-29 family plays an important antifibrotic role and is downregulated in models of cardiac ischemia and hypertrophy [ 43 , 61 ] .
the mirna-29 family comprises three members mirna-29a , 29b , and 29c , which are all predominantly expressed in fibroblast within the heart .
knockdown of mirna-29 resulted in the upregulation of collagens in the heart , suggesting that mir 29 indeed acts an inhibitor of cardiac fibrosis .
unpublished data from our laboratory indicate that early t3 replacement after ischemia / reperfusion in rat induces the expression of mirna-29c which is associated to the maintenance of cardiac performance .
globally , these results suggest that the downregulation of mirna-29 contributes to cardiac fibrosis and that strategies to maintain mirna-29 expression may be beneficial in the setting of fibrotic disease . besides cms and cfs , both endothelial cells and microvasculature play a key role in regulating and maintaining cardiac function .
insufficient angiogenesis is one of the causes of myocardial dysfunction and there is strong evidence of reduced myocardial capillary density in several human heart disease such as aortic stenosis , dilated cardiomyopathy , and ischemic cardiomyopathy .
th - induced sprouting of endothelial cells has been documented in different models such as the chick chorioallantoic membrane and cultured left ventricle tissue [ 66 , 67 ] .
the molecular mechanism of the proangiogenic action of th is initiated nongenomically at plasma membrane of endothelial cells ( ecs ) through the interaction with integrin v3 .
the transduction of the hormone signal is mediated by mitogen - activated protein kinase erk1/2 and results in downstream transcription of several angiogenesis - relevant genes such as basic fibroblast growth factor ( bfgf ) and vascular endothelial growth factor ( vegf ) .
this pro - angiogenic effect may be potentiated by a crosstalk between the integrin receptor for th and the vegf and bfgf receptors .
iodothyronines seem also to stimulate angiogenesis through enhancing the expression of hypoxia inducible factor 1 alpha ( hif-1 ) , a transcription factor important for ischemia - induced coronary artery collateralization [ 22 , 70 ] .
the proposed mechanism involves the interaction of the hormone with tr in the cytosolic compartment and the activation of pi3k signaling [ 71 , 72 ] .
the contractile component of blood vessel , the vascular smooth muscle cell , is also influenced by th action . in a previous study
, we reported that t3-induced arteriolar dilation in a nongenomic way , likely enhancing nitric oxide production . in this model
, the local conversion of t4 to t3 appears to be crucial for the dilation induced by t4 administration .
th treatment of coronary smooth muscle cell isolated from tr-ko mice decreased contractility by enhancing k channel activity .
moreover , t3 administration suppressed cytokine expression and vascular smooth muscle cell proliferation in an experimental model of coronary atherosclerosis .
this effect was , at least in part , dependent on the inhibition of angiotensin ii signalling .
the important role of th in the regulation of cardiac vasculature is further confirmed by the impaired coronary artery contraction , the reduced density of small arterioles , and the altered echocardiographic parameters reported in low thyroid function .
cr observed during progression of cardiac disease includes concentric and eccentric hypertrophy of surviving cms .
the pathological hypertrophy of the human heart has been described as the single most important independent risk factor for increased mortality [ 77 , 78 ] .
cardiac hypertrophy develops as an overall adaptive response to stress conditions ; signaling pathways activated in cms and non - cms by various noxious stimuli ( e.g. , neurohormonal stimulation and oxidative stress ) should be tightly regulated to maintain cardiac homeostasis and prevent pathological cardiac hypertrophy .
maladaptive hypertrophy is associated with impaired myocardial capillary growth , unfavourable changes in ecm composition and fibrosis .
it is sustained by elevation of angiotensin ii , tnf- , and catecholamine plasma levels and seems to involve downstream pathways such as c - jun n - terminal kinase and p38 [ 79 , 80 ] . on the contrary ,
physiological hypertrophy is characterized by parallel growth of vascular network , absence of fibrosis , and lack of fetal gene reprogramming .
th enhances many of the signaling pathways activated during cardiac physiological growth including pi3k / akt / mtor and gsk3 axes .
ths have been recognized to induce physiological growth of the heart through genomic regulation of specific target genes that encode both structural and functional proteins . in cms ,
th upregulates serca2 , -mhc , na / k - atpase , and k channel while downregulates -mhc , plb , tr1 , and na / ca exchanger .
thus , ths regulate expression of specific genes of critical importance for cardiac contractility and prevent the expression of the fetal gene program characteristic of pathological hypertrophy [ 2 , 84 ] .
the mechanisms that regulate mhc gene switching by th have been the focus of intense interest . besides to genomic action , th can affect myocardial mhc composition through epigenetic modification and mirna regulation . in hypothyroid rat , treatment with trichostatin a , an inhibitor of histone deacetylation ,
together with t3 significantly increased the transcription of -mhc , thus demonstrating a potential role for histones as cofactors in the t3 regulation of cardiac -mhc transcription .
recently , a family of mirnas ( mirna-208a , mirna-208b , and mirna-499 ) , localized within the mhc genes loci , has been shown to form an intricate regulatory network together with their host genes to regulate cardiac hypertrophy [ 86 , 87 ] .
these mirnas are coexpressed and are regulated together with their host mhc genes by the same transcriptional events and signaling pathways .
mir-208a is a cardiac - specific mirna encoded by the -mhc gene . while physiological levels of mir-208a are required for proper cardiac electrical conduction , a fourfold increase of mir-208a induces hypertrophic growth in mice heart by suppressing thyroid hormone - associated protein 1 and myostatin , which are negative regulators of muscle growth and hypertrophy .
these results clearly demonstrate the pathological consequences deriving from the perturbed expression of a single mirna .
deregulation of th signaling in cardiac disease leads to -mhc / mirna-208a inhibition while in vitro treatment with ths significantly upregulates -mhc
mirna-208b and mirna-499 play redundant role in -mhc upregulation and seems to be regulated in the same way by th .
this data suggests that physiological th concentrations are required to ensure proper mirna levels and to preserve mhc composition .
the observation that th activates many of the beneficial aspects of physiological hypertrophy has raised the possibility of their therapeutic utility in the treatment of the post - infarcted heart or in hf [ 4 , 89 , 90 ] .
experimental and clinical studies strongly support the concept that th plays a fundamental role in cardiovascular homeostasis in both physiological and pathological conditions .
increasing evidence indicates that the low th is not simply a consequence of cardiac disease evolution but a permissive state that favours adverse cardiac remodeling and failure .
as summarized in figure 1 , restoration of a normal th profile might counteract the progression of adverse cardiac remodeling by ( 1 ) inhibiting cm death pathways , ( 2 ) reducing fibrous tissue deposition , ( 3 ) improving myocardial perfusion , and ( 4 ) preventing pathological cardiac hypertrophy .
regulation of mirna expression and prevention of mitochondria - driven death pathways are emerging mechanisms of t3-mediated cardioprotection that deserve further investigation .
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### Summary:
: heart failure represents the final common outcome in cardiovascular diseases . despite significant therapeutic advances , morbidity and mortality of heart failure
remain unacceptably high . heart failure is preceded and sustained by a process of structural remodeling of the entire cardiac tissue architecture .
prevention or limitation of cardiac remodeling in the early stages of the process is a crucial step in order to ameliorate patient prognosis .
acquisition of novel pathophysiological mechanisms of cardiac remodeling is therefore required to develop more efficacious therapeutic strategies . among all neuroendocrine systems
, thyroid hormone seems to play a major homeostatic role in cardiovascular system . in these years
, accumulating evidence shows that the low triiodothyronine syndrome is a strong prognostic , independent predictor of death in patients affected by both acute and chronic heart disease . in experimental models of cardiac hypertrophy or myocardial infarction , alterations in the thyroid hormone signaling , concerning cardiac mitochondrion , cardiac interstitium , and vasculature , have been suggested to be related to heart dysfunction .
the aim of this brief paper is to highlight new developments in understanding the cardioprotective role of thyroid hormone in reverting regulatory networks involved in adverse cardiac remodeling .
furthermore , new recent advances on the role of specific mirnas in thyroid hormone regulation at mitochondrion and interstitial level are also discussed .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: class iii malocclusion is a more common clinical problem in mongolian populations than in caucasian populations.1 the prevalence of class iii malocclusion ranges from 2.3% to 16.7% in asia,234 and in a series of studies undertaken in korea 38.1% to 48.3% of the patients who visited a clinic seeking orthodontic treatment had class iii malocclusion.567 similarly , survey results pertaining to the distribution of orthodontic patients in wonkwang university dental hospital revealed that class iii patients comprised 34.2% of them , and of these , 33.6% were growing patients .
treatment of class iii malocclusions in growing patients is generally considered one of the most complex challenges in orthodontics.891011 early intervention treatment in patients with class iii malocclusion is controversial , for reasons relating to unpredictable prognoses , reduced efficiency arising from the associated long treatment times , and patient compliance problems .
11213 in view of these potential problems , the success or otherwise of several orthopedic treatments including chin caps , facemasks , and functional appliances have been studied.14151617 however , early intervention treatment of growing patients with skeletal class iii malocclusion via orthodontic or orthopedic modalities has become a common practice , favored for reasons relating to effectiveness , functional rehabilitation , and patients ' quality of life ; including their emotional development.1819 successful clinical results of functional appliance therapy have been reported by several authors.142021 examples of functional orthopedic appliances for class iii treatment are the frnkel function regulator iii , bionator iii , and class iii activator .
many previous studies have investigated the effects of class iii activator therapy.2022232425 several of these have reported short - term treatment effects , including improvement of patient profile with significant clockwise rotation of the mandible and proclination of the maxillary incisors.22232425 however , few studies have investigated the long - term effects of this treatment modality.20 in the current study , we investigated the short - term and long - term skeletodental changes associated with class iii activator treatment in growing patients .
patients in the study groups were selected from the growing korean patients diagnosed by the department of orthodontics , wonkwang university dental hospital from july 2000 to june 2011 .
this retrospective study was approved by the institutional review board of the dental hospital of wonkwang university ( approval number wkdirb201408 - 01 ) .
patients were classified into either a class iii activator treatment group ( ag ) or a class iii control group ( cg ) .
the ag was comprised of 22 patients ( 9 boys , 13 girls ) treated with class iii activator over the mandibular growth peak without combination or alteration with other growth modification appliances .
the cg was comprised of 17 patients ( 6 boys , 11 girls ) who were not treated with removable appliances that could modify growth or mandibular position ( table 1 ) .
inclusion criteria included skeletal class iii discrepancy beyond the normal range ( normal mean 1 standard deviation ) based on the anb angle and wits appraisal measurements according to individual growth state ; premandibular growth peak stage at the time of diagnosis ; no previous orthodontic treatment ; no craniofacial anomalies including cleft lip and palate ; and availability of adequate specific supplementary data including diagnostic records , treatment progress notes , and lateral cephalometric radiographs . the total treatment period was divided into three stages ; the initial stage ( t1 ) , the post - activator treatment or post - mandibular growth peak stage ( t2 ) , and the long - term follow - up stage ( t3 ) . skeletal maturation was determined by lateral cephalometric radiographs using a modified version of the cervical vertebral maturation method26 ( modified cvm method ) , independently of chronologic age .
the stages of cervical vertebral maturation were all classified by the same investigator ( hkr ) .
all patients were in pre - mandibular growth peak stage ( cs1-cs3 ) at t1 , post - mandibular growth peak stage ( cs4-cs5 ) at t2 , and more than a year after mandibular growth peak stage ( cs5-cs6 ) at t3 . in the ag ,
the construction bite of the activator was taken in the most posterior mandibular position with various heights , according to the patients ' individual conditions .
clinicians instructed the patients to wear the appliances for at least 14 hours per day .
all ag patients were treated via the same protocol by the residents , under the supervision of faculty supervisors .
ag patients wore the activator consistently until post - mandibular growth peak stage ( t2 ) , at which time the second phase of fixed appliance treatment was recommended .
after activator treatment , comprehensive orthodontic treatment was initiated in 18 patients ( 8 boys , 10 girls ) .
orthodontic treatment combined with orthognathic surgery was recommended to 4 other patients ( 1 boy , 3 girls ) .
four girls were observed for habit control , 5 patients ( 3 boys , 2 girls ) were treated with removable appliances for dental alignment , and 8 patients ( 3 boys , 5 girls ) initially underwent fixed appliance treatment on only their maxillary dentition . after t2 , 13 patients ( 5 boys , 8 girls ) received comprehensive orthodontic treatment and 4 patients ( 1 boy , 3 girls ) were observed regularly for possible further growth of the jaws . orthodontic treatment combined with orthognathic surgery
lateral cephalometric radiographs were acquired via a planmeca pm 2002 cc proline ceph ( planmeca , helsinki , finland ) until october 31st 2002 , then via a planmeca promax dimax3 ceph ( planmeca ) thereafter , with the patients in natural head position .
lateral cephalometric radiographs were taken at t1 , t2 , and t3 in each patient .
original films taken with the planmeca pm 2002 cc proline ceph were scanned and converted into a digital format via a vidar diagnostic pro plus film digitizer ( vidar systems corporation , herndon , va , usa ) .
all lateral cephalometric radiographs were digitized and calculated using v - ceph 6.0 software ( osstem implant co. , ltd . ,
cephalometric analysis was conducted to identify the changes in 20 measurements ( figures 1 and 2 , and table 2 ) . prior to statistical analysis ,
evaluations of stages of the cervical vertebrae were performed twice , 2 weeks apart , and there was no difference . also , 10 randomly selected radiographs were retraced and remeasured by the same investigator ( hkr ) 2 weeks after the initial analysis
, the paired t - test was used to compare the two data - sets of registrations , and there was no significant difference between the data - sets ( p > 0.05 ) .
all of the intraclass correlational coefficients were above 0.904 , suggesting that intra - examiner agreement was excellent .
descriptive statistics were calculated for all cephalometric measurements at t1 , t2 , and t3 in the ag and cg .
statistical analyses were performed using the statistical package for social sciences software ( version 12.0 ; spss , chicago , il , usa ) .
shapiro - wilks test revealed that the calculated measurements were not normally distributed , thus , the nonparametric mann - whitney u - test , friedman test , and wilcoxon signed rank test with bonferroni correction for post - hoc comparisons were used .
patients in the study groups were selected from the growing korean patients diagnosed by the department of orthodontics , wonkwang university dental hospital from july 2000 to june 2011 .
this retrospective study was approved by the institutional review board of the dental hospital of wonkwang university ( approval number wkdirb201408 - 01 ) .
patients were classified into either a class iii activator treatment group ( ag ) or a class iii control group ( cg ) .
the ag was comprised of 22 patients ( 9 boys , 13 girls ) treated with class iii activator over the mandibular growth peak without combination or alteration with other growth modification appliances .
the cg was comprised of 17 patients ( 6 boys , 11 girls ) who were not treated with removable appliances that could modify growth or mandibular position ( table 1 ) .
inclusion criteria included skeletal class iii discrepancy beyond the normal range ( normal mean 1 standard deviation ) based on the anb angle and wits appraisal measurements according to individual growth state ; premandibular growth peak stage at the time of diagnosis ; no previous orthodontic treatment ; no craniofacial anomalies including cleft lip and palate ; and availability of adequate specific supplementary data including diagnostic records , treatment progress notes , and lateral cephalometric radiographs .
the total treatment period was divided into three stages ; the initial stage ( t1 ) , the post - activator treatment or post - mandibular growth peak stage ( t2 ) , and the long - term follow - up stage ( t3 ) . skeletal maturation was determined by lateral cephalometric radiographs using a modified version of the cervical vertebral maturation method26 ( modified cvm method ) , independently of chronologic age .
the stages of cervical vertebral maturation were all classified by the same investigator ( hkr ) .
all patients were in pre - mandibular growth peak stage ( cs1-cs3 ) at t1 , post - mandibular growth peak stage ( cs4-cs5 ) at t2 , and more than a year after mandibular growth peak stage ( cs5-cs6 ) at t3 .
in the ag , the construction bite of the activator was taken in the most posterior mandibular position with various heights , according to the patients ' individual conditions .
clinicians instructed the patients to wear the appliances for at least 14 hours per day .
all ag patients were treated via the same protocol by the residents , under the supervision of faculty supervisors .
ag patients wore the activator consistently until post - mandibular growth peak stage ( t2 ) , at which time the second phase of fixed appliance treatment was recommended .
after activator treatment , comprehensive orthodontic treatment was initiated in 18 patients ( 8 boys , 10 girls ) .
orthodontic treatment combined with orthognathic surgery was recommended to 4 other patients ( 1 boy , 3 girls ) .
four girls were observed for habit control , 5 patients ( 3 boys , 2 girls ) were treated with removable appliances for dental alignment , and 8 patients ( 3 boys , 5 girls ) initially underwent fixed appliance treatment on only their maxillary dentition . after t2 , 13 patients ( 5 boys , 8 girls ) received comprehensive orthodontic treatment and 4 patients ( 1 boy , 3 girls ) were observed regularly for possible further growth of the jaws . orthodontic treatment combined with orthognathic surgery
lateral cephalometric radiographs were acquired via a planmeca pm 2002 cc proline ceph ( planmeca , helsinki , finland ) until october 31st 2002 , then via a planmeca promax dimax3 ceph ( planmeca ) thereafter , with the patients in natural head position .
lateral cephalometric radiographs were taken at t1 , t2 , and t3 in each patient .
original films taken with the planmeca pm 2002 cc proline ceph were scanned and converted into a digital format via a vidar diagnostic pro plus film digitizer ( vidar systems corporation , herndon , va , usa ) .
all lateral cephalometric radiographs were digitized and calculated using v - ceph 6.0 software ( osstem implant co. , ltd . ,
cephalometric analysis was conducted to identify the changes in 20 measurements ( figures 1 and 2 , and table 2 ) .
evaluations of stages of the cervical vertebrae were performed twice , 2 weeks apart , and there was no difference . also , 10 randomly selected radiographs were retraced and remeasured by the same investigator ( hkr ) 2 weeks after the initial analysis
, the paired t - test was used to compare the two data - sets of registrations , and there was no significant difference between the data - sets ( p > 0.05 ) .
all of the intraclass correlational coefficients were above 0.904 , suggesting that intra - examiner agreement was excellent .
descriptive statistics were calculated for all cephalometric measurements at t1 , t2 , and t3 in the ag and cg .
statistical analyses were performed using the statistical package for social sciences software ( version 12.0 ; spss , chicago , il , usa ) .
shapiro - wilks test revealed that the calculated measurements were not normally distributed , thus , the nonparametric mann - whitney u - test , friedman test , and wilcoxon signed rank test with bonferroni correction for post - hoc comparisons were used .
no statistically significant differences were found between the skeletodental measurements of the ag and cg at t1 ( table 3 ) .
descriptive data and statistical comparisons for cephalometric measurements at t1 , t2 , and t3 are presented in table 4 . in the ag ,
the following measurements changed statistically significantly during the periods : sna , anb , wits appraisal , mx . length , mn .
length , a - n perpend , pog - n perpend , convexity of a , gonial angle , u1 to sn , and u1 to fh . of these measurements , sna ,
anb , convexity of a , u1 to sn , and u1 to fh increased significantly between t1 and t2 .
perpendicular gradually increased throughout the t1 , t2 , and t3 periods . in the cg , the following measurements changed statistically significantly during the periods : snb , wits appraisal , mx .
length , mn . length , pog - n perpend , and convexity of a. of these measurements , snb , and
pog - n perpend significantly increased between t1 to t2 , while convexity of a significantly decreased .
descriptive data and statistical comparisons of the cephalometric changes in both groups in each period are presented in table 5 . from t1 to t2 , snb of the ag increased significantly less than that of the cg , resulting in an increase in anb in the ag that was greater than that of the cg .
plane angle , y - axis angle , and odi also increased significantly more in the ag than in the cg .
there was a significantly larger decrease in apdi in the ag than in the cg .
u1 to sn , and u1 to fh increased significantly more in the ag than in the cg .
there were no significant differences in the changes from t2 to t3 . from t1 to t3 , anb , wits appraisal , and convexity of
compared with the cg , a significantly larger change in u1 to fh was observed in the ag .
descriptive data and statistical comparisons for cephalometric measurements at t1 , t2 , and t3 are presented in table 4 . in the ag , the following measurements changed statistically significantly during the periods : sna , anb , wits appraisal , mx .
length , a - n perpend , pog - n perpend , convexity of a , gonial angle , u1 to sn , and u1 to fh . of these measurements , sna ,
anb , convexity of a , u1 to sn , and u1 to fh increased significantly between t1 and t2 .
perpendicular gradually increased throughout the t1 , t2 , and t3 periods . in the cg , the following measurements changed statistically significantly during the periods : snb , wits appraisal , mx .
length , pog - n perpend , and convexity of a. of these measurements , snb , and pog - n perpend significantly increased between t1 to t2 , while convexity of a significantly decreased .
descriptive data and statistical comparisons of the cephalometric changes in both groups in each period are presented in table 5 . from t1 to t2 , snb of the ag increased significantly less than that of the cg , resulting in an increase in anb in the ag that was greater than that of the cg .
plane angle , y - axis angle , and odi also increased significantly more in the ag than in the cg .
there was a significantly larger decrease in apdi in the ag than in the cg .
u1 to sn , and u1 to fh increased significantly more in the ag than in the cg .
there were no significant differences in the changes from t2 to t3 . from t1 to t3 , anb , wits appraisal , and convexity of
compared with the cg , a significantly larger change in u1 to fh was observed in the ag .
the objective of this study was to determine short - term and long - term skeletodental changes in growing patients in a class iii ag and a class iii cg .
this study revealed that class iii activator therapy resulted in an improved jaw relationship and proclinated maxillary incisors ( table 4 ) . compared with the cg , skeletodental changes in the ag occurred during the activator treatment period , and remained without substantial changes during the long - term follow - up period ( table 5 )
the study has some limitations , most of which stem from the small sample size and retrospective study design .
it was difficult to obtain control data from completely untreated patients with equivalent skeletodental conditions , due to ethical considerations .
accordingly , patients treated with habit control and/or dental alignment excluding orthopedic treatment were included in the cg .
furthermore , radiographic images were obtained by two methods , from original films and digital images , increasing the possibility of discrepancies in the cephalometric analysis . in a previous study comparing scanned lateral cephalograms with corresponding original radiographs ,
some distortions were found.27 however , the authors considered that the use of scanned cephalograms was valid , because the relatively small discrepancies were deemed clinically insignificant .
the appraisal of skeletal maturity in both groups was performed by evaluating stages of the cervical vertebrae , using a modified cvm method.26 in this study , the modified cvm method was applied to assess whether the pubertal spurt in mandibular growth had started or finished .
the appraisal of skeletal maturity based on cervical vertebral maturation can be helpful for determining the completion of active growth in studies dealing with the long - term effects of orthopedic treatment strategies.26 previous studies have shown that the greatest increase in mandibular length occurs during the pubertal growth spurt , which happens during the interval between cs3 and cs4.2829 the pubertal peak of patients with skeletal class iii malocclusion reportedly lasts longer than that of patients with skeletal class i malocclusion.30 therefore , growth modification treatment should be maintained during this growth period , and a long period of observation of patients should be undertaken , until the active growth period is over .
accordingly , patients undergoing maintained activator treatment over the mandibular growth peak were included in this study ( table 1 ) .
the significant changes in sna , and a - n perpend were associated with forward displacement of the maxilla in the ag ( table 4 ) . nonetheless , these treatment changes in the ag did not result in significant differences between the ag and cg ( table 5 ) .
the insignificant mean change in the measurements related to maxillary growth also suggests that maxillary growth could be caused by both natural factors and activator treatment .
this point of view corresponds with previous studies.2024 based on assessment of the effect of class iii activator treatment on mandible changes , undertaken by comparing the skeletal changes within each group , we could assume that it prevents the anteroposterior relationship of the mandible from getting worse ( table 4 ) .
in particular , the measurements that differed significantly between both groups , such as snb , anb , wits appraisal , convexity of a , and ab to mn . plane angle were associated with significantly greater improvement of the jaw relationship in the ag ( table 5 ) .
similar findings relating to class iii activator treatment have been described in other studies.222325 the clockwise rotation of the mandible in the ag had the additional effect of repositioning of the chin downward and backward , and developed facial convexity that contributed to improvement of profile .
dental changes in the maxilla such as u1 to sn , and u1 to fh were evident in the ag ( table 4 ) .
the proclination of the maxillary incisors caused by class iii activator treatment was similar to the dental compensation seen in class iii malocclusion .
furthermore , in terms of dental compensation , proclination of the maxillary incisors contributed to a positive overjet in untreated skeletal class iii patients.31 the observed changes associated with class iii activator therapy , particularly clockwise rotation of the mandible and proclination of maxillary incisors , facilitate the formation of a positive overjet by the class iii camouflage treatment .
moreover , some authors believe that early correction of an anterior crossbite has a positive effect on the sagittal development of the maxilla , developing the bone according to its endogenous growth potential.20 in this respect , correction of the jaw relationship by class iii activator therapy induces harmonious skeletal and dental changes . from t1 to t3 , measurement differences between the ag and cg reflecting skeletal changes , such as anb , wits appraisal , and convexity of a showed that the treatment effects were maintained during the second phase of treatment ( table 5 ) .
this showed that helpful skeletal outcomes induced by class iii activator during the activator treatment period were generally maintained until the long - term follow - up period of the post - mandibular growth peak stage .
favorable skeletal changes were achieved by class iii activator therapy that started before the mandibular growth peak and continued over the mandibular growth peak .
these treatment effects of class iii activator therapy were generally maintained during the long - term follow - up period of the post - mandibular growth peak stage .
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### Summary:
: objectivethe purpose of this retrospective study was to investigate short - term and long - term skeletodental outcomes of class iii activator treatment.methodsa class iii activator treatment group ( ag ) comprised of 22 patients ( 9 boys , 13 girls ) was compared with a class iii control group ( cg ) comprised of 17 patients ( 6 boys , 11 girls ) .
the total treatment period was divided into three stages ; the initial stage ( t1 ) , the post - activator treatment or post - mandibular growth peak stage ( t2 ) , and the long - term follow - up stage ( t3 ) .
cephalometric changes were evaluated statistically via the mann - whitney u - test and the friedman test.resultsthe ag exhibited significant increases in the sna angle , anb angle , wits appraisal , a point - n perpendicular , convexity of a point , and proclination of the maxillary incisors , from t1 to t2 . in the long - term follow - up ( t1-t3 ) , the ag exhibited significantly greater increases in the anb angle , wits appraisal , and convexity of a point than the cg.conclusionsfavorable skeletal outcomes induced during the class iii activator treatment period were generally maintained until the long - term follow - up period of the post - mandibular growth peak stage .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: dna damage caused by oxidative stress plays an important role in the initiation and carcinogenic process of mutation .
a recent study has suggested that oxidative stress leads to increased mutation frequency in a murine model of myelodysplastic syndrome ( mds ) ( 1 ) , and that additional mutations lead to the progression of mds .
patients with mds often require blood transfusions and consequently develop iron overload , and the excess iron causes oxidative stress .
therefore , antioxidant therapy and iron chelation are thought to be suitable for the management of mds patients ( 2 - 4 ) .
furthermore , a correlation has been reported between the serum ferritin level and serum reactive oxygen species ( ros ) levels in mds patients ( 2 - 5 ) .
mds is a heterogeneous group of clonal hematopoietic stem cell disorderd that transforms to acute myeloid leukemia ( 6 ) .
azacitidine was the first dna - hypomethylating agent that demonstrated superiority over conventional therapy in the treatment of mds in patients unable to undergo allogeneic stem cell transplantation ( 8) . a relationship between oxidative stress and hypermethylation has also been reported ; oxidative stress is likely involved in the mds disease progression ( 9 ) . in the present study , we evaluated the changes in the oxidative stress markers derivatives of reactive oxidative metabolites ( drom ) in serum and 8-hydroxy-2'-deoxyguanosine ( 8-ohdg ) in urine in an mds patient treated with azacitidine .
the ethics committee of toho university approved this study , and we obtained informed consent from the patient .
a 69-year - old japanese man with exertional chest pain was transported to our hospital by ambulance in april 2014 .
he had a history of unstable angina and had undergone percutaneous coronary intervention at the left anterior descending artery in november 2013 . on admission ,
however , his white blood cell ( wbc ) count was 1,970 /l without blast cells ; his hemoglobin ( hb ) level was 5.0 g / dl ; and his platelet count was 16.610/l .
a bone marrow examination revealed myelodysplastic syndrome classified as refractory anemia with excess blasts-1 ( raeb-1 ) .
initially , metenolone enanthate and menatetrenone were administered , and red blood cell transfusion was performed to treat his anemia . he was admitted to our hospital at the end of july 2014 , and azacitidine therapy was started on day 3 after admission .
after the fourth course of azacitidine therapy , he developed acute myocardial infarction on day 140 .
azacitidine therapy was discontinued , and therapy with the iron chelator deferasirox 500 mg / day was started on day 192 .
a total of 28 units of blood had been transfused before starting administration of the iron chelator .
we evaluated the levels of drom in serum by colorimetry ( 10 ) and 8-ohdg in spot urine by elisa method ( 11 ) , ferritin in serum , and wilms ' tumor 1 ( wt1 ) gene in the peripheral blood throughout the course of treatment ( fig .
the level of wt1 was 14,000 copies/g rna before the first course of azacitidine treatment , which was reduced markedly to 400 copies/g rna after the second course .
however , the level of wt1 increased sharply around the fourth course of azacitidine therapy , and pancytopenia with blasts in the peripheral blood became gradually apparent .
the respective serum drom and urinary 8-ohdg levels were 418 u.carr and 10.2 ng / mg creatinine before the first course of azacitidine treatment and 360 u.carr and 11 ng / mg creatinine after the first course .
the serum drom level decreased transiently with each course of azacitidine therapy until the third course . from the fourth course , accompanying an increase in wt1 , drom increased simultaneously while 8-ohdg and ferritin began to increase later .
aza : azacitidine , wt1 : wilms tumor 1 , drom : derivatives of reactive oxygen metabolite , 8-ohdg : 8-hydroxy- 2-deoxyguanosine , fer : ferritin , ( ) : red blood cell transfusion 2 units .
normal ranges : serum drom ; < 300 u. carr , urinary 8-ohdg ; < 15.7 ng / mg creatinine ferritin ; 18.6 - 261 ng / dl , lactate dehydrogenase ( ldh ) ; 120 - 240 iu / l
.
around day 190 after the first admission , the wbc count of the peripheral blood showed 30% blasts , and the serum drom , urinary 8-ohdg , and serum ferritin levels were elevated markedly to peak levels of 685 u.carr , 25.3 ng / mg creatinine , and 4,440.3 ng / ml , respectively .
he was admitted to the hospital on day 192 due to a deterioration of general condition .
he received circulatory support and blood transfusion and was treated with deferoxamine mesilate at 500 mg / day .
his disease progressed , and he eventually died of multi - organ failure on day 210 .
we analyzed the associations among drom , 8-ohdg , ferritin , and wt1 levels during his clinical course by pearson 's test .
the serum drom levels correlated significantly with wt1 ( r=0.80 , p=0.03 ) and ferritin ( r=0.85 , p=0.002 ) ( fig . 2a and b ) .
in addition , the urinary 8-ohdg levels also correlated significantly with wt1 ( r=0.96 , p=0.002 ) and ferritin ( r=0.92 , p=0.000 ) ( fig . 2c and d ) .
the serum drom levels also correlated significantly with 8-ohdg ( r=0.69 , p=0.01 ) ( fig .
2e ) . on analyzing the correlation among c reactive protein ( crp ) , drom , and 8-ohdg
, crp did not correlate with drom ( r=0.45 , p=0.138 ) or 8-ohdg ( r=-0.21 , p=0.534 ) ( fig .
2f and g ) .
the relationship among the derivatives of reactive oxygen metabolite ( drom ) , 8-hydroxy-2-deoxyguanosine ( 8-ohdg ) , ferritin ( fer ) , and wilms tumor 1 ( wt1 ) .
the correlation between ( a ) drom and wt1 , ( b ) drom and fer , ( c ) 8-ohdg and wt1 , ( d ) 8-ohdg and fer , ( e ) 8-ohdg and drom , ( f ) drom and c reactive protein ( crp ) , and ( f ) 8-ohdg and crp .
we encountered a patient with mds classified as raeb-1 , in whom we measured oxidative stress markers , ferritin , and wt1 over the course of disease . to our knowledge , this is the first report of azacitidine therapy in an mds patient with serial measurements of oxidative stress , ferritin , and wt1 values during therapy .
a marked decline in the wt1 expression in peripheral blood showed that three courses of azacitidine therapy were effective in suppressing disease progression .
oxidative stress and ferritin are considered important biological factors in the pathogenesis of mds ( 2 - 4 ) . however , as shown in fig .
1 , the wt1 expression started to increase before the administration of the fourth course of azacitidine therapy , and the serum drom levels began to rise earlier than ferritin during disease progression .
1 shows that the response of drom appeared to be more sensitive than that of 8-ohdg .
we also evaluated the correlation among drom , 8-ohdg , and the total number of blasts in the peripheral blood .
while the drom levels and the blast count correlated significantly , the 8-ohdg levels showed no correlation with the blast count ( data not shown ) .
on investigating the source of the increased ros expression during disease progression in this patient by examining the ros in each fraction of wbcs , the results suggested that the oxidative stress markers were produced by tumor - derived cells ( data not shown ) .
furthermore , to clarify the relationship between the elevation of the oxidative stress markers with the deterioration of the general condition , we also analyzed the correlation among crp , drom , and 8-ohdg .
however , in the present case , we observed a transient decreasing trend in the drom levels after each course of azacitidine treatment before the progression of mds , and the transition of drom was not related to the iron overload caused by blood transfusion .
recently reported a relationship between oxidative stress and dna methylation , suggesting that oxidative stress modulates the enzyme activity of dna methylation and the deacetylation of histones ( 9 ) .
it remains unclear whether the increase in oxidative stress is a cause or a result of disease progression .
the increase in the oxidative stress markers may have not only caused the iron overload but also resulted in the disease progression of mds .
we herein reported an mds patient who showed simultaneous increases in oxidative stress markers and wt1 during disease progression while being treated with azacitidine .
we speculate that the increase in the oxidative stress markers is a cause of not only iron overload but also the disease progression of mds .
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### Summary:
: oxidative stress is closely related to iron overload in myelodysplastic syndrome ( mds ) and induces dna damage . we evaluated the oxidative stress markers derivatives of reactive oxidative metabolites ( drom ) and 8-hydroxy-2'-deoxyguanosine ( 8-ohdg ) during azacitidine treatment in an mds patient .
simultaneous with an increase in the expression of wilms ' tumor 1 ( wt1 ) gene in the peripheral blood , the serum drom level was elevated , and this increase was observed earlier than the increases in ferritin and 8-ohdg . throughout the clinical course , drom and
8-ohdg correlated significantly with wt1 and with ferritin , suggesting that changes in the oxidative stress marker levels reflect not only iron overload but also disease progression of mds .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
:
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### Summary:
: actinobacteria generate a large number of structurally diverse small molecules with potential therapeutic value .
genomic analyses of this productive group of bacteria show that their genetic potential to manufacture small molecules exceeds their observed ability by roughly an order of magnitude , and this revelation has prompted a number of studies to identify members of the unknown majority . as a potential window into this cryptic secondary metabolome ,
pairwise assays for developmental interactions within a set of 20 sequenced actinomycetes were carried out .
these assays revealed that amycolatopsis sp .
aa4 , a so - called rare actinomycete , produces a novel siderophore , amychelin , which alters the developmental processes of several neighboring streptomycetes . using this phenotype as an assay
, we isolated amychelin and solved its structure by nmr and ms methods coupled with an x - ray crystallographic analysis of its fe - complex .
the iron binding affinity of amychelin was determined using edta competition assays , and a biosynthetic cluster was identified and annotated to provide a tentative biosynthetic scheme for amychelin .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: appendiceal mucocele ( am ) is defined as a gross dilatation of the vermiform appendix , caused by the accumulation of mucus material within the appendiceal lumen secondary to inflammatory or neoplastic proliferation of the appendiceal mucosa .
am is a rare clinical entity comprising 8% of all appendiceal tumors , and it is seen in 0.2 - 0.3% of all appendectomy specimens .
histologically , ams have been divided into four subtypes : ( 1 ) simple mucocele or retention cyst accounting for 20% of the cases , considered to be due to the obstruction of the appendiceal lumen , ( 2 ) focal or diffuse mucosal hyperplasia ( 20% ) , ( 3 ) mucinous cystadenoma ( 50% ) , and ( 4 ) mucinous cystadenocarcinoma ( 10% ) .
mucinous cystadenoma is the most common histological subtype of mucocele mostly exhibiting epithelial villous adenomatous changes with some degree of atypia .
although synchronous colorectal neoplasia has been reported in 3 - 5% of the cases , the synchronous occurrence of mucinous cystadenoma of the appendix and colorectal cancer is a rare clinical entity .
we describe a case of simultaneous occurrence of mucinous cystadenoma of the appendix , colon adenocarcinoma , and tubulovillous colon adenoma .
diagnostic evaluation and management of the patient are discussed along with a review of the relevant literature .
a 67-year - old caucasian male presented with a 10-day history of gradually worsening abdominal pain , associated with nausea and episodes of vomiting .
physical examination revealed a distended abdomen with a palpable tender mass in the left lower quadrant .
the hemogram revealed anemia with a hemoglobin level of 9.5 g / dl and a hematocrit of 30% .
biochemical investigations revealed elevated serum levels of c - reactive protein 49 mg / l ( reference range < 5 ) and significantly elevated serum carcinoembryonic antigen levels 500 ng / ml ( reference range 0 - 10 ) .
colonoscopy revealed an ulcerative mass obstructing the lumen of the sigmoid colon located 25 cm from the anal verge and a mass located 9 cm from the anal verge .
scans showed significant thickening of the sigmoid colon along with hepatic and pulmonary nodules suggestive of secondary lesions . at laparotomy
, a large tumor was found obstructing the sigmoid lumen with infiltration of the serosal fat .
a hartman 's procedure ( sigmoid colectomy with end colostomy and oversewing of the rectal stump ) along with appendectomy was performed .
histopathological examination showed a moderate to poorly differentiated sigmoid adenocarcinoma with extensive infiltration of the pericolic fat tissue .
histology of the rectal tumor showed a tubulovillous rectal adenoma with high - grade dysplasia measuring 1.8 cm 1.5 cm whereas the histopathological findings of the appendiceal tumor were suggestive of mucinous cystadenoma [ figure 1 ] .
although the patient received adjuvant chemotherapy , he unfortunately died of progressive disease 5 months later .
( a ) metastatic nodal infiltration by a colon adenocarcinoma ( h and e , 100 ) .
( b ) extensive infiltration of the pericolic fat tissue by a moderate to poorly differentiated colon adenocarcinoma ( h and e , 100 ) . ( c )
note the absence of atypia and mitotic figures ( h and e , 100 ) .
( d ) a tubulovillous adenoma with high - grade dysplasia ( h and e , 400 )
the appendix is embryologically derived from the large intestine and has a similar mucosal pattern to the colon .
therefore , any neoplastic change of the colon can affect the appendix . apart from sporadic cases , there are no enough published data about the incidence of synchronous appendiceal tumors in patients with colorectal cancer .
khan and moran reported an incidence of 4.1% of synchronous appendiceal epithelial tumors among 169 patients who underwent colorectal surgery and removal of the appendix .
the authors suggested that appendectomy should be performed in all patients undergoing surgery for colorectal cancer , given that appendectomy is unlikely to be an additional infective risk in major bowel resection and that the appendiceal mucosa is inaccessible in postoperative surveillance colonoscopy , which is mandatory in this group of patients . in another retrospective study ,
lohsiriwat et al . found that only one ( 0.3% ) out of 293 patients who underwent surgery for colorectal cancer had a synchronous epithelial appendiceal tumor .
the authors ' explanation for the very low incidence of synchronous primary appendiceal tumor in patients with colorectal cancer was that tumorigenesis of the appendix and other parts of the large bowel is not the same because the appendiceal mucosa , unlike large bowel mucosa , is not directly exposed to carcinogens in fecal material .
larger prospective studies are necessary to accurately determine the incidence of synchronous appendiceal tumors and colorectal cancer . regarding the synchronous occurrence of am , colon adenocarcinoma , and tubulovillous colon adenoma , we found only one case reported in the literature so far .
ams occur most frequently in middle - aged patients with a higher incidence in women .
. some patients may be asymptomatic and the lesions are incidentally detected during imagings or operations performed for unrelated reasons . in some cases , however , the appendix may be macroscopically normal with even no intraoperative suggestion of an appendix tumor .
in our case , the appendix was mildly distended with a maximum diameter of 1.3 cm on histology report . in a retrospective study including 129 patients with mucocele , stocchi et al .
found that the presence of symptoms such as abdominal pain and weight loss was associated more frequently with malignancy . on ultrasonography , am can appear as an anechoic or hypoechoic structure depending on mucus composition . on ct scan , which is the diagnostic modality of choice
the main differential diagnoses of am include periappendicular abscess , enteric duplication cysts , mesenteric cysts , and ovarian masses .
the treatment of am is surgical resection to eliminate any malignant potential , whereas the type and extend of surgery primarily depend on the dimensions and histological features .
thorough inspection of the peritoneal cavity is essential during surgery to detect a concomitant colon or ovarian tumor .
special care should be taken when mobilizing the tumor to avoid rupture of mucocele and subsequent spillage of mucin into the peritoneal cavity , resulting in the development of pseudomyxoma peritonei .
follow - up is of utmost importance after surgery not only for the appendicular disease but also due to the possibility of synchronous or metachronous tumors .
the synchronous occurrence of mucinous cystadenoma of the appendix , colon adenocarcinoma , and tubulovillous rectal adenoma is an extremely rare clinical entity .
thorough preoperative and perioperative evaluations are mandatory in patients with colorectal cancer to exclude a synchronous colon or appendiceal primary tumor .
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### Summary:
: appendiceal mucocele ( am ) is a rare clinical entity comprising 8% of all appendiceal tumors , and it is seen in 0.2 - 0.3% of all appendectomy specimens .
apart from sporadic cases , there are no enough published data about the incidence of synchronous appendiceal tumors in patients with colorectal cancer .
we describe a very rare case of synchronous occurrence of am , colon adenocarcinoma , and tubulovillous adenoma of the rectum and review the relevant literature .
we conclude that thorough preoperative and perioperative evaluations are mandatory in patients undergoing surgery for colorectal cancer to exclude a synchronous colon or an appendiceal primary tumor .
larger prospective studies are necessary to accurately determine the incidence of synchronous appendiceal tumors and colorectal cancer .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: regardless of gender , race , or economic status , trauma remains a leading cause of death worldwide .
most deaths occur early after hospital admission and are mainly caused by early exsanguinating hemorrhage .
this potentially preventable cause of mortality is compounded by a distinct coagulopathy of trauma that sets in the early stage of injury .
patients with posttraumatic coagulopathy have a 34fold higher mortality in comparison to those who have normal coagulation process and are up to eight times more likely to die on the 1 day posttrauma .
this coagulopathy complicates resuscitation and is the target of massive transfusion protocols ( mtp ) consisting of multiple blood products .
recent military and civilian experience has brought about many shifts in the principles and practice of early trauma resuscitation .
prioritizing aggressive blood component therapy has replaced long institutionalized , crystalloid dependent fluid resuscitation .
there is an increased emphasis on the early and liberal use of balanced blood product ratio , including packed red blood cell ( prbc ) , fresh
frozen plasma , and platelets ( plt ) . despite the widespread implementation of mtps for trauma
, there is still considerable controversy regarding the optimal ratio of plt : prbc in trauma patients requiring mtp .
this growing debate has evolved to include the time at which the best ratio is achieved .
the attainment of high resuscitation ratio as early as 4 h postinjury and their impact on patient outcomes have not been adequately analyzed to date .
this report aims to describe the effect of attaining a high plt : prbc ratio ( 1:1.5 ) within 4 h postinjury on the outcomes of trauma patients receiving mtp .
all trauma patients who received blood transfusion were retrospectively identified from the blood bank database , and their detailed records were reviewed from the trauma registry of the hamad trauma center , hospital , the national level i trauma referral center in qatar between january 2010 and december 2011 .
all adult ( 18 years ) traumatic injury patients who received massive blood transfusion were included in the study .
blood bank records were used to identify patients who received > 6 units prbc and particularly screened patients who received > 10 units prbc in the first 24 h of injury .
we defined massive transfusion as the infusion of 10 units of prbc over the initial 24 h postinjury .
patient 's data regarding demographics , mechanism of injury , initial vitals , laboratory findings , injury severity score ( iss ) , revise trauma score ( rts ) , glasgow coma score , hospital length of stay , complications , and outcomes were collected from the trauma registry database .
based on plt : prbc ratio , patients who received at least a plt : prbc ratio of 1:1.5 within 4 h from the time of injury was categorized into the high ratio group ( hmtp ) .
those who received a plt : prbc ratio of 1:1.5 within 4 h postinjury represented the low ratio group ( lmtp ) .
the ratio was then calculated based on the blood products given from 4 to 24 h postinjury .
one mtp shipment was defined as the infusion of 6 units of prbc , 6 units of plasma , and 6 units of plts .
patients were followed up to 30-day post injury , hospital discharge , or death ; whichever occurred first .
outcomes measured included overall mortality , postshipment international normalized ratio ( inr ) , multiple organ failure ( mof ) , and infectious complications .
the diagnosis of ventilator associated pneumonia ( vap ) was based on quantitative culture threshold of 10 cfu / ml for bronchoalveolar lavage specimens .
related blood stream infections were confirmed by positive peripheral cultures with the identical organism obtained from either a positive semi quantitative culture ( 15 cfu / segment ) or positive quantitative culture ( 10 cfu / segment ) from a catheter segment specimen .
the diagnosis of urinary tract infection was confirmed if there was 10 organisms / ml in the urine specimens .
this study was approved by the medical research center at hamad medical corporation , qatar ( irb # 11153/11 ) .
data were presented as proportions , mean standard deviation , or median as appropriate .
baseline demographic , clinical characteristics , laboratory findings , and outcomes were compared according to plt : prbc ratio at 4 h ( hmtp vs. lmtp ) using chi
the fisher 's exact test was used if the expected cell frequencies were below 5 . a two sided p < 0.05 was considered to be statistically significant .
data analysis was carried out using the statistical package for social sciences version 18 ( spss inc . ,
a total of 3244 trauma admissions were admitted during the 2-year study period , of which 100 ( 2.06% ) patients met inclusion criteria . of these , plt : prbc ratio was attainable in 58 patients as the remaining 42 patients did not receive plts within the first 4 h postinjury .
the mean age of the patients was 32.3 10.7 years ; majority were males ( 89.6% ) who sustained severe injuries with a high mean iss of 31.9 11.5 and rts of 5.1 2.2 .
demographic , epidemiologic , and clinical presentation of massive transfusion protocol patients who received platelet transfusion ( n=58 ) table 2 demonstrated the baseline comparison of clinical characteristics and injury severity according to plt : prbc ratio ( hmtp vs. lmtp ) within 4 h postinjury .
both the groups were comparable for age , gender , type of injury , presenting hemoglobin level , inr , iss , and rts except initial ph .
table 3 shows the comparison of complications and outcomes of patients based on hmtp and lmtp within the first 4 h. no significant differences were noted for the incidence of hyperkalemia , hypomagnesemia , hypocalcemia , abdominal compartment syndrome , blood stream , catheter related , and urinary tract infections between the two groups .
the frequency of mof was significantly lower in the hmtp group ( 42.1% vs. 87.2% , p = 0.001 ) .
however , the rate of vap ( 38.9% vs. 10.8% ; p = 0.02 ) and wound infection ( 50% vs. 10.8% ; p = 0.002 ) were significantly higher in the hmtp group .
the overall mortality was 69% , and the number of deaths was significantly lower in hmtp ( 36.8% , vs. 84.6% p = 0.001 ) in comparison to lmtp within the first 30 days .
baseline comparison of clinical characteristics and injury severity by platelet to packed red blood cell ratio within 4 h postinjury comparison of complications and clinical outcomes by platelet to packed red blood cell ratio within first 4 h postinjury similarly , in the later period ( 424 h ) , the frequency of mof ( 69.2% vs. 56.0% ; p = 0.29 ) and overall mortality ( 44% vs. 61.5% , p = 0 .
0.207 ) was nonsignificantly lower in hmtp group as compared to lmtp [ figure 1 ] .
moreover , no significant difference was observed for the incidence of vap , wound infection , blood stream infection , catheter related , or urinary tract infections between the two groups in the later period ( 424 h ) .
mortality and multiple organ failure rates by time and platelet : packed red blood cell ( pbbc ) ratio
our study demonstrates the significance of obtaining a high plt : prbc ratio early in the resuscitation phase . at the beginning of the 80s , kashuk et al .
had proposed the formation of the lethal triad of coagulopathy , hypothermia , and acidosis secondary to bleeding , cellular shock , and tissue injury .
coagulopathy post traumatic injury was once regarded as a secondary outcome , due to dilution and consumption , but now it is recognized as a primary consequence of trauma . about a third of severely injured patients are coagulopathic on arrival .
abnormal coagulation noticed in the exsanguinating trauma victims is often multifactorial in nature with hemodilution , consumption of coagulation factors , activation of inflammatory cascade , hyperfibrinolysis , and plt dysfunction secondary to acidosis / hypothermia identified as causative or contributory factors .
in addition , activated plts promote secondary hemostasis by offering an excellent catalytic surface for the aggregation of enzyme complexes required for optimizing coagulation .
most guidelines that indicate the use of plts in massively bleeding trauma patients are count based .
thrombocytopenia witnessed in massively transfused patients is often misleading as both consumptive factors and dilution can contribute to the same .
plt dysfunction , which is more critical than plt count , is unaccountable due to lack of a diagnostic test .
despite recent studies that cite the role of point - of - care testing in titrating hemostatic resuscitation efforts , these practical difficulties have led to the continued use of a structured blood product ratio in the form of mtps .
current literature favors early and aggressive plt use to attain plt : prbc transfusion ratio as high as 1:1 .
however , early attainment of such high ratio in clinical practice is challenging as evidenced from civilian data . both military and civilian experience of late have supported the use of higher plt : prbc transfusion ratio to improve survival rates in these patients . there
currently exists no consensus on what constitutes a relevant cutoff between an adequately high and low plt ratio .
table 4 compares a few recent studies and their outcomes with varied plt : prbc cutoff ratio .
our study demonstrated a significant improvement in overall mortality and mof in patients received hmtp within the initial 4 h [ figure 1 ] .
a similar survival benefit has been documented in other studies that have tested high plt : prbc ratio .
however , attainment of this ratio within the first 4 h postinjury and their impact on patient outcome have not been analyzed adequately to date . in our series , the significant improvement in mortality and mof rates in hmtp group during the early period ( < 4 h ) was not observed during the later period ( 424 h ) .
hence , we believe the survival benefit of attaining hmtp ratio , documented in other studies , when the ratio was calculated at 24 h is possibly secondary to its early rather than late attainment . regardless of how
this early high plt : prbc transfusion ratio is achieved , its survival benefit is noteworthy and a call for a rigidly attained and implemented mtp that can ensure the timely availability and transfusion of these components that are associated with increased survival in trauma patients is in need of mtp .
usually , earlier studies have categorized patients into high and low plt : prbc ratio groups based on the blood products consumed with initial 24 h [ table 4 ] . the prommtt study conceded 60% of hemorrhagic deaths to have occurred within 3 h of admission .
dutton et al . revealed that 80% of those who will bleed to death have done so within 6 h of admission .
thus , 24 h ratio inadequately accounts for and addresses the underlying cause of early deaths .
first raised the question of survival bias in this subgroup of patients as opposed to a survival benefit with a high transfusion ratio .
our meticulous review of records supplemented by blood bank and trauma registry data enabled an hourly blood product transfusion data , describing exactly when prbcs and plts were infused .
we were able to account for early deaths by calculating blood product ratio as early as 4 h , whereby reducing possibilities for survival bias . in the present study ,
both hmtp and lmtp groups were well matched in terms of initial coagulopathy , injury severity , and demographic characteristics .
these results add to our current understanding of the benefits associated with aggressive plt use to prevent and treat early onset coagulopathy after injury .
these could be explained by the potential for a contributory immunodeficiency from this type of resuscitative practice .
our study is limited to a retrospective analysis of major transfusion practices for an adult and predominantly blunt trauma population from a single center .
however , the hamad trauma center is the national trauma referral center in qatar ; as such this is a nationally representative sample of severely injured patients who are the beneficiaries of a single system and consistent standard of trauma care .
small sample size and power are another limitations of our study as it is a single center study .
we have minimized the confounding effect from this bias by calculating blood product ratio at 4 h , unlike other studies that have calculated ratio at 24 h.
the mortality risk associated with lmtp ( plt : prbc < 1:1.5 ) may occur very early , possibly secondary to ongoing coagulopathy , plt dysfunction , and hemorrhage . aggressive attainment of high plt : prbc ratio as early as 4 h postinjury can substantially improve coagulopathy and reduce mortality and mof rates but increases the rate of infectious complications .
these results suggest that the survival advantage of hmtp for trauma patients is realized early .
we believe that there is a need to conduct a large prospective multi institutional study to further confirm the significance of attainment of high plt ratio and improved survival of trauma patients targeted within the first 4 h postinjury .
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### Summary:
: introduction : the optimal ratio of platelets ( plts ) to packed red blood cell ( prbc ) in trauma patients requiring massive transfusion protocol ( mtp ) is still controversial .
this report aims to describe the effect of attaining a high plt : prbc ratio ( 1:1.5 ) within 4 h postinjury on the outcomes of trauma patients receiving mtp.methods:over a 24-month period , records of all adult patients with traumatic injury who received mtp were retrospectively reviewed .
data were analyzed with respect to plt : prbc ratio ( [ high - mtp 1:1.5 ] [ hmtp ] vs. [ low - mtp < 1:1.5 ] [ lmtp ] ) given within the first 4 h postinjury and also between ( > 4 and 24 h ) .
baseline demographic , clinical characteristics , complications , and outcomes were compared according to hmtp and lmtp.results:of the total 3244 trauma patients , plt : prbc ratio was attainable in 58 ( 1.2% ) patients who fulfilled the inclusion criteria .
the mean age was 32.3 10.7 years ; the majority were males ( 89.6% ) with high mean injury severity score ( iss ) : 31.9 11.5 and revise trauma score ( rts ) : 5.1 2.2 .
there was no significant association between age , gender , type of injury , presenting hemoglobin , international normalized ratio , iss , and rts .
the rate of ventilator associated pneumonia ( 38.9% vs. 10.8% ; p = 0.02 ) and wound infection ( 50% vs. 10.8% ; p = 0.002 ) were significantly higher in the hmtp group . however
, hmtp was associated with lower rate of multiple organ failure ( mof ) ( 42.1% vs. 87.2% , p = 0.001 ) and mortality ( 36.8% vs. 84.6% , p = 0.001 ) within the first 30 days postinjury.conclusions:our study revealed that early attainment of high plt / prbc ratio within 4 h postinjury is significantly associated with lower mof and mortality in trauma patients .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: infection with oncogenic types of human papilloma virus ( hpv ) is regarded as the main causal factor of cervical cancer .
there is evidence to suggest that genetic factors affecting an individual 's susceptibility to hpv infection may influence the risk to develop cervical cancer .
however , the genes involved and mutations or variants in those genes remain to be fully established . as in other cancers ,
although cervical cancer is the third most common cancer in women worldwide , reports on familial cases of cervical cancer , on calculated tumour risks for relatives and , even more so , on its clinical implications are relatively rare . due to
the national screening programme cervical cancer is not a common disease in the netherlands , and no reports have been published on familial clustering of cervical cancer in dutch patients . in this paper
we report three dutch families with multiple cases of cervical cancer and cervical intraepithelial neoplasia ( cin ) , we review the literature on familial occurrence of cervical ( pre)neoplastic disease and discuss present and possible future practical clinical implications .
recently members of three non - related families ( pedigrees shown in figures 1 , 2 and 3 , respectively ) were referred to our clinic with questions regarding the possible hereditary nature of cervical cancer in their families and possible preventive options .
because family histories of cancer may be inaccurate , we verified the cervical cancer cases whenever possible .
pedigrees of the three families with three or four women with cin or cervical cancer . diagnosis or cause of death and age of diagnosis or death
= individual referred for genetic counselling black symbols = diagnosis confirmed by medical records grey symbols = medical records unavailable hatched symbols = non - cervical cancer reported by family numbers in the symbols = number of individuals .
cc = cervical cancer , scc = squamous cervical cancer , acc = adenocarcinoma of the cervix , asc = adenosquamous carcinoma of the cervix , ccc = clear cell carcinoma of the cervix , cis = cervical cancer in situ , cin = cervical intraepithelial neoplasia , dm = diabetes mellitus , cva = cerebro - vascular accident , ?
pedigrees of the three families with three or four women with cin or cervical cancer . diagnosis or cause of death and age of diagnosis or death
= individual referred for genetic counselling black symbols = diagnosis confirmed by medical records grey symbols = medical records unavailable hatched symbols = non - cervical cancer reported by family numbers in the symbols = number of individuals .
cc = cervical cancer , scc = squamous cervical cancer , acc = adenocarcinoma of the cervix , asc = adenosquamous carcinoma of the cervix , ccc = clear cell carcinoma of the cervix , cis = cervical cancer in situ , cin = cervical intraepithelial neoplasia , dm = diabetes mellitus , cva = cerebro - vascular accident , ? = unknown medical history family 3 .
pedigrees of the three families with three or four women with cin or cervical cancer . diagnosis or cause of death and age of diagnosis or death
= individual referred for genetic counselling black symbols = diagnosis confirmed by medical records grey symbols = medical records unavailable hatched symbols = non - cervical cancer reported by family numbers in the symbols = number of individuals .
cc = cervical cancer , scc = squamous cervical cancer , acc = adenocarcinoma of the cervix , asc = adenosquamous carcinoma of the cervix , ccc = clear cell carcinoma of the cervix , cis = cervical cancer in situ , cin = cervical intraepithelial neoplasia , dm = diabetes mellitus , cva = cerebro - vascular accident , ?
= unknown medical history in family 1 , the index - patient ( iv-3 ) was diagnosed with a clear cell carcinoma of the cervix , figo stage ib1 at the age of 24 .
the maternal great - grandmother ( i-1 ) was reported by the family to have been diagnosed with cervical cancer before the age of 50 . the maternal grandmother ( ii-5 )
had been diagnosed with adenosquamous carcinoma of the cervix and had died at the age of 46 . the paternal aunt ( iii-6 )
had been diagnosed with squamous carcinoma in situ ( cis ) of the cervix at the age of 35 , which was treated with a conization .
the diagnoses in iv-3 , ii-5 and iii-6 could be confirmed by checking the medical records .
the question on referral was what advice should be given to young girls of 16 and 15 years of age ( iv-4 and iv-5 , respectively ) . in family 2
, the proband ( iii-7 ) was referred because three of her sisters ( iii-1 , 5 and 9 ) had been diagnosed with cervical cancer or cin , all confirmed by medical records .
they were under the impression that their mother ( ii-6 ) had been diagnosed with cervical cancer as well .
however , checking medical records revealed that she had been diagnosed with hyperplasia of the endometrium instead . in family 3 , three cases of cervical cancer or cis , all squamous cell carcinoma , confirmed by medical recorts occurred in two generations ( ii-1 , iii-3 and 8) .
the medical history of two half - sisters ( iii-1 and 2 ) was unknown . hpv status was unknown in families 1 , 2 and 3 and the patterns of non - cervical cancer types reported in these families were not suggestive of any known hereditary cancer syndrome .
familial clustering of cervical cancer might be coincidental , the result of shared exogenic risk factors , shared genetic risk factors or a combination of these factors .
all these risk factors may well be shared within families because of possible shared lifestyles .
presently there is neither indication that cervical cancer which presents in familial clusters develops at a significantly earlier age than sporadic cervical cancer ( considered to be a hallmark of hereditary cancer ) nor that its clinical behaviour differs from that of sporadic cervical cancer .
the heritability of cervical cancer has been estimated between 22% and 46% [ 12 - 14 ] .
as publications on cases of familial clustering of cervical cancer are relatively rare , more data are needed to arrive at more precise estimations [ 15 - 17 ] . reported figures on familial cervical cancer in situ ( cis ) and cervical cancer are summarized in table 1 . apart from three studies ( the study by brinton et al , who investigated the family history of women with cervical cancer in a north american population , the study by fischer et al in a german population and furgyik et al in a swedish population ) , all other available studies used the swedish national cancer registers .
not surprisingly the results of the swedish studies are similar , although different methods to investigate familial risks were used and results were expressed in different types of risk units [ 12 - 14,21,22 ] . in these studies ,
a ( familial ) relative risk of about 1.5 - 2.3 for cis and/or cervical cancer for first degree relatives of affected women was reported , although some histological subtypes might be associated with a higher risk .
only one study included patients with severe dysplasia ( cin iii ) of the cervix .
because cin iii and ii are considered to be the precursors of cervical cancer , a genetic predisposition to cervical cancer might manifest itself as not only cervical cancer and cis , but also as cin ii and iii .
familial clustering of cin ii and iii as well as cis and cervical cancer might therefore be expected to occur , as shown in our family 2 .
therefore , it would be logical to include cin ii and iii in the analysis of familial cervical cancer risk , although cin will not be readily observed in populations without cervical cancer screening .
reported familial risks and heritability of cervical cancer rr = relative risk = the risk of cases compared with the risk of control frr = familial relative risk = the risk to the relatives of cases divided by the risk to the relatives of controls heritability = the proportion of total variance due to genetic variance cin = cervical intra - epithelial neoplasia cis = carcinoma in situ scc = squamous cell carcinoma asc = adenosquamous carcinoma so far , familial cervical cancer risk has been investigated in mother - daughter and sister - sister relationships only , but the pedigree of family 1 might suggest a paternally inherited genetic susceptibility for ( pre)neoplastic cervical disease .
future studies should include the history of ( cervical ) cancer in the paternal branch of the family , as ignoring the possibility of paternally transmitted genetic susceptibility will underestimate the genetic contribution to cervical cancer risk .
the familial risks reported so far are in the same order of magnitude as the familial relative risks ( frrs ) found in cancers with an identified hereditary component such as breast , ovarian and colon cancer .
in contrast to these tumour types , there is no evidence yet for the existence of a highly penetrant cervical cancer predisposition gene . to identify candidate genes associated with genetic susceptibility to cervical cancer , genes currently under investigation are the hla genes and other genes that are involved in cell - mediated immunity like il-10 , tp53 and genes involved in the detoxification of carcinogens found in tobacco smoke [ 2,3,25 - 30 ] .
the products of most of these genes are known to interact with the exogenic agents mentioned earlier .
no definitive conclusion about genetic predisposition in familial clusters of cervical cancer , including those that we have reported , will be possible before genes responsible for such predisposition have been convincingly identified .
what medical advice should we give to women with a family history of cervical cancer and/or its precursor lesions ?
for practical purposes , and based on current literature , close female relatives of the affected women in the families we have reported and similar ones can be presumed to have a moderately increased risk to develop cervical cancer .
this raises the issue of possible primary and secondary preventive options . in these women ,
adherence to screening programmes and reducing exposure to known exogenic risk factors might therefore be especially important . as hpv is the main causal factor for developing cervical cancer and this virus
is spread mainly through sexual contact , use of condoms might be advisable , since the use of condoms at least prevents hpv re - infection and thus can be a way to lower cervical cancer risk . another well - studied risk factor for cin and cervical cancer is smoking [ 32 - 34 ] .
smoking has been suggested to be a confounding effect caused by the association of smoking with a lifestyle with an increased risk of hpv infection , but in other studies , adjusted for hpv , smoking appeared to be an independent risk factor .
thus , smoking cessation should be advised , particularly in women at an increased risk for cervical cancer .
recently new guidelines for the early detection of cervical neoplasia and cancer were published by the american cancer society ( acs ) .
these recommendations include more frequent cervical screening for women who have been reported to be at a higher risk for cervical cancer , because they are either immunocompromised ( by organ transplantation , chemotherapy or chronic corticosteroid treatment ) or have a history of in utero exposure to diethylstilbestrol ( des ) .
interestingly , no recommendations were included for women with a positive family history of cin or cervical cancer although the relative risk for cervical cancer due to des exposure is comparable to that for cervical cancer due to familial clustering .
similar relative risks have lead to recommendations for increased surveillance in women with a family history of cancer of the breast , ovaries or colon . taken together
, it appears to be consistent to consider a more intensive screening policy for women with a family history of cervical cancer as well . in our opinion
, this screening could follow the acs guidelines for other groups of women with an increased cervical cancer risk , in particular those listed for women with a history of in utero exposure to des ( table 2 ) , until more data become available .
however , whether this annual screening should continue beyond the age of 70 ( acs does not mention a fixed upper age limit for the des - exposed group ) is questionable . as in other familial cancer screening programmes , the benefits and costs ( physical , psychological and economic ) of increased surveillance in women with a family history of cervical cancer would need to be established and this surveillance should therefore be monitored in a research setting .
summary of the american cancer society guidelines on screening for cervical cancer diagnosis of condition associated with compromised immune system or start of immunocompromising therapy , respectively when in the future genetic predisposition to cervical cancer can be identified at a molecular level , presymptomatic genetic testing will become an option .
the identification of such predisposition might stimulate compliance to screening programmes , or , in regions without population screening , it might make cervical cancer screening available to the women involved
. moreover , when genetic susceptibility indeed turns out to act through a decreased host response to hpv infection , then women with this particular susceptibility might be good candidates for prophylactic hpv vaccination .
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### Summary:
: we report three dutch families with familial clustering of ( pre)neoplastic cervical disease , review the literature on familial risks of cervical intraepithelial neoplasia ( cin ) and cervical cancer , and discuss possible practical guidelines for women with a family history of cervical cancer .
daughters and sisters of women with cervical cancer have been reported to have a relative risk of 1.5 - 2.3 to develop this type of cancer . from a practical clinical point of view
, we suggest that as in women with an increased non - genetic risk to develop cervical cancer ( e.g. because of immunosuppressive therapy ) increased surveillance to detect this tumour should be considered in women with an increased risk based on family history .
cessation of smoking should be advised .
as the use of condoms at least prevents hpv re - infection its use can be recommended as a way to lower the cervical cancer risk .
future studies to determine the genetic contribution to the development of cervical cancer should include the paternal family history of cancer and , because genetic predisposition might express itself as a higher risk to develop precursors of cervical cancer , carcinoma in situ and cin grade ii - iii .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: choroid plexus papillomas ( cpp ) and choroid plexus carcinoma ( cpc ) ) comprise 2 - 4% of all pediatric brain tumors . of these ,
these tumors produce symptoms even when small because of their strategic location as well as cerebrospinal fluid ( csf ) over production .
diagnosis rests on imaging studies that show the mass in the location of third ventricle with typical imaging features on sonography , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) .
radiosurgery can achieve reduction in size as well as vascularity of lesion to allow less complicated procedure .
a 7-week - old full term male presented with downward gaze since birth and progressive increase in head size . on examination , the child was normal weight but was lethargic .
cranial sonography revealed dilatation of bilateral lateral ventricles with an echogenic lobulated mass in the third ventricle with internal vascularity [ figure 1 ] .
no evidence of hemorrhage was noted in the ventricular system or brain parenchyma . a diagnosis of choroid plexus papilloma was suspected and a contrast - enhanced ct ( cect ) confirmed the enhancing mass in the third ventricle with hydrocephalus [ figure 2 ] . to know the exact extent of the tumor as a part of preoperative evaluation , a contrast - enhanced mri was performed .
a lobulated mass was noted in the third ventricle , hypointense on t1 weighted and mildly hyperintense on t2 with intense enhancement on administration of intravenous gadolinium .
contrast - enhanced images depict the extension into the fourth ventricle [ figure 3 ] .
child underwent a shunt procedure followed by right frontal craniotomy with transcortical transforaminal total excision of the tumor .
the main feeding vessel was posterior choroidal artery that was identified after debulking of the tumor .
the histopathology of the resected tumor showed tumor cells arranged in multiple papillae with fine fibrovascular core , confirming the diagnosis of cpp [ figure 4 ] .
postoperative course of the child was uneventful and follow - up ct revealed no residual tumor .
sagittal ( a ) , coronal ( b ) , and axial ( c ) cranial sonographic images reveal an echogenic lobulated mass obstructing the third ventricle .
doppler evaluation ( d ) reveals vascularity within the lesion axial computed tomography ( ct ) images reveal an enhancing lobulated mass in the third ventricle ( a ) with dilatation of the lateral ventricles ( b ) axial magnetic resonance ( mr ) images ( a and b ) reveal the tumor with hypointensity on t1-weighted images and mild hyperintensity relative to gray matter on t2-weighted images .
contrast - enhanced axial ( c ) and sagittal ( d ) images reveal avid enhancement within the tumor and extension into fourth ventricle low power ( a ) and high power ( b ) photomicrograph reveal tumor cells arranged in multiple papillae with fine fibrovascular core
cpp is a rare tumor of neuroectodermal origin , accounting for less than 1% of all intracranial neoplasms .
it has predilection for lateral ventricle in infants and children and fourth ventricle in adults .
however , description of third ventricular cpp is limited to a few case reports or case series .
these tumors typically present in neonatal or early childhood period , pointing to their congenital origin .
these lesions typically manifest with increasing head size , lethargy , decreased activity , and poor psychomotor milestones in infants .
as the child grows older , visual impairment and gait disturbances are noted , related to raised intracranial pressure ( icp ) and direct pressure caused by the tumor .
hydrocephalus and raised icp in these cases is caused by two mechanisms : direct obstruction of csf pathway and overproduction of csf by tumor cells that tend to maintain function of choroid plexus .
because of their strategic location , very small lesions can be symptomatic and likely to be missed . most of the lesions that have been described measure between 2 and 5 cm .
razzaq and chishti , reported a giant cpp of third ventricle measuring 8 5 5 cm .
certain interesting symptoms and signs associated with third ventricle cpps have been described in literature .
bobble - head doll syndrome and drop attacks were noted in a 2-year - old child by pollack et al .
this was hypothesized to be due to intermittent obstruction of foramen of monro and proximal aqueduct .
behavioral disturbances have also been described , probably related to proximity and pressure by the tumor on hypothalamus .
imaging plays a vital role in differentiating between obstructive ( noncommunicating ) and nonobstructive ( communicating ) hydrocephalus .
ultrasonography ( usg ) is the modality of choice in utero , neonatal , and early infancy .
one of the greatest strengths of usg is the monitoring of hydrocephalus in the most noninvasive way , with no need for sedation and without the use of radiation .
besides , usg can detect small intraventricular lesions that can sometimes be missed on noncontrast ct , as was the case in our patient .
ct plays an important role in older children where the acoustic window for usg is lost .
the degree of heterogeneity is variable depending on the size of tumor , and if marked , should lead to a suspicion of cpc , particularly if there is adjacent invasion .
mri is the modality of choice in older children for central nervous system imaging in general and intracranial tumors in particular .
this is related to the ability of mri to provide multiparametric imaging affording a preoperative typing and grading of tumor , planning of biopsy , and surgery .
complete removal of tumor is advocated and various approaches to third ventricular tumors include transcallosal , transfrontal , transforaminal routes , and supracerebellar infratentorial . in individual cases ,
the surgical approach would depend on location , size , vascularity , and extension of tumor through the foramen of monro .
another innovative therapy is radiosurgery that aims at avascular necrosis and shrinkage of tumor and is best as an adjunct to surgery .
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### Summary:
: choroid plexus papilloma ( cpp ) represents an uncommon pediatric brain tumor with an overall incidence less than 1% of all intracranial tumors .
most of these tumors occur in the lateral ventricles in neonates .
third ventricular location is uncommon , limited to a few case reports .
these highly vascular tumors retain the physiological function of choroid plexus and thus lead to overproduction of cerebrospinal fluid ( csf ) , besides obstructing the csf pathway .
imaging is fairly sensitive and specific in affording the diagnosis of this tumor .
surgical approaches differ according to the site of tumor and aim is complete removal of tumor .
we present an interesting report of an infant who presented to our department for cranial sonography that lead to suspicion of this tumor , later confirmed by other imaging modalities and histopathology .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: specific mutations of epidermal growth factor receptor ( egfr ) play a key role in the development of non - small cell lung cancer ( nsclc ) , primarily adenocarcinomas , and specific egfr - targeted therapy has revolutionized the treatment of the subset of adenocarcinomas harboring an egfr - activating mutation.1,2 several small molecules , egfr - tyrosine kinase inhibitors ( tkis ) , such as gefitinib , erlotinib and afatinib , have been demonstrated to significantly improve clinical outcomes ( response rate [ rr ] , progression - free survival [ pfs ] and overall survival ) in patients with advanced egfr - mutated nsclc , when used in the first-,3 second- or third - line treatment.4,5 erlotinib is not only approved as a single agent in nsclc that has egfr - activating mutations , but it is also approved in chemorefractory nsclc regardless of egfr mutation status and maintenance therapy after platinum - based doublets .
it is known that , in addition to the mutation in the egfr gene , patient characteristics of being asian , having an adenocarcinoma , being female and being a nonsmoker give an increased response to egfr inhibitors , but erlotinib activity in egfr wild - type squamous carcinoma is still highly debated.6 here , we describe a prolonged and unexpected clinical response to erlotinib in a male former heavy cigarette smoker with wild - type egfr squamous - cell cancer .
we report a case of a 65-year - old caucasian man , a former heavy smoker , with a medical history of arterial hypertension , cardiac arrhythmia and chronic obstructive pulmonary disease ( copd ) .
a whole - body computed tomography ( ct ) scan showed an area of parenchymal consolidation in the anterior segment of the right upper lobe and infiltration of the pulmonary vein . in october 2009 ,
histopathology and mutation testing ( pcr in real time ) showed an egfr wild - type squamous non - small cell lung cancer ( sqnsclc ) involving the cava vein and the mediastinum with no metastasis in 13 lymph nodes removed ; whole - body ct scan did not show evidence of metastasis ( tnm 2009 : pt4 pn0 ) .
the first post - surgery ct scan evaluation performed in january 2010 revealed a solid nodule of 2.6 cm in diameter in the mediastinum , a paraesophageal lesion and spleen metastases .
the patient received a first - line chemotherapy regimen , four cycles of carboplatin and gemcitabine , but subsequent ct scan evidenced disease progression .
the patient was started on a second - line weekly docetaxel chemotherapy ; this treatment was interrupted after the first cycle for grade 4 neutropenia and atrial fibrillation .
the clinical history of this patient ( short disease progression - free interval and limiting bone marrow toxicity ) ruled out the use of a new chemotherapy regimen .
the patient presented a grade 3 skin rash within 1 month from the beginning of erlotinib treatment .
skin toxicity was treated with topic and systemic therapies , and an erlotinib dose reduction was needed ( 100 mg / die ) .
the first posttreatment evaluation ct scan showed a stable disease according to recist criteria ( reduction of paraesophageal lesion from 50 mm to 40 mm ) .
the patient continued erlotinib 100 mg once daily with a persistent grades 23 skin toxicity .
four months later , a new ct scan highlighted a persistent stable disease with continuing reduction of the paraesophageal lesion ( 35 mm vs 40 mm ) .
the paraesophageal lesion continued to decrease : a ct scan performed on may 2 , 2013 , showed a reduction of paraesophageal lesion ( 18 mm [ figure 1b ] vs 35 mm [ figure 1a ] ) .
several ct scans performed every 4 months confirmed stable disease , but due to g4 skin toxicity , erlotinib treatment was stopped in march 2016 , 5 years after the beginning of the therapy .
however , 2 months later , in may 2016 , the first posttreatment ct scan documented lung disease progression .
written informed consent has been provided by the patient to publish the case and the image .
approximately 10% of caucasian and up to 50% of asian patients with nsclc are positive for egfr mutations , and this is predictive for response to the egfr - tkis.1 several randomized trials showed that tkis provide an improved rr and pfs , with a good tolerability of treatment and a better quality of life ( qol ) , when compared with cytotoxic treatment as a first - line chemotherapy in egfr - mutated advanced nsclc patients.3 conversely , for the first - line treatment of locally advanced or metastatic nsclc with wild - type or unknown egfr status , platinum - based doublet chemotherapy ( with agents such as taxanes , gemcitabine , vinorelbine and etoposide ) remains the standard of care.7 regarding the second- or third - line therapies , the br.21 trial demonstrated that erlotinib is superior to the best supportive care for the treatment of patients with egfr wild - type nsclc , including squamous - cell cancer.8 these results were confirmed by a phase iv study ( tarceva lung cancer survival treatment [ trust]).9 however , a randomized trial comparing erlotinib and docetaxel as a second - line treatment for egfr wild - type nsclc ( tarceva italian lung optimization trial [ tailor ] ) demonstrated the superiority of using docetaxel in a second - line therapy.10 similarly , a japanese randomized phase iii trial of erlotinib versus docetaxel as a second- or third - line therapy ( docetaxel and erlotinib lung cancer trial [ delta ] ) indicated the superiority of using chemotherapy in an egfr wild - type subpopulation.11 however , despite the evidence of the tailor and delta trials , for patients in whom no alternative is recommended , erlotinib might be considered.12 furthermore , in the br.21 trial , the survival benefit with erlotinib was maintained in male smokers with squamous histology and in the tailor trial , there was similar os in the sqnsclc patients treated with erlotinib or docetaxel .
although activating mutations in the egfr gene are the most important predictive biomarkers for egfr - tki treatment , the clinical benefits can not only be explained by these mutations.13,14 in patients with progressive egfr wild - type sqnsclc after failure of standard chemotherapy , comorbidities and a low probability of a response to a chemotherapy , egfr - tkis are a valid treatment option over placebo or best supportive care .
in fact , although in the second - line setting , survival outcomes are comparable between cytotoxic treatment and egfr - tki treatment in a meta - analysis , the latter is more tolerable as a second - line therapy.15 in the present case report , the choice of erlotinib exhibited an unexpected prolonged response , unexpected because the clinical characteristics of our patient did not correlate with those predictive of response .
in fact , despite the absence of egfr mutation , squamous histology , male sex and heavy smoking history , the clinical efficacy of tki continued for 5 years .
our patient has already reached a pfs of 62 months and os not yet reached , with a global objective response of 64% .
moreover , our case report shows how important it is to maintain egfr inhibition overtime .
in fact , when skin toxicity forced to stop therapy , the disease has quickly progressed . in conclusion
, this case report confirms that egfr - tki therapy may confer benefit in terms of pfs and os , regardless of the mutation status , histological subgroup and other clinical characteristics .
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### Summary:
: several small molecules , epidermal growth factor receptor ( egfr)-tyrosine kinase inhibitors ( tkis ) , such as gefitinib , erlotinib and afatinib , have been demonstrated to significantly improve clinical outcomes in patients with advanced egfr - mutated non - small cell lung cancer ( nsclc ) , but erlotinib activity in egfr wild - type squamous carcinoma is still highly debated . here , we describe a prolonged and unexpected clinical response to erlotinib in a male former heavy cigarette smoker with wild - type egfr squamous - cell cancer .
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### Summary:
: aging is characterized by clonal expansion of myeloid - biased hematopoietic stem cells and by an increased risk of myeloid malignancies .
exome sequencing of 3 elderly females with clonal hematopoiesis demonstrated by x - inactivation analysis identified somatic tet2 mutations .
recurrence testing found tet2 mutations in 10 out of 182 individuals with x - inactivation skewing .
tet2 mutations were specific to individuals with clonal hematopoiesis without hematologic malignancies and were associated with alterations in dna methylation .
