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type 2 diabetes mellitus ( t2 dm ) has become a worldwide epidemic . it is estimated that 60% of the world 's diabetic population is in asia .
this rapid increase in the prevalence of t2 dm in asia has been fuelled by urbanization , economic growth and associated changes in diet and physical activity levels .
specifically , in korea , data derived from the korean national health and nutrition examination survey shows that t2 dm prevalence has increased moderately over the last decade from 7.7% in 2001 to 10.2% in 2010 .
the most recent data report that the overall prevalence of diabetes in adults aged 30 years or older is 12.4% . in general , asian populations tend to develop diabetes at a younger age and at lower body mass index ( bmi ) levels than has been observed in caucasian populations .
recent research in korea has , however , revealed differences between both gender and age groups : prevalence had decreased amongst women aged 30 to 59 years ( 6.9% in 2001 to 4.5% in 2010 ) , but increased amongst males aged 60 years or more ( 15.9% in 2001 to 21.6% in 2010 ) . a similar age - related trend is also reflected in data from the korean health insurance review and assessment database , in which there were significant ( p<0.001 ) decreases in annual incidence rates in younger adults ( 20 to 49 years ) and increases in the elderly ( 70 to 79 years ) .
in recognition of the progressive nature of t2 dm , the current korean clinical practice guidelines ( korean diabetes association ) suggest a stepped interventional approach .
lifestyle modification ( which incorporates diet , exercise , and education ) remains the foundation of the current clinical guidelines .
however , because lifestyle modification alone does not enable glycemic goals to be achieved or maintained in most patients , metformin monotherapy should be added at , or soon after , diagnosis , unless there are contraindications or intolerance .
ultimately the addition of a basal insulin either alone or in combination with other agents will be required to maintain glucose control .
glycosylated hemoglobin ( hba1c ) plays an important role in monitoring and targeting glycemic control and is the gold standard index for follow - up of glycemic control .
after lifestyle modification , treatment options are stratified according to the individual 's hba1c status ; patients whose hba1c is < 8% would initially receive oral hypoglycemic agents only , those in whom it is 8% to 10% would receive oral hypoglycemic agent combination therapy with basal insulin and those whose hba1c is > 10% would receive insulin with or without oral hypoglycemic agents .
patients not at target ( ideally 6.5% ) within 2 to 6 months would receive an additional oral hypoglycemic agent ( e.g. , triple therapy ) or a multiple - component insulin regimen again with or without oral hypoglycemic agents . within these guidelines
glucagon - like peptide-1 receptor agonists ( glp-1ras ) are recognized as providing a moderate ( 0.5% to 1.0% ) reduction in hba1c , with no hypoglycemia and no weight gain . however , there are some concerns with respect to gastrointestinal ( gi ) side effects and the lack of long - term established safety profile . in this paper
we review recent data on glp-1ras to help better understand their current risk / benefit profile and provide primary care practitioners with guidance on how this evidence can inform their clinical practice .
glp-1 is an incretin hormone secreted from gi l - cells early ( within 10 minutes ) after an ingested meal .
glp-1 stimulates insulin secretion in a glucose - dependent manner , suppresses glucagon secretion , delays gastric emptying , and suppresses appetite .
this combination of effects makes a significant contribution to glucose homeostasis , particularly the control of postprandial glucose .
the incretin response is markedly attenuated in people with t2 dm , making the incretin based system an attractive therapeutic target .
however , the short plasma half - life ( 1 to 2 minutes ) of glp-1 limits its suitability as a therapeutic agent for diabetes .
incretin enhancers , including dipeptidyl - peptidase 4 ( dpp4 ) inhibitors and glp-1ras have therefore emerged as a novel class of glucose - lowering therapy . a systematic review aimed at evaluating the comparative effectiveness of these two drug classes has found both to be effective , with hba1c reductions of 0.59% to 0.90% with dpp4 inhibitors and 0.77% to 1.62% with glp-1ras .
here we focus only on the glp-1ras , which mimic the effects of endogenous glp-1 , thereby stimulating pancreatic insulin secretion in a glucose dependent fashion , supressing glucagon out - put , slowing gastric emptying and decreasing appetite .
overall , the clinical advantages of glp-1ras are established in the literature ; they provide significant improvements in glycemic control with a relatively low risk of hypoglycemia and are associated with moderate , but significant , weight loss ( up to 3 kg in some clinical trials ) . efficacy has been confirmed in a meta - analysis of 17 randomized trials comparing glp-1ras ( exenatide , liraglutide , albiglutide , taspoglutide , lixisenatide ) with placebo or an active comparator ( insulin glargine , dpp4 inhibitor , thiazolidinedione , sulfonylurea ) in patients with t2 dm and suboptimal control on one or two oral hypoglycemic agents ( metformin and/or sulfonylurea ) . although the duration of the individual trials ranged from 8 to 30 weeks , overall , in comparison to placebo , all glp-1ras reduced hba1c by approximately 1% ( treatment difference 0.47% to 1.56% ) .
body weight reduction with glp-1ras has been confirmed in a meta - analysis of 21 industry - sponsored trials comparing glp-1ras with placebo , no intervention , or other diabetes medications in overweight patients with or without diabetes ( weighted mean difference , -2.9 kg ; 95% confidence interval [ ci ] , -3.6 to -2.2 ) .
however , long - terms data on the durability of this effect are currently lacking .
in addition , glp-1ras have beneficial effects on cardiovascular ( cv ) risk factors ( body weight , blood pressure , postprandial lipids , markers of oxidative stress ) with no suggestion of increased risk of cv disease : for example , exenatide cv disease - extended events , relative risk ( rr ) 0.69 ( 95% ci , 0.46 to 1.04 ) ; liraglutide major adverse cv events , incidence ratio 0.73 ( 95% ci , 0.38 to 1.41 ) . in a recent ultrasonography study , 3 months treatment with
a glp-1ra induced a redistribution of adipose tissue deposits , which may contribute to a better cv risk profile in patients with t2 dm . there are no long - term data to assess durability of weight loss , clinically important health outcomes ( cv events , mortality ) , or safety of glp-1ras .
the results of the liraglutide effect and action in diabetes : evaluation of cardiovascular outcome results ( leader ) study , formally assessing the cv safety of liraglutide , are awaited .
control of postprandial hyperglycemia is an important aspect of overall glycemic control and may contribute to lower cv morbidity and mortality .
data suggest that reduction of postprandial hyperglycemia may be particularly beneficial for patients early in the progression of diabetes .
the relevance of postprandial hyperglycemia is heightened as hba1c levels decrease ; thus , focusing on strategies to control it may increase a patient 's ability to achieve their hba1c target .
recognizing the importance of postprandial glucose , international organizations ( american association of clinical endocrinologists / american college of endocrinology , international diabetes federation , american diabetes association ) now provide 2-hour postprandial glucose targets of between 7.8 to 10.00 mmol / l ( 140 to 180 mg / dl ) in their treatment guidelines .
data from the diabetes epidemiology : collaborative analysis of diagnostic criteria in asia ( decoda ) and diabetes epidemiology : collaborative analysis of diagnostic criteria in europe ( decode ) studies suggest that the prevalence of post - challenge hyperglycemia is higher in asian patients than it is in caucasians . in a study of 68 korean patients with well - controlled t2 dm , glycemic variability and total glucose exposure were both mostly explained by postprandial glucose .
it has been suggested that the high carbohydrate diet eaten by asians , including koreans , may increase postprandial glucose more rapidly than does a lower carbohydrate diet . in the authors ' experience , postprandial hyperglycemia continues to present a problem when determining choice of treatment in patients with t2 dm ; in korea anecdotal reports suggest that 50% to 60% of patients may be affected by postprandial glucose excursions . when lifestyle interventions have failed , the addition of oral anti diabetic agents ( glucosidase inhibitors or meglitinides ) , a dpp4 inhibitor , short- or prandial - acting glp-1ra , or short - acting insulin ( for patients already on basal insulin ) may be of benefit .
the glp-1ra class can be subdivided into two types : short - acting agonists and long - acting agonists , which each have different mechanisms of action and result in different effects on fasting and postprandial glucose levels .
short - acting glp-1ras ( exenatide twice - daily and lixisenatide once a day ) act intermittently to activate the glp-1 receptor and slow gastric emptying exerting their effects predominantly on postprandial plasma glucose .
phase ii data and the results from randomised controlled clinical trials consistently demonstrate that these glp-1ras reduce postprandial plasma glucose more effectively than they do fasting plasma glucose .
for example , adding exenatide twice - daily to basal insulin ( alone or in combination with metformin and/or pioglitazone ) , lead to significant reductions in morning and evening 2-hour postprandial excursions ( both p<0.001 vs. placebo ) whilst lixisenatide 20 g once a day has been shown to significantly reduce postprandial glucose when compared with placebo after breakfast ( p<0.0001 ) , lunch ( p<0.001 ) , and dinner ( p<0.05 ) .
it is because of this effect on postprandial glucose that short - acting glp-1ras may be considered as prandial - acting glp-1ras . in contrast
long - acting glp-1ras ( liraglutide , dulaglutide , exenatide long - acting release [ lar ] ) lead to sustained activation of the glp-1 receptor and have a greater impact on fasting plasma glucose and a lesser impact on gastric emptying owing to tachyphylaxis . to date
there have been five studies directly comparing short- and long - acting glp-1ras , each demonstrating that the latter have a lesser effect on postprandial glucose . in duration-1 ,
exenatide 10 g twice - daily had a greater effect on postprandial glucose than did exenatide lar 2 mg once weekly ( least - squares meanstandard error -6.9 mmol / l vs. -5.30.5
similar results were found in the liraglutide effect and action in diabetes ( lead ) 6 study ; the estimated difference in postprandial glucose reduction was higher with exenatide 10 g twice - daily than with liraglutide 1.8 mg once a day , when measured at breakfast ( 1.33 mmol / l , p<0.0001 ) and at dinner ( 1.01 mmol / l , p<0.0005 ) . in a study comparing lixisenatide 20 g once a day and
liraglutide 1.8 mg once a day in patients with t2 dm insufficiently controlled on metformin , more patients in the lixisenatide group achieved a 2-hour postprandial plasma glucose of < 7.8 mmol / l ( 69% vs. 29% ) . in a study
comparing once - daily lixisenatide with liraglutide in patients on optimised insulin glargine with or without metformin therapy improvement in postprandial glucose levels was significantly greater with lixisenatide 20 g once a day versus liraglutide 1.2 and 1.8 mg once a day ( p<0.0001 for both comparisons ) .
most recently , the assessment of weekly administration of ly2189265 ( dulaglutide ) in diabetes-1 ( award-1 ) study , comparing dulaglutide 0.75 and 1.5 mg once weekly with exenatide 10 g twice - daily , demonstrated significantly greater reductions in the mean of all 2-hour postprandial glucose excursions with exenatide than in the two dulaglutide dose groups ( p<0.001 for both comparisons ) .
recently published data have demonstrated differences in the efficacy of glp-1ras between asian and non - asian populations . whilst this meta - analysis included data from studies with liraglutide and exenatide
, it demonstrated a significant difference in the hba1c - lowering efficacy of glp-1ras in asians ( weighted mean difference -0.32 [ -0.64 ; 0.01 ] ; p=0.044 ) and a trend for more asian patients to achieve target hba1c goals .
nevertheless , data from a population with a significant proportion of korean patients are rare . amongst the studies included in the recent meta - analysis three included only japanese patients whilst one included patients of asian descent , of which 17% were korean .
in addition to the study by gao et al . , four other studies , have reported efficacy data for glp-1ras in korean patients with t2 dm ( table 1 ) .
evaluated the efficacy of exenatide in asian patients with t2 dm inadequately controlled with oral agents , showing improved glycemic control ( mean hba1c reduction : -1.2% vs. -0.4% , p<0.001 ) , and greater weight reduction ( -1.2 kg vs. -0.1 kg ) compared with placebo .
shin et al . reported the results of a retrospective cohort analysis of 52 korean patients ; 6 months ' therapy with exenatide had significantly reduced hba1c ( 8.5% to 6.7% , p<0.001 ) and body weight ( 82.3 to 78.6 kg , p<0.001 ) ; no serious adverse events were reported . of note
the patients were young ( mean age 45.1 years ) and obese ( mean bmi 30.0 kg / m ) .
2011 included chinese , indian , and korean ( 167/929 , 18% ) patients ; it demonstrated liraglutide to have a similar efficacy profile in asian patients to that seen in caucasian , african american and hispanic populations in other global liraglutide phase iii trials . in the global harmony-7 study , five of the 162 study sites were in korea .
patients who received liraglutide once a day had greater reductions in hba1c than did those who received albiglutide once weekly . of note
the patients were older ( mean age 55 years ) , had a long duration of diabetes ( mean 8.4 years ) and were obese ( mean bmi > 30.0 kg / m ) .
lixisenatide , 20 g once a day , as add - on therapy in patients with t2 dm insufficiently controlled on basal insulin ( with or without sulfonylurea ) significantly improved hba1c vs. placebo and enabled more patients to achieve hba1c target ( < 7% and < 6.5% ) .
although there was a trend towards weight loss for lixisenatide ( p=0.0857 ) in this study , the observed weight losses were small ( -0.38 kg and + 0.06 kg in the lixisenatide and placebo groups , respectively ) possibly due to the low mean baseline bmi ( 25 kg / m ) and body weight ( 66 kg ) .
fear of hypoglycemia is a major barrier to optimizing glycemic control in patients with t2 dm . hypoglycemic events increase the direct and indirect economic burden of diabetes and also have a negative impact on a patient 's quality of life and confidence in diabetes self - management ability .
preventing hypoglycemic events and the early detection of patients at high risk for hypoglycemia are important aspects of clinical care . in the recent meta - analysis by kim et al .
, there was no significant difference in the rr of hypoglycemia with glp-1ras between asian and non - asian populations ( 2.8 vs. 1.5 , respectively ; p=0.164 ) . in the study by gao et al .
the incidence of symptomatic hypoglycemia was higher in exenatide - treated patients than those who received placebo ( 36% and 9% , respectively ; p<0.001 ) .
hypoglycemia rates ( events / patient - year ) for patients taking exenatide with metformin alone were 1.8 and this increased to 4.7 amongst those taking metformin plus a sulfonylurea .
there were no reported incidences of hypoglycemia in the retrospective cohort analysis 52 korean patients after 6 months ' therapy with exenatide .
, only two subjects in the glimepiride group reported major hypoglycemia while there were none in the liraglutide groups . in the getgoal - l - asia study 42.9% of lixisenatide - treated patients and
23.6% of those receiving placebo reported symptomatic hypoglycemia ; none of these events was severe . when patients also receiving a sulfonylurea
were excluded the incidence of hypoglycemia in the lixisenatide group was comparable to that in the placebo group ( 32.6% vs. 28.3% ) . the most frequently reported adverse events with glp-1ras are gi related and include nausea , vomiting , and diarrhea .
for example , a study of lixisenatide versus liraglutide found that the incidences of nausea ( 23% vs. 22% ) and vomiting ( 10% vs. 7% ) were similar in both groups , whereas diarrhea was more common in the liraglutide group ( 16% vs. 3% ) . a recent meta - analysis sought to interrogate the data from randomised controlled trials with glp-1ras ( liraglutide and exenatide ) and assess the impact of different doses on gi adverse events .
pairwise random - effects meta - analyses and mixed treatment comparisons found that , relative to placebo , exenatide 10 mg twice - daily is significantly more likely to cause nausea and vomiting whilst liraglutide 1.2 mg once a day is significantly more likely to cause diarrhea .
however the meta - analysis included only liraglutide and exenatide and did not provide specific data on asian patients . as with efficacy , there is limited data on the gi tolerability of glp-1ras in asian populations and even more so in korean patients .
the recent meta - analysis found that the rr of nausea ( 13.8 vs. 3.3 , p=0.100 ) and vomiting ( 21.0 vs. 5.2 , p=0.205 ) were numerically higher in asians than in non - asians but were not statistically significantly different .
conversely , the rr of diarrhea was numerically , but not statistically , higher in non - asians ( 1.7 vs. 2.6 , p=0.275 ) . in a korean retrospective cohort analysis of patients who used exenatide , 23/143 ( 16.1% ) had nausea and 17 ( 11.9% ) suffered from vomiting .
only eight patients stopped taking exenatide due to anorexia or vomiting , all had above target hba1c levels and were receiving two oral hypoglycemic agents but not insulin in the 3 months prior to study inclusion .
reported a similar safety profile with the use of exenatide in asians as in non - asian patients ; nausea , which was generally mild - to - moderate , as the most common adverse event with exenatide ( 25% vs. 1% with placebo ) .
a total of 64 subjects ( 7% ) withdrew from the study by yang et al .
adverse gi events , predominantly diarrhea , nausea , and vomiting led to the majority of these withdrawals . amongst those gi adverse events most occurred within the first 4 weeks , were transient and decreased over time . in the harmony-7 study patients in the albiglutide group had more injection - site reactions and fewer gi adverse events than did those in the liraglutide group .
in the getgoal - l - asia study 61% of patients in the lixisenatide group reported having had a gi adverse event versus 14.6% in the placebo group ; nausea occurred most frequently ( 39.6% of patients ) , followed by vomiting ( 18.9% ) and diarrhea ( 6.5% ) .
in this exclusively asian population , the frequency of nausea was slightly higher than had been observed in global populations ( 39.6% vs. 22% to 25% ) .
potential explanations for the higher incidence of gi adverse events amongst asian patients include higher drug exposure due to the lower bmi of the participants ( vs. non - asian studies ) .
gi adverse events are generally transient ; they tend to be higher during the first 2 weeks of treatment and often decrease with ongoing use . nevertheless , when such events are experienced in the early stages of treatment they have the potential to impact patient compliance . to help with this
, patients should be informed of the potential for these effects , including their likely duration , before treatment commences .
fear of hypoglycemia is a major barrier to optimizing glycemic control in patients with t2 dm . hypoglycemic events increase the direct and indirect economic burden of diabetes and also have a negative impact on a patient 's quality of life and confidence in diabetes self - management ability . preventing hypoglycemic events and the early detection of patients at high risk for hypoglycemia are important aspects of clinical care . in the recent meta - analysis by kim et al .
, there was no significant difference in the rr of hypoglycemia with glp-1ras between asian and non - asian populations ( 2.8 vs. 1.5 , respectively ; p=0.164 ) . in the study by gao et al .
the incidence of symptomatic hypoglycemia was higher in exenatide - treated patients than those who received placebo ( 36% and 9% , respectively ; p<0.001 ) .
hypoglycemia rates ( events / patient - year ) for patients taking exenatide with metformin alone were 1.8 and this increased to 4.7 amongst those taking metformin plus a sulfonylurea .
there were no reported incidences of hypoglycemia in the retrospective cohort analysis 52 korean patients after 6 months ' therapy with exenatide .
, only two subjects in the glimepiride group reported major hypoglycemia while there were none in the liraglutide groups . in the getgoal - l - asia study 42.9% of lixisenatide - treated patients and 23.6% of those receiving placebo reported symptomatic hypoglycemia ; none of these events was severe . when patients also receiving a sulfonylurea
were excluded the incidence of hypoglycemia in the lixisenatide group was comparable to that in the placebo group ( 32.6% vs. 28.3% ) .
the most frequently reported adverse events with glp-1ras are gi related and include nausea , vomiting , and diarrhea .
for example , a study of lixisenatide versus liraglutide found that the incidences of nausea ( 23% vs. 22% ) and vomiting ( 10% vs. 7% ) were similar in both groups , whereas diarrhea was more common in the liraglutide group ( 16% vs. 3% ) . a recent meta - analysis sought to interrogate the data from randomised controlled trials with glp-1ras ( liraglutide and exenatide ) and assess the impact of different doses on gi adverse events .
pairwise random - effects meta - analyses and mixed treatment comparisons found that , relative to placebo , exenatide 10 mg twice - daily is significantly more likely to cause nausea and vomiting whilst liraglutide 1.2 mg once a day is significantly more likely to cause diarrhea .
however the meta - analysis included only liraglutide and exenatide and did not provide specific data on asian patients . as with efficacy , there is limited data on the gi tolerability of glp-1ras in asian populations and even more so in korean patients .
the recent meta - analysis found that the rr of nausea ( 13.8 vs. 3.3 , p=0.100 ) and vomiting ( 21.0 vs. 5.2 , p=0.205 ) were numerically higher in asians than in non - asians but were not statistically significantly different .
conversely , the rr of diarrhea was numerically , but not statistically , higher in non - asians ( 1.7 vs. 2.6 , p=0.275 ) . in a korean retrospective cohort analysis of patients who used exenatide , 23/143 ( 16.1% )
only eight patients stopped taking exenatide due to anorexia or vomiting , all had above target hba1c levels and were receiving two oral hypoglycemic agents but not insulin in the 3 months prior to study inclusion .
reported a similar safety profile with the use of exenatide in asians as in non - asian patients ; nausea , which was generally mild - to - moderate , as the most common adverse event with exenatide ( 25% vs. 1% with placebo ) .
a total of 64 subjects ( 7% ) withdrew from the study by yang et al .
adverse gi events , predominantly diarrhea , nausea , and vomiting led to the majority of these withdrawals . amongst those gi adverse events most occurred within the first 4 weeks , were transient and decreased over time . in the harmony-7 study patients in the albiglutide group had more injection - site reactions and fewer gi adverse events than did those in the liraglutide group . in the getgoal - l - asia study 61% of patients in the lixisenatide group
reported having had a gi adverse event versus 14.6% in the placebo group ; nausea occurred most frequently ( 39.6% of patients ) , followed by vomiting ( 18.9% ) and diarrhea ( 6.5% ) . in this exclusively asian population ,
the frequency of nausea was slightly higher than had been observed in global populations ( 39.6% vs. 22% to 25% ) .
potential explanations for the higher incidence of gi adverse events amongst asian patients include higher drug exposure due to the lower bmi of the participants ( vs. non - asian studies ) .
gi adverse events are generally transient ; they tend to be higher during the first 2 weeks of treatment and often decrease with ongoing use . nevertheless , when such events are experienced in the early stages of treatment they have the potential to impact patient compliance .
to help with this , patients should be informed of the potential for these effects , including their likely duration , before treatment commences .
the available data , in global and asian populations , support a number of benefits with the use of glp-1ras .
these include a reduction hba1c levels , weight maintenance or loss , and a relatively low risk of hypoglycemia .
these benefits are offset against the propensity for gi adverse effects early in the course of treatment and a lack of long - term outcomes data .
population statistics suggest that increasing age and obesity are key factors contributing to t2 dm in korea . in a cohort study involving 34,999 korean men and women aged 30 to 59 years who were free of diabetes at baseline , metabolic health status , obesity , and weight change
were all shown to be independently associated with increased incidence of t2 dm over 5 years of follow - up .
whilst the overall prevalence of diabetes in adults 30 years and older is 12.4% , it increases with age such that it affects 23.2% of koreans aged 60 to 69 years and 25.9% of those aged 70 years or more .
half of all koreans with t2 dm are obese ( when defined as a bmi 25.0 kg / m and a waist circumference > 90 cm in men and > 85 cm in women ) with data showing the highest prevalence of obesity ( 63.6% ) and abdominal obesity ( 57.6% ) amongst those aged 40 to 49 years . given these statistics , treatment approaches that contribute to weight management such as the glp-1ras are becoming more relevant in the current management of t2 dm in korea .
the inclusion of a prandial glp-1ra into the treatment regimen of overweight patients with high hba1c due to postprandial hyperglycemia is reasonable .
other clinical factors that may aid in treatment choice include uncontrolled hba1c due to high postprandial ( rather than fasting ) blood glucose levels , need to control weight particularly amongst patients who already have a basal insulin as part of their regimen , patients at increased risk of hypoglycemia and patients with or at high risk of cv comorbidities , such as hypertension and fatty liver .
the different pharmacodynamic features of glp-1ras may help guide the decision to administer a glp-1ra with basal insulin ; using short - acting lixisenatide or exenatide twice - daily in patients with prandial or postprandial hyperglycemia after near normalisation of fasting blood glucose and long - acting exenatide lar , liraglutide , albiglutide or dulaglutide if the main concern is elevated fasting blood glucose levels .
whilst korean - specific data are limited , much progress has been made in understanding the full scope of their utility in the management of t2 dm .
gaps in the literature that would warrant further research in the korean population include the impact of glp-1ra treatment on the recovery of insulin secretion , their effects on -cell function and insulin sensitivity , whether they modulate improvements in endothelial dysfunction , their impact on lipid metabolism and any associations between their glucose lowering efficacy and gi side effects .
in concluding , we ask the question how does the above data help inform the optimal management of t2 dm in korea ? the global data , in general , demonstrate that glp-1ras can contribute to patient management , providing beneficial reduction in hba1c , without weight gain or hypoglycemia .
more data are needed to examine efficacy , safety , and optimal dosing of glp-1ras in korean patients with t2 dm . however , based on the data currently available , their use is appropriate in several patient groups , including patients whose hba1c is uncontrolled , especially if this is due to postprandial glucose excursions and patients who are overweight or obese due to dietary problems ( e.g. , appetite control ) .
whether or not the potential for gi adverse events is increased in asian patients , the possibility that it might occur should be explained to patients at treatment initiation to help maintain patient compliance . | glucagon - like peptide-1 receptor agonists ( glp-1ras ) reduce glycosylated hemoglobin ( hba1c , 0.5% to 1.0% ) , and are associated with moderate weight loss and a relatively low risk of hypoglycemia .
there are differences between asian and non - asian populations .
we reviewed available data on glp-1ras , focusing on korean patients , to better understand their risk / benefit profile and help inform local clinical practice .
control of postprandial hyperglycemia is important in asians in whom the prevalence of post - challenge hyperglycemia is higher ( vs. non - asians ) .
the weight lowering effects of glp-1ras are becoming more salient as the prevalence of overweight and obesity among korean patients increases .
the higher rate of gastrointestinal adverse events amongst asian patients in clinical trials may be caused by higher drug exposure due to the lower body mass index of the participants ( vs. non - asian studies ) .
data on the durability of weight loss , clinically important health outcomes , safety and optimal dosing in korean patients are lacking .
use of glp-1ras is appropriate in several patient groups , including patients whose hba1c is uncontrolled , especially if this is due to postprandial glucose excursions and patients who are overweight or obese due to dietary problems ( e.g. , appetite control ) .
the potential for gastrointestinal adverse events should be explained to patients at treatment initiation to facilitate the promotion of better compliance . |
a recent survey showed that oral cancer , which accounts for 2.8% of all malignant tumors in europe , is the most common cause of maxillofacial defects .
moreover , the incidence of oral cancer in china is relatively high compared with the rest of the world .
severe deficiencies in appearance , pronunciation and swallowing occur when maxillofacial defects cause oral nasal transport .
, prosthodontists have tried to help these patients by separating the oral and the nasal cavity using obturator prostheses to improve deglutition , articulation , pronunciation and facial appearance , and in some cases to support the orbital contents to prevent enophthalmos and diplopia .
the absence of teeth , and the size and configuration of the maxillary defect , may influence the masticatory function of patients wearing an obturator prosthesis and may lead to a poor rehabilitation outcome .
moreover , the adequate retention of these prostheses is difficult to achieve , especially for large - sized defects . for many years
, prosthodontists have tried different ways to achieve better retention for cases who have undergone extensive maxillectomy .
schmidt et al . used zygomatic and standard endosseous implants to retain prostheses in near - total and total maxillectomy patients .
reported the case of a partial maxillectomy patient with an obturator that was supported by five mini - dental implants .
the author concluded that mini - dental implants can be used when there is a lack of adequate bone tissue for conventional implant placement in the region of the resection .
however , for some patients , these implants are not acceptable due to their general health , personal inclination or economic reasons . finding
payne and welton designed a latex rubber balloon attached to a denture that was inflated with air to fill the surgical defect , but its clinical usage has not been reported . in the last decade ,
this article is the first to describe the restoration of maxillary defects with inflatable obturator prostheses based on silicone , which also represented an alternative technique for maxillofacial rehabilitation .
a 20-year - old male patient diagnosed with fibrous dysplasia was referred to the department of prosthodontics , peking university school and hospital of stomatology .
his chief complaints were deficiencies in speaking and swallowing after extensive resection of the maxilla 3 months previously ( figure 1 ) .
the patient had undergone bilateral sub - total maxillectomy , which resected most of the maxillary structures ; only a portion of the maxillary tuberosity remained . no surgical reconstruction or any graft for the defect had been applied .
clinical investigation showed an extensive defect of the palatal area and crest ridge , which was classified as a class iv defect according to aramany 's classification .
transport between the oral cavity and the nasal cavity was observed , and the surgical site showed tolerable healing with the formation of a scab and no sign of recrudescence ( figure 2 ) . as the maxillectomy defect included almost the whole palatal area , the amount of undercut area available to provide retention for a prosthesis was very limited , making adequate retention by traditional methods extremely difficult .
however , the use of endosseous implants in pathological bone has not been thoroughly investigated and some commentaries suggest avoiding it for cases of fibrous dysplasia . furthermore , the patient did not want to accept implant therapy for economic reasons .
sectional maxillary prosthesis , including a complete denture and an inflatable obturator , was used to restore the extensive maxillary defect .
the definitive maxillary denture ( luciton 199 denture base material ; dentsply , york , pennsylvania , usa ) was made according to the mandibular dentition with proper occlusion contact .
on the intaglio side of the denture , an appropriate undercut that imitated an edentulous ridge contour was made so that the complete denture could be retained afterwards ( figure 3 ) .
then , impression material ( rapid putty soft ; coltene / whaledent , altsttten , switzerland ) was placed into the intaglio surface of the denture .
this material is easy to control and it is not likely to enter deep undercut areas under appropriate pressure .
when the impression was made , we ensured that the maxillary denture had a good occlusal contact with the mandibular dentition .
the jaw position was guided to the appropriate occlusal vertical dimensions by measuring the distance of the points marked on the patient 's face at rest , and was maintained in this position until the impression material had set ( figures 3 and 4 ) .
the impression was poured with dental stone ( die - stone type iii ; heraeus , south bend , indiana , usa ) to form the working cast , and a one - way air valve ( botou shengwei factory , botou city , hebei province , china ) was fixed on the palatal surface of the obturator when the obturator prosthesis was made .
odontosil ( dreves , unna , germany ) silicone material was used to process the hollow silicone obturator prosthesis in the laboratory ( figure 5 ) .
the obturator prosthesis and the definitive denture were then trialed by the patient , and an adequate vertical dimension and retention of the prosthesis was demonstrated ( figure 6 ) .
when the patient tried the sectional maxillary prosthesis by himself , the compressed obturator was firstly seated in the defect , and the patient could inflate it using a portable air pump ( figure 7 ) to connect the installed air - valve on the palatal surface of the obturator .
when inflated , the obturator was well - fitted with extending evenly into the undercuts .
the denture was then seated on the inflated obturator by engaging the suitable undercuts on the palatal surface of the obturator formed during the manufacture of the denture . when the patient wishes to remove the obturator ,
the compressed air pump is connected directly with the valve to deflate it after the denture is removed .
a 68-year - old male complained of deficiencies in speaking , swallowing and mastication after bilateral total maxillectomy 3 months previously ( figure 8) .
a hollow inflatable silicone obturator prosthesis combined with a complete denture was prescribed for the patient according to the procedure described above ( figures 9 and 10 ) . when inflated , the obturator was well - fitted and provided adequate support and retention for the complete denture .
a 20-year - old male patient diagnosed with fibrous dysplasia was referred to the department of prosthodontics , peking university school and hospital of stomatology .
his chief complaints were deficiencies in speaking and swallowing after extensive resection of the maxilla 3 months previously ( figure 1 ) .
the patient had undergone bilateral sub - total maxillectomy , which resected most of the maxillary structures ; only a portion of the maxillary tuberosity remained . no surgical reconstruction or any graft for the defect had been applied .
clinical investigation showed an extensive defect of the palatal area and crest ridge , which was classified as a class iv defect according to aramany 's classification .
transport between the oral cavity and the nasal cavity was observed , and the surgical site showed tolerable healing with the formation of a scab and no sign of recrudescence ( figure 2 ) . as the maxillectomy defect included almost the whole palatal area , the amount of undercut area available to provide retention for a prosthesis was very limited , making adequate retention by traditional methods extremely difficult .
however , the use of endosseous implants in pathological bone has not been thoroughly investigated and some commentaries suggest avoiding it for cases of fibrous dysplasia . furthermore , the patient did not want to accept implant therapy for economic reasons .
sectional maxillary prosthesis , including a complete denture and an inflatable obturator , was used to restore the extensive maxillary defect .
the definitive maxillary denture ( luciton 199 denture base material ; dentsply , york , pennsylvania , usa ) was made according to the mandibular dentition with proper occlusion contact .
on the intaglio side of the denture , an appropriate undercut that imitated an edentulous ridge contour was made so that the complete denture could be retained afterwards ( figure 3 ) .
then , impression material ( rapid putty soft ; coltene / whaledent , altsttten , switzerland ) was placed into the intaglio surface of the denture .
this material is easy to control and it is not likely to enter deep undercut areas under appropriate pressure .
when the impression was made , we ensured that the maxillary denture had a good occlusal contact with the mandibular dentition .
the jaw position was guided to the appropriate occlusal vertical dimensions by measuring the distance of the points marked on the patient 's face at rest , and was maintained in this position until the impression material had set ( figures 3 and 4 ) .
the impression was poured with dental stone ( die - stone type iii ; heraeus , south bend , indiana , usa ) to form the working cast , and a one - way air valve ( botou shengwei factory , botou city , hebei province , china ) was fixed on the palatal surface of the obturator when the obturator prosthesis was made .
odontosil ( dreves , unna , germany ) silicone material was used to process the hollow silicone obturator prosthesis in the laboratory ( figure 5 ) .
the obturator prosthesis and the definitive denture were then trialed by the patient , and an adequate vertical dimension and retention of the prosthesis was demonstrated ( figure 6 ) .
when the patient tried the sectional maxillary prosthesis by himself , the compressed obturator was firstly seated in the defect , and the patient could inflate it using a portable air pump ( figure 7 ) to connect the installed air - valve on the palatal surface of the obturator .
when inflated , the obturator was well - fitted with extending evenly into the undercuts .
the denture was then seated on the inflated obturator by engaging the suitable undercuts on the palatal surface of the obturator formed during the manufacture of the denture . when the patient wishes to remove the obturator ,
the compressed air pump is connected directly with the valve to deflate it after the denture is removed .
a 68-year - old male complained of deficiencies in speaking , swallowing and mastication after bilateral total maxillectomy 3 months previously ( figure 8) .
a hollow inflatable silicone obturator prosthesis combined with a complete denture was prescribed for the patient according to the procedure described above ( figures 9 and 10 ) . when inflated , the obturator was well - fitted and provided adequate support and retention for the complete denture .
in case 1 , a large palatal defect was evident after surgery for fibrous dysplasia .
oral nasal transport and the resultant functional deficiencies greatly affected the patient 's quality of life .
however , the lack of structures to provide retention made it extremely difficult to construct a conventional prosthesis for him .
a recent report of the use of endosseous implants has been described in a patient with fibrous dysplasia ; however , osseointegration in the pathological bone at a microscopic level has not yet been proven .
the inflatable hollow obturator prosthesis may be a suitable alternative in patients such as this one who did not want to undergo oral implant treatment for medical and/or economic reasons .
when dealing with extensive defects , the prosthesis requires greater extension into the defect and is therefore heavier . as described in the literature , the continuous stress placed on the remaining tissues by a large , heavy obturator jeopardizes their health , compromises the function of the prosthesis and can cause discomfort to the patient .
the use of a hollow maxillary obturator may reduce the weight of the prosthesis by up to 33% , depending on the size of the maxillary defect .
shaker used medical - grade silicone to fabricate the obturator part of the prosthesis with mushroom - like extensions .
it was retained by this resilient material which engaged the soft tissue undercuts within the defect . however , the extension into the undercut by this obturator has to be limited , or else its insertion may cause discomfort and bleeding .
our inflatable obturator can be easily placed in undercuts when compressed and extends further when inflated , thereby improving retention without causing obvious discomfort .
compared with the semi - customized balloon - shaped inflatable latex obturator designed by payne and welton , this new hollow and inflatable obturator is completely customized according to the configuration and size of defects and has been proven clinically . with this advantage , an obturator with the same shape as the defect can extend into the undercut area and engage the undercut more evenly .
the inflatable hollow obturator is lightweight , can be retained well and creates an adequate oral
deglutition , pronunciation and mastication can be improved and the psychological impact of extensive maxillectomies on patients can therefore be minimized . when faced with patients with microstomia after surgery , an inflatable obturator may also be an alternative option as the obturator can be seated into the oral cavity while deflated and compressed . in cases of extensive maxillectomy in which implants can not be used , as in the two cases in this report
, the inflatable obturator makes prosthetic treatment more acceptable as good retention is achieved by engaging more undercut areas after inflation . in cases of partial maxillectomy , the use of an inflatable obturator may minimize deleterious forces on the remaining structures as further support is gained from surrounding tissues .
silicone - based inflatable obturators offer a simple technique for the reconstruction of maxillary defects , can be used by most prosthodontists and offer a treatment alternative for patients who may not be able to receive conventional implants .
however , the extent to which the inflatable obturator improves pronunciation , deglutition and mastication compared with traditional prostheses , and the long - term serviceability of this prosthesis , requires further investigation . | the presence of a large palatal or maxillary defect after partial or total maxillectomy for tumor , trauma or congenital deformation poses a challenge to prosthodontists , particularly when the use of an implant can not be considered .
this case report described the use of an air valve in a hollow silicone obturator to manufacture an inflatable obturator that could be extended further into undercut area to retain itself .
the inflatable obturator exhibited adequate retention , stability and border sealing , thereby improving the masticatory , pronunciation and swallowing functions of patients .
it may be a suitable alternative treatment option to an implant - retained obturator . |
administrative qof data describing performance of family practices under the program were analyzed for the years 20042008 ( 3 ) .
data for each family practice included the number of registered diabetic subjects , the proportion of eligible subjects who achieved the targets , and the proportion of diabetic subjects excluded from evaluation of each target as exceptions .
exceptions arise because practices are permitted to identify some individuals as ineligible for evaluation if the target is regarded as clinically inappropriate ( 4 ) .
the targets included in this report were the percent of diabetic subjects with the last a1c 7.5% , with last blood pressure 145/85 mmhg , or with the last measured total cholesterol 5 mmol / l .
we estimated the total number of registered diabetic subjects , the total number excluded as ineligible , and the number ( and percent ) of subjects who achieved the target after allowing for exclusions .
the linear association between outcomes and year was estimated using robust standard errors to allow for repeated measures .
data were analyzed for family practices in england that remained independent and had more than 750 registered patients or more than 500 patients per doctor , in the study year .
data were analyzed for 8,423 practices in 20042005 , 8,264 in 20052006 , 8,192 in 20062007 , and 8,255 in 20072008 , representing 98% of all practices .
the median number of registered diabetic subjects per practice increased from 181 ( interquartile range [ iqr ] 107284 ) in 20042005 to 218 ( iqr 130342 ) in 20072008 ( table 1 ) .
the estimated resident population of england is 51 million ( 5 ) , giving an overall prevalence of 4% .
the median practice - specific proportion of diabetic subjects declared ineligible for the a1c target was 9.4% in 20042005 but declined to 8.7% in 20072008 ( p < 0.001 ) .
the median proportion excluded for the blood pressure target was 6.3% in 20042005 declining to 5.7% in 20072008 ( p < 0.001 ) and for cholesterol was 9.0% in 20042005 declining to 8.4% in 20072008 ( p < 0.001 ) .
the median practice - specific proportion achieving the a1c target of 7.5% increased from 59.1% in 20042005 to 66.7% in 20072008 ( table 1 ) .
the proportion achieving the blood pressure target of 145/85 mmhg increased from 70.9% in 20042005 to 80.2% in 20072008 .
the proportion achieving the cholesterol target of 5 mmol / l increased from 72.6% in 20042005 to 83.6% in 20072008 . the estimated annual increase in percent of diabetes subjects achieving targets was 3.03% ( 95% ci 2.953.10 ; p
< 0.001 ) for the a1c target , 3.26% ( 3.183.34 ; p < 0.001 ) for the blood pressure target , and 3.99% ( 3.924.07 ; p < 0.001 ) for the cholesterol target .
the total number of diabetic subjects in england achieving the a1c target , after allowing for exclusions from assessment , increased by 341,173 between 20042005 and 20072008 , representing 16% of diabetic subjects registered in 20072008 . over the same period ,
the number achieving the blood pressure target increased by 453,785 ( 22% of 20072008 registrations ) , and the number achieving the cholesterol target increased by 452,347 ( 22% of 20072008 registrations ) .
practices were classified as low performing if they achieved less than the 25th centile for the a1c target across all practices in 20062007 .
there were 57% of practices classified as low performing in 20042005 . among the 10 english regions , 69.9% of practices were low performing in london compared with
the overall proportion of low - performing practices declined to 47.4% in 20052006 , 25.0% in 20062007 , and 26.0% in 20072008 . in 20072008 , the proportion of low - performing practices ranged from 37.5% in london to 11.6% in the north east .
in the u.k . , the care of subjects with type 2 diabetes is increasingly undertaken outside of specialist clinics by family physicians and practice nurses in primary care . this has led to concerns that some patients may experience poor - quality care ( 6 ) . the new national contract for family practices introduced in 2004 appears to have achieved favorable results in its initial year ( 4,7 ) and may have contributed to reducing socioeconomic inequalities in care ( 8,9 ) .
lower - performing practices have shown the greatest improvements , and regional variations in care have reduced .
there has been a substantial increase in the proportion of all diabetic subjects achieving intermediate outcome targets . in our previous report ( 7 ) , we analyzed clinical data from individual patient records for 26 practices during the period of 20002003 that gave results consistent with administrative data from the qof .
two other reports , including data from the first or second years of qof , suggest that qof data are consistent with audits of individual patient records ( 10,11 ) . in a single group study , without any control practices ,
it is not possible to conclude that pay - for - performance incentives caused the observed changes .
there was already evidence of improving quality of care before the introduction of qof ( 7,12 ) .
the qof targets are designed for audit rather than best practice , and practitioners may be utilizing clinical practice guidelines that recommend more stringent targets .
recommendations for a widespread use of statins were introduced in many countries at the start of this period , leading to improvements even in the absence of pay - for - performance .
the greater improvement of low - performing practices may , in part , be accounted for by a ceiling effect , which restricted the potential improvement in high - performing practices .
we caution that it is not clear that proposed benefits from pay - for - performance would be observed if this model is adopted in systems with different organizational arrangements and models of practitioner remuneration . | objective to evaluate diabetes outcomes under a national pay - for - performance
program.research design and methods data were analyzed for 98% of all english family practices .
for each practice , the proportion of diabetic subjects with a1c 7.5% , blood pressure 145/85 mmhg , and cholesterol 5 mmol / l was determined .
practices achieving less than the 25th centile for the a1c target for 20062007 were classified as low performing.resultsthe proportion achieving the a1c target at the median practice increased from 59.1% ( interquartile range [ iqr ] 51.765.9 ) in 20042005 to 66.7% ( iqr 60.672.7 ) in 20072008 , blood pressure from 70.9% in 20042005 to 80.2% in 20072008 , and cholesterol from 72.6% in 20042005 to 83.6% in 20072008 . in 20042005 ,
57% of practices were low performing ( range by region 42.469.9 ) . in 20072008 ,
26% of practices were low performing ( range 11.637.5).conclusions introduction of pay - for - performance may be one factor contributing to increasing achievement of targets and reducing problems of low performance . |
polycystic ovary syndrome ( pcos ) is a heterogeneous syndrome , involving a growing number of women in reproductive age , diagnosed on the basis of three different factors : oligo- or anovulation , clinical / biochemical hyperandrogenism , and polycystic ovary , with the presence on ultrasound of 12 follicles in each ovary measuring 2 9 mm in diameter and/or increased ovarian volume ( > 10 ml ) [ 1 , 2 ] . pcos affected patients that had menstrual irregularity , followed in many cases by infertility [ 3 , 4 ] and mood disorders , such as anxiety and depression .
though pcos pathogenesis still remains unclear , insulin resistance ( ir ) and the consequential hyperinsulinemia are considered primary triggers , both in obese and in lean women with this syndrome [ 68 ] .
indeed , hyperinsulinemia induced by ir occurs in roughly 80% of pcos obese women , as in 3040% of pcos lean women , suggesting that ir is independent but also exacerbated by obesity ; this latter considered an enhancing factor that positively correlates with the multifactorial syndrome [ 1012 ] .
it was hypothesized that in patients with pcos altered insulin signaling may generate the ir which in turn causes abnormal ovarian steroidogenesis [ 13 , 14 ] , so that several insulin - sensitizing compounds have been proposed as possibly safe and efficacious long - term treatment of pcos . among these drugs ,
metformin resulted to be the most used and studied drug , even if this molecule is predominantly associated with gastrointestinal discomforts consisting of bloating , nausea , and diarrhea [ 14 , 16 ] .
interesting and promising results have been obtained focusing on two inositol stereoisomers , such as myo - inositol ( mi ) and d - chiro - inositol ( dci ) , acting like insulin mediators [ 1719 ] .
as insulin second messengers , both these molecules are involved in increasing insulin sensitivity of different tissues to improve metabolic and ovulatory functions . in particular , at low dosage , dci restores normal insulin sensitivity in the typical insulin target tissues , reducing the circulating insulin and androgens and inducing an enhancement in ovulation frequency . on the contrary
, mi exerts its beneficial effects mainly at the ovary level , where it is highly concentrated , both enhancing insulin pattern and also directly acting on a number of ovarian functions , including steroidogenesis .
some authors postulated that pcos affected women , with ir , presenting in the ovary a misbalance in mi / dci ratio , resulting in dci overproduction and in turn in a deficiency in mi , which would explain the excessive androgen biosynthesis .
other authors , instead , proposed that the increased androgen levels in pcos patients might be linked to a decreased mi / dci . in a recent study ,
facchinetti et al . discovered that the physiological mi / dci ratio was 40 : 1 and , based on this finding , as well as on the specific behavior of both stereoisomers , we investigated the effects of a therapy that combines mi plus dci in the ratio of 40 : 1 , versus placebo , in order to improve some clinical outcomes in pcos young overweight women .
this randomized controlled trial enrolled 46 obese women with bmi > 30 who were affected by pcos according to rotterdam criteria [ 1 , 2 ] .
all the women were enrolled at the department of clinical and experimental medicine , university of pisa .
patients with diabetes , smokers , and alcohol users were ruled out from the study .
after all patients subscribed their written informed consent to be involved into the study , they were randomly assigned to two groups , a and b. at baseline , patients in groups a and b did not differ significantly . in group
a , 21 women received mi plus dci combined treatment at the ratio of 40 : 1 ( the physiologic ratio of the two isomers in the body ) in soft gel capsule containing 550 mg of mi , 13.8 mg of dci , and 200 g of folic acid ( inofolic combi , lo.li.pharma ) twice a day .
group b , with 25 women , received the same amount of folic acid ( 200 g ) as placebo twice a day .
all patients were evaluated for fsh , lh , 17-beta - estradiol ( e ) , sex hormone binding globulin ( shbg ) , androstenedione , free testosterone , and dehydroepiandrosterone sulphate ( dheas ) levels at the baseline and after the six months of therapy with mi plus dci association or with placebo .
fsh and lh serum levels were detected by immune - enzymatic assay ( access immunoassay system , hlh , hfsh , beckman coulter , brea , ca , usa ) .
estradiol levels were measured by competitive immunoassay ( access immunoassay system , estradiol , beckman coulter , brea , ca , usa ) .
shbg levels were detected by immunoassay ( access immunoassay system , shbg , beckman coulter , brea , ca , usa ) .
serum levels of androstenedione were measured by conventional immune - enzymatic assay ( access immunoassay system , androstenedione , beckman coulter , brea , ca , usa ) .
free testosterone serum levels were measured by immune - enzymatic assay ( access immunoassay system , free testosterone , beckman coulter , brea , ca , usa ) .
dheas was measured by conventional immunoassay ( access immunoassay system , dheas , beckman coulter , brea , ca , usa ) .
insulin resistance was measured by means of homeostasis model assessment ( homa ) in addition to determining fasting glucose and insulin with the same timeline and modalities .
blood samples , taken at the baseline and after the six - month treatment period under similar conditions , were separated by centrifugation at 2000 g for 15 minutes at 4c , and the serum obtained was stored at 20c within one hour of collection . before the analysis , all the serum samples were thawed and entirely mixed .
data reported indicate mean values standard deviation ( sd ) . paired t - test
was used to identify the differences between variables at baseline and after six months of treatment with mi plus dci or with placebo , respectively .
the goal of this study was to investigate if the therapy combining mi and dci in the ratio of 40 : 1 could improve the endocrine profile and the insulin resistance of obese women with a pcos diagnosis . to address this issue ,
46 young obese patients affected by this syndrome , whose characteristics are summarized in table 1 , were randomly included in two groups and then treated with mi plus dci at the ratio of 40 : 1 with or placebo for six months .
insulin resistance , evaluated as homa index , fasting insulin , and fasting glucose , and also hormonal parameters were determined at the baseline and after the six - month therapy . as shown in table 2
, we observed that , with respect to the baseline values , only the combined therapy of mi plus dci significantly rebalanced the endocrine and metabolic profiles of these patients , ameliorating their insulin resistance and the ovulatory function , as successfully recorded by ultrasound . as a matter of fact , lh and free testosterone levels decreased after the combined treatment , downregulating the hyperandrogenism , and even homa index and fasting insulin , markers of insulin resistance , resulted to be significantly reduced . on the other hand , e and shbg significantly increased , showing restoring in ovulation capability .
no relevant changes in these sex hormones were reported in group b , treated with placebo , and no significant modifications were observed after the treatment in both groups a and b for what concerns bmi , fsh , androstenedione , dheas , and fasting glucose .
importantly , no relevant side effect was recorded during the combined therapy with mi plus dci .
overall , these results demonstrated the clinical importance of a combined therapy of mi plus dci to correct the pcos metabolic and reproductive aspects and they are largely in agreement with the issues discussed on the two international consensus conferences on mi , dci , and their link with pcos [ 25 , 26 ] .
pcos is a syndrome whose pathogenesis remains still largely unclear , even though several etiological factors are demonstrated to be involved .
compelling evidences claimed the pivotal role of insulin resistance and/or compensatory hyperinsulinemia in this syndrome [ 9 , 2729 ] ; indeed they tightly contribute both directly ( increasing the ovarian production of androgens ) and indirectly ( modulating the hepatic shbg synthesis ) to hyperandrogenism development , one of the main features of those patients affected by pcos [ 30 , 31 ] , especially in case of overweight women .
nevertheless , literature findings consistently demonstrated that a deficiency in the tissue availability and/or usage of mi and/or dci in women diagnosed with pcos could likely concur to the ir typical of this syndrome [ 22 , 23 ] .
the two inositol stereoisomers , mi and dci , acting as insulin - sensitizers , have been demonstrated to positively influence the clinical history of pcos patients , ameliorating their endocrine and metabolic profile both alone and in combination [ 19 , 3337 ] .
dci alone , at low dosage , may restore normal insulin sensitivity in the typical insulin target tissues , inducing an enhancement in ovulation frequency which could be ascribed to the general improved insulin sensitivity and to the reduced circulating insulin and androgens . on the contrary
, mi exerts its beneficial effects mainly at the ovary level , both enhancing insulin pattern and also directly acting on a number of ovarian functions , including steroidogenesis .
the ability of both inositol stereoisomers to regulate glucose metabolism in a different manner ( dci promotes glycogen synthesis , while mi may support glucose cell intake ) is mirrored by their different concentration in the tissues : while dci is highly concentrated in glycogen storage tissues ( liver , muscles , and fat ) , mi is more abundant in those tissues that need a large amount of glucose , such as brain , heart , or ovary . from this knowledge , a combined therapy with mi plus dci in their physiological plasma ratio ( mi / dci 40 : 1 ) seems to be the most appropriated clinical approach to integrate the positive effects exerted by both inositol stereoisomers .
the data reported are encouraging and they offer therapeutic options to the first - line treatments in pcos women with moderate or severe hyperandrogenism and/or menstrual abnormalities , which are represented by metformin as well as by oral contraceptives .
these compounds effectively suppress lh release and the consequent androgen production from the ovary ; also they increase the sex hormone binding protein synthesis , lowering the levels of circulating free androgens .
unfortunately , if the patient aims to restore ovulation in order to conceive , contraceptives are not the clinical strategy to follow .
furthermore , prolonged use of contraceptives may increase homocysteine levels after six months of treatment , as well as the risk of venous thromboembolism . for what concerns metformin , several side gastrointestinal effects ( diarrhoea , nausea , vomiting , and abdominal bloating ) and metabolic complications have been evidenced after a long - term treatment .
for all these reasons , even though more studies on a higher number of patients and with greater statistical significance are needed to confirm these striking posttreatment outcomes , safe combined use of inositol stereoisomers should be largely suitable and it might represent a valid clinical approach in pcos management . | introduction . we evaluated the effects of a therapy that combines myo - inositol ( mi ) and d - chiro - inositol ( dci ) in young overweight women affected by polycystic ovary syndrome ( pcos ) , characterized by oligo- or anovulation and hyperandrogenism , correlated to insulin resistance . methods .
we enrolled 46 patients affected by pcos and , randomly , we assigned them to two groups , a and b , treated , respectively , with the association of mi plus dci , in a 40 : 1 ratio , or with placebo ( folic acid ) for six months .
thus , we analyzed pretreatment and posttreatment fsh , lh , 17-beta - estradiol , sex hormone binding globulin , androstenedione , free testosterone , dehydroepiandrosterone sulphate , homa index , and fasting glucose and insulin .
results .
we recorded a statistically significant reduction of lh , free testosterone , fasting insulin , and homa index only in the group treated with the combined therapy of mi plus dci ; in the same patients , we observed a statistically significant increase of 17-beta - estradiol levels . conclusions .
the combined therapy of mi plus dci is effective in improving endocrine and metabolic parameters in young obese pcos affected women . |
chronic mastitis ( cm ) is a group of diseases characterized by chronic inflammation of the breast , affecting mainly women of reproductive age in their fourth decade [ 13 ] .
cm is histopathologically defined as inflammation of the breast , with the microabscess formation and/or the presence of granulomas .
this disease generally involves the breast unilaterally and may affect every quadrant region except for the subareolar area .
cases mainly present with a breast mass , which may involve the overlying skin or penetrate the underlying pectoralis muscle with nipple retraction , sinus formation , and axillary lymphadenopathy .
other symptoms may include galactorrhea , inflammation , pain , peau d'orange , tumorous indurations , nipple retraction and/or discharge , diffuse heaviness and enlargement , and ulcerations of the skin .
due to this variable clinical presentation and these similarities in symptoms as well as clinical and radiological findings with inflammatory breast cancer , diagnosis is difficult and must be confirmed histopathologically after surgical excision or core biopsy .
diagnosis of cm should be done after exclusion of other causes of infective lesions of the breast including brucellosis , filariasis , actinomycosis , sarcoidosis , histoplasmosis , wegner 's granulomatosis , giant - cell arteritis , duct ectasia , fat necrosis , and breast cancer [ 2 , 4 ] . known etiologies of cm are diverse and include diabetes , lupus erythematosus , mycobacterium tuberculosis , staphylococcus aureus , and candida albicans , as well as several species of corynebacterium and other rare syndromes and infections [ 35 ] .
several other suspected predisposing or related diseases have been mentioned in the literature , including weber - christian disease , sjogren 's syndrome , hyperprolactinemia , igg4 sclerosing disease , immune response to local trauma , and even cat scratch disease [ 610 ] .
however , many cases of cm are idiopathic , presenting difficulties for determining the etiology and optimal treatment .
nearly all published case series on cm find that the average age of women at presentation is in their fourth decade [ 14 , 1114 ] .
studies have suggested risk factors involving parity , breastfeeding habits , contraception use , socioeconomic status , and treatment patterns ; however , all of these stated risks are based on physicians ' observations in small case series reports and have not been studied epidemiologically .
the prevalence of cm is generally reported at less than 1% worldwide among women presenting with breast problems in studied hospitals ; however , reports vary greatly by country and ethnic group . reports from pakistan show prevalence at less than 1% among women undergoing biopsy for breast diseases , and a study of granulomatous mastitis ( gm ) in the united states demonstrated a prevalence of less than 1% among women who underwent biopsy for breast diseases . in the uk , periductal mastitis
a second us - based study of gm found a prevalence of 2.4/100,000 women ; however , the prevalence was 12 times higher among hispanic women .
a study in saudi arabia found idiopathic granulomatous mastitis ( igm ) , one form of chronic mastitis , to be 1.8% of cases presenting with breast diseases ; likewise , a study in turkey found that 6.8% of patients undergoing surgery for benign breast diseases had gm .
initial reports from hospitals in morocco and egypt , as well as our review of chronic mastitis diagnoses at the department of pathology at the national cancer institute of cairo university , indicate that the disease may be less rare than in the developed countries , with prevalence rates estimated between 1% and 10% .
however , due to the lack of access to specialized care , cases captured in tertiary hospitals and cancer centers are likely to be a very low estimate of total prevalence within these countries . given the rarity of cm worldwide , little is known about etiology , risk factors , and treatment .
a thorough literature review of pubmed , scopus , and isi web of knowledge using the search term breast disease or mastitis or chronic mastitis or granulomatous mastitis and covering 1960 through 2011 revealed that almost all published studies are hospital - based case - series reports .
the only exception is one small retrospective case - control study of 18 cases of gm .
therefore , a major gap seems to exist in the literature regarding epidemiological study of chronic mastitis .
the current state of knowledge on cm is inadequate to inform treatment protocols , prevention efforts , or patient education .
no risk factors are statistically shown to be associated with cm , severely limiting the potential prevention efforts .
the lack of information , combined with the relatively high estimated prevalence in north africa , created an optimal setting for the study of a neglected rare disease .
the aim of this study was to identify potential risk factors for cm to inform future clinical research and possible prevention interventions .
based on a previous case - series work , we hypothesized that breastfeeding and reproductive factors would have important associations with diagnosis of cm .
we preformed a retrospective hospital - based case - control study and identified 85 cases of cm and 112 controls from 5 hospitals in morocco and egypt .
the hospitals in morocco included l'hopital ibn rochd , hassan ii university , in casablanca , and l'hopital ibn tofail in marrakech .
the hospitals in egypt were cairo university medical school in cairo , the mansoura university oncology center in mansoura city ( in the east nile delta region ) , and the tanta cancer center in tanta , in the center of the nile delta region .
cases were defined as any female patient with histopathological diagnosis of chronic mastitis ( periductal , lobular , granulomatous , lymphocytic , and duct ectasia with mastitis ) seen at the study hospitals between 2008 and 2011 .
controls were women with histopathologic diagnoses of other forms of benign breast diseases , excluding mastitis and malignancy , and including fibroadenoma , benign phyllodes , and adenosis , diagnosed at the same hospitals during the same period .
cases and controls identified from pathology records of each collaborating hospital were frequency age - matched within 5 years .
pathological records of the study subjects were linked to their medical records , and both were abstracted for relevant information .
any contact information available from medical records was used to contact and obtain consent from patients , as well as to conduct a follow - up interview .
the consent process and interview were generally conducted in tandem by an arabic - speaking interviewer over the telephone . at the mansoura study site in egypt
, interviews were conducted in a face - to - face format at the hospital .
no incentive was provided to interview , and the overall response rate among those contacted was 89% .
the 20082011 databases of the pathology departments of the participating hospitals included 204 cases and 419 controls meeting the study criteria ( figure 1 ) .
of these subjects originally identified , 165 cases and 251 controls were linked to existing medical records .
seventy - four cases and 79 controls had working contact information and were contacted for interviews .
an additional 19 cases and 34 controls had sufficient information contained in their records to complete the interview questions and were included in the analysis , for a total of 85 cases and 112 controls .
interviewer - administered questionnaire included questions on demographics , reproductive history , breastfeeding history , hormone use , menstrual and menopause history , and occupational history , as well as a section on description of symptoms and treatment for their breast diseases .
this questionnaire was a shortened version of a risk - factor assessment that was pilot - tested for reliability and validity in morocco and egypt and is currently in use in our ongoing study of the epidemiology of breast cancer in north africa .
the study questionnaire was translated from english into arabic by an experienced native - speaking translator .
the study protocol was approved by the institutional review board at the university of michigan , as well as by ethics boards at all collaborating institutions .
data were compiled and imported into sas , version 9.1 , statistical software ( sas institute , car , nc , usa ) for statistical analysis .
crude associations were assessed by frequency tables and descriptive statistics , and differences between cases and controls were tested by chi - square tests or fisher 's exact tests for categorical variables and by student 's t - tests for continuous variables , with a two - sided significance level of = 0.05 .
bivariate analysis was performed using logistic regression , adjusted for age and hospital site in the model to control matching factors , to assess significance of variables as risk factors for case status . due to the small sample size and the large number of strata created in the presence of two matching factors , conditional logistic regression was not used .
the dataset was then imputed twenty times to allow for complete - case analysis by logistic regression .
imputation was carried out using iveware , imputation and variance estimation version 0.2 , survey methodology program ( survey research center , institute for social research , university of mi , usa ) .
following imputation , the complete set of twenty datasets was used to conduct bivariate analyses and multivariate analyses , adjusted for age and study site , to model predictors of the dichotomous outcome cases status and create an integrated predictive model .
in the crude modeling of variables , 10 factors were shown to be significant at alpha 0.05 .
these variables were then included in an adjusted model , and all but 1 variable remained significant .
this variable was breastfed at least one child , as its significance was accounted for in the variables breastfed all children and did not breastfeed .
additionally , 3 interaction terms were shown to significantly predict case status in the fully adjusted model , and these were included in the final analysis .
table 1 provides information on the sociodemographic characteristics of the study population , divided by case status and compared by chi - square or fischer 's exact tests .
the median age of cases at presentation was 33 ( range : 1759 years ) .
cases and controls had similar rates of urban versus rural residence ( 63% and 61% , resp . ) ; however , controls were significantly more likely to be employed outside home ( 32% of controls versus 11% of cases , p = 0.005 )
. significantly larger numbers of cases ( 96% ) were parous , compared to only 67% of controls ( p < 0.0001 ) . additionally , cases had a higher average parity of 3.46 births than controls , 2.30 births ( p = 0.0002 ) .
median ages at first birth ( 22 years ) and last birth ( 29 years ) were the same for cases and controls .
similar frequencies of miscarriage ( 12% ) , irregular menstruation ( 18% ) , and infertility ( 13% ) were seen among cases and controls . a significantly higher proportion of cases reported past use of contraceptive methods ( p = 0.005 ) , but when broken down by type of contraception used , no difference was seen .
for those for whom information was available , 76% of cases were pre - menopausal ; the distribution between menopausal statuses did not differ significantly for cases and controls . however , average age at menopause was 1.25 years lower for cases than for controls .
, no significant differences were seen between cases and controls regarding the number of those who breastfed all of their children or who breastfed at least one child .
however , significant differences were seen in the history of always using both breasts in feeding , with significantly lower rates among cases ( 51% ) than controls ( 83% ) ; p = 0.0019 .
cases were less likely to wear a bra ( 78% ) than controls ( 90% ) ; p = 0.071 .
table 4 depicts information on the characteristics of the breast problems experienced by subjects in the study .
cases were more likely to experience their breast problem in the left breast ( 55% ) than the right breast ( 34% ) ; however , this information was reported for too few of the cases for the finding to be significant .
the majority of the cases were granulomatous mastitis ( n = 37 , 43.5% ) , one of the types of chronic mastitis included . in terms of temporal proximity to lactation ( defined as problem within 6 months of lactation ) , no significant differences were seen between cases and controls ; however , information was missing for the majority of both cases and controls .
the main symptom of the breast problem was most likely to be swelling for both cases ( 40.0% ) and controls ( 74.6% ) , but cases were more likely to report abscess , secretions , and redness .
treatment administered to cases was most likely to be antibiotics or radiation ; controls reported other treatments or radiation as most common .
results from the adjusted bivariate analysis and fully adjusted logistic regression model , completing postimputation , are shown in table 5 .
the bivariate analysis found 10 variables to be significant : 9 of these were included in the fully adjusted model after stepwise addition . in the multivariate analysis ,
several potentially important predictors were found to be significantly associated with an outcome of cm .
parity was significantly associated with an outcome of cm ; cases experienced a 75% increase in odds of each additional birth .
cases were more likely to have a history of contraception use ( or = 2.73 , 2.073.61 ) , but this significance could not be related to type of contraception .
postmenopausal status was found to be protective against chronic mastitis when compared to premenopausal status ( or = 0.62 , 0.490.79 ) .
a case of cm was 3.76 times more likely to have breastfed all of her children than a control , and cases who breastfed were 4.40 times more likely to have not alternated breasts when breastfeeding .
cases wore a bra less often than controls ( or = 0.56 , 0.470.67 ) .
temporal proximity of problem to pregnancy and lactation was also found to be significant in the final model , with cases being 67% more likely than controls to have experienced their breast problem during pregnancy , lactation , or within 6 months after lactation .
a woman with cm was found to have 0.71 times lower odds of maintaining employment outside home than a control subject .
additionally , variables associated with reporting mice in the study subject 's neighborhood or household were found to be risk factors ; cases had a 47% increase in odds of reporting a campaign to control mice in their neighborhoods and a 63% increase in odds of reporting attempts to control mice in their households .
interactions between parity and contraception were found to be significant ; likewise , the effect of whether or not a woman breastfed all of her children varied across levels of parity .
both effects were less than additive ( or = 0.86 , 0.800.93 , and or = 0.80 , 0.730.87 , resp . ) , with the effect of breastfeeding all children decreasing with each additional birth .
use of only one breast as a risk factor appeared to have a significant interaction with whether or not the breast problem was associated with lactation ( or = 0.32 , 0.220.46 ) .
previous studies have suggested risk factors for cm involving parity , breastfeeding habits , contraception use , socioeconomic status , and treatment patterns ; however , ours is the first study to present statistically significant findings [ 3 , 17 , 22 ] .
our results indicate that the strongest measured risk factors for cm are breastfeeding all children and not alternating breasts when breastfeeding .
these risks match observations made by clinicians and reported in published case - series [ 3 , 4 , 17 ] .
several additional factors in this study appear to be associated with chronic mastitis , including premenopausal status , employment , higher parity , use of contraceptives , not wearing a bra , and reporting mice in one 's household .
some of these coincide with factors that have been previously identified as potential risk factors , and all can potentially lead to hypothesis - based testing for mechanisms of etiology .
history of contraception has been suggested in previous studies as a potential risk factor [ 4 , 7 , 11 , 22 ] . in our study ,
any history of contraceptive use was found to be statistically more common in cases than controls , adding evidence to the importance of contraceptive use as a risk factor for cm .
however , individual types of contraceptives could not be associated with case outcome , indicating that further research should be done to establish risk behavior .
suspected associations with pregnancy have been reported previously in case - series [ 3 , 4 , 7 , 11 , 14 , 17 ] . even after controlling for breastfeeding ,
parity was associated with cm in our study , indicating that it has a statistical significance outside its relation to breastfeeding .
interactions between parity and any history of contraception were found to be significant , indicating that the effect of history of contraception use varied across levels of parity . specifically , the effects of parity and contraception were less than additive ( or = 0.86 ) .
this indicates that there is only so much explanatory power that can be attributed to contraception with each increasing birth for each additional child that a woman has , the added importance of history of contraception use decreases .
this concept of less - than - additive interaction is illustrated in figure 2(a ) .
breastfeeding has previously been correlated with cm in several case - series [ 4 , 7 , 17 , 22 ] ; however , in another study , no association was found . in our study , breastfeeding all children was found to be very significant in the full model , accounting for the significance of breastfeeding at least one child .
this finding is probably related to the fact that cases were more likely than controls to experience their breast problem during or within 6 months after lactation , although it was independently significant .
however , this strong effect of breastfeeding all children decreases with increasing parity , as the interaction between parity and breastfeeding all children is less than additive ( or = 0.80 , figure 2(b ) ) .
the association of breastfeeding all children is seen to decrease with each additional birth , tempering the effect of breastfeeding all children in the presence of high parity .
one of the main expected risk factors , as communicated by clinicians and reported in the literature , is the failure to alternate between breasts when breastfeeding .
this practice was found to be highly significant as a predictor of cm in our study .
however , among those with a problem during lactation , the importance of using both breasts or not breastfeeding at all decreases in explaining case status .
this interaction indicates that the present study can not separate cause and effect whether a woman experiences a problem because she is breastfeeding from only one breast or whether she breastfeeds in this pattern due to a breast problem .
this problem has been described elsewhere , in the use of mastitis - related terms such as milk stasis , retention , obstruction , or
breast engorgement , since these processes are considered as both predisposing factors and direct consequences of the disease .
nearly all published reports [ 14 , 1114 ] on cm find that the average age of women at presentation is in their fourth decade .
additionally , menopausal status was also found to be significant in the final model , with postmenopausal status seen to be protective .
this finding correlates with the view of cm as a reproductive - age problem often associated with pregnancy and lactation .
other protective factors seen in this study include being employed outside home and wearing a bra , both of which could potentially be related to higher socioeconomic status .
we included the condition of having mice in one 's household on the basis of a hypothesis relating mouse mammary tumor virus ( mmtv ) to breast cancer .
the results from the modeling of this variable indicate that there may be a dose - response relationship with the odds of case status increase .
therefore , exposure to mice may be related to an important risk factor for disease .
however , the relationship to mice could also indicate lower socioeconomic status of cases , which is unlikely .
while presentation and treatment were not included in the final predictive models , due to temporal occurrence after presentation with disease , differences in presentation and treatment between cases and controls were seen .
the main symptom of the breast problem was most likely to be swelling for both cases and controls , but cases were more likely to report abscess , secretions , and redness .
treatment administered to cases was most likely to be antibiotics or radiation ; controls reported other treatments or radiation as most common . from the published literature , the optimal treatment of cm is still unclear [ 18 , 25 ] . reported case - series have described treatment with antibiotics , steroids , abscess drainage , wide surgical resection , and even mastectomy , with variable results [ 2 , 2530 ] .
there are currently insufficient data to make definitive recommendations , but conservative treatment with steroids and surgery only if necessary is generally agreed upon , following the exclusion of infection [ 1 , 2 , 11 , 25 ] .
successful treatment with prednisolone , methotrexate , and azathioprine has been reported [ 13 , 2529 ] .
antibiotics should only be used in the treatment of cm if a bacterial infection is identified , despite the prevalent use of antibiotics seen in this study and reported elsewhere .
no information was available on the identification of infection before prescription of antibiotics . in the presence of abscess
data and prior studies on cm are extremely limited ; however , when compared with the previously published associations , our results reveal similar findings .
the majority of published papers and case reports have come from the developing countries , which has been hypothesized to be due to lower prevalence of cm in the developed countries , overdiagnosis in the developing countries , or underdiagnosis of tuberculosis mastitis in the developing countries [ 3 , 7 ] .
a study in the usa found that hispanic women had 12 times the risk of cm as non - hispanic women , and a study in the uk found that women with cm were significantly less likely to be caucasian .
this indicates that the disease may be more common in minority or developing - country settings , an idea that correlates with our findings in morocco and egypt .
conclusions drawn from this study therefore require an assumption of nondifferential missingness patterns between cases and controls , along with an assumption that those who could be contacted were a representative sample of all cases and controls .
the case definition used in this study was wider than that commonly used to study cm , as the vast majority of the published literature studies specific types of cm . due to the rarity of the disease and the commonalities in presentation and treatment , as well as a general lack of knowledge
while recall bias is often a concern in case - control studies , the fact that both our case and control populations experienced past breast disease potentially mitigates this concern .
there is still some temporal ambiguity ; however , many of the potential risk factors surround pregnancy and lactation , and dates of pregnancy were known , eliminating some ambiguity . despite these limitations , this study is a landmark picture of the epidemiology of cm .
clinical observations long reported in the literature but never studied broadly now have statistical significance , opening the way for future studies .
little is known about the etiology or risk factors for the disease . because studies on risks , etiologies , and treatments are lacking ,
women experiencing cm are often incorrectly treated . despite its relatively severe presentation and lack of standard treatment protocol
, we are unaware of any controlled analytic studies modeling risk factors for cm to date .
therefore , we believe that we have conducted the largest case - control study of cm in the literature , investigating the epidemiology and potential risk factors for the disease .
our findings indicate that the disease typically occurs in the fourth decade of life and has a statistically significant association with pregnancy / parity , lactation , breastfeeding patterns , menopausal status , employment status , history of use of contraception , having mice in one 's household , and not wearing a bra .
further studies are required to parse out understandings of mechanisms , establish temporality , and further understand risks and etiologies of chronic mastitis . | chronic mastitis is a prolonged inflammatory breast disease , and little is known about its etiology .
we identified 85 cases and 112 controls from 5 hospitals in morocco and egypt .
cases were women with chronic mastitis ( including periductal , lobular , granulomatous , lymphocytic , and duct ectasia with mastitis ) .
controls had benign breast disease , including fibroadenoma , benign phyllodes , and adenosis .
both groups were identified from histopathologically diagnosed patients from 2008 to 2011 , frequency - matched on age .
patient interviews elicited demographic , reproductive , breastfeeding , and clinical histories .
cases had higher parity than controls ( or = 1.75 , 1.621.90 ) and more reported history of contraception use ( or = 2.73 , 2.073.61 ) .
cases were less likely to report wearing a bra ( or = 0.56 , 0.470.67 ) and less often used both breasts for breastfeeding ( or = 4.40 , 3.395.72 ) .
chronic mastitis cases were significantly less likely to be employed outside home ( or = 0.71 , 0.600.84 ) and more likely to report mice in their households ( or = 1.63 , 1.361.97 ) .
this is the largest case - control study reported to date on risk factors for chronic mastitis .
our study highlights distinct reproductive risk factors for the disease .
future studies should further explore these factors and the possible immunological and susceptibility predisposing conditions . |
despite the explosive increase in cancer genomics data , the actual application of genomics data and approach to clinics offers big challenges [ 1 , 2 ] .
the main reason for the delay in the application to clinics is non - connectivity between bioinformaticians , non - clinical scientists , and clinicians .
clinicians are not confident in how much additional information might be given through genomics technology compared to conventional tools , and non - clinical scientists and bioinformaticians are not aware of what clinical problems should be solved with priority .
cancer clinicians expect novel diagnostic , prognostic , and therapeutic values of this new technology .
now , the clinical field is aware that multiple assays using next - generation sequencing ( ngs ) has some advantage over single genetic tests [ 1 , 3 ] .
a recent cancer guideline from the national comprehensive cancer network ( nccn ) pointed out that epidermal growth factor receptor ( egfr ) and/or anaplastic lymphoma kinase ( alk ) testing should be conducted as part of multiplex / ngs in non - small - cell lung cancer ( nsclc ) .
the feasibility of an ngs diagnostic platform at cancer clinics was validated by cancer centers in the united states and europe .
one platform showed that potential hurdles , time , cost , purity , and ethical problems could be overcome in implementing ngs using low - coverage whole - genome sequencing , whole - exome sequencing ( wes ) , and whole - transcriptome sequencing .
another platform sequenced clinical samples , including formalin - fixed , paraffin - embedded ones , and showed that this approach is practical and can give some benefits to the patients with lung cancer .
the wes approach in mendelian disorders showed that ~25% of sequencing can give a specific diagnosis based on the germline genetic make - up from the data of 250 probands , mostly consisting of neurological phenotypes in real practice .
the clinical application of cancer genomics should be considered in the context of unmet clinical needs to maximize the value of this novel paradigm . here , we dealt with the potential clinical application of cancer genomics in the aspect of practical management of cancer patients , along with a clinical scenario covering the whole course of cancer management .
we overviewed the potential of cancer genomics in the context of real cancer practice through this approach .
he quit smoking 10 years ago and had smoked an average of one pack per day for 30 years .
he had regular check - ups every year and had the last check - up with no abnormality 6 months ago .
he got a percutaneous needle aspiration ( pcna ) and biopsy but did not have any tumor cells .
he repeated the pcna and biopsy but developed pneumothorax ( air leakage from lung ) related to the diagnostic procedure .
after a staging work - up with a computed tomography ( ct ) scan , positron - emission tomography ( pet ) scan , brain magnetic resonance imaging scan , and bronchoscopy , his tumor was revealed as t2n0 clinical stage .
he got a surgery based on the result of the pathology and staging work - up . in this situation , we face unmet clinical needs of early cancer diagnosis .
he was diagnosed with symptoms related to the cancer , despite his regular check - up .
if we detect his malignancy before the symptoms , we can get a better result compared with the symptomatic case .
this imaginary patient was diagnosed with operable cancer , but as many as 60% to 70% of lung cancer patients are diagnosed with inoperable , advanced disease . to diagnose the cancer sooner ,
lots of trials failed to show an actual benefit of screening strategy in lung cancer [ 10 , 11 ] , but recently , a screening trial using low - dose chest ct of lung cancer was proven to be successful .
low - dose chest ct reduced the mortality from lung cancer through early detection in people with a history of at least 30 pack - years of smoking . despite this good approach to detect early lung cancer ,
this advantage should be counterbalanced with the potential harmful effects of radiation exposure or false positivity
many biomarkers were investigated for the early detection of lung cancer - for example , circulating tumor dna and rna .
but , false positivity and false negativity of single tests prevent these tests from clinical usage .
if we use multiple biomarkers using cancer genomics for the detection of early cancer , the false positives and negatives could be reduced to the level of clinical usage .
another effective approach of early diagnosis of cancer is to select a high - risk population for screening tests .
many genome - wide association studies identified some germline single - nucleotide polymorphism ( snps ) that are associated with the risk of cancers [ 14 - 16 ] , and these findings suggested important potential germline markers for the definition of high - risk populations .
mardis told the case of mike snyder as an example of an actual application of a germline genome test .
he sequenced his own genome and found a higher risk of type 2 diabetes ; since that time , he checked his glucose level regularly , and he detected high glucose levels at the very beginning .
the concept of early diagnosis , like the story of mike snyder , can be applied to the early detection of cancer , too .
if we define a high - risk population with germline genetic tests , we could focus on the use of screening tests in this population .
this
complication gives pain and discomfort of the sensation of dyspnea , prolongs the period of admission , and increases the cost of cancer management .
this problem originated from the invasive nature of the diagnostic procedure - pcna and biopsy using a needle , moving back and forth through the patient 's fragile lung tissue .
new diagnostic tools using cancer genomics - for example , identification of cancer - specific genetic alterations from circulating tumor dna - can avoid this type of complication from invasive procedures .
one pioneering study showed that circulating tumor dna could be combined with genomic technology to diagnose and monitor the status of cancer very safely .
his final pathological diagnosis was adenocarcinoma and revealed the involvement of some mediastinal lymph nodes ; therefore , the final pathological stage was t2n2 , iiia .
this stage required adjuvant chemotherapy , and he was referred to the department of medical oncology . a medical oncologist prescribed adjuvant chemotherapy - a navelbine and cisplatin regimen ( np ) for 4 cycles .
np 4 cycles has been prescribed to patients with high - risk clinical stage ib , iia , iib , iiia , and iiib , regardless of other clinicopathological factors .
the actual benefit from this treatment is only a 4% long - term survival advantage , with some hematological and non - hematological toxicities [ 19 , 20 ] .
the way to improve the risk - to - benefit ratio of adjuvant treatment is further selection of a patient population that could benefit from this treatment .
stage - based risk calculation - the current practice - cannot predict an accurate risk of relapse and attenuates the value of adjuvant treatment .
single patient - based risk calculation should be done to identify the adequate patients for adjuvant treatment .
cancer genomics will provide useful tools for a single patient - based risk calculation model .
this personalized adjuvant model will definitely increase the proportion of patients who benefit from it .
if he had been diagnosed as stage iiia preoperatively , he might have chosen a no - surgery option , such as concurrent chemoradiation , because no definite benefit of surgery was demonstrated until now , although this is controversial [ 21 , 22 ] .
we have relatively sensitive diagnostic methods , such as ct , pet , and endobronchial ultrasonography , but the sensitivity and specificity are still not perfect .
one focus of genomics technology should be a more accurate prognosis preoperatively , in addition to postoperatively .
prediction issues in cancer patients are not limited to survival , recurrence , and response to specific treatment .
the goal of cancer care is improvement of survival and , more importantly , quality of life .
predictive markers of bone metastasis or bone - related events , such as fracture or paralysis , are very important to improve the quality of life in cancer patients .
an unbiased approach is needed to discover good biomarker for these events , because we have very limited clues to dig into these problems .
the most important advantage of genomics approach is that is " unbiased " with regard to any hypotheses or theories .
that is why clinical problems with low - depth knowledge are a good candidate for the application of cancer genomics .
the connection between clinicians and genomic scientists can facilitate the development of clinical problems that are solved with a genomic approach .
he got 4 cycles of adjuvant chemotherapy and then followed up regularly with a physical examination , blood test , ct scan , and/or pet scan . at 6 months since the completion of adjuvant chemotherapy
, he presented with right - side chest pain , and his ct scan showed tumor relapse in the right lung and pleura .
he planned systemic anticancer treatment for life prolongation and palliation of symptom , not curative .
the role of ct scan and/or pet scan is not defined , but many clinicians use this high - cost image for monitoring relapse .
the reason for the popular use of this high - cost image is that there is no other alternative of monitoring cancer status .
recent studies showed the possibility of circulating tumor cell and cell - free dna as a tool for monitoring tumor status [ 18 , 25 ] .
cancer genomics enables circulating tumor cell or cell - free dna to detect relapse earlier than image findings or tumor markers .
the delicate delineation of circulating dna with ngs technologies is an essential part of this success story .
no tool is available with a definite clinical benefit for the monitoring of cancer ; therefore , we absolutely need new , non - invasive tools for it . because it takes at least several years to prove the benefit of monitoring
, we should start a well - designed prospective study using cancer genomics to prove it as soon as possible .
the medical oncologist who was in charge of him tested for egfr mutation and alk by fluorescence in situ hybridization to see rearrangements of alk .
he chose cytotoxic chemotherapy with pemetrexed and cisplatin according to the double - negative results of the egfr and alk tests .
the ultimate goal of the clinical application of cancer genomics should be therapeutics . there are increasing gene lists for nsclc that have matching targeted agents [ 26 , 27 ] , but many patients do not have genetic alterations that can be a target .
even for the same type of cancer - for example , lung adenocarcinoma - the genetic make - up is very heterogeneous .
the accurate definition of the genetic make - up of each patient will be the first step for personalized medicine .
many target panels have been developed by many vendors and hospitals for commercial or non - commercial use , but the usage is still very limited . as more cancer genome data are available [ 28 , 29 ] , the list of target panels will increase and be specified to tumor types .
now , we have two targeted agents to prolong the survival of lung cancer patients : the egfr tyrosine kinase inhibitor ( tki ) erlotinib or gefitinib for egfr mutations and the alk tki crizotinib for alk translocations . for this case
, there was no targeted agent , because he did not have egfr or alk alterations .
based on the histology of his tumor , he was treated with pemetrexed and cisplatin . despite the substantial toxicity of cytotoxic chemotherapy , many patients are prescribed these drugs with the individualized possibility of this agent not known .
this decision relies on population data from large - scale prospective trials . because clinical trials targeting specific genetic alterations are increasing , the clinical need for more genetic information is also increasing .
if the patient was diagnosed as stage iv initially , the amount of tissue for genetic testing is very limited .
the amount of tissue is enough for 2 or 3 genetic tests . for these patients ,
multiplexing or ngs technologies have important advantages to overcome the limitation of tissue amounts in the clinic .
this patient got adjuvant chemotherapy with np for 4 cycles but progressed 6 months after the completion .
the way to increase the benefit of adjuvant treatment could be the application of variable adjuvant treatment that is selected from individualized factors .
personalized cancer genomic analysis can show different and private profiles of individual cancers , and accumulation of these genomic data enables different adjuvant treatment strategies for each patient . despite the theoretical promise of personalized cancer genomic analysis for the selection of the right drug ,
one of important example is the story of lukas wartman , a doctor at washington university in st .
he suffered from a second relapse of acute lymphoblastic leukemia and sequenced the tumor dna and rna .
he found very high expression of flt3 and then figured out that sunitinib , a kidney cancer drug , could block tumor survival signals .
sunitinib induced complete remission in him , and thereafter , he received allogeneic stem cell transplantation and is now in remission . another story from the fox chase cancer center was reported on kit mutations from a pancreatic neuroendocrine tumor .
the patient had an advanced pancreatic neuroendocrine tumor and sequenced his tumor using a cancer panel .
he had a kit mutation and was treated with imatinib , a well - known kit inhibitor that is not approved for his tumor , for more than 2 years . if he had been in in routine practice without cancer panel sequencing , he would have died without imatinib treatment .
a pharmacogenomic approach could be used to minimize toxicity to cancer treatment , too . in this case
if the patient has a homozygous polymorphism in the ugt1a1 gene - the * 28 variant - he should be prescribed with a dose reduction to avoid severe , sometimes fatal , toxicity from irinotecan .
a genomic approach is required to properly assess pharmacogenomics in association with drug toxicity . a commercial chip , the affymetric dmet chip , covers 25 - 32% of genes of pharmacogenomics , and it is an important question whether more coverage with wes will be more beneficial .
he was treated with pemetrexed and cisplatin for 4 cycles , and his tumor shrank with the treatment .
but , his tumor rebounded , with a complaint of dyspnea and cough , 4 months after the completion of treatment .
after 4 cycles of second - line chemotherapy with docetaxel , he had stable disease . even in the case of a dramatic response to the first - line treatment
, nearly all cancers rebound with various relapse - free durations [ 30 , 34 ] .
the choice of second - line treatment depends on clinical guidelines , clinical parameters , and clinicians ' experiences .
any of the parameters may not be best if a specific patient 's situation is not considered .
we do not know the exact mechanism of resistance to the previous treatment and do not have any good way to assess the mechanism of resistance .
if cancer genomics is adopted in this situation , we can profile the tumor tissue that was resistant to the previous treatment .
the profile can help clinicians decide on a second - line treatment option and improve the individual 's outcome .
the resistance mechanism could vary , depending on multidimensional factors , such as the patient , tumor biology , and previous treatment .
especially , this situation should be approached individually with the concept of true personalized medicine .
he was in hospice care and died 1 year after the initial diagnosis of relapse .
after his death , his tumor was sequenced by whole - transcriptome analysis , and his tumor showed a ros1 translocation .
the median overall survival of metastatic lung cancer patients is 1 year if there is no genetic alteration for which targeted agents are available [ 30 , 34 ] , as in this case .
if the patient has an egfr - sensitive mutation or alk translocation , the median overall survival time reaches 2 years [ 35 , 36 ] .
a ros1 targeting agent , another example of successful targeting of oncogene addiction , is now available after his death .
if the ros1 translocation was known for his tumor and he was treated with a ros1 inhibitor , he could have a chance to live an additional one year or more .
tumors have very heterogeneous profiles [ 37 , 38 ] , and resistant tumors have even more heterogeneous ones .
this is why tumors should ultimately be approached on an individual basis , rather than a population basis .