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: the online version of this article ( doi:10.1007/s00280 - 013 - 2209 - 7 ) contains supplementary material , which is available to authorized users .
an increasing number of nanomedicines , including liposomes , polymer conjugates , block copolymer micelles , nanoparticles and other complex hybrid technologies , are being developed as anticancer therapeutics , imaging agents and theranostics ( reviewed in [ 14 ] ) .
several products are already in routine clinical use ( e.g. doxil , abraxane ) with a growing number of technologies including polymer therapeutics in clinical development [ 2 , 3 ] . intravenously ( i.v . )
administered long - circulating nanosized constructs have long been known to exhibit passive tumour targeting due to the enhanced permeability and retention ( epr ) effect [ 5 , 6 ] .
this phenomenon has been demonstrated in many in vivo tumour models and the features of polymer conjugates [ 7 , 8 ] , nanoparticles and block copolymer micelles governing extravasation , as well as tumour characteristics regulating the efficiency of the process have been discussed .
liposomal and polymer conjugate - based gamma camera imaging probes have also demonstrated the epr effect in some tumours in patients [ 1113 ] . a diverse array of tumour pathophysiological features regulate the efficiency of epr - mediated tumour targeting including heterogeneity of intratumoural blood flow , angiogenic vascular permeability , tumour microenvironment including extracellular matrix features , interstitial pressure and lymphatic drainage , and thus , no single biomarker adequately predicts tumour targeting and/or antitumour activity . as a functional epr effect is the primary factor governing nanomedicine tumour access ( fig . 1 ) , it is surprising that tumour models used to evaluate pharmacokinetics ( pk ) and/or antitumour activity have been rarely pre - calibrated for this characteristic
this makes comparison of results , both between experiments and between laboratories , difficult and prevents effective benchmarking of emerging technologies against those for which there is now a considerable clinical database .
moreover , although many constructs have been designed for intratumoural activation , particularly intralysosomal drug release ( using low ph or lysosomal enzymes such as cathepsin b ) ( fig . 1 ) , few tumour models are calibrated with respect to these activation mechanisms .
therefore , the aim of this study was to quantify systematically the early - phase epr - mediated tumour targeting using a panel of murine and human xenograft tumours .
the albumin - binding dye evans blue , widely used as a physiological marker of vascular permeability , and n-(2-hydroxypropyl)methacrylamide ( hpma ) copolymer gflg dox ( fce28068 ) ( fig . 1 )
, a conjugate that has undergone phase i / ii clinical trials [ 11 , 12 ] , were selected as probes of the epr effect in tumour models . in certain experiments , doxorubicin ( dox ) alone was used as a reference control . to establish the comparability of evans blue and fce28068 as epr probes
b16f10 model previously used widely to document the pk and antitumour activity of polymer conjugates and a s.c . l1210 model known to be sensitive to dox .
fce28068 was then used to quantify the effect of tumour size on early - phase ( 1 h ) tumour accumulation in a panel of murine and human xenograft tumours ( table 1 ) and to study the effect of tumour size on passive targeting .
in addition , as cathepsin b mediates dox liberation from fce28068 , and this enzyme is also responsible for activation of polyglutamic acid anticancer conjugates currently in clinical trials ( e.g. paclitaxel poliglumex ( ppx ) ) , the extent of dox released at 1 h was also quantified . it should be noted that the 1 h time point was selected for this study as it minimises additional kinetic effects introduced by differences in blood clearance rate , probe- or tumour model - dependent tumour efflux rates and/or inter - tumoural differences in the degradation rate of biodegradable probes , e.g. proteolysis of albumin over time [ 8 , 17].fig .
1structure of a fce28068 , evans blue and a schematic representation of pendant dox molecules conjugated to a nanomedicine and b a schematic diagram showing the key steps in tumour targeting and lysosomotropic activationtable 1summary of the tumour models usedcodetype of tumourmousemurine modelsl1210lymphocytic leukaemiadba2b16f10melanomac57 blkmac 15aadenocarcinomanmrimac
26adenocarcinomanmrimeta 7lung tumourbalb / chuman xenograftsrxf 1220renal cell carcinomanu / nurxf 486renal cell carcinomanu / nupaxf 546pancreatic carcinomanu / numexf
carcinomanu / nucor l23human non - small cell lung carcinomanu / nusk - n - shneuroblastomascidimr 32neuroblastomascidsk - n - dzneuroblastomascid and for others [ 17 , 29 ] structure of a fce28068 , evans blue and a schematic representation of pendant dox molecules conjugated to a nanomedicine and b a schematic diagram showing the key steps in tumour targeting and lysosomotropic activation summary of the tumour models used and for others [ 17 , 29 ]
hpma copolymer gflg dox ( fce28068 ; also known as pk1 ) ( mw ~ 30,000 g / mol ; mw / mn = 1.3 ; total dox 68 wt% ; free dox < 1 % in respect of total ) and dox were a gift from pharmacia and upjohn , italy .
meta-7 and cor l23 cells were from european collection of cell cultures ( ecacc ) , wiltshire , uk .
c57 black , dba2 , balb - c and nu / nu mice were supplied by bantin and kingman ltd . , uk .
all animal experiments were performed in accordance with the united kingdom co - ordinating committee on cancer research ( ukcccr ) guidelines for the welfare of animals in experimental neoplasia ( 1998 ) and with uk home office guidelines .
models ( see table 1 and [ 14 , 15 , 17 ] for full details ) .
generally , cells were implanted into the posterior right flank of mice , and the studies were initiated when tumour reached 25289 mm ( product of two diagonal width ) .
evans blue ( 2 mg / ml in 0.9 % nacl ; 10 mg / kg ) , fce28068 or free dox ( 1 mg / ml in pbs ; 5 mg / kg dox - equiv ) was administered i.v . via the tail vein .
fce28068 was given the higher dose of 40 mg / kg dox - equiv to nmri mice bearing the murine adenocarcinomas mac 15a and mac 26 to enable comparison with efficacy experiments in this model .
after 1 h , all animals were humanely killed , and tumour was carefully removed , washed with pbs , weighed and snap - frozen in liquid nitrogen before extraction and analysis .
it should be noted that most of this study was conducted according to the 2nd edition of the ukcccr guidelines for the welfare of animals in experimental neoplasia , which states that the maximum tumour burden should not exceed 10 % of the host animal s normal body weight or 2.5 g. the initial study involving measurement of evans blue levels in the s.c .
l1210 tumour model was conducted prior to the 2nd edition of ukcccr guidelines , thus accounting the use of tumours in excess of 2.5 g. evans blue was extracted from the tumour samples using a method adapted from harada et al .
the homogenate ( 1,900 l ) was mixed with aqueous sodium sulphate solution ( 0.5 % w / v , 3 ml ) and acetone ( 7 ml ) in polypropylene tubes and then incubated at room temperature ( 20 c ) overnight before centrifugation ( 1,000g , 30 min , 20 c ) to precipitate the tissue .
the concentration of evans blue in the supernatant was quantified spectrophotometrically at 590 nm using a standard curve prepared by spiking foetal bovine serum ( fbs ) samples with known concentrations of evans blue .
administration of free dox , drug in tumour samples was quantified using a previously described hplc method [ 20 , 21 ] . throughout the extraction procedure
, all samples were maintained at 4 c and kept in the dark ( wrapped in foil ) .
briefly , tumour samples were thawed , homogenates were prepared in pbs , and samples ( 900 l ) were placed in polypropylene tubes .
a daunomycin ( dnm ) standard ( donated by rhne - poulenc , france ) ( 100 ng , 100 l ) in pbs was added as an internal standard , followed by ammonium formate buffer ( 1 m ; ph 8.5 ; 100 l ) and chloroform / propan-2-ol ( 4:1 v / v , 5 ml ) . after thorough vortex - mixing ( 3 10 s ; within 30 min ) , the tubes were centrifuged ( 1,000g ; 30 min , 10 c ) .
the remaining organic phase was evaporated to dryness using a sample concentrator under constant nitrogen gas ( n2 ) flow over 20 min .
the samples obtained were re - dissolved in hplc grade methanol ( 100 l ) by vortex - mixing ( 30 s ) and centrifuged ( 1,000g , 10 min , 10 c ) to remove any remaining biological materials .
samples ( 100 l ) were then analysed by hplc using a c18 bondapak column eluted with propan-2-ol in water ( 29 % v / v ) adjusted to ph 3.2 with orthophosphoric acid with a fluorimetric detector ( excitation 480 nm , emission 560 nm ) . data were collected and analysed using a powerchrom integrator and software programme ( powerchrom v 2.0.7 ) .
as hpma copolymer dox does not extract directly into the organic solvent , free dox present in the tumour samples could be extracted and quantified as described above [ 20 , 21 ] .
dox , a previously described acid hydrolysis method [ 20 , 21 ] was used to first liberate the dox aglycone from the conjugate as follows .
tumours were homogenised in pbs ( 2,000 l ) . then to duplicate samples ( 900 l ) , dnm was added as an internal standard ( 500 ng dnm ; 100 l in pbs ) followed by hcl ( 2 m , 1 ml ) before heating at 80 c for 20 min .
samples were neutralised by the addition of naoh ( 2 m ; 1 ml ) , ammonium formate buffer ( 1 m ; ph 8.5 ; 1.5 ml ) and then chloroform / propan-2-ol ( 4:1 v / v , 5 ml ) were added , and the samples were extracted .
hplc analysis was conducted exactly as described above for free dox . to ensure quantitation , hpma copolymer
data describing tumour accumulation of evans blue or hpma copolymer dox were expressed as either ( i ) the % administered dose per tumour ( for the conjugate the value is given as % dox - equiv dose administered ) or ( ii ) the administered dose % dose / g tumour ) .
statistical significance was calculated using student s t test for comparison of the mean of two small samples .
statistical differences of at least p < 0.05 were considered statistically significant . to allow comparison between
small and large tumours , the three smallest and three largest tumours available were grouped according to the classifiers for small and large described in the figure legends where tumour size as a variable is examined .
hpma copolymer gflg dox ( fce28068 ; also known as pk1 ) ( mw ~ 30,000 g / mol ; mw / mn = 1.3 ; total dox 68 wt% ; free dox < 1 % in respect of total ) and dox were a gift from pharmacia and upjohn , italy .
meta-7 and cor l23 cells were from european collection of cell cultures ( ecacc ) , wiltshire , uk .
c57 black , dba2 , balb - c and nu / nu mice were supplied by bantin and kingman ltd . , uk .
all animal experiments were performed in accordance with the united kingdom co - ordinating committee on cancer research ( ukcccr ) guidelines for the welfare of animals in experimental neoplasia ( 1998 ) and with uk home office guidelines .
models ( see table 1 and [ 14 , 15 , 17 ] for full details ) .
generally , cells were implanted into the posterior right flank of mice , and the studies were initiated when tumour reached 25289 mm ( product of two diagonal width ) .
evans blue ( 2 mg / ml in 0.9 % nacl ; 10 mg / kg ) , fce28068 or free dox ( 1 mg / ml in pbs ; 5 mg / kg dox - equiv ) was administered i.v . via the tail vein .
fce28068 was given the higher dose of 40 mg / kg dox - equiv to nmri mice bearing the murine adenocarcinomas mac 15a and mac 26 to enable comparison with efficacy experiments in this model .
after 1 h , all animals were humanely killed , and tumour was carefully removed , washed with pbs , weighed and snap - frozen in liquid nitrogen before extraction and analysis . it should be noted that most of this study was conducted according to the 2nd edition of the ukcccr guidelines for the welfare of animals in experimental neoplasia , which states that the maximum tumour burden should not exceed 10 % of the host animal s normal body weight or 2.5 g. the initial study involving measurement of evans blue levels in the s.c .
l1210 tumour model was conducted prior to the 2nd edition of ukcccr guidelines , thus accounting the use of tumours in excess of 2.5 g.
evans blue was extracted from the tumour samples using a method adapted from harada et al .
the homogenate ( 1,900 l ) was mixed with aqueous sodium sulphate solution ( 0.5 % w / v , 3 ml ) and acetone ( 7 ml ) in polypropylene tubes and then incubated at room temperature ( 20 c ) overnight before centrifugation ( 1,000g , 30 min , 20 c ) to precipitate the tissue .
the concentration of evans blue in the supernatant was quantified spectrophotometrically at 590 nm using a standard curve prepared by spiking foetal bovine serum ( fbs ) samples with known concentrations of evans blue .
administration of free dox , drug in tumour samples was quantified using a previously described hplc method [ 20 , 21 ] . throughout the extraction procedure
, all samples were maintained at 4 c and kept in the dark ( wrapped in foil ) .
briefly , tumour samples were thawed , homogenates were prepared in pbs , and samples ( 900 l ) were placed in polypropylene tubes .
a daunomycin ( dnm ) standard ( donated by rhne - poulenc , france ) ( 100 ng , 100 l ) in pbs was added as an internal standard , followed by ammonium formate buffer ( 1 m ; ph 8.5 ; 100 l ) and chloroform / propan-2-ol ( 4:1 v / v , 5 ml ) .
after thorough vortex - mixing ( 3 10 s ; within 30 min ) , the tubes were centrifuged ( 1,000g ; 30 min , 10 c ) .
the remaining organic phase was evaporated to dryness using a sample concentrator under constant nitrogen gas ( n2 ) flow over 20 min .
the samples obtained were re - dissolved in hplc grade methanol ( 100 l ) by vortex - mixing ( 30 s ) and centrifuged ( 1,000g , 10 min , 10 c ) to remove any remaining biological materials .
samples ( 100 l ) were then analysed by hplc using a c18 bondapak column eluted with propan-2-ol in water ( 29 % v / v ) adjusted to ph 3.2 with orthophosphoric acid with a fluorimetric detector ( excitation 480 nm , emission 560 nm ) . data were collected and analysed using a powerchrom integrator and software programme ( powerchrom v 2.0.7 ) . for each experiment , a dox standard curve was prepared in parallel .
as hpma copolymer dox does not extract directly into the organic solvent , free dox present in the tumour samples could be extracted and quantified as described above [ 20 , 21 ] . to quantify hpma copolymer
dox , a previously described acid hydrolysis method [ 20 , 21 ] was used to first liberate the dox aglycone from the conjugate as follows .
tumours were homogenised in pbs ( 2,000 l ) . then to duplicate samples ( 900 l )
, dnm was added as an internal standard ( 500 ng dnm ; 100 l in pbs ) followed by hcl ( 2 m , 1 ml ) before heating at 80 c for 20 min .
samples were neutralised by the addition of naoh ( 2 m ; 1 ml ) , ammonium formate buffer ( 1 m ; ph 8.5 ; 1.5 ml ) and then chloroform / propan-2-ol ( 4:1 v / v , 5 ml ) were added , and the samples were extracted .
hplc analysis was conducted exactly as described above for free dox . to ensure quantitation , hpma copolymer
data describing tumour accumulation of evans blue or hpma copolymer dox were expressed as either ( i ) the % administered dose per tumour ( for the conjugate the value is given as % dox - equiv dose administered ) or ( ii ) the administered dose % dose / g tumour ) .
statistical significance was calculated using student s t test for comparison of the mean of two small samples .
statistical differences of at least p < 0.05 were considered statistically significant . to allow comparison between
small and large tumours , the three smallest and three largest tumours available were grouped according to the classifiers for small and large described in the figure legends where tumour size as a variable is examined .
to assess evidence for the operation of the epr effect , intratumour levels of evans blue , fce28028 and dox detected at 1 h following i.v .
b16f10 or l1210 tumours are shown in figs . 2 and 3 . when the tumour levels were expressed as % dose for evans blue ( fig .
2a , c ) , tumour levels increased in an approximately linear fashion with increasing tumour size up to a maximum of 46 % dose / tumour for the b16f10 model and ~15 % dose / tumour for the l1210 model ( fig .
3a ) were quantitatively similar across the b16f10 tumour size range , as expected for agents requiring permeable blood vessels for distribution .
administration of free drug were consistently much lower ( < 0.8 % dose per tumour ; fig .
3c ) , and expression in terms of % dose / g tumour tissue showed dox localisation to be constant across all tumour sizes studied ( in the range 1.12.5 % dose / g tumour ; fig .
3b ) levels expressed as % dose / g tumour decreased as tumour size increased ( figs .
values fell to 0.43.7 % dose / g in the largest tumours ( > 600 mg ; fig .
3b ) . the observed tumour size dependence of evans blue accumulation while still evident was less marked in the l1210 model ( fig .
figure 4 summarises data with respect to size of b16f10 tumours in relation to accumulated systemically administered evans blue , fce28028 and dox.fig .
a , c tumour levels expressed as % dose per tumour and b , d tumour levels expressed as % dose / g / tumour .
a , c tumour levels expressed as % dose per tumour , and b , d tumour levels expressed as % dose / g / tumour .
4comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) .
. not significantly different levels of evans blue detected in s.c . b16f10 and l1210 tumours at 1 h after i.v . administration .
a , c tumour levels expressed as % dose per tumour and b , d tumour levels expressed as % dose / g / tumour .
each point represents a single tumour sample levels of fce28068 and free dox detected in s.c .
a , c tumour levels expressed as % dose per tumour , and b , d tumour levels expressed as % dose / g / tumour .
each point represents a single tumour comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) . statistical significance
. not significantly different in contrast to the results obtained in the b16f10 models , when fce28068 was administered to mice bearing mac 15a ( 8.212.6 % dose / g ) , mac 26 adenocarcinoma ( 6.910.8 % dose / g ) or meta-7 lung ( 3.54.7 % dose / g ) tumours ( fig . 5 ) , the amount of conjugate in the tumour did not show statistically significant changes as tumour size increased .
while the mac tumours accumulated fce28068 to a similar degree as the b16f10 model , approximately two to three lower conjugate levels were seen in the meta-7 lung model ( fig .
5d).fig . 5fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v
c individual tumour samples and d a comparison of the levels after measured in the three smallest and three largest tumours .
the distribution of fce 28608 for the b16f10 model is shown for comparison . in fig
. 5 small b16f10 , mac 15a mac 26 and meta-7 tumours were < 100 , < 470 , < 480 and < 150 mg , respectively . large b16f10 , mac 15a , mac 26 and meta-7 tumours were > 400 , > 900 , > 1,000 and > 300 mg , respectively
0.02 ; ns not significantly different fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v .
c individual tumour samples and d a comparison of the levels after measured in the three smallest and three largest tumours .
the distribution of fce 28608 for the b16f10 model is shown for comparison . in fig .
5 small b16f10 , mac 15a mac 26 and meta-7 tumours were < 100 , < 470 , < 480 and < 150 mg , respectively . large b16f10 , mac 15a , mac 26 and meta-7 tumours were > 400 , > 900 , > 1,000 and > 300 mg , respectively . statistical significance * * p < 0.02 ; ns not significantly different we therefore examined a series of additional models , as a tumour size - dependent nanomedicine delivery has been incompletely explored , and the existence of this phenomenon has important implications for the design of pre - clinical and clinical studies with nanomedicines .
the mexf 276 , maxf 449 and rxf 486 human xenograft models displayed a tumour size - dependent pattern of fce28068 accumulation ( supplemental fig . 1 ) .
although paxf 546 showed a similar trend , it should be noted that the three smallest and the three largest tumours of paxf 546 pancreatic carcinoma tumours did not give statistically significance different values . in contrast , the rxf 1220 , cor l23 , imr 32 , sk - n - sh and sk - n - dz tumours ( supplemental fig .
( only two sk - n - dz tumours were available , but the data are included for completeness ) .
comparison of summary mean values obtained for fce28068 accumulation in all the tumour xenografts ( fig . 6 ) revealed the highest values in the non - small cell lung cancer cor l23 ( 4.712.2 % dose / g ) and the lowest values in the larger ( 0.20.4 g ) maxf 449 tumours ( 1.0 0.1 % dose / g ) .
early - phase tumour levels of fce28068 across all the xenograft tumours ( and sizes ) examined displayed ~12-fold variation in magnitude.fig .
6comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se , n = 3 ) .
tumour model size cut - offs ( mg small , mg large ) are as follows : mexf276 ( < 50 , > 200 ) ; maxf449 ( < 50 , > 200 ) , paxf 546 ( < 100 , > 500 ) , rxf 486 ( < 100 , > 600 ) , rxf 1220 ( < 100 , > 400 ) , corl 23 ( < 100 , > 400 ) , imr32 ( < 120 , > 220 ) . statistical significance * p < 0.05 * * * p < 0.01 , ns not significantly different . for the full data set , see supplementary figs . 1 and 2 comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se ,
tumour model size cut - offs ( mg small , mg large ) are as follows : mexf276 ( < 50 , > 200 ) ; maxf449 ( < 50 , > 200 ) , paxf 546 ( < 100 , > 500 ) , rxf 486 ( < 100 , > 600 ) , rxf 1220 ( < 100 , > 400 ) , corl 23 ( < 100 , > 400 ) , imr32 ( < 120 , > 220 ) .
0.05 * * * p < 0.01 , ns not significantly different . for the full data set , see supplementary figs . 1 and 2 after administration of fce28068 , the free dox detected in all tumour samples at 1 h expressed as percentage of total dox ( i.e. conjugated drug + free ) is shown in fig .
only the mac 15a , mac 26 murine tumours and the cor l23 xenograft showed tumour size dependency in terms of dox liberation at 1 h. this is interesting as none of these tumour models displayed tumour size - dependent fce28208 levels at 1 h. dox release was greater in the smaller tumours , and the difference is particularly striking for the smaller ( < 100 mg ) cor l23 tumours .
notably , the extent of dox release in both mac tumours was very low ( supplemental fig . 3 ) .
overall , there were a > 200-fold difference in the free dox levels seen at 1 h in the murine models studied and a 30-fold variation in the xenograft models .
the fastest dox release was observed in b16f10 ( 22.9 1.2 % at 1 h ) and the smaller in cor l23 tumours ( 30.1 5.3 % ) with the slowest release rate observed in the larger mac 26 tumours ( 0.06 0.01 % ) and the sk - n - sh neuroblastoma ( 1.0 0.1 % ) .fig .
statistical significance : * * p < 0.02 ; * * * p < 0.01 ; ns not significantly .
panel ( b ) human xenograft models ( mean se , n = 3 ; except sk - n - sh , n = 5 and sk - n - dz , n = 2 ) .
tumour size classification as per figs . 4 and 6 comparison of the dox released from fce28068 in the different tumour models .
statistical significance : * * p < 0.02 ; * * * p <
panel ( b ) human xenograft models ( mean se , n = 3 ; except sk - n - sh , n = 5 and sk - n - dz , n = 2 ) .
statistical significance : * * p < 0.02 ; ns not significantly different .
to assess evidence for the operation of the epr effect , intratumour levels of evans blue , fce28028 and dox detected at 1 h following i.v .
b16f10 or l1210 tumours are shown in figs . 2 and 3 . when the tumour levels were expressed as % dose for evans blue ( fig .
2a , c ) , tumour levels increased in an approximately linear fashion with increasing tumour size up to a maximum of 46 % dose / tumour for the b16f10 model and ~15 % dose / tumour for the l1210 model ( fig .
3a ) were quantitatively similar across the b16f10 tumour size range , as expected for agents requiring permeable blood vessels for distribution .
administration of free drug were consistently much lower ( < 0.8 % dose per tumour ; fig .
3c ) , and expression in terms of % dose / g tumour tissue showed dox localisation to be constant across all tumour sizes studied ( in the range 1.12.5 % dose / g tumour ; fig .
3b ) levels expressed as % dose / g tumour decreased as tumour size increased ( figs .
values fell to 0.43.7 % dose / g in the largest tumours ( > 600 mg ; fig .
3b ) . the observed tumour size dependence of evans blue accumulation while still evident was less marked in the l1210 model ( fig .
figure 4 summarises data with respect to size of b16f10 tumours in relation to accumulated systemically administered evans blue , fce28028 and dox.fig .
a , c tumour levels expressed as % dose per tumour and b , d tumour levels expressed as % dose / g / tumour .
a , c tumour levels expressed as % dose per tumour , and b , d tumour levels expressed as % dose / g / tumour .
4comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) .
. not significantly different levels of evans blue detected in s.c . b16f10 and l1210 tumours at 1 h after i.v . administration .
a , c tumour levels expressed as % dose per tumour and b , d tumour levels expressed as % dose / g / tumour .
each point represents a single tumour sample levels of fce28068 and free dox detected in s.c .
a , c tumour levels expressed as % dose per tumour , and b , d tumour levels expressed as % dose / g / tumour .
each point represents a single tumour comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) . statistical significance
in contrast to the results obtained in the b16f10 models , when fce28068 was administered to mice bearing mac 15a ( 8.212.6 % dose / g ) , mac 26 adenocarcinoma ( 6.910.8 % dose / g ) or meta-7 lung ( 3.54.7 % dose / g ) tumours ( fig . 5 ) , the amount of conjugate in the tumour did not show statistically significant changes as tumour size increased .
while the mac tumours accumulated fce28068 to a similar degree as the b16f10 model , approximately two to three lower conjugate levels were seen in the meta-7 lung model ( fig .
5d).fig . 5fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v . administration . the data in a
c individual tumour samples and d a comparison of the levels after measured in the three smallest and three largest tumours .
the distribution of fce 28608 for the b16f10 model is shown for comparison . in fig . 5 small b16f10 , mac 15a mac 26 and meta-7 tumours were < 100 ,
< 470 , < 480 and < 150 mg , respectively . large b16f10 , mac 15a , mac 26 and meta-7 tumours were > 400 , > 900 , > 1,000 and > 300 mg , respectively .
statistical significance * * p < 0.02 ; ns not significantly different fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v . administration . the data in a
c individual tumour samples and d a comparison of the levels after measured in the three smallest and three largest tumours .
the distribution of fce 28608 for the b16f10 model is shown for comparison . in fig .
5 small b16f10 , mac 15a mac 26 and meta-7 tumours were < 100 , < 470 , < 480 and < 150 mg , respectively .
large b16f10 , mac 15a , mac 26 and meta-7 tumours were > 400 , > 900 , > 1,000 and > 300 mg , respectively .
0.02 ; ns not significantly different we therefore examined a series of additional models , as a tumour size - dependent nanomedicine delivery has been incompletely explored , and the existence of this phenomenon has important implications for the design of pre - clinical and clinical studies with nanomedicines .
the mexf 276 , maxf 449 and rxf 486 human xenograft models displayed a tumour size - dependent pattern of fce28068 accumulation ( supplemental fig . 1 ) .
although paxf 546 showed a similar trend , it should be noted that the three smallest and the three largest tumours of paxf 546 pancreatic carcinoma tumours did not give statistically significance different values .
in contrast , the rxf 1220 , cor l23 , imr 32 , sk - n - sh and sk - n - dz tumours ( supplemental fig . 2 ) displayed size - independent accumulation of fce28068 .
( only two sk - n - dz tumours were available , but the data are included for completeness ) .
comparison of summary mean values obtained for fce28068 accumulation in all the tumour xenografts ( fig .
6 ) revealed the highest values in the non - small cell lung cancer cor l23 ( 4.712.2 % dose / g ) and the lowest values in the larger ( 0.20.4 g ) maxf 449 tumours ( 1.0 0.1 % dose / g ) .
early - phase tumour levels of fce28068 across all the xenograft tumours ( and sizes ) examined displayed ~12-fold variation in magnitude.fig .
6comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se ,
tumour model size cut - offs ( mg small , mg large ) are as follows : mexf276 ( < 50 , > 200 ) ; maxf449 ( < 50 , > 200 ) , paxf 546 ( < 100 , > 500 ) , rxf 486 ( < 100 , > 600 ) , rxf 1220 ( < 100 , > 400 ) , corl 23 ( < 100 , > 400 ) , imr32 ( < 120 , > 220 ) . statistical significance * p < 0.05 * * * p < 0.01 , ns not significantly different . for the full data set , see supplementary figs . 1 and 2 comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se , n = 3 ) . tumour model size cut - offs ( mg small , mg large ) are as follows : mexf276 ( < 50 , > 200 ) ; maxf449 ( < 50 , > 200 ) , paxf 546 ( < 100 , > 500 ) , rxf 486 ( < 100 , > 600 ) , rxf 1220 ( < 100 , > 400 ) , corl 23 ( < 100 , > 400 ) , imr32 ( < 120 , > 220 ) . statistical significance * p <
0.05 * * * p < 0.01 , ns not significantly different . for the full data set , see supplementary figs . 1 and 2
after administration of fce28068 , the free dox detected in all tumour samples at 1 h expressed as percentage of total dox ( i.e. conjugated drug + free ) is shown in fig .
only the mac 15a , mac 26 murine tumours and the cor l23 xenograft showed tumour size dependency in terms of dox liberation at 1 h. this is interesting as none of these tumour models displayed tumour size - dependent fce28208 levels at 1 h. dox release was greater in the smaller tumours , and the difference is particularly striking for the smaller ( < 100 mg ) cor l23 tumours .
notably , the extent of dox release in both mac tumours was very low ( supplemental fig . 3 ) .
overall , there were a > 200-fold difference in the free dox levels seen at 1 h in the murine models studied and a 30-fold variation in the xenograft models .
the fastest dox release was observed in b16f10 ( 22.9 1.2 % at 1 h ) and the smaller in cor l23 tumours ( 30.1 5.3 % ) with the slowest release rate observed in the larger mac 26 tumours ( 0.06 0.01 % ) and the sk - n - sh neuroblastoma ( 1.0 0.1 % ) .fig .
panel ( a ) murine tumour models ( mean se , n = 3 ) .
statistical significance : * * p < 0.02 ; * * * p < 0.01 ; ns not significantly .
panel ( b ) human xenograft models ( mean se , n = 3 ; except sk - n - sh , n = 5 and sk - n - dz , n = 2 ) .
statistical significance : * * p < 0.02 ; ns not significantly different .
tumour size classification as per figs . 4 and 6 comparison of the dox released from fce28068 in the different tumour models .
statistical significance : * * p < 0.02 ; * * * p < 0.01 ; ns not significantly .
panel ( b ) human xenograft models ( mean se , n = 3 ; except sk - n - sh , n = 5 and sk - n - dz , n = 2 ) .
statistical significance : * * p < 0.02 ; ns not significantly different .
in vitro and in vivo pre - clinical models used to screen novel low molecular weight anticancer agents and biologics have evolved significantly to better mimic the clinical disease setting ( reviewed in [ 22 , 23 ] ) .
while these advances give hope of lead compounds with an increased probability of a successful clinical outcome , many of the in vitro and in vivo methods / models used today are not optimal for nanomedicine evaluation given their very different cellular and whole - body pharmacokinetics compared to low molecular weight agents ( discussed in ) .
administration , low molecular weight drugs distribute rapidly throughout the body with little or no tumour selectivity ( evidenced here for dox in fig .
typically < 0.1 % of the administered drug dose is recoverable in the circulation , usually in the form of metabolites and/or protein - bound drug .
in contrast , nanomedicines ( including liposomes , polymer conjugates and nanoparticles ) are retained within the bloodstream due to the tight vascular endothelial barrier present in most organs and limitation of cellular uptake to the endocytic pathways ( discussed in ) . in the absence of receptor - mediated targeting
, nanomedicine blood clearance is largely governed by the rate of reticuloendothelial system ( res ) uptake and/or renal elimination .
1b ) , and a low epr effect is typically seen when a nanocarrier is cleared rapidly by professional phagocytes .
however , it widely recognised that thereafter vasculature complexity ( e.g. different classes of angiogenic vessels , vessel disorganisation and heterogeneity of tumour perfusion ) and intratumoural biological barriers ( e.g. high interstitial pressure , the extracellular matrix , coupled with the presence or absence of lymphatic drainage , etc .
[ 2 , 10 , 26 ] ) play an important role in passive targeting ultimately achieved in any particular tumour mass . in the 1950s and 1960s ,
unknowingly , these imaging agents were already capitalising on the epr effect for tumour selectivity , as do all chemotherapeutic and phototherapy agents that bind to plasma proteins . using an evans blue albumin complex , matsumura and maeda visualised and quantified tumour - specific passive accumulation in a sarcoma 180 model ( 32 % dose / g tumour at 48 h ) . in the experiments reported here ,
evans blue and fce28068 accumulation by l1210 and b1f10 ( figs . 2 , 3 )
tumours was in the range ~122 % dose / g at 1 h. there was good correlation between evans blue and the nanomedicine fce 28068 across the tumour size range .
the data are in agreement with the previous reports describing evans blue , hpma copolymer fractions ( molecular weight from 4.5 to 800 kda ) , pamam dendrimer - dox and liposomal doxorubicin tumour accumulation .
rapid passive targeting gave similar tumour values ( % dose / g ) irrespective of polymer molecular weight or construct architecture [ 2831 ] .
a significant observation in these experiments is a 12-fold variation in functional epr , with many models displaying tumour size - dependent accumulation .
the higher tumour localisation was noticeably in the smaller tumours , and this maybe a reflection of greater angiogenic activity in smaller tumours , reduced or absent tumour blood supply in hypoxic regions of larger necrotic tumours , an increase in tumour interstitial fluid pressure as the tumour grows , or a combination of all of these factors .
kb human head and neck squamous cell xenografts also displayed tumour levels inversely correlated with tumour size : 15.1 10.8 % dose / g in tumours of < 0.1 g and 3.0 1.3 % dose / g in tumours of > 1 g . in this regard ,
the current results of experiments raise the possibility of a novel development strategy for nanomedicines , focusing on systemic delivery of such constructs in the adjuvant setting or with small - volume
whatever the mechanism , there is a growing body of evidence that nanosized vectors show greatest targeting to the smaller tumours and this could provide an important opportunity to localise to those micrometastases so difficult to diagnose and treat effectively .
use of in - dtpa - labelled pegylated liposomes to image 17 patients with locally advanced cancers gave positive tumour images in 15/17 patients and tumour levels in the range of ~0.53.5 % dose at 72 h. highest liposome localisation was observed in patients with head and neck cancers ( ~0.033 % injected dose / g tumour ) ( also noted for fce2068 in phase i clinical studies ) and lower levels in breast cancers ( ~0.005 % injected dose / g ) .
the breast tumours were larger than the lung tumours , supporting a relationship between small tumour site and improved localisation , but the relationship was not unequivocal as it was noted that some tumours of similar size but different histological type displayed different levels of liposomal uptake . using tc - dtpa - labelled pegylated liposomes that localised to lung and head and neck cancers , others have suggested a correlation between tumour targeting and microvessel density as defined by anti - cd31 staining .
although tumour vessel density ( vegf expression ) does correlate with tumour vascular permeability ( evans blue ) in some human tumour xenografts , other studies have indicated that a more complex multifactorial mechanism underpins epr - mediated targeting and efficacy , e.g. inflammatory status . at this time , it is difficult to predict tumour localisation / antitumour activity using a single biomarker .
overexpression of lysosomal thiol - dependent proteases in many human tumours has been well documented , and these enzymes play an important role in tumour progression ( e.g. metastasis and angiogenesis ) . a growing number of polymer conjugates
are being designed for lysosomotropic delivery [ 34 , 35 ] and cathepsin b - mediated drug release [ 1 , 2 ] .
the wide variation in early - phase dox release from fc28068 ( ~200-fold ) observed here illustrates the need to characterise tumour models around the basis for delivery to tumours and response of tumours to nanomedicines as part of evolving an optimal clinical strategy for their development .
for example , retrospective analysis of clinical data from ppx phase iii trials in advanced lung cancer patients showed improved survival in female but not in male patients , and it has been postulated that patient oestradiol level might play a pivotal role as oestrogens are known to induce cathepsin b activity .
b16f10 tumours [ 15 , 37 ] indicated an ~30 min time lag before dox release began , supporting the hypothesis of drug release following endocytic internalisation .
however , it should be noted that other factors may play a role in controlling the rate of drug release : ( i ) rate of conjugate diffusion through the tumour interstitium , ( ii ) the rate of endocytic internalisation ( endocytic gateways and intracellular trafficking pathways are often dysregulated in cancer ) and ( iii ) exposure to cathepsin b in the extracellular milieu .
this complexity underlines the importance of functional tumour calibration in terms of drug release and also the future role of the real - time tumour imaging techniques that are emerging for quantitation of proteolysis per se and polymer degradation .
the wide variation in passive targeting ( % dose / g ) and dox liberation observed here ( 1 h ) highlights the need to calibrate in vivo tumour models in respect of these parameters before they are used to define pharmacokinetics and/or antitumour activity of nanomedicines .
this would provide more clinically relevant models for optimisation of lead candidates and benchmarking performance against anticancer nanomedicines already in routine clinical use .
evans blue is a useful tool for routine evaluation of passive targeting , and the 1 h time point enables comparison of different nanocarriers minimising complications arising due to different blood clearance rate , tumour efflux and intratumoural degradation of the probe over time .
these observations also emphasise the potential to select patients for early clinical trial involving nanomedicine therapy that are most likely to respond , i.e. by use of clinical imaging to verify functional epr , and monitoring of tumour biopsy samples for biomarkers relevant to activating conditions , e.g. in this case cathepsin b.
fig . 1 fce28068 levels in mexf 276 , paxf 546 , maxf 449 and rxf 486 tumours at 1 h after i.v . administration . the data in panels ( a ) - ( d )
( ppt 165 kb ) fig . 2 fce28068 levels in rxf 1220 , imr 32 , cor l23 , sk - n - sh and sk - n - dz tumours at 1 h after i.v .
administration . the data in panels ( a ) - ( e ) show individual tumours .
( ppt 178 kb ) fig . 3 summary of tumour uptake of fce28068 and the rate of dox release in the mac tumours ( mean se , n = 11 ) .
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### Summary:
: purposeintravenously ( i.v . )
administered nanomedicines have the potential for tumour targeting due to the enhanced permeability and retention ( epr ) effect , but in vivo tumour models are rarely calibrated with respect to functional vascular permeability and/or mechanisms controlling intratumoural drug release . here
the effect of tumour type and tumour size on epr - mediated tumour localisation and cathepsin b - mediated drug release was studied.methodsevans blue ( 10 mg / kg ) and an n-(2-hydroxypropyl)methacrylamide ( hpma ) copolymer doxorubicin ( dox ) conjugate ( fce28068 ) ( 5 mg / kg dox - equiv ) were used as probes and tumour levels ( and dox release ) measured at 1 h after i.v .
administration in a panel of murine and human xenograft tumours.resultsevans blue and fce28068 displayed similar tumour levels in the range of 218 % dose / g at 1 h for b16f10 and l1210 .
approximately half of the tumour models evaluated exhibited tumour size - dependent accumulation of fce28068 ; smaller tumours had the highest accumulation .
administration of free dox ( 5 mg / kg ) produced tumour levels of < 2.5 % dose / g independent of tumour size . whereas the degree of epr - mediated targeting showed ~12-fold difference across the tumour models evaluated , dox release from fce28068 at 1 h displayed ~200-fold variation.conclusionsmarked heterogeneity was seen in terms of epr effect and dox release rate , underlining the need to carefully calibrate tumour models used to benchmark nanomedicines against known relevant standard agents and for optimal development of strategies for late pre - clinical and clinical development.electronic supplementary materialthe online version of this article ( doi:10.1007/s00280 - 013 - 2209 - 7 ) contains supplementary material , which is available to authorized users .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: nicotine [ 1-methyl-2-(3-pyridyl - pyrrolidine ) , c10h14n2 ] is the principal component of tobacco .
the predominant effects of nicotine include rise in blood pressure , heart rate , respiratory rate ; increase in the level of catecholamines in blood ; increase in level of free fatty acids , mobilization of blood sugar , and it was also found to disturb the antioxidant defense mechanisms [ 26 ] .
there is strong evidence that smokeless tobacco use leads to oral mucosal lesions [ 7 , 8 ] including oral pre - cancerous lesions , gingival recession , cardiovascular risk factors and disease , diabetes , reproductive health effects , and overall mortality .
nicotine rapidly crosses the placenta and has toxic effects on the foetus [ 10 , 11 ] .
india 's share of the global burden of tobacco - induced disease and death is substantial .
india has one of the highest rates of oral cancers in the world ; 65% of all cancers in men and 33% of all cancers in women are tobacco related .
annual incidence of oral cancer is said to be 10 per 1,00,000 per males . in india
tobacco products are popular as a dentifrice in different parts of india and children also use such dentifrices . among female smokeless tobacco users ,
the dominating form is tobacco toothpowder ( 41.3% ) . among men it was khaini ( 57.1% ) followed by tobacco toothpowder ( 8.8% ) [ 16 , 17 ] .
many companies take advantage of this misconception and exploiting the addictive nature of nicotine by packaging and positioning their products as dental care products without explicitly stating so .
there was an amendment in drugs & cosmetics act , 1940 vide notification published in the gazette of india vide g.s.r .
443(e ) and 444(e ) dated 30.04.1992 , mentioning that manufacture and sale of all cosmetics and all ayurvedic drugs licensed as toothpowders / toothpastes containing tobacco have been prohibited [ 6 , 18 ] .
however , recent studies reported the presence of nicotine in some toothpowders [ 6 , 18 ] .
the present studies were motivated by an interest in examining the presence of nicotine in these dentifrices to evaluate the presence of nicotine in same brands of toothpowders ( dant manjans ) as reported in previous study .
furthermore , this year we also find it interesting to evaluate the presence of nicotine in toothpastes as well .
eight brands of particular batches of commonly used toothpowders ( dant manjans ) and toothpastes were selected for the study from the local market .
one sample from each batch was kept sealed in our laboratory as a reference sample .
the toothpowders selected are coded as follows : dabur red - m1 ( 8652 ) , vicco - m2 ( 008 ) , musaka gul - m3 ( not mentioned ) , yunadent - m4 ( 01 ) , baidyanath - m5 ( 903 ) , mdh - m6 ( 98 ) , gumtone - m7 ( gt509 ) , and payokil - m8 ( 1503 ) .
the toothpastes selected for study were as follows : dabur red - p1 ( bd0698 ) , vicco - p2 ( 065 ) , arodent - p3 ( 64 ) , babool - p4 ( 1498 ) , herbodent - p5 ( 20 ) , colgate herbal - p6 ( b24cp ) , ipco - p7 ( not mentioned ) , and dentobac - p8 ( not mentioned ) .
mumbai , chloroform and methanol of hplc grade from merck ltd . , mumbai , and ( )-nicotine of synthesis grade from merck - schuchardt , hohebrunn , germany .
extraction and isolation of nicotine from toothpowders were done as described by agrawal and rajagopal .
extraction and isolation of nicotine from toothpastes were done by taking 25 g of toothpaste in a conical flask , 15 ml nh3 ( strong ) was added to completely humidify the sample followed by 250 ml chloroform , and the conical flask was closed with a cap .
after that , 2 min hand shaking and then chloroform extract is then filtered out and dried .
the yield values of alkaloids obtained are 0.154 , 0.44 , 0.22 , 0.12 , 0.14 , 0.24 , 0.56 , and 0.37 , respectively .
the ion source was operated in the electron ionization mode ( ei ; 70 ev , 150c ) .
full - scan mass spectra ( m / z 40450 ) were recorded for analyte identification .
separation was achieved with a bp fused - silica capillary column with 5% diphenyl - poly ( dimethylsiloxane ( bp 5 ) , ~30 m length , 0.25 mm i.d .
samples were injected in the split mode with a split ratio of 1 : 10 .
the gc operating parameters were as follows : injector temperature , 250c ; transfer line temperature , 280c ; oven temperature , programmed from 100c ( held for 3 min ) at 10c / min to 280c ( held for 3 min ) .
the presence of nicotine in toothpowders ( dant manjans ) and toothpastes were confirmed by gas chromatography - mass spectroscopy ( gc - ms ) in full scan mode and selective ion mode ( sim ) . out of the eight brands of toothpowders included in the present study only ,
a comparison of retention time , molecular ion peak , and base peak shows the presence of nicotine in these dant manjans ( m2 and m3 ) .
nicotine presence was identified by comparing the rt values of toothpowder extracts with standard nicotine .
the primary target ion of nicotine has a mass of 162 m / z .
the molecular ion peak at 162 m / z and base peak at 84 m / z corresponds to the fragmentation of nicotine .
the rt of m2 and m3 was found to be 16.99 and 16.94 , respectively .
this was further confirmed by mass spectrum ( figures 1 , 2 , 3 , 4 , 5 , and 6 ) .
similarly , out of the eight brands of toothpastes analysed , four brands were found to contain nicotine .
the rt of p1 , p3 , p7 , and p8 was found to be 17.05 , 17.16 , 16.93 , and 16.91 , respectively as compared to that of nicotine standard 17.00 and was confirmed by its molecular ion peak and base peak ( figures 7 , 8 , 9 , 10 , 11 , 12 , 13 , and 14 ) . the dant manjan extracts which indicated the presence of nicotine were also quantified with gcms .
the nicotine contents determined in gcms of m1 and m2 were 12.95 mg / g and 216.10 mg / g respectively .
similarly , the nicotine content in toothpaste extracts of p1 , p3 , p7 , and p8 were found to be 5.752 mg / g , 2.093 mg / g , 123.82 mg / g , and 119.13 mg / g , respectively .
abuse of tobacco , like drug and alcohol abuse , is a worldwide public health problem .
india 's tobacco problem is more complex than probably that of any other country in the world , with a large consequential burden of tobacco - related disease and death .
various tobacco products are popular as a dentifrice in different parts of india [ 16 , 22 ] .
tobacco was not , perhaps , used for cleansing the teeth , but more so to get kick out of the contents of tobacco , and many companies exploit the addictive nature of nicotine by packaging and positioning them as dentifrice . researches comparing herbal toothpastes with conventional dentifrices , however , suggest that there is no specific advantage of herbal - based pastes over conventional pastes [ 2325 ] .
indian legislation prohibits the use of tobacco as an ingredient in dental care products [ 6 , 15 ] . after the passage of a law banning the use of tobacco in dental care products , the listing of tobacco as an ingredient was stopped .
these products , however , continue to be available openly in the market , often without mentioning tobacco as one of the ingredient [ 6 , 16 , 17 ] .
nicotine is a cns stimulant , which leads to habit formation subsequently dependence on its users .
nicotine causes withdrawal symptoms such as anger , anxiety , depressed mood , difficulty in concentration , and craving for tobacco .
there is strong and compelling evidence that oral tobacco use leads to oral mucosal lesions including oral precancerous lesions , gingival recession , oral cancer [ 27 , 28 ] , and overall mortality
the present studies were motivated by an interest in examining the presence of nicotine in these dentifrices and to endow with an overview of the tobacco problem in indian dentifrices , from public health challenges to policy responses . with this background , an attempt was made to further curb the menace of tobacco in commercially available dentifrices in india and to provide a credible basis for evolving future tobacco regulation in dentifrices .
our team again analysed the same brands as of yesteryears with an objective to review that whether the regulations with respect to tobacco / nicotine in toothpowders have been adequately implemented or not .
also it was thought to estimate nicotine in toothpastes of herbal / ayurvedic origin . in consistent with the previous report , the presence of nicotine in four out of eight brands of dant manjans analysed
, this year only two bands were found to contain nicotine and is quantified as m2 ( 12.95 mg / g ) and m3 ( 216.10 mg / g ) , respectively .
this year out of the eight most commonly available herbal toothpastes selected for the study , four brands were found to contain nicotine and was quantified as p1 ( 5.32 mg / g ) , p3 ( 2.093 mg / g ) , p7 ( 119.13 mg / g ) , and p8 ( 123.82 mg / g ) , respectively .
moreover , these nicotine - containing toothpowders ( dant manjans ) and toothpastes lack pictorial warning which implies that these manufactures are disguising it as a dentifrice .
a person on a regular oral hygiene uses 2 - 3 g of toothpowder / paste each day .
the ld50 of nicotine in mice , rats , and human is 3 mg , 50 mg , and 4060 mg , respectively .
thus , the present study indicates that a considerable amount of nicotine ( 2216 mg ) could enter the oral cavity if these toothpowders and toothpastes were used .
children may retain a much greater percentage of dentifrice than adults , because of incomplete rinsing thus resulting in ingestion . once a person is addicted to nicotine
, quitting is very difficult no matter whichever form he uses it . in a survey
thus the presence of nicotine in these dentifrices even in low concentrations could considerably lead to addiction and oral mucosal lesions due to its routine use .
hence this epidemic of addiction to nicotine among young people may have enormous implications in public health .
therefore , this study underscores the importance of support of prevention of tobacco use in developing countries especially india where population is roaring ; tobacco use threatens to contribute to a growing proportion of the burden of disease .
thus , even in low concentrations , one would expect tobacco to be present in these dentifrices to exert its addictive influence , which emphasizes the need for strong economic , legislative , and educational means to eradicate this menace .
however , much more needs to be done including possibly enacting legislation to stop the manufacturing of such dentifrices .
the findings of this study will help to design , implement , and evaluate tobacco control and prevention programmes .
thus , our study clearly confirmed the presence of nicotine in few dentifrices , and regulations have not been implemented properly even after 17 years .
with a population of over 1 billion people , tobacco use contributes to india 's and global public health threat .
thus even after 17 years of amendment of drugs and cosmetic act banning tobacco toothpowders and toothpastes , nicotine is still present in some dentifrices which clearly shows that the law is not strictly enforced and implemented .
hence regulatory authorities in india and internationally should take adequate measures to stop the addition of nicotine or tobacco to the dentifrices to provide nicotine-/tobacco - free dentifrices
. however , continued surveillance of such dental products is necessary in order to better inform and educate the public regarding the risks associated with respect to this public health concern .
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### Summary:
: the use of tobacco products as dentifrices is still prevalent in various parts of india .
tobacco use in dentifrices is a terrible scourge which motivates continued use despite its harmful effects .
indian legislation prohibits the use of nicotine in dentifrices .
nicotine is primarily injurious to people because it is responsible for tobacco addiction and is dependence forming .
the present study was motivated by an interest in examining the presence of nicotine in these dentifrices .
our earlier report indicates the presence of nicotine in toothpowders . to further curb the menace of tobacco , our team again analysed the toothpowder brands of previous years and in toothpastes as well .
eight brands of commonly used toothpastes and toothpowders were evaluated by gas chromatography - mass spectroscopy . on the whole , there are a few successes but much remains to be done .
our findings indicated the presence of nicotine in two brands of dant manjans and four brands of toothpastes .
further our finding underscores the need for stringent regulations by the regulatory authorities for preventing the addition of nicotine in these dentifrices .
hence government policy needs to be targeted towards an effective control of tobacco in these dentifrices and should be properly addressed .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: choledochal cysts , although rare in adulthood , are now correctly diagnosed more frequently due to recent improvement in biliary imaging techniques ( 1 ) . there have been numerous descriptions and classifications based on the location and anatomical structure of choledochal cyst . among these , the most practical classification is proposed by alonso - lej et al .
( 2 ) , which is modified by todani et al . ( 3 ) in 1977 .
the major point of this classification is that the gallbladder is usually not distended and arises from a small cystic duct , usually not at the upper end of the cyst ( 4 ) .
initial reports suggested that the following treatments provided satisfactory results : t - tube choledochocystomy with internal or external drainage , cystojejunostomy , modified cystojejunostomy with t - tube external drainage , cyst excision and hepaticoduodenostomy , cystoduodenostomy , jejunal segment interposition with cystoduodenostomy , and jejunal segment interposition with hepaticojejunostomy .
however , it is now recognized that these procedures are associated with an unacceptably high rate of late complications .
late complications of internal cyst drainage may result in a high rate of cholangiocarcinoma arising from the cyst ( 1 ) , as well as the development of cholangitis and biliary stone disease ( 1 , 5 - 8 ) .
surgical excision of cyst and hepaticojejunostomy are now considered as the treatment of choice for choledochal cysts ( 1 , 5 - 8 ) . in this paper
seventy - two patients ( 59 females and 13 males ) with ages ranging from 15 to 63 ( mean age=31 ) yr were treated for choledochal cyst by the department of surgery , the second affiliated hospital of harbin medical university , between january 1 , 1985 and december 31 , 2002 .
follow - ups were done on all of the patients until december 31 , 2002 .
we analyzed the clinical outcome in patients who underwent the following treatments : cyst excision with hepatojejunostomy ( fig .
there were 49 ( 68.1% ) type i or type iv - b , one ( 1.3% ) type ii , 21 ( 29.3% ) type iv - a , and one ( 1.3% ) type v choledochal cysts . among the patients , the most common symptom was abdominal pain , which occurred in 64 of the 72 patients ( 88.9% ) .
jaundice was present in 40 patients ( 55.6% ) , and abdominal mass was present in 21 patients ( 29.2% ) .
only eight patients ( 11.1% ) presented with the classic triad of abdominal pain , jaundice , and abdominal mass . the biochemical measurement showed anomalies in ast , which appeared in 22 of the 72 patients ( 30.6% ) , alt in 48.6% , -gt in 31.9% , and total bilirubin in 34.7% of all the patients .
one or more of the following test procedures established the diagnosis of choledochal cyst : ultrasonography ( us ) ( n=49 ) , magnetic resonance imaging ( mri ) ( n=4 ) , computed tomography ( ct ) ( n=13 ) and endoscopic retrograde cholangiopancreatography ( ercp ) ( n=14 ) .
the choledochal cysts were associated with other diseases including the pancreaticobiliary duct not filled in one case , anomalous junction of the pancreatic duct and common bile duct in one case , dilatation of pancreatic duct in one case , retention of stones in ampulla of vater in one case , ectopic pancreas within jejunum in one case , right hepatic duct anomaly in one case , cyst of liver in two cases , coexistent choledocholithiasis in 12 cases , secondary cholestatic liver cirrhosis , which occurred in one of the 5 cases of intrahepatic cholestasia , cholecystitis in 54 cases , infection of biliary tract in 29 cases and secondary chronic pancreatitis in 5 cases .
women with choledochal cyst were diagnosed during pregnancy ( jaundice , abdominal pain ) in 3 cases ( table 1 ) .
the definite surgical procedures were cyst excision with hepaticojejunostomy in 25 cases for type i or type iv - b and complete excision of extrahepatic choledochal cyst from the hepatic hilum with hepaticojejunostomy in 8 cases for type iv - a , extrahepatic cyst excision with modified hepaticojejunostomy in 2 cases for type iv - a , choledochocystomy with internal or external drainage in 3 cases for type i or type iv - b and 1 case in type iv - a , cyst excision and the biliary tract reconstruction by jejunal segment interposition with hepaticojejunostomy ( fig .
3 ) in 3 cases for type i or type iv - b and extrahepatic cyst excision and the biliary tract reconstruction by jejunal segment interposition with hepaticojejunostomy in 2 cases for type iv - a , the biliary tract reconstruction by jejunal segment interposition with cystoduodenostomy in 2 cases for type i or type iv - b , cystoduodenostomy in 2 cases for type i or type iv - b and 1 case in type iv - a , cyst excision and hepaticoduodenostomy in 2 cases for type i or type iv - b and 1 case for type ii , cystojejunostomy in 4 cases for type i or type iv - b and 5 cases in type iv - a , modified cystojejunostomy with t - tube external drainage for 1 case in type iv - a ( table 2 ) .
27 out of 62 with received operation cases were performed non - cyst excision with or without hepaticojejunostomy in type i , ii , iv - a , iv - b . because of choledochal cyst adhered with surrounding tissue extensively , we can not dissect choledochal cyst from the round ligament in 27 cases .
the early postoperative morbidity and mortality rates were 16.1% ( 9/62 ) and 6.5% ( 4/62 ) respectively .
the incidence of carcinoma was 6.9% ( 5/72 ) , while the incidence of cholangiocarcinoma with non - cyst excision or non - operated congenital choledochal cyst was 10.8% ( 4/37 ) .
early postoperative complications included wound infection , septic shock after cystojejunostomy in one case , acute septic peritonitis after cyst excision with hepatojejunostomy in one case , abdominal pain , pancreatic ductal dilatation with retrograde cholangitis after cyst excision with hepatojejunostomy in one case , which was resolved after a few days with conservative antibiotics treatment .
two cases required reoperative revision in the early postoperative period due to anastomotic leakage after cyst excision with roux - en - y hepaticojejunostomy .
unfortunately , one patient died of brain infection within one week and the other suffered from anastomotic leakage and intra - abdominal hematoma formation after cystoduodenostomy and eventually died of multiorgan failure within one week . during the immediate postoperative period , one patient with fistula formation after cystoduodenostomy died of hepatic failure within 72 hr . in two cases , a 44 yr old male patient and a 37 yr old female , both with type i or
type iv - b , were admitted to the hospital with ( postoperative ) diagnosis of well - differentiated cholangiocarcinoma . within sixth month
for one pregnant 27 yr old ( type iv - a ) , a roux - en - y cystojejunostomy with t - tube external drainage was performed .
however , primigravida presented at 20 weeks ' gestation , and the patient died of septic shock and multiorgan failure on postoperative day 25 .
the long - term complications : of the 6 patients who were re - hospitalized , two patients developed retrograde cholangitis after cystoduodenostomy with internal drainage .
second patient suffered from recurrent retrograde cholangitis and subsequently developed secondary sclerosing cholangitis 10 yr after cystojejunostomy ; this was resolved by cyst excision with hepatojejunostomy .
one patient presented with progressive obstructive jaundice , bile duct stones , and strictures after t - tube choledochocystostomy with external drainage but was cured by cyst excision and hepatojejunostomy .
one case presented with anastomotic stricture after jejunal segment interposition with cystoduodenostomy and successfully treated by cyst excision and modified hepatojejunostomy .
another patient presented with postoperative abdominal pain as well as retrograde cholangitis 5 yr after cystojejunostomy , which was treated with cyst excision with hepatojejunostomy .
one 25 yr old female patient with type iv originally received cyst excision with hepaticojejunostomy , but developed primary hepatocellular carcinoma and died eight years after the original procedure . a 26 yr old multiparous woman presented at 28 weeks ' gestation with choledochal cyst with a type i or type iv - b .
this patient received roux - en - y cystojejunostomy after a vertex delivery by induced labor at 28 weeks ' gestation .
she developed intermittent retrograde cholangitis within 10 yr , and ultimately died of well - differentiated congenital cholangioadenocarcinoma within 1 month after re - operation with exploratory biopsy at the age of 36 yr .
one patient received cystojejunostomy for type iv - a with development of cholangiocarcinoma in 12 yr after operation .
she received cyst excision with rox - en - y hepaticojejunostomy , but died after one year at age 60 yr .
finally , one patient with type iv - a died of carbon monoxide toxicosis within six years following cystojejunostomy . out of 72 patients , only 10 cases did not receive operation . over the course of the study , two patients with type i or type iv - b died of natural causes ( table 3 )
long after the first pathological description of a choledochal cyst by vatero in 1723 , and the first clinical report by douglas in 1852 , the aetiology of this anomaly remains controversial ( 9 ) .
two main theories emerged . in 1936 , yotsuyanagi ( 9 ) suggested that choledochal cysts arise from inequality in the vacuolization of the biliary tract in early embryonic life .
the common channel theory proposed by babbitt ( 9 ) in 1969 is presently the most widely accepted idea .
this theory is based on an anomaly of the pancreaticobiliary junction and the formation of an abnormally long common channel ( greater than 15 mm ) outside the control of the sphincters of boyden .
the pancreatic enzymes then lead to constant inflammation , epithelial denudation , thinning of the bile duct wall and distal obstruction , and eventually leading to cyst formation .
although the incidence of choledochal cysts was reported to be low ( from 1 in 13,000 to 1 in 2 million patients ) ( 1 , 9 ) , they are now diagnosed more frequently with the use of improved diagnostic techniques such as us , ct , and direct cholangiography ( ptc and ercp ) .
it is necessary to classify the type of cyst and to recognize the presence of an anomalous pancreaticobiliary duct junction , visualization of both the biliary tree and pancreatic duct . for this purpose , direct cholangiography , especially ercp ,
however , intraoperative cholangiography is not sensitive enough to detect the presence of an anomalous pancreaticobiliary duct junction .
radiographic visualization of both the biliary tree and pancreatic duct prior to surgery is helpful for the surgical manipulation and complete excision of the cyst ( 1 , 10 , 11 ) . in our review
ercp was only shown the pancreaticobiliary duct not filled , an anomalous junction of the pancreatic duct and common bile duct , dilatation of pancreatic duct and retention of stones in the ampulla of vater ( table 1 ) .
therefore , no conclusion can be justified concerning the presence or absence of a common channel in our experience .
other pathologic features of choledochal cysts include acute and chronic mucosal inflammation , mucosal dysplasia , and a relative absence of smooth or elastic fibers . a true mucosal lining may be hard to find .
there is usually a large amount of fibrosis , some of which may be involved in luminal stenoses .
pathologic complications include biliary obstruction , cholangitis , hepatic abscess , rupture , or development of cancer .
however , in our study we observed very different preoperative pathologic complications , as listed in table 1 . in our experience , the combination of thorough history takings along with understanding of mechanism of manifestation and pathology of disease , it is possible to correctly diagnose and treat congenital choladochal cyst in adults .
early reports suggested that internal or external drainage of a choledochal cyst by choledochocystojejunostomy or t - tube choledochocystomy was a satisfactory method of treatment ; however , with longer follow - up period , it has become clear that complications such as suppurative cholangitis , lithiasis , pancreatitis , secondary biliary cirrhosis , portal hypertension , and intrahepatic abscess may manifest in up to 40 percent of cases ( 9 ) . that compares favorably with our results . in our review , two cases were resolved by cyst excision with hepatojejunostomy after t - tube choledochocystomy .
one case appeared with progressive obstructive jaundice , bile duct stones , and strictures after t - tube choledochocystomy with external drainage , and then re - treated by cyst excision with hepatojejunostomy . the other case presented with anastomosis stricture after jejunal segment interposition with cystoduodenostomy ;
this patient was successfully treated by cyst excision and modified hepatojejunostomy the increased risk of bile duct carcinoma in choledochal cysts is well characterized ( 9 ) . the reported incidence of biliary tract carcinoma in choledochal cysts varies from 2.5% to 17.5% , which is significantly higher than that found in the general population , which ranges from 0.01% to 0.05% ( 9 ) .
the incidence of cancer in those who have undergone enteric drainage without cyst excision is much higher than that of carcinoma of the bile ducts in the general population .
one 44 yr old male and one 37 yr old female with type i or type iv - b carried postoperative diagnosis of a well - differentiated cholangiocarcinoma .
these patients were admitted to the hospital but died within sixth months after cyst excision with hepaticojejunostomy . in these two cases ,
the problem might have been treated or avoided entirely through early diagnosis and early cyst excision ( 9 ) .
the age - related incidence of cyst - associated cancer has been shown to increase from 0.7% in the first decade of life to 14.3% after age 20 .
this means the favorable outcome in congenital choledochal cyst patients may be due to earlier detection and treatment ( 9 ) , which may be the most important issue in the successful treatment of congenital choladochal cyst .
some authors have reported that cyst excision does not completely eliminate the risk of intrahepatic cholangiocarcinoma after the excision of a type i cyst ( 9 , 12 - 18 ) . in our experience ,
one 25 yr old female patient with type iv - a received cyst excision with hepaticojejunostomy but developed primary hepatocellular carcinoma within 8 yr of postoperation and died .
however , there is insufficient evidence in this one case to suggest whether congenital choledochal cyst was a direct causative factor .
one gestation women with type - a after cystojejunostomy showed repetitive intermittent retrograde cholangitis over a 10 yr period , then developed well - differentiated cholangioadenocarcinoma and died at the age 36 yr .
one patient with type iv - a received cystojejunostomy developed cholangiocarcinoma in within 12 yr postoperation then received extrahepatic cyst excision from the hepatic hilum with roux - en - y hepaticojejunostomy .
( 9 ) were the first to report the increased incidence of carcinoma arising in choledochal cysts and to advocate primary cyst excision .
moreover , the advances in diagnostic and therapeutic procedures and increased operative experience have lowered the mortality rate to 0 - 7% ( 9 ) .
rouxen - y choledochojejunostomy has replaced choledochoduodenostomy as the preferred operative procedure due to the high morbidity rate from cholangitis and the frequent need for re - operation ( 9 ) . in our review
, we also noted the same complications ; the patients received cyst excision with hepatojejunostomy .
excision of type i , ii and iv choledochal cysts is now a widely accepted procedure because of the lowered incidence of postoperative complications .
in contrast to cyst enterostomy , cyst excision with hepaticojejunostomy has demonstrated satisfactory results ( table 3 ) . in type
i cysts , total cyst excision showed ideal long - term follow - up results except for one patient who developed hepatolithiasis secondary to stenosis of the hepaticojejunostomy .
although the occurrence of intrahepatic cholangiocarcinoma after the excision of a type i cyst has been reported ( 12 , 13 ) , cyst excision remains the primary treatment of choice for type i cyst .
if this procedure can not be undertaken , operative sphincteroplasty with transduodenal cyst excision may be attempted ( 1 ) .
sphincteroplasty done either endoscopically or surgically are a satisfactory method according to venu et al .
either excision of the extrahepatic cyst alone ( 1 ) or total cyst excision including hepatectomy ( 15 ) has been recommended .
we have performed excision of the cyst with hepatojejunostomy in 23 cases for type i or iv - b and excision of the extrahepatic cyst with hepatojejunostomy in 8 cases for type iv - a , extrahepatic cyst excision and modified hepatojejunostomy in 2 cases in type iv - a ( table 2 , 3 ) .
although we did not perform hepatectomy in type iv - a cases for complete excision of intrahepatic cyst , the intrahepatic cyst did not developed to cholangiocarcinoma in our review , only two cases due to leakage of anastomosis after cyst excision with roux - en - y hepaticojejunostomy were performed reoperation in the early postoperative days . and
other three cases died of non - cyst excision with hepatojejunostomy or non - cyst excision with modified hepatojejunostomy . in our experience ,
the early postoperative morbidity and mortality rate were 16.1% ( 9/62 ) , and 6.5% ( 4/62 ) .
the incidence of carcinoma was 6.9% ( 5/72 ) , but the incidence of biliary tract carcinoma with non - cyst excision or non - operated congenital choladochal cyst was 10.8% ( 4/37 ) .
it has carried considerable number of complications as well as incidence of biliary tract carcinoma . however , in our experience , we noted that the incidence of complications and incidence of biliary tract carcinoma appeared higher in non - cyst excision with or non - hepatojejunostomy than in cyst excision with hepatojejunostomy or cyst excision with modified hepatojejunostomy .
complete excision of the extrahepatic bile duct from the hepatic hilum to the pancreaticobiliary duct junction is the treatment choice for type iv cysts and i ( 16 - 19 ) .
however , it should be mentioned that since complete excision seems to be difficult in some cases , pancreaticoduodenectomy or hepatic resection should also be considered . in such cases , the distal choledochus is resected just above the pancreaticobiliary duct junction with the aid of preoperative ercp and intraoperative us to avoid injury to the pancreatic duct . in the hepatic hilum
, the hepatic duct must be resected at the hilum , and hepaticojejunostomy with a wide opening by plastic of both the hepatic ducts is necessary . in conclusion ,
we recommend that 1 ) complete excision of choledochal cyst for types i , ii , iv - b and 2 ) complete excision of extrahepatic choledochal cysts from the hepatic hilum in type iv - a with hepaticojejunostomy or modified hepaticojejunostomy .
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### Summary:
: this study was undertaken to analyze and evaluate the diagnosis and principal treatment methods for congenital choledochal cyst , focusing on various surgical procedures and clinical outcome . a comprehensive
, retrospective study was conducted on 72 adult patients who presented with choledochal cyst from 1985 to 2002 .
surgical procedures were cyst excision with hepaticojejunostomy in 25 cases for type i or type iv - b , extrahepatic cyst excision with hepaticojejunostomy in 8 cases for type iv - a , extrahepatic cyst excision with modified hepaticojejunostomy in 2 cases for type iv - b , non - cyst excision with or without hepaticojejunostomy in 27 cases for types i , ii , iv - a , iv - b .
the early postoperative morbidity and mortality rate were 16.1% ( 9/62 ) and 6.5% ( 4/62 ) respectively , and the complication rate related to surgical procedure was 30.6% ( 19/62 ) .
the incidence of cholangiocarcinoma with non - cyst excision or non - operated congenital choledochal cyst was 10.8% ( 4/37 ) .
one patient died of primary hepatocellular carcinoma after cyst excision with hepatojejunostomy . in conclusion , our results showed that complete exci - sion of choledochal cyst for types i , ii , and iv - b and complete excision of extra - hepatic choledochal cyst from the hepatic hilum in type iv - a with hepaticojejunostomy or modified hepaticojejunostomy are the treatment of choice for choledochal cyst in adult patients .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: recurrent aphthous ulcers ( rau ) are the most common oral disease characterized by the repeated development of painful ulcers with unknown etiology and an incidence of up to 20% of the general population .
a large variety of local , hematological , gastrointestinal , immunologic , genetic , nutritional , and medication reactions have been identified as the probable trigger factors of rau .
iga is the principal immunoglobulin isotype found in the external secretions including saliva and tears .
mucosal infections can induce specific salivary iga immunological responses in the absence of detectable serum antibody ; hence , iga plays an important role in defence against infections .
two subclasses of iga are found in humans ; iga1 and iga2 based on the antigenic differences .
there is a relatively high proportion of iga2 in saliva because of the preferential transport of iga2 produced in the distant tissues into saliva , and partly on the relatively increased number of iga2 containing plasma cells in exocrine tissues .
most of the findings have been limited to the determination of total iga either in serum or saliva . in the light of these factors
this study was undertaken to investigate the possible alterations in salivary iga1 and iga2 in patients with recurrent aphthous ulcerations .
thirty clinically confirmed untreated ( with steroids ) cases of rau and 30 age and sex matched controls were included in the study . after a detailed case history and thorough clinical examination ,
2 ml of saliva was collected in both acute and remission phases of the disease .
the obtained saliva samples were subjected to quantification of iga1 and iga2 levels using the rid kit .
unstimulated whole saliva was collected in a sterile bottle by asking the patient to expectorate into it gradually over a period of 510 min .
the obtained saliva samples were stored in deep freezer at 20c until further test was carried out .
the salivary iga subclasses levels were estimated using the rid kit ( human iga subclass nlcombikit , the binding site , uk ) .
appropriate volumes ( 10 l for iga1 and 10 l for iga2 ) of nondiluted saliva as well as of appropriately diluted calibrator and control sample were placed into agarose gel immunodiffusion plates containing the appropriate monospecific antibody .
the plates were then tightly closed , resealed in their original pouches and placed in the moist chamber at 37c in the incubator .
after 96 h incubation , the precipitin ring diameters were measured using a tripartisan ruler .
the antibody concentrations were read from the reference table enclosed , and expressed in mg / l of saliva .
the ring diameters of the calibrator and control serum were always in the expected range , that is , their concentrations were always within 10% of the concentrations stated on the vial label .
the obtained salivary iga1 and iga2 levels in both study and control group were tabulated and statistical analysis was calculated using mann - whitney u test and students paired and unpaired t test was done .
using spss 11 version package a p value of < 0.05 was taken as significant .
unstimulated whole saliva was collected in a sterile bottle by asking the patient to expectorate into it gradually over a period of 510 min .
the obtained saliva samples were stored in deep freezer at 20c until further test was carried out .
the salivary iga subclasses levels were estimated using the rid kit ( human iga subclass nlcombikit , the binding site , uk ) .
appropriate volumes ( 10 l for iga1 and 10 l for iga2 ) of nondiluted saliva as well as of appropriately diluted calibrator and control sample were placed into agarose gel immunodiffusion plates containing the appropriate monospecific antibody .
the plates were then tightly closed , resealed in their original pouches and placed in the moist chamber at 37c in the incubator .
after 96 h incubation , the precipitin ring diameters were measured using a tripartisan ruler .
the antibody concentrations were read from the reference table enclosed , and expressed in mg / l of saliva .
the ring diameters of the calibrator and control serum were always in the expected range , that is , their concentrations were always within 10% of the concentrations stated on the vial label .
the obtained salivary iga1 and iga2 levels in both study and control group were tabulated and statistical analysis was calculated using mann - whitney u test and students paired and unpaired t test was done .
using spss 11 version package a p value of < 0.05 was taken as significant .
the results of this study show that there was a statistically significant increase in the levels of iga1 in an acute phase ( p<0.001 ) as compared to remission phase and controls .
since iga1 is susceptible to the proteases , it appears that its levels diminish in the remission phase .
there was a significant rise in the iga2 levels in both acute and remission phases of the disease as compared to controls ( p<0.001 ) .
a genderwise comparison showed that females had higher levels of iga1 in an acute phase ( p<0.05 ) .
for iga2 , levels were increased both in acute and remission phases as compared to males ( p<0.001 ) .
tables 1 and 3 illustrate that the comparison between mean iga1 levels in acute phase of the study group and controls was statistically significant ( p<0.001 ) .
iga1 levels in acute and remission phases in the study group the comparison between mean iga1 levels in remission phase of the study group and controls .
tables 2 and 3 illustrate that the comparison between mean iga2 levels in acute phase of the study group and controls was statistically significant ( p<0.001 ) .
iga2 levels in acute and remission phases in the study group iga1 and iga2 levels in controls the comparison between mean iga2 levels in remission phase of the study group and controls was statistically significant ( p<0.001 ) .
recurrent aphthous ulceration or recurrent aphthous stomatitis ( ras ) is an intriguing condition known to humanity since ages and was first described by hippocrates ( 460370 bc ) .
aphthai , a greek word meaning small ulcer , to describe the disorders of the mouth .
secretory iga ( s - iga ) help maintain the integrity of the oral surfaces by limiting microbial adherence to epithelial and tooth surfaces ; by neutralizing enzymes , toxins , and virus ; or by acting in synergy with other antibacterial factors such as lysozyme , lactoferrin , salivary peroxidase , and mucins .
the literature search gives the evidence that levels of iga in ( serum / saliva ) gets altered in oral mucosal diseases such as rau.[81113 ] most of the studies have been limited to the estimation of the whole salivary / serum iga levels that have given contradictory results .
hence , this study was aimed to evaluate and compare salivary iga1 and iga2 levels in patients with minor rau .
this is in accordance with study of porter et al . , who stated that minor aphthous stomatitis affects 80% of the population .
in this study , 41.7% ( seven cases ) were affected by rau during the time of their menstruation .
this finding substantiates the previous reports that menstruation may be a triggering factor for rau .
this may be due to hormone induced alterations in the microbiota of oral mucosa leading to inflammation .
out of 30 subjects , 15 cases , that is , 50% gave a positive history of stress .
the high incidence of rau seen in our study may be due to academic stress and anxiety faced by the students .
the nicotine in tobacco has been found to be beneficial in rau and its effects may result from influences on nerve functions , although these may also exert direct anti - inflammatory effects .
numerous studies have reported an increase in the s - iga levels at the acute period and its reduction in the periods of remission .
these results suggest a strong correlation between s - iga and the lesion mechanisms in rau .
conversely many authors found no differences in the levels of salivary iga in either acute or remission phases of rau .
the mean iga1 and iga2 levels in acute and remission phases showed a statistically significant increase ( p<0.001 ) [ table 1 ] .
[ the comparison of mean iga1 levels in the study group in the acute phase and controls [ graph 1 ] showed significantly raised levels while the iga1 levels in the remission phase ( 684.8 mg / l ) returned to the values not different from those in healthy controls ( 617.9 mg / l ) [ tables 1 and 3 ] .
this finding is in contrast to a study sistig et al . , 2002 in which only iga2 levels were significantly raised .
mean values for iga1 in acute , remission phases of study groups compared with control on comparison of acute and remission phases of aphthous patients with controls , it was found that the mean iga2 levels remained high in both the phases [ tables 2 and 3 ] ( p<0.001 ) .
graph 2 illustrates consistently raised iga2 levels in both the phases , being highest in the acute phase ( 545.9 mg / l ) compared to controls . the consistent rise in iga2 levels in our study suggests that minute variations in iga subclasses in rau could not be elicited by simple determination of total iga .
the results of this study are compatible with the study of sistig et al . , with one difference that in our study iga2 levels were raised both in acute and remission phases .
they suggested that these changes could be related to the chronic antigen stimulation because of higher concentration of the specific microorganism that could be involved in the pathogenesis of rau .
mean values of iga2 in acute and remission phase in study groups compared with control changes in the iga1 and iga2 concentrations in saliva may be the result of different antigen stimulation in the oral cavity that preferentially induces either iga1 or iga2 antibody production from plasma cells .
furthermore , the iga subclass ratio may change during an active immune response ( crago et al . ,
so the decrease seen in the iga1 levels in the remission phase to the same as healthy controls could be due to its reduced sustainability because of its degradation by enzymes . a genderwise comparison in the levels of salivary iga1 and iga2 was made .
the mean comparison of iga1 levels in the acute phase between males and females showed statistically significant difference ( p<0.05 ) with higher levels seen in females , that is , 1667.7 mg / l as compared to males 1121.15 mg / l .
the mean iga2 levels in both acute and remission phases in females showed a statistically significant increase ( p<0.05 ) as compared to males .
the reason for the increased levels in females as compared to males is not clear and further studies are required to confirm the present finding .
the iga subclasses levels in female patients did not show any association ( p>0.05 ) with menstrual cycle . in a study on rau ,
showed that salivary iga levels were affected by menstruation . in the current study , results are not conclusive as the sample size in the two categories of females is very small .
effect of microbiological load in the oral cavity has not been studied in this study .
immunoglobulin a is an important parameter to assess the status of mucosal immune system that can be measured by noninvasive methods and without patient discomfort .
this study was an attempt to determine the iga subclasses levels in acute and remission phases of the disease rau using rid kit .
considering the limitations of the current study following conclusion can be drawn
iga subclass ratio may be changed during the active immune response , reflecting the generation of specific antibodies to various antigens of mucosal pathogens.determination of salivary iga using rid is a reliable , quick and a noninvasive means to assess the immune status in patients with rau.iga subclasses play an important role in the pathogenesis and defense mechanism of rau .
iga subclass ratio may be changed during the active immune response , reflecting the generation of specific antibodies to various antigens of mucosal pathogens .
determination of salivary iga using rid is a reliable , quick and a noninvasive means to assess the immune status in patients with rau .
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### Summary:
: context : recurrent aphthous ulcer ( rau ) is an inflammatory disease characterized by recurrent , painful oral ulcers .
it is of multifactorial etiology .
salivary immunoglobulins have important role in the protection of mucosal surfaces.aim:the aim of this study was to determine salivary immunoglobulin a1 ( iga1 ) and iga2 in acute and remission phases of the disease.materials and methods : thirty clinically confirmed cases of rau and 30 age - and sex - matched controls were included in the study . after detailed case history and thorough clinical examination ,
2 ml of saliva was collected in both acute and remission phases of the disease .
the obtained saliva samples were subjected to quantification of iga1 and iga2 levels using rid kit.results:the mean iga2 level was significantly higher ( p<.001 ) in both acute and remission phase of the study group .
the mean iga1 level also showed a significant increase in the acute phase compared to remission as well as controls ( p<.05 ) .
females exhibited a higher level in acute phase for iga1 and in both phases for iga2 ( p<.05).conclusion : the results associated with clinical observations suggest that acute phase is characterized with increase in iga2 that might reflect increased immune response as a possible result of the microbial stimulation seen in the acute phase in comparison to the remission period .
iga plays an important role in the pathogenesis of rau and it can be used as a parameter to assess the mucosal immune status
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: the denaturant urea is among those
osmolytes and can occur as a stressor , e.g. , in the kidney , but it is also used against osmotic stress . in either case , urea is found in mixtures with
at least one other osmolyte , typically a methylamine , such as trimethylamine - n - oxide ( tmao ) .
this compound
has generated considerable research interest , not only because it
is the strongest known protein stabilizer among the natural osmolytes and a crystallizing agent ; it also has been found to correct medicinally significant
issues , such as prion aggregation and
cellular folding defects .
it has
remained somewhat elusive what the mechanism of tmao s
action on protein is , and how it counters the denaturing effect of
urea .
the range of opinions is broad and includes suggestions that
tmao stabilizes proteins by classical preferential exclusion or by altering the structure of water and that tmao counteracts
urea by directly interacting with urea , by urea - independent
preferential exclusion from the protein , or by reverting changes of the structure of water caused by urea . since many of these ideas have to do with the
bulk solution , it is expedient to focus on ternary mixtures of tmao
with urea in water . here
we report thermodynamic experiments
that are combined with
rigorous statistical mechanics to derive
structural properties of the solution .
buff approach provides a link between
thermodynamics and preferential interactions on the one side and the
structure of the solution on the other .
the basic idea of this approach
is that deviations from random distribution of molecules around each
other produce characteristic changes in the partial molar volumes
and chemical activities of the solution components , and that conversely knowledge of these thermodynamic properties
allows us to derive information about the proximity of molecules in
solution .
buff
integrals ( integrated pair correlation functions ) are a measure of
the excess or deficit of one type of molecule around another . using this kirkwood buff approach
,
we find that , in aqueous
urea , tmao behaves as if it were a hard - sphere gas and the effective
size of the hard - sphere depends on its occupancy with either water
or urea molecules at two mandatory solvation sites .
this result gives
a rationale for the strong exclusion of tmao from peptide groups , which is substantially enhanced by the water / urea spacers intercalated
between tmao and the peptide group .
it also explains the experimentally
observed ( though small ) synergy between
urea and tmao , which is caused by the increased exclusion of tmao
from the peptide group , as small spacers ( water ) are replaced by larger
ones ( urea ) .
the primary goal here is to derive the solvation
behavior of aqueous
mixtures of urea and tmao , which requires partial molar volumes and
chemical activities of the solution components ( see eqs 16 and 17 ) .
the partial molar volumes are shown in figure 1 , where the horizontal lines represent the limiting
values in plain water .
the partial molar volumes of both urea ( top
panel ) and tmao ( bottom panel ) depend very little on the concentration
of the respective other osmolyte within the range of concentrations
used in the vapor pressure experiments ( 3 m ) .
our data
on urea compare favorably with previous data reported by lee and chalikian ( points in the top panel of figure 1 ) .
the partial molar volume of tmao comes very close to the
data reported by di michele et al .
this could be due to deviations between
the assumed and actual concentrations of tmao .
di michele et al . used
a very hygroscopic preparation of anhydrous
tmao without further treatment , and their data become identical with
ours if we assume that their tmao contained 34% water .
partial molar volumes of urea and tmao
in their ternary mixtures
with water .
top panel : urea partial molar volume ( thick
lines ) in 0 m , 2 m , 4 m , and 6 m tmao ( bottom to top ) . propagated fitting
errors are given by thin lines , and points represent previous data .
bottom panel : same for tmao in 015 m urea ( 3 m steps from bottom to top ) .
the parameters
from a fit of the density data to eq 7 are given
in table 1 .
the osmotic
coefficient data are shown in figure 2 along
with the fit ( eq 12 ) .
our urea data ( thin open
circles ) compare well with previous data ( thick open circles ) .
the osmotic coefficient of tmao ( filled circles )
is symptomatic for molecules that have repulsion between each other .
we decided to sample the osmotic coefficient
of a 2:1 mixture of urea : tmao ( triangles ) , because this is a ratio
found in living organisms . at this ratio ,
the
osmotic coefficient remains close to unity ; i.e. , the water behaves
close to ideally in the molality scale .
the molar activity coefficients
can be described as previously by1where i stands for either
urea ( u ) or tmao ( t ) .
the parameters c1,i and c2,i are the inverse effective volumes occupied by a single osmolyte
and a pair of osmolyte molecules , and g2,i is an interaction parameter .
the parameter 0 accounts for the effect of the second osmolyte on the infinite
dilution activity coefficient of the first one .
all parameters depend
on the other osmolyte s concentration , e.g.,2the parameters are given in table 2 .
lines are the
fit : blue , addition of urea to water ( solid ) or 1 m tmao ( dashed ) ; red , addition of tmao to water ( solid ) or 1 m urea ( dashed ) ; magenta , urea / tmao = 2/1 . data : thin open
circles , urea in water ( this work ) ; thick open circles , urea in water ; open squares , urea in 1 m tmao ;
filled circles , tmao in water ; filled squares , tmao in 1 m urea ; triangles , urea / tmao = 2/1 .
( bottom panel ) molar activity coefficients
as a function of the tmao concentration : red lines , c of tmao ; blue lines , c of urea .
the successively
shorter lines that are offset relative to unity correspond to 03
m urea , spaced by 0.5 m increments .
the resulting molar activity coefficients ( figure 2 , bottom panel ) depend primarily on the molarity
of tmao ,
and to a much lesser degree on the molarity of urea .
for example ,
the change in c , u ( blue lines ) as a function of
tmao ( going from left to right ) is several fold larger than as a function
of urea ( going from the uppermost curve to the lowest ) .
for this reason ,
c is plotted as a function of the tmao concentration .
both activity coefficients increase upon addition of the respective
other osmolyte , suggesting mild mutual exclusion .
this effect is , however , small compared to the strong increase
of the activity coefficient of tmao with its own concentration ( see
bottom panel of figure 2 ) . from the combined volumetric and osmometric
data we derived the kirkwood buff integrals ( kbis ) of all components
in the ternary mixture , using eq 16 .
remember
that the kbis are a measure of the excess or deficit of one type of
molecule around another type .
the first observation is that again , as in the
case of the activity coefficients ( figure 2 ) , all concentration dependencies are almost exclusively due to tmao .
that is , the change along the tmao axis is generally larger than the
urea - dependent offset within each group of curves .
only the self - solvation
kbis of the osmolytes uu and tt depend slightly on the concentration
of urea .
thus the cluster of lines corresponding to increasing urea
concentration becomes visible in these two cases .
the urea concentration
increases from the highest line ( 0 m ) to the lowest
one ( 3.6 m ) in both cases .
the overall level of the
tmao self - solvation , tt , is at least 3 times more
negative than expected for an osmolyte of its size .
but as expected , the hydration terms wt , wu , and ww are small in comparison . solvation
in aqueous mixtures of urea with tmao .
buff
integrals ij for hydration
of urea ( cyan ) , tmao ( blue ) , and water ( black ) are given separately
( top panel ) to avoid overlap with uu .
the data are given as a function
of the tmao molarity in clusters of curves that correspond to increasing
urea concentrations ( starting form zero , offset in increments of 0.4 m ) .
the propagated fitting error is smaller than 0.001 l / mol
for hydration , and smaller than 0.01 l / mol for osmolyte
figure 4 demonstrates that tmao closely follows a solvation pattern
expected for a hard - sphere gas , i.e. , a system in which the one and
only interaction between molecules is hard - core repulsion ( molecules
can not overlap each other ) . at low concentrations , such spheres occupy
the space randomly . as the concentration increases ,
their proximity
is determined by the question of how well they can be packed .
the
lower panel shows tt at 0 m urea ( continuous red
line ) , along with three sets of predictions according to the carnahan
starling
equation for hard - spheres ( eq 23 ) , using the
partial molar volumes for tmao shown in figure 1 .
tmao actually behaves like a hard - sphere of the size of a doubly
hydrated tmao molecule .
this solvation pattern makes sense , because
it is known that tmao is strongly hydrated by two water molecules . naturally , though tmao has a
very compact shape , it is not literally a perfect sphere .
but tmao
shows a remarkable similarity to the solvation expected for an ideal
hard - sphere . the hard - sphere like behavior
can be further tested
by investigating the tmao hydration , wt , shown in the upper panel
of figure 4 .
also this kbi perfectly matches
the model of a hydrated hard - sphere that has the size of a doubly
hydrated tmao molecule ( eq 24 ) .
the same behavior
is found with the water self - hydration , ww ( figure 4 , top panel ) .
there is no straightforward way to calculate the other ,
urea - related kbis from the hard - sphere model .
the upper panel shows the tmao
hydration , wt ( blue ) , and the water self - hydration , ww ( gray ) , at 0 m urea , along
with the hard - sphere behavior calculated for 0 to 2 hydration waters ,
using eqs 24 and 25 .
the
lower panel contains the same information for the tmao self - solvation , tt ( red ) , at 0 m urea ( continuous
red line ) and in the limit of 0 m tmao ( dashed red line ) .
the latter
is shown as a function of the urea molarity , and the dash - dotted line
is calculated according to eq 5 .
the deviations
between the best prediction and the data are smaller than the uncertainty
in the data ( 10 ml / mol ) .
much has been written about the interactions
among tmao , urea ,
and water in solution , and the main focus has been on the water interactions .
we start by first considering the hydration of tmao , and then examine
the other ( previously more neglected ) binary interactions in solution ,
and close with a discussion of water structure .
our finding that
aqueous tmao behaves similarly to a hard - sphere of the size of tmao
dihydrate strongly suggests that tmao has two mandatory hydration
waters .
anhydrous
tmao can not be produced by evaporating the two hydration waters ; rather ,
tmao has to be sublimed at high temperature.ab initio calculations also show tightly coordinated
water .
dielectric relaxation finds tmao
as a dihydrate in solution , and in the same research the very large
enthalpy of h - bond formation was pointed out .
based on nmr it was found that all tmao in water is present as dihydrate .
raman
spectroscopy indicates that tmao has at least three h - bonds from neighboring
water , but the mere existence of h - bonded
waters does not necessarily imply that all ( or even any ) of them are
mandatory hydration waters .
the spectroscopic observation that the
solvation shell of tmao contains slower moving water molecules may be at least in part due to the solvation
sites at the tmao oxygen .
significantly , small angle scattering measurements
found that both water oxygen and urea nitrogen are found around the
tmao oxygen at hydrogen - bonded distances , with one hydrogen atom between .
thus , both urea and water snap into hydrogen - bonded positions as
they approach the tmao oxygen .
it has been found both by raman spectroscopy and neutron / x - ray scattering that tmao does not self - aggregate .
we can add now the significant
finding to which degree tmao does not aggregate ; viz .
, its self - solvation tt is indistinguishable from
that of a hard - sphere gas there is no significant attraction .
according to the effective size of the hard - sphere , the tmao must
be doubly hydrated , as discussed above .
upon addition of urea , these
waters are progressively replaced by urea molecules , and this should lead to an increase of the effective hard - sphere
size .
indeed , tt changes upon urea addition
toward more negative values , which correspond to larger sphere diameters
( figures 3 and 4 ) , as
discussed in the following around eq 5 .
argued that urea binds to tmao with an affinity that is comparable
to the interaction of urea with peptide groups .
however , they also pointed out that their analysis was
assuming a binding model , rather than the proper exchange model .
therefore we reanalyze their data , as shown
in figure 5 as continuous lines .
we focus on
the first two reported binding events , because a potential third
the
reaction model is3where t , u , and w represent
tmao , urea , and water , respectively , and k1 and k2 are the exchange constants for
the two reactions .
the populations for each species were calculated
and fit to the data numerically . the resulting affinities
are quite weak , so that the midpoint of the binding
of the first urea is around 7.5 m urea . in comparison ,
the midpoint
of the binding of urea to a peptide nh is around 4 m. in the face of such weak affinities the question arises ,
how do the exchange curves in figure 5 compare
to random occupancy with water and urea ? if we take the volume fraction
as an approximate model for the likelihood that a site is randomly
occupied by a species , we first need
to consider that direct tmao tmao interaction at the oxygen
site does not occur ( see previous paragraph ) .
so , we normalize the
volume fractions of water and urea to refer to the non - tmao volume
( 1 t ) , and obtain for the populations of
the three species4the resulting curves are shown as dashed lines
in figure 5 .
the population of the dihydrate , pww , closely follows the exchange curve , and
thus double occupancy with water is practically random . for the other
two species
, there is a marginal deviation from random distribution ,
such that the single urea species is populated slightly less than
random , and the double urea species slightly more .
this view of nearly
random interaction is also supported by recent molecular dynamics
simulations , where tmao was found to be equally h - bonded to water
and urea at about 35% by volume of each urea and water , and a rough equivalence of both was noted .
the idea of random interaction of water and
urea with tmao is also consistent with the spectroscopic finding that
there is no specific urea - tmao binding . a computational finding that both urea and water interact strongly
with tmao
should then be taken to mean
that though both are strongly interacting with tmao ( judged by the
fact that the solvation sites are mandatory ) , they are about equally
strongly interacting ( judged by the random interaction ) . binding
of urea to tmao .
continuous lines are global
fits of model eq 3 to the data ( k1 = 4.6 , k2 = 2.5 ) . dashed
lines are random encounters calculated according to volume fractions
( eq 4 ) . from the concept of random occupancy we can also
predict the change
in tt upon addition of urea that
we observe in figures 3 and 4 . if we take the three populations of differently sized hard - spheres
( eq 4 ) as weights for the kbis for the three
sphere sizes,5we get very good agreement between the data
and the prediction as shown in figure 4 ( lower
panel , red dashed and black dash - dotted lines ) . from neutron and x - ray diffraction measurements meersman
et al . derived the radial distribution function between a urea nitrogen
and the tmao oxygen as shown in figure 6 ( blue
line ) .
although there is a clear first
solvation peak corresponding to direct h - bonding , this peak does not
exceed unity much , which is indicative of massive steric exclusion .
this makes sense , considering that urea can approach the tmao oxygen
only from that hemisphere that is not blocked by the nitrogen and
three methyl groups .
radial distribution function of urea nitrogen around tmao
oxygen :
blue line , data for about 2 m each of urea and tmao ; red line , effect of direct steric exclusion between urea
and tmao ( eq 6 ) .
top : schematic illustration
of the origin of the peaks for ( a ) direct h - bonding of urea to tmao
and ( b ) solvent - separated h - bonding .
how do the data compare to a situation where urea
is excluded from
tmao but randomly distributed beyond the limits of the tmao molecule
( in a urea continuum ) ?
analytic integration of a 3d space with a tmao - sized
spherical hole gives the contribution of the direct steric exclusion
to the radial distribution function as6where rmin is
the minimal distance between urea and the tmao oxygen ( about 1.2 ) , and rt is the effective
radius of dry tmao ( 2.66 ) .
the
resulting curve is shown in red in figure 6 . comparing the data ( blue ) with the steric contribution ( red ) reveals
more clearly the second solvation peak around 5.5 , which may
come from both the second urea nitrogen and water - separated urea
significantly , the blue scattering curve integrates within error to
the same value as the red curve , viz . , the measured ut
90 ml / mol at 2 m tmao .
this result highlights the
dominance of steric exclusion , where the h - bonding related deviations
of the actual g(r ) from eq 6 merely have an alignment effect that averages out
to zero .
previous findings support our observation that the hydration of the
osmolytes , tw and uw , hardly depends on osmolyte
concentration ( except for the nonspecific hard - sphere - like behavior
of tw as a function of ctmao ) .
near - infrared spectra do not show any evidence
that the hydration of either urea or tmao changes upon mixing .
also nmr , combined with molecular dynamics ,
showed that the hydration shell structure of tmao is maintained over
a wide concentration range .
there are two schools of thought on
the origin of the behavior of osmolytes , viz .
either direct interaction
of water / osmolyte with target molecules ( preferential interactions ) or indirect effects through alterations
of the structure of water .
typical uses of the
term water structure refer to just one small aspect
of the properties of water .
thus it is not surprising that one can
reach diametrically opposite conclusions regarding the effect of osmolytes
merely by choosing how the term water structure is
used .
it has been suggested that the
term water structure may be even undefinable in view
of such issues .
it is therefore expedient
to refer to the actual observations ( e.g. , h - bond angles ) rather than
to use a word with multiple disparate uses .
what is the appropriate
definition of water structure when we want to understand
the thermodynamics of the solution ? perhaps the most general definition
is given by the kbis , which provide a rigorous link between the positions
of molecules in solution and thermodynamics .
the kbis are directly linked with preferential interactions , which means
that the school of thought that osmolytes act through preferential
interaction has a rigorous statistical mechanic backing .
does that
mean there is no place for the other school of thought ? to answer
this question , we have to remember that the kbis are integrated radial
distribution functions , so they are thermodynamically valid descriptions
of solution structure as well . and all the other measures of water
structure can be equally valid thermodynamically , to the degree to
which they explain the form of the kbi .
there are two general
aspects of water structure : the bulk water
structure ( related to ww ) and the hydration structures
of the osmolytes ( related to tw and uw ) .
we already discussed some
of the latter above , and numerous more could be added .
the point is that it is by no means obvious how those findings could
be predictive of a significant change in any of the hydration kbis , ww , tw , and uw .
specifically , figure 3 shows that the water self - hydration , ww , changes very little as osmolyte
is added
. the effect may be significant in terms of the precision
of the data ( see figure 4 ) , but negligibly
small compared to the changes seen in the other kbi .
moreover , it
has been pointed out that ww does not even appear in the
kirkwood buff expressions that describe the impact of osmolytes
on protein stability .
the other two hydration
kbi do explicitly occur in those expressions , but tw seems to be governed by mere
packing , as judged by its hard - sphere like behavior .
we have used a combination of our current
experimental volume and
chemical activity data with kirkwood buff theory to elucidate
the behavior of tmao in aqueous urea . a second look on scattering
data provided a strong confirmation and some additional insight .
the
picture that emerges shows a dominant role of hydrogen bonding along
with simple steric exclusion .
tmao is a roughly spherical molecule
and behaves as such to a degree that its self - solvation and hydration
resemble the properties of a hard - sphere gas .
however , it has two
mandatory solvation sites that can be occupied by water or urea , substantially
enlarging the effective hard - sphere diameter .
the hydrogen bonding
at these sites is sufficiently strong to prevent both water and urea
from approaching the oxygen with anything else other than their hydrogen
atoms .
it is well known that tmao is strongly excluded from peptide groups . such
preferential exclusion ( also termed
osmophobicity ) favors the native state because the denatured
state exposes more surface , leading to more exclusion .
aqueous tmao appears to interact with the peptide groups
only through its hydration waters .
thus , the
h - bonds donated to the tmao from peptide groups must be significantly
weaker than those donated from water and urea .
so , in the presence
of tmao but absence of urea , the peptide groups will be preferentially
hydrated , with at least one water layer between peptide and tmao
just
because tmao will only hydrogen - bond to the peptide through an intercalated
water .
what is then expected upon addition of urea is that the
situation
does not fundamentally change .
the only difference is that now tmao
has the option to interact with the peptide group through either an
intercalated urea , or an intercalated water .
the mechanism of stabilization
by tmao does not change as urea is added . only the spacing between
tmao and the peptide groups
that is , tmao should be more excluded and become
a more potent protein stabilizer in the presence of urea than in its
absence .
m - values of tmao in the presence of urea has been observed ( m - values are a measure of
how potent an osmolyte is in stabilizing a protein ) .
conversely , if
tmao becomes a more potent stabilizer in the presence of urea , then
urea must become less effective as a denaturant for
symmetry reasons ( maxwell relations ) .
a similar
trend to synergy has been found with mixtures of sarcosine ( another
methylamine ) with urea , raising the question
whether similar principles apply to other osmolytes as well .
tmao dihydrate from sigma and ultrapure
urea from usb were dried at 60 c for at least 20 h. the masses
used to prepare the solutions were measured using a mettler toledo
at20 analytical balance equipped with an antistatic device .
the densities
of the solutions were measured in an anton paar dma 5000 m density
meter , using automatic viscosity correction .
the tmao dihydrate used for
the osmotic coefficient measurements was freshly synthesized and recrystallized .
the water vapor pressure was measured in a wescor
vapro 5520 osmometer with modifications described previously .
the measured osmolality values were divided by the total molality
of urea and tmao to obtain the osmotic coefficient , which is a measure
of the deviation of the water chemical activity from ideal behavior .
the density of the urea tmao
mixtures was measured to obtain the partial molar volumes .
using wolfram
mathematica , the data were fit to the relation7where the numerator is the mass of the solution
per kg of water and the denominator the volume per kg of water ( note
that the molecular weights mi have to be given in kg / mol ) . among the parameters , 0 is the density of plain water , v1,0 and v0,1 are the limiting
partial molar volume in plain water ( of urea and tmao , respectively ) ,
and the vi , k terms in general are mixed derivatives of the volume with
respect to the molalities of urea and tmao , mu and mt .
the partial molar volumes
of urea and tmao are then89and the partial molar volume of water10directly follows , knowing the molar concentrations
of urea and tmao ( cu and ct ) and the molecular weights of all species ( 18.015 , 60.06 ,
and 75.11 g / mol ) , and considering11 the vapor pressure data
were used to derive the chemical activities of urea and tmao through
the method of schnert .
the osmotic
coefficient data were fitted to12and it was found that terms up to second order
are sufficient .
there are additional isopiestic data available on
the dependence of on urea concentration in the absence of
tmao . including these data requires terms
up to seventh order in urea concentration .
equations for the chemical
activities of water , urea , and tmao follow directly:131415 solvation properties
can be calculated from the partial molar volumes and chemical activities
as follows .
the excess or deficit of molecules of type i and j around each other ( relative to the bulk )
is given by integrated pair correlation functions , the so - called kbis,16where the elements of matrix a are given by17here , is the compression coefficient ,
i , j the kronecker
delta , t the absolute temperature , r the gas constant , |a| the determinant of a , and |a|i , j denotes a cofactor of the matrix .
after eq 17 is substituted into eq 16 , has little impact on ij .
this is because is then not in an isolated position
in the denominator any more , and is negligibly small for our purposes , so we just set it to its value in plain water . in order to link the derivatives in this equation
back to the vapor pressure results , they are rewritten as either18for all cases except when i = k = w , or19by use of the gibbs duhem relation .
as
shown in the results section , tmao behaves
like a hard - sphere gas in urea solution . calculating the kbis for
hard - spheres
is conveniently done through the carnahan starling
equation . though this relation is for
a single - component hard - sphere gas , it is possible to infer the implicit
properties of the water as follows . in a two - component solution
( here ,
water and tmao ) , the kbis are20and21for the self - solvation of tmao and self - hydration
of water , and22for the hydration of tmao , where aii =
( ln ai/ ln ci ) and ii = ( ln ai/ ci ) .
taking
into account ww = ( aww 1)/cw allows use of eqs 20 to 22 to obtain ww and wt from tt , if att and the partial molar volumes are known:232425 multiplying the carnahan
starling
equation for a hard - sphere gas by the
concentration cs and
performing a derivative with respect to cs gives ass(63 ) as follows:26where = csvs is the volume fraction of the
hard - sphere , cs its molarity , and vs its partial molar volume .
equation 26 can then be used in place of att to calculate the various kbis ( eqs 2325 ) . when the size of the hard - spheres
is different from vt , we change vt in eq 23 accordingly .
that is , the partial molar volume of tmao mono- or dihydrate is larger
than that of tmao itself .
however , when it comes to the partial molar
volume in the context of wt , there is no such thing as
a mono- or dihydrate .
this is because wt considers the spacing between
tmao and the surrounding water molecules independently of whether
or not they are mandatory hydration waters .
accordingly , we do not
change vt in eq 24 when the size of the hard - spheres is assumed to be different
from vt . note that in the
limit of cs0 ,
we recover the classically expected result for the excluded
volume ( equaling the second virial coefficient ) , wt tt = 4vs .
|
### Summary:
: trimethylamine - n - oxide ( tmao ) and urea
represent
the extremes among the naturally occurring organic osmolytes in terms
of their ability to stabilize / destabilize proteins .
their mixtures
are found in nature and have generated interest in terms of both their
physiological role and their potential use as additives in various
applications ( crystallography , drug formulation , etc . ) .
here we report
experimental density and activity coefficient data for aqueous mixtures
of tmao with urea . from these data
we derive the thermodynamics and
solvation properties of the osmolytes , using kirkwood
buff
theory
. strong hydrogen - bonding at the tmao oxygen , combined with
volume exclusion , accounts for the thermodynamics and solvation of
tmao in aqueous urea . as a result
, tmao behaves in a manner that is
surprisingly similar to that of hard - spheres .
there are two mandatory
solvation sites . in plain water
, these sites are occupied with water
molecules , which are seamlessly replaced by urea , in proportion to
its volume fraction .
we discuss how this result gives an explanation
both for the exceptionally strong exclusion of tmao from peptide groups
and for the experimentally observed synergy between urea and tmao .
|
<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: in the 24 years since the discovery of hiv-1 1 , with the impetus to cure aids , there have been an unprecedented number of publications examining retroviral polymorphisms .
these have led to significant advances in understanding mechanisms generating them . in particular , when a provirus replicates along with host cellular dna during mitosis , progeny are extremely homogenous .
a case in point is clonal expansion of htlv-1 2 , resulting in small variability over centuries 2 - 5 .
this can be attributed to the high fidelity of mammalian cellular dna replication , mediated by the 3'-5 ' exonuclease proofreading activity of dna polymerase 6 .
when a provirus replicates exogenously by infection , progeny can be extremely heterogeneous 4 , 7 .
since exogenous retroviral replication involves transcription and reverse transcription , both could contribute towards generating polymorphisms .
therefore , for exogenously replicating retroviruses , such as hiv-1 , polymorphisms are primarily generated during error prone reverse transcription ( fig .
in particular the mis - incorporations produced by rna - templated dna synthesis and dna - templated dna synthesis ( fig .
1 ) , have been proven to be exacerbated by dntp imbalances both in vitro and in vivo 11 - 14 .
proof of the existence of these dntp imbalances in a proportion of retroviral permissive cells 13 has provided a mechanistic explanation for observed retroviral g to a hypermutations in vivo 11 , 13 - 17 .
these mis - incorporations result in polymorphisms , since reverse transcriptase lacks 3'-to-5 ' exonuclease proof - reading activity 18 , provided that the mutations result in replication competent virus that is naturally selected in vivo .
more recently , rna editing , through apobec enzymes , that deaminate cytosine to uracil , have also been proposed as a mechanism for retroviral g to a hypermutation 19 .
in fact , the newest role for the hiv-1 vif protein , is to counter the effect of apobec 20 , 21 .
interestingly , vif may also be exploiting the mutation potential afforded by apobec , in generating more hiv-1 diversity 21 , rather than simply providing immunity from the innate host apobec defense mechanism .
importantly , both templated and non - templated miss - incorporations , as well as slippage
therefore , in addition to point mutations , in vivo sampled retroviruses also frequently harbor deletions and insertions . here , among the most probable mechanistic explanations , in addition to , or in combination with the above mentioned non - templated additions ,
is forced copy - choice strand switching during reverse transcription followed by mis - alignment and continued polymerization on the new template 23 - 28 .
in essence this is a forced emulation of the reverse transcription " jumps " depicted in figure 1 29 .
this mechanism reconciles the fact that retroviruses can synthesize cdna with only one rna genomic copy 30 whereas viral particles carry 2 copies .
the second would then serve the failsafe function of providing new template to " jump to " when reverse transcriptase encounters a nick .
in addition , forced copy choice strand - switching provides a probable mechanistic explanation for phylogenetic revelations of inter - subtype recombinant hiv isolates 15 , 31 .
the implication is that chimeric exogenous retroviruses , containing transcribed genomic rna from two different proviruses , are produced from doubly or multiply infected cells .
in fact , recombination has now been shown to be the primary driving force for generating hiv-1 variants 32 , 33 , despite cellular retroviral superinfection resistance mechanisms 34 .
these mechanisms are shared by the larger family of elements capable of retro - transposition 7 , 35 .
however , hiv-1 undergoes such rapid exogenous viral replication and turnover 36 - 38 , that natural selection generates enormously heterogeneous populations termed quasispecies 16 , 39 , or swarms , which have either temporally adapted to cell - specific microenvironments , or exist because their specific polymorphisms are neutral .
the proviral hiv-1 ltr sequences first investigated , were from the initial isolate 40 and rapidly became the prototypical ltr .
these sequences orchestrate the cellular rna polymerase ii transcription machinery , in conjunction with viral proteins , and include a tata - box , 3 sp1 sites , 2 nfkb enhancer elements , an rbeiii site and an upstream usf site ( fig .
importantly , the extensive hiv-1 polymorphism detected in vivo , extends to the ltr ( 15 , 42 and references therein ) .
in particular , a length polymorphism immediately 5 ' of the enhancer region , has been detected , investigated , or referred to by a growing list of publications 42 - 60 .
this polymorphism was initially detected as a 24 base pair duplication by golub , li and volsky , looking for ltr variants ; but no regulatory cis - acting motifs were described within it , despite a correlation with increased transcription 43 . blumberg and co - authors , described a similar duplication , in adult and pediatric patients , while looking for variant nef sequences , but again did not find any motifs that might explain functional significance 44 .
koken and co - authors , were the first to refer to this duplication as the
ctg-motif , and noted that it encompassed a repeat of the sequence 5'-actgctga-3 ' , and further found that a single ctg-motif virus , could out - compete an isogenic virus containing the duplication , in co - culture experiments , in 42 days 45 .
these authors also noted similarities between the duplicated region and the recently - described tcf-1 ( hlef ) binding site , and pointed out that hivant 70 , as well as sivcpz , have the duplicated ctg-motif ( 45 and references therein ) .
these authors also pointed out that nakanishi , masamune and kobayashi , had described the
in fact , in their paper , nakanishi and co - authors designated this region ( -157 to -121 ) the upstream regulatory element ( ure ) and showed that it bound a transcription factor ( designated ure - binding factors c1 and c2 , by emsa - specific analysis with nuclear extracts ) distinct from ap-1 61 .
furthermore , koken and co - authors 45 noted that zeichner , kim and alwine , had published data suggesting positive regulation in the
soon afterwards , kim , gonzales , zeichner and alwine , published another paper , showing a more profound effect of mutations in the ure - region ( ctg-motif ) , in the context of chromatin and viral replication , but attributed the effect to mutation of the tcf-1 ( hlef ) binding - site 63 . in another paper by koken and co - authors
ctg-motif duplicated isogenic viruses , are out - competed in cell culture assays by the monomeric isomeric virus 46 .
these authors also showed that a circa 64kd protein , binds with specificity to the 5'-actgctga-3 ' sequence , duplicated in the
ctg-motif duplications and obtained similar emsa results to those of nakanishi and co - authors , but further reported no correlation between the ctg-motif duplication and disease stage or viral phenotype 46 , 61 .
so , by 1994 , it was already clear that a cis - element , named ure , enhanced viral replication , contained a putative binding site for tcf-1 ( hlef ) , could specifically bind proteins from nuclear extracts , and was encompassed in a frequent nef / ltr duplication ( ctg-motif duplication )
ctg-motif duplication , in tissue culture assays with isogenic viruses , no dramatic effects were observed .
further reports on this region of the hiv-1 ltr , included a paper from michael and co - authors , that detected the tcf-1 duplication in uncultured samples from patients , and suggested it may mediate a repressive effect 47 , and a paper by ait - khaled , also detecting the tcf-1 duplication , in post - mortem samples 48 .
however , in 1996 , in a cross - sectional analysis of nearly 500 hiv-1 ltrs , we published that the ctg-motif or ure region duplications , could best be aligned as a single larger polymorphism , which we named the most frequent naturally - occurring length polymorphism ( mfnlp ) of the hiv-1 ltr 42 .
furthermore , we proposed that the mfnlp encompassed a binding site for an unpublished nuclear factor named rbf-2 , instead of tcf-1 42 .
this transcription factor was defined in a subsequent publication 64 , by a tga - specificity for the 5'-actgctga-3 ' sequence , in crude nuclear extracts , and correlated with a region of the hiv-1 ltr required for optimal responsiveness to ras signaling through the mapk pathway , in tissue culture transient transfections .
strikingly , a subsequent publication by linqi zhang and co - authors , reported a tcf-1 duplication in a patient that progressed towards aids , despite the lack of nfb binding 49 .
interestingly , these authors suggested a compensatory role for the tcf-1 duplication , in the face of nfb binding - site abrogation , yet they found very little effect of the duplication in tissue culture 49 .
soon after , kirchhoff and co - authors , reported on the presence of the mfnlp sequence in clinical samples , but did not point out that they duplicated rbeiii sequences , responsive to ras 50 .
similarly , zhang and co - authors , reported on more clinical samples with tcf-1 duplications , for which they found no phenotype , but failed to point out that the mfnlps duplicated rbeiii 51 .
quinones - mateu , reported similar detection of sequence duplications , which we called mfnlps 53 . in 1998 , we further expanded our knowledge about the rbeiii and mfnlp - binding proteins , by showing : ( i ) that hlef ( tcf-1 ) can footprint on the wild type site but not on all mfnlps or rbeiii sites ; ( ii ) that all mfnlps bind rbf-2 ; and ( iii ) that a small repressive effect was mediated through the mfnlp in transient transfections 65 . in 1999 naghavi and co - authors also reported tcf-1 duplications in clinical samples 55 , 66 . at this time , we had already attempted to purify the components of rbf-2 by chromatography , including an oligo - affinity step 67 .
purified rbf-2 included proteins of approximately 120 kd and 45kd , however our repeated attempts to obtain peptide sequences from large scale purifications were inconclusive 67 .
importantly , in the lab of robert roeder , naghavi and i began doing some experiments to confirm her sequencing data that predicted various hiv-1 subtypes would not bind to the nre usf site , since the sequence was not highly conserved , as i had shown for samples from the long - standing vancouver lymphadenopathy study ( vlas ) cohort 42 .
while studying the role of usf-1 in hiv-1 ltr - directed transcription and viral replication , we used a double stranded rbeiii site and a mutant rbeiii site , as a non - specific competitor in an emsa with labeled double stranded e - box targets , encompassing the upstream usf-1 site in the prototypical lav hiv-1 ltr sequence , and nuclear extracts ( nre b usf site , fig 2b , lane1 ) .
surprisingly , we found that at 100x molar excess , rbeiii competed with the band shift , but the rbeiii mutant did not ( fig 2b , lane 9 versus lane 10 ) .
although we at first assumed we had made a mistake , we immediately tested our new hypothesis that rbf2 contains usf-1 , by competing a standard nuclear extract rbf2-rbeiii emsa , with the e - box unlabelled target ( fig .
the hallmark of the rbf2 emsa ( fig 2c , lane 1 ) is competition with 100x wild type rbeiii ( fig .
2c , lane 5 ) in the face of no competition with 100x mutant rbeiii ( fig . 2c ,
the specific rbf2 bands were further tested with the non - specific 100x wtnfb sequences from the prototype lav hiv-1 ltr ( fig .
this important result indicated that our initial observation was true , and suggested that rbf2 contains usf-1 .
we therefore compared the emsa mobility of rusf-1 versus nuclear extracts , with an rbeiii target ( fig .
the standard rbf-2 emsa ( fig . 2d , lanes 1 - 4 , lower arrow ) appears to have a specific band that is lower in mobility than the specific band forming with rusf-1 ( fig .
importantly , although the mobility is slightly different , both bands are specific , since the nuclear extract band can be competed with 100x molar excess of wtrbeiii ( lane 2 ) but not with 100x mut rbeiii ( lane 3 ) and the rusf-1 band can be competed with 100x wtrbeiii ( lane 8) but not 100x mutrbeiii ( lane 9 ) .
additionally , antibodies to rusf-1 appear to supershift both rbf-2 and rusf-1 ( lanes 4 and 10 ) , consistent with usf-1 being in rbf-2 . since our focus is on
the mfnlp and mfnlps encompass rbeiii sequences , the above results strongly suggested that mfnlps would also bind usf-1 .
however , the canonical e - box canntg site , recognized by usf-1 , is not present in rbeiii sites .
indeed target detection analysis ( tda ) , with random targets , using usf-1 , does not select an rbeiii site 68
. however , we noted that in the purified rbf-2 preparations there was a circa 120 kd component 67 .
it was then robert roeder who immediately suggested to me that rbf-2 was comprised of usf and tfii - i . because tfiii and usf-1 bind the initiator / e - box region of the hiv-1 ltr , and rbf-2 had been shown to bind an rbei site ( in addition to the rbeiii site ) that overlaps with the initiator sequences ( fig.2a ) 64 , the hypothesis that rbf-2 was comprised of usf and tfii - i seemed plausible .
in fact , i found this was indeed the case , for at least 2 naturally occurring rbeiii hiv-1 ltr sequences ( from clones pmce36.1 and pmce9.104 , described elsewhere 65 ) , one of which is encompassed in an mfnlp ( pmce9.104 ) ( fig .
2d ) , in addition to the prototype hiv-1 ltr lav sequences ( above ) . importantly , since binding of rusf-1 alone ( fig .
2d , lanes 1 and 2 ) and binding of rtfiii alone ( lanes 5 and 6 ) is significantly weaker than when usf-1 is combined with tfiii ( lanes 3 and 4 ) , it is likely that cooperative binding was occurring .
indeed , this important result suggested that usf-1 and tfiii , when integrated at an rbeiii site , exert a stronger effect than individually , thus potentially explaining why their individual binding to an rbeiii site has not been previously observed .
subsequent to our discovery , we asked the laboratory of ivan sadowski to confirm our results .
martin hirst detected both usf and tfii - i in rbf-2 purifications 67 , confirming our results .
all the above initial findings were excluded from our publication about the nre usf subtype sites 69 , in order to better develop them into a clearer picture of the function of usf / tfii - i at the rbeiii sites , in collaboration with the lab of ivan sadowski ( see below ) 70 .
importantly , jeeninga and co - authors , confirmed in 2000 , that rbeiii sites are absolutely conserved , in ltr subtypes a through g 66 .
a publication in 2000 by gomez - roman and co - authors , detected mfnlp sequences , duplicating rbeiii , in a long term non progressor ( ltnp ) , but no deletions to rbeiii sites were found 56 .
these ltnp are clinically distinct groups with lower viral replication and a slower or non - progressive form of hiv-1 infection ( see below ) . in 2000
, chen and co - authors found that an mfnlp from one of their patient samples , created a new ap-1 binding site , and identified c - fos and junb as binding proteins for their mfnlp 57 .
birch and co - authors described in 2001 , ltnp from the sydney blood bank cohort ( sbbc ) , that are accumulating progressively more ltr / nef deletions , and some of these persons harbor rbeiii duplications in the form of mfnlps 58 .
similarly , another ltnp study appears to have ltr / nef sequences progressively accumulating greater deletions over time 60 .
however it has not been at all clear if the ltr or the nef deletions are responsible for the ltnp phenotype . in 2002 , hiebenthal - millow and kirchhoff , found that in primary t cells , the mfnlp appears not to confer a major effect upon viral replication in culture 59 .
a noteworthy observation about this paper is that these authors actually found enhanced transcriptional activity occurred when both the rbeiii site and mfnlps were deleted , and that the mfnlp slightly reduced transcriptional activity .
perhaps the most important paper about the role of the rbeiii site and the mfnlp is that from chen and co - authors 70 .
this paper expands on our initial discovery that rbf-2 is composed of tfiii and usf , based on the tga specificity of the emsa used by bell and sadowski 64 .
rbf-2 appears to be a heterodimer of usf-1/usf-2 ; and tfii - i appears to load usf-1/ usf-2 onto the rbeiii site .
importantly , this paper shows that rbeiii mutant ltrs are unresponsive to cross - linking of the t cell receptor or stimulation with pma and ionomycin .
thus it appears that the phenotype of the rbeiii site and hence the compensatory mfnlp role , is to provide a target for ras / mapk activation of the hiv-1 ltr , in the context of chromatin 70 , 71 .
importantly , the most recent sbbc ltnp study , indicates that there is a convergence in ltr / nef deletions for these ltnps 72 .
these deletions appear to leave the sequences downstream of the nfb sites intact , yet appear to be abrogating rbeiii sites , since they are deleted , without mfnlps .
importantly , there are publications indicating that nef can activate the mapk pathway 73 and increase t cell activation 74 .
therefore nef could be actually acting through the nef / ltr cis - acting rbeiii site , which is abrogated in the ssbc ltnps 72 .
ltrs with normal rbeiii sites and a normal nef sequence , could contribute to high viral replication and progress to aids ( fig 3a ) .
the presence of mfnlps in the context of normal rbeiii sites would simply provide a failsafe function .
however , ltrs with mutated rbeiii site would require an mfnlp , as a compensatory mechanism ( fig 3 b ) . if however , ltrs receive difficult - to - revert nef deletions , either after an rbeiii mutation ( fig .
3d ) , then the selective pressure to maintain the rbeiii site and the mfnlp disappears .
the result of this model , would explain the convergent deletions observed in the ltr / nef ssbc ltnp study , as well as being consistent with the role for rbf-2 , rbeiii and mfnlps . whether my model about ltnp is true or not , the conserved nature of the rbeiii sites in vivo , makes it an important potential target for delivery of chimeric transcription factor repressors , which could emulate the ltnp phenotype , by blocking activation through this highly conserved site 75 .
this is illustrated in figure 4 , where a chimeric transcription factor encompassing a repressor ( such as mcef75 , 76 ) and a dna binding partner ( rbf-2 ) , might have the potential to both block tat - transactivation and interfere with the function of rbeiii .
( a ) synthesis of minus strong stop dna , begins with t - rna ( clover - leaf structure ) binding to the primer binding site ( pbs ) , followed by rna - templated polymerization of minus strong stop dna ( dashed arrow ) .
rnase h degrades the template rna after polymerization ( arrows ) ; ( b ) template switching to the other rna genome copy or to the same rna , occurs via complementary sequences in the repeat region ( r ) . during the template switch
, slippage can occur ; ( c ) minus strand extension could combine with slippage , templated and non - templated polymerization , resulting in duplication of the ure / rbeiii / partial e - box region ( single white box , in rna u3 region ) , generating a first copy of the mfnlp ( two white boxes in u3 region ) , in the minus dna .
( d ) initial synthesis of plus strand dna begins from the central polypurine tract ( cppt ) and from the 3'-polypurine tract ( 3'-ppt ) , resulting in synthesis of a plus strand mfnlp ( four white boxes ) ; ( e ) annealing of the free pbs , further extension and rearrangements , are followed by nuclear import and integration of the proviral hiv-1 sequences .
( f ) the final integrated proviral sequences contain a 5'-ltr and a 3'-ltr with u3 , r , u5 sequences . in this model , both the 5 ' and 3'-ltr would contain the mfnlp . note the nef orf in the 3'-ltr and the nef / ltr sequences in the 5'-ltr .
integrity of the nef tga stop codon ( actgctga ) is critical for rbf-2 binding to the mfnlp .
( a ) schematic representation of the hiv-1 ltr . some transcription factors landmarks ( nfat , usf-1 , rbf2 , nfkb , sp-1 ) known to interact with target sequences on the hiv-1 ltr are indicated in their relative positions .
the -121 position where the mfnlps occur is indicated , with the mfnlp above , bound by rbf2 at the mfnlp rbeiii site . the -453 and
the + 1 site indicates the start of transcription and the + 98 site , the end of tar sequences .
the dashed circles represent the approximate positions of two nucleosomes ( nuc-0 and nuc-1 ) .
a p32-labelled wt rbeiii ds oligo was used as target in emsa with nuclear extracts from hela - s cells ( ne , lanes 1 - 5 ) .
the target site that the respective competitor ds oligo sequences encompass , are designated with a + sign above each lane , and are listed on the right top of the panel .
( d ) rusf-1 specifically binds to rbeiii , but results in a higher mobility emsa than rbf2 .
a p32-labelled wt rbeiii ds oligo was used as target in emsa with nuclear extracts from hela - s cells ( ne , lanes 1 - 4 ) or with rusf-1 ( lanes 5 - 10 ) .
the target site that the respective competitor ds oligo sequences encompass , are designated with a + sign above each lane , and are listed on the right top of the panel .
( d ) rtfiii and rusf-1 bind cooperatively to mfnlp and wt rbeiii sites from 2 patients . a p32-labelled ds oligonucleotide target , encompassing the rbeiii site of either clone pmce36.1 ( lanes 1 , 3 , 5 ) or pmce9.104 ( lanes 2 , 4 , 6 ) was used as target in emsa with either rusf-1 , tfiii , or both , as indicated with a + sign above each lane , and listed at the right top of the panel .
( b to e ) the nre , rbeiii , usf , nfb sequences and emsa conditions have been previously described 55 , 64 , 65 , 67 , 69 .
mfnlps compensate for rbeiii mutations , by maintaining functional coupling , of nef and rbf-2 . in this schematic representation
four states are considered . in a & b , normal progressive disease with high viral titer
would develop . in c & d , difficult - to - revert - mutations to nef or
the rbeiii site , would result in ltnp , and deletions of both rbeiii ltr sequences and nef sequences .
this is because , in our model , nef can activate the mapk pathway , and exert its effects through rbeiii .
delivery of a repressor with the ability to interfere with tat - transactivation and bind to the rbeiii/ mfnlp is hypothesized .
the rna stem - loop structure termed tar , can form a ternary complex with hiv-1 ta and p - tefb , to hyperphosphorylate the carboxy terminal domain ( ctd ) of rna polymerase ii ( polii ) . because mcef interacts with p - tefb and represses hiv-1 tat - transactivation , if tethered to the rbeiii/ mfnlp site , mcef could interfere with the binding of rbf-2 ( 3 ) and tat transactivation ( 1 and 2 ) .
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### Summary:
: although the prototypical hiv-1 ltr sequences were determined 22 years ago from the initial isolate , elucidating which transcription factors are critical to replication in vivo , has been difficult .
one approach has been to examine hiv-1 ltrs that have gone through the gamut of in vivo mutation and selection , in search of absolutely conserved sequences . in this vein ,
rbeiii sequences are virtually 100% conserved in naturally occurring hiv-1 ltrs .
this is because when they are mutated , the mfnlp recreates an rbeiii site .
here , i enumerate some retroviral mutation mechanisms , which could generate the mfnlp .
i then review the literature corresponding to the mfnlp , highlighting the discovery in 1999 , that rbeiii and mfnlp sequences , bind usf and tfii - i cooperatively , within the context of earlier and later work that suggests a role in hiv-1 activation , through t - cell receptor engagement and the mapk cascade .
one exception to the nearly absolute conservation of rbeiii , has been a group of long term non progressors ( ltnp ) .
these patients harbor deletions to the nef gene .
however , the nef gene overlaps with the ltr , and the ltnp deletions abrogate rbeiii , in the absence of an mfnlp .
i suggest that the mfnlp retains functional coupling between the mapk - mediated effects of nef and the hiv-1 ltr , through rbeiii .
i propose that difficult - to - revert - mutations , to either nef or rbeiii , result in the convergent ltnp nef / ltr deletions recently observed .
the potential exploitation of this highly conserved protein - binding site , for chimeric transcription factor repression ( ctfr ) of hiv-1 , functionally striving to emulate the ltnp deletions , is further discussed .
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: succinylcholine , a neuromuscular blocking agent ( nmba ) with the fastest onset and recovery time , is considered the drug of choice for rapid sequence induction of anaesthesia .
traditionally , the dose of succinylcholine recommended for this purpose is 1 mg / kg . the effective dose ( ed ) 95 of succinylcholine is less than 0.30 mg / kg .
doses equivalent to twice the ed 95 are generally considered to be the appropriate dose of non - depolarizing nmbas for intubation .
recovery of spontaneous respiration following 1.0 mg/ kg succinylcholine administration may not occur rapidly enough to prevent haemoglobin desaturation in patients whose ventilation is not assisted .
based on a mathematical model of haemoglobin desaturation during apnoea , benumof et al . predicted that in the large majority of patients with 1 mg / kg succinylcholine - induced apnoea , significant to life - threatening haemoglobin desaturation will occur when
heier et al . reported that significant haemoglobin desaturation ( spo2 < 80% ) occurred in one - third of the volunteers during the period of apnoea induced by 1 mg / kg succinylcholine .
decreasing the dose of succinylcholine would allow a more rapid recovery of spontaneous ventilation , thereby proving a greater margin of safety in airway management .
the aim of this prospective , randomized , double - blind study was to compare tracheal intubating conditions and the duration of apnoea following administration of 0.4 , 0.6 and 1.0 mg / kg of succinylcholine during simulated rapid sequence induction of anaesthesia .
the study was approved by the hospital ethics committee and written informed consent was obtained from all patients .
i and ii adult patients , aged 18 - 60 years , scheduled for elective surgery requiring general anaesthesia and tracheal intubation , were included in this study .
patients with cardiac , pulmonary , neuromuscular disease , hepatic or renal impairment , and those with body mass index > 28 kg / m were excluded from the study , as were pregnant women .
the airway was clinically assessed ( mouth opening , mallampati class , thyromental distance , range of neck movement , any obvious head or neck pathology ) to exclude those in whom difficulty with intubation was anticipated .
was established [ electrocardiogram ( ecg ) , non - invasive blood pressure , pulse oximetry and capnography ) and baseline ( pre - induction ) measurements were recorded .
patients were randomly allocated to one of three groups ( 23 patients each ) according to the dose of succinylcholine to be administered intravenously ( 0.4 , 0.6 or 1.0 mg / kg ) using a computer - generated random number table ( sealed envelopes ) : group 0.4 received 0.4 mg / kg of succinylcholine ; group 0.6 received 0.6 mg / kg of succinylcholine ; and group 1.0 received 1.0 mg / kg of succinylcholine .
succinylcholine was taken in a 2 ml syringe and saline 0.9% was added to make a volume of 2 ml .
all drugs were prepared by an anaesthetist not involved with the study to keep the study investigator blinded .
pre - oxygenation using a tight - fitting mask was performed for 3 min with 100% oxygen .
anaesthesia was induced with propofol 2 mg / kg followed by application of cricoid pressure and administration of the designated dose of succinylcholine .
laryngoscopy was performed 50 s after the administration of succinylcholine ( size 3 macintosh blade ) , aiming to intubate the trachea at 60 s. cuffed tracheal tubes of 7 and 8 mm size were used in female and male patients , respectively .
tracheal intubation and grading of the intubating conditions was performed by an experienced anaesthetist unaware of the dose of succinylcholine given .
the cormack and lehane grade of laryngoscopy was recorded : grade 1=complete visualization of the vocal cords ; grade 2=visualization of the inferior portion of the glottis ; grade 3=visualization of only the epiglottis ; grade 4=non - visualized epiglottis .
intubating conditions were evaluated by a qualitative scoring system described by viby - mogensen et al . which includes ease of laryngoscopy , position and movement of vocal cords , coughing and movement of the limbs [ table 1 ] .
laryngoscopy was considered as easy ( jaw relaxed , no resistance to laryngoscope blade ) , fair ( jaw not fully relaxed , slight resistance to blade ) or difficult ( poor jaw relaxation , active resistance of the patient to laryngoscopy ) .
intubating conditions were considered as excellent ( all variables were excellent ) , good ( all variables were either excellent or good ) or poor ( the presence of a single variable listed under poor ) . excellent or good intubating conditions were regarded as clinically acceptable ; poor intubating conditions were regarded as clinically not acceptable .
duration of laryngoscopy ( time from start of laryngoscopy until tracheal intubation and removal of laryngoscope blade from the mouth ) was noted . blood pressure heart rate ( hr ) and spo2 were recorded before induction of anaesthesia ( pre - induction ) , after induction ( post - induction ) and then every 1 min after tracheal intubation for 5 min ( time 1 - 5 ) . in the event of bradycardia ( hr < 50 beats per min ) ,
atropine 0.6 mg was administered . in the event of a decrease in mean arterial pressure ( map )
adverse events such as laryngospasm , bronchospasm , masseter spasm or muscle rigidity were recorded .
assessment of intubating conditions after tracheal intubation , ventilation ( 0.6% isoflurane and 66% nitrous oxide in oxygen using carbon dioxide absorption ) was gently assisted manually to maintain an etco2 between 35 and 40 mmhg .
apnoea time was defined as the time from iv succinylcholine administration to the time to first visible diaphragmatic contraction that coincided with reservoir bag movement .
time to resumption of spontaneous breathing was taken as the time from iv succinylcholine administration to the time to regular reservoir bag movements that produced a well - formed end - tidal co2 waveform .
when the trachea could not be intubated because of inadequate relaxation , the lungs were ventilated by face mask , and rocuronium 0.6 mg / kg was administered iv and another attempt was made 1 min later .
descriptive statistics in the form of mean , standard deviation , frequency and percentages have been calculated for interval and categorical variables , respectively . to see a significant difference among the groups , one way analysis of variance ( anova ) with post - hoc bonferroni test
statistical package for social sciences ( spss ) 18.0 statistical software has been used for the analysis .
descriptive statistics in the form of mean , standard deviation , frequency and percentages have been calculated for interval and categorical variables , respectively . to see a significant difference among the groups , one way analysis of variance ( anova ) with post - hoc bonferroni test
statistical package for social sciences ( spss ) 18.0 statistical software has been used for the analysis .
sixty - nine eligible patients were enrolled in the study , 23 in each group .
two patients in group 0.4 mg / kg and three patients in group 1.0 mg / kg were excluded from the study due to protocol violation .
there were no significant differences in patient characteristics among the three groups [ table 2 ] .
the modified mallampati class of pharyngeal structures , cormack grade of laryngoscopy and the duration of laryngoscopy in the three groups was comparable .
excellent intubating conditions were obtained in 52.4% , 95.7% and 100% of the patients after 0.4 , 0.6 and 1.0 mg / kg succinylcholine , respectively ; p<0.001 . the 0.6 and 1.0 mg /
kg groups were similar with regard to the incidence of excellent intubating conditions ; p>0.05 .
overall intubating conditions were regarded as acceptable ( excellent and good grade combined ) in 66.7% , 100% and 100% of the patients after 0.4 , 0.6 and 1.0 mg / kg succinylcholine , respectively .
comparable intubating conditions were achieved after 0.6 and 1.0 mg / kg succinylcholine ; p>0.05 .
patients receiving 0.4 mg / kg succinylcholine had a frequent incidence ( 33.3% ) of poor tracheal intubating conditions .
laryngoscopy was easy in all patients except one patient in group 0.6 mg / kg succinylcholine .
. a greater number of patients ( four of 21 patients ) had sustained coughing in the group receiving 0.4 mg / kg succinylcholine compared with patients receiving 0.6 and 1.0 mg / kg succinylcholine , p=0.04 ) .
five patients in group 0.4 mg / kg succinylcholine had vigorous limb movement compared with none in the other two groups ; p<0.001 [ table 4 ] . intubating conditions and requirement for rocuronium details of intubating conditions apnoea time and the time to regular spontaneous breathing ( end tidal co2 waveform )
apnoea time and time to resumption of regular spontaneous breathing haemodynamic responses to induction and intubation are shown in tables 6 and 7 .
there was no statistically significant difference in mean hr and map between the three groups at any time point .
heart rate response to induction and intubation mean arterial pressure response to induction and intubation oxygen saturation before as well as following induction of anaesthesia and tracheal intubation ranged between 97% and 100% .
no episodes of laryngospasm , bronchospasm , masseter spasm , or generalized rigidity were observed .
this study demonstrates that satisfactory tracheal intubating conditions can be achieved 1 min after succinylcholine administration , with doses much less than the traditionally recommended dose of 1.0 mg/ kg .
the intubating conditions following 0.6 mg/ kg succinylcholine were similar to those obtained after the 1.0 mg / kg dose .
doses of 0.4 mg / kg did not often result in acceptable intubating conditions and therefore can not be recommended for rapid sequence tracheal intubation .
the effectiveness of small doses of succinylcholine in achieving satisfactory intubating conditions has been previously described.[41012 ] stewart et al . reported that 26 ( 96% ) of 27 patients receiving 1.5 mg/ kg succinylcholine and 30 ( 94% ) of 32 patients receiving 0.5 mg / kg had acceptable intubating conditions .
however , in patients with a full stomach or in those with raised intracranial pressure , excellent intubating conditions are warranted . in our study
the administration of 1.0 and 0.6 mg / kg succinylcholine was associated with 100% and 95.7% incidence of excellent tracheal intubating conditions .
in contrast , stewart et al . reported that , after induction of anaesthesia with 5 mg / kg thiopental , 23 ( 85% ) of 27 patients receiving 1.5 mg / kg succinylcholine , and 18 ( 56% ) of 32 patients receiving 0.5 mg / kg succinylcholine had excellent intubating conditions at 60 s. naguib et al . also found the incidence of excellent intubating conditions following induction with 2 g / kg fentanyl and 2 mg / kg propofol to be 0.0% , 43.3% , 60.0% , 63.3% , 80.0% and 86.7% of patients after 0.0 , 0.3 , 0.5 , 1.0 , 1.5 , and 2.0 mg / kg succinylcholine , respectively .
our results indicate that apnoea time and time to resumption of regular spontaneous breathing ( end tidal co2 waveform ) were dose - dependent . in all the three groups ,
start of regular spontaneous breathing occurred approximately 1 min after the detection of the first diaphragmatic movement .
there was a statistically and clinically significant difference in apnoea time between 0.6 mg / kg and 1 mg / kg dose of succinylcholine ( 4.30.9 min versus 8.23.4 min , respectively ) .
return of regular spontaneous breathing occurred significantly earlier when the 0.6 mg / kg dose of succinylcholine was used compared with the 1 mg/ kg ( 5.51.1 min and 8.93.5 min , respectively ) dose .
although initial spontaneous and regular breathing may not reflect complete functional recovery , this may still prevent haemoglobin desaturation that would ensue if the patient remained apneic .
thus , critical haemoglobin desaturation may not occur with the use of the 0.6 mg / kg dose , especially in healthy adults . because we did not allow our patients to desaturate
, we can not comment on the effect of these doses in a can not ventilate , can not intubate situation .
however , naguib et al . found that , compared with the traditional intubating dose of 1.0 mg / kg succinylcholine , a reduction in succinylcholine dose to 0.56 mg / kg was associated with a 20% absolute decrease and a 50% relative decrease in the incidence of haemoglobin desaturation ( spo2 < 90% ) in asa physical status i patients anaesthetized with 2 g / kg fentanyl and 2 mg / kg succinylcholine .
our results with regard to duration of apnoea are in contradistinction with that reported by others .
el - orbamy et al . noted that the mean time ( sd ) to spontaneous diaphragmatic movements after 0.6 mg/ kg and 1.0 mg / kg succinylcholine were 3.410.6 and 5.38.0 min , respectively ( p<0.05 ) .
reported that the recovery of diaphragmatic movements occurred 4.7 ( sd1.5 - 2.0 ) min after the administration of 1 mg / kg succinylcholine in patients with 1 g / kg fentanyl and 3 - 7 mg / kg thiopentone . similarly in volunteers who received 5 mg / kg thiopentone and 1 mg / kg succinylcholine , heier et al .
noted diaphragmatic activity 5.2 ( 1.5 - 2.0 ) min after succinylcholine and stewart et al . reported a 3.8 min apnoea time after 0.5 mg / kg .
an interesting finding of our study is the striking dissimilarity in the intubating conditions ( better ) obtained and the duration of apnoea ( longer ) observed following administration of three doses of succinylcholine compared with that reported previously in the literature .
reported that the neuromuscular blocking effect of succinylcholine ( 1 mg / kg ) in adults was shorter in london ( 9.12.9 min ) than in new york ( 14.63.6 min ) .
reported that the recovery of spontaneous ventilation after succinylcholine was 35% slower in asian patients than in european patients .
. showed that irish subjects ( 7.820.14 u / ml ) had more serum cholinesterase activity than iranian subjects ( 4.220.90 u / ml )
. these inter - racial differences in drug response could be due to differences in drug kinetics or sensitivity .
thus , ethnic and geographic differences could possibly explain the differences found in our results in the indian population .
the quality of intubating conditions without nmbas is less predictable and the incidence of failed tracheal intubations is higher .
we felt that intubation without nmba is unlikely to be used in a rapid sequence induction scenario .
second , we did not study the onset and recovery times of different doses of succinylcholine by neuromuscular monitoring . in studies assessing the neuromuscular effects ,
succinylcholine is administered after calibration of the response to stimuli after induction of anaesthesia , and the results of intubating conditions so obtained can not be applied to the clinical situation of rapid sequence induction .
moreover , monitoring the adductor pollicis is not a useful measure for evaluating the neuromuscular block at the laryngeal , diaphragm , and masseter muscles as the diaphragm recovers faster than hand muscles after succinylcholine .
demonstrated that diaphragm recovery occurs 2 min earlier than adductor pollicis recovery at all levels of twitch height recovery .
increasing age , obesity and pre - existing lung disease would make patients more vulnerable to desaturation .
there are clinical situations in which acceptable conditions for tracheal intubation may not be ideal , e.g. a patient with increased intracranial pressure and a full stomach . in such patients , decreasing the dose of succinylcholine to less than 1 mg / kg might increase morbidity .
the faster return to spontaneous ventilation with the 0.6 mg / kg dose increases the margin of safety in the event of a can not intubate , can not ventilate situation compared with the 1.0 mg / kg dose .
to conclude , a reduction in succinylcholine dose to 0.6 mg / kg provided equally acceptable intubating conditions at 60 s compared with the use of 1 mg / kg succinylcholine during rapid sequence induction of anaesthesia , with the advantage of a shorter apnoea time and time to regular spontaneous breathing .
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### Summary:
: background : the aim of this prospective , randomized , double - blind study was to compare tracheal intubating conditions and the duration of apnoea following administration of 0.4 , 0.6 and 1.0 mg / kg of succinylcholine during simulated rapid sequence induction of anaesthesia.methods:anaesthesia was induced with fentanyl 2 g / kg and propofol 2 mg / kg followed by application of cricoid pressure .
patients were randomly allocated to three groups according to the dose of succinylcholine administered ( 0.4 , 0.6 or 1.0 mg / kg ) .
intubating conditions were assessed at 60 s after succinylcholine administration .
time to first diaphragmatic contraction ( apnoea time ) and time to resumption of regular spontaneous breathing were noted.results:excellent intubating conditions were obtained in 52.4% , 95.7% and 100% of the patients after 0.4 , 0.6 and 1.0 mg / kg succinylcholine , respectively ; p<0.001 .
acceptable intubating conditions ( excellent and good grade combined ) were obtained in 66.7% , 100% and 100% of the patients after 0.4 , 0.6 and 1.0 mg/ kg succinylcholine , respectively ; p<0.001 .
apnoea time and resumption of regular spontaneous breathing were dose - dependent .
apnoea time was 3.81.1 min , 4.30.9 min and 8.23.4 min in groups 0.4 , 0.6 and 1.0 mg / kg , respectively ; p<0.001 .
time to regular spontaneous breathing was 5.31.2 min , 5.51.1 min and 8.93.5 min in groups 0.4 , 0.6 and 1.0 mg / kg , respectively ; p<0.001.conclusion : a dose of 0.6 mg / kg succinylcholine can be used for rapid sequence induction of anaesthesia as it provides acceptable intubating conditions with a shorter apnoea time compared with a dose of 1 mg / kg .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
:
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### Summary:
: we
present a novel approach to reversibly control the assembly of liposomes
through an anchored multistimuli responsive dna oligonucleotide decorated
with an azobenzene moiety ( azo - on1 ) .
we show that liposomes
assembly can be simultaneously controlled by three external stimuli :
light , mg2 + , and temperature .
( i ) light alters the interaction
of azo - on1 with liposomes , which influences dna coating
and consequently liposomes assembly .
( ii ) mg2 + induces
the assembly , hence variation in its concentration enables for reversibility .
( iii ) double - stranded azo - on1 is more efficient than single - stranded
azo - on1 in triggering the assembly of liposomes and temperature
has been used for controllable assembly through dna thermal denaturation .
our multiresponsive azo - on1 represents a unique example
in which multiple stimuli can be simultaneously applied to regulate
the reversible assembly of liposomes .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: bisphosphonates are a group of drugs widely recommended and used for the treatment of moderate and severe hypercalcemia associated with cancer , for osteolytic lesions associated with metastases of breast cancer , prostate cancer , or multiple myeloma in combination with other chemotherapeutic agents .
they are also used in the prevention and therapy of osteoporosis in postmenopausal women and also in the treatment of paget 's disease [ 1 , 2 ] .
these drugs are completely resistant to the hydrolytic cleavage , whereby this is the reason why they accumulate in the bone tissue and have a long half - life .
their rapid uptake in bone matrix allows an accumulation that goes from 30 to 70% of the administered intravenous dose or that absorbed after oral intake , while the remaining fraction is excreted unchanged into urine .
the accumulation of bisphosphonates in the bone , in particular in maxillary bones , is not reversible .
their toxic effect on osteoclasts depends on both the dose administered and the duration of therapy .
the intravenous administration of high doses of aminobisphosphonates ( n - bf ) , that is , the bisphosphonates of last generation containing nitrogen in the side chains , can cause the onset of necrosis of the jaw bone and/or of the mandibular bone [ 3 , 4 ] .
this pathology was identified with the acronym bronj ( bisphosphonates related osteonecrosis of the jaw ) .
it is a pathological condition described for the first time in 2007 and in 2009 the aaoms underlined that the presence of bronj is also discernible in the absence of bone exposure clinically detectable , by introducing a new stage of the disease : stage 0 . in 2012
bedogni et al . defined the bronj as an adverse reaction that is drug related , characterized by the destruction and necrosis of the jaw / maxillary bone in subjects treated with aminobisphosphonates , with no previous radiation treatment .
on the basis of the recommendations published by the sicmf - sipmo 2013 stage 0 was deleted by replacing in the other stages all cases without bone exposure .
unequivocally effective treatments have not been reported , and discontinuation of therapy with n - bf does not involve the healing of necrotic disease .
the surgical approach , when indicated , is very aggressive and sometimes can cause a widening of the areas of bone exposure and amplify the symptoms .
the preventive approach is certainly the best way to avoid the onset of the disease . particularly important in the prevention of bronj
is the cofactors evaluation , that in the absence of bisphosphonates do not lead to the onset of the disease .
the knowing of bronj risk factors can be very helpful in planning a protocol . as suggested in the recommendations of the sicmf - sipmo ( italian society of maxillofacial surgery and italian society of pathology and oral medicine ) , we do not yet have definitive data ; certainly , taking the molecule n - bf is an high risk factor as well as the way of the administration : indeed , the risk increases in proportion to the dose administered intravenously . besides the cancer disease ,
which requires the recruitment of the molecule , seems to have a correlation with the increase of the risk .
( even if steroids are not able to produce osteonecrosis , they are undoubtedly cytotoxic and have an effect on the synthesis of collagen and then consequently wound healing .
the local risk factors have also a relevant role ; it is just in their knowledge that many of prevention strategies is based . on the basis of the data reported in the literature
the dentoalveolar surgery is the most important risk factor followed by the osteointegrated implants ; the dentoperiodontal or peri - implant pathology is only the third one . among the local risk factors ,
it is an inflammatory process induced by bacteria , causing an alveolar bone remodeling ; it strikes the adult population with a frequency of 90% . in the case of recruitment of n - bf
a recent study with rats showed that , after administration of a dose of zoledronic acid , corresponding to the one accumulated in humans oncology therapies , and after inducing experimentally periodontal disease with sterile ligatures , the periodontal diseases , associated with the recruitment of zoledronic acid , are a necessary and sufficient condition to develop bronj .
the aim of this work has been to reduce the level of risk of bronj in patients with cancer and in therapy with aminobisphosphonates , before the recruitment , through a protocol targeted in a particular way at control of periodontal disease and the maintenance of oral health .
the recruitment of patients occurred at the surgical and special stomatology of the neurological medical sciences department , in the ospedali riuniti hospital of ancona , in the period from january 2012 to october 2012 . since 2001
the structure adopts a protocol for the prevention of osteonecrosis ( table 1 ) [ 12 , 13 ] in cooperation with the oncology , surgery , clinical medicine , and endocrinology division as synthetically reported : dental treatment before the therapy ( phase i),dental treatment during the therapy , without bone disease ( stage ii ) , with bone necrosis ( phase iii),follow - up to 1 month6 months.this protocol is similar to the one proposed in the sicmf - sipmo recommendations updated to 2013 on the basis of the latest scientific evidences , in which it is possible to identify different paths depending on the type of patient and on the time in which it was intercepted . in the case of patients that have yet to start the recruitment of the drug it provides a path comparable to that described in table 1 for phase i. dental treatment before the therapy
( phase i ) , dental treatment during the therapy , without bone disease ( stage ii ) , with bone necrosis ( phase iii ) , follow - up to 1 month6 months .
one of the main aspects , that comes out in all stages of this path , is the professional and the home oral hygiene care aimed at achieving and maintaining a state of health .
the protocol has received the approval of the marche region ethics committee and is carried out in accordance with the ethical standards approved by the declaration of helsinki in 1964 . in 2012
43 oncology patients taken in care presented the following : 14% with lung cancer , 42% breast cancer , 23% multiple myeloma , 7% prostate cancer , and 7% bone metastases .
the remaining 7% included oral carcinoma and cancer of the bladder , kidney , and colon .
all patients read and signed , after careful and detailed verbal explanation , an informed consent included in the protocol of the department . in this standard format
is also specified a consent to any use of the clinical data collected for scientific purposes .
all the patients were subjected to a dental visit ( anamnesis ; objective examination of intraoral and extraoral environment ; assessment of removable prostheses ; radiographic examinations ) and were informed on the issues relating to the risk of the occurrence of bronj in relation to the level of oral health . carrying out
for this purpose , each patient was subjected to questions about the diagnosis of cancer , the type and dosage of the drug administered , duration of therapy , and the presence of other drugs associated with the dental history and the oral habits ( table 2 ) . for the present prospective study ,
patients were selected within 43 oncology patients , taken in care in 2012 , and by considering the following inclusion criteria : people of both sexes , patients who must begin therapy with n - bf due to cancer or metastases , adults above the age of 30 years , complete or partial teeth , no manifestation of osteonecrosis , no radiotherapy of cervicofacial district.patients with the following were excluded : total edentulous , precarious conditions of general health ( elderly patients very debilitated , patients undergoing recent surgical therapies , patients with nutritional deficiencies , patients with immune deficiency , and people who have cardiac and/or respiratory serious compromises),lack of collaboration , bisphosphonates therapy in act ( phase ii),clinical manifestation of bronj , no oncological diseases.a decisive inclusion criterion of the study was the possibility to follow the patient throughout the period of observation at the hospital .
in fact in most cases , once the phase i , the patient is entrusted to the territory for monitoring and maintenance .
people of both sexes , patients who must begin therapy with n - bf due to cancer or metastases , adults above the age of 30 years , complete or partial teeth , no manifestation of osteonecrosis , no radiotherapy of cervicofacial district .
precarious conditions of general health ( elderly patients very debilitated , patients undergoing recent surgical therapies , patients with nutritional deficiencies , patients with immune deficiency , and people who have cardiac and/or respiratory serious compromises ) , lack of collaboration , bisphosphonates therapy in act ( phase ii ) , clinical manifestation of bronj , no oncological diseases . after the visit ( t0 ) , all the patients were subjected to the following.assessment of the visible plaque index ( in this text abbreviated with the acronym fmps , i.e. , full mouth plaque score , so called by tonetti and his collaborators in 2002 ) and of the dichotomous bleeding index ( abbreviated form now on as fmbs ) , both drafted , as suggested by the international scientific literature , noting the positive sites and putting them in relationship with all of the sites examined.assessment of the clinical attachment level ( cal ) ; involvement of furcations ; degree of dental mobility.professional oral hygiene care.all patients were instructed to perform correctly the oral hygiene at home , with particular attention to use nontraumatic tools and their association with mouthwashes that are alcohol - free .
assessment of the visible plaque index ( in this text abbreviated with the acronym fmps , i.e. , full mouth plaque score , so called by tonetti and his collaborators in 2002 ) and of the dichotomous bleeding index ( abbreviated form now on as fmbs ) , both drafted , as suggested by the international scientific literature , noting the positive sites and putting them in relationship with all of the sites examined .
assessment of the clinical attachment level ( cal ) ; involvement of furcations ; degree of dental mobility .
there were also addressed the issues related to hyposialism caused by the imminent pharmacology therapy : salivary substitutes , feeding and risk of caries , on the basis of a clinical protocol already existing .
three sessions of maintenance and monitoring of oral health were made : ( a ) during therapy ( t1 ) ; ( b ) at the end of the treatment with n - bf ( t2 ) in which patients were subjected again to a session of professional oral hygiene care and to a reinforcement of education on oral hygiene care at home ; ( c ) after 18 months from the start of therapy with n - bf ( t3 ) .
the last phase included new probing and cal , fmps and fmbs reevaluation , and tissues and clinical signs control to exclude the occurrence of bronj .
for ethical reasons it was not possible to form a group of patients for the control .
out of 43 patients , 15 patients , belonging to phase 1 in 2012 and satisfying the criteria described above , were included . due to a subsequent aggravation of the general state of health
the 10 remaining patients , 7 females and 3 males , were aged between 38 and 78 years ( 50% over 70 years , one person less than 40 years , and 40% between 38 and 70 years ) and all were to start therapy with n - bf for metastasis .
the primary systemic pathology was breast cancer in 70% of the cases ( 7 women ) ; two persons showed metastasis on colon and bladder .
eight patients had to begin the periodic administration of intravenous zoledronic acid ( zometa ) , from 3 to 5 cycles every 28 days ; 2 patients had to begin the ibandronic acid ( bondronat ) by oral administration .
six people have received the dose of 4 mg of zoledronate , pharmaceutically acceptable as a reconstituted and further diluted infusion ( diluted with 100 ml of saline 0.9% w / v solution or glucose ( 5% w / v ) ) , in at least 15 minutes for 3 administrations every 28 days ; 1 patient received 4 doses every 28 days , and 1 person received 5 administrations of the drug with the same dosage and frequency .
two patients have received an ibandronate daily dose of 2.5 mg per oral administration throughout the observation period ( table 3 ) .
in addition 2 patients were also subjected to chemotherapy , 2 patients were subjected to administration of corticosteroids , and 4 patients have carried out radiotherapy , at the end .
the main preexisting dental pathology proved to be the generalized chronic periodontitis and , in fact , it is present in 70% of patients . in one case
the initial level of risk of the subjects is described in table 4 : all patients were considered at high risk of developing bronj .
this evaluation was carried out on the basis of the high dosage of drug taken during the period of observation and on the conditions of oral health detected during the first visit . in the first visit ( t0 ) ,
4 patients out of 10 had a level of oral hygiene , expressed with the index fmps , higher than 90% , 5 showed percentages ranging between 40 and 70% , and only one patient had a visible plaque index of 24% ; the average index was 73% .
nine people needed a tooth extraction and all were subjected to one or more sessions of professional oral hygiene before starting therapy with n - bf . in the second control ( t1 ) , 3 months after the start of therapy , the average of the fmps has suffered a considerable reduction coming to 50% .
only two patients have participated after 6 months in a further follow - up ( t2 ) expressing an average percentage of 36% fmps .
the last control , performed 18 months from the beginning of therapy ( t3 ) , has been detected on 9 patients because of a supervening death .
the gingival inflammation , expressed through the fmbs , shows a sample less homogeneous with respect to the oral hygiene level . at the first visit two patients had a fmbs greater than 50% ; six out of 10 people had a percentage lower than 20% and the average is 24% .
subsequent checks showed , at 3 months , a fmbs average of 16% ; at 6 months the two patients monitored had an average less than 5% and at 18 months the average of 9 people was 15% .
the periodontal exam has highlighted the presence of a periodontal impairment with loss of clinical attachment ( cal ) in all the patients : range of 2 to 4.5 mm , average of 3.15 mm . at
the first follow - up the cal average dropped to 2.9 mm and in the last control ( 18 months ) it was 2.8 mm ( figure 3 ) .
the last visit ( follow - up at 18 months ) was made through clinical examination and radiographic examination and revealed the total absence of signs of osteonecrosis in all patients .
the bronj are complications that affect 2.8% of patients who receive n - bf for bone metastases of breast cancer .
the sample selected for this study , although small , is therefore representative of the most risk of osteonecrosis . on the basis of the first reports , the literature identified bronj only in relation to oral surgical access to the maxillary bones ( extractions ) [ 16 , 17 ] .
today it tends to emphasize the importance of the presence of periodontal disease , latent or not fully treated , such as infection triggers of bronj [ 1820 ] . in all cases of bronj treated by marx et al . , the 25% of the lesions were found to be arising spontaneously , while 75% were engendered by some type of dental invasive procedure .
more precisely , marx indicates that , in 152 patients with bronj , more than a third , a trigging factor was due to tooth extractions . of these ,
about half , was caused by periodontal disease , of which 26% was represented by untreated parodontitis , and in 25% of the cases , it seemed to be a manifestation of the osteonecrosis which the author calls spontaneous .
the latter confirmed the hypothesis that there is no doubt that the subclinical osteonecrosis also exists even if there is no bone exposure .
this justifies the assertion of many authors that the prevalence of bronj has not yet been established and its pathogenesis is not entirely clarified . in the present study , the first visit revealed in all the patients the presence of oral preexisting diseases and the most popular is periodontitis .
the presence of this disease , manifest or latent , associated with bacterial plaque and calculus and inadequate oral hygiene ; it can certainly be regarded as a serious risk factor for the onset of bronj .
the risk of developing bronj for these patients , in phase 1 of the protocol ( t0 ) , was judged to be very high especially in relation to the high dose of the drug taken during the period of observation and to the conditions of oral health detected during the first visit .
optimizing oral health should therefore be the primary objective ; teeth that are not treated or teeth with a poor prognosis must be extracted by delaying the start of therapy with n - bf at least 46 weeks to ensure complete healing of the tissues .
patients should be instructed on the importance of good hygiene at home and motivated to undergo regular checks of monitoring and maintenance .
after the first preventive intervention ( t0 ) figure 1 shows a general progressive reduction of the plaque index .
it was possible to confirm a general improvement in the level of oral hygiene even if the educational intervention in these patients is very difficult , not only because of the age but also because often their interest is focused on pain , on the therapies that must be undergone , on emotional factor that comprises the concern for the sick , and on the outcome of care .
most patients , during the administration of the drug , have suffered from fever , severe joint pain , general malaise , and gastrointestinal problems with consequent general debilitation .
such symptoms are immediately manifested after administration and are attenuated during the following days . in this context to speak about toothbrush and proxabrush may seem irrelevant . a correct psychological approach and respect of each patient 's limits
the fmps and fmbs percentages decreased , except some exceptions . in two cases the bleeding index , in the second control , resulted higher than those on the first check ; it is not to exclude an effect of the drug on gingival tissue . as regards the cal , in the subsequent controls , differences are not significant ( figure 3 ) but they show the slight packaging of tissues following the periodontal therapy .
it could indicate a constant maintenance of the level of periodontal health and the absence of periodontal pockets or latent osteonecrosis . in three patients showing a greater reduction of cal from t0 to t1
, it is reasonable to assume a reduction in the depth following the professional oral hygiene .
there seems to be no difference between patients who were taking n - bf intravenous and by oral administration .
the data collected show that patients observed in t0 showed a high level of risk disease ; this risk was significantly reduced once included in the protocol of prevention of bronj .
these considerations justify the result reached after 18 months , when the follow - up evaluation shows patients with good oral health and total absence of bronj .
bronj represents an unwanted complication of n - bf and its prevention begins with the close cooperation of the following specialists : oncologist , rheumatologist , maxillofacial surgeon , dentist , and dental hygienist . in the light of the data and clinical observations reported in the present study ,
it is conceivable that the protocol applied and described above has been important in cancelling the incidence of the disease in the group of patients examined , that is , group considered at high risk of bronj .
today the occurrence of bronj is calculated on the bases of retrospective studies putting it in a range from 8% to 11% , but these percentages are increasing .
the low number of observed patients and the lack of a group of control ( excluded from protocol for ethical reasons ) call for further depths even if this work suggests the big importance of the preventive approach .
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### Summary:
: objectives . bisphosphonates related
osteonecrosis of the jaw ( bronj ) is a pathological condition characterized by bone exposure or latent infection in patients treated with the drug .
the aim of the study is to monitor the bronj level of risk health in patients with cancer , according to a preventive clinical protocol , which is firstly aimed at reducing risk factors such as the periodontal infections .
materials and methods .
10 patients participated in the protocol and were evaluated at baseline and after 3 and 18 months of treatment with bisphosphonates , through full mouth plaque and bleeding scores ( fmps and fmbs ) , clinical attachment level ( cal ) measurement , and the occurrence of osteonecrosis .
results .
the mean plaque and bleeding were reduced and the cal has not shown significant changes and in no cases was there manifestation of bronj .
conclusion .
the protocol proved crucial for the maintenance of good oral health conditions by eliminating the risk of bronj during the observation period .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: in their most recent work , giamarellos - bourboulis and colleagues examine sepsis - related immune dysfunction by assessing the proinflammatory cytokine production capacity in septic patients and healthy volunteers following ex vivo stimulation .
a critical examination of their article not only reveals new insights into the possible mechanisms underlying sepsis - related immune system dysfunction , but also highlights the difficulties inherent in this line of inquiry .
the authors report that septic patients ' isolated leukocytes produced lower amounts of the proinflammatory cytokines il-1 , il-6 and , to a lesser extent , tnf when stimulated within 24 hours of gram - negative sepsis onset .
results were similar in healthy volunteers challenged with lipopolysaccharide , indicating an early shift to an immune - suppressed phenotype following systemic inflammation . in our current understanding ,
an early anti - inflammatory phenotype following close on the heels of a severe inflammatory challenge ( sepsis , trauma , burn injury , and so forth ) acts as a counterweight to the systemic inflammatory response syndrome and restores immune homeostasis . when short - lived and self - limited , this anti - inflammatory surge is best termed the compensatory anti - inflammatory response syndrome ( cars ) . in some patients , however , cars is pathologically exaggerated and prolonged ( beyond 48 hours ) .
this persistent state - known as immunoparalysis - is associated with increased rates of nosocomial sepsis , multiorgan dysfunction syndrome and death in critically ill patients [ 3 - 5 ] .
although the features are similar , the distinction between cars and immunoparalysis is important when contemplating therapy .
the issue is one of timing : to reverse physiologic cars too early in the course of illness risks further inflammatory insult .
the literature , however , suggests that pathologic immunoparalysis is reversible , to the benefit of the patient , without iatrogenic hyperinflammation [ 6 - 8 ] . although the identification of immunoparalysis , its impact on outcome , and its potential reversibility are becoming increasingly understood , relatively little is known about the cellular and molecular mechanisms driving the process .
previous authors have investigated the mechanisms of immunoparalysis at the transcriptome level [ 9 - 11 ] .
in addition to ex vivo cytokine production , giamarellos - bourboulis and colleagues examined levels of caspase-1 , a protease that cleaves pro - il-1 into its active form . in their septic patients ,
reduced il-1 production capacity was associated with markedly diminished caspase-1 levels . in their healthy volunteers ,
injection with lipopolysaccharide resulted in lower levels of caspase-1 , as well as impaired production of il-1. heretofore , immunoparalysis research has largely centered on the canonical proinflammatory cytokine tnf. ( indeed , assessing ex vivo lipopolysaccharide - induced tnf production capacity by isolated monocytes is a commonly used method for diagnosing immunoparalysis . ) by introducing altered il-1 biology as a contributory process in the development of cars / immunoparalysis , the authors lay the ground work for additional avenues of research into the cause(s ) or diagnosis of these conditions .
further research is necessary to confirm a causal link between impaired caspase-1 function and il-1 production .
it is also important to note that , like tnf , il-1 is one of myriad proinflammatory cytokines whose production and regulation may be altered in immunoparalysis . despite these new and interesting contributions , placing the current work by giamarellos - bourboulis and colleagues into proper context
the early ( within 24 hours ) sampling window , without additional longitudinal samples , makes it impossible to distinguish patients with physiologic cars from those with pathologic immunoparalysis .
previous evidence has shown that only patients unable to reverse an early anti - inflammatory phenotype by 3 to 4 days are at increased risk of poor outcome .
an early anti - inflammatory phenotype ( possible cars ) is thus apparently not as concerning as prolonged immune suppression ( probably immunoparalysis ) .
unfortunately , the mechanisms or predispositions mediating the transition from cars to immunoparalysis are currently unknown .
the work by giamarellos - bourboulis and colleagues , and that of many other researchers , seeks to uncover the events surrounding sepsis - related immune system dysfunction . by understanding the mechanisms involved , such as altered caspase-1 action
, novel therapeutic targets may be identified . through careful targeting of therapy to those patients with true immunoparalysis , the best chance for improved outcomes will be realized .
cars : compensatory anti - inflammatory response syndrome ; il : interleukin ; tnf : tumor necrosis factor .
wjf receives funding from the child health research career development award k12 hd043372 - 09 , and from the research institute at nationwide children 's hospital .