now is the right time to think about how we can use cancer genomics for individual patients .
functional annotation of sequencing results , the way to integrate multi - dimensional data , and the exploration of the clinical meaning of sequencing variants for the routine clinical usage of cancer genomics . defining unmet clinical needs is as important as overcoming technological hurdles to maximize the benefit from cancer genomics .
early detection , non - invasive molecular diagnosis , risk prediction of relapse and tumor - related events , monitoring of disease status , matching good drugs , and finding out personal grade resistance mechanisms are good examples of unmet clinical needs that should be solved with cancer genomics .
it would speed up and optimize the application of genomics to cancer clinics if cancer biologists , bioinformaticians , and cancer clinicians cooperate towards one goal of achieving it . | the large amount of data on cancer genome research has contributed to our understanding of cancer biology .
indeed , the genomics approach has a strong advantage for analyzing multi - factorial and complicated problems , such as cancer .
it is time to think about the actual usage of cancer genomics in the clinical field .
the clinical cancer field has lots of unmet needs in the management of cancer patients , which has been defined in the pre - genomic era .
unmet clinical needs are not well known to bioinformaticians and even non - clinician cancer scientists
. a personalized approach in the clinical field will bring potential additional challenges to cancer genomics , because most data to now have been population - based rather than individual - based .
we can maximize the use of cancer genomics in the clinical field if cancer scientists , bioinformaticians , and clinicians think and work together in solving unmet clinical needs . in this review ,
we present one imaginary case of a cancer patient , with which we can think about unmet clinical needs to solve with cancer genomics in the diagnosis , prediction of prognosis , monitoring the status of cancer , and personalized treatment decision . |
atherosclerosis represents a major factor of coronary heart disease characterized by the formation of fat - laden plaque in large and medium - sized vessels . during atherogenesis
, the endothelial layer of the vessels is constantly confronted with a range of stress signals .
elevated levels of circulating oxidized low - density lipoprotein ( ox - ldl ) , increased turbulent blood flow , and excessive inflammation all contribute to endothelial injury .
regardless of the nature of the stress cue , the transcriptome of vascular endothelial cells is profoundly altered. for instance , down - regulation of enos transcription and simultaneous up - regulation of et-1 transcription in endothelial cells precedes the impairment of vasodilation and rhythmic vessel tone .
transcriptional activation of adhesion molecules , on the other hand , enables circulating leukocytes to attach to the endothelium and establish a pro - inflammatory microenvironment .
therefore , elucidation of the mechanisms underlying these characteristic transcriptional events will potentially further our understanding of atherogenesis and yield druggable targets for the intervention and prevention of atherosclerosis .
recent advances in the transcriptional regulation of atherosclerosis suggest a growing involvement of the epigenetic machinery , .
this mini - review is a modest attempt to summarize the current state of research on the epigenetic regulation of endothelial disorder in atherosclerosis and to provide an outlook .
unlike prokaryotic organisms , eukaryotic transcription takes place on nucleosome - wrapped chromatin . in order for the basic transcription machinery to be recruited to promoter region and initiate transcription
the epigenetic machinery , composed of histone modifying enzymes , dna modifying enzymes , chromatin remodeling proteins , non - coding regulatory small rnas , and histone variants , serves as an intricate regulatory layer for eukaryotic transcription by altering chromatin structure . due to space constraints
, we only give a brief overview of histone modifications and chromatin remodeling , which will be the focus of discussion in this review .
the n - terminal tails of core histones , h3 and h4 in particular , can be post - translationally modified . the term
histone code was coined to correlate a specific set of histone modifications to a predictable transcriptional outcome .
though a subject of constant controversy and debate , it is generally believed that acetylation of histones h3 and h4 surrounding the promoter region is synonymous with transcriptional activation . whereas methylation of histone h3 lysine 4 ( h3k4 ) may herald activation , h3k9 methylation often marks repressed chromatin . in order for sequence - specific transcription factors and the basic transcription machinery to access the dna and initiate transcription ,
this is achieved by moving the nucleosomes along the dna at the expense of atp hydrolysis .
initially identified and characterized in yeast , , the chromatin remodeling proteins represent a most conserved branch of the epigenetic machinery during evolution .
the mammalian chromatin remodeling complex is a multi - subunit mega - protein conglomerate that contains a catalytic component .
brahma related gene 1 ( brg1 ) and brahma ( brm ) are the best studied chromatin remodeling proteins with atpase activity .
brg1 and brm have been known to participate in both transcriptional activation and repression depending on the specific chromatin environment and the transcription factors they interact with .
the n - terminal tails of core histones , h3 and h4 in particular , can be post - translationally modified . the term histone code was coined to correlate a specific set of histone modifications to a predictable transcriptional outcome .
though a subject of constant controversy and debate , it is generally believed that acetylation of histones h3 and h4 surrounding the promoter region is synonymous with transcriptional activation . whereas methylation of histone h3 lysine 4 ( h3k4 ) may herald activation , h3k9 methylation often marks repressed chromatin .
in order for sequence - specific transcription factors and the basic transcription machinery to access the dna and initiate transcription , the chromatin has to be unwound and loosened .
this is achieved by moving the nucleosomes along the dna at the expense of atp hydrolysis .
initially identified and characterized in yeast , , the chromatin remodeling proteins represent a most conserved branch of the epigenetic machinery during evolution .
the mammalian chromatin remodeling complex is a multi - subunit mega - protein conglomerate that contains a catalytic component .
brahma related gene 1 ( brg1 ) and brahma ( brm ) are the best studied chromatin remodeling proteins with atpase activity .
brg1 and brm have been known to participate in both transcriptional activation and repression depending on the specific chromatin environment and the transcription factors they interact with .
under physiological conditions , the vessel wall is free from the attachment of circulating leukocytes . under certain pro - atherogenic conditions , such as turbulent shear stress , oxidative stress , intermittent hypoxia , and excessive nutrition , endothelial cells up
- regulate the transcription of adhesion molecules ( cam ) including icams , vcams , and selectins , which consequently allow a much stronger interaction between leukocytes and the endothelium perpetuating a pro - inflammatory niche , .
diabetes is one of the leading causes for vasculopathies including atherosclerosis . a seminal study by brownlee and colleagues examined the effect of transient hyperglycemia on endothelial function . of great intrigue
, these authors have found that exposure to high glucose ( hg ) for a short period time ( 16 hours ) induced a prolonged activation of vcam-1 in bovine aortic endothelial cells even when these cells were switched to and maintained in low glucose ( lg ) for additional 6 days , a phenomenon dubbed as metabolic memory .
chromatin immunoprecipitation ( chip ) revealed an uptick of h4k4 monomethylation on the proximal promoter of the p65 gene mediated by the histone methyltransferase set7/9 .
transient hg , these authors propose , leaves an epigenetic dent on the p65 gene such that even in the absence of the original stimulus endothelial dysfunction will sustain .
circulating oxldl presents a major risk to atherosclerosis in part by promoting the expression of adhesion molecules .
have recently reported a novel epigenetic mechanism underlying the induction of icam-1 in human endothelial cells .
oxldl activated the transcriptional modulator mrtf - a , which in turn was recruited to the icam-1 promoter by p65 and synergistically stimulated icam-1 transcription with oxldl .
depletion of mrtf - a erased h3/h4 acetylation and h3k4 dimethylation but restored h3k9 trimethylation on the icam-1 promoter . since mrtf - a is known to engage the epigenetic machinery in regulating transcription within the vasculature , it is conceivable that mrtf - a may serve as the critical link of endothelial injury bringing histone modifying enzymes to the chromatin .
alternatively , kim et al . propose that the regulatory subunit of the napdh oxidase complex p66shc mediates the induction of icam-1 by ldl .
ldl induced histone acetylation but inhibited dna methylation of the p66shc promoter to up - regulate the transcription of p66shc . of note , oxldl has been reported to elicit epigenetic changes on a host of gene promoters in endothelial cells , although a genome - wide survey is lacking .
chronic hypoxia has emerged as an independent risk factor of atherosclerosis . as a result of intermittent low oxygen supply ,
the transcriptome of endothelial cells undergoes marked changes that include an increase in the transcription rate of adhesion molecules .
our laboratory has recently uncovered a potential role for brg1 and brm in hypoxia induced endothelium - leukocyte interaction .
expression of brg1 and brm in vitro was up - regulated in cultured endothelial cells exposed to 1% o2 and in vivo in pulmonary arteries isolated from mice kept in a low - oxygen chamber for 4 weeks .
introduction of brg1 and brm into a brg1/brm - negative cell line ( sw-13 ) greatly potentiated hypoxia - induced cam transactivation whereas silencing of brg1/brm in endothelial cells crippled the effect of hypoxia .
brg1 and brm formed a dynamic interaction with p65 on the cam promoters where p65 recruits brg1/brm and brg1/brm reciprocally stabilizes p65 .
brg1/brm influenced cam transactivation by altering histone h3/h4 acetylation and h3k4 methylation creating a friendly chromatin structure for the basic transcription machinery .
more important , endothelial - specific targeting of brg1 and brm normalized cam expression and attenuated hypoxia induced leukocyte adhesion in mice . of intrigue ,
a similar strategy also alleviated the development of atherosclerotic lesions in apoe mice , indicating that brg1 and brm might be able to orchestrate endothelial injury in response to a range of different pro - inflammatory stimuli ( unpublished observation ) .
endothelium derived no plays a critical role in maintaining vascular integrity , the disruption of which contributes to atherogenesis .
oxldl decreases acetylation of h3 and h4 and dimethylation of h3k4 while simultaneously increasing h3k9 trimethylation surrounding the enos promoter consistent with the repression of enos transcription in endothelial cells .
again , mrtf - a appears to be the key coordinator of these epigenetic changes .
in addition , there is a decrease of enos expression in mice deficient in lsd1 , an h3k4/k9 demethylase , highlighting the role of histone methylation in fine - tuning enos transcription .
fish et al . conducted a comprehensive survey of histone modifications on the enos promoter region in endothelial cells challenged with anoxia . in the proximal region of the enos promoter ( -166/-26 ) ,
h3/h4 acetylation and h3k4 dimethylation declined as early as 1 hour following exposure to low oxygen .
in contrast , these signature changes were not observed on the distal enos promoter ( -891/-797 and -488/-398 ) .
a closer examination revealed that acetylation levels of specific lysine residues fluctuated with distinct patterns .
for instance , 1 hour after hypoxia , only h3k14 and h4k5 acetylation plummeted significantly , which was joined by a decrease in h3k9/h4k8/h4k12 acetylation at 2 hours with h4k16 acetylation unaltered .
interestingly , histone h2a.z was evicted from the proximal enos promoter during hypoxia thereby creating a closed chromatin conformation and rendering a repressed transcription state .
consistently , a recent study has correlated decreased enos expression with high levels of dna methylation on the proximal enos promoter in patients with obstructive sleep apnea ( osa ) , a typical pathology of hypoxia . paradoxically , enos expression has been observed to increase , rather than decrease , in endothelial cells in response to hypoxia probably as means of compensation .
the up - regulation of enos transcription is accompanied by increased h3 and h4 acetylation across the enos promoter region ( -4501/+23 ) .
therefore , while it remains debatable how enos transcription responds to hypoxia , suffice it to say that a specific epigenetic code is intimately associated with the transcription status .
the vessel wall , particularly the endothelial layer , is subject to the pressure caused by various hemodynamic forces .
whereas laminar shear stress ( lss ) is considered atheroprotective and stimulates enos transcription , turbulent blood flow creates shear stress that damages the vascular endothelium especially at the sites of arterial branches . among the detrimental effects exerted by pro - atherogenic shear stress
are accelerated turnover of endothelial cells , increased adhesion of leukocytes , accumulation of reactive oxygen species , and decreased synthesis of no stemming from enos repression. illi et al .
have reported that lss increased global h3 and h4 acetylation levels in endothelial cells through activating histone acetyltransferases ( hats ) .
in addition , lss augmented h3 and h4 acetylation on c - jun and c - fos promoters .
since c - jun / c - fos is considered essential for enos transactivation , increase ap-1 activity likely explains elevated enos transcription in response to lss .
impaired vessel relaxation during atherogenesis is also rooted in the enhanced expression of endothelin , a potent vasoconstrictor .
accumulating evidence has provided a clear link between histone modification and et-1 transactivation . in diabetic rats , increased et-1 release was accompanied by an up - regulation of the histone acetyltransferase p300 .
silencing of p300 completely abolished et-1 activation by high glucose thought it remained unclear whether p300 acted through histone or non - histone factors .
have demonstrated that h4 acetylation was increased on the et-1 promoter surrounding a conserved p65 binding element in endothelial cells treated with two pro - inflammatory stimuli , tnf- and ifn- , suggesting a potential role for a hat like p300 . in rats with intrauterine growth retardation ( iugr ) where hypoxia plays a determining role , h3k9/k18 and h4 acetylation increased on the et-1 promoter .
recently , our investigation has led to the identification of an mrtf - a - centered epigenetic complex on the et-1 promoter in response to hypoxia . under hypoxic conditions ,
mrtf - a interacted with and was recruited by the sequence specific transcription factor srf to the proximal et-1 promoter ( -81/+150 ) . upon the joining of brg1/brm , this epigenetic complex altered histone acetylation and h3k4 methylation to facilitate the binding of rna polymerase ii thereby activating et-1 transcription .
the turn - of - the - century saw a boom in research on epigenetics with the initiation of several epigenomics projects .
relying on high - fidelity chromatin immunoprecipitation ( chip ) coupled with high - throughput sequencing techniques , these projects aim to decode the epigenetic information bringing insights for the prevention and intervention of human diseases .
several exciting findings have provided clues for such basic biological events as adipogenesis , macrophage activation , and cell cycle progression .
so far , there has been a lack of effort in deciphering the epigenetic code on a genomewide scale in any of the major cardiovascular diseases including atherosclerosis .
since single - gene based epigenetic analysis tends to give very limited and often biased knowledge to the understanding of atherogenesis , long considered a multifactorial disease , basic scientists and clinicians like will benefit from an undertaking that unveils a comprehensive picture of epigenetic regulation of endothelial injury in atherosclerosis . | atherosclerosis is a progressive human pathology that encompasses several stages of development .
endothelial dysfunction represents an early sign of lesion within the vasculature .
a number of risk factors for atherosclerosis , including hyperlipidemia , diabetes , and hypertension , target the vascular endothelium by re - programming its transcriptome .
these profound alterations taking place on the chromatin rely on the interplay between sequence specific transcription factors and the epigenetic machinery . the epigenetic machinery , in turn , tailor individual transcription events key to atherogenesis to intrinsic and extrinsic insults dictating the development of atherosclerotic lesions .
this review summarizes our current understanding of the involvement of the epigenetic machinery in endothelial injury during atherogenesis . |
removal of third molars is a common procedure in oral surgery and it is associated with
postoperative complications such as pain , swelling and trismus . therefore , several
medications such as corticosteroids and non - steroidal anti - inflammatory drugs are often
recommended despite their common side effects such as bleeding , gastrointestinal irritation
and allergic reactions , which can evoke discomfort and health risks in patients1 . in terms of strategies
used to minimize tissue damage in oral surgeries , the low - level
laser therapy ( lllt ) seems to offer many benefits in the stimulation of the healing process
and control of the inflammatory process by reducing the swelling and pain , with no reports
of adverse effects2 . according to medeiros et
al.3 , the
effects of lllt are more evident in the early stages of wound healing .
the use of polarized
light helps in resolution of inflammation and in increasing the collagen deposition as well
as the number of myofibroblasts
however , previous studies have reported that in teenagers , lllt reduces
pain intensity without modifying the healing process4 .
on the other hand , lllt showed better results in controlling
swelling and trismus when compared to ozone therapy5 .
however , lopez - ramirez et al.2 found that lllt showed no positive effects in reducing pain ,
swelling and trismus after the removal of third molars .
these differences in the effects of lllt may be explained by the variability in the
protocols and surgical procedures performed1 , 2 , 6 .
the lllt protocol should be determined considering the type of light used as well as the
tissue layers that need to be irradiated . in this context , the red spectra in the visible to
near - infrared spectra ( 620830 nm ) have the potential to stimulate photoreceptors in the
plasma membranes of cells .
the higher wavelengths are absorbed by the deeper tissues and are
less active in the surface tissues7 .
therefore , this study aimed to evaluate the efficacy of intraoral application of lllt
( 660 nm ) in controlling pain intensity , swelling and interincisal opening in oral
surgery .
this study included 10 patients ( 7 women , 3 men , mean age 23 6 years ) and it was
initiated after obtaining the approval of the ethics in research committee ( protocol number :
512.476 ) .
the
exclusion criteria were as follows : presence of local infections , various systemic diseases ,
smoking habit , use of oral contraceptives , pregnancy and lactation .
all patients underwent the same surgical protocol standardized by the same surgeon
( r.l.n . ) .
however , the values
recorded for pain , swelling and trismus were assessed by a different surgeon in order to
conduct a blind experiment . before the laser application , the mean power of the equipment
was measured and calibrated using the laser check power meter instrument .
intraoral lllt was applied using continuous scan mode at four different points , covering
the entire suppurative surgical area , which was divided into four quadrants , each of
1 cm area at a distance of 1 cm .
preoperatively , all patients used a mouthwash with chlorhexidine ( 0.12% ) and received a
topical application of povidone - iodine ( 1% ) .
postoperatively , all patients received
amoxicillin 500 mg , every 8 hours , for 7 days , ibuprofen 600 mg , every 8 hours , for 3 days ,
and paracetamol 500 mg , every 6 hours as rescue medication for pain during the first 72
hours . in cases with penicillin allergy ,
clindamycin was selected as the antibiotic at a
dose of 300 mg , every 8 hours , for 7 days . in this study ,
, amparo , sp , brazil ) with a pen station beam area of 0.035 cm , algalnp
visible diode with continuous wave ( 35 mw and wavelength of 660 nm ) was used .
the energy
applied at each point was equal to 5 j / cm / dose , continuous rate for 8 seconds ,
as specified by the manufacturer .
the formula below was used to calculate the energy density ( d ) , where the area ( a :
cm ) corresponds to the broadcast area of the pen tip , power ( p : w ) corresponds
to the maximum and constant power ( 20 mw or w 0.020 ) and the time ( t ) is measured in
seconds . after applying the values supplied by the manufacturer in the formula , the energy density
applied at each point was calculated to be 4.57 j / cm multiplied by 4 quadrants of
1 cm , resulting in a total of 18.28 j / cm in each session .
considering previous reports of several authors , a dose of 3 j / cm up to
6 j / cm2 , 8 ,
9 has a regenerative action .
four consecutive daily sessions , with the first session occurring 24 hours after surgery ,
were applied .
this protocol was chosen based on the fact that the process of wound repair is
composed of 3 phases : inflammatory , proliferative , and remodeling phases involving the
formation of collagen10 , and lllt is more
evident in the initial stages of healing2 .
the outcome variables assessed were pain , interincisal opening , facial swelling , and presence
of late infection .
the experimental times considered in this protocol were as follows :
t0 ( initial time ) , t1 ( 24 h ) , t2 ( 48 h ) , t3 ( 72
h ) , t4 ( 96 h ) , t5 ( 120 h ) , t6 ( 144 h ) , and t7 ( 7
days after the surgery ) .
the interincisal opening was assessed by measuring with a caliper of maximum opening between
the right upper and lower central incisors before surgery and in all subsequent sessions11 . to evaluate swelling , a measurement from one tragus to the opposite one
all patients were instructed to register the daily intensity of pain , beginning 24 hours
after surgery , for seven postoperative days , using a visual analogue scale ( vas ) of 100 mm , in
which the score for lack of pain was 0 ( zero ) , and 100 ( one hundred ) was the score used for
the worst imaginary pain .
all data were analyzed using graphpad 5.0 ( graphpad software , la jolla , california , usa ) .
the
level of significance was set at p < 0.05 with a confidence interval of 95% .
this study included 10 patients ( mean age : 23 6.03 years ) with asymptomatic third molars
( no pain observed on the day prior to the surgery , as assessed by vas ) .
all data on the interincisal opening , swelling and
pain intensity are shown in table 1table 1.all data on the interincisal opening , swelling , and pain intensitypost - operatory evaluationt0t1*t2t3t4t5interincisal openning ( mm)25th .
quartile24.525.223.013.76.0median49.040.028.523.016.575th . quartile70.254.045.029.725.2 * statistically different from t0 , friedman test , p<0.05 . * * statistically
different from t0 , friedman test , p<0.05 . * * * statistically different from t0 ,
friedman test , p<0.05 .. * statistically different from t0 , friedman test , p<0.05 . * * statistically
different from t0 , friedman test , p<0.05 . * * * statistically different from t0 ,
friedman test , p<0.05 . with regard to the interincisal opening , a statistical decrease
was observed 24 hours after
the surgery ( friedman test , p = 0.0001 ) .
similarly , an increase in swelling was observed
when comparing the initial condition and the first postoperative day ( friedman test , p =
0.01 ) .
therefore , it may be noted that both the swelling and interincisal opening returned
to normal condition 24 hours after the first lllt application .
concerning the pain intensity ( assessed by vas ) , no differences were observed between the
first and second postoperative days ( median : 49 versus median : 40 ) .
however , a reduction in pain intensity was observed immediately after the third
postoperative day , reinforcing the possible effect of lllt on pain ( friedman test , p =
0.001 ) .
additionally , on the seventh day , the patients were asymptomatic . even though the
patients with a greater surgical duration had a slight increase in pain intensity on the
first day , it was not statistically significant ( spearman s correlation , p > 0.05 )
in this case series , we have focused on the local postoperative clinical factors after
removal of third molars , such as pain , swelling and trismus . despite the proven efficacy of
lllt in the prevention of these factors , its mechanism of action
thus , studies with positive as well as negative results have been reported , due mainly to a
poorly calculated dosimetry and lack of uniformity in laser application1 , 2 , 6 .
sezer et al.13 found that only the diode
laser with 808 nm wavelength were effective in reduction of trismus , whereas applying a
laser diode with 660 nm wavelength was ineffective .
carrillo et al.14 showed that the incidence of trismus in the laser group
( helio neon , 633 nm with energy density of 10 j / cm ) was significantly lower than
that in the placebo group seven days after extraction of third molars .
aras and
gngrm15 , 16 found that irradiation with diode laser with wavelength of 808 nm
and 4 j / cm significantly decreases trismus .
moreover , lopez - ramirez et al.2 reported
that lllt ( gaalas , 810 nm , energy density of 5 j / cm ) demonstrated no benefits in
reducing pain and trismus after removal of impacted third molars .
similar results were
described by roynesdal and colleagues17
when applying lllt with 830 nm 40 mw laser at 6 j. according to gngrm and aras15 , 16 , extraction of teeth can cause spasms of
certain muscles , especially the masseter ; therefore , applying an intraoral laser on a small
area would not affect this muscle directly . however , in this study , favorable results were verified , as the reduction in trismus was
probably related to the relaxation effect of lllt on other masticatory muscles such as
masseter as well as the medial pterygoid .
lpez - ramirez and colleagues2 reported
that lllt was not statistically effective in the reduction of trismus .
amarillas - escobar et
al.18 conducted a similar study , but in
order to assess the cumulative effect of the therapeutic laser , they applied multiple daily
intraoral doses to the patients , immediately after surgery , and three sessions
post - operatively ( 24 , 48 , and 72 h ) , using lllt ( 660 nm ) .
the results of this study showed
no significant differences in reducing pain , swelling , or trismus between the laser and the
control group , although better conditions were noted in the laser group 24 hours after the
application .
however , sezer and colleagues13 found
that only the diode laser ( 808 nm ) was effective in biostimulation , whereas the diode laser
( 660 nm ) was ineffective .
however , the positive result observed at this study is probably due
to the onset of laser application .
although lllt is considered to have analgesic effects , especially in neuralgic and muscular
pain , its effects on pain related to inflammation are controversial2 , 15,16,17 , 19 .
there is evidence suggesting that lllt can have significant pharmacological effects on the
synthesis , release and metabolism of a series of biochemical mediators20 , such as increase in the production of serotonin and
acetylcholine in the central nervous system as well as histamine and prostaglandins
peripherally21 , 22 .
it has also been shown that lllt induces analgesia by stimulating the synthesis of
endogenous endorphins ( -endorphin ) , decreasing the activity of bradykinin and c fibers and
changing the pain threshold21 , 22 .
lllt induces morphological changes of neurons , reduces
the power of the mitochondrial membrane and blocks nerve conduction23 , 24 . a steady decrease in pain
was observed during the application days in this study , which is
in agreement with the results of a previous study by markovi and todorovi25 , who reported pain reduction by
application of a gaalas laser after surgical removal of third molars , suggesting a
dose - dependent effect .
moreover , it is important to state that there is a lack of knowledge concerning
the use of this wavelength in oral surgery .
a recently published meta - analysis and
systematic review of the literature concluded that previous studies have important
methodological differences that prevent establishment of the exact dose and ideal parameters
for lllt26 , 27 , considering that there is a lack of standardization in the
parameters as well as assessment tools employed27 . in conclusion , considering the limitations of this case series , these data suggest that
oral application of lllt has anti - inflammatory and analgesic effects , which consequently
reduces postoperative complications of oral surgeries .
further studies are needed ,
especially randomized controlled clinical trials , to help elucidate the impact of laser
therapy after third molar extraction and establish the ideal parameters for lllt as an
assessment tool in this promising therapy field . | [ purpose ] this study aimed to evaluate the anti - inflammatory and analgesic effects of
intraoral application of low - level laser therapy ( 660 nm ) to control pain , swelling and
interincisal opening following the extraction of mandibular third molars .
[ subjects and
methods ] ten patients underwent removal of lower third molars using the same surgical
protocol and pharmacological approach . in the postoperative period , all patients received
four consecutive daily sessions of low - level laser therapy , beginning 24 hours after the
surgery .
intraoral applications using the diode laser with 660 nm wavelength in the
continuous scan mode were performed covering the entire surgical area , which was divided
into four quadrants , each of 1 cm2 area at a distance of 1 cm .
the energy
applied at each point was 5 j / cm2 during 8 seconds .
[ results ] the swelling and
interincisal opening returned to normal 24 hours after the first low - level laser therapy
application ( friedman test ) .
moreover , the pain intensity was reduced on the third
postoperative day , according to the friedman test .
[ conclusion ] low - level laser therapy
( 660 nm ) , at the dosimetry used in this study , was effective in reducing postoperative
pain and swelling following oral surgery . |
the transversus abdominis plane ( tap ) block provides effective post - operative analgesia after abdominal surgery .
rafi first described tap block , who injected local anesthetic through the ilio - lumbar triangle of petit within the tap between the internal oblique muscle and transversus abdominis muscle utilizing the double - loss of resistance technique .
virtually , the ultrasound - guided tap block is highly effective and easy technique for rendering analgesia for post anterior abdominal incision as supported by literature ; however , it can also be used as a sole anesthetic technique for abdominal surgeries where the autonomic innervation is partly or not involved .
a 67-year - old woman , 55 kg was admitted to hospital with a 3-day history of abdominal pain , nausea and vomiting .
she was diagnosed to have chronic obstructive pulmonary disease ( copd ) 5 years ago and had four episodes of prior hospitalization secondary to acute exacerbation .
she often required nebulisers and had an exercise tolerance of approximately 100 yards . in the week prior to admission she had received oral antibiotics and steroids for an infective exacerbation of copd . on admission to the hospital she had signs of peritonitis and her abdomen
examination revealed reduced bi - basal air entry with crepitations all over the chest and chest x - ray showed consolidation of the right middle and lower lobe .
her arterial blood gas revealed type i respiratory failure ( fio2 0.60 , ph 7.37 , po258 mmhg , pco245 mmhg ) .
intravenous acetaminophen was administered for pain relief , but despite this she could not cough or take deep inspiration due to ongoing pain .
she was categorized as american society of anesthesiologists ( asa ) physical status grade iv / e and was planned for emergency laparotomy .
we planned to avoid general , epidural or spinal anesthesia , so we opted for tap block under ultrasound guidance as a sole anesthetic technique .
after proper explanation to the patient about the technique of tap block , she was taken to operation theatre table and monitors attached .
hence oxygen supplementation was carried out with venturi mask with oxygen flow at 6 l / min .
saturation improved to 92% . after securing an iv access with 18 g ( gauge ) needle on the right dorsum of the hand ,
she was sedated with intravenous dexmedetomidine infusion at 4 mcg / kg / min to make her comfortable , cooperative , and pain free for performing the block .
then she was laid supine with the anterolateral abdominal wall exposed bilaterally from the iliac crest to the sub - costal margins and scrubbed aseptically .
the block was performed using an aseptic ultrasound guided in - plane technique ( s - nerve sonosite , hfl38 ( company brand name of the 6.0 to 13.0 mhz linear probe ) probe , 100 mm sonoplex needle ) . when the needle tip position is within the tap neuro - fascial plane a mixture of 20 ml of 0.25% bupivacaine , 20 ml of 1% lidocaine and 0.2 mg adrenaline
a remarkably good clinical effect was achieved within 30 min with almost complete resolution of pain .
she made a slow post - operative recovery to be discharged from hospital 14 days after admission .
the use of tap blocks for control of post - operative analgesia has been described following a variety of abdominal operations such as appendectomy , hernia repair , caesarean section , abdominal hysterectomy , and prostatectomy etc .
bilateral blocks can be given for midline incisions or laparoscopic surgery with careful safe dosing .
to our knowledge , the use of ultrasound - guided bilateral tap for a sole anesthetic technique for upper abdominal surgery in a very high - risk patient has been rarely reported . the skin , muscles and parietal peritoneum of the anterior abdominal wall are innervated by the lower six thoracic nerves and the first lumbar nerve .
after leaving the respective intervertebral foramina the anterior rami ( sensory afferents ) of these nerves course around the transverse process , then pierce the musculature of the lateral abdominal wall to course through a muscle neurovascular plane superficial to the transversus abdominis . in the midaxillary line ,
the sensory afferents branch out as a lateral cutaneous branch and continue within the tap to perforate anteriorly supplying the skin as far as the midline .
the tap plane thus provides a space into which the local anesthetic can be deposited to achieve myocutaneous sensory blockade .
whilst early studies showed a t7 to l1 spread with a single posterior injection making the block suitable for midline abdominal incisions , other studies , however , failed to demonstrate a spread cephalad to t10 making it more suited for lower abdominal surgery . in a small cadaveric study , t11 , t12 , and l1 were most consistently present in the tap while t10 was present in 50% of the cases .
however , augmentation with a subcostal injection will help attain a higher block up to t7 which is a modification of the original technique in which the ultrasound probe is placed just beneath the costal margin and parallel to it .
the needle is then introduced from the lateral side of the rectus muscle in the plane of the ultrasound beam and 10 ml of local injected into the tap to extend the analgesia provided by the posterior tap block above the umbilicus . in our patient bilateral ,
sub - costal tap block under ultrasound guidance resulted in highly effective myocutaneous sensory blockade .
as the patient was categorized as asa iv / e general anesthesia or central neuraxial blockade for the emergency operation for such a high - risk case would have resulted in untoward and fatal complications both intra - operatively and postoperatively .
the abdominal wall sensory afferents , which course through the tap plane could be blocked successfully and effectively by abdominal field blocks or tap block under ultrasound guidance then the abdominal incision and operation could be carried out without patient 's discomfort .
pain caused by visceral stimulation of the celiac plexus may still challenge intraabdominal surgical success which is a major limitation .
when the surgeons manipulated the intestine , the patient complained of pain due to stimulation of autonomic nervous system through celiac plexus ( vagus ) , but dexmedetomidine infusion helped in relieving the pain or retching sensation .
anatomically , sympathetic and somatic innervation are closely related near the neuraxis , but become separated peripherally .
thus , spinal , epidural or paravertebral blocks will cause significant sympathetic block , resulting in major cardiovascular changes and other physiological effects . on the other hand ,
if complete denervation of viscera is required , vagal afferents have to be blocked by celiac plexus block .
the extent and spread of the local anesthetic solution in the tap affecting anterior abdominal wall sensory afferents depend on time factor .
it seems that the full effect of analgesia takes at least 20 - 25 min after injection of local anesthetic solution .
mcdonnell et al . suggest that local anesthetic spreads within the tap plane progressively over the several hours and an early assessment of the extensive tap block may be missed .
as ultrasound - guided bilateral transversus abdominais neurofascial plane block is quite simple , quick , safe , and effective especially for a very high - risk patient with multi - medical problems and geriatric patients needing an elective or emergency abdominal surgery , the surgeons and anesthesiologists should encourage this technique , even in this advanced era , when it is deemed suitable . | although transversus abdominis plane ( tap ) block is an effective way of providing analgesia in post - operative abdominal surgery patients ; however , it can be considered as an anesthetic technique in high - risk cases for surgery .
we report a case of a geriatric female with chronic obstructive pulmonary disease in the respiratory failure , hypotension , posted in an emergency with old perforation leading to peritonitis .
the surgery was successfully conducted under bilateral tap block , which was used as a sole anesthetic technique .
tap block can be considered as an anesthetic technique for abdominal surgery in moribund patients . |
a retrospective review of clinical records of late postpartum eclampsia patients who had presented with acute onset of visual and/or neurological symptoms to the emergency department of a medical college hospital in south india between april 2009 and may 2013 was done .
all patients had underwent mri with diffusion weighted imaging ( dwi ) and apparent diffusion coefficient ( adc ) mapping to differentiate vasogenic from cytotoxic cerebral edema .
patient records were analyzed for presence of visual symptoms , peak systolic and diastolic blood pressures , associated neurological symptoms , location of mri lesions and time taken for recovery .
the mr images were graded for extent and severity of cortex and white matter vasogenic edema , degree of confluence , mass effect and ventricular distortion on a scale from 1 to 5 .
statistical analysis was performed using chi - square test and a p value of less than 0.05 was considered to be statistically significant .
clinical and imaging findings are summarized in table 1 . clinical profile of patients with pres the 10 patients ranged in age from 21 to 32 years ( average-26 3.49 years ) .
mean peak systolic and diastolic blood pressures were 144 21.71 and 93 9.19 mm of hg , respectively .
bilateral visual loss was noted in five patients of whom three patients were able to perceive hand movements and two patients had only perception of light .
the most common abnormality noted on mri included bilateral symmetrical hyperintensities on t2-weighted images and fluid attenuated inversion recovery ( flair ) sequences in the parieto - occipital regions [ figs . 1 and 2 ] .
dwi showed high signal intensity with no areas of restricted diffusion while adc mapping did not show corresponding low signal intensity thus suggesting vasogenic edema .
axial mr image ( fluid attenuated inversion recovery ) showing confluent edema in subcortical and deep white matter ( grade 4 ) seen as hyperintensities in bilateral parieto - occipital lobes ( patient 6 ) axial mr image ( fluid attenuation inversion recovery ) showing edema in subcortical white matter ( grade 2 ) seen as hyperintensities in bilateral occipital lobes ( patient 9 ) five patients had additional abnormalities in the cerebellar hemispheres and three patients had involvement of frontal lobes . average time taken for visual recovery was 2.6 1.34 days .
follow - up mri in four patients after 1 month revealed complete resolution of these abnormalities . in the remaining , clinical symptoms and signs
follow - up scans were not performed due to financial constraints . the extent of cerebral edema in patients with and without visual loss was compared and results summarized in table 2 .
posterior reversible encephalopathy syndrome ( pres ) is a rare neurotoxic state that presents with altered mental status , headache , seizures , and visual disturbances along with neuroimaging features of vasogenic edema involving the posterior cerebral circulation .
although several cases of pres have been reported in neurology and neuroradiology literature , exposure in ophthalmic literature has been rather limited .
the causes of pres are diverse and include pre - eclampsia , eclampsia , renal insufficiency , solid organ transplantation and immunosuppressive therapy .
hypertensive encephalopathy is said to be the cause of this syndrome which has been demonstrated by various clinical and experimental studies .
patients with hypertensive encephalopathy have the same clinical signs as those with pres and they also have rapid resolution of clinical and imaging abnormalities once the blood pressure is lowered .
the most widely accepted theory states that sudden elevation of blood pressure causes failure of autoregulation in the cerebral blood vessels leading to hyperperfusion , breakdown of blood brain barrier , and vasogenic edema . the posterior circulation is preferentially affected since it has less sympathetic innervation than the carotid circulation , thus rendering it less able to adjust to blood pressure fluctuations . however , this theory is not comprehensive because pres can affect normotensives .
high degree of suspicion is required in these patients of late postpartum eclampsia because it occurs between 48 hours postpartum and 1 month after delivery frequently in women who have had a normal pregnancy and delivery and no signs of a pre - eclamptic syndrome .
alternative theory implicates endothelial dysfunction as the cause for occurrence of pres in eclampsia and sepsis .
this is supported by the fact that elevations in markers of endothelial dysfunction such as lactate dehydrogenase and abnormal red blood cell fragmentation have been reported in pres .
vasospasm with subsequent ischemia has also been hypothesized to cause loss of integrity of the blood brain barrier in eclampsia .
although reversible by definition early recognition and prompt treatment is essential to prevent secondary complications like intracerebral hemorrhage and infarction .
a large retrospective study of pres in diverse clinical situations revealed the occurrence of visual symptoms in 20% and headache in 26% while seizures was observed in 74% of cases .
the visual disturbances reported with pres include cortical blindness , visual neglect , homonymous hemianopia and blurred vision .
cortical visual loss was observed in five of our patients ( 50% ) while the remaining denied any visual disturbances .
our findings are in line with liman et al . , who observed that visual disturbances like cortical blindness , blurred vision and hemianopia are more common in eclampsia related pres ( 50% ) .
patients with pres due to other etiologies more often present with severe symptoms like altered mental status or neurological deficit and lesser visual disturbances ( 27.8% ) .
roth et al . , have also reported a higher percentage of disturbed vision in pre - eclampsia - eclampsia - related pres .
this could be because of the younger age and lesser associated comorbidities in these patients as compared to patients with pres due to other etiologies .
all patients ( with / without visual loss ) demonstrated bilateral symmetrical hyperintensities in the parieto - occipital regions on t2-weighted images .
this feature along with predominant involvement of the white matter distinguishes this syndrome from bilateral posterior cerebral artery territory infarction .
although several studies have reported visual symptoms of varying degrees in pres , to the best of our knowledge no study has attempted to correlate the visual symptoms with imaging abnormalities .
the limitations of our study include its retrospective design , and smaller sample size of postpartum eclampsia patients .
we conclude that there is a higher prevalence of cortical visual loss in patients with pres associated with late postpartum eclampsia .
patients with higher degree of vasogenic edema of the posterior cerebral white matter might present with visual loss .
ophthalmologists should be aware of this clinical entity since it is reversible and readily treated by controlling the blood pressure .
further prospective studies comprising larger sample size and different etiologies of pres are warranted in this regard . | the purpose of this study was to determine the prevalence of visual disturbances in patients with posterior reversible encephalopathy syndrome ( pres ) associated with late postpartum eclampsia .
we retrospectively reviewed the clinical records of late postpartum eclampsia patients with features of pres for the presence of visual disturbances and location of radiological abnormalities .
we found a higher prevalence of cortical visual loss in patients with pres associated with late postpartum eclampsia .
bilateral symmetrical vasogenic edema of the parieto - occipital lobe was the most common magnetic resonance imaging ( mri ) abnormality noted .
no significant differences were observed in the extent of edema in patients with and without visual loss . |
isotretinoin is a systemic synthetic retinoid used to treat moderate to severe nodulocystic acne vulgaris that is not responding to other therapies , as well as a number of other dermatologic diseases , including psoriasis , hidradenitis suppurativa , ichthyosis , lesions associated with lupus erythematosus , some cases of severe acne rosacea , and various skin cancers , or even adjunctive therapy for acute promyelocytic leukemia.1 among other adverse effects , isotretinoin has repeatedly been associated with depression , suicidality , and psychotic symptoms , although the link between isotretinoin use and psychiatric events remains controversial.2 remarkably , when affective psychosis and obsessive - compulsive disorder ( ocd ) occur following isotretinoin treatment , associated psychopathology is generally severe and eventually poorly responsive to conventional treatment , as highlighted by recent , large , retrospective studies.3 also , although patients suffering from bipolar disorder may be at increased risk for mood symptom exacerbations due to isotretinoin therapy , including suicidal ideation , even with a concurrent antimanic therapy,3 less is known about the risk for developing ex novo affective psychosis and antidepressant manic vulnerability when isotretinoin treatment is used during adolescence . furthermore , acne itself often occurs during adolescence , and can be eventually induced by lithium , a standard treatment for bipolar disorder , which , in turn , usually has its onset at the same age .
a 25-year - old caucasian male came to his first psychiatric consultation at the age of 23 years at the suggestion of his general practitioner due to complaints about intrusive obsessive thoughts starting about one year earlier .
obsessive symptoms essentially consisted of self - reproachment about his ineptitude in sitting academic examinations and consequent overwhelming feelings of guilt toward his parents .
there were no associated physical problems , although he reported a history of severe acne vulgaris that had been successfully treated by a dermatologist with isotretinoin 10 mg / day since the age of about 16 years .
initial evaluation led to a diagnosis of ocd , after excluding a personal or family history for psychiatric disorders .
diagnosis was also made on the basis of the mini international neuropsychiatric interview4 and yale - brown obsessive - compulsive scale,5 the total score for which was 26 ( 24 indicates severe ocd ) .
he was prescribed fluvoxamine 100 mg / day , having agreed to the proposal of dose augmentation within the following weeks .
two weeks later he had an unexpected precocious response with a marked ego - dystonic reaction , so current therapy was maintained .
more surprisingly , two weeks later , intrusive thoughts reappeared , now associated with loss , concomitant intractable delusions of grandeur , flight of ideas , and an increase in goal - directed activity ( eg , a sudden desire to start guitar lessons and to leave law school to become a theatrical agent ) .
a diagnosis of mixed state with psychotic features was made on the basis of a young mania rating scale6 total score of 24 ( 12 indicates mania , although mixed state assessment may be roughly estimated ) .
therapy was then revised , halving the antidepressant dose and adding 1200 mg / day of carbamazepine ( lithium , a neuroprotective antisuicidal drug having the potential to worsen psoriasis , and typical antipsychotic augmentation options being disregarded due to a likely lack of compliance and the patient s concerns about potential dermatologic recurrences ) . despite the proposal of cognitive behavioral therapy augmentation or other nonpharmacologic interventions , the patient refused to take advantage of any therapy likely to be free of side effects , apparently for compliance reasons . after a further month
the patient had a relapse of obsessive symptoms , with a substantial persistence of elation ( despite considerably high carbamazepine serum levels , usually increasing by up to 60% in case of fluvoxamine coadministration ) , so he was prescribed fluvoxamine 25 mg / day and olanzapine 5 mg / day .
doses were then increased every three weeks , reaching a final amount of 300 mg / day and 15 mg / day , respectively .
both obsessive and mood symptoms progressively remitted until almost complete resolution was observed six months later .
patients with ocd and related disorders , eg , body dysmorphic disorder , may have excessive concern about minor variations in skin color or texture , ordinary moles , or thinning hair.7 a minority of cases , eg , some patients with severe psychotic body dysmorphic disorder and acne vulgaris , may abuse dermatologic drugs and cosmetics to inhibit normal seborrhea , as in the dorian gray syndrome ( dream of eternal youth ) , eventually increasing the risk of long - term development of other psychiatric disorders , including affective psychosis.8 on the other hand , acne , alopecia areata , atopic dermatitis , and psoriasis may be associated with psychiatric conditions such as depression and suicidal ideation , as well as with the use of some psychotropic medications , possibly suggesting a substantial immunoneuroendocrine overlap.9 nonetheless , when a psychiatric comorbidity is diagnosed , patients seeking dermatologic care may interpret a suggestion to see a psychiatrist as indicating that the dermatologist thinks they are crazy or wants to get rid of them , thereby further reducing the chance of appropriate treatment . concerning the relationship between isotretinoin and the potential for subsequent onset of psychopathology ,
positive dechallenge and rechallenge cases have been reported , prompting physicians to look out for the onset or worsening of psychiatric symptoms , including bipolar disorder.10,13 nonetheless , the neurotoxicity of the synthetic retinoic acid analogs is supported by a considerable body of literature .
in fact , retinoids may lead to a decrease in dopaminergic orbitofrontal functioning via their effect on the hippocampus , which modulates dopaminergic function in the medial prefrontal cortex .
retinoic acid - induced deficits in hippocampal function may lead to a downstream effect on orbitofrontal function .
however , evidence regarding the effects of retinoic acid in the serotonin pathways is controversial , although higher doses have resulted in decreased or complete loss of serotonergic neurons.1 this finding has psychiatric implications because the prefrontal dopaminergic imbalance may be closely associated with depression .
recent evidence from positron emission tomography studies in human subjects showed isotretinoin to be associated with a decrease in orbitofrontal metabolism , which is known to play a major role in the symptomatology of both ocd and bipolar disorder .
in fact , the molecular components required for retinoic acid signaling are expressed in the adult brain , and the overlap of brain areas implicated in retinoic acid function , stress , and depression suggests that retinoids could play a role in affective and anxiety disorders,14 which are often comorbid . in this case , the choice of the selective serotonin reuptake inhibitor fluvoxamine was based on its proven efficacy in ocd and on its modulatory effects on -1 receptors , which might increase the antipsychotic effect of atypical antipsychotic drugs , possibly contributing to the early psychotic resolution showed by the patient described here.15,16 naturally occurring neurosteroids , as well as many psychotropic drugs , bind to -receptors , which may mediate some neuroprotective action , including mood and cognition improvement.17,18 although olanzapine and other atypical antipsychotic augmentation strategies for selective serotonin reuptake inhibitor - refractory , psychotic ocds and/or concomitant mood disorder are supported by an increasing body of literature,19,20 the choice of a single agent is often difficult.21 given their higher affinity for 5-ht2a receptors than for dopamine receptors , it has been speculated that atypical antipsychotics may induce ocd , even at low doses , due to high 5-ht2a antagonism , whereas improvement is thought to occur only at high doses in response to high dopaminergic antagonism.22 the choice of olanzapine augmentation for fluvoxamine therapy was also suggested by pharmacokinetic considerations .
fluvoxamine increases the olanzapine plasma concentration by approximately two - fold via cyp1a2 inhibition . on the other side , the alternative strategy of adding carbamazepine ( even at low doses ) to fluvoxamine and olanzapine
was discounted for compliance reasons . moreover , the patient did not originally show a satisfactory response to this antiepileptic drug even at 1200 mg / day ( whereas olanzapine was indeed more effective , especially in reducing psychotic features ) .
there were also pharmacokinetic considerations in the decision not to add carbamazepine , because it increases the renal clearance of olanzapine by about 45% and reduces the half - life of olanzapine by about 20%.23 in this case , the unusually rapid exacerbation on antidepressants , as well as the presence of plausible iatrogenic - induced depression with flights of ideas,24 pointed to a bipolar diathesis related to previous treatment with isotretinoin , suggesting the switch to an atypical antipsychotic that is associated with a rapid positive antipsychotic response .
indeed , antidepressant lability could occur even without any previous or current isotretinoin exposure , and it may be difficult to discriminate whether mood switches and rapid exacerbation phenomena are due to isotretinoin or not .
it is noteworthy that the patient had no prior familial or personal predictors of bipolar disorder , including during adolescence , which is the usual age of onset .
moreover , in this case it is remarkable that even a considerable dose of carbamazepine was not able to prevent the mood switch . also ,
the use of antidepressants such as fluvoxamine in the course of mixed states is debatable , although they are apparently well established in the management of ocd symptoms .
although the demonstration of a def initive causal relationship between isotretinoin use and increased risk for subsequent ocd development characterized by iatrogenic vulnerability for affective psychosis is beyond the scope of this report , the modern pathogenic concept of the vulnerability stress model25 ( with isotretinoin considered an essential stress factor ) of psychiatric disease may prompt further controlled research on this association .
finally , in addition to the suggestion of a relationship between isotretinoin and psychiatric disorders , this case report suggests that we should consider carefully both the course of illness and psychopharmacologic choices for patients reporting long - term use of isotretinoin during adolescence and complicated by ocd . | isotretinoin , a drug used for moderate to severe acne , has been repeatedly associated with various psychiatric complications , although a definitive causal relationship has not been established to date .
this case report describes a 25-year - old male who developed obsessive - compulsive disorder at the age of 23 years following isotretinoin treatment for acne ( 1020 mg / day ) since the age of 16 years .
although standard treatment for obsessive - compulsive disorder caused mood swings , the combination of fluvoxamine 300 mg / day and olanzapine 15 mg / day significantly improves the clinical picture .
although rare , severe adulthood psychiatric complications may occur following isotretinoin treatment , requiring management which is individually tailored to the patient . |
gentamicin ( gm ) is one of the amino glycoside drugs used in gram - negative infections .
nephrotoxicity is specified by renal dysfunction , which is distinguished by increasing serum levels of blood urea nitrogen ( bun ) and creatinine ( cr ) .
compounds such as lycopene , metformin , garlic , vitamin e , probucol , and erythropoietin could prevent renal damage induced by gm .
furthermore , several studies have suggested that supplementations of herbal extracts such as ginkgo biloba , bauhinia variegata , pongamia pinnata flower , and grape seed may attenuate gm - induced nephrotoxicity .
althaea officinalis ( marshmallow , marshmallow , or a common marshmallow ) , the member of malvaceae family , is well - known for its medicinal properties .
it is demonstrated that a. officinalis has potential therapeutic benefits in lipomia , inflammation , gastric ulcer , and platelet aggregation .
the pharmacological and antioxidant activities of a. officinalis refer to various compounds such as polysaccharides and flavonoids present in the plant . in the present study
, we attempted to investigate the effect of a. officinalis flower extract ( aofe ) as an antioxidant against nephrotoxicity - induced by gm in male rats .
adult male wistar rats ( animal centre , isfahan university of medical sciences ) were used in this study .
this research was approved in advance by the isfahan university of medical sciences ethics committee . dried violet flowers of a. officinalis
preparation of the extract was fulfilled in two steps ; first , 600 ml ethanol 70% was added to 150 g prepared powder and the total mixture was shaken for 24 h at the temperature of 23 - 25c .
, 600 ml ethanol 96% was added to the material remained from the first step and again the total mixture was shaken for 24 h at the temperature of 23 - 25c . the extract obtained after filtration in this step was mixed with the yield of the first step .
then , the total extract was incubated at the temperature of 50c for 48 h and finally 100% dried extract was obtained .
group 1 ( n = 6 ) as negative control group received aofe 250 mg / kg / day for 9 days , and saline was added from day 3 ongroups 2 ( n = 5 ) as positive control group received saline during the study and gm ( 100 mg / kg / day ) was added from day 3 ongroup 3 ( n = 6 ) received aofe 50 mg / kg / day for 9 days , and gm ( 100 mg / kg / day ) was added from day 3 on . groups 4 ( n = 7 ) and 5 ( n = 6 ) had the same regimen of group 3 except aofe dose which were 250 mg / kg / day and 500 mg / kg / day , respectively . all administrations were done intraperitoneally . at the end of the experiment ,
blood samples were obtained via heart puncture , and the serum was kept at 20c to measure the serum levels of bun and cr . finally , the animals were killed .
the kidneys were removed and weighed immediately . left kidney was fixed in formalin and staining was performed to detect the tissue damage .
group 1 ( n = 6 ) as negative control group received aofe 250 mg / kg / day for 9 days , and saline was added from day 3 on groups 2 ( n = 5 ) as positive control group received saline during the study and gm ( 100 mg / kg / day ) was added from day 3 on group 3 ( n = 6 ) received aofe 50 mg / kg / day for 9 days , and gm ( 100 mg / kg / day ) was added from day 3 on .
groups 4 ( n = 7 ) and 5 ( n = 6 ) had the same regimen of group 3 except aofe dose which were 250 mg / kg / day and 500 mg / kg / day , respectively .
blood samples were obtained via heart puncture , and the serum was kept at 20c to measure the serum levels of bun and cr . finally , the animals were killed .
the kidneys were removed and weighed immediately . left kidney was fixed in formalin and staining was performed to detect the tissue damage .
the left kidney was fixed in 10% neutral formalin and embedded in paraffin . after slicing , hematoxylin and eosin staining
was performed to examine tissue damage including tubular atrophy , cast , debris , and necrotic materials in the tubular lumen .
intensity of tubular lesion was scored from 1 to 4 , while zero score was assigned to normal tissue without damage .
the two groups were compared with regard to the serum levels of bun and cr , and kidney weight ( kw ) by independent student 's t - test .
the parameters were analyzed by one - way anova followed by least significant difference test among the groups .
the kidney tissue damage score ( ktds ) was compared using kruskal wallis or mann whitney tests .
preparation of the extract was fulfilled in two steps ; first , 600 ml ethanol 70% was added to 150 g prepared powder and the total mixture was shaken for 24 h at the temperature of 23 - 25c .
, 600 ml ethanol 96% was added to the material remained from the first step and again the total mixture was shaken for 24 h at the temperature of 23 - 25c . the extract obtained after filtration in this step was mixed with the yield of the first step .
then , the total extract was incubated at the temperature of 50c for 48 h and finally 100% dried extract was obtained .
group 1 ( n = 6 ) as negative control group received aofe 250 mg / kg / day for 9 days , and saline was added from day 3 ongroups 2 ( n = 5 ) as positive control group received saline during the study and gm ( 100 mg / kg / day ) was added from day 3 ongroup 3 ( n = 6 ) received aofe 50 mg / kg / day for 9 days , and gm ( 100 mg / kg / day ) was added from day 3 on .
groups 4 ( n = 7 ) and 5 ( n = 6 ) had the same regimen of group 3 except aofe dose which were 250 mg / kg / day and 500 mg / kg / day , respectively .
blood samples were obtained via heart puncture , and the serum was kept at 20c to measure the serum levels of bun and cr . finally , the animals were killed .
group 1 ( n = 6 ) as negative control group received aofe 250 mg / kg / day for 9 days , and saline was added from day 3 on groups 2 ( n = 5 ) as positive control group received saline during the study and gm ( 100 mg / kg / day ) was added from day 3 on group 3 ( n = 6 ) received aofe 50 mg / kg / day for 9 days , and gm ( 100 mg / kg / day ) was added from day 3 on .
groups 4 ( n = 7 ) and 5 ( n = 6 ) had the same regimen of group 3 except aofe dose which were 250 mg / kg / day and 500 mg / kg / day , respectively .
blood samples were obtained via heart puncture , and the serum was kept at 20c to measure the serum levels of bun and cr . finally , the animals were killed .
the kidneys were removed and weighed immediately . left kidney was fixed in formalin and staining was performed to detect the tissue damage .
the left kidney was fixed in 10% neutral formalin and embedded in paraffin . after slicing , hematoxylin and eosin staining
was performed to examine tissue damage including tubular atrophy , cast , debris , and necrotic materials in the tubular lumen .
intensity of tubular lesion was scored from 1 to 4 , while zero score was assigned to normal tissue without damage .
the two groups were compared with regard to the serum levels of bun and cr , and kidney weight ( kw ) by independent student 's t - test .
the parameters were analyzed by one - way anova followed by least significant difference test among the groups .
gentamicin itself induced nephrotoxicity , which was confirmed by increasing in the serum levels of bun and cr as well as elevating in ktds and kw ( p < 0.05 ) [ table 1 ] .
administration of various doses of aofe accompanied with gm did not attenuate the serum levels of bun and cr ; rather it increased the values ( p < 0.05 ) .
high dose of aofe aggravated renal damage induced by gm in comparison with other groups ( p < 0.05 ) [ figure 1 ] .
sample images from group 1 treated with aofe alone and group 5 treated with gm plus high dose of aofe are demonstrated in figure 2 .
bun and cr serum levels , ktds , and kw g/100 g body weight in the experiment groups blood urea nitrogen ( bun ) and creatinine ( cr ) serum levels , kidney tissue damage score ( ktds ) , and kidney weight g/100 g of body weight in the experiment groups .
gentamicin ( gm ) , althaea officinalis flower extract ( aofe ) 50 + gm , aofe
250 + gm , and aofe 500 + gm groups received saline , aofe 50 mg / kg / day , aofe 250 mg / kg / day , and aofe 500 mg / kg / day for 9 days , respectively , and gm ( gm ; 100 mg / kg / day ) was added from day 3 on .
indicates significant difference from aofe 250 + gm group sample images from group 1 treated with althaea officinalis flower extract ( aofe ) alone ( left ) and group 5 treated with gm plus high dose of aofe ( right ) .
the aim of this study was to investigate whether aofe could ameliorate nephrotoxicity induced by gm in the male rat .
we observed that aofe administration did not ameliorate nephrotoxicity induced by gm ; rather it intensified renal failure .
gm induces renal dysfunction , which is characterized by increase in levels of cr , uric acid , and bun .
in addition , it is accompanied with tissue alterations such as glomerular congestion , disruption of glomerular capillaries , vacuolar degeneration of tubular epithelial cells , and hyaline cast formation .
furthermore , the present study indicated that gm enhanced normalized kw probably due to edema caused by tubular necrosis .
useful properties of a. officinalis flower were documented in the literature , but we did not obtain positive results in the administered doses . it is demonstrated that administration of 50 mg / kg dose of a. officinalis flower result in a significant increase in serum hdl cholesterol level . also , antiinflammatory and antiulcerogenic effects of the extract were observed at doses of 50 , 100 , and 250 mg / kg .
in contrast , we observed that doses of 50 and 250 mg / kg of aofe aggravated the increased levels of bun and cr induced by gm .
in addition , aofe at the dose of 500 mg / kg aggravated both renal dysfunction and tissue damage .
it has reported that increasing the dose of aofe to 500 mg / kg significantly decreased stool water content .
it is also reported that high doses of some antioxidants provide a harmful effect on survival of the subjects .
therefore , it is possible that aofe at the doses lower than 50 mg / kg may ameliorate nephrotoxicity induced by gm .
although aofe acts as an antioxidant , doses of aofe used in the current study did not ameliorate nephrotoxicity induced by gm , and it is necessary to test doses lower than 50 mg / kg . | background : gentamicin ( gm ) is used as antibiotic for gram - negative infections , but its administration is limited due to a side - effect of nephrotoxicity . it was attempted to investigate the effect of althaea officinalis flower extract ( aofe ) against nephrotoxicity induced by gm in male rats.methods:30-year-old male wistar rats were divided into five groups .
group 1 as a negative control group received aofe 250 mg / kg / day .
groups 2 - 5 received saline , aofe 50 mg / kg / day , aofe 250 mg / kg / day , and aofe 500 mg / kg / day for 9 days , respectively , and gm ( 100 mg / kg / day ) was added from the 3rd day on . at the end of the experiment , blood samples were obtained , animals were sacrificed , and the kidneys were removed immediately.results:gentamicin ( in group 2 ) significantly increased serum levels of blood urea nitrogen and creatinine as well as the pathological damage score ( p < 0.05 ) when compared with group 1 .
low dose of aofe did not decrease the nephrotoxicity induced by gm while the high dose of aofe aggravated renal toxicity ( p < 0.05).conclusions : although aofe acts as an antioxidant , at the doses used in the current study did not ameliorate nephrotoxicity induced by gm . |
institutional review board approval was obtained from the wake forest university , and informed consent forms were completed for all participants .
items for four concepts identified in the literature were glucose control , well - being and side - effects , lifestyle burden , and treatment complexity and convenience and were evaluated in a series of five focus groups made up of five to eight patients drawn from an evaluation study of community diabetes clinics in north carolina ( 11 ) .
participants were male and female and white and nonwhite with simple and complex medication regimens and a1c levels that ranged from well controlled to uncontrolled .
the resulting 35-item prototype instrument was administered by mail to a convenience sample of 75 patients ( the exploratory sample ) , who were treated with diabetes medications at our study community - care site , to assess item reliability , mean and distribution , redundancy or uniqueness , skewness , and construct validity .
also examined were item correlations with a1c level , the multidimensional diabetes questionnaire ( 12 ) lifestyle interference scale , the medical outcomes studies ( mos ) health worries scale score ( 13 ) , and global items assessing extent that blood glucose has been unacceptably high or low . an item performance score was constructed ( 0 , weak ; 1 , moderate ; or 2 , ideal performance ) to guide item retention .
fifty - five ( 73% ) patients completed the survey , and nine items were removed based on skewness or redundancy ( r > 0.75 ) with other items . in the initial test sample
, patients of a large family - medicine practice treated for diabetes with a recent a1c value within the last 3 months ( the evaluation sample ) were invited to complete the study survey packet including the revised 26-item instrument and validation instrument described above .
medication complexity was assessed using a score of 0 or 1 ( no / yes ) for common diabetes medications and a score of 0 or 2 ( no / yes ) for insulin , a more demanding regimen .
self - reported adherence to medications was by recall of skipped or missed doses over the last 10 days .
packets were mailed to patients with instructions and a voucher for a 25 usd gift certificate .
exploratory factor analysis ( efa ) of the dmsat items was conducted using sas ( version 8 ; sas , cary , nc ) to assess whether the common factor model was appropriate ( 14 ) based on kaiser 's sampling adequacy , scree plot , and model fit .
discriminant validity of the dmsat was examined by comparing means across levels of a1c ( < 8% and 8% ) , treatment complexity ( low and high ) , self - reported adherence , and mos health worries . for the final test sample ,
another sample of patients from our community diabetes care clinics ( 11 ) and from an academic medical center was recruited to conduct and evaluate confirmatory factor analysis of the dmsat and confirm validity
in the evaluation sample , 194 ( 63% ) of 307 eligible patients returned the survey packet ; of these , 140 reported current medication use .
participants had a mean age of 63 years , and most had completed high school ( 77% ) and had been diagnosed with diabetes at least 5 years previously ( 61% ) .
one - third ( 2939% ) were taking one , two , or three medications for diabetes , with 16% taking insulin ; 14% had a recent a1c > 8.0% , and 19% rated their adherence to their medication regimen in the last 10 days as less than complete .
ten items displayed high inter - item correlations ( > 0.75 ) and were removed .
initial factor analysis of the reduced 16-item questionnaire identified a four - factor structure consistent with our domains of lifestyle , medical control , convenience , and well - being and explained 75% of the total variance .
as shown in table 1 , dmsat scales and total score discriminated ( p < 0.05 ) between high and low levels of treatment complexity , self - rated glucose control , mos health worries scale score , and clinical value for recent a1c ( < 8% vs. 8% ) in the expected direction .
correlation of the dmsat scores with continuous a1c values was 0.24 ( p = 0.0049 ) . in the final , confirmatory sample ,
as shown in table 1 , dmsat scales and total scores discriminated between validity groups as in the previous sample and were highly correlated with the dtsq ( r = 0.68 ; p < 0.001 ) . unlike the dmsat , the dtsq total score did not discriminate between levels of treatment complexity and clinical a1c value .
the dmsat is intended as a brief measure of diabetes medication treatment satisfaction and discriminates between important correlates of patient management .
it performed as well as the dtsq in detecting self - rated glucose control and health worries but showed superior properties in correspondence with treatment complexity and a1c .
note that appraisals of cost of medications or specific side effects that may be caused by diabetes or its treatment , such as diminished sexual functioning , bloating , or weight gain , are not separately assessed and may require assessment elsewhere .
, we believe that the 16-item dmsat offers a comprehensive assessment of satisfaction with diabetes therapy and may aid in individualizing patient diabetes treatment . | objective to develop and test a patient questionnaire on treatment satisfaction with diabetes regimens.research design and methods survey items were developed from community clinic focus groups , pretested in patients with diabetes , and examined in two samples of treated patients.resultssixteen items performed well in assessing treatment experiences : ease and convenience , lifestyle burdens , well - being , and medical control .
construct validity was supported by associations ( p < 0.05 ) with treatment complexity , self - rated glucose control , health worries , and a1c .
internal consistency ranged from 0.89 to 0.95.conclusionsthe diabetes medication satisfaction tool offers a comprehensive assessment of patient acceptability , with diabetes therapy useful for individualizing therapeutic decision making . |
the incidence of infective endocarditis ( ie ) remained relatively stable from 1950 through 2000 at about 3.6 to 7.0 cases per 100,000 patient - years.1 in selected areas , the incidence may rise because of the concentration of populations at uniquely high risk of infection , specifically intravenous ( iv ) drug abusers .
the risk of ie among iv drug abusers , 2 to 5% per patient - years , is several - fold greater than that for patients with rheumatic heart disease or prosthetic valves .
ie is located on the tricuspid valve in 46 to 58% of patients with iv drug abuse .
staphylococcus aureus causes more than 50% of ie occurring in iv drug abusers overall and 60 to 70% of infection involving the tricuspid valve .
the clinical manifestation of ie in iv drug abusers depends on the valve involved and , to a lesser degree , on the infecting organism .
the sensitivity of transthoracic echocardiography ( tte ) for the detection of vegetations in patients with native valve endocarditis ( nve ) is approximately 65% , whereas that of transesophageal echocardiography ( tee ) in these patients is 85 to 95%.2 tee is the preferred approach in patients in whom tte is technically suboptimal and is the procedure of choice for imaging the pulmonic valve.3 when initial tee is negative and the clinical suspicion of ie remains , repeating tee within 7 to 10 days is advocated.4 perivalvular abscess or intracardiac fistula formation occurs in 10 to 14% of patients with nve.6 persistent , otherwise unexplained , fever despite appropriate antimicrobial therapy in patients with ie suggests infection extending beyond the valve leaflet .
tee is superior to tte for detecting invasive infection in patients with nve and prosthetic valve endocarditis ( pve ) .
cardiac surgery should be considered to debride abscesses , allowing the eradication of uncontrolled infection , and to reconstruct cardiac structures , restoring homodynamic and alleviating congestive heart failure .
a 21-year - old man , who was an iv drug abuser , presented with fever and dyspnea .
tte showed highly mobile , large vegetation on the anterior leaflet of the tricuspid valve and no vegetation on the aortic valve , mitral valve , and pulmonic valve .
the patient was admitted to the cardiology ward for antibiotic therapy and received vancomycin and gentamycin .
the blood culture of the patient became positive for staphylococcus areus 3 times in 24-hour intervals . despite the antibiotic therapy for 10 days
tee revealed severe aortic insufficiency , moderate tricuspid regurgitation , and an echo - lucent space between the tricuspid and aortic valves ( figures 1 ) .
color doppler demonstrated a flow within the echo - lucent space and a connection between that and the left ventricle , suggesting a perivalvular abscess of the tricuspid valve opening in the left ventricle ( figure 2 ) .
after consultation with a cardiac surgeon , the patient was transferred to the operating room , where he unfortunately expired due to severe bleeding and disseminated intravascular coagulation .
ie is a serious complication of iv drug abuse , with a reported mortality of 5 to 10%.1 endocarditis in iv drug abusers commonly involves the tricuspid valve , and staphylococcus aureus is the most common causative organism .
dyspnea , cough , and chest pain are the common complaints of iv drug users .
this is likely related to the predominance of tricuspid valve endocarditis in this group and secondary embolic showering of the pulmonary vasculature . within a week after the initiation of effective antimicrobial therapy , almost 70% of patients with nve or pve are afebrile and 90% have defervesced by the end of the second week of treatment.5 persistence or recurrence of fever more than 10 days after the initiation of antibiotic therapy identifies patients with increased mortality rates and with complications of infection or therapy.5 patients with a prolonged or recurrent fever should be evaluated for intracardiac complications .
perivalvular infection beyond the valve leaflet results in abscesses in the annulus or adjacent structures , intracardiac fistulas , and purulent pericarditis .
periannular extension is common , occurring in 10 to 40% of all native valve ie and complicates aortic valve endocarditis more commonly than mitral or tricuspid valve endocarditis.3 intra - cardiac fistulas are rarely seen and they are estimated to account for < 1% of all cases of ie.6 fistulization of the paravalvular abscess has been found in 6 to 9% of all cases.7 perivalvular abscess and intracardiac fistula of the tricuspid valve is very rare .
ie is a lethal disease if not treated aggressively with parental antibiotics , often in combination with surgery .
cardiac surgery should be considered in patients with perivalvular abscess and intracardiac fistula to debride abscesses and to reconstruct cardiac structures , restoring homodynamic and alleviating congestive heart failure .
| infective endocarditis is a serious complication of intravenous ( iv ) drug abuse , with a reported mortality of 5 to 10% . a 21-year - old man , who was an intravenous drug abuser , presented with fever and dyspnea .
transthoracic echocardiography showed a highly mobile , large vegetation on the anterior leaflet of the tricuspid valve . despite antibiotic therapy for ten days , the patient remained febrile .
transesophageal echocardiography revealed severe aortic regurgitation and an echo - lucent space between the tricuspid and aortic valves .
color doppler demonstrated a flow within the echo - lucent space and a connection between that and the left ventricle , suggesting a perivalvular abscess of the tricuspid valve opening in the left ventricle .
the patient was transferred to the operating room , where he unfortunately expired . |
six hundred officers were recruited from the police departments of new york , ny , and oakland and san jose , calif , usa . fifty - two percent of the sample was caucasian .
the mean age was 36,50 years ( standard deviation [ sd])=6.96 ) . years in the police force everaged 12.37 ( sd=6.78 ) .
number of exposure to critical incidents ranged from 2 to 670 ( mean [ m]= 171.27 , sd=130.93).the incident selected for completing the questionnaires had occured on average 6.50 ( sd= 5.11 ) years ago .
the peritraumatic distress scale ( pds ) was used to assess emotional , cognitive , and physical reactions occuring during a critical incident and immediatly after .
dissociation at the time of the incident was measured with the peritraumatic dissociative experience questionnaire ( pdeq).the impact of event scale - revised ( ies - r ) was used to measure ptsd symptoms in the last 7 days .
the mississippi scale ( mcs ) was used to measure ptsd and associated symptoms since the critical incident .
we conducted a cronbach alpha reliability analysis and an oblique principal factor analysis with promax rotation on the items of the pds .
two series of hierarchical multiple regression analyses were conducted using sociodemographics ( gender , ethnicity , years of service ) , exposure , the pdeq and pds as predictors of either the mcs or the ies - r
six hundred officers were recruited from the police departments of new york , ny , and oakland and san jose , calif , usa . fifty - two percent of the sample was caucasian .
the mean age was 36,50 years ( standard deviation [ sd])=6.96 ) . years in the police force everaged 12.37 ( sd=6.78 ) .
number of exposure to critical incidents ranged from 2 to 670 ( mean [ m]= 171.27 , sd=130.93).the incident selected for completing the questionnaires had occured on average 6.50 ( sd= 5.11 ) years ago .
the peritraumatic distress scale ( pds ) was used to assess emotional , cognitive , and physical reactions occuring during a critical incident and immediatly after .
dissociation at the time of the incident was measured with the peritraumatic dissociative experience questionnaire ( pdeq).the impact of event scale - revised ( ies - r ) was used to measure ptsd symptoms in the last 7 days .
the mississippi scale ( mcs ) was used to measure ptsd and associated symptoms since the critical incident .
we conducted a cronbach alpha reliability analysis and an oblique principal factor analysis with promax rotation on the items of the pds .
two series of hierarchical multiple regression analyses were conducted using sociodemographics ( gender , ethnicity , years of service ) , exposure , the pdeq and pds as predictors of either the mcs or the ies - r
the pds scores ranged from 0.10 to 3.57 and the mean was 1.37 ( sd=0.56 ) .
the scale was internally consistent ( =0.80 ) and showed strong convergent validity with the pdeq , r(599)=0.55 , p<0.001 .
the pds factor solution is presented in table i items defining factor 1 included dysphoric emotions such as helplessness , sadness and grief , frustration and anger , and horror .
factor 2 was mostly defined by items related to loss of safety and arousal , such as being afraid , thinking one might die , and having intense bodily reactions ( sweating , shaking , heart - pounding ) .
items loading on factor 3 were related to the loss of positive beliefs about the self and others , such as thinking that one had done all he or she could during the critical incident , not felling prepared by one 's experience , and not believing tha others understood .
we labeled the factors negative emotions , perceived life threat and bodily arousal , and appraisal .
the sum of communality estimates was 7.58 , explaining 38% of the communality estimates was 7.58 , explaining 38% of the total variance and 93% of trace .
intercorrelations among the pds factors were low , ranging from -0.25 to 0.12 ( p<0.05 ) .
the low pds factor intercorrelation coupled with correlations of 0.17 to 0.42 ( p<0.001 ) with the outcome measures ( ies - r and mcs ) suggest that various forms of peritraumatic distress , as captured by the pds , can lead to the development of ptsd symptoms .
two stepwise regression analyses ( not fully reported here ) were conducted . in predicting the mcs and ies - r ,
the pdeq , entered in the second step , explained 20% and 16% of unique variance on the mcs and ies - r , respectively . entering the pds in step 3 explained 11% and 8% unique variance on the mcs and ies - r , respectively .
we repeated this set of analyses with the inclusion order of the pdeq and pds reversed .
entered in the second step , the pds explained 29% and 17% of unique variance on the mcs and ies - r , respectively . entered in the third step , the pdeq explained 3% of unique variance on both the mcs and the ies - r .
the items and factors of the pds provide insight as to what some of the salient peritraumatic dimensions may be , in addition to peritraumatic dissociation . in this study ,
the pds explained a significant amount of variance over and above peritraumatic dissociation which is currently considered among the mos powerful predictors of ptsd symptoms .
test - retest data for the pds is currently being gathered as well as data from individuals not working in the police . in future
, it would be useful to investigate prospectively the power of the pds in predicting ptsd diagnosis rather than symptoms , as well as other trauma - related disorders . | posttraumatic stress disorder ( ptsd ) occurs when significant intrusion , avoidance , and hyperarousal symptoms are manifest for at least 1 month following exposure to a traumatic event , with at least 1 month following exposure to a traumatic event , with at least 1 month elapsed betwenn the event and the diagnossis ( diagnostic and statistical manual of mental disorders , 4th edition , 1994 [ dsm - iv]).1 however , such symptoms are not necessarily manifest in the immediate aftermath of the trauman,2 nor does their intial presence strongly predict who will develop ptsd.3 one immediate response to trauma which has been convicingly linked to ptsd symptoms is peritraumatic dissociation.4 in this poster , we briefly introduce a new scale assessing immediate responses distinct from peritraumatic dissociation , and we examine its power to predict ptsd symptoms . |
the most important goal of health systems is improving quality , effectiveness and efficiency of healthcare services ( 1 ) .
however , scattered health information has a negative effect on healthcare services and would impose more expenses to the healthcare system ( 2 ) .
electronic health record ( ehr ) as a modern method of storing and processing health information is a solution for imp - roving quality , safety and efficiency of patient care and health system .
the basis of ehr is clinical information ; however , it is used not only by clinicians , but also by those who manage healthcare operations , quality , risk , research , education and payment .
however , iso / tr 20514 defined it as a repository of information regarding the health status of a subject of care in computer processable form , stored and transmitted securely , and accessible by multiple authorized users .
it has a standardized or commonly agreed logical information model which is independent of ehr systems .
its primary purpose is the support of continuing , efficient and quality integrated healthcare and it contains information which is retrospective , concurrent , and prospective ( 4 ) .
the benefits of ehr have been examined by considering clinical , organizational , and social outcomes .
however , social outcomes include more ability for conducting research and improving population health ( 5 ) .
it should even be capable of integrating data across providers and from personal medical records to form a longitudinal health record .
however , ehr requires a significant investment of time and money , while many of healthcare providers have very limited access to capital .
that is why the cost benefit issue and the application of open source software ( oss ) have been taken into consideration in some ehr projects ( 3 ) .
the present study identifies the potential impact of application of open source software in developing national electronic health record in iran .
open source software ( oss ) , free and open source software ( foss ) and free , libre and open source software ( floss ) -
although are not exactly the same- are alternative terms to describe free software ( 6 ) .
there are basic principles for open source software including free redistribution , inclusion of source code , allowing for modification , integrity of the author s source code , no discrimination against any person or group , no discrimination against fields of usage , no need for an additional license
. also the license should not be specific to a product or technology and should not restrict other software ( 7 ) .
the promise of open source software is better quality , higher reliability , more flexibility , lower cost , and an end to predatory vendor lock - in .
open source technology is one of the most effective mechanisms for technology transfer which could foster the national economy .
this is because the source codes and licensing of the software are free of charge and open for inspection and usage ( 9 ) .
today , a wide range of open source applications is accessible through the web including web servers , web browsers , databases , programming languages , image editors and email clients ( 1015 ) .
oss could provide competition in commercial market , encourage innovation and promote compatibility and interoperation in health domain .
this would guarantee better quality and lower cost . also it would make the systems more responsive to changing clinical and organizational requirements ( 16 ) .
several communities work on developing open source ehr systems to reduce the costs of ehr deployment and maintenance ( 17 ) . in america , many systems deployed by the u.s .
nowadays , a large number of health information systems are available as open source evidence based guideline and decision support system ( egadss ) is an open source clinical decision support system ( cdss ) project began in may 2004 to provide patient specific point of care reminders .
egadss would respond to requests from electronic medical records to provide patient specific clinical guidance based on its internal guidelines .
it operates on decision algorithms , medical knowledge , and specific patient data to generate recommendations and guidance for clinicians in order to aid them provide high quality care ( 19 ) .
care2x is a hospital information system through which the authorized staff like physicians can access patients data even from outside the hospital without using specialized software .
open source cluster application resources ( oscar ) is open source electronic medical record ( emr ) which was started by the department of family medicine at mcmaster university in 2001 .
it contains healthcare information with a lot of functions including complete scheduling , e - charting , prescribing , billing , lab downloads , tables and graphs , chronic disease management tools , disease registry , automated customizable forms , sophisticated antenatal care record and planner , tabulation of outstanding preventative and intervention practices , and also research tools .
oscar is used by healthcare professionals including physicians , nurses , midwives , social workers , psychologists and physiotherapists ( 21 ) .
open source ehr project directed by the american academy of family physicians ( aafp ) is an attempt to lower the cost of ehr by sharing the source code ( 3 ) another example is orca ( open receipt computer advanced ) which is a project of information and communication infra - structure based on oss to overcome problems of high costs , inflexibility and restrictions associated with proprietary systems . about 14,000 medical providers in japan are using products of orca ( 22 ) .
the advantages of an open source ehr outweigh the costs of a proprietary ehr system ( 23 ) .
open source technology makes the opportunity for advanced innovation in the health information sector of low income countries ( 24 ) . in iran several scattered attempts to design
these systems were produced by different companies and applied in different regions . as they did not follow particular standards , integrating their outputs
however , development of a set of standards is always a necessary requirement for integrating different information systems ( 26 ) . in iran ,
several studies on medical information systems have been done focusing on various domains especially on usability , efficacy , standards , architecture , structure and content . in 2001 ,
the usability of software packages in medical records departments of several hospitals in tehran was compared .
the findings showed that the overall usability of medical records software packages in the hospitals was 55% which indicated the available packages did not have the necessary capabilities to meet the requirements of medical records departments ( 27 ) . in another study nine hospital information systems applied in a number of hospitals in iran
the results showed that total average of compatibility of systems with the general requirements of his was 65.4% , ( 28 ) . in 2006 , an object - oriented model for some key messages was designed to support ehr through comparing weaknesses and strengths of hl7 and openehr standards . according to the study ,
it could ensure open , future - oriented electronic health records in iran ( 29 ) . in 2007 , ehr content , structure , and terminology standards in selected organizations were investigated in order to design an appropriate model of ehr for iran .
the study suggested a national model for ehr based on the weakness and capabilities of standards provided by the investigated organizations ( 2 ) . according to the evidences ,
a wide range of information systems are used in iranian hospitals , clinics , pharmacies and offices .
however , the majority of these systems are originally designed as non - shareable . to design an integrated ehr system , all components of the health system
therefore , the available clinical data on the existing systems in iran have neither been applied in decision making nor served as a basis for evidence based medicine .
this situation addresses the need for developing an integrated shareable information system of ehr in iran .
such a system will not only help using the collected data for different purposes , but also serves as an infrastructure for a variety of e - health activities in the country ( 25 ) .
the evidences also showed that financial limitation is one of the obstacles to implement electronic health records in developing countries .
therefore , establishment of an open source ehr system capable of modifications according to the national requirements seems to be inevitable in iran .
establishment of a national ehr requires a wide cooperation and coordination among involved ministries and organizations .
the prerequisites for a national ehr can be summarized as follows :
electronic record in a national extent including health data centerfacilities for electronic recording and transferring information produced in health data centers including hardware and software equipments and also cultural infrastructuresdeveloping standards for coding and data exchangedevelopment of information technology infrastructures with sufficient band - width and connections to meet the present and future needs of national health systemdevelopment plan for communication and messaging services to support new information cycles ( 25 ) .
electronic record in a national extent including health data center facilities for electronic recording and transferring information produced in health data centers including hardware and software equipments and also cultural infrastructures developing standards for coding and data exchange development of information technology infrastructures with sufficient band - width and connections to meet the present and future needs of national health system development plan for communication and messaging services to support new information cycles ( 25 ) .
the project of iranian electronic health record ( sepas ) supported by iranian ministry of health is the greatest information technology project in the health sector in the country . at the present time , the infrastructure of sepas is being processed to provide a platform for data exchange among health centers all over the country .
health information of all citizens from birth to death will be recorded in their individual ehrs . as a result
the key values of sepas include providing citizen rights , justice , data confidentiality and information security .
the main objectives of this project are :
integration of citizens health information;improving the quality of health services;equitable distribution of health resources;improving the management of health resources and improving healthcare system based on quick access to correct and accurate information;expediting and facilitating production and management of biomedical knowledge;contribution to the development of evidence based medicine;reducing complications resulted from medical errors;reducing healthcare costs;increasing security of health information;strengthening the monitoring role of the health system;improving the quality of medical education;providing new electronic services;improving the industry of healthcare in iran ( 30 ) .
integration of citizens health information ; improving the quality of health services ; equitable distribution of health resources ; improving the management of health resources and improving healthcare system based on quick access to correct and accurate information ; expediting and facilitating production and management of biomedical knowledge ; contribution to the development of evidence based medicine ; reducing complications resulted from medical errors ; reducing healthcare costs ; increasing security of health information ; strengthening the monitoring role of the health system ; improving the quality of medical education ; providing new electronic services ; improving the industry of healthcare in iran ( 30 ) . according to the architecture plan for national ehr in iran
, information systems would be categorized in three levels which are peripheral , intermediate and central .
these systems are independent and connected to one another via certain standard protocols . as a result ,
information would flow upwards through the following levels and circulate throughout the whole network :
information system level 1 ( peripheral level ) which is connected to medical equipments or portable devices such as smart cards or pdas .
it can simply be used at intermediate health providing centers for the ease of data entry and transfer.information system level 2 ( intermediate level ) which is installed and utilized in health providing centers .
such systems are customized according to the needs and specification of healthcare providing centers.information system level 3 ( central level ) which will be installed at the ministry of health and medical universities .
information system level 1 ( peripheral level ) which is connected to medical equipments or portable devices such as smart cards or pdas .
it can simply be used at intermediate health providing centers for the ease of data entry and transfer . information system level 2 ( intermediate level ) which is installed and utilized in health providing centers .
information system level 3 ( central level ) which will be installed at the ministry of health and medical universities .
development of electronic health record in iran would increase :
public access to healthcare services especially in deprived regions and evenly distribution of healthcare services throughout the country;modification of the national health management system based on the evidence and precise data;utilization of healthcare resources;delivery of modern electronic services such as teleappointing , teleconsulting , reminder and alerting systems;provision of a proper background for production and management of medical knowledge;supervision , evaluation and audition of healthcare services;establishment of statistical registry system;community cooperation in public health;individual accessibility to personal health information to choose the appropriate method of diagnosis and treatment;provision of necessary tools for clinical decision making;the work flow of the whole system through reengineering the process and performance of different units;the income cycle;citizens ` satisfaction;quality of care through developing a standard method for collection and interpretation of data;time and cost effectiveness and efficiency;medical knowledge by creating a suitable structure for retrospective as well as prospective medical researches;documentation and audition of documents;following the medical guidelines through sending alerts by the system;management of health centers;and the accessibility of medical information in cases of crisis and natural disasters .
public access to healthcare services especially in deprived regions and evenly distribution of healthcare services throughout the country ; modification of the national health management system based on the evidence and precise data ; utilization of healthcare resources ; delivery of modern electronic services such as teleappointing , teleconsulting , reminder and alerting systems ; provision of a proper background for production and management of medical knowledge ; supervision , evaluation and audition of healthcare services ; establishment of statistical registry system ; community cooperation in public health ; individual accessibility to personal health information to choose the appropriate method of diagnosis and treatment ; provision of necessary tools for clinical decision making ; the work flow of the whole system through reengineering the process and performance of different units ; citizens ` satisfaction ; quality of care through developing a standard method for collection and interpretation of data ; time and cost effectiveness and efficiency ; medical knowledge by creating a suitable structure for retrospective as well as prospective medical researches ; documentation and audition of documents ; following the medical guidelines through sending alerts by the system ; management of health centers ; and the accessibility of medical information in cases of crisis and natural disasters . on the other hand , developing electronic health record in iran could decrease :
medical errors and the consequent complications;mortality rate caused by medical errors;drug adverse effects through recording individuals previous reactions;need for more human resources especially in accounting and medical records departments;cost of healthcare by cutting repeated or unnecessary services;cost of stationary materials e.g. paper charts and radiology films and fixed expenses for equipments;accounting , documentation and archiving expenses;required time for retrieving and analyzing medical data;waiting time for admission;hospitalization time ( 31 ) .
medical errors and the consequent complications ; mortality rate caused by medical errors ; drug adverse effects through recording individuals previous reactions ; need for more human resources especially in accounting and medical records departments ; cost of healthcare by cutting repeated or unnecessary services ; cost of stationary materials e.g. paper charts and radiology films and fixed expenses for equipments ; accounting , documentation and archiving expenses ; required time for retrieving and analyzing medical data ; waiting time for admission ; hospitalization time ( 31 ) .
open source technology is one of the most effective mechanisms for technology transfer which could foster the national economy .
this is because the source codes and licensing of the software are free of charge and open for inspection and usage ( 9 ) .
many organizations and countries face various problems in using proprietary software which could be resolved through using oss . among them , the cost of licensing , the difficulty of choosing a system capable of being modified according to the changing needs with the ability to integrate with the existing and future systems are major barriers ( 22 ) .
open source solution provides many potential benefits for the establishment of national ehr in iran .
it allows quick implementation of the tools , while reducing medical errors and costs in a fast and easy process ( 32 ) .
the advantages of utilizing oss in developing national ehr include high quality , reliability and security of system resulting from continuous and extensive peer review of software ; availability of source code which enables any modification in response to the changing missions and threats ; avoidance of vendor lock - in ; freedom to use ; and low total cost of ownership ( 18 ) .
the approach of open source is similar to the peer - review scientific process through which one can see and reproduce results to understand and promote what is already shared ( 33 ) . in healthcare systems ,
continuous and broad peer review of source code could make the system secure and reliable .
this will improve data confidentiality and will also be useful for quality assessment ( 34 , 35 ) .
evaluation of health information system would improve the performance of the system ( 36 ) . at present time , developing countries are well - positioned to take advantage of the open - source movement especially in health sector to overcome health inequity ( 37 ) .
open source software would create a mutually beneficial relationship between vendor and customer while proprietary software generally lacks this characteristic .
open source is a collaborative process that is improved by the coordinated sharing of information ( 38 ) .
in other words , cooperative development of high quality software is one of the main advantages of open source approach .
well - designed open source software could enable other developers to adopt and modify the software according to their requirements .
finally , they can build their own tools based on the contribution of other expert s experiences ( 39 ) .
millard and colleagues believe that a new millennium development goal should include the creation of a universal open - source health informatics platform that will allow the collection , management and delivery of clinical and population data that will guide decision processes at the local , regional and global levels ( 40 ) . by using open source systems ,
institutions are not locked in to specific vendors and have more control over the application s features .
some believe that the reduced total cost of ownership is the major benefit of oss ( 35 ) .
however , this is highly dependent on the financial situations of the country and its total income . for a developing country like iran
, the application of oss in the establishment of national ehr and other health information systems would create a great opportunity to improve national healthcare setting without paying major costs for development and maintenance of health information systems .
open source software ( oss ) , free and open source software ( foss ) and free , libre and open source software ( floss ) - although are not exactly the same- are alternative terms to describe free software ( 6 ) .
there are basic principles for open source software including free redistribution , inclusion of source code , allowing for modification , integrity of the author s source code , no discrimination against any person or group , no discrimination against fields of usage , no need for an additional license .
also the license should not be specific to a product or technology and should not restrict other software ( 7 ) .
the promise of open source software is better quality , higher reliability , more flexibility , lower cost , and an end to predatory vendor lock - in .
open source technology is one of the most effective mechanisms for technology transfer which could foster the national economy .
this is because the source codes and licensing of the software are free of charge and open for inspection and usage ( 9 ) .
today , a wide range of open source applications is accessible through the web including web servers , web browsers , databases , programming languages , image editors and email clients ( 1015 ) .
oss could provide competition in commercial market , encourage innovation and promote compatibility and interoperation in health domain .
this would guarantee better quality and lower cost . also it would make the systems more responsive to changing clinical and organizational requirements ( 16 ) .
several communities work on developing open source ehr systems to reduce the costs of ehr deployment and maintenance ( 17 ) . in america , many systems deployed by the u.s .
nowadays , a large number of health information systems are available as open source evidence based guideline and decision support system ( egadss ) is an open source clinical decision support system ( cdss ) project began in may 2004 to provide patient specific point of care reminders .
egadss would respond to requests from electronic medical records to provide patient specific clinical guidance based on its internal guidelines .
it operates on decision algorithms , medical knowledge , and specific patient data to generate recommendations and guidance for clinicians in order to aid them provide high quality care ( 19 ) .
care2x is a hospital information system through which the authorized staff like physicians can access patients data even from outside the hospital without using specialized software .
open source cluster application resources ( oscar ) is open source electronic medical record ( emr ) which was started by the department of family medicine at mcmaster university in 2001 .
it contains healthcare information with a lot of functions including complete scheduling , e - charting , prescribing , billing , lab downloads , tables and graphs , chronic disease management tools , disease registry , automated customizable forms , sophisticated antenatal care record and planner , tabulation of outstanding preventative and intervention practices , and also research tools .
oscar is used by healthcare professionals including physicians , nurses , midwives , social workers , psychologists and physiotherapists ( 21 ) .
open source ehr project directed by the american academy of family physicians ( aafp ) is an attempt to lower the cost of ehr by sharing the source code ( 3 ) another example is orca ( open receipt computer advanced ) which is a project of information and communication infra - structure based on oss to overcome problems of high costs , inflexibility and restrictions associated with proprietary systems .
the advantages of an open source ehr outweigh the costs of a proprietary ehr system ( 23 ) .
open source technology makes the opportunity for advanced innovation in the health information sector of low income countries ( 24 ) .
in iran several scattered attempts to design health information systems software has been made so far .
as they did not follow particular standards , integrating their outputs is not possible ( 25 ) .
however , development of a set of standards is always a necessary requirement for integrating different information systems ( 26 ) . in iran ,
several studies on medical information systems have been done focusing on various domains especially on usability , efficacy , standards , architecture , structure and content . in 2001 ,
the usability of software packages in medical records departments of several hospitals in tehran was compared .
the findings showed that the overall usability of medical records software packages in the hospitals was 55% which indicated the available packages did not have the necessary capabilities to meet the requirements of medical records departments ( 27 ) . in another study
nine hospital information systems applied in a number of hospitals in iran were evaluated based on the general requirements of hospital information system ( his ) .
the results showed that total average of compatibility of systems with the general requirements of his was 65.4% , ( 28 ) . in 2006 , an object - oriented model for some key messages was designed to support ehr through comparing weaknesses and strengths of hl7 and openehr standards . according to the study ,
it could ensure open , future - oriented electronic health records in iran ( 29 ) . in 2007 ,
ehr content , structure , and terminology standards in selected organizations were investigated in order to design an appropriate model of ehr for iran .
the study suggested a national model for ehr based on the weakness and capabilities of standards provided by the investigated organizations ( 2 ) . according to the evidences ,
a wide range of information systems are used in iranian hospitals , clinics , pharmacies and offices .
to design an integrated ehr system , all components of the health system should be engaged through an organized shareable information system .
therefore , the available clinical data on the existing systems in iran have neither been applied in decision making nor served as a basis for evidence based medicine .
this situation addresses the need for developing an integrated shareable information system of ehr in iran .
such a system will not only help using the collected data for different purposes , but also serves as an infrastructure for a variety of e - health activities in the country ( 25 ) .
the evidences also showed that financial limitation is one of the obstacles to implement electronic health records in developing countries .
therefore , establishment of an open source ehr system capable of modifications according to the national requirements seems to be inevitable in iran .
establishment of a national ehr requires a wide cooperation and coordination among involved ministries and organizations .
the prerequisites for a national ehr can be summarized as follows :
electronic record in a national extent including health data centerfacilities for electronic recording and transferring information produced in health data centers including hardware and software equipments and also cultural infrastructuresdeveloping standards for coding and data exchangedevelopment of information technology infrastructures with sufficient band - width and connections to meet the present and future needs of national health systemdevelopment plan for communication and messaging services to support new information cycles ( 25 ) .
electronic record in a national extent including health data center facilities for electronic recording and transferring information produced in health data centers including hardware and software equipments and also cultural infrastructures developing standards for coding and data exchange development of information technology infrastructures with sufficient band - width and connections to meet the present and future needs of national health system development plan for communication and messaging services to support new information cycles ( 25 ) .
the project of iranian electronic health record ( sepas ) supported by iranian ministry of health is the greatest information technology project in the health sector in the country . at the present time , the infrastructure of sepas is being processed to provide a platform for data exchange among health centers all over the country .
health information of all citizens from birth to death will be recorded in their individual ehrs . as a result
the key values of sepas include providing citizen rights , justice , data confidentiality and information security .
the main objectives of this project are :
integration of citizens health information;improving the quality of health services;equitable distribution of health resources;improving the management of health resources and improving healthcare system based on quick access to correct and accurate information;expediting and facilitating production and management of biomedical knowledge;contribution to the development of evidence based medicine;reducing complications resulted from medical errors;reducing healthcare costs;increasing security of health information;strengthening the monitoring role of the health system;improving the quality of medical education;providing new electronic services;improving the industry of healthcare in iran ( 30 ) .
integration of citizens health information ; improving the quality of health services ; equitable distribution of health resources ; improving the management of health resources and improving healthcare system based on quick access to correct and accurate information ; expediting and facilitating production and management of biomedical knowledge ; contribution to the development of evidence based medicine ; reducing complications resulted from medical errors ; reducing healthcare costs ; increasing security of health information ; strengthening the monitoring role of the health system ; improving the quality of medical education ; providing new electronic services ; improving the industry of healthcare in iran ( 30 ) . according to the architecture plan for national ehr in iran
, information systems would be categorized in three levels which are peripheral , intermediate and central .
these systems are independent and connected to one another via certain standard protocols . as a result
, information would flow upwards through the following levels and circulate throughout the whole network :
information system level 1 ( peripheral level ) which is connected to medical equipments or portable devices such as smart cards or pdas .
it can simply be used at intermediate health providing centers for the ease of data entry and transfer.information system level 2 ( intermediate level ) which is installed and utilized in health providing centers .
such systems are customized according to the needs and specification of healthcare providing centers.information system level 3 ( central level ) which will be installed at the ministry of health and medical universities .
information system level 1 ( peripheral level ) which is connected to medical equipments or portable devices such as smart cards or pdas .
it can simply be used at intermediate health providing centers for the ease of data entry and transfer .
information system level 2 ( intermediate level ) which is installed and utilized in health providing centers .
information system level 3 ( central level ) which will be installed at the ministry of health and medical universities .
development of electronic health record in iran would increase :
public access to healthcare services especially in deprived regions and evenly distribution of healthcare services throughout the country;modification of the national health management system based on the evidence and precise data;utilization of healthcare resources;delivery of modern electronic services such as teleappointing , teleconsulting , reminder and alerting systems;provision of a proper background for production and management of medical knowledge;supervision , evaluation and audition of healthcare services;establishment of statistical registry system;community cooperation in public health;individual accessibility to personal health information to choose the appropriate method of diagnosis and treatment;provision of necessary tools for clinical decision making;the work flow of the whole system through reengineering the process and performance of different units;the income cycle;citizens ` satisfaction;quality of care through developing a standard method for collection and interpretation of data;time and cost effectiveness and efficiency;medical knowledge by creating a suitable structure for retrospective as well as prospective medical researches;documentation and audition of documents;following the medical guidelines through sending alerts by the system;management of health centers;and the accessibility of medical information in cases of crisis and natural disasters .
public access to healthcare services especially in deprived regions and evenly distribution of healthcare services throughout the country ; modification of the national health management system based on the evidence and precise data ; utilization of healthcare resources ; delivery of modern electronic services such as teleappointing , teleconsulting , reminder and alerting systems ; provision of a proper background for production and management of medical knowledge ; supervision , evaluation and audition of healthcare services ; establishment of statistical registry system ; community cooperation in public health ; individual accessibility to personal health information to choose the appropriate method of diagnosis and treatment ; provision of necessary tools for clinical decision making ; the work flow of the whole system through reengineering the process and performance of different units ; citizens ` satisfaction ; quality of care through developing a standard method for collection and interpretation of data ; time and cost effectiveness and efficiency ; medical knowledge by creating a suitable structure for retrospective as well as prospective medical researches ; documentation and audition of documents ; following the medical guidelines through sending alerts by the system ; management of health centers ; and the accessibility of medical information in cases of crisis and natural disasters . on the other hand , developing electronic health record in iran could decrease :
medical errors and the consequent complications;mortality rate caused by medical errors;drug adverse effects through recording individuals previous reactions;need for more human resources especially in accounting and medical records departments;cost of healthcare by cutting repeated or unnecessary services;cost of stationary materials e.g. paper charts and radiology films and fixed expenses for equipments;accounting , documentation and archiving expenses;required time for retrieving and analyzing medical data;waiting time for admission;hospitalization time ( 31 ) .
medical errors and the consequent complications ; mortality rate caused by medical errors ; drug adverse effects through recording individuals previous reactions ; need for more human resources especially in accounting and medical records departments ; cost of healthcare by cutting repeated or unnecessary services ; cost of stationary materials e.g. paper charts and radiology films and fixed expenses for equipments ; accounting , documentation and archiving expenses ; required time for retrieving and analyzing medical data ; waiting time for admission ; hospitalization time ( 31 ) .
open source technology is one of the most effective mechanisms for technology transfer which could foster the national economy .
this is because the source codes and licensing of the software are free of charge and open for inspection and usage ( 9 ) .
many organizations and countries face various problems in using proprietary software which could be resolved through using oss . among them , the cost of licensing , the difficulty of choosing a system capable of being modified according to the changing needs with the ability to integrate with the existing and future systems are major barriers ( 22 ) .
open source solution provides many potential benefits for the establishment of national ehr in iran .
it allows quick implementation of the tools , while reducing medical errors and costs in a fast and easy process ( 32 ) .
the advantages of utilizing oss in developing national ehr include high quality , reliability and security of system resulting from continuous and extensive peer review of software ; availability of source code which enables any modification in response to the changing missions and threats ; avoidance of vendor lock - in ; freedom to use ; and low total cost of ownership ( 18 ) .
the approach of open source is similar to the peer - review scientific process through which one can see and reproduce results to understand and promote what is already shared ( 33 ) . in healthcare systems ,
continuous and broad peer review of source code could make the system secure and reliable .
this will improve data confidentiality and will also be useful for quality assessment ( 34 , 35 ) .
evaluation of health information system would improve the performance of the system ( 36 ) . at present time , developing countries are well - positioned to take advantage of the open - source movement especially in health sector to overcome health inequity ( 37 ) .
open source software would create a mutually beneficial relationship between vendor and customer while proprietary software generally lacks this characteristic .
open source is a collaborative process that is improved by the coordinated sharing of information ( 38 ) . in other words , cooperative development of high quality software
well - designed open source software could enable other developers to adopt and modify the software according to their requirements .
finally , they can build their own tools based on the contribution of other expert s experiences ( 39 ) .
millard and colleagues believe that a new millennium development goal should include the creation of a universal open - source health informatics platform that will allow the collection , management and delivery of clinical and population data that will guide decision processes at the local , regional and global levels ( 40 ) . by using open source systems ,
institutions are not locked in to specific vendors and have more control over the application s features .
some believe that the reduced total cost of ownership is the major benefit of oss ( 35 ) .
however , this is highly dependent on the financial situations of the country and its total income . for a developing country like iran , the application of oss in the establishment of national ehr and other health information systems would create a great opportunity to improve national healthcare setting without paying major costs for development and maintenance of health information systems .
as most developing countries like iran suffer from insufficient financial resources to purchase and support ehr commercial software , the application of open source ehr with the ability to be customized based on the national needs seems inevitable .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors . | abstractelectronic health record ( ehr ) is a repository of patient health information shared among multiple authorized users . as a modern method of storing and processing health information ,
it is a solution for improving quality , safety and efficiency of patient care and health system .
however , establishment of ehr requires a significant investment of time and money .
while many of healthcare providers have very limited capital , application of open source software would be considered as a solution in developing national electronic health record especially in countries with low income .
the evidence showed that financial limitation is one of the obstacles to implement electronic health records in developing countries .
therefore , establishment of an open source ehr system capable of modifications according to the national requirements seems to be inevitable in iran .
the present study identifies the impact of application of open source software in developing national electronic health record in iran . |
oral lichen planus ( olp ) , a common mucocutaneous disease , was first described by wilson in 1869 .
poor described the formation of cavities in lichen planus of the mucosa , corresponding in character to subepithelial bullae and gougerot described the reticular , annular , plaque , ulcerated , dotted and sclerotic forms of lichen planus .
olp is considered to be a t - cell mediated autoimmune disease in which cytotoxic cd8 + t - cells trigger the apoptosis in oral epithelial cells .
the lichen planus antigen has not been definitely identified and it may be a self - peptide . in most of the studies , olp is considered to be a t - cell - mediated chronic inflammatory oral mucosal disease .
it is mainly observed among women and symmetrically affects the buccal mucosa , tongue , gingiva , floor of the mouth , lips , and palate .
the role of angiogenesis in the pathogenesis of chronic inflammatory diseases is of considerable interest . a positive feedback loop , in which an inflammatory state promotes angiogenesis and the angiogenesis in turn facilitates chronic inflammation , has been found in some inflammatory diseases .
there is increasing evidence that chronic inflammation is tightly linked to diseases associated with endothelial dysfunction , and plays a role in the induction of aberrant angiogenesis .
microvessel density ( mvd ) evaluation is commonly applied for the estimation of tumor angiogenesis and is widely accepted to play a role in the pathogenesis of some inflammatory conditions also .
cd34 monoclonal antibody can be used to highlight the microvessels in inflammatory or neoplastic disorders because of its capability of staining the vascular endothelial cells ( membranous staining pattern ) .
the purpose of this study was to evaluate the role of angiogenesis in the pathogenesis of olp , using cd34 stain to highlight the blood vessels for measuring the mvd .
better understanding of the etiopathological mechanism underlying olp will help in the development of new treatment strategies , as well as to manage persistent inflammation in patients showing poor response to conventional immunosuppressive drug regimes .
this study included 40 cases , categorized into two groups : group i and group ii .
group i , the control group , consisted of 15 specimens of normal oral mucosa ( buccal mucosa / gingival tissue ) from individuals who underwent extractions as part of orthodontic treatment and from the paraffin blocks available with the archives of the department of oral and maxillofacial pathology and microbiology , m. m. college of dental sciences and research , mullana .
group ii consisted of 25 diagnosed cases of olp ; these cases were subdivided into two groups : group iia and group iib .
group iia consisted of 18 diagnosed cases of reticular olp and group iib consisted of 7 diagnosed cases of erosive olp .
these cases were obtained either from the blocks of previously diagnosed cases from the archives of the department of oral and maxillofacial pathology and microbiology , m. m. college of dental sciences and research , mullana , or from fresh cases identified at the department of oral medicine and radiology . case history pertaining to the lesion was obtained and recorded .
sections of 4-m thickness were cut from the paraffin blocks and were used for immunohistochemical staining .
briefly , endogenous peroxidase activity was blocked with 0.6% h2o2 and sections were incubated at room temperature with 100 l of prediluted primary cd34 antibody ( dako ) .
after washing in phosphate - buffered saline ( pbs ) , secondary antibody was applied on the section and then washed with pbs .
after counterstaining with hematoxylin , the sections were mounted with dibutyl phthalate xylol ( dpx ) and the slides were observed under a nikon research microscope .
negative and positive controls were used simultaneously to assess the specificity and reliability of the staining process .
pbs was substituted for the primary antibodies of cd34 as a negative control , and placental tissue sections with known cd34 positivity were used as the positive control .
microvessels were highlighted on the sections of olp and normal oral mucosal tissue by immunostaining with anti - cd34 monoclonal antibody [ figures 13 ] .
any single brown - stained cell or cluster of endothelial cells that were clearly separated from adjacent microvessels , histiocytes , and other connective tissue elements were considered as a single vessel .
cd34-stained brown - colored blood vessels in normal oral mucosal tissue cd34-stained brown - colored blood vessels in reticular oral lichen planus cd34-stained brown - colored blood vessels in erosive oral lichen planus cd34-stained sections were scanned at low magnification to identify the most vascular areas ( hot spot areas ) , which were almost exclusively localized within the inflammatory infiltrate .
a maximum of five fields were selected and the images were captured on the computer under the 20 objective of a nikon research microscope ( y - thr - l ; japan ; 0132508 ) for counting of the number of blood vessels in hot spot areas .
briefly , endogenous peroxidase activity was blocked with 0.6% h2o2 and sections were incubated at room temperature with 100 l of prediluted primary cd34 antibody ( dako ) . after washing in phosphate - buffered saline ( pbs ) ,
after counterstaining with hematoxylin , the sections were mounted with dibutyl phthalate xylol ( dpx ) and the slides were observed under a nikon research microscope .
negative and positive controls were used simultaneously to assess the specificity and reliability of the staining process .
pbs was substituted for the primary antibodies of cd34 as a negative control , and placental tissue sections with known cd34 positivity were used as the positive control .
microvessels were highlighted on the sections of olp and normal oral mucosal tissue by immunostaining with anti - cd34 monoclonal antibody [ figures 13 ] .
any single brown - stained cell or cluster of endothelial cells that were clearly separated from adjacent microvessels , histiocytes , and other connective tissue elements were considered as a single vessel .
cd34-stained brown - colored blood vessels in normal oral mucosal tissue cd34-stained brown - colored blood vessels in reticular oral lichen planus cd34-stained brown - colored blood vessels in erosive oral lichen planus cd34-stained sections were scanned at low magnification to identify the most vascular areas ( hot spot areas ) , which were almost exclusively localized within the inflammatory infiltrate .
a maximum of five fields were selected and the images were captured on the computer under the 20 objective of a nikon research microscope ( y - thr - l ; japan ; 0132508 ) for counting of the number of blood vessels in hot spot areas .
in the present study , of the difference between mean mvd in group i ( 44.47 ) and that in group ii ( 97.24 ) was statistically highly significant ( p=.001 ) [ table 1 ] .
a comparison of the mean mvd in group iia ( 84.61 ) with that in group iib ( 129.71 ) also showed a statistically significant difference ( p=.015 ) [ table 2 ] .
comparison of microvessel density between group i and group ii comparison of mvd between group iia and group iib
various concepts have been quoted by different authors but the differences have not yet been sorted . as an autoimmune disease with inflammatory origin and chronic progression ,
macrophages and the other cells of the immune system produce vascular endothelial growth factor ( vegf ) , which stimulates the degradation , proliferation , and migration of endothelial cells .
tnf - alpha , il-1 alpha , interleukin-6 , and interleukin-8 also upregulate the expression of vegf , and these factors have been found in the oral fluids of patients with olp . also , olp shows significantly increased vegf as compared to normal oral mucosa .
proangiogenic and angiogenic factors such as histamine , heparin , chymase , bfgf , vegf , and tgf - beta are produced by mast cells , and an increase in the number of mast cells has been observed in olp .
a significant correlation between mast cell density and mvd has been found during the evolution of oral squamous cell carcinoma from normal oral tissue through premalignant lesion with various degrees of dysplasia to carcinoma .
the malignant potential of olp remains controversial , and different research groups have proposed different approaches and interpretations .
it appears advisable to carry out a meticulous follow - up of patients with olp for early detection of the malignant transformation of suspected lesions .
malignant transformation of olp may be related to , or dependent on , a series of molecular stimuli originating in the inflammatory infiltrate .
some molecules and radicals generated by inflammatory cells can act as mutagenic agents for epithelial cells or influence important cell cycle regulation mechanisms , e.g. , apoptosis , cell cycle arrest , and cell proliferation , among others .
a source of possible mutation in olp derives from the action of cyclooxygenase-2 ( cox-2 ) that is produced by inflammatory infiltrating cells .
apart from its other actions , cox-2 also intervenes in the metabolism of arachidonic acid , generating the carcinogenic metabolite malondialdehyde , which can damage dna .
attraction of lymphocytes to a particular site would require cytokine - mediated upregulation of adhesion molecules on endothelial cells and concomitant expression of receptor molecules by circulating lymphocytes .
in olp , there is in fact increased expression of several vascular adhesion molecules ( known by the acronyms elam-1 , icam-1 , and vcam-1 ) and infiltrating lymphocytes that express reciprocal receptors ( known as l - selectin , lfa-1 , and vla4 ) , supporting the hypothesis that there is activation of a lymphocyte homing mechanism in lichen planus .
thus , if angiogenesis is increased , it will lead to more recruitment and retention of lymphocytes or inflammatory infiltrate , or progression of disease or recurrence of the lesions ; the inflammatory infiltrate can further lead to carcinogenesis .
cd34 , a myeloid progenitor cell antigen present in endothelial cells , is detectable in all types of endothelium .
the monoclonal antibody against cd34 reacts with the endothelium of arteries and venules and has been found to stain capillary endothelium most intensely , because it is highly sensitive for endothelium and produces lowest background staining .
therefore , in the present study , mvd was estimated by cd34 immunostaining . in the present study , comparison of mean mvd between group
i ( 44.47 ) and group ii ( 97.24 ) showed a statistically highly significant result .
our study suggests that angiogenesis is significantly increased in olp as compared to normal oral mucosa [ figure 1 ] , also in erosive olp [ figure 3 ] as compared to reticular olp [ figure 2 ] ; this suggests that angiogenesis is one of the main contributing factors in the progression of olp .
malignant transformation in olp is said to occur in 0.3%3% of cases and is more common in the erosive form .
thus , the increased mvd in erosive olp as compared to reticular olp in our study could be suggested as one of the factors contributing to this higher malignant potential .
angiogenesis has long been known to be closely linked to chronic inflammation , and it is a component of various chronic inflammatory diseases . however , the exact etiopathological mechanism of olp is still not clear .
most of the studies have not been able to demonstrate a direct relation between angiogenesis and olp .
if antiangiogenic drugs can be demonstrated to be beneficial in olp patients , it would reduce the dependency on corticosteroid drugs . considering the role of angiogenesis in the inflammatory lesions of olp , and the fact that some olp patients respond poorly to conventional immunosuppressive drugs
, angiogenesis could be a possible therapeutic target to reverse the persistent and stubborn inflammation .
from this study , we conclude that angiogenesis can be considered to play a role in both the etiopathogenesis and progression of olp .
our findings could be helpful for formulating new treatment strategies for olp utilizing antiangiogenic medications . | background : the etiology of oral lichen planus ( olp ) is not fully understood .
it is generally considered to be a t - cell mediated chronic inflammatory oral mucosal disease .
there is increasing evidence that chronic inflammation is linked to the diseases associated with endothelial dysfunction and is involved in the induction of aberrant angiogenesis.aim:our aim was to evaluate the role of angiogenesis in the pathogenesis of olp by immunohistochemistry , using the cd34 antibody.materials and methods : forty tissue sections ( 7 of erosive lichen planus , 18 of reticular oral lichen planus , and 15 of normal oral mucosa ) , were assessed for microvessel density ( mvd ) in five selected areas of high inflammatory infiltrate by immunohistochemistry for the expression of cd34 antibody.results and conclusion : the mean mvd was 44.47 in the control group ( normal oral mucosa ) and 97.24 in the olp group , showing that there is increased angiogenesis in the latter .
reticular olp had mean mvd of 84.61 and erosive olp had mean mvd of 129.71 , showing relatively greater angiogenesis in erosive olp as compared to reticular olp .
thus , angiogenesis can be considered to play a role in both the etiopathogenesis and the progression of olp . |
age - related structural and functional deterioration of sensory and motor mechanisms , especially of the vestibular system , is a leading cause of dizziness and imbalance in older adults ( 1 , 2 ) .
it is well established that older individuals with a history of imbalance and dizziness are at higher risk of fall related injuries , loss of independence , and even death ( 1 ) , all depicting the importance of determining the effect of aging on the vestibular system ( 3 ) .
the peripheral vestibular apparatus consists of the labyrinth ( semicircular canals and otoliths ) , vestibular nerve , and a central component consisting of the vestibular nuclei ( 4 ) .
the otoliths ( saccule and utricle ) register forces related to linear acceleration ( 2 ) .
vestibular evoked myogenic potential ( vemp ) is a reliable clinical tool to assess the accuracy of the sacculo - collic reflex ( 5 ) , first introduced by colebatch and halmagyi ( 1992 ) and further described by colebatch et al .
vemp is a short - latency myogenic response evoked by loud sounds and recorded using surface electrodes placed over cervical muscles ( 6 ) .
the vemp response consists of a positive peak ( p1 ) in the ipsilateral sternocleidomastoid ( scm ) followed by a negative peak ( n1 ) ( 6 ) .
the effect of aging on vemp response has been reported in previous studies ( e.g. su et al . ( 7 ) , lee et al .
the evidence is accumulated that the aging influences on p1 and/or n1 latencies , although a new study by nguyen et al . ( 10 ) did not report any significant difference between latency measurement and aging .
furthermore , the effects of aging on vemp amplitude has also been reported in previous studies ( 3 , 5 , 9 , 10 )
( 8) did not find any effect of aging on vemp amplitude at the threshold level , though amplitude was negatively correlated with age in suprathreshold level .
age- dependent anatomical and physiological changes in vestibular system has also been reported in previous studies ( 11 - 19 ) .
as age - related loss of vestibular otolith - ocular function is associated with increased mediolateral measures of sway ( 20 ) , characterizing the impact of aging on saccular function is important .
however , little is known about the influence of aging on the function of otolith organ . in recent years
, vemp has become a standard clinical test of otolith ( mainly saccular ) function ( 6 ) .
the vemp is an easy - to - use test that can be performed in a relatively short time , though some previous studies have contradictory recommendations ( 3 , 5 , 7 - 10 ) .
the aim of the present study was to evaluate the impact of aging on saccular function using cvemp response .
sixty - two subjects were including , 31 young adults ( 62 ears ) with a mean age of 22.15 1.93yr ( range : 19 - 26yr ) comprising 27 females and 4 males as control group , and 31 old adults ( 50 ears ) with a mean age of 69.76 5.14yr ( over age 60 ) comprising 26 females and 5 males , as test group .
the mean pure tone average at 500 , 1000 , and 2000hz was 25db hl for both groups .
inclusion criteria were as follows : participants with no history of middle ear pathology , and no vestibular , cervical and neurological disorders .
the study protocol was approved by the ethics committee of shahidbeheshti university of medical sciences , and informed consent was obtained from all participants .
adherence to inclusion criteria was confirmed by having all volunteers complete a case history , berg balance test ( to evaluate total balance status ) , otoscopy and acoustic immittance evaluation ( to test middle ear health using an impedance - interacoustic at235 audiometer , denmark ) , and pure tone and speech audiometry ( to evaluate the integrity of auditory pathway - interacoustic ac40 audiometer , denmark ) .
participants also underwent a cervical vemp ( cvemp ) test performed using electrophysiologic equipment ( ics charter ep , usa , software version 6.20 ) . to activate the scm muscle ,
participants were asked to sit in a chair and turn their head toward the contralateral side shoulder .
for cvemp recording , 150 responses to air - conducted 500 hz tone bursts with two - cycle rise / fall and no plateau ( blackman gated ) were presented monaurally with rarefaction polarity via an insert receiver .
the responses were amplified ( 5000 ) and band - pass filtered ( 10 hz-2000hz ) .
the skin was prepared and the active electrode was placed at the midpoint of the scm muscle belly ; the reference electrode was set on the sternoclavicular junction , and the ground electrode was placed on the forehead .
the cvemp responses were obtained at threshold level ( vemp threshold was defined as the lowest level at which vemp parameters were repeatable and distinctive ) and at 95db nhl intensity level .
vemp response thresholds were obtained using a down 10 , up 5 db step manner .
each recording was repeated twice to assure reproducibility . to avoid distortion of the results by neck muscle fatigue ,
the vemp threshold , p1 and n1 latency , p1/n1 amplitude , and asymmetry ratio were determined for all participants after the vemp responses were recorded .
the asymmetry ratio ( ar ) between the right and left ears was computed according to the following formula ( 21 ) .
the data was analyzed using a paired - samples t - test and independent - samples t - test .
adherence to inclusion criteria was confirmed by having all volunteers complete a case history , berg balance test ( to evaluate total balance status ) , otoscopy and acoustic immittance evaluation ( to test middle ear health using an impedance - interacoustic at235 audiometer , denmark ) , and pure tone and speech audiometry ( to evaluate the integrity of auditory pathway - interacoustic ac40 audiometer , denmark ) .
participants also underwent a cervical vemp ( cvemp ) test performed using electrophysiologic equipment ( ics charter ep , usa , software version 6.20 ) . to activate the scm muscle ,
participants were asked to sit in a chair and turn their head toward the contralateral side shoulder .
for cvemp recording , 150 responses to air - conducted 500 hz tone bursts with two - cycle rise / fall and no plateau ( blackman gated ) were presented monaurally with rarefaction polarity via an insert receiver .
the responses were amplified ( 5000 ) and band - pass filtered ( 10 hz-2000hz ) .
the skin was prepared and the active electrode was placed at the midpoint of the scm muscle belly ; the reference electrode was set on the sternoclavicular junction , and the ground electrode was placed on the forehead .
the cvemp responses were obtained at threshold level ( vemp threshold was defined as the lowest level at which vemp parameters were repeatable and distinctive ) and at 95db nhl intensity level .
vemp response thresholds were obtained using a down 10 , up 5 db step manner .
each recording was repeated twice to assure reproducibility . to avoid distortion of the results by neck muscle fatigue ,
the vemp threshold , p1 and n1 latency , p1/n1 amplitude , and asymmetry ratio were determined for all participants after the vemp responses were recorded .
the asymmetry ratio ( ar ) between the right and left ears was computed according to the following formula ( 21 ) .
the data was analyzed using a paired - samples t - test and independent - samples t - test .
to examine the effect of aging on saccular function , cvemp results for the older group were compared with the controls .
there was no significant difference between vemp parameters for the right and left ears , therefore , the results for both ears were combined .
the vemp responses revealed that 23 participants had bilateral vemp response and 4 participants had unilateral vemp response ( in 112 of 124 ears in the 62 participants ) .
1 shows the cvemp waveforms obtained at 95 db nhl from the right ear for a young and an old adult .
the independent - samples t - test demonstrated a significant difference in vemp response threshold between the two groups ( p<0.001 ) .
there was also a significant difference in p1 wave latency between the two groups ( p<0.001 ) .
no significant difference existed for n1 wave latency between the two groups ( p=0.06 ) .
the independent t - test showed a significant difference in p1/n1 amplitude ( p<0.001 ) , and asymmetry ratio of p1/n1 amplitude showed a significant difference between the two groups ( p=0.01 ) .
the standard deviation , mean threshold level , latency , amplitude , and asymmetry ratio of p1/n1 amplitude of vemp for
the two groups are shown in table 1 .
the cervical vestibular evoked myogenic potentials recorded in the right ear of a young and an old adult at 95 db nhl and threshold intensity level .
sd : standard deviation , ms : millisecond , v : microvolt , and db nhl : decibel normalized hearing level .
dizziness and imbalance are common complaints by older adults . although , in recent years , research has demonstrated the role of otolith function in postural stability ( 20 ) , few studies have investigated age - related loss of otolith organ function .
the purpose of this study was to examine the effect of aging on saccular function in the elderly .
the cvemp measurements were recorded using tone burst 500 hz stimuli delivered through air conduction and compared between the two study groups .
unilateral or bilateral omission of cvemp response in some of the elderly has also been reported in the previous studies ( 7 , 8 , 22 ) , which might be due to age- dependent changes in the course of producing the required response .
effects of age on cvemp threshold : the results showed that cvemp thresholdsincreases with an increase in the age , which is in association with previous studies ( 8 , 22 , 23 ) .
ochi and ohashi ( 23 ) reported the effects of age on vemp response in 60 healthy adults .
welgampola and colebatch ( 22 ) investigated the influence of aging on the vestibule - collic reflex in response to click stimuli and demonstrated that vemp thresholds increased as age increased .
janky and shepard studied 46 normal adults ranging in age from 20 to 76 yr .
cvemp responses were recorded at threshold with a click for 250 , 500 , 750 , and 1000 hz tone burst stimuli and at a suprathreshold level for a 500 hz tone burst stimuli .
the increase in cvemp threshold may be caused by age - related functional changes in the sensory and neural components involved in the projection of cvemp responses .
in fact , aging may affect the cvemp response pathway from the saccule , scarpa ganglion , inferior vestibular nerve , lateral vestibular nucleus and lateral vestibule - spinal tract to the scm muscle .
effects of age on cvemp latency : the present research demonstrated a difference for p1 wave latency between the two study groups where p1 wave latency increased as age increased .
analysis of the data also indicated that there was no n1 wave latency difference between the two study groups .
this may be because the spread of central transmission and the time required to activate the vestibulo - collic reflex that causes the vemp response changed as age increased .
previous studies have reported inconsistent results on age - related changes in the latency of cvemp responses .
the findings of the present study are in agreement with those of janky and shepard ( 8) who reported no difference between groups for n23 latency in response to click and tone burst stimuli .
su et al ( 7 ) reported that the p13 latency increased as age increased , although this difference was not significant .
these results , however , are in disagreement with those of nguyen et al . ( 10 ) , who found no correlation between latency of vemp response and age .
possible explanations for this discrepancy are differences in recording techniques , such as variations in stimuli rise / fall time , and filter setting , for instance , in nguyen et al . , tone bursts stimuli with a linear envelope and 1ms rise / fall time and 2 ms plateau time were used .
age - dependent variations observed in latency of cvemp response might probably be due to the created changes in processing messages from otolith organs by central nervous system but might not be related to reduced function in peripheral vestibular system .
effects of age on cvemp p1/n1amplitude :
the results of the present study showed a difference in p1/n1 amplitude between the two groups . in other words , the p1/n1 amplitude decreased as age increased .
these findings confirm the results of other studies ( 3 , 5 , 7 , 22 - 25 ) .
this decrease may be caused by several factors , such as age - dependent anatomical and physiological changes in the vestibular end organ and central pathways or age - related changes in the tonic scm muscle electromyogram level .
effects of age on cvempasymmetry ratio : the results of the present study also showed no difference between left - right cvemp parameters , which again confirms the results of the previous research ( 5 , 8 , 22 , 23 ) . although the mean asymmetry ratio for p1/n1 amplitude in each group was within normal range , the mean asymmetry ratio showed a significant difference between the two groups .
the cvemp response is an easy - to - use test that can be performed in a short time .
thus , cvemp response can be used as an objective screening tool to identity the first symptoms of vestibular disorders .
pathologies affecting vemp parameters include : acute vestibular neuritis ( 26 ) , benign positioning vertigo ( 27 ) , meniere s disease ( 28 ) , vestibular schwannoma ( 29 ) , otosclerosis ( 30 ) , middle ear disease and hearing loss ( 31 , 32 ) , superior canal dehiscence syndrome ( 33 ) , gentamicin toxicity ( 34 ) , central vestibular disorders ( 35 - 37 ) .
it is proposed that audiology clinics establish normative data for different age groups using standard protocols for recording vemp responses similar to auditory evoked potential and vestibular - ocular reflex function tests .
a limitation of the present study was inability of the device to record the emg level , which required maintaining muscle tone using the feedback method ( 38 ) .
the results of the present study showed that aging can influence the parameters of the vemp response .
the cvemp response threshold , latency ( especially p1 ) , and asymmetry ratio of p1/n1amplitude increased , while the p1/n1amplitude decreased .
vemp abnormalities observed in healthy older adults indicated the sensitivity of this test in identifying the first signs of vestibular dysfunction .
the vemp response is easy to use , can be performed in a short time and only requires equipment for evaluating auditory evoked brainstem responses .
therefore , the cvemp response can be used as a selective objective screening test to detect vestibular disorders .
the results of the present study can provide normative data for correct interpretation of vemp response results in the identification of saccular disorders , which are representative of vestibular disease .
| background : aging can cause loss of balance , which may lead to physical and psychological problems . as the
role of the otolith organs in maintaining postural stability has been emphasized in recent years , the present study
investigated the effect of aging on saccular function using cervical vestibular evoked myogenic potentials
( cvemp ) .
methods : the participants were assigned into two groups ; group one included 31 young adults with a mean
age of 22.15 ( range : 19 - 26 yr ) and group two consisted of 31 old adults with a mean age of 69.76 years ( range :
61 - 79 yr ) .
all participants hearing sensitivity was normal with no history of balance problems .
vemp was recorded
for all subjects using tone burst 500 hz stimuli at the threshold level and 95 db nhl intensity level
through air - conduction stimulation via an insert receiver .
results : there was a significant difference in the cvemp response threshold ( p < 0.001 ) , p1 wave latency
( p<0.001 ) , p1/n1 amplitude ( p < 0.001 ) , and asymmetry ratio of p1/n1 amplitude ( p < 0.05 ) between the two
groups .
no significant difference was found between the left and right ears or in n1 wave latency between the
two groups .
conclusion : vemp abnormalities observed in healthy older adults showed the sensitivity of this test in identifying
early signs of vestibular dysfunction .
vemp is an easy - to - use test that requires a short time to be performed .
therefore , it can be used as a selective objective screening test to detect vestibular disorders |
this retrospective , cohort study includes patient data collected at 5 etus operated by international medical corps ( imc ) in liberia and sierra leone from 15 september 2014 to 15 september 2015 as part of its comprehensive response to the west african evd epidemic .
all patients who presented to the study etus with symptoms concerning for evd were triaged to ensure that they met the suspect case definition , which was created based on world health organization ( who ) and ministry of health guidelines from each country ( supplementary appendix 1 ) .
after triage , patients who met the suspect case definition were admitted to the etu suspect ward and had a blood sample drawn within 24 hours for evd and malaria diagnostic testing .
patients with an initial negative evd test remained as inpatients for repeated testing after 2 days . patients with a second negative evd result were discharged or transferred to another healthcare facility .
all patients aged < 18 years who were admitted at any of imc s 5 etus with a positive evd test and outcome data were included for analysis .
all patients were treated according to standard treatment protocols based on guidelines developed by the who , mdecins sans frontires , and local ministries of health , as permitted by local resources .
empiric treatment included antimalarial medications , broad - spectrum antibiotics , and nutritional supplementation , as well as focused supportive treatment ( appendix 1 ) .
patients were cared for by trained medical staff who recorded clinical data on paper forms .
these data were digitized at each etu and unified into a database , as described previously .
laboratory data , including evd real - time polymerase chain reaction ( rt - pcr ) and malaria testing , were linked to clinical data for analyses .
rt - pcr testing was performed by the us naval medical research center for etus in liberia , by public health england laboratories for lunsar and makeni etus in sierra leone , and by the nigerian laboratory for the kambia etu in sierra leone .
cycle threshold ( ct ) values , which are based on rt - pcr of the same ebola virus ( ebov ) zaire locus , are presented in this study as surrogates of viral load .
a ct > 40 was considered negative in all cases , though rna extraction procedures differed slightly between laboratories .
malaria testing ( conducted in sierra leone only ) was performed at each laboratory site using the commercially available binaxnow rapid diagnostic test , which identifies 4 plasmodium species : falciparum , malariae , vivax , and ovale . throughout hospitalization ,
patients were cared for by trained local or international nurses , physician assistants , or physicians , who rounded at least daily and recorded vital signs , clinical data , and laboratory results on paper forms .
patient temperatures were measured with infrared or oral thermometers . for children who were unable to communicate history and who had no accompanying guardians ,
a random sampling audit concluded that > 99% of the data in imc s unified database was accurate .
the variables of interest included demographic and clinical characteristics , malaria status , initial evd rt - pcr ct values , and mortality .
length of stay was calculated as the number of days from date of admission to date of discharge .
age was categorized into the following groups : < 2 years , 24 years , 59 years , 1014 years , and 1517 years . for descriptive statistics , median values with interquartile ranges ( iqrs )
were calculated for nonnormally distributed continuous data , and proportions were calculated for categorical data .
bivariate analyses were performed to determine the associations between mortality , the primary outcome of interest , and independent covariates using the mann - whitney u test for continuous variables and or fisher exact test for categorical variables where appropriate .
covariates that had a p value < 0.1 from bivariate analyses were entered in a backward stepwise logistic regression model to determine which variables independently predicted mortality .
we used kaplan - meier curves and the log - rank test to compare survival among children by age group , hemorrhagic features , and viral loads estimated by ct values .
the sierra leone ethics and scientific review committee , the university of liberia pacific institute for research & evaluation institutional review board , and the lifespan ( rhode island hospital ) institutional review board provided ethics approval for this study and exemption from informed consent .
this retrospective , cohort study includes patient data collected at 5 etus operated by international medical corps ( imc ) in liberia and sierra leone from 15 september 2014 to 15 september 2015 as part of its comprehensive response to the west african evd epidemic .
all patients who presented to the study etus with symptoms concerning for evd were triaged to ensure that they met the suspect case definition , which was created based on world health organization ( who ) and ministry of health guidelines from each country ( supplementary appendix 1 ) .
after triage , patients who met the suspect case definition were admitted to the etu suspect ward and had a blood sample drawn within 24 hours for evd and malaria diagnostic testing .
patients with an initial negative evd test remained as inpatients for repeated testing after 2 days . patients with a second negative evd result were discharged or transferred to another healthcare facility .
all patients aged < 18 years who were admitted at any of imc s 5 etus with a positive evd test and outcome data were included for analysis .
all patients were treated according to standard treatment protocols based on guidelines developed by the who , mdecins sans frontires , and local ministries of health , as permitted by local resources .
empiric treatment included antimalarial medications , broad - spectrum antibiotics , and nutritional supplementation , as well as focused supportive treatment ( appendix 1 ) .
patients were cared for by trained medical staff who recorded clinical data on paper forms .
these data were digitized at each etu and unified into a database , as described previously .
laboratory data , including evd real - time polymerase chain reaction ( rt - pcr ) and malaria testing , were linked to clinical data for analyses .
rt - pcr testing was performed by the us naval medical research center for etus in liberia , by public health england laboratories for lunsar and makeni etus in sierra leone , and by the nigerian laboratory for the kambia etu in sierra leone .
cycle threshold ( ct ) values , which are based on rt - pcr of the same ebola virus ( ebov ) zaire locus , are presented in this study as surrogates of viral load .
a ct > 40 was considered negative in all cases , though rna extraction procedures differed slightly between laboratories .
malaria testing ( conducted in sierra leone only ) was performed at each laboratory site using the commercially available binaxnow rapid diagnostic test , which identifies 4 plasmodium species : falciparum , malariae , vivax , and ovale .
throughout hospitalization , patients were cared for by trained local or international nurses , physician assistants , or physicians , who rounded at least daily and recorded vital signs , clinical data , and laboratory results on paper forms .
patient temperatures were measured with infrared or oral thermometers . for children who were unable to communicate history and who had no accompanying guardians ,
a random sampling audit concluded that > 99% of the data in imc s unified database was accurate .
the variables of interest included demographic and clinical characteristics , malaria status , initial evd rt - pcr ct values , and mortality .
length of stay was calculated as the number of days from date of admission to date of discharge .
age was categorized into the following groups : < 2 years , 24 years , 59 years , 1014 years , and 1517 years . for descriptive statistics , median values with interquartile ranges ( iqrs )
were calculated for nonnormally distributed continuous data , and proportions were calculated for categorical data .
bivariate analyses were performed to determine the associations between mortality , the primary outcome of interest , and independent covariates using the mann - whitney u test for continuous variables and or fisher exact test for categorical variables where appropriate .
covariates that had a p value < 0.1 from bivariate analyses were entered in a backward stepwise logistic regression model to determine which variables independently predicted mortality .
we used kaplan - meier curves and the log - rank test to compare survival among children by age group , hemorrhagic features , and viral loads estimated by ct values .
the sierra leone ethics and scientific review committee , the university of liberia pacific institute for research & evaluation institutional review board , and the lifespan ( rhode island hospital ) institutional review board provided ethics approval for this study and exemption from informed consent .
among the 547 pediatric cases admitted to imc etus in liberia and sierra leone during the study period , 123 had pcr - confirmed evd .
one patient , whose outcome was unknown , was transferred to another facility , leaving 122 patients available for analysis . included in this number were 7 patients who were previously admitted briefly with suspected evd but whose diagnosis was confirmed during second admissions .
children were divided into 5 age categories : younger than 2 years ( 15% ) , 24 years ( 21% ) , 59 years ( 25% ) , 1014 years ( 26% ) , and 1517 years ( 13% ) .
the median length of stay was 9 days ( iqr , 615 ; range , 131 ) .
almost all children ( 94% ) had a history of contact with documented cases of evd . at triage ,
more than 50% of children had fever , anorexia , and weakness ( table 1 ) . the most commonly observed signs and symptoms during hospitalization were weakness , fever , diarrhea , and anorexia ( table 1 ) .
clinical characteristics of ebola virus disease patients observed at triage and during hospitalization at ebola treatment units in liberia and sierra leone abbreviation : nd , no data available .
self - reported at triage ; measured during hospitalization at least daily ( range 16 times per person per day ) .
malaria testing was available at the sierra leone sites only ; 68 of 84 ( 81% ) children were tested , of whom 27 ( 40% ) were positive for plasmodium parasitemia .
the differences in incidence by age group were significant ( p = .024 ) , with the most cases reported among children aged < 5 years ( 56% ) compared with 59 years ( 50% ) , 1014 years ( 25% ) , and 1517 years ( 9% ) .
there was no difference in rates of plasmodium parasitemia in children with or without fever ( 38% vs 43% , respectively ; p = 1.0 ) .
as shown in table 2 , median length of stay was significantly longer for those who survived compared with those who died ( 16 vs 6 days ; p < .001 ) . the median age of patients who died was significantly younger than of patients who survived ( 4 vs 11 years ; p < .001 )
. stratified by age , the cfr was highest ( 89% ) for children aged < 5 years compared with other age groups ( 59 years , 43% ; 1014 years , 41% ; 1517 years , 25% ; p < .001 ; figure 1 ) .
in addition to age , other significant predictors of mortality included evidence of bleeding at any time during hospitalization ( 58% in children who died vs 27% in survivors ; p = .003 ; figure 2 ) and median initial pcr ct values ( 19.2 in children who died vs 25.0 in survivors ; p < .001 ) .
overall , a pcr ct cutoff value of 20 predicted mortality across age groups better than a cutoff value of 25 , except for children aged < 2 years ( figure 3 , supplementary figure 1 ) .
a diagnosis of plasmodium parasitemia did not influence mortality in aggregate ( p = .76 ) or by age stratified analyses ( data not shown ) .
comparison of demographic and clinical characteristics in children who survived vs those who died
number ( % ) unless otherwise stated .
differences remained significant when stratified by age ( < 5 , 59 , 1014 , 1517 years ; p < .02 for each comparison of outcomes ) .
kaplan - meier curve for overall survival among 122 ebola virus disease patients stratified by age .
children aged < 5 years had significantly greater mortality than older children ( log rank test , p < .001 ) .
kaplan - meier curve for overall survival among 112 ebola virus disease patients stratified by bleeding status .
children with evidence of bleeding at any time during their hospitalization had significantly greater mortality ( log rank test , p = .003 ) .
kaplan - meier curve for overall survival among 95 ebola virus disease ( evd ) patients based on initial evd real - time polymerase chain reaction ( rt - pcr ) cycle threshold ( ct ) cutoff of 20 , stratified by age .
overall , mortality of children with initial rt - pcr ct values 20 was significantly greater than for those with higher values ( log rank test , p < .001 ) .
the youngest and oldest groups of children accounted for most of the difference in outcome .
the difference in death rates among afebrile hospitalized children stratified by age ( < 5 years , 2 ; 59 years , 2 ; 1014 years , 2 ) was not significant ( p = .6 )
. fifty percent of the afebrile children died , but no distinguishing clinical features or rt - pcr ct values predicted these deaths .
significant variables from the bivariate analyses ( table 2 ) were entered in backward stepwise logistic regression models . in the first model that excluded pcr tests ( n = 112 ; goodness of fit , 0.68 ) , the odds of mortality were 20.1-fold higher for patients aged < 5 years compared with older children ( 95% confidence interval [ ci ] , 5.969.0 ) and 5.9-fold higher if there was evidence of bleeding at any time during hospitalization ( 95% ci , 2.215.6 ) . when evd rt - pcr ct values were added to the model ( n = 86 ; goodness of fit , 0.53 ) , the odds of mortality were 14.8-fold higher for patients aged < 5 years compared with older children ( 95% ci , 3.562.1 ) , 5.2-fold higher if the rt - pcr ct value was 20 ( 95% ci , 1.518.3 ) , and 5.0-fold higher if there was evidence of bleeding at any time during hospitalization ( 95% ci , 1.615.8 ) .
this is one of the largest cohort studies of children and adolescents with evd published to date .
the odds of death were 20-fold higher in evd patients aged < 5 years compared with older children .
this finding is consistent with other data reported from the west african evd epidemic [ 4 , 21 , 27 , 28 ] .
days of hospitalization before death for half of those aged < 2 years ranged from 6 to 12 days .
this range is comparable to that reported by shah et al [ 21 , 28 ] .
hemorrhagic features developed in fewer than half of pediatric patients ; when present , they were associated with a 5-fold higher odds of mortality .
this observation is in line with other reports that bleeding was not a prominent finding among all evd patients during this epidemic [ 19 , 21 , 22 , 29 ] .
it is also noteworthy that 1 in 5 children presented without fever and , of these , 27% never developed fever , which is similar to findings from one study but not another .
our findings support the mounting evidence that fever is not a sensitive screening tool for evd [ 17 , 28 ] .
in fact , the low frequency of hemorrhagic features and the conspicuous absence of fever at presentation in up to 20% of patients during the west african epidemic questions the utility of referring to evd as a disease characterized primarily as viral hemorrhagic fever , which may mislead clinicians .
we confirmed that a high viral load estimated by a low rt - pcr ct value ( < 20 ) at the time of presentation reliably predicted mortality in infected children who had 5-fold higher odds of mortality .
the fact that pcr ct values did not differ between survivors and fatal cases in those aged < 2 years ( unlike the older age groups ) suggests that factors other than viral load , such as immunological or physiological dysregulation , contributed to death in the youngest age group .
therefore , semiquantitative molecular diagnostic testing at the time of initial evaluation is a simple and effective method for screening for evd as well as for prognosticating outcomes ( especially in children aged > 2 years ) if ct values are below a predefined threshold and should be used routinely .
a diagnosis of plasmodium parasitemia did not impact the risk of mortality or presence of fever in a subpopulation of children who had testing performed , which differs from a recent report in which a diagnosis of malaria reduced the risk of evd mortality .
considering that children s body surface area to volume ratio is higher than that of adults and some children are malnourished , children are particularly susceptible to life - threatening dehydration and nutritional decompensation .
chertow et al made the observation that large volumes of diarrhea caused by evd were not unlike that of cholera .
treatment protocols for future evd outbreaks could potentially improve outcomes by instituting early and aggressive rehydration strategies .
this along with serum electrolyte balance and nutrition could be the most important treatments to reduce mortality in those aged < 5 years in a low - resource setting .
furthermore , prompt , accurate diagnosis and isolation precautions are critically important to reduce transmission both in the community and in healthcare facilities .
the first and historically most often associated reason is multiorgan failure secondary to coagulopathy associated with a cytokine storm and immune dysregulation .
the odds of mortality in our cohort were significantly higher in children with the classic presentation of viral hemorrhagic fever .
the large volumes of diarrhea often accompanied by vomiting and exacerbated by weakness and inability to drink fluids quickly depletes the younger children . in the low - resource settings of west africa ,
the scarcity of healthcare providers to administer oral fluids frequently or appropriate continuous intravenous ( iv ) fluids probably resulted in numerous deaths that could have been prevented with more resources .
the third reason for death is cardiac arrest from electrolyte disturbances . in patients treated in high - resource settings , abnormal serum levels of potassium and magnesium
were observed , along with cardiac arrhythmias ; similar electrolyte findings were noted in children in west africa [ 3436 ] .
the abnormal electrolyte levels may result from profuse diarrhea , prerenal kidney dysfunction from dehydration , or direct acute kidney injury ( aki ) from evd . beyond the direct effects of the virus on the kidney ,
evd has been associated with rhabdomyolysis , which can cause aki and subsequent electrolyte abnormalities .
this study had limitations in common with others conducted during the west african evd epidemic .
environmental conditions made it challenging for clinicians to collect patient data , given that they were working in full evd personal protective equipment in extreme heat , which limited the time with each patient .
patients temperatures were measured with an infrared digital thermometer gun or oral thermometer , which may be less accurate than the gold standard rectal thermometer .
frequently , clinicians were unable to collect dependable medical histories because of patients severity of illness , absence of accompanying guardians , varying skills of clinicians , or language and cultural barriers . because accurate nutritional assessments were not performed , the additional impact of malnutrition was not taken into account . without clinicians ability to confirm the length of illness
prior to arrival at etus , we were limited in determining the total duration of illness and in assessing accurately patients clinical courses .
a few of the patients included in this analysis received an experimental antiviral treatment as part of a separate trial , which may have impacted their outcomes .
in addition , it is quite likely many patients either died or recovered from evd without seeking medical attention , thereby potentially introducing ascertainment bias .
two important interventions that were not accounted for in the analyses could have impacted mortality rates .
first was whether or not patients received iv fluids , which typically was inconsistent among etus because of varying practices relating to the use of iv catheters .
the second intervention that was not taken into consideration was whether young children had constant bedside caregivers , either family members or healthcare workers , who administered and encouraged oral rehydration and nutrition and provided critical emotional support .
practices varied among etus because some parents were incapacitated or did not want to assume the risk of nosocomial infection and caregivers were not always continuously available . in conclusion
, we found that 3 clinical and virologic factors , after adjustment , were significant poor prognostic factors for children with evd : aged < 5 years , bleeding at any time during hospitalization , and high viral loads as estimated by rt - pcr ct values .
there is an urgent need to investigate the mechanisms of disease to understand how these risk factors as well as malnutrition , dehydration , and electrolyte imbalance contribute to severe outcomes .
the goal is to identify simple , scalable , and relatively inexpensive measures that can improve health outcomes substantially in children in resource - limited settings .
these fundamental interventions will effectively complement vaccines and antiviral agents that are currently undergoing human trials .
consisting of data provided by the author to benefit the reader , the posted materials are not copyedited and are the sole responsibility of the author , so questions or comments should be addressed to the author .
acknowledgements . the authors thank the governments of liberia , sierra leone , and guinea for contributing to international medical corps(icm) humanitarian response .
we also thank all of our generous institutional , corporate , foundation , and individual donors who placed their confidence and trust in international medical corps ( icm ) and made our work during the ebola epidemic possible .
we thank the us naval medical research center , public health england , the european union mobile laboratory , and the nigerian laboratory for providing laboratory data to our ebola treatment units ( etus ) .
we further acknowledge the many contributions made by all members of our research review committee and other technical teams that contributed to this research , including rabih torbay , ann canavan , dennis walto , dan rodman , yoav rappaport , samuel grindley , syed hassan , erin shedd , ryan burbach , saikrishna madhireddy , benedict adjogah , melody xie , nadezda sekularac , inka weissbecker , sean casey , farrah zughni , natalie sarles , and august felix .
we also thank medical directors vanessa wolfman and kassahun gebrehiwot and monitoring and evaluation staff including annie abbate , razia laghari , allison stewart , alex tran , matthew siakor , david mansaray , lamin bangura , sorie sesay , and joseph fangawa as well as all other data collection officers at our etus .
finally , we thank the medical and support staff , both local and ex - patriot , who set up and operated international medical corps ( icm) etus and cared for the patients during the ebola epidemic in west africa . financial support .
icm was supported by grants from the national institutes of health , national institute of allergy and infectious diseases ( k08 ai100997 ) and national institute of general medical sciences ( p20gm 104317 ) .
conflicts that the editors consider relevant to the content of the manuscript have been disclosed .
| summary.in a large , retrospective cohort of ebola virus infected children aged < 18 years , the odds of death were 5-fold higher if patients were aged < 5 years , had evidence of bleeding , or real - time polymerase chain reaction cycle threshold value 20 . |
gastrointestinal perforations constitute one of the commonest surgical emergency encountered by surgeons [ 1 , 2 ] .
management of these patients continues to be highly demanding despite the advances made in diagnosis and surgical therapy .
the etiological spectrum of perforation peritonitis in india differs significantly from its western counter parts [ 35 ] .
our study was carried out to highlight the spectrum of perforation peritonitis ( diffuse ) in a single unit at lok nayak hospital , a tertiary care hospital in delhi .
the retrospective study was conducted at the department of surgery , maulana azad medical college and associated lok nayak hospital , delhi , from may 2010 to june 2013 .
the study population included 400 patients of perforation peritonitis ( diffuse or localized ) presenting to the surgical emergency of lok nayak hospital , delhi , who underwent exploratory laparotomy .
cases were studied with respect to clinical features at the time of presentation , comorbid conditions , radiological investigations , operative findings , and postoperative course . after establishing the clinical diagnosis of perforation peritonitis
on performing exploratory laparotomy , the operative findings were noted and the source of peritonitis was found and managed accordingly .
all patients were then treated in the postoperative ward initially under the cover of parenteral broad spectrum antibiotics and fluids ; orals were started on the appearance of bowel sounds .
98% patients presented with the history of abdominal pain , 62.5% with altered bowel habit , 41.5% with nausea and vomiting , and 28% with abdominal distention .
15% patients had positive history of nsaid intake for more than 6 months ( table 1 ) . in our study ,
the commonly associated comorbidity was chronic obstructive pulmonary disease followed by renal disease , diabetes , and hypertension .
multiple air fluid levels on abdominal x - ray in erect position were present in 28% patients .
electrolyte imbalances included hyponatremia in 21% , hypokalemia in 19% and elevated serum creatinine in 18% patients .
most of the patients were operated within 24 hours of presentation under the cover of broad spectrum antibiotics after adequate resuscitation and correction of electrolyte imbalances .
the commonest cause of perforation peritonitis in our study was gastroduodenal perforation due to acid peptic disease ( 45% ) followed by appendicitis ( 18.5% ) , typhoid fever ( 12% ) , tuberculosis ( 10% ) , and trauma ( 9% ) , ( table 2 ) .
patients of peptic ulcer perforation usually had a short history of pain starting in epigastrium followed by generalized tenderness .
175 such were managed by an omental pedicle repair , in the other 4 cases a feeding jejunostomy was also done due to the large size of the perforation .
8% of these patients were managed by a limited resection with ileo - ascending anastomosis due to associated unhealthy caecum .
patients of typhoid perforation had an initial history of high grade fever prior to abdominal complaints .
of the 40 patients of tubercular perforation , 60% had previous history of tuberculosis and 50% of these patients took antitubercular therapy for < 6 months . in cases of traumatic perforation ,
the most common site was jejunum ( 49% ) followed by ileum ( 42% ) .
the most commonly performed procedure was omental pedicle closure of peptic ulcer perforation ( 43.75% ) , followed by exteriorization of the gut in the form of ileostomy or colostomy ( 22.5% ) .
appendectomy was the third most common procedure ( 17% ) , ( table 2 ) .
the most common complication was wound infection followed by dyselectrolytaemia , abdominal collection , and respiratory complications .
the morbidity rate was higher in the patients with intestinal perforation ( 58% ) than those with gastroduodenal perforation ( 32% ) .
factors involved in death included septicemia due to fecal peritonitis , respiratory complications , pulmonary embolism , and late presentation .
it is commonly seen in a younger age group in the tropical countries ( mean age in our study was 37.8 years ) as compared to the studies in the west [ 79 ] .
more commonly the perforations involve the proximal part of the gastrointestinal tract ; [ 1013 ] this being in contrast to studies from the western countries , where perforations are common in the distal part [ 1416 ] .
etiological factors also show a wide geographical variation . according to a study from india ,
infections formed the most common cause of perforation peritonitis , around 50% cases in this study were due to typhoid . in our study 22% of the cases were due to typhoid and tuberculosis .
in contrast to this , noon et al . from texas in their study reported only 2.7% cases due to infections .
also studies from the west have shown that around 1520% cases are due to malignancy [ 19 , 20 ] , this being in stark contrast to our study where malignancy was ascertained to be the cause of perforation peritonitis in only 3% of the cases .
this shows that malignancy is not a common cause of perforation peritonitis in our setup as compared to our western counterparts .
adequate preoperative resuscitation ( with fluids , etc . ) , correction of electrolyte imbalances followed by an early surgical intervention , to remove the source of infection and stop further contamination , is imperative for good outcomes minimizing morbidity and mortality .
peptic ulcer perforation , perforating appendicitis , typhoid , and tubercular perforations are the major causes of gastrointestinal perforations .
early surgical intervention under the cover of broad spectrum antibiotics preceded by adequate aggressive resuscitation and correction of electrolyte imbalances is imperative for good outcomes minimizing morbidity and mortality . | background .
perforation peritonitis is the one of the commonest emergency encountered by surgeons .
the aim of this paper is to provide an overview of the spectrum of perforation peritonitis managed in a single unit of a tertiary care hospital in delhi
. methods .
a retrospective study was carried out between may 2010 and june 2013 in a single unit of the department of surgery , lok nayak hospital , delhi .
it included 400 patients of perforation peritonitis ( diffuse or localized ) who were studied retrospectively in terms of cause , site of perforation , surgical treatment , complications , and mortality .
only those patients who underwent exploratory laparotomy for management of perforation peritonitis were included . results .
the commonest cause of perforation peritonitis included 179 cases of peptic ulcer disease ( 150 duodenal ulcers and 29 gastric ulcers ) followed by appendicitis ( 74 cases ) , typhoid fever ( 48 cases ) , tuberculosis ( 40 cases ) , and trauma ( 31 ) .
the overall mortality was 7% .
conclusions .
perforation peritonitis in india has a different spectrum as compared to the western countries .
peptic ulcer perforation , perforating appendicitis , typhoid , and tubercular perforations are the major causes of gastrointestinal perforations . early surgical intervention under the cover of broad spectrum antibiotics preceded by adequate aggressive resuscitation and correction of electrolyte imbalances is imperative for good outcomes minimizing morbidity and mortality . |
the term isotopic response connotes the occurrence of a new skin disorder at the site of another , unrelated , and already healed skin disease . since the introduction of this phenomenon in dermatology , a variety of dermatoses
we hereby describe a young girl who developed lesions of lichen planus pemphigoides ( lpp ) over preexisting atrophic scars , an example of wolf 's isotopic response . to the best of our knowledge , such a clinical presentation of lichen planus ( lp ) pemphigoides is not documented yet in the english literature .
an 18-year - old girl presented with multiple itchy hyperpigmented papules and plaques , along with intermittent blisters over the lower limbs and buttocks for last 3 months [ figures 1 and 2 ] .
past history was significant for painful ulcers over the lower limbs and buttocks 6 years back .
the diagnosis of pyoderma gangrenosum was made ( as evident from documents available with patient ) , and she was treated for a year , resulting in complete resolution of ulcers .
the bulla was noted over both lichenified plaques and otherwise normal appearing skin of scars .
interestingly , most of the lichenified plaques and bullae were found over preexisting scars only [ figure 3 ] .
skin biopsy was done from multiple sites , for both histopathological examination and direct immunofluorescence ( dif ) .
the histological features of lichenified plaque were orthokeratotic hyperkeratosis , acanthosis , wedge shaped hypergranulosis , basal layer degeneration and band - like of infiltrate of lymphocytes in the papillary dermis ; thus corroborating with a diagnosis of lp [ figure 4a c ] .
histopathology of bulla showed a subepidermal bulla with eosinophilic infiltrate ; which were suggestive of bullous pemphigoid ( bp ) [ figure 5a c ] .
the scar tissue was characterized histologically by the presence of abundant collagen fibers without any features of inflammation [ figure 6 ] .
dif from plaque showed a linear pattern of deposition of igg and c3 along the basement membrane zone [ figure 7 ] .
dif from the perilesional skin surrounding a bulla showed linear igg and c3 along basement membrane zone ; a feature suggestive of bp [ figure 8 ] .
thus , in conjunction with the clinical , histopathological and dif findings , the final diagnosis for our case was lp pemhigoides developing over preexisting scars , as an isotopic phenomenon .
atrophic scars with overlying lichenoid papules and plaques ; and tense bulla on lower limbs atrophic scars with overlying lichenoid papules and plaques on buttocks close - up of lesions on the leg .
lesions are restricted to area of scar only histopathology from plaque showing hyperkeratosis , hypergranulosis , saw toothing of rete ridges , band - like infiltration in upper dermis ( a ) ( h and e , 40 ) and ( b ) ( h and e , 100 ) with basal layer degeneration ( c ) ( h and e , 400 ) histopathology from bulla showing partial loss of epidermis including basal layer ( a ) ( h and e , 40 ) and ( b ) ( h and e , 100 ) and plenty of eosinophils in split ( c ) ( h and e , 400 ) histopathology from scar showing unremarkable epidermis , increased collagen in dermis and no inflammatory cells ( h and e , 40 ) direct immunofluorescence from plaque showing linear deposition of ig g and c 3 along basement membrane zone direct immunofluorescence from perilesional skin of bulla showing linear deposition of ig g and c3 along basement membrane zone
lpp is an uncommon variant of bp , clinically manifested with bullous lesions over papules and plaques of lp , as well as on the uninvolved skin .
it was first described by kaposi in 1892 as a classical case of lp complicated by extensive bullous dermatosis ; which was termed as lpp .
it can be precipitated by certain medications like cinnarizine , captopril , ramipril , psoralen and ultraviolet a ( puva ) , simvastatin , and antituberculous medications .
igg autoantibodies against both bpag2 and bpag1 are thought to play a pivotal role in the development of the dermatosis .
the widely accepted mechanism proposed is that lp or sometimes , puva causes damage to basal cells leading to unmasking of hidden antigens ; thus paving the pathway for the formation of antibodies and development of lesions of bp .
a report described the correlation of levels of antibodies against bpag2 with the disease activity in lpp .
the salient points of difference between lpp and bullous lp have been summarized in table 1 .
dif testing show a smooth linear distribution of immunoreactants ( c3 , igg , igm , and iga ) along the basement membrane zone of dermoepidermal junction .
however , a unique 200 kd antigen has been recognized on the epidermal side of salt split skin of patients with lpp and thus , lpp may represent a clinically distinct entity and not merely , a co - existence of lp and bp .
effective treatment modalities include topical and systemic steroids , tetracycline , nicotinamide , dapsone , isotretinoin , methotrexate and other immunosuppressive drugs .
this could be explained by the hypothesis that lpp has a unique antigenic target and thus , represents a distinct entity from bp .
lpp has been reported in association with keratoacanthoma and colon adenocarcinoma . a unilateral presentation has also been reported .
the occurrence of a different or unrelated dermatological disease at the site of the healed disease is termed as an isotopic phenomenon .
the proposed etiologies of isotopic response include viral , immunologic , neural , vascular and locus minoris resistentiae ( a site of lessened resistance ) .
however , development of lpp over preexisting scars is extremely unique and to the best of our knowledge , is not described in the english literature .
it is a well - known fact that bulla formation in lpp is mediated by immunological damage against bp antigen along basement membrane zone .
the predilection of the blisters to develop on the lower extremities could be explained by sluggish circulation in those regions . in our case ,
scars involving the dermis and subcutaneous tissue with largely unaffected epidermis might have altered the blood circulation in that particular area .
this could explain the preferential deposition of immunoglobulins in the scar area and subsequent bulla formation .
wolf 's isotopic response in lpp has probably not been described earlier in the medical literature .
wolf 's isotopic response in lpp has probably not been described earlier in the medical literature .
wolf 's isotopic response in lpp has probably not been described earlier in the medical literature . | an 18-year - old girl presented with multiple itchy hyperpigmented papules and plaques , along with tense blisters over the lower limbs and buttocks for last 3 months .
these papules , plaques , and bullae were mostly localized to preexisting scars .
the histopathological findings from papule and bulla were consistent with lichen planus ( lp ) and bullous pemphigoid , respectively .
direct immunofluorescence ( dif ) of perilesional skin around bulla showed linear deposition of igg and c3 .
considering clinical , histopathological and dif findings , diagnosis of lp pemphigoides ( lpp ) was made .
the preferential localization of lpp lesions over preexisting scars was a very interesting finding in our case an extremely rare instance of the isotopic phenomenon . |
charcoal was originally used for localizing a non - palpable tumor mass ( 1 ) , however , it is being increasingly used for localizing suspicious metastatic cervical lymph nodes or recurrent tumors in the thyroid bed after surgery ( 2).although , charcoal is known to be safe and stable when injected subcutaneously for preoperative tumor localization , it may cause foreign body reactions and granuloma , if it remains in situ for more than 6 months ( 1 ) . until recently ,
charcoal granuloma in the neck was not reported in the literature . under the approval of the institutional review board of our hospital
, we reported a case of a charcoal granuloma that was suspected to be a recurrent tumor on ultrasonography ( us ) and f - fluorodeoxyglucose ( fdg)-positron emission tomography / computed tomography ( pet / ct ) , but was correctly identified after us - guided core biopsy .
a 47-year - old woman was referred to our department for us - guided biopsy due to a persistent hypermetabolic nodule in the right level iv nodal station .
she was diagnosed with invasive ductal carcinoma of the right breast and cervical lymph node metastasis at the right level iv ( tnm stage ; t2n3m0 ) .
she had undergone breast - conserving surgery and excision of a metastatic lymph node ( fig .
before excision of cervical lymph node metastasis , us - guided localization had been performed using charcoal .
after surgery , she received chemotherapy and radiotherapy ( up to 50.4 gy ) for the right breast and the right lower cervical area . on her first follow - up fdg - pet / ct ( fig .
1d ) , which was performed 6 months after completion of treatment , a focal hypermetabolic lesion ( maximum standardized uptake value [ suvmax ] = 4.0 ) was noted at the lateral aspect of the previous operative bed in the right level iv .
the second follow - up pet / ct examination , performed 1 year after the first follow - up pet / ct scan , showed a persistent hypermetabolic lesion ( suvmax = 4.3 ) at the same site ( fig
the lesion was ovoid , measured 9.8 mm in its maximal longitudinal diameter , and had an indistinct margin from the adjacent sternocleidomastoid muscle ( fig .
on pre - contrast ct , the image noise and suspicious beam hardening artifact related to overlapped clavicles and shoulder girdles , rendered identification of the lesion difficult .
however , the lesion seemed to be slightly hyperdense relative to the adjacent sternocleidomastoid muscle ( mean attenuation : 89 - 100 hounsfield unit ) and did not significantly enhance after contrast administration ( fig .
1 g ) . since there was no evidence of recurrence at any other site of the body except the right side of the neck , and given the conflicting results of the previous cytological and pet / ct or chest ct examinations were , we decided to closely monitor and follow - up again 6 months later .
six months later , us showed a change in the echogenicity of the nodule with no change in size , but a slight change in shape ( fig .
as the morphology of the nodule changed , we could not rule out the possibility of a recurrent tumor .
thus , we decided to perform a core - needle biopsy , as the previous us - guided fine - needle aspiration did not yield sufficient tissue for an accurate diagnosis .
us - guided core - needle biopsy was performed thrice using an 18-g semi - automatic biopsy needle ( tsk stericut ; tsk laboratory , soja , japan ) .
microscopically , these dark - pigmented specimens were foreign body granulomas that comprised extensive deposits of charcoal and multinucleated giant cells , as well as fibrosis ; no tumor cells were detected ( fig .
patients who undergo surgery for malignant lesions in the neck may subsequently show benign conditions mimicking recurrent tumors . these include reactive lymph nodes , traumatic neuroma , postoperative fibrosis , and suture granuloma ( 3 , 4 , 5 ) .
charcoal is originally used for localization of the non - palpable breast mass ( 1 ) .
charcoal is known to be a biologically inert material when injected subcutaneously and may remain in situ for up to 60 days without triggering a foreign body reaction ( 1 ) . however , in an experimental study , charcoal particles were ingested by macrophages and very slowly carried away from the injection site , causing inflammatory processes ( 6 ) .
charcoal particles that remained in the surgical bed might have facilitated an inflammatory reaction during the healing process after surgery in our patient .
our patient also received radiotherapy after excision of the metastatic lymph node , possibly resulting in synergistic effects leading to fibrotic charcoal granuloma .
the us features of charcoal granuloma in our report were characterized by ill - defined irregular shaped lesion with initial hyperechogenicty that changed over time , and posterior shadowing .
indistinct margin and posterior shadowing of the charcoal granuloma in our patient was in accordance with the finding of charcoal granuloma in the breast ( 7 ) .
conversely , our lesion was hyperechoic at first , and decreased in echogenicity over time .
initial hyperechogenicity of our lesion might be a helpful finding for differentiating a tumor recurrence from a non - tumorous lesion , such as a charcoal granuloma .
tumor recurrence was reported to be hypoechoic to markedly hypoechoic in the surgical bed in the post - thyroidectomy neck ( 8) .
although the size of the nodule was not helpful to differentiate recurrence from benign postoperative change at one time point ( 8) , the interval change of the nodule size could become one of the important imaging findings to differentiate recurrence from benign lesion .
in our case , the shape and echogenicity of the lesion was changed over time , however , the lesion did not significantly increase in size during a follow - up , which suggested a benign status .
ct and fdg - pet imaging findings in our patient were consistent with those of charcoal granulomas in the peritoneal cavity related to intraoperative , intraperitoneal chemotherapy ( 9 ) .
the lesions showed high attenuation on precontrast ct with no remarkable enhancement on post - contrast scans .
higher attenuation of the lesion compared to the adjacent muscles might be attributed to the high concentration of carbon particles ( 9 ) .
both suture granulomas and charcoal granulomas can present hypermetabolic lesions due to increased utilization of glucose by activated multinucleated giant cells ( 9 , 10 ) .
therefore , such foreign body granulomas can be a common cause of false positive findings in the evaluation of tumor recurrence in the postoperative neck .
ultrasonography is the first line of imaging follow - up in patients with neck dissection or thyroid surgery .
suture granulomas as a result of the knotted suture materials after thyroid surgery , usually had characteristic irregularly shaped , multiple or paired central or paracentral echogenic foci > 1 mm , and heterogeneous hypo- to isoechoic lesions on us ( 4 ) .
although us alone is not easy to distinguish tumor recurrence from other benign lesions , initial hyperechogenicity might be a feature of a non - tumorous condition .
ct may be helpful to differentiate the 2 entities because of the hyperattenuation of the charcoal granuloma .
following imaging workup , us - guided fine - needle aspiration biopsy is generally indicated for the evaluation of recurrent nodal metastases . however , as in our patient , charcoal granuloma with charcoal materials may not be diagnosed properly with fine needle aspiration cytology alone .
cases suspected of charcoal granuloma are better diagnosed by us - guided core - needle biopsy . in summary , we reported us , ct , and fdg - pet / ct findings of a charcoal granuloma in the neck after excision of a metastatic lymph node resulting from breast cancer .
these findings included an irregular , heterogeneous hyperechoic nodule with subsequent decreased echogenicity and development of posterior acoustic shadowing on us , a high attenuating nodule on precontrast ct and a hypermetabolic lesion on pet / ct .
charcoal granulomas should be included in the differential diagnosis of incidentally detected mass - like lesions at the site of presurgical localization using charcoal . | charcoal can be used for preoperative localization of metastatic lymph nodes in the neck .
charcoal remains stable without causing foreign body reactions during as hort period .
however , foreign body reactions may develop if charcoal is left in situ for more than 6 months .
we reported a case of charcoal granuloma mimicking local recurrence on fluorodeoxyglucose - positron emission tomography / computed tomography and ultrasonography in a 47-year - old woman who had cervical lymph node dissection due to metastatic invasive ductal carcinoma of the breast . |
lesch - nyhan syndrome was first recognized and clinically characterized in 1964 by lesch and nyhan , observing two affected brothers .
lns is a rare x - linked recessive disorder , resulting from a mutation on the gene encoding for the enzyme hypoxanthine - guanine phosphoribosyltransferase ( hgprt ) , located on the long arm of the x chromosome between xq26 and xq27 .
the hgprt is an enzyme involved in purine metabolism , which catalyses the condensation reaction which combines phosphoribosyl pyrophosphate ( prpp ) and the purine bases hypoxanthine and guanine to form the respective nucleotides , inosinic acid , and guanylin acid .
the hgprt deficiency causes a backlog of guanine and hypoxanthine that oxidize in uric acid and an increase of intracellular prpp with a consequent major production of new purine .
it results in an overproduction and accumulation of uric acid , which , if untreated , usually leads to renal failure and death in early childhood .
one of the first symptoms of the disease is the presence of orange sand - like crystals of uric acid in the diapers of the affected infant .
overproduction of uric acid may lead to the development of uric acid crystals or stones in the kidneys ( nephrolithiasis ) , ureters , or bladder , causing hematuria and increasing the risk of urinary tract infection , of nephrite , and of renal failure .
another potential consequence of untreated hyperuricemia is gouty arthritis caused by uric acid crystal deposition in articular cartilage .
the affected children appear to be normal for the first few months of life , but hypotonia and development delay are evident from age three to six months .
later extrapyramidal involvement with dystonia , choreoathetosis , opisthotonos , dysarthria , dysphagia , and sometimes ballismus becomes evident .
the patients also develop signs of pyramidal involvement as spasticity , hyperreflexia , and extensor plantar reflexes .
the motor disability is so extensive that most individuals never walk and are confined to a wheelchair .
the cognitive disturbs , attention deficit problems , and mental retardation are also present in patients with lesch - nyhan syndrome .
the affected children appear normal for the first months of life ; with the eruption of deciduous teeth , they begin to show self - mutilative behavior , biting their oral / perioral tissue and their fingers .
the finger nails may be completely ripped , and the self - destructive process involves the bone . in some cases , extensive lesions
the secondary infection complicates and retards the healing of traumatic lesions [ 4 , 5 ] .
nyhan ( 1997 ) reported that children with lns feel pain and remorse when they mutilate themselves , but they are unable to control and to stop their action .
they are usually relaxed when they have physical restraints , instead when it is removed the patients become agitated and their compulsive actions increase .
the relationship between the enzyme deficiency and the neurological manifestations in lesch - nyhan syndrome is not so clear .
those disorders may be associated with deficits in dopaminergic activity in the basal ganglia ; neurochemical studies of lns patients ' brains show large reductions in da levels , elevated numbers of da receptors , and decreased levels of da transporters . the therapy with allopurinol , a xanthine oxidase inhibitor which blocks the metabolism of hypoxanthine and xanthine into uric acid , can control the overproduction of uric acid , reducing the risk of nephrolithiasis , gouty arthritis , and correlated diseases .
the dose of allopurinol has to maintain the uric acid within normal limits ; an excessive allopurinol therapy results in the accumulation of hypoxanthine and xanthine , which could result in xanthine stones .
the pharmacological treatment of self - injurious behavior is addressed to control the deregulation in the dopaminergic , opiatergic , or serotonergic systems , as a possible cause of compulsive self - mutilating behavior .
the administration of baclofen or benzodiazepines helps in muscle relaxation and keeps the patient calm .
the use of hydroxytryptophan together with decarboxylase inhibitor has been proven effective in reducing self - mutilation for a short time .
recent therapeutic options also include gabapentin , botulinum toxin a , injected into bilateral masseters , and deep brain stimulation in globus pallidus .
the self - destruction behavior should be managed by a combination of physical restraints , behavior modifications , and pharmaceutical therapy , according to olson and houlihan 2000 .
in the literature various therapeutic approaches to manage self - destruction behavior are described , but to date the treatment to solve the source of the problem has not been found yet .
pharmacological treatment of dopaminergic dysfunction , the possible opiate , and/or serotonin system dysfunction have shown variable successes , but there are disadvantages to pharmacologic treatment , as it usually requires chronic use of the drug that often places the patients in a state of chronic stupor . the use of botulinum toxin a ( btx - a ) should be considered as a good treatment for self - mutilating behavior in lns , but the action of btx - a in decreasing self - inflicted injury is not clear .
btx - a may be acting directly on peripheral nerve endings , inhibiting the release of acetylcholine and indirectly on the basal ganglia , resulting in muscle weakness and in decreased behavior .
inquiries on btx - a nowadays are not enough and a further study is necessary to define the mechanism of action and the therapeutic results [ 9 , 10 ] .
the deep - brain stimulation has been performed on a few patients with lesch - nyhan syndrome and some patients experienced a decrease in spastic self - injurious symptoms .
published literature suggests that the role of dentist is crucial to prevent and control self - mutilation . in several cases , the extraction of deciduous teeth is adopted in young patient , but the extraction of all teeth is necessary also in permanent dentition .
the extraction of only anterior permanent teeth ( incisors and canines ) , as recommended by rosenbloom and other authors , is not effective in preventing self - injury and so all permanent teeth are involved .
the extraction of all teeth represents an extreme solution that may cause functional problems and requires a treatment under general anesthesia . to avoid the extractions , a series of intraoral appliance
budnick studying a lesch - nyhan syndrome patient , unable to chew his lips for lack of anterior occlusion , decided to create an acrylic splint firstly cemented in the lower arch and secondly in the upper one .
he experimented a new technique by covering the posterior teeth to create anterior open bite , eliminating wounds caused by incisors .
in the most collaborative patients with few self - mutilations he proposed only normal care of preventive odontology ; in patients showing such a grade of collaboration and of spasticity to allow the intraoral alginate impression , he suggested the use of soft splint without cemented retention which caused hygienic and demineralization problems .
finally , in patients with insufficient collaboration the only solution was represented by the extractions of all teeth . a hard acrylic chin - cup with lip extension fitted by means of orthodontic headgear was proposed .
the appliance was well tolerated by patients and it guaranteed the health of the oral tissue but increased the dribbling habit with a consequent fungal infection of chins dermis . to solve fungal inflammation the appliance had to be removed .
a thin soft resin mouth guard was employed to prevent finger and lower lip self - mutilation in combination with an arm restraint . in six days of treatment and after three months the patient self - mutilations disappeared and he stopped biting himself , also without the mouthguard . in order to prevent dermatitis a soft resin mouthguard not covering the lip was built .
it was first cemented and then to increase the retention he preferred heat - cured acrylic resin due to adequate rigidity and he added head strap ; it had to rebuild to be adapted to development of teeth and it was not indicated in periodontal patients . in some resistant casese orthognathic surgery was proposed to create open bite and when the conservative options of treatment failed he suggested the amputation of teeth , as an alternative method to extraction of all teeth to preserve the alveolar bone . the upper and the lower incisors , canines , and the first premolars
were performed with formocresol pulpotomies , the crowns were amputated on gingival level , and the dental root canals were filled with light curing glass ionomer cement and polished with softlex discs . the patient was sedated using choral hydrate and hydroxane , but in case of poor cooperation the general anesthesia was indicated [ 1719 ] .
a removable acrylic dental appliance was made to create anterior open bite , orthodontic acrylic resin covered the palate , and occlusal surface of posterior teeth and adam 's claps on first upper molar increased the retention . a resin mouthguard thick in posterior part was combined with a lip bumper in order to open anterior bite and to protrude lower lip from the teeth , but this treatment had minimal to partial success .
the acrylic resin bite plate 's retention was increased by a combination between adam 's clasps on the premolars and ball clasps among incisors .
the operators of the orthodontic ward of gaslini hospital realized a new orthodontic device to preserve oral and perioral tissue of lns patients . at first irreversible hydrocolloid impressions of maxillary and mandibular arches
these impressions were finally taken in spite of patient 's lack of compliance ; operators ' persuasive capacity , family , and operators ' efforts to change the negative behavior of m.c . were crucial . on these impressions
a plaster model was built , the undercuts were eliminated ( figures 1 and 2 ) , and the orthodontic dispositive was realized by thermoforming disc materials ( with a hard copolyster outer layer and a soft polyurethane inner layer ) ( figure 3 ) , using a positive - pressure thermoformer ( ministar ) ( figures 2 and 5 ) .
the hard side was placed in contact with teeth to increase friction and retention ; on the contrary the soft one was placed in contact with antagonist dental arch to prevent traumatic occlusion .
the first devices were removed by patients ' tongue , so it was decided to change the dispositive , reducing mucosal flange to prevent the mouthguard 's removal ( figure 4 ) . at the beginning , patients used the device only for few hours , but time by time they were persuaded to wear it longer . at
first a thick lower bite was made to produce an anterior open bite ; then it was substituted by a thinner maxillary and mandibular mouthguards well tolerated by the patient also during the meals .
after these treatments , the patients were able to wear the device for 24 hours and it guaranteed the health of the oral and perioral tissue .
the most common disadvantages of preventive intraoral devices are the design that may cause fungal infections , extensive laboratory time , and difficulty in daily oral care . also frequent adjustments are required and heavy biting forces added to poor cooperation may interfere with impression taking .
these devices extend to the vestibule area and the splint that creates an open bite increases the drooling habit with a consequent fungal infection of chin dermis .
cemented splint requires an extensive laboratory work , daily hygiene care is difficult , and the demineralization of the teeth is frequent .
some patients with lns show a degree of collaboration to allow the intraoral alginate impression , in other lns patients is impossible to have a degree of cooperation on making the intraoral impression ; in fact sedation is sometime necessary . the use of putty - type vinyl polysiloxane impression material or quick alginate to overcome the insufficient patient 's collaboration that makes the intraoral impression very difficult is suggested .
it is possible to obtain greater patient 's collaboration thanks to behavioral modification and persuasive capacity of family and operators .
the simple intraoral device presented by gaslini operators is easy to fabricate ; it is a mouthguard realized by thermoforming disc and characterized by two different matrices : hard and soft .
the hard matrix guarantees a good retention , so during the cementation , the head straps , the splint extension to vestibule area , and the metal clasps are not necessary to give stability .
the double matrix device is readily removed for everyday cleaning and the placement is also comfortable .
the bite is very thin , it permits that the patient can wear the mouthguard all day , also during the meals , and consequently the drooling habit is not accentuated .
the device is built on the patient plaster model ; it can be customized for each individual case .
heavy biting force and poor patient 's collaboration may interfere with impression taking . in the case
described here the first impression to create an individual impression trays was taken and a second one to fabricate the device .
also the work was simplified by the use of quick alginate . in a second moment
the efficacy of this device was observed during a period of about 7 years . during these years
the major difficulties encountered during this therapy are as follows : necessity of modifying the dispositive to adapt it to the mouth changes , risk of small cracks on the occlusal resin bite that must be often repaired
. necessity of modifying the dispositive to adapt it to the mouth changes , risk of small cracks on the occlusal resin bite that must be often repaired .
in conclusion the literature analysis demonstrates that the intraoral appliances , despite some limitations , can represent a good alternative option to invasive treatment and a good choice of therapy to limit and to prevent self - mutilative behavior in lns patients .
the thin resin mouthguard described was successfully employed to prevent self - mutilation of oral and perioral structures .
it represented a conservative solution and a good alternative option to invasive treatment of extracting the teeth or of orthognathic surgery .
in addition , the device is easy to build and is well accepted by patient . a good choice of treatment for improving the quality of life of these patients can be represented . | lesch - nyhan syndrome , described in 1964 by lesch and nyhan , is a x - linked recessive disorder , occurring in 1 : 100000 to 1 : 380000 live births .
lns is characterized by a decrease in activity of hypoxanthine guanine phosphoribosyl transferase , an enzyme involved in purine metabolism , resulting in overproduction of uric acid .
hyperuricemia and neurological features including choreoathetoid spasticity , self - mutilation , and mental retardation clinically characterize this syndrome . in lns patients
the typical feature is loss of tissue from biting themselves with partial or complete amputation of fingers , lips , and tongue .
the self - mutilation compares with the eruption of the deciduous teeth .
several drugs trials have been administered to improve self - destructive behavior and invasive treatment approaches , such as extractions of teeth and orthognathic surgery , have been suggested with variable effectiveness . nowadays prevention is , therefore , the standard of care .
the role of dentistry is essential in the management of the self - mutilating behavior , because the teeth represent the main self - injury instrument .
this report presents a revision of various therapeutic approaches to manage self - destruction , highlighting the effectiveness of a preventive treatment .
it describes a new technique : a resin mouthguard , realized at gaslini hospital , to obtain immediate healing of the oral lesions , confirmed in the follow - up period . |
this review will focus on scorpion venom and its major toxins and their functions in excitable cells .
it well also compares some of the research done on scorpions from different parts of the world to highlight open areas of interest .
these animals are found in all continents except antarctica , and are known to cause problems in tropical and subtropical regions .
they are adapted to survive in a wide variety of habitats including tropical forests , rain forests , grasslands , savanna , temperate forests , caves , and even snow covered mountains .
actually these animals are represented by 16 families and approximately 1500 different species and subspecies which conserved their morphology almost unaltered [ 1 , 2 ] .
the scorpion species that present medically importance belonging to the family buthidae are represented by the genera androctonus , buthus , mesobuthus , buthotus , parabuthus , and leirus located in north africa , asia , the middle east , and india .
centruroides spp are located in southwest of united states , mexico , and central america , while tityus spp are found in central and south america and caribbean . in these different regions of the world
the scorpionism is considered a public heath problem , with frequent statements that scorpion stings are dangerous .
accidents caused by scorpion stings are a relatively common event in subtropical and tropical countries and can cause lethal envenomation in humans , especially in children .
the signs of the scorpion envenomation are determined by the following : ( a ) scorpion species , ( b ) venom composition , and ( c ) the victim 's physiological reaction to the venom .
the symptoms of the sting start immediately with a few minutes after the sting and usually progress to a maximum severity within 5 hours . at this period the massive release of neurotransmitters results in sweating , nausea , and vomiting .
the victims usually have the major signs , with the most common being mydriasis , nystagmus , hypersalivation , dysphagia , and restlessness .
they may exhibit signs and symptoms involving the central nervous system , stimulation of the autonomic nervous system , and occasionally , respiratory and heart failure , and even death .
after stings by dangerous scorpions from different parts of the world the signs and symptoms are similar .
the victims of scorpion envenoming that presented multi - system - organ failure characterized by changes in hormonal environment with a massive release of counter - regulatory hormones , such as catecholamine , glucagon , cortisol , angiotensin - ii , and with decreased levels of insulin and an increase blood glucose level .
the grading of these scorpions envenomation depends on local signs and whether or not neurological signs predominant .
the local signs observed in victims can present effects that can separate in a neurotoxic and cytotoxic local ( table 1 ) .
central nervous system signs are sympathetic , parasympathetic , somatic , cranial , and peripheral nervous system and their major characteristics are shown in table 2 .
the nonneurological signs which include cardiovascular , respiratory , gastrointestinal , genitourinary , hematological , and metabolic signs . with respect the neurological signs ,
most of the symptoms are due to either the release of catecholamine from the adrenal glands or the release of acetylcholine from postganglionic parasympathetic neurons .
table 3 shows the summarized characteristics of the different grade of envenomation caused by scorpion venom .
scorpion uses its venom for both prey capture and defense . the venom is constituted by mucopolysaccharides , hyaluronidase , phospholipase , serotonin , histamine , enzyme inhibitors , and proteins namely neurotoxic peptides [ 7 , 8 ] .
the signs of the scorpion envenomation are determined by the symptoms presented by victims of scorpion envenomation are usually complex in nature and can be attributed mainly to hyperactivity of the autonomic nervous system .
the venom contain neurotoxic peptides which are responsible for the symptoms that present during envenomation by interacting with ion channels and have the potential to cause massive damage to nervous system of both vertebrates and invertebrates .
ion channels are gated pores of which gating may be intrinsic or regulated by binding or changes in the voltage gradient .
this gradient across is responsible by excitation of the nerve and muscle , hormonal secretion , cell proliferation , sensory transduction , the control of salt and water balance and regulation of blood pressure .
scorpion toxins presents specificity and high affinity and have been used as pharmacological tools to characterize various receptor proteins involved in normal ion channel functionating , as abnormal channel functionating in disease - states [ 10 , 11 ] .
advanced methods of fractionation , chromatography , and peptide sequencing have made it possible to characterize the components of scorpion , snakes and spiders venoms [ 1214 ] .
the venoms can be characterized by identification of peptide toxins analysis of the structure of the toxins and also have proven to be among the most and selective antagonists available for voltage - gated channels permeable to k , na , and ca [ 1517 ] .
the neurotoxic peptides and small proteins lead to dysfunction and provoke pathophysiological actions , such as membrane destabilization , blocking of the central , and peripheral nervous systems or alteration of smooth or skeletal muscle activity .
the comparison of pharmacological characteristics with the different structures of scorpion toxins is important to understand and the mechanisms of action [ 17 , 19 , 20 ] .
these toxins are responsible to overcome the defensive systems of the hosts such as proteases and/or significant local ph variations that may result from inflammation states induced by the animal bite itself [ 21 , 22 ] .
scorpion toxins are classified according to their structure , mode of action , and binding site on different channels or channel subtypes [ 1719 ] .
each class consists of several peptides isolated from the venom of different species of scorpion and are based on their pharmacological action and also agrees well with the structural properties of this peptide family .
the long - chain toxins affecting sodium channels have been subdivided primarily into two major subtypes , - and -toxins [ 23 , 24 ] . the -toxins bind to receptor site 3 of the voltage - gated na channels of vertebrates in a membrane - dependent manner .
the major effects of -toxins induce a prolongation of the action potential of nerves and muscles by fast inactivation of sodium channels receptor affinity dependent upon membrane potential [ 17 , 19 , 2532 ] .
the -toxins are isolated from american scorpions , bind to receptor site 4 on vertebrate na channels and producing a shift to a more negative membrane potential [ 3338 ] .
several studies described the mode of action of these toxins that are related with the increment of sodium [ 36 , 3942 ] .
the -scorpion toxin cssiv obtained from centruroides suffussus scorpion venom is believed to specific bind in sodium channel [ 23 , 24 , 33 , 34 ] .
other toxin also described such as ts1 is also known as ts which is a major toxic component obtained of the venom from the brazilian scorpion tityus serrulatus .
this toxin has also been classified as a -scorpion toxin based on its structural homology , competitive binding assay , and its site of action [ 4143 ] .
the effect of these toxins has been described in different cell types [ 4143 ] .
the -scorpion toxin is believed to bind , to only one of the four voltage sensors of the sodium channel [ 34 , 36 , 42 , 44 ] . in accordance to the classical models of sodium channel gating ,
the voltage sensors of the sodium channel activate independently , and at least three of them have to be in an activated position for the channel to open [ 4548 ] .
however , if one of them is activated by the -toxin , the threshold of activation is unlikely to shift significantly since other voltage sensors remain unaffected .
voltage - gated sodium channels are critical for generation and propagation of action potentials initiation and propagation in excitable cells [ 25 , 33 , 49 ] .
these channels are targeted for neurotoxins present a large variety of chemically distinct compounds that bind to several receptor sites on the pore - forming -subunit [ 34 , 35 , 49 ] . with respect to scorpion toxins
have been observed that they show a preference for distinct sodium channels subtypes of mammals or insects [ 20 , 3335 , 50 ] .
potassium channels are part of a large variety of biological processes and also are involved in an increasing number of human pathologies [ 9 , 51 ] . the diversity of potassium channel blockers and their therapeutic value to overcome in the potential treatment of a number of specific human diseases specially autoimmune disorders , inflammatory neuropathies and cancer .
scorpion toxins that target kchannels ( ktx ) are short - chain peptides cross - linked by three or four disulfide bridges .
the -ktx family constitutes by more than 50 different -ktx have been reported and listed in more than 18 families [ 18 , 5254 ] .