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### Summary:
: sepsis is now understood to affect a variety of changes in the host , chief among them being alterations in immune system function .
proper immune function involves a competent proinflammatory response to stimuli as well as a regulated counteracting force to restore homeostasis and prevent systemic inflammation and organ dysfunction .
broad - spectrum suppression of the inflammatory response has not been shown to be beneficial for patients suffering from septic disease .
in fact , sepsis - related immune suppression has become increasingly recognized as an important contributor to late morbidity and mortality in the critically ill .
giamarellos - bourboulis and colleagues detail the impaired ability of septic patients to produce proinflammatory cytokines upon ex vivo stimulation , and introduce altered caspase-1 activity as potentially contributory to this process .
proper understanding of the cellular and molecular events resulting in immune suppression following sepsis is important in the identification of new strategies for treatment and the ideal timing of therapy .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: the simplest interacting quantum dot ( qd ) system is a qd molecule ( qdm ) composed of two closely spaced qd [ 1 - 3 ] ] .
qdm are receiving much attention both as playground for studying coupling and energy transfer processes between
artificial atoms and as new systems , which substantially extend the range of possible applications of qd . in such systems ,
the coupling involves tunneling of electrons and holes between two adjacent dots separated by a thin intermediate barrier layer .
in particular , it has been observed that for a 4-nm - thick intermediate tunneling layer , the coupling strength is optimized for the spectroscopic observation of large electron anti - crossing energy splitting .
it is , however , technologically challenging to obtain resonant quantum mechanical coupling due to size , composition , and strain inhomogeneities inherent in self - assembled qd . in this sense , great efforts have been dedicated to the formation of vertical coupled qd by self - assembling processes .
the most used fabrication process leading to vertically aligned qd is based on the formation of one layer of self - assembled qd , followed by a thin spacer layer . then , upon further inas deposition , a second layer of qd is formed , on top of the buried qd , by strain - driven processes . by this growth procedure ,
tuning of the emission energy of one of the qd at the same time that is maintained unaltered the emission properties of the other , although not impossible , is difficult to achieve due to the lack of control in size for self - assembled qd .
the tuning effect of the emission energy of one of the qd forming the molecule respect to the other would permit to build symmetric vertical qdm , in which the energy emission of both qd forming the molecule matches , and asymmetric vertical qdm , in which the energy emission of one qd is blue - shifted or red - shifted respect to the other [ 5 - 7 ] . apart from this lack of control in size of the qd forming the qdm by a self - assembling process , there is an intrinsic inter - dependent nature between the qd size and density that makes difficult the design of qd of a desired size and with a low enough density for its spectroscopic study at the single nanostructure level [ 8 - 10 ] . in this sense , here , we present a process that optimizes the growth of coupled structures in vertical arrangement by the use of a low - density nanoholes template fabricated by droplet epitaxy .
in particular , after the deposition of the designed amount of inas on the template that permits the formation of a first layer of size controlled nanostructures , a thin gaas layer is grown to create a carrier tunneling barrier that separates a second layer of inas nanostructures formed , via a strain - driven process , on top of the first ones .
thus , the growth procedure starts with the filling of previously formed nanoholes with inas material .
as detailed in , the nanoholes appear with a density of 2 10 cm , and they are 4.4 0.8 nm in depth with a mean diameter of 43 3 nm at their half maximum . on this pattern , three samples with different amount of inas , 1.2 , 1.4 and 1.5 monolayers ( ml ) , have been grown at a substrate temperature of 500c , growth rate rg ( inas ) = 0.01 ml / s , and as4 beam equivalent pressure bepas4 = 510 torr . once formed the first layer of qd ( qd1 hereinafter ) , a gaas intermediate barrier with a thickness of 4 nm
the growth of this layer is performed by atomic layer molecular beam epitaxy ( almbe ) growth technique at a substrate temperature ts = 450c , growth rate rggaas = 0.5 ml / s , and beam equivalent pressure bepas4 = 2 10 torr . at this moment , for completing the molecule structure , a second layer of qd ( qd2 hereinafter ) , located above the qd1 , is grown by the deposition of 0.9 ml of inas at the same conditions used for the underlying nanostructures . with the aim of studying the optical emission of the resulting qd molecule structures , a 155-nm - thick gaas layer is finally grown . a schematic diagram of these samples is shown in fig .
the first layer of nanostructures ( qd1 ) , is formed after depositing 1.2 , 1.4 , and 1.5 ml of inas into gaas nanoholes previously formed by droplet epitaxy . after the growth of 4-nm - thick gaas barrier layer , a second layer of nanostructures ( qd2 ) is formed on top of qd1 by a stress induced growth process when 0.9 ml of inas is deposited transmission electron microscopy images have been obtained to know the structural configuration of the resulting molecules .
figure 2a and 2b shows 002 dark field ( cross - sections ) transmission electron microscopy ( tem ) images for the samples with 1.4 and 1.2 ml of inas deposited into the nanoholes forming the qd1 layer .
contrast in this image is due to changes in composition , dark areas indicating the presence of in in in(ga)as layers .
it can be observed the formation of the double structure that consists of two inas qd separated by the 4-nm - thick gaas barrier layer . their respective wetting layers ( wls ) are also clearly observed .
it is noticeable that as a difference to the strain - driven formation of qd2 , or in general , in a self - assembling process , qd1 forms at a lower level than the wl , clearly indicating a formation mechanism that involves preferential nucleation of inas material into previously fabricated gaas nanoholes by droplet epitaxy .
a 002 dark field ( cross - section ) transmission electron microscopy ( tem ) images of two vertically aligned qd formed after depositing1.4 ml of inas into a gaas nanohole ( qd1 ) , the growth of 4-nm - thick gaas barrier acting as tunneling layer and a final deposition of 0.9 ml of inas ( qd2 ) .
b tem image of the formed structure when 1.2 ml of inas is deposited to form the qd1 nanostructures . in this case
, it can be observed that qd1 is now quite similar in size to qd2 in the case of depositing 1.4 ml of inas ( fig .
2a ) , qd1 clearly presents a larger size than qd2 nucleated on top of it . on the other hand , when 1.2 ml of inas are deposited ( fig .
2b ) , qd1 is more similar in size to qd2 . according to these results , the aimed tuning effect on the size of qd1 by the use of nanoholes has been successfully obtained . on the other hand , despite this engineered change in size of qd1 , the nucleation of qd2 takes place on top of the buried qd1 with same density of qd1 layer and similar dimensions in all cases .
these results show that the size of the strain driven formed qd2 is controlled by the amount of inas deposited .
moreover , the strain gradient at the surface is large enough to promote preferential nucleation of inas just on top of the buried qd1 , even for inas deposited thickness of 0.9 ml , which is much lower than the critical thickness for qd formation in absence of local strain fields . concerning the optical properties of these paired nanostructures , fig .
3 shows the pl signal for the three different cases . in particular , the black , red , and blue lines in the figure correspond to qd pair structures obtained by depositing 1.2 , 1.4 , and 1.5 ml of inas for the formation of qd1 layer , respectively .
the evolution of these two pl peaks with increasing excitation power ( not shown ) reveals that they correspond to two different qd families , as they do not show any relative saturation effects corresponding to one qd family with ground and excited states . combining these optical emission results with those obtained by tem structural analysis
, it can be established a direct correspondence of the different emission energies to the recombination of carriers at the upper and lower qd layers of the molecule structure .
figure 4a shows as filled areas the three different pl emissions that would correspond to the qd1 family for the three different qd paired structures .
as expected from previous results , the effect of the different amount of inas deposited in the first layer of nanostructures permits the emission energy of the qd1 to be tuned in a wide range .
black , red , and blue lines correspond to samples in which the qd1 nanostructure layer is formed by depositing 1.2 , 1.4 , and 1.5 mlof inas , respectively .
the arrows in the figure point out the two families of qd formed in each sample photoluminescence emission ( filled areas ) ascribed to qd1(a ) and qd2(b ) nanostructures for 1.2 , 1.4 , and 1.5 ml of inas deposited to form the qd1 layer .
it can be clearly shown in ( a ) a tuning effect on the emission energy of qd1 .
on the other hand , ( b ) shows that the emission corresponding to qd2 layer remains almost unaltered despite the difference in size in qd1 table 1 lists the different values of the emission energies ascribed to qd1 and qd2 in the molecule structures .
the arrow highlights the increase in pl emission peak energy of qd1 with inas deposited .
pl emission peak energies ascribed to qd1 and qd2 layers the qd1 layer emission energy decreases as a function of the amount of inas deposited .
an arrow in the table accounts for this tuning effect . on the other hand ,
the emission energy of the second layer of nanostructures ( qd2 ) remains almost constant for the three different structures in a similar way , fig .
4b shows the emission of the remaining three pl peaks that would correspond to the qd2 family in the qd paired structures .
it can be observed that the emission energies are similar in all the cases showing that the size of the nanostructures formed in the qd2 layer remains almost invariable despite the different amount of inas deposited underneath ( see pl energy values in table 1 ) .
more experimental work is needed to demonstrate if these paired qd structures show electronic coupling as corresponding to qd molecules .
altogether , these results show that by varying the amount of inas material deposited at a nanoholes template formed by droplet epitaxy , the emission of a first layer of qd can be tuned to obtain either a symmetric or an asymmetric vertically coupled qdm . as a characteristic when using a droplet epitaxy patterning process
, the density of qd molecules obtained is low enough ( 2 10 cm ) to permit their integration as active elements in advanced photonic devices where spectroscopic studies at the single nanostructure level are mandatory .
the authors wish to acknowledge to spanish micinn through projects consolider - ingenio 2010 qoit ( csd2006 - 0019 ) and imagine ( csd2009 - 00013 ) , naninpho - qd ( tec2008 - 06756-c03 - 01/03 and 02 ) , the junta de andaluca ( pai research group tep-120 and project p08-tep-03516 ) and cam 2010 q&clight ( s2009esp-1503 ) .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .
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### Summary:
: in this work , we present results on the formation of vertical molecule structures formed by two vertically aligned inas quantum dots ( qd ) in which a deliberate control of energy emission is achieved .
the emission energy of the first layer of qd forming the molecule can be tuned by the deposition of controlled amounts of inas at a nanohole template formed by gaas droplet epitaxy .
the qd of the second layer are formed directly on top of the buried ones by a strain - driven process . in this way
, either symmetric or asymmetric vertically coupled structures can be obtained . as a characteristic when using a droplet epitaxy patterning process , the density of quantum dot molecules finally obtained is low enough ( 2 108 cm2 ) to permit their integration as active elements in advanced photonic devices where spectroscopic studies at the single nanostructure level are required .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: all participants were enrolled in the present study retrospectively on the basis of research protocols that were approved by the institutional review board at okayama university ( approval no .
a total of 133 clinically confirmed patients with sporadic probable or possible msa and 200 non - neurodegenerative disease controls were included in the present study .
the diagnosis of sporadic msa was made on the basis of the current consensus criteria .
of the 133 patients with msa , 87 were diagnosed as having opca ( msa - c ) , 28 as having snd ( msa - p ) , and 18 as having sds .
non - neurodegenerative disease controls ( n = 200 ) included patients with peripheral neuropathy ( n = 51 ) , cns infection ( n = 25 ) , collagen neurologic disease ( n = 23 ) , stroke ( n = 19 ) , myopathy ( n = 18 ) , cervical or lumbar spondylosis ( n = 12 ) , neurosis ( n = 8) , epilepsy ( n = 7 ) , headache ( n
= 5 ) , and other neurologic diseases without neurodegenerative disease ( n = 32 ) .
genomic dna was extracted from peripheral lymphocytes obtained from the patients with msa and controls .
dna sequence analysis was performed for exons 1 , 2 , 6 , and 7 of all 133 patients with msa after amplification by pcr using the primers for coq2 .
we used sequence scanner software to align and detect variations against the human reference genome ( figure 1 ) .
( a c ) direct dna sequencings detect g21s ( a ) , l25v ( b ) , and x422k ( c ) variations .
( d ) g21s dna variation confirmed by pcr restriction fragment length polymorphism ( rflp ) with the arrow as normal band and the arrowhead as variant band .
( e ) l25v dna variation confirmed by allele - specific ( as)-pcr with the variant case positive both for common forward ( cf ) plus normal reverse ( nr ) primers and cf plus variant reverse ( vr ) primers .
( f ) x422k dna variation confirmed by pcr - rflp with the variant allele ( arrowhead ) . to verify the specificity of the sequence analysis and dna variations , pcr restriction fragment length polymorphism ( pcr - rflp ) analysis method
was implemented to check the dna samples of patients with msa carrying variations and all 200 controls .
the selectivity of restriction enzyme , which can cut dna at the variant site , was achieved using takara cut - site navigator online .
bfa , alu , and ase were applied for g21s , v393a , and x422k , respectively .
twenty microliters of the pcr mixture was digested in a final volume of 25 l with 1- to 3-l units of enzyme and 2.5-l units of buffer for more than 4 hours .
digested samples were then resolved by 3% or 5% agarose gel electrophoresis in tris - borate - edta ( tbe ) buffer at 50 or 100 v for approximately 15 hours . because the corresponding restriction enzyme site was not found to confirm l25v and p157s variations , allele - specific pcr ( as - pcr )
was performed for the patients ' dna samples carrying these variations and for all controls .
a common forward primer was designed about 200 base pairs upstream from the variation sites ( 5-ggggctgcaagtcaccac-3 for l25v and 5-gagcctctctattccttttagg-3 for p157s ) . as for the reverse primer sets ,
2 reverse primers were designed for detecting both the normal and variant sequences at single pcr , matching only 1 of the biallelic single - nucleotide substitution ( 5-cagcctggcagactagggtt-3 as normal reverse primer and 5-cagcctggcagactaggcgt-3 as variant reverse primer for l25v and 5-gctgaaccaggttgtttacc-3 as normal reverse primer and 5-gctgaaccaggttgttgttc-3 as variant reverse primer for p157s ) .
the underlined amino acids of the above primers are complementary to the normal site or variant site .
double underlined amino acids of the above primers enhance the specificity of the pcr by destabilizing the extension of the doubly mismatched primers .
the amplification products are separated on a 3% agarose gel and run in tbe buffer at 100 v for 30 minutes ( figure 1 ) .
all variations in all 133 patients with msa were investigated in the single nucleotide polymorphism database , human genetic variation database , and human gene mutation database . for g21s , l25v , v66l , p157s , v393a , and x422k , statistical analysis
was performed using text and fisher exact test to calculate the significance of the difference in the number of variation carriers and noncarriers between patients with msa and controls , with contingency tables and standard methods used to calculate odds ratios and the corresponding 95% confidence intervals .
all participants were enrolled in the present study retrospectively on the basis of research protocols that were approved by the institutional review board at okayama university ( approval no .
a total of 133 clinically confirmed patients with sporadic probable or possible msa and 200 non - neurodegenerative disease controls were included in the present study .
the diagnosis of sporadic msa was made on the basis of the current consensus criteria .
of the 133 patients with msa , 87 were diagnosed as having opca ( msa - c ) , 28 as having snd ( msa - p ) , and 18 as having sds .
non - neurodegenerative disease controls ( n = 200 ) included patients with peripheral neuropathy ( n = 51 ) , cns infection ( n = 25 ) , collagen neurologic disease ( n = 23 ) , stroke ( n = 19 ) , myopathy ( n = 18 ) , cervical or lumbar spondylosis ( n = 12 ) , neurosis ( n = 8) , epilepsy ( n = 7 ) , headache ( n = 5 ) , and other neurologic diseases without neurodegenerative disease ( n = 32 ) .
genomic dna was extracted from peripheral lymphocytes obtained from the patients with msa and controls .
dna sequence analysis was performed for exons 1 , 2 , 6 , and 7 of all 133 patients with msa after amplification by pcr using the primers for coq2 .
we used sequence scanner software to align and detect variations against the human reference genome ( figure 1 ) .
( a c ) direct dna sequencings detect g21s ( a ) , l25v ( b ) , and x422k ( c ) variations .
( d ) g21s dna variation confirmed by pcr restriction fragment length polymorphism ( rflp ) with the arrow as normal band and the arrowhead as variant band .
( e ) l25v dna variation confirmed by allele - specific ( as)-pcr with the variant case positive both for common forward ( cf ) plus normal reverse ( nr ) primers and cf plus variant reverse ( vr ) primers .
( f ) x422k dna variation confirmed by pcr - rflp with the variant allele ( arrowhead ) .
to verify the specificity of the sequence analysis and dna variations , pcr restriction fragment length polymorphism ( pcr - rflp ) analysis method was implemented to check the dna samples of patients with msa carrying variations and all 200 controls .
the selectivity of restriction enzyme , which can cut dna at the variant site , was achieved using takara cut - site navigator online .
bfa , alu , and ase were applied for g21s , v393a , and x422k , respectively .
twenty microliters of the pcr mixture was digested in a final volume of 25 l with 1- to 3-l units of enzyme and 2.5-l units of buffer for more than 4 hours .
digested samples were then resolved by 3% or 5% agarose gel electrophoresis in tris - borate - edta ( tbe ) buffer at 50 or 100 v for approximately 15 hours .
because the corresponding restriction enzyme site was not found to confirm l25v and p157s variations , allele - specific pcr ( as - pcr ) was performed for the patients ' dna samples carrying these variations and for all controls .
a common forward primer was designed about 200 base pairs upstream from the variation sites ( 5-ggggctgcaagtcaccac-3 for l25v and 5-gagcctctctattccttttagg-3 for p157s ) . as for the reverse primer sets ,
2 reverse primers were designed for detecting both the normal and variant sequences at single pcr , matching only 1 of the biallelic single - nucleotide substitution ( 5-cagcctggcagactagggtt-3 as normal reverse primer and 5-cagcctggcagactaggcgt-3 as variant reverse primer for l25v and 5-gctgaaccaggttgtttacc-3 as normal reverse primer and 5-gctgaaccaggttgttgttc-3 as variant reverse primer for p157s ) .
the underlined amino acids of the above primers are complementary to the normal site or variant site .
double underlined amino acids of the above primers enhance the specificity of the pcr by destabilizing the extension of the doubly mismatched primers .
the amplification products are separated on a 3% agarose gel and run in tbe buffer at 100 v for 30 minutes ( figure 1 ) .
all variations in all 133 patients with msa were investigated in the single nucleotide polymorphism database , human genetic variation database , and human gene mutation database . for g21s , l25v , v66l , p157s , v393a , and x422k , statistical analysis
was performed using text and fisher exact test to calculate the significance of the difference in the number of variation carriers and noncarriers between patients with msa and controls , with contingency tables and standard methods used to calculate odds ratios and the corresponding 95% confidence intervals .
among our 133 patients with probable or possible msa , direct dna sequencing of the coq2 gene found the following 6 variations : g21s ( figure 1a ) , l25v ( figure 1b ) , v66l , p157s , v393a , and x422k ( figure 1c ) .
g21s and x422k were new variations that have never been reported and were confirmed by pcr - rflp for g21s ( figure 1d ) and x422k ( figure 1f ) . as - pcr also confirmed the genetic variation of l25v in patients with msa ( figure 1e ) .
p157s and v393a were also confirmed by as - pcr and pcr - rflp , respectively .
five dna variations ( g21s , l25v , p157s , v393a , and x422k ) were also simultaneously examined by pcr - rflp or as - pcr in 200 control participants .
v66l was not examined because all 133 patients with msa carried v66l variation in the direct dna sequence ( the allele frequency of heterozygous v66l is 0.07 and of homozygous v66l is 0.93 ) , which suggested that v66l variation was common in the japanese participants . among the 5 dna variations ,
g21s , l25v , and v393a were also found in controls by as - pcr or pcr - rflp .
one control carried homozygous v393a and another carried a combinational variation ( g21s / v393a ) .
coq2 variants found in patients with msa and controls the frequencies of l25v and v393a variation in patients with msa were larger than other variations of coq2 gene except for v66l variation .
although there was no difference in the allelic l25v frequencies between 133 patients with msa and 200 controls ( 2.63% and 1.00% , respectively ) , the allelic l25v variation in patients with opca showed a 3.44-fold greater relative risk of developing the disease ( 3.44% , p = 0.04 vs control ) compared with controls ( 1.00% ) ( table 2 ) . on the other hand , snd showed no such difference in the allelic l25v frequencies ( 1.79% in patients with snd vs 1.00% in controls ) .
as for allelic v393a frequencies , there was no difference in total msa , opca , snd , or sds in patients compared with controls ( 2.63% , 2.78% , 2.87% , 1.79% , and 3.00% , respectively ) .
g21s , p157s , and x422k variations were detected in 1 chromosome in each of 3 patients with opca , but frequencies of chromosomes were not different compared with controls ( data not shown ) .
association between the coq2 l25v and v393a variants and msa the amino acid sequences of coq2 gene in humans were compared with those of different species ( figure 2 ) .
although g21s and l25v were conserved in human , gorilla , and monkey , they were not located in coq2 gene of other species .
although variations of v66l and v393a were conserved as valine among primates , other mammals of lower order showed different amino acids .
x422k was conserved as x in human , gorilla , cattle , and sheep but not in monkey and the other mammals .
among our 133 patients with probable or possible msa , direct dna sequencing of the coq2 gene found the following 6 variations : g21s ( figure 1a ) , l25v ( figure 1b ) , v66l , p157s , v393a , and x422k ( figure 1c ) .
g21s and x422k were new variations that have never been reported and were confirmed by pcr - rflp for g21s ( figure 1d ) and x422k ( figure 1f ) . as - pcr also confirmed the genetic variation of l25v in patients with msa ( figure 1e ) .
p157s and v393a were also confirmed by as - pcr and pcr - rflp , respectively .
five dna variations ( g21s , l25v , p157s , v393a , and x422k ) were also simultaneously examined by pcr - rflp or as - pcr in 200 control participants .
v66l was not examined because all 133 patients with msa carried v66l variation in the direct dna sequence ( the allele frequency of heterozygous v66l is 0.07 and of homozygous v66l is 0.93 ) , which suggested that v66l variation was common in the japanese participants . among the 5 dna variations ,
g21s , l25v , and v393a were also found in controls by as - pcr or pcr - rflp .
one control carried homozygous v393a and another carried a combinational variation ( g21s / v393a ) .
the frequencies of l25v and v393a variation in patients with msa were larger than other variations of coq2 gene except for v66l variation .
although there was no difference in the allelic l25v frequencies between 133 patients with msa and 200 controls ( 2.63% and 1.00% , respectively ) , the allelic l25v variation in patients with opca showed a 3.44-fold greater relative risk of developing the disease ( 3.44% , p = 0.04 vs control ) compared with controls ( 1.00% ) ( table 2 ) . on the other hand
, snd showed no such difference in the allelic l25v frequencies ( 1.79% in patients with snd vs 1.00% in controls ) .
as for allelic v393a frequencies , there was no difference in total msa , opca , snd , or sds in patients compared with controls ( 2.63% , 2.78% , 2.87% , 1.79% , and 3.00% , respectively ) .
g21s , p157s , and x422k variations were detected in 1 chromosome in each of 3 patients with opca , but frequencies of chromosomes were not different compared with controls ( data not shown ) .
the amino acid sequences of coq2 gene in humans were compared with those of different species ( figure 2 ) .
although g21s and l25v were conserved in human , gorilla , and monkey , they were not located in coq2 gene of other species .
although variations of v66l and v393a were conserved as valine among primates , other mammals of lower order showed different amino acids .
x422k was conserved as x in human , gorilla , cattle , and sheep but not in monkey and the other mammals .
our 133 patients with sporadic msa showed 6 dna variations in coq2 gene : g21s ( figure 1a ) , l25v ( figure 1b ) , v66l , p157s , v393a , and x422k ( figure 1c ) .
g21s ( figure 1a ) and x422k ( figure 1c ) variations have not been reported previously but did not show differences in the allelic frequencies between 133 patients with msa and 200 controls ( table 1 ) . on the other hand ,
the allele frequency of l25v ( figure 1b ) in patients with opca ( 3.44% ) was significantly higher than that in the controls ( 1.00% ) , with a relative risk of 3.44 ( table 2 ) .
all 133 patients with msa carried v66l variation , and both p157s and v393a showed no difference between patients with msa and controls ( table 2 ) .
amino acid sequences of coq2 gene in different species showed that the above 6 variations were relatively conserved in primates but not in the lower order mammals ( figure 2 ) .
we performed genetic analyses for exons 1 , 2 , 6 , and 7 because previous reports showed that the dna variations of coq2 were located in exons 1 , 2 , 6 , and 7 .
another japanese group also reported 3 variations ( v66l , p157s , and v393a ) in japanese patients with msa .
v66l variation was also found in british and american patients with msa , p157s in european patients with msa , and v393a in european , korean , and chinese patients with msa . among the previous reports ,
only the japanese report suggested a risk of msa - c ( opca ) for v393a , but this was not proven in the patients with msa considered in the present study ( table 2 ) . in the current study
, l25v was found to be a risk factor for opca corresponding to msa - c , which has not been reported in the previous reports from japan , the united states , europe , korea , or china .
most of the patients with msa and controls included in the present study were living in the same chugoku area of japan , and both the groups may have similar genetic backgrounds . although the patients with msa included in the present study were diagnosed as having probable or possible msa , the number of participants in our study was small and inhomogeneous between opca ( n = 87 ) , snd ( n = 28 ) , and sds ( n = 18 ) .
there were no studies containing patients with msa and controls in the same pedigree and the segregation study was not done .
therefore , further studies with additional numbers and containing first - degree relatives will be needed to confirm this result .
coq2 is a mitochondrial enzyme that is essential for the biosynthesis of antioxidative coenzyme q10 in the mitochondrial respiratory chain .
a primary deficiency of coenzyme q10 caused by coq2 gene mutations was associated with infantile - onset multisystem disorder , renal disease , and cardiovascular failure .
the decrease of coenzyme q10 induced by coq2 variations can cause the decline of atp level in mitochondria .
the mitochondrial dysfunction characterized as an increased fission , an impaired fusion , and a fragmentation of mitochondria is associated with the pathogenesis of several neurodegenerative diseases , such as parkinson disease , alzheimer disease , huntington disease , amyotrophic lateral sclerosis , and charcot - marie - tooth disease .
however , to confirm the influence for coq10 expression due to l25v coq2 variation , further studies should be done . in the present study , l25v variation in the coq2 gene
was newly found as a risk factor of msa - c ( opca ) , but v393a variation was not .
although previous reports showed no association of variations of coq2 with msa ( except for the japanese report ) , our study could suggest a race - specific pathologic role of coq2 gene variations in msa .
abe received funding for this study from grand - in - aid for scientific research ( b ) 2529320216 , ( c ) 24591263 , and challenging research 24659651 , and by grants - aid from the research committees ( mizusawa h , nakano i , nishizawa m , sasaki h , and aoki m ) from the ministry of health , labour and welfare of japan .
koji abe has received research support from the ministry of health , labour and welfare in japan .
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### Summary:
: objective : the aim of this study was to analyze the association between the variations of coenzyme q2 4-hydroxybenzoate polyprenyltransferase gene ( coq2 ) and japanese patients with multiple system atrophy ( msa).methods : we investigated the genetic variations in exons 1 , 2 , 6 , and 7 of the coq2 gene in 133 japanese patients with msa and 200 controls and analyzed the association between the variations and msa.results:six dna variations ( g21s , l25v , v66l , p157s , v393a , and x422k ) were found in the 133 patients with msa , and g21s and x422k were new variations that had never been reported .
v66l was a common variation that was found in all 133 patients with msa .
g21s , p157s , v393a , and x422k did not show gene frequency differences between patients with msa and controls . on the other hand , l25v
was newly proven to be the only risk factor of sporadic msa with predominant olivopontocerebellar ataxia.conclusions:the present study suggests l25v variant of coq2 gene as a genetic risk factor in japanese patients with msa with cerebellar ataxia .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: metal - on - polyethylene and ceramic - on - polyethylene articulations are common in total hip replacements .
the success of these devices depends heavily upon the harder femoral heads remaining smooth over time , since increased roughness of the counterface may dramatically accelerate abrasive polyethylene wear [ 13 ] .
approximately 3% of total hip arthroplasties are complicated by dislocation within two years of implantation , however , which can result in metal transfer and surface roughening when the dislocated or subluxed femoral head contacts the rim of the metallic shell of a modular acetabular component [ 5 , 6 ] .
entrapment of delaminated porous coating materials or broken prongs within the polyethylene acetabular liner has also been associated with metal transfer in vivo .
titanium ( ti ) and cobalt - chromium - molybdenum ( cocr ) alloys are the primary components in metallic acetabular shells , while porous coatings are often commercially pure titanium or cocr beads . in either case , ti or cocr
may transfer to the surface of the harder femoral head material and increase its roughness , leading to increased severe scratching and abrasive wear of polyethylene [ 912 ] .
mller et al . reported that ti transfer was abraded away in ceramic - on - ceramic heads ; however , for alumina - on - polyethylene it was suggested that the transfer was more harmful , retaining its increased roughness and accelerating polyethylene wear .
the aims of the present study were ( 1 ) to study retrieved femoral heads and compare them with nonimplanted controls , and ( 2 ) due to the concern for increased wear , to perform an experiment with artificially scratched and nonscratched femoral heads .
surface roughness parameters were quantified in regions of confirmed metal transfer on retrievals for comparison with unused controls , and on intentionally altered femoral heads before and after controlled wear simulations .
scanning electron microscopy and electron dispersive spectroscopy were used to study visual characteristics and the chemical composition of the adherent transfer elements .
sixteen retrieved femoral heads from four independent manufacturers ( biomet , depuy , johnson and johnson , richards ) were obtained from the university of alabama orthopedic retrieval laboratory with approval from the uab institutional review board ( irb no .
six cocr and ten zro2 heads were selected for visual presence of transfer , a known history of dislocation , wear - through of the polyethylene , or a combination of all three . the extent of damage on the retrievals varied from localized to widespread .
this group represented the entire collection of components in our retrieval collection that met the criteria at the time of the study .
available implant and donor data portrayed 28 mm ( n = 13 ) and 32 mm ( n = 3 ) femoral heads implanted between 010 years in patients ranging from 2775 years old ( table 1 ) .
sterilized specimens were ultrasonically cleaned in acetone to remove any biologic remains and mounted for imaging via a scanning electron microscope with an electron dispersive spectrometer ( sem - eds , philips 515 , amsterdam , the netherlands ) .
the eds was used to confirm the presence of ti or other transfer metals on the surface of each femoral head .
the retrieved specimens were profiled using a form talysurf stylus - based contact profilometer ( taylor - hobson , uk ) . for each specimen ,
three 5-mm scans were made at 45-degree increments crossing a randomly selected region of transfer . each 5-mm scan
was then broken down into 1-mm segments , where the surface texture parameters could be determined for isolated regions of transfer .
surface texture parameters including roughness average , ra , root mean square , rq , and skewness , rsk , were calculated according to the equations described in the appendix .
the skewness is a measure of symmetry of the amplitude distribution about the mean line , such that a positive rsk indicates a trend toward peaked surface asperities , while negative rsk is associated with more troughs and flattened asperities .
eight nonimplanted control heads ( 4 cocr , 4 zro2 ) were profiled using 5 mm traces without isolating particular regions .
analysis of variance ( anova ) with two factors ( head type : cocr , zro2 ; and treatment : transfer , control ) was used to compare the roughness parameters , ra , rq , and rsk , for transfer regions on the retrieved femoral heads and for the unaltered controls .
twelve nonimplanted 32 mm femoral heads were obtained ( six cocr and six zro2 ) from three different manufacturers ( biomet , richards , zimmer ) .
six specimens were selected for the induction of transfer using an orthopod friction and wear tester ( amti , watertown , mass , usa ) .
each specimen was fit with a tapered three - centimeter aluminum stem for fixation to the orthopod ( figure 2 ) and programmed to trace a four - centimeter perimeter square pattern on a fixed ti disc at 100 n for 10 cycles at 0.2 hertz . following this articulation ,
after ultrasonication in an acetone bath , the six femoral heads were imaged with sem .
surface profilometry was performed using three 5 mm scans at 45 degree increments across the transfers . in this case 2
medical grade ultrahigh molecular weight polyethylene ( uhmwpe ) discs ( piedmont plastics , birmingham , ala , usa ) , which were machined from ram - extruded bar stock and gamma sterilized ( 25 kgy ) in air served as the counterface material .
the selected material represents a uniform grade of uhmwpe that was the standard in the 1980s and 1990s , which is the time period from whence the retrievals were collected .
the discs were soaked in bovine serum solution for at least 24 hours prior to testing in order to minimize fluid absorption .
each disc was five centimeters in diameter and 15 millimeters thick and secured to the base plate ( disc portion ) of the orthopod .
wear tests were then performed in which four specimens were tested simultaneously : two control heads with no transfer ( one zro2 , one cocr ) and two test heads with induced ti transfer ( one zro2 , one cocr ) , along with one unloaded soak - control disc . all tests were performed in a 30% bovine serum solution , with 0.3% sodium azide and 20 mm edta added as antibacterial and decalcifying agents , respectively .
the solution and test components were kept at 37 2c with an external circulation heater ( neslab instruments , portsmouth nh , usa ) for the duration of all tests .
the articulating femoral heads were programmed to trace a figure - eight pattern onto the surface of the respective polyethylene discs ( approximately 50 mm sliding distance / cycle ) for 100 000 cycles at 200 n at a rate of 1 hz .
traditional hertz equations predicted mean stresses of approximately 25 mpa . prior to and after each test
, the polyethylene discs were weighed with a mettler toledo ag245 microbalance ( columbus , ohio , usa ) to the nearest ten - thousandths of a gram . subtracting the mean control disc weight gain from the difference in individual test disc weights ,
as indicated in astm f-732 , the wear loss of each polyethylene specimen was calculated and volumetric wear rates determined .
volumetric wear was derived as vn = wn/ , where wn represents the net weight loss calculated as
( 1)wn=(w1w2)+(s2s1 ) ,
where w1 , w2 , s1 , and s2 were the pretest , posttest , pretest control , and posttest control polyethylene weights , respectively .
the bulk density of the polyethylene was provided by the manufacturer as = 0.930 g / cm .
after wear testing , surface profilometry was performed using three 5 mm scans at 45 degree increments across the transfer region .
again , 2 mm regions were isolated to quantify surface parameters of the transfer .
the roughness parameters and the volumetric wear were compared ( pre- and posttest ) to investigate changes associated with the articulation of the induced transfer against polyethylene .
the sem images showed gross topographical alterations of the retrieved femoral heads due to the presence of transfer , as compared to undamaged control surfaces ( figures 3 and 4 ) .
residual polishing scratches were visible running underneath the adherent transfers and occasional polyethylene fragments were found lodged in the transfer surface
. means and standard deviations were determined for roughness parameters , ra , rq , and rsk , associated with the transfer regions on the retrieved femoral heads , and for nonimplanted controls .
roughness averages for the control surfaces were similar at ra = 0.0120.013 m ( table 2 ) .
mean ra values for the transfers were an order of magnitude greater than the control values ( p = .01 ) , however , with ra = 0.38 m on cocr and ra = 0.294 m on zro2 , ra was 29% higher on average for the transfer regions on cocr as compared to zro2(p = .86 ) .
similar trends were observed for rq , with lower average values for controls ( rq = 0.017 m ) as compared to transfer regions ( p = .01 , table 2 ) .
rq was 49% higher for transfer on cocr ( rq = 0.54 m ) as compared to zro2 ( rq = 0.363 m , p = .09 ) .
rsk values were negative for control heads and positive for the transfer specimens ( p = .01 , table 2 ) . on average
, rsk was 2.34 times greater for transfer regions on the zro2 heads than for transfers on the cocr heads ( p = .08 ) .
pretest profilometry indicated that the average roughness values , ra , were an order of magnitude greater for regions of induced transfer ( ra = 1.21 m for cocr ; ra = 1.02 m for zro2 ) , as compared to the transfer regions on the retrieved devices ( table 3 ) and two orders of magnitude greater than the surfaces of nonimplanted controls ( recall table 2 ) .
a similar trend was observed for the root mean square roughness of the transfers , with rq = 2.21 m for cocr ; rq = 1.69 m for zro2 .
skewness values were strongly positive for the transfers , with rsk averaging 3.5 and 3.4 for cocr and zro2 , respectively .
femoral heads with induced transfer were associated with more polyethylene wear than unaltered controls .
polyethylene wear of 2.9 mg and 2.5 mg was measured , respectively , for cocr and zro2 heads subjected to articulation against polyethylene for 100 000 cycles ( figure 5 ) .
these values were significantly higher ( p < .003 ) as compared to those obtained for the controls .
wear associated with the unaltered cocr and zro2 specimens averaged 0.23 mg and 0.12 mg , respectively , however , these differences were not statistically significant ( p > .05 ) . the negative net losses associated with the unaltered cocr heads indicated that their respective polyethylene discs gained weight during the simulation , which was attributed to fluid absorption during testing .
post - test sem images of the cocr and zro2 samples subjected to the present wear simulation provided visual evidence of adherent transferred metal that closely resembled that of the zirconia retrievals ( figure 6 ) .
mean roughness measures , ra , rq , and rsk decreased in magnitude following wear testing for all three cocr heads ; however , ra and rq actually increased for the zro2 heads ( table 3 ) .
the results of this study demonstrated that metal transfer increased the surface roughness above standard manufacturing parameters as seen in nonimplanted cocr and zro2 femoral heads .
sem evaluation of retrieved cobalt - alloy and zirconia specimens with eds - confirmed transfer demonstrated highly altered surface topographies .
the average ra values for the present retrieved devices of around 300 nm compare reasonably with the 181 nm roughness averages reported by kim et al . for
the negative values of rsk measured for the control heads were expected for polished surfaces , which are typically extremely flat with residual polishing grooves , that is , valleys .
the strongly positive rsk values for transfer regions reflected a change in surface topography towards an increased percentage of peaking asperities , which would produce a more abrasive surface than the originally finished component .
one limitation of the present retrieval analysis is that there was no accurate way to calculate the amount of time the transfers underwent articulation .
were one of the first to report transfer of metallic debris to a zirconia femoral head from contact with an acetabular shell following hip dislocation .
others have confirmed that ti or cocr transfer increases the surface roughness of femoral heads , leading to increased scratching and abrasive wear of polyethylene [ 11 , 12 ] .
how well the transfer adheres to the femoral head and maintains its roughness during subsequent articulation is likely to affect the long - term wear rates and may depend upon the femoral head material . while ti transfer may abrade away in ceramic - on - ceramic heads , such transfer retains its increased roughness in ceramic - on - polyethylene devices accelerating polyethylene wear . while schuh et al .
reported no adherent ti on a scratched cocr retrieval , the present results suggested that titanium may remain adherent to cocr and zirconia surfaces in contact with polyethylene counterfaces , thereby increasing surface roughness and potentially increasing polyethylene wear .
the present in vitro wear simulation demonstrated more polyethylene wear with femoral surfaces roughened by metal transfer than with control surfaces , consistent with general conclusions made for lubricated wear couples [ 1 , 2 ] .
these results demonstrated significant contributions of the transfer element to wear for both cocr and zro2 femoral heads , consistent with those of kim et al . for alumina heads .
roughness measures for the induced transfer regions were an order of magnitude higher than for transfers found on retrievals , however , indicating more severely roughened surfaces among the induced transfer specimens as compared to the retrievals .
this finding indicates a possible limitation of the present in vitro study , which appears to represent a much more severe condition than encountered in - vivo .
another limitation of the present study is that the femoral head - on - disk wear test that was employed is , at best , a test that demonstrates the potential for increased uhmwpe wear when a cocr or zirconia counterface is artificially modified with metal transfer .
the tests were conducted for an extremely limited number of cycles ( 100 000 ) and did not accurately replicate hip joint conditions ; therefore , these results should be considered as a preliminary step to in vitro hip wear simulator tests .
lowered rsk values demonstrated some reduction in peak heights for regions of transfer after wear simulation ; however , ra and rq were nearly unchanged suggesting that the transfer elements remained rough with only a slightly less irregular ( sharp ) morphology .
thus the surface profiles for tested pieces would still be considered abrasive by clinical standards .
it is difficult to estimate the clinical significance of such femoral head damage ; however , greater polyethylene wear can be expected to increase the occurrence of osteolysis [ 1618 ] .
clinically , the overall results of the present study suggest that cocr and zirconia femoral heads that have experienced dislocation or subluxation should be considered suboptimal wear surfaces and should be exchanged for new femoral heads if revision is necessary . because metal transfer may not be as visibly apparent on cocr heads , clinicians should take extra care to avoid impingement of these devices against the metallic backing during operative procedures .
surgeons treating patients with recurrent subluxation or dislocation should closely monitor the patients with regular x - rays in order to detect and intervene if accelerated wear or osteolysis should appear .
it is the arithmetic mean of the vertical departures ( both above and below ) from the centerline of the segment of profile under examination and is determined by integrating the profile function and dividing by the length of the scan :
( a.1)ra=1l|y(x)|dx ,
where l is the length of the scan and y is the vertical displacement from the centerline as a function of position , x. the root mean square parameter , rq , is defined as the square root of the mean of the squares of the ra values for a particular scan .
it is derived from the scan mathematically in similar fashion ,
( a.2)rq=1l y(x)2dx .
while these are the most widely utilized parameters for surface roughness , they do not recognize valleys from peaks over the course of the scan . in order to more accurately approximate the surface roughness as a function of peak height and positive deviations from the centerline , rsk parameters were tabulated for each scan .
rsk represents the skewness of the profile and is a measure of symmetry of the amplitude distribution about the mean line , or
( a.3)rsk=1nrq3(yiy)3 ,
where n serves as the number of coordinate values made ( y values ) and y is the numerical value of the mean line .
a positive skewness measure denotes asymmetry above the mean line representing a greater number of peaking asperities .
negative skewness values represent asymmetry below the mean line , or greater troughs in the profile .
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### Summary:
: metal transfer to femoral heads may result from impingement against the metallic acetabular shell following subluxation / dislocation , or when metallic debris enters the articulation zone .
such transfers roughen the head surface , increasing polyethylene wear in total hip replacements . presently , we examined the surface roughness of retrieved femoral heads with metallic transfer .
profilometry revealed roughness averages in regions of metal transfer averaging 0.380 m for cocr and 0.294 m for zro2 which were one order of magnitude higher than those from non - implanted controls .
scanning electron microscopy ( sem ) revealed adherent transfers on these retrievals , with titanium presence confirmed by electron dispersive spectroscopy . due to the concern for
increased wear , metal transfer was induced on non - implanted heads , which were then articulated against flat polyethylene discs in multidirectional sliding wear tests .
increased polyethylene wear was associated with these specimens as compared to unaltered controls .
sem imaging provided visual evidence that the transfers remained adherent following the wear tests .
pre- and post - test roughness averages exceeded 1 m for both the cocr and zro2 heads .
overall , these results suggest that metal transfer increases the surface roughness of cocr and zro2 femoral heads and that the transfers may remain adherent following articulation against polyethylene , leading to increased polyethylene wear .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: stridor is a common presentation in children defined as high pitched monophonic sound associated with breathing .
vocal cord palsy is the second most common congenital laryngeal abnormality , accounting for approximately 6%23% of cases and can be further sub classified , anatomically , into unilateral or bilateral paralysis .
syringomyelia is the term given to define a fluid filled cavity laying within the spinal cord , most commonly occupying space between the cervicothoracic vertebral levels .
it is commonly associated with a chiari i malformation and may be an incidental finding . although the association between vocal cord palsy and syrinx with arnold chiari has been described in the literature , it is rarely the only sign .
we present a case where an incidental note of stridor lead to the diagnosis of syringomyelia with chiari i malformation .
a three year old female presented to hospital for further investigation into her speech and language delay .
incidental note was made of her high pitched breathing , which began suddenly two months prior to presentation and had been treated as a viral upper respiratory tract infection leading to the exacerbation of asthma .
family members noted that despite her cough and rhinorrhea subsiding and otherwise being well , her stridor had progressed and become louder and consistently audible . she had been treated with multiple courses of antibiotics as well as salbutamol and various steroid inhalers with minimal relief .
she had normal voice , snoring but no apneas and had not had any cyanotic episodes or any evidence of respiratory distress .
a semi urgent microlaryngoscopy revealed a bilateral vocal cord palsy her vocal cords were paramedical with no active abduction and paradoxical movement with respiration .
she was admitted to the paediatric intensive care unit for further investigation and ongoing investigative oximetry .
a nasogastric tube was inserted for feeding as she was deemed a high risk patient for aspiration ( table 1 , table 2 ) .
an mri brain and spine was ordered to further investigate a cause of the bilateral vocal cord palsy and revealed a large syrinx formation extending cranially from the c2 vertebrae to the t10 vertebrae caudally .
the cerebellar tonsils were low lying , projecting 8 mm below the foramen magnum , indicating a chiari i malformation .
she was subsequently treated operatively with a posterior fossa decompression with improvement but not resolution of her stridor and discharged home with outpatient follow up .
this patient was followed up in the outpatient clinic six weeks post - operatively with further improvement in her inspiratory stridor at rest but persistence during sleep ( fig 1 , fig .
4 ) . due to the nocturnal stridor and the association of central apneas with arnold chiari malformations ,
this reealed mixed sleep disordered breathing with borderline gas exchange , high work of breathing as well as sleep fragmentation .
airway evaluation at three months showed no resolution of cord mobility but clinically she has no audible stridor at rest or with exertion .
stridor , originating from the latin term , stridere , meaning to creak , is a common presentation in the paediatric population .
however the multitude of underlying pathologies associated with stridor means that it may be difficult to distinguish a clear cause .
, emphasis must be placed on the acuity of the symptom in the history as well as pattern of stridor , i.e. whether it is an inspiratory or expiratory stridor .
this may be due to a foreign body aspiration , burn , epiglottitis , laryngotracheitis or an abscess formation , being either retropharyngeal or peritonsillar abscess .
it is the chronic stridor which is more likely in accordance with a structural abnormality .
common causes include laryngomalacia , the most common cause of stridor in the neonatal population , as well as vocal cord palsy , subglottic stenosis subglottic cysts , vascular rings , respiratory papilloma and tumor causing compression of the airway .
chronic stridor can be misdiagnosed for long periods as asthma or bronchiolitis and hence mismanaged as in the case presented above .
vocal cord palsy is one of the leading causes of stridor , the most common being laryngomalacia .
de gaudamar et al . found similar rates of unilateral to bilateral vocal cord palsy .
out of the 113 patients evaluated with a vocal cord palsy , 61 had a unilateral palsy versus 52 who had a bilateral palsy .
unilateral cord paralysis can often be idiopathic although can also be attributed to mechanical injury to the left recurrent laryngeal nerve during cardiac and neck surgery as well as birth trauma .
de gaudamar also described that out of the 113 cases of vocal cord palsy , the aetiology varied , with 37% being idiopathic , 25% being secondary to a central nervous system dysfunction ( most commonly an arnold chiari malformation ) , 13% due to obstetric complication and approximately 5% iatrogenic post cardiac surgery .
presentation at age three in unusual and bilateral cord palsy as the etiology for new onset stridor is an unexpected diagnosis .
all children with bilateral vocal cord immobility , and those with unexplained unilateral lesions should undergo an mri to exclude this and other central causes .
in addition to stridor , the child with vocal cord palsy may present with a weak / soft cry , recurrent aspiration pneumonia or respiratory distress .
in the case presented above , it is common that a syringomyelia and/or chiari i malformation in an otherwise well patient may not yet be diagnosed .
nohria et al . showed that the average age of diagnosis of a chiari i malformation is 18 .
in addition to this , previous studies have estimated that up to 30% of cases of chiari
30% of cases of chiari i malformation have an associated syrinx with the most common presentation being a headache , likely as a result of meningeal irritation .
a neurological examination will usually reveal other focal neurological signs although , however we emphasize that this is not always the case .
arnold chiari malformation is the name given to a group of anatomic variances which describe an inferiorly displaced cerebellum / brainstem into the spinal cord .
chiari i malformation , the most common of the chiari subtypes , defines a low lying cerebellar tonsil , beneath the foramen magnum .
it is this displacement , which causes compression of the cranial nuclei , resulting in vocal cord palsy .
the risk of aspiration with vocal cord paralysis is well proven and we do recommend the insertion of a nasogastric tube as a tool of prevention and speech therapy review . up to one third of vocal cord palsy cases reviewed by bhattacharyya et al
. showed radiologic evidence of aspiration , regardless of the cause of the paralysis .
the association between central sleep apnea and type i chiari malformation is also well known .
hershberger and chidekel showed that sleep disordered breathing can be the only manifestation of a type i chiari malformation .
after neurological decompression , this patient had improvement but not resolution of her sleep disordered breathing .
tools such as the children s sleep habits questionnaire by owens ja , spirito a et al .
a sleep study should be considered in all individuals with a diagnosis of arnold chiari .
this again portrays the variable presentations of chiari malformations and high clinical suspicion required in cases such as this .
daya et al . found that over 50% of cases of bilateral vocal cord palsy will require a tracheostomy to maintain a safe airway .
posterior fossa decompression remains as the mainstay of therapy for symptomatic chiari i malformation although variances do remain in management . in the presence of hydrocephalus , ventricular shunting
it is still unclear over the best timing of surgery , with early operative management now being favored .
although despite this treatment , it is difficult to predict outcomes post - surgical intervention .
past literature shows that prognostically , bilateral palsy has worse outcomes compared to unilateral palsy .
73% of unilateral vocal cord palsy cases will resolve spontaneously compared to only 52% of bilateral vocal cord palsy estimated to resolve .
stridor is a common symptom in young patients which can often be mistaken for reactive airways disease .
syrinx formation and chiari i malformation , although uncommon in presenting solely as stridor , should certainly be considered .
a posterior fossa decompression resulted in improvement of symptoms but not resolution of vocal cord immobility .
this case highlights the importance of mri as a tool for diagnosis in children with stridor and advise that is should be highly considered for all patients with vocal cord palsy of unknown etiology .
i , saif yousif , would like to state that i am not the recipient of a research scholarship and this paper is not based on a previous communication to a society or meeting .
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### Summary:
: highlightswe present the case of a three year old female who had a long standing stridor , previously being incorrectly managed as asthma by family practitioner.she was found to have a bilateral vocal cord palsy causing her stridor , as a result of a large syrinx formation and arnold chiari malformation.we highlight the importance of mri as a diagnostic tool in the evaluation of vocal cord palsy and stridor .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: angiogenesis , the development of new blood vessels from preexisting vessels , is critical for a wide array of complex normal and pathological processes including morphogenesis , wound healing , and tumor growth . under normal physiologic conditions ,
angiogenesis is well controlled by the local balance between endogenous angiogenesis stimulators and angiogenesis inhibitors , although the regulatory mechanism is still not clearly defined .
judah folkman pioneered the strategy of stopping tumor growth and metastasis by blocking tumor angiogenesis . with the 2004 fda approval of bevacizumab ( avastin ) , a humanized monoclonal antibody against vascular endothelial growth factor ( vegf ) , to treat metastatic colorectal cancer in combination with 5-fluorouracil ( 5-fu ) , antiangiogenesis therapy has emerged as an essential new strategy for cancer treatment .
angiogenesis is a highly regulated event that involves complex , dynamic interactions between microvascular endothelial cells and ecm proteins . in developing capillary sprouts
, endothelial cells digest the surrounding extracellular matrix ( ecm ) and invade the matrix as a cylindrical aggregate of cells .
these events clearly require an integrated response of endothelial cells to angiogenic factors and ecm proteins .
alteration of ecm composition and architecture is a hallmark of wound clot and tumor stroma .
ecm matrices induce multiple dynamic interactions with endothelial cells and stimulate the transduction of signals by cross - linking integrin receptors on endothelial cells .
initially viewed as merely a physical barrier , the ecm is now recognized as having a profound effect on the angiogenic phenotype
. however , the integrated regulatory mechanism of microvascular endothelial cell response to ecm and angiogenic factors is poorly defined [ 4 , 5 ] .
in addition , numerous evidences indicate that the in vitro cellular regulations of many cell types in 2d environment are significantly different than those of cells in 3d environment .
since 3d environment is more close to the in vivo microenvironment of cell functions , it suggests that reproducible and quantifiable in vitro 3d assays play an important role to study the regulation of cellular behaviors during physiological and pathological processes .
it has been reported that fibrin enhances angiogenesis of wound healing in vitro and in vivo .
in contrast , type i collagen is a major component of normal dermis which has minimal angiogenesis activities , although some in vitro studies demonstrate that type i collagen gel supports angiogenesis as well as fibrin gel .
the results of these in vitro studies are not consistent with the in vivo data reported by dvorak et al .
expression of integrin alpha v beta 3 is one of the hallmark features of sprout angiogenesis . remarkably , integrin beta 3 expression was highly upregulated in vascular endothelial cells found in fibrin rich but not in collagen rich matrix environment in vivo and in vitro .
we recently demonstrated that fibrin and collagen differentially regulated integrin expression in human dermal microvascular endothelial cells ( hdmec ) and in human dermal fibroblasts .
in particular , fibrin , but not collagen , increased the expression of integrin alpha v beta 3 in hdmec . since integrin alpha v beta 3 expression is differentially regulated by ecm and it is required for an angiogenic response to certain angiogenic factors , such as vegf and bfgf , we hypothesized that fibrin and collagen differentially regulate angiogenesis .
angiogenesis is a tightly regulated event , which visually includes endothelial invasion , migration , capillary tube formation , and capillary network formation .
it is essential to have a reproducible and quantifiable in vitro assay of human sprout angiogenesis to investigate the integrated response of human microvascular endothelial cells to angiogenic factors and 3d ecm .
using a modified microcarrier - based 3d angiogenesis assay [ 1113 ] , we demonstrated in vitro that fibrin and collagen differentially regulate sprout angiogenesis .
our in vitro data also indicated that fibrin was essential for sprout angiogenesis of human microvascular endothelial cells in response to angiogenic factors .
this was consistent with our in vivo data that there was a close correlation between fibrin presence and sprout angiogenesis occurrence in porcine wound .
in addition , for the first time we demonstrated that integrin alpha v beta 3 ( receptor for fibrin ) and integrin alpha 2 beta 1 ( receptor for collagen ) differentially but synergistically regulate sprout angiogenesis .
gelatin - coated microcarrier beads ( cytodex-3 ) were purchased from pharmacia ( uppsala , sweden ) .
dimethyl dichlorosilane , aprotinin , dibutyryl cyclic amp , hydrocortisone , trypsin , soybean trypsin inhibitor , and edta were obtained from sigma chemical co. ( st . louis , mo , usa ) .
cyclic rgd , gly - pen - gly - arg - asppro - cys - ala ( gpengrgdspca ) , vitronectin specific gpengrgdspca peptides ( rgd ( vn ) ) , and inactive sham control peptide , gly - arg - ala - asp - ser - pro ( gradsp ) , from life technologies ( carlsbad , ca , usa ) .
endothelial cell basal medium ( ebm ) , endothelial cell growth medium bulletkit-2 ( egm-2 bulletkit ) , bovine brain extract , and epidermal growth factor were obtained from clonetics corp .
vascular endothelial cell growth factor ( vegf ) was purchased from peprotech ( rocky hill , nj , usa ) .
propidium iodide ( pi ) was obtained from molecular probes ( eugene , or , usa ) .
disintegrin elp12 was a kindly gift from cezary marcinkiewicz of temple university , pa , usa .
echistatin was obtained from sigma chemical co. ( st . louis , mo , usa ) .
human dermal microvascular endothelial cells ( hdmec ) were isolated from human neonatal foreskins as previously reported .
briefly , after initial harvest from minced trypsinized human foreskins , microvascular endothelial cells were further purified on a percoll density gradient .
hdmec were cultured on collagen type 1 coated tissue culture flasks in egm ( endothelial cell growth medium ) consisting of ebm supplemented with 10 ng / ml epidermal growth factor , 0.4% bovine brain extract , 17.5 microgram / ml dibutyryl cyclic amp , and 1 microg / ml hydrocortisone in the presence of 30% normal human serum .
endothelial cell cultures were characterized and determined to be > 99% pure on the basis of formation of typical cobblestone monolayers in culture , positive immunostaining for factor viii - related antigen , and selective uptake of acetylated low density lipoprotein .
approximately 80,000 sterile microcarrier beads were washed , resuspended in egm , and added to approximately 4.5 million endothelial cells ( hdmec ) .
the beads and cells were mixed by gentle swirling , incubated at 37c for 6 hr , and then rotated for 2436 hr on an orbital mixer in a 37c oven to generate endothelial cell - loaded microcarrier beads ( ec - beads ) .
a microcarrier in vitro angiogenesis assay previously designed to investigate bovine pulmonary artery endothelial cell angiogenic behavior in bovine fibrin gels [ 1113 ] was modified for the study of human microvascular endothelial cell angiogenesis in different ecm environment ( figure 1(a ) ) .
briefly , human fibrinogen , isolated as previously described , was dissolved in m199 medium at a concentration of 1 mg / ml ( ph 7.4 ) and sterilized by filtering through a 0.22-micron filter .
pepsin - solubilized bovine dermal collagen dissolved in 0.012 m hcl was 99.9% pure containing 9598% type i collagen and 25% type iii collagen ( vitrogen 100 , collagen biomaterials , palo alto , ca , usa ) .
an isotonic 1.5 mg / ml collagen solution was prepared by mixing sterile vitrogen 100 in 5x m199 medium and distilled water .
ph was adjusted to 7.4 by 1 n naoh . in certain experiments , angiogenic stimulators and/or inhibitors , such as vegf , bfgf , rgd peptides , elp12 , and echistatin , were added to the fibrinogen or collagen solutions ( figure 1 ) .
the angiogenic response was monitored visually and recorded by video image capture . specifically , capillary sprout formation was observed and recorded with a nikon diaphot - tmd inverted microscope ( nikon inc . ,
melville , ny , usa ) , equipped with an incubator housing with a nikon np-2 thermostat and sheldon # 2004 carbon dioxide flow mixer .
the microscope was directly interfaced to a video system consisting of a dage - mti ccd-72s video camera and sony 12 pvm-122 video monitor linked to a macintosh g3 computer .
the effect of angiogenic factors on sprout angiogenesis was quantified visually by determining the number and percent of ec - beads with capillary sprouts .
one hundred beads ( five to six random low power fields ) in each of triplicate wells were counted for each experimental condition .
all experiments were repeated at least three times . to locate the nucleus of hdmec ,
the fibrin or collagen gel was fixed by methanol / acetone ( 1 : 1 ) and stained by 0.001% pi .
porcine cutaneous wounds were harvested at various times and then immunoprobed for expression of integrin receptors as previously described .
briefly , full - thickness wounds were made with an 8 mm punch on the backs of white yorkshire pigs and harvested at the times indicated .
specimens were bisected ; one half was fixed in formalin and stained with masson trichrome ; the other half was frozen in liquid nitrogen for immunofluorescence studies .
all antibodies were used at dilutions that gave maximal specific fluorescence and minimal background fluorescence on frozen tissue specimens .
bound antibody was detected by the avidin - biotin - complex ( abc ) technique .
stained specimens were observed and photographed using a nikon microphot fxa epifluorescence microscope equipped with a nikon fx-35dx 35 mm camera .
confocal microscopy was done at the university microscopy imaging center , health sciences center , suny at stony brook , to confirm that sprouts emanating from the ec - beads formed tubes and to investigate the expression of integrin alpha v beta 3 on endothelial surface in 3d ecm . for these studies ec - beads ( 150 ) were suspended in a three - dimensional fibrin gel with vegf ( 30 ng / ml ) and bfgf ( 25 ng / ml ) in a 4-well lab - tek chambered coverglass and incubated for 5 days . for experiments of tube formation , samples were washed in 2xpbs and then fixed in 2% paraformaldehyde . with the kind assistance of david colflesh at the university microscopy imaging center , images were sequentially obtained ( 1 micron contiguous tangential cross sections ) using a noran laser scanning confocal system ( odyssey ; noran instruments inc . , middleton , wi , usa ) attached to a nikon diaphot - tmd microscope .
a silicon graphics iris workstation was used for processing digitized micrographs and assembling three - dimensional renditions from confocal images using voxelview software ( voxelview ; vital images ) . for experiments of immunostaining , 23c6 , a monoclonal antibody to integrin beta 3 , was used .
to understand the relationship between angiogenesis and different ecm components during granulation tissue formation of wound repair , we analyzed tissue specimens from 5- , 7- , and 10-day porcine wounds ( figure 2 ) .
the 5-day wounds are mainly composed of a fibrin - rich provisional matrix , whereas 7-day wounds have a substantial organized collagen fiber network , and 10-day wounds have developed a compacted contracted collagen scar [ 15 , 16 ] .
staining of 5- , 7- , and 10-day wound specimens with masson trichrome ( figures 2(d ) , 2(e ) , and 2(f ) ) and with antilaminin ( figures 2(a ) , 2(b ) , and 2(c ) ) revealed that the fibrin - rich early granulation tissue in 5-day wounds is filled with newly formed vessels ( figures 2(a ) and 2(d ) ) .
these neovessels consistently stained weakly for laminin , most likely as a result of blood vessel immaturity .
such weak staining for laminin in immature blood vessels was previously observed by us in the microvasculature of human fetal skin . by 7 days ,
the maturing blood vessels form an organized vertical array as collagen accumulates in the wound ecm ( figures 2(b ) and 2(e ) . at 10 days , as the collagen bundles thicken to produce scar , many blood vessels are regressing ( figures 2(c ) and 2(f ) ) .
there is a close correlation between fibrin presence and sprout angiogenesis occurrence in porcine wound .
sprout angiogenesis mainly occurred in day 5 when wound clot was mainly filled with fibrin ( figures 2(a ) and 2(d ) ) .
inversely , when fibrin was almost totally replaced by collagen on day 10 , sprout angiogenesis regressed ( figures 2(c ) and 2(f ) ) .
thus during wound repair in vivo , the angiogenic neovessels in early granulation tissue invade the fibrin clot as capillary sprouts , mature , and then regress as fibrin is replaced by collagen in the wound space . to delineate why angiogenesis of early wound granulation tissue started by invading the fibrin clot as capillary sprouts
, we studied the regulatory effect of fibrin microenvironment on sprout angiogenesis . in our modified microcarrier - based in vitro 3d human sprout angiogenesis system , when vegf ( 30 ng / ml ) and bfgf ( 25 ng / ml ) were added to serum free fibrin gel containing ec - beads , hdmec formed capillary sprouts , which projected from the surface of the ec - beads and invaded into the fibrin gel within 48 hr ( figure 3(b ) ) . by 5 days the endothelial sprouts had elongated and in some cases formed branching capillary sprouts ( figure 3(c ) ) .
local capillary networks formed by branching and fusion of capillary sprouts from the same bead ( figure 3(d ) ) , and wild capillary networks formed by fusion of capillary sprouts from adjacent beads ( figures 3(e ) and 3(f ) ) . in contrast , without the addition of an angiogenesis factor , no significant hdmec sprout formation occurred from the surface of ec - beads in fibrin gel , despite the presence of 20% normal human serum in the medium above the gels ( figure 3(a ) ) .
fluorescence of cell nuclei staining clearly revealed that 5-day capillary sprouts ( figure 4(a ) ) were composed by multiple cells ( figure 4(b ) ) . to demonstrate whether capillary tube - like structures ( figure 4(c ) ) had lumina , we used reflective confocal microscopic analysis .
computer - assisted sectioning clearly revealed the presence of a lumen ( figure 4(d ) ) in the capillary - like structure shown in figure 4(a ) . as the tube was cut through by a series of 1 micron contiguous tangential cross sections ,
first the top and then the central lumen with two walls become visible ( figure 4(d ) ) . to determine whether ecm environment , fibrin and collagen in particular , regulates human sprout angiogenesis , a modified in vitro 3d angiogenesis system , which can differentiate sprouting angiogenesis from invasive migration , was used to compare the angiogenic response of hdmec in 3d fibrin and collagen gels .
ec - beads were embedded in fibrin gel or in collagen gel , with or without presence of vegf ( 30 ng / ml ) and bfgf ( 25 ng / ml ) for 48 hr . in the absence of angiogenic stimulators ,
hdmec remained on the surface of ec - beads and did not invade either fibrin gel ( figure 5(a ) ) or collagen gel ( figure 5(b ) ) . in the presence of vegf in fibrin gel , hdmec formed capillary - like sprouts from the surface of ec - beads and invaded and migrated into the surrounding fibrin ( figure 5(c ) ) . in contrast ,
when angiogenic factors were added to collagen gel , hdmec invaded and migrated into the surrounding collagen as individual cells but did not form sprouts ( figure 5(d ) ) .
after 5 days , the capillary sprouts in fibrin elongated and fused into networks ( figure 5(e ) ) , but in collagen individual hdmec randomly migrated without capillary sprouts and networks ( figure 5(f ) ) .
nucleus staining by pi confirmed that in fibrin the capillary tubes were composed of multiple cells ( figure 5(g ) ) , but in collagen hdmec randomly distributed in the gel ( figure 5(h ) ) . to determine if presence of fibrin in collagen gels will enable angiogenic factors to induce capillary sprouts in collagen gels , we embedded ec - beads in collagen admixed with fibrin and compared the angiogenic response to that in either pure fibrin or pure collagen gels . combination of vegf ( 30 ng / ml ) and bfgf ( 25 ng / ml ) induced individual cell invasion and migration in pure collagen gels ( figure 6(a ) ) and induced capillary sprouts in pure fibrin gels ( figure 6(d ) ) as previously observed .
20% fibrin , and figure 6(c ) , 30% fibrin ) , tube - like capillary sprout formation occurred together with individual cell invasion and migration .
thus the presence of fibrin appeared to be essential for hdmec sprout angiogenesis induced by angiogenic factors .
we recently demonstrated that integrin alpha v beta 3 is essential for sprout angiogenesis and integrin beta 3 expression is highly upregulated in fibrin rich but not in collagen rich matrix environment in vitro and in vivo [ 4 , 13 ] . using the modified in vitro
sprout angiogenesis model , we demonstrated that gpengrgdspca peptide ( rgd ( vn ) ) , which is a specific antagonist for integrin alpha v beta 3 , significantly blocked sprout angiogenesis of hdmec in fibrin compared to the control rge peptide ( figure 7 ) .
echistatin ( 10 microgram / ml , 3 microgram / ml , 1 microgram / ml ) , a disintegrin specific for alpha v beta 3 , dose dependently inhibits sprout angiogenesis of hdmec in fibrin ( data not shown ) .
in contrast , ecl12 , inhibitor to collagen receptor integrin alpha 3 beta 1 , had no inhibitory effect on sprout angiogenesis of hdmec in fibrin , even at a concentration 10 microgram / ml ( figure 8) . at a concentration of 0.1 microgram / ml
however , the combination of 0.1 microgram / ml echistatin and 10 microgram / ml ecl12 completely inhibited sprout angiogenesis of hdemc in fibrin induced by vegf ( 30 ng / ml ) and bfgf ( 25 ng / ml ) .
these data indicates that integrin alpha v beta 3 and integrin alpha 2 beta 1 differentially but synergistically inhibit sprout angiogenesis of hdmec in 3d fibrin .
alteration of fibrin and type i collagen matrices in the ecm microenvironment is a hallmark feature of wound clot and tumor stroma ; however its role in the regulation of sprout angiogenesis is still poorly defined . using in vitro 3d angiogenesis assay and in vivo wound healing model ,
we demonstrate in this study that robust angiogenic vessels invaded the fibrin clot of early wounds but matured and regressed as fibrin was replaced by type i collagen ( figure 2 ) .
these results are consistent with the finding that fibrin deposition is commonly observed in angiogenesis associated with wound healing and tumor growth .
it has been reported that fibrin enhances angiogenesis of wound healing in vitro and in vivo [ 7 , 8 ] .
in contrast , type i collagen is a major component of normal dermis which has minimal angiogenesis activities . in our
in vitro 3d angiogenesis assay , angiogenic stimulators induce endothelial cell migration but not sprout and capillary network formation in 3d type i collagen matrices ( figure 5 ) .
interestingly once fibrin was added to type i collagen gel , the sprout angiogenesis is restored ( figure 6 ) .
dvorak and colleagues also demonstrate that implanted fibrin gels themselves induce an angiogenic response in the subcutaneous space of guinea pigs in the absence of tumor cells or platelets , while type i collagen or agarose does not induce new blood vessel formation . after tissue injury , type i collagen
is removed with normal dermis and fibrinogen leaks from ligated blood vessels to form the fibrin clot , which fills the wound space [ 1618 ] . using in vivo wound healing model , we demonstrated that after a 3-day lag , endothelial cells migrate from the periphery of the wound and invade the fibrin clot as sprout angiogenesis to form nascent granulation tissue ( figure 2 ) .
type i collagen accumulation begins as the granulation tissue matures , and in small excisional wounds , type i collagen replaces fibrin in the wound space by 7 days and form contracted wound scar in 10 days [ 19 , 20 ] .
we demonstrate here a substantial correlation between sprout angiogenesis and presence of fibrin matrices and the regression of sprout angiogenesis when fibrin is replaced by type i collagen . however , type i collagen supports sprout angiogenesis in the presence of fibrin fibrils .
recently two teams of researchers demonstrated that an amino terminal peptide of angiocidin binds type i collagen and inhibits tumor growth and angiogenesis [ 21 , 22 ] .
although fibrin is actively involved in regulating angiogenesis , we demonstrated that pure fibrin matrices themselves do not induce sprout angiogenesis without presence of angiogenic factors ( figure 5(a ) ) .
demonstrated that platelet - rich fibrin matrix improves wound angiogenesis via inducing vascular endothelial cell proliferation .
lafleur and colleagues demonstrated that membrane - type - matrix metalloproteinases ( mt - mmps ) are essential for endothelial angiogenesis in fibrin matrix .
it has been argued that if fibrin itself was sufficient to induce angiogenesis , it must perform at least two functions : ( 1 ) providing a three - dimensional matrix that supports cell migration and ( 2 ) expressing selective chemotactic and/or chemokinetic activity such that endothelial cells migrate into fibrin clot .
it is not surprising that fibrin might provide a three - dimensional matrix capable of supporting cell migration .
however , it is unexpected that fibrin might directly induce cell migration without the presence of any soluble chemotactic factors . greiling and clark demonstrated that human dermal fibroblasts fail to migrate from a collagen matrix into a fibrin gel in the absence of platelet releasate or pdgf - bb .
our in vitro data also demonstrated that there was no sprout angiogenesis of hdmec in fibrin without presence of angiogenesis growth factors , such as vegf and bfgf , in the matrix .
thus fibrin is essential to support sprout angiogenesis of microvascular endothelials induced by angiogenic stimulators in 3d environment .
expression of integrin alpha v beta 3 is one of the hallmark features of sprout angiogenesis .
we recently demonstrated that fibrin and type i collagen differentially regulated integrin expression in human dermal microvascular endothelial cells ( hdmec ) and in human dermal fibroblasts . in particular , fibrin , but not collagen , increased the expression of integrin alpha v beta 3 in hdmec .
since integrin alpha v beta 3 is the marker for sprout angiogenesis associated with wound healing and tumor growth [ 14 , 25 ] , this suggests that fibrin and collagen differentially regulate angiogenesis , in part , by altering endothelial cell integrin expression .
compared to highly regulated angiogenesis in wound healing , angiogenesis persists in solid tumor growth , as does the blood vessel leak of fibrinogen and resultant interstitial clotting .
numerous studies indicated that integrin alpha v beta 3 has potential to be a novel target for cancer treatment [ 2729 ] .
our results indicate that gpengrgdspca peptide ( rgd ( vn ) ) , which is a specific antagonist for integrin alpha v beta 3 , significantly blocked sprout angiogenesis of hdmec in fibrin induced by vegf ( figure 7 ) .
currently many new investigational cancer drugs based on rgd peptide are tested in clinical trials .
integrin alpha 2 beta 1 is the integrin receptor for native collagens which mediate many important cellular functions , such as adhesion , migration , invasion , and contraction of collagen lattices .
the role of integrin alpha 2 beta 1 in angiogenesis is controversial and is still not clearly defined . using in vitro 2d models , senger and colleagues demonstrated that blocking antibodies for integrin alpha 2 beta 1 inhibit endothelial cell migration on type i collagen .
in addition , they demonstrated that blocking antibodies to integrin alpha 2 beta 1 also potently inhibit vegf induced angiogenesis using matrigel transplant in mice skin .
in contrast , zweers and colleagues found strong enhancement of angiogenesis in cutaneous wound and implanted sponges in alpha 2 null mice .
furthermore , zutter and colleagues demonstrated that poor expression of integrin alpha 2 beta 1 might play an essential role in cancer progression .
this is confirmed by ramirez and colleagues in spontaneous mouse model of breast cancer showing alpha 2 beta 1 integrin suppresses metastasis .
haidari and colleagues recently reported evidence that integrin alpha 2 beta 1 mediates tyrosine phosphorylation of vascular endothelial cadherin induced by invasive breast cancer cells , which is essential for transendothelial migration ( tem ) of invasive cancer cells . in our
in vitro 3d angiogenesis assay , antagonists for integrin alpha 2 beta 1 demonstrate minimal inhibitory effect on sprout angiogenesis of hdmec in 3d fibrin matrices .
interestingly , when combined with echistatin , a disintegrin antagonist specific for integrin alpha v beta 3 and antagonists for integrin alpha 2 beta 1 synergistically inhibit sprout angiogenesis of hdmec in fibrin . taken together , these results suggest that fibrin and collagen differentially but synergistically regulate sprout angiogenesis through controlling integrin alpha v beta 3 and integrin alpha 2 beta 1 functions .
this study indicated that growth factor - rich fibrin is essential to promote sprout angiogenesis .
fibrin and type i collagen 3d matrices differentially but synergistically regulate sprout angiogenesis in wound healing and solid tumors .
thus blocking both integrin alpha v beta 3 and integrin alpha 2 beta 1 might be a novel strategy to synergistically block sprout angiogenesis in solid tumors .
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### Summary:
: angiogenesis is a highly regulated event involving complex , dynamic interactions between microvascular endothelial cells and extracellular matrix ( ecm ) proteins .
alteration of ecm composition and architecture is a hallmark feature of wound clot and tumor stroma .
we previously reported that during angiogenesis , endothelial cell responses to growth factors are modulated by the compositional and mechanical properties of a surrounding three - dimensional ( 3d ) extracellular matrix ( ecm ) that is dominated by either cross - linked fibrin or type i collagen .
however , the role of 3d ecm in the regulation of angiogenesis associated with wound healing and tumor growth is not well defined .
this study investigates the correlation of sprout angiogenesis and ecm microenvironment using in vivo and in vitro 3d angiogenesis models .
it demonstrates that fibrin and type i collagen 3d matrices differentially but synergistically regulate sprout angiogenesis . thus blocking both integrin alpha v beta 3 and integrin alpha 2 beta 1 might be a novel strategy to synergistically block sprout angiogenesis in solid tumors
.