2000 described that / scaffold formed by an -helix and a two- or three - stranted -sheet linked by two bridges .
however the / fold is shared by a variety of polypeptides with diverse functions , such as toxins active on na channels .
the neurotoxin -ktx 12.1 initially named as tstx - iv was isolated from the t. serrulatus venom which is constituted with four disulfide - bridged described by several studies [ 18 , 53 , 5659 ] .
the voltage - gated potassium channel has been shown to play a role in immune responsiveness .
previous in vitro studies described that the blockade of the channel leads to diminution of t cell activation and delayed type hypersensitivity .
butantoxin which is present in the venoms of three brazilian scorpions t. serrulatus , t. bahiensis , and t. stigmurus has shown to reversibly block the potassium channels and inhibit the proliferation of t cells and il-2 production .
ca ions play important roles in regulating a variety of cellular functions such as second messenger - coupling - receptor to active many cellular processes that including cellular excitability , neurotransmitter release , intracellular metabolism , and gene expression .
the increment of ca concentration is mediated by voltage - gated ca channels that regulate ca influx across the plasma membrane and control the release of ca from intracellular stores .
the ca channels are widely distributed in the body such as heart muscle , smooth muscle , skeletal muscle neurons , and endocrine cells [ 9 , 25 ] .
scorpion venom consists of numerous peptides that may interfere with the activity of ion channels and modulate their functional properties .
various studies have been shown that scorpion toxins are used in insecticides , vaccines , cancer treatment , and protein engineering scaffolds .
the inflammatory response is triggered by cascade that includes systems , cell elements , and release of mediators .
after exposure to antigen vertebrates respond by antibodies production through a series of events involving multiple cellular interaction .
initially , antigen presenting cells is recognized by a t cell antigen receptor . at the end ,
b cells produce antibodies that are able to specifically recognize the antigen that provoked their formation . in between the two antigen - specific events t cells help b cells to make antibody by producing cytokines and/or cell - cell interaction .
the two subpopulations of th cells which are named as th1 and th2 that differ in the effectors functions that differ mainly in the repertoire of cytokine secreted in response to antigenic stimulation [ 62 , 63 ] .
th1 type cells secrete mainly interleukin-2 ( il-2 ) , tumor necrosis factor ( tnf- ) , and interferon- ( ifn- ) which are responsible for macrophages activation and promote cell - mediated immune responses against invasive intracellular pathogens or the presence of toxins .
the other subpopulation , th2 type cells are important in the defense against extracellular parasites by induction of the ige and iga and produce a variety of cytokines such as il-4 , il-5 , il-6 , and il-13 . both th1 and
th2 cells secrete lesser amounts of tnf- , granulocyte - macrophage colony - stimulating factor ( gm - csf ) , and il-13 .
mutual cross - inhibition between th1 and th2 type cytokines polarize functional th cell responses into cell - mediated or humoral immune responses [ 6267 ] . in accordance with their actions or properties
pro - inflammatory cytokines such as il-1 , il-6 , and tnf are primarily responsible for initiating an effective defense against exogenous pathogens .
however , the overproduction of these mediators can be harmful and may ultimately lead to shock , multiple organ failure , and death ( table 4 ) [ 68 , 69 ] .
by contrary , anti - inflammatory cytokines include il-4 , il-5 , il-6 , and il-10 are crucial for downregulating the exacerbated inflammatory process and maintaining homoeostasis for proper functionating of vital organs , but excessive anti - inflammatory response may also result in the suppression of body immune function ( table 4 ) [ 7073 ] .
the balance between pro- and anti - inflammatory activities determines the degree and extent of inflammation , and thus can lead to different clinical effects [ 7476 ] .
anti - inflammatory cytokines counteract the effects of proinflammatory cytokines and therefore the relative concentration of a cytokine to its inhibitor or antagonist will determine its final effect .
cytokines imbalances mediate the development of organ damage and lethality during severe sepsis and envenomation , a lethal syndrome that can develop after infection or injury .
health requires that cytokine production is balanced ; low levels are required to maintain homeostasis .
overproduction of some cytokines causes diseases that span the range of severity from mild to lethal .
in addition , the setting of local infection or tissue injury , a well - adapted anti - inflammatory response may contain local inflammation and prevent it from evolving into systemic inflammation .
systemic inflammation may be divided as acute or chronic ; in acute systemic inflammation such as in sepsis , trauma , burns , and surgery is characterized by rapid and increments in plasma - levels of proinflammatory cytokines [ 77 , 78 ] . in contrast , chronic low - grade inflammation is characterized by a modest but sustained increment in cytokines and acute phase reactants , usually of two to three folds and may be a key player in the pathogenesis of most chronic noncommunicable diseases [ 79 , 80 ] .
much evidence supports the role of cytokines in scorpion envenomation seem that both pro- and anti - inflammatory cytokines levels are overproduced in sepsis syndrome . their clinical significance and prognostic value have not been elucidated [ 76 , 8186 ] . the production of cytokines in the envenomation has previously been referred to as a cascade ( table 5 ) . with respect to the pathogenesis of tissue injury is complex and can not be attributed to a single agent .
tissue injury occurs during inflammation and is a progressive process which may eventually lead to organ dysfunction failure .
the categorization of cytokines into pro- and anti - inflammatory response is essential for structural and functional repair of injured tissue , but excessive generation of proinflammatory signals can aggravate tissue damage because of the products derived from inflammatory cells .
il-1 is a prototypic proinflammatory cytokine that exists in two forms named as il-1 and il-1 , both of which exert similar but not completely overlapping biological functions mediated through the il-1 receptor ( il-1r ) . after binding to il-1r , il-1 induces the production of a high spectrum of cytokines and chemokines as well as the expression of adhesion molecules on endothelial cells , thus leading to the recruitment of inflammatory cells .
in addition , il-1 also contributes to the development of vascular damage by stimulating cell proliferation and differentiation and the release of matrix - degrading enzymes .
il-1r antagonist ( il-1ra ) is a structural homologue of il-1 that binds to il-1r but does not induce any cellular responses and is therefore a natural inhibitor of il-1 activity . both are synthesized as precursor molecules ( pro - il-1 and pro - il-1 ) by many different cell types .
pro - il-1 is biologically active and needs to be cleaved by calpain to generate the smaller mature protein .
in contrast , pro - il-1 is biologically inactive and requires enzymatic cleavage by caspase-1 in order to become active .
il-1 is primarily bound to the membrane , whereas il-1 is secreted and thus represents the predominant extracellular form of il-1 .
the levels of il-1 in serum from human and mice injected with brazilian scorpion t. serrulatus and/or its majors toxins are characterized by rapid increments of this proinflammatory cytokine .
high levels of these cytokines were observed in supernatants of macrophage from mice exposed to t. serrulatus venom and its major toxins [ 91 , 92 ] . increased levels of il-1 were determined in plasma from patients moderately or severely envenomed by t. serrulatus sting .
high levels of il-1 and il-1 were observed in sera from mice exposed to mexican scorpion centruroides noxius .
the role of il-1 in scorpion envenomation has been investigated through influencing its level or activity .
table 5 shows the presence of this cytokine production after accident caused by different scorpions .
il-6 is a multifunctional cytokine that regulates various aspects of the immune response , acute - phase reaction , and haematopoiesis .
il-6 is inducible by il-1 and consequently concentrations in serum are often a reflection of il-1 in vivo activity .
contrarily other cytokines , the il-6 is a pleiotropic cytokine that exerts its proinflammatory effects is produced by a variety of cells including b and t lymphocytes , monocytes , fibroblasts , keratinocytes , endothelial cells , mesenchymal cells , and certain types of tumor cells .
high levels of il-6 were observed in sera from mice exposed to centruroides noxius and t. serrulatus scorpion venoms [ 76 , 84 ] . increased levels of il-6
were also observed in plasma from patients with different grade of envenomation by t. serrulatus [ 81 , 82 ] . the cytokine production caused by different scorpions
it downregulates the synthesis of il-1 and tnf- and also inhibits the production of gm - csf , ifn- , and mip-2 [ 9598 ] .
scorpion venoms can stimulate the neuroendocrinal - immunological axis by its ability to release catecholamines , corticosteroids , bradykinin , and prostaglandins and all these agents proved to induce the release of immunological mediator cytokines .
there is now accumulating evidence to suggest a causal relationship between overproduction of certain cytokines such as il-1 and il-6 and morbility and mortality associated with critically ill patients .
sofer 1995 , , was the first that reported the involvement of the inflammatory systems after scorpion envenomation in humans . in this work
is documented the increment of il-6 levels in the serum of 8 of 10 children severely envenomed by the scorpions l. quinquestriatus and b. judaicus .
the cytokines were measured on admission to the hospital and 1 to 3 hours after the sting il-6 levels gradually returned to normal values at 12 and 24 hours measurements , but remained above control levels in all measurements .
these results were quite similar to those found by others authors that described the cytokine production after sting caused by tityus serrulatus scorpion in humans [ 81 , 82 ] . with respect the experimental animal high levels of cytokines
were found in serum from mice injected with centruroides noxius and t. serrulatus venom [ 76 , 84 ] . in all
works the authors concluded that the activation and release of cytokines may play an important role in the pathophysiology of envenomation after stings and may be responsible for some systemic inflammatory manifestations and organ failure .
more human and experimental animal studies are required to determine the contribution of the inflammatory system in the genesis of scorpion envenomation .
il-8 as pivotal mediator of cerebral reperfusion is increased in brain tissues and a neutralizing anti - il-8 antibody significantly reduced brain oedema and infarct size in comparison to rabbits receiving a control antibody . increased levels of il-8 were observed in serum from patients with different grade of envenomation caused by t. serrulatus and leiurus quinquestriatus scorpions ( table 5 ) [ 82 , 89 , 90 ] .
tnf is a pleiotropic cytokine that exerts potent proinflammatory effects on envenomed and other metabolic and inflammatory disorders which are also risk factors for cardiovascular diseases .
lymphocytes and macrophages orchestrate a lot of the inflammation in envenomation , mainly the production of tnf- that exerts its proinflammatory effects through increased production of il-1 and il-6 , expression of adhesion molecules , proliferation of fibroblasts , and procoagulant factors , as well as initiation of cytotoxic , apoptotic , and acute - phase responses [ 87 , 100 , 101 ] .
tnf- is a major inflammatory cytokine due to its ability to stimulate the synthesis of nitric oxide and other inflammatory mediators that drive chronically delayed hypersensitivity reaction [ 102 , 103 ] . with respect to tnf-
, il-1 seems to be important in the pathogenesis of envenoming because of its immunological upregulatory and proinflammatory activities [ 76 , 84 , 87 ] .
the il-1 system consists of il-1 and il-1 , both of which are produced by various cell types through the initiation of cyclooxygenase type 2 , phospholipase a , and inducible nitric oxide synthase [ 102 , 103 ] .
high levels of tnf- were observed in human and mice serum and also in mice macrophages supernatant ( table 5 ) .
ifn-
: the pleiotropic cytokine ifn- is a proinflammatory mediator that is expressed at high levels in envenomation by various cells , including monocytes / macrophages , th 1 cells , and natural killer t cells ( nk ) .
high levels of ifn- were observed and documented during the envenomation caused in human and experimental animals by different scorpion venoms centruroides noxius and tityus serrulatus ( table 5 ) [ 76 , 8284 ] .
il-4 is a highly pleiotropic cytokine that is able to influence th cell differentiation , its early secretion leads to polarization of th cell differentiation toward th2 like cells .
th2 cells secrete their own il-4 , and subsequent autocrine production of il-4 supports cell proliferation .
the th2 cell secretion such as il-4 and il-10 has marked inhibitory effects on the expression and release of the inflammatory cytokines .
il-4 is considered to be able to block or suppress the monocyte - derived cytokines , including il-1 , il-6 , il-8 , tnf- , and macrophage inflammatory protein 1 alpha [ 104 , 105 ] .
overall anti - inflammatory cytokines whose roles are less well characterized in envenomation include il-4 which has a stimulatory molecule for b and t cells , and has known immunosuppressive effects in the intestine .
t - cell receptor chain - deficient mice ( tcr-/- ) treated with anti - il-4 monoclonal antibody showed a decrease in th2 cells mrna cytokine production and an increase in expression of ifn- , suggesting that il-4 plays a major role in inducing th2 type cd4 + cells in the gut to shift towards a th1 response .
increment of il-4 production was observed in serum from rats exposed to androctonus australis hector scorpion ( table 5 ) .
il-10 is produced by several cell types including cd4 + and cd8 + , t cells , macrophages , monocytes , b cells , dendritic cells , and epithelial cells .
il-10 is the most important anti - inflammatory cytokine found within the human immune response .
it is the potent inhibitor of th1 cytokines , including ifn- , tnf- , il-1 , il-2 , il-6 , il-8 , and il-12 [ 63 , 108 ] .
in addition , a major stimulus for the production of il-10 is inflammation itself and il-1 and tnf- can stimulate il-10 production directly .
the one other property of il-10 is to suppress free oxygen radical release and nitric oxide activity of macrophages and the production of prostaglandins . in serum from patients envenomated with tityus serrulatus scorpions and in experimental animals exposed to androctonus australis hector , centruroides noxius , and t. serrulatus venoms were observed modified levels of il-10 ( table 5 ) .
the pathophysiology of envenomation is complex , but there is little doubt that injection often progresses from systemic inflammatory response to severe envenomation . in humans
envenomed or experimental animal exposed to venom crude and/or purified toxins from different scorpions is the primary event in this sequence . during the interaction of venom components with cells and serum proteins to initiate a series of reactions that generally may lead to cell injury and death .
the discovery that cytokines have the capacity to cause disease focused a new field of investigation on the physiological control mechanisms that maintain health by restraining on counter regulation cytokine release .
systemic effects of the cytokines have been shown to induce fever and increase symptoms . in the local action
the balance between pro- and anti - inflammatory cytokines determines the degree and extent of inflammation , and thus can lead to different clinical effects .
anti - inflammatory cytokines counteract the effects of proinflammatory cytokines and therefore the relative concentration of a cytokine to its inhibitor or antagonist will determine its final effect . following the injection of venoms , a variety of proinflammatory cytokines are released , along with counter - regulatory or anti - inflammatory cytokines , and the outcome of an inflammatory response is dictated by a variety of factors , that including the duration of the stimulus , and the balance between the proinflammatory and anti - inflammatory response .
an excessive proinflammatory response is thought to be important in the pathogenesis of septic shock .
in contrast , an excessive anti - inflammatory response could result in failure to clear a venom action , with equally deleterious effects [ 73 , 85 , 110 ] .
however the prolonged compensating anti - inflammatory response syndrome may be associated with excess mortality and morbidity because of increased risk for envenomation ( figure 1 ) . with respect to the functions of many cytokines , and frequently multiple antagonists for any given agonist ,
the ability to compensate for a certain amount of divergence in production of individual cytokine is significant .
cytokines are important for regulation of inflammatory response . during the local and systemic responses
are observed the release of proinflammatory cytokines , arachidonic acid metabolites proteins of the contact phase and coagulation system , complement factors ; it is defined as systemic inflammatory response .
the massive production of the cytokines causes that spam the range of severity mild to lethal .
however , in parallel , anti - inflammatory mediators which produce an imbalance of these dual immune responses seems to be responsible for organ dysfunction .
the pathogenesis of tissue injury is complex and can not be attributed to a single agent .
tissue injury occurs during inflammation and is a progressive process which may eventually lead to organ dysfunction and failure .
the systemic inflammatory response is the overproduction inflammatory reaction resulting from systemic mediator release that may lead to multiple organ dysfunctions ( figure 2 ) .
excessive generation of proinflammatory signals can aggravate tissue damage because of the products derived from inflammatory cells .
the increment of the levels of proinflammatory cytokines leads to activation of macrophages , neutrophils , nk cells , t cells , and b cells [ 67 , 73 , 111 ] .
the inflammatory response is essential for structural and functional repair of injured tissue . among the clinical features observed in severely envenomed patients , mainly children , the gastrointestinal symptoms such as vomiting , excessive salivation , and abdominal pain
based on these and others clinical observations , several studies have been carried out to improve our understanding of the effects of scorpion venom on the gastrointestinal system .
experimental studies have shown that the injection of whole venom and purified toxins from the venom of scorpions can cause profuse salivation , increased gastric and pancreatic injuries as well as disorders of intestinal motility [ 113117 ] .
most of these effects have been related to the acute autonomic disturbances triggered by the venom , which can provoke both the activation and delayed inactivation of neuronal sodium channels , where they modulate the release of neurotransmitters , that leads to a variety of adverse effects which include respiratory failure , lung edema , arrythmias , tachycardia followed by bradycardia , skeletal muscle stimulation , lacrimation , convulsions , and enlarged pupils , among others [ 112 , 116124 ]
. however , the role of other members of il - family in envenomation is increasingly appreciated , and in the present work are summarized all currently available information from human and experimental studies . with respect to the scorpion envenomation the immune response
also is triggered by cascade that including the released of mediators such as nitric oxide and complement system .
nitric oxide ( no ) is a free radical generated by the conversion of the aminoacid l - arginine to no and l - citrulline by the enzyme no synthase and is the key endothelium - derived releasing factor implicated in the regulation of vascular tone and vasomotor function .
no is also considered as a second messenger for a number of physiological in processes including neurotransmission , maintenance of vasodilator tone , and arterial pressure and it has been suggested that cytokine - mediated circulatory shock is caused by activation of the inducible isoform of nos .
oneb of the isoforms , the endothelial nos , plays an important role in determining and maintaining aspects of normal renal function , for instance proximal tubule sodium reabsorption . in biological systems such as cardiovascular and nervous ,
the no has important functions by its implication as vasodilator and neurotransmitter . in some instances
the no decomposes to nitrite and nitrate , and cytokine - mediated increases in concentrations of nitrite / nitrate .
several inducers of insulin resistance that including proinflammatory cytokines and oxidative stress are capable to cause modifications in the levels of nitrite and nitrate production have been associated with several conditions : severe envenomation , septic shock , and hypertension [ 83 , 84 ] .
no plays a pivotal role in the pathophysiology and pathology in various systems including the envenomation caused by scorpion venom .
the presence of no in serum from patients envenomed with l. quinquestriatus and mice exposed to t. serrulatus venom , and supernatants of macrophage treated with t. serrulatus venom and/or and its toxins are shown in table 5 .
the oxidant and anti - oxidant balance is an important determinant of immune cell function , not only for maintaining the integrity and functionality of membrane lipids , cellular proteins , and nucleic acids , but also for the control of signal transduction and gene expression in immune cells .
the cells of the immune system are sensitive to changes in the balance of the oxidant / antioxidant because high levels of ros are produced as part of their normal function .
one other effect is associated with complement - activating components in the venoms that indirectly contribute to tissue damage .
bertazzi et al . , 2003 , described the effects of t.serrulatus venom and tstx1 gamma on lytic activity of the complement system . in this
study the authors showed that the complement system is involved in inflammatory process induced by the venom or toxin and consequently in the lung edema , hemolysis , leukocytosis , among other clinical manifestation of severe envenomation .
the effects of androctonus australis hector venom on the lytic activity of the complement system were also studied and reported .
scorpion envenoming essentially results in a syndrome of fuel - energy deficits and causes an inability to utilize the existing metabolic substrates by vital organs causing multi - organ system failure and death .
the effect of stings of scorpion venoms in humans and experimental animals exposed to venom , such as rabbits , rats , and mice were studied in serum biochemical parameters and are presented in table 6 .
2009 shown that the brazilian scorpion t. serrulatus resulted in increased lung , kidney , liver , and heart inflammation , characterized by an increased density of mononuclear cells after injection in rats .
these authors concluded that this venom leads to acute lung injury , characterized by altered lung mechanisms and increased pulmonary inflammation .
the primary pathological process is pulmonary capillary endothelial dysfunction resulting in interstitial and alveolar oedema of protein and phagocytic immune cell rich exudative fluid .
the symptoms associated with pulmonary oedema are variable but may be rapid . depending on the severity and duration of the renal dysfunction this accumulation
is accompanied by disturbances such as metabolic acidosis and hyperkalemia , changes in blood fluid balance , and effects on may other organ systems .
the scorpion venom caused a great increase in renal oedema which is related to the decreased glomerular filtration rate and urinary flow .
the t. serrulatus venom also affects haemodynamics probably by a direct vasoconstrictor action leading to increased renal flow in mice [ 136 , 137 ] .
acute renal failure is a rapid loss of renal function due to damage to the kidneys , resulting in retention of nitrogenous such as creatinine and non - nitrogenous waste products that are normally excreted by the kidney . in various works
the authors described that scorpion venom caused a great increase in renal oedema , which is related to the decreased glomerular filtration rate and urinary flow . during the acute renal failure
has been reported to occur after scorpion stings [ 127 , 138 , 139 ] . in normal state
the kidney maintains renal blood flow and glomerular filtration through autoregulation dependent on the tone of the afferent and efferent arterioles .
the cytokine - induced systemic vasodilation and relative hypovolaemia in sepsis are responsible for renal hypoperfusion .
the renal vasculature has been shown to participate variably to mediators of systemic vasodilation and renal blood .
in particular , the arachidonic acid metabolites of thromboxane and leukotrienes both reduce renal blood flow and antagonists of these substances have been shown to have renal protective effects .
the kidney is susceptible to leukocyte mediated tissue injury with neutrophil aggregation in response to production of proteases and ros .
fatality after scorpion envenomation may be the result of cardiovascular failure complicated by pulmonary oedema as well as by respiratory arrest .
both of heart and the blood vessels are sensitive to the effects of proinflammatory cytokines as well as vasoactive substances present in excessive amounts in envenomation .
a direct effect of scorpion venom on myocardium has been shown in several studies [ 128 , 130 , 131 , 133 , 140142 ] .
aspartate aminotransferase ( ast ) is distributed to all parts of the body but mostly concentrates in the liver and the heart .
the activity of alanine aminotransferase ( alt ) is similar to ast that is considered a liver - specific enzyme .
therefore , the increase in ast and alt levels may be due to a direct action of the venom on the liver and the heart . envenoming by different scorpions has showed an increase of circulating enzymes levels those are succinate dehydrogenase , creatine phosphokinase , lactate dehydrogenase ( ldh ) , glucose-6-phosphate dehydrogenase ( g6pd ) , alt , and ast ( table 6 ) . following venom injection
the highest toxin concentration has been found in the kidneys , liver , heart , and lungs [ 128134 , 140148 ] .
the alt and ast activities have reported in the serum change during scorpion envenomation [ 128134 , 140149 ] .
scorpion venom also acts on the sodium - potassium pump , producing from local symptoms to systemic problems which lead to changes in the victim 's life .
various studies described that the mechanisms of action toxins on cell membranes that producing changes on the transmembrane action potential and permeability changes in calcium and potassium channels altering the release of neurotransmitters such as acetylcholine .
scorpion venom increases the membrane permeability to sodium by opening the voltage sensitive sodium channels , which is accompanied with calcium entry , and blockade of calcium - activated potassium channels resulting in relative hyperkalemia who induces the release of catecholamines .
one of main metabolic changes produced by scorpion stings is hyperkalemia . in human victims of the mexican scorpion
these authors described that hyponatremia could be explain abdominal distention caused by hypokalemic intestinal paralysis and hypernatremia , the cause of irritability .
one factor that contributes and aggravates the hyperkalemia condition is that the potassium influx causes the pronounced and prolonged hyperglycemia , enhanced glycolysis , and inhibited glycogenesis from decreased insulin secretion .
some researchers have emphasized that blood glucose levels increase after envenomation resulting in hyperglycemia in animal models .
also several studies have reported that this might be to a massive release of catecholamines , increased glucagon and cortisol levels , changes in thyroid hormone levels and changes in insulin secretion [ 84 , 87 , 149 ] .
the elevation of circulatory catecholamines and angiotensin result in intense vasoconstriction and cardiac stimulation , increased myocardial oxygen requirement , and alterations in myocardial perfusion and metabolism , with hyperglycaemia and an increase in circulating free fatty acids .
murthy and hase in 1994 described that the insulin has a primary metabolic role in preventing and reversing the cardiovascular , haemodynamic , and neurological manifestations and pulmonary oedema induced by scorpion envenoming .
insulin is a pleiotropic hormone which has diverse functions including stimulation of nutrient transport into cells regulation of gene expression modification of enzymatic activity and regulation of energy homeostasis actions .
rabbits and dogs following scorpion envenomation present a reduction of insulin , hyperglycemia , and enlace glycogenolisis in heart , liver , and skeletal muscle [ 149 , 150 ] .
various studies described the electrolytes changes such as occurrence of hyponatremia accompanied by normal to increased levels of serum potassium and lowered serum calcium after injection of scorpion venom from different species .
the production and/or release of cytokines may also play a role in the development of hyperglycemia , in particular tnf- has been demonstrated to induce insulin resistance in animals models .
recent studies have showed that the proinflammatory cytokines , such as il-1 , tnf- , and ifn- , are putative mediators of the progressive loss of pancreatic -cells in type diabetes mellitus .
these cytokines are released by macrophages and t cells in infiltrated islets of langerhans and cause impaired function and ultimately cell death by apoptosis or necrosis . with respect to anti - inflammatory cytokines in particular il-4 , il-10 , and il-13 ,
cytokines acting alone or in combination induce various transcription factors and signal transduction pathways within -cells .
one of the most important signaling events is the activation of transcription factor , nuclear factor kappa b .
these factors play the role of a master switch in -cells , activating transcription of a number of genes involved in cytokine - mediated toxicity . of great importance for cytokine toxicity in -cells are in particular the generation of nitric oxide via induction of the inducible nitric oxide synthase ( inos ) and production of reactive oxygen species .
cytokine - induced nitrosative and oxidative stresses trigger eventually -cells death [ 153 , 156 , 157 ] .
although the action of anti - inflammatory cytokines has been studied in different cell types during recent years , little is known about the effects of these cytokines in pancreatic -cells [ 158161 ] .
it is known from the studies in different cell models that il-4 is able to counteract many of the il-1 effects , and reduced no production has been considered an important element for this beneficial effect [ 159 , 162 , 163 ] .
the biological effects of il-13 may be achieved through binding to the il-4 receptor and therefore it is generally assumed that these two cytokines overlap in the biological effects .
the effects of il-10 on the nitric oxide pathway are unclear and the opposite findings have been reported . some studies on anti - inflammatory cytokine action in insulin - producing cells have been published recently , but opposite effects with respect to -cell survival have been reported and the underlying molecular mechanisms of the action of anti - inflammatory cytokines still remain unknown .
the mediators affecting inflammatory processes may be released after scorpion envenomation including kinins , ecosanoids , platelet activating factor , permeability increasing factor , nitric oxide , and cytokines .
this released of cytokines and other mediators may account for several of inflammatory manifestations observed such as acute respiratory of inflammatory manifestations observed such as acute respiratory distress syndrome , systemic inflammatory responses syndrome and multiple organ failure .
the cytokines regulate and amplify the immune response , induce tissue injury and mediate complications of the inflammatory response .
equilibrium between pro- and anti - inflammatory is essential to maintain the homeostasis in the system .
dysregulations of the pro- versus anti - inflammatory are involved in the pathogenesis of envenomation in humans and experimental animals .
the balance between proinflammatory and anti - inflammatory cytokines in envenomation determines the degree and extent of inflammation which can lead to major clinical effects such as cardiac dysfunction , pulmonary edema and shock . in line with the findings , high levels of tnf- , il-1 , il-6 and il-8 | scorpion venoms consist of a complex of several toxins that exhibit a wide range of biological properties and actions , as well as chemical compositions , toxicity , and pharmacokinetic and pharmacodynamic characteristics .
these venoms are associated with high morbility and mortality , especially among children .
victims of envenoming by a scorpion suffer a variety of pathologies , involving mainly both sympathetic and parasympathetic stimulation as well as central manifestations such as irritability , hyperthermia , vomiting , profuse salivation , tremor , and convulsion .
the clinical signs and symptoms observed in humans and experimental animals are related with an excessive systemic host inflammatory response to stings and stings , respectively .
although the pathophysiology of envenomation is complex and not yet fully understood , venom and immune responses are known to trigger the release of inflammatory mediators that are largely mediated by cytokines . in models of severe systemic inflammation produced by injection of high doses of venom or venoms products , the increase in production of proinflammatory cytokines significantly contributes to immunological imbalance , multiple organ dysfunction and death .
the cytokines initiate a cascade of events that lead to illness behaviors such as fever , anorexia , and also physiological events in the host such as activation of vasodilatation , hypotension , and increased of vessel permeability . |
cytokines are known to play essential roles in hematopoiesis such as the regulation of differentiation and production of progenitor cells and mature blood cells .
the knowledge of cytokine function has not only contributed to the development of supportive therapies ( i.e. , erythropoietin ( epo ) ) , but dysregulation of cytokines also argues in the diagnosis of some hematopoietic disorders . for example , one of the minor criteria of polycythemia vera ( pv ) according to who 2008 classification is the subnormal serum epo level .
recently , clinical trials with janus kinase ( jak ) inhibitors have confirmed the presence of aberrant production of inflammatory cytokines and highlighted their role in the pathophysiology of philadelphia negative myeloproliferative neoplasms ( ph mpns ) .
indeed , clinical impact of jak inhibitors on the functional symptoms and splenomegaly in patients were concomitant with a significant effect on the plasma levels of many cytokines [ 3 , 4 ] . the first experimental data that showed elevations of serum and/or plasma cytokines in ph mpn date back to more than 15 years . in the 90s , changes in serum levels of interleukin ( il ) such as il6 [ 5 , 6 ] , il2 and its soluble receptor , and of tumor necrosis factor ( tnf )
the study of hermouet et al . in 2002 has expanded this panel , showing elevated serum levels of il8 and il11 in patients with pv , compared to healthy subjects .
elevated serum concentrations of il11 and il8 were observed in 30% and 100% , respectively , of the pv but not in controls .
this high concentration of these two cytokines was also observed in the bone marrow plasma in 48% and 100% of pv patients , respectively , concerning il11 and il8 .
the authors have also shown that the stimulation of stromal cells with il1 induced an increase in the production of these two cytokines , suggesting that bone marrow stromal cells regulate il11 and il8 production .
this study also described an elevation of il8 both in sera and in bone marrow plasma among patients classified as idiopathic erythrocytosis ( in the absence of endogenous erythroid colonies ) . in 2005 , panteli et al .
measured the serum levels of il1 , il1 , il2 , il6 , soluble il2 receptor alpha ( sil2-ra ) , and thrombopoietin ( tpo ) in 25 primary myelofibrosis ( pmf ) , 40 essential thrombocytemia ( et ) , and 8 pv in comparison with a group of 27 healthy subjects and a subgroup of 10 chronic myeloid leukemia ( cml ) patients .
the interest issue of this study was to show that all ph mpns mpns ( pmf , et , and pv ) had significant increased serum levels of il2 and its soluble receptor , compared to healthy subjects .
the cml patients showed the same increases compared to the healthy subjects , but with significantly lower values than pmf .
similarly , pv and et patients had significantly lower levels of il2 compared to pmf ones . overall , pmf patients displayed a gain of all cytokines measured in this study , with the exception of il1 and il1 , compared to healthy subjects and cml , pv , and et patients .
the profiles of et and pv patients were relatively similar with no significant difference reported between these 2 subgroups , although the rate of il2 and its receptor were higher in pv ( but not significant ) . concerning all the patients evaluated ( ph mpn and cml ) , the authors did not find any significant increase of il1 nor il1 compared to healthy subjects . regarding tpo results ,
the authors found a significant increase in tpo serum compared to controls only for patients with pmf .
et and pv patients , despite moderately higher median levels , had no significant overexpression of tpo ( versus controls ) , although high levels of tpo in et have previously been reported [ 10 , 11 ] . no difference between pv and et could be demonstrated in this study .
the moderate increase of tpo levels must be interpreted in view of the decreasing rates of epo reported in several studies , in correlation with epo independent growth of hematopoietic progenitors in mpns . in particular , a multicenter study on a cohort of 116 pv reported a significant reduction in rates of epo in 85% of patients compared to secondary polycythemia , confirming the interest of the diagnostic assessment of serum epo in pv . in the study of panteli et al .
, the observed changes do not suggest that the assay of tpo can serve as a diagnostic marker of et .
indeed , increasing levels of tpo were not correlated to platelet count or bone marrow megakaryocyte to clumping . in 2011 , tefferi et al . , using a multiplex assay using magnetic nanobeads coupled with flow cytometry , have assessed plasma levels of 30 cytokines including several growth factors such as granulocyte colony - stimulating factor ( g - csf ) , vascular endothelial growth factor ( vegf ) , and hepatocyte growth factor ( hgf ) , in a cohort of 127 patients with pmf .
firstly , this study confirmed the wide deregulation of cytokine expression described in pmf patients .
in fact , 20 of the 30 cytokines tested in plasma showed significant differences , compared to healthy subjects .
the authors approved the previously described increases of il2 , il6 , and il8 but also found significant increases in il10 , il12 , il13 , il15 , tnf , and interferon alpha ( inf ) .
contrary to previous results , the authors found elevated levels of il1 and il1. this difference is probably due to the different techniques / antibodies used ( conventional elisa versus multiplex assay ) . in this inflammatory profile , additional deregulations of hematopoietic growth factors such as g - csf , hgf , and vegf were observed .
of the 127 patients included in this study , 90 patients had a blood sample taken at diagnosis before any treatment , showing that inflammatory conditions characterized by a cytokine overproduction play an integral part in the disease .
were able to study the cytokine profiles of another cohort of 65 pv compared to the results obtained in their cohort of 127 pmf and 35 controls . in this study , they showed that several plasma cytokines were abnormally expressed in pv compared to normal controls , but pv patients presented a different pattern to pmf patients .
compared to normal controls , pv patients demonstrated significantly higher levels of il1ra , il5 , il6 , il7 , il8 , il12 , il13 , ifn , granulocyte - macrophage colony - stimulating factor ( gm - csf ) , macrophage inflammatory protein 1 and 1 ( mip-1 and mip-1 ) , hgf , ifn inducible protein 10 ( ip-10 ) , monokine induced by ifn ( mig ) , monocyte chemotactic protein-1 ( mcp-1 ) , and vegf .
conversely , levels of epidermal growth factor ( egf ) and regulated on activation normal t - cell expressed and secreted ( rantes ) were lower in pv compared to normal controls . differences between pv and pmf were numerous .
levels of the following cytokines were significantly higher in pmf compared to pv : il-1 , il1ra , il-2r , egf , il10 , basic fibroblast growth factor ( b - fgf ) , il12 , ifn , and rantes . in contrast , levels of il7 , ifn , gm - csf , mip-1 , ip-10 , mig , and vegf were significantly higher in pv compared to pmf . using the same multiplex assay technology ,
these results were improved by pourcelot et al . , who studied the plasma concentrations of 13 cytokines in the plasma of 17 pv and added data on 21 et .
this study firstly permitted to highlight a significant elevation of these 13 cytokines in pv and in et .
as in the study of vaidya et al . , the authors found previously reported increases in il6 , il8 , il12 , ifn , gm - csf , hgf , and vegf in pv .
moreover , the study of pourcelot et al . found a significant increase in plasma levels of il4 , il10 , mcp-1 , tnf ( not significant in the study of vaidya et al . ) , and platelet derived growth factor ( pdgf - bb ) ( not determined in the study of vaidya et al . ) .
interestingly , the authors showed that pv and et patients differ by their plasma cytokine profiles .
et patients had higher levels of il4 , il8 , gm - csf , ifn , mcp-1 , pdgf , and vegf compared to pv .
it is interesting to note that cytokines evaluated in this study were higher in et patients than in pv .
all these studies confirmed the existence of an inflammatory context associated with mpns ; gangemi et al . have focused on the evaluation of il22 , il23 , and il10 circulating cytokines which are considered as markers for the activation of t helper lymphocytes .
however , et patients did not show any changes in these 3 cytokines compared with controls and no difference between pv and et could be demonstrated . from all of these studies ,
several comments emerge : ( i ) ph mpns ( pv , et , and pmf ) are all characterized by a significant change in the cytokine production objectified by increased plasma levels of many inflammatory cytokines ( i.e. , il1 , il2 , il6 , il8 , il12 , tnf , and ifn ) , several growth factors ( e.g. , gm - csf , g - csf , hgf , pdgf , and egf ) , and angiogenic factors ( i.e. , vegf ) ( table 1 ) ; ( ii ) deregulations also concern anti - inflammatory cytokines such as il4 and il10 ; ( iii ) there are differences in levels and cytokine profiles among mpns but no particular continuum between these diseases could be objectified ( figure 1 ) .
some cytokines are overexpressed in pmf versus pv ( i.e. , il1 , il1ra , il2-ra , egf , and il10 ) .
conversely , some are overexpressed in pv versus pmf ( i.e. , il7 , ifn , gm - csf , mip-1 , ip-10 , and mig ) and , finally , et also have higher rates than pv concerning il6 , il8 , il12 , ifn , gm - csf , and hgf ; ( iv ) on a technical level , in the absence of standardization of methods between different studies , it is difficult to compare these results to each other ; ( v ) the investigation of a large cohort of mfp , pv , and et using the same technology would clarify these differences and allows to better define the existence of specific profile of each disease ; ( vi ) interpretation of cytokine levels should take into account other factors that may limit the ability to use cytokines in everyday practice .
consequently , in healthy patients , age was shown to increase the measurement of il6 and interferon - gamma inducible chemokines ( mig and ip-10 ) and conversely to decrease il2 , egf , and egfr measurements [ 17 , 18 ] .
in the normal bone marrow , the stroma cells comprise fibrocytes / fibroblasts , endothelial cells , osteocytes / osteoblasts as well as osteoclasts .
fibrosis is the result of collagen production by fibroblasts and its deposition in the extracellular space and in parallel scar tissue formation . in general ,
megakaryocytic clustering can be regarded as surrogate for increased megakaryocytic proliferation . in mpns , clonal (
mutated ) neutrophilic or erythroid cells are morphologically indistinguishable from their polyclonal counterparts but the increase in bone marrow granulocytic progenitors and neutrophils is the morphological surrogate for aberrantly increased proliferation .
erythropoiesis is usually not increased in pmf and patients can have normal hemoglobin values or anemia .
the shape of the megakaryocytic nuclei and the emphasis of granulocytic proliferation are matters of dogmatic debate , in particular cloud - like ( pmf ) versus staghorn - like ( et ) megakaryocytic nuclei [ 2123 ] .
the fundamental question is , why do fibroblasts start to produce more fibers and why are these fibers not degraded ?
all bone marrow cells ( hematopoietic and nonhematopoietic ) communicate with each other via direct cell - cell contact and via cytokine - receptor signaling . in his editorial article some speculations on the myeloproliferative syndromes , william dameshek raised the hypothesis that hormonal signaling factors ( myelostimulatory factors ) may lead to these myeloid diseases . nowadays , although we have found clonal markers , aberrant expression of cell signaling molecules , and regulatory micrornas [ 2527 ] , we still speculate on the causes of these diseases and in particular why progressive and prognostic adverse myelofibrosis develops in these patients .
the fact that prefibrotic pmf has similar megakaryocytic atypia as fibrotic - stage pmf and that megakaryoblastic acute myeloid leukemia ( aml ) is frequently associated with fibrosis make it likely that megakaryocytes could be the neoplastic cell subtype which predominantly forces fibroblasts to produce fibers .
in contrast to et megakaryocytes , pmf megakaryocytes form a more dense net of proplatelets within the bone marrow .
two central fibrosis - related cytokine / receptor pairs in mpns are pdgf and its receptor ( pdgfra ) and transforming growth factor beta 1 ( tgf1 ) and the tgf type ii receptor ( tgfbr2 ) [ 2931 ] .
however , the link between the production of pdgf and tgf1 and mpn - associated mutations remains unclear .
the jak2v617f mutation does not directly result in fibrosis although it was observed that , in pmf , the mutant allele frequency is high .
in fact , jak2v617f is mutated in 5060% of pmf ( including mf0 - 3 ) as well as 5060% of et , but in almost all cases of pv [ 25 , 32 ] .
jak2v617f , through its association with pdgfra and tgfbr2 , may contribute to enhance their signaling , mimicking the action of their ligands .
it is more likely a progressive and long - lasting shift of the cytokine microenvironment towards fibrosis rather than one single genetic trigger .
several matrix modulating factors are increased , in particular thrombospondins ( thbs ) 1 and 2 and matrix metalloproteinases ( mmps ) such as mmp14 which are produced by megakaryocytes ( figure 2 ) [ 33 , 34 ] .
mmps degrade fibers while tissue inhibitors of metalloproteinase ( timp ) contribute to fiber accumulation but , in the most advanced stage pmf , timps are not increased . therefore , increased expression of mmp14 could reflect a higher turnover of the extracellular matrix .
profibrogenic and simultaneously antiangiogenic thbs are matricellular factors which are not involved in structuring the extracellular matrix , but which regulate other factors of the extracellular matrix .
it is thought that angiogenesis plays a critical role in the pathogenesis of pmf . increased thbs acts ineffectively against exaggerated angiogenesis ( e.g. , mediated by increased pdgfra levels ) [ 29 , 36 ] .
data from bone marrow histopathology suggest an increase in the microvessel density ( mvd ) and vegf .
reported that pmf patients were characterized by a significant increase of mvd , particularly microvessels assessed by cd105 expression , compared to pv , et , and controls .
similarly , post - pv and post - et myelofibrosis harbored significantly higher numbers of microvessels compared to pv and et , respectively . in parallel to the increasing density of microvessels , a significant increase expression of vegf has been observed in pmf patients compared to et , pv , and mds / mpn .
gianelli et al . confirmed high levels of vegf expression in pmf compared to et or controls but failed to objectify differences between pmf and pv .
both receptors were weakly expressed , mainly in megakaryopoietic and erythropoietic progenitors , with heterogeneous intensity .
mvd have been shown to correlate with a high jak2v617f allele burden ( 55% ) in mutated patients suggesting that angiogenesis may be influenced by allele burden in these patients , keeping in mind that about half of pmf are jak2 wild type which clearly indicate that other factors ( yet unknown mutations or aberrant cytokine expression ) mediate microvessel proliferation .
one of the first issues is the existence of a correlation between the level of circulating cytokines and the intensity of hematopoietic production and/or the existence of specific cytokine overproduction correlated to a cell type overproduction .
the study of tefferi et al . showed , in pmf , the existence of a correlation between the rate of sil2-ra , hgf and ip-10 ( only sil2-ra in multivariate analysis ) , and leukocytosis . in pv , correlation of il-1 , il2 , il7 , b - fgf , and hgf with leukocytosis has been described .
reported a correlation between il6 , tnf , and the number of lymphocytes in pv , and a correlation between hgf , il6 , il12 , gm - csf , and vegf with the numbers of neutrophils in et .
regarding erythrocyte production , significant correlations were reported in pv between il12 and hematocrit ( ht ) , tnf , and ht and between il4 or mcp-1 and hemoglobin ( hb ) . moreover , pourcelot et al .
these results suggest a relative specificity of the plasma levels of some cytokines with the deregulation of the red cell mass . regarding the platelet count ,
no cytokine plasma levels were correlated with platelet count in et patients in the literature . however , vaidya et al . in 2012 reported a significant reduction of inf and in patients with a platelet number greater than 450 , and a significant increase of il6 , capable of regulating the platelet count , has been reported for et patients in several studies .
the difficulty in interpretation of these data is related to the fact that the mechanisms of cytokine production can be multiple ( bone marrow stroma cells , tumor cells , and extra - hematopoietic cells ) and the plasma cytokine changes may also be linked to chronic inflammation associated with mpns and therefore not only reflect myeloproliferation .
surprisingly , there are few data on cytokine plasma levels correlated to jak2v617f status . in their study on pmf , tefferi et al . found a correlation in multivariate analysis between the presence of the mutation and il1ra , ip-10 , and il2-ra rates .
the study of pourcelot et al . showed a correlation between the presence of the mutation and the plasma concentration of tnf and pdgf - bb in pv and et , respectively .
the impact of the jak2v617f mutation on cytokine secretion seems to be restricted , suggesting that the regulation of cytokine production is done by both phenomena jak2v617f - driven and not driven .
on the other hand , cytokines which appear jak2v617f - driven differ between pmf , pv , and et .
this suggests that there is a difference in the cytokine impact of the mutation according to the pathology or conversely that these differences of cytokine profiles could contribute to the phenotypic differences within the jak2 mutated mpn .
indeed , when comparing jak2v617f mutated et and pv patients , significant differences in the expression of some cytokines such as il4 , il8 , ifn , and pdgf - bb have been demonstrated . to summarize , il4 rates were also correlated with ht in this study .
for example , tnf levels were reported previously to be correlated with jak2v67f allelic burden .
this may explain why in pv patients who express the highest levels of jak2 mutated , a correlation between tnf and the presence of the mutation was observed .
this suggests in pv a direct link between plasma levels of tnf , jak2v617f allelic burden , and increased red cell mass .
the increase of pdgf in et patients and its correlation with the presence of jak2v617f mutation probably reflect the impact of the mutation on the regulation of megakaryopoiesis via tpo and the deregulation of this pathway in mutated patients .
this suggests that , in et patients , pdgf assay may be a functional marker of jak2v617f allelic load and indirectly a marker of jak2 activation level .
thus , the plasma level of pdgf could identify et patients for whom a jak2 inhibitor therapy would be the most fruitful . to our knowledge , no studies have until now described any correlation between calr or mpl mutations and circulating cytokines .
fibrosis progression during the chronic phase of myeloid neoplasms is regarded as an indirect surrogate of the aggressiveness of the clonal disease .
the mechanisms which lead to myelofibrosis , primary or secondary , are still an enigma .
it is likely that in bone marrow cytokines secreted by mpn cells , in particular megakaryocytes , could induce activation of fibroblasts and endothelial cells . moreover , circulating cytokines ( not originating from bone marrow ) could by themselves influence the bone marrow microenvironment and thereby contribute to the development of myelofibrosis . hence , circulating cytokines represent an interesting opportunity of simple , accessible , and easily measurable biomarkers for the evaluation of the disease at diagnosis ( in addition to usual genetic and clinical markers ) , but also the determination of prognosis . in tefferi
, the follow - up of patients naive to treatment assessed the prognostic value of some of these biomarkers . in particular , the rate of il8 , il10 , il12 , and il15 and sil2-ra levels were independent predictors of low survival , correlated with dipss categories .
the prognostic value of these biomarkers was confirmed retrospectively on 127 patients , including those who received a therapeutic treatment , proving their clinical interests .
plasma levels of il8 and sil2-ra allowed a prognostic classification of patients as they showed an increase of one or two of these cytokines .
patients with elevation of at least one of these markers displayed a significantly decreased survival among both treatment - naive patients and those who had already received therapy at the time of cytokine explorations .
moreover , the study of distribution of patients according to their prognosis has shown a concomitant increase in the frequency of patients with elevation of one or two cytokines and the severity of the pathology .
thus , there were more frequently patients with an increase in one of these two markers within intermediate risk groups 1 and 2 ( classified according to dipss plus ) .
in addition , patients with 2 elevated markers were only found in risk group 2 patients . in pv and et ,
the study of gangemi et al . found a link between increased levels of il2 and its soluble receptor with progression to myelofibrosis .
furthermore , the prognostic value of high levels of 13 cytokines significantly associated with a lower survival in pv patients has also been reported . in univariate analysis ,
fibrotic transformation was significantly associated with high levels of the following cytokines : il-1 , il5 , il6 , il10 , il12 , il15 , il17 , and ip-10 .
however , in multivariate analysis , only mip-1 remained significant even when age and leukocytosis were added as covariates .
the ability to predict the evolution towards this serious complication by a circulating marker would allow early therapeutic management of these patients with a very poor prognosis .
showed that pmf patients who evolved into acute leukemia had elevated il8 and sil2-ra levels .
in particular , the elevation of il8 levels was significantly correlated with decrease in leukemia - free survival and an increase in the incidence of transformation in acute leukemia .
the predictive value of il2 rate and its soluble receptor was also highlighted in the study of gangemi in pmf patients progressing into acute leukemia .
the prognostic value of plasma levels of il8 and sil2-ra has been reported in other hematological tumors [ 40 , 41 ] or solid cancer ( head , neck , and esophagus ) . to our knowledge , there is no data in the literature on predictive cytokine markers of leukemic evolution concerning pv or et .
assessment of vascular risk , particularly thrombosis , is a key element in the therapeutic management of patients for prescription of cytoreductive and antithrombotic treatments .
this assessment of vascular risk is important because it could cause inappropriate exposure of patients to potentially leukemogenic drugs .
this assessment is still based on indirect criteria such as age , a history of stroke , or the presence of vascular risk factors .
the existence of an inflammatory context in thrombotic events has long been demonstrated , suggesting the importance of the evaluation of inflammatory cytokines in the evaluation of thrombotic risk .
however , little is known about the existence of cytokine dysregulation associated with thromboembolic events in mpn and their potential predictive values . in the study of pourcelot et al .
comparison of both subgroups did not show significant statistical difference for age , jak2 mutational status , and biological parameters ( leukocytes , platelets , numbers of neutrophils and lymphocytes , red cells , hb , and ht ) . except for il12 ( p70 ) which was increased in patients with vascular complications , there were no significant differences in other cytokine levels between patients with or without vascular complications .
comparison of vascular complications within pv and et revealed a significant difference of il12 and gm - csf in the pv subgroup .
the decrease of il12 has been previously reported in patients presenting a thrombotic event without any mpn diagnosis suggesting that this cytokine is a specific marker of the occurrence of thromboembolic events independent of mpn pathogenesis .
however , these results must be interpreted with caution since some thrombotic events occurred before diagnosis , and therefore the cytokine evaluation was done after the accident .
it would be necessary to assess changes in cytokine levels at multiple times , before and after thrombosis , to identify more specifically predictive biomarkers of vascular complications .
a study of barbui et al . has focused on the interest of c - reactive protein ( crp ) and pentraxin 3 ( ptx3 ) as markers of thrombotic risk . in this study of 244 et and pv patients , a difference in prognostic value
high thrombosis risk patients were characterized by a significant increase ( > 3rd percentile ) of crp .
prognostic stratification based on serum levels of these two inflammation markers has shown that patients with high crp and low ptx3 levels had a significantly higher risk of thrombotic stroke ( or = 2.66 , p = 0.045 ) .
in addition , the levels of these two markers were correlated with the mutational status of patients and with an allelic load greater than 50% .
in this review , we highlight the usefulness of cytokines as potent markers of prognosis . but in which way do treatments especially jak inhibitors modify cytokines and cytokine levels ?
treatment with ruxolitinib , the first jak inhibitors approved in myelofibrosis , leads to a rapid and sustained downregulation of cytokine levels in myelofibrosis patients .
another proof of the action of ruxolitinib on cytokine levels is the withdrawal syndrome consistent with cytokine storm observed after its discontinuation .
independently of jak status or of mpn subtype ( myelofibrosis or pv ) , several cytokines were reduced after ruxolitinib treatment such as il1ra , il6 , il8 , tnf , and bfgf [ 3 , 4 ] .
reduction of il16 , il18 , vegf , and mip-1 was also reported more specifically in myelofibrosis and reduction of sil2ra , sil6r in pv . moreover ,
a correlation between symptomatic reduction of the spleen size in myelofibrosis and reductions of il-1ra , mip-1 , il6 , and tnf was observed .
data reported on mouse models and on supernatants of in vitro cultures of mononuclear cells confirmed reductions of il6 and tnf after jak1/2 inhibitors [ 47 , 48 ] .
showed that ruxolitinib treatment normalizes cytokine levels in mice transplanted with jak2v617f - mutant as well as those transplanted with mplw515l - mutant cells . beyond mpn , there is a rising interest in jak inhibitors for other disorders such as autoimmune diseases , solid cancers , or other hematopoietic malignancies [ 5052 ] .
reduction of cytokines was also observed in experimental models of those disorders [ 53 , 54 ] .
nowadays , the only jak inhibitor approved for the treatment of primary and secondary myelofibrosis is ruxolitinib , which inhibits not only jak2 but also jak1 . nevertheless , more selective jak2 inhibitors ( i.e. , fedratinib , lestaurtinib , pacritinib ) are in clinical development and differences between selective jak2 inhibitors and jak1/jak2 inhibitors could be observed .
on one hand , more selective jak2 inhibitors appear to have a less pronounced anticytokine effect and , on the other hand , they induce a more pronounced antierythropoiesis effect . for example , no consistent changes in levels of proinflammatory cytokines ( il6 , il2 , il8 , and tnf ) relative to baseline were observed during the course of fedratinib treatment ; however a rapid and durable improvement of symptoms concomitantly with an impact on jak2v617f allele burden was induced .
similarly , santos et al . studied effects of lestaurtinib ( cep701 ) , a selective jak2 inhibitor , on the levels of 19 cytokines ( il-1 , il-1ra , il2 , il6 , il8 , il9 , il10 , il12 , il13 , il15 , bfgf , gm - csf , ifn , ip-10 , mip-1 , mip-1 , rantes , tnf- , and vegf ) . in the same way
in contrast to selective jak2 inhibitors , momelotinib ( cyt387 ) , a jak1/jak2 inhibitor , normalized inflammatory cytokines in jak2v617f - transduced mice . even if jak inhibitors affect cytokine levels , recent studies suggest that cytokine regulation by jak inhibitors is not enough by itself to fully abort this aberrant inflammatory cytokine production .
. showed in myelofibrosis , pv , or et patients receiving either ruxolitinib or fedratinib a significant decrease of cytokines after the first month ( ifn , ifn , il10 , il2r , il4 , and il17 ) but a weak rise in cytokine levels after six months .
this fact argues a possible therapeutic failure but also supports the interest for drug associations in mpn treatment targeting other molecular pathways implicated in inflammatory response .
moreover , the role of tumor microenvironment in hematopoietic neoplasms development is essential ; not only malignant cells but also nonmalignant cells induce cytokine dysregulation .
altogether , those studies suggest the interest of synergistic associations of jak inhibitors with others drugs to normalize aberrant cytokine production in mpn .
the cytokine profiles of mpn patients involving deregulation of proinflammatory and anti - inflammatory cytokines as well as growth factors suggest that the impact of these deregulations is involved in hematological but also extra - hematological manifestations of these pathologies .
these deregulations confirm the existence of an inflammatory reaction in mpns that may contribute to the initiation and progression of the disease . in this context
, circulating cytokine levels could be useful markers of mpns for their characterization at diagnosis but could also be interesting in prognostic evaluation of these patients .
moreover , the impact of jak2 inhibitors on plasma concentrations of inflammatory cytokines suggests that circulating cytokine assays could be useful to monitor therapeutic efficacy of these molecules .
long - term treatment with jak2 inhibitors may also raise the question of patient compliance with their treatment . as a result ,
the significant reduction of cytokines by mechanisms of inhibition of jak2 , and most likely jak1 , could serve as an indirect marker for evaluation of therapeutic compliance in case of absence or inadequate therapeutic response . | cytokines are well known mediators of numerous physiological and pathological processes .
they contribute to the regulation of normal hematopoiesis but increasing data suggest that they also have a clinical impact in some hematopoietic malignancies . in particular , there is evidence that cytokines are implicated in the functional symptoms of philadelphia negative myeloproliferative neoplasms ( ph mpns ) , suggesting that evaluation of circulating levels of cytokines could be of clinical interest for the characterization of patients at the time of diagnosis and for disease prognosis . in this review , we present the current knowledge on alteration of circulating cytokine profiles in mpns and their role in myelofibrosis pathogenesis .
phenotypic correlation , prognostic value of cytokines , and impact of jak inhibitors are also discussed . |
the study was conducted at the microbial containment complex , national institute of virology ( niv ) , pashan , pune , india .
infected mosquitoes were kept in plastic jars inside mosquito cages and all these experiments were carried out inside a bio - safety level-2 laboratory .
normal as well as infected mosquitoes were maintained at 282c with 805 per cent relative humidity and 12:12 h light : dark cycle .
this isolate has undergone six passages in baby hamster kidney ( bhk-21 ) cell line .
the virus at p-7 ( stock ) had a titre of 5.0 log10 50 per cent tissue culture infective dose ( tcid50)/ml.(ii)jev - strain no .
057434 , a human isolate obtained from gorakhpur , india , in 2005 ( niv , unpublished data ) has undergone several passages in mice and two passages in vero e6 cell line . the stock virus prepared in vero e6 cell line
had a titre of 7 log10 tcid50/ml.(iii)wnv - the prototype strain of wnv ( eg101 ) procured from niv virus repository was used in the study .
the strain has undergone several passages in mouse brain and passaged once in vero e6 cell line for stock preparation .
this isolate has undergone six passages in baby hamster kidney ( bhk-21 ) cell line .
the virus at p-7 ( stock ) had a titre of 5.0 log10 50 per cent tissue culture infective dose ( tcid50)/ml .
057434 , a human isolate obtained from gorakhpur , india , in 2005 ( niv , unpublished data ) has undergone several passages in mice and two passages in vero e6 cell line . the stock virus prepared in vero e6 cell line had a titre of 7 log10 tcid50/ml .
wnv - the prototype strain of wnv ( eg101 ) procured from niv virus repository was used in the study .
the strain has undergone several passages in mouse brain and passaged once in vero e6 cell line for stock preparation .
virus stock preparation : bhk-21 cell line was preferred for stock preparation and virus titration based on the high virus yield in the cell line ( unpublished data ) . to prepare virus stock , bhk-21 cells grown in 225 cm bottles were infected with 1moi ( multiplicity of infection ) of bagv as described earlier1 .
the cultures were observed daily for appearance of cytopathic effect ( cpe ) and when 70 - 80 per cent cells showed cpe , cultures were harvested .
virus was extracted from cells by freeze - thawing thrice and collecting the supernatant after centrifugation at 2790 g in a refrigerated ( + 4c ) centrifuge ( hettich , germany ) for 20 min .
one ml aliquots of the suspension was made in sterile vials and stored at -86c .
one of the aliquots was titrated in the same cell line and virus titre was determined as mentioned earlier .
bhk-21 cell line was maintained in minimum essential medium ( mem ) supplemented with 10 per cent foetal bovine serum ( fbs ) and was passaged at every 3 - 4 days .
both mem and fbs used for cell line maintenance were procured from invitrogen , usa . insect flavivirus free mosquitoes were procured from the niv insectary maintained at 282c with 805 per cent relative humidity and 12:12 h light : dark cycle .
mosquito larvae were fed on a mixture of yeast powder and dog biscuit ( 3:1 w / w ) while adults were maintained on a diet of 10 per cent glucose .
female mosquitoes were provided with 5 - 6 wk old chickens for blood meal on alternate days .
infection of mosquitoes with virus : infection of mosquitoes was carried out by intrathoracic inoculation ( it ) and oral feeding . for it inoculation ,
50 mosquitoes for each experiment were immobilized by keeping them in ice for 5 - 10 min and inoculated intrathoracically with 100 plaque forming units ( pfu ) of bagv inside a bsl-2 laboratory following the method described by rosen and gubler8 . for oral feeding , mosquitoes ( n=50 for each experiment )
were starved for 12 h and allowed to feed on blood virus mixture through a chicken membrane as previously described1 .
infected mosquitoes ( both it inoculated and oral fed ) were secured in plastic mosquito holding jars inside double walled mosquito cages and incubated at 282c with 70 - 80 per cent humidity .
after completion of the experiment , mosquitoes of each day post - infection ( pi ) were triturated in 1 ml mem containing 2 per cent fbs using a chilled mortar and pestle .
the mosquito suspension was centrifuged ; millipore filtered ( pore size=0.22 m ) , diluted serially ( ten - fold ) and titrated in bhk-21 cells in quadruplicate .
the cultures were observed daily , readings ( cells with cytopathic effects ) were scored , stained with amido black and virus titre of each day pi was determined9 .
determination of jev / wnv replication in bagv infected mosquitoes : the influence of bagv in replication of jev and wnv was studied using two methods . in the first method , mosquitoes were infected simultaneously with a mixture of bagv and jev or wnv by it inoculation . in the second method , mosquitoes were infected with bagv , incubated for eight days and superinfected with jev / wnv .
the infections were made by it inoculation . in the oral infection method , mosquitoes were fed blood - virus mixture as described earlier . in all the cases , mosquitoes were incubated further , harvested on days 7 and 14 of infection with jev / wnv , titrated and determined virus titre as described earlier .
jev / wnv yield in bagv infected mosquitoes was compared with virus yield of mosquitoes infected with jev / wnv alone and determined the influence of bagv in replication of jev / wnv .
determination of effect of bagv in oviposition and life cycle of mosquitoes : culex quinquefasciatus and cx .
tritaeniorhynchus mosquitoes ( n=50 each ) were infected orally with bagv and allowed to oviposit .
the number of egg rafts obtained , duration of oviposition , hatching rate and duration to complete the larval and pupal stages , duration of emergence and adult survival were recorded .
similar data from an equal number of uninfected adults were also recorded and analyzed to determine the effect of bagv in mosquito survival and oviposition .
tritaeniorhynchus mosquitoes : culex tritaeniorhynchus mosquitoes were inoculated with bagv as described earlier1 , incubated for seven days and fed on normal infant mice .
fully engorged mosquitoes were collected , placed in double walled mosquito holding cages and allowed to lay eggs .
the eggs , larvae and adults were screened for presence of bagv by tissue culture assay1 .
this isolate has undergone six passages in baby hamster kidney ( bhk-21 ) cell line .
the virus at p-7 ( stock ) had a titre of 5.0 log10 50 per cent tissue culture infective dose ( tcid50)/ml.(ii)jev - strain no .
057434 , a human isolate obtained from gorakhpur , india , in 2005 ( niv , unpublished data ) has undergone several passages in mice and two passages in vero e6 cell line . the stock virus prepared in vero e6 cell line
had a titre of 7 log10 tcid50/ml.(iii)wnv - the prototype strain of wnv ( eg101 ) procured from niv virus repository was used in the study .
the strain has undergone several passages in mouse brain and passaged once in vero e6 cell line for stock preparation .
this isolate has undergone six passages in baby hamster kidney ( bhk-21 ) cell line .
the virus at p-7 ( stock ) had a titre of 5.0 log10 50 per cent tissue culture infective dose ( tcid50)/ml .
057434 , a human isolate obtained from gorakhpur , india , in 2005 ( niv , unpublished data ) has undergone several passages in mice and two passages in vero e6 cell line . the stock virus prepared in vero e6 cell line
wnv - the prototype strain of wnv ( eg101 ) procured from niv virus repository was used in the study .
the strain has undergone several passages in mouse brain and passaged once in vero e6 cell line for stock preparation .
virus stock preparation : bhk-21 cell line was preferred for stock preparation and virus titration based on the high virus yield in the cell line ( unpublished data ) . to prepare virus stock , bhk-21 cells grown in 225 cm bottles were infected with 1moi ( multiplicity of infection ) of bagv as described earlier1 .
the cultures were observed daily for appearance of cytopathic effect ( cpe ) and when 70 - 80 per cent cells showed cpe , cultures were harvested .
virus was extracted from cells by freeze - thawing thrice and collecting the supernatant after centrifugation at 2790 g in a refrigerated ( + 4c ) centrifuge ( hettich , germany ) for 20 min .
one ml aliquots of the suspension was made in sterile vials and stored at -86c .
one of the aliquots was titrated in the same cell line and virus titre was determined as mentioned earlier .
bhk-21 cell line was maintained in minimum essential medium ( mem ) supplemented with 10 per cent foetal bovine serum ( fbs ) and was passaged at every 3 - 4 days .
both mem and fbs used for cell line maintenance were procured from invitrogen , usa . insect flavivirus free mosquitoes were procured from the niv insectary maintained at 282c with 805 per cent relative humidity and 12:12 h light : dark cycle .
mosquito larvae were fed on a mixture of yeast powder and dog biscuit ( 3:1 w / w ) while adults were maintained on a diet of 10 per cent glucose .
female mosquitoes were provided with 5 - 6 wk old chickens for blood meal on alternate days .
infection of mosquitoes with virus : infection of mosquitoes was carried out by intrathoracic inoculation ( it ) and oral feeding . for it inoculation ,
50 mosquitoes for each experiment were immobilized by keeping them in ice for 5 - 10 min and inoculated intrathoracically with 100 plaque forming units ( pfu ) of bagv inside a bsl-2 laboratory following the method described by rosen and gubler8 . for oral feeding , mosquitoes ( n=50 for each experiment ) were starved for 12 h and allowed to feed on blood virus mixture through a chicken membrane as previously described1 .
fully engorged mosquitoes were separated and used for the study . infected mosquitoes ( both it inoculated and oral fed ) were secured in plastic mosquito holding jars inside double walled mosquito cages and incubated at 282c with 70 - 80 per cent humidity .
after completion of the experiment , mosquitoes of each day post - infection ( pi ) were triturated in 1 ml mem containing 2 per cent fbs using a chilled mortar and pestle .
the mosquito suspension was centrifuged ; millipore filtered ( pore size=0.22 m ) , diluted serially ( ten - fold ) and titrated in bhk-21 cells in quadruplicate .
the cultures were observed daily , readings ( cells with cytopathic effects ) were scored , stained with amido black and virus titre of each day pi was determined9 .
determination of jev / wnv replication in bagv infected mosquitoes : the influence of bagv in replication of jev and wnv was studied using two methods . in the first method , mosquitoes were infected simultaneously with a mixture of bagv and jev or wnv by it inoculation . in the second method , mosquitoes were infected with bagv , incubated for eight days and superinfected with jev / wnv .
the infections were made by it inoculation . in the oral infection method , mosquitoes were fed blood - virus mixture as described earlier . in all the cases , mosquitoes were incubated further , harvested on days 7 and 14 of infection with jev / wnv , titrated and determined virus titre as described earlier .
jev / wnv yield in bagv infected mosquitoes was compared with virus yield of mosquitoes infected with jev / wnv alone and determined the influence of bagv in replication of jev / wnv .
determination of effect of bagv in oviposition and life cycle of mosquitoes : culex quinquefasciatus and cx .
tritaeniorhynchus mosquitoes ( n=50 each ) were infected orally with bagv and allowed to oviposit .
the number of egg rafts obtained , duration of oviposition , hatching rate and duration to complete the larval and pupal stages , duration of emergence and adult survival were recorded .
similar data from an equal number of uninfected adults were also recorded and analyzed to determine the effect of bagv in mosquito survival and oviposition .
tritaeniorhynchus mosquitoes : culex tritaeniorhynchus mosquitoes were inoculated with bagv as described earlier1 , incubated for seven days and fed on normal infant mice .
fully engorged mosquitoes were collected , placed in double walled mosquito holding cages and allowed to lay eggs .
the eggs , larvae and adults were screened for presence of bagv by tissue culture assay1 .
this isolate has undergone six passages in baby hamster kidney ( bhk-21 ) cell line .
the virus at p-7 ( stock ) had a titre of 5.0 log10 50 per cent tissue culture infective dose ( tcid50)/ml.(ii)jev - strain no .
057434 , a human isolate obtained from gorakhpur , india , in 2005 ( niv , unpublished data ) has undergone several passages in mice and two passages in vero e6 cell line . the stock virus prepared in vero e6 cell line
had a titre of 7 log10 tcid50/ml.(iii)wnv - the prototype strain of wnv ( eg101 ) procured from niv virus repository was used in the study .
the strain has undergone several passages in mouse brain and passaged once in vero e6 cell line for stock preparation .
this isolate has undergone six passages in baby hamster kidney ( bhk-21 ) cell line .
the virus at p-7 ( stock ) had a titre of 5.0 log10 50 per cent tissue culture infective dose ( tcid50)/ml .
057434 , a human isolate obtained from gorakhpur , india , in 2005 ( niv , unpublished data ) has undergone several passages in mice and two passages in vero e6 cell line . the stock virus prepared in vero e6 cell line
wnv - the prototype strain of wnv ( eg101 ) procured from niv virus repository was used in the study .
the strain has undergone several passages in mouse brain and passaged once in vero e6 cell line for stock preparation .
virus stock preparation : bhk-21 cell line was preferred for stock preparation and virus titration based on the high virus yield in the cell line ( unpublished data ) . to prepare virus stock , bhk-21 cells grown in 225 cm bottles were infected with 1moi ( multiplicity of infection ) of bagv as described earlier1 .
the cultures were observed daily for appearance of cytopathic effect ( cpe ) and when 70 - 80 per cent cells showed cpe , cultures were harvested .
virus was extracted from cells by freeze - thawing thrice and collecting the supernatant after centrifugation at 2790 g in a refrigerated ( + 4c ) centrifuge ( hettich , germany ) for 20 min .
one ml aliquots of the suspension was made in sterile vials and stored at -86c .
one of the aliquots was titrated in the same cell line and virus titre was determined as mentioned earlier .
bhk-21 cell line was maintained in minimum essential medium ( mem ) supplemented with 10 per cent foetal bovine serum ( fbs ) and was passaged at every 3 - 4 days .
both mem and fbs used for cell line maintenance were procured from invitrogen , usa . insect flavivirus free mosquitoes were procured from the niv insectary maintained at 282c with 805 per cent relative humidity and 12:12 h light : dark cycle .
mosquito larvae were fed on a mixture of yeast powder and dog biscuit ( 3:1 w / w ) while adults were maintained on a diet of 10 per cent glucose .
female mosquitoes were provided with 5 - 6 wk old chickens for blood meal on alternate days .
infection of mosquitoes with virus : infection of mosquitoes was carried out by intrathoracic inoculation ( it ) and oral feeding . for it inoculation ,
50 mosquitoes for each experiment were immobilized by keeping them in ice for 5 - 10 min and inoculated intrathoracically with 100 plaque forming units ( pfu ) of bagv inside a bsl-2 laboratory following the method described by rosen and gubler8 . for oral feeding , mosquitoes ( n=50 for each experiment ) were starved for 12 h and allowed to feed on blood virus mixture through a chicken membrane as previously described1 .
fully engorged mosquitoes were separated and used for the study . infected mosquitoes ( both it inoculated and oral fed ) were secured in plastic mosquito holding jars inside double walled mosquito cages and incubated at 282c with 70 - 80 per cent humidity .
after completion of the experiment , mosquitoes of each day post - infection ( pi ) were triturated in 1 ml mem containing 2 per cent fbs using a chilled mortar and pestle .
the mosquito suspension was centrifuged ; millipore filtered ( pore size=0.22 m ) , diluted serially ( ten - fold ) and titrated in bhk-21 cells in quadruplicate .
the cultures were observed daily , readings ( cells with cytopathic effects ) were scored , stained with amido black and virus titre of each day pi was determined9 .
determination of jev / wnv replication in bagv infected mosquitoes : the influence of bagv in replication of jev and wnv was studied using two methods . in the first method , mosquitoes were infected simultaneously with a mixture of bagv and jev or wnv by it inoculation . in the second method , mosquitoes were infected with bagv , incubated for eight days and superinfected with jev / wnv .
the infections were made by it inoculation . in the oral infection method , mosquitoes were fed blood - virus mixture as described earlier . in all the cases , mosquitoes were incubated further , harvested on days 7 and 14 of infection with jev / wnv , titrated and determined virus titre as described earlier .
jev / wnv yield in bagv infected mosquitoes was compared with virus yield of mosquitoes infected with jev / wnv alone and determined the influence of bagv in replication of jev / wnv .
determination of effect of bagv in oviposition and life cycle of mosquitoes : culex quinquefasciatus and cx .
tritaeniorhynchus mosquitoes ( n=50 each ) were infected orally with bagv and allowed to oviposit .
the number of egg rafts obtained , duration of oviposition , hatching rate and duration to complete the larval and pupal stages , duration of emergence and adult survival were recorded .
similar data from an equal number of uninfected adults were also recorded and analyzed to determine the effect of bagv in mosquito survival and oviposition .
tritaeniorhynchus mosquitoes : culex tritaeniorhynchus mosquitoes were inoculated with bagv as described earlier1 , incubated for seven days and fed on normal infant mice .
fully engorged mosquitoes were collected , placed in double walled mosquito holding cages and allowed to lay eggs .
the eggs , larvae and adults were screened for presence of bagv by tissue culture assay1 .
bagv yield in bhk-21 cell line : bagv growth kinetics in bhk-21 cell line showed maximum yield of 5.5 and 5 log10 tcid50/ml in cells and tissue culture fluid ( tcf ) , respectively at 36 h pi .
however , virus titre in tcf was maintained upto 84 h pi while a sharp decline in titre was detected in cells ( fig .
, cells were harvested at 60 h pi and the titre was determined as 5 log10 tcid50/ml .
growth kinetics of bagv in bhk-21 cell line and tissue culture fluid ( tcf ) .
bagv infection alters the replication of jev / wnv during simultaneous infection : in the pilot study carried out by intrathoracic inoculation of cx .
tritaeniorhynchus mosquitoes , bagv was found to play a significant role in inhibition of replication of both jev and wnv ( fig .
jev replication was found affected both by simultaneous infection as well as in mosquitoes previously infected with bagv . however , the influence was more prominent in the latter case as approximately 4 log reduction in virus titre was obtained both at 7 and 14 day pi ( fig .
2b ) while the reduction in the simultaneous infection was only 2 log10 tcid50/ml ( fig .
0 day was approximately 2 log virus ( 100 pfu ) in all the experiments . in the case of wnv replication in cx .
tritaeniorhynchus , simultaneous infection or super - infection with wnv yielded almost identical results ( fig . 2c ,
d ) despite the initial titre of 2 log tcid50/ml on 0day in all the experiments . at 7 day pi ,
reduction in wnv yield was prominent ( 4 log ) while on 14 day pi , the difference in wnv yield was approximately 2 log10 tcid50/ml in either mode of infection .
tritaeniorhynchus mosquitoes ( intrathoracic inoculation method ) ; ( a ) differential yield of jev in mosquitoes infected simultaneously with bagv to mosquitoes infected with jev alone ; ( b ) differential yield of jev in mosquitoes previously infected with bagv to mosquitoes infected with jev alone ; ( c ) differential yield of wnv in mosquitoes infected simultaneously with bagv to mosquitoes infected with wnv alone ; ( d ) differential yield of wnv in mosquitoes previously infected with bagv to mosquitoes infected with wnv alone .
both the methods of infection , reduction in jev yield in presence of bagv was approximately 1 log10 tcid50/ml only ( fig .
no difference in virus yield was found on 14 day pi in simultaneously infected mosquitoes as identical titres were obtained in bagv infected mosquitoes and normal mosquitoes ( fig .
3a ) though a difference of 1 log was detected in mosquitoes previously infected with bagv ( fig .
however , susceptibility to wnv was found to be altered as remarkable reduction was observed in both simultaneously infected and mosquitoes previously exposed to bagv ( fig .
the reduction in virus yield was more prominent at day 7 pi than day 14 pi as the differences in titre of 3 log10 and 1log10tcid50/ml were observed , respectively . influence of bagv infection in replication of jev and wnv in cx .
quinquefasciatus mosquitoes ( intrathoracic inoculation method ) ; ( a ) differential yield of jev in mosquitoes infected simultaneously with bagv to mosquitoes infected with jev alone ; ( b ) differential yield of jev in mosquitoes previously infected with bagv to mosquitoes infected with jev alone ; ( c ) differential yield of wnv in mosquitoes infected simultaneously with bagv to mosquitoes infected with wnv alone ; ( d ) differential yield of wnv in mosquitoes previously infected with bagv to mosquitoes infected with wnv alone .
effect of bagv in orally fed mosquitoes : oral feeding experiments were conducted only with jev in cx .