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: substance abuse is a common phenomenon in the world and has invaded the human society as the most important social damage .
substance abuse is a nonadaptive model of drug use , which results in adverse problems and consequences , and includes a set of cognitive , behavioral , and psychological symptoms .
iran also , due to its specific human and geographic features , has a relatively high degree of contamination .
the world health organization 's report in 2005 shows that there are about 200 million opiate addicts in the world , reporting the highest prevalence in iran and the most frequency in the 2535 year - age group .
the onset of drug use is often rooted in adolescence , and studies show that substance abuse is often related to cigarette and alcohol consumption in adolescence .
results of studies indicate that age , being male , high - risk behavirs , and the existence of a cigarette smoker in the family or among friends , the experience of substance abuse , inclination and positive thoughts about smoking have relationship with adolescent cigarette smoking .
studies also confirm that the chance of becoming a cigarette smoker among males and females is almost equal ( 11.2% ) ; however , the prevalence of regular alcohol consumption in males ( 22.4% ) is slightly higher than in females ( 19.3% ) .
few studies have been conducted in iran on adolescents patterns of substance abuse , producing various data on the prevalence and the type of consumed drugs , but there is currently no known specific pattern of substance abuse in this age group ; therefore , this review study has studied drug consumption prevalence in the student population of the country by collecting various data .
this article is a narrative review focusing on studies conducted in iran . in this research ,
all articles related to substance abuse and its patterns among high school students , which were conducted in iran and published in domestic and international journals , were investigated .
the articles were acquired from academic medical journals , research periodicals and the scholar google , magiran , irandoc , and medlib .
this study explored articles in the past 10 years ( 20012011 ) about iranian high school students .
the full texts of the articles were often accessible in the scientific information database and magiran websites , but the full text of the article about gilan province was obtained after contacting the journal 's office .
correspondence was made with the author of the article about mahriz city to obtain the article as it was not published in the toloee behdasht journal .
these articles provide information about the consumed drug type , its prevalence in terms of the sex and age , and the experience of at - least - once consumption in the adolescent 's life .
some articles had only pointed to drug consumption , which was also included in this research . some had attended to substance abuse in general terms without distinguishing different kinds of drugs , and in some articles only psychoactive drug use , was mentioned .
the cases , in which the sample volume was not sufficient , or were not in the studied age groups , were excluded from the study . due to different categorizations in these articles regarding the long - term prevalence of substance abuse or the experience of at - least - once consumption , in this study the shared aspect of these articles ,
some articles had addressed the students predisposing factors for drug abuse , in addition to drug use prevalence , which were not included in this study for being scattered .
an initial search into the data bases yielded 11 articles , two of which were related to years before the study time frame ( 1997 and 1998 ) .
furthermore , two articles were ignored , one because of its different age group ( a lower age ) and the other because it had addressed a particular district in tehran with a small sample size . these results are based on 7 articles .
all studies were about the 1419 years old group , and only three studies had distinguished between the sexes .
a study was conducted in 2003 on 500 students , from 142 high schools and vocational schools in zahedan city , using a multi - stage cluster sampling method . in total , from the total of 259 females and 216 males who completed the questionnaire , the following results were obtained .
the first experience of smoking was most often seen at the age of 14 ( 26.2% ) .
a study was conducted in 2009 on 610 students of kerman 's male pre - university centers , in which the prevalence of each drug was reported , but the total consumption prevalence was not mentioned . a study in gilan province in 20042009 on 1927 high school students , including 46% females and 54% males , showed that the percentage of at - least - once use , including and excluding cigarette , was 23.7 and 12.8 , respectively . a study in karaj city in 20092010 on 447 high school students , including
239 females and 208 males , showed that 57% had at - least - once experience of drug use , including cigarette , of this number 56.1% were male and 43.9% were female . a study in nazarabad city in 2007 on 400 3 year high school students , including 204 females and 196 males with the mean age of 17.3 , showed that drug use prevalence , including and excluding cigarette , was 24.5% and 11.1% , respectively .
a study was performed in lahijan city in 2004 on 2328 high school students , including 42.2% females and 57.8% males .
a descriptive study was conducted in 2008 on a 285-member sample of male high school students . a research on kerman 's male pre - university students yielded the following results .
the consumption prevalence of hookah was 15.5% , sedatives ( without medical prescription ) 40.7% , alcohol 37.7% , cigarette 34.6% , strong analgesics 10.2% , nas 9.7% , opium 8.7% , hashish 6.7% , ecstasy 6.6% , and heroin 4.9% .
consumption prevalence for each drug type in gilan : the prevalence was 20% for cigarette , 10.5% for alcohol , 2.4% for opium , 1.2% for ecstasy , 2% for hashish , and 0.3% for heroin . in karaj city ,
the consumption prevalence was 53% for hookah , 24.8% for cigarette , 13.6% for alcohol , 2% for ecstasy , 2% for opium , 1.1% for hashish , 0.4% for crystal , and 0.2% for heroin .
in nazarabad city , the consumption prevalence was found to be 23.1% for cigarette , 2% for opium , 1% for amphetamines and ecstasy , 0.5% for heroin , 0.3% for hashish and cocaine .
the male and female drug consumption was 69.7% and 36.2% , respectively , representing a significant statistical difference ( p < 0.05 ) .
a study in lahijan city showed that the consumption prevalence was 14.9% for cigarette , 2.4% for ecstasy , 4.1% for other drug types
( with the highest rate of consumption for opium and hashish ) . in the mahriz city of yazd , the consumption prevalence among the male 3 year high school students in 2008 was reported 6.8% for alcohol and 3% for psychoactive substances [ table 1 ] .
the comparison of the prevalence of at - least - once drug use for each drug type in each studied region a study in zahedan also reported that at - least - once drug use prevalence was 1.6% and 8% , respectively , among females and males ; and at - least - once cigarette smoking prevalence was 7.8% and 25.2% , respectively , for females with the mean age of 15.8 and males with the mean age of 16 . in gilan , drug use , excluding cigarette , was reported 19.1% and 5.3% , respectively , for males and females , representing a significant statistical difference ( p < 0.05 ) .
furthermore , cigarette and drug use prevalence was 31.3% and 14.8% in males and females , respectively , showing that this rate was significantly higher in males ( p < 0.05 ) .
cigarette use prevalence was 25.9% and 3% , respectively , for male and female students .
opium consumption was 3.3% and 1.5% among males and females , respectively , which was a significant statistical difference ( ) .
drug consumption , excluding cigarette , was 19.1% and 5.3% , respectively , for males and females , pointing to a statistically significant difference ( p < 0.05 ) .
ecstasy use prevalence was reported 3% and 1.1% , respectively , for males and females , pointing to a statistically significant difference ( p < 0.00081 ) ; 0.5% of males and 0.1% of females were heroin consumers , lacking any significant statistical difference ( p > 0.05 ) . in karaj city ,
drug consumption prevalence was studied for each sex and drug type [ table 2 ] . the comparison of the prevalence of at - least - once drug consumption for each sex in each studied region as the study conducted on students with the mean age of 16 in zahedan showed that the highest incidence of the first experience of cigarette smoking belonged to the age of 14 . a study in kerman on students with the mean age of 17.9 about the age at the first experience yielded the following results for each drug type : 14 for cigarette , 14.6 for alcohol , 13.9 for hookah , 13.1 for sedatives , 15.3 for analgesics , 17 for ecstasy , 16.7 for hashish , 16.7 for heroin , 16.7 for opium , and 15.3 for naswar .
a study in gilan indicated that drug and cigarette consumption had significantly increased in males aged 19 and above ( 88.9% of males aged 19 and above ) ( p < 0.05 ) . according to a study in nazarabad , the highest drug use onset was at the age of 1516 .
a study was conducted in 2003 on 500 students , from 142 high schools and vocational schools in zahedan city , using a multi - stage cluster sampling method . in total , from the total of 259 females and 216 males who completed the questionnaire , the following results were obtained .
the first experience of smoking was most often seen at the age of 14 ( 26.2% ) .
a study was conducted in 2009 on 610 students of kerman 's male pre - university centers , in which the prevalence of each drug was reported , but the total consumption prevalence was not mentioned . a study in gilan province in 20042009 on 1927 high school students , including 46% females and 54% males , showed that the percentage of at - least - once use , including and excluding cigarette , was 23.7 and 12.8 , respectively . a study in karaj city in 20092010 on 447 high school students , including
239 females and 208 males , showed that 57% had at - least - once experience of drug use , including cigarette , of this number 56.1% were male and 43.9% were female . a study in nazarabad city in 2007 on 400 3 year high school students , including 204 females and 196 males with the mean age of 17.3 , showed that drug use prevalence , including and excluding cigarette , was 24.5% and 11.1% , respectively .
a study was performed in lahijan city in 2004 on 2328 high school students , including 42.2% females and 57.8% males .
a descriptive study was conducted in 2008 on a 285-member sample of male high school students .
the consumption prevalence of hookah was 15.5% , sedatives ( without medical prescription ) 40.7% , alcohol 37.7% , cigarette 34.6% , strong analgesics 10.2% , nas 9.7% , opium 8.7% , hashish 6.7% , ecstasy 6.6% , and heroin 4.9% .
consumption prevalence for each drug type in gilan : the prevalence was 20% for cigarette , 10.5% for alcohol , 2.4% for opium , 1.2% for ecstasy , 2% for hashish , and 0.3% for heroin . in karaj city ,
the consumption prevalence was 53% for hookah , 24.8% for cigarette , 13.6% for alcohol , 2% for ecstasy , 2% for opium , 1.1% for hashish , 0.4% for crystal , and 0.2% for heroin .
in nazarabad city , the consumption prevalence was found to be 23.1% for cigarette , 2% for opium , 1% for amphetamines and ecstasy , 0.5% for heroin , 0.3% for hashish and cocaine .
the male and female drug consumption was 69.7% and 36.2% , respectively , representing a significant statistical difference ( p < 0.05 ) .
a study in lahijan city showed that the consumption prevalence was 14.9% for cigarette , 2.4% for ecstasy , 4.1% for other drug types
( with the highest rate of consumption for opium and hashish ) . in the mahriz city of yazd , the consumption prevalence among the male 3 year high school students in 2008 was reported 6.8% for alcohol and 3% for psychoactive substances [ table 1 ] .
the comparison of the prevalence of at - least - once drug use for each drug type in each studied region
a study in zahedan also reported that at - least - once drug use prevalence was 1.6% and 8% , respectively , among females and males ; and at - least - once cigarette smoking prevalence was 7.8% and 25.2% , respectively , for females with the mean age of 15.8 and males with the mean age of 16 .
in gilan , drug use , excluding cigarette , was reported 19.1% and 5.3% , respectively , for males and females , representing a significant statistical difference ( p < 0.05 ) .
furthermore , cigarette and drug use prevalence was 31.3% and 14.8% in males and females , respectively , showing that this rate was significantly higher in males ( p < 0.05 ) .
cigarette use prevalence was 25.9% and 3% , respectively , for male and female students .
opium consumption was 3.3% and 1.5% among males and females , respectively , which was a significant statistical difference ( ) .
drug consumption , excluding cigarette , was 19.1% and 5.3% , respectively , for males and females , pointing to a statistically significant difference ( p < 0.05 ) .
ecstasy use prevalence was reported 3% and 1.1% , respectively , for males and females , pointing to a statistically significant difference ( p < 0.00081 ) ; 0.5% of males and 0.1% of females were heroin consumers , lacking any significant statistical difference ( p > 0.05 ) . in karaj city ,
drug consumption prevalence was studied for each sex and drug type [ table 2 ] .
the comparison of the prevalence of at - least - once drug consumption for each sex in each studied region
as the study conducted on students with the mean age of 16 in zahedan showed that the highest incidence of the first experience of cigarette smoking belonged to the age of 14 . a study in kerman on students with the mean age of 17.9 about the age at the first experience yielded the following results for each drug type : 14 for cigarette , 14.6 for alcohol , 13.9 for hookah , 13.1 for sedatives , 15.3 for analgesics , 17 for ecstasy , 16.7 for hashish , 16.7 for heroin , 16.7 for opium , and 15.3 for naswar
a study in gilan indicated that drug and cigarette consumption had significantly increased in males aged 19 and above ( 88.9% of males aged 19 and above ) ( p < 0.05 ) . according to a study in nazarabad , the highest drug use onset was at the age of 1516 .
exploring the mft performed in the usa on the 10 graders showed that drug use had increased from 11% to 34% during 19921996 . in 1998 , 12.10% of the 8 year and 12.5% of the 10 graders and 25.611 % had experienced illegal drug use in the previous month . it was shown that hashish , followed by opium and alcohol , is the most commonly used illicit drug .
the immediate necessity of planning for reducing the consumption of these drugs among students , and consequently among university students , has become increasingly important .
investigating addictive drugs prevalence among university students showed the prevalence in the following order : hookah ( 74.5% ) , cigarette ( 67.5% ) , opium ( 6.1% ) , alcohol ( 13.5% ) , psychoactive pills ( 5.26% ) , hashish and heroin .
results of a meta - analysis showed that 7% of iranian adolescents regularly smoke , and 27% had experienced smoking .
the increased cigarette use prevalence among iranian adolescents is a major public health concern . paying attention to healthy recreations for adolescents and
the cross - sectional prevalence of drug use in 1997 among american 1217 years old adolescents was reported 11.4% , which was close to drug use prevalence , excluding cigarette .
another study showed that 56% of male and 42% of female university students were drug users , which accords with the present research with regard to the higher number of the males .
since , the addiction problem is an old problem in other countries , it might be better to use the solutions practiced by them to speed up our reaction in cases which adhere to our culture and customs . at - least - once alcohol use prevalence among
the 8 year american students in 2005 and 2006 was 27% and 20% , respectively , increasing to 88% among the 12 year students .
the history of hashish consumption among the 8 , the 10 , and the 12 year students was 10% , 23% , and 36% , respectively , representing a remarkable difference with our country 's students . about 0.5% of the 8 year and 10% of the 12 year students consumed cocaine , and the consumption of amphetamines by the 12 year students was 1.5% , being almost close to the consumption rate of iranian students .
the open consumption of hashish is common in france by almost one - third of the population ( nearly 30% ) , compared with the average rate of 19% in european countries ; also the consumption of ecstasy and cocaine has increased over 20002005 , although it is 4% but yet remarkable .
a study on students knowledge of narcotics in rafsanjan and yazd cities showed that 5.6% of yazdian and 10% of rafsanjanian students had at least one addicted person in their families .
also , 2.23% of the yazdian and 7% of the rafsanjanian students held that narcotics could also be useful .
the important issue here is the existence of an addicted relative and his or her leadership role in this regard ; therefore , this point suggests the further importance of the sensitivity of this age group with regard to their dependence on narcotics .
it is noteworthy that kerman city , compared to other studied cities , has received higher rates of drug use , such that opium and heroin consumption in this city has been , respectively , almost 4 and 5 times that of other cities .
these statistics also hold true clearly with regard to ecstasy and alcohol consumption , each being almost 3 times that of karaj and gilan .
hashish consumption in the pre - university stage in this city is also higher than in other cities , which might be related to easier drug access in kerman . in the cities , in which sex - distinct studies were conducted , drug consumption by males had been , with no exception , far higher than by the females , which is , almost 4 times except for hookah and then cigarette .
of course , it is not possible to judge firmly about drug use general prevalence as a result of the few studies in this field ; however , the important point is the relatively high drug use among the adolescent and effective group of the society , which deserves particular attention for education and intervention in this group .
it has been observed that adolescent and young crystal users , compared to nonusers , show clinical symptoms , have less control and affection in their families , with excitable , aggressive and anxious personalities , and low accountability ; on the other hand , behavioral problems and friend influence are among the strongest risk factors of drug consumption among adolescent consumers . nevertheless , it is not clear to what extent the adolescent can manage the effect of behavioral problems and peer group interaction for refusing invitations for drug consumption .
it has been stated that using software programs would assist in the prevention and increasing the youth 's skills for reducing drug use .
it has been shown that adolescent inclination to and consumption of drugs decrease significantly in the 1 year of educational intervention .
on the other hand , studies indicate that there is a relationship between the borderline personality disorder and the extent of drug abuse . therefore , prevention programs for harm reduction , treatment and consultation as the main objective of the intervention structure should apply to consumers .
also , emphasis should be laid upon the relationship between schools and parental care as important protective factors for adolescents health .
adolescence is a growth period which is associated with a relatively high rate of drug use and its related disorders .
accordingly , recent progress in evaluating drug abuse among adolescents would continue for information sharing in the field of clinical and research services .
therefore , attention to this group through coherent planning for damage prevention would still remain in priority .
drug use is relatively high in the adolescent and effective group of the society , which requires particular attention and prompt and immediate intervention .
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### Summary:
: background : as the first experience of substance abuse often starts in adolescence , and studies have shown that drug use is mainly related to cigarette and alcohol consumption , an initial exploration of substance abuse prevalence , including cigarette and alcohol , seems to be the first step in preventing and controlling drug consumption .
this study aimed to explore studies on drug use among high school students by investigating articles published in the past decade in iran.methods:in this study , the databases inside the country were used to access articles related to substance abuse by students during 20012011 , among which 7 articles on 1419 years old high school students were studied.results:the seven studied articles showed that the highest drug use prevalence pertained to cigarette and hookah , followed by alcohol , opium , ecstasy , hashish and heroin .
opium and heroin use in kerman city were , respectively , about 4 and 5 times of their use in other studied cities.conclusions:drug use is relatively high in the adolescent and effective group of the society , which requires particular attention and prompt and immediate intervention .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: obesity is a global epidemic and a major risk factor in the development of metabolic syndrome and diabetes associated complications such as cardiovascular disease , kidney disease , hypertension , and neuropathies [ 13 ] .
epoxyeicosatrienoic acids ( eets ) are arachidonic acid derived metabolites generated by a family of cytochrome p450 ( cyp ) monooxygenases and epoxygenases [ 4 , 5 ] .
eets are rapidly hydrolyzed by ros and by soluble epoxide hydrolase ( seh ) to their respective dihydroxyepoxytrienoic acids [ 68 ] .
eet agonists prevent both adiposity and vascular complications both in vitro and in vivo and obesity - induced adipose tissue expansion impairs the cyp epoxygenase pathway and the generation of eet in vivo [ 912 ] .
mitochondrial biogenesis , oxygen consumption , and oxidative phosphorylation are regulated by peroxisome proliferator - activated receptor gamma coactivator 1-alpha ( pgc-1 ) , which in turn is regulated by posttranslational modifications , including phosphorylation and deacetylation by ampk and sirt1 , respectively .
pgc-1 activates several key components of the adaptive thermogenesis program , including the stimulation of energy uptake , and mitochondrial fatty acid oxidation .
transgenic mice with mildly elevated muscle levels of pgc-1 are resistant to age - related obesity .
furthermore , mice that are lacking pgc-1 in adipose tissue and fed hf diet develop insulin resistance and have increased circulating lipid levels .
additionally , the uncoupling proteins 13 ( ucp13 ) are located in the mitochondrial intramembranous space and play a key role in thermogenesis .
ucp-1 is highly expressed in brown adipose tissue in a pgc-1-dependent manner to increase energy expenditure and oxygen consumption .
heme oxygenase-1 ( ho-1 ) is a stress response enzyme which in rodents and humans degrades heme to carbon monoxide , iron , and the potent antioxidant and anti - inflammatory molecule biliverdin , which is subsequently degraded to bilirubin [ 18 , 19 ] , thereby offering increased protection against obesity - induced ros and hypertension .
a strong case has been made for the existence of a positive feedback loop between eet and ho-1 .
reported that eet - mediated vascular dilation is dependent on ho-1 expression and eets increase ho-1 protein levels and ho activity in vitro [ 21 , 22 ] .
a decrease in ho-1 levels increases adipocyte hypertrophy contributing to elevated liver fat content and steatohepatitis that is associated with mitochondrial dysfunction .
the majority of cellular ros that contributes to increased adipogenesis is generated by the mitochondria and contributes to energy metabolism .
one of the seven mammalian sirtuins , sirtuin 3 ( sirt3 ) , a mitochondrial deacetylase , was recently reported to be the target of pgc-1 and impact mitochondrial processes , such as mitochondrial biogenesis , suppression of ros , and energy metabolism , including mitochondrial fatty acid oxidation .
mitochondrial energy and metabolic demands as well as viability are tightly linked to mitochondrial network morphology and depend greatly on quality control and a balanced relationship between mitochondrial fusion ( the merge of dysfunctional to functional ) and fission ( budding and isolation of dysfunctional mitochondria ) processes .
mitochondrial fission is orchestrated by the dynamin - related protein 1 ( drp1 ) and the mitochondrial fission 1 ( fis1 ) protein [ 25 , 26 ] , while the fusion process is controlled by the autosomal dominant optic atrophy 1 ( opa1 ) protein , located on the mitochondrial inner membrane , together with the mitochondrial fusion proteins mitofusins 1 and 2 ( mfn 1 and 2 ) , located on the mitochondrial outer membrane [ 27 , 28 ] . studies of the balance between fission and fusion have shown that development of obesity and insulin resistance is associated with a reduction in mitochondrial fusion [ 26 , 29 , 30 ] and increased mitochondrial fission . given the regulatory role of pgc-1 on adipogenesis and mitochondrial function , we hypothesize that the eet - mediated modulation of adiposity and the subsequent increase of mitochondrial fusion , oxidative phosphorylation , and ho-1 expression is dependent upon pgc-1. further , the effect of pgc-1-deficiency on metabolic parameters and mitochondrial biogenesis , function , and fusion potential was performed using lentiviral gene delivery that is effective for 9 months .
all animal experiments followed a pla , general hospital , beijing , china , and nymc iacuc institutionally approved protocol in accordance with the nih guidelines .
two separate experiments ( a and b ) were performed . in the first experiment ( a ) we investigated the effect of pgc-1 ablation and short - term eet - a treatment of mice fed a hf diet for 8 weeks . in the second experiment ( b ) we examined the effect of an eet - a regimen in mice fed a hf diet for 24 weeks : for experiment ( a ) with pgc-1-deficient mice , animals were divided into 4 groups : ( 1 ) lean , ( 2 ) hfd , ( 3 ) hfd+eet - analog [ eet - a is sodium ( s , z)-2-(13-(3-pentylureido)tridec-8-enamido)succinate ] , and ( 4 ) hfd+eet - a+pgc-1 lentivirus .
lean mice ( group 1 ) were fed ad libitum a normal chow diet containing 11% fat , 62% carbohydrate , and 27.0% protein with total calories of 12.6 kj / g .
the remaining animals ( groups 2 , 3 , and 4 ) were fed a hf diet containing 58% fat ( from lard ) , 25.6% carbohydrate , and 16.4% protein with total calories of 23.4 kj / g ( harlan , teklad lab animal diets , us ) for 8 weeks .
mice were treated as follows : group ( 1 ) was fed normal chow diet , group ( 2 ) was fed hf diet , group ( 3 ) was injected with eet - a , intraperitoneally , every other day for 4 weeks at a dose of 1.5 mg/100 gm of body weight , and group ( 4 ) received a 2-bolus injection of pgc-1(sh ) lentivirus ( dharmacon , lafayette , co ) injected into the retroorbital vein at a concentration of 4070 10
tu / mouse in 80100 l and received eet - a injections identical to group 3 . for the second experiment ( long - term effect of eet - agonist )
( b ) mice were divided into three groups similar to groups 1 , 2 , and 3 in experiment ( a ) but were maintained on the diets for 24 weeks and group ( 3 ) received injections with eet - a every other day for the last 8 weeks of the experiment . for fasting blood glucose level measurement mice were fasted for 6 h with access to water , after blood was obtained from tail and blood glucose concentration was determined with the accuchek advantage glucometer ( roche , madison , wi ) . at the end of the experimental period , mice were anesthetized with sodium pentobarbital ( 65 mg / kg , i.p . ) and , at the time of sacrifice , body weight was measured .
3t3-l1 murine preadipocytes were purchased from atcc ( atcc , manassas , va ) . after thawing ,
3t3-l1 cells were cultured at 37c in a 5% co2 incubator in -minimal essential medium ( -mem , invitrogen , carlsbad ca ) supplemented with 10% heat inactivated fetal bovine serum ( fbs , invitrogen , carlsbad , ca ) and 1% antibiotic / antimycotic solution ( invitrogen , carlsbad , ca ) .
the medium was changed after 48 h and every 3 - 4 days thereafter as described previously . for adipogenesis studies
the medium was replaced with adipogenic medium ( dulbecco 's modified eagle medium ( dmem ) ) with high glucose ( invitrogen ) , supplemented with 10% ( v / v ) fbs , 10 g / ml insulin ( sigma - aldrich , st .
louis , mo ) , 0.5 mm dexamethasone ( sigma - aldrich ) , and 0.1 mm indomethacin ( sigma - aldrich ) and the cells were cultured for an additional 8 days .
cells were cultured in the absence and presence of eet - a at a dose of 10 m . at the experimental endpoints ,
cells were collected by trypsinization , washed once with pbs , and then lysed for protein measurements and for rna extraction .
smart vector lentiviral shrna - ppargc1a or scrambled rna ( dharmacon , lafayette , co ) was applied to 3t3-l1 cells to establish a stably transduced cell line .
briefly , 1 10 cells were seeded in 6-well plates 1 day prior to transduction . on the day of transduction ,
the transduction medium was made by 1 10 transducing units ( tu ) of lentiviral particles with 0.5 ml -mem growth medium being applied to each well and incubated for 3 h to maximize the contact between each cell and lentiviral particles .
cells were also treated with the transduction medium without lentiviral particles which served as untransduced control .
growth medium ( 1.5 ml ) was then added to each well in the presence of 8 g / ml polybrene ( final concentration ) .
after 48 h incubation , antibiotic selection medium ( -mem growth medium with 10 g / ml puromycin ) was used to kill all the untransduced cells .
total rna was extracted from 3t3 cells using trizol ( ambion , austin , tx ) and from frozen adipose tissue by rneasy lipid tissue ( qiagen ) , as per instructions provided by the manufacturers .
rna was determined by measuring the absorbance at 260 nm ( a260 ) with a biotek plate reader and the take3 plate ( biotek , winooski , vt ) , and assessed by the a260/a280 ratio .
cdna was synthesized from total rna using the high capacity cdna reverse transcription kit ( applied biosystems ) , after real - time pcr was performed using taqman fast universal master mix ( 2x ) , on a 7500 ht fast real - time pcr system ( applied biosystems ) .
specific taqman gene expression assays probes for mouse ho-1 , pgc1 , ucp1 , cox - iv ( cytochrome c oxidase subunit - iv ) , adiponectin , tnf , il-6 , mfn1 , mfn2 , drp1 , fis1 , opa1 , ap2 , sirt3 , c / ebp , and gapdh were used as previously described .
frozen mouse adipose tissue was ground under liquid nitrogen and suspended in homogenization buffer ( mmol / l : 10 phosphate buffer , 250 sucrose , 1.0 edta , 0.1 pmsf , and 0.1% v / v tergitol , ph 7.5 ) . for in vitro western blot analysis pelleted cells were lysed with lysis buffer supplemented with protease and phosphatase inhibitors ( complete mini and phosstop , roche diagnostics , indianapolis , ia ) .
immunoblotting for ho-1 , sirt1 , ucp1 , tfam , ap2 , peg1/mest , mnsod , ampk , pampk , akt , pakt , fas , and pgc-1 and phosphorylation of insulin receptors ( ir ) irp972 , irp1146 , cox - iv , adiponectin , -actin , and gapdh were performed as previously described [ 8 , 34 ] .
oxygen consumption ( vo2 ) and carbon dioxide production ( vco2 ) were measured using the oxylet gas analyzer and air flow unit ( panlab - bioseb , vitrolles , france ) .
animals were allowed to acclimatize in the oxygen consumption chambers for 2 hours three times a week for 3 weeks prior to the recording of data .
mice were placed in the oxylet chamber and the flow rate was adjusted and the dco2 maintained between 0.4 and 0.8 as per the instructions provided by the manufacturer , after the hourly respiratory quotients were calculated based on the vco2 and vo2 obtained by the gas analyzer .
the data for vo2 are expressed as the consumed volume of oxygen per kilogram body weight per minute ( ml / kg / min ) .
significance of difference in mean values was determined using one - way analysis of variance followed by the bonferroni posttest for comparison between groups .
to examine whether eet - a can modulate the mitochondrial fusion - to - fission ratio through pgc-1 we knocked down pgc-1 in 3t3-l1 adipocyte cells using lentivirus vector carrying target specific shrna .
rt - pcr data show more than 80% inhibition of pgc1 mrna levels after transduction with pgc-1 shrna ( figure 1(a ) ) and a concomitant decrease in ho-1 mrna expression ( figure 1(b ) ) .
pgc-1 knockdown resulted in increased mrna expression of fis1 ( p < 0.05 ) as compared to wt cells .
the mrna expression of drp1 was not affected by pgc-1 silencing . while eet - a did not affect fission related expression in wt cells ( figures 1(c ) and 1(d ) ) , the mfn2 levels were elevated ( p < 0.05 ) in wt cells treated with eet - a as compared to wt control cells .
the mrna levels of opa1 were not increased by eet - a ( figure 1(e ) ) .
pgc-1 silencing decreased ( p < 0.05 ) mfn2 and opa1 mrna expression as compared to wt control cells .
importantly , eet - a had no effect on mfn2 or opa1 mrna expression levels in pgc-1 deficient cells ( figures 1(e ) and 1(f ) ) .
the mrna expression levels of sirt3 and cox - i were decreased ( p < 0.05 ) in the pgc-1 knockdown cells as compared to wt cells . while eet treatment had only moderate effect on the expression of sirt1 , sirt3 , and cox - i in wt cells , the expression of these genes were not affected by eet - a treatment of pgc-1 deficient cells ( figures 1(g ) , 1(h ) , and 1(i ) ) .
we examined the effect of eet - a on body weight , blood pressure , fasting blood glucose , o2 consumption , and the ratio of co2/o2 in mice fed a hf diet for 8 and 24 weeks ( figure 2 ) .
as expected , mice on a hf diet displayed a decrease ( p < 0.05 ) in vo2 consumption .
however , hf diet fed mice treated with eet - a exhibited an increase ( p < 0.05 ) in oxygen consumption with concomitant lowering of vco2/vo2 .
importantly , eet - a had no effect on oxygen consumption or respiratory quotient in hf diet fed pgc-1 deficient mice ( table 1 ) .
fasting blood glucose levels and blood pressure were increased in mice fed a hf diet as compared to control animals ( table 1 , p < 0.05 ) .
importantly , fasting blood glucose levels were normalized in mice fed a hf diet and treated with eet - a ( p < 0.01 ) when compared to lean mice .
the eet - a mediated decrease in fasting blood glucose levels was prevented in pgc-1 deficient animals fed a hf diet .
importantly , regardless of the duration , eet - a treatment normalized all four metabolic parameters ( table 1 ) .
pgc-1 mrna expression in hf diet fed mice treated with vehicle ( veh ) solution ( h2o ) was reduced ( p < 0.05 ) as compared to lean mice .
importantly eet - a treatment nullified the inhibitory effect of hf diet on pgc-1 expression .
eet - a treatment of pgc-1-deficient mice had no effect on pgc-1 mrna expression ( figure 3(a ) ) .
the mrna expression levels of mitochondrial fission related drp1 and fis1 in adipose tissue were increased ( p < 0.05 ) by a hf diet .
interestingly , eet - a treatment of mice on a hf diet decreased ( p < 0.05 ) the adipose tissue levels of both drp1 and fis1 as compared to hf diet fed mice .
inversely , drp1 and fis1 mrna levels vat of pgc1-deficient mice were increased ( p < 0.05 ) as compared to hf diet fed mice treated with eet - a alone ( figures 3(b ) and 3(c ) ) .
the expression levels of fusion related mfn1 and mfn2 were significantly decreased by a hf diet in adipose tissue .
prominently , eet - a treatment of mice on a hf diet increased ( p < 0.05 ) the expression levels of both mfn1 and mfn2 as compared to hf diet fed mice ( figures 3(d ) and 3(e ) ) .
the observed increase was pgc-1-dependent as the levels of both mfn1 and mfn2 in adipose tissue from pgc1-deficient mice reduced ( p < 0.05 ) as compared to hf diet fed mice treated with eet - a alone ( figures 3(d ) and 3(e ) ) .
mice fed a hf diet had a decrease ( p < 0.05 ) in the protein expression of irp - tyr 972 , irp - tyr 1146 , pampk , and pakt .
mice fed a hf diet and treated with eet - a displayed an 8.2-fold increase ( p < 0.001 ) in irp - tyr 972 and a 7.3-fold increase in irp - tyr 1146 ( p < 0.01 ) as compared to mice fed a hf diet alone ( figure 4 ) .
likewise , eet - a increased the pakt / akt and pampk / ampk ratio ( p < 0.05 ) when compared to mice fed a hf diet alone ( figure 4 ) .
western blot analysis demonstrated that mice fed a hf diet for 24 weeks had decreased levels of ho-1 protein expression ( figure 5 ) as compared to mice fed a normal chow diet .
treatment with eet - a for 8 weeks resulted in a 16-fold increase ( p < 0.01 ) in ho-1 protein expression as compared to mice fed a hf diet alone ( figures 5(a ) and 5(e ) ) .
visceral adipose tissue obtained from mice fed a hf exhibited a decrease ( p < 0.05 ) in pgc-1 , sirt1 , and adiponectin expression as compared to lean mice fed a regular chow diet , while eet - a increased ( p < 0.05 ) pgc-1 , sirt1 , and adiponectin expression levels as compared to the mice fed a hf diet alone ( figure 5 ) .
our analysis demonstrated a decrease ( p < 0.05 ) in the ucp1 , tfam , and mnsod expression levels in adipose tissue of mice fed a hf diet as compared to lean mice fed a regular chow diet .
eet - a increased ( p < 0.05 ) mnsod , ucp1 , and tfam expression as compared to mice fed a hf diet alone ( figure 6 ) .
mrna expression of cox - iv was elevated in obese mice treated with eet- a ( group 3 ) ( p < 0.05 ) ( figure 6 ) .
eet - a - treatment visibly reduced weight gain in mice fed hfd ( figure 2(a ) ) , an effect that was prevented in pgc-1-deficient mice .
the final weights after 4 or 8 weeks of vehicle or eet - a treatment are presented in table 1 .
the eet - a - mediated reduction of weight gain was observed as early as after 4 weeks . as seen in figure 2(b ) , visceral fat in control mice fed hfd was decreased by eet - a - treatment , but this eet - a - mediated effect was prevented in pgc-1-deficient mice .
a hf diet increased ( p < 0.05 ) the expression of adipogenic marker proteins fas , ap2 , and mest ( figures 7(a)7(d ) ) as compared to lean mice fed a regular chow diet .
mice treated with eet - a had decreased protein levels of fas , mest , and ap2 in adipose tissue as compared to mice fed a hf diet alone ( figures 7(a)7(d ) ) ( p < 0.05 ) . a hf diet was associated with an increase ( p < 0.05 ) in the adipose tissue expression of ap2 and c / ebp mrna levels as compared to lean mice fed a regular chow diet ( figures 7(e ) and 7(f ) ) . however , mice fed a hf diet and treated with eet - a exhibited decreased ap2 and c / ebp mrna levels in adipose tissue as compared to mice fed a hf diet ( figures 7(e ) and 7(f ) ) ( p < 0.05 ) . adipose tissue of mice fed a hf diet presented with an increase ( p < 0.05 ) in the mrna expression levels of the inflammatory mediators , tnf and il-6 , in adipose tissue as compared to lean mice .
administration of eet - a attenuated ( p < 0.01 ) the hf diet induced increase in tnf and il-6 levels in mouse adipose tissue ( figures 7(g ) and 7(h ) ) .
this study demonstrates that eet participates in the regulation of mitochondrial function in adipocyte cells in vitro and in adipose and hepatic tissues in vivo by influencing the levels of pgc-1 and ho-1 expression .
we show that eet is upstream of pgc-1 and that contributes to increased mitochondrial biogenesis , function , and fusion potential , leading to an improvement of metabolic parameters in obese mice that is pgc-1 dependent .
eet was effective in the restoration of mitochondrial integrity in the early stage of obesity ( 8 weeks ) as well as in chronic obesity ( 24 weeks ) .
firstly we show that lentiviral vector - mediated knockdown of pgc-1 cultured murine adipocytes in vitro increases the mitochondrial fission potential ( drp1 and fis1 ) at the expense of mitochondrial quality control and fusion potential ( opa1 , mfn2 ) as well as reducing the expression of genes involved in oxidative phosphorylation and suppression of ros ( cox - i , sirt1 , and sirt3 ) .
while treatment of wt adipocytes with eet - agonist had a beneficial effect on the mitochondrial function and fusion potential , these effects were absent in pgc-1-deficient adipocytes . in vivo ,
a hf diet precipitated increased expression of the genes regulating mitochondrial fission , while concomitantly reducing the expression of the genes responsible for mitochondrial quality control and fusion processes in visceral adipose tissue as compared to mice fed a regular chow diet .
eet - agonist treatment of mice fed a hf diet resulted in a reduction in mitochondrial fission potential and a normalized or an enhanced expression of mitochondrial fusion - associated genes . moreover , while the expression of pgc-1 in obese mice was reduced when compared to lean mice , the levels of pgc-1 in hf diet fed mice treated with an eet - agonist were higher than in both lean mice and mice fed a hf diet alone .
importantly , global knockdown of pgc-1 in mice fed a hf diet nullified the eet - a - mediated beneficial effects on mitochondrial fusion and ros suppressing potential in visceral adipose tissue . increasing hf intake has been shown to enhance free fatty acid generation and increase mitochondrial dysfunction and ros [ 3538 ] . together
, these results clearly indicate that a recruitment of pgc-1 is crucial to the beneficial effects of the eet - agonist on mitochondrial function and on the reduction of fission and increase of fusion - associated processes in both adipose and hepatic tissues .
our observation linking eets to mitochondrial viability and fusion potential is supported by the demonstration that eets protect neurons from oxidative damage while stimulating mitochondrial biogenesis .
approximately 90% of cellular ros are generated by the mitochondria and contribute to the mitochondrial energy metabolism .
sirt3 is important in ros suppression , mitochondrial biogenesis , and metabolic homeostasis , including mitochondrial fatty acid oxidation , and the target of pgc-1 [ 23 , 24 ] .
multiple studies have reported the importance of mitochondrial function and dynamics in health and aging ; for review see . knockout mice of mfn1 , mfn2 , and opa1 are all embryonic lethal and mutations in opa1 in humans are associated with hereditary blindness , while mfn2 mutations are the cause of charcot - marie - tooth disease [ 27 , 40 , 41 ] . since mitochondrial integrity encompasses the balance between mitochondrial fusion and fission [ 42 , 43 ] , an increase of mfn1 and mfn2 or decrease in fission drp1 and fis is an indication of mitochondrial integrity and less oxidative stress that is responsible for mitochondrial damage and subsequent clearance by mitophagy .
this is in agreement with our data that showed liver - specific knockout of mfn2 in mice is linked to impaired glucose metabolism , to decreases in sirt1 , sirt3 , and insulin signaling resulting in hepatic steatosis .
thus , we show a pgc-1-dependent increase of ho-1 expression , thereby potentiating the beneficial effect of pgc-1 itself .
these results are supported by the demonstration that doxorubicin - induced oxidative stress increased mitochondrial fragmentation and fis1 expression , causing mitophagy and mitochondrial fragmentation , an effect that was prevented by increased levels of ho-1 favoring expression of mfn1 and mfn2 .
furthermore , we report that short- and long - term treatment with eet - a normalized impaired insulin receptor phosphorylation and decreased fasting blood glucose , bw , vo2 , and rq in mice fed a hf diet .
the increase in vo2 may be related to an eet - mediated alternate metabolic strategy of hf - fed mice , that is , to increase the metabolic rate in response to increased eet - a - mediated pgc-1 expression and mitochondrial function leading to a reprogramming of adipose stem cells to brown - like cells , thus allowing the recovery of homeostatic control and the subsequent decrease in body fat , increase in insulin sensitivity , and normalization of oxygen consumption .
further , in agreement with our results an increase of pgc-1 is associated with the mechanism controlling mitochondrial biogenesis and integrity and increased insulin sensitivity .
increase of insulin receptor phosphorylation as a result of activation of ho-1 gene expression by eet - agonist is responsible for control of adiposity and insulin sensitivity ( reviewed in [ 18 , 48 ] ) .
this supports the hypotheses linking eet - a , the increase of pgc-1 , mitochondrial fusion , sirt1 , pampk , and ho-1 expression to a metabolic adipocyte reprogramming that may influence adipocyte hemostasis and delay terminal differentiation as evidenced by a decrease in peg1/mest and an increase in insulin receptor phosphorylation and adiponectin downstream signaling , including pampk .
ampk is as a master regulator of energy expenditure and performs its actions based on changes in the intracellular amp / atp ratio . in metabolic organs , such as the liver , adipose tissues , and skeletal muscle , activated
ampk stimulates catabolic processes , while inhibiting anabolic processes . as ampk through its energy expenditure regulation
also impacts the cellular redox balance of nad and nadph it can also activate sirt1 deacetylation - activity , which is dependent on cellular nad levels ; meanwhile several actions of ampk have been shown to be dependent on sirt1 activity .
ampk and sirt1 regulate pgc-1 activity through phosphorylation and deacetylation , respectively , and together ampk , sirt1 , and pgc-1 control a wide range of metabolic processes , including insulin receptor signaling and gluconeogenesis in the liver , adipocyte browning and thermogenesis , and mitochondrial biogenesis [ 50 , 51 , 53 ] .
eet - a increased pampk and sirt1 levels in both adipose and liver tissues of hf diet fed mice corroborating the eet - mediated increase in total body oxygen consumption .
the importance of pgc-1 in adipose tissue has been established in mice lacking pgc-1 specifically in adipocytes which develop insulin resistance and have increased circulating lipid levels when fed a hf diet .
pgc-1 control of energy homeostasis suggests a role as a target for both antiobesity and diabetes drugs .
the finding that eet - a restored oxygen consumption and respiratory quotient to levels found in lean mice is supported by the beneficial increase in pgc-1 , sirt1 , pampk , and cox - iv ( a unit of the mitochondrial oxphos complex ) in the adipose tissue of mice fed a hf diet and treated with eet - a , thus highlighting the role of eets in mitochondrial energy production and expenditure .
eet - a increased pgc-1 expression in both adipose and hepatic tissue of mice fed a hf diet as compared to obese mice .
pgc-1 is abundantly expressed in tissues with high energy demand [ 13 , 47 ] , and its induction , along with ampk and sirt1 , regulates mitochondrial oxidative metabolism as well as mitochondrial biogenesis [ 52 , 53 , 55 ] .
thus , pgc-1 is critical in the activation of mitochondrial function including quality control , fusion potential , thermogenesis , and energy expenditure .
pgc-1 , ucp1 , tfam , and ho-1 levels were increased in adipose tissue of mice treated with eet - a , thus signifying a role of eets in thermogenesis and browning of adipose tissue , which is masterly regulated via pgc-1 activation [ 52 , 53 , 56 ] .
eet - mediated induction of pgc-1 and ho-1 favors an increase in mitochondrial fusion , biogenesis , and oxphos , thereby providing metabolic protection in hf diet induced obesity in mice ; this finding is supported by recent reports by us and others showing that ho-1 regulates mitochondrial function , quality control , biogenesis , and dynamics [ 46 , 57 , 58 ] .
regardless of the duration of treatment , eet - a reduced hf diet induced weight gain while normalizing blood glucose levels , thus confirming the beneficial eet - mediated effects in diet - induced obesity [ 8 , 11 , 59 ] .
the restoration of the metabolic parameters was mirrored by the normalization of the adipogenic markers fas , ap2 , peg1/mest , and c / ebp , as well as the reduction of inflammation ( tnf and il-6 ) in visceral fat obtained from eet - a - treated mice fed a hf diet as compared to the obese control mice . the normalization of adipogenic and inflammatory markers in hf diet fed mice treated with eet - a was coupled with increased levels of adiponectin and ho-1 , both key to the maintenance of adipocyte maturation in favor of early - stage adipocyte differentiation , that is , healthy adipocytes [ 8 , 58 , 6062 ] .
the present study is of substantial interest from both a basic and a clinical science perspective , defining the existence of an eet - pgc-1-ho-1 regulatory axis that can ameliorate the deleterious effects of hf diet - induced metabolic abnormalities , including insulin resistance , and prediabetic conditions involving adipose tissues .
a deeper understanding of the mode of action of eet - pgc-1 axis necessary to maintain mitochondrial dynamics by favoring fusion over fission , increased insulin sensitivity , and improved energy expenditure will , we believe , provide new approaches in the treatment of obesity and metabolic syndrome associated complications , including insulin resistance , and subsequent cardiovascular complications .
we consider the metabolically favorable interaction of lipid mediators generated by cyp450 epoxygenase that provide the potential of therapeutic targeting of the mitochondria dynamics in order to increase fusion over fission potential and other mitochondrial associated parameters that are dependent upon pgc-1 expression .
this approach offers a portal to the treatment of metabolic diseases and hypertension with the result of an improved outcome in what is acknowledged to be a global epidemic of obesity .
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### Summary:
: background / objectives . obesity and metabolic syndrome and associated adiposity are a systemic condition characterized by increased mitochondrial dysfunction , inflammation , and inhibition of antioxidant genes , ho-1 , and eets levels .
we postulate that eets attenuate adiposity by stimulating mitochondrial function and induction of ho-1 via activation of pgc-1 in adipose and hepatic tissue .
methods .
cultured murine adipocytes and mice fed a high fat ( hf ) diet were used to assess the functional relationship among eets , pgc-1 , ho-1 , and mitochondrial signaling using an eet - agonist ( eet - a ) and pgc-1-deficient cells and mice using lentiviral pgc-1(sh ) . results .
eet - a is a potent inducer of pgc-1 , ho-1 , mitochondrial biogenesis ( cytochrome oxidase subunits 1 and 4 and sirt3 ) , fusion proteins ( mfn 1/2 and opa1 ) and fission proteins ( drp1 and fis1 ) ( p < 0.05 ) , fasting glucose , bw , and blood pressure .
these beneficial effects were prevented by administration of lenti - pgc-1(sh ) .
eet - a administration prevented hf diet induced mitochondrial and dysfunction in adipose tissue and restored vo2 effects that were abrogated in pgc-1-deficient mice .
conclusion .
eet is identified as an upstream positive regulator of pgc-1 that leads to increased ho-1 , decreased bw and fasting blood glucose and increased insulin receptor phosphorylation , that is , increased insulin sensitivity and mitochondrial integrity , and possible use of eet - agonist for treatment of obesity and metabolic syndrome .
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: pregnancy is a hypercoagulable state due to an addition in coagulation factors , a reduction in anticoagulants and a deficiency in fibrinolysis ( 1 ) .
recurrent miscarriage ( rm ) is an important clinical problem with many etiologies such as genetic , anatomic , endocrine , and infectious diseases as well as immune defects .
further - more , pregnancy complications such as rm and recurrent implantation failure have been found to be related to thrombophilic gene mutations ( 27 ) .
rm is defined as the loss of two or more successive pregnancies before 20th week of gestation and affects 1% of couples ( 8) .
methylenetetrahydrofolate reductase ( mthfr ) is an essential enzyme to the folic acid pathway . in this regard ,
insufficiencies of folic acid or defects in mthfr have demonstrated dna hypomethylation and abnormal biochemical and/or phenotypic changes in animal models ( 9 , 10 ) , cell culture ( 11 , 12 ) and humans ( 1216 ) .
mthfr is involved in the synthesis of 5-methylenetetrahydrofolate , which is a cofactor in the enzymatic formation of methionine from homocysteine ; and therefore plays a critical role in early embryonic development .
the mthfr c677 t polymorphism results in an amino acid replacement ( alanine to valine ) , which causes the decrease in the activity of the encoded enzyme at 37c or more ( 17 ) . in the mthfr a1298c allele , a polymorphism in exon 7 results in an amino acid replacement ( glutamate to alanine ) .
the activity of the encoded enzyme is decreased , although less than that in c677 t allele ( 18 ) .
most studies show the association of these two polymorphisms with rm in women ( 6 , 1921 )
. however , in couples suffering from rm , the role of mthfr gene polymorphisms in the male partner has not been well defined .
the prevalence of the mthfr mutation in combination with other thrombophilia mutations in rm couples in comparison with controls has been reported ( 22 , 23 ) . however , these studies reported no significant difference in the prevalence of inherited thrombophilia between the two groups .
the aim of this study was to analyze the frequencies of mthfr c677 t and a1298c polymorphisms in male partners of rm couples whose female partners suffered from extensive mutations in mthfr , compared to those in control couples .
two hundred and twenty five couples with at least three successive pregnancy losses below 20th week of gestation , who were referred to avicenna fertility center , tehran , iran , were selected as the case group . all females in the case group were carrying either double heterozygotic polymorphisms of mthfr c677 t and mthfr a1298c or had at least a homozygotic polymorphism at either mthfr gene loci ( these conditions are termed mthfr high risk in this study ) .
the control group included 100 couples with at least two live births and no history of pregnancy complications such as miscarriage , stillbirth , small for gestational age fetuses or pre - eclampsia .
the exclusion criteria for the case group included anatomic disorders , infectious diseases , single heterozygotic or normal genotype of mthfr gene at positions 677 and 1298 .
the mean age of the different gender groups were 32.46.0 and 35.24.5 years in females and 37.58.4 and 39.87.9 years in males , in case and control groups , respectively .
all couples entered in the study after signing informed written consent and the study was also approved by the ethical committee of avicenna research institute .
five milliliters of venous blood were collected from each individual ( both men and women ) in tubes containing edta , and dna was extracted from the samples using salting out method .
polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) amplifications of the dna samples for mthfr c677 t and a1298c genes were performed using 5-tgaaggagaa ggtgtctgcggga-3 and 5-caaggaggag ctgctgaaga-3 as forward and 5-aggacg gtgcggtgagagtg-3 and 5-ccactccag catcactcact-3 as reverse specific primers , respectively ( 24 ) .
the amplification products were analyzed on 1.5% ethidium bromide - stained agarose gels and then subjected to enzymatic digestion using hinf i and mbo ii restriction enzymes for mthfr c677 t and a1298c snps , respectively .
the genotype distributions of each snp between the case and the control groups were compared by mann - whitney u - test .
heterozygous and homozygous mutations of mthfr c677 t and a1298c and also distribution of them were compared between the case and the control groups using chi - square test .
two hundred and twenty five couples with at least three successive pregnancy losses below 20th week of gestation , who were referred to avicenna fertility center , tehran , iran , were selected as the case group . all females in the case group were carrying either double heterozygotic polymorphisms of mthfr c677 t and mthfr a1298c or had at least a homozygotic polymorphism at either mthfr gene loci ( these conditions are termed mthfr high risk in this study ) .
the control group included 100 couples with at least two live births and no history of pregnancy complications such as miscarriage , stillbirth , small for gestational age fetuses or pre - eclampsia .
the exclusion criteria for the case group included anatomic disorders , infectious diseases , single heterozygotic or normal genotype of mthfr gene at positions 677 and 1298 .
the mean age of the different gender groups were 32.46.0 and 35.24.5 years in females and 37.58.4 and 39.87.9 years in males , in case and control groups , respectively .
all couples entered in the study after signing informed written consent and the study was also approved by the ethical committee of avicenna research institute .
five milliliters of venous blood were collected from each individual ( both men and women ) in tubes containing edta , and dna was extracted from the samples using salting out method .
polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) amplifications of the dna samples for mthfr c677 t and a1298c genes were performed using 5-tgaaggagaa ggtgtctgcggga-3 and 5-caaggaggag ctgctgaaga-3 as forward and 5-aggacg gtgcggtgagagtg-3 and 5-ccactccag catcactcact-3 as reverse specific primers , respectively ( 24 ) .
the amplification products were analyzed on 1.5% ethidium bromide - stained agarose gels and then subjected to enzymatic digestion using hinf i and mbo ii restriction enzymes for mthfr c677 t and a1298c snps , respectively .
the genotype distributions of each snp between the case and the control groups were compared by mann - whitney u - test .
heterozygous and homozygous mutations of mthfr c677 t and a1298c and also distribution of them were compared between the case and the control groups using chi - square test .
pcr - rflp was carried to analyze the mthfr genotype composition of the male partners of rm couples whose females were suffering from mthfr high risk . to achieve this goal ,
225 rm women with mthfr high risk snps were selected and their male partners were genotyped for mthfr a1298c and c677 t snps .
genotype distributions in the maternal and paternal control groups of mthfr c677 t polymorphism and also maternal control group of a1298c polymorphism were in hardy - weinberg equilibrium ( p>0.05 ) .
the numbers and frequencies of mthfr c677 t and a1298c snps in males and females of the case and control groups are shown in table 1 .
the individuals in both groups were further categorized based on gender , rm / normal and the type of mthfr snps .
combined heterozygosity of the two polymorphisms in the males of the two groups is presented as a common phenomenon , 52 ( 23.1% ) and 14 ( 14% ) of males in rm and control groups , respectively .
rare combinations of a1298c / c677 t snps including homo / hetero - zygote or hetero/ homo - zygote variants were observed in males and females of the both groups ( table 1 ) .
a major observation of this study was the absence of combined homozygosity for both snps in all studied groups / genders .
the pattern of individual grouping in table 1 allowed us to further group them into mthfr high risk and low risk groups .
the data was summarized in table 2 where the males were statistically compared depending on their degree of rm risk .
surprisingly , a statistically significant difference in the frequency of mthfr high risk group among the male partners in the rm group was detected compared to that in the normal males ( p=0.019 ) .
combination of mthfr a1298c and c677 t snps in rm and normal groups rare combination of a1298c and c677 t snps including homo / hetero- or hetero / homo -zygotes ; rm : recurrent miscarriage frequency of individuals with high and low risk mthfr genotypes in different groups p=0.019 , males in mthfr high and low risk groups were compared using chi - square test ; high risk : individuals who carried either combined heterozygous c677t
/ a1298c mthfr snps or at least one homozygous snp ; low risk : individuals who carried no c677t / a1298c mthfr snps or had just one heterozygous snp ; rm : recurrent miscarriage
pcr - rflp was carried to analyze the mthfr genotype composition of the male partners of rm couples whose females were suffering from mthfr high risk . to achieve this goal ,
225 rm women with mthfr high risk snps were selected and their male partners were genotyped for mthfr a1298c and c677 t snps .
genotype distributions in the maternal and paternal control groups of mthfr c677 t polymorphism and also maternal control group of a1298c polymorphism were in hardy - weinberg equilibrium ( p>0.05 ) .
the numbers and frequencies of mthfr c677 t and a1298c snps in males and females of the case and control groups are shown in table 1 .
the individuals in both groups were further categorized based on gender , rm / normal and the type of mthfr snps .
combined heterozygosity of the two polymorphisms in the males of the two groups is presented as a common phenomenon , 52 ( 23.1% ) and 14 ( 14% ) of males in rm and control groups , respectively .
rare combinations of a1298c / c677 t snps including homo / hetero - zygote or hetero/ homo - zygote variants were observed in males and females of the both groups ( table 1 ) .
a major observation of this study was the absence of combined homozygosity for both snps in all studied groups / genders .
the pattern of individual grouping in table 1 allowed us to further group them into mthfr high risk and low risk groups .
the data was summarized in table 2 where the males were statistically compared depending on their degree of rm risk .
surprisingly , a statistically significant difference in the frequency of mthfr high risk group among the male partners in the rm group was detected compared to that in the normal males ( p=0.019 ) .
combination of mthfr a1298c and c677 t snps in rm and normal groups rare combination of a1298c and c677 t snps including homo / hetero- or hetero / homo -zygotes ; rm : recurrent miscarriage frequency of individuals with high and low risk mthfr genotypes in different groups p=0.019 , males in mthfr high and low risk groups were compared using chi - square test ; high risk : individuals who carried either combined heterozygous c677t
/ a1298c mthfr snps or at least one homozygous snp ; low risk : individuals who carried no c677t / a1298c mthfr snps or had just one heterozygous snp ; rm : recurrent miscarriage
this study was aimed to survey the possible role for paternal mthfr snps in rm . in this
regard , mthfr c677 t and a1298c snps in male partners of rm couples whose female partners had mthfr high risk genotypes were compared to that in normal counterparts .
the results suggest that rm male mthfr genotypes could also be involved in the pregnancy outcome .
few studies on mthfr polymorphism variants and rm have been performed in both male and female partners . in this
studied factor v leiden , mthfr c677 t and prothrombin g20210a snps in males and females of rm couples and compared their results with those in normal male and female counterparts ( 22 ) .
they concluded that multiple genetic thrombophilic mutations , but not single mutations , in either partner , significantly increased the risk of miscarriage ( 22 ) . in a similar study , toth et al . reported no association between paternal thrombophilia and rm ( 23 ) . in the study performed by yenicesu et al .
, 12 thrombophilic gene polymorphisms including mthfr c677 t and a1298c in rm and normal couples were considered ( 5 ) .
they reported significantly higher frequency of mthfr c677 t homozygous mutations in rm males ( 5 ) .
role of male mthfr in rm has also been addressed through the analysis of mthfr gene promoter hypermethylation , where rotondo et al .
elegantly showed the significant increase in the promoter hypermethylation in rm males ( 25 ) .
this is in line with the fact that hypermethylation of gene promoters can suppress the transcription process leading to silencing of gene expression ( 26 ) .
lack of mthfr activity due to presence of its polymorphic variants will reduce the availability of methyl group donors that are needed for methylalation of dna ( 9 ) which may adversely affect the global genome methylation and also the genomic imprinting of paternal genes in spermatogenetic cells . on the other hand , genomic imprinting which is involved in silencing of genes at the level of imprinted loci is highly regulated by dna methylation ( 27 ) . in this regard , impaired methylation imprints have been shown to lead to generation of poorly developed placenta that carried trophoblasts with proliferation , apoptosis and differentiation abnormalities ( 2831 ) .
these data suggest that a global hypomethylation of sperm dna following hypermethylation of mthfr promoter can cause pregnancy loss through male factor in rm couples ( 25 ) .
such an effect of mthfr polymorphism may also account for repeated implantation failure in infertile couples undergoing in vitro fertilization treatments .
considering the nature of our study , significantly increased mthfr snps were found in rm males compared to that in males of normal couples .
interestingly , no combined homozygosity for c677 t and a1298c snps was found among the individuals in this study and thus our data can not exclude the possibility that inheritance of such combination might predispose the product of conception to embryonic / fetal death .
it is also well documented that dna hypermethylation directly affects chromosomal instability ( 32 ) .
it is thus suggested that folate deficiency and dna hypomethylation following increased frequency of mthfr polymorphisms may be associated with chromosomal instability ( 33 ) which may lead to production of aneuploid gametes and embryos .
previous reports ( 6 , 19 , 35 ) as well as the data presented in this study are in favor of an increased number of mthfr polymorphic variants donated by both male and female partners of rm couples to the embryo
. the mthfr gene composition of the embryo may thus be adversely affected by dna hypomethylation that may increase the risk of embryonic / fetal death and be reflected as recurrent pregnancy loss in the couples . in this
regard , mthfr genotyping of couples with multiple pregnancy losses or repeated implantation failures who remain unresponsive to treatments , followed by selection of ivf embryos with the least frequencies of mthfr polymorphic variants by preimplantation genetic diagnosis ( pgd ) may play a significant role in increasing the survival chance of embryo / fetus .
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### Summary:
: background : methylenetetrahydrofolate reductase ( mthfr ) single - nucleotide polymorphisms ( snps ) c677 t and a1298c have been described as strong risk factors for idiopathic recurrent miscarriage ( rm ) . however ,
very few studies have investigated the association of paternal mthfr snps with rm . the aim of the present study was to evaluate the prevalence of paternal c677 t and a1298c snps among iranian rm couples.methods:the study subjects comprised 225 couples with more than three consecutive pregnancy losses , and 100 control couples with no history of pregnancy complications .
all females in the case group had mthfr polymorphisms ; and genotype snps were analyzed by pcr - rflp .
groups were statistically compared using mann whitney u - test and chi - square statistical tests .
the p<0.05 were considered significant.results:statistically significant difference was detected in the frequency of mthfr snps in male partners of the two groups ( p=0.019 ) .
combined heterozygosity of mthfr polymorphisms was a common phenomenon in the males ; 52 ( 23.1% ) and 14 ( 14% ) of males in rm and control groups , respectively .
absence of combined homozygosity for both snps in all studied groups / genders was observed.conclusion:the mthfr gene composition of male partners of rm couples may contribute to increased risk of miscarriage .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: burnout may be defined as a state of physical , emotional and mental exhaustion that results from long - term involvement in work situations that are emotionally demanding .
a great deal of research has been devoted to the understanding of factors contributing to burnout and to its consequences for individuals and their health .
research indicates that stress and burnout are significant factors in the development of both physical and psychological illness .
moreover , other studies have indicated the association of burnout with different self - reported measures of distress . apart from the fact that several studies have been conducted regarding the assessment of burnout , there are a limited number of studies in greece investigating the influence of social support on nurses burnout and their quality of life ( qol ) .
the present study aims to : i ) assess burnout and qol in nursing staff working in hospitals in the broader area of athens ; ii ) examine possible correlations between burnout as well as qol and social environment ; iii ) investigate differences with regards to the levels of burnout and qol in nursing professionals working in general and mental hospitals respectively .
a cohort of 100 nurses will be recruited from general and mental hospitals located within the broader area of athens .
the inclusion criteria are i ) 18 years of age and older ; ii ) ability to speak and read greek ; and iii ) being a registered nurse or nurse , working in the public or private sector .
the data will be obtained with a greek version of the maslach burnout inventory human services survey ( mbi - hss ) .
these items are answered on a seven point likert - scale , ranging from 0 ( never ) to 6 ( everyday ) . according to maslach et al . , the instrument is made up of three subscales : personal accomplishment ( pa ) ( 8 items ) , emotional exhaustion ( ee ) ( 9 items ) , and depersonalization ( dp ) ( 5 items ) .
the specific tool is comprised of 36 questions which provide an eight - scale profile of functional health and well - being scores ( physical function , role function , bodily pain , general health , vitality , social functioning , emotional well - being and mental health ) as well as physical and mental health measures .
the sf-36 has been used in many population surveys . during the last years , researchers have shown a great interest in the use of the multidimensional scale of perceived social support ( mspss ) in order to assess perceived social support in clinical and social context .
the sum of 4 items under each sub - scale gives the sub - scale score , while the sum of all sub - scale scores gives the overall scale score .
mspss has shown satisfactory psychometric properties with high internal consistency and test - retest reliability .
the data will be obtained with a greek version of the maslach burnout inventory human services survey ( mbi - hss ) .
these items are answered on a seven point likert - scale , ranging from 0 ( never ) to 6 ( everyday ) . according to maslach et al . , the instrument is made up of three subscales : personal accomplishment ( pa ) ( 8 items ) , emotional exhaustion ( ee ) ( 9 items ) , and depersonalization ( dp ) ( 5 items ) .
the specific tool is comprised of 36 questions which provide an eight - scale profile of functional health and well - being scores ( physical function , role function , bodily pain , general health , vitality , social functioning , emotional well - being and mental health ) as well as physical and mental health measures .
, researchers have shown a great interest in the use of the multidimensional scale of perceived social support ( mspss ) in order to assess perceived social support in clinical and social context .
the sum of 4 items under each sub - scale gives the sub - scale score , while the sum of all sub - scale scores gives the overall scale score .
mspss has shown satisfactory psychometric properties with high internal consistency and test - retest reliability .
the aim of the present study is to examine levels of burnout as well as qol in nursing staff in greece .
we believe that the findings will contribute to the scientific community demonstrating the importance of social network in the evaluation of nurses qol and burnout .
moreover , the findings of the present study can be used in the development of health care services and health professionals management .
qol in particular may play an important role and could therefore be identified as a new area for psychological intervention among nurses .
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### Summary:
: burnout has received increased research attention in recent years .
the aim of the present study is to examine levels of burnout as well as quality of life ( qol ) in nursing staff in greece .
the association of social support with burnout and qol is also investigated .
one - hundred individuals working in mental and general hospitals in the broader area of athens will participate in this study .
the measurement tools include i ) the maslach burnout inventory , ii ) the sf-36 health survey and iii ) the multidimensional scale of perceived social support .
burnout and qol are expected to be related to the evaluation of social environment .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: taylor et al . first described the possibility of raising a free flap in the groin region , consisting of an osteomuscular section and the overlying skin , nourished by the deep vascular system ( deep circumflex iliac artery perforator flap , dciap flap ) .
compared to the superficial circumflex iliac artery perforator flap ( scip flap ) , the dciap flap is superior in providing nourishment to the bone and adjacent muscle [ 1 , 2 ] .
dependable vascular supply to the osteomyocutanous complex is one of the reasons why the dciap flap is a good option in reconstructive surgery involving , in particular , composite mandibular , maxillary , and facial defects .
one of the main problems we encountered while harvesting a dciap flap including a vascularized iliac crest as a compound flap was raising the overlying skin island which is attached without great mobility to the bone . the overlying skin is also often limited in size and thus does not provide adequate soft tissue coverage of the defect , which should be treated . in 1993 , koshima et al .
introduced the chimeric composite flaps , consisting of two or more flaps , connected by microanastomoses .
these flaps offered greater mobility and variable skin size and thus represented a satisfactory solution .
however , this technique is highly complex , time consuming , and due to the several microanastomoses required , highly risky . in the case described below , it was possible to achieve complex reconstruction with restoration of the upper jaw and partial bony restoration of the midface and orbit as well as coverage of a huge soft tissue defect with a single flap .
a 76-year - old woman presented to our department , requiring facial reconstruction following radical resection of a widespread basal cell carcinoma , involving the right orbit , the anterior base of the skull with infiltration of the dura mater , the cheek , the whole nose , upper lip , right maxilla , the pterygopalatine fossa , and the right epipharynx ( figs . 1 and 2 ) .
the patient had a longstanding history of local tumor recurrence , treated surgically several times since 2000 .
our primary intention was to raise a conventional osteomusculocutaneous dciap flap as described by taylor et al . and if necessary to perform a flap supercharging by including and anastomosing the superficial circumflex iliac artery ( scia ) to increase the size of the soft tissue flap .
general anatomy of the perforators of the deep circumflex iliac artery ( dcia ) and of the scia was confirmed using doppler ultrasonography .
but intraoperative preparation of the proximal dcia vessels revealed a very large ascending branch about 1 cm medial to the anterior superior iliac spine ( asis ) .
careful surgical preparation and reevaluation with doppler ultrasonography confirmed the presence of a skin paddle , 15 cm long , and about 10 cm wide , superolaterally to the asis , which was vascularized by a perforator originating from this ascending branch . due to this large
ascending branch with its large perforator , flap supercharging was not necessary anymore and decision was made to raise this skin paddle together with about 10 4 cm osseous flap from the full thickness of iliac crest along with the adherent internal oblique muscle .
the osteomuscular portion was vascularized by perforators , originating from the deep circumflex iliac artery itself . due to the superolateral location of the skin paddle
, it was possible to mobilize a 15 cm vascular pedicle together with the bone and muscle , located proximally ( figs . 3 and 6 ) .
after preparation and flap harvest at the donor site , reconstruction and defect closure started . at the donor site , primary wound closure was achieved without complications .
intraoral soft tissue reconstruction was performed , using the internal oblique muscle , to induce secondary epithelization .
the huge epidermal defect , also involving the right orbit due to loss of the eye , was covered with the highly mobile skin paddle ( fig .
postoperatively , the patient developed venous thrombosis of the flap after being administered desmopressin for idiopathic diabetes insipidus on day 5 .
after removal of it , she was able to eat a normal semi - solid diet and after wound healing ( about 4 weeks postoperatively ) also speech function recovered and verbal communication was possible , even though the speech was unclear .
the patient also received an epithesis including the nasal and right orbital area and had an excellent functional and aesthetic result ( figs . 5 , 7 and 8) .
taylor et al . first described the classic dcia perforator flap and its use in facial ( mandibular ) reconstruction .
the use has been limited because of the unnecessary bulk of its obligatory muscle cuff and the tethering of the skin to the bone .
. tried to reduce the adjacent soft tissue of the flap and achieved good results .
reported in 1997 that the skin paddle of the dcia flap is supplied in 30 % of the cases by a dominant cutaneous branch of the dcia .
the authors concluded that the adjacent soft tissue , which had been routinely harvested together with the skin to ensure adequate perfusion , was not necessary in these cases .
soft tissue bulk could thus be reduced and the problem of skin tethering to the bone could be overcome .
identified a long dcia perforator vessel in 10 of the 12 cases in a cadaveric study and described an anatomical situation quite similar to the one we observed in our patient .
the authors raised the flap without the iliac crest and described its use for local treatment of urogenital defects and its use as a free flap for covering a chronic wound on the lower leg , but they did not consider using the flap for the treatment of facial defects . in the case of our patient , it was necessary to raise a flap with a bony part due to the large maxillectomy defect .
bulkiness , a disadvantage often mentioned in connection with a dciap flap , was not a problem ; on the contrary , we required a large volume of adjacent soft tissue because of the extensive defect .
a few years ago , shaw and brown demonstrated the high value of dcia perforator flaps for facial reconstruction .
one of the advantages they mentioned was the skin pigmentation , which , although in fact not ideal , is much better than that in forearm or scapula flaps .
our dciap flap variant is in complete agreement with the description of their anatomic observation .
they reported that either one or two perforators originate from the ascending branch of the dcia 511 cm posterior to the asis .
they did not raise a flap larger than 6 8 cm centered on these perforators but found that the vascularity was excellent . in 2009 , ting et al . evaluated 44 hemiabdominal walls with computed tomography angiography ( cta ) before dcia flap surgery .
they observed high variability and concluded that exact preoperative evaluation is only possible with cta , but that the osteomusculocutanous dciap flap is a good option for facial reconstruction in general .
bergeron et al . also performed an anatomical study of the groin region and found perforators originating from the dcia in 92 % of the cases investigated .
although we did not use cta , because ultrasound confirmed the availability of the standard dcia anatomy , we would recommend doing it preoperatively for exact surgical planning of raising the best flap available in the unique anatomical situation of each patient , as we have learned in this case .
the authors also strengthen the importance of preoperative cta and also introduce advanced imaging modalities and other technologies such as image - guided stereolithographic biomodeling for preoperative flap planning .
the observed anatomy in our patient has already been described , but such a dciap free flap has never been raised and reported in literature before ( large skin paddle , long vascular pedicles , and osteomuscular part ) .
the literature analysis also revealed that nowadays preoperative cta or mra should be standard for a dciap flap harvest ( figs . 1 , 2 , 3 , 4 , 5 , 6 , 7 and 8) .
1preoperative view , a 76-year - old woman with widespread basal cell carcinoma involving the whole right and central midface preoperative view , a 76-year - old woman with widespread basal cell carcinoma involving the whole right and central midface fig .
a 76-year - old woman with widespread basal cell carcinoma involving the whole right and central midface intraoperative photo during radical resection . a 76-year - old woman with widespread basal cell carcinoma involving the whole right and central midface fig .
3the dcia perforator flap variant shown intraoperatively , with its long vascular pedicle connecting the osteomyocutanous part and the skin paddle the dcia perforator flap variant shown intraoperatively , with its long vascular pedicle connecting the osteomyocutanous part and the skin paddle fig .
5the patient several weeks after surgery , wearing her epithesis of the nose and right eye the patient several weeks after surgery , wearing her epithesis of the nose and right eye fig .
6schematic illustration of the intraoperative anatomical situation of the groin region in this unique case schematic illustration of the intraoperative anatomical situation of the groin region in this unique case fig .
7preoperative computer tomography scan showing complete destruction of the right maxilla and hard palate and bony infiltration of the orbital floor , zygomatic bone and ethmoidal cells preoperative computer tomography scan showing complete destruction of the right maxilla and hard palate and bony infiltration of the orbital floor , zygomatic bone and ethmoidal cells fig .
8postoperative computer tomography scan showing the bony reconstruction of the zygomatic contour and the maxilla and partial reconstruction of the bony orbit postoperative computer tomography scan showing the bony reconstruction of the zygomatic contour and the maxilla and partial reconstruction of the bony orbit permission for publication obtained .
the authors have no financial or personal relationships with other people or organizations that could inappropriately influence ( bias ) this work .
none of the authors has a financial interest in any of the products , devices , or drugs mentioned in this manuscript .
the authors have no financial or personal relationships with other people or organizations that could inappropriately influence ( bias ) this work .
none of the authors has a financial interest in any of the products , devices , or drugs mentioned in this manuscript .
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### Summary:
: summarybackgroundanatomical variance of the deep circumflex iliac artery is of high clinical value in facial reconstruction using a deep circumflex iliac artery perforator ( dciap ) flap.methodswe present the case of a 76-year - old woman treated with an osteomyocutaneous dciap flap variant for facial reconstruction .
we also review here the literature on dcia perforator flaps and the different anatomical variances , which might bring clinical benefits.resultsthe observed anatomy in our patient offered the possibility to raise a free flap variant with high mobility of a large skin paddle and a long vascular pedicle combined with a variable osteomuscular portion.conclusionthe characteristics of the flap thus raised help overcome the disadvantages of the conventional dciap flap and offer excellent options for facial reconstruction .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: more than one million of necessitate hemodialysis ( hd ) and
renal transplantation all over the world for the year2 . as renal function
the usual consequences of
uremic syndrome include decreased physical ability ( by approximately 50% ) , diminished
quality of life ( qol ) , and increased cardiovascular disease6,7,8,9 .
dialysis interventions are generally performed three times per week for 46 hours per
session .
this causes hd patients to have a monotonous and restricted daily life , limiting
their activities after treatment onset as well as contributing to and supporting sedentary
habits , functional disability , and inactivity6 .
activity limitations are often caused by muscle atrophy , which
affects 1880% of patients undergoing dialysis10,11,12,13 .
muscle wasting is
another remarkable problem and is a significant anticipator of morbidity and mortality14 .
muscle quantity is strongly correlated
with muscle strength , muscle oxygen extraction , and functional ability in hd patinets10 , 11 , 15 , 16 . in rare cases ,
the quadriceps tendon and patellar ligament are
susceptible to rupture17 . establishing a physical activity routine for patients on dialysis has advantages such as
greater willingness to engage in training , convenient scheduling , reduced boredom of
dialysis sessions , and better ease of medical care .
in addition , physical activity in hd
patients is related with improved physical capacity and reduced blood pressure , and also
contributes to improved qol18 , 19 .
however , the physical condition of hd patients limits
their ability to perform high - intensity training such as resistance training6 .
recent investigations have promoted
intra - dialytic training as a beneficial intervention to enhance compliance , improve
motivation , and promote the medical monitoring3 , 19 .
therefore , this study assessed the impacts of
individualized aerobic training programs supervised and carried out during dialysis sessions
on body composition , muscle function of the knee joint , physical performance , and qol in hd
patients .
ten ckd patients who were undergoing maintenance dialysis in the outpatient hd unit were
enrolled .
they were evaluated for
eligibility based on review of medical history and physical examination .
the inclusion
criteria contains : older patient ; during dialysis for more than three months ; approval from
physician ; independent ambulation for at least 50 m , and properly dialyzed and stable during
dialysis .
a single - group interventional study design was used to confirm the influence of aerobic
training in hd patients .
body composition , muscle function of the knee joint , physical
performance , and qol were evaluated .
after these assessments , the patients underwent
supervised aerobic capacity during dialysis sessions for 12 weeks .
training consisted of a warm - up , aerobic training on a stationary bicycle , and a cool - down .
for the warm - up , patients performed passive stretching of the lower limbs and aerobic
activity on the bicycle at 0.5 km / h ( approximately 35 rpm ) , which is the lowest load
applicable to the ergometer ( 700r recumbent bike , cybex , boston , usa ) used , for 5 minutes .
after the warm - up , the conditioning phase included 30 minutes of training during the first
two hours of dialysis to avoid physical stress in the second half of the session when
patients hemodynamic conditions are unfavorable .
heart rate was constantly monitored using
a heart rate monitor ( h1 heart rate sensor , polar , ny , usa ) , and blood pressure was measured
at rest every five minutes during training and after the cool - down period . the cool - down
consisted of two minutes on the bicycle with minimal load at 35 rpm followed by passive
stretching of the lower extremity .
the prescribed training intensity was based on borg s perceived exertion scale7 . according to this scale ,
every 5 minutes during the
aerobic training , patients were asked about the score they would assign to their fatigue at
that moment , and the bicycle load was adjusted to achieve sufficient intensity to maintain a
fatigue score between 11 and 13 points , which corresponds to an intensity of mild to
quite hard . the rotation speed of the pedals remained close to 35 rpm to achieve stable
training intensity .
the inbody 720 ( biospace , seoul , korea ) was used to confirm body composition calculated
from weight , mass of skeletal mass , mass of body fat , percentage of body fat , and body mass
index which was measured by the electrical resistance to four frequencies in each body part .
the electrical resistance was measured body composition by attaching sensors on the patient
in standing position .
maximum peak torque in the knee joint was assessed using the isokinetic digital cybex
system ( humac norm , ny , usa ) ; this is a noninvasive method that uses the isokinetic mode and
enables reliable measurement .
the test relies on flexion and extension of the legs at
the knee joint with maximum force and measures maximum flexion and extension peak torque at
an arc speed of 60/s ( five movements with a one - minute rest between each set ) .
training testing was performed using a branching treadmill protocol with modified
bruce7 that started at a brisk walking
speed ; speed increased gradually every two minutes to assure that at least four measurements
were obtained between rest and peak training .
when the patient was unable to keep pace with the treadmill or requested
to stop , cardiac output was measured before stopping .
the electrocardiogram was measured
constantly for oxygen uptake ( vo2 in l / min ) , and cardiac output were estimated at
each stage .
the six - minute walk test ( 6mwt ) was used as an index of physical endurance and was
conducted before and after training .
the 6mwt is reliable for establishing the physical
capacity of patients with heart and respiratory insufficiency as well as patients with
chronic renal failure20 .
this test can be
used to assess the influence of rehabilitation , particularly for subjects in whom maximal
training testing is not possible , such as elderly patients .
patients walked unassisted for 25 m down the hospital
corridor accompanied by a physiotherapist ; they were permitted to stop to rest .
the distance
( m ) was recorded for each patient21 . to evaluate qol ,
this questionnaire has 36 items
divided into eight divisions : physical function , role of physical , body pain , general
health , vitality , social function , role of emotional , and mental health .
the shapiro - wilk test was verified normality distribution of data . after verifying that the
data were normally distributed ,
it was
considered significantly difference when the p value was < 0.05 using spss version 21.0
( spss inc . ,
mean age was 56.50 4.48 years ( males : 55.50 7.96 , females : 57.17 5.88 ) , and mean
height was 158.08 2.72 years ( males : 167.23 1.74 , females : 151.98 1.53 ) .
the number of complications ranged from one to five
( table 1table 1.general characteristics in the subjectsage(years)genderheight ( cm)complications142male167.6diabetes , hypertension , anemia242male162.7diabetes , hyperlipidemia , anemia , autoimmune disease362female156.7hypertension , arthritis , anemia , autoimmune disease473male167.4diabetes , hypertension , anemia547female154.3hyper - tension , anemia634female154.3diabetes , hypertension , anemia , autoimmune disease ,
tuberculosis762female146.6diabetes865male171.2diabetes , hypertension , anemia , autoimmune disease , spinal
stenosis963female149.9diabetes , hypertension , arthritis , anemia,1075female150.1hypertension , arthritis ) .
the goal of 30 minutes of tolerance to training was achieved around the fourth week .
it is
worth noting that no clinical complications occurred during training ; there was only one
episode of absence without leave , and training was resumed at the next dialysis session .
there were no difference between before and after training in weight , muscle mass , body fat
mass , fat percentage , body mass index , and peak torque of knee extension and flexion . each
post - training variable
( 97.4% to 103.0% ) was quite similar to pre - training values ( table 2table 2.pre- and post - training on body composition and knee joint muscle functionvariablepre - trainingpost - trainingbody compositionweight ( kg)59.611.859.811.4muscle mass ( kg)22.74.422.54.1body fat mass ( kg)17.27.017.76.8fat percentage ( % ) 28.26.829.07.1body mass index ( % ) 23.94.523.84.5muscle function of knee jointright knee extension ( peak torque)50.425.351.924.2right knee flexion ( peak torque)23.513.326.011.8left knee extension ( peak torque)45.726.444.524.4left knee flexion ( peak torque)23.615.723.112.9no significant difference between pre- and post - training ) .
no significant difference between pre- and post - training the average pre - training distance traveled in the 6mwt was 390.80 78.62 m , compared to a
post - aerobic training value of 419.00 93.68 m. thus , the distance traveled after the
aerobic training increased significantly ( p < 0.05 ) .
there were no significant changes in
resting metabolic rate , lactate threshold , vo2max , or qol subscale scores .
however , each of these variables increased after training ( table 3table 3.pre-and post - training on physical performance and qolvariablepre - trainingpost - training6mwt ( m)390.878.6419.093.7rmr ( kcal / day)895.2272.4977.8396.7lt ( ml / min)703.8450.6738.2421.5vo2max ( ml / min)965.0439.8993.2385.7qol ( score)1,392.0494.21,596.9647.8*p < 0.05 , 6mwt : six - minute walk test , rmr : resting metabolic rate , lt : lactate
threshold , vo2max : maximum oxygen uptake qol : quality of life ) . *
p < 0.05 , 6mwt : six - minute walk test , rmr : resting metabolic rate , lt : lactate
threshold , vo2max : maximum oxygen uptake qol : quality of life
there are no known data concerning physical fitness and training tendency in korean hd
patients , but similar data from other countries including the u.s .
have showed 45% of
dialysis patients seldom training , and only 8% of patients among entirety hd patients did
training at least once a week6 . in this study
, we observed that aerobic training sessions held during dialysis were
associated with or maintained or increased physical performance , qol , control of body
composition and muscle function of the knee joint , and increased safe participation in
physical activity .
muscle wasting is caused by a variety of
reasons and process begins early in the disease23 ,
24 .
the resistance training of
high - intensity was to improve the muscle strength of hd patients25 , 26 .
similar
trials27 , 28 showed to improve muscle strength by regimens with lower - intensity
strength training .
resistance training highly increases metabolism of protein synthesis ,
leading to increased cross - sectional volume of muscle fibers .
in contrast , another study was
no changes of lower extremity strength following six weeks of dialysis period29 . however , this is not unpredictable
considering that aerobic training is not the preferred intervention for improving muscle
strength6 . in comparison ,
while aerobic training
alone is not enough to activate hypertrophy , it can maintain skeletal muscle mass29 .
in the our study , there were no
significant change on body composition and muscle function of the knee joint .
however , each
of these variables was quite similar between before and after - training ( 97.4103.0% of the
respective pre - training values ) .
this study used the 6mwt , because it is most commonly used test to estimate physical
performance , is easy to use , inexpensive , and is representative of daily activities30 .
this test is widely used in patients with
cardiac and lung diseases , but its application in hd patients in clinical research is often
limited .
the average distance achieved in the 6mwt in the pre - training phase was comparable
to that achieved in other studies evaluating hd patients3 , 25 , 31 .
after aerobic training , the distance covered in the 6mwt increased
significantly indicating considerable improvement in physical endurance .
these findings were
coherent with previous study observed an improve of physical performance in patients
undergoing dialysis according to the 6mwt after 20 weeks of training31 .
in the our study , there were no significant changes on
resting metabolic rate , lactate threshold , vo2max , or qol , although each of these
variables tended to increase after training .
hd patients was can significantly increase
their vo2max 1723% by performing aerobic training during dialysis27 .
in contrast , a little studies reported no
significantly changes of vo2max following aerobic training , for that reason attributed to
prescribing low intensity32 or short
duration aerobic training33 , several
studies of training in hd patients have used sf 3634 scores as a measurement of qol .
these findings suggest the benefit of a supervised training program for improvement of
physical performance and qol .
people who persistently have a sedentary lifestyle have a greater risk of adverse cardiac
events than those who progressively improve their physical activity level35,36,37,38 .
in comparison with risks associated with vigorous activities ,
thus , training according to
these recommendations incurs a lower cardiovascular risk than vigorous training .
as required
training test before starting a low - intensity physical activity program would constitute a
substantial barrier to participation for many patients and could consequently contribute to
an increased risk of remaining sedentary .
pre - training testing is unnecessary , because the
recommendations are for gradually increasing low - intensity activity and because the
recommendations are made according to the individual s ability with regard to chronic
conditions and lower energy levels34 . in
our study , patients were willing to participate in 95% of the training sessions , and the
goal of 30 minutes of tolerance to training was achieved by patients around the fourth week .
no relevant clinical complications occurred during training ; there was only one instance of
absence without leave , with a resumption of training at the next dialysis session . in summary , aerobic training during dialysis sessions
is a safe and tends to be associated
with improvements in physical performance and qol .
it also contributes to the maintenance of
body composition and muscle function of the knee joint in hd patients .
therefore , aerobic
training can increase participation in physical activity and appears to be a safe approach
to initiating training .
further research is required to resolve the composition of
interventions including type , duration , intensity , frequency and these data will require
confirmation in samples with a larger number of patients .
|
### Summary:
: [ purpose ] we assessed the influences of individualized aerobic training on body
composition , knee joint muscle function , physical performance , and quality of life in
chronic kidney disease patients .