. simultaneous infection of the viruses was not conducted in the study ( oral feeding ) . in both species of mosquitoes ,
bagv was found to be playing an important role in inhibiting jev and wnv ( fig .
jev replication was found restricted to approximately 2 log10 tcid50/ml in bagv infected mosquitoes upto 9 day pi in comparison to jev yield in normal mosquitoes which yielded approximately 5 log10 tcid50/ml during the same period ( fig .
however , from 9 day pi onwards , enhanced replication of jev was observed yielding titres comparable to jev yield in normal mosquitoes . in cx .
quinquefasciatus mosquitoes the influence of bagv was found prominent in inhibiting wnv replication upto 15 day pi ( fig .
the difference in virus yield was prominent during 5 to 13 day pi which showed 2 - 3 log10 tcid50/ml reduction in wnv titre in comparison to wnv yield in the controls .
tritaeniorhynchus mosquitoes and their susceptibility to jev : persistent infection of bagv was established in cx .
presence of bagv was detected in eggs ( 1.7 log10 tcid50/ml ) laid by the 3 generation of mosquitoes .
the progeny of the 4 generation adults , when infected with jev showed a gradual decline and maintained at a titre of 2 log10tcid50/ml upto 9 day pi demonstrating a reduction of approximately 3 log10 tcid50/ml in comparison to jev yield in normal mosquitoes ( fig . 5 ) .
however , an increase in jev titre to approximately 5 log10 was detected on 11 day pi , which was maintained upto 14 day pi .
tritaenior - hynchus mosquitoes : during the establishment of persistent infection , it was observed that egg laying and hatching of eggs was affected by bagv . when a virus titre of 4.7 log10 tcid50/ml was used to infect mosquitoes by oral feeding , only three egg rafts could be obtained from 43 fully engorged mosquitoes . however , none of the eggs hatched .
the virus concentration was lowered 10- and 100-folds which resulted in higher number of egg rafts ( table ) .
however , the number of eggs laid was comparatively lower than what was obtained in normal mosquitoes ( table ) .
in the present study , we explored the potential of bagv in influencing replication of jev and wnv in cx .
earlier studies have shown that mosquito cell line or a susceptible mosquito , once infected with an arbovirus , does not get infected from subsequent infection with another virus of the same group1011121314151617 .
it was observed that mosquito cell lines persistently infected with sindbis virus altered the productivity of other togaviruses in the cell lines upon subsequent infection .
hobson - peters et al19 demonstrated suppression of wnv and murray valley fever encephalitis viruses in mosquito cell cultures infected with palm creek virus , a new flavivirus isolated from coquillettidia xanthogaster mosquitoes .
contradictory to the above findings , kent et al20 could not demonstrate suppression of wnv replication in cx .
they could not find any significant impact either on virus replication or transmission of wnv in the mosquitoes previously infected with the mosquito flavivirus .
using intrathoracic inoculation of mosquitoes , suppression of replication of both jev and wnv was observed in mosquitoes on 7 and 14 day pi either infected simultaneously or sequentially .
tritaeniorhynchus infected sequentially with bagv rather than simultaneous infection . unlike jev , significant reduction in wnv titre was observed in both cx .
quinquefasciatus mosquitoes in presence of bagv especially on 7 day pi . in the oral feeding experiment , which is the natural route of infection , the findings were almost identical to that of inoculation method .
this has significance in nature as there is a possibility of mosquitoes being infected with different insect viruses and these viruses might be playing an important role in the control of pathogenic viruses21 .
the major limitation of the present study was the inability to determine the individual titres of bagv , jev and wnv in the co - infected mosquitoes as we could not standardize real time rt - pcr for bagv . that would have given the exact picture of the reduction in the titres of jev or wnv .
quinquefasciatus mosquitoes showed that the maximum titre bagv yielded in the mosquitoes never exceeded 5 log10 and 4 log10 tcid50/ml , respectively in repeated experiments .
that substantiated the fact that the observed reduction in the titres of jev and wnv in the present study in co - infected mosquitoes could be due to these viruses only and not bagv .
another important finding of the present study was that mosquitoes infected with 4.7 log of bagv , did not oviposit , but retained eggs in the ovaries .
oviposition was observed when lower concentrations of virus were administered ; however , majority of the eggs did not hatch .
this shows that infection with bagv alters oogenesis and oviposition depending on the virus titre .
this is also an important observation which has importance in the natural control of mosquitoes .
however , laboratory and field conditions are quite different and more studies will be needed before using bagv or other insect viruses in the natural control of mosquitoes . the findings of the present study pointed towards the role of bagv in the inhibition of jev and wnv replication in two important vector mosquitoes though we could not determine the exact quantity of reduction in virus titres individually due to the lack of quantitative assay for bagv .
bagv and other novel flaviviruses are associated with mosquitoes naturally without causing pathogenicity in the hosts .
tritaeniorhynchus mosquitoes which affected oviposition and hatching of eggs that clearly showed that bagv was not a co - evolved symbiont of cx .
other studies have demonstrated the isolation and characterization of a number of novel flaviviruses , of which , a few are known to enhance / suppress subsequent infection with flaviviruses of public health importance192021 .
the precise mechanism of inhibition of replication of a second member of the same family is not yet understood fully . to understand the host competition by different viruses , studies at the receptor and cellular levels are warranted . with the advancements made in molecular virology , it will be possible to identify the factors in the near future .
the unfolding of the mechanism , therefore , would have great epidemiological significance as viruses of less pathogenicity could be used as a tool to control / limit the spread of highly pathogenic mosquito borne viruses such as jev , wnv and other encephalitis causing viruses . | background & objectives : studies have shown that certain flaviviruses influence susceptibility of mosquitoes by inhibiting / enhancing replication of important flaviviruses .
hence , a study was designed to determine whether bagaza virus ( bagv ) , a flavivirus isolated from culex tritaeniorhynchus mosquitoes in india , alters susceptibility of cx .
tritaeniorhynchus and cx .
quinquefasciatus mosquitoes to japanese encephalitis ( jev ) and west nile viruses ( wnv).methods : jev and wnv infection in cx .
tritaeniorhynchus and cx .
quinquefasciatus mosquitoes in the presence of bagv was carried out by intrathoracic ( it ) inoculation and oral feeding methods .
mosquitoes were infected with bagv and wnv / jev either simultaneously or in a phased manner , in which mosquitoes were infected with bagv by it inoculation followed by super - infection with jev / wnv after eight days post - infection ( pi ) .
jev and wnv yield on 7th and 14th day pi after super - infection was determined by 50 per cent tissue culture infective dose ( tcid50 ) method.results:in cx .
tritaeniorhynchus mosquitoes , prior infection with bagv significantly reduced jev and wnv replication while in cx .
quinquefasciatus , bagv influence was only seen with wnv .
reduction in virus titre was observed in it inoculated and oral fed mosquitoes irrespective of the infection mode .
jev replication was also found reduced in cx .
tritaeniorhynchus mosquitoes persistently infected with bagv at passage four.interpretation & conclusions : bagv infection in cx .
tritaeniorhynchus and cx .
quinquefasciatus mosquitoes altered their susceptibility to jev and wnv producing low virus yield .
however , the role of bagv in inhibiting jev / wnv replication in field mosquitoes needs further investigations . |
signature peptide based quantification of proteins using liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) is increasingly used to measure endogenous or therapeutic proteins because it is a high - throughput , selective , and sensitive method when compared with other quantitative proteomics and immunoblotting methods . in this approach , the analyte protein
is first digested , the peptides are separated by lc , and the unique peptide representing target protein is analyzed by ms in multiple reaction monitoring ( mrm ) mode [ 2 , 3 ] . the protein abundance is typically measured based on calibration curve generated using the unique synthetic peptide as an external standard and the stable isotope labeled peptide ( sil ) as an internal standard .
although this approach has been validated for lc - ms / ms variability [ 2 , 3 ] , variation in trypsin digestion is not addressed by this approach .
conventionally , trypsin digestion conditions ( e.g. , incubation time and trypsin : protein ratio ) that ensure maximum protein digestion are established to minimize the influence of such variability [ 57 ] . however , even under these circumstances , the sample or process dependent factors can cause variability in protein digestion . as a result , relying on the sil peptide as an internal standard can be questioned . in such cases , in theory ,
the use of the whole stable isotope labeled protein ( silac ) as an internal standard would be considered to be superior to the use of sil peptide [ 4 , 913 ] .
therefore , we investigated if the use of silac protein as an internal standard would result in greater assay precision than using the sil peptide .
we compared these methods using four hepatic drug transporter proteins , that is , organic anion transporter polypeptide ( oatp ) 1b1 , oatp1b3 , oatp2b1 , and p - glycoprotein ( p - gp ) .
it is important to note here that our method differs in a fundamental way from the absolute silac method proposed by hanke et al . .
in that method , the labeled silac protein is used as a calibrator while it is used as an internal standard in our method .
because hanke et al . used the silac protein as a calibrator , the quantity of the protein used must be known and therefore it must be purified before use . while this approach is ideal and takes into consideration variability in trypsin digestion , it can not be used to quantify expression of membrane proteins ( e.g. , transporters ) because purified recombinant versions of these proteins are extremely difficult to routinely obtain . therefore , our approach utilizes synthetic pure peptides as calibrators and the labeled silac protein as the internal standard .
since the silac protein is used only as an internal standard , it does not need to be pure though it must be completely or almost completely labeled ( i.e. , isotopically pure ) .
heavy amino acids , c6n2-lysine and c6n4-arginine , were obtained from sigma - aldrich ( st . louis , mo ) .
low - glucose dulbecco 's modified eagle 's medium ( dmem ) , phosphate - buffered saline ( pbs ) , penicillin / streptomycin , and geneticin were purchased from invitrogen , carlsbad , ca . fetal bovine serum ( fbs ) and
silac dmem ( without l - lysine and l - arginine ) was purchased from thermo fisher scientific , ma , usa . the proteoextract native membrane protein extraction kit was purchased from calbiochem ( temecula , ca ) . the protein quantification bca kit and
the in - solution digestion kit were from pierce biotechnology ( rockford , il ) .
pure synthetic unlabeled peptides , nvtgffqsfk , nvtgffqslk , vlavtdspar , and nttgalttr , with purity determined by quantitative amino acid analysis , were obtained from new england peptides ( boston , ma ) .
the sil internal standards , nvtgffqsf[c6n2]k , nvtgffqsl[c6n2]k , vlavtdspa[c6n4]r , and nttgaltt[c6n4]r , were from thermo fisher scientific ( rockford , il ) .
hplc - grade acetonitrile and other solvents were purchased from fischer scientific ( fair lawn , nj ) and formic acid was purchased from sigma - aldrich ( st .
twenty human liver samples ( hl1-hl20 ) were obtained from human liver bank of the university of washington ( uw ) school of pharmacy .
all the subjects were caucasians ; age 2567 yr ; 11 female and 9 male .
procurement , characteristics , and storage of these liver samples has been previously described [ 14 , 15 ] . due to the anonymous nature of these samples ,
their use was classified as non - human subjects research by the uw human subjects division .
human oatp1b1 , oatp1b3 , and oatp2b1 expressing hek293 cells were kindly provided by dr .
the p - gp - expressing llcpk - mdr1 cells were grown in dmem supplemented with 10% fbs , 100 u / ml penicillin , and 100 g / ml streptomycin at 37c under 5% co2 and 95% humidity .
the individual oatp1b1 , oatp1b3 , and oatp2b1 expressing hek cells were grown using pdl - coated flasks for better cell adhesion and growth .
geneticin ( g-418 sulfate , invitrogen ) ( 750 g / ml ) was used as selection antibiotic in the media .
briefly , stock solutions of c6n2-l - lysine and c6n4-l - arginine were prepared in pbs at concentrations of 146 mg / ml and 84 mg / ml , respectively . 0.5 ml of the stock solutions was then added to 1 liter silac dmem containing fbs and antibiotics as discussed above .
the silac labeling was performed using exactly the same conditions as used for normal cell culture except the normal medium was replaced with the silac medium . after approximately five doublings of the cells , ~10 cells were isolated and subjected to membrane extraction .
trypsin digestion was performed of the membrane fraction to quantify the extent of labeling by comparing ms response for three labeled and unlabeled peptides in full scan ms mode for each protein .
the cells were exposed to the silac medium until more than > 95% of the protein was labeled .
later , the cells were harvested and cell pellet was stored at 80c before membrane extraction . the liver tissue ( ~100 mg )
was processed to isolate the membrane fraction as per manufacturer 's instructions ( calbiochem , temecula , ca ) and the previously described method [ 1517 ] .
briefly , the tissue was subjected to homogenization in 2 ml extraction buffer i ( eb - i ; proteoextract native membrane protein extraction kit ) plus protease inhibitor cocktail of the kit and incubated with gentle shaking for 10 min .
the homogenate was centrifuged at 16,000 g for 15 min and the supernatant was removed .
the pellet was resuspended in 1 ml eb - ii ( containing surfactants ) from the kit plus 10 l of protease inhibitor cocktail .
the suspension was incubated with gentle shaking for 30 min at 4c followed by centrifugation at 16,000 g for 15 min at 4c .
total membrane protein concentration in the isolated membrane fraction ( i.e. , supernatant ) was determined using the bca protein assay kit and diluted to a working concentration of 2 g/l .
similar to the tissues , the labeled cell pellet ( 25 10 cells ) was processed as discussed above except that the cells were washed twice with pbs before adding 2 ml of the eb - i to the cells followed by gently shaking for 10 min .
the signature peptides were selected based on online in silico prediction tools as well as the literature [ 4 , 15 ] ( table 1 ) .
briefly , peptides susceptible to degradation , that is , containing methionine , cysteine , histidine , and tryptophan , were not selected .
peptides within the transmembrane regions or containing single nucleotide polymorphism and posttranslational modifications were not considered .
continuous sequences of r and k ( rr , rk , kr , kk ) were avoided in the region of trypsin digestion to avoid miscleavages .
a genome wide blast search was also performed to ensure that the peptide was selective for the protein .
only one peptide per protein was selected in this study because our goal was to compare the precision of sil peptide versus silac protein internal standards . the indicated peptide ( table 1 ) was selected over other possible signature peptides , because it yielded the best signal to noise ratio in the liver membrane samples .
slightly different sample preparation conditions were used for sil versus silac internal standard methods prior to lc - ms analysis as shown in figure 1 . for trypsin digestion , the optimized protein : trypsin ratio
was maintained at 25 : 1 for both methods . before trypsin digestion ,
the labeled cell membrane extracts were assessed for protein expression and pooled to prepare a cocktail of labeled oatp1b1 , oatp1b3 , oatp2b1 , and p - gp . for the silac method
, 20 l of 2.0 g/l of tissue membrane fraction and 12 l of 0.67 g/l of labeled cell membrane fraction were denatured with 4 l dithiothreitol ( 100 mm ) and alkylated with 4 l iodoacetamide ( 200 mm ) in 10 l ammonium bicarbonate digestion buffer ( 50 mm , ph 7.8 ) using the previously outlined protocol [ 15 , 17 ] .
the protein samples were digested by trypsin ( 10 l ) in a final volume of 60 l at 37c for 24 h and the reaction was quenched by 30 l of quenching solvent ( 70% acetonitrile in water containing 0.1% formic acid ) .
samples were centrifuged at 4000 g for 5 min and supernatant was used for lc - ms / ms analysis .
for the sil internal standard method , the synthetic sil peptide was used as internal standard .
20 l of 2.0 g/l total membrane protein was trypsin digested as described above except that eb ii was used instead of the silac protein .
the reaction was quenched by 20 l of sil peptide internal standard cocktail ( prepared in the quenching solvent described above ) and 10 l of the neat quenching solvent .
the samples were centrifuged as described above . for both methods ( sil and silac ) ,
the calibration curves were generated by using 20 l of eb ii instead of 20 l of the tissue membrane protein in samples and the neat quenching solvent above was replaced with the signature peptide cocktail .
the uhplc - ms / ms system consisting of agilent 6460a triple - quadrupole mass spectrometer coupled to agilent 1290 infinity lc system ( agilent technologies , santa clara , ca ) was operated in esi positive ionization mode . for quantification ,
the dynamic mrm algorithm was used to maximize dwell time on each transition to allow multiplexed quantification .
approximately 22.5 g of the digest ( 5 l ) was injected onto the column ( kinetex 2.6 m , c18 , 100 3 mm , phenomenex , torrance , ca ) and eluted at 0.4 ml / min by a mobile phase with initial conditions of 97% a ( water containing 0.1% v / v formic acid ) and 3% b ( acetonitrile containing 0.1% v / v formic acid ) held for 4 min , followed by seven steps of linear gradient of mobile phase b concentration of 3% to 12.5% , 12.5% to 18% , 18% to 19.5% , 19.5% to 20% , 20% to 35% , 35% to 50% , and 50% to 90% over 48 min , 811 min , 1113.5 min , 13.516 min , 1618 min , 1818.4 min , and 18.418.6 min .
then , the column was washed using 90% mobile phase b for 1.6 min followed by a reequilibration period of 4.8 min . the doubly charged parent to singly charged product transitions for the analyte peptides and their respective labeled peptides were monitored .
sil internal standard method was validated for lower limit of quantification , linearity , range , accuracy , precision , and stability .
the calibration curve was generated using 7 calibrators , ranging 0.16.0 fmol/g of total digested protein for oatp1b1 , oatp1b3 , oatp2b1 , and p - gp , respectively .
assay accuracy and precision was performed in triplicate at three different quality control ( qc ) concentrations ( low , middle , and high ) of each peptide across the calibration range .
two different matrices were used , that is , pooled human liver membrane protein ( n = 50 ) or eb ii . although mrm data were acquired using three different transitions , only the two most intense transitions ( table 1 ) were processed by integrating the peak areas generated from the reconstructed ion chromatograms for the analyte peptides and their respective internal standards using the masshunter software ( agilent technologies , santa clara , ca ) .
the average peak areas of these two mrm transitions were used for the calibrators and internal standard ( sil or silac ) to construct the calibration line and to estimate the transporter protein concentration in the unknown samples .
the impact of freeze - thaw stress on protein quantification was assessed by exposing liver tissue membrane extracts ( n = 3 ) to zero or three freeze and thaw cycles before trypsin digestion .
similarly , the effect of bench - top stability on protein quantification was investigated by storing the membrane preparation at ambient temperature for 6 h prior to trypsin digestion .
additionally , the autosampler stability of the peptide was determined by repeating analysis of the extracted samples , stored in the lc - ms autosampler ( at 6c ) , over 48 hr .
the silac internal standard method was also validated for all the parameters described for sil internal standard method except stability , which was common for both methods . additionally , to ensure maximum trypsin digestion , membrane fraction isolated from a pooled human liver sample was subjected to digestion in triplicates up to 24 h ( 1 , 2 , 5 , 16 , and 24 h ) as described above .
after 24 h , fresh trypsin was added to the samples and incubated for another 24 h. the magnitude of protein digestion in all samples was expressed relative to that in the 24 h samples .
finally , protein expression in 20 liver samples was determined in triplicate using the two internal standard methods .
since 100% silac labeling is rarely achieved , the concentration of silac protein as internal standard was kept low to minimize the effect of endogenous unlabeled protein on quantification .
in addition , the endogenous unlabeled protein response of silac protein was taken into consideration by subtracting it from the analyte response .
similarly , the endogenous protein expression in qc samples , which were prepared by spiking standards into the pooled liver membrane matrix , was also taken into consideration .
the precision of the two methods ( silac versus sil ) was compared by the paired t - test analysis of the standard deviation of protein expression in the three independently trypsin digested samples from each liver . prior to statistical analysis ,
the data were log transformed as they were found to be log - normally distributed .
the individual and population mean sd protein expression across the 20 livers was also computed .
lc - ms / ms chromatograms ( figure 2 ) show the specificity of the analytical method .
the calibration curves generated using both sil and silac internal standard methods showed linear response throughout the range . the lower limit of quantification , defined as the lowest concentration of spiked peptides in pooled human liver membrane fraction with error and precision less than or equal to 25% , was 0.13 , 0.08 , 0.05 , and 0.10 fmol/g digested protein for oatp1b1 , oatp1b3 , oatp2b1 , and p - gp , respectively . accuracy and precision ( % coefficient of variance ( % cv ) ) in the quantification of the qc samples
were found to be acceptable ( table 2 ) at the three different concentrations using both the mrm transitions ( table 1 ) as per fda bioanalytical method validation guideline for proteins immunoquantification .
rate of protein digestion of unlabeled versus silac oatp1b1 , oatp1b3 , oatp2b1 and p - gp was parallel ( figure 3 ) .
optimum trypsin digestion was confirmed by comparing peptide recovery at 24 h versus 48 h. the peptide recovery after 48 h did not change significantly ( figure 3 ) .
it is important to note here that maximum digestion may not be equivalent to complete digestion .
test of complete digestion is possible only when pure protein standards for these transporters are available .
although three of the four peptides selected contain a potentially labile asparagine residue at the n - terminus , the peptide response was stable even when sample was exposed to three freeze - thaw cycles and at bench - top for 6 h ( table 3 ) .
this indicates that sample processing variables do not affect quantification of these transporters using selected peptide approach .
the mean and range of standard deviation of triplicate measurements of oatp1b1 , oatp1b3 , and p - gp in 20 liver tissue samples were marginally ( but significantly ) larger with the silac versus the sil internal standard method ( figure 4 , table 4 ) .
in other words , the sil method was slightly more precise than the silac method when quantifying the hepatic expression of oatp1b1 , oatp1b3 , and p - gp .
for quantification of oatp2b1 , the precision of the two methods was not significantly different ( figure 4 , table 4 ) .
as assessed by the paired student 's t - test , the population mean ( sd ) protein expression ( fmol/g of membrane protein ) in twenty livers estimated by the sil versus silac method was not significantly different for oatp1b3 ( 0.97 0.47 versus 0.94 0.54 ) or p - gp ( 0.34 0.23 versus 0.34 0.25 ) but was marginally different for oatp1b1 ( 1.86 0.68 versus 2.07 0.77 ) and oatp2b1 ( 1.91 0.50 versus 2.10 0.73 ) .
hence , in contrast to the theoretical prediction , we found that the sil internal standard method was either marginally more precise than the silac method or the precision of the two methods was not significantly different ( figure 4 and table 4 ) .
consistent with this observation , the variability in the qc samples was also higher when using the silac versus sil internal standard approach ( table 2 ) .
this was perhaps due to the small ms response of these low abundant transporters , where variability , introduced due to the additional biological matrix present in the silac internal standard method , exceeded the variability in trypsin digestion .
the latter was consistent across samples as measured by the standard deviation of the lc - ms / ms response of the labeled peptide originating from silac protein .
the above statement is supported by our observation that the precision in determination of expression of the highly ms responsive protein , oatp2b1 ( table 4 ) , was not different between the two methods .
surprisingly , the estimate of the mean value of transporter expression in each liver sample was also found to be marginally but significantly different ( see above ) .
the silac internal standard method reported marginally higher expression for two of the four transporters ( oatp1b1 , oatp2b1 ) , perhaps due to the unlabeled protein in the silac internal standard .
based on these data , when trypsin digestion is maximized and all other constituents are the same ( e.g. , the matrix ) and the conditions are strictly controlled ( e.g. , protein concentration ) , we predict that sil internal standard method will perform as good or better than the silac internal standard method .
the population mean transporter expression in human liver in our study was comparable or modestly lower than those reported by other groups [ 1922 ] .
however , unless confirmed by pure protein standards , the limitation of these peptide based lc - ms / ms quantification methods is the assumption of complete trypsin digestion . to address the latter
, we recently validated the surrogate peptides based protein quantification method using the only available purified transporter available to us , namely , p - gp . when this purified transporter was used as qc sample ,
the recovery of quantity in these p - gp quality control samples was within 73125% of the actual value .
the mean expression and variability of individual transporters by the two methods are presented in figures 4(e)to 4(h ) .
metabolic labeling of proteins by labeled amino acids , that is , silac , is an established technique for relative quantification of proteins [ 2326 ] and has also been used for quantification of proteins [ 4 , 911 ] . as outlined in the introduction , use of labeled protein generated using silac approach
is considered better in peptide based lc - ms / ms protein quantification because the sil method can not take into account any variability in trypsin digestion . since trypsin digestion is expected to be affected by different sample and process dependent variables , the sil method is theoretically expected to be less precise than the silac method . however , the accuracy of the two methods is expected to be equivalent as the two methods differ only in the internal standard used . here , we tested for the first time these theoretical predictions .
our study concludes that if trypsin digestion is consistent across samples , the precision of the sil method in quantification of proteins is similar to silac internal standard method .
thus , we recommend that the sil method be used for quantification of proteins when a single matrix is used . whether this conclusion will remain the same when different biological matrices ( e.g. , liver versus kidney or different sample extraction buffers ) are used remains to be tested .
quantification of transporter expression in various human tissues , including the interindividual variability in expression due to age , sex , or genotype , will be invaluable for prediction of in vivo disposition of drugs from in vitro data . | we studied the precision of quantification of organic anion - transporting polypeptide 1b1 ( oatp1b1 ) , oatp1b3 , oatp2b1 , and p - glycoprotein ( p - gp ) in human livers by surrogate peptide based lc - ms / ms approach using two different internal standards : stable isotope labeled peptide ( sil ) versus stable isotope labeled protein ( silac ) .
the sil peptides were procured commercially and the silac proteins were generated in - house by labeling arginine and/or lysine residues in cells expressing these transporters .
liver tissue ( n = 20 ) was homogenized and the membrane fraction was isolated .
the membranes were trypsin digested and the peptides were analyzed using lc - ms / ms under optimized conditions .
the precision in the quantification of proteins in three independently trypsin digested samples from each liver was calculated as the standard deviation of the log transformed protein concentration .
the precision of the sil internal standard method was either slightly ( p < 0.05 , paired t - test ) better than that of the silac method ( oatp1b1 , oatp1b3 , and p - gp ) or not different ( oatp2b1 ) .
trypsin digestion , as measured by the response of the labeled peptide derived from the silac protein , was consistent across liver samples .
these results indicate that when maximum trypsin digestion is ensured , the sil internal standard method can be used with confidence for quantification of drug transporters . |
advances in treatment have led to an increase in survival in people with cystic fibrosis ( cf ) . as patients age with cf , they experience an increased rate of complications , including cystic fibrosis related diabetes ( cfrd ) .
cfrd results from the progressive inflammatory destruction of the pancreas [ 2 , 3 ] and leads to the development of insulin insufficiency with varying levels of insulin resistance during acute illness .
the ferret cf model also shows that early inflammation leads to pancreatic cell destruction and replacement with fibrosis .
while patients with cf have not been observed to develop macrovascular complications of diabetes , microvascular complications are seen .
importantly , diagnosis of cfrd is associated with poor nutrition status and decreased lung function and survival [ 711 ] .
recommendations for diagnosis , screening , and management of cfrd were written in a jointly created clinical care guideline by the cystic fibrosis foundation ( cff ) , the american diabetes association ( ada ) , and the pediatric endocrine society ( pes ) . based on the guideline , screening for cfrd
should be performed annually on patients with cf > 10 years of age using the 2-hour 75-gram oral glucose tolerance test ( ogtt ) .
patients with 2 h ogtt plasma glucose > 200 mg / dl meet criteria for the diagnosis of cfrd . because of
the time - consuming nature of the test , and requirement that patients fast prior to testing , adherence to the guidelines has been challenging ; approximately one - quarter of adults with cf were tested in 2014 .
this result suggests that an alternative means of evaluation is needed for the timely diagnosis of cfrd .
although infrequently used in us cf centers , cgm has been validated in children and adolescents with cf [ 13 , 14 ] .
additionally , two small studies showed that early glucose abnormalities are detectable by cgm in children and adults with cf and are associated with historical decline and contemporaneously worse health outcomes [ 15 , 16 ] .
one study in children with cf < 18 years of age showed that abnormal cgm was the strongest predictor of abnormal glucose metabolism at 2.5-year follow - up .
however , these studies did not answer the question of whether abnormal cgm predicts future health outcomes in adults with cf .
our objectives for this study were to determine whether abnormal cgm results are associated with subsequent development of cfrd , lung function , and bmi decline and increased rate of cf pulmonary exacerbations .
to evaluate the correlation between cgm and ogtt for detection of cfrd in adult patients with cf , we conducted a prospective single center trial in 2009 - 2010 in which 21 adult patients due for routine ogtt underwent simultaneous 3-day cgm and 2-hour 75 g ogtt . subsequently , to determine whether abnormal cgm predicted health decline , we performed a retrospective observational study . medical records and data from medical charts and
the cff registry were reviewed from 2008 to 2015 to obtain clinical information ( diagnosis of cfrd , pulmonary function , bmi , and pulmonary exacerbations ) .
the original protocol was approved by the national jewish health institutional review board ( njh irb ) , and subjects provided written informed consent . prior to the conduct of the retrospective review , the new proposal underwent expedited review and approval by the njh irb .
, the following definitions were used : fasting glucose > 126 mg / dl or 2-hour glucose > 200 mg / dl indicated cfrd , fasting glucose 100125 mg / dl indicated impaired fasting glucose ( ifg ) , and 2-hour glucose 140199 mg / dl indicated impaired glucose tolerance ( igt ) [ 12 , 19 ] . in order to explore the correlation between 1-hour ogtt glucose levels and cgm results , 1-hour ogtt results were also interpreted using the 2-hour ogtt definitions . for subjects without diabetes who did not have ogtt in the observation period ( n = 5/10 ) ,
the alternative guideline criteria were used to determine the development of cfrd : hemoglobin a1c ( a1c ) 6.5% or rpg 200 mg / dl in conjunction with polyuria and polydipsia .
one touch glucometer and medtronic cgm system were used to obtain cgm data . a board - certified endocrinologist ( jj ) who was blinded to the ogtt results interpreted cgm data .
although there are no officially defined criteria for interpreting cgm , for our pilot study , a priori we used the american diabetes association criteria for ogtt to define the following criteria to interpret cgm , all values were required on two separate dates : fasting glucose > 126 mg / dl or random glucose > 200 mg / dl indicated cfrd ; random glucose > 140 mg / dl indicated igt ; fasting glucose > 100 mg / dl indicated ifg .
each subject 's highest lung function and bmi for the year prior to ogtt / cgm testing , and for each of the 5 years following ogtt / cgm testing , were extracted from the cf registry .
the number of exacerbations each subject had for each of the 5 years following ogtt / cgm testing was also extracted from the cf registry , and the average number of exacerbations per year was calculated . during the period of retrospective review
, three subjects died and three patients moved away from the center ; all available data points were included for these subjects .
six patients began chronic use of cystic fibrosis transmembrane conductance regulator ( cftr ) modulators during the period of retrospective review ( two patients began ivacaftor , and four patients began lumacaftor / ivacaftor ) .
the highest fev1 and bmi for the year prior to initiation of cftr modulators were used for analysis of decline in these parameters over time .
means and standard deviations are reported for demographic information ( graph pad prism v6.07 ) .
agreement between test methods ( ogtt and cgm ) was evaluated using both pearson correlation and the bland - altman method .
test was used to evaluate whether abnormal cgm predicts subsequent development of cfrd . using two - sample t - tests , average lung function , bmi , and average exacerbation rate were compared between subjects who developed cfrd and those who did not develop cfrd .
standard error of the mean is reported for group means compared by t - tests .
pearson correlation was calculated to evaluate the relationship between maximum cgm and lung function and bmi decline .
the average age of the subjects was 32.4 years ( sd 13.1 ; median 27.0 , range 2065 ) .
fifty - two percent ( 11/21 ) of subjects were homozygous for the f508del mutation ; 90% ( 19/21 ) of subjects had at least one copy of the f508del mutation .
average percent of predicted fev1 and average bmi of subjects at the time of enrollment was 68.8 ( sd 5.5 ; range , 22109 ) and 21.6 ( range 17.129.1 ; sd 0.72 ) , respectively ( table 1 ) .
one patient did not have adequate data for ogtt interpretation due to improper collection of the timed samples .
two patients did not have adequate data for interpretation by cgm due to failure of the device and/or data retrieval .
for these 18 patients ( mean results for test methods shown in figure 1 ) , there was a modest correlation between interpreted results from ogtt and cgm ( r = 0.52 , 95% ci 0.07 to 0.79 , p = 0.03 ; not shown ) ; only 3/18 ( 17% ) comparisons yielded the same result ( two subjects with impaired glucose tolerance and one subject with cfrd ) . in 14 of 15 ( 93% ) subjects for whom results differed between the two tests
one - hour ogtt glucose interpretation yielded the same result as that of cgm in 11/17 ( 65% ) subjects .
there was a strong positive correlation between one - hour ogtt interpretation and that of cgm ( r = 0.77 , 95% ci 0.46 to 0.91 , p = 0.0003 ; not shown ) . in 5/6 ( 83% ) of the subjects for whom results differed between the two tests ,
we also compared maximum cgm glucose to blood glucose based on 1-hour and 2-hour ogtt using the bland - altman method ( figure 2 ) .
the comparison between ogtt and maximum cgm values showed poor agreement between the two methods ( bias 96.06 , sd 56.91 , 95% limit of agreement 207.6 to 15.49 ) .
in contrast , the comparison between 1-hour ogtt and maximum cgm values showed some agreement ( bias 32.81 , sd 41.18 , 95% limit of agreement 113.5 to 47.9 ) .
one subject met criteria for cfrd by ogtt ( as well as by the cgm criteria defined for this study : glucose > 200 mg / dl on two dates ) at the time of the prospective study . of those remaining subjects with complete cgm and ogtt data ( n = 17 ) , 7/17 ( 41% ) subjects were diagnosed with cfrd within 4 years of their study enrollment ( 5 subjects were diagnosed by symptoms in combination with rpg > 200 mg / dl , and 2 subjects were diagnosed by subsequent ogtt ) ( table 2 ) .
all of the subjects who were subsequently diagnosed with cfrd had abnormal cgm results at the time of the original study ; cgm results were interpreted as consistent with igt in 3 subjects and with cfrd in 4 subjects .
one hundred percent of the subjects whose cgm results were consistent with cfrd went on to develop cfrd in the follow - up study period .
abnormal cgm ( glucose > 200 mg / dl on two dates ) correctly identified those subjects who would subsequently be diagnosed with cfrd ( = 17.27 , df = 2 , p = 0.0002 ) .
two of the 7 subjects had normal ogtts at the time of the original study ; the remainder of the subjects had ifg / igt by ogtt .
one - hour glucose results were normal in 2 subjects , but the remaining 5 subjects had 1-hour ogtt results consistent with cfrd .
thus , the 1-hour ogtt result was also useful in determining which subjects would develop cfrd ( = 13.26 , df = 2 , p = 0.001 ) .
of the 10 subjects who have not met criteria for cfrd since study enrollment ( two of these subjects died , and 3 moved from the center ) , 5 subjects ( 50% ) had at least one ogtt in the 4 - 5 years subsequent to study enrollment .
although 3 of 5 of the subjects had normal ogtts at enrollment ( cgm results indicated igt in all three subjects ) , those 3 subjects have subsequently been diagnosed with igt by ogtt .
all 10 subjects who have not met criteria for cfrd since study enrollment have had at least one rpg and/or a1c measured in the 4 - 5 years subsequent to enrollment .
none of the 10 subjects met criteria for cfrd based on an a1c 6.5% or rpg 200 mg / dl in conjunction with polyuria and polydipsia . using current glucose metabolism as the outcome ( table 2 )
, we calculated the sensitivity , specificity , and positive ( ppv ) and negative predictive values ( npv ) for cfrd of subjects ' initial testing ( cgm , 1-hour ogtt and 2-hour ogtt results ) .
the sensitivity , specificity , ppv , and npv of cgm for subsequent diagnosis of cfrd were 0.63 ( ci 0.30.86 ) , 1.0 ( ci 0.711.0 ) , 1 ( ci 0.541.0 ) , and 0.77 ( ci 0.490.92 ) , respectively . the sensitivity , specificity , ppv , and npv of 1-hour ogtt for subsequent diagnosis of cfrd were 0.75 ( ci 0.40.92 ) , 1.0 ( ci 0.691.0 ) , 1 ( ci 0.591.0 ) , and 0.82 ( ci 0.520.95 ) , respectively .
finally , the sensitivity , specificity , ppv , and npv of 2-hour ogtt for the subsequent diagnosis of cfrd were 0.13 ( ci 0.030.48 ) , 1.0 ( ci 0.711.0 ) , 1 ( ci 0.151.0 ) , and 0.59 ( ci 0.350.78 ) , respectively .
there was no difference in baseline lung function between the patients who went on to develop cfrd and those who did not ( 67.1 10.7 versus 66.3 7.9 , p = 0.95 ) .
there was a trend towards a correlation between maximum cgm values and decline in lung function over time in the group as a whole ( r = 0.45 ; 95% ci 0.01 to 0.76 , p = 0.06 ) , but there was no correlation between these values in those later diagnosed with dm ( r = 0.03 , 95% ci 0.69 to 0.72 , p = 0.9416 ) .
there was no difference in baseline bmi between the patients who went on to develop cfrd and those who did not ( 20.80 1.08 versus 22.83 1.13 , p = 0.22 ) .
there was no correlation between maximum cgm and decline in bmi over time in the group as a whole ( r = 0.33 , 95% ci 0.160.69 , p = 0.18 ) , although there was a trend towards decline in bmi in those later diagnosed with cfrd ( r = 0.67 , 95% ci 0.93 to 0.07 , p = 0.07 ) .
there was no difference between rate of exacerbations in subjects who went on to develop cfrd versus those who did not ( 1.21 0.29 versus 1.71 0.26 , p = 0.22 ) .
there was no correlation between maximum cgm and rate of exacerbations in the group as a whole ( r = 0.34 , 95% ci 0.70 to 0.15 , p = 0.16 ) , nor in those who later were diagnosed with cfrd ( r = 0.59 , 95% ci 0.20 to 0.91 , p = 0.12 ) .
the goal of the prospective pilot study was to compare the ability of the gold standard ogtt versus cgm to evaluate glucose metabolism in cf .
subsequently , we sought to determine if glucose abnormalities detected by cgm predicted decline in health . in our small study population , abnormal cgm results were not associated with decline in lung function or bmi or increased rate of exacerbations over the 5 years following the study .
however , abnormal glucose metabolism ( glucose > 200 mg / dl on two dates ) identified by cgm accurately identified those who would later be diagnosed with cfrd .
the sensitivity of cgm was similar to that of 1-hour ogtt , and both were better than that of 2-hour ogtt for subsequent development of cfrd .
numerous studies have shown that development of cfrd is associated with lung function and bmi decline [ 4 , 8 , 9 , 20 ] ; these adverse health effects can begin in the years prior to diagnosis .
a recent review of diabetes - related mortality in cf showed that the overall mortality for patients with cfrd was 1.8 per 100 person - years , compared with 0.5 in patients with cf without diabetes ( p = 0.0002 ) .
importantly , the increased morbidity and mortality can be improved with use of insulin therapy in patients with cfrd [ 4 , 2127 ] .
in addition to those patients who are diagnosed with cfrd , 1020% of patients with cf who do not meet criteria for diagnosis of cfrd have impaired glucose tolerance when it is evaluated .
other investigators have shown that patients with elevated 1-hour ogtt values are at increased risk of developing cfrd [ 28 , 29 ] .
furthermore , patients with one - hour ogtt glucose greater than 140 mg / dl have been shown to be at risk for lung function decline directly related to the degree of one - hour glucose elevation . while treatment of patients with igt is not well studied ,
2 case series have shown reversal of decline in lung function and bmi decline with use of insulin therapy in cf patients with igt [ 31 , 32 ] .
thus , impaired glucose metabolism is a prevalent issue leading to increased morbidity and mortality that can be reversed with treatment when it is diagnosed .
although rates of screening for cfrd using ogtt have increased since the guidelines were published , rates of screening are still quite low . in 2014 , only approximately 54% of children between 10 and 17 years of age and 28% of adults 18 years of age underwent cfrd screening with the recommended ogtt .
at our center in 2014 , approximately 36% of patients 10 years of age underwent ogtt screening .
because of the difficulty in obtaining annual screening ogtt , it would be ideal to use a sensitive test that does not require fasting ( in a patient population that suffers from malnutrition ) and an additional 2 - 3 hours in clinic .
although aic is quick and easy to assess , it is not recommended for cfrd screening because it demonstrates a low degree of correlation with ogtt and is insensitive for the diagnosis of cfrd [ 33 , 34 ] .
in contrast to the performance of a1c against ogtt , we showed a strong correlation with cgm and 1-hour ogtt and a moderate degree of correlation with 2-hour ogtt results .
cgm detected a greater degree of glucose excursions > 140 mg / dl than ogtt . because there are no dietary restrictions during cgm , cf patients ' recommended diet
however , a glucose > 140 mg / dl occurs very rarely in nondiabetic individuals tested by cgm .
furthermore , the test can be performed with relative ease , with a small increase in overall cost of diabetes screening .
o'riordan and colleagues sought to validate the use of cgm in a prospective cohort of children and adolescents with cf .
they demonstrated that cgm performed on two occasions over a 12-month period was reliable when compared with ogtt ( bland - altman agreement 0.81 mmol / l ; 95% ci for bias 2.90
nineteen of the twenty - one patients in our study had adequate data for analysis .
subsequent to the validation of cgm in children and adolescents with cf , other researchers have examined whether abnormal glucose metabolism by cgm predicts clinical outcomes .
prospectively studied 33 children with cf who were scheduled to undergo ogtt as part of routine health screening .
they found that the amount of time a patient experienced elevated blood glucose , specifically a blood glucose of > 7.8 mmol / l ( 140.4 mg / dl ) for 4.5% of the time , was associated with declining nutritional status and lung function in the preceding 12 months .
additionally , in a single center cross - sectional study in stable adult and pediatric patients with cf undergoing routine annual ogtt , investigators performed cgm and evaluated the effects of cgm glucose > 11 mmol / l ( 198 mg / dl ) on health outcomes . patients with cgm glucose > 11
mmol / l had lower lung function and increased prevalence of pseudomonas aeruginosa lung infection .
these two studies show that early glucose abnormalities are detectable by cgm in children and adults with cf and are associated with historical decline and contemporaneous worse health outcomes .
obtained ogtt and cgm on 17 children ( mean age approximately 13 years ) with cf followed by repeat ogtt in 2.5 years .
they found that altered glucose metabolism measured by cgm was the best predictor of future glucose metabolism abnormalities .
these studies did not answer the question of whether abnormal cgm predicts future health outcomes in adults with cf .
we were unable to show that maximum cgm value predicted declines in lung function and / bmi or increased rate of exacerbations . because this study was a pilot study , it was underpowered to detect such differences .
limitations .
although ogtt and cgm were collected prospectively , information regarding subsequent diagnosis of cfrd , lung function , and bmi was collected retrospectively . in the original study ,
cgm data was interpreted in a blinded fashion ; however , knowledge of cgm results might have led to bias / more sensitivity to symptoms by clinicians . on the other hand , abnormal ogtt results ( i.e. , impaired fasting glucose and/or impaired glucose tolerance ) should also alert clinicians / caregivers to increase monitoring of a patient 's glucose metabolism .
although a formal study of the impact of ivacaftor on glucose metabolism is underway ( nct02039986 ) , to date only a case report and a case series have been published [ 36 , 37 ] .
based on these reports , it is possible that cftr modulation may have improved glucose metabolism in our six patients who received them , thus influencing their current state of glucose metabolism .
in adult patients with cf , cgm identified a greater degree of impaired glucose metabolism than the gold standard 2-hour ogtt .
furthermore , glucose > 200 mg / dl on two dates by cgm correctly identified subjects who developed cfrd over time .
the sensitivity and negative predictive values were similar for both cgm and 1-hour ogtt for subsequent development of cfrd , and both exceeded those values for the traditional ogtt measurements .
our findings suggest that either including a 1-hour ogtt measurement or using cgm may provide a better tool for identifying patients who are at highest risk for the development of cfrd .
larger studies are required to test this hypothesis and to more accurately evaluate the ability of cgm data to predict changes in clinical endpoints such as lung function , nutritional status , and rate of pulmonary exacerbations .
impaired glucose metabolism is clearly associated with poor health outcomes in patients with cf ; therefore , such investigation may be important as we look for better ways to quickly identify and intervene in subjects at greater risk of clinical decline . | rationale . cystic fibrosis related diabetes ( cfrd ) is the most common comorbidity in patients with cf . in spite of increased screening , diagnosis , and treatment of cfrd , the mortality rate in patients with cfrd still far exceeds the mortality rate in those without cfrd .
guidelines suggest that screening for cfrd be performed annually using the 2-hour 75-gram oral glucose tolerance test ( ogtt ) .
adherence to recommended screening has been poor , with only approximately one - quarter of adults with cf undergoing ogtt in 2014 .
use of continuous glucose monitoring ( cgm ) for diagnosis may become an alternative . objectives . our objective was to determine whether abnormal cgm predicts subsequent development of cfrd , lung function , and body mass index ( bmi ) decline and increased rate of cf pulmonary exacerbations in adults with cf .
methods . in a prospective single center pilot trial from september 2009 to september 2010 ,
21 adult patients due for routine ogtt were recruited to complete simultaneous 3-day cgm and 2-hour 75 gram ogtt .
subsequently , clinical information was reviewed from 2008 to 2015 .
conclusions .
there was a moderate correlation between interpreted results of 2-hour ogtt and cgm ( p = 0.03 ) ; cgm indicated a greater level of glucose impairment than ogtt .
glucose > 200 mg / dl by cgm predicted development of cfrd ( p = 0.0002 ) . |
.
the baff / april axis plays an important role in the pathogenesis of various autoimmune diseases including sle ; inhibitors of this axis are therefore useful in treatment of these disordersresults from bliss-52 and bliss-76 trials have shown belimumab to be efficacious in treating patients with sleserious adverse effects noted with belimumab such as progressive multifocal leukoencephalopathy , depression , suicidal tendency , and malignancy are to be watched for .
the baff / april axis plays an important role in the pathogenesis of various autoimmune diseases including sle ; inhibitors of this axis are therefore useful in treatment of these disorders results from bliss-52 and bliss-76 trials have shown belimumab to be efficacious in treating patients with sle serious adverse effects noted with belimumab such as progressive multifocal leukoencephalopathy , depression , suicidal tendency , and malignancy are to be watched for .
.
the baff / april axis plays an important role in the pathogenesis of various autoimmune diseases including sle ; inhibitors of this axis are therefore useful in treatment of these disordersresults from bliss-52 and bliss-76 trials have shown belimumab to be efficacious in treating patients with sleserious adverse effects noted with belimumab such as progressive multifocal leukoencephalopathy , depression , suicidal tendency , and malignancy are to be watched for .
the baff / april axis plays an important role in the pathogenesis of various autoimmune diseases including sle ; inhibitors of this axis are therefore useful in treatment of these disorders results from bliss-52 and bliss-76 trials have shown belimumab to be efficacious in treating patients with sle serious adverse effects noted with belimumab such as progressive multifocal leukoencephalopathy , depression , suicidal tendency , and malignancy are to be watched for . | belimumab is the only approved biological agent for the treatment of systemic lupus erythematosus ( sle ) .
it is a fully humanized igg1 monoclonal antibody directed against soluble b lymphocyte stimulator ( blys ) .
it is indicated as an add - on therapy for the treatment of adult patients with active , autoantibody - positive sle , who are receiving standard therapy .
belimumab is generally well - tolerated , common adverse effects include infections , infusion reactions , hypersensitivity , headache , nausea , and fatigue . psychiatric events including suicidal tendency , progressive multifocal leukoencephalopathy and malignancies too have been reported .
apart from sle , the drug is also being tried for other autoimmune disorders . |
ovarian cancer affects close to 25,000 women yearly and most patients have extensive metastatic disease at the time of diagnosis.ovarian cancer metastasis is thought to result from exfoliation of tumor cells from the ovary and/or direct extension onto the peritoneal surfaces , the omentum , and the surface of organs such as the liver and bowel .
a standard approach to therapy is to surgically remove surgically as much of the tumor(s ) as possible , a process known as surgical cytoreduction .
this technique , which leaves only microscopic residual disease , is used in conjunction with chemotherapy .
these dismal statistics show the need for improved understanding of the process of metastatic colonization , the final step in metastasis , in which cancer cells undergo progressive growth at secondary sites [ 2 , 3 ] ( see figure 1 ) .
while invasion and adhesion have been well studied , mechanisms regulating metastatic colonization are largely unknown .
remarkably , in 2000 when our laboratory began working on metastasis suppressors in ovarian cancer , there were only a handful of papers that specifically addressed aspects of ovarian cancer metastasis .
not surprisingly , research in the molecular underpinnings of ovarian cancer metastasis continues to lag behind other cancer types .
in addition to fundamental aspects of metastasis , there are promising developments in the area of therapeutic application of metastasis suppressors .
dan theodorescu ( university of virginia ) demonstrates the feasibility of taking metastasis suppressors into the clinic ( reviewed in ) .
the following sections describe our approach to using the jnkk1/mkk4 metastasis suppressor to dissect molecular events governing omental metastatic colonization in the skov3ip.1 model .
it is our goal to encourage others to examine metastasis suppressors in clinical and experimental ovarian cancer metastases .
this observation prompted the hypothesis that molecular processes regulating tumorigenicity and metastasis are distinguishable and could be targeted therapeutically .
to identify events specifically involved in metastasis regulation , our laboratory and others hypothesized that genes and their encoded proteins that specifically regulate metastasis formation could be functionally identified [ 47 ] .
metastasis suppressors are operationally defined as genes which , when ectopically expressed in metastatic cells , can inhibit the development of spontaneous overt metastases without significantly affecting primary tumor growth . this definition has been extended to include genes and their encoded proteins which specifically inhibit metastatic colonization ( i.e. , experimental metastasis formation using intravenous or intraperitoneal injection ) .
identification of metastasis suppressors requires in vivo testing since in vitro assays generally do not model the process of metastasis .
when efforts to find metastasis suppressors were initiated , it was expected that their utility would be in predicting disease outcome ; however , robust in vivo studies have showed that metastasis suppressors can control the growth of cancer cells at metastatic sites [ 4 , 8 ] . as a result there now is evidence that metastasis suppressors can influence the interaction of disseminated cells with the microenvironment of distant organs and impair metastatic colonization .
interestingly , other investigators , working on completely different questions , also identified metastatic colonization as a rate - limiting step in metastasis formation [ 8 , 9 ] .
to date our laboratory and others have identified 23 bona fide metastasis suppressors , many of which would not have been predicted a priori based on their previously known function(s ) [ 4 , 5 ] .
determining how metastasis suppressors modulate cancer cell - microenvironmental interactions will shed light on their function in metastatic colonization , a clinically tractable therapeutic target [ 2 , 10 ] .
our laboratory identified c - jun nh2-terminal kinase ( jnk ) kinase 1/mitogen - activated protein kinase ( mapk ) kinase 4 ( jnkk1/mkk4 ) as a prostate cancer metastasis suppressor in 1999 and subsequently as an ovarian cancer metastasis suppressor in 2002 .
map kinases occupy a central position in cell growth , differentiation , and transformation . to date , three map kinase modules have been well characterized : extracellular signal - regulated protein kinase ( erk ) , c - jun nh2-terminal protein kinase ( jnk ) , and p38 .
the jnk signaling cascade consists of two map2ks , jnkk1 , and mkk7 , while the p38 signaling cascade map2ks includes jnkk1 , mkk3 , and mkk6 .
jnkk1/mkk4 is a dual - specificity kinase which , in response to extracellular stimuli , can become activated and in turn can phosphorylate and activate the jnk and p38 mapks ( figure 2 [ 24 ] ) .
in contrast , the mkk7 map2k can only phosphorylate jnk , while the mkk3 and mkk6 map2ks can only phosphorylate p38 . downstream targets of mapk signaling include components of the ap-1 transcription factor complex .
the biological outcome of mapk activation can depend , in part , on the transcriptional regulation of target genes .
specificity depends on factors such as cell type , cell environment , signal strength and duration , and the particular composition of the transcription factor , such as ap-1 .
while conventional wisdom stipulates that the jnk and p38 pathways mediate viability to stresses , increasing evidence from several model systems indicates a role for both of these mapks in cell cycle and consequent proliferation .
for instance , reports demonstrate important functions for jnk in the g1/s transition , g2/m progression , and/or cytokinesis .
similarly , p38 can activate the g2/m and spindle assembly checkpoints in mammalian cells and delay entry into mitosis or may prevent anaphase entry when the mitotic spindle is damaged [ 16 , 17 ] . in sum ,
the biological and biochemical functions of jnkk1 were consistent with its putative role in metastasis suppression ; however , there were no published studies testing its function in complex and dynamic pathological processes such as metastasis .
jnkk1/mkk4 protein levels were significantly decreased in metastases as compared to normal ovarian surface epithelium . profiling studies identified high jnkk1/mkk4 expression as a significant predictor of improved response to surgical cytoreduction . in vivo functional studies used skov3ip.1 human ovarian cancer cells , which form metastatic deposits of a serious papillary histology and produce highly reproducible numbers of metastases on the omentum , liver , and bowel .
after intraperitoneal injection of 1 10 parental skov3ip.1 or skov3ip.1-vector control cells into female immunodeficient mice , the cells adhere to target organs and by 30 days post injection ( dpi ) animals have 30 metastases .
skov3ip.1 cells have low endogenous levels of jnkk1/mkk4 but retain physiologic levels of other components of its signaling cascade .
ectopic jnkk1/mkk4 decreased the number of skov3ip.1 metastases by 88% ( p < .0001 ) and increased the animal lifespan by 70% ( wilcoxon , p = .0045 ) .
its metastasis suppressor function is kinase - dependent and studies showed that selective activation of p38 by ectopic mkk6 reduced skov3ip.1 metastasis formation by 70% ( p = .0082 ) , while selective activation of jnk by ectopic mkk7 had no effect ( p = .43 ) ( figure 3 , 3(a ) ) .
these data further defined jnkk1/mkk4 's metastasis suppressor activity and prompted the question what is the biological mechanism of jnkk1/mkk4-mediated metastasis suppression ?
jnkk1/mkk4-mediated metastasis suppression could be due to decreased adhesion of cells , increased apoptosis of cells , or inhibition of cell proliferation .
quantitative real time pcr showed that there was not a significant difference between the numbers of vector - only and jnkk1/mkk4-expressing cells present on the omentum at 3 dpi ( p = .06 ; ) .
this showed rare apoptotic cells ( < 1% ) in both groups ( p = .43 , figure 4(a ) ) .
these data were confirmed by morphological assessment as well as immunohistochemistry ( ihc ) for cleaved caspase 3 , which is an early marker of apoptosis . to determine if skov3ip.1-jnkk1/mkk4 cells were deficient in proliferation , incorporation of brdu ( a marker of s - phase cells ) and endogenous levels of phospho - histone h3 ( ( ph3 ) , a marker of m - phase cells )
these studies showed that brdu incorporation was decreased in skov3ip.1-jnkk1/mkk4 cells ( figure 4 ; 6% versus 19% positive cells , p < .0001 ) .
similarly , ph3 staining showed decreased numbers of mitotic skov3ip.1-jnkk1/mkk4 cells ( average of 0.7% versus 2.5% positive cells in the skov3ip.1-vector cells , p = .004 ) .
the decrease in brdu incorporation and ph3-staining in skov3ip.1-ha - jnkk1/mkk4 microscopic lesions suggested that fewer cells were traversing s- and subsequently m - phase compared to controls .
this prompted the examination of cell cycle inhibitory proteins , including p21 and p27 , using ihc .
this showed a nearly 10-fold increase in p21 in skov3ip.1-jnkk1/mkk4 microscopic lesions in vivo as compared to controls ( average 9% versus 1% , p < .0001 , figure 4(c ) ) .
since only a portion of the total population of skov3ip.1 cells is in cell cycle at any point in time ( with 19% entering s - phase in a 4-hour window ) , the observed increase in p21 ( 9% of the population ) is biologically relevant .
the observation that jnkk1/mkk4 activation inhibits disseminated cell growth prompted us to examine the extent and duration of this suppression . despite the reduction in the number of skov3ip.1-jnkk1/mkk4 metastases at 30 dpi and extension of survival ,
a mathematical analysis of the rates of overt metastasis formation suggested that suppression and outgrowth of jnkk1/mkk4 cells are due to the behavior of the population and not selection of a subset of cells , as would occur with increased apoptosis or differential adhesion to the omentum [ 20 , 21 ] .
molecular analyses showed that overt metastases still express functional jnkk1/mkk4 , supporting the notion that metastasis formation was not due to selection for cells that have permanently altered their jnkk1/mkk4 signaling status .
our accumulated data support a model in which binding of cells to the omentum results in the activation of jnkk1/mkk4 and induction of a cell cycle arrest . in order to determine what cellular and molecular signals activate jnkk1/mkk4 and how overt metastases ultimately form , we must consider the microenvironment in which suppression is taking place .
in essence we are ahead of ourselves and need to step back and consider what is known about the structure , function , and morphology of the omentum and integrate this knowledge into our current understanding of jnkk1/mkk4-mediated suppression of metastatic colonization .
the omentum , the primary site for ovarian cancer metastases , is a fatty peritoneal fold that covers most of the abdominal organs and serves as a storage site for lipids , as a regulator of fluid exchange , and as a reservoir for immune cells . despite its importance
, prevailing views of ovarian cancer metastasis formation do not consider the potentially dynamic and specialized functions that the omentum may contribute to this process .
historically , the omentum is viewed as being somewhat of an inert , black box
the implication is that ovarian cancer metastasis formation is the result of uncontrolled growth of cancer cells and not a regulated process which is in part controlled by the omental microenvironment . a review of the literature challenges the view that the omentum plays a passive role in ovarian cancer metastasis formation .
the human and murine omenta are structurally similar , being composed of both adipose - rich and translucent membranous tissues covered by a mesothelial layer .
mesothelial cells share characteristics of both epithelial and mesenchymal cell types and range from flattened to cuboidal in shape , depending on the body site or state of activation [ 23 , 24 ] .
it is well established that omenta from a wide variety of animals , including immunodeficinet rodents , contain aggregates of immune cells known as milky spots .
these were first described by von recklinghausen in 1863 and termed milky spots by ranvier in 1874 . in the omentum
, these structures are specialized to enable mobilization of immune cells for migration into the peritoneal cavity .
they may also facilitate reentry of immune cells from the peritoneum into the connective tissue ( and therefore bloodstream ) [ 18 , 2230 ] .
remarkably , physiologic functions of milky spots , or even their existence , have not been integrated into generally accepted models of ovarian cancer metastasis .
this is a crucial oversight , as it does not consider the possibility that ovarian cancer cells may exploit a highly regulated physiologic system in order to adhere , survive , and grow into metastases .
there is a limited amount of published data that suggests that cancer cells can specifically interact with milky spot structures [ 31 , 32 ] .
interestingly , in our studies , lotan et al . found the association of skov3ip.1-vector and skov3ip.1-jnkk1/mkk4 cells with immune aggregates which we now suspect that they are milky spot structures ( figure 5 ) .
our laboratory is currently investigating the potential role for milky spot interactions in jnkk1/mkk4-mediated suppression of metastatic colonization .
we hypothesize that disseminated skov3ip.1 cells interact with milky spots in the omentum , and these interactions contribute to the microenvironmental context - dependent activation of jnkk1/mkk4 , resulting in impaired metastatic colonization .
evidence for specific interactions of ovarian cancer cells with milky spot structures immediately identifies a target for mechanism - based studies of ovarian metastatic colonization .
therapeutic leads may be discerned by determining why disseminated cancer cells , which have molecular modifications that should enable their growth at distant sites , often lodge at target organs and persist as undetectable , or dormant disease .
our data to date support the hypothesis that activated jnkk1/mkk4 impairs proliferation of cells early in the course of metastatic colonization .
it is remarkable that few , if any , studies have been conducted that specifically examine growth control of cells during metastatic colonization . from the standpoint of translational science ,
the crucial yet underexplored question is how disseminated cells ultimately bypass suppression and form progressively growing metastases .
historically , the fundamental tenets of metastasis biology dictate that acquisition of metastatic ability is the result of the drive of malignant cells towards growth .
thus it was predicted that bypass of suppression is simply the result of mutation - selection cycles which permanently inactivate jnkk1 or members of its signaling cascade .
challenge this paradigm and suggest that jnkk1-mediated suppression may be due to a reversible cell cycle arrest concomitant with changes in jnkk1 activation status [ 20 , 21 ] .
these findings demonstrate a crucial need to reexamine important but scattered literature on population - dependent behaviors of metastatic cells , which have heretofore been refractory to mechanistic study [ 3336 ] .
this also presents an opportunity to examine the interaction of ovarian cancer cells with their microenvironment of the omentum during metastatic colonization .
given the rich literature on the bidirectional communication between cancer cells and their microenvironments , it is important that we consider microenvironmental functions and adaptations as we examine the population - dependent behaviors of cancer cells .
ultimately such studies can lay the foundation for the development of adjuvant therapies that can be used in conjunction with local therapy to delay the onset of disease recurrence , extend survival , and improve quality of life for patients with ovarian cancer . | ovarian cancer affects approximately 25,000 women in the united states each year and remains one of the most lethal female malignancies . a standard approach to therapy is surgical cytoreduction , after which the remaining microscopic residual disease is treated with chemotherapy .
the vast majority of patients have disease recurrence , underscoring the crucial need for approaches to control the regrowth , or colonization , of tissues after local treatment .
improved therapies require mechanistic information about the process of metastatic colonization , the final step in metastasis , in which cancer cells undergo progressive growth at secondary sites .
studies of metastasis suppressors are providing insights into events controlling metastatic colonization .
this paper reviews our laboratory 's approach to the identification , characterization , and functional testing of the jnkk1/mkk4 metastasis suppressor in ovarian cancer metastatic colonization .
specifically , we demonstrate that interaction of ovarian caner cells with the omental microenvironment activates jnkk1/mkk4 resulting in decreased proliferation without affecting apoptosis .
the potential role of the omental microenvironment , specifically milky spot structures , is also described .
it is our goal to provide this work as a usable paradigm that will enable others to study metastasis suppressors in clinical and experimental ovarian cancer metastases . |
imaging techniques play an important role in the diagnosis , staging , and follow - up of cancer patients . while standard imaging techniques , such as computed tomography ( ct ) , are based on differences in the anatomical structure of the tissues , positron emission tomography ( pet )
uses radiolabeled molecular probes to assess differences in biological or biochemical properties of tissues .
the most commonly used tracer for pet is the glucose analogue 2-[f]fluoro-2-deoxy - d - glucose ( [ f]fdg ) , which provides an estimate of tissue glucose utilization .
today , [ f]fdg pet is widely used in clinical diagnoses of cancer , including lung cancer , non - hodgkin lymphoma , and glioblastoma .
because of this increased utility in oncology [ 57 ] , [ f]fdg pet is being evaluated as a tool to assess antitumor effects of standard or novel anticancer drugs in both human subjects and in animal models of cancer .
pet imaging may provide evidence of biological responses of novel anticancer compounds , which , in turn , can facilitate the transition of compounds from preclinical to clinical investigation .
one such novel compound is enzastaurin , which was developed as a protein kinase c - beta ( pkc- ) inhibitor .
the family of protein kinase c ( pkc ) has been implicated in processes that control tumor growth , survival , and progression .
in particular , pkc- activation has been recognized as an important contributor to malignant growth in diffuse large cell b cell lymphoma and in glioblastoma .
recently , enzastaurin has shown antitumor activity in xenograft models of the colorectal cancer cell line hct116 and in the glioblastoma cell line u87 mg . while enzastaurin was designed as a selective pkc- inhibitor , recent studies suggest that its antitumor activity is modulated by activation of gsk-3 and the pi3k / akt pathway .
in this study we evaluated the extent to which [ f]fdg pet imaging can accurately characterize the antitumor activity of enzastaurin in two different mouse xenograft tumor models .
nonobese diabetic / severe combined immunodeficient ( nod / scid ) mice were raised and cared for by the indiana university simon cancer center transplant and xenograft core following the institutional guidelines of animal care .
the glioblastoma cell line , u87 mg , and burkitt 's lymphoma cell line , raji , were obtained from the american type culture collection ( atcc , manassas , va , usa ) and cultured as recommended by atcc .
as previously published , u87 mg and raji cells express high levels of pkc- [ 12 , 13 ] .
injection , cells were resuspended in a 1 : 1 ratio of tumor cells : matrigel ( bd biosciences , bedford , mass , usa ) and burkitt 's lymphoma cell line , raji was resuspended in a 1 : 2 ratio of tumor cells : matrigel .
each mouse was injected s.c . in the right flank with 5 10 cells .
enzastaurin treatment was initiated when the tumors reached a volume of at least 150 mm .
mice with similar tumor sizes were matched in the control and enzastaurin treated groups .
enzastaurin was suspended in 10% acacia ( fisher scientific , fair lawn , nj , usa ) in water and dosed by gavage twice daily at 75 mg / kg based upon weekly body measurements for each treated group .
each animal was anesthetized with acepromazine ( 1 - 2 mg / kg i.m . ) and torbugesic ( 2 mg / kg i.m . ) and placed on a custom bed for imaging .
a static 15-minute pet study was performed using the indypet ii scanner [ 14 , 15 ] at 45 minutes posttracer injection . following the pet study ,
the animal bed was moved to and mounted on an evs rs9 microct scanner , and a volumetric image that encompassed the tumor volume was imaged at approximately 90 micron spatial resolution .
fdg utilization estimatesfdg uptake estimates are generated by placing rois on pet images acquired over the time period from 4560 minutes posttracer administration .
indices of tumor fdg uptake were generated by calculating a ratio of the tumor to muscle uptake and by calculating the standardized uptake value ( suv ) :
( 1)suvfdg=[4560ct(t)dt](id)/m .
in ( 1 ) ct(t ) is the concentration of [ f-18 ] in the tumor from the pet image , m is the mass of the animal , i d is the dose of the tracer injected into the animal , and is tissue density .
[ f]fdg was prepared by the hamacher method using a commercial synthesis unit provided by petnet / cti ( knoxville , tenn , usa ) .
fdg uptake estimates are generated by placing rois on pet images acquired over the time period from 4560 minutes posttracer administration .
indices of tumor fdg uptake were generated by calculating a ratio of the tumor to muscle uptake and by calculating the standardized uptake value ( suv ) :
( 1)suvfdg=[4560ct(t)dt](id)/m .
in ( 1 ) ct(t ) is the concentration of [ f-18 ] in the tumor from the pet image , m is the mass of the animal , i d is the dose of the tracer injected into the animal , and is tissue density .
[ f]fdg was prepared by the hamacher method using a commercial synthesis unit provided by petnet / cti ( knoxville , tenn , usa ) .
endpoints included tumor volume via calipers , tumor volume via ct , pet - determined measures for tumor , muscle , tumor / muscle ratio , and a standardized measure of uptake .
changes from baseline for each endpoint were analyzed for each cell line ( u87 mg , raji ) and each time point ( week 2 , week 3 ) using nonparametric statistical methods ( wilcoxon rank - sum test ) to compare treated versus control animals . due to the exploratory nature of the analyses , p - values were used to indicate trends and potential future research hypotheses , rather than to test and confirm prespecified hypotheses .
we first investigated whether two tumor cell lines provided acceptable tumor growth in nod / scid mice to allow reproducible imaging with [ f]fdg pet ( figure 1 ) .
we used u87 mg cells as a representative cell line for solid tumors , and raji as a model for hematologic cancers .
both the u87 mg ( figure 1(a ) ) and the raji ( data not shown ) tumor types grew consistently in nod / scid mice prior to drug treatment and [ f]fdg pet imaging reliably detected glucose uptake in xenografts as determined by standardized uptake value ( suv ) ( see materials and methods ) . in these feasibility studies
, we also found that suv correlated with u87 mg tumor size as measured by ct ( figure 1(b ) ) . based on these preliminary observations of initial tumor growth , we elected to use both tumor cell lines to evaluate enzastaurin - induced metabolic changes as detected by [ f]fdg uptake .
enzastaurin - induced metabolic changes were evaluated using [ f]fdg uptake in two independent experiments for each tumor cell line , u87 mg and raji , respectively ( table 1 ) .
consistent with previous studies in nude mice , enzastaurin induced tumor growth delay in nod / scid mice implanted with u87 mg and raji ( figures 2(a ) and 2(b ) ) .
u87 mg cells grew slower than raji cells , and enzastaurin had a tumor growth delay mainly in the u87 mg tumor cell model ( figures 2(a ) and 2(b ) ) .
a significant tumor growth delay was seen in u87 mg after treatment with enzastaurin over the period of 3 weeks ( figure 2(a ) ) . in the raji xenograft model
, there was a trend in the tumor growth delay , but not a statistically significant difference between vehicle and enzastaurin - treated groups ( figure 2(b ) ) .
[ f]fdg pet imaging was evaluated using suv ( figures 2(c ) and 2(d ) ) and tumor / muscle ratio ( figures 2(e ) and 2(f ) ) .
enzastaurin administration did not alter tumor glucose metabolism as measured by suv changes in u87 mg xenografts ( figure 2(c ) ) .
compared to raji , the suv changes in u87 mg xenografts occurred at smaller signal intensities , and thus contributed to an overlap of suv measurements between vehicle- and enzastaurin - treated mice ( compare figures 2(c ) and 2(d ) ) . in raji , suv uptake appeared to be increased at weeks 2 and 3 in enzastaurin - treated compared to vehicle - treated mice ( p < .10 at weeks 2 and 3 ; figure 2(d ) ) .
it is possible that metabolic heterogeneity of the tumor glucose metabolism in different areas of the tumor may have caused this difference in suv uptake .
in addition to suv , we also used tumor / muscle ratio to determine the metabolic effect of enzastaurin in tumors .
the tumor / muscle ratio uses muscle tissue with its low metabolic rate to normalize the tumor tissue [ f]fdg uptake . based on this analysis
there was no clear evidence of a metabolic change induced by enzastaurin compared to vehicle treatment ( figures 2(e ) and 2(f ) ) .
however , for u87 mg there was a trend in fdg uptake in enzastaurin - treated mice ( p < .10 at week 3 ) , which was not observed in raji xenografts ( figures 2(e ) and 2(f ) , resp . ) .
after enzastaurin treatment , we observed a trend in tumor size reduction for raji xenografts but not for u87 mg xenografts at week 2 ( p
while the collective analysis of all animals from 2 independent experiments did not reveal a clear distinction between enzastaurin- and vehicle - treated animals , there were some animals that did show a metabolic change after enzastaurin treatment .
in these cases we saw a decline only after two weeks of treatment that was essentially undetectable after the third week of treatment ( figures 3(a)3(f ) and figures 4(a)4(f ) ) .
the changes observed in u87 mg were different from changes observed in raji . in u87 mg xenografts , [ f]fdg uptake declined after the 2nd week of treatment .
the raji tumors were fast growing and had large areas of necrotic tissue and some areas with newly increased [ f]fdg uptake ( figure 5 ) .
the growth pattern of these tumors is partly responsible for the intertumoral heterogeneity seen in this study and may contribute to the lack of detecting enzastaurin - induced changes in the tumor .
in this study we used a specialized pet imaging approach which was developed to assess activity of anti - cancer agents in small animals .
changes in [ f]fdg uptake were generally found to correlate with reduction in tumor size as determined by caliper measurements [ 17 , 18 ] .
our study uses a metabolic kinase inhibitor to compare drug - mediated tumor growth reduction with metabolic alterations in vivo . while previous imaging studies evaluated anti - tumor activity of cytotoxic agents , it is not clear how [ f]fdg pet imaging can help determine antitumor activity of kinase inhibitors .
in addition to its cost , the use of pet imaging may be limited due to the spatial resolution of current pet scanners .
thus , few studies have been published which evaluate the use of small animal imaging in drug discovery .
for the first time , we assessed the anti - tumor activity of the serine / threonine kinase inhibitor enzastaurin by [ f]fdg uptake in mice . because we used nod / scid instead of conventional nude mice
, we first confirmed that [ f]fdg pet images could reproducibly be obtained in xenografts of glioblastoma and lymphoma .
this feasibility assessment was important to establish the tumor size at which [ f]fdg uptake is detectable in mice .
tumors had to be at least 150 mm in volume to be visualized by the scanner , and the best assessment was observed in tumors that were more than 400 mm ( figure 1 ) .
this is consistent with other studies which reported on the need of specific tumor sizes for scanner assessment .
the subsequent studies with enzastaurin treatment did not provide clear evidence of enzastaurin - induced metabolic changes in either of the two tumor types examined .
however , there are several possibilities why enzastaurin - induced changes were not detected by [ f]fdg pet imaging .
first , it is possible that enzastaurin may not have a homogenous impact on the metabolic rate in the tumor tissue .
although pkc isoenzymes have been implicated in cell proliferation , their specific role during glucose metabolism is still not understood .
on one hand , glucose can induce pkc- expression , and on the other hand , overexpression of pkc- reduces glucose uptake in cells . hence , selective pkc- inhibitors have been investigated as potential treatments for diabetes . whether such a pkc--dependent glucose regulation exists in tumor cells is not known .
considering the observation of this study , in which tumor growth delay and glucose metabolism are not correlated with enzastaurin activity in xenograft tumors , it is possible that enzastaurin is not able to modulate the complex glucose regulation in the tumor cells .
recently , the antiangiogenic kinase inhibitor azd2171 was also evaluated in a small animal study for its metabolic change in tumors .
compared to [ f]fdg pet imaging , only [ f ] fluoromethane proved as a useful tool to assess the anti - tumor activity of azd2171 .
therefore , it appears that only some kinase inhibitors will have metabolic changes in tumors , which can be assessed by [ f]fdg pet imaging .
implanted tumors tend to grow initially along the skin surface and infiltrate the underlying tissue to a lesser extent .
this observation might explain why perhaps the caliper measurements can be used to demonstrate anti - tumor effects while the imaging of the deeper tissue by [ f]fdg or ct is not able to detect differences of treatment effect . because of the inability to delineate clearly the borders of the infiltrating tumor tissue , the current study may underestimate the metabolic change and thus lead to false - negative imaging results . in some animals , we used contrast dye to delineate better the tumor border in mice .
, future studies will need to be conducted with contrast imaging techniques to approximate the anatomical borders of the tumor .
tumor weight did not correlate with caliper measurements , because ct scans or caliper measurements were taken during the course of the study , while tumor weight was collected only at the end of the study , when animals were sacrificed , and tumors had become heterogeneous . finally , the observed [ f]fdg uptake signals are considerably lower relative to what is measured in humans .
the low baseline level in the present xenograft study negatively impacted the ability to assess early changes in metabolism associated with treatment effect .
other factors influencing the low uptake signal are heterogeneity of glucose uptake in tumors , possible emergence of drug - resistant tumor cells , variability of pharmacokinetic exposure of the inhibitor , and the diffusion of tracer to the tumor site . in summary ,
our data are consistent with the hypothesis that shrinking tumor size ( assessed by caliper measurements ) does not equate to reduction in overall tumor metabolism .
in fact , pockets of drug - resistant tumor cells with increased glucose uptake and growth kinetics may become prevalent in certain areas of the tumor , while glucose reduction will be present in other areas .
additional longitudinal pet imaging studies will have to examine in detail the variables which affect the accurate measurement of tumor response to drug treatment .
our study implies that [ f]fdg pet imaging will be useful only in a select type of tumors and perhaps detect antitumor activity only for a limited number of drugs . | background .
the use of 2-[18f]fluoro-2-deoxy - d - glucose ( [ 18f]fdg ) may help to establish the antitumor activity of enzastaurin , a novel protein kinase c - beta ii ( pkc-ii ) inhibitor , in mouse xenografts .
methods .
the hematologic cell line raji and the solid tumor cell line u87 mg were each implanted in nod / scid mice .
standard tumor growth measurements and [ 18f]fdg pet imaging were performed weekly for up to three weeks after tumor implantation and growth .
results .
concomitant with caliper measurements , [ 18f]fdg pet imaging was performed to monitor glucose metabolism .
heterogeneity of glucose uptake in various areas of the tumors was observed after vehicle or enzastaurin treatment .
this heterogeneity may limit the use of [ 18f]fdg pet imaging to measure enzastaurin - associated changes in xenograft tumors . conclusion .
[ 18f]fdg pet imaging technique does not correlate with standard caliper assessments in xenografts to assess the antitumor activity of enzastaurin .
future studies are needed to determine the use of [ 18f]fdg pet imaging in preclinical models . |
review articles in the scientific literature can be classified as a general review article , a systematic review ( sr ) , or meta - analysis ( ma ) .
the purpose of a review article is to provide readers with a summary of published research in a particular field
. reviews usually focus on areas of progress over the recent past , for example five years .
a general review article attempts to summarize all the relevant , published literature and provide some analysis of the controversial areas of the field or topic .
in addition , it may suggest some novel ways to advance the field further 1 .
such review articles provide a concise analysis of a large body of literature and hence are important for readers from a variety of fields .
articles in pubmed , for example , can be searched based on whether they are classified as review articles .
in contrast , srs seek to be more rigorous and comprehensive in addition to providing an opinion about outcomes or practice .
for example , in medical science , srs are used in hopes of ascertaining whether treatment a is superior to treatment b. why is such an analysis necessary ?
unfortunately , few research protocols are perfect so there may be controversy surrounding treatment options even after numerous studies . therefore
however , there is another reason explained by greenhalgh : experts , who have been steeped in a subject for years and know what the answer ' ought ' to be , are less able to produce an objective review of the literature in their subject than non - experts .
this would be of little consequence if experts ' opinions could be relied on to be congruent with the results of independent systematic reviews , but they can not 2 . one of the premises upon which the practice of srs is based , is the inability of informed scientists to evaluate , without bias , the controversies in their own field .
this is a reflection of human nature and is unlikely to change anytime in the near future 3 .
a sr requires clearly stated objectives and rigorous criteria for what studies can and can not be included , should be reproducible , include all relevant studies , seek to detect bias , and attempt to make determinations 4 , 5 .
srs are acknowledged as being an integral component of evidence based medicine , where the goal is to analyze the evidence gained from the best scientific studies that qualify for consideration in order to make a determination regarding clinical intervention .
the sr is thus the conscientious , explicit , judicious use of current best evidence in making decisions about the care of individual patients 6 .
the term meta - analysis was coined in 1980 by smith , glass and miller and involves a statistical analysis of the topic of a sr .
greenhalgh stated : a meta - analysis is a mathematical synthesis of the results of two or more primary studies that addressed the same hypothesis in the same way 2 .
while the purpose of any scientific literature review is to summarize and evaluate relevant articles in a scholarly and rigorous manner , the review must also consider relevant research in other disciplines of science consilience as well as the scientific underpinnings of the topic under consideration .
for example , any sr of research articles regarding acupuncture should take place in light of the fact that no mechanisms have been discovered that would allow scientists to expect success from using acupuncture in order to alleviate objective pathology 8 , 9 .
in contrast , the germ theory of disease supports a sr of the efficacy of antibacterial use for preventing complications from , or shortening the course of , ear infections in children .
an example of this concept comes from oncological surgeon david gorski who criticized the national center for complementary and alternative medicine ( nccam ) for spending resources to study : treatment modalities that are inherently unscientific , being as they are based on prescientific or demonstrably incorrect understandings of human physiology and disease 10 .
an example of knowledge from other fields of science affecting how a sr might be conducted can be found in homeopathy .
knowledge from chemistry and physics vis - - vis how to apply avogadro 's number when calculating dilutions , should inform scientists seeking to evaluate homeopathy by conducting a sr 11 .
finally , the fact that conclusions drawn from srs and mas have been shown to be wrong should also be considered when evaluating a treatment or other practice being evaluated by a sr or ma .
for example , a meta - analysis by the cochrane group reported that albumin increased deaths in critically ill patients 12 .
however , a large randomized study in australia later revealed no such effects 13 . in summary , srs and mas
are a valuable tool in assessing what is currently known regarding a subject but , like any tool , can fail .
because nonhuman animal models ( hereafter referred to as animal models or animals ) have on multiple occasions been unsuccessful in predicting human response to drugs and disease ( we will address this claim in depth ) , many have called for srs in order to improve the models 14 - 24 .
an example of this predicament would be the animal models used to determine which drugs to develop in an attempt to diminish neurological damage from ischemia events of the central nervous system ( cns ) 17 , 25 - 30 . by analyzing animal - based research with srs , flaws in the methodology
this would ostensibly also lead to better predictive values for humans ( see table 1 for calculating such values ) .
bracken supports this , stating : one reason why animal experiments often do not translate into replications in human trials or into cancer chemoprevention is that many animal experiments are poorly designed , conducted and analyzed .
another possible contribution to failure to replicate the results of animal research in humans is that reviews and summaries of evidence from animal research are methodologically inadequate 18 .
further evidence that srs are expected to transform the predictive value of animal - based research comes in the form of the 1 international symposium and workshop on systematic reviews in laboratory animal science that was held at the radboud university nijmegen medical centre on february 9 - 10 , 2012 .
the workshop celebrated 5 years of the 3r [ the 3r here refers to reduce , refine , and replace animals used in research ] research centre ( 3rrc ) and stimulating an international discussion and collaboration between animal and clinical researchers on systematic reviews ( srs ) of animal studies31 .
malcolm macleod , the keynote speaker , discussed , the transforming potential of the systematic evaluation of laboratory research .
the use of srs for the optimisation of animal testing is still rare which can lead to waste in funding and harm to patients and research volunteers .
the 3rrc encourages the use of srs in animal studies as they improve scientific quality , lead to implementation of the 3rs principles , improve translational research and help in determining the value of animal studies to human health 31 .
while we do not dispute the value of srs to improve the quality of research and perhaps increase acceptance of the 3rs , we strongly contest the notion that srs will allow scientists to develop animal models that are predictive modalities for human responses to drugs and disease .
claims such as those above by bracken and the organizers of the symposium ( and more we will cite below ) regarding the benefit of using animal models in translational research however , directly assumes predictive ability .
32 highlighted some of the potential flaws when using animal models , including : variations in drug dosing schedules and regimens that are of uncertain relevance to the human condition.variability in the way animals are selected for study , methods of randomization , choice of comparison therapy ( none , placebo , vehicle ) , and reporting of loss to follow up.small experimental groups with inadequate power , simplistic statistical analysis that does not account for potential confounding , and failure to follow intention to treat principles.nuances in laboratory technique that may influence results may be neither recognized nor reported , e.g. methods for blinding investigators.selection of a variety of outcome measures , which may be disease surrogates or precursors and which are of uncertain relevance to the human clinical condition.length of follow up before determination of disease outcome varies and may not correspond to disease latency in humans 32 .
variations in drug dosing schedules and regimens that are of uncertain relevance to the human condition .
variability in the way animals are selected for study , methods of randomization , choice of comparison therapy ( none , placebo , vehicle ) , and reporting of loss to follow up .
small experimental groups with inadequate power , simplistic statistical analysis that does not account for potential confounding , and failure to follow intention to treat principles .
nuances in laboratory technique that may influence results may be neither recognized nor reported , e.g. methods for blinding investigators .
selection of a variety of outcome measures , which may be disease surrogates or precursors and which are of uncertain relevance to the human clinical condition .
length of follow up before determination of disease outcome varies and may not correspond to disease latency in humans 32 .
hooijmans et al 14 have called for a gold standard for research involving animals that includes stating the specifics regarding housing , species , randomization , cage size and bedding among other parameters . other checklists and suggestions aimed toward improving standardizations
note that even here however , hooijmans et al link standardization to prediction of human response stating : in addition , an improved experimental design contributes to a better translation to the clinic and increases patient safety 14 .
many reviews and opinions have echoed the above reasons for translational failure or predictive failure and have suggested ways to improve the likelihood of successfully predicting human responses to drugs and disease .
the arrive ( animals in research : reporting in vivo experiments ) guidelines for reporting animal research 39 consist of a 20 item checklist containing : the minimum information that all scientific publications reporting research using animals should include , such as the number and specific characteristics of animals used ( including species , strain , sex , and genetic background ) ; details of housing and husbandry ; and the experimental , statistical , and analytical methods ( including details of methods used to reduce bias such as randomization and blinding ) 39 .
another example of such an effort is the camarades group ( the collaborative approach to meta - analysis and review of animal data in experimental studies ) .
for example , the camarades group identified significant sources of bias in a sample of almost 5,000 animal studies .
these shortcomings included a frequent lack of blinding , randomization , and sample size calculation , in addition to overstatement of treatment efficacy due to unpublished studies .
while some scientists are more modest in their claims for the value of srs and the standardization of protocols , clearly there are high hopes for what srs can accomplish regarding the predictive value of animal models .
we will now examine , in more depth , the reasons for the above concerns regarding the predictive value of animal models .
the use of animals in science and research can be categorized per table 2
40 .
while all uses of sentient animals are cause for ethical concern 41 - 43 , the use of animal models to predict human response to drugs and disease appears to be the main focus of the scientific community when attempting to justify animal use to society 44 - 52 [ 53 p3 ] .
this is consistent with giles , writing in nature , who stated : in the contentious world of animal research , one question surfaces time and again : how useful are animal experiments as a way to prepare for trials of medical treatments in humans ?
the issue is crucial , as public opinion is behind animal research only if it helps develop better drugs .
consequently , scientists defending animal experiments insist they are essential for safe clinical trials , whereas animal - rights activists vehemently maintain that they are useless 54
. statements from advocates for animal - based research acknowledge the importance society places on animal models being able to predict human response to drugs and disease .
for example , cheng stated : animal tests are necessary for some research , such as testing drugs for toxicity .
it would be , in my opinion , improper to release drugs for human use without animal testing 55 .
heywood likewise stated : animal studies fall into two main categories : predictive evaluations of new compounds and their incorporation into schemes designed to help lessen or clarify a recognised hazard 56 .
vassar agrees , stating : chronic dosing in mice and monkeys is necessary to show the efficacy and safety of the antibody before it 's taken into humans 51 .
the council for international organizations of medical sciences implies prediction when they state : clinical testing must be preceded by adequate laboratory or animal experimentation to demonstrate a reasonable probability of success without undue risk 45 .
rudczynski wrote : the basic research model used by yale university and its peer institutions is scientifically valid and predictive of human disease 57 .
the animal - based research community clearly stresses the importance and validity of using animals to predict human response to drugs and disease .
the above claims are , however , in direct opposition to those advocating for srs in order to improve the predictive ability of animal - based research .
before we survey the literature for empirical confirmation and present views of other scientists that strongly disagree with the above , we need to first define the term predict and refresh the reader 's memory of how it is used in science .
several confirmations of the hypothesis , by predictions that are found to be true , strengthen the hypothesis while one failed prediction may neccesitate revising the hypothesis or even destroy it altogether .
this is standard science based on the hypothetico - deductive method and we have no issues with using the term predict in this manner .
animal use involving categories 5 , 7 , and 9 in table 2 would employ this use of predict .
the second manner predict is used is when discussing the predictive value of a modality or practice .
an example outside of biomedical science would be when italian geologists were asked whether a series of small quakes in the area meant that residents should evacuate their houses because a major earthquake was likely forthcoming .
the geologists stated that a major earthquake was unlikely and this was consistent with current knowledge of earthquakes . nevertheless , the italian legal system convicted the scientists on charges that essentially said they were negligent in failing to warn the residents to evacuate 58 .
this was a cause for concern in the scientific community as an analysis revealed that small quakes forecast a major quake only 2% of the time 59 , 60 .
clearly , a practice or modality that correctly calculates the answer only 2% of the time does not qualify as predictive . exactly what percentage is necessary to qualify will vary with the field of study .
finding a method that will result in the correct answer 51% in the field of gambling , in blackjack for example , would be very productive and probably qualify as meeting the criteria for being a predictive practice . using instruments to fly an aircraft on the other hand ,
requires that the instruments correctly communicate the exact location of the aircraft 100% of the time . while medical science does not require predictive values of 100% ,
tests that correlate with reality even 70% of the time are not very useful . just as important as what the word predict means in terms of predictive value and how ppv and npv are calculated
for example , a single example of correlation does not qualify a model as predictive or indicate a high ppv or npv .
a modality or practice must be evaluated based on its history of correlating with reality .
moreover , one must be very precise when defining what is being evaluated for predictive value .
if one wishes to evaluate animal models in general then all the wrong answers by all species must be included in the calculation as well as all the correct answers .
if one is calculating ppv for a specific animal model , say using beagles in hepatotoxicity testing , then all correct and incorrect answers for beagles should be included but not outcomes from different species or even different breeds .
with that background we can now evaluate the claims of animal models being , or not being , predictive for human response to drugs and disease .
the assumption that animal models are predictive of human outcome is foundational for much of their use in biomedical research and for justifying animal - based research in general . whether this assumption is true
is a separate issue from that of methodology and study design although methodology may influence predictive value .
the prevailing view within the animal model community among those calling for standardization and srs , per above , is that animal models would perform better , meaning they would have a higher ppv and npv for humans , if researchers adhered to strict criteria with respect to study design and methodology 61 .
it is important to note that the potential validity of the animal model per se for predicting human response to drugs and disease is not questioned , at least in most of the literature that addresses srs and standardization .
we acknowledge that animals can successfully be used in categories 3 - 9 in table 2 and that srs could positively impact on such use and that some calling for srs and standardization advocate for such on this basis .
however , it appears that the main emphasis among those calling for srs and standardization is to improve predictive value .
therefore we consider it appropriate to explore whether a proper understanding of evolutionary biology and complexity science allows for the use of one species to predict responses to drugs and diseases for another , even under ideal circumstances 40 , 41 , 62 - 66 .
if the practice per se is not viable , then srs will be of little value .
we will now present the empirical evidence and later seek to place it within the context of complexity science and evolutionary biology .
empirical evidence regarding the predictive value of animal models comes in the form of research amenable to quantification via table 1 and examples of multiple failures over many years in the same subject .
examples of the latter would include the search for a vaccine against hiv and neuroprotective drugs .
approximately 100 vaccines have been shown effective against an hiv - like virus in animal models , however , none have prevented hiv in humans 67 , 68 .
even if an hiv vaccine came from animal - based research tomorrow , the animal model per se would not be predictive for humans as the ppv would be somewhere in the 0.01 area .
likewise , up to one - thousand drugs have been shown effective for neuroprotection in animal models but none have been effective for humans 23 - 25 , 29 , 38 , 61 , 69 - 71 . the predictive value is again minimal even if a successful drug is currently in development .
the animal model has failed as a modality for predicting neuroprotection . along the same lines , of twenty two drugs tested on animals and shown to be therapeutic in spinal cord
injury , none were effective in humans 72 . as we are attempting to prove that animal models are not predictive such examples are important .
relatively few failures can disqualify a practice from being of predictive value while proving the opposite requires a large number of successes .
the success of the animal model in basic research can also be questioned based on the fact that , according to one report , only 0.004% of basic research papers in leading journals led to a new class of drugs 41 , 73 and the fact that the success rate for target identification is similarly dismal 74 - 78 .
for example , in part because the targets derived from animal models are not predictive for humans , the percentage of new drugs in development , after initial evaluation , that ultimately make it to market is somewhere in the area of 0.0002% 79 , 80 .
we acknowledge that the goals of basic research differ from the goals of applied research where predictive values are most often evaluated .
however , because of funding challenges , research that would have historically been considered basic is now being promoted as applied and hence should be judged accordingly 41 .
the empirical evidence from research outcomes quantifiable by the calculations in table 1 also supports our position that animal models can not currently predict human response .
litchfield 81 studied rats , dogs , and humans in order to evaluate responses to six drugs .
the rat model demonstrated a ppv of 0.49 while the dog model demonstrated a ppv of 0.55 .
a ppv around 0.5 is not sufficient to qualify a modality as predictive in medical science .
medical science demands values of 0.8 or higher if the modality is to be used for anything that will intersect with patient care .