[ subjects ] ten chronic kidney disease patients undergoing
dialysis . [ methods ]
overall physical function and quality of life before and after 12
weeks of aerobic training were evaluated by body composition , the six - minute walk test ,
cardiopulmonary exercise tests , and short form 36-item questionnaire .
[ results ] the
six - minute walk test distance increased significantly after 12 weeks aerobic training .
resting metabolic rate , lactate threshold , maximum oxygen uptake , and quality of life
tended to increase after training .
post - training weight , muscle mass , body fat mass , fat
percentage , body mass index , and peak torque of right and left knee extension and flexion
did not change significantly .
[ conclusion ] intra - dialytic training can a safe approach to
maintain or improve physical performance and quality of life of chronic kidney disease
patients undergoing hemodialysis without adverse events or negative cardiovascular
responses . aerobic training may prevent a decline in body composition and knee joint
muscle function due to inactivity in chronic kidney disease patients .
clinically , aerobic
training may initially be adapted to maintain overall physical function or improve quality
of life in chronic kidney disease patients undergoing hemodialysis .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: org , physicist adam frank , speaking specifically of climate change but using it as but one example of many science - relevant policy issues , said , but when it comes to telling us what we should do about climate change a question that hinges on values as much as on data scientists are not in a privileged position ( 2 ) .
science ethicsinclude , in addition to global climate change , ethical issues such as the creation of transgenic organisms , the use of reproductive , genetic , and biomedical enhancement technologies , the conduct of research with human and animal subjects , the allocation and prioritization of research dollars , and so on .
scientists are generally aware that a good science education has to engage these sorts of issues .
indeed , i will mostly assume for the remainder of this article that scientists agree on the importance of including these issues in a good education .
after all , as adam frank points out in his article , scientists typically do not have training in ethics , and they are not in any kind of privileged position with respect to issues in science ethics .
these issues are not resolved by gathering more data because the data do not indicate , apart from ethical argument , what we ought to do with what we know . in this article
, i will argue for collaborative science ethics education , drawing on the separate but equally valuable strengths of two very different domains and methods of inquiry .
in particular , i will argue that science ethics teaching should combine the strengths of scientists and philosophically trained ethicists , ideally those with training and experience in practical ethics . as i hope to show , science ethics involves at least two domains of knowledge .
rather than assume that one professor has expertise in both areas , scientists and ethicists should combine forces in the classroom to provide the best science ethics education for students .
i understand my proposal to be relevant to undergraduate , graduate , and post - graduate education . before making my argument
first of all , i am assuming some sort of meaningful distinction between the disciplines of science and ethics .
while i will not go into any detail on how this distinction might be made , i do offer the following rough characterization .
the sciences deal with description of reality , as best we can know it at this time .
there are some very different methodologies used within these two disciplines , but i take the descriptive / normative distinction to be fundamental .
some of the recommendations i make later concern what might be thought of as metaethical claims .
the distinction between metaethics and normative ethics is not entirely clear and is sometimes contested ( 4 ) .
but even when i refer to metaethical concepts , i do so by way of making normative recommendations about how they ought to be handled in teaching ethics .
policies are also normative in nature , but not all science ethics issues need be expressed in the form of policy , just as not all ethical norms need be codified in law .
science policy represents , in most cases , a consensus view , whereas many issues in ethics remain contested ( 10 ) . to anticipate a possible confusion ,
that is , social scientists might investigate the question of what individuals views are on ethical questions .
this should not be confused with normative ethics , which raises the question , for instance , of whether the majority view is the best or the right one .
social scientists who study the ethical views of others , in fact , might consider my proposal here as applying to them as well .
their students , too , would likely benefit from collaborative education on issues at the intersection of social science and ethics .
consider this scenario : a professor with a ph.d . in philosophy , hired by a university philosophy department , who proposes to teach a science course .
i have heard of universities that support a department s right to control who can teach courses in the department s area .
there may even be in some places formal policies related to this patrolling of disciplinary boundaries .
setting aside university politics , there seem to be some good reasons to prevent a philosopher from teaching a science course . in short ,
different disciplines have different methodologies and bodies of knowledge . while someone with advanced graduate education in one area might be able to learn some new area ,
it is doubtful that in the typical case they can learn it as thoroughly as others whose training and experience in that area go back many years . at the least ,
such an individual would be comparatively underprepared to teach the course . in making my case
, i will hereafter assume the qualifier that it is unlikely that someone taking up a new discipline could do so as well as someone whose training and experience are in that area .
there will be exceptions , but this does not undermine the more general claim . as a matter of efficiency , we might ask whether it makes sense to learn some new discipline in which the university already has faculty expertise . scientists who hear of this philosophy professor s plans would have good reason to question whether the philosopher is properly qualified to teach in their area for other reasons as well .
scientists might worry about someone who lacks qualification misrepresenting the state of knowledge in a way that parallels the potential misrepresentation of scientific discoveries in the popular media .
students might be drawn away from a better course offered by a scientist with training in the relevant area .
it would also be unhelpful for students to have the impression that they have learned those issues and thereby be less inclined to seek further , better training in the area . more positively
the modern university has to be concerned about the quality of education students receive . ensuring that the person charged with teaching a given course is properly credentialed to do so is a matter of some concern in a university eager to show the value of its degrees . to summarize , there are reasons related to disciplinary competence , budgetary and university efficiency , the representation of a discipline to students and others , and the quality of students educational experience that could be used to support opposition to this proposal .
when we reverse the scenario and think about scientists teaching ethical issues related to their discipline , i want to argue that these and other reasons support the opposite claim that it would be better to have someone who has disciplinary training in ethics teaching ethics .
but as i noted when indicating my proposal above , because ethical issues in science generally involve both scientific and philosophical / ethical claims and issues , the best approach will be a collaborative one in which both a scientist and an ethicist participate .
if philosophers ought not generally teach science courses , scientists ought not teach ethics courses . if scientists ought not teach whole courses on ethics , what about incorporating ethics into their courses ?
for instance , a course in biology might include some discussion of research ethics or look at the ethical implications of genetics .
a graduate program might incorporate ethics into courses or occasional seminar presentations on professional issues , such as the responsible conduct of research ( 8) . in each of these contexts , i believe it would be best to have someone with the relevant training in ethics involved in the teaching .
why not bring in someone whose training and experience best position them to teach the normative aspects of the issue
? scientists might respond to this proposal with the following : we do not intend to start teaching ethics courses , but we do believe it is important to look at the social impacts of the science we teach .
while philosophers should teach the subject of ethics , we reserve the right to teach science ethics because it is directly linked to our discipline . to this i respond that we should recognize that science ethics involves disciplinary expertise in at least two areas .
but there are also the normative issues , captured by questions like can genetic testing be done in a way that does not foster discriminatory attitudes ? hence , i offer a collaborative proposal .
the view in opposition to my proposal is essentially that scientists can cover both the empirical claims and the normative issues .
to return to the claim that science ethics is distinct from philosophical ethics and can be adequately covered by scientists alone , let us consider a particular issue to highlight the value of collaborative teaching .
the ethics of embryonic stem cell research has generated an enormous amount of public discussion .
it has been the subject of reflection by at least two presidentially - appointed bioethics councils ( 5 , 6 ) . and
one of the most fundamental ethical issues of embryonic stem cell research is the moral status of human lives at the earliest stages of development .
the question of when moral status for humans begins probably can not be answered in ignorance of the science of embryonic development .
it is possible for individuals to attempt to defend views that are based on counterfactual claims about embryos .
but the science alone will not yield an answer to the question of when moral status begins . a claim about moral status and an argument for that claim will also require the use of normative concepts .
it is not enough to know about particular biological changes that occur during development . to make an argument for moral status beginning here as opposed to there
, one must also argue for why that particular change is relevant to moral status .
for instance , a view that says that not all humans are persons and that being a person is a necessary feature of having moral status must meet a number of objectives to secure that claim .
we need an account of why these person - making features ground moral status ( 3 ) . addressing this goes beyond the relevant science . and it would not be enough to talk only about one position on the issue . even if one had a handle on the personhood view , a responsible presentation of the ethics of this issue should also explain and offer arguments for respectable alternative views
. indeed it would be an ethical failing on the part of a teacher not to teach alternative views where there are respectable ones , as is the case with moral status ( 1 ) .
further , good ethics education would involve attempts to bring , as much as possible , respectable alternative views into conversation with one another in the classroom . if there is active work among proponents of alternative views who are responding to one another s objections and offering further reasons to believe their own view is the best one , this ought to be discussed . to do
my aim here is to show that it would take more than training in biology to teach the ethics of moral status .
the moral status of early human lives is a perfect example of the importance of collaborative teaching in which both relevant disciplines are properly represented . in the latter paragraphs of this section ,
it sets an ambitious agenda but also gives a sense of what responsible ethics education should seek to achieve .
some aims of science ethics education . to raise awareness of the fact that there are ethical issues to be considered ( and to become aware of implicit ethical commitments in one s practices / views ) to reflect on realistic cases and draw meaningful comparisons to other cases to separate descriptive from normative claims ( and avoid assuming the former are the latter without appropriate arguments ) to avoid errors of ethical reasoning more generally to carefully evaluate rhetorical appeals , and to discern what normative claims are truly valuable in supporting a position to identify a broad range of considerations that might be relevant to the intellectual or practical resolution of an issue to weigh various ethical considerations and assign relative importance to some over others , based on personal , professional , or social values to learn to articulate internally consistent reasons to support normative judgments and to hold views that are consistent across issues to understand the ethical significance of seeing the sciences as professions to appreciate how the motivation to do a good job as a scientist is intrinsically linked to certain character traits like integrity , persistence , intellectual humility , and others ( 7 ) to come to a relatively unified understanding of the place of ethics in successful scientific research and the professions ( 9 ) to appreciate that the fact of moral disagreement does not necessarily entail that the other view is irrational to learn from engagements with representatives of views other than one s own to be able to explain why someone might disagree with one s own view and to offer responses to objections and clarify why one continues to hold one s own view
scientists bring to the classroom expertise in the empirical data and the scientific method and experience with these .
when it comes to teaching science ethics , i believe the best education will come from bringing both sets of strengths into the classroom together .
below , i offer some models for implementing collaborative teaching . but let me address one possible response here .
however , i believe it is better to have ethicists visiting in science classes and scientists visiting in philosophy classes .
this shows that the scientist , who generally has disciplinary credibility with students , recognizes the importance of ethical issues .
they are not something to be discussed somewhere else , but have a place in the midst of a science curriculum
. for ethicists teaching science - related philosophy classes , inviting in scientists signals a commitment to working with the best possible empirical information when discussing ethical issues .
i offer one example with which i am familiar , but also discuss a number of other possible forms of implementation . each fall
after a couple of weeks introducing ethics as a discipline , the course looks at a variety of specific issues : the use of human embryos , reproductive and genetic technologies , plant biotechnologies , neuroscience , the use of human / animal research subjects , the responsible conduct of research , etc .
when we discuss , for example , the ethics of reproductive technologies , i invite in a scientist whose research and teaching are in the area of reproductive science .
he presents for about 20 to 30 minutes , before the remainder of the class is devoted to discussion of ethical issues related to that area of science .
the visiting scientist stays and participates in the discussion , sometimes addressing questions about the science , sometimes offering views on how different ethical positions match with current scientific practice , etc .
having a professor visit another s class allows the visiting professor to demonstrate their approach to the content .
if the subject is sufficiently interdisciplinary ( e.g. , biology in society ) , the ethical and scientific issues might be so interwoven throughout that it makes sense to have both professors active in each discussion .
the push is toward interdisciplinary activity in the contemporary university , and courses that bridge scientific and ethical issues would be among the more important ones in this respect .
i have argued here for collaborative science ethics teaching , drawing on the strengths of the sciences and ethics to promote better education for students .
not only does such collaboration promote good pedagogy , it also fosters collegiality across disciplines .
teaching together allows professors to appreciate one another s strengths rather than give the impression that they can do each other s work .
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### Summary:
: i offer a normative argument for a collaborative approach to teaching ethical issues in the sciences .
teaching science ethics requires expertise in at least two knowledge domains the relevant science(s ) and philosophical ethics . accomplishing the aims of ethics education ,
while ensuring that science ethics discussions remain grounded in the best empirical science , can generally best be done through collaboration between a scientist and an ethicist .
ethics as a discipline is in danger of being misrepresented or distorted if presented by someone who lacks appropriate disciplinary training and experience .
while there are exceptions , i take philosophy to be the most appropriate disciplinary domain in which to gain training in ethics teaching .
science students , who must be prepared to engage with many science ethics issues , are poorly served if their education includes a misrepresentation of ethics or specific issues .
students are less well prepared to engage specific issues in science ethics if they lack an appreciation of the resources the discipline of ethics provides .
my collaborative proposal looks at a variety of ways scientists and ethicists might collaborate in the classroom to foster good science ethics education .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: recent developments and modern technology have improved vastly the safety and efficacy of laparoscopic surgery , and the availability of smaller instruments has expanded the application of this modality to the treatment of children and infants . moreover ,
antenatal ultrasonography has also developed , and the incidence of neonatal intra - abdominal cystic lesions has increased .
we employed laparoscopy - assisted surgery ( las ) to treat two female infants in whom intra - abdominal cystic lesions had been diagnosed by antenatal ultrasonography , with excellent results .
two female infants with congenital intra - abdominal cystic lesions that had been diagnosed by antenatal ultrasonography underwent las at 47 - 51 days of age ( table 1 ) .
ultrasounds performed on the fetuses at 32 and 28 weeks gestation revealed abdominal cystic masses , respectively .
the cystic lesions of both patients were 4 to 5 cm in diameter and contained some solid matter , hemorrhage , or mucinous fluid and had the indications for surgery .
postoperative examination confirmed that one of the two patients had an omental cystic lesion with torsion , and the other one had the same lesion and a left ovarian cyst .
with the patient under general anesthesia , a nasogastric tube and a foley 's bladder catheter were inserted .
the patient was placed in the supine position , and a laparoscope was inserted through a 10-mm trocar immediately below the umbilicus by an open method .
we examined the peritoneal cavity and diagnosed that the cystic lesions with torsion had no connection with the normal reproductive organs and was attached to the greater omentum with pedicle ( figure 1 ) .
a soft - cup aspirator set ( hakko co. , tokyo , japan ) was inserted , and the cyst was pulled out while its contents were aspirated ( figure 2 ) .
the cystic lesion was pulled up to the umbilical wound and completely resected with a pedicle . in the patient with a left ovarian cyst ,
the cystic lesions in both patients were accurately diagnosed by laparoscopy , and no intraoperative complications were encountered .
the operating time of las was 87 minutes in one patient and 140 minutes in the other patient with left ovarian cyst .
no wound infection was encountered , and the postoperative course was uneventful for both patients .
oral intake was recommenced on postoperative day 2 in the patients who had undergone las ( table 1 ) .
the cosmetic results of the wound following las were excellent in the opinion of the parents of both patients ( figure 3 ) .
laparoscopic surgery has become widely employed throughout the world and is performed in pediatric patients .
higashimoto et al reported that the merits of las were that it provided an accurate diagnosis and treatment , it was minimally invasive , it required short hospitalization , and normal activities were able to be resumed rapidly .
cohen et al reported that video - assisted , minimally invasive surgery is suitable for the treatment of adnexal pathology in children .
waldschmidt et al reported that laparoscopy will find more general application in pediatrics and pediatric surgery for both diagnostic and operative purposes .
van der zee et al reported that using a laparoscopic approach for ovarian cysts avoids the disadvantages of large scars , adhesion formations , and even gonadal loss in cases of infant surgery . saw et al reported that laparoscopic resection of a giant mesenteric cyst has some theoretical advantages , including less postoperative pain and shorter convalescence .
we employed las to treat two infants in whom intra - abdominal cystic lesions had been diagnosed by antenatal ultrasonography .
in fact , we diagnosed omental cystic lesions with torsion and ovarian cyst accurately and performed accurate operative procedures in both infants , with excellent results . on the other hand , a number of complications of pediatric laparoscopic surgery have been reported . according to chen et al , these complications included hemorrhage , umbilical hernia , cellulitis , pelvic abscess and small bowel obstruction .
higashimoto et al reported the cases of two neonates who developed temporary decreased percutaneous saturation of the oxygen concentration following las for intra - abdominal cysts .
esposito et al recommended that the open approach with a blunt trocar should be used to prevent the complications of pediatric laparoscopy .
our experience of adult patients who have suffered endoabdominal organ perforation or vascular injury with the veress needle leads us to agree with his comments .
thus , we performed the open approach method of trocar insertion in both infants , and both our patients encountered no complication .
moreover , nezhat et al reported that the benefits of laparoscopy for the surgical removal of dermoid cysts in adults were the video augmentation and the minimal cyst content spillage .
van der zee et al reported that the transabdominal puncture and percutaneous gastrostomy introduction set are used for the aspiration of cyst contents .
we used the aspirator kit known as the soft cup aspirator set , which was very easy to use and proved useful for preventing spillage of the cystic contents .
we recommend that this aspirator kit be used when intra - abdominal cystic lesions are removed by las .
it leads to preventing the risk of bowel obstruction secondary to remnant pedicle as an adhesive band .
the number of cases of neonatal and infantile intra - abdominal cystic lesions being diagnosed antenatally has increased in accordance with the prevalence of antenatal ultrasonography . in neonates ,
ovarian cysts are frequent , whereas mesenteric or omental , lymphatic and duplication cysts are few and splenic , hepatic and pancreatic cysts are extremely rare .
the surgical indications for neonatal intra - abdominal cystic lesions are the presence of symptoms , such as vomiting and abdominal pain ; the possibility of torsion ; and a lesion greater than 5 cm in diameter without any reduction , especially ovarian cysts .
both patients had indications for surgery because of lesions that measured 4 to 5 cm in diameter and because of the possibility of torsion . regarding the operative procedure for intra - abdominal cystic lesions , extirpation of cystic lesions with a pedicle
the further development of miniaturized laparoscopic apparatus for pediatric use and improvements in surgical techniques might result in better laparoscopic procedures for neonates and infants with all intra - abdominal cystic lesions .
we conclude that las is suitable for the management of omental cystic lesions with torsion and ovarian cysts in infants .
an aspirator is useful for preventing the spillage of cystic contents in patients with intra - abdominal cystic lesions .
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### Summary:
: objectives : we report two cases of infants found to have congenital omental cystic lesion with torsion and ovarian cyst treated by laparoscopy - assisted surgery ( las).methods : a laparoscope was inserted by an open method . the intra - abdominal pressure was maintained at 8 mm hg . we diagnosed the cystic lesions with torsion .
an aspirator was used to aspirate the cystic content and remove the cyst.results:no intra- or postoperative complications were encountered .
oral intake was commenced on postoperative day 2 in both patients .
the cosmetic results following las were excellent.conclusions:we conclude that las is suitable for the management of omental cystic lesions with torsion and ovarian cyst in nursing infants .
an aspirator is useful in preventing spillage of cystic contents in patients with intra - abdominal cystic lesion .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: synovial or ganglion cysts arising from the capsules of facet joints or from yellow ligament are uncommon disease entity3,4,10 ) .
these cysts are usually encountered in the lumbar spine , but can occur in the cervical or cervicothoracic spine5,8 ) .
although rare , a synovial cyst or ganglion cyst can form in the cervical spinal canal , usually near an articular facet or in a yellow ligament .
the majority of these cysts are asymptomatic , but occasionally , they can induce neurologic symptoms7 ) .
moreover , brown - squard syndrome caused by a synovial cyst is very rare and no previous report of a synovial cyst presenting with brown - squard syndrome has been previously issued in the english literature .
we experienced a rare case of brown - squard syndrome caused by a cervical synovial cyst , and here , report this unique case with a review of the literature .
a 64-year - old man , who had experienced sudden weakness and numbness in the left lower extremity for 4 weeks , visited our emergency department for evaluation and treatment . over several days , his gait disturbance gradually worsened and the numbness had extended to the left upper chest region .
there was no evident traumatic episode before admission . a physical examination conducted at presentation revealed gait disturbance with spasticity .
in addition , sensory disturbances of vibration and positional sensation were present on the left side below the t4 dermatome .
impaired perception of pain and temperature stimuli were also noted on the right side below the t4 dermatome .
sagittal t2-weighted mr images revealed a left - sided cystic extradural lesion at the c7-t1 junction ( fig .
1a ) , and axial images showed that the extradural cystic lesion markedly compressed the spinal cord ( fig .
the lesion appeared homogenously isointense on t1 weighted images and exhibited heterogeneous high signal intensity on t2 weighted images . under a presumptive diagnosis of a cystic lesion in the lower cervical spine ,
emergent surgery was performed . because there were additional osteophytes causing cord compression at c4 - 5 and c5 - 6 , modified expansive laminoplasty of c5 - 6 and a laminectomy of c7 followed by complete removal of the extradural cyst
the lesion was homogeneous , bright yellow in color , firm in consistency , and slightly adherent to dura mater , and contained a small amount of serous fluid .
pathological examination showed the cyst to be composed of fibrous connective tissue and to have a distinct synovial cell lining with focal calcification ( fig .
, he was able to walk without any assistance and returned to work . at his 8-month follow - up ,
these cysts are often located at lumbar facet joints , and less frequently , at cervical facet joints1,12 ) .
synovial cysts and ganglion cysts have similar clinical and radiological features but are histopathologically differentiable .
although several hypotheses have been proposed to explain the development of epidural juxtafacet cysts , their pathogenesis remains unclear .
synovial cysts are believed to develop from degenerative diseases of joints and to represent herniations of joint synovium , but no joint communication may be evident by imaging . on the other hand , ganglion cysts are believed to originate from mucinous degeneration within periarticular dense fibrous connective tissue2,7 ) .
generally , synovial cysts contain clear , serous fluid , whereas ganglion cysts contain gelatinous , viscous fluid .
however , this distinction is not clear , and their differential diagnosis based on contents is difficult .
furthermore , distinguishing between these two entities does not appear to be meaningful from clinical perspective because the treatments afforded are identical .
for this reason , kao et al.9 ) grouped synovial and ganglion cysts together and proposed the term " juxtafacet cyst " .
other pathologies that should be included in the differential diagnosis include all types of space - occupying lesions that may occur in the spinal canal .
mri is most helpful at demonstrating free fluid in a cyst , whereas ct can reveal gas contents in a cyst , the presence of which strongly suggests that the cyst communicates with a facet joint or a degenerated disc space . the clinical progression of patients with a degenerative juxtafacet cyst is variable .
synovial cysts of the spine are usually asymptomatic unless acute hemorrhage occurs after trauma or a significant increase in size causes nerve root or cord compression . in cases without neurological deterioration ,
close observation could be recommended because spontaneous reduction or resolution of a cyst have been reported during conservative treatment6,11 ) .
in addition , a less invasive treatment , that is , intra - articular local anesthetic and steroid injection , has been proposed when pain is the exclusive symptom . however , this management is accompanied by risks of neurologic complications in the cervical region .
our patient also had associated brown - squard syndrome and combined spondylotic spinal stenosis , and thus , spinal cord decompression by open door laminoplasty was indicated in addition to complete cyst removal .
although rare , a synovial cyst in the lower cervical spine should be taken into consideration as a possible cause of brown - squard syndrome in patients with a degenerated cervical spine .
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### Summary:
: synovial cysts are recognized as an uncommon cause of radicular and myelopathic symptoms .
they are most frequently found in the lumbar region . the cervical spine or cervicothoracic junction is a rare location for a degenerative intraspinal synovial cyst as compared with the lumbar spine . at given cervical spinal levels , synovial cysts probably share clinical features with disc herniation and stenosis .
however , the pathogenesis of synovial cysts remains still controversial .
here , we report a rare case of a synovial cyst in the lower cervical spine presented as brown - squard syndrome and include a brief review of the literature . to the best of our knowledge ,
no previous report has been issued in the english literature on a synovial cyst presenting with brown - squard syndrome .
neurologic function recovered completely after complete removal of the cyst and expansive laminoplasty .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: acronyms were most useful when letters and reports were hand written or when one needed to save the ink on the typing ribbon .
alzheimer 's disease and alzheimer 's dementia are not synonymous , but when an author uses the acronym ad , it either suggests that the author believes ( wrongly ) that they are , or we ( as the readers ) are left in doubt as to which condition they were referring to .
alzheimer 's disease is a pathological diagnosis using a series of standardised criteria ( e.g. , khachaturian , consortium to establish a registry for alzheimer 's disease and reagan [ 13 ] ) whereas alzheimer 's dementia is a clinical syndrome that is possibly or probably as a consequence of alzheimer 's disease .
alzheimer 's dementia is most commonly defined in a research setting using the nincds - adrda criteria which compared to results at autopsy , detects alzheimer 's disease with a sensitivity of 9198% .
however , there are a group of clinical conditions probably mediated by alzheimer 's disease that do not satisfy the criteria for alzheimer 's dementia and these have been described as a premanifest or incipient alzheimer 's dementia .
these conditions that will inevitably precede dementia are often indistinguishable from those caused by different pathologies and have therefore all been loosely referred to as mild cognitive impairment ; of which at least 16 prognostically weak definitions currently exist . in huntington 's disease research ,
the availability of an accurate diagnostic test has allowed investigation of the clinical correlates of the disease whilst the person is asymptomatic .
work of this nature has already , from baseline data alone , demonstrated neuroimaging and motor changes .
premanifest , according to the track - hd programme a large collaborative european research programme which is investigating the earliest clinical and biomarker changes ( plasma proteomics and neuroimaging ) in this population .
the overarching aim of their work is to be able to define means of identifying changes in the premanifest population that will act as surrogate markers of an intervention 's efficacy at secondary prevention .
accordingly these interventions ' efficacy can be measured reliably before the patients manifest overt signs of illness . in alzheimer 's dementia research
population ( save for a small proportion of patients with fully penetrant , autosomal dominant mutations ) and urgently need to be able to do so .
the main aims of the cochrane collaboration 's diagnostic test accuracy workstream within the dementia and cognitive impairment group are threefold .
firstly , to scrutinise the research of tests purporting to have diagnostic accuracy for alzheimer 's disease in vivo , secondly , using well rehearsed statistical techniques to derive a value for the test 's diagnostic accuracy , and finally to highlight the deficiencies ( where they exist ) of extant research to improve the quality and direction of future primary research of diagnostic tests for alzheimer 's disease .
it should be made clear from the outset that the reviews can only reflect the primary research .
whilst the first few reviews are all of single tests or domains of tests ( episodic memory , csf and plasma a levels , volumetric mri , and apoe status ) , the reviews will not be limited exclusively to single tests , be they clinical , genetic , or biomarker .
we are aware that the optimal and only realistic way to make a diagnosis of premanifest dementia will be made by combining tests and such combination of tests ( e.g. , the csf - tau / a ratio ) can also be reviewed by authors registering an interest with the cochrane collaboration dementia and cognitive impairment group .
it is hoped also that by demonstrating the diagnostic accuracy of single tests through these reviews , this will help researchers to assemble test batteries and biomarker panels in the future from the strongest possible evidence base . finally , the groups work is not limited to alzheimer 's disease , and we will welcome reviews on other forms of neurodegenerative disease both in their premanifest stages as well as a test or test batteries ability to distinguish between different types of dementia . with that said ,
the following section describes the assumptions and observations that we have had to make and the methodology that will be enacted in an upcoming series of diagnostic test accuracy reviews by our group and its collaborators .
this methodology describes the methodology for identifying alzheimer 's disease in patients before alzheimer 's dementia develops and is applicable to both single tests as well as scrutiny of combinations of tests .
it is assumed that every patient who develops alzheimer 's dementia has to pass through a prodromal or incipient stage of mild cognitive impairment as described by various authors using several definitions .
the most common presentation for a patient with premanifest alzheimer 's dementia is a subjective episodic memory complaint , which is then diagnosed as mild cognitive impairment after an objective battery of neuropsychological tests demonstrate age - matched deficiencies .
however as matthews et al . described , these definitions hold little prognostic significance with considerable variations in the level of impairment and outcome at two years .
most definitions of mild cognitive impairment are cross sectional which over time may develop along one of five clinical courses . though these courses are not necessarily specific to any one disease or illness trajectory .
for example , depression or other psychiatric illness may exhibit courses 3 , 4 , and 5 below .
conversion to another subtype of dementia , for example , lewy body or vascular dementia . continued deterioration in cognition which does not go on to satisfy criteria for a dementia .
there has been high quality evidence produced of late that suggests modification of identified epidemiological risk factors for dementia ( e.g. , decreased oestrogen levels in the menopause [ 10 , 11 ] ) have little benefit and indeed may have safety concerns as treatments once dementia has developed .
this and other similar observations have led to the belief that there may well be a critical window , earlier in the course of the underlying disease , for intervention or risk factor modification which may include optimising cardiovascular health , increasing exercise , minimising the risk of depression , improving diet , increasing intellectual activity and reducing the risks for diabetes and stroke .
people who have capacity and knowledge that they have premanifest alzheimer 's dementia will be in a position to create lasting powers of attorneys , advanced directives and engage in financial , social and lifestyle planning whilst well . therefore , it is further assumed that there is merit for the patient in identifying alzheimer 's disease prior to dementia developing .
common sense dictates the earlier one starts a course of treatment for any disease the better , though being mindful of any side effects that may outweigh the initial benefits .
no potentially disease modifying therapies for alzheimer 's disease have demonstrated clear efficacy in late stage clinical trials in patients with alzheimer 's dementia , though several still retain promise . whilst this may be due to the drugs simply not working , the targets being wrong or the tools to measure effect being insensitive to change ; it is also possible that these drugs will only work when the disease process is less advanced .
this concept has been described in multiple sclerosis , where disease modifying therapies are ineffective in patients whose disability has progressed to a more severe level .
in essence , testing this hypothesis will require recruiting a population who definitely have early stage disease to be able to develop disease modifying drugs and thereafter use this population as the target for the clinical intervention
. such intervention may lead to the secondary prevention of alzheimer 's dementia by manipulating the progression of the predementia pathology ; with great human , social , and economic benefit .
the development of a new diagnostic test for any condition is predicated on the ability to determine the accuracy of the new or index test against the gold or reference standard .
this creates problems specific to alzheimer 's disease in that there is no in vivo gold standard , currently its premortem presence is probable or possible based on an array of symptoms described as alzheimer 's dementia and neuroimaging findings .
it is worth considering that the applicability and acceptability of clinical criteria such as nincds - adrda are partly based on their concordance with pathological studies .
brain biopsy ( the means to achieving the gold standard ) is not ethical for this purpose unlike other invasive gold standard tests ( e.g. , liver biopsy to determine the accuracy of fibroscan for cirrhosis ) .
as the aim of the cochrane reviews are to accurately diagnosis disease before dementia develops , the fact that a patient develops alzheimer 's dementia ( nincds - adra criteria ) is taken as ipso facto proof that at the point of testing , disease was present .
we recognise that an alternative reference standard is needed to be used and this choice was subject to much debate and discussion by the cochrane dementia and cognitive impairment editors .
we hope that over time a better reference or gold standard will emerge , until such time though and with inherent limitations , the conversion to nincds - adrda alzheimer 's dementia will be used as the gold standard .
accepting the assumption that there is a group of patients at the predementia stage of alzheimer 's disease manifesting cognitive impairment and that there are currently no means to affect the trajectory of their disease then conventionally , it is assumed that the development of alzheimer 's dementia represents the necessary level of proof that at the pre - dementia stage they had alzheimer 's disease . if this is accepted , then this overcomes the problem of the index test and reference test being separated over time .
again , although not ideal , it is at this stage considered the optimal solution to the definition of a gold standard .
accordingly , the reference standard is the development of clinically diagnosed alzheimer 's dementia or other dementias depending upon the aims of specific reviews .
this , however , does create problems considering whether the test is actually diagnostic or prognostic .
if it is the latter , then we are asking whether the test at baseline predicts a pathway of decline , stability or improvement .
however in the context of the cochrane diagnostic test accuracy reviews we are only interested in the ability of the test to identify underlying alzheimer 's disease and therefore be associated with the development of alzheimer 's dementia .
it has no bearing what the prognosis ( clinical , behavioural or functional ) of the alzheimer 's disease is for any given individual who returns a positive test but rather whether or not they have alzheimer 's disease in their brain confirmed by the test in question .
there are three stages in developing clinically applicable diagnostic tests or criteria for premanifest alzheimer 's dementia ( table 1 ) .
the testing of single or individual diagnostic tests which may be laboratory based , clinical , genetic or radiological in nature . applying diagnostic test accuracy statistical enquiry on a battery or collection of the above tests ( e.g. , devanand et al . )
consensus statement by clinical specialists using what the assembled and invited experts deem to be the most promising imaging , neuropsychological and laboratory tests .
for example those described by tabert et al . and dubois et al . [ 8 , 9 ] .
the remainder of this paper will focus on the efforts of the cochrane collaboration to determine the accuracy of a single diagnostic test .
it is recognised that it is highly unlikely that one test for a complex disorder will have sufficient diagnostic accuracy however , each test will be a constituent of a battery that may be developed from the strongest candidates be they biomarker , genetic , or clinical . over time
the same methodology will be applied to test batteries as and when they arise . as the cochrane collaboration is widely known for its reviews of randomised controlled trials ( which helped in the drive towards the consort criteria for the reporting of clinical trials ) the diagnostic test accuracy review will have the following quality features as set out in the cochrane collaborations dta handbook .
systematic searching . making comparisons between tests concerning their global accuracy . estimating the accuracy of a test operating at a particular threshold .
the purpose of a cochrane diagnostic test accuracy review is to use meta - analysis techniques to establish a confidence around test accuracy which informs diagnosis of the condition in question .
this is done by using a meticulous , objective search strategy in order to identify data from high quality studies which can be combined in meta - analysis .
the principle of this diagnostic test accuracy review methodology is to look for studies which document the test of interest in patients with any of the definitions of mild cognitive impairment and then compare the results of the test between those who convert to alzheimer 's dementia and those with different outcomes , in order to determine its diagnostic accuracy when applied at the mild cognitive impairment stage .
the undertaking of a successful diagnostic test accuracy review requires a clear description of the studies suitable for inclusion before the search , to keep bias to a minimum and to ensure only the studies of highest quality are analysed . in order to achieve the objectives of a dta review , three major points have to be defined .
the test to be assessed
this can include plasma , urine or cerebrospinal fluid biomarkers and genetic , radiological and well defined neuropsychological assessments . as well as combinations of these .
this can include plasma , urine or cerebrospinal fluid biomarkers and genetic , radiological and well defined neuropsychological assessments . as well as combinations of these .
the population to study
we concluded that the tests should have been applied in any of the referenced definitions of cognitive impairment believed to be due to neurodegeneration . only by including all such definitions
can we define the tests ability to identify alzheimer 's disease from the absence of neurodegenerative disease , but also its ability to identify which type of neurodegenerative disease is present .
we concluded that the tests should have been applied in any of the referenced definitions of cognitive impairment believed to be due to neurodegeneration . only by including all such definitions
can we define the tests ability to identify alzheimer 's disease from the absence of neurodegenerative disease , but also its ability to identify which type of neurodegenerative disease is present .
the condition under interest
it is a dilemma that there is no gold standard
post - mortem provides an apparently definitive diagnosis however in the extant literature , few baseline biomarker levels are recorded in those individuals who then have a post mortem . for future studies , the reference standard will be conversion to alzheimer 's dementia based on the nincds - adrda criteria , which is probably or possibly a result of alzheimer 's disease .
this of course implies a restricted view of the outcome to one particular form of dementia .
it could be argued that a broader concept of dementia would be acceptable as this is what is ( in effect ) most relevant to the patient , their carer and their doctor .
this is acceptable as long as each review explicitly states the final condition of interest .
the cochrane collaboration dementia and cognitive impairment dta group is not only interested in dta 's for alzheimer 's disease but also reviews for all other dementias and neurodegenerative disease associated with cognitive impairment .
post - mortem provides an apparently definitive diagnosis however in the extant literature , few baseline biomarker levels are recorded in those individuals who then have a post mortem . for future studies , the reference standard will be conversion to alzheimer 's dementia based on the nincds - adrda criteria , which is probably or possibly a result of alzheimer 's disease .
this of course implies a restricted view of the outcome to one particular form of dementia .
it could be argued that a broader concept of dementia would be acceptable as this is what is ( in effect ) most relevant to the patient , their carer and their doctor .
this is acceptable as long as each review explicitly states the final condition of interest .
the cochrane collaboration dementia and cognitive impairment dta group is not only interested in dta 's for alzheimer 's disease but also reviews for all other dementias and neurodegenerative disease associated with cognitive impairment . a sensitive search strategy is then employed to form the basis of the systematic review .
the analysis of dta reviews is more challenging than with intervention reviews due to the binary classifications of sensitivity and specificity and the tradeoff between the two measures .
the statistical method behind combining the results is described in each review and the data from the two - by - two tables will be used to calculate the sensitivity , specificity , positive and negative likelihood ratios , and diagnostic odds ratio for each study .
individual study results are presented graphically by plotting estimates of sensitivities and specificities in receiver operating characteristic ( roc ) space . if more than one threshold is reported by the study authors , then the two - by - two table for one threshold
the main difficulties to be faced are threefold the use of clinical criteria occurring after the test as a gold standard . the reliability of the criteria for participant inclusion in studies . the relevance of the test of interest and the defined population
. the most accurate reference standard would be to use alzheimer 's disease diagnosed by brain biopsy in order to combat the current lack of an ante - mortem gold standard test .
however , this is currently unethical in the live patient purely for the purpose of research . also important are the multiple definitions for the premanifest stage of alzheimer 's dementia . the criteria to diagnose mci vary and are not uniformly applied , resulting in a dissimilar predictive power for progression to alzheimer 's dementia between definitions . in other words , each definition of a clinical state
this will lead to heterogeneity in the review which will be documented and explained where possible .
an accurate diagnostic test for alzheimer 's disease in the patient with premanifest alzheimer 's dementia may be imminent .
however based on the methodology of the diagnostic test accuracy review this will only be demonstrable in a defined population with diagnosed cognitive impairment . there is an argument that those with no symptoms what - so - ever ( truly premanifest ) should be targeted because the benefit of potential treatments or risk modification will be augmented .
this will hopefully be the next step if accurate diagnosis of disease can be achieved in the patients already exhibiting and complaining of cognitive impairment .
the objective of this paper is to ensure that in future years the cochrane collaboration will have reviewed and constructed a list of accurate diagnostic tests and test batteries , such that clinicians will be able to accurately identify and intervene with best practice in impaired patients with an earlier disease stage prior to the development of disabling dementia .
secondly , researchers will have a means to define a premanifest alzheimer 's disease population to direct disease modifying therapies towards and , finally , we hope that in being critical of current diagnostic test methodologies we will have some impact on generally improving the quality of primary research of diagnostic tests in dementia over time .
we believe that there are many approaches and dilemmas to consider , which can only be overcome by using dispassionate , unbiased , stringent , and evidence - based methods . using these methods ( which may evolve over time ) , we can optimise the chances of achieving accurate diagnosis of alzheimer 's disease at an earlier stage than is currently possible when the patients and their carers can derive maximal benefit from future treatments , psychosocial interventions , as well as lifestyle changes and risk modification .
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### Summary:
: there has been a large increase in the amount of research seeking to define or diagnose alzheimer 's disease before patients develop dementia . if successful , this would principally have clinical benefits both in terms of treatment as well as risk modification . moreover , a better method for diagnosing predementia disease would assist research which seeks to develop such treatments and risk modification strategies .
the evidence - based definition of a diagnostic test 's accuracy is fundamental to achieve the above goals and to address this , the cochrane collaboration has established a diagnostic test accuracy group dedicated to examining the utility and accuracy of proposed tests in dementia and cognitive impairment .
we present here the assumptions and observations underpinning the chosen methodology as well as the initial methodological approach decided upon .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: the
mechanisms at the origin of surface - enhanced raman scattering
( sers ) are now , to a certain extent , rather well established . after almost twenty years of debate
, it is nowadays admitted that
the major part of the enhancement arises from the amplification of
the electromagnetic ( em ) field near a metal surface .
this so - called
electromagnetic mechanism involves
the excitation of localized surface plasmon resonances ( lspr ) which
entails an enhancement of the absorption and scattering cross sections
of subwavelength particles occurring in the visible or near - infrared
regions for silver and gold metals . in the sers enhancement
process
, the particles may be considered
to act as antenn for the molecules since the particles enhance
both the in - coming em fields and also the out - going raman shifted
radiation . under some specific conditions , this raman enhancement
factor ( ef )
can be expressed as ef = |eloc / e0| where e0 is the incident field and eloc is the local field at the molecule . an
additional chemical contribution to the enhancement process has also
been invoked arising from resonant molecule / metal charge transfer
complex .
recently , a. t. zayak et al . quantified
this contribution for benzenethiol molecule chemisorbed on gold substrates
and connected a strong modification of raman spectra to changes in
the electronic structure of the metal - adsorbate interface ; but if the sers effect is understood as the cooperative
effect of both mechanisms , there is a strong predominance of the electromagnetic
mechanism . among the great variety
of systems that have been used as sers
substrates , like roughened electrodes , colloids , metal islands films , nanospheres lithography , or arrays
of lithographically designed particles
, the latter appears to be the most suitable to study the influence
of localized surface plasmon resonances on the enhancement factor .
for example , regular arrays of nearly identical metal nanoparticles
obtained by electron beam lithography ( ebl ) have received considerable
attention in the past two decades , mainly for their ability to support
very narrow lspr , and for the ease with which the lspr wavelength
can be tuned ( through the change of the particle s material ,
size and shape ) . in these structures ,
the raman enhancement factor ( ef ) , typically of about 10 , is distributed almost equally over all particles .
thanks to the improvements in the uniformity of ebl nanoparticle - based
sers substrates , it was possible to verify experimentally the validity
of the em enhancement model in sers and tackle basic aspects of the
sers effect ( optimal spectral position of the lspr , sers polarization
dependence , ... ) . on substrates consisting
of particles with a large size and distance
distributions ( e.g. , colloids or metal island films ) ,
the ef was found
to originate mainly from specific local areas called hot spots . a closer analysis of this kind of substrates revealed that hot spots
are located within the gap of aggregates of closely spaced particles .
the strong electromagnetic coupling taking place between such close
spaced particles leads to giant inhomogeneous em fields within the
gap .
these fields are considered to be
responsible for the huge sers effect , with ef of the order of 10 to 10 , thus opening the route to single molecule
detection by sers .
although ebl structures
appear to be suitable substrates for fundamental
sers investigations , a crucial aspect has often been overlooked concerning
this kind of samples : the fabrication technique ( thermal evaporation
and the lift off process itself ) generates nanometer scale surface
roughness ( nsr ) which is likely to play a role in the optical response
of the as - fabricated structures . the poor degree of crystallinity
and the surface roughness contrast with the single - crystalline nature
of the particles fabricated by chemical synthesis .
the presence of nsr features can be critical since they
may change the near - field distribution of the em field , which can not
be witnessed by simple far - field measurements .
this aspect
has rarely been addressed , though being of key importance in the context
of nano - optics or molecular sensing applications .
it was shown that the rougher
the gold particles are , the higher the sers activity is , demonstrating
the important role of nsr on the near - field optical response .
this boost of the sers signal was assigned to the presence of hot - spots
on the particles surface , rather than to an increase of the surface
area due to corrugation .
however , these
experiments were performed on colloidal particles in solution or deposited
as a single layer on a dielectric substrate ; and one knows that this kind of sers - active substrates often leads
to particle aggregation with large size distribution and broad plasmon
resonances , thus preventing any detailed and systematic approaches
for an in - depth analysis of the influence of nsr on sers . to
our knowledge , this issue has not been investigated in detail
on ebl structures , despite its important implications in plasmonics .
the purpose of this article is then to study how sers can be modified
by the presence of roughness and/or protrusions located at the surface
of lithographically designed structures .
choosing fabricated arrays
of gold stripes with subwavelength widths for their optical response
is quite simple .
if the polarization
of the incident radiation is set perpendicular to the stripes ( i.e. ,
transverse polarization ) , a localized resonance can be observed in
the visible to the near - infrared spectral range , while under longitudinal
polarization no localized resonance is excited ; no propagating surface
plasmon polariton can be launched due to our excitation scheme .
thus ,
any sers effect obtained with a longitudinal polarization can not be assigned to resonant plasmon excitation . in order to study the influence of nsr on the sers activity of
the particles ,
the surface roughness and crystallinity parameters
were modified through thermal annealing . as the annealing temperature
is increased ( up to 200 c in our case ) , the grain size increases
and the surface roughness decreases . the comparison of the experiments
carried out on annealed and nonannealed ebl stripes raised four important
questions : ( i ) is the sers intensity significantly
governed by the nsr located on the particles ?
( ii ) can we quantitatively estimate the impact of nsr when the excitation
wavelength is set within the plasmon resonance of the structures ?
( iii ) do the nsr features give rise to a sers activity for
an off resonance excitation ? ( iv ) what is the amount of off resonance hot spots enhancement
and how does it depend on the incident wavelength ?
after a short
description of the experimental and numerical techniques ,
we begin the discussion by describing the optical properties of the
different samples investigated in this work .
next we turn to the comparison
of the sers activity of stripes arrays with different nsr and crystallinity
parameters .
we then move to the impact of nsr on the sers intensity ,
by changing the excitation polarization and wavelength .
we systematically
compared the experimental results to numerical calculations based
on the discrete dipole approximation dda technique .
we finally conclude on the importance of surface roughness of ebl
structures in sers experiments .
.
both sets are fabricated with identical parameters , but set a is annealed
( reduced nanoscale roughness ) whereas set b is not .
figure 2a depicts typical extinction spectra of a nonannealed
( b1 ) and an annealed ( a1 ) stripe array with nearly identical transversal
plasmon resonances , which both are characterized by a pronounced extinction
peak for excitation polarized perpendicular to the stripe axis ( see
also the figure si3 displaying the extinction
spectra of all stripe arrays of set b ) .
in contrast , when the
electric field is set parallel to the stripes
( longitudinal polarization ) , the extinction spectrum shows the same
features as expected for a smooth , thin gold film .
the annealing process
results in a blue shift of the lspr wavelength of ca .
2030
nm , together with a reduction of the full - width at half - maximum of
the spectra and a higher extinction , as recently shown by j. c. tinguely
et al .
therefore , in order to compare
the sers efficiency on equivalent stripes before
and after annealing , pairs of stripes from set a and b must be selected
in such a way that the plasmon resonance wavelength and the optical
extinction of the array before annealing is nearly identical to those
of the other array after the annealing process , which is well fulfilled
for array a1 ( annealed , width 150 nm ) and b1 ( not annealed , width
120 nm ) ( see figure 2a ) . for qualitative
comparison , we plot in figure 2b the dda - simulated
extinction spectrum of a smooth target
( width 110 nm ; height 40 nm ) in transversal and parallel polarizations .
in the calculation , the unit cell length of the stripe has been chosen
to 110 nm , which allows us to match the lspr wavelength ( located at
ca .
this is necessary due to incertainties in the dielectric functions
of gold and ito .
( a ) extinction spectra of the samples a1 and b1 in perpendicular
( transverse ) and parallel ( longitudinal ) polarizations ; ( b ) dda calculation
of the extinction cross section of a smooth unit cell reproduced infinitely
using periodic boundary conditions ( width : 110 nm , length : 120 nm ;
height : 40 nm ) , in both polarizations . note that the extinction cross
section is indicated in nm per unit cell ( length 120 nm ) .
the purpose is
to show the influence of the nsr features of the ebl samples on the
sers efficiency by comparing measurements on annealed ( smooth ) gold stripes ( set a ) and nonannealed ( rough ) ones ( set b ) ( details of the geometrical
parameters of samples investigated in this work are given in supporting information ( si ) table 1 ) .
figure 3 presents typical sers spectra of mb adsorbed on
gold stripes a1 and b1 , respectively , for a laser polarization set
perpendicular ( transverse ) and parallel ( longitudinal ) to the stripes
( both plasmon resonances are close to the 633 nm laser line ) .
the 1618 cm raman peak used to estimate the sers intensity is assigned to a
c n stretch .
note that the sers signal is accompanied by a
broad background attributed to the fluorescence ( possibly modified
by the lspr ) of mb molecules .
two
main observations can be pointed out : ( i ) in
transverse polarization , the sers signal of mb is more intense on
the nonannealed array ( b1 ) than that of the annealed one ( a1 ) .
the
decrease of the sers signal after the annealing process has been systematically
observed for all the samples studied in this work ( see the sers spectra
of the different samples in the figure si.4 ) .
this result is also in agreement with previous results we recently
published , using gold circular dots as sers - substrates.(ii ) when the incident polarization
is parallel to the stripes , the mb sers signal almost disappears on
the sample a1 as expected since no lspr is excited , while a strong
unexpected sers signal appears in the case of the nonannealed sample
b1 .
sers of mb molecules ( concentration 10 m ) on
samples a1 ( red spectra ) and b1 ( black spectra ) in longitudinal ( dot
lines ) and transverse ( solid line ) polarizations .
excitation : 633
nm ; acquisition time : 5 s ; back scattering configuration .
note that
the sers spectra are vertically shifted for more clarity . in order to confirm and explain these experimental
results , we
compare them to the optical near - field enhancements calculated at
an incident wavelength of 633 nm for a rough and smooth stripe .
figure 4a displays the distribution of the near - field enhancement
ef = |eloc / einc| on a unit cell length
of a smooth stripe excited with a transverse polarization .
as
expected , the three - dimensional mapping of the optical near - field
( figure 4a ) shows variations only in the cross - sectional
area perpendicular to the stripe and is constant along the stripe
length . however , one interesting observation is that the enhancement
is mainly located on the side of the stripe at the ito / gold interface ,
as previously observed on silver nanocubes using the same illumination
conditions .
a transverse cross section
( figure 4b ) allows for the quantification of
the enhancement along a line : almost no enhancement on top of the
stripe and a maximum value of ef of 9 10 at the
ito / stripe interface . in the case of the rough target
, the mapping
of the electromagnetic
distribution clearly reveals a strong variation over the surface of
the stripe ( figures 5a and 5b ) .
compared to the case of a smooth stripe , the electric
field on a rough stripe is significantly enhanced on its top and reduced
on its sides .
the presence of the nsr indeed gives rise to an near - field
enhancement exceeding ef = 2 10 for a cross - section
located at z 10 nm and y = 1 nm ( figure 5b ) .
so the calculation
of the local fields distribution enabled us to ( i ) understand the
higher sers signals observed for mb molecules on nonannealed samples
and ( ii ) suggest that the enhancement of the raman scattering is due
to both the dipolar lspr excitation and also the hot - spots created
by the nsr features on top of the stripes .
smooth target : ( a ) mapping
of the near - field enhancement on a unit
cell length of a 110 nm wide stripe ; ( b ) transverse section profile
corresponding to the 3d mapping ( a ) of the nf enhancement in transverse
polarization . as a direct consequence
of the nsr features ,
the near - field enhancement
is no longer expected to follow the extinction spectrum . to highlight
this , and taking into account that the sers intensity reflects the
near - field enhancement , we compared the value of the extinction of
the different samples at 633 nm ( the excitation wavelength ) with the
integrated intensity of the 1618 cm raman band
of mb molecules ( figure 6 ) . for samples from
set a , we observe sers intensities which are proportional to the excitation
strength of the transversal plasmon resonance ( red circular dots in
figure 6 ) . for samples from set b ,
no correlation
between the sers intensity and the extinction values can be spotted ,
which we attribute to the fact that the near - field enhancement , partially
arising from the nsr features , is in this case not fully connected
to the dipolar plasmon resonance sustained by the stripe .
an
important feature of the stripes is the strong polarization
dependence of the plasmon resonance and related near - field enhancement .
in theory
if the polarization is changed from transversal to parallel
to the stripes , the near - fields are not enhanced but , contrarily ,
are reduced due to the absorption of gold . in the case of the annealed
stripes a1 , a strong decrease of the sers signal
is observed when
the incident polarization is set along the stripes length , as expected
( figure 3 ) .
note that the residual sers signal
of mb observed can be assigned to the fact that the stripes a1 , after
the annealing process , are not perfectly smooth ( figure 1c and d but only show reduced roughness . in contrast , for
the stripes b1 , the cross - sectional nonuniformities and the larger
surface roughness cause greater deviations from the ideal behavior ,
including 3-dimensional field variations . although in the absence
of lspr excitation , a significant sers signal is still observed in
longitudinal polarization ( figure 3 ) , in accordance
with dda calculations of the near - field distribution ( figure 7 ) .
in the simulations , the enhancements arising
from the nsr features under longitudinal polarization remain quite
moderate , reaching locally ef = 10 .
rough target : ( a ) mapping
of the near - field enhancement on a unit
cell length of a 110 nm wide stripe ; ( b ) transverse section profile
corresponding to the 3d mapping ( a ) of the nf enhancement in transverse
polarization .
the dash - dotted line indicates the average height i.e.
the height of the smooth surface . in order to quantify the contribution of
the nsr features
versus
the dipolar lspr character of the stripes on the sers effect , we performed
sers measurements at 633 nm , under different polarization angles for
samples a1 and b1 ( figure 8) . in both cases ,
the intensity of the sers spectra strongly varies with the excitation
polarization angle , as displayed in figure 8a , showing that sers is maximum for a polarization
perpendicular to the stripes and minimum for a polarization parallel
to the stripes . in the case of the annealed sample a1 ( figure 8b ) , the sers intensities have been fitted by a cos law : the sers signal remarkably
follows the far - field dipolar lspr character of the stripes ( see also figure si.5 displaying the polar graph of the
extinction spectrum of the sample ) .
this cos dependence suggests that sers emission enhancement is independent
of the excitation polarization . in the case of the sample b1 ,
the plot of isers( ) clearly deviates from expectations : the sers intensity does not
follow anymore the cos behavior
due to the contribution of nonresonant enhancement from the roughness
features .
the plot of isers( ) for sample b1 can be compared to the one of an evaporated gold
film , for which the sers signal of mb molecules originates solely
from the nsr features ( generated by the thermal evaporation process ) ,
and is independent of the incident angle of polarization ( see figure si.2 ) .
optical extinction measured at 633 nm
for samples of sets a and
b ( horizontal axis ) , compared to the sers integrated intensity ( excitation
at 633 nm ) of the 1618 cm raman peak of mb molecules
( vertical axis ) . in the case of the annealed samples ( set a - red
dots ) , the sers intensity is proportional to the excitation strength
of the transversal plasmon resonance , in contrast with the nonannealed
samples ( set b - blue dots ) .
the sers intensities isers( ) have been compared with the calculation of the
optical near - field enhancement ef( ) on a smooth
and a roughened target , using the dda method ( figure 8c ) .
both polar calculated curves show a cos dependence , instead of a cos dependence for the experimental data .
this
cos dependence is explained
by the fact that , in the calculations , we assume , for the sake of
simplicity , a |e| enhancement . nevertheless , the polarization dependence of
the calculated electromagnetic enhancement is in qualitative agreement
with the experimental sers intensities .
the calculated enhancement
factor ef is systematically lower for the ideal stripe compared to
the roughened target , whatever the incident angle of polarization
is ( the field enhancement ef cos , for the roughened target , does not fall to zero the minimum
is ca .
rough
target : ( a ) mapping of the near - field enhancement on a unit
cell length of a 110 nm wide stripe ; ( b ) transverse section profile
corresponding to the 3d mapping ( a ) of the nf enhancement in longitudinal
polarization .
the dash - dotted line indicates the average height , i.e. ,
the height of the smooth surface ( please note the linear scale for
the field enhancement ) .
this first set of sers and extinction experiments clearly
indicates
that important deviations are observed between the far - field and the
near - field response in the case of the nonannealed rough nanostripes
of set b. this is in accordance with recent observations on colloidal
sers substrates .
strong sers enhancement
was observed from gold aggregates , at excitation wavelengths far from
the lspr , with a maximum of enhancement observed in the red part of
the visible range , in contrast to the plasmon band peaking in the
blue spectral range . assuming that , in the present case , the sers
signal of mb partially originates from hot - spots due to surface roughness
on top of the stripes , the sers intensity should show little dependence
on the far - field lsp resonance according to the observations on gold
nanoparticle aggregates .
( a ) : sers signal of mb molecules on stripes
b1 at exc = 633 nm , for two incident
angles of = 0 degree ( transverse polarization )
and = 90 degrees ( longitudinal polarization ) ;
( b ) : polar graph of the
sers intensity of mb molecules : for sample a1 ( red dots ) , the data
are fitted with a cos curve .
in the case of sample b1 ( black dots )
the experimental
data are fitted by the function i( ) = 9 10 cos + 2.910 ; ( c ) calculation of the optical near - field enhancement
ef( ) on a smooth ( red circular dots ) and a
roughened target ( black square ) , using the dda method . in order to validate this assumption and analyze
the influence
of the excitation wavelength on the optical near - field enhancement
,
we performed sers experiments on samples a1 and b1 using a 785 nm
laser line as excitation source , which is far from the lspr wavelength .
for the annealed stripe a1 , we could not detect any sers signal for
this excitation condition , regardless of the polarization .
in contrast ,
for array b1 , sers signal of mb molecules was obtained although the
excitation was lying off any lspr ( figure 9a ) .
note that for all the arrays of set b , we did
observe a sers signal of mb molecules with the 785 nm excitation ;
for arrays from set a , a low sers signal could be obtained only on
the stripes with the larger widths and lspr closer to the 785 nm ( see figure si.6 ) .
the observation of a sers signal off lspr is in accordance with the mapping of the optical
near - field enhancement at 785 nm ( see figure si.7 ) . as for the excitation at 633 nm
, it clearly reveals a strong variation
in the cross - sectional area perpendicular and parallel to the stripe .
the em field is mainly enhanced where the roughness lies , while it
is minimum on the sides of the structure ( similar to the mapping at
633 nm ( figure 5 ) , with maxima close to ef
= 10 ( see the section profiles in the figure si.7 ) .
the weak correlation between near - field enhancement
and resonant excitation of the transverse plasmon mode is further
supported by sers measurements with different polarization angles
of the excitation for the sample b1 . as shown in figure 9b ,
the intensity of the sers spectra is almost independent
of the incident polarization angles for sample b1 , contrasting with
the cos dependence for the 633
nm excitation .
this experimental sers result is in quite good qualitative
agreement with the dda calculated polar graph of the enhancement factor
ef , displaying no dependence with the polarization of the exciting
light .
an interesting and remarkable feature is the elliptic shape
observed for both polar graphs of figure 9b
and 9c , with similar aspect ratio isers0/isers90 1.3 which point toward a small residual nonresonant
contribution of the em enhancement of the transverse plasmon mode .
( a ) extinction
spectrum of sample b1 and sers signal of mb molecules
at exc = 785 nm ; ( b ) polar graph
of the sers intensity of mb for sample b1 at 785 nm ; ( c ) dda calculation
of the optical near - field enhancement efmax vs the incident
angle , at 785 nm , in the case of a roughened target .
extinction calculated cross section of a roughened target
( red
curve ) , together with the near - field enhancement : for a smooth stripe
in transverse polarization ( black square ) ; for a roughened target
in transverse polarization ( black circle ) , in longitudinal polarization
( empty circle ) . in the case of the smooth target , the nf enhancement
remarkably follows the far - field extinction cross section . on the
other hand , the nf enhancement is maximum in the red part of the visible
spectral range for the roughened target , in both polarizations .
since the sers intensity is not
systematically correlated to the
far - field extinction spectrum for the nonannealed samples , one can
wonder where is located the region of the maximum enhancement .
we
thus calculated the enhancement factor as a function of the incident
wavelength , as shown in figure 10 , for four
incident wavelengths 633 , 785 , 900 , and 1000 nm in both transverse
and longitudinal polarizations .
the calculations show that , as the
excitation wavelength is red - shifted , the enhancement factor increases ,
reaching a maximum in the near - infrared range ( ca .
a physical insight has been proposed to understand
the presence of a maximum for the enhancement factor in regions where
extinction is the lowest , in the case of trimers of gold particles .
it was shown that destructive interferences arising from plasmon excitation
of coupled particles at 820 nm may not manifest themselves in the
far - field spectrum , although resulting in a high sers signal .
further studies are needed to find out if this
argument can be extended to random nsr features on lithographically
designed nanoparticles .
in this
study , the sers efficiency of mb molecules was investigated
on nonannealed and annealed lithographic gold stripes .
we showed that
the sers intensity is not tightly linked to the far - field response
of the nonannealed stripes .
this mismatch between the far - field and
near - field response is attributed to a significant contribution of
the nsr features to the sers intensities . the annealing process of
the stripes , which decreases the nsr , results in a weakening of the
sers signal of the adsorbed molecules .
the sers intensity remarkably
follows the far - field dipolar response on the annealed structures ,
which is not the case for the nonannealed samples .
a remarkable result
is the observation of a sers signal off any lspr
excitation , for nonannealed samples , with a nearly complete loss of
polarization anisotropy inherent to ideal stripe structures .
all our
results are in agreement with the results of the dda calculations .
the results further suggest that the sers effect can be more pronounced
in the red part of the visible range , far from the plasmon resonance
of the structures under study .
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### Summary:
: in this article , we investigate the
surface - enhanced raman scattering
( sers ) efficiency of methylene blue ( mb ) molecules deposited on gold
nanostripes which , due to their fabrication by electron beam lithography
and thermal evaporation , present various degrees of crystallinity
and nanoscale surface roughness ( nsr ) . by comparing gold nanostructures
with different degrees of roughness and crystallinity ,
we show that
the nsr has a strong effect on the sers intensity of mb probe molecules .
in particular , the nsr features of the lithographic structures significantly
enhance the raman signal of mb molecules , even when the excitation
wavelength lies far from the localized surface plasmon resonance ( lspr )
of the stripes .
these results are in very good agreement with numerical
calculations of the sers gain obtained using the discrete dipole approximation
( dda ) .
the influence of nsr on the optical near - field response of
lithographic structures thus appears crucial since they are widely
used in the context of nano - optics or / and molecular sensing .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: during the seventies , strong and jako proposed the use of endoscopic laser surgery in the treatment of laryngeal lesions.1
2 since then , the transoral laser microsurgery ( tlm ) has been gaining ground in the world of laryngeal oncology , establishing itself as an effective option in the management of malignant tumors of the glottis , supraglottis , and hypopharynx .
the treatment 's advantages , such as magnification generated by the microscope , allows us to make limited resections to differentiate healthy tissue from that which has been affected , thus , preserving adjacent disease - free areas . in turn
, it has allowed a decrease in the number of tracheotomies and in the use of nasogastric tubes.3
4
5 moreover , it is possible to achieve oncological benefits similar to those obtained through open surgery in patients with early and properly selected tumor stages.5 the results reported in the literature show all patients that underwent this treatment presented relatively low rates of complications .
therefore , the aim of this study is to describe the complications that occurred in a group of patients treated for glottic and supraglottic carcinomas in all stages by tlm in a tertiary hospital .
a retrospective analysis of the patients diagnosed with squamous cell carcinoma ( scc ) of the glottis and supraglottic for all stages ( t1 , t2 , t3 , t4 ) , n /+ , m /+ was performed according to the criteria of the union internationale contre le cancer ( uicc ) and the american joint committee on cancer ( ajcc ) in a tertiary hospital .
we included in the study patients treated with tlm between january 2009 and march 2012 .
we identified cases by searching the medical records of our department using the codes from the international classification of diseases ( icd ) 9 .
the ethics committee of our institution approved this study . by reviewing medical records we obtained demographic data ( age , gender ) , medical history , tumor stage , diagnostic tests information , histological findings , complications , and type of surgery . in the final analysis , only complications strictly associated with the surgical technique were included .
we excluded complications associated with anesthesia or exacerbation of chronic diseases that were not possible to associate with the use of this technique .
the type of complications were classified according to the stage of presentation : intra - operatively or post - operatively and immediate or delayed . before the surgery , an interdisciplinary committee specialized in head and neck tumors discussed each case .
patients with lesions suspicious for malignancy were scheduled for laryngeal microsurgery with biopsy ; those that were positive underwent glottic or supraglottic co2 laser resection .
after surgery , the committee reviewed ptnm and assessed the need for reoperation or additional treatment with radiotherapy ( rt ) .
for the surgical procedure , all of the patients received general anesthesia with orotraqueal intubation with special tubes for laser surgery ( covidien - mallinckrodt , dublin , ireland ) .
the anesthetist reduced their o2 level using narcotic gases , and protected the balloon tube in its distal portion with cotton sponges moistened with saline .
the resection was performed using a lumenis co2 laser device ( yokneam , israel ) , with a power setting of 48 w , used in superpulsed mode and continuous setting , varying in size and shape of the spot according to the moment of the surgery by using the micro- manipulator acublade ( lumenis ) .
the type of cordectomy was classified according to the european laryngological society ( elsoc ) proposal for glottic6 and supraglottic endoscopic resection.7 in the case of small tumors ( t1a , t1b ) , whenever possible , en bloc resection was attempted . after resection ,
the piece was pinned and oriented on a corkboard . in bulky tumors ( t2 , t3 , or t4 ) , most cases required piecemeal resections .
laser vestibulectomy was performed when the lateral or anterior portion of the tumor was hidden by a ventricular fold . in all cases ,
the surgeons tried to achieve a margin of healthy tissue of 23 mm , trying to preserve the functions without affecting the oncological radicality of the procedure . in our department , patients treated for head and neck malignant tumors are followed for at least five years at the department of head and neck oncology . for this study , however , we considered a group of patients that had been followed for a minimum of 36 months .
we conducted statistical analysis using spss for windows , version 20.0 ( spss , inc . ,
we applied the chi - square test to study differences in hospital stay , the need to place sng , or dysphagia , depending on t stage .
from a total of 131 interventions ( 98 primary , 33 reoperations ) , 98 patients met the inclusion criteria .
ninety - four ( 95.9% ) patients were men and 4 ( 4.1% ) patients were women .
the mean age of the study group was 64.2 years ( 10.7 years = min 45 / max 88 ) .
of these , 22 ( 22.4% ) were diabetic , 40 ( 40.8% ) were hypertensive , 93 ( 94.9% ) were smokers , and 41 ( 41.8% ) consumed alcoholic beverages . regarding to tumoral stage , 45 ( 45.9% )
patients were classified as pt1 , 32 ( 32.7% ) as pt2 , 15 ( 15.3% ) as pt3 , and 6 ( 6.1% ) as pt4 ( table 1 ) .
eighty - seven ( 88.8% ) patients were classified as n0 , 6 ( 6.1% ) as n1 , 4 ( 4% ) as n2a , and 1 ( 1% ) as n2b . in the sample of patients included in the study
the mean follow - up was 40.1 months ( 14 = min 5 / max 72 ) . as for most frequent type of cordectomy , in glottis tumors , type 4
was performed in 28 ( 28.6% ) patients , whereas , for supraglottic tumors , type ivb was performed in 12 patients ( 12.2% ) ( table 2 ) .
the results will be presented in subgroups as glottis ( gtg ) , supraglottic ( stg ) , and transglottic tumors ( ttg ) .
the mean hospital stay was 3.6 days ( 8.3 = min 1 / max 66 ) for glottic carcinoma , 13.2 days ( 25.5 = min 2 / max 149 ) for stg group , and 38 days ( 54.8 = min 2 / max 116 ) for ttg group , being statistically significant when comparing the periods for different locations ( p < 0.0001 ) .
abbreviations : m , distant metastases ; n , regional lymph node patient groups ; pt , pathological tumoral stage .
abbreviations : elsoc , european laryngological society ; gls , glottic laser surgery ; pt , pathological tumoral stage ; sgl , supraglottic laser surgery . in the group that had intraoperative complications , one patient with a transglottic tumor ( ptiva ) suffered ignition of the airway , which the surgical team was able to control without vital impact for the patient , while another lost a tooth after surgery ( 2/98 = 2.04% ) .
there were no skin burns or eye injuries in any of the patients nor laser injuries on operating room personnel .
immediate post - surgical complications occurred in 6 patients ( 6/98 = 6.1% ) , five of them presented post - surgical bleeding and required clipping of the superior laryngeal pedicle .
two patients in the stg ( 1pt3 , 1 ptiva ) , two patients in the gtg ( 1 ptiva pt3 ) , and another patient from the ttg ( ptiva ) , a total of five patients , required urgent tracheotomy postoperatively .
another patient from the stg ( 1pt2 ) needed urgent tracheostomy due to acute dyspnea secondary to airway edema ( table 3 ) .
abbreviations : min , minimum ; max , maximum . moreover , 42 ( 42.9% ) patients required the use of a nasogastric feeding tube ( nft ) in the immediate postoperative period , 13 ( 20.6% ) in the gtg ( pt1 2 , 4 pt2 , 4pt3 , and 3 pt4a ) , 25 ( 80.6% ) in the group of stg ( 1 pt1 , 10 pt2 , pt3 13 , ptiva 1 ) , and 4 ( 100% ) in the group of ttg ( 1 ptiva pt2 and 3 ) ( p < 0.0001 ) .
the mean duration of nft was 0.9 days ( 2.2 days = min 0 / max 13 ) in the group of patients treated for glottic tumors , 6 days ( 5.7 days = min 0 / max 29 ) in the group with supraglottic tumors , and 15.5 days ( 19.2 days = min 2 / max 44 ) in the group of patients treated for transglottic tumors .
thus , the difference between the mean duration of the nft in patients treated for supraglottic and transglottic tumors regarding patients by glottic tumors was statistically significant ( p < 0.0001 ) ( table 4 )
. abbreviations : min , minimum ; max , maximum ; nft , nasogastric feeding tube . during post - surgical follow - up , 13 ( 13/98 = 13.2% )
eight ( 8.1% ) patients had aspiration pneumonia after surgery , 7 patients in the stg ( 2 pt2 and 5 pt3 ) and one in the gtg ( ptiva ) .
two ( 2.04% ) patients had a cervical abscess , both in the stg ( pt2 and pt3 ) , and one of them complicated by mediastinitis
. two ( 2.04% ) patients from the stg group ( pt3 ) suffered laryngeal stenosis after treatment with tlm , and up to 3 interventions were required to achieve local control for both .
one ( 1% ) patient on whom the surgeon had to expose the thyroid cartilage during tumor resection required accurate hospitalization for medical treatment due to chondritis ( ca .
fourteen ( 14.2% ) patients required tl : 6 ( 6.1% ) of them secondary to aspiration pneumonia , 4 in the stg ( 3 pt3 , 1 pt4a ) , and 2 in ttg ( 2pt4a ) . for the rest of tl ,
the surgical team had to achieve local control of the disease , which they were not able to achieve by tlm .
post - surgical delayed complications were more frequent in the ttg group , affecting 50% ( 2/4 ) of patients , and in the stg group , affecting 41% ( 13/31 ) of patients ( p < 0.001 ) .
furthermore , complications were statistically more frequent in larger tumors for all locations ( p < 0.001 ) ( table 3 ) . during follow - up , 40 ( 40.8% )
twelve ( 20.04% ) from the gtg group ( 3pt1 , 3 pt2 , pt3 , and pt4a 2 ) , 25 ( 80.64% ) from the stg group ( 2 pt1 10 pt10 12 pt3 1 pt4a ) , and 4 ( 100% ) in the ttg had some type of dysphagia , most of them improving after recovery from surgical site and through swallowing rehabilitation .
nonetheless , it is important to note that we do not have a standardized swallowing rehabilitation protocol in patients treated with partial laryngeal surgery . in 6 ( 6.1% )
patients , catheterization percutaneous gastrostomy ( peg ) was necessary ( 4 pt3 supraglottic , 1 pt4a glottis , and 1 ca .
alcohol consumption and snuff consumption were statistically related to the occurrence of complications ( p < 0.001 ) ; however , we found no statistical correlation between diabetes ( p = 0.863 ) and the occurrence of complications . in this study ,
authors have previously classified complications from laser surgery as intraoperative and postoperative ( immediate or delayed ) , also being divided into minor and major , minor referring to complications that resolve spontaneously or can be treated in the office under local anesthesia without major consequences for the patient , while major complications are those requiring intensive medical treatment and even revision surgery.3
in this retrospective study , we analyzed the results of a group of 98 patients and 131 co2 laser interventions ( 98 primary , 33 reoperations ) due to laryngeal tumors ( pt1 , pt2 , pt3 , and pt4 ) treated by primary intervention by tlm .
we found that incidence of intraoperative complications was low , affecting only 2% of patients ; whereas immediate postoperative complications occurred in 6.1% and delayed in up to 13.2% of patients .
none of them were fatal . among the various types of complications that can affect patients ,
post - surgical bleeding is the most feared . in our series , this complication represents 5.1% of the immediate post - surgical complications , affecting 6.4% of patients in the stg , 3.2% of gtg , and 25% of ttg ( ) .
vilaseca et al3 reported 8% of bleeding in a series of 275 patients , of which 6.9% were in the group with supraglottic tumors and 2.9% had glottic tumors .
similarly , steiner and ambrosch8 reported bleeding rate of 7% in supraglottic tumors and 0% in glottic tumors ; whereas authors like peretti et al,9 remacle et al,10 and canis et al11 reported 4 , 4.4 , and 9% of episodes of post - surgical bleeding from treatment of supraglottic tumors . in our series , no late bleedings were evident ; however , we believe these complications should be considered , as they are even more dangerous because the patient is at home and , thus , the possibility of a fatal outcome is higher.12 for this reason , some authors recommend performing prophylactic electrocoagulation of blood vessels in the laryngeal pedicle . as for associating tlm with neck dissection ,
some authors suggest ligation of the laryngeal branches of the external carotid artery.12 moreover , ellies and steiner , in a study that included 1,528 patients treated for tlm , showed 4.7% ( 72 patients ) incidence of post - surgical bleeding .
external carotid ligation was required on 7 of such cases.13
in our study , 8.1% of patients suffered aspiration pneumonia after surgery .
our finding was lower than that described by roh et al,14 who reported an incidence of 11.5% of aspiration pneumonia after tlm and higher than that described by vilaseca et al,3 who reported a rate of 6.1% of pneumonia in the treatment of glottic , supraglottic , and hypopharyngeal tumors , and 2% described by canis et al11 after treatment with tlm of supraglottic tumors .
six ( 6.1% ) patients included in our study , all of them over 65 years old , had to undergo tl due to recurrent episodes of aspiration pneumonia ; 4 of them received treatment for extended supraglottic tumors , confirming the limited indication of these cases . regarding cervical complications , 2 ( 2% ) patients had cervical abscess formation , one complicated by mediastinitis .
no case of emphysema or cervical fistula was evident . however , vilaseca et al3 reported development of cervical emphysema in 3 patients and cervical fistula in one patient in their study , while peretti et al9 describe two other cases of persistent cervical fistula after performing temporary tracheotomy in their patients . another 2% of our patients had stenosis of the laryngeal vestibule after extensive laser resection of supraglottic tumor , a higher incidence compared with 0.4% stenosis found in canis et al11 only one ( 1% ) patient included in the study required treatment for chondritis in the thyroid cartilage .
these findings corroborate those described by vilaseca et al,3 showing 0.72% of cases of thyroid cartilage chondritis after tlm . in our study , 42% of patients required nft ; 20.6% of these were from the gtg group , 80.6% from the stg , and 100% of those patients treated in the ttg .
it is important to note that , in most cases , the placement of nft follows a security principle due to the width of resection . during admission ,
swallowing was evaluated and the nft was removed after achieving adequate swallowing ; hence , the difference in duration between the various groups . in 6.1% of patients , on the other hand , peg placement was necessary due to deglutition complications .
bernal - sprekelsen et al,15 in a study in of 210 patients treated for tumors of the larynx and hypopharynx in stage t2 to t4 , report an average use of nft of 23.2% in patients with small tumors and 63% in advanced tumors . of these patients , 6.2% required gastrostomy and another 3.8% required tracheostomy due to deglutition difficulties .
moreover , canis et al11 reported nft averaging 74% during the first 14 days after surgery , requiring that the nft be kept during 30 days in 18% of cases , of which 6% would require keeping it between 31 and 90 days .
conducted their study in patients with supraglottic tumors and that most of these patients had advanced tumoral stages ( iii and iv ) .
finally , of all patients in the study by canis et al,11 8 ( 3% ) required additional measures due to swallowing failure , 5 patients would require gastrostomy , and 3 other total laryngectomy . in our sample ,
air ignition was evident in one case , which we were able to treat immediately without conditioning further consequences for the patient .
this occurred despite using special orotracheal tube for laser surgery and relying on the invaluable collaboration from anaesthetists to reduce levels of o2 , and taking surgical precautions such as placing cottonoid sponge with saline .
nonetheless , it is clear that the risk of ignition is always present , even if every precaution is taken . finally , it is important to highlight that the complication rate for glottic , supraglottic , and hypopharyngeal tumors , at all stages , that have been operated on by experienced surgeons ranges between 3% and 19%.3
12
16
17 this hypothesis may be confirmed by comparing tumor size , tumor location , and level of surgeon experience with the occurrence of complications.3 moreover , we must emphasize the limitations of our study . given that it is a retrospective study
, there is a possible risk of bias in our results , as it relies on the number of incidents reported by the surgeon in the surgical protocol and the rate of complication reported in the medical reports during follow - up .
previous studies have compared the use of tlm showing good oncologic results and low complication rates , compared with traditional open surgery during the intervention , in the immediate and delayed postoperative period and in the long term , with respect to rt.11
18 however , it is important to consider that life - threatening complications such as bleeding , dyspnea , or ignition of the air19 may appear in this type of surgery , requiring immediate attention and close post - surgical follow - up due to the effects on these patients . in the long term ,
follow - up is also important to assess the occurrence of recurrent pneumonia and swallowing disorders among such patients , as thee are complications that will put the patient 's life at risk or reduce their quality of life .
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### Summary:
: introduction transoral laser microsurgery ( tlm ) has established itself as an effective option in the management of malignant tumors of the glottis , supraglottis , and hypopharynx . nonetheless , tlm is not a harmless technique .
complications such as bleeding , dyspnea , or ignition of the air may appear in this type of surgery .
objective the aim of this study is to describe the complications that occurred in a group of patients treated for glottic and supraglottic carcinomas in all stages by tlm .
methods this study is a retrospective analysis of patients diagnosed with squamous cell carcinoma of the glottis and supraglottis for all stages ( t1 , t2 , t3 , t4 ) , n -/ + , m -/+ treated with tlm between january 2009 and march 2012 in a tertiary hospital .
results ninety - eight patients met the inclusion criteria , which had undergone a total of 131 interventions .
ninety - four ( 95.9% ) patients were male and 4 ( 4.1% ) were female .
the mean age was 64.2 years ( 10.7 years = min 45 ; max 88 ) .
the presence of intraoperative complications was low , affecting only 2% of patients .
immediate postoperative complications occurred in 6.1% , whereas delayed complications affected 13.2% of patients , without any of them being fatal .
conclusion tlm has shown good oncologic results and low complication rate compared with traditional open surgery during intervention , in the immediate and delayed postoperative period and in the long - term with respect to radiotherapy .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: intracranial hypertension ( icht ) management in patients who have suffered a severe tbi remains a challenge . despite improvements in monitoring and medical treatment , morbidity and mortality remains high in patients with icht refractory to therapy .
initially one should try to manage the patient 's icht after a severe tbi with medical treatment ( elevation of the bed side , sedation , relaxation , osmolar therapy , hyperventilation , cerebrospinal fluid drainage , and barbiturate coma ) .
however , in 10 to 15% of the patients , icht is refractory to medical treatment , being in these cases a dc , an effective measure for reducing intracranial pressure , in up to 80% of the cases .
there is a growing body of literature supporting the efficacy of dc , especially after tbi .
it has been significantly demonstrated that in patients with icht , craniectomy alone lowered it , but opening the dura in addition to the bony skull resulted in an average decrease in intracranial pressure .
although it is associated with a shorter stay in intensive care units , it is unclear whether dc improves the functional outcome in adult patients with severe tbi and icht refractory to treatment .
moreover , it should be noted that this technique is not without complications , and those can appear following a sequence , in time .
one of the first things that can occur is the expansion of a hemorrhagic contusion , followed by the appearance of a new subdural hematoma on the contralateral side , seizures , loss of cerebrospinal fluid through the scalp , and external brain herniation .
the appearance of a subdural hematoma and postoperative infections are complications that can develop even four weeks after surgery . over time
, some changes have been implemented in the manner in which the technique is carried out , by trying to find better surgical approaches , to avoid further complications . comparing the effect of standard trauma craniectomy ( stc ) versus limited craniectomy ( lc )
, the stc significantly improves the outcome in severe tbi , with refractory icht resulting from unilateral frontotemporoparietal contusion , with or without intracerebral or subdural hematoma .
this suggests that stc , rather than lc , be recommended for such patients . at the moment
it seems that the tendency has been to recommend unilateral frontotemporoparietal dc for the treatment of cerebral hemisphere injury or edema , where bifrontal dc is indicated if there is diffuse swelling .
we present two cases of patients with tbi , who required dc and who suffered complications secondary to the technique .
a 15-year - old female patient was admitted after a severe tbi . the axial computed tomography ( ct )
scan showed a frontoparietal subarachnoid hemorrhage , right frontal subdural hematoma , cerebral edema , and important bilateral uncal herniation .
seventeen days after admission , when objectified 11 points in glasgow coma score ( gcs ) ( 4 - 1 - 6 ) , and obeying simple commands , the patient suffered a neurological deterioration to 3 points on the gcs and pupil anisocoria .
the ct scan showed that frontal intraparenchymal hemorrhagic lesions opened to the ventricular system [ figure 1 ] .
the patient died in a state of severe brain damage , within the context of nosocomial infection , 35 days after admission .
( a ) decompressive craniectomy performed after head injury and ( b ) the subsequent appearance of a frontal hemorrhage a 60-year - old man who suffered a severe tbi with a right subdural effusion , with a mass effect on the midline of 19 mm , ventricular compression on the same side , uncal and transfalcine herniation with effacement of cisterns and compression on the temporal horn , had a right frontotemporoparietal dc performed , and hematoma evacuation was done .
the patient remained with 10 points ( 3 - 2 - 5 ) in the gcs , but with no connection to the environment .
after 20 days of admission , a right subdural hygroma appeared , which required surgery and titanium cranioplasty [ figure 2 ] .
the patient was discharged home with a 3-point gcs , with severe disability , conscious , but with basic family - support for daily activities , after 28 days of admission .
( a ) subdural hygroma next to the craniectomy area and ( b ) after evacuation of the hygroma and titanium cranioplasty
a 15-year - old female patient was admitted after a severe tbi . the axial computed tomography ( ct )
scan showed a frontoparietal subarachnoid hemorrhage , right frontal subdural hematoma , cerebral edema , and important bilateral uncal herniation .
seventeen days after admission , when objectified 11 points in glasgow coma score ( gcs ) ( 4 - 1 - 6 ) , and obeying simple commands , the patient suffered a neurological deterioration to 3 points on the gcs and pupil anisocoria .
the ct scan showed that frontal intraparenchymal hemorrhagic lesions opened to the ventricular system [ figure 1 ] .
the patient died in a state of severe brain damage , within the context of nosocomial infection , 35 days after admission .
( a ) decompressive craniectomy performed after head injury and ( b ) the subsequent appearance of a frontal hemorrhage
a 60-year - old man who suffered a severe tbi with a right subdural effusion , with a mass effect on the midline of 19 mm , ventricular compression on the same side , uncal and transfalcine herniation with effacement of cisterns and compression on the temporal horn , had a right frontotemporoparietal dc performed , and hematoma evacuation was done .
the patient remained with 10 points ( 3 - 2 - 5 ) in the gcs , but with no connection to the environment .
after 20 days of admission , a right subdural hygroma appeared , which required surgery and titanium cranioplasty [ figure 2 ] .
the patient was discharged home with a 3-point gcs , with severe disability , conscious , but with basic family - support for daily activities , after 28 days of admission .
( a ) subdural hygroma next to the craniectomy area and ( b ) after evacuation of the hygroma and titanium cranioplasty
it is generally reported that the outcome of patients younger than 50 years or with an initial gcs score of 6 or more is significantly better than that for older patients or those with an initial gcs score lower than 6 .
the factor having the greatest influence on the outcome is the time from injury to surgery .
patients who undergo dc within the first four hours following trauma have a better outcome compared to those who undergo dc after four hours .
although the dc is a simple technique , it can be risky , and risks must be considered , as they can alter the prognosis .
complications are more frequent in patients with a low score on the glasgow scale and in those over 60 years . up to 50% of the patients
the expansion of hemorrhagic contusions is the most common complication of neurological impairment and death in patients who have had a period of lucidity after a severe tbi , as in the cases that we have reported .
the explanation for this phenomenon is that when performing the dc , the hemostatic effect is lost when removing the bone and this facilitates the expansion of the effusion on the same side , and is contralateral in a few cases .
the dc seems to be useful in icht treatment and in reducing the mortality rate .
it is relatively common and it can be developed in up to 50% of the cases .
the main cause is a disruption between the dura and arachnoids , when performing the dc . among their most common locations
are the subdural , subgaleal or the interhemispheric , being more common where the craniectomy it is site .
it is rare that effusions present clinical expression and most of them are reabsorbed spontaneously , but when its expansion produces neurological impairment it is necessary to have an aggressive treatment such as surgical excision cranioplasty being a definitive solution , as in the case that we have reported .
the presence of interhemispheric , subdural or subgaleal hygroma is a predictive radiographic sign of hydrocephalus in patients undergoing dc .
hemorrhagic collections with mass effect are usually extra - axial collections , and are usually located at some distance from the craniectomy .
its etiology can be explained by the sudden decrease in intracranial pressure , so they rapidly appear .
its incidence is up to 25% and it is caused due to edema induced by cerebral high perfusion and due to the increase of the hydrostatic gradient from the capillaries , after decompression .
when this complication occurs , it can generate brain parenchymal lacerations and can compromise the cortical venous flow against the edges of the bone defect , causing ischemia and necrosis . performing a wide craniectomy
the dc is also a risk factor for the development of hydrocephalus in up to 30% of cases , and can complicate the prognosis . its appearance may be because this technique may induce alterations in the circulation of the cerebrospinal fluid ( csf ) .
it is usually solved after placing the bone ; however , it can persist for a long time despite the replacement .
the surgical emergency of the procedure can damage the superficial temporal artery , and reduce flow to the flap , or also can accidentally open the frontal sinus , contaminating the area .
the wound infection decreases its incidence due to the administration of antibiotics during surgery , which remains at around 7% .
moreover , the sealing of the dura prevents csf leaks , which can cause meningitis . replacing the bone
is also associated with higher rates of infection , increasing when the bone flap is preserved for long periods of time under freezing conditions .
the dc is a useful technique for the treatment of tbi with icht resistant to medical treatment .
it increases the patient 's survival , but its role in the functional prognosis of patients is still not defined .
early bi - frontotemporoparietal dc decreases the intracranial pressure and the length of stay in the icu , but is associated with more unfavorable outcomes . undertaking this decision
requires that the clinicians involved take appropriate account of having used all the conventional weapons against icht and the absence of data indicating an already negative outcome .
in addition , it is a technique that is not free of complications , which themselves can further increase the worst prognosis of these patients .
just as more studies are needed to define the type of patient who can benefit from a dc , and to decide which technique is most appropriate , all the complications derived from this technique must be evaluated .
we also recommend further studies to monitor brain oxygenation , because the normalization of intracranial pressure does not mean that the brain perfusion has been adequate .
strategies to improve cerebral oxygenation suggest the benefit of multiple approaches to monitor these patients .
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### Summary:
: decompressive craniectomy ( dc ) is a useful technique for the treatment of traumatic brain injuries ( tbi ) with intracranial hypertension ( icht ) resistant to medical treatment , increasing survival , although its role in the functional prognosis of patients is not defined .
it is also a technique that is not without complications , and may increase the patient 's morbidity and mortality .
we report two cases of patients with tbi who required dc and suffered complications from the technique
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: fny s ( figure 1 ) have long been used to probe enzyme reaction
mechanisms , protein environmental perturbation of pka s , and protein structures in biology .
prime examples of the former can be found in recent studies on ketosteroid
isomerase , which has three y s and conserved waters in its
active site environment with one y with a pka of 6.3 , and protein y kinases in which
the y pka within a polypeptide substrate
is perturbed and the rate of its phosphorylation is examined .
3-fluorotyrosine ( 3-fy ) has been incorporated
into the oxygen evolving core complex of photosystem ii to probe the
mechanism of proton - coupled electron transfer ( pcet ) operative in
yz oxidation to yz over a broad ph range .
the high sensitivity of nmr analysis to the f nucleus , in conjunction with its large window and sensitivity
to environment , have been used extensively
to study conformational changes in proteins and the interaction of
proteins with other biological molecules .
recent technology has allowed site - specific incorporation of fny ( n = 2 or 3 , figure 1 ) into any protein
of interest . in our case ,
these fny s have played an important role in studying the mechanism
of the 35 radical transfer in the e. coli ribonucleotide
reductase ( rnr ) , a process which involves one stable and three transient
y s ( figure s1 ) . in this manuscript
we report the general method of fny insertion into proteins using the small 2
subunit of rnr as an example and the characterization of each fny-2 by x - ray crystallography and of
each oxidized fny by multifrequency
electron paramagnetic resonance ( epr ) and electron nuclear double
resonance ( endor ) spectroscopy methods .
this work provides the foundation
for others to use these tools to investigate enzyme mechanisms proposed
to involve stable or transient y s in their catalytic
mechanism or to determine if hole hopping through y and w chains protect redox
active proteins from oxidative damage .
ep represents the potential difference vs the
y/y couple at ph 7.5 as determined by differential pulse voltammetry
( dpv ) studies performed on the n - acetyl fluoro - l - tyrosinamides .
rnrs catalyze the conversion of nucleotides to
deoxynucleotides
in all organisms ( scheme 1 ) .
although the mechanism of nucleotide reduction is
conserved , the mechanism by which the complex radical reaction is
initiated is dependent on the metallo - cofactor .
the class i rnrs are composed of two subunits ,
and , which in e. coli form an active 22
complex .
a y in the subunit is used to oxidize the active
site cysteine to a thiyl radical in the subunit .
this oxidation
occurs over a very long distance and involves a specific pathway ( figure s1 ) . since in the best studied e. coli rnr ,
the rate - limiting step is a conformational change that masks
all the chemistry , altering this step
by a variety of methods has been crucial to our current understanding
of this process .
the most successful approach has been to use the
suppressor trna / rs methodology to incorporate y analogs in place of
four y s in the pathway .
recently we evolved an rs to incorporate ( 2,3,5)f3y and
found that it was polyspecific ; i.e. , it was able to incorporate a
variety of fny s ( figure 1 ) .
these fny s have been shown to
have peak potentials ( ep s ) that
range from 30 mv easier to oxidize than y to 130 mv harder to oxidize
at ph 7.5 in addition to having altered pka s .
these perturbations are ideal for investigating the multistep
redox chemistry that involves a number of pcet steps , required to
initiate nucleotide reduction . in the original paper
, we also demonstrated
that when the stable y122 in 2 is replaced
with ( 2,3,5)f3y , it could be oxidized to the ( 2,3,5)f3y. the electron paramagnetic
resonance ( epr ) spectrum of this radical exhibited hyperfine features
at the low - field and high - field sides of the spectrum associated with
the f ( i = 1/2 ) nucleus that allowed
detection of this radical even in the presence of overlapping unlabeled
y signals . from these early studies and the chemical properties
of the fny s themselves , it was clear that these compounds ( figure 1 ) might provide a useful tool
to study enzymatic mechanisms involving stable and transient y s .
a pair of cysteines
in the
active site of 2 are oxidized to a disulfide on each turnover .
multiple turnovers require re - reduction of the disulfide by the thioredoxin
( tr ) , thioredoxin reductase ( trr ) , nadph system .
in this paper we report the general optimized methodology for incorporation
of these fny s in place of y122 in 2 .
we also report the methodology used to generate
each fny at this position , taking
advantage of self - assembly of the essential diferric - y cofactor
subsequent to the addition of fe , o2 and reductant
to apo-2 or its assembly during recombinant expression .
a potential
issue with the asymmetric fny s
( 2 , 3 , 4 , figure 1 ) is whether they adopt multiple
conformations in 2 either during the folding process or due
to rotation around the c-c bond .
the structures of fny-2 and fny have been determined by x - ray crystallography and multifrequency
epr and endor methods , respectively .
the results support a model where
the fluorine substitution minimally perturbs the overall protein conformation
of 2 , although in several cases , multiple conformers of the
fny itself are observed by both paramagnetic
and crystallographic methods .
the epr spectra of each of the radicals ,
the simulation of these spectra , and the calculated spin density are
reported and reveal the detailed f hyperfine parameters .
in conjunction with the range of reduction potentials spanned by these
analogs ( figure 1 )
, this information will be generally useful to investigate redox active
y s reported in a number of metabolically important enzymes .
( his)6 wt-2 ( specific activity
2600 nmol / min / mg ) was expressed from pet28a - nrda and purified using the standard laboratory protocol .
e. coli thioredoxin ( tr , 40 u / mg ) and thioredoxin reductase ( trr , 1400 u / mg )
were purified following previously published methods .
e. coli top10
chemically competent
cells were transformed with pbad - nrdb122tag and pevol - fnyrs - e3 and grown
at 37 c on lb - agar plates containing 100 g / ml ampicillin
( amp ) and 35 g / ml chloramphenicol ( cm ) .
a single colony was
used to inoculate a starter culture ( 5 ml ) that was grown until saturation
( 37 c , 1216 h ) .
this culture was diluted 100-fold into
fresh 2xyt medium supplemented with amp and cm .
after 16 h at 37 c ,
the culture was diluted 100-fold into 4 2 l of 2xyt containing
the antibiotics and variable concentrations of fny ( 0.5 mm ( 3,5)f2y , 0.7 mm ( 2,3,5)f3y ,
1.5 mm ( 2,3)f2y , or 1.5 mm ( 2,3,6)f3y ) . fnyrs and nrdb were both induced at an od600 of 0.6 with
0.05% ( w / v ) l - arabinose . in the case of ( 2,3,5)f3y-2 and ( 2,3,6)f3y-2 , 100 m o - phenanthroline was added to chelate the iron 30 min prior
to induction with l - arabinose . in all cases ,
growth was continued
for an additional 5 h and the cells were harvested by centrifugation
( 3500 g , 15 min ) .
apo ( 2,3,5)f3y-2
and ( 2,3,6)f3y-2 and holo ( 3,5)f2y-2
and ( 2,3)f2y-2 constructs were isolated by anion - exchange
chromatography as previously described
. as - isolated ( 3,5)f2y-2 and ( 2,3)f2y-2 were deoxygenated and
taken into an anaerobic chamber ( 4 c ) . solid sodium dithionite
( na2s2o4 ) and methyl viologen ( mv )
were also taken into the chamber where stock solutions of each ( 400
mm and 11 mm respectively ) were prepared in 50 mm n-2-hydroxyethylpiperazine - n-ethanesulfonic
acid ( hepes ) ph 7.6 , 5% glycerol . na2s2o4 and mv were added to the protein at final concentrations
of 20 mm and 110 m , respectively .
the resulting blue solution
was stirred in the anaerobic chamber for 1 h prior to addition of
ferrozine ( final concentration 20 mm ) .
the purple mixture was taken
out of the chamber , and the ( ferrozine)3fe complex was
removed by sephadex g-25 chromatography to obtain apo ( 3,5)f2y-2 and ( 2,3)f2y-2 .
apo fny-2s
( either isolated or generated using the protocol described above )
were reconstituted as previously described .
after a 15 min incubation period with 5 equiv of ferrous ammonium
sulfate , the iron - loaded proteins were treated with 3.5 equiv of o2 in the form of o2 saturated 50 mm hepes ph 7.6 ,
5% glycerol .
the reconstituted proteins ( fny-2s ) were analyzed by x - band epr spectroscopy to quantitate
the radical yield .
the methods followed previous protocols to study y122 formation with wt-2 and are
described in more detail in the supporting information ( si ) .
the specific activities of fny-2s were determined by the spectrophotometric
and radioactive assays utilized for wt rnr . for the spectrophotometric assay , 0.5 m fny-2 , 2.5 m wt-2 ,
1 mm cdp , 3 mm
atp , 30 m tr , 0.5 m trr , and 0.2 mm nadph in 50 mm hepes
ph 7.6 , 15 mm mgso4 , and 1 mm edta were combined in a total
volume of 300 l . in the case of ( 3,5)f2y-2 ,
the concentrations of 2 and 2
the reaction was monitored for 1 min at
340 nm for consumption of nadph . for the radioactive assays ,
[ h]-cdp was utilized ( 0.5 mm , 20004000 cpm / nmol ) and
the amount of nadph was increased to 1 mm .
the reactions were performed
in a total volume of 170 l , aliquots were quenched at different
time points by heat denaturation , and the samples were worked up as
previously described .
all 2s
were prepared at 250 m ( 21 mg / ml )
in assay buffer ( 50 mm hepes ph 7.6 , 15 mm mgcl2 , 1 mm
edta ) for crystallization . wt-2 was reduced with hydroxyurea in order to ensure all of the protein was in
the met state ( y-reduced diferric state ) .
initial crystallization
conditions were identified for wt-2 in 96-well , sitting drop ,
sparse matrix screens ( hampton research , qiagen ) dispensed by a phoenix
pipetting robot ( art robbins ) at room temperature ( 23 c ) .
optimization of the initial conditions was carried out in 24-well ,
hanging or sitting drop , vapor diffusion trays at room temperature .
the final ,
optimized conditions contained 1.1 m ( nh4)2so4 , 0.91.2 m kcl , and 0.1 m tris ph 8.0 .
protein and
precipitant were mixed in a 1:1 ratio with a final drop volume of
2 l .
crystals of each of the fny-2 variants grew in the same conditions as identified for
wt-2 and typically appeared after 2448 h and grew to
maximum size over 12 weeks .
most crystals appeared as pale
green hexagons or pyramids , but crystal size and morphology did not
greatly influence the quality of the resulting data set .
all crystals
were transferred to a cryoprotection solution containing 60% saturated
li2so4 , 0.4 m kcl , 0.1 m bicine ph 8.59.5
for 13 h prior to cryocooling by plunging in liquid n2 .
the length of the soak did not alter the quality of the
data set .
data sets for fny-2
variants were collected at the advanced photon source beamline 24id - c
on a pilatus 6 m detector ( dectris ) or 24id - e on a q315 ccd detector
( asdc ) .
the raw diffraction images for each data set have been deposited
in the sbgrid data bank .
data were indexed in space group p6122 , integrated , and scaled in hkl2000 with cell dimensions of a = b = 91 , c = 206 .
a similar
crystal form has been reported for the dimanganese(ii)-containing e. coli 2 ( pdb i d 2alx ) ; the structure
was solved by molecular replacement in the phenix implementation of
phaser using this structure as the search
model .
a single monomer is found in the asymmetric unit of
this crystal form with the physiological dimer generated by crystallographic
symmetry .
the highest resolution structure , ( 3,5)f2y-2 ,
was rebuilt and refined first using reciprocal- and real - space positional
refinement in phenix.refine .
atomic displacement
parameters ( b factors ) were optimized for each chain
with translation / libration / screw refinement .
water molecules were
added automatically in coot and checked
manually against composite omit electron density maps .
the entire
model was checked for geometry and fit to the density in coot .
this
completed model was used as the basis for refining the other fny- and wt-2 structures after removal
of the uaa .
restraints for each fny residue were
generated in phenix elbow , and the residues were placed in fo
fc omit
density . for ( 3,5)f2y-2 , this residue was placed
into unambiguous omit density in a single conformation . the ( 2,3 ) ,
( 2,3,6 ) , and ( 2,3,5)f3y models contain some fraction of
a second orientation of the fny , as described
below .
for angle measurements , hydrogen atoms were added in pymol ,
and the dihedral angle to the ring plane ( hcc1c2/6 ) was measured .
was calculated by subtracting the dihedral to the ring plane
from 90. the final structures for all of the models were
verified with composite
omit electron density maps . in each case the final model contains
residues 2349 out of 375 and residue 1 , the n - terminal methionine ,
is not visible in any structure .
the final models have been deposited
in the protein data bank ( 5ci0 , 5ci1 , 5ci2 , 5ci3 , and 5ci4 ) .
all software used
for crystallographic data processing and refinement was compiled and
maintained by the sbgrid consortium .
all reconstituted fny-2s
were transferred to the appropriate
epr tubes and frozen in liquid n2 prior to epr spectroscopy
at a specific observation temperature . in the case of ( 2,3,6)f3y-2
only , the sample was aged for 2 min following
addition of o2 during reconstitution of the active cofactor
and then frozen in liquid n2 .
all
9 ghz ( x - band ) continuous - wave ( cw ) epr spectra were collected at
a temperature of 80 k under slow - passage , nonsaturating conditions
using a bruker ( billerica , ma ) elexsys e500 x - band spectrometer equipped
with an oxford instruments esr900 cryostat and an itc-503 temperature
controller .
all
pulsed epr and endor
spectroscopic studies were performed at a temperature of 30 k using
a recently redesigned home - built 130 ghz epr spectrometer ( vide infra )
equipped with an oxford - cf935 liquid helium cryostat and an itc-503
temperature controller .
the spectrometer is also equipped with an
8 t cryogen - free magnet ( cryogenic limited , uk ) , and all data were
acquired using a te011 mode cylindrical resonant cavity
designed and manufactured by hf epr instruments , inc .
( v. krymov ,
new york ) equipped with radio frequency ( rf ) coils for endor .
magnetic field - swept echo - detected
epr spectra were acquired using the hahn echo pulse sequence : /2echo .
the magnetic field axis for each field -
swept spectrum was calibrated
using mgo with an mn impurity ( 95+% fused mgo , aldrich )
with a g - value of 2.00100(5 ) and a mn
hyperfine coupling constant of 243.6(5 ) mhz , as determined
previously .
each field swept echo - detected
epr absorption spectrum was modified using a pseudomodulation function
( modulation amplitude = 0.3 g ) to approximate the effect of field
modulation and produce the cw - like first derivative spectrum .
rfs for davies endor experiments were generated
by an hp 8656b signal generator under gpib control , and pulses were
formed by a ttl - driven mini - circuits ( brooklyn , ny ) zaswa-2 - 50dr+
rf switch .
the rf pulses were then amplified by either a 300 w bruker
blax300 rf amplifier ( 6250 mhz dynamic range ) or a 500 w kalmus
rf amplifier ( 200330 mhz dynamic range ) .
endor spectra were
acquired using the pulse sequence trfrf trf/2echo ,
where rf is the optimized rf pulse length and trf is a fixed delay separating mw and rf pulses .
endor spectra spanning 80 to 250 mhz were acquired using the blax300
amplifier with rf = 24 s , and those spanning
from 200 to 330 mhz were acquired using the kalmus amplifier with
rf = 18 s . the resultant spectra were stitched
together by first normalizing each to the most intense -h signal near 220 mhz and then splicing them together at 212.5
mhz to give the resultant endor spectrum shown in each figure .
the
specific parameters for field positions , microwave frequencies , and
pulse and delay lengths are given in the captions of each figure .
the transmitter section of the microwave bridge ( hf epr instruments ,
inc . , v. krymov , new york ) of our laboratory - built 130 ghz spectrometer was heavily modified in order to increase microwave
pulse power and operational stability ( figures s2 and s3 ) .
the topology of our upgraded 130 ghz microwave
transmitter is very similar to that described in smith et al .
as in the original bridge , a single phase - locked
dielectric resonator oscillator at 7.647 ghz ( output power = 20 dbm ,
nexyn , inc . ) serves as the base frequency , preserving the cw epr functionality .
this base frequency
is then mixed and multiplied up to 32.5 ghz and
provides 0 dbm ( 1 mw ) input power to the high - power side of the transmitter
arm .
the dual - channel impatt diode ( cascaded ) amplifiers of the original
bridge were replaced with a single channel amplifier
multiplier
chain ( amc ) fabricated by virginia diodes , inc . , ( vdi ) of charlottesville ,
va .
the amc consists of a set of cascaded varactor multipliers , in
our case an x2 2 design , with a spacek laboratories input preamplifier
and a separate power supply module .
the amc pulse output power is
23.2 dbm ( 210 mw ) as measured by vdi at 130 ghz .
a synthesizer channel
( herley - cti xs-7314 ) was added so that two - frequency experiments such
as deer and ednmr could also be conducted .
further details on these
modifications to the bridge can be found in the si ( figure s2 and table s1 ) .
all spectral simulations
were performed in the matlab 8.1.0 ( r2013a ) software package ( the
mathworks inc . ,
a nucleus exhibiting a large anisotropic hyperfine interaction ( a ) will have an orientation relative to the static b0-field at which the isotropic and anisotropic
contributions to the hyperfine tensor are of equal magnitude and opposite
sign .
this condition leads to a cancellation of the hyperfine field
for this nucleus , i.e. , a = 0 for this orientation .
the theoretical endor spectrum of such a nucleus would possess spectral
intensity at the corresponding larmor frequency . in practice , however ,
the davies endor spectrum suffers from a lack of endor response as
the hyperfine interaction approaches zero , a
0 .
fortunately , this behavior is easily modeled by convolving simulated
endor spectra by the detectability function:1where a is the
hyperfine
coupling in mhz and tp is the duration of the initial microwave -pulse in the davies
sequence in s .
the breadth of this blind spot is inversely
proportional to the length of the initial preparation pulse . in the case of each of the fny
species investigated here
, this suppression effect must be taken into
account in order to accurately simulate the endor intensities arising
from all hyperfine - coupled f nuclei whose a - tensors are highly anisotropic owing to the unpaired spin populating
the fluorine 2pz orbital
( vide infra ) .
all dft calculations
were performed on a desktop computer using the orca 2.9.1 suite of
software .
y and fny derivatives were approximated as the corresponding phenol
( i.e. , a methyl group replaced the -methine and amino acid ) .
sham method with becke s three - parameter
hybrid functional for exchange combined with the lee yang
parr
correlation functional ( b3lyp / g ) using
the default 20% hartree fock exchange .
all atoms were treated
with the 6 - 31g*(d , p ) basis set , and all
computations were completed using a polarizable solvent dielectric
field ( = 10 d ) . the size of the integration
grid used in all cases was 5 .
single - point calculations on the
converged geometries were performed with the hybrid functional b3lyp
using barone s eprii basis set on all atoms .
epr properties were computed using coupled - perturbed
self - consistent field ( cp - scf ) theory with the origin of the gauge
dependence set at the center of electronic charge .
fny-2s were
expressed from the pbad - nrdb122tag and pevol - fnyrs - e3 plasmids .
the effect
of the concentration of fny and media
ph to optimize fny uptake into cells ,
and the induction time for protein production were examined in an
effort to maximize the yield of each full length fny-2 .
the optimized yields for ( 3,5 ) and ( 2,3,5)f3y-2 were obtained with 0.50.7 mm fny , whereas for ( 2,3 ) and ( 2,3,6)f3y-2 ,
the concentrations were 1.5 mm .
the ph had minimal effect in all cases
and induction of fny-2 at an od600 of 0.6 followed by 45 h of growth , proved optimal
for protein expression .
isolation of fny-2 with high yields of fny required extensive experimentation and the results
are summarized in table s2 . in the case
of ( 2,3,5 ) and ( 2,3,6)f3y-2 expression was carried
out in the presence of 100 m o - phenanthroline
in the growth media prior to protein induction as well as inclusion of o - phenanthroline
in the lysis buffer prior to purification of the apo protein . on the
other hand , ( 3,5 ) and
( 2,3)f2y-2 were expressed
and purified in the holo form , with addition of ferrous ammonium sulfate
to crude cell extracts . by this protocol , the concentrations of the
( 3,5 ) and ( 2,3)-f2y were low and
consequently , subsequent
to purification , the iron was removed from these proteins with dithionite ,
mv and ferrozine . in each case , the resulting apo
protein was then reconstituted with 5 equiv of fe and
3.5 equiv .
although the basis for the
requirement for distinct optimization procedures remains unclear ,
in all cases the fny-2 had
3 fe/2 and 1 fny/2 ( table s2 ) .
the stability of y122 in wt-2 is 4
days at 25 c , whereas that of the
nitrotyrosyl radical ( no2y ) , a much stronger oxidant ,
was recently found to be only 40 s. x - band
epr spectroscopy was used to assess the stability of fny in each 2 at 20 s , 2.5 min , and 20
min subsequent to cluster assembly .
no changes in the total spin or
the spectrum appearance were observed for any of the fny-2s except for ( 2,3,6)f3y-2 . in this case , 40% spin loss was observed by 2 min
( 0.7 fny/2 ) .
to investigate
the basis for this observation with one possibility being that the
asymmetric fluorine distribution on the aromatic ring results in multiple
( 2,3,6)f3y conformations during the protein folding
process , the assembly of the diferric ( 2,3,6)f3y
was studied in more detail by rfq - epr spectroscopy ( figure s4 ) . the spectra between 23 and 256 ms ( figure s4a c ) reveal that intermediate
x , the fe / fe core responsible for the oxidation
of y to y , is completely formed within 23 ms and that it had
disappeared within 256 ms .
the 256 ms time point showed 1.2 f3y/2 ( figure s4c ) ,
which decayed to 0.7 f3y/2 over the subsequent
60 s and then remained unchanged ( figure s4d ) .
our current model to explain this observation is that ( 2,3,6)f3y exists in at least two distinct conformations generated
during 2 folding .
one gives rise to a rapidly decaying radical ,
whereas the other results in a radical that is substantially stabilized .
all epr data discussed subsequently were collected on the stable
( 2,3,6)f3y. although , we have not investigated the
reconstitution of the other fny-2s
by rfq - epr spectroscopy , no spin loss was observed with any of them
up until 23 h after reconstitution or with multiple freeze thaw
cycles .
subsequent to optimization of [ fny ]
to 1 fny/2 for all fny-2 , their activities were measured
and the results are summarized in table s2 . all fny-2s are active
with activities that vary between 5% to 85% of wt-2 and that
scale with radical concentration . the turnover number for ( 2,3,6)f3y-2 is measured for the stable
radical , and even for this species , activity is lost as a function
of time ( 50% loss within 2 min ) . thus ,
this 2 variant which has the largest reduction potential is ,
perhaps not surprisingly , the least stable , a result that parallels
our observations with no2y-2 ( no2y at position 122 in 2 ) .
all of the met - fny-2s and met - wt-2 proteins were crystallized
under similar conditions at ph 8.0 at room temperature ( 23
c ) .
the details of the refinement are described in the methods , and
the results are summarized in table s3 .
an
important issue in the refinement process was that in three of the
four fny s , the fluorines are asymmetrical
with respect to the aromatic ring and can thus potentially have multiple
conformations resulting from a 180 rotation around the cc
bond of the aromatic ring . our previous structural studies on no2y-2 showed a single conformation
in which the no2 group resided on the side away from d84
and was embedded within the hydrophobic pocket composed of l77 , q80 , i125 , n227 , i231 , and i234 ( figure s5 ) . in
the case of all the fny-2 structures ,
these residues adjacent to fe1 are identical to wt-2 and refined
to an all - atom rmsd of 0.1 ( figure s5 ) .
( a ) the structure of wild - type met-2 contains a diferric cluster
( fe1 and fe2 , ball and stick ) that coordinates two water molecules
( red spheres ) .
iron ligation by protein residues ( green ) or water
is represented by black dashes .
y122 ( light blue ) is positioned adjacent
to fe1 , but not within hydrogen bonding distance ( white
dash ) .
the electron density for y122 is shown inset ( 2fo fc at
1 ; green : fo fc at 3 ; red : fo fc at 3 ) .
( b e ) each fny-2 is shown
as in ( a ) . for ( 2,3)f2y , ( 2,3,5)f3y , and ( 2,3,6)f3y two conformations
the in conformation ( yellow )
in each structure places the fluorine atoms on carbons 2 and 3 adjacent
to d84 .
the out conformation ( orange ) is flipped 180.
analysis of the occupancy of these conformations ( figure s6 ) suggests the in conformation is typically dominant .
several additional features of
the fe1 in the cluster are thought
to play an important role in active cofactor assembly and in initiation
of radical transfer ( rt ) between the and subunits .
both d84 and the water ligand to fe1 may be involved in
the redox chemistry of y122 . in all the structures ,
d84 in fny-2 is almost identical
to that in wt-2 and is always monodentate ; the distances for
its two oxygens to the fe1 are 2.0 and 2.8 .
the distance between the phenolic oxygen of y122 and the water in wt-2 is 3.9 ( figure 2a ) .
this distance is comparable to the 3.95/3.88
distance in pdb 1mxr , a previously reported
1.42 resolution structure . in the initial structure ( pdb 1r1b(79 ) )
however , these
structures contained hg atoms in a number of positions that could
alter the cofactor environment .
as observed in figure 2c e , two conformations of the phenol
ring in ( 2,3)- , ( 2,3,5)- , and ( 2,3,6)f3y-2 are modeled
to interact with the water with distances between 3.8 and 4.5 .
finally the iron occupancy in each site of the cluster appears complete ,
despite the fact that there are only 33.5 fe/2 measured
biochemically .
the presence of different fluorine substitution
patterns in each
of the fny at position 122 as noted has
given rise to multiple conformations of the aromatic ring relative
to the fe1 site .
as will be discussed subsequently , published structures
with 3-fy and 2-fy globally incorporated into different proteins show
that the fluorine conformation(s ) are protein environment - dependent
and , in most cases , not predictable .
multiple
conformations of fny s can arise
either from complete flipping of the aromatic rings ( a 180 rotation
around the cc bond of y ) or from a subtle shift
in their positions . in the case of ( 2,3)- , ( 2,3,5)- , and ( 2,3,6)f3y-2
, the electron density can not be modeled well by
a single conformation of the aromatic ring but instead is more consistent
with two conformations that differ by a 180 rotation around
the cc bond and a slight shift within the hydrophobic
pocket ( figure s6 ) .
the precise ratio of
these two conformations is uncertain due to the modest resolution
of these structures , but refinement of a single conformation or complete
omission of fny yields difference density
clearly indicating a second conformation is present ( figure s6 ) . in all cases , the favored conformation has two
fluorine atoms packed against d84 , which we will now call
the in conformer .
the alternate conformation , in which
c2 and c3 fluorines are away from d84 , is accordingly denoted out . finally , the
c - terminal tail ( residues 340375 ) of 2 ,
which provides the interaction site between the and
subunits , is disordered in all published 2 structures . here
all of the fny-2 structures have
an additional 9 residues ( 341349 ) that are observed although
they are moderately disordered ( average b factors of 100150
) and have no specific , stabilizing contacts with
the exterior of the protein .
residues 360375 have been previously
visualized in structures of the inactive 44 complex and in structures of 2 in complex with
a 20-mer peptide corresponding to residues 355375 of 2 ; these residues , however , are completely absent
in wt-2 and all fny-2 structures .
although observation of residues 341349 is unprecedented ,
these residues are highly flexible in our current model and are likely
to adopt a different conformation in the proposed 22
active conformation , as they would bridge the intersubunit interface .
the magnetic
properties of natural y have been shown to be sensitive reporters
of the surrounding protein environment .
for example , the magnitude
of the gx - shift ( figure 3 , right ) is correlated
to the strength and number of hydrogen bonds to the phenoxy oxygen .
the hyperfine couplings of the ring protons report on the spin distribution
about the -system and the magnitude of the hyperfine couplings
to the two -methylene protons , which arise from their interaction
with the -electrons ( figure 3 , left ) , can be interpreted to give the degree to which
the y ring is rotated relative to the peptide backbone . from left
to
right : depiction of interaction between -electrons
and protons as a function of angle ( see discussion
on mcconnell relations ) ; the numbering of tyrosine ring atoms and
typical approximate spin densities at selected atoms in y , and
the alignment of the g - tensor relative to the molecular frame for
y and fy. although epr spectroscopy has previously been used to examine
fny s installed in both photosystem
ii and class ia rnr , a spectroscopically validated electronic
structure description of these non - natural amino acid radicals has
not yet been developed . to this end , we have produced rnrs with one
of four different fny s installed
at position 122 of 2 and characterized the oxidized fny ( fny ) using multifrequency
epr and orientation - selected pulsed endor spectroscopies at high field
( figures 4 , 6 , 7 , and s7s10 ) . we hope that the detailed analysis of these
spectra will encourage the use of fny
as a redox - tunable radical probe for the local protein environment .
in what follows , we will illustrate the process of interpreting these
multiple data sets using the epr and endor spectra of ( 3,5)f2y-2 as an example .
x - band cw epr ( panel a ) and d - band pseudomodulated
ese - epr spectra
( panel b ) and simulations of ( 3,5)f2y in rnr 2 .
field positions of g - tensor principle components
are shown with vertical dotted lines .
resolved hyperfine splittings
from f and h are denoted with the solid angled
brackets .
acquisition parameters : x - band cw epr : temperature = 80
k ; mw frequency = 9.395 ghz ; mw power = 317 w ; modulation amplitude
= 1.5 g ; modulation frequency = 100 khz ; conversion time = 50 ms .
d - band ese - epr : temperature = 30 k ; mw frequency = 129.996 ghz ; /2mw = 37.5 ns ; = 200 ns ; shot rep time ( srt ) = 10 ms . the epr spectrum of ( 3,5)f2y-2 was acquired
at both x - band ( 9.4 ghz ) and d - band ( 130 ghz ) excitation frequencies
( figures 4a and 4b , respectively ) .
the multifrequency epr spectra
for all four fny-2s are presented
in the si ( figures s710 ) . at the
relatively low magnitude of the applied magnetic field ( b0 = 330 mt ) at x - band , the g - anisotropy
of ( 3,5)f2y-2 is not well resolved ( see vertical
dashed lines , figure 4a ) .
instead , the spectral line shape is determined by the largest h and f hyperfine interactions ( figure 5 and table 1 ) . in the case of ( 3,5)f2y-2 ,
a splitting pattern composed of a triplet of doublets
these doublets
are caused by a h hyperfine interaction that is relatively
large and isotropic ( a 53 mhz)properties
that preclude the signals from being assigned to the ring protons
and point instead to a strongly coupled -methylene proton , h1 , as the source ( figure 3 and table 1 ) .
the triplet pattern results from the hyperfine interaction
of the two nearly equivalently coupled f nuclei at the
3 and 5 positions .
if the intensity pattern of the triplet were simply
1:2:1 , this ratio would indicate that these hyperfine interactions
are nearly isotropic . that the observed intensity pattern is closer
to 1:6:1 is instead indicative of an axial hyperfine interaction in
which the largest component ( az ) is approximately 157 mhz and the splittings along gx and gy
values of
each component of the hyperfine tensor are presented in order as ax , ay , az .
parameters determined from simulations of the combined epr and
endor data for each position are presented on top in red , and the
parameters from dft calculations are presented on the bottom in black .
experimental values for wt y are from an endor study by hoganson
et al .
computed using trssa with b
= 162 mhz ( svistunenko et al . ) .
x - ray diffraction ( xrd ) angles measured
manually in pymol after addition of hydrogen atoms .
the in and out conformations differ
by an approximate 180 rotation of the tyrosine ring about cc ; here we report the reduced
angles ( e.g. , for > 180 , subtract 180 ) .
no signal attributable to the out
conformation was observed . by going to a much higher applied field using our 130 ghz spectrometer
( ca .
4630 mt ) , the g - anisotropy becomes apparent
( see vertical dashed lines in figure 4b and g - values in table 2 ) .
the strong nuclear hyperfine
interactions of h1 , 3-f ,
and 5-f still contribute to the spectrum , and we observe
that the largest component of the two f hyperfine tensors
is aligned along gz ( ca .
4639 mt ) , where the familiar triplet of doublets splitting pattern
from the two equivalent f nuclei and h1 is centered .
again , along gx ( ca . 4625 mt ) , only a single splitting arising from
the isotropically coupled h1 is resolved .
thus , far , with just these two multifrequency epr
spectra , the
molecular g - values can be determined from the high - field
d - band spectrum and the largest components of f and h hyperfine tensors can be estimated , first by inspection
of both spectra in figure 4 , then refined by performing least - squares optimization of
simulations of the x and d - band epr data . however , endor spectroscopy
is necessary to determine the smaller hyperfine couplings of the f tensors and those from the ring protons at the 2 and 6
positions as the corresponding hyperfine splittings are lost in the
inhomogeneously broadened lines of the field - swept epr spectrum .
high - field
endor at 130 ghz is particularly helpful in identifying the smaller
couplings arising from h vs f because their
respective nuclear larmor frequencies , and thus the center of their
respective endor spectra , are separated by a much greater extent ( h i = 197 mhz , f i = 186 mhz ) than at x - band ( h i = 14.3 mhz , f i = 13.4 mhz ) or q - band
( h i = 51.6 mhz , f i = 48.6 mhz ) . again returning to our exemplar data for
the symmetrically fluorinated
( 3,5)f2y-2 sample , we show in figure 6a the central region ( i.e. , within 40 mhz of the h larmor frequency ) of the endor spectra collected at three
field positions across the epr envelope . at 130 ghz/4630 mt , all hyperfine
interactions for these radicals fall within the weak coupling limit
( |i| > |a|/2 ) ,
meaning that each individual nuclear coupling can be expected to produce
an endor spectrum that consists of a pair of peaks centered about
|i| and split by |a| .
because the individual g - values are resolved
at these high fields ( figure 4b ) , changing the field at which the endor experiment is performed
allows us to select only a subset of possible orientations of the
f2y , allowing for unambiguous determination of the
principal components ( e.g. , ax , ay , az ) of each hyperfine tensor .
from these data , the smaller couplings from h s
at the 2 and 6 positions as well as the second , more weakly coupled
-methylene proton ( h2 ) can be
determined ( figure 6a ) , with assignments guided by comparison to dft - predicted values
for a simplified ( 3,5)f2-phenoxy radical model ( figure 5 ) .
a modest asymmetry
in the intensity of the h endor patterns likely arises
from a combination of hyperfine enhancement ( for the more strongly coupled protons ) and a small amount of saturation
of the endor transitions .
the complete
endor spectrum ( 100300 mhz ) for the gz component is shown in figure 6b .
this spectrum reveals that the f hyperfine interactions from the fluorines at positions 3 and 5
are extremely large ( 157 mhz ) and broad .
thus , by combining analysis
of the multifrequency epr spectra , high - field ( hf ) , orientation - selected
endor , and insights from dft calculations , all relevant magnetic couplings
and g - tensor elements can be rigorously assigned .
this same methodology was used for evaluating the other three asymmetric
fny-2 radicals and the combined
x - band , d - band and d - band endor data sets with simulations can be
found in the si in figures s7s10 .
( a ) field - dependent
d - band davies endor of ( 3,5)f2y
in rnr 2 in the frequency range from 160233 mhz .
experimental
data are shown in black , the total simulated spectrum is in red , and
isolated contributions from h and f nuclei
are in blue and green , respectively . resolved peaks and assigned nuclei
are denoted with black brackets .
( b ) full endor spectrum acquired
at b0 gz ( 4643 mt ) .
broad signal associated with
the f3,5az component is denoted with the black bracket below the experimental
spectrum .
acquisition parameters : temperature = 30 k ; mw frequency
= 129.996 ghz ; /2mw = 37.5 ns ; rf = 24 s ; = 300 ns ; shot rep time ( srt ) = 10 ms .
the parameters obtained from the
simulations of the multifrequency
epr data reveal a great deal of information on the electronic structure
of each fny-2 variant through
changes in the g - tensor and hyperfine couplings at
each ring position , as well as yielding structural information regarding
the rotational orientation of the y side chain through the hyperfine
coupling from the protons on the carbon .
analysis of the g - tensors for each radical ( table 2 ) reveals that the gx values of all of the fny-2s are very similar to each
other ( 2.00822.0085 )
though rather different from the gx value of the wt-2 ( 2.00912 ) previously determined by
hoganson et al . in the case of wt-2
,
the gx element of the g - tensor is aligned along the c o bond of y
( figure 3 ) and its
value is often invoked as a measure of the strength of hydrogen bonding
or a sensor of the electrostatic environment of the o of the phenoxy
group .
all of the fny-2s exhibit smaller gx values than wt-2 that could be indicative
of differences in their electrostatic environments .
however , the dft - predicted
magnetic parameters using fluorinated phenoxyl radicals indicate that
o4 has moderately reduced spin populations compared to those for the
nonfluorinated y ( table 3 ) , which would also affect gx .
in contrast to gx and gz , the addition of fluorines appears to have a larger
effect on the value of gy , a trend also predicted by dft ( table 2 ) .
importantly , the orientation of the predicted g - tensor relative to the molecular frame is conserved in
all cases , even with the asymmetrical fny s . despite the strong electronegativity of fluorine ,
the
changes in the spin density distribution remain roughly symmetrical
about the c2 rotational axis of the phenoxyl ring ; thus , gx remains aligned along the c o
bond and gz is oriented
orthogonal to the plane of the ring ( figure 3 ) . for
each fny-2 , the hyperfine
tensors for
each
of the ring protons and fluorine nuclei ( figure 5 ) were determined primarily by fitting the
field - dependent d - band endor spectra ( figure 7 ) .
the exceptions
were the az components
of the f hyperfine tensors of positions 3 and 5 which
were constrained by large splittings observed in the x- and d - band
epr ( figure 4 ) .
the
fit was of particular importance for determination of the magnitude
of the smaller h and f couplings at the 2
and 6 positions , which have only minor effects on the appearance of
the field - swept epr spectra .
in addition , results from the orientation - selected
endor experiments allowed the small elements of the hyperfine tensors
for the strongly coupled but highly anisotropic nuclei to also be
determined .
comparison of the endor spectra of each radical acquired
at the center of each spectrum reveals substantial differences ( figure 7 ) .
though the magnitude
of the largest element of the most strongly coupled f
nucleus was not as well resolved in the endor spectrum for all of
the fny s examined due to
the considerable breadth of this endor feature , this parameter is
constrained by the span of the x - band cw spectrum for each variant
( panel a of figures s7s10 ) .
the f hyperfine tensors , particularly in the 3 and 5 positions ,
show strong , axial anisotropy due to the unpaired spin contained in
the fluorine 2pz orbital .
this value is
estimated by dft to be up to 2% to 3% for fluorine in the 3 or 5 position
and 0.3% to 0.8% for fluorine in the 2 or 6 position .
comparison of d - band
davies endor acquired at the spectral center
( b0 gy ) of each fny in
rnr 2 .
experimental data are shown in black , total simulated
spectrum is in red , and isolated contributions from h
and f nuclei are in blue and green , respectively . resolved
peaks and assigned nuclei are denoted with black brackets .
acquisition
parameters : temperature = 30 k ; mw frequency = 129.996 ghz ; /2mw = 37.5 ns ; rf = 24 s ; =
300 ns ; shot rep time ( srt ) = 10 ms . in the case of ( 3,5)f2y-2 , the only
symmetric
fny we studied , the two f
hyperfine tensors are essentially identical ( figure 5 ) .
this result suggests that any differences
in the protein environment near f3 and f5 are either small or do not
influence the magnetic properties .
however , to best fit the weaker h couplings required slightly different tensors for h2 and
h6 .
these differences in this symmetric fny may be due to the rotation of the side chain about the -carbon ,
as this rotation has been shown to have modest effects on the hyperfine
couplings of ring protons in y models in a previous dft study . when the symmetric ( 3,5)f2y-2
is compared
to the asymmetric fny s ,
the az of the most strongly
coupled f displays an upward trend in magnitude between
variants , with ( 3,5)f2y < ( 2,3)f2y
< ( 2,3,5)f3y < ( 2,3,6)f3y
( figure 5 ) . for ( 2,3)f2y and ( 2,3,6)f3y , the f at the 3 position
is predicted to exhibit the largest coupling ,
while in ( 2,3,5)f3y the largest f coupling
is predicted to occur at the 5 position .
this trend in the magnitude
of the f hfi for each variant is matched by estimations
of the mulliken spin populations for f atoms at these positions , as
well as for the adjacent spin - carrying c3 and c5 atoms by dft ( table 3 ) .
the changes in
spin localization about the tyrosine ring that are reflected in the
experimentally observed f and h couplings
and calculated spin populations on the ring atoms are primarily caused
by inductive effects of the electron - withdrawing , highly electronegative
fluorine atoms .
the sign and magnitude of the ring atom hyperfine
tensors for each
fny are qualitatively predicted
by dft calculations ( figure 5 ) , although the anisotropy of all f tensors appear
to be slightly overestimated , thus precluding quantitative accuracy
compared to the experimental results .
dft calculations predict that ax and ay for f at the 3 and 5 position
are negative , but az is
positive , which is consistent with the simulations of the d - band epr
and endor when the davies suppression hole is included as a 0 . for each
ring
f or h atom , the relative
orientation of the coordinate system of the nuclear hyperfine tensor a to the molecular g - tensor must also be
considered , as related by the euler rotation angles , ,
and . dft calculations predict that the a tensors
for each ring atom for all fny variants
are rotated by angle about the ring plane normal ( corresponding
to gz ) such that ax is aligned approximately
along the chemical bond vector , with minor deviations not exceeding
15 ( table 4 ) ,
with no appreciable rotations about gx or gy .
the angles predicted by dft were used in our simulations
of the multifrequency epr and hf endor for each variant and appear
to be in excellent agreement with the experimental data .
in addition to hyperfine
couplings arising from the ring h and f nuclei ,
we also observe relatively large isotropic proton couplings of approximately
4555 mhz ( table 1 ) , as well as very small isotropic couplings of less than 3 mhz which
reside near the h larmor frequency ( n 197 mhz at 4634 mt , figures 67 ) .
these isotropic
proton couplings arise from the two unequivalent protons of c ( figure 3 ) .
the magnitude of these couplings is dependent on the degree of
hyperconjugation of the ch bonding orbital
with the -system of the aromatic ring ( see figure 3 ) as a function of the dihedral
angle between the ch bond and the
normal vector of the y ring plane .
this interaction is maximized when
the ch bond is aligned perpendicular to
the y ring plane such that the dihedral angle = 0 and is minimized
when this bond is parallel to the ring plane .
the observed aiso for each proton can be mathematically
correlated to the dihedral angle between the ch
bond and the vector normal to the ring plane using the mcconnell relations:2here a1 and a2 are the isotropic hyperfine
couplings of the two respective h nuclei , c1 is the spin density on the c1 atom , and b is a constant ( b = 162
mhz ) .
this relationship allows for facile estimation
of both the spin density on c1 and
through the use of the tyrosyl radical spectral simulation
algorithm ( trssa ) developed by svistunenko and cooper .
this analysis has been utilized to estimate
these parameters for each of the fny
variants examined here ( table 1 ) , with comparison to the calculated for each fny refined in the xrd data . for
the symmetric ( 3,5)f2y , only a single large h coupling with aiso = 53 mhz
this value is near the maximum
of the theoretical limit for a h1 coupling
where c1 is in the range 0.350.42
that is typically observed for y ; thus we assign the very small isotropic coupling observed with aiso = 1.7 to the other
proton , h2 . using these values
, trssa
analysis indicates a dihedral angle for h1 of 20.3 , which is in excellent agreement with the modeled
orientation from the xrd of 19.7 ( table 1 ) .
the presence of only a single strong h coupling is indicative of the presence of a single , conserved
rotational orientation of the y side chain within all proteins in
the sample , which is also consistent with the refined position of
( 3,5)f2y in the x - ray structure presented here .
though
the c1 value of 0.387 obtained
from this analysis is higher than the mulliken spin population estimate
of 0.338 from dft ( table 3 ) , it should be noted that the dft models are simplified phenoxy
radicals with only protons on c and , thus , would
not exhibit effects from the rotation of the carbon relative to the
ring plane that may affect c1 . for each of the asymmetric fny s ,
there is also at least one clearly resolved large isotropic h coupling assigned to a -h .
for the ( 2,3)f2y , there appears to be only a single large -h coupling with an aiso of 54.3 mhz , with a paired weakly coupled -h with aiso = 1.7 mhz , indicating a
of 21.7 for h1 , which is very
similar to the ( 3,5)f2y. this result is consistent
with the x - ray refinement of ( 2,3)f2y , in which the in
conformer , with the fluorines of c2 and c3 packed
against the side chain of d84 , predominates ( figure s6 ) .
interestingly , for ( 2,3,5)f3y and ( 2,3,6)f3y , two distinct large
isotropic couplings are clearly
resolved with different relative spectral intensities ( figure 7 , figures s9c and s10c ) . here , the high - field endor is invaluable in
identifying these distinct couplings , as in the x - band cw and d - band
epr spectra the inhomogeneous line broadening largely obscures these
subtleties . in both cases ,
these two values are too large to represent
the two protons of a single orientation of f3y
due to the constraints imposed from the mcconnell relations described
above .
rather , these couplings must arise from two distinct , conserved
populations of rotational conformers of f3y with
different h1 dihedral angles , each with
their own associated weakly coupled h2 ( table 1 ) . by using
weighted multicomponent simulations of these two subpopulations , where
all g - values and nuclear couplings were held fixed
except the h1 values , a rough estimate
of the relative abundance of each conformer was achieved by comparison
to the observed spectral intensity of these
strongly coupled -h peaks in the d - band endor ( figures s9c and s10c ) .
the weighting of the two individual components was
optimized to fit the experimental data and yielded the estimates of
the relative abundance of each conformation presented in table 1 .
as the in conformation
observed by xrd displayed a smaller h1 , we assigned this conformation to the larger h1 coupling according to the mcconnell relation
described above . likewise , the smaller h1 coupling is produced by the out conformation .
interestingly , the
proportions of these rotational conformers seem to be reversed between
these two f3y variants , with the in conformation
exhibiting the larger h1 coupling being
dominant ( 0.80 ) in ( 2,3,5)f3y , whereas the out conformation
exhibiting the smaller h1 coupling is
more abundant in ( 2,3,6)f3y ( 0.75 ) . while the crystal
structures also indicate the presence of two conformations for both
( 2,3,5 ) and ( 2,3,6)f3y side chains , the favored one in
both cases has the fluorines at positions 2 and 3 pointing toward
d84 ( the in position )
. these differences
may be due to the fast - decaying component observed for ( 2,3,6)f3y by rfq - epr during cluster assembly ( figure s4 ) .
as described in the materials
and methods , the ( 2,3,6)f3y-2 samples
used for the epr analysis in this paper were aged for 2 min to represent
the stable ( 2,3,6)f3y. the apparent
change in the relative abundance of the in and out conformations of
( 2,3,6)f3y relative to that of the reduced ( 2,3,6)f3y present in the crystal structure may indicate that the in
conformation corresponds to this rapidly decaying population observed
by rfq - epr .
fny s have long been used to
study protein structure and function , as they perturb the electronic
properties of y , while minimally perturbing its size ( van der waals
radius of f is 1.42 compared to 1.27 for h ) .
global
incorporation of fy and site - specific incorporation of fny by expressed protein ligation ( epl ) have been the
most widely used methods . in the former
case , a y auxotroph is grown with 2- or 3-fy in medium that is depleted
of y. thus , all y s in the protein of interest are replaced
by the y analog .
efforts to incorporate fny with n > 1 by this method , however , typically
result in lower incorporation efficiency . in the case of epl ,
site - specific incorporation of fny involves ligation of a chemically synthesized peptide
containing fny with a recombinantly expressed
protein to generate the intact mutant protein . in this case , only
a designated y is replaced with fny ( n = 2 , 3 , or 4 ) .
the epl method is limited , as the residue
of interest must be synthetically accessible and , hence , localized
to either the c- or n - terminus of the protein .
however , epl can be mechanistically informative when the fny properties are compared to each other , as the pka of fny s
range from 5.6 to 10.0 and their reduction
potentials cover a range of 300 mv .
the
incorporation of these analogs has provided investigators with the
tools to study the mechanisms of phosphorylation of y in a peptide
and of proteins by tyrosine kinases , the importance
of h - bonding networks , and pka perturbation
within these networks in ketosteroid isomerase and to study pcet in the oxygen evolving complex as a function of
ph .
recently , li et al . and minnihan
et al . have used a third method to site - specifically
incorporate fny analogs , the amber suppressor
method developed by peter schultz that utilizes an evolved , orthogonal
trna / rs pair . in the former case
, the
method has been used to study the oxidase activity of an engineered
myoglobin model of cytochrome oxidase proposed to involve a transient
y intermediate . in the latter case
, the unique
properties of the e. coli rnr system to self - assemble
the active diferric - fny cofactor
from diferrous - fny , o2 , and
reducing equivalents have allowed investigation
of y equilibration and assessment of the thermodynamic landscape
of the rt pathway ( figure s1 ) .
the crystallographic and multifrequency epr
and hf endor spectroscopic studies reported in this paper have allowed
characterization of the ground state and oxidized state of fny within the protein environment providing a benchmark
for other protein systems .
we have successfully incorporated
fny in place of all four y s in
the rt pathway ( figure s1 ) .
the
unique positioning of y122 , adjacent to the diiron cluster
in 2 , allows generation of fny122 and investigation of their spectroscopic properties
( figures 4 , 6 , 7 , and s7s10 ) .
the f nucleus provides distinct
features in the low- and high - field regions of the epr spectra relative
to y. simulation of these epr spectra is critical to the deconvolution
of spectra involving multiple y s , as the features of
most y s are narrow and superimposable . our ability to
site - specifically incorporate ( 3,5)f2y in place of y731 or y730 of 2 has allowed detection of
radical equilibration between the three transient y s
in the rt pathway between the two rnr subunits .
when no2y-2 is incubated with 2 ,
cdp , and atp ( effector ) , the y356 is generated concomitant
with reduction of no2y to no2y phenolate
( no2y ) .
the inability of y356 to
reoxidize no2y to no2y allows y356 to equilibrate with the pathway y s in 2 .
unfortunately , this equilibration can not be observed due to substantial
overlap in the epr spectra of y356 , y731 , and y730. when the experiment was repeated
with ( 3,5)f2y731- or ( 3,5)f2y730-2 , the unique f hyperfine features
allowed observation and quantitation of y356 and
( 3,5)f2y demonstrating , for the first time , the equilibration
between the pathway y s .
although the majority of the spin
was localized to either y356 or no2y
( > 85% ) , we were able to detect ( 3,5)f2y at each
position .
a second example of the usefulness of the method was
recently demonstrated
with the myoglobin system engineered to model cytochrome c oxidase . in studies of o2 reduction ,
an epr active species
was observed , postulated to be a y. site - specific incorporation of ( 3,5)f2y in place of y
allowed unambiguous assignment of the new radical based on the observation
of the f hyperfine interactions .
furthermore , site - specific incorporation of several y analogs with
altered pka s and reduction potentials
provided insight into the mechanism of the model oxidase .
interpretation
of these results , however , requires an understanding of the perturbation
of the pka s and reduction potential
of each uaa within the protein environment as well as an understanding
of the rate - limiting steps in the overall reaction .
recently , liu et al . reported the design of a genetically
encoded
photoinduced electron transfer ( pet ) sensor starting with a flavoprotein
ilovu optimized for its fluorescence properties by engineering of
a y adjacent to the fmn binding site .
fny and clny were incorporated in place of y as their lowered pka s allowed them to function as excellent pet quenchers
relative to y. the charge neutral ( i.e protonated ) fny and clny are not efficient pet
donors , and the sensor ilovu is in the fluorescence on state .
the
deprotonated fny and clny are much more rapidly oxidized than the neutral phenols and
are better pet donors .
similar to protonation , the y state results
in a reduction in the pet rate , turning on fluorescence .
pet from
cl2y to the fmn of ilovu was supported by detection of
cl2y. the authors also described several interesting
examples for the use of this pet sensor .
in addition to covering
a range of reduction potentials , fny s
also offer a range of pka s that
can be mechanistically informative .
by replacing each of the pathway y s in rnr with no2y and by taking advantage of the visible properties of the no2y ,
we have previously shown that the pka is minimally perturbed at y356 , y731 , and y730 , whereas it is shifted > 3 units at y122 ( figure s1 ) .
given that y oxidation requires loss of a proton and an
electron ,
the protein environment plays a very important role in this process .
if reversible redox chemistry is occurring at a specific y , it is
not clear with the fny analogs what the
fate of the proton will be subsequent to oxidation . in the case of
the cofactor assembly process generates the no2y that then initiates rt into the active site .
initiation of
rt with no2y , however , generates the phenolate and
not the phenol , in contrast with y122 . during reverse rt ,
y356 can not reoxidize the phenolate , and thus , no2y-2 only catalyzes one turnover . radical initiation
with ( 2,3,5)f3y-2 catalyzes multiple turnovers ; however , it is not known if this radical gets transiently reduced
to the phenolate or the phenol . a method to measure
the pka s of the fny s
in the different environments and linking this information to the
mechanism is thus important
. this conclusion will definitely be protein
environment dependent , and one can not assume that the pka of the analog is not perturbed . from our studies ,
a number of issues may be encountered when using
this site - specific incorporation methodology especially if 100 mg
quantities of homogeneous protein are required for mechanistic studies .
we have had experience with fny incorporation
at positions 122 and 356 within 2 of rnr and at positions 731
and 730 within 2 .
we have also incorporated
fny into a small three helix bundle protein
( 3x , 65 amino acid ) containing a single buried y that we have
used to measure the reduction potentials of fny s . in general ,
incorporation of the same y analog into a different position and into
distinct proteins requires optimization .
the vectors used , the timing
of induction of the trna and rs expression relative to the expression
of the protein of interest , and the growth conditions , all need careful
study .
the timing of addition of fny to
the growth media and uptake into the cell also need optimization .
in our hands , the purification of the proteins can also be challenging .
in all cases one obtains truncated and full - length proteins that must
be separated , the relative amounts of which depend on the level of
suppression .
in addition , in the case of proteins that are multimeric ,
2 in our case , full length and truncated hetero- and homodimers
are obtained .
their separation depends on charge and size and may
require considerable effort or , in the worst - case scenario , may not
be successful .
finally , in the case of fny-2s , assembly of the active cofactor also required optimization
with different protocols for each 2 .
our evolved fny trnr / rs is polyspecific ,
and thus a single trna / rs pair could be used to incorporate all of
the fny s .
we note that f4y is missing from the incorporated analogs ( figure 1 ) . in our hands
the suppression levels were
poor , we think as a consequence of the inability of the predominant
anionic form ( pka 5.6 ) to cross the membrane ,
resulting in low concentrations inside the cell and consequently poor
suppression efficiency .
although an engineered myoglobin containing
f4y was presented in the recent paper of liu et al . , sds - page gels in the si show poor expression of the full - length protein ( i.e. , low suppression
efficiency ) . a second issue that requires comment is that both
our crystallographic
analyses and the epr simulations require that some of the fny analogs exist in multiple conformations at position
122 in 2 .
an examination of 28 crystallographically distinct
glutathione s - transferase structures containing globally incorporated
3-fy reveal that , even with this minimal
perturbation , multiple conformations are observed in distinct sites .
from these studies ,
since multiple fny conformations can give rise to mechanistic complexity ,
this possibility is important to consider in any kinetic analyses .
an additional possibility that we considered is that the aromatic
ring of the fny s can flip within
the protein environment .
our structures , however , reveal that fny s at position 122 fit snugly in
a very hydrophobic cavity .
flipping would require a sizable activation
volume as well as coordinated movement of these hydrophobic residues ,
which seems unlikely given the structures shown in figure s5 .
only recently have rate constants for aromatic
ring dynamics within different environments been measured using c relaxation dispersion measurements .
thus , we can not say whether the two conformations that we observe
of these side chains were established during protein folding or if
they represent a slow flipping process that occurs after protein folding
is complete .
this paper provides
new tools to investigate the mechanistic role
of proposed y s in catalysis .
a growing list of proteins
including photosystem ii , cytochrome
oxidase that catalyzes the reduction
of o2 to water , prostaglandin synthase , the target of nonsteroidal anti - inflammatory agents , dna
repair photolyases , and fatty acid oxidases utilize y s in their mechanisms .
in addition , y s have been proposed to be involved in
the repair of off pathway oxidations with oxidoreductases having increased
levels of y s and w s adjacent to the metallo - cofactor
site .
the tools described in this paper may be useful in
unraveling the mechanisms of these systems and other enzymes that
have yet to be discovered .
|
### Summary:
: fluorinated tyrosines
( fny s , n = 2
and 3 ) have been site - specifically incorporated into e. coli class ia ribonucleotide reductase ( rnr ) using the
recently evolved m. jannaschii y - trna synthetase / trna
pair .
class ia rnrs require four redox active y s , a stable
y radical ( y ) in the subunit ( position 122 in e. coli ) , and three transiently oxidized y s ( 356
in and 731 and 730 in ) to initiate the radical - dependent
nucleotide reduction process .
fny ( 3,5 ;
2,3 ; 2,3,5 ; and 2,3,6 ) incorporation in place of y122-
and the x - ray structures of each resulting with a diferric
cluster are reported and compared with wt-2 crystallized under
the same conditions .
the essential diferric - fny cofactor is self - assembled from apo fny-2 , fe2 + , and o2 to produce 1
y/2 and 3 fe3+/2 .
the fny are stable and active in nucleotide
reduction with activities that vary from 5% to 85% that of wt-2 .
each fny-2 has been characterized
by 9 and 130 ghz electron paramagnetic resonance and high - field electron
nuclear double resonance spectroscopies .
the hyperfine interactions
associated with the 19f nucleus provide unique signatures
of each fny that are readily distinguishable
from unlabeled y s .
the variability of the abiotic fny pka s
( 6.4 to 7.8 ) and reduction potentials ( 30 to + 130 mv relative
to y at ph 7.5 ) provide probes of enzymatic reactions proposed to
involve y s in catalysis and to investigate the importance
and identity of hopping y s within redox active proteins
proposed to protect them from uncoupled radical chemistry .
|
<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: colorectal cancer ( crc ) is the third leading cause of cancer - specific death in females and the fourth in males worldwide.1 the incidence and mortality of crc increases with advancing age , and the global burden of the disease is expected to grow further over the next few decades due to the increase in life expectancy.2 in the usa , approximately 60% of new cases of crc and 70% of crc - related deaths occur in patients aged 65 years and older.3 about 50% of patients diagnosed with early - stage disease will ultimately develop metastatic crc ( mcrc ) in spite of radical surgery and adjuvant therapies , whereas 25% of patients present with de novo mcrc at diagnosis . unfortunately , when metastatic disease occurs , the prognosis remains poor , even if survival has improved , often exceeding 24 months , due to the introduction of targeted agents.4 despite the high prevalence of the disease in the elderly , this patient population has been historically excluded or underrepresented in most clinical trials because of stringent inclusion criteria . moreover ,
elderly patients enrolled in trials are highly selected , and generally fitter than the average patients observed in daily clinical practice . as a result , there is not sufficient evidence on the appropriate management of elderly patients with mcrc .
the safety and efficacy of standard chemotherapy in older patients seem to be comparable with that reported for younger individuals,5 but limited data are available about the tolerance and effectiveness of biologic agents in the elderly population .
bevacizumab ( avastin , genentech , south san francisco , ca , usa ) is a recombinant , humanized monoclonal antibody that specifically targets vascular endothelial growth factor.6 bevacizumab improves progression - free survival ( pfs ) and overall survival ( os ) of mcrc patients when added to chemotherapy in the first - line and second - line setting and beyond the first disease progression when given with a chemotherapy backbone different from that used in the front line.79 bevacizumab has a particular toxicity profile including : cardiovascular events , such as hypertensive crisis , chronic heart failure , myocardial infarction , venous thromboembolic events , and arterial thrombotic events ; cerebrovascular events , such as transient ischemic attack , stroke , and central nervous system hemorrhage ; gastrointestinal perforation ; fistula formation ; wound healing complications ; and nephrotic syndrome.10,11 these adverse events may be of some concern for medical oncologists when choosing treatment for elderly patients with mcrc , and an accurate risk - benefit balance should be carried out before planning the therapeutic strategy on an individual basis .
the aim of this paper is to present the available evidence on the efficacy and safety of bevacizumab in elderly patients with mcrc , in order to help clinicians select patients who are potentially good candidates for receiving a bevacizumab - based therapy .
the currently available evidence on the effectiveness and tolerability of bevacizumab in older patients derives from retrospective analyses of subgroups enrolled in clinical trials without an upper age limit , pooled analyses of randomized trials , observational community - based studies , and prospective , randomized clinical trials specifically assessing the efficacy and safety of bevacizumab in elderly patients . for this paper ,
the pubmed database was searched for papers published before january 2014 using bevacizumab and colorectal cancer and elderly as search terms .
in the avf2107 g study , a landmark , randomized , placebo - controlled , phase iii clinical trial , the addition of bevacizumab to an irinotecan - based chemotherapy as first - line treatment for mcrc significantly improved os ( 20.3 versus 15.6 months , hazard ratio [ hr ] 0.66 ) , pfs ( 10.6 versus 6.2 months , hr 0.54 ) and response rate ( 44.8% versus 34.8%).7 subgroup data analysis suggested a comparable survival benefit ( hr 0.61 ) for patients aged 65 years , who represented 32% of the entire population enrolled in the study . in this trial , bevacizumab did not exacerbate chemotherapy - related toxicities , but was associated with an increased incidence of grade three hypertension ( 11% versus 2.3% ) and gastrointestinal perforation ( 1.5% versus 0% ) .
the incidence and grade of bevacizumab - related adverse events were not reported by age group , and concerns remained regarding the safety profile of bevacizumab in elderly patients.7 more information on the tolerability of bevacizumab in older patients was provided by the randomized , double - blind phase ii avf2192 g study evaluating the addition of beva - cizumab to fluorouracil / leucovorin in patients with a poor prognosis and deemed unfit for first - line irinotecan - based chemotherapy.12 the design of this study was based on a retrospective , exploratory analysis of a small randomized phase ii trial ( avf0780)13 comparing 5-fluorouracil / folinic acid alone or combined with two different bevacizumab doses ( 5 or 10 mg / kg ) , which suggested a particularly meaningful clinical benefit from anti - vascular endothelial growth factor therapy in a subset of patients with poor prognostic features , including age 65 years , eastern coast oncology group performance status 1 , and albumin 3.5 g / dl.13 as a result of the eligibility criteria used for the avf2192 g trial , 80% of patients were aged 65 years , and the mean age was 70.7 and 71.3 years in the fluorouracil - leucovorin arm and fluorouracil - leucovorin plus bevacizumab arm , respectively.12 patients receiving bevacizumab had a significantly longer median pfs ( 9.2 versus 5.5 months ; hr 0.5 ; p=0.0002),12 but the study did not meet the primary endpoint of demonstrating a 39% reduction in risk of death in the bevacizumab arm .
however , the median os was 3.7 months longer for patients treated with bevacizumab and chemotherapy in comparison with those who received chemotherapy plus placebo ( 16.6 versus 12.2 months ; hr 0.79 ; p=0.16).12 despite its use in a population at high risk for serious adverse events , bevacizumab was well tolerated .
grade three hypertension was more frequent with bevacizumab ( 16% versus 3% ) but was manageable with anti - hypertensive drugs , and did not lead to any discontinuation of treatment .
there was no difference between the two groups in terms of incidence of bleeding and venous thromboembolic events , but an increased rate of arterial thrombotic events was observed in the bevacizumab arm when compared with the placebo arm ( 10% versus 4.8%).12 further information on use of bevacizumab in the elderly population derives from subgroup analyses of other randomized studies in patients not selected for age . in the phase
iii bicc - c ( bolus infusional , or capecitabine with camptostar - celecoxib ) study , 430 mcrc patients were randomized to receive first - line folfiri , modified irinotecan / fluorouracil / levoleucovorin ( mifl ) , or irinotecan plus capecitabine.14 after approval of bevacizumab , the study protocol was amended and an additional 117 patients , 24.8% of whom were elderly , were randomized to folfiri or mifl plus bevacizumab .
the subgroup analysis did not show any significant difference in terms of efficacy and safety of bevacizumab plus chemotherapy for patients 65 years compared with those < 65 years.14 the randomized phase iii cairo-2 study ( clinicaltrials.gov identifier nct00208546 ) investigated the addition of the anti - epidermal growth factor receptor monoclonal antibody cetuximab to bevacizumab plus capecitabine and oxaliplatin ( capox ) in previously untreated mcrc patients.15 the study reported negative results , since the combination of the two monoclonal antibodies with chemotherapy determined a shorter pfs and a poorer quality of life . a subgroup analysis performed in the control arm showed that the effect of bevacizumab plus capox was similar in terms of median pfs across ages ( 10.6 versus 9.6 versus 13.5 months for patients aged < 75 , 7075 , and > 75 years , respectively ; p=0.908 ) , but a trend toward a worse median os
was observed in elderly patients ( 20.3 versus 17.6 versus 13.1 months for patients aged < 75 , 7075 , and > 75 years , respectively ; p=0.063).16 the incidence of grade three and four adverse events did not differ significantly among the three age groups .
however , older patients discontinued treatment for unacceptable toxicity more frequently than younger patients , and it is not clear whether bevacizumab was the main reason responsible for early discontinuation in elderly patients , since the evaluation of bevacizumab - specific toxicity among the three age groups was not an object of the analysis.16 the australasian gastrointestinal trials group max study was a phase ii / iii trial comparing capecitabine versus capecitabine plus bevacizumab versus capecitabine and mitomycin plus bevacizumab as first - line therapy for mcrc patients.17 the addition of bevacizumab to chemotherapy resulted in an improved pfs in comparison with capecitabine alone ( 5.7 months for capecitabine , 8.5 months for capecitabine and bevacizumab , and 8.4 months for capecitabine , mitomycin and bevacizumab ; capecitabine versus capecitabine plus bevacizumab , hr 0.63 ; p<0.001 ) .
patients aged > 75 years , who comprised 21% of the overall population enrolled in the study , achieved a comparable benefit in terms of pfs when treated with bevacizumab ( hr 0.53 for capecitabine versus capecitabine plus bevacizumab ; p=0.001).18 the effect of bevacizumab was not different between patients > 75 and 75 years , as suggested by an interaction test between treatment and age groups for response rate , pfs , and os .
interestingly , no significant differences in bevacizumab - related toxicity were reported by age . however , in elderly patients receiving bevacizumab and chemotherapy , an increased rate of hand - foot syndrome was observed , and this finding may reflect the longer exposure to treatment in the bevacizumab - containing arm.18
subgroup data analyses for age are limited to the small number of elderly patients enrolled in each trial , and generally lack the power to detect any significant difference in outcome between younger and older patients . pooling data from different studies increases the sample size for the analysis , producing more reliable results
. a pooled analysis of data from the avf2107 g and avf2192 g studies included 439 patients aged 65 years ( 276 patients 70 years ) , and showed that bevacizumab was associated with a statistically significant and clinically meaningful improvement in median pfs ( 9.2 versus 6.2 months ; hr 0.52 ; p<0.0001 ) and os ( 19.3 versus 14.3 months ; hr 0.70 ; p=0.006 ) in this elderly population , with a similar benefit observed also for patients aged 70 years.19 the incidence of bevacizumab - associated adverse events was comparable with that observed in the overall study populations in the two trials , and did not result in a higher rate of treatment discontinuation .
cassidy et al20 pooled data from the avf2107 g and avf2192 g trials with data from two randomized phase iii studies of oxaliplatin - based chemotherapy with or without bevacizumab in the first - line and second - line setting , respectively , ie , the no16966 trial and the e3200 trial . in this analysis ,
data from 1,142 patients aged 65 years and 712 patients 70 years were compared with those from 1,864 patients < 65 years .
this analysis confirmed a remarkable survival advantage for older patients from the addition of bevacizumab to standard chemotherapy in comparison with chemotherapy alone .
indeed , bevacizumab prolonged median pfs from 6.7 , 6.9 , and 6.4 months to 9.5 ( hr 0.59 ; p<0.0001 ) , 9.3 ( hr 0.58 ; p<0.0001 ) , and 9.2 months ( hr 0.54 ; p<0.0001 ) , and median os from 16.5 , 15 , and 14.1 months to 19.9 ( hr 0.77 ; p<0.0001 ) , 17.9 ( hr 0.85 ; p=0.015 ) , and 17.4 months ( 0.79 ; p=0.005 ) for patients aged < 65 , 65 , and 70 years , respectively.20 as shown by the difference among hrs for progression and for death ( table 1 ) , the effect of bevacizumab on median os was less pronounced than its effect on median pfs .
regarding the safety profile of bevacizumab , the pooled analysis revealed that most of the bevacizumab - associated adverse events in the elderly population were not more frequent than those observed in younger patients .
however , the incidence of arterial thrombotic events was higher in elderly patients receiving bevacizumab than in those treated with chemotherapy alone ( 5.7% versus 2.5% for patients 65 years , 6.7% versus 3.2% for patients
avex ( avastin in the elderly with xeloda ) was the first trial evaluating the efficacy and safety of the addition of bevacizumab to chemotherapy specifically in an elderly population ( table 1).21 in this open - label phase iii study , 280 patients aged 70 years with previously untreated mcrc and deemed not candidates for oxaliplatin - based or irinotecan - based chemotherapy were randomized to receive capecitabine alone or capecitabine with bevacizumab .
patients were excluded from the study if they had clinically significant cardiovascular disease or a history of thromboembolic events within the previous 6 months .
the median age was 77 years in the control group and 76 years in the bevacizumab group , and about 50% of patients had an eastern coast oncology group performance status 1 .
the response rate and disease control rate were significantly higher in the combination arm ( 19.3% versus 10% , respectively , p=0.042 ; and 74.3% versus 57.9% , p=0.005).21 moreover , the addition of bevacizumab to capecitabine led to a 4-month gain in median pfs ( 9.1 versus 5.1 months ; hr 0.53 ; p<0.001 ) , which was the primary endpoint of the study .
the effect of bevacizumab on pfs was consistent across all age groups , with hrs of 0.52 , 0.60 , and 0.36 for patients aged 7074 , 7579 , and 80 years , respectively .
os was longer for the bevacizumab arm compared with the capecitabine alone arm ( 20.7 versus 16.8 months ) , although the difference was not statistically significant.21 however , as the authors pointed out , the study was not powered to detect differences in os .
treatment - related toxicities of grade three occurred more frequently in the bevacizumab - containing arm than in the control group ( 40% and 22% , respectively ) , and treatment discontinuation because of adverse events was more common for patients treated with bevacizumab and capecitabine compared with those receiving capecitabine alone ( 41% versus 26% ) .
furthermore , venous thromboembolic events and hand - foot syndrome were more frequent in the combination arm than in the capecitabine arm .
however , the overall incidence of thromboembolic events was not different from that reported for nonelderly patients in previous trials with bevacizumab .
they are often fit and without relevant comor - bidities , and are different from the general population of elderly patients managed in daily clinical practice , who often have poorer performance status and additional risk factors .
observational studies provide an interesting opportunity to evaluate the efficacy and safety of bevacizumab in older patients outside of the clinical trials setting .
brite ( bevacizumab regimens : investigation of treatment effects and safety ) was a prospective , observational study evaluating the efficacy and tolerability of bevacizumab and chemotherapy in a community - based setting.22 a subgroup analysis for age groups in this study showed that median pfs times were 9.6 , 10 , and 8.6 months for patients aged 6574 , 7580 , and > 80 years , comparable with the median pfs observed in younger patients ( 9.8 months).23 in contrast , the median os decreased with age , from 26 months for patients < 65 years to 16.2 months in patients > 80 years . however , elderly patients receive all the active drugs for mcrc less frequently than younger patients , and it has been demonstrated that median os is longer with exposure to all effective therapies.24 the safety profile of bevacizumab in the brite study was similar for older and younger patients , with the exception of thromboembolic events , which increased significantly with age , occurring in 1.4% of patients aged > 65 years , 4% of those aged 7580 years , and 4% of those aged > 80 years.23 in the prospective , observational aries ( avastin registry : investigation of effectiveness and safety)25 study , there was no difference between patients aged < 70 years and 70 years in terms of pfs both in first line ( 10.3 versus 9.9 months ; hr 1.11 ) and second line ( 7.9 versus 7.9 months ; hr 0.96 ) .
however , in this study , for patients receiving bevacizumab in first line , median os was longer for younger patients ( 25.1 versus 19.6 months , hr 1.23).25 a cohort of 2,526 mcrc patients aged 65 years treated from 2002 to 2007 with a fluoropyrimidine - based chemotherapy regimen as first line was identified by meyerhardt et al within the surveillance , epidemiology and end results ( seer ) medicare database.26 all the patients received a fluoropyrimidine - based chemotherapy regimen as first line .
addition of bevacizumab in 903 of these patients significantly prolonged median os ( 19 versus 15.9 months ; hr 0.87 ; p=0.003 ) , but the benefit was limited to patients receiving irinotecan .
interestingly , patients treated with chemotherapy alone and those treated with chemotherapy and bevacizumab experienced a comparable rate of thromboembolic events within the first 6 months of treatment ( 17.1% and 16.4% ) , and the rate of cardiac events was higher in the chemotherapy group than in the bevacizumab group ( 14.5% versus 11.5% ) .
however , stroke occurred more frequently in patients receiving bevacizumab ( 4.9% versus 2.5%).26 the seer database was also searched by shankaran et al27 who analyzed data from patients aged 65 years and diagnosed with mcrc between 2001 and 2007 . of 6,821 patients , 3,282 ( 48% ) were diagnosed in 20052007 , and 19% of them received first - line bevacizumab .
in contrast with other reports , adverse event incidence rates in this analysis were comparable between patients in 20012003 receiving first - line chemotherapy ( 135 events per 100,000 person - days ) and patients in 20052007 receiving first - line chemotherapy with bevacizumab ( 141 events per 100,000 person - days ) .
the optimal management of elderly patients with mcrc is still challenging . assessment of geriatric patients considering only the chronological age may be inadequate to make a decision on the treatment strategy .
indeed , patients in the same age groups are often heterogeneous with respect to overall health status .
geriatric medicine has developed specific tools to detect some issues that can be missed by routine clinical and physical examination .
a comprehensive geriatric assessment ( cga ) includes social parameters , functional status and falls history , cognitive and psychological status , nutritional status , comorbidities , and polypharmacy.28 in elderly patients with cancer , several domains of cga were demonstrated to correlate with oncological outcomes .
therefore , multiple organizations , including the international society of geriatric oncology,29 have recommended that all older patients should undergo a cga at the time of diagnosis and in the decision - making process .
however , a cga might be resource - consuming and time - consuming in daily practice , and rapid screening tests have been developed for a brief geriatric evaluation.30 although cga and rapid screening tests are prognostic tools , they have not been widely used in clinical trials as inclusion or stratification criteria . also , in the avex trial , the only available phase iii study of bevacizumab in elderly patients , formal geriatric and comorbidity assessments were not performed.21 another important issue when treating elderly patients with mcrc , given the palliative intent of the therapy , is quality of life ( qol ) .
only limited data are available on the effect of bevacizumab on qol in older patients . in the subgroup analysis of the cairo-2 study ,
qol was not significantly different across age groups of patients treated with capox plus bevacizumab ; in this study , a 10-point increase in qol was achieved in 17% , 15% , and 33% of patients aged < 75 , 7075 , and > 75 years , respectively.16
data from retrospective subgroup analyses and pooled analyses of randomized clinical trials , taken together with results from observational cohort studies , suggest a meaningful benefit from the addition of bevacizumab to standard chemotherapy for elderly patients with mcrc ( table 1).31 the effectiveness of bevacizumab in older patients was comparable with that observed in younger patients .
recently , results from the avex trial confirmed a significant improvement of median pfs with the addition of bevacizumab to capecitabine.21 bevacizumab is well tolerated , but its use may increase the risk of thromboembolic events , and the avex and the australasian gastrointestinal trials group max studies showed that it can also exacerbate hand - foot syndrome when given with capecitabine.18,21 therefore , age alone should not be a barrier to use of bevacizumab , but treatment should be individualized , carefully selecting patients on the basis of medical history , with adequate assessment of clinically relevant comorbidities or risk factors , such as recent cardiovascular events , uncontrolled hypertension , or unstable angina .
furthermore , during treatment with bevacizumab , particular attention should be paid to monitoring and treating side effects in order to optimize the risk - benefit ratio .
when patients are not properly selected , toxicity related to bevacizumab may be potentially more than expected .
it should also be kept in mind that elderly patients treated outside of clinical trials are often frail , and that preservation of qol is crucial in this setting .
further research should investigate the role of bevacizumab in elderly mcrc patients , with the incorporation of cga , qol assessment , and geriatric endpoints in clinical trials .
|
### Summary:
: metastatic colorectal cancer ( mcrc ) , like many cancers , is primarily a disease of elderly people . despite this prevalence
, such patients are often excluded from randomized trials or represent a minority of enrolled patients . moreover ,
the criteria for establishing benefit or side effects of treatment strategies in this population are uncertain and not well recognized .
bevacizumab improves the outcome of mcrc when used in combination with standard first - line and second - line chemotherapy and beyond the first disease progression when given with a chemotherapy backbone different from that used in the precedent line .
the particular toxicity profile of this antiangiogenesis agent ( in particular hypertension , thromboembolic events , hemorrhage , and renal failure ) may discourage its use in elderly patients with comorbidities .
data from subgroup analyses of randomized trials and the results of recent cohort studies suggest a significant benefit from the addition of bevacizumab to standard chemotherapy for elderly patients comparable with that observed in younger patients , except for the increased risk for thromboembolic events .
age alone should not be a barrier to use of bevacizumab , and further research with a more complete geriatric assessment should investigate the role of bevacizumab in elderly patients with mcrc to avoid undertreatment of this patient population due to a historical conservative approach .
|
<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: it infects humans and many species of endothermic animals
with a great infection rate , achieving nearly 100% in some local populations
.
the course , symptoms , and consequences of t. gondii infection depend strongly on the virulence and inoculum
size of the parasite and the genetic background and immune status of the
infected host . in general ,
most postnatal cases of toxoplasmosis are mild and
subclinical , with lifelong persistence of the parasite in the host .
however , in immunosuppressed beings ,
both primary infection and reactivation of chronic infection could occur very
serious , causing even death .
primary infection acquired during pregnancy can
lead to an abortion and neonatal malformations . unfortunately , despite many
efforts there are no satisfactory immunoprevention methods against
toxoplasmosis .
the only accepted vaccine against toxoplasmosis ( toxovax , ovilis
toxovac ) contains live tachyzoites of incomplete nonpersistent s48 strain and
is used to prevent congenital toxoplasmosis in sheep .
the vaccine causes a
decrease in abortion cases frequency but not complete eradication of the
parasite .
successful parasitism by t. gondii is based
on its ability to form within the host cell a replication permissive niche
this growing
niche is delimited from
the host cell cytoplasm by pv membrane ( pvm ) , a unique and dynamic organelle
found only in infected cells which prevents t.
gondii degradation by the host cell endocytic machinery and enables
parasite 's intracellular propagation .
unique to the apicomplexan protozoa
including toxoplasma gondii are also three types of electrodense
secretory organelles : micronemes , rhoptries , and dense granules , carrying their
characteristic proteins : mics , rons , rops , and gras , respectively ; see table 1 .
host cell
invasion is mediated by the sequential secretion of the contents of all three
organelles , which are exocytosed at the apical region when the parasite invades
the host cell .
the processes , critical in the establishment of a productive
infection , are highly regulated at possibly two levels : physical separation of
exocytosis sites in the parasite 's plasma membrane and different trigger
mechanisms [ 3 , 4 ] .
micronemes are involved in the attachment and penetration of t. gondii , while rhoptries are required for creating a transient
enigmatic structure , moving junction and
then the establishment of pv .
host cell entry of the parasite is completed in
1520 seconds [ 5 , 6 ] .
the
secretion process coincides with the formation of intravacuolar network and
continues during the intracellular residence of t. gondii [ 3 , 4 ] .
unlike most secretory and lysosomal granules in
mammalian cells , which are of a semispheroidal shape , the mature rhoptries are club shaped with a bulbous
base and an extended duct ( neck ) , connected to the extreme apical pole of the
parasite . the rhoptries in the number of 812 per cell
occupy 1030% of the total
cell volume .
they are the only known acidified organelles in t. gondii ;
ph of immature rhoptries is 3.55.5 and of mature
rhoptries is 5.07.0 .
current findings suggest that rhoptries are
most analogous to secretory lysosomal granules because they receive material
from both biosynthetic and endocytic cell pathways . in the exit site of
endoplasmic reticulum , the rhoptry proteins ( in a form of proproteins )
are
loaded into coated vesicles and then travel to the golgi apparatus where they
are sorted to an immature rhoptry using the specific sorting signals . both
tyrosine - based and dileucine sorting motifs were detected within cytoplasmic
tails of the predominant rop2 family proteins .
proproteins undergo
proteolytic cleavage of an n - terminal prodomain , which occurs in post - golgi
compartment , probably in immature rhoptries .
in contrast to all known
prominent rop2 family members , a new described rop5 protein is not processed
during trafficking but is synthesized in the mature form .
some rhoptry
cargo is delivered by endosomal pathway via
multivesicular body and immature rhoptry .
immature rhoptries evolve into
mature rhoptries characterized by specific morphology and
subcompartmentalization of their contents : rop proteins are located in the bulb
body whereas rons in the neck of the rhoptry .
a proteomic analysis revealed over 30 rhoptry
proteins , all localized in the contents of the rhoptries , although many of the
identified proteins have putative transmembrane domains .
as shown for
rop2 , a prototype protein of rop2 protein family , apart from an n - terminal
signal sequence , the molecule is characterized by the high abundance of charged
amino acids and proline residues , n- terminal arginine - rich stretches and
hydrophobic sequence in c - terminus considered as transmembrane domain [ 14 , 15 ] .
the rop2 protein was shown to be inserted in pvm during the invasion of a host
cell and its n - terminal domain is exposed to host cytosol mediating the
association of parasitophorous vacuole with host cell mitochondria [ 14 , 16 ] .
targeted depletion of rop2 resulted in multiple effects , such as : ( i ) impairments
of rhoptry biogenesis and cytokinesis , ( ii ) reduction in the association of
host cell mitochondria with pvm of the parasite , and a reduced sterol uptake
from the host cell , and ( iii ) reduced capacity to invade and replicate in vitro
in human fibroblasts and attenuation of virulence in mice .
besides ,
anti - rop2 antibodies caused a decrease in the invasion ability of t. gondii rh tachyzoites in vitro .
the observed effects of
rop2 depletion or inactivation and simultaneous synthesis of several related
proteins ( rop2 family ) suggest that they serve crucial biological functions .
all
members of the rop2 family contain a protein kinase - like domain but only some of them are
catalytically active .
the exocytosis of rhoptry contents coincides with
the formation of pv but the biological trigger of rhoptry secretion has not
been identified .
mobilization of intracellular ca by ionophores
triggers the discharge of micronemes but not rhoptries and dense granules .
in contrast to other secretory organelles , rhoptry proteins and lipids are
exocytosed through the duct , whereas delimiting membrane is retained in the
cell and the rhoptry is transiently empty .
secreted rhoptry proteins are
subsequently discharged into nascent parasitophorous vacuole and localized in
its membrane [ 14 , 20 ] . as mentioned earlier , certain rhoptry proteins
are preferentially localized in the neck of the rhoptry . during invasion ,
ron4
relocalizes at the moving junction and participates in the formation of this short living and very
enigmatic structure between the host cell and the parasite , sliding over the
parasite as it invades .
ron4 , ron2 , and ron5 ( ts4705 ) form protein trio
that associates with adhesion microneme protein tgama1 ( t. gondii apical membrane protein 1 ) forming an unusual complex
derived from two distinct secretory organelles : rhoptries and micronemes .
moving junction functions as a molecular sieve for the quick selective sorting
of parasite surface components ( like rons - tgama1 complex mentioned above ) as
well as surface proteins of the host cell .
because of the exclusion of most
host cell membrane proteins , developing parasitophorous vacuole is not
recognized by the host cell fusogenic machinery enabling the vigorous parasite
proliferation in this safe intracellular compartment [ 5 , 23 ] .
despite the very high genetic homology
achieving ~98% , t. gondii strains
express dramatic differences in many phenotype aspects , including virulence .
the majority of t. gondii isolates from europe and north america
belong to three clonal lines ( types ) significantly differing in their
virulence , which has been well characterized in the mouse model .
type i
isolates have an ld100 of a single organism and lead to rapid death of the infected
mice ( 610 days ) , whereas
types ii and iii ( called nonvirulent or avirulent ) typically have ld100 of 10 organisms ; mice which survive acute phase of toxoplasmosis remain chronically
infected and highly seropositive .
dramatic
differences in acute virulence between three major types of t. gondii enabled the mapping of
virulence genes by an analysis of f1 progeny from genetic crosses between three
toxoplasma types , using an experimental mouse model to test virulence .
the
method applied to types i iii recombinants revealed first two closely
adjacent virulence loci on parasite chromosome viia and then single gene
encoding rop18 protein as a major virulence component .
type iii and type i
alleles of rop18 differed significantly in nucleotide sequence and expression
level .
the contribution of rop18 to virulence was confirmed by the transfection
of the virulent rop18 allele into nonpathogenic type iii parasite strain .
the
obtained transformants showed an increased growth rate in vitro and an enhanced mortality by 4 to 5 logs in cd1 mice ,
as compared to wild nontransformed strain .