( drug development is a clear example of a product or modality intersecting with patient care . )
a similar study reported in 1990 , examined six drugs in animal models , the side effects of which were already known from human data .
the study found that at least one species demonstrated 22 side effects , but the models incorrectly identified 48 side effects that did not occur in humans , while missing 20 side effects that did occur in humans .
a similar study - reported in 1990 - examined drugs abandoned during clinical trials secondary to toxicity . in 16 out of 24 cases ,
a 1994 study revealed that only six of 114 drug toxicities had animal correlates [ 84 p57 - 67 ] . while the data do not allow the calculations in table 1 to be made , obviously these numbers fall far short of qualifying as a predictive medical modality or test .
these examples could be easily multiplied ( for example , see 85 [ 86 p67 - 74 ] 38 , 87 - 93 ) . moreover , in 1995 , lin compared pharmacologically important parameters in different species and pointed out that many examples of animal models predicting human response were in fact retrospective and hence not predictive at all 94 .
first , the animal model per se is simply not predictive of human response to drugs and disease .
( for more on the failure of animal models of human disease to correlate with humans , see 62 , 64 - 66 , 96 - 100 . ) second , perhaps the proposed srs and standardization will allow for correction of methodological problems that have resulted in animal models failing to be of predictive value .
, the following questions must be addressed : is there an all - encompassing explanation for the failure of animal models to be of predictive value regardless of methodology ?
is there a theory or law in science that explains the empirical evidence we presented ?
we propose that the fact that all animals are examples of evolved complex systems constitutes a scientific theory explaining why animal models fail to be predictive modalities for human response to drugs and disease .
in addition , this theory requires us to question whether an animal model will ever be a predictive modality for humans at the level of organization where disease and drug response occurs , regardless of methodological improvements .
the assumption that animal models are predictive of human outcome is foundational for much of their use in biomedical research and for justifying animal - based research in general . whether this assumption is true
is a separate issue from that of methodology and study design although methodology may influence predictive value .
the prevailing view within the animal model community among those calling for standardization and srs , per above , is that animal models would perform better , meaning they would have a higher ppv and npv for humans , if researchers adhered to strict criteria with respect to study design and methodology 61 .
it is important to note that the potential validity of the animal model per se for predicting human response to drugs and disease is not questioned , at least in most of the literature that addresses srs and standardization .
we acknowledge that animals can successfully be used in categories 3 - 9 in table 2 and that srs could positively impact on such use and that some calling for srs and standardization advocate for such on this basis .
however , it appears that the main emphasis among those calling for srs and standardization is to improve predictive value .
therefore we consider it appropriate to explore whether a proper understanding of evolutionary biology and complexity science allows for the use of one species to predict responses to drugs and diseases for another , even under ideal circumstances 40 , 41 , 62 - 66 .
if the practice per se is not viable , then srs will be of little value .
we will now present the empirical evidence and later seek to place it within the context of complexity science and evolutionary biology .
empirical evidence regarding the predictive value of animal models comes in the form of research amenable to quantification via table 1 and examples of multiple failures over many years in the same subject .
examples of the latter would include the search for a vaccine against hiv and neuroprotective drugs .
approximately 100 vaccines have been shown effective against an hiv - like virus in animal models , however , none have prevented hiv in humans 67 , 68 .
even if an hiv vaccine came from animal - based research tomorrow , the animal model per se would not be predictive for humans as the ppv would be somewhere in the 0.01 area .
likewise , up to one - thousand drugs have been shown effective for neuroprotection in animal models but none have been effective for humans 23 - 25 , 29 , 38 , 61 , 69 - 71 . the predictive value is again minimal even if a successful drug is currently in development .
the animal model has failed as a modality for predicting neuroprotection . along the same lines , of twenty two drugs tested on animals and shown to be therapeutic in spinal cord
injury , none were effective in humans 72 . as we are attempting to prove that animal models are not predictive such examples are important .
relatively few failures can disqualify a practice from being of predictive value while proving the opposite requires a large number of successes .
the success of the animal model in basic research can also be questioned based on the fact that , according to one report , only 0.004% of basic research papers in leading journals led to a new class of drugs 41 , 73 and the fact that the success rate for target identification is similarly dismal 74 - 78 .
for example , in part because the targets derived from animal models are not predictive for humans , the percentage of new drugs in development , after initial evaluation , that ultimately make it to market is somewhere in the area of 0.0002% 79 , 80 .
we acknowledge that the goals of basic research differ from the goals of applied research where predictive values are most often evaluated .
however , because of funding challenges , research that would have historically been considered basic is now being promoted as applied and hence should be judged accordingly 41 .
the empirical evidence from research outcomes quantifiable by the calculations in table 1 also supports our position that animal models can not currently predict human response .
litchfield 81 studied rats , dogs , and humans in order to evaluate responses to six drugs .
the rat model demonstrated a ppv of 0.49 while the dog model demonstrated a ppv of 0.55 .
a ppv around 0.5 is not sufficient to qualify a modality as predictive in medical science .
medical science demands values of 0.8 or higher if the modality is to be used for anything that will intersect with patient care .
( drug development is a clear example of a product or modality intersecting with patient care . )
a similar study reported in 1990 , examined six drugs in animal models , the side effects of which were already known from human data .
the study found that at least one species demonstrated 22 side effects , but the models incorrectly identified 48 side effects that did not occur in humans , while missing 20 side effects that did occur in humans .
a similar study - reported in 1990 - examined drugs abandoned during clinical trials secondary to toxicity . in 16 out of 24 cases ,
a 1994 study revealed that only six of 114 drug toxicities had animal correlates [ 84 p57 - 67 ] . while the data do not allow the calculations in table 1 to be made , obviously these numbers fall far short of qualifying as a predictive medical modality or test .
these examples could be easily multiplied ( for example , see 85 [ 86 p67 - 74 ] 38 , 87 - 93 ) . moreover , in 1995 , lin compared pharmacologically important parameters in different species and pointed out that many examples of animal models predicting human response were in fact retrospective and hence not predictive at all 94 .
first , the animal model per se is simply not predictive of human response to drugs and disease .
( for more on the failure of animal models of human disease to correlate with humans , see 62 , 64 - 66 , 96 - 100 . ) second , perhaps the proposed srs and standardization will allow for correction of methodological problems that have resulted in animal models failing to be of predictive value .
, the following questions must be addressed : is there an all - encompassing explanation for the failure of animal models to be of predictive value regardless of methodology ?
is there a theory or law in science that explains the empirical evidence we presented ?
we propose that the fact that all animals are examples of evolved complex systems constitutes a scientific theory explaining why animal models fail to be predictive modalities for human response to drugs and disease .
in addition , this theory requires us to question whether an animal model will ever be a predictive modality for humans at the level of organization where disease and drug response occurs , regardless of methodological improvements .
science as a discipline can arguably be dated to newton and descartes , both of whom accepted a mechanistic , deterministic universe amenable to study by reductionism 101 , 102 . because the systems under examination at that time were simple systems that were no more than the sum of their parts , exhibited predictable behavior with few interactions and feedback loops , and
hence could be intuitively understood , linear cause and effect relationships were the order of the day . because of the nature of the universe , such systems are amenable to laws while complex systems are usually described using statistics .
hence biological complex systems are more likely to be described by theories than laws 103 - 105 .
moreover , outcomes are usually described as involving a causal chain as opposed to a linear cause and effect relationship 105 .
ecosystems , climate , financial markets , and the us power grids are examples of complex systems , while humans and animals are examples of evolved complex systems .
reductionism has been of value in the study of complex systems but because of the nature of complex systems , reductionism alone is inadequate to fully describe the system 106 - 108 .
van regenmortel states : the reductionist method of dissecting biological systems into their constituent parts has been effective in explaining the chemical basis of numerous living processes . however , many biologists now realize that this approach has reached its limit .
biological systems are extremely complex and have emergent properties that can not be explained , or even predicted , by studying their individual parts . the reductionist approach
although successful in the early days of molecular biology underestimates this complexity and therefore has an increasingly detrimental influence on many areas of biomedical research , including drug discovery and vaccine development 109 .
complex systems have very specific characteristics that influence the ability of one complex system to predict the response of another 102 , 106 , 107 , 109-127.complex systems are more than the sum of their parts , thus reductionism will yield an incomplete analysis of a complex system . as animal modeling
is based in large part on reductionism 65 , 105 , 109 , 120 , 125 , 128 - 132 , this portends problems.complex systems exhibit emergence , meaning that new properties of a complex system arise from the interactions of the parts .
these new properties can not be determined even in light of full knowledge of the component parts , thus compromising reductionism even further.complex systems are resistant to changes and exhibit redundancy in their components .
this again complicates extrapolation between complex systems.complex systems exhibit self-organization.complex systems demonstrate responses to perturbations that are nonlinear.complex systems are very dependent upon initial conditions ( for example , genetic make - up ) .
for example , strains of mice have been noted to respond very differently to gene deletion 133 , 134 and groups of humans , such as sexes 135 - 140 and ethnic groups 141 - 149 , respond differently to drugs and disease .
monozygotic twins have also been discovered to respond differently to perturbations because of small differences in genetic make - up 150-154.complex systems are composed of many components , which can be grouped into modules that interact with each other.complex systems have hierarchal levels of organization ( different levels can even respond oppositely to the same perturbation).complex systems have feedback loops.complex systems interact with their environment are dynamic.complex systems are nonsimulable 155 - 158 .
complex systems are more than the sum of their parts , thus reductionism will yield an incomplete analysis of a complex system . as animal modeling
is based in large part on reductionism 65 , 105 , 109 , 120 , 125 , 128 - 132 , this portends problems .
complex systems exhibit emergence , meaning that new properties of a complex system arise from the interactions of the parts .
these new properties can not be determined even in light of full knowledge of the component parts , thus compromising reductionism even further .
complex systems are very dependent upon initial conditions ( for example , genetic make - up ) .
for example , strains of mice have been noted to respond very differently to gene deletion 133 , 134 and groups of humans , such as sexes 135 - 140 and ethnic groups 141 - 149 , respond differently to drugs and disease .
monozygotic twins have also been discovered to respond differently to perturbations because of small differences in genetic make - up 150 - 154 .
complex systems are composed of many components , which can be grouped into modules that interact with each other .
complex systems have hierarchal levels of organization ( different levels can even respond oppositely to the same perturbation ) .
koch describes the problems of studying complex systems : such systems [ like the human brain ] are characterized by large numbers of highly heterogeneous components , be they genes , proteins , or cells .
these components interact causally in myriad ways across a very large spectrum of space - time , from nanometers to meters and from microseconds to years .
a complete understanding of these systems demands that a large fraction of these interactions be experimentally or computationally probed .
fields as diverse as neuroscience and cancer biology have proven resistant to facile predictions about imminent practical applications .
improved technologies for observing and probing biological systems has only led to discoveries of further levels of complexity that need to be dealt with .
we are far away from understanding cell biology , genomes , or brains , and turning this understanding into practical knowledge 159 . in summary ,
complex systems are very different from the simple systems described so well by newtonian physics and which are routinely studied by reductionism .
complex systems are best described by partial differential equations and many of the values of the variables are unknown . hence predicting intra - complex system response is difficult and predicting inter - complex system response is essentially impossible at higher levels of organization .
the fact that the complex systems under study have evolved is also significant ( see figure 2 ) . while all of the characteristics of a complex system influence inter - system extrapolation , we will illustrate the importance of evolution on just one characteristic
evolution has used numerous mechanisms to match species to niche and all of these mechanisms affect initial conditions
. even among humans , very small differences in genetic makeup can result in dramatically different outcomes to perturbations such as drugs and disease .
for example , copy number variants ( cnvs ) in monozygotic twins can influence outcomes 150 .
single nucleotide polymorphisms ( snps ) among family members and/or other humans 161 - 163 , pleiotropy 164 , alternative splicing 165 , the fact that different genes and molecules can accomplish the same purpose , and that the same gene can be used for different purposes 166 all influence response to drugs and disease .
changes in initial conditions such as the presence of different alleles , snps , cnvs and so forth negate the similarities between complex systems in terms of predicting response to perturbations that occur at higher levels of organization such as where drug and disease response occurs .
the reality is even more complicated however , as gene regulation and expression account for the major changes in evolution 167 , 168 .
theoretically , by varying the regulation and expression of the same genes , a new species could evolve with the same structural genes of its ancestor .
gene expression varies greatly in humans 169 - 172 and in animals 173 - 176 .
studied gene expression in the brains of humans , chimpanzees , and macaques and discovered accelerated evolution of gene expression in the human prefrontal cortex 177 thus casting doubt on the ability to extrapolate inter - species research for that area .
puente et al discovered at least twenty genes implicated in human cancers that differ significantly from chimpanzees 178 .
in addition , chimpanzees are essentially immune to hiv , hepatitis b , and common malaria and they respond differently to other human pathogens 179 - 182.h according to caldwell , it has been obvious for some time that there is generally no evolutionary basis behind the particular - metabolizing ability of a particular species .
indeed , among rodents and primates , zoologically closely related species exhibit markedly different patterns of metabolism 183 . festing
stated : there is substantial genetic variation in the response of laboratory rats to xenobiotics , and this variation has important implications for toxicologic research and screening .
articles published in the journal toxicology and applied pharmacology from 1979 to 1999 . in a majority of the articles , the authors did not specify which rat strain was being used 184 .
the above has profound consequences for using animal models to predict human response to drugs and disease .
it is important to note here that many of the scientists quoted above do not take the position that animal models will never be predictive modalities . while we do not want to speculate as to their reasons
, we must point out that the fact that animals and humans are evolved complex systems that are differently complex and this leads us to our conclusion that animal models will fail as predictive modalities .
the fact , and implications , of models as differently complex is not addressed by most animal modelers quoted above and we suspect this may , in part , explain their position .
this brings us to the logical conclusion of our animals as evolved complex systems argument .
it is also perhaps our best reason against expecting animal models to ever be capable of predicting human response to drugs and disease : the concept of personalized medicine .
personalized medicine is perhaps best illustrated by allen roses , then - worldwide vice - president of genetics at glaxosmithkline ( gsk ) , who stated : the vast majority of drugs - more than 90% - only work in 30 or 50% of the people 185 .
physicians have long recognized intra - species variation in response to drugs and disease 186 , 187 .
it is now understood that the variations in response are caused by variations in the genome ( see tables 3 and 4 ) including epigenetic changes .
for example , because of differences in genes , like snps , some children are not protected by a vaccine 162 , 163 .
king states : between 5 and 20 per cent of people vaccinated against hepatitis b , and between 2 and 10 per cent of those vaccinated against measles , will not be protected if they ever encounter these viruses 163 . in the future , such children may be able to receive a personalized vaccine .
personalized medicine will result in medical practice resembling the outline in figure 3 whereas today medical practice is more often
the fact there is such variation among humans and that this variation causes so much concern 188 - 197 should cast doubts on the ability of another species to predict human response to drugs and disease 63 , 65 . also illustrative of the problems of extrapolation between complex systems , and in line with the basis for personalized medicine , is the fact that the sexes respond differently to drugs and diseases 135 - 140 , 198 , as do ethnic groups 141 - 149 .
moreover , monozygotic twins respond differently to drugs and disease 74 , 199 - 204 .
if monozygotic twins respond differently to perturbations such as drugs and disease , then expecting even genetically modified animals to be of predictive value seems nave .
indeed genetically modified animals have failed to be of predictive value 74 , 199 - 204 .
our position , and apparently the position of scientists calling for standardization of animal protocols and srs , that animal models do not currently qualify as predictive modalities for human response to drugs and disease is supported by experts in various fields of science .
for example , alan oliff , then - executive director for cancer research at merck research laboratories stated :
the fundamental problem in drug discovery for cancer is that the model systems are not predictive at all 210 .
an editorial in nature reviews drug discovery states : clearly , one part of the problem [ of drug research ] is poorly predictive animal models . . .
ellis and fidler echo this staing : preclinical models , unfortunately , seldom reflect the disease state within humans 212 .
horrobin addressed the use of animal models stating : does the use of animal models of disease take us any closer to understanding human disease ? with rare exceptions , the answer to this question is likely to be negative 98 .
fliri pointed out that : currently , no method exists for forecasting broad biological activity profiles of medicinal agents even within narrow boundaries of structurally similar molecules 213 .
speaking of toxicity trials for new drugs in humans , an unnamed clinician was quoted in science as stating : if you were to look in [ a big company 's ] files for testing small - molecule drugs you 'd find hundreds of deaths 214 .
frances collins , director of nih , has also spoken out on the poor predictive value of animal models 215 , 216 .
neuzil et al state : animal testing is not ideal either , as the predictive value of such tests is limited owing to metabolic differences between humans and animals , and many ethical issues are raised by the testing 217 .
cook et al state : over many years now there has been a poor correlation between preclinical therapeutic findings and the eventual efficacy of these [ anti - cancer ] compounds in clinical trials 218 , 219 . . .
the development of antineoplastics is a large investment by the private and public sectors , however , the limited availability of predictive preclinical systems obscures our ability to select the therapeutics that might succeed or fail during clinical investigation .
220 seidle 221 reported on the conclusions of a conference of experts in toxicology from pharmaceutical companies , contract research companies and others .
the consensus was that : the information obtained from conventional acute toxicity studies is of little or no value in the pharmaceutical development process 222 .
this statement was subsequently considered and endorsed by regulators and scientists from the eu , us and japan at a workshop in november 2006 222 .
a survey at the conference 223 revealed that:100% of respondents found data from acute toxicity studies of little or no use and only used the information in dose setting for other studies in exceptional circumstances.100% of respondents agreed that they would not carry out acute toxicity testing if it were not a regulatory requirement.100% of respondents agreed that acute toxicity studies were not used to identify target organs.100% of respondents never use acute toxicity data to help set the starting dose in man.81% of respondents thought the data obtained from acute toxicity studies was of no use to regulators or clinicians .
221 100% of respondents found data from acute toxicity studies of little or no use and only used the information in dose setting for other studies in exceptional circumstances .
100% of respondents agreed that they would not carry out acute toxicity testing if it were not a regulatory requirement .
100% of respondents agreed that acute toxicity studies were not used to identify target organs .
100% of respondents never use acute toxicity data to help set the starting dose in man .
81% of respondents thought the data obtained from acute toxicity studies was of no use to regulators or clinicians .
221 sharp and langer summarized the current situation : the next challenge for biomedical research will be to solve problems of highly complex and integrated biological systems within the human body .
predictive models of these systems in either normal or disease states are beyond the capability of current knowledge and technology 224 .
we note that the above scientists have not , to the best of our knowledge , agreed with us that animal models are incapable of being predictive modalities .
we again attribute this to the fact that the discussion regarding evolved complex systems is relatively new .
we also again note that srs and standardization may contribute to the use of animals in categories 3 - 9 of table 2 .
we do not deny that animals can be successfully used for such endeavors in science and research and recognize the value of srs in improving such uses .
however , we have presented a case against expecting animal models to ever be predictive modalities for human response to drugs and disease regardless of improvement in methodology . even if methodological issues were to prove the problem in some of the studies that reveal ppvs of ~0.5 , the lack of studies revealing any animal model to be predictive modality ( for example in teratogenicity , carcinogenicity , hepatotoxicity , efficacy for a class of drugs , mechanisms of a class of diseases ) is consistent with our theory .
animal models have historically been unable to predict human response to drugs and disease and animal - based research has historically displayed methodological problems that make srs difficult .
one proposed solution that would address both problems is standardization of protocols thus permitting srs of animal models , which would in turn improve the models thus possibly allowing accurate predictions , via high ppv and npvs , for human response to drugs and disease .
we have argued that even if the methodology for animal models could be standardized and subject to srs , animal models would still fail to be predictive modalities for human response to drugs and disease because of considerations from complexity theory and evolutionary biology .
we also reject the notion that a combination of the results of several studies in a sr or meta - analysis may produce information relevant for judging the safety and efficacy of drugs that is not directly visible in the individual animal studies ( such as significant side effects or overall efficacy ) .
the problem is that animal models are not predictive modalities , not that animal models fail to reveal side effects .
many side effects from drugs in development are already observed in animal models but there is no predictive value for humans .
as we discussed , srs are only useful if there is scientific validity to the assumptions or axioms underlying the research .
there is no reason to conduct srs of homeopathy nor does complexity theory and evolutionary biology offer any reason to expect srs of animal models to be productive .
regardless of how the problem is approached , animal and humans will always be differently complex .
one reason srs are necessary is that experts are unreliable for evaluating controversies in their own field .
we would extend that concept to include the fact that human nature is also problematic when questioning assumptions is required .
tradition , the status quo , we always do it that way , resistance to change both individually and in the form of institutional inertia , all combine to challenge those who ask epistemological questions .
add to all of this the fact that the axioms underlying such practices are not usually discussed among scientists ( being i the realm of philosophy of science ) and the result is that challenging the axioms upon which these practices are based becomes almost impossible .
nevertheless it is vital to do so in order for science in general , and medical science in particular , to advance . | systematic reviews are currently favored methods of evaluating research in order to reach conclusions regarding medical practice .
the need for such reviews is necessitated by the fact that no research is perfect and experts are prone to bias . by combining many studies that fulfill specific criteria ,
one hopes that the strengths can be multiplied and thus reliable conclusions attained .
potential flaws in this process include the assumptions that underlie the research under examination
. if the assumptions , or axioms , upon which the research studies are based , are untenable either scientifically or logically , then the results must be highly suspect regardless of the otherwise high quality of the studies or the systematic reviews .
we outline recent criticisms of animal - based research , namely that animal models are failing to predict human responses .
it is this failure that is purportedly being corrected via systematic reviews .
we then examine the assumption that animal models can predict human outcomes to perturbations such as disease or drugs , even under the best of circumstances .
we examine the use of animal models in light of empirical evidence comparing human outcomes to those from animal models , complexity theory , and evolutionary biology .
we conclude that even if legitimate criticisms of animal models were addressed , through standardization of protocols and systematic reviews , the animal model would still fail as a predictive modality for human response to drugs and disease . therefore ,
systematic reviews and meta - analyses of animal - based research are poor tools for attempting to reach conclusions regarding human interventions . |
cementoblastoma in the current world health organization classification of odontogenic tumor , is in the category of tumors of mesenchyme and/or odontogenic ectomesenchyme with or without odontogenic epithelium .
it generally occurs in young persons , comprises < 1 - 6.2% of all odontogenic tumor and is characterized as being attached to the roots , most frequently associated with first permanent molar .
the majority of these tumor are radiopaque , but radiolucent tumor may occur in rare instances .
histologically , it presents as a well - circumscribed tumor composed of cementum like tissue surrounded by a fibrous capsule .
this was a case report of a 16-year - old male patient who reported to the department of oral medicine , gdc , ahmedabad with the chief complaint of swelling and mild pain in the right side of the jaw since 6 months , with a history of extraction of 46 because of pain before 6 months in a private dental clinic . on clinical examination , there was diffuse bony hard swelling present in the right body of mandible with normal overlying skin .
intra - orally , there was diffuse bony hard swelling in 46 region with normal overlying mucosa and expanded buccal and lingual cortical plates [ figure 1 ] .
adjacent teeth were immobile and undisplaced . radiological examination revealed a well - defined round radiopacity with radiolucent rim in the right body of mandible [ figure 2 ] .
he had pre - extraction intraoral periapical radiography ( iopa ) radiograph of 46 region , which showed a well - defined radiopacity surrounded by radiolucent rim attached to roots of 46 [ figure 3 ] .
the typical radiographic presentation of radiopacity attached to root with radiolucent rim suggested of benign cementoblastoma with differential diagnosis of hypercementosis , osteoblastoma , periapical cemental dysplasia and condensing osteitis .
histopathologically it showed a well - circumscribed tumor composed of cementum like tissue surrounded by a fibrous capsule [ figure 4 ] .
intra - oral view showing diffuse swelling in right lower quadrant with missing 46 and expansion of buccal and lingual cortical plates right lateral oblique of mandible showing well defined radiopacity surrounded by radiolucent zone in body of mandible with missing 46 pre - extraction radiograph showing radiopaque lesion attached to roots of 46 microphotograph showing sheet of cementum like tissue with intervening loose fibrovascular connective tissue stroma
the male to female ratio of the prevalence has been reported to be 2.1:1 with a mean age of 20.7 years .
benign cementoblastoma is also reported in the maxillary sinus and associated with deciduous and unerupted permanent tooth and multiple teeth . clinical sign and
the radiographic appearance of benign cementoblastoma is well - defined radiopacity with radiolucent zone . though it is a benign tumor , but
some cases reported in the literature exhibited signs of local aggressiveness and destruction , including bony expansion , erosion of cortical plates , displacement of adjacent teeth , maxillary sinus involvement , invasion of the pulp chamber and root canals and extension to and incorporation of adjacent teeth .
expansion , pain and erosion or perforation of bony cortex were seen in a higher percentage of recurrent tumors , but were also seen in non - recurrent tumors .
the differential diagnosis for a periapical radiopacity include cementoma , osteoblastoma , periapical cemental dysplasia , condensing osteitis and hypercementosis .
histologically , this tumor presents sheets of cementum like tissue , which may contain a large number of reversal lines with active cementoblasts .
cementoblastoma and osteoblastoma are essentially identical histologically and the only distinguishing feature is attachment of cementoblastoma to the root of a tooth .
histologically , the cementoblast in cementoblastoma may be plump with pleomorphic and hyperchromatic nuclei , however , mitotic figures are not seen in cementoblastoma .
in contrast to osteoblastoma , the cementoblastoma is an odontogenic tumor that recapitulates cementum deposition similar to that during root formation in the later stages of odontogenesis .
furthermore , the cementoblastoma is continuous with the cemental layer of the apical third of the tooth root and remains separated from bone by a continuation of the periodontal ligament , all of which supports an odontogenic origin . whereas osteoblastoma arises in the medullary cavity of bones .
the treatment of choice is complete removal of the lesion with extraction of associated tooth , followed by thorough curettage and peripheral ostectomy .
cases have been also reported of endodontically preserving the tooth while surgical removal of benign cementoblastoma is done .
we presented a rare case of benign cementoblastoma in mandible and it should be considered in differential diagnosis of bony swellings of mandible . when extraction is attempted in such cases leaving the lesion behind
makes the clinical diagnosis difficult . though the patient had pre - extraction records , which helped in formulating the diagnosis . | we present a rare case of radiopaque lesion with radiolucent rim in the right body of mandible , with history of extraction of involved tooth , which made diagnosis confusing unless pre - extraction intraoral periapical radiography radiograph was recovered , finally diagnosed as benign cementoblastoma .
it was surgically managed , with no recurrence of the lesion more than 2 years of follow - up .
benign cementoblastoma is a rare , benign tumor of odontogenic ectomesenchymal origin , usually associated with roots of first mandibular molar . |
understanding the physical interactions governing the structure and dynamics of ribonucleosides should improve the accuracy of simulations of ribonucleic acid ( rna ) molecules .
methods for simulating biological systems include residue - centered force fields ( coarse - grained),(1 ) atom - centered force fields ( amber,(2 ) charmm , gromos ) , approximate quantum mechanics , and mixed quantum mechanics / molecular mechanics methods ( qm / mm ) . with advances in computer power , it is possible to run simulations at least as long as milliseconds and microseconds with coarse - grained and atom - centered potentials , respectively .
they have provided satisfactory descriptions of structural and thermodynamic properties for some rna and deoxyribonucleic acid ( dna ) systems , while some challenging systems still provide difficulty .
predictions for the individual ribonucleosides have not been extensively used as benchmarks for amber force fields .
a fundamental understanding of nucleosides is crucial to simulate the behavior of residues in single strands , noncanonical base pairs , and hairpins .
due to limitations of computer power , small model systems were used to parametrize the glycosidic dihedral angle in the amber94 force field.(2 ) in this article , the glycosidic dihedral angle , , of ribonucleic acids is reparameterized by extending the quantum mechanical ( qm ) fitting protocol , and new parameters are used in a revised force field , amber99. structural and thermodynamic results are extracted from molecular dynamics ( md ) simulations using amber99(33 ) and amber99 force fields .
previous experimental work on nucleosides and nucleotides has classified the behavior of individual torsion angles . structures of modified and unmodified nucleosides / nucleotides have been interrogated by one - dimensional ( 1d ) h nuclear magnetic resonance ( nmr ) and steady - state 1d h nuclear overhauser effect ( noe ) difference spectroscopy ( ssnoe ) . in this work ,
transient 1d h noe spectroscopy(51 ) and sugar proton coupling constants extracted from 1d h nmr spectra for cytidine ( c ) and uridine ( u ) are used to quantitatively deduce the preferred conformations of the glycosidic dihedral angle and the sugar pucker , respectively .
the amber99 force field overestimates the fraction of syn conformations for the base orientation and of c2-endo sugar puckering of the pyrimidines , while the results of amber99 are more consistent with that of the experimental nmr data .
simulations on adenosine ( a ) and guanosine ( g ) show that amber99 prefers high anti conformations around 310 , while amber99 prefers anti conformations around 185. the latter is more consistent with qm energy profiles and is the typical anti region seen in crystal structures of nucleic acids .
solutions of 0.2 , 1 , and 5 mm nucleosides were made in h2o with an nmr buffer consisting of 80 mm nacl , 10 mm sodium phosphate , and 0.5 mm disodium edta at a ph of 7.0 .
two lyophilizations were performed on each sample , reconstituting each time with 99.9% d2o ( cambridge isotopes laboratories ) .
one final lyophilization was performed , and each sample was reconstituted with 99.990% d2o ( sigma aldrich ) .
chemical shift data were extracted from 1d h nmr ( see supporting information ) . for a , the chemical shifts of h8 , h2 , h1 , and h2 protons vary with concentration , implying that there is base stacking and/or base pairing interactions ( see supporting information).(52 ) the 5-guanosine monophosphate is known to form quadruplex structures and other kinds of aggregates in solution , and presumably , guanosine does the same .
thus , the nmr spectra for nucleosides of a and g were not interpreted , except that j spinspin couplings of 0.2 mm samples were measured as a function of temperature ( see supporting information ) . for c and u ,
transient 1d noe measurements were performed with a selective inversionrecovery experiment in which the frequency of the selective inversion pulse was alternated between on resonance with the h6 proton and 2000 hz downfield , where no resonances are present .
the on / off resonance spectra were subtracted , and the integral of the resulting noe peaks was divided by peak integrals in a 1d spectrum to obtain percent enhancement .
steady - state 1d noe spectra were acquired in a similar manner with the inversionrecovery replaced by low - power irradiation for 10 s that was on / off the h6 resonance .
initial geometries were chosen to represent experimental conformations . the dihedral angle ( o5c5c4c3 ) was set to 54 , which is the observed value for a - form rna .
the dihedral angle ( c5c4c3o3 ) was set to either 140 or 81 , which is c2- or c3-endo sugar pucker , respectively .
the o4c1c2c3 dihedral was set to either 32 or 24 to force the sugar pucker to stay in c2- or c3-endo conformations , respectively . in ribonucleosides , there are three oh groups ( 5 , 3 , and 2 ) that are free to rotate in solution .
the 3 oh group will not interact with the base as much as the 5 and 2 oh groups .
thus , different conformations of 5 and 2 oh groups were included in the fitting . for each nucleoside ( figure 1 ) , four different sugar conformations ( table 1 ) were chosen for qm calculations with gaussian03.(56 ) for each sugar conformation , a potential energy surface ( pes ) scan was done around the glycosidic dihedral angle with increments of 5 , yielding 4 72 = 288 conformations for each nucleoside .
for each conformation in the pes scan , the structures were first optimized with hf/6 - 31 g * level of theory . during the optimization ,
most dihedrals were frozen in order to have a smooth energy profile with respect to the torsion angle ( see supporting information ) .
then , qm energies , eqm , were calculated with mp2/6 - 31 g * level of theory .
atom notations of nucleosides : ( a ) cytidine , ( b ) uridine , ( c ) adenosine , and ( d ) guanosine . for c and u ,
is the dihedral angle defined by o4c1n1c2 , and for a and g , is defined by o4c1n9c4 .
the molecular mechanics ( mm ) energies , emm(nochi ) , of each conformation were calculated by restraining the dihedral angles to the values of the optimized qm geometries with a force constant of 1500 kcal / mola using the amber99(33 ) force field parameters , except torsion parameters were set to zero ( see supporting information ) .
amber9(57 ) was used to calculate the mm energies , which use the default 14 vdw and electrostatic screening factors of 2.0 and 1.2 , respectively .
the energy difference , eqm emm(nochi ) , represents the potential energy due to torsion :
for each nucleoside , the 4 72 = 288 data points from eq 1 were fitted by linear least - squares to the fourier series shown in eq 2 .
here , 1 and 2 are the dihedral angles of o4c1n1c6 ( o4c1n9c8 ) and c2c1n1c6 ( c2c1n9c8 ) , respectively .
vn1 and vn2 are the potential energy barriers of o4c1n1c6 ( o4c1n9c8 ) and c2c1n1c6 ( c2c1n9c8 ) torsions . for each nucleoside , a separate fitting
each structure was created with the xleap module of amber9.(57 ) two conformations were used as initial structures : c3-endo sugar puckering with base orientations of anti or syn .
c , u , a , and g were solvated with tip3p water molecules(58 ) in a truncated octahedral box , having 458 , 451 , 427 , and 430 water molecules , respectively .
the structures were minimized in two steps : ( i ) with the nucleoside held fixed with a restraint force of 500 kcal / mol , steepest descent minimization of 500 steps was followed by a conjugate gradient minimization of 500 steps .
( ii ) with all restraints removed , steepest descent minimization of 1000 steps was followed by a conjugate gradient minimization of 1500 steps .
after minimization , two steps of pressure equilibration were done with the sander module in amber9 : ( i ) nucleosides were held fixed with a restraint force of 10 kcal / mol .
a total of 20 ps of md were run with a 2 fs time step .
( ii ) the above conditions were chosen , except the constant pressure dynamics with isotropic position scaling was turned on .
a total of 100 ps of md were run with a 2 fs time step .
the production run was similar to the second step of the pressure equilibration described above .
, a total of 30 ns of md were run with a 1 fs time step .
for cytidine with amber99 force field , the simulation time was 120 ns for convergence purposes . in production runs ,
simulations were carried out with the pmemd module in amber9.(57 ) trajectory files were written at each 250 fs time step .
simulations were performed for systems prepared with the amber99 and amber99 force fields . for c , u , a , and g , and each force field
, 10 separate simulations of 30 ns each were run at 300 k yielding a total of 300 ns of explicit solvent md simulation ( see supporting information ) .
five of the 10 md simulations had a starting structure of anti type , while the other five had a starting structure of syn type ( see supporting information ) .
for c with amber99 force field , the simulations were extended to 11 separate simulations with 120 ns each ( see supporting information ) .
the fractions of anti and syn conformations observed were essentially independent of the starting structure as were values obtained for c when the time for each of the 11 simulations was extended from 30 to 60 ns and 120 ns ( see supporting information ) .
ultrasonic relaxation studies in aqueous solution revealed a relaxation time of 3 ns for a and no relaxation signal for pyrimidines .
evidently , 300 ns of md simulations of the nucleosides is sufficient to sample adequately the synanti transformation .
in solution , nucleosides have two important regions that describe their structures : ( i ) the glycosidic dihedral angle , and ( ii ) the sugar pucker .
nmr noe experiments were done to analyze the structures of c and u. the magnitudes of noes are proportional to 1/(rij ) , where rij is the distance between the protons of i and j. when the base of a pyrimidine is oriented in an anti conformation , the h6 proton is about 3.5 from the h1 proton , essentially independent of sugar pucker.(62 ) thus , irradiation of h6 yields a moderate noe to h1. when the base of a pyrimidine is oriented in a syn conformation , however , the h6 proton is about 2.1 from h1 , yielding a strong noe to h1 when h6 is irradiated.(62 ) in pyrimidines , the distance between the h5 and h6 protons is constant at 2.48 , which can be used as a reference for calculating interproton distances from noesy or transient noe experiments according to eq 3:(63 )
here , noeij is the noe between protons i and j , noeh5h6 is the noe between h5 and h6 protons , and rh5h6 is the distance between the h5 and h6 protons , i.e. 2.48 .
transient noe spectroscopy(51 ) with different mixing times was used to quantitatively analyze the preferences for anti / syn populations , and the results are presented in table 3 ( also see supporting information ) .
transient noe is similar to noesy nmr except that it is 1d . to minimize spin diffusion effects and maximize signal - to - noise ratio , mixing times in the linear region of intensity vs mixing time plots were used to estimate distances between protons ( see supporting information ) .
a two - state model described by the following equation , which assumes that the structure is in either syn or anti conformations , was used to determine the proportions of each conformation :
for a transformation of ab , gab = rtln(k ) , where r = 1.987 cal k mol , t is the temperature in kelvins , and k is the ratio of the concentrations of each species , [ b]/[a ] ( see supporting information ) .
measurements of the syn / anti proportions of pyrimidines were extracted from transient noe experiments at 10 c , while the simulations were done at 300 k ( 27 c ) .
nmr spectra for c at 2 and 10 c indicate essentially no temperature dependence for the synanti equilibrium ( see supporting information ) , so all g s were calculated at 300 k. these values are for 30 c ( see supporting information ) .
these values are for 0.2 mm samples of a and g at 30 c where there may be some association ( see supporting information ) .
these values represent populations of high anti conformations with 310 ( see supporting information and figure 7 ) .
these values represent populations of anti conformations with 185 ( see supporting information and figure 7 ) . here
, noeh1h6 is the noe between the protons of h1 and h6 , fanti and fsyn are the fractions of anti and syn conformations satisfying fanti + fsyn = 1 , rh1h6,anti and rh1h6,syn are the distances between the protons of h1 and h6 when the structures are in anti and syn conformations , respectively , which are 3.48 and 2.12 , corresponding to the distances extracted from the minimum energy structures of the pes scans for c and u ( see methods section ) .
as can be seen from table 3 , the anti orientation is favored over syn .
comparison of nmr results for c at 2 and 10 c show that the fraction of anti base orientation is essentially independent of temperature ( supporting information ) .
higher temperature could not be used because of the overlap of the h1 and h5 peaks ( see supporting information ) .
ssnoe spectroscopy confirms that anti is favored over syn base orientation ( see supporting information ) .
sugar proton coupling constants extracted from 1d h nmr spectra were used to determine the sugar puckering on the basis of the following equation:(64 )
where j12 and j34 are j spinspin couplings between h1 and h2 and between h3 and h4 protons , respectively .
the proportion of c2-endo sugar puckering is equal to ( 1 fraction of c3-endo ) .
sugar pucker ( 2% ) is independent of temperature from 5 to 40 c ( supporting information ) , and results at 30 c are presented in table 3 .
figures 25 show the qm , mmamber99 , mmamber99 , and mmode(65 ) energy profiles with respect to the glycosidic dihedral angle of all the structures used in the fitting protocol for the nucleosides , where amber99 , amber99 , and ode(65 ) force fields were used to calculate mmamber99 , mmamber99 , and mmode energies , respectively . in all the plots ,
energy profiles of the amber99 force field describe the qm energy profiles best , although the ode force field s energy profile is also similar to the qm energy profiles .
the differences between the predictions of the amber99 and ode force fields is likely due to the ode force field using ch3 , h2cch3 and h2coch3 as model systems to represent the sugar , while the amber99 force field used the entire ribose with four different sugar conformations to calculate the torsional parameters .
, both ode and amber99 force fields should provide similar predictions for structural and/or dynamical properties of rna .
comparisons of the force fields to qm calculations on eight sugar conformations not included in the fitting showed that amber99 also describes those qm energy profiles better than amber99 and ode force fields ( see supporting information ) .
total energy ( in kcal / mol ) vs o4c1n1c6 of cytidine with amber99 ( black ) , amber99 ( red ) , qm ( green ) , and ode force field ( blue ) for : ( a ) sc 1 , ( b ) sc 2 , ( c ) sc 3 , and ( d ) sc 4 ( see table 1 ) . for visualization purposes , minimum energies of each curve
anti , high anti , and syn base orientations correspond to x - axis ranges of 070 , 100180 , and 200300 , respectively , because the x - axis is + 180 to be consistent with the amber94 force
field.(2 ) total energy ( in kcal / mol ) vs o4c1n1c6 of uridine with amber99 ( black ) , amber99 ( red ) , qm ( green ) , and ode force field ( blue ) for : ( a ) sc 1 , ( b ) sc 2 , ( c ) sc 3 , and ( d ) sc 4 ( see table 1 ) . for visualization purposes , minimum energies of each curve
anti , high anti , and syn base orientations correspond to x - axis ranges of 070 , 100180 , and 200300 , respectively , because the x - axis is + 180 to be consistent with the amber94 force
field.(2 ) total energy ( in kcal / mol ) vs o4c1n9c8 of adenosine with amber99 ( black ) , amber99 ( red ) , qm ( green ) , and ode force field ( blue ) for : ( a ) sc 1 , ( b ) sc 2 , ( c ) sc 3 , and ( d ) sc 4 ( see table 1 ) . for visualization purposes , minimum energies of each curve
anti , high anti , and syn base orientations correspond to x - axis ranges of 070 , 100180 , and 200300 , respectively , because the x - axis is + 180 to be consistent with the amber94 force field.(2 ) total energy ( in kcal / mol ) vs o4c1n9c8 of guanosine with amber99 ( black ) , amber99 ( red ) , qm ( green ) , and ode force field ( blue ) for : ( a ) sc 1 , ( b ) sc 2 , ( c ) sc 3 , and ( d ) sc 4 ( see table 1 ) . for visualization purposes , minimum energies of each curve are set to zero .
anti , high anti , and syn base orientations correspond to x - axis ranges of 070 , 100180 , and 200300 , respectively , because the x - axis is + 180 to be consistent with the amber94 force field.(2 ) for comparison with nmr results , predictions of population distributions of dihedral angle and sugar pucker were analyzed for c , u , a , and g using the combined trajectories of the 10 individual md simulations with amber99 and amber99 force fields ( see methods ) .
population distribution plots in 2d of dihedral and pseudorotation angles for each nucleoside are shown in figures 6 and 7 .
table 3 shows the force field predictions of base orientation and sugar pucker for each nucleoside ( also see table 4 and supporting information ) .
analyses of the individual md simulations show at least seven synanti transformations for each ( see supporting information ) .
population distribution of cytidine and uridine using amber99 ( a and b , respectively ) and amber99 ( c and d , respectively ) force fields .
pse ( y - axis ) and chi ( x - axis ) stand for the pseudorotation and dihedral angles .
pse angles of 18 and 162 correspond to c3-endo and c2-endo sugar pucker , respectively .
angles of 200 , 300 , and 60 correspond to anti , high - anti , and syn conformations , respectively.(70 ) population distribution of adenosine and guanosine using amber99 ( a and b , respectively ) , and amber99 ( c and d , respectively ) force fields .
pse ( y - axis ) and chi ( x - axis ) stand for the pseudorotation and dihedral angles .
table 4 shows the predicted populations of ( i ) , ( ii ) , ( iii ) , ( iv ) , ( v ) , and ( vi ) .
pse angles of 18 and 162 correspond to c3-endo and c2-endo sugar pucker , respectively .
angles of 200 , 300 , and 60 correspond to anti , high - anti , and syn conformations , respectively.(70 ) regions of ( i ) syn / c2-endo , ( ii ) syn / c3-endo , ( iii ) anti / c2-endo , ( iv ) anti / c3-endo , ( v ) high anti / c2-endo , and ( vi ) high - anti / c3-endo ( figures 6 and 7 ) .
table 3 shows the experimental results for c and u as well as the predictions of amber99 and amber99 force fields of the base orientation and the sugar pucker for c , u , a , and g. for the synanti equilibrium of c and u , nmr indicates 87% and 93% anti conformation , respectively , corresponding to gsynanti of 1.07 and 1.45 kcal / mol .
the amber99 force field predicts 30% and 28% anti conformation , respectively , corresponding to gsynanti of 0.49 and 0.55 kcal / mol . in comparison ,
the amber99 force field predicts 66% and 83% anti conformation , respectively , corresponding to gsynanti of 0.45 and 0.95 kcal / mol , closer to the nmr results .
evidently , amber99 overestimates the syn conformations of c and u ( see figure 6 ) . for the c2-endoc3-endo equilibrium of c and u
, nmr indicates 60% and 56% c3-endo sugar puckering at 30 c , respectively , corresponding to free energy differences , gc2c3 , of 0.24 and 0.15 kcal / mol ( table 3 ) .
the percentages are essentially independent of temperature from 5 to 40 c ( see supporting information ) .
the amber99 force field predicts 27% and 35% c3-endo sugar pucker at 27 c , respectively , corresponding to gc2c3 of 0.58 and 0.36 kcal / mol . in comparison ,
the amber99 force field predicts 54% and 55% c3-endo sugar pucker at 27 c , respectively , corresponding to gc2c3 of 0.11 and 0.13 kcal / mol , which is close to the experimental values .
evidently , amber99 underestimates c3-endo sugar puckering of c and u ( see figure 6 ) .
the amber99 force field predicts a and g to have 13% and 24% anti conformation ( table 3 ) , respectively , with a dihedral angle around 185 , which is consistent with qm calculations and typical of the anti region seen in crystal structures of rna.(66 ) the amber99 force field predicts 15% and 11% anti conformation ( table 3 ) , respectively , but with a dihedral angle around 310 ( figure 7 ) , which is the high anti region .
qm pes scans did not find any minimum around 310 but rather between 180250 for three different sugar puckers for a and g ( figures 45 and supporting information , where the x - axis , however , is + 180 ) .
the concentration dependence of chemical shifts for a and g indicated aggregation at concentrations required to determine noes with enough signal - to - noise to determine the base orientation quantitatively .
pioneering studies of 2- and 3- amp and gmp at high concentrations , however , indicated syn populations well over 50% . the amber99 force field predicts a and g to have 24% and 35% c3-endo sugar puckering , respectively , while amber99 predicts 32% and 54% .
chemical shift data of 0.2 , 1.0 , and 5.0 mm a implies base stacking that differs with concentration .
the differences of the chemical shifts between 0.2 and 1.0 mm samples are small , however .
therefore , the 0.2 mm samples of a and g were used to calculate j spinspin couplings to estimate the sugar puckering ( see supporting information ) . at room temperature , the c3-endo sugar puckering of a and g is about 40% ( table 3 and supporting information ) . for a , both force fields predictions are similar to the experimental results . for g ,
it is known , however , that guanosine monophosphate forms quadruplex structures and other aggregates in solution .
thus , it is not conclusive whether 0.2 mm g can be used to reveal the sugar puckering of monomer g. there are several reasons why the amber99 force field improves predictions for nucleosides .
when the torsions were parametrized for amber99 , model systems for adenosine and thymidine were used , and the results were generalized for all dna / rna residues.(2 ) moreover , the model systems mimicked deoxyribose c2-endo sugar puckering . at that time ,
qm calculations were limited by computer power and only 89 data points were used in the qm fitting . also , in the amber99 force field , the original cornell force field parameters for torsions were changed without doing any fitting .
the v2 term of torsion parameters was zeroed to improve the c2-endo sugar puckering phase angle for dna residues.(69 ) this effect , however , changes the whole predicted potential energy surface of the nucleosides , which , therefore , does not represent the qm energy surface well .
for the amber99 force field , the torsions of c , u , a , and g were reparameterized individually .
a multiconformational fitting that included the entire nucleoside with different sugar puckering was done to provide the torsion parameters . in the pes scan ,
a total of 4 72 = 288 data points were used in the fitting protocol for each nucleoside .
the new parameter set was tested on 12 different sugar conformations ( four separate conformations for each of c2-endo , c3-endo , and o4-endo sugar puckering ) for each nucleoside and shown to predict well the qm energy surface for these conformations ( see figures 25 and supporting information ) .
the shape of the qm energy surfaces of these conformations is also predicted well by the ode force field,(65 ) although not quite as well as by amber99 ( see figures 25 and supporting information ) . as a result , there should not be any big difference between amber99 and ode force field(65 ) predictions for structural and thermodynamic properties of nucleosides .
many reasonable combinations of parameters were tested for approximating the qm pes representing the four major conformations of each nucleoside .
for instance , we tried fitting to two dihedrals with three cosine terms , four dihedrals with two cosine terms , and four dihedrals with three cosine terms .
two dihedrals with four cosine terms provided excellent fits , and more terms gave minimal improvement .
as a comparison , the ode force field(65 ) uses 3 dihedrals ( a total of 13 vi parameters ) to represent the torsions , while we use 2 dihedrals ( a total of 8 vi parameters ) , but comparisons of the force fields to the qm potential energy surfaces shown in figures 25 and supporting information reveal that amber99 provides a better fit .
it is crucial to use a force field that appropriately models the true behavior of rna systems .
otherwise , during md simulations , sampling space will include unphysical regions , which will cause errors in predictions . with the amber99 modification
, significant improvements are seen in the structural and thermodynamic predictions for cytidine and uridine in solution ( table 3 ) .
this modification should be particularly important for non - watsoncrick regions and terminal base pairs because sampling will not include unrealistic populations of syn conformations or of c2-endo sugar puckering .
in watsoncrick regions , the torsion is restricted by hydrogen bonding in base pairs , so little effect should be seen there . thus , the amber99 force field should improve structural and thermodynamic predictions for rna . | a reparameterization of the torsional parameters for the glycosidic dihedral angle , , for the amber99 force field in rna nucleosides is used to provide a modified force field , amber99. molecular dynamics simulations of cytidine , uridine , adenosine , and guanosine in aqueous solution using the amber99 and amber99 force fields are compared with nmr results . for each nucleoside and force field , 10 individual molecular dynamics simulations of 30 ns
each were run .
for cytidine with amber99 force field , each molecular dynamics simulation time was extended to 120 ns for convergence purposes .
nuclear magnetic resonance ( nmr ) spectroscopy , including one - dimensional ( 1d ) 1h , steady - state 1d 1h nuclear overhauser effect ( noe ) , and transient 1d 1h noe , was used to determine the sugar puckering and preferred base orientation with respect to the ribose of cytidine and uridine .
the amber99 force field overestimates the population of syn conformations of the base orientation and of c2-endo sugar puckering of the pyrimidines , while the amber99 force field s predictions are more consistent with nmr results .
moreover , the amber99 force field prefers high anti conformations with glycosidic dihedral angles around 310 for the base orientation of purines .
the amber99 force field prefers anti conformations around 185 , which is more consistent with the quantum mechanical calculations and known 3d structures of folded ribonucleic acids ( rnas ) .
evidently , the amber99 force field predicts the structural characteristics of ribonucleosides better than the amber99 force field and should improve structural and thermodynamic predictions of rna structures . |
fructose is a monosaccharide that is widely available in natural food sources such as fruits and honey .
however , in most countries the main source of fructose is from sucrose , a disaccharide composed of equal portions of fructose and glucose . in the united states another major source of fructose is high - fructose corn syrup ( hfcs ) , which is a commercial liquid product consisting of fructose and glucose in varying proportions , but which in soft drinks is usually 55% fructose and 45% glucose .
fructose intake has increased markedly over the last 2 centuries , primarily due to the increasing intake of sucrose and hfcs [ 13 ] . in particular ,
the introduction of hfcs in the 1970s resulted in an accelerated intake of added sugars in the usa , in part because hfcs was inexpensive and could be easily mixed in with processed foods .
it has been suggested that the increase in added sugars worldwide may partially explain the marked increase in frequency of overweight and obese humans and may explain the rising frequency of metabolic syndrome , diabetes , hypertension , and cardiovascular diseases ( coronary artery disease , congestive heart failure , stroke , and chronic kidney disease ) [ 13 ] .
fructose is absorbed into the intestine enterocyte by the glut-5 specific transporter . while some fructose is metabolized in the small intestinal wall , much of it
is passed via the portal vein to the liver , with perhaps 20 to 30% escaping into the systemic circulation [ 5 , 6 ] . within the hepatocyte ,
fructose is phosphorylated to fructose-1-phosphate by fructokinase . because this reaction has no negative feedback system ,
this results in the generation of amp which is metabolized by amp deaminase to inosine monophosphate and eventually to uric acid .
the transient atp depletion has some similarities to ischemia and can result in arrest of protein synthesis with the induction of oxidative stress and inflammation [ 79 ] .
circulating fructose is taken up by a variety of cell types , including endothelial cells , but also is excreted into the urine where it is absorbed via the glut-5 transporter into the s3 segment of the proximal tubule .
this cell also expresses fructokinase ; as such , the metabolism of fructose by this proximal tubular cell can also lead to local oxidative stress and inflammation [ 8 , 10 ] .
fructose is known to stimulate fat accumulation in the liver by both increasing synthesis and blocking fat oxidation [ 11 , 12 ] .
perhaps not surprisingly , clinical studies have linked the intake of excessive fructose with the development of nonalcoholic fatty liver disease in humans , and the amount of fructose ingested correlates with the risk for progression to cirrhosis [ 15 , 16 ] .
fructose is also known to stimulate glycogen accumulation in the liver [ 17 , 18 ] , which primarily appears to be due to inhibiting glycogenolysis due to inhibition of glycogen phosphorylase .
fructose intake has been shown to induce insulin resistance in rats [ 20 , 21 ] ; fructose can also induce insulin resistance in humans .
the proposed mechanisms are complex but may include a consequence of hepatic lipid deposition , with diacylglycerol accumulation leading to activation of protein kinase [ 22 , 23 ] or via the hepatic stimulation of pgc-1 . some of the effects may also be due to fructose - induced hyperuricemia with effects on endothelial and adipocyte function [ 14 , 25 ] .
however , the lowering of uric acid with allopurinol did not prevent the development of insulin resistance in subjects administered large doses ( 200 g / d for two weeks ) of fructose
. fructose intake may also accelerate the development of type 2 diabetes in rats , possibly by accelerating islet dysfunction via induction of mild islet inflammation and oxidative stress [ 12 , 27 ] .
one study suggests that this might be due in part to the effects of systemic uric acid that increase in response to fructose ( or sucrose ) ingestion .
fructose may also stimulate the production of advanced glycation end products ( ages ) that have been shown to be toxic in diabetes .
indeed , chronic fructose ingestion has been associated with the accelerated formation of cataracts in diabetic rats .
intake of sugary soft drinks has also been associated with the development of obesity and diabetes . [ 30 , 31 ] indeed , a recent meta - analysis by malik et al . found a strong independent relationship between the intake of sugar - containing soft drinks with the subsequent development of diabetes .
fructose does not acutely stimulate leptin or insulin release and hence may not trigger normal satiety responses .
in addition , added sugars such as sucrose have been found to trigger dopamine responses in the ventral and dorsal striatum , which chronically may lead to downregulation of the d2 receptors and sugar bingeing .
fructose intake can also induce a central leptin resistance in rats , leading to increased food intake and the development of visceral obesity .
interestingly , rats on a high - fructose diet may not show an increase in overall body weight unless it is associated with diets high in fat .
fructose intake from added sugars is also associated with elevated blood pressure in humans , and diets low in added sugar have been reported to lower blood pressure .
furthermore , the acute ingestion of fructose ( 60 g ) can increase systolic blood pressure in humans , and this is not seen in subjects given the same dose as glucose .
in addition , in one study , overweight men were administered 200 g fructose daily for two weeks and sustained a significant increase in ambulatory blood pressure .
interestingly , the increase in blood pressure in response to fructose is better observed by tail cuff measurement as opposed to intra - aortic telemetry [ 40 , 41 ] .
however , blood pressure rises in response to sucrose or fructose diet by telemetry during the first hours of feeding [ 12 , 42 ] .
the mechanism of hypertension in response to fructose is complex but appears to be mediated by increased sodium absorption in the intestine , by inhibition of systemic endothelial function , and by stimulation of the sympathetic nervous system [ 14 , 43 , 44 ] ( figure 1 ) .
in addition , some of the effect of fructose to increase blood pressure may be the consequence of fructose - induced increases in intracellular and serum uric acid .
first , fructose - induced hypertension in rats is largely ameliorated by lowering uric acid levels .
second , in one study in overweight men , the rise in ambulatory blood pressure in response to 200 g of oral fructose per day for two weeks was blocked in those subjects concomitantly administered allopurinol .
fructose and sucrose are also known to induce renal hypertrophy and tubulointerstitial disease in rats [ 10 , 45 , 46 ] .
first , the rise in uric acid in response to uric acid may cause an afferent arteriolopathy resulting in glomerular hypertension .
second , fructose may also be filtered into the urine where it is taken up in the s3 segment of the proximal tubule , leading to local intracellular generation of uric acid with oxidative stress and local inflammation .
the administration of fructose to rats with reduced renal function ( the remnant kidney model ) can accelerate the progression of renal disease , resulting in worse proteinuria , glomerulosclerosis , and tubulointerstitial fibrosis . fructose intake also impairs calcium absorption and reduces 25-oh vitamin d and 1,25-dihydroxy vitamin d levels in this model . furthermore , the intake of sugary soft drinks in humans is associated with increased prevalence of albuminuria .
our group has also recently administered a low - fructose diet to subjects with stable chronic kidney disease for a period of 6 weeks . while we observed no effect on renal function during the period of the study
, we did observe a reduction in inflammatory markers and a fall in blood pressure in subjects with the dipper physiology ( i.e. , those subjects whose blood pressure spontaneously falls at night during sleep ) .
clearly further studies are needed to determine if limiting added sugars may benefit subjects with kidney disease .
while much work has focused on fructose as driving obesity , insulin resistance , and cardiorenal disease , not all fructose sources may be the same .
thus , natural fruits also are rich in antioxidants , ascorbate , polyphenols , potassium , and fiber that may counter the effects of fructose [ 13 , 50 ] . indeed , forman et al .
reported that fructose intake did not correlate with elevated blood pressure in a population in which much of the fructose intake was from fruit , whereas jalal et al . found a strong association of fructose intake with blood pressure when the fructose content from natural fruits was excluded .
while there is increasing evidence for a role for fructose as a contributory factor to obesity and metabolic syndrome , most of the data has relied on epidemiological studies , experimental models , and cell culture .
in contrast , studies in which fructose or sucrose is administered to subjects have shown variable effects on metabolic parameters .
in general , few metabolic effects are observed with fructose when it is given to young , healthy , and lean subjects [ 5254 ] .
this contrasts with studies in overweight / obese or insulin - resistant subjects in which metabolic effects from fructose or sucrose are commonly observed [ 22 , 26 , 5558 ] .
one potential explanation may relate to the absorption of fructose , which is known to increase with fructose exposure [ 59 , 60 ] .
fructose effects are also potentiated by glucose [ 61 , 62 ] , and most studies have only examined fructose alone .
most studies also show , both in animals and humans , that the effects of fructose are greater on postprandial lipids , fatty liver , and insulin resistance rather than on weight gain per se . indeed , the effects of fructose may be more on inducing leptin resistance , and it may require the addition of high - fat diet to show the weight gain .
nevertheless , the evidence that excessive intake of fructose may have multiple adverse effects on human health seems to be mounting .
there are likely multiple mechanisms driving the current epidemic of obesity , diabetes , and cardiorenal disease .
future studies should investigate the effect of reducing fructose intake or blocking the metabolic effects of fructose as a means for preventing or treating these important diseases . | fructose is a sugar present in sucrose , high - fructose corn syrup , honey , and fruits .
fructose intake has increased markedly in the last two centuries , primarily due to increased intake of added sugars .
increasing evidence suggests that the excessive intake of fructose may induce fatty liver , insulin resistance , dyslipidemia , hypertension , and kidney disease .
these studies suggest that excessive intake of fructose might have an etiologic role in the epidemic of obesity , diabetes , and cardiorenal disease . |
on february 23 , 2010 , a military service member preparing for deployment received smallpox vaccination and was counseled by the us department of defense about postvaccination care and infection control . on february 27 ,
the index patient wrestled 2 persons in a semiprofessional match , during which the dressing covering the vaccination site was detached . within 3 days , skin lesions developed in both contacts .
one of these 2 wrestlers participated in another wrestling match on march 5 , exposing a third person in whom lesions on the chest developed . a fourth contact
, a household member of a wrestler from the february 27 match , had lesions develop on the face .
all were examined , and test results of specimens were negative for vacv ( figure 1 ) . *
timeline of investigation of secondary and tertiary transmission of vaccinia virus from a us military service member , new york , usa , 2010 .
nysdoh , new york state department of health ; ph , public health ; vig , varicella immune globulin ; pmd , private physician ; derm , dermatologist . on march 11 ,
a 26-year - old male wrestler with no noteworthy medical history visited a dermatologist after being referred by his private physician who had prescribed trimethoprim / sulfamethoxazole for presumed methicillin - resistant staphylococcus aureus from lesions on his face , neck , and chest ( figure 2 ) .
molluscum contagiosum and impetigo were included in the differential diagnosis , and a culture was sent to a local laboratory to test for methicillin - resistant s. aureus .
the dermatologist notified the local health department of the patient s possible exposure to vacv .
a nysdoh public health physician evaluated the patient on march 12 . on examination , he had clinically compatible vacv lesions , including papular , umbilicated lesions with overlying vesicles and a few pustules .
vaccinia lesions in patients with secondary and tertiary cases , new york , usa , 2010 .
top row , case - patient 1 ; bottom row , case - patient 3 . on march 11
, a 24-year - old male wrestler with no noteworthy medical history was contacted by the nysdoh because he was a wrestling contact of the vaccinee .
he reported lesions on his face , neck , chin , and left eye that developed several days after the wrestling event and were associated with substantial pruritus , exudate , and erythema . at his public health evaluation on march 12 , he had numerous papular lesions that were umbilicated with overlying vesicles , several of which were draining serous fluid , and vacv was clinically diagnosed .
he received slit - lamp examination by a local ophthalmologist , who after consultation with cdc and nysdoh , treated him with trifluridine ophthalmic solution ( 4 ) .
the blepharitis and eyelid erythema resolved within 48 hours after initiation of trifluridine . on march 10 , a 25-year - old male wrestler and roommate of the vaccinee sought treatment for vesicular lesions on his trunk and chest .
he reported that several pruritic papules had developed 2 days after wrestling case - patient 1 .
the physician thought the lesions appeared to be molluscum contagiosum , but given the patient s history , consulted an infectious disease physician , who observed lesions compatible with vacv . at the public health clinical evaluation on march 12 , the patient had grouping of vesicular lesions with central umbilication on mildly erythematous bases on his trunk ( figure 2 ) , with a solitary lesion near his left areola and on the volar aspect of his right forearm .
vacv was clinically diagnosed . on march 9 , lesions developed along the mandible of the 29-year - old household contact of case - patient 1 . on march 11
, she was evaluated by a dermatologist , who performed a punch biopsy for suspected herpes . over the next several days ,
she was seen by the nysdoh public health physician on march 16 , and vacv was clinically diagnosed .
the most plausible route of exposure was a shared hand towel with case - patient 1 . over the next several days , her lesions started draining and became substantially more erythematous and painful .
after consultation between her physician , nysdoh , and cdc , varicella immune globulin was released from the strategic national stockpile for administration because of the new mucosal involvement ( 5 ) .
varicella immune globulin was administered with no complications on march 19 at a local hospital .
samples were obtained by unroofing vesicles , collecting the tissue , performing slide touch preps of the unearthed base of each vesicle , and obtaining viral swabs by using pox collection kits that had been distributed through nysdoh .
all patient specimens were tested at the nysdoh wadsworth center ( albany , ny , usa ) by real - time pcr , and preliminary results indicated vacv .
vacv subsequently was confirmed in all 4 patients by real - time pcr at the nysdoh wadsworth center .
the complete hemagglutinin gene was sequenced by cdc for 3 of the samples ( cases 1 , 3 , 4 ) and was identical to that of acam2000 ( 6 ) .
nysdoh provided appropriate transmission precautions and wound care instructions to all 4 case - patients ( 7 ) .
on march 11 , a 26-year - old male wrestler with no noteworthy medical history visited a dermatologist after being referred by his private physician who had prescribed trimethoprim / sulfamethoxazole for presumed methicillin - resistant staphylococcus aureus from lesions on his face , neck , and chest ( figure 2 ) .
molluscum contagiosum and impetigo were included in the differential diagnosis , and a culture was sent to a local laboratory to test for methicillin - resistant s. aureus .
the dermatologist notified the local health department of the patient s possible exposure to vacv .
a nysdoh public health physician evaluated the patient on march 12 . on examination , he had clinically compatible vacv lesions , including papular , umbilicated lesions with overlying vesicles and a few pustules .
vaccinia lesions in patients with secondary and tertiary cases , new york , usa , 2010 .
top row , case - patient 1 ; bottom row , case - patient 3 .
on march 11 , a 24-year - old male wrestler with no noteworthy medical history was contacted by the nysdoh because he was a wrestling contact of the vaccinee .
he reported lesions on his face , neck , chin , and left eye that developed several days after the wrestling event and were associated with substantial pruritus , exudate , and erythema . at his public health evaluation on march 12 , he had numerous papular lesions that were umbilicated with overlying vesicles , several of which were draining serous fluid , and vacv was clinically diagnosed .
he received slit - lamp examination by a local ophthalmologist , who after consultation with cdc and nysdoh , treated him with trifluridine ophthalmic solution ( 4 ) .
on march 10 , a 25-year - old male wrestler and roommate of the vaccinee sought treatment for vesicular lesions on his trunk and chest .
he reported that several pruritic papules had developed 2 days after wrestling case - patient 1 .
the physician thought the lesions appeared to be molluscum contagiosum , but given the patient s history , consulted an infectious disease physician , who observed lesions compatible with vacv . at the public health clinical evaluation on march 12 , the patient had grouping of vesicular lesions with central umbilication on mildly erythematous bases on his trunk ( figure 2 ) , with a solitary lesion near his left areola and on the volar aspect of his right forearm .
on march 9 , lesions developed along the mandible of the 29-year - old household contact of case - patient 1 . on march 11 , she was evaluated by a dermatologist , who performed a punch biopsy for suspected herpes . over the next several days ,
she was seen by the nysdoh public health physician on march 16 , and vacv was clinically diagnosed .
the most plausible route of exposure was a shared hand towel with case - patient 1 . over the next several days , her lesions started draining and became substantially more erythematous and painful .
after consultation between her physician , nysdoh , and cdc , varicella immune globulin was released from the strategic national stockpile for administration because of the new mucosal involvement ( 5 ) .
varicella immune globulin was administered with no complications on march 19 at a local hospital .
samples were obtained by unroofing vesicles , collecting the tissue , performing slide touch preps of the unearthed base of each vesicle , and obtaining viral swabs by using pox collection kits that had been distributed through nysdoh .
all patient specimens were tested at the nysdoh wadsworth center ( albany , ny , usa ) by real - time pcr , and preliminary results indicated vacv .
vacv subsequently was confirmed in all 4 patients by real - time pcr at the nysdoh wadsworth center .
the complete hemagglutinin gene was sequenced by cdc for 3 of the samples ( cases 1 , 3 , 4 ) and was identical to that of acam2000 ( 6 ) .
nysdoh provided appropriate transmission precautions and wound care instructions to all 4 case - patients ( 7 ) .
in 2003 , members of the military , selected health care workers , public health personnel , and first responders began receiving smallpox vaccinations as part of bioterrorism preparedness ( 8) . although smallpox vaccination campaigns directed toward health care workers and public health officials ended in january 2008 ( 9 ) , smallpox vaccinations continue for military service members .
this case report illustrates the need to ensure that military vaccinees understand the risk associated with contact transmission .
regions with active military smallpox vaccination programs need to maintain awareness among community medical providers , health departments , and laboratories to facilitate recognition , correct diagnosis , and appropriate response to inadvertent inoculation of vaccinia virus to help limit further transmission . especially in areas with ongoing smallpox vaccination programs ,
finally , updates are needed on identification of vacv cases , along with the notification and involvement of public health . | during february and march 2010 , the new york state department of health investigated secondary and tertiary vaccinia contact transmission from a military vaccinee to 4 close contacts .
identification of these cases underscores the need for strict adherence to postvaccination infection control guidance to avoid transmission of the live virus . |
strontium titanate
( srtio3 , sto ) , the prototypical perovskite
oxide , has long been known to show interesting effects taking place
on its surfaces .
for example , it is commonly used as a substrate for
the growth of complex oxides , and it supports
the formation of a two - dimensional electron gas on its surfaces .
furthermore , srtio3 was one of the first materials to
show a higher photocatalytic water - splitting efficiency than the classical photocatalyst tio2 .
what makes a material a good
photocatalyst are an appropriate band
gap allowing harvesting most of the solar spectrum ( maximum intensity
at around 2.4 ev ) and a suitable alignment of the valence band maximum
( vbm ) and conduction band minimum ( cbm ) with respect to the redox
potentials of water . as was found by scaife , for most semiconducting transition metal oxides , the ( h2o / h2 ) redox potential of water lies around 3 v above the
vbm , i.e. , inside the band gap for srtio3 ( eg = 3.2 ev ) .
this means that band engineering toward an
efficient photocatalyst should decrease the magnitude of the band
gap while retaining the position of the cbm .
such band engineering
was already successfully demonstrated by
konta et al . by doping sto with rhodium .
the rhodium dopants introduce an impurity level located 2.3 ev below
the cbm of sto .
visible light can therefore excite electrons from
the dopant levels into the conduction band of sto , and the photocatalytic
process is initiated .
recently , we have studied the adsorption
of water on the pristine
srtio3(110)-(4 1 ) surface .
this reconstruction is formed to compensate for the polarity of
the sto(110 ) surface .
the structure consists of six-
and ten - membered rings of tio4 tetrahedra , sitting on a
bulk - truncated sto(110 ) plane , in which titanium is octahedrally coordinated .
we found that this surface is remarkably inert toward the interaction
with water , providing a very good starting point for studying the
effect of promoters .
the photocatalytic
activity of the srtio3:nio system
has already been demonstrated successfully by townsend and co - workers . in their study ,
townsend et al . showed that
niox - sto is more likely a three - component
ni - sto - nio catalyst , in which sto absorbs the light , ni reduces protons ,
and nio oxidizes water . the present study is intended to provide
fundamental information on the srtio3:nio system .
we have
performed x - ray absorption near - edge structure ( xanes ) , x - ray and
ultraviolet photoelectron spectroscopy ( xps / ups ) , and scanning tunneling
microscopy ( stm ) to determine the effects of adsorption of nio onto
the sto(110)-(4 1 ) surface .
nio was prepared by either ( i )
postoxidation of submonolayer amounts of ni deposited by molecular
beam epitaxy ( mbe ) in ultrahigh vacuum or ( ii ) reactive mbe growth
of metallic ni in a molecular oxygen background .
stm reveals that
the nanometer - sized nio patches that develop on sto(110)-(4
1 ) distort the local surface structure independently of the preparation
method .
consistently , xanes indicates a distortion / transformation
of the surface tio4 tetrahedra upon nio growth .
band bending
is observed in both xps and ups , which increases with the amount of
nio .
the onset of the vbm is effectively shifted upward , while minimal
changes are observed on the cbm , as seen in the xanes results . combining
xps , ups , and low - energy ion scattering spectroscopy ( leis ) , a clear
signature of dissociative adsorption of water
nb - doped ( 0.5 wt % ) srtio3 single crystals
with polished
( 110 ) surface were purchased from mateck ( germany ) and crystec ( germany ) .
the sto(110)-(4 1 ) surface was prepared by cycles of sputtering
( ar , 1 kev , 5 a , 10 min ) and annealing ( 900 c ,
3 10 mbar o2 , 1 h ) .
the samples
were heated either by passing alternating current ( 7.6 v , 1.75 a )
through the samples or by bombarding them with electrons from the
back ( 900 v , 15 ma ) .
the sto(110 ) surface exhibits a series
of well - defined reconstructions , which can be tuned by the sr / ti stoichiometry
in the near - surface region . for the present
investigation
the surface structure was adjusted to yield the 4
1 reconstruction , as checked by low - energy
electron diffraction ( leed ) .
nickel metal ( purity of 99.999% ) was
deposited onto the surface from an electron beam evaporator ( omicron
efm3 ) , with a typical growth rate of 0.1 / min , as measured in
uhv via a home - built quartz - crystal microbalance .
the oxidation of
metallic ni was achieved either by reactively depositing ( rd ) ni in
5 10 mbar of o2 at different
temperatures ( room temperature [ rt ] , < 300 c , and 355 c )
or by postoxidizing ( po ) the submonolayer ni amounts ( deposited in
ultrahigh vacuum at rt ) in 5 10 mbar of
o2 for 20 min . in the latter case ,
the substrate temperature
was adjusted to be slightly below the onset of the ir pyrometer reading
( 300 c ) , unless otherwise stated . in the following ,
synchrotron
radiation photoemission spectroscopy ( pes ) and x - ray absorption spectroscopy
( xanes ) experiments were performed at beamline i311 at the max - lab
laboratory .
the base pressure of the
beamline end station was below 1 10 mbar .
all synchrotron - based photoemission spectra ( scienta ses200 analyzer )
were acquired with a takeoff angle for the photoelectrons of approximately
55 away from the surface normal and with suitable kinetic energies
( see spectra for photon energies ) to ensure highest surface sensitivity .
only one set of photon energy measurements was carried out for each
sample treatment by measuring the fermi - edge position on the ta sample
holder .
therefore , uncertainties of binding energies are stated for
the shown synchrotron - based spectra .
work functions ( ) were
determined from a straight line tangent to the leading edge of the
low kinetic - energy cutoff of the secondary electrons , as measured
at normal emission with a sample bias of 10 v. x - ray absorption
was performed in two different modes .
auger - electron yield ( aey ) spectra
were acquired by measuring the intensity of the ti l2m2,3m2,3 auger peak , while sweeping the photon energy
across the ti l2,3 edge . in secondary - electron yield ( sey )
,
the increase of low - kinetic - energy electrons ( ekin 60 ev ) was measured as an indication of the absorption
of x - rays .
stm experiments were performed in a separate uhv system
with a specs aarhus stm at room temperature ( rt ) , using electrochemically
etched w tips .
all stm images were taken in constant - current mode
with approximately + 2 v sample bias voltage ( empty states ) and a tunneling
current of around 0.2 na .
xps ( omicron x - ray source with al anode , h = 1486.7 ev ) and leis ( specs iq12/38 , 1000 ev he ions , 5.8 10 he/[cms ] , and total scattering angle = 127 )
experiments were performed with a specs phoibos 100 hemispherical
analyzer in the same analysis chamber with a base pressure below 6
10 mbar . because of the small uncertainties
in the photon energy calibration for the experiments performed at
max - lab synchrotron radiation facility , measurements of band bending
as a result of different surface treatments were repeated in the home
laboratory , providing a well - defined photon energy .
such band bending
was derived from the binding energy of fitted xps core - levels ( o 1s
and ti 2p3/2 ) . samples were prepared in a connected preparation
chamber with a base pressure of 3 10 mbar .
deionized water and isotopically labeled h2o ( sigma - aldrich ) were cleaned by repeated freeze pump
thaw
cycles and dosed onto the sample in the preparation chambers through
high - precision leak valves . peaks in ion scattering spectra have been
assigned by calculating the kinetic energy of he ions
elastically scattered by o , o , ti , ni , and
sr atoms , considering a 127 total scattering angle .
figure 1a shows
photoemission spectra ( raw data ) of the valence band region of differently
treated srtio3(110)-(4 1 ) surfaces .
the inset shows
a zoom into the gap region with the spectra aligned at the o 2s peak at eb 22 ev
( not shown ) .
vbm were determined from the intersection of the horizontal
( zero counts ) line with a straight segment tangent to the leading
edge of the ups spectra , as represented in the inset of figure 1a . for the clean surface ( black
curve ) ,
the onset of the valence band is located at approximately
3.2 ev below the fermi level , in nice agreement with the n - type doping
of the sto crystals and a reported band gap of 3.2 ev , i.e. , a flat - band situation .
the gap - region
of the pristine surface does not show any detectable density of states ,
meaning that no in - gap states are introduced by the nb dopants in
the sto single crystal .
black , red , and blue spectra correspond to the
pristine surface , the surface after deposition of 0.1 ni , and
after postoxidizing the same ni amount at 300 c , respectively .
the inset shows a zoom into the gap region
with the spectra aligned to the o 2s feature at 22
ev .
( b ) ni 2p core levels before and after oxidizing 0.1 ni . ( c )
the insets show the o 1s high binding - energy region ( c ) and the ti 2p3/2 low binding - energy region ( d ) after aligning the spectra to the
corresponding main peak .
( e ) band bending as measured from the o 1s
and ti 2p peak positions with al k radiation as a function
of the rd - deposited nio deposition amount ( quartz crystal microbalance
readings assuming ni density ; error bars represent 99% confidence
intervals on the fitted peak positions ) .
evaporation of ni ( red curve ) shifts all spectral features
to lower
binding energies ( by 0.22 0.15 ev ) and an in - gap state is formed .
the adsorption of single ni adatoms at the srtio3(110)-(4
1 ) surface has been studied recently , showing that isolated ni adatoms are formed up to a coverage of
0.05 [ see figure 2b ] . upon postoxidizing the ni adatoms , the valence band shifts
to even
lower binding energies by 0.38 0.15 ev compared to the pristine
surface , and the in - gap state is shifted downward , forming a shoulder
of the valence band . in the case of 0.1
ni postoxidized , the
onset of the valence band is shifted to lower binding energies by
around 1.5 ev [ inset of figure 1a ] .
the upward bending of the energy bands as a result of
nio adsorption is consistent with the higher workfunction of nio compared
to the pristine sto surface ( sto(110)-(41 ) 4.9 ev , nio = 6.26.7 ev ) and the measured increase of the workfunction
of the nio - loaded surface ( sto+0.1 nio 5.3 ev ) .
figure 1b shows
the ni 2p core - level spectra measured on as - deposited 0.1 ni
( red ) and after postoxidizing the sample ( blue ) .
the as - deposited
ni exhibits the typical metallic ni 2p core - level features with a
binding energy of the j = 3/2 component of 853.6
0.3 ev and a 17.1 ev spin orbit - split ( sos ) 2p1/2 peak ( compared to 2p3/2 photoemission from pure metallic
ni at eb = 852.7 ev with a sos of 17.3
ev ) . upon postoxidizing the nickel ,
the
ni 2p core - level features change to the typical nio line shape , with
a binding energy of the j = 3/2 component of 855.9
0.3 ev and a sos of 17.6 ev ( compared to literature values
of eb = 853.8 ev of 2p3/2 and
sos = 17.5 ev for bulk nio ) .
the
o 1s core - level [ figure 1c ] shows only a very weak change .
the presence of ni adatoms
causes the main o 1s spectral feature to be damped and a small shoulder
at higher binding energy is formed .
this shoulder is slightly diminished
upon postoxidation [ see inset of figure 1c , in which the spectra are shifted for clarity ,
aligning the main o 1s feature ] .
the effect of nickel adatoms binding
to the surface oxygen can also be seen in the ti 2p core - level spectra
in figure 1d . here ,
the pristine surface ( black ) shows the commonly observed ti 2p features
of ti ions ( ti 2p3/2eb = 459 0.15 ev with a sos of 5.7 ev ) . upon deposition
of ni adatoms , a shoulder at lower binding energies
is formed , as
it can be seen in the inset of figure 1d .
such a shoulder is normally assigned to reduced
ti species , as described elsewhere . upon oxidation , this shoulder vanishes , and the spectrum
resembles the pristine surface , except for a rigid shift of 0.2
0.15 ev to lower be .
band bending is found to progressively increase
with the successive deposition of nio , attaining a saturation value
of approximately 0.8 ev at deposited amounts above 1 nio [ see figure 1e ] .
the ( 4
1 ) reconstructed surface of srtio3(110 ) ,
having a unit cell size of 15.6 5.52 , appears
in stm images as periodic double lines of weak maxima along the [ 110 ]
direction [ see figure 2a ] .
these maxima correspond to tio4 tetrahedra , which
form six- and ten - membered rings via corner - sharing oxygen atoms .
bright protrusions on the ( 4 1 ) rows [ a
few marked by green dots in figure 2a ] are assigned to single strontium adatoms , which
are stabilized on antiphase domain boundaries of the reconstruction ,
ensuring polarity compensation .
we have recently investigated the adsorption of nickel on this
surface , showing that isolated single
ni adatoms [ red stars in figure 2b ] are stabilized when submonolayer ni amounts are
deposited on sto at rt .
ni adatoms can adsorb either at the center
of ( 4 1 ) rows ( six - membered tio4 rings ) or on their
sides , in proximity of the dark trenches ( ten - membered tio4 rings ) . upon postoxidizing 0.1
ni in 5
10 mbar of o2 at 375 c , the ni adatoms coalesce into
irregularly shaped patches [ highlighted by the dashed blue line in figure 2c ] , extending over
several adjacent ( 4 1 ) lines , and not showing any apparent
preferential adsorption site .
no clear ordering of atomic - scale features
could be observed on such patches , likely indicating the formation
of a locally defective nio structure .
similar morphological and structural
features are observed when growth is carried out by reactively depositing
ni in o2 atmosphere [ figure 2d , e ] , and no significant improvement of the measurable
atomic - scale structure is obtained upon increasing the deposited ni
amount [ cf .
the area of nio patches progressively extends with increasing
the deposited amount , and 0.3 nio ( not shown ) nearly fully
cover the sto surface , with only little ( 4 1 ) structure visible
in stm .
leed images acquired on the samples at different nio coverages
( not shown ) do not exhibit any additional features in the diffraction
pattern , but an overall increase of the inelastic background upon
increasing the deposited material .
stm images [ ( a d ) 20 20
nm , ( e ) 16
16 nm , ( f ) 30 30 nm ] of differently
treated srtio3(110)-(4 1 ) surfaces .
( b ) pristine
surface after deposition of 0.05 ni . panels c and d show 0.1 ni postoxidized ( po ) and reactively
deposited ( rd ) , respectively .