. mapped
virulence in f1 progeny derived from crosses between type ii and type iii
strains .
five virulence loci were identified , and two of them coded rhoptry
proteins : rop18 and rop16 , both hypervariable protein kinases that are
responsible for the high virulence of certain strains of the parasite .
contrary
to rop2 and several other members of this family that lost detectable kinase
activity and retained molecular fossils
, newly described rhoptry
proteins rop16 and rop18 are true kinases , the substrates of which are unknown .
both kinases presumably disrupt some signaling processes and alter
intracellular environment in a way that favors parasite growth . the work of el
hajj et al .
the protein showed a
very strong tropism to pvm and during the invasion it was translocated from
rhoptry to pvm .
the mature recombinant rop18 was able to phosphorylate two as
yet unknown parasite 's ( but not host 's ) proteins , a major one of 70 kda and a
minor one of 68 kda .
the overexpression of rop18 protein promoted dramatically
parasite 's proliferation , whereas the mutation ( d394a ) in rop18 molecule
resulted in the loss of both the enzyme activity and accelerated growth . in
contrast to rop18 ,
the second recently described rhoptry protein , rop16 is
translocated to the host cell nucleus like phosphatase 2c , subverts
stat3/6 signaling , and , in consequence , il-12 production in infected host
cells .
allele shared by types i and iii of the parasite induce prolonged
phosphorylation of stat3 ( stat3-po4 ) which results in a much lower
level of il-12 secretion , as compared to type ii allele .
the observed
type - specific effects show the key role of rop kinases in t. gondii virulence and could explain different disease outcomes
seen with type i , ii , and iii strains .
the role of rops in virulence suggests that they could be
promising vaccine candidates against toxoplasmosis .
in contrast to rop18 and
rop16 , another novel rhoptry protein , bradyzoite rhoptry protein 1 ( brp1 ) , is
expressed in bradyzoites but absent from tachyzoites .
bradyzoites are a
low - replicating , inactive developmental stage , sequestered in tissue cysts ,
which are a typical marker of chronic toxoplasmosis . the biological function of
brp1 is not defined yet .
this protein does not play an essential role in the
development of the bradyzoite stage , brain cyst formation , and oral infection
of new hosts .
as mentioned earlier , the only vaccine against
toxoplasmosis has been licensed for use in sheep in europe and new zealand .
this conventional vaccine contains live attenuated tachyzoites of the
nonpersistent t. gondii s48 strain
and it can not be considered for an application in humans because of the risk of
reverting to tissue cyst formation . from both humanitarian and economic points
of views , an effective anti - t .
taking into
account the main sources and transmission routes of t. gondii , host groups of a particularly high infection risk and
consequences of congenital infection , as well as postnatal infections in
immunocompromised individuals , targets for vaccination strategy should include ( i )
preventing tissue cyst formation in consumption animals ( to avoid parasite
transmission to humans and animals ) and in people ( to protect against
reactivation of infection in immunosuppression state of persistently infected
individuals ) , ( ii ) reducing oocyst shedding in cats ( to limit environmental
contamination and infection risk for all intermediate hosts ) , and ( iii )
preventing development of parasitemia in pregnant women and farm livestock ( to
avoid transplacental transmission to the fetus and congenital toxoplasmosis )
.
vaccine candidates should induce protective
cellular th1 and humoral responses as well , both at the level of intestinal
mucosa ( local ) and whole organism ( systemic ) .
many studies have shown that
protective immunity to t. gondii is
characterized by the development of cell - mediated immunity dominated by the
production of ifn-
by t lymphocytes ( a th1 type response ) . whereas immune cd8 + lymphocytes are
cytotoxic for t. gondii - infected host
cells , cd4 + lymphocytes exert their protective activity primarily by the
production of many proinflammatory cytokines ( ifn- ,
tnf- ,
il-12 ) and providing growth factor , il-2 .
specific antibodies limit
multiplication of t. gondii by
killing of extracellular tachyzoites , either by activating complement , or
opsonizing the parasites for phagocytosis and next killing by macrophages .
the major challenge in novel vaccine construction is a selection of relevant t.
gondii antigens and then their presentation to host immune system in an
appropriate manner to induce strong and long - lasting protective immunity .
because of the complex life cycle of the parasite , the expression of numerous
common and stage - dependent antigenic epitopes and host - dependent immune responses ,
the vaccination with multiantigen preparation comprising antigens from
different life cycle stages is likely to be more efficacious than with a single
antigen .
previous and current vaccine approaches focus not only on classic
formulations ( live attenuated or mutant t.
gondii strains ) [ 2 , 34 , 35 ] but more often on the defined subcellular
components of the parasite such as biosynthetic ( recombinant ) antigens and
naked t. gondii dna or rna .
nonpathogenic recombinant microorganisms with the expression of
immunoprotective antigens of t. gondii ( live
vector vaccines ) and synthetic
peptides have also been tested as interesting antitoxoplasmic vaccine
alternatives .
these new generation subunit vaccines require an addition of
potent adjuvants enhancing the intensity of the immune response and inducing
the desired th1 profile .
several new compounds , for instance unmetylated cpg
nucleotides , present satisfactory adjuvant th1 activity .
most of the current vaccine
candidates for t. gondii and other apicomplexa
are either surface or secreted antigens that appear to be essential for the
invasion process , rhoptry proteins among them . until now
garcia et al . used
crude native rhoptry antigens incorporated in the immunostimulating
complexes ( iscoms ) to protect pigs against an infection with oocysts of t.
gondii veg strain ( of type iii ) .
two - fold subcutaneous immunization
procedure led to a partial protection against cysts formation in muscles and
brain during chronic infection ( as confirmed by bioassay in mice ) but it was
not protective against acute infection , probably because of the lack of
intestinal immunity after subcutaneous immunization .
taking into account
epidemiological role of cats as an important source of t. gondii dissemination by shedding millions of oocysts in feces , the authors tested the
same antigen material in domestic cats .
the cats were immunized
intranasally three times ( 0 , 21 , 42 day ) with crude rhoptry proteins adjuvanted
with quil - a and then challenged with tissue cysts of the t. gondii veg
strain .
this vaccination procedure resulted in a partial protection only two from three
immunized cats did not shed oocysts .
till now vaccine trials with individual rops of t. gondii have mostly focused on the
rop2 antigen , prominent member of rop2 protein family . in majority ,
the
experiments were performed with dna vaccines which are believed to elicit a
predominant th1 response .
vercammen showed that the immunization with rop2
gene protected mice against lethal challenge with a highly virulent rh strain ,
but the observed protection was mouse strain dependent
inbred c3h mice
were protected , whereas c57bl/6 and balb / c were not .
similar results were obtained by leyva et al . in mice of three
inbred strains ( cba / j , c57bl/6 , and balb / c ) , vaccinated with plasmid encoding
antigen rop2 .
the mice did not resist a challenge with rh strain but the
protection was achieved by vaccination with live tachyzoites of the t. gondii nonpersistent thermosensitive
ts-4 strain .
in the mice immunized with rop2-plasmid , a mixed th1/th2 immune
profile instead of a desirable th1 profile and a significant variability in the
response between individual mice was observed . using similar vaccine material ,
that is , rop2-plasmid , wei et al .
reported both a high immune response and
significant immunoprotection against a lethal dose of t. gondii rh
strain in balb / c mice . recently , live nonpathogenic microbes ( viruses
or bacteria ) were used as vectors of selected rop genes .
recombinant vaccinia
virus carrying rop2 gene induced in mice a specific humoral response very similar ,
in intensity and profile , to the thermosensitive ts-4 strain of t. gondii .
because the ts-4 strain as vaccine was able to fully protect mice against the
challenge with the highly virulent rh strain , the authors conclude by analogy
that rop2-vaccinia virus could be equally effective in toxoplasmosis
immunoprotection .
mishima et al . obtained recombinant feline
herpesvirus 1 ( fhv1 ) encoding rop2 and evaluated its ability to induce antitoxoplasmic
activity and protective immune responses .
antisera of rop2-fhv1-immunized cats
reduced significantly the invasion capacity of t. gondii rh tachyzoites in
vitro .
the number of parasites in the brains of immunized cats lowered , however
oocyst shedding intensity was not reduced .
tested
the efficacy of recombinant mycobacterium bovis bcg expressing rop2 gene
as a vaccine , using mouse experimental toxoplasmosis model .
bcg , widely applied
in immunoprophylaxis of tuberculosis , is a particularly attractive vector for
delivering heterologous antigens because mycobacteria elicit th1-mediated
immune response without an additional adjuvant .
vaccine rop2-bcg induced
strong specific humoral and cellular ( il-2 and ifn-
in supernatants of splenocytes cell cultures ) immunity . a significant delay in
mortality
the use of recombinant rop2 antigen , instead of
rop2-plasmid , revealed again in mouse experimental model that the protective
activity of rop2 protein is mouse strain - dependent ; rop2 vaccination resulted in the partial
protection ( decrease in tissue cyst burden ) only in c3h mice but not c57bl/6
mice .
the efficacy of rop2 protein vaccine significantly increased by
including new immunostimulatory components , leishmania
infantum heat shock protein 83 ( hsp83 ) fused with the rop2 .
immunization with this fusion protein elicited a predominant th1 response in
all mouse strains used ( balb / c , c57bl/6 , and c3h ) , whereas rop2 alone or in
mixture with hsp83 induced a mixed th1/th2 profile .
multiple endogenous and exogenous parameters
can influence the type and intensity of vaccine - induced immune response .
the
results obtained on inbred mouse strains proved that the protection induced by
rop2 and other vaccine antigens of t.
gondii is strongly dependent on the host genetic background [ 43 , 50 , 52 ] .
in relation to the vaccine itself
, the composition seems a very important
factor in the context of both selected rop antigens and adjuvants used . in the
light of our current knowledge on rhoptry antigens , rop18 protein , described
recently as a major component of pathogenicity of t. gondii , seems a very promising candidate
for a vaccine , as a single antigen or , more possibly , accompanied by other t.
gondii antigens because many previous studies showed greater effectiveness
of combined than single - antigen vaccines .
however , the usefulness of rop18 as a
vaccine antigen for toxoplasmosis needs to be experimentally confirmed ,
especially in relation to the polymorphism of this protein .
whether protection
across t. gondii species could be
achieved with that polymorphic antigen remains an open question .
the advantages of combined vaccines as compared
to single - antigen vaccines were recently emphasized by zhang et al . who
constructed a multiantigenic dna vaccine consisting of plasmid encoding both
rop2 and surface antigen 1 ( sag1 )
antigens and inoculated it to balb / c mice with or without plasmid encoding
murine il-12 .
multiantigenic ( cocktail ) vaccine elicited stronger antibody and
cellular responses than single - gene vaccines and , additionally , coimmunization
with il-12 gene significantly enhanced the intensity of immune responses . the
most immunostimulating vaccine , psag1+rop2+pil-12 , delayed about three times
the survival time of mice after a lethal challenge with rh tachyzoites . as
found by guo et al .
another genetic adjuvant , plasmid carrying ifn- ,
revealed also a potent stimulatory activity , enhancing the cellular immune
response induced in mice by rop1-plasmid . in summary , all the above presented
experimental vaccination strategies with rop antigens still remain suboptimal
but the vaccination results appear promising and therefore future research on
rop vaccine seems rational .
toxoplasmosis , widely dispersed in the
populations of humans and endothermic animals all over the world , is a serious
problem of human and veterinary medicine in the case of immunocompromised
individuals ( fetuses , aids patients , etc . ) .
the consequences of primary
infection in pregnant females and the possibility of reactivation of latent
infection in immunodepressed individuals cause that both immunoprophylactic and
immunotherapeutic vaccines are needed .
the persistence of the memory t lymphocytes , stimulated by
the regular rupture of t. gondii tissue
cysts or by recurrent contact with infected food , prevents a reinfection in
most individuals .
the effective and safe vaccine should mimic the immune
response observed in natural infections and protect the hosts against t. gondii infection , however without the
potential risk of tissue cyst generation in intermediate hosts and development
of oocysts in cats , associated with the vaccination using live attenuated or
incomplete parasite strains . in this
context , for t.
gondii rop antigens as key molecules in biogenesis of parasite
intracellular niche in the host cells and their association with host
organelles , major virulence factors showing satisfactory immunogenicity
seem particularly valuable vaccine candidates for toxoplasmosis prophylactic
approaches but their vaccine potential needs experimental confirmation .
it is
worth mentioning that rhoptry proteins are also vaccine candidates in other apicomplexa ,
including rhoptry - associated protein 1 ( rap1 )
and rap2 proteins of plasmodium
falciparum .
several rhoptry proteins are polymorphic , with t and b
epitopes that are conserved among strains but not across species and over
species , as found for instance for plasmodium and eimeria .
the question , if it would be possible to construct
universal anti - t .
the results of
the studies on rop - vaccine presented in this paper are promising and could be a
good starting point to develop an effective vaccine not only for the prevention
of toxoplasmosis but also other parasitoses caused by apicomplexan protozoa .
recent advances in bioinformatics and experimental tools will make the
selection of vaccine candidates easier .
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### Summary:
: toxoplasma gondii is an obligate intracellular protozoan parasite classified in the phylum apicomplexa , which includes numerous notable human and animal pathogens ( plasmodium species ,
cryptosporidium species , neospora caninum , etc . ) .
the invasive stages of apicomplexans are characterized by the presence of an apical complex composed of specialized cytoskeletal and secretory organelles , including rhoptries .
rhoptries , unique apical secretory organelles shared exclusively by all apicomplexan parasites , are known to be involved in an active parasite 's penetration into the host cell associated with the biogenesis of specific intracellular compartment , parasitophorous vacuole in which the parasite multiplies intensively , avoiding intracellular killing . due to the key biological role of rhoptries ,
rhoptry proteins have recently become vaccine candidates for the prevention of several parasitoses , toxoplasmosis among them .
the article presents current data on t. gondii rhoptries biology and new approaches to the development of effective vaccines against toxoplasmosis using rhoptry antigens .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: dapsone hypersensitivity reaction was reported as early as 1950 by lowe and was so named by allday and barnes .
hypersensitivity to dapsone is thought to be the major cause of this syndrome . with the advent of who multidrug therapy for leprosy , more cases of dapsone syndrome
a 34-year - old man who was on multidrug treatment for lepromatous leprosy for the past two months presented with history of high - grade fever of two - week duration associated with pruritic rash . on admission , his temperature was 40c , blood pressure was 110/70 mm hg , and heart rate was 112 beats / min .
he had pallor and pruritic maculopapular exanthematous rash on the extremities and face and hepatosplenomegaly .
investigations revealed initially hemoglobin - 7.3 g% ; white blood cell count - 4 000/mm ( neutrophils , 76% ; lymphocytes , 20% ; and eosinophils , 4% ) , erythrocyte sedimentation rate of 30 mm in the first hour and a platelet count
after two days , his hemoglobin dropped to 4.9 g% , the total leukocyte count to 2 000/mm , and the platelet count to 1 lakh / mm .
his liver function tests were abnormal with a direct bilirubin of 1.6 mg / dl , indirect bilirubin of 2.0 mg / dl , aspartate aminotransferase of 69 u / l , alanine aminotransferase - 28 u / l , alkaline phosphatase - 145 u / l , serum albumin - 2.3 g / dl , and prothrombin time - 13 seconds .
viral hepatitis serology ( igm antibody to hepatitis a antigen , hepatitis b surface antigen , and hepatitis c antibody ) were negative .
the levels for urea , creatinine , uric acid , and electrolytes were within normal limits .
abdominal ultrasound showed uniform liver enlargement with a slight increase in echo texture , and there was no evidence of portal hypertension or biliary obstruction .
bone marrow trephine biopsy showed multiple granulomas with adjacent scattered hematopoietic elements showing erythroid precursors with normoblastic and megaloblastic maturation , few myeloid maturing cells and megakaryocytes , and grade 2 myelofibrosis .
a diagnosis of dhs ( dapsone hypersensitivity syndrome ) was thus based on the patient 's treatment history , presence of fever prutitic maculopapular erythematous rash , hepatosplenomegaly and deranged liver function tests , and evidence of bone marrow suppression .
dapsone was stopped and corticosteroids were given both orally ( prednisolone 30 mg / d ) and topically .
his clinical condition improved after two weeks and laboratory test results returned to normal levels within 3 weeks .
a repeat bone marrow was done after 8 weeks of starting steroids , which showed persistence of lepra bacilli .
disappearance of granulomas on repeat biopsy supports the diagnosis of drug - induced bone marrow granuloma .
he did not get a recurrence of fever or bone marrow suppression after stopping steroids .
dapsone ( 4 , 4-diamino - diphenyl sulfone ) is the parent compound of sulfone drugs . though synthesized in 1908
dapsone has been used as a first - line treatment for leprosy since the 1950s .
dapsone is absorbed well from the gut and primarily metabolized through n - acetylation and n - hydroxylation ( oxidation ) .
the hydroxylamine metabolite and other hydroxylated metabolites are potent oxidants and have been thought to cause the hematologic adverse effects associated with dapsone , including methemoglobinemia and hemolytic anemia .
it has a long elimination half life , between 24 to 30 hours on the average .
this is important to keep in mind incase adverse reactions emerge after a long metabolite impact period .
it differs from other drug reactions in that it can begin after prolonged exposure to the offending agent and can occur up to 6 months or more after exposure .
the features include fever , eosinophilia , mononucleosis , jaundice , hepatosplenomegaly , and cutaneous manifestations .
cutaneous manifestations may occur in the form of erythroderma , maculopapular eruption , erythema multiforme , toxic epidermal necrolysis , and stevens - johnson syndrome .
the eruption , which is often initially morbilliform , may develop into overt exfoliative dermatitis .
agranulocytosis occurs rarely , but a dose - related hemolysis and methemoglobinemia are more common ; hemolysis occurs especially in patients with glucose-6-phosphate dehydrogenase deficiency .
two cases have been reported of dapsone - induced neutropenia with bone marrow suppression in patients with ocular cicatricial pemphigoid and granuloma annulare .
hypoalbuminemia present in our patient is also a feature of dapsone hypersensitivity , which is probably due to binding of dapsone to the circulating serum albumin .
hyperbilirubinemia present in dapsone syndrome may partly be due to hemolysis in addition to hepatotoxicity .
hepatocellular injury is characterized by elevated transaminases , with liver biopsy showing predominantly eosinophilic lobular and portal infiltration .
cholestatic pattern may have less severe course and is characterized by high alkaline phosphatase level and modest transaminases level
the mechanism of injury , including hepatotoxicity in dapsone syndrome , seems to be hypersensitivity reaction .
granulomatous inflammation may be seen in the bone marrow in a diverse group of processes such as infections , neoplasms , and hypersensitivity reactions where the commonly implicated drugs include sulfonamide , procainamide , and phenytoin .
disappearance of the granuloma on a subsequent biopsy supports the diagnosis of drug - induced granuloma .
richardus and smith have mentioned the following criteria to diagnose a case of dapsone hypersensitivity :
the symptoms appear within 8 weeks after commencement of dapsone and disappear after the discontinuation of the drug.the symptoms can not be ascribed to any other drug given simultaneously with dapsone.the symptoms are not attributable to lepra reaction.no other disease liable to cause similar symptoms is diagnosed .
the symptoms appear within 8 weeks after commencement of dapsone and disappear after the discontinuation of the drug .
the reaction was classified as dapsone syndrome when symptoms start between the second and eighth week of treatment and at least two of the following signs or symptoms are present : fever , skin eruption , lymphadenopathy , liver pathology ( hepatomegaly , jaundice , and/or abnormal liver function tests . as
hypersensitive reactions have become more frequent in both multi- and paucibacillary cases on mdt , combination with rifampicin may be an important factor in contributing to the rise of hypersensitivity reactions , as suggested by richards and smith .
they also considered other factors like increased awareness , low dose of dapsone administered before 1976 , and changes in manufacturing of dapsone being responsible for the observed rise .
the long elimination half - life of dapsone averaging between 24 and 30 hours is thought to be due to significant enterohepatic recirculation of the drug .
strong protein binding of the drug itself ( 70 - 90% ) and its major metabolite , monoacetyl dapsone ( 99% ) , contribute to that long half - life .
systemic corticosteroids have been used to treat dhs . since dapsone persists up to 35 days in organs through protein binding and enterohepatic recirculation ,
slow tapering off of the corticosteroid therapy over at least one month with close monitoring of organ function is required .
generally , dhs is a self - limiting drug reaction and most patients recover following cessation of dapsone therapy and starting treatment with oral corticosteroid ; deaths , however , have been reported .
physicians should be aware of this infrequent but potentially fatal severe form of adverse reaction that can mimic other conditions .
the treatment regime followed in such cases is as follows . for pb leprosy , give monthly rifampicin pulse and daily clofazimine therapy . and
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### Summary:
: dapsone , a potent antiparasitic and anti - inflammatory compound , is mainly used in the treatment of leprosy and a variety of blistering skin diseases .
it may cause a severe adverse drug reaction with multiorgan involvement known as dapsone hypersensitivity syndrome .
we report an unusual case of dapsone hypersensitivity , manifesting as bone marrow suppression and peripheral pancytopenia in addition to fever , rash , and hepatosplenomegaly .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: ischemic stroke as a complication of cerebral cavernous angioma has rarely been described , and hemorrhage after ischemic wallenberg 's syndrome has not been reported before .
a 45-year - old woman presented with perioral numbness , hoarseness , dysphagia , and worsening of her previous sensory symptoms . the patient had been taking aspirin for 3 years after suffering from ischemic wallenberg 's syndrome with left paresthesia as a residual symptom .
we presume that cerebral cavernous angioma was responsible for both the ischemia and the hemorrhage , and we also cautiously speculate that the aspirin contributed to the development of hemorrhage in the previously infarcted area .
medullary hemorrhage has been attributed to hypertension,1 anticoagulant therapy,2 hemangioblastoma,3 and vascular malformation.4 - 6 although hemorrhagic transformation after lateral medullary infarction has been reported,7 there has been no report of medullary hemorrhage in the old infarction .
we report herein a case of hemorrhage in a previously infarcted area that appeared during oral aspirin treatment for previous ischemic wallenberg 's syndrome in a patient who was subsequently diagnosed as having a cavernous angioma in the right medulla .
an otherwise healthy 45-year - old woman presented with dizziness and dysarthria lasting for 3 days .
neurological examination revealed fragmented wallenberg 's syndrome including hoarseness , hypesthesia on the left hemibody ( sparing the face ) , and gait disturbance .
magnetic resonance imaging ( mri ) of the brain demonstrated ischemic infarction of the right lateral medulla ( fig .
1a , b and c ) , but no evidence of parenchymal hemorrhage , intramural hematoma of the arteries , or leukoaraiosis in the periventricular white matter .
extensive diagnostic studies including transesophageal echocardiography revealed no cardiac or paradoxical source of embolism , and there were no abnormalities on coagulation work - up . oral aspirin treatment ( 100 mg / day )
was started , and cilnidipine ( 10 mg / day ) was added before discharge because the patient was found to be hypertensive .
three years later the patient developed sudden onset of perioral numbness , followed by worsening of the previous sensory symptoms , vomiting , hoarseness , and dysphagia .
neurological examination revealed ocular lateropulsion to the left , gaze - evoked counterclockwise torsional and down - beat nystagmus , hypesthesia on the left hemibody including the face , glossopharyngeal paresis , tongue deviation to the right , and marked gait ataxia , but no horner 's syndrome , diplopia , facial weakness , hearing loss , or limb weakness . computed tomography of the brain revealed an acute medullar hematoma in the previously infarcted area ( fig .
ataxia improved after conservative treatment including rehabilitation , and the patient was discharged on hospital day 35 .
a follow - up mri performed approximately 3 months after the initial hemorrhage produced findings suggestive of cavernous angioma in the right dorsomedial medulla ( fig .
even though pathological confirmation was not achievable , this case probably represented cerebral cavernous angioma , and not hemorrhagic metastasis , which was another consideration .
however , benign hemorrhage is characterized by a complete hemosiderin ring and lack of surrounding edema.8 other diagnostic possibilities include hypertensive hemorrhage in association with cerebral microbleeds , hemorrhagic infarction , and arteriovenous malformation .
cerebral microbleeds , which are tiny extravasations of blood from lipohyalinized small penetrating cerebral arterioles , may be of importance because these lesions are associated with spontaneous hemorrhage.9 we do not know whether our patient had these lesions in the medulla prior to the ischemic episode . although it has been demonstrated that hypertension is a risk factor for cerebral microbleeds,10 the patient had no hypertensive changes such as retinopathy , left ventricular hypertrophy , or small - vessel changes on mri .
the patient did have high blood pressure during the hemorrhage , but her blood pressure had since been controlled adequately .
the possibility of hemorrhagic infarction by reperfusion injury seems quite unlikely given the time interval between the infarction and the hemorrhage .
although there was no evidence of cavernous angioma in initial mri , we presume that the ischemia was caused by the cavernous angioma based on the initial ischemic lesion of this patient being located more medially than is typical for ischemic wallenberg syndrome caused by cardioembolic , large - artery , or small - vessel diseases , and the hemorrhage developing in the same area in which the ischemia had occurred previously . in patients
suspected to have an arteriovenous malformation , mra or conventional angiography would be helpful in detecting lesions and demonstrating blood flow within those lesions .
the main complication of cerebral cavernous angioma is hemorrhage , but ischemic stroke has been described in the form of wallenberg 's syndrome as the presenting symptom of cavernous angioma.11 cavernous angiomas present a dynamic balance between intracavernous bleeding and thrombosis .
an increase in the intracavernous pressure may cause bleeding , and a reduced blood flow may be responsible for thrombosis.11 disturbance of this balance may lead to either ischemia or hemorrhage .
cantu et al.12 reported that the nonlobar location of cavernous angiomas confers a higher risk of hemorrhage , and that the hemorrhagic rate was 2.33% per patient per year for brainstem angiomas , but 1.22% per patient per year for lobar angiomas .
the largest lesions showed a reticulated pattern , the smallest lesions had a primarily hemosiderin pattern , and medium - sized lesions usually exhibited a target pattern , as in the present case .
our patient received no anticoagulant therapy , and extensive study demonstrated no hemorrhagic diathesis , but she had been taking aspirin since the initial ischemic episode that had occurred 3 years previously .
prior use of antithrombotic drugs is known to be a risk factor for hemorrhage after ischemic stroke.13,14 although aspirin is widely used for the prevention of stroke , and the benefits outweigh its hemorrhagic risks , prolonged use of aspirin is associated with an increased risk of hemorrhage.15 hemostatic mechanisms are impaired in patients treated with antithrombotic agents , and a small leakage may result in a large hematoma .
although the medulla is not a typical site for microbleeds , aspirin - associated hemorrhages have been described in association with cerebral microbleeds .
we presume that cerebral cavernous angioma was responsible for both the ischemia and the hemorrhage in the present case , and we also cautiously speculate that aspirin contributed to the development of a hemorrhage in the previously infarcted area by rendering the brain prone to bleeding .
this case may represent an unusual complication of cerebral cavernous angioma , showing delayed hemorrhage after ischemic wallenberg 's syndrome .
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### Summary:
: backgroundthe main complication of cerebral cavernous angioma is hemorrhage .
ischemic stroke as a complication of cerebral cavernous angioma has rarely been described , and hemorrhage after ischemic wallenberg 's syndrome has not been reported before.case reporta 45-year - old woman presented with perioral numbness , hoarseness , dysphagia , and worsening of her previous sensory symptoms .
the patient had been taking aspirin for 3 years after suffering from ischemic wallenberg 's syndrome with left paresthesia as a residual symptom .
brain computed tomography revealed an acute medullary hematoma in the previously infarcted area .
follow - up magnetic resonance imaging revealed a cavernous angioma in the right medulla.conclusionswe presume that cerebral cavernous angioma was responsible for both the ischemia and the hemorrhage , and we also cautiously speculate that the aspirin contributed to the development of hemorrhage in the previously infarcted area .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: paclitaxel ( ptx ) is one of the most important compounds which has recently been obtained from a natural source .
it is approved for the treatment of ovarian , breast , nonsmall cell lung carcinomas and aids - related kaposi 's sarcoma . the currently available pharmaceutical formulation of ptx ( such as taxol , bristol - myers squibb , new york , ny , usa ) contains 30 mg ptx dissolved in 5 ml 50% cremophor el ( polyethoxylated castor oil , crel ) and 50% dehydrated ethanol ( 1 : 1 , v / v ) to enhance the solubility of ptx in water . however , a large number of studies have reported various side effects of crel , such as hypersensitivity , neurotoxicity , and neuropathy [ 2 , 3 ] .
the premedication schedule includes corticosteroids , diphenhydramine or chlorpheniramine , h2-receptor antagonists and antiemetics . despite such premedication , minor reactions ( flushing and rash ) still occur in 4144% of all patients and major , potentially life threatening , reactions in 1.53% [ 46 ] .
although crel has been used to administer other drugs , such as cyclosporine and teniposide , the amount of crel necessary to deliver the required doses of taxol is significantly higher than that used with any other commercially available drugs .
therefore , current approaches to improve this situation are focused mainly on the development of formulations that contain no crel .
these strategies include increasing the aqueous solubility of ptx by cosolvency , emulsification , microemulsification , drug complexation with cyclodextrins , carrier mediation using liposomes , nanoparticles , and structural modification to obtain analogues and prodrugs .
it has been reported that some crel - free or reduced crel ptx formulations , such as genexol - pm , ai-850 , or genetaxyl , have been developed and evaluated in clinical trials [ 811 ] .
now , a novel crel - free albumin - bound nanoparticle formulation of ptx ( abi-007 ) has been approved by the us food and drug administration for pretreated metastatic breast cancer patients .
this formulation is prepared by high - pressure homogenization of ptx in the presence of serum albumin , resulting in a nanoparticle colloidal suspension .
the albumin concentration is 3 - 4% , which is similar to the albumin concentration in blood .
microemulsions are isotropic , thermodynamically stable transparent ( or translucent ) systems of oil , water , and surfactant , frequently in combination with a cosurfactant with a droplet size usually in the range of 20200 nm [ 13 , 14 ] .
the combination of surfactants with oils to form microemulsions offers an advantage over a micellar or cosolvent system in terms of the drug solubilization capacity for lipophilic compounds , because of the extra solubilization provided by the oil phase . also , because of the small droplet diameter
the obvious benefits of microemulsions as drug delivery systems have led to the development of several systems for intravenous administration of ptx in recent years .
recently , two crel - free paclitaxel microemulsions were developed for intravenous administration [ 17 , 18 ] .
although ptx was first discovered in the early 1960s , its clinical development were delayed until the early 1980s , one reason being its poor solubility .
after much investigation , crel , and dehydrated ethanol usp ( 1 : 1 , v / v ) was identified as the most suitable option for the solvent system employed in the commercial formulation of ptx , taxol . until now , although several reports have linked this solvent to alterations in the pharmacokinetic profile of ptx and the production of side effects , crel is still used as an excipient for taxol . in the present study ,
the in vivo antitumor activity of the ptx microemulsion was evaluated in tumor - bearing mice and the safety of the ptx microemulsion was also evaluated .
cremophor el ( crel ) was purchased from basf corporation of germany ( local agent in shanghai , china ) .
egg phosphatidylcholine ( lipoid e 80 ) was obtained from lipoid gmbh ( local agent shanghai toshisun enterprise co. , ltd in shanghai , china ) .
ptx injection was commercially available from a local hospital in beijing ( taxol , bristol myers squibb co. , princeton , nj , usa , for pharmacokinetic and biodistribution experiments ; ptx injection , beijing shiqiao biological pharmaceutical co. , ltd . , beijing , china , for antitumor and allergic reaction experiments ) , and the ptx injection formulation contained 30 mg ptx in 5 ml 50% crel ( v / v ) and 50% dehydrated ethanol ( v / v ) . medical products for premedication were also commercially available from a local hospital in beijing .
sprague - dawley ( sd ) rats weighing 200 20 g , new zealand albino rabbits weighing 1.82.0 kg and beagle dogs weighing 812 kg were all obtained from the experimental animal center of peking university health science center .
female balb / c nude mice weighing 1822 g ( 68 weeks ) were purchased from the academy of military medical sciences .
all care and handling of animals were performed in accordance with the institutional authority for laboratory animal care of peking university . a human ovarian cancer cell line ( ovcar-3 ) and a human nonsmall - cell lung cancer cell line ( a 549 ) were obtained from atcc .
the ptx microemulsion used in the experiment was prepared in our laboratory with some modification of the published method . in brief , to prepare ptx microemulsion concentrated solution , ptx was dissolved in dehydrated alcohol and then mixed with lipoid e 80 , crel and soybean oil . when this concentrated solution was mixed with 5% glucose infusion
the volume of 5% glucose solution used for diluting the ptx concentration solution was 24 ml ( the final concentration of ptx in microemulsion was 1 mg / ml ) . for particle size analysis , this microemulsion
was further diluted with 5% glucose solution to a concentration of ptx about 0.03 mg / ml . the particle size of the ptx microemulsion was measured by photon correlation spectroscopy using malvern zeta sizer nano - zs instrument ( malvern , uk ) at 25c .
the morphological examination of ptx microemulsions was performed on a jeol jem-200cx instrument at an acceleration voltage of 200 kv by transmission electron microscope ( tem ) .
briefly , the tem samples were prepared by dropping one drop of ptx microemulsion ( about 0.03 mg / ml , ptx concentrated solution was diluted with 5% glucose solution ) onto a 300-mesh formvar - coated copper grid .
the grids were negatively stained at room temperature with freshly prepared and sterile - filtered 2% ( w / v ) uranyl acetate aqueous solution .
ptx injection or microemulsion concentrated solution was diluted with 5% glucose injection at a concentration of 1 mg / ml , respectively .
these ptx formulations were all injected into the tail vein at a single dose of 5 mg / kg .
blood samples ( 0.5 ml ) were collected via the orbital venous plexus at 0 , 0.17 , 0.33 , 0.67 , 1 , 1.5 , 2 , 3 , 4 , 6 , and 8 h after injection .
after centrifugation at 3000 g for 5 min , the obtained plasma was stored at 20c until required for determination .
ptx injection or microemulsion concentrated solution was diluted with 5% glucose injection at a concentration of 1 mg / ml .
these ptx formulations were all given intravenously via the tail vein at a dose of 5 mg / kg .
after drug administration , rats were exsanguinated through the femoral artery at 0.167 , 1 , 4 , and 8 h ( three rats for each time point ) . the heart , liver , spleen , lung , kidney , brain , and ovaries were collected .
tissue samples were blotted with paper towel , rinsed in saline , blotted to remove excess fluid , weighed , and stored at 20c .
the female balb / c nude mice were inoculated subcutaneously in the right flank with 0.1 ml of an ovcar-3 cell suspension ( 4 10 ) .
administration was started at day 14 after inoculation when the tumor size reached approximately 150 mm . on that day , mice were randomly divided into a control group , a ptx injection treatment group , and a ptx microemulsion treatment group .
injection of 0.1 ml of an a549 cell suspension ( 5 10 ) into the right flank of the mice .
treatments were started when the tumor size reached approximately 150 mm , about 7 days after inoculation . on that day
, mice were randomly divided into a control group , a ptx injection treatment group and a ptx microemulsion treatment group .
each group consisted of 6 tumor - bearing mice . in the antitumor activity experiment , ptx injection or microemulsion
concentrated solution was diluted with 5% glucose infusion to give a concentration of 1.5 mg / ml . each group was treated with 5% glucose infusion , ptx injection ( 15 mg / kg , i.v . , q4d4 , every 4 days for four doses ) , or ptx microemulsion ( 15 mg / kg , i.v . , q4d4 , every 4 days for four doses ) , respectively .
tumor volumes were calculated from the formula : v = length ( cm ) width ( cm ) 0.5236 .
once an animal in any group died , all the other animals in the experiment were sacrificed .
hemolysis test : rabbit blood was used to test the hemolytic effect of the ptx microemulsion .
briefly , 10 ml rabbit blood was obtained from the arteria cruralis and the fibrinogen was removed by stirring with a glass rod .
then , 10 milliliter 5% glucose infusion was added to the fibrinogen - free blood sample and supernatant was removed after centrifugation at 3000 rpm for 5 min .
the erythrocyte pellet at the bottom of centrifuge tube was washed four times ( centrifugation followed by re - dispersion ) with 5% glucose infusion . finally , after repeated washing and centrifugation
, a suitable amount of 5% glucose infusion was added to the erythrocyte pellet to give a 2% erythrocyte standard dispersion which was stored at 4c .
the ptx microemulsion concentrated solution was diluted with 5% glucose infusion to give a concentration of 1 mg / ml , and different amounts of microemulsion in volumes of 0 , 0.1 , 0.2 , 0.3 , 0.4 , and 0.5 ml were added to six tubes each tube containing 2.5 ml 2% erythrocyte dispersion .
then , suitable amounts of 5% glucose infusion were added to every tube to obtain a final volume of 5 ml .
a positive control was prepared by addition of 2.5 ml distilled water to 2.5 ml 2% erythrocyte dispersion instead of 5% glucose infusion .
after vortexing , the tubes were incubated at 37c and observed microscopically from 15 min to 4 h. intravenous irritation assessment : one male and two female rabbits weighing 1.82.0 kg were used for this study .
ptx microemulsion concentrated solution was diluted with 5% glucose infusion to give a concentration of 1 mg / ml . each rabbit ( three animals ) received a daily dose of 5 mg / kg ptx microemulsion into the vein at the edge of the left ear at an injection rate of 1 ml / min for 3 days .
an equivalent volume of 5% glucose infusion was injected into the right ear - border vein of these three animals as a control .
the rabbits were sacrificed by exsanguination 24 h after the last administration , and the ears were cut and fixed in 10% formaldehyde for histological examination . at sites 3 cm from the injection site to the proximal part , histological sections were prepared for histopathological examination .
allergic reaction : in the allergic reaction experiment , ptx injection or microemulsion concentrated solution was diluted with 5% glucose infusion to give a concentration of 0.6 mg / ml . the beagle dogs were divided into five groups ( n = 6 ) : a control group , a ptx injection treatment group , a ptx microemulsion treatment group , a ptx injection premedication treatment group , and a ptx microemulsion premedication treatment group . in the control group ,
in the ptx formulation treatment groups , the diluted ptx formulations were administered by infusion at a dosage of 165 mg / m ( 8.25 mg / kg ) .
infusion was initiated at a rate of 30 ml / h for 10 minutes .
if no allergic reaction was noted in the first 10 minutes , the rate of infusion was increased to 60 ml / h for 10 additional minutes , and if no reaction was noted , it was increased again to 90 ml / h for the rest of the infusion .
, the animals were initially premedicated with prednisone ( 1 mg / kg po ) , diphenhydramine ( 4 mg / kg i m ) , cimetidine ( 4 mg / kg iv ) , and dexamethasone na phos ( 2 mg / kg iv ) according to a previous report , and then they received the ptx formulations by infusion as the ptx formulation treatment groups .
the allergic reactions exhibited by the animals were observed and ranked according to table 1 .
after administration , animals were continuously monitored for at least 14 days to further evaluate their mental state , food intake , and defecation .
the body weights were recorded on day 4 , 8 , and 12 , and full blood cell counts were carried out on day 4 and 8 .
briefly , an aliquot of 100 l plasma ( or blood ) , 50 l docetaxel solution ( 10 g / ml , as an internal standard ) , and 2.5 ml acetonitrile were mixed using a vortex mixer for 30 s. the mixture was centrifuged at 3000 g for 10 min , then 2.0 ml supernatant was collected , and dried under a gentle stream of nitrogen gas at 50c in a water bath .
the residue was reconstituted using the mobile phase below , and assayed by hplc using a waters system consisting of a 1525-pump , and a 2487-ultraviolet detector ( waters co. , inc . , westerville , oh , usa ) . the mobile phase consisted of methanol - water - tetrahydrofuran ( 70 : 27.5 : 2.5 , v / v / v ) delivered at a flow rate of 1 ml / min .
chromatographic separation was performed on a phenomenex ods3 column ( 250 4.6 mm , 5 mm , torrance , ca , usa ) , and the detector wavelength was set at 230 nm .
the peak areas of ptx ( ap ) and docetaxel ( ad ) were recorded , and the concentration of ptx was calculated from the ratio of ap / ad .
briefly , tissue samples were homogenized in saline and the homogenate ( 0.7 ml ) was mixed with 50 l docetaxel solution ( 10 g / ml , as an internal standard ) , and 2.5 ml acetonitrile .
after vortex mixing for 30 s , the mixture was centrifuged at 3000 g for 10 min , 2.0 ml supernatant was collected , and dried under a gentle stream of nitrogen gas at 50c in a water bath .
the residue was reconstituted using the mobile phase and then determined by hplc as described above for the measurement in plasma .
one - way analysis of variance ( anova ) was used to determine significance among groups , after which post - hoc tests with the bonferroni correction were used for comparison between individual groups .
nonparametric test ( 2 independence samples ) was used to determine significance in allergic reaction data .
the concentration of ptx in the microemuslion concentrated solution was 5 mg / ml .
the average particle size of the ptx microemulsion after the concentrated solution was diluted with 5% glucose infusion was approximately 142.4 1.2 nm with a polydispersity of 0.267 0.07 . the typical particle size and distribution
tem was used to examine the morphology of the ptx microemulsion ( figure 1(b ) ) .
the plasma concentration - time profiles of ptx after intravenous administration of the ptx formulations ( 5 mg / kg ) were characterized in rats and illustrated in figure 2 .
data fitting was conducted and the result showed that the pharmacokinetic behavior of the ptx microemulsion and ptx injection fitted a two - compartment model .
as shown in figure 2 , the curve for the ptx microemulsion group was similar to that of the ptx injection group .
the main pharmacokinetic parameters of ptx are summarized in table 2 . as shown in table 2 , the values of auc08 in the ptx microemulsion group and the ptx injection group were 3174.9 592.4 and 3770.9
the bioavailability of ptx in the microemulsion group compared with that in the injection group was 84.2% .
meanwhile , there was no significant difference in the main parameters between the ptx microemuslion group and the ptx injection group ( table 2 ) .
the tissue distribution profiles of ptx in the ptx microemulsion group and the ptx injection group were compared in rats .
the concentration of ptx in each tissue was determined by hplc analysis . as shown in figure 3 ,
ptx was widely and rapidly distributed into most tissues following intravenous administration of ptx formulations , and the highest concentration of ptx was found in liver , followed by kidney and lung at 0.167 h after administration .
there was no significant difference in the tissue distribution of ptx between the ptx microemulsion group and the ptx injection group .
the in vivo antitumor activity of the ptx microemulsion was investigated in ovcar-3 and a549 tumor - bearing mice . as shown in figures 4(a ) and 4(b ) ,
ptx microemulsion markedly inhibited the growth of ovcar-3 and a-549 tumors ( p < .01 ) .
a similar result was observed in the ptx injection treatment group ( p < .01 ) .
there was no significant difference in the antitumor activity between the microemulsion group and the injection group ( p > .05 ) . in the ovcar-3 tumor - bearing mice groups , the mean tumor sizes at day 33 after implantation in the ptx injection , and the microemulsion group were 131 112 and 63 49 mm , respectively , compared with 690 206 mm in the control group ( p < .01 ) .
the corresponding tumor growth inhibition in the ptx injection group and in the ptx microemulsion treated group was 81.0% and 90.9% , respectively .
similar results were also observed in the a 549 tumor - bearing mice groups , the mean tumor sizes at day 32 after implantation in the ptx injection group and the microemulsion group were 659 228 and 528 316 mm , respectively , compared with 1746 520 mm in the control group ( p < .01 ) .
the corresponding tumor growth inhibition in the ptx injection group and in the ptx microemulsion group was 62.3% and 69.7% , respectively . as shown in figures 5(a ) and 5(b ) , the average tumor weight in the 5% glucose infusion , ptx injection , and ptx microemulsion groups at day 33 after ovcar-3 cell implantation was 362 105 , 76 94 , and 40 59 mg , respectively .
the average tumor weight in the 5% glucose infusion , ptx injection and ptx microemulsion groups at day 32 after a 549 cell implantation was 927 286 , 353 142 , and 268 143 mg , respectively .
there was no significant difference in body weight among the ptx microemulsion group , ptx injection group , and control group ( data not shown ) .
hemolysis test : complete hemolysis was observed in the positive control tube at 15 min , the solution was red clearly diaphanous , and no erythrocytes survived at the bottom of the tube .
the erythrocytes were precipitated at the bottom of the other six tubes and dispersed after shaking , while the supernatant was achromatic and transparent over the observation period of 4 h. these results demonstrated that the ptx microemulsion at a concentration of 1 mg / ml did not cause hemolysis and erythrocyte agglutination at 37c .
intravenous irritation : after a 3-day administration of pacltaxel microemulsion , 5% glucose infusion , no erythema , and edema were observed at the injection sites .
the histopathologic examination of the rabbit ear - border vein indicated that the vessel wall and endothelial cell structures were unimpaired .
furthermore , no angiectasia and thrombus formation was observed in the lumen of the vein .
there were no significant pathological changes , inflammatory cell infiltrate , hemorrhage epimorphosis , and necrosis in the vessel walls and surrounding tissues .
the histopathologic examination results of the ears from rabbits given the microemulsion were similar to those of the control group .
all the results demonstrated that ptx microemulsion at a dose of 5 mg / kg produced no irritation of the rabbit ear veins .
allergic reaction : the ranking of the allergic reactiona is shown in table 4 . in the ptx injection group , the rank of the allergic reaction was grade 3/+++ in all animals ( 6/6 ) .
most animals in the ptx microemulsion group ( 5/6 ) and the ptx injection premedication group ( 5/6 ) experienced grade 2/++ .
the rank of the allergic reaction in the ptx microemulsion premedication group was grade 1/+ ( 6/6 ) . in the control group ,
the main observed allergic reactions were transient flushing and rash , licking the nose , drowsiness , rash , angioedema , mucous membrane hyperemia , vomiting , gatism , mania , and dyspnea .
the allergic reaction in paclitaxel injection treatment group was significantly serious than that in ptx microemulsion treatment groups or paclitaxel injection premedication treatment groups ( p < .01 ) .
similar results were also found in ptx microemulsion treatment group versus ptx microemulsion premedication treatment group ( p < .01 ) or paclitaxel injection premedication treatment group versus ptx microemulsion premedication treatment group ( p < .01 ) .
there were no significant differences observed in ptx microemulsion treatment group versus paclitaxel injection premedication treatment group ( p > .05 ) .
after administration of ptx formulations , the body weight decreased slightly compared with that of the control group and but gradually returned to normal during the observation phase .
the commercially available ptx injection is a clear to colorless formulation supplied in 5 ml vials containing ptx 6 mg / ml , crel 527 mg / ml , and dehydrated ethanol 396 mg / ml .
therefore , the amounts of ptx , crel , and dehydrated ethanol in a vial of ptx injection are 30 mg , 2.6 g and 2.4 g , respectively .
the amount of crel in the ptx microemulsion concentrated solution was markedly reduced compared with that in the ptx injection ( from 2.6 g to 0.8 g ) .
when this microemulsion concentrated solution was diluted with 5% glucose infusion , the particle size of the formed ptx microemulsion was about 150 nm . in the present study
the curve of ptx in the microemulsion group was similar to that in the ptx injection group ( figure 2 ) .
the auc value in the ptx microemulsion group was slightly lower than that in the ptx injection group .
the tissue distribution profiles of ptx in the ptx microemulsion and the injection groups were compared in rats .
there were no significant differences in ptx in the main tissues between the microemulsion group and the injection group ( figure 3 and table 3 ) .
these results indicate that the pharmacokinetic and distribution properties of ptx in the microemulsion were similar to those in the ptx injection .
in addition , the toxic effect of ptx microemulsion to mice or rats will be further investigated in future since the pharmacokinetics behaviors of ptx may be influenced by the toxicity of its vehicles .
it has been reported that ptx is commonly used to treat ovarian , breast , and nonsmall cell lung cancers . in the present study
, we selected a human ovarian cancer cell line ( ovcar-3 ) and a human nonsmall cell lung cancer cell line ( a 549 ) as animal tumor models .
the in vivo antitumor studies indicated that the antitumor efficacy of the ptx microemulsion in ovcra-3 and a 549 tumor - bearing animals was similar to that of the ptx injection ( figures 4 and 5 ) .
the results of injection safety test of the ptx microemulsion indicated that the ptx microemulsion did not cause hemolysis , erythrocyte agglutination , and simulative reactions , showing that it was safe to inject .
the allergic reaction studies showed that all animals in the ptx formulation treatment groups experienced allergic reactions , but to a varying degree .
the allergic reactions in the ptx microemulsion premedication group were not as severe as those in the ptx injection premedication group ( p < .01 ) . also , the allergic reactions in the ptx microemulsion group were milder than those in the ptx injection group ( p < .01 ) , similar to those in the ptx injection premedication group .
it has been reported that crel and its formulated micelles can cause allergic reactions [ 25 , 26 ] and dogs are known to be particularly sensitive to agents that cause allergic reactions [ 25 , 27 ] .
therefore , in the present study , we chose beagle dogs as an animal model to evaluating the allergic reactions caused by ptx formulations . due to the amount of crel necessary to deliver the required doses of cyclosporine or teniposide being much lower than that in the ptx injection ,
the reported side effects linked with crel are limited [ 28 , 29 ] . considering the formulation of the present ptx microemulsion , the amount of crel was markedly reduced compared with that in the ptx injection ( 0.8 g in microemulsion versus 2.6 g in injection ) .
our allergic reaction results indicated that the incidence and degree of allergic reactions in the ptx microemulsion group , with or without premedication , were significantly lower than those in the ptx injection group .
similar results were also observed in guinea pigs ( data not shown ) , indicating that the potential allergic reaction induced by ptx microemulsion was significantly decreased compared with that in taxol .
therefore , we suggest that this ptx microemulsion containing less crel significantly reduces the allergic side effects associated with crel . now , this ptx microemulsion concentration solution has been approved for phase i trials by the china state food and drug administration ( no .
2010l01157 ) . the antitumor efficacy and safety of the ptx microemulsion will now be further evaluated in volunteers .
in summary , a novel ptx microemulsion delivery system containing a reduced amount of crel was developed and characterized as an effective alternative to commercially available ptx injection .
the ptx microemulsion had similar pharmacokinetic and antitumor efficacy to the ptx injection , but significantly reduced the allergic reactions caused by crel .
these results indicate that ptx microemulsion overcomes the disadvantages of conventional ptx injection and is one possible approach to avoid the limitations of the current injection product and to produce a desirable therapeutic efficacy .
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### Summary:
: the purpose of this study was to prepare a novel paclitaxel ( ptx ) microemulsion containing a reduced amount of cremophor el ( crel ) which had similar pharmacokinetics and antitumor efficacy as the commercially available ptx injection , but a significantly reduced allergic effect due to the crel .
the pharmacokinetics , biodistribution , in vivo antitumor activity and safety of ptx microemulsion was evaluated .
the results of pharmacokinetic and distribution properties of ptx in the microemulsion were similar to those of the ptx injection .
the antitumor efficacy of the ptx microemulsion in ovcra-3 and a 549 tumor - bearing animals was similar to that of ptx injection .
the ptx microemulsion did not cause haemolysis , erythrocyte agglutination or simulative reaction .
the incidence and degree of allergic reactions exhibited by the ptx microemulsion group , with or without premedication , were significantly lower than those in the ptx injection group ( p < .01 ) . in conclusion ,
the ptx microemulsion had similar pharmacokinetics and anti - tumor efficacy to the ptx injection , but a significantly reduced allergic effect due to crel , indicating that the ptx microemulsion overcomes the disadvantages of the conventional ptx injection and is one way of avoiding the limitations of current injection product while providing suitable therapeutic efficacy .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: it increases heart rate and cardiac mass , promotes cardiac arrhythmias , and elevates systolic blood pressure .
it also shortens the ejection period and impairs diastolic function.1)2 ) rarely , pulmonary hypertension has been reported in association with hyperthyroidism.3)4 ) we report a patient with isolated pulmonary arterial hypertension accompanied by hyperthyroidism due to graves ' disease .
a 54-year - old woman was admitted to the emergency room with dyspnea and generalized edema .
physical examination revealed diaphoresis , jaundice , distended jugular veins , hepatomegaly , ascites , ankle edema , and decreased breath sounds in both lower lung fields .
laboratory tests revealed leukocytosis without neutrophilia , normal cellular liver enzymes { aspartate aminotransferase ( ast)/alanine aminotransferase ( alt ) of 16/12
iu / l } , mild bilirubinaemia ( total bilirubin of 3.4 mg / dl ) , a prolonged prothrombin time of 17.7 seconds { international normalized ratio ( inr ) 1.7 } , and a normal partial prothrombin time of 28 seconds .
the patient was given oxygen through nasal cannula and was administered intravenous furosemide for pulmonary congestion .
she underwent a transthoracic echocardiogram , which revealed a dilated right ventricle , reduced right ventricular systolic function , a severely elevated systolic pulmonary arterial pressure of 80 mmhg , and moderate tricuspid valvular regurgitation .
the left ventricular chamber was normal sized , with a reasonable ejection fraction of 0.75 ( fig .
serum d - dimer was elevated at 2,460 ng / ml ( normal , 0 - 500 ng / ml ) . a chest computed tomogram obtained to rule out pulmonary embolism revealed no evidence of pulmonary embolism .
a ventilation - perfusion pulmonary scan suggested a low probability of pulmonary embolism , as well .
2 ) . further laboratory tests revealed a decreased serum thyroid - stimulating hormone ( tsh ) level ( less than 0.08 uiu / ml ) , an increased serum free thyroxine ( ft4 ) level ( 4.45 ng / dl ; normal , 0.89 - 1.76 ng / dl ) , and an increased serum total triiodothyronine ( tt3 ) level ( 3.03 ng / ml ; normal , 0.60 - 1.81 ng / ml ) .
a right heart catheterization performed the following day demonstrated severely elevated systolic / mean pulmonary artery hypertension of 80/60 mmhg .
the pulmonary capillary wedge pressure was normal at 10 mmhg , without evidence of intracardiac shunt .
anti - thyroid peroxidase antibodies were positive at 54.7 u / ml . the patient was confirmed to have idiopathic pulmonary artery hypertension and hyperthyroidism due to grave 's disease and was started on bosentan , amlodipine , and methimazole . during the following days
, the patient 's clinical condition gradually improved , and her body weight returned to baseline .
the patient was discharged in good clinical condition without signs or symptoms of heart failure .
tsh was still low ( < 0.08 uiu / ml ) , and tt3 was slightly elevated ( 2.53 ng / ml ) , but ft4 was within the normal laboratory range ( 1.63 ng / dl ) .
approximately four weeks later , a transthoracic echocardiogram revealed a normal sized right ventricle with good systolic function , improved systolic pulmonary artery pressure of 35 mmhg , and trivial tricuspid valvular regurgitation ( fig .
tt3 improved ( 2.28 ng / ml ) , and ft4 remained in the normal range ( 1.34 ng / dl ) .
we decided to discontinue bosentan , which is capable of masking the effect of the euthyroid state .
after discontinuation of bosentan , the patient remained in good condition , without symptoms or signs of right - sided heart failure .
many of the clinical manifestations of hyperthyroidism are attributable to the ability of thyroid hormones to alter cardiovascular hemodynamics .
the usual manifestations occur secondary to high cardiac output causing left - sided heart failure .
however , there is an increasing body of evidence suggesting that hyperthyroidism might cause reversible pulmonary arterial hypertension ( pah ) , resulting in right - sided heart failure .
this phenomenon might be underdiagnosed , because pulmonary artery pressure usually returns to the normal range after a euthyroid state is achieved.5 ) the potential pathogenic mechanisms of hyperthyroidism - related pah remain unclear .
it has been postulated that the pathogenesis of pah in hyperthyroid patients is similar to that of other autoimmune diseases related to pah.6 ) however , siu et al.7 ) reported that there was no significant difference in the prevalence of positive autoimmune antibodies between patients with pah and those without pah , or in the resolution of pah after successful antithyroid treatment regardless of the underlying etiology of hyperthyroidism .
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### Summary:
: we describe a 54-year - old woman with isolated pulmonary arterial hypertension accompanied by hyperthyroidism due to graves ' disease .
her pulmonary artery hypertension resolved spontaneously after restoration of euthyroidism .
this case suggests that hyperthyroidism should be considered a reversible cause of pulmonary arterial hypertension .
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<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
: thyroid hormone secreted from the thyroid gland is essential for maintenance of normal
metabolism1 . while the effects of most
hormones occur in specific tissues ; thyroid hormone occurs in most tissues and affects
energy metabolism , heat generation , glycometabolism and lipid metabolism2 .
reduced thyroid hormone levels due to the
insufficient production of thyroid hormone is called hypothyroidism3 .
hypothyroidism is a disease that progresses very slowly ,
and is usually not diagnosed as quickly as other diseases .
its clinical signs vary depending
on the progression of the disease ; however , patients generally experience fatigue , weakness ,
sensitivity to coldness , weight gain , lethargy , feebleness , memory deterioration , loss of
appetite , muscle pain , arthralgia , and hypohidrosis4 .
thyroid
hormone insufficiency especially affects neuronal differentiation and hippocampus
development , leading to deterioration of memory and spatial learning ability6 .
most
symptoms of hypothyroidism are permanent and patients require lifetime hormone
supplementation . most patients with hypothyroidism take oral thyroid hormones , but hormone
overdose increases ventricle wall thickness and the risk of atrial fibrillation in elderly
patients and may also cause osteoporosis in women after menopause8 . therefore ,
several studies have proposed exercise as a
method to decrease these side effects and manage health problems associated with thyroid
hormone .
both aerobic and anaerobic exercises have been suggested as effective methods to
recover thyroid function9,10,11 .
however , most
studies lack coherence and are limited to analyzing the effects of one session of
exercise12,13,14,15,16,17,18 .
although research has
reported the effects of exercise on activation of energy metabolism and hormone levels ,
research on the effects of exercise therapy on lethargy and memory deterioration are
insufficient . in this context , the current study analyzed changes in spatial learning
ability deterioration and lethargy caused by hypothyroidism following aerobic and anaerobic
exercise .
eleven male sprague - dawley rats were divided into spf ( specific pathogen free ) animals ,
infected microorganism blockade clean system and green animal by using barrier system .
during the purification process and pre - experimental period ,
they were raised in a
polycarbonate box called with fixed temperature ( 2022c ) , relative humidity ( 3070% ) ,
ventilation ( 1015
they were allowed to feed
freely with a maximum 60 g of solid feed ( samyang co. korea ) with a specific amount of feed
intake ( daily feed consumption per rat : 1530 g or 56 g per 100 g weight ) and 150 ml water
( daily water consumption per rat : 2460 ml or 1012 ml per 100 g weight ) . to induce
hypothyroidism , 0.05 g 6-propyl-2thiouracil ( ptu , sigma , mo , usa )
was dissolved in 100 ml
saline solution ; 10 ml were administered to the rats daily for 6 weeks .
the hypothyroid
status of two normal and two rats who had received ptu were confirmed by inhalation
anesthetization and blood analysis .
the remaining seven rats were divided into normal ( n=2 )
group , thyroid control ( n=1 ) , aerobic exercise ( n=2 ) , and anaerobic exercise ( n=2 ) groups .
they were made to exercise for 30 minutes , 5 days per week , for 4 weeks .
aerobic exercise
was performed on a treadmill ( 10 m / s for 5 minutes , followed by and 25 m / s for 25 minutes
for a total of 30 minutes of exercise ) .
anaerobic exercise consisted of climbing a 1-m
ladder 8 times with a mass 50% of weight attached to their tail .
the rats were given 2
minutes to rest each time they performed the exercise .
the thyroid control group was left to
do nothing while the rats in the other groups exercised .
lethargy and memory deterioration
of white rats with hypothyroidism were analyzed using open field and morris water maze
tests .
the research was approved by the animal test ethics committee of busan catholic
university .
after administration of ptu ( the starting date of the experiment ) , weights were
recorded twice weekly and analyzed according to group .
the weight was measured using an
electronic scale and 23 batches were repeated each time the weight was measured .
weights
were measured in grams and in a steady state . for biochemical analysis of blood , the rats
were lightly anaesthetized and the abdomen was incised .
a 25-gauge syringe was used to
collect 510 ml of blood in a heparinized tube ( franklin lakes , nj07417 , usa ) .
the collected
blood was immediately centrifuged for 10 minutes a 3,000 rpm to collect plasma for
biochemical analysis and was quickly frozen ( 196 c ) in liquid n2 gas and kept
frozen until further analysis .
open field tests were performed to evaluate emotional
behavior and exercise ability of the rats with induced hypothyroidism .
the open equipment
used in the research was a box without a lid , 78 cm wide , 78 cm long , and 30 cm tall .
the
boxes were monitored with a video camera set up in the middle of the box .
water maze measuring equipment and data analyzer
( kinovea 0.8.22 ) were used to measure spatial learning ability .
a stress - reducing
environment was created in a cylindrical water tank 120 cm in diameter and 50 cm tall .
the
tank was filled with 30 cm of water 13 c lower ( 26 2 c ) than the body temperature of
rats ( 3937.4 c ) to separate the rats from external disturbance .
a circular platform 15 cm
in diameter and 35 cm high was fixed and water level was adjusted so that it was not visible
be seen 0.5 cm below the surface of water .
the water maze was divided into four equal quadrants ; northeast ( ne ) , southeast ( se ) , and
southwest ( sw ) .
a video camera was also set
up in the upper middle part of the water maze ceiling to record the rats movement and time
to find the platform . as a method to train the rats during the experimental period ,
a visual
spatial cue was artificially created around the tank and 10 practice repetitions were
performed in water without coloring by making the rats start from the rest quadrants so that
they could remember and find their way .
the next day , harmless water coloring was dissolved
to hide the surface of the fixed platform from the rats visibility . the time for rats to
reach the target point from each quadrant and
first , thyroid hormone blood analysis revealed reduced initial levels of t3 , t4 , ft4 , and
thyroid hormone in the group of rats who received ptu , compared to levels in the normal
group .
blood analysis after the experiment showed that thyroid hormone levels of all groups
( thyroid control , thyroid aerobic exercise , and thyroid anaerobic exercise groups ) were
equal to those of the normal group .
weights measured before the experiment and the results
of open field and morris water maze tests after the experiment are shown in table 1table 1.changes in body weight ( g ) , o.f.t ( cm ) , and m.w.m.t ( sec ) before and after the
experimentbeforeafterweightnormal351 15.5380.5 9.9ptu - control268 19.8298 5.6ptu - aerobic ex.262.5
14.8317.5 4.2ptu - resistance ex.258.5 17.6335 4.7o.f.t.normal2,269.2 100.42,312.1 120.3ptu - control1,773.1
25.31,860.3 70.3ptu - aerobic ex.1,647.2 24.41,920.1 110.2ptu - resistance ex.1,821.4 24.62,439.2 85.5*m.w.m.t.normal52 11.117.1 9.7ptu - control89 9.675 13.9ptu - aerobic ex.120
: morris water maze test . before the experiment , hypothyroidism - induced rats were heavier than normal
rats . as a result of aerobic and anaerobic exercise in the hypothyroidism - induced rats ,
their weight increased compared to that of the control group , an effect
more clearly seen in
the anaerobic exercise group than in the aerobic exercise group . even in the open field
test , hypothyroidism - induced rats showed less movement ; as a result of the exercise
intervention , increased movement was observed compared to the control group ; this increase
was more pronounced in the anaerobic exercise group .
the morris water maze test results
revealed that , initially , the normal group found the platform in time , whereas
hypothyroidism - induced rats either could not find the platform in time or took more than one
minute to find it .
however , after exercise intervention , the rats showed reduced time , with
the anaerobic exercise group showing greater time reductions .
the current study determined the effect of aerobic and anaerobic exercise on rats with
spatial learning ability deterioration and lethargy due to hypothyroidism .
after the
intervention , thyroid hormone levels had returned to normal levels in all groups .
lethargy
assessed by the open field test revealed that the group of rats that performed exercise had
higher measurements compared to those of the control group , with greater increases of
observed in the anaerobic exercise group compared to measurements in the aerobic exercise
group .
is performed , adenosine monophosphate - activated protein kinase ( ampk ) is
activated , which accelerates insulin secretion due to increased fatty acid oxidation and
glucose absorption19 .
the results of the morris water maze test , used to assess memory and
learning ability , showed that the exercise group had decreased times compared to those of
the control group , with greater reductions in the anaerobic exercise group compared to those
of the aerobic exercise group .
exercise appeared to have a positive effect on general memory
deterioration in both anaerobic and aerobic exercise groups20 , 21 . according to cassihans
et al . in 2007 , resistance exercise in white rats resulted in insulin growth factor-1
( igf-1 ) secretion and improved memory22 .
during aerobic exercise , brain - derived neurotrophic factor ( bdnf ) , tropomyosin receptor kinase b ( trkb ) , and beta- calmodulin - dependent protein kinase
ii ( camk ii ) are secreted , and are also reported to improve memory23 .
however , the results of the current study indicated that
the effect was stronger in rats that underwent anaerobic exercise than those who underwent
aerobic exercise rats ; the reason for this observation may be the relationship between igf-1
secretion induced by anaerobic exercise and memory deterioration caused by hypothyroidism .
in addition , it is generally reported that , despite the normality of hypothyroidism
patients , their igf-1 blood concentration decreases24,25,26 . however , the thyroid control group that did not perform any
exercise appeared to have natural recovery from the weight increase , lethargy , and memory
deterioration , although the changes were much lower compared to those in the exercising
groups .
both aerobic and anaerobic exercise had positive effects on lethargy and memory
deterioration in induced - hypothyroidism rats , but anaerobic exercise appeared to be more
effective than aerobic exercise .
however , the research was performed for only eight weeks
blood was analyzed only before and after the experiment ; therefore , it was not possible to
determine precisely when the recovery of thyroid hormone occurred .
only male rats were used
for the experiment and they were fed freely ; thus , the weight change could not be
restricted .
even water with dissolved ptu administered freely ; therefore , the doses were not
standardized .
this study assessed the effects of aerobic and anaerobic exercise on rats with
spatial learning ability deterioration and lethargy caused by hypothyroidism .
the exercises
resulted in function recovery in induced - hypothyroid rats with these symptoms , and , compared
to aerobic exercise , anaerobic exercise was more beneficial in alleviating the symptoms .
|
### Summary:
: [ purpose ] this pilot study analyzed the degradation of spatial learning ability caused by
hypothyroidism using aerobic and anaerobic exercise .
[ subjects and methods ] the
experiments were performed on 11 , four - week - old male sprague - dawley rats .
hypothyroidism - induced rats receiving propylthiouracil ( ptu ) treatment were divided into
aerobic exercise , anaerobic exercise , and control groups .
each group performed exercise
and rest for four weeks .
changes in lethargy , memory deterioration , and thyroid function
were measured in each group by blood analysis and open field and morris water maze tests .
[ results ] after four weeks , blood analysis revealed that the thyroid hormone levels had
returned to normal in the aerobic exercise , anaerobic exercise , and control groups ,
whereas the open field and morris water maze tests showed that the aerobic and anaerobic
exercise groups had faster recovery compared to that of the control group .
in addition ,
comparison of aerobic and anaerobic groups showed that the anaerobic exercise group had
faster recovery compared to that of the aerobic group .
[ conclusion ] the findings of this
study suggest that exercise helped to improve lethargy and deteriorated spatial learning
ability caused by hypothyroidism and to recover function in rats .
anaerobic exercise was
more beneficial than aerobic exercise in alleviating symptoms .
|
<s> [INST] <<SYS>> You a helpful assistant capable of providing accurate and concise responses. You will assist me in generating Summary.<</SYS>> [/INST] <s> ### Text:
:
|
### Summary:
: murine erythroleukemia ( mel ) cells are erythroid progenitors whose programs of erythroid differentiation has been interrupted by transformation with the friend virus complex . as a result of the ability of certain chemicals such as dimethylsulfoxide ( dmso ) to induce terminal erythroid differentiation ,
the cells have been used as a model for understanding the molecular basis of cellular differentiation .
recent work on mel cells as well as other differentiating systems indicates that expression of cellular protooncogenes is implicated in chemically mediated differentiation . in mel cells the expression of the c - myc protooncogene undergoes unusual biphasic changes following inducer treatment .
levels of c - myc mrna decrease 10- to 20-fold between 1 and 2 hr and are then reexpressed between 12 and 24 hr .
these changes occur as a result of complex transcriptional and posttranscriptional regulatory events .
recent dna transfection experiments , in which mel cells were transfected with myc expression vectors , indicate that both the early decrease in c - myc expression and its subsequent reexpression are important events in the differentiation pathway .
the work on mel cells , as well as on other models of differentiation , is directed at understanding the molecular basis of leukemogenic transformation and cellular differentiation .
the ability of c - myc , as well as other protooncogenes , to influence both of these events indicates that cellular protooncogenes play a central role in their regulation.imagesfigure 1.figure 2.figure 3.figure 4.figure 5 .
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