( e ) reactively deposited 0.2
ni . ( f ) 0.1 ni after postoxidation at 600 c .
tunneling
parameters for panels a f : us =
+ 2.0 to + 2.5 v , it = 0.02 to 0.3 na .
it is worth pointing out that
a local distortion of the ( 4
1 ) rows is visible in stm in close proximity of the nio patches .
this
is mostly evident wherever nio patches extend up to the dark trenches
separating adjacent dotted rows , as indicated by the white arrows
in figure 2c , d . in
such regions
the dark trenches appear wider in the in - plane direction ,
and a deeper corrugation ( by 1020 pm ) is measured in stm .
figure 2f shows
the sample surface with 0.1 nio postoxidized at 600 c .
the nio patches coalesce into rectangular islands , and several defect
structures are introduced in the sto substrate [ orange solid lines
in figure 2f ] .
one
type of defect appears as periodic , bright dots centered within the
( 4 1 ) structure .
another type of defect is a cross - shaped vacancy
centered on one of the two rows of periodic dots , which build up the
( 4 1 ) structure .
finally , localized depressions occasionally
appear next to nio patches and possibly correspond to missing units
of the surface structure .
it should be mentioned that despite the
relatively high temperature of 600 c , only a small decrease
( < 6% ) of the ni 2p xps intensity was measured ( not shown ) , excluding
an extended intermixing of ni atoms into the sto crystal .
electrons from occupied core levels get excited into
unoccupied conduction band states , e.g. , ti 2p ti 3d transitions .
as has been already pointed out
, the ( 4 1 ) surface consists
of tetrahedral tio4 units residing on bulk srtio3 consisting of octahedral tio6 units .
the spectral features
corresponding to these two configurations can be disentangled in the
xanes line shape , as has been recently shown [ wang et al . ,
the ti 3d levels split into subsets with t2g / eg or t / e symmetry in an octahedral ( oh ) or tetrahedral ( td ) crystal
field , respectively .
figure 3a shows xanes spectra of the ti l2,3 absorption
edge for differently treated srtio3(110 ) surfaces .
common
to all spectra are a strong absorption peak at a photon energy of
approximately 458 ev ( ti 2p3/2 ti 3d oh - t2 g ) followed by two overlapping peaks at 459 ev ( ti 2p3/2
ti 3d td - e ) and 460 ev ( ti 2p3/2
ti 3d oh - eg ) and two broad overlapping features at 463
and 465 ev ( ti 2p1/2 ti 3d
oh - t2 g and
ti 2p1/2 ti 3d oh - eg ) .
note the
small shifts of the ti l2,3 peak at 458 ev
for different treatments of the surface .
these can not be ascribed
to band bending , which is caused by a local difference in electrostatic
potential and should therefore affect all core levels and conduction
band states equally .
the shifts are therefore related to changes in
the conduction band , i.e. , in the ti 3d levels .
this indicates that the cbm is only
minimally altered upon adsorption of nio , as compared to the change
in the vbm [ see inset of figure 1a ] .
a strong decrease of the tetrahedral signature
after adsorption
of ni and nio is visible in figure 3a .
this indicates that either the tetrahedral information
is damped by the adsorbates or that the latter distort the tio4 tetrahedra .
the latter possibility is consistent with the
distortion , and disruption of the surface structure , which was visible
in stm after preparing a nio - loaded surface at 300 c [ white
arrows in figure 2c , d ]
and 600 c [ figure 2f ] , respectively . in general ,
ti l2,3 xanes spectra acquired
in sey mode ( not shown ) show the same absorption peaks as aey with
the relative peak ratios dominated by the octahedral coordination
of ti in bulk sto .
the spectrum appears
similar for all surface treatments and acquisition modes ( aey and
sey ) , showing only minimal variations in the relative intensities
of the main features . because of the low signal , related to the small
amounts of ni deposited , the ni l2,3 xanes spectrum [ figure 3c ] was acquired in
secondary electron yield mode ( see materials and
methods section )
therefore , the use of sey mode is not
limiting the surface sensitivity of the measurement .
the photon energy
at which adsorption at the l3 edge occurs ( approximately
853 ev ) is consistent with previous reports for both metallic ni and
nio samples .
in the case of postoxidized
samples the main features of the characteristic line shape of nio
are observed .
namely , the intensity of the l2,3 white lines
with respect to the background is increased due to the oxidation of
metallic ni , resulting in an increased number of d holes for oxidized
ni species .
x - ray absorption spectra
of the pristine surface , with 0.1
ni adatoms , and after postoxidation to nio ( black , red , and blue curve ,
respectively ) .
features related to ti coordinated in tetrahedral or octahedral environment
are indicated by td and oh , respectively .
here we studied the interaction of h2o with
the nio - loaded srtio3(110)-(4 1 ) surface by means
of ups / xps and leis .
the valence states of the adsorbed molecule allow
distinguishing whether water is adsorbed molecularly or dissociatively .
molecularly adsorbed h2o is characterized by its 1b1 , 3a1 ,
and 1b2 valence orbitals , with typical binding energies of 7 , 10 ,
and 14 ev , respectively . on the other
hand ,
oh species are characterized by their 1 ( 6 ev )
and 3 ( 11 ev ) valence orbitals .
figure 4 shows valence band and o 1s core level photoemission spectra before
( blue ) and after ( green ) dosing 50 langmuirs ( 1 langmuir = 1.33
10 mbar s ) of h2o on a ( 4 1 )
surface with 0.1 nio .
an increased density of states is found
in regions commonly assigned to adsorbed oh , i.e. , the oh 3
state at 11 ev binding energy [ see inset of figure 4a , in which the spectra are aligned to the
o 2s peak at eb 22 ev to highlight
differences ] , and the high binding - energy shoulder of the o 1s core - level
[ inset of figure 4b ,
again with aligned o 1s peak energies ] . after dosing 50 langmuirs
of h2o ,
( a ) valence band and ( b ) o 1s photoemission
spectra of nio - loaded
surfaces before ( blue ) and after ( green ) dosing 50 langmuirs of water
at rt .
the inset of ( a ) shows a zoom into the oh 3 region ( spectra
aligned to the o 2s peak at 22 ev ) .
inset of ( b ) shows the o 1s high
binding - energy region after aligning the spectra to the corresponding
main peak .
a different approach for detecting
adsorbed water is low - energy
he ion scattering ( leis ) .
leis is considered one of the
most surface sensitive spectroscopic techniques with the ability of
resolving small mass differences by choosing a suitable primary ion
mass .
figure 5 shows
ion scattering spectra of differently treated srtio3(110)-(4
1 ) surfaces .
the spectrum of the pristine ( 4 1 ) surface
( black curve ) shows peaks related to he ions scattered
at o ( 400 ev ) , ti ( 710 ev ) , and sr ( 795
ev ) atoms .
it should be mentioned that upon impingement of 1000 ev
he ions a progressive degradation of the surface is observed ,
most likely due to intermixing of the first few surface layers . in
particular ,
an inversion of the apparent ti to sr ratio is visible
already after 15 scans ( each scan takes about 1 min ; the ion currents
ranged from 15 to 23 na , which translates into a total ion fluence
of ( 1.11.7 ) 10 ions / cm for
each spectrum ) .
in order to limit beam damage as much as possible
while still retaining good statistics , we used only the sum of the
first four scans for each leis spectrum in figure 5 . to investigate the adsorption of water
on the nio - loaded srtio3(110)-(4 1 ) surface
, we
performed leis after dosing up to 100 langmuirs ( 1.3 10 mbar 1000 s ) of isotopically labeled water
( h2o ) . dosing up to 100 langmuirs of h2o onto the pristine surface does not introduce
any further features in the leis spectrum , consistent with the inert
nature of this surface .
we successively
deposited increasing amounts of nio onto the surface
and subsequently dosed up to 100 langmuirs of h2o , and each spectrum in figure 5 corresponds to a freshly prepared sample .
nickel introduces
a peak in leis spectra at a kinetic energy of approximately 770 ev .
this
peak is assigned to he ions scattered at o belonging to adsorbed water .
the spectra acquired on samples with
different amounts of nio clearly show that water binds to the nio - loaded
surface , in agreement with the photoemission data in figure 4 .
the existence of a larger o peak in the case of 0.2 nio compared to the case
of 0.3 nio ( close to full coverage ) possibly indicates that
either h2o adsorption or dissociation takes place at the
nio / srtio3 boundary .
low - energy he ion scattering
spectra acquired on the
pristine srtio3(110)-(4 1 ) and after depositing
different amounts of nio with subsequent dosing of 100 langmuirs of
h2o .
the combination of various spectroscopic
techniques and direct - space
imaging provides new insights into the nio / sto system .
ups and xanes
results indicate that the deposition of nio onto the sto(110)-(4
1 ) surface effectively reduces the band gap of the system from 3.2
to approximately 1.7 ev .
this change is visible in the altered onset
of the valence band when nio is present at the surface as well as
in the relatively stable positions ( within 0.2 ev ) of ti l2,3 xanes peaks ( probing the cbm ) with and without nio . assuming that
this vbm shoulder is hybridized with the o 2p valence band , these
results suggest a considerable reduction of the band gap of the system
nio / sto , allowing harvesting of visible light ( 1.7 ev corresponding
to 729 nm , i.e. , near - infrared ) .
although the present data do not
provide information on hybridization of these states , it is known
from experiments with n - doped tio2 that such vbm shoulders can considerably
enhance the visible - light response in photocatalysis experiments .
in addition , a pronounced ( up to 0.8 ev ) upward
band bending is observed for the nio - loaded surface .
photogenerated
excitons can effectively dissociate in the electrostatic potential
gradient related to the band bending , which drives electrons toward
the bulk of the substrate and holes to the very surface , provided
that extended states exist to allow charge transport before electron
the excess holes located at the surface can
then participate in the catalytic splitting of adsorbed h2o . combining stm and xanes , a
disruption of the reconstructed surface
lattice
the resulting defects
probably represent favorable adsorption sites for water , which is
also consistent with ion scattering results .
leis shows a higher water
signal for the surface with 0.2 nio compared to the nearly
full coverage obtained upon depositing 0.3 nio .
this indicates
that the adsorption may take place at the circumference of the nio
patches , i.e. , at the triple phase boundary nio / sto / uhv .
no interdiffusion
of ni was detected ; i.e. , the two oxide phases are well separated .
it should be mentioned that the patches observed in stm possibly consist
of a mixed ni
ti oxide phase , since the amount of reactively
deposited ni resulting in a nearly full coverage ( 0.3 ) approximately
corresponds to 33% of a single nio(110 ) layer .
such a mixed oxide
phase might contribute to the damping of the tetrahedral feature in
xanes . in a previous study
, we argued about the inertness of
the pristine
( 4 1 ) surface to be caused by the undistorted tetrahedra forming
the surface reconstruction . therefore ,
the distortion or disruption of these tetrahedra could be a reason
for the enhanced reactivity of the surface toward water adsorption .
we have studied the morphology and electronic structure of the
nio - modified srtio3(110)-(4 1 ) surface .
an in - gap
state , formed by the deposition of metallic ni onto this surface ,
is transformed into a valence band shoulder upon oxidation of the
ni into nio .
this effectively increases the vbm with the cbm being
unchanged , as seen in xanes , and therefore decreases the band gap
of the system .
furthermore , xanes results indicate that
surface tio4 tetrahedra get considerably distorted or disrupted
upon bonding with nio , as is also evident from stm images of nio - covered
surfaces .
ups and leis experiments confirm that the nio activates
the surface toward dissociative adsorption of h2o , possibly
involving sites at the interface between nio patches and the substrate . | nickel
oxide ( nio ) , deposited onto the strontium titanate ( srtio3 ) ( 110)-(4 1 ) surface , was studied using photoemission
spectroscopy ( pes ) , x - ray absorption near edge structure ( xanes ) ,
and low - energy he+ ion scattering ( leis ) , as well as scanning
tunneling microscopy ( stm ) .
the main motivation for studying this
system comes from the prominent role it plays in photocatalysis .
the
( 4 1 ) reconstructed srtio3(110 ) surface was previously
found to be remarkably inert toward water adsorption under ultrahigh - vacuum
conditions .
nickel oxide grows on this surface as patches without
any apparent ordered structure .
pes and leis reveal an upward band
bending , a reduction of the band gap , and reactivity toward water
adsorption upon deposition of nio .
spectroscopic results are discussed
with respect to the enhanced reactivity toward water of the nio - loaded
surface . |
according to the world health organization 's classification of glomerular diseases , both thrombotic microangiopathy ( tma ) and fibrillary glomerulonephritis ( fgn ) consist of glomerular lesions , the former in the context of a vascular disease and the latter in the setting of a metabolic disease .
anti - glomerular basement membrane ( gbm ) antibody glomerulonephritis is considered to be a glomerular nephritis of a systemic disease . here ,
we report an anti - gbm antibody - positive patient with fgn associated with tma .
our patient was a 54-year - old male who presented with anuria , fever , and lower - leg edema .
on june 3 , 2012 , the patient visited the urology department of a local community hospital with macroscopic hematuria and dysuria .
he was diagnosed with acute prostatitis and began treatment with levofloxacin hydrate , loxoprofen sodium hydrate , and cernitin pollen extract . however
, his condition did not improve , and he subsequently developed lower extremity numbness , bilateral flank pain , and diarrhea , followed by anuria .
on june 11 , he visited another hospital in his community and was eventually transferred to our hospital on june 13 for inpatient treatment of possible acute kidney injury . physical examination on admission revealed the following : height 175 cm ; body weight 80 kg ; systolic and diastolic blood pressure values 179 and 102 mm hg , respectively ; pulse 98 beats / min ( regular ) , and body temperature 37.9c .
g / l ) , blood ( + + + ) , pyuria , and a fractional sodium excretion rate of 20.3% .
hematological analyses of peripheral blood yielded a white blood cell count of 9.61 10 cells / l ( neutrophils 85.0% , lymphocytes 8.5% ) , a red blood cell count of 3.44 10 cells / l , a hemoglobin level of 100
g / l , the presence of fragmented red blood cells , and a platelet count of 90 10 cells / l .
g / l , the prothrombin time was 45.6 s , and the fibrinogen level was 4.1
serum biochemistry tests yielded a blood urea nitrogen level of 58.1 mmol / l and a creatinine level of 2,044 mol / l , suggesting significant renal impairment .
iu / l , and the haptoglobin level was below 1.2 mol / l . the patient had hypoproteinemia , as evidenced by a total serum protein level of 58
serum samples were negative for anti - nuclear antibodies as well as myeloperoxidase- and proteinase 3-specific antineutrophil cytoplasmic autoantibodies , although anti - gbm antibody levels were high at 230 elisa units .
the patient reported a history of cervical disc herniation diagnosed at the age of 52 years .
he had no noteworthy family history and was not taking any medications on a regular basis at the time of admission to our hospital .
changes in platelet counts , lactate dehydrogenase levels , and mean blood pressure over time during the 119-day hospital stay are schematically represented in figure 1 , along with major therapeutic interventions . computed tomography ( ct ) imaging conducted on admission indicated renal atrophy and ascites , but no collapse of the inferior vena cava .
these findings negated the possibility of prerenal and postrenal failure and suggested an acute aggravation of chronic kidney disease rather than a typical acute kidney injury .
since the patient had not undergone regular medical checkups , it was not clear whether proteinuria had existed in the past . during the early hospitalization period , we administered -globulin , diuretics , and hemodialysis , under the assumption that the patient had either sepsis- or drug - induced acute kidney injury .
peripheral blood smears revealed the presence of fragmented red blood cells ( hospital day 2 ) , suggesting hemolytic uremic syndrome ( hus ) resulting from food poisoning caused by escherichia coli o-157:h7 or other pathogenic strains .
however , the patient had no diarrhea and was negative for intestinal pathogens and verotoxins .
epistaxis developed on hospital day 4 , platelet counts and hemoglobin levels decreased to 29 10 cells / l and 55
g / l , respectively , and the lactate dehydrogenase level rose to 1,253 iu / l .
the patient 's anemia was unresponsive to erythropoiesis - stimulating therapy , and frequent blood transfusions were required ( i.e. , a total of 16 units of irradiated red cell concentrates ) .
the patient was diagnosed with tma [ thrombotic thrombocytopenic purpura ( ttp ) or hus ] based on the following findings : thrombocytopenia , hemolytic anemia evidenced by anemic signs and elevated lactate dehydrogenase levels , signs of renal impairment , fever , bleeding episodes ( i.e. , epistaxis ) , neuropsychiatric manifestations ( i.e. , headache ) , direct and indirect coombs test results , and haptoglobin levels below 1.2
assays conducted several days later indicated an adamts13 activity of 31.6% ( reference range 70 - 120% ) , and adamts13 inhibitors were negative .
these findings led us to administer plasma exchange therapy with a total of 30 units of fresh frozen plasma , starting on hospital day 8 . on hospital day 15 , steroid pulse therapy ( 3 methylprednisolone doses of 1,000 mg / day ) was initiated to lower the elevated anti - gbm antibody level . following the completion of this 3-day course ,
the patient was given oral prednisolone at a starting dose of 50 mg / day , which was gradually tapered and discontinued .
after this , platelet counts rose to 197 10 cells / l , and kidney biopsy was conducted on hospital day 22 ( fig .
seventeen glomeruli examined by optic microscopy all showed destructive patterns with characteristic architecture ranging from atypical proliferative changes to global sclerosis .
renal arterioles , both afferent and efferent , showed extensive endothelial cell edema and swelling , indicative of glomeruloid changes .
electron microscopy showed that the loss of glomerular structure observed under optic microscopy was the result of an extensive and dense extracellular deposition of fibrillar components , which were larger in diameter than amyloid fibers and aggregated to form large bundles .
immunofluorescence analysis for immunoglobulin g and third component ( c3 ) deposits was not carried out because of glomerular collapse .
available findings supported the diagnosis of fgn involving severe glomerular destruction . on hospital day 29 , a second course of steroid pulse therapy was started due to significantly decreased platelet counts . on hospital day 46 , the mean blood pressure values started to increase gradually . although the target body weight was lowered and arotinolol hydrochloride and methyldopa hydrate were added to nifedipine and candesartan cilexetil , these measures did not achieve successful blood pressure control . on hospital day 56 , the patient had a mild seizure .
t2-weighted fluid - attenuated inversion recovery ( flair ) magnetic resonance scans of the bilateral cerebellum , occipital lobe , and parietal lobe showed multiple high - intensity areas , suggesting a diagnosis of reversible posterior leukoencephalopathy syndrome ( fig .
carvedilol , nifedipine , and nitroglycerin infusion , hypertension persisted , and the patient 's headache continued . on hospital day 61 , generalized convulsions recurred , and head ct images showed multiple hemorrhagic lesions in the right temporal , right parietal , and right frontal lobes ( fig .
blood pressure levels gradually decreased with the increase in platelet counts , starting around hospital day 65 . on hospital day 72 , the patient started rehabilitation exercises .
fgn and immunotactoid glomerulonephritis are characterized by electron - dense deposits of fibrillary structures in the glomerular extracellular matrix . for both conditions , optical microscopy of renal biopsies
fgn is characterized by randomly oriented nonbranching fibrils with a mean diameter of 15 - 25 nm , a size comparatively larger than that of amyloid fibrils ( 8 - 10 nm ) . in our case , although immunofluorescence analysis of immunoglobulin g and c3 was not conducted because of glomerular collapse , the diagnosis of fgn was established based on the negative congo red staining and electron microscopy findings .
the term tma designates pathological conditions characterized by microangiopathic hemolytic anemia , thrombocytopenia resulting from peripheral destruction of platelets , and organ damage due to platelet thrombi ( notably , renal impairment ) .
the differential diagnoses include disseminated intravascular coagulation syndrome , idiopathic thrombocytopenic purpura , heparin - induced thrombocytopenia , paroxysmal nocturnal hemoglobinuria , and systemic lupus erythematosus .
analyses of adamts13 activity and adamts13 inhibitors and further investigation suggested that idiopathic ttp or hus , autoimmune diseases or malignant tumors were highly probable diagnoses . considering that adamts13 activity and observed clinical signs did not meet the diagnostic criteria for atypical hus , we judged that atypical ttp was more likely than atypical hus .
they are present at high levels in patients with anti - gbm nephritis and goodpasture 's syndrome , a condition often culminating in rapidly progressive glomerulonephritis .
the likelihood of goodpasture 's syndrome was rejected in our case because the patient did not present with any pulmonary disorders .
while admitting the possibility that high levels of anti - gbm antibodies had a complicating impact on tma or fgn , we considered it unlikely that anti - gbm nephritis was the primary cause given the renal biopsy findings . with all of the above taken into consideration , we concluded that the patient had fgn associated with tma and high levels of anti - gbm antibodies .
our literature search identified a patient with fgn who was diagnosed as having anti - gbm glomerulonephritis based on immunofluorescence microscopy findings .
this patient was found to be negative for circulating anti - gbm antibodies 1 week after the start of treatment .
this example illustrates the possibility that the anti - gbm antibody status could shift from positive to negative over time in patients with anti - gbm nephritis , suggesting that anti - gbm antibodies may have aggravated the fgn in our patient . a literature search conducted in 2011 identified 11 reported cases of tma patients positive for anti - gbm antibodies .
due to tma - induced glomerular collapse , we did not attempt to diagnose anti - gbm nephritis via immunofluoroscopy examination of immunoglobulin g or c3 depositions .
the fact that our patient was positive for anti - gbm antibodies may suggest that he had anti - gbm nephritis .
deficiency of factor h induces uncontrolled activation of the alternative pathway and c3 consumption , resulting in endothelial injury .
research suggests that factor h mutations confer a predisposition to the development of hus , and factor h deficiency has been reported in anecdotal cases of fgn and membranoproliferative glomerulonephritis type ii .
these findings suggest that decreased levels of factor h contributed to the co - occurrence of fgn and tma . however , the direct relation of fgn and tma through factor h was not accepted because the factor h was within normal levels in our case
. there may be a relationship between fgn and tma through anti - gbm antibody in our case .
reversible posterior leukoencephalopathy syndrome ( rpls ) is characterized by neurologic conditions resulting from hypertensive cerebral edema . to the best of our knowledge ,
6 cases of antineutrophil cytoplasmic autoantibody - negative anti - gbm disease with central nervous system involvement have been reported in the literature .
it is likely that rpls was caused by hypertension , which developed from the combination of anti - gbm antibody- and tma - induced vasculitis , judging from uncontrolled rises in blood pressure and the onset of numbness and seizures .
our medical opinion was supported by a retrospective analysis of 46 acute ttp patients by burrus et al . , which showed that renal function was the only variable highly associated with the occurrence of rpls on brain magnetic resonance imaging in acute ttp patients .
plasma exchange therapy can effectively improve atypical ttp through the following mechanisms : elimination of ultralarge von willebrand factor multimers ; supplementation of normal - size von willebrand factor multimers , which play an important role in blood coagulation ; removal of proinflammatory cytokines from the circulation ; elimination of adamts13 inhibitors , and supplementation with adamts13 .
a previous study reported a case of fgn in which repeat plasma exchange therapy resulted in remission of refractory nephrotic syndrome .
these previous findings suggest that plasma exchange therapy contributed to improving both tma and fgn in our case .
| we present the first report of a case of fibrillary glomerulonephritis ( fgn ) associated with thrombotic microangiopathy ( tma ) and anti - glomerular basement membrane antibody ( anti - gbm antibody ) .
a 54-year - old man was admitted to our hospital for high fever and anuria . on the first hospital day
, we initiated hemodialysis for renal dysfunction .
laboratory data revealed normocytic - normochromic anemia with schistocytes in the peripheral smear , thrombocytopenia , increased serum lactate dehydrogenase , decreased serum haptoglobin , and negative results for both direct and indirect coombs tests .
based on these results , we diagnosed tma .
assays conducted several days later indicated a disintegrin - like and metalloprotease with a thrombospondin motif 13 ( adamts13 ) activity of 31.6% , and adamts13 inhibitors were negative .
we started plasma exchange using fresh frozen plasma and steroid pulse therapy .
anti - gbm antibody was found to be positive .
renal biopsy showed fgn .
blood pressure rose on the 46th hospital day , and mild convulsions developed . based on magnetic resonance imaging of the head
, the patient was diagnosed with reversible posterior leukoencephalopathy syndrome .
hypertension persisted despite administration of multiple antihypertensive agents , and the patient experienced a sudden generalized seizure . computed tomography of the head showed multiple cerebral hemorrhages .
however , his blood pressure subsequently decreased and the platelet count increased .
tma remitted following 36 plasma exchange sessions , but renal function was not restored , and maintenance hemodialysis was continued .
the patient was discharged on the 119th day of hospitalization . in conclusion , it was shown that tma , fgn and anti - gbm antibody were closely related . |
traditionally , the presence of specific clinical signs and symptoms coupled with abnormalities in cardiacspecific measurements and estimates of endorgan dysfunction generates an integrated view of an individual patient 's risk profile .
recently , the broad acceptance of novel bloodbased biomarkers has emerged in the arena of risk stratification , even though the true incremental value of such measures in routine clinical practice has been challenged in populationbased studies .
the ability to determine functional capacity has relied on subjective assessment based on direct patient interviews during the history and physical examination and objectively by quantifying measures identified from standardized exercise testing . standardized instruments to assess functional capacity and/or health status have been developed , but their clinical adoption has been challenged by both logistical hurdles and an emphasis on hard clinical end points in the determination of treatment responses . remarkably , few studies have undertaken the prospective validation of the functional capacity measures in the prediction of future major adverse cardiac outcomes beyond allcause mortality alone or direct comparisons with clinically available biomarkers . here , we sought to determine the prognostic value of estimating functional capacity using a simple selfadministered assessment tool in a contemporary stable cardiac patient population .
the genebank study was conducted via prospective screening of patients aged 18 years who underwent elective coronary evaluation with coronary angiography or computed tomography between 2001 and 2007 .
consecutive patients were screened and considered for enrollment without intentional exclusion . at the time of enrollment , we excluded patients who had known acute coronary syndrome within 30 days of enrollment and those who were unable to comply with or unwilling to follow study protocol .
all potentially eligible patients were approached , and those who agreed to participate were provided with written informed consent .
all clinical characteristics were determined by patient selfreport and confirmed by clinical histories from electronic medical record . coronary artery disease ( cad )
was defined as any clinical history of myocardial infarction ( mi ) , percutaneous coronary intervention , or coronary artery bypass graft surgery . peripheral artery disease ( pad )
was defined as a selfreported history of noncad cardiovascular disease and/or history of or repair of aortic dissection / aneurysm .
selfreported degree of angina was quantified by canadian cardiovascular society ( ccs ) angina classification . the duke activity status index ( dasi )
questionnaire is a selfassessment tool to estimate functional capacity , which includes 12 activities representative of major aspects of physical function ( personal care , ambulation , household tasks , sexual function , and recreational activities ) .
a score is calculated based on weighted answers from 12 questions related to daily activities of living , for which each item is weighted by its known metabolic cost , and weights of positive terms are summed to form the individual patient dasi score .
the possible scores range from 0 ( all no answers ) to 58.2 ( all yes answers ) .
the dasi assessment was prospectively performed in this study as a predefined assessment of signs and symptoms related to activities of daily living at the time of enrollment and was performed by study personnel in a systematic manner whereby only definitive answers were scored . specifically , trained research personnel approached each patient shortly after informed consent to administer the dasi questionnaire .
estimated peak oxygen consumption was calculated according the original formula from hlatky and colleagues : peak vo2 ( in mlkgmin)=0.43dasi+9.6 , whereas metabolic equivalent ( met ) was calculated as : 1 met=3.5 mlkgmin vo2 .
all 8987 patients were followed prospectively over the ensuing 3 years by telephone contact , mailing , and medical record review by designated research personnel independent of study investigators .
all major adverse cardiovascular events ( mace ) , defined as death , nonfatal mi , or nonfatal stroke after enrollment , were adjudicated with source documentation .
significantly obstructive cad was defined as any clinical history of mi , percutaneous coronary intervention , coronary artery bypass graft surgery , or angiographic evidence of cad ( 50% stenosis ) in 1 major coronary artery .
subsequent mi was adjudicated from source documentation with supporting evidence including elevated cardiac enzymes , significant qwave definitive electrocardiographic evidence of new infarction , the presence of myocardial wall akinesis or scar on imaging , or treatment with thrombolytic agents or direct percutaneous intervention .
stroke was defined as documented loss of neurologic function caused by an ischemic event with residual symptoms continuing 24 hours after onset ( not to include transient ischemic attacks , microvascular infarcts , or amarosis fugax ) .
blood samples were obtained either via venipuncture ( in the case of coronary computed tomography patients ) or during diagnostic coronary angiographic procedure right after arterial sheath access but before any heparin administration .
the biomarker subset included 4805 patients , for whom stored samples were randomly selected to undergo biomarker testing in a central core laboratory .
specifically , highsensitivity creactive protein ( hscrp ) , btype natriuretic peptide ( bnp ) , creatinine , fasting lipid profile , apolipoprotein a1 ( apoa1 ) , and apolipoprotein b ( apob ) were all measured in the architect ci8200 platform ( abbott laboratories ) .
myeloperoxidase ( mpo ) was measured by using the cardiompo assay kit ( cleveland heart lab ) .
total leukocyte count was measured by using the advia 120 hematology system ( siemens medical systems ) .
we used the student 's t test or wilcoxon rank sum test for continuous variables and test for categorical variables for betweengroup comparisons .
cox proportional hazards regression was used for timetoevent analysis to determine hazard ratio ( hr ) and 95% confidence intervals ( cis ) for mace in comparing the highest with the lowest dasi quartiles for each cohort of interest .
adjustments were made for individual traditional cardiac risk factors ( including age , sex , lowdensity and highdensity lipoprotein cholesterol , systolic blood pressure , former or current cigarette smoking , and diabetes mellitus ) to predict incident 3year mace risk . the survival curve was derived using kaplan meier survival analysis .
a cubic spline term of dasi was fitted in the cox model to assess how the spectrum of dasi scores related to the hr of mace .
a nomogram for mace based on survival models was constructed ( details of the nomogram construction methods are listed in data s1 as previously published ) to visually display the predicted probability of a mace event from a multivariate cox model .
net reclassification analysis was performed to quantify improvement in models with and without dasi scores . in cutoff values for net reclassification index estimation , we used a ratio of 6:3:1 for low , medium and highrisk categories .
comparison between dasi score and adult treatment panel iii score , which estimates the 10year risk of developing cad outcomes ( mi or coronary death ) , was performed by resampling ( 250 bootstrap samples from the whole cohort [ n=8987 ] ) .
all data analyses , including receiver operator characteristic analyses and area under the curve ( auc ) determinations , were separately recalculated at each resampling , and the aucs calculated from the bootstrap samples were compared .
the genebank study was conducted via prospective screening of patients aged 18 years who underwent elective coronary evaluation with coronary angiography or computed tomography between 2001 and 2007 .
consecutive patients were screened and considered for enrollment without intentional exclusion . at the time of enrollment , we excluded patients who had known acute coronary syndrome within 30 days of enrollment and those who were unable to comply with or unwilling to follow study protocol .
all potentially eligible patients were approached , and those who agreed to participate were provided with written informed consent .
all clinical characteristics were determined by patient selfreport and confirmed by clinical histories from electronic medical record . coronary artery disease ( cad )
was defined as any clinical history of myocardial infarction ( mi ) , percutaneous coronary intervention , or coronary artery bypass graft surgery . peripheral artery disease ( pad )
was defined as a selfreported history of noncad cardiovascular disease and/or history of or repair of aortic dissection / aneurysm .
selfreported degree of angina was quantified by canadian cardiovascular society ( ccs ) angina classification .
the duke activity status index ( dasi ) questionnaire is a selfassessment tool to estimate functional capacity , which includes 12 activities representative of major aspects of physical function ( personal care , ambulation , household tasks , sexual function , and recreational activities ) .
a score is calculated based on weighted answers from 12 questions related to daily activities of living , for which each item is weighted by its known metabolic cost , and weights of positive terms are summed to form the individual patient dasi score .
the possible scores range from 0 ( all no answers ) to 58.2 ( all yes answers ) .
the dasi assessment was prospectively performed in this study as a predefined assessment of signs and symptoms related to activities of daily living at the time of enrollment and was performed by study personnel in a systematic manner whereby only definitive answers were scored . specifically , trained research personnel approached each patient shortly after informed consent to administer the dasi questionnaire . estimated peak oxygen consumption was calculated according the original formula from hlatky and colleagues : peak vo2 ( in mlkgmin)=0.43dasi+9.6 , whereas metabolic equivalent ( met ) was calculated as : 1 met=3.5 mlkgmin vo2 .
all 8987 patients were followed prospectively over the ensuing 3 years by telephone contact , mailing , and medical record review by designated research personnel independent of study investigators .
all major adverse cardiovascular events ( mace ) , defined as death , nonfatal mi , or nonfatal stroke after enrollment , were adjudicated with source documentation . significantly obstructive cad was defined as any clinical history of mi , percutaneous coronary intervention , coronary artery bypass graft surgery , or angiographic evidence of cad ( 50% stenosis ) in 1 major coronary artery .
subsequent mi was adjudicated from source documentation with supporting evidence including elevated cardiac enzymes , significant qwave definitive electrocardiographic evidence of new infarction , the presence of myocardial wall akinesis or scar on imaging , or treatment with thrombolytic agents or direct percutaneous intervention .
stroke was defined as documented loss of neurologic function caused by an ischemic event with residual symptoms continuing 24 hours after onset ( not to include transient ischemic attacks , microvascular infarcts , or amarosis fugax ) .
blood samples were obtained either via venipuncture ( in the case of coronary computed tomography patients ) or during diagnostic coronary angiographic procedure right after arterial sheath access but before any heparin administration .
the biomarker subset included 4805 patients , for whom stored samples were randomly selected to undergo biomarker testing in a central core laboratory .
specifically , highsensitivity creactive protein ( hscrp ) , btype natriuretic peptide ( bnp ) , creatinine , fasting lipid profile , apolipoprotein a1 ( apoa1 ) , and apolipoprotein b ( apob ) were all measured in the architect ci8200 platform ( abbott laboratories ) .
myeloperoxidase ( mpo ) was measured by using the cardiompo assay kit ( cleveland heart lab ) .
total leukocyte count was measured by using the advia 120 hematology system ( siemens medical systems ) .
we used the student 's t test or wilcoxon rank sum test for continuous variables and test for categorical variables for betweengroup comparisons .
cox proportional hazards regression was used for timetoevent analysis to determine hazard ratio ( hr ) and 95% confidence intervals ( cis ) for mace in comparing the highest with the lowest dasi quartiles for each cohort of interest .
adjustments were made for individual traditional cardiac risk factors ( including age , sex , lowdensity and highdensity lipoprotein cholesterol , systolic blood pressure , former or current cigarette smoking , and diabetes mellitus ) to predict incident 3year mace risk .
a cubic spline term of dasi was fitted in the cox model to assess how the spectrum of dasi scores related to the hr of mace . a nomogram for mace based on survival models was constructed ( details of the nomogram construction methods are listed in data s1 as previously published ) to visually display the predicted probability of a mace event from a multivariate cox model .
net reclassification analysis was performed to quantify improvement in models with and without dasi scores . in cutoff values for net reclassification index estimation , we used a ratio of 6:3:1 for low , medium and highrisk categories .
comparison between dasi score and adult treatment panel iii score , which estimates the 10year risk of developing cad outcomes ( mi or coronary death ) , was performed by resampling ( 250 bootstrap samples from the whole cohort [ n=8987 ] ) .
all data analyses , including receiver operator characteristic analyses and area under the curve ( auc ) determinations , were separately recalculated at each resampling , and the aucs calculated from the bootstrap samples were compared .
table 1 shows the baseline characteristics of the study population , which is representative of a contemporary patient population undergoing elective diagnostic coronary angiography ( see data s2 ) . the reasons for angiography included history of positive or indeterminate stress test ( 45% ) , evaluation for possible ischemic causes of symptoms ( 63% ) , preoperative evaluation ( 15% ) , and history of cardiomyopathy ( 4% ) .
the median dasi score was 38.2 ( iqr 24.2 to 50.7 ) , which corresponded to an estimated peak oxygen consumption of 26 ml / kg per minute ( iqr 20 to 31 ml / kg per minute ) .
as expected , patients with a lower dasi score were more likely to be older and female and to have an underlying history of hf , previous mi , or pad ( table 1 ) .
we observed the association between lower dasi score with higher likelihood of underlying cad ( quartile 4 versus quartile 1 of dasi score , unadjusted odds ratio [ or ] 2.99 , 95% ci 2.59 to 3.46 , p<0.01 ) or pad ( unadjusted or 5.65 , 95% ci 4.74 to 6.73 , p<0.01 ) .
after adjustment for traditional risk factors , the association remained statistically significant ( cad : adjusted or 2.65 , .95% ci 2.22 to 3.15 , p<0.01 ; pad : adjusted or 4.61 , 95% ci 3.73 to 5.71 , p<0.01 ) .
baseline characteristics for whole cohort and across quartiles of duke activity status index ( dasi ) score cad indicates coronary artery disease ; mi , myocardial infarction ; hf , heart failure ; pad , peripheral artery disease ; lvef , left ventricular ejection fraction ; bmi , body mass index ; ldl , lowdensity lipoprotein ; hdl , highdensity lipoprotein ; hscrp , highsensitivity creactive protein ; egfr , estimated glomerular filtration rate ; mpo , myeloperoxidase ; bnp , btype natriuretic peptide ; wbc , total leukocyte count ; apoa1 , apolipoprotein a1 ; apob , apolipoprotein b ; ace , angiotensinconverting enzyme ; arb , angiotensin ii receptor blocker .
the relationship between dasi score and incident cardiovascular risk is illustrated in figure 1 , in which the kaplan
meier analysis revealed that lower dasi score ( across quartiles of dasi score as well as estimated mets ) is associated with a greater risk of future development of mace . over a 3year prospective followup period
, there were a total of 829 deaths , 404 mis , and 178 strokes in our study cohort .
we observed that a lower dasi score was associated with a higher risk of future death , mi , or stroke ( quartile 4 versus quartile 1 of dasi score , unadjusted hr 4.76 , 95% ci 4.03 to 5.61 , p<0.01 ) .
the prognostic value of dasi score was preserved when adjusted for traditional risk factors ( adjusted hr 3.77 , 95% ci 3.15 to 4.51 , p<0.01 ) or even plus history of heart failure and history of pad ( adjusted hr 2.89 , 95% ci 2.39 to 3.50 , p<0.01 ) and in cohorts with and without significantly obstructive cad ( table 2 ) .
as illustrated by the cubic spline curve , the risk for future mace appeared to be steep and linear , with hr significantly above unity , particularly for dasi scores < 38 ( or the median , figure 2 ) .
lower dasi scores also predicted higher future risk of mace at 3 years regardless of age , sex , body mass index , diabetes mellitus , hypertension , lipid status , hscrp status , or prior mi ( all p<0.01 , figure 3 ) .
the prognostic value of dasi score was also consistent regardless of the clinically assessed ccs angina class ( see data s3 ) .
use of dasi score over traditional risk factors was also shown to reclassify patients ( net reclassification index 15% , p<0.001 ; integrated discrimination improvement 13% , p<0.001 ; cstatistics 66.5% versus 71.5% , p<0.001 ) .
direct headtohead comparison also demonstrated that dasi score provided more robust prediction of future development of mace over a 3year period than did the adult treatment panel iii score ( auc : 0.67 [ 95% ci 0.66 to 0.69 ] versus 0.59 [ 0.57 to 0.60 ] , p<0.001 ) in our study population . combining traditional risk factors and comorbidities with dasi score , a nomogram is constructed with a point scoring system indicative of prospective risk for future mace at 3 years ( figure 4 ) .
kaplan meier analysis of duke activity status index ( dasi ) scores in predicting future risk of major adverse cardiac events in stable cardiac patients undergoing elective coronary angiography according to dasi quartiles ( a ) and estimated metabolic equivalents ( mets , b ) .
ranges of dasi and mets are the same as for groups in tables 2 and 3 .
unadjusted and adjusted hazard ratio ( hr ) for major adverse cardiac events at 3 years stratified according to quartiles of dasi score model 1 : adjusted for traditional risk factors including age , sex , systolic blood pressure , lowdensity lipoprotein cholesterol , highdensity lipoprotein cholesterol , smoking , and diabetes mellitus .
model 2 : adjusted for model 1 plus history of heart failure and peripheral artery disease .
cad indicates coronary artery disease ; dasi , duke activity status index . unadjusted and adjusted hazard ratio ( hr ) for major adverse cardiac events at 3 years stratified according to previously reported metabolic equivalents ( mets ) categories model 1 : adjusted for traditional risk factors including age , sex , systolic blood pressure , lowdensity lipoprotein cholesterol , highdensity lipoprotein cholesterol , and smoking , diabetes mellitus .
model 2 : adjusted for model 1 plus history of heart failure and peripheral artery disease .
cubic spline curve for hazard ratios for major adverse clinical events ( mace ) at 3 years with duke activity status index ( dasi ) scores .
subgroup analysis of duke activity status index ( dasi ) scores and future risk of major adverse cardiac events ( mace ) according to standard cardiac biomarkers
. hazard ratio ( xaxis ) of 3year mace similar to that presented in table 2 ( quartile 4 vs quartile 1 of dasi score ) but across subgroups of different biomarker cutoffs .
apoa1 indicates apolipoprotein a1 ; apob , apolipoprotein b ; bnp , btype natriuretic peptide ; egfr , estimated glomerular filtration rate ; hdl , highdensity lipoprotein ; hscrp , highsensitivity creactive protein ; ldl , lowdensity lipoprotein ; mpo , myeloperoxidase ; wbc , white blood cell count .
nomogram for estimating risk of major adverse cardiac events ( mace ) incorporating duke activity status index ( dasi ) score with traditional risk factors
hdl indicates highdensity lipoprotein ; ldl , lowdensity lipoprotein ; sbp , systolic blood pressure . in the subset of patients with biomarkers measured ( n=4805 ) , there were no significant differences in baseline characteristics compared with those without biomarker assessment .
lower dasi scores remained prognostically significant in both high and low levels of fasting lipid profiles , hscrp , and bnp , as well as total leukocyte count and mpo .
in fact , the predictive value of dasi score in predicting future mace ( auc : 0.67 [ 95% ci 0.66 to 0.69 ] ) appears to be comparable with commonly used prognostic cardiac biomarkers such as bnp ( auc : 0.68 [ 95% ci 0.66 to 0.70 ] or hscrp ( auc : 0.61 [ 95% ci 0.59 to 0.64 ] ) . when hscrp , bnp , and estimated glomerular filtration rate were all added to the multivariate model that included traditional risk factors , lower dasi score ( quartile 4 versus quartile 1 ) still demonstrated a significant 2fold increased risk in future mace at 3 years ( adjusted hr 2.00 , 95% ci 1.55 to 2.58 , p<0.01 ) .
the key finding of this study is the strong prognostic value of functional capacity estimated by using a simple 12question evaluation tool in a large , contemporary cohort of stable cardiac patients undergoing coronary angiography .
we observed that the functional capacity estimated with the dasi score is incremental and superior to that of traditional cardiovascular risk factors , with or without underlying heart failure .
we further established the association between functional capacity estimated by dasi score and markers of inflammation and oxidative stress in a subset of patients . taken together , our findings underscore the relative importance of objectively determining the functional capacity for purposes of risk stratification in cardiac patients . although the dasi questionnaire was developed over 2 decades ago , the primary focus in using this selfassessment tool thus far has been to estimate functional capacity in the setting of preoperative evaluation or prediction of underlying ischemia .
while the original objective of dasi was to estimate functional capacity , the responses can also be used to assess physical limitations relevant to an individual 's quality of life and to potentially uncover significant problems related to patients who experienced cad , pad , or heart failure .
the importance of prognostication of dasi in our study , particularly with its strong incremental value to traditional risk factors and cardiac biomarkers , underscores the need to systematically assess how dasi score can be of clinical utility .
indeed , the findings of our study may not be limited to dasi or even to cad patients , in that other instruments that assess daily physical activity such as the rand physical limitation scale or the kansas city cardiomyopathy questionnaire for heart failure have also demonstrated their incremental prognostic utilities and responses to therapeutic interventions .
these latter instruments aim to assess health status and have not been evaluated as to whether they correlate directly with functional capacity .
interestingly , the design of dasi queries an individual 's selfperception of his or her functional capacity ( ie , what an individual is able to do ) rather than a direct recall of prior physical activities and/or limitations .
therefore , the dasi questionnaire may potentially overestimate the actual functional capacity as illustrated in the validation studies , yet this further underscores an even greater importance for a standardized instrument such as the dasi questionnaire to estimate functional capacity when the ability to reclassify risk may be up to 15% as shown in our study . while the prognostic value for functional capacity as determined by standardized exercise testing has been well established
, the requirements of testing skills , equipment , and experience often hinder broad adoption . at the other end of the spectrum ,
simple grading systems such as new york heart association classification and ccs angina classification have aided physicians in communicating the degrees of symptomatic severity , yet they are often subjective and imprecise . hence , the ability for a series of questions that can be collected at the bedside ( or adapted to remote monitoring devices or portals ) to risk stratify patients for future mace above and beyond established prognostic cardiac biomarkers speaks to the enduring importance of bedside evaluation of patientcentered reporting of functional limitations in the era of biomarker testing .
our findings also highlight the limitations of any clinical or biochemical measure to adequately quantify functional limitations that may be more meaningful to patients . based on our findings ,
the quest is to examine what treatment strategies ( eg , the ability to detect impaired functional capacity as a target of physical training and cardiac rehabilitation ) can be effectively incorporated in those patients with low dasi scores to improve their perceived or objective functional capacity and to determine whether such interventions can modify the natural history of the disease .
few studies have directly examined the potential use of dasi to predict longterm adverse cardiovascular risk in a broad patient population with stable cardiac disease . indeed , the women 's ischemia syndrome evaluation ( wise ) study was one of the first studies to identify that functional impairment estimated by the dasi correlates with indeterminate exercise test results and is associated with an adverse prognosis among women with suspected myocardial ischemia .
surgical studies also illustrated the finding that poor functional capacity estimated by dasi following cardiac surgery identifies patients who are at risk for reduced longterm survival .
of note , the relationship between dasi score and risk was linear and extended to the higher functional capacity end ( higher score ) .
thus , those who achieved a maximum baseline dasi demonstrated better riskadjusted longterm survival ( hr 0.64 ; 95% ci 0.50 to 0.83 ; p=0.0005 ) .
such a finding is consistent with improved overall survival conferred by improved functional capacity achieved during cardiac rehabilitation in the post cardiothoracic surgical setting . in the setting of heart failure or chronic kidney disease , the ability to assess functional status and prognosis has been demonstrated .
recent data from a large heart failure cohort further implied that those with changes in dasi scores over 1 year demonstrated stronger association with longterm outcomes than objective assessment ( 6minute walk distance ) .
these findings are supportive of the ability of improving dasi score as a potential therapeutic target . future investigations into potential benefits of using dasi scores ( especially those with dasi < 38 ) to triage cardiac evaluation and
guide therapeutic interventions , such as more indepth evaluation of disability , more aggressive secondary prevention strategies , more intensive cardiac rehabilitation , or closer outpatient followup , are therefore warranted for this higherrisk population . despite one of the largest studies conducted to date on the prognostic value of dasi and functional capacity in the cardiovascular population , our study has several limitations .
the large sample size is reassuring regarding how the results can be generalized , although it may not be completely generalizable beyond the setting of coronary evaluation due to referral and ascertainment bias at the point of recruitment and study ( ie , cardiac catheterization laboratory ) .
first , while dasi has been validated in the past as a measure of metabolic capacity and function , there is no objective validation of functional capacity measurement in our large cohort besides angina class , and the ability to respond to the questions is required ( thus excluding those that were unable to complete the questionnaires due to their underlying diseases or functional or communication limitations ) .
we did not systematically collect baseline or interim information regarding history of atrial fibrillation and stroke in those enrolled .
second , we only have biochemical analyses on roughly half of the study cohort , although there is still adequate power to determine the incremental prognostic value of the dasi score .
third , although we have provided subgroup assessment in the non heart failure and nonpad cohorts ( potential confounding effect due to underlying cardiac and vascular insufficiency ) , we can not exclude other reasons that affect an individual 's functional limitations .
nevertheless , this also speaks to the power of this simple questionnaire that can depict risks that are otherwise unaccounted for , such as frailty , deconditioning , or psychosocial issues .
last , the fact that there is only a single timepoint assessment may require further studies to determine if improvement in dasi score can be associated with improvement in short and longterm prognosis as implied in the postsurgical or heart failure literature or with treatment strategies such as cardiac rehabilitation .
we would also like to emphasize that the nomogram is largely derived to highlight the relative clinical contributions of dasi to the overall prognostic value when other factors of functional capacity are under consideration .
all patients were used in deriving the score , and further validation of this score is warranted in an independent study population .
the dasi , a selfassessment tool of functional capacity , provides strong independent and incremental prognostic value for longterm adverse clinical events that is comparable to cardiac biomarkers in stable cardiac patients undergoing coronary evaluation .
| backgroundfew studies have investigated functional capacity selfassessment tools in either prediction of future major adverse cardiac outcomes beyond allcause mortality or direct comparisons with clinically available biomarkers.methods and resultswe estimated functional capacity using the duke activity status index ( dasi ) questionnaire in 8987 sequential stable patients without acute coronary syndrome who were undergoing elective diagnostic coronary angiography with 3year followup of major adverse cardiac events ( death , nonfatal myocardial infarction , or stroke ) . a low dasi score provided independent prediction of a 4.8fold increase in future risk of incident major adverse cardiac events at 3 years ( quartiles 1 versus 4 hazard ratio [ 95% ci ] 4.76 [ 4.03 to 5.61 ] , p<0.001 ) , and a 3.8fold increased risk after adjusting for traditional risk factors ( 3.77 [ 3.15 to 4.51 ] , p<0.001 ) .
the prognostic value of the dasi score was evident in both primary and secondary prevention cohorts , with and without heart failure , as well as high and low creactive protein and btype natriuretic peptide levels .
the dasi score reclassified 15% of patients ( p<0.001 ) beyond traditional risk factors in predicting future mace.conclusiona simple selfassessment tool of functional capacity in stable patients undergoing elective diagnostic cardiac evaluation provides independent and incremental prognostic value for prediction of both significant coronary angiographic disease and longterm adverse clinical events . |
emergency peripartum hysterectomy ( eph ) is a major surgical venture invariably performed in the setting of life threatening hemorrhage during or immediately after abdominal and vaginal deliveries [ 15 ] . despite advances in medical and surgical fields
, post partum hemorrhage continues to be the leading cause of maternal morbidity and mortality .
eph is the most dramatic operation in modern obstetrics and is generally performed when all conservative measures have failed to achieve haemostasis in the setting of life threatening hemorrhage . the unplanned nature of the surgery and the need for performing it expeditiously , compound matters .
moreover the acute loss of blood renders the patient in a less than ideal condition to undergo emergency surgical intervention .
the predominant indications for eph are placenta previa / accreta and uterine atony and eph in some of them is unavoidable . however recognizing and assessing patients at risk and
appropriate and timely intervention would go a long way in ensuring a better outcome in this otherwise difficult situation .
hysterectomy following cesarean section ( cs ) was first described by porro , and was used to prevent maternal mortality due to post partum hemorrhage .
the reported incidence of eph varies from 0.24 to 8.9 per 1000 deliveries [ 16 ] , ranging from 0.33(netherland ) , 0.2 ( norway ) , 0.3 ( ireland ) , 0.5 ( israel ) , 0.63 ( saudi arabia ) and 1.2 to 2.7 per 1000 deliveries in usa [ 25810 ] . a difference in the incidence of eph is noted following vaginal delivery and cesarean section .
while the incidence of eph after vaginal delivery varies from 0.1 to 0.3/1000 deliveries and is rather constant between european and us studies , the incidence of eph following cs varies widely between 0.17 and 8.7/1000 deliveries .
this is attributed to the proportion of women with previous cs with the concomitant risk of placenta previa andaccreta [ 16 ] .
the risk factors for post partum hemorrhage include coagulopathies , uterine atony , retained products of conception , precipitate or prolonged labor , fetal macrosomia or multiparity , maternal obesity and previous primary post partum hemorrhage [ 16814 ] .
according to one report the indication for eph which was uterine atony in 43.45% and placenta previa or accreta in 33.9% cases in 1984 changed 9 years later to placenta accreta ( 45% ) and uterine atony in 20% of the cases .
similar findings are reported by others with abnormal placentation as the predominant indication , the incidence ranging from ( 45 to 73.3% ) ; and uterine atony in ( 26.6% to 35.6% ) [ 18815 ] . in recent years
, abnormal placentation has become a more common indication due to the greater number of pregnant women with previous cesarean section deliveries [ 16 ] .
the incidence of previous cesarean section ranges between 59.8% in patients with adherent placenta and 75% in patients with placenta previa . in view of several such reports ,
association between abnormal placentation and cesarean delivery has been suggested and the high incidence of eph is directly related to the increasing number of cesarean sections [ 1616 ] .
this was further substantiated by another report where the incidence of placenta previa which was 1.9/1000 after one previous cs , increased by 47 fold to 91/1000 in patients with four previous cs .
patients with placenta previa and scarred uterus had 16% risk of undergoing eph compared to 3.6% in patients with unscarred uterus .
advancing age and parity are also reported to be important risk factors in developing placenta previa and accrete .
the incidence of eph was higher in patients with placenta previa and accreta than in patients with placenta previa alone .
the combination of factors including high parity , number of previous cesarean sections , abortion , previous curettage , strongly increased the likelihood of placenta previa and increased risk of abnormal adherent placenta .
therefore , it appears prudent for the obstetrician to prepare for the possibility of eph for massive hemorrhage in patients undergoing cesarean section with these risk factors . of concern
however , is the limited experience of performing emergency hysterectomy among the younger obstetricians as according to one report from netherlands , the average chance of performing one eph is once in 11 years .
the decreasing rate of abdominal hysterectomy for gynecological conditions in recent years does not help matters with regard to gaining this valuable experience .
this implies that more effort should be undertaken to recognize the potential risk of patients requiring eph and the need for involvement of an experienced obstetrician in the management at an early stage .
eph being performed by an experienced surgeon is reported to significantly reduce the operating time , number of units of blood transfusion and hospital stay .
the predisposing risk factors can be determined to a certain extent by performing antenatal ultrasound with color doppler and magnetic resonance imaging ( mri ) [ 1820 ] .
however , the limiting factor is the high cost of mri and extensive experience needed .
the measures include uterotonic drugs , uterine or hypogastric artery embolisation , hemostatic sutures , uterine or internal iliac artery ligation .
conservative management is of particular importance in patients who are young , have low parity and who are haemodynamically stable .
however while there are reports of 96% success rate following uterine artery ligation there are others who have achieved success in only 39.4% of these cases .
the choice between conservative management and eph should be individualized . in situations where conservative treatment is likely to fail or has failed , there should be no further delay in performing eph as delay leads to increase in blood loss , transfusion requirement , operative time , dic , and increased possibility of admission to icu [ 16 ] .
uterine atony is an indication for eph in 20.6% to 43% of the cases [ 16817 ] .
while this was traditionally the leading cause for eph the incidence has reduced due to the use of newly developed pharmacologic treatment strategies including prostaglandins .
multiparity and oxytocin use for uterine stimulation were found to be the risk factors for uterine atony requiring eph [ 16 ] .
combs et al in their large case control study of patients with post partum hemorrhage reported that pre - eclampsia , nulliparity , twins , induction , prolonged labor and augmentation were all identified as independent risk factors for uterine atony .
patients with uterine rupture as an indication for eph ranged from 11.4% to 45.5% . the risk factor for
this would be multiple previous cesarean sections with a scarred uterus ( figure 1 ) .
total hysterectomy is the recommended surgical method of eph due to the potential risk of malignancy developing in the cervical stump and the need for regular cytology and other associated problems such as bleeding or discharge associated with the residual cervical stump .
currently the proportion of subtotal hysterectomy performed for eph ranges from 53% to 80% .
the proponents of subtotal hysterectomy report a lesser blood loss , a reduced need for blood transfusion , reduced operating time and reduced intra and postoperative complications .
total hysterectomy should however be considered when active bleeding occurs from lower uterine segment as the cervical branch of uterine artery may remain intact .
the final decision to perform subtotal or total hysterectomy would be influenced by patient 's condition .
hence , while total abdominal hysterectomy is a more convenient procedure , subtotal eph may be a better choice in certain conditions where surgery needs to be completed in a shorter time .
uterine atony is an indication for eph in 20.6% to 43% of the cases [ 16817 ] .
while this was traditionally the leading cause for eph the incidence has reduced due to the use of newly developed pharmacologic treatment strategies including prostaglandins .
multiparity and oxytocin use for uterine stimulation were found to be the risk factors for uterine atony requiring eph [ 16 ] .
combs et al in their large case control study of patients with post partum hemorrhage reported that pre - eclampsia , nulliparity , twins , induction , prolonged labor and augmentation were all identified as independent risk factors for uterine atony .
the risk factor for this would be multiple previous cesarean sections with a scarred uterus ( figure 1 ) . uterine rupture with life threatening hemorrhage managed by peripartum hysterectomy .
total hysterectomy is the recommended surgical method of eph due to the potential risk of malignancy developing in the cervical stump and the need for regular cytology and other associated problems such as bleeding or discharge associated with the residual cervical stump . currently the proportion of subtotal hysterectomy performed for eph ranges from 53% to 80% .
the proponents of subtotal hysterectomy report a lesser blood loss , a reduced need for blood transfusion , reduced operating time and reduced intra and postoperative complications .
total hysterectomy should however be considered when active bleeding occurs from lower uterine segment as the cervical branch of uterine artery may remain intact .
the final decision to perform subtotal or total hysterectomy would be influenced by patient 's condition .
hence , while total abdominal hysterectomy is a more convenient procedure , subtotal eph may be a better choice in certain conditions where surgery needs to be completed in a shorter time .
uterine atony is an indication for eph in 20.6% to 43% of the cases [ 16817 ] .
while this was traditionally the leading cause for eph the incidence has reduced due to the use of newly developed pharmacologic treatment strategies including prostaglandins .
multiparity and oxytocin use for uterine stimulation were found to be the risk factors for uterine atony requiring eph [ 16 ] .
combs et al in their large case control study of patients with post partum hemorrhage reported that pre - eclampsia , nulliparity , twins , induction , prolonged labor and augmentation were all identified as independent risk factors for uterine atony .
the risk factor for this would be multiple previous cesarean sections with a scarred uterus ( figure 1 ) .
total hysterectomy is the recommended surgical method of eph due to the potential risk of malignancy developing in the cervical stump and the need for regular cytology and other associated problems such as bleeding or discharge associated with the residual cervical stump . currently the proportion of subtotal hysterectomy performed for eph ranges from 53% to 80% .
the proponents of subtotal hysterectomy report a lesser blood loss , a reduced need for blood transfusion , reduced operating time and reduced intra and postoperative complications .
total hysterectomy should however be considered when active bleeding occurs from lower uterine segment as the cervical branch of uterine artery may remain intact .
the final decision to perform subtotal or total hysterectomy would be influenced by patient 's condition .
hence , while total abdominal hysterectomy is a more convenient procedure , subtotal eph may be a better choice in certain conditions where surgery needs to be completed in a shorter time .
the complications included blood transfusion ( 88% ) , febrile episodes ( 26.5% ) , perinatal death ( 22.8% ) , bladder injuries ( 8.8% ) , wound infection , dic , ileus , vaginal cuff bleeding and adnexectomy .
the maternal mortality ranged from 0 to 12.5% with a mean of 4.8% [ 168192124 ] .
although no risk assessment system can predict all instances where cesarean delivery will be needed , a significant percentage of the patients who are at high risk for severe hemorrhage and the subsequent need of emergency hysterectomy can be identified before surgery .
the presence of preoperative risk factors should facilitate consultation , referral or transfer of patients before surgery to a tertiary care facility . due to the complexity of the surgery and decision making ,
proper surgical measures such as hemostatic sutures or uterine or hypogastric artery ligation or embolization are options in attempting uterine conservation particularly in patients who are young and in whom future fertility is important and who are relatively haemodynamically stable .
when conservative treatment is not feasible or has failed , prompt eph is performed failing which the delay would contribute to the maternal morbidity and in unfortunate cases mortality . | background : peripartum hysterectomy is a major operation and is invariably performed in the presence of life threatening hemorrhage during or immediately after abdominal or vaginal deliveries.material and methods : a medline search was conducted to review the recent relevant articles in english literature on emergency peripartum hysterectomy .
the incidence , indications , risk factors and outcome of emergency peripartum hysterectomy were reviewed.results:the incidence of emergency peripartum hysterectomy ranged from 0.24 to 8.7 per 1000 deliveries .
emergency peripartum hysterectomy was found to be more common following cesarean section than vaginal deliveries .
the predominant indication for emergency peripartum hysterectomy was abnormal placentation ( placenta previa / accreta ) which was noted in 45 to 73.3% , uterine atony in 20.6 to 43% and uterine rupture in 11.4 to 45.5 % .
the risk factors included previous cesarean section , scarred uterus , multiparity , older age group .
the maternal morbidity ranged from 26.5 to 31.5% and the mortality from 0 to 12.5% with a mean of 4.8% .
the decision of performing total or subtotal hysterectomy was influenced by the patient 's condition.conclusion:emergency peripartum hysterectomy is a most demanding obstetric surgery performed in very trying circumstances of life threatening hemorrhage .
the indication for emergency peripartum hysterectomy in recent years has changed from traditional uterine atony to abnormal placentation .
antenatal anticipation of the risk factors , involvement of an experienced obstetrician at an early stage of management and a prompt hysterectomy after adequate resuscitation would go a long way in reducing morbidity and mortality . |
after obtaining institutional ethical committee approval and written informed consent , 50 asa physical status i - iii patiets , aged from 30 to 75 years , undergoing elective major abdominal surgery were included in this prospective , randomized , double blind study .
patients with cardiac desease , uncontrolled hypertension , hypovolemia , cronically hepatic or renal disease , significant electrolyte disorder , restrictive or obstructive respiratory disease , acute intermittent porphyria , neurological disorder , bleeding or coagulation test abnormalities , diabetes , allergy to local anesthetics or opioids and psychological disorders were excluded .
the anesthetists who performed the epidural catheterisation and collected the datas were blinded to the solutions .
i ( n=25 ) , 250 mg bupivacaine 0.5% and 400 g fentanyl in 150 cc saline 0.9% and in group ii ( n=25 ) , 250 mg levobupivacaine 0.5% and 400 g fentanyl in 150 cc saline 0.9% was infused via epidural pca .
the concentration of the solution was 0.125% for bupivacaine and levobupivacaine , 3 /ml for fentanyl as used in previous studies.1415 the device was consisted of a syringe pump and activated by a hand switch that delivered the demand dose of solution epidurally .
the instructions about pca ( grasebay 3300 ) , holter mashines ( dms300 - 12l ) and vas scores were given during the preoperative patient visits .
the degree of motor blockade was assessed using the modified bromage scale : 0 = free movement of legs and feet ; 1 = inability to raise extended leg but able to move knees and feet 2 = unable to flex knees but with free movement of feet 3 = unable to move feet or knees.16 maximum cephalad sensory blockade to pinprick , cold and touch was measured .
would mean no pain and 10 would mean worst possible pain. vas values were recorded at 0 , 1 , 3 , 5 , 7 , 11 , 15 , 19 and 23 hour in the postoperative period .
0.25% bupivacaine in 10 ml for group i and 0.25% levobupivacaine for group ii was injected via epidural catheter following the record of vas value immediatly after the surgery in the recovery room .
infusion rate was set as 4 ml / h and bolus dose was set as 5 ml with lockout 20 minutes .
when the vas value of the patient was higher than 3 , additional 5 ml bolus dose was given and recorded .
the datas were compared as excessive analgesic requirement . before epidural catheterisation , a peripheral 18 g
mean arterial blood pressure was measured at 5 minutes interval before and during the procedure .
18 g tuohy needle was introduced in the midline at the lumbar 3 - 4 or 4 - 5 interspace with the patients undergoing low anterior resection for rectum cancer and at the thoracal 7 - 8 or 6 - 7 interspace with the patients undergoing total gastrectomy for stomach cancer .
20 g epidural catheter was settled via tuohy cannula and 60 mg lidocaine test dose was performed .
then the patients were premedicated by 0.03 mg / kg intravenous ( iv ) midazolam .
after completion of epidural block , anesthesia was induced with iv penthotal 5 mg / kg and iv fentanyl 3 g / kg .
patients lungs were mechanically ventilated with 65% nitrous oxide and 35% oxygen with a fresh gas flow of 5 lt / min . a heart rate under 45 beat / min was accepted as bradycardia and treated with atropine 0.5 mg iv .
mean arterial pressure under 50 mmhg was considered as hypotension and treated by 500 ml iv colloid infusion .
if colloid did not rise the blood pressure , 10 mg iv ephedrine was administered .
the patients were again moniterized after the surgery in recovery room and the values of arterial systolic and diastolic pressure , heart rate , vas , oxygen saturation and respiratory rates were recorded .
the sedation scores were determined by ramsay sedation scale :
agitated or restless patientco - operative , oriented and tranquil patientresponds to commands onlybrisk response to a light glabellar tap or auditory stimulusdoes not respond to mild prodding or shakingexhibits no response
agitated or restless patient co - operative , oriented and tranquil patient responds to commands only brisk response to a light glabellar tap or auditory stimulus does not respond to mild prodding or shaking the patients who were awake and hemodynamically stable were transported to intensive care unit ( icu ) .
nausea and vomiting were treated by metoclopramid 10 mg iv and pruritus by difenhydramine 10 mg iv and were repeated as necessary .
statistical package for social sciences ( spss ) for windows programe ( ver.10 ) was used for statistical analyses .
for categorical variables , proportions of the variances in two groups were compared by the chi - square test .
group size was selected using proportions sample size estimates ( = 0.05 , = 0.09 ) .
statistical package for social sciences ( spss ) for windows programe ( ver.10 ) was used for statistical analyses .
for categorical variables , proportions of the variances in two groups were compared by the chi - square test .
group size was selected using proportions sample size estimates ( = 0.05 , = 0.09 ) .
statistical package for social sciences ( spss ) for windows programe ( ver.10 ) was used for statistical analyses .
for categorical variables , proportions of the variances in two groups were compared by the chi - square test .
group size was selected using proportions sample size estimates ( = 0.05 , = 0.09 ) .
one patient in bupivacaine group was excluded from the analyses because of failure to perform the epidural blockade and the patient was treated with iv pca .
this left 25 patients in group i. 16 female and 34 male patients were studied .
no significant difference was obtained in systolic or diastolic pressure values between groups ( figure 1 ) .
thirty two patients ( 64% ) underwent low anterior resection operation for rectum cancer and 18 ( 36% ) patients underwent total gastrectomy for stomach cancer .
eleven patients who had low anterior resection and four patients who had total gastrectomy needed blood transfusion .
mean duration of surgery for rectum cancer and stomach cancer was 128 21 min and 79 12 min respectively .
the data analysis showed that the incidence of pruritus was not clinically and statistically different in patients receiving epidural bupivacaine compared with patients receiving levobupivacaine ( p > 0.05 ) .
postoperative satisfaction with the epidural analgesia was similar with median scores of 69 ( levobupivacaine ) and 73 ( bupivacaine ) ( vas ; 100 mm = extremely satisfied ) in the first 24 hour after operation .
demographic characteristics systolic and diastolic arterial pressure group i : 0.125% bupivacaine and 3 /ml fentanyl pca was used in major abdominal surgery .
group ii : 0.125% levobupivacaine and 3 /ml fentanyl pca was used in major abdominal surgery .
p > 0.05 there was no significant difference between groups for heart rate ( figure 2 ) , arterial oxygen saturation ( figure 3 ) , vas values ( figure 4 ) and postoperative analgesic requirements .
total analgesic consumption was 145 ml for group i and 150 ml for group ii ( p = 0.091 ) .
additional analgesic need was 25 ml for group i and 30 ml for group ii ( p = 0.185 ) .
supraventricular arrhythmia ( sva ) incidence for the postoperative period was significantly higher in bupivacaine group ( p < 0.05 ) .
total number of pca demands was 14 and 15 for group i and group ii , respectively .
however the incidence increased to 20 times in bupivacaine group and 9 times in levobupivacaine group .
the type of supraventricular arrhythmias included atrial fibrillation ( 0% ) , atrial flutter ( 0% ) , paroxysmal supraventricular tachycardia ( 92% ) and wolf - parkinson - white syndrome ( 8% ) .
postoperative incidence of ventricular arrhythmia ( va ) , conduction abnormalities and pauses longer than two seconds were similar in both preoperative and postoperative period ( p > 0.05 ) .
additionally , the heart rate of patients in bupivacaine group increased during first postoperative three hours but this result was not statistically significant .
group i : 0.125% bupivacaine and 3 /ml fentanyl pca was used in major abdominal surgery .
group ii : 0.125% levobupivacaine and 3 /ml fentanyl pca was used in major abdominal surgery .
p > 0.05 pulse oxygen saturation group i : 0.125% bupivacaine and 3 /ml fentanyl pca was used in major abdominal surgery .
group ii : 0.125% levobupivacaine and 3 /ml fentanyl pca was used in major abdominal surgery .
p > 0.05 group i : 0.125% bupivacaine and 3 /ml fentanyl pca was used in major abdominal surgery .
group ii : 0.125% levobupivacaine and 3 /ml fentanyl pca was used in major abdominal surgery .
p > 0.05 two patients had hypotension ( mean arterial pressure under 50 mmhg ) after epidural catheterisation .
they were treated with colloid infusion but one of them needed 10 mg ephedrine after liquid infusion .
there were no clinically meaningful differences between treatment groups in physical findings or laboratory parameters .
the levels of sensory and motor block did not differ among groups at all time .
bromage scores ( n , 0/1/2/3 ) of group i were 21/4/0/0 patients and of group ii were 22/3/0/0 patients .
asa categorization ( n , 1/2/3 ) of group i was 15/6/4 and of group ii was 12/8/5 patients .
no cases of cardiac depression or central nervous system toxicity caused by vascular absorption or direct intravascular injection of local anesthetic occurred .
our postoperative repeated visits for sedation and respiratory rate monitorization was a precaution for early detection of respiratory depression and provides increased patient satisfaction .
the present study demonstrates that levobupivacaine , the pure s(_)-enantiomer of racemic bupivacaine , is as effective as bupivacaine in epidural analgesia when used with fentanyl for major abdominal surgeries .
racemic bupivacaine has been compared to levobupivacaine for epidural , spinal or infiltration anesthesia and for supraclavicular brachial plexus block .
the comparisons of these two local anesthetics were planned for lower abdominal surgeries , lower limb surgeries or gynecologic surgeries.1317 no significant difference for the quality of analgesia was recorded between these local agents and all of them provided efficient clinical anesthesia.1819 morphine or fentanyl was used in order to rise the quality of analgesia in the postoperative period .
however there are no comparative studies of efficacy for patient controlled epidural analgesia with fentanyl addition in both lower and upper abdominal surgeries . in a separate study ,
three of seven animals given racemic bupivacaine died from sudden onset ventricular fibrillation , whereas the same doses of levobupivacaine produced only nonfatal arrhythmias such as single premature ventricular contractions , bigeminy or couplets .
these findings spontaneously reverted to sinus rhythm.12 we found sva incidence significantly higher in our setting .
same concentrations of epidural bupivacaine and levobupivacaine with fentanyl increased the incidence of supraventricular arrhythmias but the increase in bupivacaine group was significantly higher than levobupivacaine group .
the number of patients with sva for bupivacaine group in the preoperative period was only 4 but it was recorded in 20 patients after epidural analgesia application .
when we checked the results of levobupivacaine group , sva incidence before local anesthetic application was again 4 but it has reached only up to 9 in the postoperative period .
there was not significant variability in the frequencies of va levels both in preoperative and postoperative periods .
the basic cardiac rythm status of the patients was determined first by holter machine before the operation .
then we compared the arrhythmogenic , analgesic and hemodynamic effects of bupivacaine and levobupivacaine in the postoperative period .
bupivacaine produces local anesthesia by blocking sodium channels and this action is probably the main mechanism responsible for its cardiotoxicity.18 because levobupivacaine has less potention for sodium channel blockade and produces less arrhythmias , it has been a popular local anesthetic agent.2021 it was thought that it can be used instead of bupivacaine because of its less toxic side effects to cardiovascular and central nervous system.2223 corrected qt is used to evaluate the arythmogenic potential of drugs .
levobupivacaine has also a poor influence on qrs or corrected qt.24 previously , ropivacaine was also compared with bupivacaine .
the epidural injection of a 1.0% concentration of ropivacaine produced similar sensory and motor block to 0.75% racemic bupivacaine in a similar group of gynecological surgery patients.25 casati et al . compared same volume of 0.5% levobupivacaine , bupivacaine and ropivacaine for major orthopedic surgery.14 they reported that onset , duration and quality of epidural anesthesia were similar .
however , motor block level after levobupivacaine was deeper than the others . in the current trial , levobupivacaine and
this result was similar with the study of glaser et al.13 the increase in heart rate between postoperative first and third hours was higher in group i. this result also supported that bupivacaine has more negative effects on haemodynamic parameters .
however this does nt prop up the results of the trial from burke et al.26 these physicians performed spinal anesthesia and they observed additional bradicardy .
this diversity may be also related to sympathetic or high sensorial blockade effect of spinal anesthesia . in an experimental trial , levobupivacaine and bupivacaine
this result was releated with the stimulation of sympathetic nervous system.27 the vas scores did not differ between groups .
several studies had the same result as our trial.2829 number of demand or bolus doses of pca were also similar between groups .
one limitation of our study is that we only assessed the local anesthetic toxicity according to the records of arrhythmogenic and hemodynamic side effects instead of studying the variability of blood levels of local anesthetics . to summarize
, the results of this study indicated that levobupivacaine - fentanyl and racemic bupivacaine - fentanyl show equally effective potencies for epidural analgesia .
we aimed to obtain the effects of both solutions on systolic , diastolic arterial blood pressure , periferic oxygen saturation and analgesia .
the rate of other type of arrhythmias like va , did not vary . as a result
, we concluded that same concentration of epidural levobupivacaine with fentanyl has less arrhythmogenic but similar analgesic potential than bupivacaine in major abdominal surgeries . with regard to the safety of the s - isomer of bupivacaine
au carried out the design of the study , developed and wrote the protocol , did the statistical analysis and participated in manuscript preparation .
kts provided assistance in the design of the study and participated in manuscript preparation and did the statistical analysis . as provided assistance in the design of study , collected data and participated in manuscript preparation . | background : opioid and local anesthetic infusion by an epidural catheter is widely used as a postoperative pain management method after major abdominal surgeries .
there are several agents nowadays to provide sufficient analgesia .
the agents which cause less side effects but better quality of analgesia are more valuable .
we aimed to postoperatively compare the analgesic , hemodynamic and arrhythmogenic effects of epidural levobupivacaine - fentanyl and bupivacaine - fentanyl solutions.methods:fifty patients were scheduled to undergo major abdominal surgery in this clinical trial .
the parameters were recorded pre- and post - operatively . in group
i ( n=25 ) , bupivacaine with fentanyl solution and in group ii ( n=25 ) , levobupivacaine with fentanyl solution was infused via epidural patient - controlled analgesia ( pca ) . according to the preoperative and postoperative holter recording reports ,
the arrhythmogenic effects were examined in four catagories : ventricular arrhythmia ( va ) , supraventricular arrhythmia ( sva ) , atrioventricular conduction abnormalities and pauses longer than two seconds.results:mean visual analog scale ( vas ) values of groups did not differ at all time .
they were 6 at the end of the surgery ( 0 .
min , p = 0.622 ) .
the scores were 5 in group i and 4 in group ii in 30 .
min ( p = 0.301 ) .
the frequency of sva was higher in bupivacaine group.conclusions:the results of our study suggest that same concentration of epidural levobupivacaine and bupivacaine with fentanyl provide stable postoperative analgesia and both were found safe for the patients undergoing major abdominal surgery . |
debate regarding the indications , timing and technique for tracheostomy seems to have been raging ever since the procedure itself was first described in ancient egypt .
the topic is of global interest , with research from a number of continents published during the past few months .
these recently published papers add new information to the ' round table ' discussion that often goes with this controversial and topical subject .
the first report , that by griffiths and coworkers , is a well researched systematic review and meta - analysis of five controlled studies that aimed to compare outcomes in critically ill patients undergoing artificial ventilation who received a tracheostomy early or late in their treatment .
early tracheostomy was defined as up to 7 days following intubation , and late was defined as any time thereafter , if at all .
the results showed that the duration of artificial ventilation was significantly lower in the early group , as was the length of intensive care unit ( icu ) stay .
this has obvious implications for icu service provision and patient care , assuming that patients leave the icu to make equal recoveries in the two groups .
however , it is difficult to analyze the impact of the results because there was significant heterogeneity between the inclusion and exclusion criteria of the studies .
the hospital and 30-day mortality rates were no different between the two groups , and the risk for hospital - acquired pneumonia was also unchanged .
a recent retrospective study conducted by chia - lin hsu and coworkers investigated the optimal timing of tracheostomy formation , its impact on weaning from artificial ventilation , and the outcomes in patients in a medical icu at the 1500-bedded national taiwan university hospital .
a total of 163 patients were included and divided into two groups : successful weaning and failure to wean .
interestingly , the study discusses rates of associated complications of both percutaneous tracheostomies ( pts ) and surgical tracheostomies ( sts ) .
the results showed that patients undergoing tracheostomy more than 3 weeks after intubation had higher icu mortality rates ( 28.3% versus 14.5% ) , higher rates of weaning failure ( 56.4% versus 30.2% ) and longer icu stays ( 14.2 days versus 10.8 days ) .
this study also showed no difference in hospital mortality or nosocomial pneumonia during the weaning period .
the authors concluded that tracheostomy after 21 days was associated with prolonged weaning , low weaning success rates and prolonged icu stay . in the third study , blot and melot performed a retrospective analysis of the indications , timing and techniques of tracheostomy in 152 of the 708 french icus contacted .
although the study had a relatively low reply rate ( 21.5% ) , it raised several interesting points .
first , their definition of early tracheostomy was any time during the first 3 weeks after intubation .
second , early tracheostomy was considered more often in nonteaching hospitals than in teaching hospitals . finally , sts were preferred over pts on surgical icus , and vice versa on medical icus . the authors concluded that long - term mechanical ventilation and failed extubation are the major indications for tracheostomy , and that tracheostomy is considered after a mean time of 3 weeks ( later than recommended by several consensus conferences ) .
this prospective and retrospective observational study looked into the problem of tracheal stenosis caused by both pt and st individually .
the investigators studied 29 patients presenting with tracheal stenosis to a uk national referral centre for tracheal reconstruction .
following bronchoscopy preoperatively , they were able to assess the level , length and diameter of tracheal stenosis .
this potentially life - threatening complication differs between the two groups in the above parameters , and therefore affects the treatment options available to the patient .
the results showed that , compared with st , pt caused tracheal stenosis closer to the vocal cords ( 1.6 cm versus 3.4 cm ; p = 0.04 ) and the onset of tracheal stenosis occurred significantly quicker in the pt group ( 5 weeks versus 28.5 weeks ) .
other quoted studies support the finding that pt resulted in the tracheal wall ' caving in ' due to cartilage fracture significantly more often ( 50% versus < 2% ) .
they also conclude that stenosis caused by pt occurred earlier and was more subglottic in nature compared with st .
considerable variety in the timing of tracheostomy formation and the technique employed continues to exist , with a number of other publications both supporting and opposing the reports discussed above .
intensive care practitioners clearly need further information and research to enable agreement to be reached on optimum tracheostomy care . from these studies ,
first , early tracheostomy ( < 7 days ) reduces the duration of artificial ventilation .
third , patients undergoing tracheostomy after 3 weeks have a higher mortality , longer duration of ventilation , reduced successful weaning and longer icu stay .
finally , pt causes more subglottic stenosis , with a quicker onset than with st .
the much anticipated tracman study is now underway in the uk and will hopefully shed much more light on some of these issues .
the vast majority of acute care hospitals in the uk are without on - site neurosurgical and neurointensive care facilities , necessitating expedient assessment of the brain - injured patient . those with severe traumatic brain injury ( tbi ; i.e. those with glasgow coma scale [ gcs ] score < 9 ) should undergo prompt transfer to a neurosurgical centre unless the prognosis is deemed to be hopeless . for those with mild ( gcs score 1415 ) and moderate ( gcs score 913 ) tbi , with no indication for immediate surgery
, it may well be preferable that they stay in the admitting non - neurosurgical centre , given the demand on the all too few neurosurgical intensive care beds in the uk .
subsequent monitoring , and therefore management , of this group of patients for secondary neurological deterioration is often suboptimal , given the inability to institute ' gold standard ' techniques such as intracranial pressure and jugular venous saturation monitoring , relying largely on clinical deterioration and computed tomography ( ct ) .
a study by jaffres and coworkers may represent a glimmer of light in this otherwise dark tunnel . in a prospective cohort study ( n = 78 ) set in the emergency room of a french district hospital , consecutive patients admitted with mild or moderate tbi underwent both ct and transcranial doppler ( tcd ) studies within 12 hours of admission .
patients were then assessed , based on objective predefined criteria , for neurological deterioration 7 days after admission .
the study attempted to correlate deterioration with initial measured variables : tcd , ct of the head , and biochemical , haematological and clinical measures , including a variety of composite scoring systems .
seven ( 17% ) patients from the mild tbi group suffered deterioration . using univariate analysis
the investigators demonstrated a significant difference in both ct findings and pulsatility index ( pi ) , as measured using tcd , in this subgroup compared with those who did not deteriorate .
the injury severity score and maximal head abbreviated injury scale score were also significantly different . in
pi and ct scoring were again significantly different in this group , along with the scoring systems mentioned above , initial gcs score and use of vasoactive drugs .
jaffres and coworkers go on to discuss the mechanisms by which pi is inversely related to cerebral perfusion pressure , and suggest that an appropriate combination of ct classification of tbi and measurement of pi on admission may be used to identify those at risk for subsequent deterioration .
however , they point out that the feasibility and practicalities of early tcd are not inconsequential and that no threshold value for pi was identified in this small study .
less invasive does not necessarily mean less useful , and cuschieri and coworkers including dr e rivers , who utilized central venous oxygen saturations in early goal - directed therapy for severe sepsis investigated the correlation between central venous arterial carbon dioxide difference ( cv aco2 ) , mixed venous arterial carbon dioxide difference ( mv aco2 ) and cardiac index ( ci ) in a group of mechanically ventilated patients with various diagnoses .
for inclusion , patients needed to have a pulmonary artery catheter in situ . simultaneous arterial , mixed venous and central venous blood samples were obtained , along with measurement of cardiac indices using the thermodilution technique .
the group found excellent correlation between cv aco2 and mv aco2 , with a correlation coefficient across all diagnoses and circulation types ( high , low and normal ) of 0.978 .
furthermore , both were found to be inversely related to ci , with similar magnitudes of correlation , as compared with thermodilution - derived values .
although the relationship between mv aco2 and ci has long been recognized , as derived from the fick principle , this study suggests an easier and less invasive way to apply the same concept and , in conjunction with centrally derived arteriovenous oxygen differences , this may be very useful in the early assessment of global tissue hypoxia . finally , a thought - provoking review ,
although one that is not terribly helpful on a practical level , was recently published in chest .
hennessy and colleagues carried out a thorough literature search on post - icu outcomes of elderly patients .
the elderly population , variously defined as > 65 years , > 70 years , > 75 years or > 85 years , is set to grow massively , and this will undoubtedly have major implications for service provision .
although many studies have scrutinized mortality rates from critical illness in the elderly , little is known about health - related quality of life ( hrqol ) and functional status in the survivors .
the investigators identified only 16 studies ( involving a total of 3247 patients ) , only one of which was multicentred , addressing this issue .
encouragingly , the majority of these studies reported good hrqol and functional status post - icu , although some discordance was evident , suggesting a change in conceptualization of quality of life following critical illness .
however , the authors were unable to pool results and draw any significant conclusions because of poor quality study designs and lack of consensus on how to measure hrqol .
they urge the need for further research , which must be well designed , prospective and use validated , reliable and responsive measures of hrqol .
aco2 = central venous arterial carbon dioxide difference ; gcs = glasgow coma scale ; hrqol = health related quality of life ; icu = intensive care unit ; mv
aco2 = mixed venous arterial carbon dioxide difference ; pi = pulsatility index ; pt = percutaneous tracheostomy ; st = surgical tracheostomy ; tbi = traumatic brain injury ; tcd = transcranial doppler .
| tracheostomies have been around for close to 3000 years , so one would hope that the controversies might have been thrashed out by now , but apparently not .
judging by some recent publications it would appear that we still do not know when or how to insert them .
monitoring is fundamental to critical care ; two papers describe novel / modified techniques for assessing traumatic brain injury and cardiac output .
the intensive care unit imposes a heavy treatment burden , particularly on the elderly .
what impact does this have on the lives of the survivors ? |
at indiana universitypurdue university indianapolis ( iupui ) , we have developed a concept called distributed drug discovery ( d ) .
herein is reported the first example of a key component of d : the distributed rehearsal of reagents and their subsequent use in real and virtual catalog production .
the premise of d is that the availability of simple , inexpensive equipment and procedures for each of the core scientific drug discovery disciplines ( computational chemistry , synthetic chemistry , and biochemical screening ) will enable large drug discovery problems to be broken down into manageable smaller units to be solved at multiple sites throughout the developing and developed world .
the recombined and coordinated results of these resources can inexpensively accelerate the identification of leads ( primarily for developing world and other neglected disease targets ) in the early stages of the drug discovery process .
in addition , this effort provides educational and job opportunities in both the developed and developing worlds while building cultural and economic bridges for the common good .
combinatorial virtual catalogs , realistically accessible by globally distributed synthesis , are key to the implementation of d. in classical combinatorial chemistry , reagent rehearsal increases the likelihood that any individual member of an enumerated virtual library can be synthesized from each unique combination of rehearsed reagents .
however , it imposes additional strong constraints on reagent - rehearsed virtual catalogs : any member of the library must be accessible by rehearsed or precedented synthesis , anywhere in the world , using simple , inexpensive equipment and procedures .
part i reports the first successful global reagent rehearsal , conducted by undergraduate and graduate students in the united states , poland , russia , and spain . in part ii , a virtual d catalog based on this work
one of the most common types of intermediates used in combinatorial chemistry is the resin - bound natural or unnatural -amino acid 1 ( scheme 1 ) .
the -amino acid substructure occurs in 28% of the structures cited in a recent comprehensive survey of combinatorial library synthesis .
scheme 1 gives examples of a few of the published libraries based on 1 . with the exception of proline ,
the methodology for the introduction of side chains by carboncarbon bond construction has expanded considerably the availability of these unnatural -amino acids . of the existing routes to such molecules
thus , introduction of an unnatural side chain onto the -position of a suitable glycine or higher amino acid derivative by alkylation can provide , respectively , -substituted or ,-disubstituted amino acid products .
because of the central role of 1 in combinatorial chemistry , and the ability to greatly expand its numbers through alkylation chemistry , we chose to focus on rehearsing alkylating agents r1x for our first exemplification of d synthesis . in the process
, we wished to show the likelihood that undergraduate or graduate students would be able to make any given member of a resulting virtual d catalog , further demonstrating the potential of distributed drug discovery .
we modified our published synthesis of 1(17c ) and incorporated the rehearsal into a student combinatorial chemistry laboratory that transformed 1 to the acylated derivatives 5 ( scheme 2 ) . this generic structure 5 would then become the basis of our first virtual d catalog . the time ranges shown in scheme 2 accommodated the details of laboratory scheduling in academic institutions at different geographic locations .
simple , inexpensive bill - board 6-pack equipment ( figure 1 ) was used to carry out this procedure , six reactions at a time .
every bill - board 6-pack had six reaction vessels in a grid of two rows ( a and b ) by three columns ( 1 , 2 , and 3 ) , and reactions were correspondingly identified by row , column ( e.g. , a1b3 ) . the equipment was designed to enable the essential steps common to most solid - phase synthetic procedures : cycles of reaction and filtration workup , followed by product cleavage , and collection .
bill - board components and 2 3 experimental grid . in this first demonstration of the distributed process , twenty - four alkylating agents r1x were rehearsed , often in quadruplicate , sometimes at geographical locations remote from one another ( indianapolis , in ; lublin , poland ; moscow , russia ; and barcelona , spain ) . in all cases
the quality of the crude products was assessed by lc / ms to obtain a marker of the suitability of a given reagent in combinatorial library generation .
this analytical measurement of quality is not definitive because uv detection can overlook , among other things , poor absolute recovery , the presence of insoluble impurities , or soluble impurities not revealed by the detection system .
nevertheless , the lc / ms data permits elimination , from a rehearsed virtual library , products based on alkylating agents that are clearly problematic . follow up synthesis and purification conducted at iupui allowed the isolation and characterization of all the unnatural amino acids reported in this distributed reagent rehearsal study . since reagent rehearsal was done by students with little to no prior experience in either organic synthesis or solid - phase chemistry , two key elements were incorporated into the laboratory : ( 1 ) students worldwide conducted the same control alkylation reaction , using r1x = benzyl bromide ( 6 ) in column 1 .
( 2 ) the evaluation of new alkylating agents was replicated by at least one other student or student team .
this worldwide distributed process permits the assessment of both the quality of the work carried out by any given student / team and the reproducible efficacy of the evaluated reagents .
the first synthetic rehearsals were conducted at iupui , utilizing four concurrent laboratory sections , to pilot the process of replicated synthesis in multiple students hands .
there were up to 20 students in each section . a total of 65 students operated in 32 teams of two per bill - board ( 192 reactions ) , with one extra bill - board used for the solo student .
the control alkylating agent , benzyl bromide ( 6 ) , was used by all students in column 1 ( figure 1 ) .
the new alkylating agents were rehearsed in columns 2 and 3 . in these experiments ,
the acylating agents used in rows a and b of the bill - board were always fmoc chloride and 2-naphthoyl chloride , respectively .
in this first extensive study , two commercial suppliers , i and ii , were commissioned to produce the starting benzophenone imine resin 2 . for comparison purposes , resin from source i was used in sections one , two , and four ( teams 18 , 1017 , and 2633 ) , and resin from source ii was used in section three ( teams 1825 ) . since all 32 teams prepared the same product ( fmocphe 5{6}a , see table 1 ) in position a1 of their bill - boards it was informative to analyze the quality , as a function of resin source , of all 32 of these replicated products .
figure 2 shows a comparison of representative products a1 from teams using resins from these two suppliers .
representative uv trace from lc / ms analysis of a1 from either resin supplier i or ii .
( a ) lc trace of product ( team 1 , al ) using resin from supplier i. ( b ) lc trace of product ( team 18 , al ) using resin from supplier ii . in the products obtained from reactions using resin from i ( figure 2a ) , there was , in addition to desired product ( 5.04 min , m + 1 = 388 , 77% ) , the universal presence of three impurities ( 2.5 min , m + 1 = 256 , 4% ; 4.8 min , m + 1 = 445 , 11% ; 5.3 min , m + 1 = 494 , 8% ) .
the ms data allowed the tentative assignment of the structures 23 , 24 , and 25 for these respective peaks ( figure 3 ) .
in contrast , there were insignificant levels of the impurities at 2.5 and 4.8 min in samples prepared from resin supplier ii ( figure 2b ) .
the supporting information contains a more complete discussion of the origin of these proposed impurities 23 and 24 in resin from i , and 25 in both resins from either i or ii . proposed structures for impurities produced from resin source i. although resins from i and ii were used in the initial laboratories at iupui , because of its higher quality , only resin from commercial source ii was used in subsequent laboratories at iupui , and in laboratories conducted in poland , russia , and spain .
replicated alkylations ( 24 replicates ) , using the same resin and alkylating agent , gave strikingly similar results ( table 1 ) .
individual results rarely differed from the average by more than 3% . as expected , because of the impurities present in the starting resin from i ( see earlier discussion and supporting information ) the purity of alkylation products from this resin was lower ( by 1520% ) than those from ii ( compare , for example , the results for the fmoc derivatives 5{7}a through 5{12}a from alkylating agents 7 through 12 , where the first two results for each compound were from resin source i , and the second two results were from resin source ii ) . nevertheless , from the same resin source the alkylation results are very reproducible . in rare cases ,
this is to be expected when so many different researchers ( students ) are performing this work for the first time .
for example , team 28 obtained none of the expected products 5{15}b or 5{17}b , whereas three other teams using the same reagent combinations ( teams 12 , 13 , 29 and 11 , 12 , 27 ) were successful and had similar results ( 63% 3% and 80% 8% , respectively ) for each of these compounds in the appropriate bill - board positions .
encouraged by the success of the iupui student alkylating agent rehearsal , we tested the ability to further distribute this process to students outside of the united states . using the same procedures
( with minor modifications ) , equipment , and resin from source ii , the laboratory was first replicated in spain . in 36 separate reactions ,
teams at the university of barcelona ( two or three students per bill - board ) evaluated control benzyl bromide ( 6 , six replicates ) and six additional alkylating agents in duplicate ( table 2 ) . with the exclusive use of starting resin purchased from supplier ii , the barcelona laboratories obtained excellent results , providing strong documentation for the ability of distributed discovery to reproducibly synthesize molecules , both within the same site , and around the world .
barcelona , team 3 ) , all the control fmocphe samples 5{6}a were synthesized in > 92% purity as determined by lc / ms ( table 2 ) .
this was consistent with the data generated in the united states at iupui , where the subset of compounds 5{6}a synthesized used starting resin from the same supplier ( ii ) produced 7 of 8 products in > 93% purity ( table 1 ) .
the barcelona laboratories also rehearsed , in duplicate , six new alkylating agents , 2631 .
the data from teams 2 and 3 for 5{28}b indicates that the alkylating agent 28 is also likely to give desired product but with moderate purity .
the methoxy - substituted reagents 29 and 31 illustrate the need for a nuanced interpretation of results .
the crude material recoveries for the methoxy derivatives 5{29}a , b and 5{31}a , b were notably lower compared to the rest of the products in this laboratory . these molecules , as anisole derivatives , are susceptible to friedelcrafts reactions .
it is well established in the peptide field that , in the absence of a powerful cation quenching agent ( such as anisole itself ) , products capable of friedelcrafts reactions can reattach themselves to the resin cation generated in the cleavage process .
accordingly , selective , cleavage - condition dependent removal of desired product ( by reattachment to the resin ) could mask a successful alkylation prior to cleavage . to further demonstrate the ability of the distributed reagent rehearsal project to provide replicated results in student laboratories across the globe ,
table 3 compares a set of molecules prepared at iupui ( united states ) , the university of barcelona ( spain ) , moscow state university ( russia ) , and the lublin school of pharmacy ( poland ) .
these results indicate that d syntheses can be carried out at multiple global locations and afford reproducible results .
the product 5{32}a ( figure 4 ) was obtained in 76% purity ( lc / ms analysis ) .
moscow single - site result . to fully characterize products from the reagent rehearsal and confirm that student - generated results can reliably predict synthetic outcomes ,
all of the listed alkylating agents were subsequently re - evaluated by a postdoctoral fellow at iupui . using resin from source ii ,
the experimental procedure used was identical to that employed in the rehearsal laboratories at iupui .
the results , summarized in table 4 , confirm the ability of student rehearsed chemistry to be replicated by a third party .
these results also resolved the questions arising from the large variability of results in the rehearsal of reagents 28 and 29 reported in table 2 .
in addition , the single result reported by the moscow site for reagent 32 was confirmed .
the supporting information provides lc / ms data for all the crude products from indianapolis , lublin , and moscow , along with nmr data for all the purified , new compounds prepared .
rl = averaged results from student rehearsal laboratories ; pd = result from postdoc synthesis at iupui .
single value , other value was 0% . in summary , these first reagent rehearsal laboratories document a key requirement of the distributed drug discovery concept : the global ability of students to evaluate reagents and reproducibly make new potential drug leads , using simple , inexpensive equipment and procedures . these reagents are now acceptable as input in the enumeration of the d accessible virtual catalogs to be described in part ii .
an essential component of distributed drug discovery is the open - access availability of databases of molecules accessible through distributed chemistry .
computational chemists can then analyze these large databases to identify and make globally available smaller databases of potential drug leads targeted for developing world diseases .
subsequently , the synthesis of the selected molecules can be carried out , in a distributed fashion , at sites across the world . the compounds ( including stereoisomers when appropriate ) in this virtual d catalog were enumerated with commercial software . to ensure that virtual library generation is not limited by cost we also enumerate libraries with alternative software currently available for free to academia .
on the basis of the distributed chemistry developed and reported in this article , two types of library enumeration were envisioned .
, a much larger , virtual d catalog ( including the compounds actually made ) can be enumerated from all combinations of rehearsed or otherwise well - precedented reagents used at the variable alkylation and acylation steps . to illustrate this process , we assembled lists of acceptable , commercially available , alkylating and acylating agents ( tables 5 and 6 , respectively ) to enumerate a representative virtual d catalog of acylated unnatural amino acids ( scheme 2 ) .
for the alkylation step extensive precedence is available , both from our solution- and solid - phase studies and those of others , to predict the reactivity of schiff base esters and related systems with these electrophiles under a variety of basic conditions . for example , even though michael addition reactions were not rehearsed in the present study , we have confidence , based on our earlier solid - phase research using resin - bound schiff bases and michael acceptors as electrophiles , that reactants 93108 ( table 5 ) can be utilized successfully .
, we plan to incorporate all rehearsed reagent information into a single database to further assist reagent selection . in the acylation step
however , we have since modified our d laboratories to use carboxylic acids to form the amides by traditional carbodiimide mediated couplings .
this makes available a much wider repertoire of inexpensive reactants for enumeration at the second variable position .
because the reactivity of carboxylic acids in forming amide bonds is extensively documented , simple chemical intuition and reactivity considerations were used to select the 100 carboxylic acids ( table 6 ) for enumeration .
on the basis of these considerations , a virtual d catalog of 24 416 acylated unnatural amino acids 5 was enumerated . as discussed in the following article , an additional 24 192 methyl esters of these acylated unnatural amino acids
were enumerated on the basis of the cleavage of resin - bound products 4 by methanolysis .
thus , a total of 48 608 unique products are available for d catalog access and searching .
the 100 commercially available alkylating agents and michael acceptors and 100 carboxylic acids used to perform these enumerations are shown in tables 5 and 6 , respectively .
the catalog of acylated unnatural amino acids contains more than the 20 000 members that would arise from a simple 100 100 combinatorial process .
this is caused by the formation of stereoisomers in the alkylation step and their combination with other stereoisomers sometimes present in the alkylating or acylating reagents .
thus , from two to eight stereoisomers appear for each structure in the virtual d catalog .
the separate listing of each of these anticipated stereoisomeric products allows appropriate computational discrimination and selection when three - dimensional modeling software is employed and chirality is important .
two stereoisomers arise when there are no other stereogenic elements in either the electrophile or the acid [ e.g. , ( r)-5{12,207 } plus enantiomer ] or when the electrophile or acid is present as a single enantiomer ( r , r)-5{22,117 } plus diastereomer ] or geometric isomer ( r , e)-5{82,208 } plus enantiomer ] .
four stereoisomers are contained in the library when a racemic or prochiral electrophile or a racemic carboxylic acid is used ( s , s)-5{18,121 } or ( r)-5{93,159 } plus three other stereoisomers for each ] .
eight stereoisomers arise when both a racemic electrophile and a racemic carboxylic acid are used ( r , r , r)-5{47,186 } plus seven other stereoisomers ] .
the synthesis of stereoisomeric mixtures of unnatural amino acid derivatives has the advantage of providing stereoisomers for evaluation .
the follow up work to identify the active stereoisomer can be accomplished , without stereoselective synthesis , through bioassay guided fractionation or chiral chromatography .
the active stereoisomer identified by these techniques can be subsequently prepared by known stereoselective synthetic routes , separation techniques , or resolution procedures .
it is important to consider potential side - reactions during the various steps of the synthesis of acylated unnatural amino acids 5 .
for example , the tert - butyl esters present in resin - bound products 4 that originated in the reactants used for alkylation , michael addition , or acylation ( 36 , 75 , 96 ) will be converted to the carboxylic acids during tfa cleavage .
likewise , the t - butyl carbamate ( n - boc ) group in reactant 83 will be cleaved to the free indole nh on tfa cleavage to products 5 . a lead reference for each of these subsequent reactions is noted with the respective reactant , and this is reflected in the enumerated product structures .
other more subtle side reactions will be incorporated into the enumeration products as they are discovered on the basis of literature precedence or rehearsal of reagents by our group or by others . to illustrate the variety of virtual molecules available through this enumeration
the open - access availability of this virtual d catalog makes possible the global computational selection of candidates for drug synthesis , while simultaneously enabling the prioritization of future reagent rehearsals . when computational analysis suggests a particular library member may be a drug lead for a developing world disease target , that molecule will be selected for distributed synthesis .
if the reagent used as input in its enumeration is unrehearsed , the synthesis will evaluate its suitability as a reagent and enable it to become a part of this expanding rehearsed reagent database , stored electronically and available through open - access .
students carry out , using simple procedures and inexpensive equipment , worldwide replicated solid - phase combinatorial chemistry , rehearsing new reagents and generating new compounds for library production . at the same time they learn and develop important synthetic skills and methodologies .
the data generated and reported here allows the construction of a globally accessible d database of well - documented alkylating agents and michael acceptors for the synthesis of resin - bound unnatural amino acids 1 .
this intermediate can then be incorporated into a wide variety of published combinatorial chemistry procedures .
virtual d catalogs based on rehearsed or well - precedented reagents and inexpensive global procedures are essential to the goals of distributed drug discovery .
accordingly , we have enumerated a 24 416-member virtual d catalog of acylated unnatural amino acids 5 based on the current work .
freely available virtual d catalogs , such as the one reported in this article , can be computationally analyzed to select potential drug leads for developing - world and other neglected diseases . in turn
, the distributed reagent rehearsal provides strong precedent and documented procedures to successfully make , through distributed synthesis , any of the molecules selected by this process .
anhydrous nmp , chloroform - d1 , 2-fluorobenzyl bromide , 4-fluorobenzyl bromide , 2-(trifluoromethyl)benzyl bromide , 4-(trifluoromethyl)benzyl bromide , 2-bromobenzyl bromide , allyl bromide , 2-nitrobenzyl bromide , 2,4-dichlorobenzyl chloride , and benzyl 2-bromoacetate and were purchased from acros organics .
btpp ( tert - butyl - imino - tri(pyrrolidino)phosphorane ) . and fmoc chloride were purchased from fluka . 5-(chloromethyl)-2-chloropyridine , n ,
n - diisopropylethylamine , benzyl bromide , 3-fluorobenzyl chloride , 3-(trifluoromethyl)benzyl chloride , 3-bromobenzyl bromide , 4-bromobenzyl bromide , 2-(bromomethyl)benzonitrile , 3-(bromomethyl)benzonitrile , 4-(bromomethyl)benzonitrile , 2-iodobenzyl bromide , 4-iodobenzyl bromide , 1-(chloromethyl)naphthalene , 2-(bromomethyl)naphthalene , 4-methoxybenzyl bromide , 3-methoxybenzyl bromide , and methanol - d4 were obtained from aldrich chemical co. all resins were purchased from midwest bio - tech laboratories ( resin i ) or polymer laboratories ( resin ii ) unless otherwise noted .
manual solid - phase organic syntheses at ambient temperature were carried out in 3.5 ml fritted glass reaction vessels equipped with polypropylene screw caps with teflon - faced silicon septa on the bill - board set , which was designed by one of us ( wls ) as inexpensive equipment to simplify and expedite multiple , manual solid - phase syntheses .
the bill - board reaction vessel components are available from chemglass : iup-0305270h for 3.5 ml reaction vessel ; iup-0305280h ( polypropylene screw cap ) ; iup-0305281h ( teflon faced silicone septa ) . analytical thin layer chromatography ( tlc )
flash column chromatography was performed on silica gel 60 ( 230400 mesh ) from silicycle chemical division .
the yields of the final compounds , after chromatographic purification , were calculated on the basis of the initial loading of the starting resins and are the overall yields of all reaction steps starting from these resins .
h nmr spectra were recorded at 200 mhz ( varian spectrometer ) using tms ( 0.00 ppm ) and chloroform - d1 mixed with methanol - d4 ( 310% ) .
electrospray ionization mass spectrometry was conducted using a pesciex api iii triple stage quadrupole mass spectrometer operated in either positive - ion or negative - ion detection mode .
the composition of reaction mixtures was determined based on the integration of nmr spectra as well as lc - ms results .
lc - ms analyses were conducted using an agilent system , consisting of a 1100 series hplc connected to a diode array detector and a 1946d mass spectrometer configured for positive - ion / negative - ion electrospray ionization .
the lc - ms samples were analyzed as solutions in ch3cn , prepared at 0.080.12 mg / ml concentration .
the lc - ms derived composition of mixtures was determined based on uv integration at 210 nm .
depending on the number of reactions to be performed , resin 2 was distributed either by weight or as aliquots from an isopycnic suspension . in the case of distribution by volume from an isopycnic suspension
, the bill - boards were placed in their drain trays , and from a neutral buoyancy suspension in ch2cl2/nmp , 50 mols of the benzophenone imine of glycine wang resin ( 2 , the resin loading is 0.7 0.9 mmol / g ) was distributed , via repeated 1 ml aliquots , to each of the six reaction vessels in a given bill - board 6-pack . during the distribution of the resin ,
when distribution was complete , residual solvent was removed with an air - push from a disposable plastic pipet ( fisher , 1371123 ) fitted with a pierced septum ( aldrich , z 127434 ) .
the resin was washed with nmp ( 3 3 ml ) ( this nmp wash was also done when resin was weighed out into reaction vessels ) .
the bottom of each reaction vessel was then capped , and a new calibrated pipet ( fisher , 13 - 711 - 24 ) was used for adding each alkylation reagent in the following step .
the first r1-x ( benzyl bromide ) [ 0.5 ml of a 0.20 m solution in nmp , 100 mols , 2 equiv ] was added to the resin in the two reaction vessels in the first column positions ( i.e. , a1 and b1 ) .
then the second r1-x [ 0.5 ml of a 0.20 m solution in nmp , 100 mols , 2 equiv ] was added to the resin in the two vessels in the second column positions ( i.e. , a2 and b2 ) .
finally , the third r1-x [ 0.5 ml of a 0.20 m solution in nmp , 100 mols , 2 equiv ] was added to the resin in the two vessels in the third column positions
( i.e. , a3 and b3 ) . base was then added [ 0.5 ml of a 0.20 m solution of btpp in nmp , 100 mols , 2 equiv ] to each of the six reaction vessels .
the tops of all reaction vessels were capped , and the bill - board was placed in a rotation apparatus .
depending on the school location and laboratory schedule , the reaction was allowed to proceed from 24 h to 2 days with rotation .
the bill - board was removed from the rotation apparatus and , after inverting the board , the bottom caps were removed .
the board was then placed , top side up , in the drain tray , the top caps were removed , and the reagents from the alkylation step were allowed to drain , followed by an air - push .
the alkylated resin - bound resin products 3 were washed with thf ( 1 3 ml ) . using 6 clean caps ,
the bottom of each reaction vessel was capped , and a 1 n aqueous hcl / thf solution ( 1:2 , 2.5 ml ) was added to each of the six reaction vessels .
the caps were then put on the top of each vessel , and the bill - board was returned to the rotator for 20 min .
the reagents and byproduct from the hydrolysis were then removed from hydrolyzed resin - bound product ( 1 ) by filtration and washing with thf ( 1 3 ml ) and then nmp ( 1 3 ml ) .
the bottom of each reaction vessel was capped , and the acylating agent , r2cocl [ 0.5 ml of a 0.30 m solution of r2cocl in nmp , 150 mols , 3 equiv ] was added to the resin 1 in each of the three vessels in row a ( a1 , a2 , and a3 ) . then the second acylating agent , r2cocl [ 0.5 ml of a 0.30 m solution of r2cocl in nmp , 150 mols , 3 equiv ] was added to the resin 1 in each of the three vessels in row b ( b1 , b2 , and b3 ) .
this was followed by addition of diea [ 0.5 ml of a 0.30 m solution in nmp , 150 mols , 3 equiv ] to each of the six reaction vessels .
the tops of the reaction vessels were capped , and the reaction was allowed to rotate overnight or up to 5 days ( again , dependent on school laboratory schedules ) .
acylated resin product 4 was washed with nmp ( 2 3 ml ) , thf ( 2 3 ml ) , and ch2cl2 ( 3 3 ml ) .
the product was cleaved from resin with tfa / h2o ( 95:5 , 2 ml ) for 30 min .
the filtrate from the cleavage reaction was collected , and the resin was washed with tfa / h2o ( 95:5 , 2 ml ) and ch2cl2 ( 1 2 ml ) . after each collection vial was swirled to thoroughly mix the cleavage and rinse solutions , a sample of each solution ( 100 l ) containing product 5 was transferred to an autosampler vial for lc / ms analysis .
designed to speed up the evaporation with a stream of nitrogen , in a contained system , while trapping the evaporating tfa in 2 n naoh .
purification of the crude product was performed via flash column chromatography , using the glass fritted reaction vessel ( 3.5 ml ) loaded with bulk silica gel ( 650700 mg ) .
the crude product was dissolved in ch2cl2 and , if necessary , meoh ( total solution volume 0.5 ml ) and applied to the column .
then a gradient of eluting solvent was applied starting with ch2cl2 to ch2cl2/meoh / h2o ( 300:10:0.5 ) .
the purified product was dried in the evaporation equipment described above at ambient temperature under a flow of nitrogen .
forty eight hours later ( wednesday ) , the reactions were worked - up ; the imine was hydrolyzed and worked - up , and the acylation reactions were begun .
five days later ( the following monday ) , the acylation reactions were worked - up ; the product was cleaved from the resin , and samples taken for tlc and lc / ms analysis .
all samples were analyzed by lc / ms in the analytical department at eli lilly & company ( indianapolis ) . at each of these locations ,
the next day the reactions were worked - up ; the imine was hydrolyzed and worked - up , and the acylation reactions were begun .
the next ( and last ) day the acylation reactions were worked - up ; the product was cleaved from the resin , and samples taken for tlc and lc / ms analysis .
the samples from moscow and lublin were taken back to indianapolis where they were analyzed by lc / ms in the analytical department at eli lilly .
6.9 mg ( 35% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 82% , tr = 5.11 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.003.14 ( m , 1h ) , 3.20 ( dd , j = 13.9 hz , j = 5.5 hz , 1h ) , 4.144.27 ( m , 1h ) , 4.284.49 ( m , 2h ) , 4.59 ( t , j = 5.8 hz , 1h ) , 7.16 ( m , 2h ) , 7.237.46 ( m , 7h ) , 7.527.62 ( m , 2h ) , 7.78 ( d , j = 6.8 hz , 2h ) ; lc / ms calcd for c24h22no4 [ m + h ] 388.1 ; found 388.1 . 8.3 mg ( 41% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 83% , tr = 5.12 min;h nmr ( 200 mhz , cdcl3/cd3od ) 3.09 ( dd , j = 14.2 hz , j = 7.7 hz , 1h ) , 3.32 ( dd , j = 14.2 hz , j = 4.9 hz , 1h ) , 4.124.23 ( m , 1h ) , 4.244.44 ( m , 2h ) , 4.61 ( t , j = 5.5 hz , 1h ) , 6.967.10 ( m , 2h ) , 7.147.45 ( m , 6h ) , 7.517.60 ( m , 2h ) , 7.76 ( d , j = 7.2 hz , 2h ) ; lc / ms calcd for c24h21fno4 [ m + h ] 406.1 ; found 406.2 . 10.0 mg ( 49% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 82% , tr = 5.17 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.08 ( dd , j = 13.8 hz , j = 6.2 hz , 1h ) , 3.19 ( dd , j = 13.9 hz , j = 5.5 hz , 1h ) , 4.20 ( t , j = 7.0 hz , 1h ) , 4.284.51 ( m , 2h ) , 4.60 ( t , j = 5.5 hz , 1h ) , 6.857.00 ( m , 2h ) , 7.177.45 ( m , 6h ) , 7.517.63 ( m , 2h ) , 7.77 ( d , j = 7.4 hz , 2h ) ; lc / ms calcd for c24h21fno4 [ m + h ] 406.1 ; found 406.3 . 7.0 mg ( 34% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 97% , tr = 5.18 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.02 ( dd , j = 13.8 hz , j = 6.0 hz , 1h ) , 3.15 ( dd , j = 14.1 hz , j = 5.3 hz , 1h ) , 4.19 ( t , j = 6.7 hz , 1h ) , 4.284.52 ( m , 2h ) , 4.56 ( m , 1h ) , 6.95 ( m , 2h ) , 7.057.15 ( m , 2h ) , 7.287.46 ( m , 4h ) , 7.527.61 ( m , 2h ) , 7.77 ( d , j = 7.2 hz , 2h ) ; lc / ms calcd for c24h21fno4 [ m + h ] 406.1 ; found 405.8 .
12.2 mg ( 54% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 94% , tr = 5.46 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.10 ( dd , j = 14.2 hz , j = 9.6 hz , 1h ) , 3.46 ( dd , j = 14.7 hz , j = 5.1 hz , 1h ) , 4.074.20 ( m , 1h ) , 4.214.36 ( m , 2h ) , 4.64 ( dd , j = 9.5 hz , j = 5.1 hz , 1h ) , 7.247.48 ( m , 7h ) , 7.54 ( t , j = 6.6 hz , 2h ) , 7.64 ( d , j = 7.4 hz , 1h ) , 7.75 ( d , j = 7.4 hz , 2h ) ; lc / ms calcd for c25h19f3no4 [ m + h ] 456.1 ; found 456.2 .
9.4 mg ( 41% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 90% , tr = 5.53 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.14 ( dd , j = 13.8 hz ,
j = 6.0 hz , 1h ) , 3.26 ( dd , j = 13.7 hz , j = 5.3 hz , 1h ) , 4.19 ( t , j = 6.6 hz , 1h ) , 4.274.51 ( m , 2h ) , 4.61 ( t , j = 5.5 hz ) , 7.287.50 ( m , 8h ) , 7.517.62 ( m , 2h ) , 7.77 ( dd , j = 7.4 hz , 2h ) ; lc / ms calcd for c25h19f3no4 [ m + h ] 456.1 ; found 456.1 .
9.4 mg ( 41% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 88% , tr = 5.55 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.12 ( dd , j = 13.7 hz , j = 5.9 hz , 1h ) , 3.26 ( dd , j = 14 hz , j = 5.6 hz , 1h ) , 4.19 ( t , j = 6.6 hz , 1h ) , 4.304.54 ( m , 2h ) , 4.63 ( t , j = 5.3 hz , 1h ) , 7.217.46 ( m , 6h ) , 7.477.61 ( m , 4h ) , 7.77 ( d , j = 7.2 hz , 2h ) ; lc / ms calcd for c25h19f3no4 [ m h ] 454.1 ; found 454.2 .
11.2 mg ( 48% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) : initial lc / ms purity 90% , tr = 5.39 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.12 ( dd , j = 13.8 hz , 9.2 hz , 1h ) , 3.44 ( dd , j = 14.1 hz , 5.3 hz , 1h ) , 4.054.40 ( m , 3h ) , 4.68 ( dd , j = 8.8 hz , 5.1 hz , 1h ) , 7.037.16 ( m , 1h ) , 7.187.45 ( m , 6h ) , 7.55 ( d , j = 7.6 hz , 3h ) , 7.76 ( d , j = 7.4 hz , 2h ) ; lc / ms calcd for c24h21brno4 [ m + h ] 466.1 ; found 468.0 .
10.7 mg ( 46% isolated yield ) following chromatographic purification ( ch2cl / meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 97% , tr = 5.49 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.05 ( dd , j = 13.8 hz , 6.6 hz , 1h ) , 3.17 ( dd , j = 13.9 hz , 5.5 hz , 1h ) , 4.154.51 ( m , 3h ) , 4.59 ( t , j = 5.5 hz , 1h ) , 7.067.21 ( m , 2h ) , 7.307.46 ( m , 6h ) , 7.527.63 ( m , 2h ) , 7.77 ( d , j = 7.4 hz , 2h ) ; lc / ms calcd for c24h21brno4 [ m + h ] 466.1 ; found 466.1 .
9.8 mg ( 42% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 98% , tr = 5.52 min ; h nmr ( 200 mhz , cdcl3/cd3od ) : 3.03 ( dd , j = 13.6 hz , j = 6.1 hz , 1h ) , 3.15 ( dd , j = 14.4 hz , j = 5.8 hz , 1h ) , 4.19 ( t , j = 6.2 hz , 1h ) , 4.284.51 ( m , 2h ) , 4.524.62 ( m , 1h ) , 7.01 ( d , j = 8.4 hz , 2h ) , 7.317.46 ( m , 6h ) , 7.537.62 ( m , 2h ) , 7.78 ( d , j = 7.0 hz , 2h ) ; lc / ms calcd for c24h21brno4 [ m + h ] 466.1 ; found 467.8 .
7.8 mg ( 38% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 94% , tr = 4.87 min ; h nmr ( 200 mhz , cdcl3/cd3od ) : 3.25 ( dd , j = 14.3 hz , j = 7.7 hz , 1h ) , 3.48 ( dd , j = 14.3 hz , j = 5.5 hz , 1h ) , 4.16 ( t , j = 6.7 hz , 1h ) , 4.224.44 ( m , 2h ) , 4.604.76 ( m , 1h ) , 7.267.64 ( m , 10h ) , 7.76 ( d , j = 7.0 hz , 2h ) ; lc / ms calcd for c25h19n2o4 [ m + h ] 413.1 ; found 413.2 .
7.8 mg ( 38% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 82% , tr = 4.81 min;h nmr ( 200 mhz , cdcl3/cd3od ) 3.09 ( dd , j = 13.8 hz , j = 6.2 hz , 1h ) , 3.22 ( dd , j = 13.5 hz , j = 5.1 hz , 1h ) , 4.19 ( t , j = 6.4 hz , 1h ) , 4.294.54 ( m , 2h ) , 4.58 ( m , 1h ) , 7.277.62 ( m , 10h ) , 7.77 ( d , j = 7.4 hz , 2h ) ; lc / ms calcd for c25h19n2o4 [ m h ] 412.1 ; found 411.3 .
7.5 mg ( 36% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 91% , tr = 4.79 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.10 ( dd , j = 13.7 hz , j = 7.2 hz , 1h ) , 3.193.33 ( m , 1h ) , 4.19 ( t , j = 6.4 hz , 1h ) , 4.314.55 ( m , 2h ) , 4.564.64 ( m , 1h ) , 7.227.46 ( m , 6h ) , 7.527.62 ( m , 4h ) , 7.78 ( d , j = 7.4 hz , 2h ) ; lc / ms calcd for c25h19n2o4 [ m h ] 412.1 ; found 412.5 .
11.4 mg ( 44% isolated yield ) following chromatographic purification ( ch2cl2-meoh - h2o , 300:10:0.5 ) ; initial lc / ms purity 97% , tr = 5.51 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.11 ( dd , j = 14.4 hz , j = 9.2 hz , 1h ) , 3.40 ( dd , j = 13.9 hz , j = 5.1 hz , 1h ) , 4.094.21 ( m , 1h ) , 4.224.39 ( m , 2h ) , 4.67 ( dd , j = 9.2 hz , j = 5.1 hz , 1h ) , 6.866.97 ( m , 1h ) , 7.227.44 ( m , 6h ) , 7.527.61 ( m , 2h ) , 7.75 ( d , j = 7.4 hz , 2h ) , 7.83 ( d , j = 7.8 hz , 1h ) ; lc / ms : calcd for c24h21ino4 [ m + h ] 514.0 ; found 514.0 .
7.1 mg ( 28% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 97% , tr = 5.61 min ; h nmr ( 200 mhz , cdcl3/cd3od ) : 3.01 ( dd , j = 14.2 hz , j = 6.4 hz , 1h ) , 3.14 ( dd , j = 13.7 hz , j = 5.3 hz , 1h ) , 4.19 ( t , j = 6.6 hz , 1h ) , 4.274.49 ( m , 2h ) , 4.504.62 ( m , 1h ) , 5.82 ( d , j = 8.4 hz , 1h ) , 6.89 ( d , j = 8.2 hz , 2h ) , 7.277.47 ( m , 4h ) , 7.517.64 ( m , 4h ) , 7.78 ( d , j = 7.4 hz , 2h ) ; lc / ms calcd for c24h21ino4 [ m + h ] 514.0 ; found 514.0 .
5.9 mg ( 27% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 86% , tr = 5.57 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.383.50 ( m , 1h ) , 3.75 ( dd , j = 14.2 hz , 5.4 hz , 1h ) , 4.084.22 ( m , 1h ) , 4.234.39 ( m , 2h ) , 4.73 ( dd , j = 7.4 hz , 5.6 hz , 1h ) , 7.237.55 ( m , 10h ) , 7.707.89 ( m , 4h ) , 8.16 ( d , j = 7.8 hz , 1h ) ; lc / ms calcd for c28h24no4 [ m + h ] 438.2 ; found 438.2 .
8.8 mg ( 40% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 85% , tr = 5.58 min;h nmr ( 200 mhz , cdcl3/cd3od ) 3.24 ( dd , j = 14 hz , j = 6.8 hz , 1h ) , 3.313.45 ( m , 1h ) , 4.124.45 ( m , 1h ) , 4.274.46 ( m , 2h ) , 4.69 ( t , j = 5.6 hz , 1h ) , 7.197.56 ( m , 10h ) , 7.64 ( m , 1h ) , 7.717.83 ( m , 4h ) ; lc / ms calcd for c28h24no4 [ m + h ] 438.2 ; found 438.2 .
10.2 mg ( 60% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 93% , tr = 4.74 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 2.452.71 ( m , 2h ) , 4.23 ( t , j = 6.7 hz , 1h ) , 4.304.49 ( m , 3h ) , 5.055.25 ( m , 2h ) , 5.73 ( m , 1h ) , 7.297.46 ( m , 4h ) , 7.61 ( d , j = 7.2 hz , 2h ) , 7.77 ( d , j = 6.6 hz , 2h ) ; lc / ms calcd for c20h20no4 [ m + na ] 360.1 ; found 360.1 .
9.6 mg ( 44% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 91% , tr = 5.00 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.25 ( dd , j = 13.2 hz , 9.4 hz , 1h ) , 3.63 ( dd , j = 13.9 hz , 5.5 hz , 1h ) , 4.064.20 ( m , 1h ) , 4.204.37 ( m , 2h ) , 4.70 ( dd , j = 8.6 hz , 5.4 hz , 1h ) , 7.277.59 ( m , 9h ) , 7.75 ( d , j = 7.2 hz , 2h ) , 7.94 ( d , j = 8.0 hz , 1h ) ; lc / ms calcd for c24h21n2o6 [ m + h ] 433.1 ; found 433.2 .
8.9 mg ( 39% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 89% , tr = 5.71 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.07 ( dd , j = 13.6 hz , 8.8 hz , 1h ) , 3.323.46 ( m , 1h ) , 4.16 ( m , 1h ) , 4.224.42 ( m , 2h ) , 4.62 ( dd , j = 8.4 hz , 5.4 hz , 1h ) , 7.16 ( s , 2h ) , 7.287.45 ( m , 5h ) , 7.497.61 ( m , 2h ) , 7.76 ( d , j = 6.8 hz , 2h ) ; lc / ms calcd for c24h20cl2no4 [ m + h ] 456.1 ; found 456.2 .
10.3 mg ( 49% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 94% , tr = 5.08 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.03 ( dd , j = 13.9 hz , 6.7 hz , 1h ) , 3.34 ( dd , j = 13.9 hz , 5.5 hz , 1h ) , 3.77 ( s , 3h ) , 4.19 ( t , j = 6.9 hz , 1h ) , 4.254.49 ( m , 2h ) , 4.56 ( t , j = 5.5 hz , 1h ) , 6.82 ( d , j = 7.6 hz , 2h ) , 7.07 ( d , j = 8.0 hz , 2h ) , 7.297.48 ( m , 4h ) , 7.527.64 ( m , 2h ) , 7.77 ( d , j = 7.2 hz , 2h ) ; lc / ms calcd for c25h24no5 [ m + h ] 418.2 ; found 418.2 .
7.9 mg ( 35% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 84% , tr = 5.25 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 2.94 ( dd , j = 16.7 hz , 4.5 hz , 1h ) , 3.05 ( dd , j = 17.2 hz , 5.2 hz , 1h ) , 4.22 ( t , j = 7.1 hz , 1h ) , 4.294.45 ( m , 2h ) , 4.62 ( m , 1h ) , 5.15 ( s , 2h ) , 6.18 ( d , j = 8.0 hz , 1h ) , 7.267.46 ( m , 9h ) , 7.60 ( d , j = 7.4 hz , 2h ) , 7.76 ( d , j = 7.4 hz , 2h ) ; lc / ms calcd for c26h24no6 [ m + h ] 446.2 ; found 446.2 .
5.6 mg ( 27% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) initial lc / ms purity 89% , tr = 5.14 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 3.003.26 ( m , 2h ) , 3.76 ( s , 3h ) , 4.144.46 ( m , 3h ) , 4.57 ( m , 1h ) , 6.736.83 ( m , 2h ) , 7.147.46 ( m , 6h ) , 7.527.62 ( m , 2h ) , 7.76 ( d , j = 7.0 hz , 2h ) ; lc / ms calcd for c25h24no5 [ m + h ] 418.2 ; found 418.2 .
5.5 mg ( 26% isolated yield ) following chromatographic purification ( ch2cl2/meoh / h2o , 300:10:0.5 ) ; initial lc / ms purity 80% , tr = 2.53 min ; h nmr ( 200 mhz , cdcl3/cd3od ) 2.753.00 ( m , 2 h ) , 3.95 ( t , j = 6.4 hz , 1 h ) , 4.064.38 ( m , 3 h ) , 6.947.22 ( m , 6 h ) , 7.32 ( d , j = 7.2 hz , 2 h ) , 7.52 ( d , j = 7.0 hz , 2 h ) , 7.90 ( s , 1 h ) ; lc / ms calcd for c23h19cln2o4 [ m h ] 421.1 ; found 421.0 . | distributed drug discovery ( d3 ) proposes solving large drug discovery problems by breaking them into smaller units for processing at multiple sites .
a key component of the synthetic and computational stages of d3 is the global rehearsal of prospective reagents and their subsequent use in the creation of virtual catalogs of molecules accessible by simple , inexpensive combinatorial chemistry .
the first section of this article documents the feasibility of the synthetic component of distributed drug discovery .
twenty - four alkylating agents were rehearsed in the united states , poland , russia , and spain , for their utility in the synthesis of resin - bound unnatural amino acids 1 , key intermediates in many combinatorial chemistry procedures .
this global reagent rehearsal , coupled to virtual library generation , increases the likelihood that any member of that virtual library can be made .
it facilitates the realistic integration of worldwide virtual d3 catalog computational analysis with synthesis . the second part of this article describes the creation of the first virtual d3 catalog .
it reports the enumeration of 24 416 acylated unnatural amino acids 5 , assembled from lists of either rehearsed or well - precedented alkylating and acylating reagents , and describes how the resulting catalog can be freely accessed , searched , and downloaded by the scientific community . |
liquid - based cytology ( lbc ) as a technique was introduced and tried on pap smears .
thereafter , this technique was performed for non - gynecological cytology , including conventional fine needle aspiration cytology ( fnac ) , guided fnac and fluid cytology .
the thin - prep ( tp ) smears were consistently devoid of obscuring elements and the cells were adequately preserved and evenly dispersed .
pitfalls noticed as cytomorphologic alterations in lbcs are small - sized cell clusters , with more single cells than sheets .
the cells are generally smaller , occasionally show spindling , chromatin detail is attenuated , nucleoli are more prominent , intranuclear inclusion is difficult to visualize , decrease of extracellular particles is seen , there is a decrease in the number of small mononuclear cells / red blood cells / myoepithelial cells and the background matrix is altered
. the pathologist should be cautious in interpreting fine needle aspirations prepared using tp if that is the only methodology employed .
the advantages of lbc , independent of diagnostic accuracy and morphology , have been well described .
although studies on lbc are documented for breast , non - gynecological specimen , thyroid , salivary gland and soft tissues , the authors supplemented bone lesions to all the above in their study .
lbc in fnac smears can be performed on aspirates from different organs like salivary gland , thyroid , lymph nodes , breast , bone and soft tissue and other usual and guided fnac samples .
processing of the samples is carried out using the tp kit , marketed by cytec corporation , malborough , ma , usa . in a comparison study , usually two passes are made .
the first pass is performed for the conventional preparation ( cp ) and the second pass is for the tp preparation .
tp smears are superior to cp smears in fnac ; this fact can be proved by performing a wilcoxon 's signed rank test , p value < 0.001 .
tp preparations are nowadays used in some sophisticated investigations like immunohistochemistry studies in breast lesions , non - hodgkin 's lymphoma ( nhl ) and hodgkin 's lymphoma ( hl ) in effusions , analysis of proliferating cell nuclear antigen ( pcna ) , cell blocks from scraping of cytosmears and comparison with conventional cell blocks .
the aim of our study was to determine the efficacy of lbc technology over conventional smear methods in fnac samples .
the study was performed on patients attending the cytology outpatient department ( opd ) of the hospital over a period of 2 years .
, samples were processed using the liquiprep processing kit and a special kit marketed by cytec corporation .
processing was performed manually in three steps , i.e. collection , concentration and cellular encapsulation .
the lp cytology processing kit contains a preservative solution and a cellular base . using the lbc processing kit ,
, the samples were pushed into a preservative solution , approximately three - times the volume of the sample , and left as such for at least 1 h. this is followed by the next step , i.e. concentration , where the preserved sample is centrifuged at 3000 rpm for 30 min . for cystic fluids , the supernatant fluid is discarded and , after adding the cleaning solution , it is again centrifuged for 15 min to increase its concentration .
a pellet is produced , the supernatant fluid is discarded and a base solution of about 50 microl is added to the pellet to make a thin , homogenous suspension .
this suspension was placed on an ethanol - cleaned glass slide and two circular smears of 1 cm diameter were made .
the second option , which uses a cellular base solution , was used in certain cases , particularly if the sample contains necrotic substance or mucin .
about 4 ml of cleansing solution was used in selected cases , along with the preservative solution , and the processing was performed in a similar fashion .
two passes were performed , the first pass was for cp and the second pass yielded material for the tp preparation , which was processed by the tp kit marketed by cytcec corporation .
different stains were used for staining the cp and lp smears , like he , pap and diff quick .
the representative cs and lb preparations were compared by a semiquantitative scoring system using several criteria [ table 1 ] .
the concentration method used in the procedure was useful in the diagnosis of cystic lesions like aneurysmal bone cysts , warthin 's tumor of salivary glands and metastatic carcinoma .
statistical analysis was performed using the wilcoxon signed rank test on the spss program ( chicago , il , usa ) .
every cytological diagnosis was recorded and tabulated and compiled to yield the p - value .
a total of 110 cases were studied , which were distributed among 30 cases of breast ( 10 fibroadenoma , four mastitis and 12 ductal carcinoma ) , 40 cases of lymph node ( 20 reactive hyperplasia , 12 granulomatous lymphadenitis , three lymphoma and five metastatic carcinoma ) , 10 cases of salivary gland ( two sialadenitis , five mixed tumor , one warthin 's tumor and two mucoepidermoid carcinoma ) , 18 cases of thyroid ( 10 colloid goiter , four thyroiditis and four carcinoma cases ) and 12 cases of soft tissue and bone lesions ( five benign and seven malignant ) .
comparison between cytological features in lbc and cs was carried out using the wilcoxon signed rank test separately for each organ in a tabular form , as shown in table 2 . in our study , in two cases of pleomorphic adenoma , one case of warthin 's tumor and two cases of colloid goiter , a support of cs was needed due to pitfalls in lbc described above .
wilcoxon signed rank test statistics of lbc versus cp for breast cases , fibroadenoma , mastitis and ductal carcinoma were better diagnosed on lbc preparations due to clarity of the nuclear features [ figure 1 ] .
comparison data for lymph nodes suggested that the rs cells were more easily diagnosed on lbc smears due to monolayering .
liquiprep cytosmear of the breast showing pleomorphic - looking ductal epithelial cells in a cluster and acinar pattern in a clear background in case of salivary gland lesions , the mixed salivary tumor was better visualized in the conventional smear because of preservation of the stromal component .
cystic neoplasms of the salivary glands were better appreciated in lbc smears because of concentration . in sialoadenitis ,
lbcs were not very useful in goiter and infectious lesions , but they proved very useful in neoplastic lesions [ figure 2 ] . in lytic bone lesions like aneurismal bone cyst ( abc ) , giant cell tumor ( gct ) and metastasis ,
liquiprep cytosmear showing papillary carcinoma of the thyroid in a clear background with nuclear grooving the study conducted by mygdakos et al .
lbc was favored over cs for evaluating gynecological specimens , which was approved by the fda in 1996 .
thereafter , lbc has been found to be useful in non - gynecological cytologic specimens like fnac and body fluids , both small and large volume , which have drawn variable conclusions . in conventional fnac
smears , cells are admixed with debris , blood and exudates , which make the interpretation difficult .
there is suboptimal preservation of cells and cellular obscuring by unwanted material , leading to a high proportion of cases reported as inadequate or unsatisfactory for assessment . to overcome this problem , the lbc technique was introduced , which preserves cells in a liquid medium and removes all debris , blood and exudates , either by filtration or density gradient interpretation
, there is even distribution of cell material , lack of obscuring factors , no drying artifact and monolayering . in our study , fnac was performed from various sites like breast , lymph node , salivary glands , thyroid and bone and soft tissue . in fibroadenoma breasts , although stromal fragments were lost , lbc proved to be useful in the diagnosis based on visualization of ductal aggregates and bipolar cells . for duct carcinomas ,
the wilcoxon signed ranked test for thyroid lesions [ table 2 ] , lbc was not useful in goiter and infectious lesions .
lbc was not useful in cystic neoplasms like warthin 's tumor , mucoepidermoid and adenoid cystic carcinoma and comparable to the study by parfitt et al . for bone and soft tissues , good results were obtained in lbc because of background clearing and concentration phenomenon . in all lesions of the lymph node ,
lbc proved to be a better option . because of the pitfalls of lbc , as described above , in some selected cases of granulomatous lymphadenitis ( lacking epitheloid cells ) , mucinous tumors ( lack of mucin ) , colloid goiter ( thin colloid fragmented ) and pleomorphic adenomas of the salivary gland ( myxoid background poor and present in droplets ) , a support on cs may prove useful .
as observed by the authors , lbc of bony lesions do not require support of cs .
tp preparations are superior to cps with regard to clear background , monolayer cell preparation and cell preservation .
it is easier and less time consuming to screen and interpret tp preparations because the cells are limited to smaller areas on clear backgrounds , with excellent cellular preservation .
however , tp preparations are more expensive than cp and require some experience for interpretation .
lbc is strongly advocated in the best interest of public health , by improving the quality of the sample and reducing the likelihood of false - negative cytology results . | background : liquid - based cytology ( lbc ) is fast becoming a useful method in evaluating both gynecological and non - gynecological preparations , including fine needle aspiration ( fna ) cytology
. even distribution of cells , decreasing obscuring background elements like blood and mucus , well preserved nuclear and cytoplasmic details and rapid fixation helps in better visualization of cells.aim:this study was conducted to asses the diagnostic accuracy of liquid based cytology versus conventional smears in fine needle aspiration samples.study design : in this prospective study , we had 110 cases , including 30 cases of breast , 40 of lymph node , 10 of salivary glands , 18 of thyroid and 12 of bone and soft tissue . in each case , two passes were performed .
the first pass was for conventional preparation ( cp ) and the second pass yielded material for thin - prep ( tp ) preparation .
both cp and tp smears were compared for cellularity , background blood and necrotic cell debris , cell architecture , informative background , presence of a monolayer of cells and nuclear and cytoplasmic details by a semiquantitative scoring system .
wilcoxon 's signed rank test on an spss program was used for statistical analysis.results:diagnostic accuracy was better in lbc smears compared with cp smears due to lack of background debris and better cell morphology , which was performed according to wilcoxon 's signed rank test , yielding a p - value of < 0.001 .
however , in some cases , because of a decrease in cell size , clustering and altered background in lbc , a support of cp was essential.conclusion:lbc performed on fna samples can be a simple and valuable technique . only in few selected cases , where background factor is an essential diagnostic clue , a combination of both cp and tp is necessary . |
eligible subjects were healthy individuals who voluntarily received routine comprehensive health check - ups , including computed tomography ( ct ) scan analyses of abdominal adipose tissue , from january to december 2008 at the seoul national university hospital healthcare system gangnam center .
data were collected through a questionnaire , anthropometric measurements , blood samples , and ct scans .
detailed descriptions of the method used for the measurement of abdominal adipose tissue have been published previously ( 8) . in brief , a 5-mm thick umbilical level abdominal section from 16-detector row ct imaging of the abdomen was obtained .
the cross - sectional area ( cm ) of the abdominal fat was calculated using rapidia 2.8 ct software ( infinitt , seoul , korea ) .
the vat area was defined as intra - abdominal fat bound by parietal peritoneum or transversalis fascia , and the sat area was calculated by subtracting the vat area from the total adipose tissue area . because of the high colinearity between sat and vat , adjustments to the regression model or stratification of vat are insufficient to control for the effects of vat on sat .
the ratio between sat and vat ( sat / vat ratio [ svr ] ) was therefore calculated and used in the current study .
mets was defined according to the modified national cholesterol education program adult treatment panel iii ( 9 ) and asia - pacific abdominal obesity criterion ( 10 ) . means with 95% ci and
numbers of participants ( % ) with metabolic risk factors were compared across the svr quintiles .
we used multivariate logistic regressions to evaluate the association between mets and svr after adjusting for age , smoking , alcohol consumption , bmi , menopausal status , and hormonal replacement therapy ( women only ) .
data are expressed as odds ratio ( or ) [ 95% ci ] ; and p for trend .
detailed descriptions of the method used for the measurement of abdominal adipose tissue have been published previously ( 8) . in brief , a 5-mm thick umbilical level abdominal section from 16-detector row ct imaging of the abdomen was obtained .
the cross - sectional area ( cm ) of the abdominal fat was calculated using rapidia 2.8 ct software ( infinitt , seoul , korea ) .
the vat area was defined as intra - abdominal fat bound by parietal peritoneum or transversalis fascia , and the sat area was calculated by subtracting the vat area from the total adipose tissue area . because of the high colinearity between sat and vat , adjustments to the regression model or stratification of vat are insufficient to control for the effects of vat on sat .
the ratio between sat and vat ( sat / vat ratio [ svr ] ) was therefore calculated and used in the current study .
mets was defined according to the modified national cholesterol education program adult treatment panel iii ( 9 ) and asia - pacific abdominal obesity criterion ( 10 ) .
means with 95% ci and numbers of participants ( % ) with metabolic risk factors were compared across the svr quintiles .
we used multivariate logistic regressions to evaluate the association between mets and svr after adjusting for age , smoking , alcohol consumption , bmi , menopausal status , and hormonal replacement therapy ( women only ) .
data are expressed as odds ratio ( or ) [ 95% ci ] ; and p for trend .
forty percent of men and 25% of women had elevated blood pressure , 31% of men and 15% of women had elevated fasting glucose , and 25% of men and 14% of women had mets .
the mean sat and vat areas were 139.8 cm and 134.4 cm in men and 173.1 cm and 80.0 cm in women , respectively .
the prevalence of elevated fasting glucose , elevated triglycerides ( tgs ) , and reduced hdl cholesterol ( hdl - c ) significantly decreased as the svr quintile increased ( 26 , 16 , and 37% in quintile 1 vs. 5 , 4 , and 11% in quintile 5 , respectively ; all p for linear trend < 0.05 ) robust linear decreases in these risk factors were also seen among men ( supplementary table 1b ) .
ors of mets significantly decreased across all 5 quintiles of the svr after adjusting for covariates in both men ( or 1.0 for quintile 1 vs. 0.5 in quintile 5 ; 95% ci 0.30.7 for quintile 5 ; p for linear trend < 0.01 ) and women ( or 1.0 for quintile 1 vs. 0.2 in quintile 5 ; 95% ci 0.10.5 for quintile 5 ; p for linear trend < 0.01 ) ( table 1 ) .
unadjusted and adjusted ors and 95% ci from logistic regressions predicting the likelihood of mets for each of the svr quintiles * adjusted ors take into consideration age , smoking , alcohol consumption , bmi , and menopausal status , hormone replacement therapy ( women only ) .
ors of mets significantly decreased across all 5 quintiles of the svr after adjusting for covariates in both men ( or 1.0 for quintile 1 vs. 0.5 in quintile 5 ; 95% ci 0.30.7 for quintile 5 ; p for linear trend < 0.01 ) and women ( or 1.0 for quintile 1 vs. 0.2 in quintile 5 ; 95% ci 0.10.5 for quintile 5 ; p for linear trend < 0.01 ) ( table 1 ) .
unadjusted and adjusted ors and 95% ci from logistic regressions predicting the likelihood of mets for each of the svr quintiles * adjusted ors take into consideration age , smoking , alcohol consumption , bmi , and menopausal status , hormone replacement therapy ( women only ) .
the purpose of this study was to assess the cross - sectional correlation of abdominal sat with mets in a korean study population .
we found that as the svr increased , mets components such as reduced hdl - c , elevated tgs , and elevated fasting glucose decreased in both sexes . furthermore , this relationship was maintained even after the adjustment for a variety of variables .
small adipocytes in sat play the role of powerful buffers , uptaking circulating free fatty acids and tgs in the postprandial period .
but if they lose their function in adipogenesis or fat storage capacity in sat , fat begins to accumulate in tissues not suited for lipid storage such as vat .
peroxisome proliferatoractivated receptor- ( ppar- ) agonists stimulate the differentiation of adipocytes , resulting in increased accumulation capacity in sat ( 12 ) .
the ppar- coactivator 1 mrna expression is significantly higher in subcutaneous fat than in omental fat ( 13).this mechanism explained the finding that treatment with thiazolidinedione improves insulin sensitivity ( 6 ) . vat and sat differ in cytokine secretion profile such as leptin , adiponectin , interleukin-6 , interleukin-8 , plasminogen activator inhibitor 1 , and angiotensin , which may play some role in the development of mets ( 14,15 ) .
longitudinal associations of vat / sat change with mets , and with leptin and/or adiponectin level should be the focus of future studies .
gluteofemoral sat , which may act as a confounding factor , was not considered in the study .
however , the current study included the analysis of a large dataset of ct - measured sat and vat .
moreover , the study population was generally healthy , which implies that the results can be applied to the general korean population . | objectivethis study aimed to assess the correlation between abdominal subcutaneous adipose tissue ( sat ) and metabolic syndrome ( mets ) in korean adults after adjusting for the effects of visceral adipose tissue ( vat).research design and methodsthe sat / vat ratio ( svr ) was calculated using abdominal computed tomography in 2,655 subjects .
we used regression analyses to assess whether the svr predicted mets.resultsfor both sexes , the prevalence of elevated triglycerides , reduced hdl , and elevated fasting glucose significantly decreased with increasing quintiles of svr ( p for trend < 0.05 ) . the prevalence and odds ratios of mets significantly decreased as the svr increased ( men : odds ratio 0.5 [ 95% ci 0.30.7 ] ; women : 0.2 [ 0.10.5 ] for comparisons of lowest vs. highest quintile ; p for trend < 0.05).conclusionsafter adjustment for vat , abdominal sat was inversely correlated with the occurrence of mets . |
recurrent aphthous stomatitis ( ras ) is the most common recurrent lesion of the oral epithelium .
the prevalence of ras was reported to be 40% in a population of children of the united states .
a prevalence of 31 - 36% has been reported for ras among medical and dental students , compared with 10 - 20% in the general population .
although few data are available regarding the etiologies and management of aphthous lesions , factors such as inheritance , hormonal effect and immunological factors have been considered .
lesions usually begin due to local trauma , stress , certain foods or drugs , hormonal fluctuations or deficiencies in the vitamin b group , folate or iron .
apart from the different causes and predisposing conditions affecting the disease , pain , recurrence , self - limitation and epithelial damage are present in almost all lesions .
recurrent aphthous lesions are divided into three groups including : minor , major and herpetiform , based on the clinical appearance and prognosis of the disease .
all the existing treatment options are based on symptomatic management , lesion count reduction and/or extending the lesion - free periods .
blood samples should then be assessed for deficiencies in serum iron , folate , ferritin and vitamin b12 .
most patients suffering from minor aphthous lesions need no special treatment or only local treatment to adequately reduce their symptoms or lesion counts as well as to increase the interval between aphthous episodes . in patients suffering from major aphthous lesions or in those susceptible to numerous minor lesions that are resistant to local treatment
the administration of local antibiotics such as tetracycline was effective in aphthous lesion management , probably due to a secondary decrease in infection and the clinical improvement of lesions .
most therapeutic approaches are based on palliative rather than curative therapy , and none of them could prevent the recurrence of the symptoms
avishan - e shirazi is a thyme - like essential oil ( eo)-bearing plant that belongs to the lamiaceae family and grows extensively wild in the central and southern parts of iran , pakistan , and afghanistan .
the dry aerial parts of the plant have been used for their flavor and preservative properties in the food products industry . in iran , zm is mainly used in traditional folk remedies for its antiseptic , analgesic , and carminative ( antiflatulence and intestine - soothing ) properties .
it also has been reported that the eos and extracts of zm can stimulate innate immunity and have antibacterial and antifungal activities .
thus , the present study was aimed to investigate the therapeutic effects of zm for treatment of oral aphthous lesions .
this triple blind clinical trial was performed on 28 patients who were randomly selected from all patients suffering from recurrent aphthous lesions who were admitted to the department of oral medicine , faculty of dentistry , babol university of medical sciences in the north of iran .
the study protocol was approved by research review board and research ethics committee of babol university of medical sciences and we registered the protocol in the iranian clinical trials registry ( irct201408076926n2 ) .
the inclusion criteria were as follows :
presence of a minor solitary aphthous lesion in labial or buccal epithelium.no usage of other treatments for aphthous lesions.no reduction in pain , pain intensity or burning sensation at the time of admission compared to the 1 days of the occurrence of the lesion.willingness to participate in the study.age between 18 and 40 years .
no reduction in pain , pain intensity or burning sensation at the time of admission compared to the 1 days of the occurrence of the lesion .
the exclusion criteria were : patients with any systemic diseases or special syndrome that aphthous ulcer is one of its symptoms ( behcet 's syndrome ) ; pregnant patients ; those with aphthous lesions older than 4 days , patients subjected to any other treatment for at least 4 weeks before the start of the study and those who declined entering the study .
diagnosis of minor aphthae was made on the basis of the patient 's health history , clinical examination and the presence of a well demarcated painful ulcer on the smooth unattached oral mucosa , which is surrounded by a light red areola . after explaining the objectives of the study ,
after that , the eligible participants were divided into zm- and placebo - treated groups randomly .
the patients received a bottle of 0.5% zm eo or a bottle of placebo ( distilled water with a cap scented with zm eo ) .
standard zm essence was obtained from barij essence pharmaceutical company ( kashan , iran ) .
all bottles were coded by a third person who did not involve in the project , so that the contents of the bottles remained unknown to the researchers and patients throughout the study . to administer the drug or placebo , 15 drops of drug / placebo were diluted into one half cup of cold water and used as a mouthwash for 2 min 3 times a day , once after each meal .
this therapy was continued until complete healing ( no ulcer and no burning sensation ) was obtained .
patients were examined by the clinician on the 1 , 5 and 7 days of treatment .
the pain intensity was measured using a scale , which the patients completed every day for 10 days ( before and after treatment ) .
in total of 14 participants in each group ( eight men and six women ) , all of them finished the study .
a history of aphthous lesions was reported for 3 patients in the placebo group and 7 patients in the zm group ( p = 0.236 ) . during the 6-month follow - up
, recurrence had occurred in 4 and 6 patients in the placebo and zm groups , respectively .
the mean ( standard deviation ) treatment durations were 8.5 1.2 and 8.4 1.2 days in the placebo and zm groups , respectively ( p = 0.765 ) . complete healing and burning sensation cut - off times ( the time in which the patient had no pain thereafter ) were significantly decreased in the zm group compared to the placebo group [ table 1 ] .
there were also significant differences between the lesion and halo diameters in the two groups [ tables 2 and 3 ] .
as illustrated in figure 1 , the peak pain score occurred between days 3 and 5 .
mean ( sd ) complete healing and burning sensation cut - off times ( days ) in the placebo and zm groups mean ( sd ) halo diameters ( mm ) in the placebo and zm groups mean ( sd ) lesion diameters ( mm ) in the placebo and zm groups mean ( standard deviation ) pain intensity according to officinale in the placebo and zataria multiflora groups .
our findings revealed that the management of ras lesion with zm could indicate a significant decrease in the characteristic of the lesion ( diameter of the lesion and halo ) , complete healing time and burning sensation of the patients .
ras is a common oral disorder of uncertain etiopathogenesis , and presently , its management is largely focused on symptomatic treatment .
ras causes considerable pain and distress for patients and presents a difficult management challenge for clinicians .
different classes of chemical and biochemical products have been reported to be of some benefit in the management of ras , but no definitive treatment is yet available .
zm eo has been reported to have antibacterial , antiviral , antioxidant , and anti - inflammatory properties .
this study was undertaken to evaluate the therapeutic effects of zm eo for treatment of recurrent aphthous lesions .
the results of the study showed that the complete healing time in the drug group was significantly shorter than that in the placebo group .
in other words , using zm led to a reduction in the time required for the lesions to heal . a review of the literature yielded few reports on the effectiveness of zm eo in treating oral aphthous lesions .
the mechanism of action that play the major role in the healing process of these lesions may due to its anti - microbial role and ant - oxidative effect on the aphtous reactivity .
nevertheless , there are many reports on the efficacy of other herbal drugs for treatment of the disease . in a study by mansoori et al .
, zm eo effectively resulted in a faster pain relief and shorter healing period . in a study conducted by jafari et al .
zm extract was a more effective treatment than myrthus communis , which is reported to be effective in the treatment of ras .
it was concluded that zm extract was an effective treatment for the management of minor aphthae .
antimicrobial and anti - oxidant activities of four tai plants ( quercus infectoria , kaempferia galanga , coptis chinensis and glycyrrhiza uralensis ) for aphthous ulcers were shown by mekseepralard et al .
previous studies have evaluated the therapeutic effects of satureja khuzestanica extract for the treatment of aphthous lesions of the oral epithelium .
these studies have demonstrated the extract to be more effective than placebo in reducing the duration of suffering from pain and burning sensation as well as the time needed for complete clinical resolution .
it has also been shown that myrtle is an effective treatment in terms of size change , pain severity , the level of erythema and exudation in ras .
moghadamnia et al . evaluated the efficacy of licorice bioadhesive hydrogel patches for controlling the pain and reducing the healing time of recurrent aphthous ulcers .
they found that licorice bioadhesive can be effective in the reduction of the pain and of the sizes of the inflammatory halo as well as the necrotic center of aphthous ulcers . in another clinical trial using herbal options muco - bioadhesive containing ginger officinale
extract was indicated relieving pain of ras but had no efficacy on ulcer diameter , inflamed halo and healing time when compared to a placebo .
our study 's limitation was the small sample size and the fact that we were not completely sure whether the patients consumed the zm as any form ( mouthwash or food ingredient ) in the follow - up period or not . within these limitations ,
herbal medicines provide a wider range of treatment options for patients and practitioners . they have limited complications and can be useful alternatives to chemical medicines .
according to our findings , zm can reduce the ras symptoms and characterisitic of the aphthous lesions compared to the placebo group .
future studies with larger sample sizes are suggested examining the role of zm in the management of ras .
the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article .
the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article . | background : aphthous lesions are one of the most common diseases of the oral cavity .
they can cause severe pain , and there is no definite treatment .
the purpose of this study was to determine the efficacy of zataria multiflora ( zm , a thyme - like plant ) essential oil for the control and treatment of aphthous lesions.materials and methods : this triple blind clinical trial study was performed on 28 patients who were divided into two groups ( eight men and six women in each group ) and given zm or placebo ( control ) .
the healing time , pain intensity , and aphthous zone diameter were recorded for each patient and followed for 6-month .
data were analyzed using mann whitney and friedman tests ( p < 0.05).results : after 6-month of follow - up , 4 patients in the placebo group and 6 patients in the zm group suffered from recurrent aphthous lesions . the average complete healing time and duration of burning sensation were significantly lower in the zm group ( p < 0.05 ) .
significant difference was observed between the two groups with regard to the diameter of lesions and halo of the lesions ( p < 0.05).conclusion : within the limitations of this study , zm shortened the healing period compared to placebo . |
many studies have demonstrated considerable effects of diabetes mellitus ( dm ) on all the layers of the cornea .
these studies have documented that diabetic cornea is vulnerable to many abnormalities such as corneal endothelial damage , recurrent corneal erosions , punctate epithelial keratopathy , persistent epithelial defects , impaired corneal sensitivity , ulcers , slowed wound repair , and superficial keratitis.13 morphological features of the corneal endothelium in diabetic patients recognized by a number of studies include a decrease in endothelial cell density ( ecd ) and polymorphism ( decrease in the percentage of hexagonal cells [ the normal percentage > 50% ] ) as well as polymegathism , which means increased coefficient of variation ( cv ) of cell area ( cv values measured between 0.22 and 0.31 are considered normal and above 0.4 are abnormal ) and increased central corneal thickness ( cct).46 em-3000 specular microscope ( cbd / tomey , phoenix , az , usa ) is a noncontact specular microscope that avoids some disadvantages of the classic contact specular microscopes such as corneal ulceration and transmission of infectious diseases .
it has the ability to assess corneal endothelium features and measure cct at the same time with accuracy of 10 m.7 the aim of this study was to compare corneal ecd , morphological features , and cct in type 2 dm with age - matched , nondiabetic control subjects using em-3000 specular microscope .
in addition , correlation between these corneal changes and the duration of dm and severity of diabetic retinopathy ( dr ) and glycemic control ( glycated hemoglobin [ hba1c ] level ) were also assessed .
it got the approval of research ethics committee of college of applied medical sciences , king saud university , and security forces hospital , riyadh , saudi arabia .
all the participants signed comprehensive consent after explanation of the possible consequences of the study prior to investigations .
the study included 57 patients ( 27 males and 30 females [ 57 eyes ] ) with type 2 dm and 45 control ( nondiabetic ) subjects ( 45 eyes [ 22 males and 23 females ] ) .
the age range of diabetic group was 4280 years ( mean : 57.088.37 years ) , and the age range of the control group was 4076 years ( mean : 50.808.39 years ) .
all patients were recruited from ophthalmology department at security forces hospital , riyadh , saudi arabia , by nonrandomized convenience sampling method from february to april 2015 .
the diagnosis of type 2 dm was based on the criteria of the world health organization ( who).8 control patients did not have diabetes was confirmed by random blood sugar test .
the mean duration of dm was 12.878.03 years ( range : 140 years ) , and the mean value of hba1c was 8.57%2.09% ( range : 6.3%17.2% ) .
exclusion criteria included presence of high myopia , history of past intraocular surgery or corneal disease or dystrophy such as fuch s endothelial dystrophy , active or previous eye infection or inflammation , previous ocular surgery or trauma , previous retinal photocoagulation , pterygium , entropion , trichiasis , contact lens wear , regular usage of any eyedrops or known tear - interfering systemic drugs ( such as hormone replacement and antihistamines ) , systemic illness such as rheumatoid arthritis and systemic lupus erythematous that are known to impair tear function , pregnancy , labor , and glaucoma .
age , gender , duration of diabetes , most recent hba1c value , status of dr , other medical illness , and current medical treatment were recorded . in this study , only the right eye of each patient was tested .
all subjects underwent a complete ophthalmic examination that included visual acuity assessment using the snellen chart , refraction using auto - refractometer ( topcon , tokyo , japan ) , and intraocular pressure measurement using air puff tonometer , slit - lamp biomicroscopy , and fundus examination . the corneal endothelial structure and cct
were examined in all eyes by noncontact specular microscopy using em-3000 specular microscope , and this examination was done by a single examiner .
the endothelial structure was studied by measuring a variety of factors , including ecd , cv , and percentage of hexagonal cells .
his / her chin was placed on the chin rest and his / her forehead rested on the special head area .
the patient was asked to fixate his / her eye for a few seconds on the red light coming from inside the device until the instrument automatically took a clear image of the corneal endothelium and measured the cct at the same time .
the diabetic eyes were classified into 3 groups according to the status of dr ( group without dr , group with nonproliferative dr [ npdr ] , and group with proliferative dr [ pdr ] ) .
also , they were divided into 2 groups according to dm duration ( group with a duration of 10 years and group with a duration of > 10 years ) and according to hba1c value ( group with hba1c 7.5% and group with hba1c > 7.5% ) .
the data were analyzed using spss 21 for windows software ( ibm , armonk , ny , usa ) .
an independent sample t - test was performed to compare the means of cct and corneal endothelium features in diabetic and control groups .
the differences among 3 or more groups were analyzed by one - way analysis of variance .
pearson correlation test was performed to find the relationship between corneal changes and dm duration , hba1c% , and dr status .
the study included 36 eyes without dr , 14 eyes with npdr , and 7 eyes with pdr .
there were 26 eyes with dm duration of 10 years and 31 eyes with dm duration of > 10 years .
also , there were 24 eyes with hba1c 7.5% and 33 eyes with hba1c > 7.5% .
ecd was significantly lower in the diabetic cornea group ( 2,491.98261.08 cell / mm ) than in the control group ( 2,629.68293.45 cell / mm ) ( p=0.014 ) .
cv was higher in the diabetic cornea group ( 0.410.07 ) than in the control group ( 0.370.08 ) ( p=0.008 , which was considered statistically significant ) .
diabetic cornea group had lower percentage of hexagonal cells ( 33.24%10.25% ) than control group ( 34.24%8.73% ) , but the difference was not statistically significant ( p=0.603 ) .
also , cornea was thicker in the diabetic group ( 545.6130.39 m ) than control group ( 539.4229.22 m ) , but not statistically significant ( p=0.301 ) .
a comparison of the mean values of cct , ecd , cv , and hexagonality between control and diabetic eyes is shown in table 3 .
eyes with dm duration of 10 years had higher ecd ( 2,509.38303.49 cell / mm ) than eyes with dm duration of > 10 years ( 2,477.38223.61 cell / mm ) ( p=0.658 ) , and they also had more hexagonality ( 35.61%9.87% ) than those with dm duration of > 10 years ( 31.26%10.29% ) ( p=0.111 ) , but the differences were not significant . also , there were no significant differences in cct ( p=0.431 ) and cv ( p=0.927 ) between the 2 groups .
a comparison of the mean values of cct , ecd , cv , and hexagonality between the diabetic groups according to the duration of dm is shown in table 4 .
specular microscope pictures taken from diabetic eyes with dm duration of > 10 years and 10 years are shown in figures 2 and 4 .
diabetic patients with hba1c% 7.5 had higher , but not statistically significant , ecd ( 2,537.62311.86 cell / mm ) than those with hba1c% > 7.5 ( 2,458.78216.06 cell / mm ) ( p=0.293 ) and higher hexagonality ( 35.58%10.09% ) than the other group ( 31.54%10.17% ) ( p=0.144 ) .
the mean of cv was significantly lower ( p=0.017 ) in diabetic patients with hba1c% 7.5 ( 0.390.05 ) than in the other group ( 0.430.07 ) .
also , there was no statistically significant difference in cct between the 2 groups ( 0.789 ) .
a comparison of the mean values of cct , ecd , cv , and hexagonality between the diabetic groups according to hba1c% is shown in table 5 .
in addition , there were no significant differences between cct , ecd , cv , and hexagonality percentage of the corneal endothelial cells in diabetic patients without dr , with npdr , and with pdr ( p=0.344 , 0.806 , 0.284 , and 0.500 , respectively ) ( table 6 ) .
pearson correlation analysis showed that dm duration , hba1c levels , and severity of dr had no significant correlations with cct , cv , hexagonality , or ecd ( table 7 ) .
diabetic cornea may give the impression that is healthy , but actually it may suffer from many morphological features affecting its function later on .
this fact was evidenced by several studies.16 in this study , we found that diabetic corneas showed a statistically significant reduction in mean corneal ecd of 5.24% ( p=0.014 ) compared to the corneas in the control group .
this was similar to the results of choo et al4 of 4.5% significant reduction in ecd ( p<0.01 ) in malaysian type 2 dm patients .
also , the results of inoue et als9 study of the corneal endothelium and cct in type 2 dm in japan showed similar results of 4.1% ( p=0.016 ) significant reduction in ecd in the diabetic group .
however , results of storr - paulsen et als6 and siribunkum et als10 studies of corneal endothelium morphology in type 2 dm according to ecd were not consistent with our findings .
the cv of corneal endothelial cells was found to be significantly increased in diabetic patients ( p=0.008 ) .
this increase indicated the presence of polymegathism , in which endothelial cells enlarge to fill the gaps between adjacent cells .
this result was concordant with those obtained by shenoy et al11 and lee et al,12 but not similar to those of sudhir et al5 and chen et al.13 also , our study demonstrated no significant difference in the percentage of hexagonal cells between the diabetic patients and the controls , which was in agreement with the results of sudhir et al,5 storr - paulsen et al,6 and inoue et al,9 and in disagreement with those of choo et al,4 and lee et al.12 many studies4,14,15 have explained the morphological features of diabetic cornea .
this was established by the assessment of polyol ( sorbitol aldose reductase ) pathway in diabetic cornea .
these studies documented that high glucose levels lead to increased activity of the aldose reductase , causing sorbitol buildup in the corneal epithelial and endothelial cells .
also , dm diminishes na k atpase activity of the corneal endothelium , resulting in morphological and permeability changes in diabetic cornea , thereby leading to corneal destruction .
furthermore , endothelial pump function was proven to be affected by decreased atp production as a result of slowing down of the krebs cycle in diabetic cornea .
regarding cct in our study , there was no significant difference between diabetic and control groups .
this result was in concordance with those of choo et al4 and sudhir et al.5 other studies , for instance , storr - paulsen et al6 and lee et al,12 demonstrated a statistically significant increase in cct of diabetic compared to control subjects .
as regards the comparison of the mean values of cct , ecd , cv , and cell hexagonality in patients with dm duration of 10 years and those with dm duration of > 10 years , no statistically significant difference was detected in all these morphological features in this study .
this finding was in agreement with altay et al,16 who reported absence of statistically significant difference in cct between the 2 groups . however , lee et al12 documented that cct and cv were significantly higher in patients with dm duration of > 10 years compared with those with dm duration of 10 years , but there was no statistically significant difference regarding ecd and hexagonality between the 2 groups . concerning the comparison of the mean values of cct , ecd , cv , and cell hexagonality in diabetic patients with hba1c 7.5% and those with hba1c
> 7.5% in our study , no significant differences were detected between the 2 groups in cct , ecd , and hexagonality , but cv showed a statistically significant difference ( p=0.017 ) .
storr - paulsen et al6 found significantly lower ecd in patients with elevated hba1c , but without any influence on cct . however , altay et al16 documented significantly thicker cct in hyperglycemic condition than in euglycemic condition in the same patient before and after successful control of hba1c . regarding the comparison of the mean values of cct , ecd , cv , and hexagonality between the 3 diabetic groups according to the status of dr , our study found no significant differences between the 3 groups .
this finding was not in accordance with that of shenoy et al,11 who demonstrated significantly lower ecd in eyes with dr than those without dr .
pearson correlation analysis reported absence of significant correlations between dm duration , hba1c levels , and severity of dr and morphological features of diabetic cornea such as cct , cv , hexagonality , or ecd .
these results were similar to those of choo et al.4 however , siribunkum et al10 found a significant correlation between dm duration and pleomorphism , polymegathism , and cct .
this study documented that type 2 dm resulted in a significant reduction of ecd and increased cv ( polymegathism ) . also , diabetic cornea had increased cct and lower percentage of hexagonal cells than in the cornea of normal subjects , but this is not statistically significant .
in addition , our study demonstrated that dm duration , hba1c levels , and severity of dr had no significant correlations with cct , cv , hexagonality , or ecd . in this study , patients with a history of laser photocoagulation were excluded due to its influence on the corneal structures.17 this exclusion produced a small sample size because most of diabetic patients who came to the clinic had undergone laser treatments .
therefore , further studies with a large sample size are recommended to verify the extent of corneal damage caused by type 2 dm . | purposethis study was conducted to compare the corneal endothelial cell density ( ecd ) , morphological features , and central corneal thickness ( cct ) in type 2 diabetes mellitus ( dm ) with age - matched , nondiabetic control subjects using em-3000 specular microscope.study designthis was a prospective , hospital - based , nonrandomized , case
control , observational , and quantitative study.subjects and methodsthe study included 57 patients ( 57 eyes ) with type 2 dm and 45 control ( nondiabetic ) subjects ( 45 eyes ) .
the corneal endothelial structure and cct were examined in all eyes by noncontact specular microscopy using em-3000 specular microscope .
the endothelial structure was studied for ecd , coefficient of variation of cell area ( cv ) , and percentage of hexagonal cells.resultsthe study included 36 eyes without diabetic retinopathy ( dr ) , 14 eyes with nonproliferative dr , and 7 eyes with proliferative dr .
there were 26 eyes with a duration of 10 years and 31 eyes with a duration of > 10 years .
also , there were 24 eyes with hba1c 7.5% and 33 eyes with hba1c > 7.5% .
ecd was significantly lower in the diabetic cornea than in control group ( p=0.014 ) .
cv was higher in diabetic cornea ( p=0.008 ) .
the diabetic cornea group had lower percentage of hexagonal cells than the control group , but the difference was not statistically significant ( p=0.603 ) .
also , diabetic cornea was thicker than control group , but not statistically significant ( p=0.301).conclusionthis study documented that type 2 dm causes a significant reduction of ecd and increased cv ( polymegathism ) .
also , diabetic cornea has increased cct and lower percentage of hexagonal cells than normal subjects , but without statistical significance . |
microbial control in the endodontic system represents a challenge to the solution of refractory infections when chemical and surgical procedures do not provide the desired response for control of infection .
the chemomechanical preparation used during endodontic therapy is the main factor responsible for eliminating the microorganisms lodged into the root canal system20 . in most clinical situations ,
however , some cases demand the application of intracanal medication as an adjuvant therapy to resolve the infection .
this occurs because microorganisms may colonize the endodontic system , demanding deeper and more guaranteed therapeutic action . in these cases , intracanal medication not only plays the role of inflammatory reaction moderator , but is also imperative to combat the remaining infection . in most cases ,
this resistance is related to microbial capacity to remain in a latent phase , at a low metabolic rate for a long period of time .
however , environmental changes , such as coronal leakage , may lead to activation of these microorganisms and their consequent proliferation6,14 .
associations of antimicrobial agents have been proposed with the goal of increasing the spectrum of action and combating the persistent infection9,11,17 .
the choice of vehicle associated with intracanal medication is also of paramount importance , as they are responsible for transporting the drug to the interior of the root canal systems and for the speed of its diffusion24 .
the purpose of this study was to investigate the antimicrobial action of ciprofloxacin hydrochloride and metronidazole , and the vehicles polyethylene glycol and natrosol , either alone or associated , supported by the fact that intracanal medication is an important adjuvant of the endodontic treatment protocol .
twenty - three microbial strains were selected from the american type culture collection ( atcc ) and the university of taubat culture collection ( ccut ) ( table 1 ) . the susceptibility of the microorganisms to the antimicrobial agents metronidazole ( henrifarma , china ) and ciprofloxacin hydrochloride ( pharmanostra , usa ) and to the vehicles polyethylene glycol ( pm 1000 ) and natrosol , was assessed , either alone and in the following associations : a ) ciprofloxacin and metronidazole ; b ) ciprofloxacin and natrosol ; c ) ciprofloxacin and polyethylene glycol ; d ) metronidazole and natrosol ; e ) metronidazole and polyethylene glycol ; f ) ciprofloxacin , metronidazole and natrosol ; and , g ) ciprofloxacin , metronidazole and polyethylene glycol .
the antimicrobial agents were submitted to filtration ( 0.22 m , millipore membrane ) and the vehicles were sterilized by gamma radiation ( co , gammacell , atomic energy of canada limited , canada ) .
the mller - hinton agar ( difco , detroit , mi , usa ) dilution method was used to determine the minimum inhibitory concentration ( mic)15 .
culture media were prepared , adding antimicrobial agents in two - fold serial dilutions , obtaining final concentrations of 16 to 0.25 g / ml of medium . for the vehicles ,
culture media containing 50 , 45 , 40 , 35 , 30 and 25% were used . for the associations ,
the substances were added to the culture media at the same concentrations and percentages used for the substances tested separately , in a total of 292 combinations . for each test ,
the selected microorganisms were plated in mller hinton agar and incubated at 37c for 24 hours . after growth and morphologic confirmation in smears stained by gram method ,
suspensions were prepared with isolated colonies and sterile saline in accordance with tube 0.5 ( approximately 1.5 10 cells / ml ) of mcfarland scale , for the bacteria , and tube 2 ( approximately 10 cells / ml ) for yeasts ( recommendation of the national committee for clinical laboratory standards12,13 ) .
next , aliquots of the suspensions were placed into the wells of the steers replicator , and then plated in the culture media .
the plates were incubated at 37c for 24 hours and the occurrence of microbial growth was evaluated .
the mic was defined at the lowest concentration of the drug that inhibited microbial growth .
minimum inhibitory concentration results for the antimicrobial agents ciprofloxacin and metronidazole , and for the vehicles ( natrosol and polyethylene glycol ) are shown in table 2 .
r = resistant at the highest concentration tested ( 16 lg / ml or 50% ) ; s = sensitive at the lowest concentration tested ( 0.25 g/ ml or 25% ) ; interval of concentration of the tested products
table 3 shows the results of the association of ciprofloxacin with the vehicles .
ciprofloxacin / natrosol association partially increased the effects of the products , also presenting effects against candida species .
s = sensitive at the lowest concentration tested ( 0.25 g / ml or 25% ) ; interval of concentration of the tested products
table 4 shows the results of the association of metronidazole with the tested vehicles .
metronidazole/ polyethylene glycol association inhibited all microbial strains , using low concentrations of the antimicrobial agent , as well as low percentages of polyethylene glycol .
resistant at the highest concentration tested ( 16 lag / ml or 50% ) s sensitive at the lowest concentration tested ( 0.25 g / ml or 25% ) interval of concentration of the tested products the mic values of ciprofloxacin / metronidazole association and these with the vehicles are expressed in table 5 .
the association of ciprofloxacin with metronidazole presented lower mic for the bacteria than ciprofloxacin alone , except for escherichia coli .
polyethylene glycol added to the formulation eliminated all bacteria , at the lowest concentration of antimicrobial agents and vehicles .
the performance of ciprofloxacin / metronidazole / polyethylene glycol association was equivalent or lowest to the association ciprofloxacin / polyethylene glycol .
r = resistant at the highest concentration tested ( 16 lag / ml or 50% ) ; s = sensitive at the lowest concentration tested ( 0.25 g/ ml or 25% ) ; > interval of concentration of the tested products . in all plates of culture media
in which the antimicrobial agents and vehicles were not added ( controls ) , there was growth of all microbial strains .
these difficulties are generally related to the anatomic characteristics of the root canal system7 , resistance to antimicrobial agents3,6 and the presence of apical biofilm18,25 . for cases of resistance to antimicrobial agents ,
the use of alternative medications , or the combination of antimicrobial agents is suggested to increase the spectrum of action and act particularly on the facultative anaerobic microorganisms . in endodontics ,
antimicrobial agents are used topically , due to the absence of blood irrigation in the root canal system at the time of therapy . in most cases ,
intracanal medication is used in aqueous solution , allowing the system to be well filled . however , the capacity to remain there and to have a prolonged action is limited and it is in contact with the healthy or contaminated periodontal fluid .
the vehicle associated with the antimicrobial agents has a strict relationship with the time of action of the antimicrobial agent19 , penetration of the medication in the root canal system2,24 , in addition to the dissociation of the principle of the drugs5,8 .
the results of the present study demonstrated , although metronidazole had no action against the tested microorganisms , which was expected , the ciprofloxacin/ metronidazole association resulted in a positive interaction , that is , there was synergism in this proposed combination .
slots , et al.21 ( 1990 ) found that the ciprofloxacin/ metronidazole association was more effective against enterobacteria collected from periodontal pockets than ciprofloxacin used alone .
all mic values for this association were lower than those of ciprofloxacin used alone or combined with metronidazole .
another advantage of the association ciprofloxacin / polyethylene glycol was the effective action on yeasts resistant to the other formulations .
all tested bacteria were sensitive to the association of ciprofloxacin and metronidazole with the vehicle polyethylene glycol , while c. albicans and c. tropicalis were eliminated with this vehicle in concentrations equal to or lower than 35% by volume of the solution .
metronidazole at 16 g / ml concentration and lower , associated with polyethylene glycol at a percentage of 30 to 50% , was responsible for eliminating all strains , including the yeasts .
as the volume of polyethylene glycol in the culture media was reduced , the number of resistant strains increased .
the results obtained were expected , since the action spectrum of metronidazole is selective for anaerobic bacteria .
therefore , the importance of the results is based on the knowledge of the interaction of metronidazole with the other studied substances .
the effectiveness of the association of ciprofloxacin , metronidazole and natrosol shows a trend towards 100% sensitivity , as the concentration of the antimicrobial agents increases .
for this combination of substances , the highest mic was 4 g / ml and 50% of natrosol .
similarity of the results with the group without the addition of metronidazole was noted . as metronidazole did not present action against the facultative anaerobic bacteria tested in this experiment , further studies are necessary with other species of microorganisms to assess the feasibility of its application .
the vehicle polyethylene glycol was capable of eliminating 100% of the microorganisms tested with mic 40% . as the volume of polyethylene glycol was diminished ( below 30% ) , some microorganisms were capable of growing , particularly c. albicans and c. tropicalis .
it is interesting to point out that polyethylene glycol ( pm 1000 ) is the vehicle that allowed greater dentinal penetration of the association calcium hydroxide , ciprofloxacin and metronidazole24 .
when studied separately , natrosol prevented the growth of some microorganisms , particularly when administered in higher percentages .
however , at no time was it capable of inhibiting the growth of all the microorganisms .
the susceptible of the microorganisms to the vehicle polyethylene glycol was similar to that of the associations used in the present study .
this suggests that the antimicrobial action demonstrated was exercised by the action of this vehicle . this may be related to the hydrophilic property of polyethylene glycol . removing water from the culture media ,
the vehicle possibly did not allow microbial growth , as a certain amount of water is required to enable them to multiply and develop .
it is suggested that further studies be conducted , to find whether this effect occurs inside the root canal system .
the microbial concentration used for the suspension was determined by using the standard 0.5 on the mcfarland scale for bacteria and 2 for the yeasts , as the latter present a greater cellular volume12,13,15,18 .
it must be emphasized that almost all clinical situations are solved by performing the simple endodontic technique , which complies with the conditional factors of good preparation of the root canal system . in spite of the usual therapeutic protocol ,
the medication here proposed can be indicated when the routine alternatives are not able of producing the desired effects .
analysis of the results obtained by the application of the experimental model allowed the following conclusions : ciprofloxacin showed antimicrobial action against all tested microbial strains , and its association with metronidazole was synergic .
ciprofloxacin associated with polyethylene glycol was the most effective combination for reducing the tested bacteria and yeasts . | in some situations , endodontic infections do not respond to therapeutic protocol . in these cases ,
it is suggested the administration of an alternative intracanal medication that presents a wide spectrum of action and has an in - depth effect on the root canal system .
the purpose of this study was to assess the antimicrobial action of ciprofloxacin , metronidazole and polyethylene glycol and natrosol vehicles with different associations and concentrations .
the minimum inhibitory concentration ( mic ) was determined by using the agar dilution method . the culture media ( mller - hinton agar )
were prepared containing antimicrobial agents at multiple two - fold dilutions of 0.25 to 16 g / ml , and with the vehicles at the concentrations of 50 , 45 , 40 , 35 , 30 and 25% .
twenty - three microbial strains were selected for the study .
metronidazole was not capable of eliminating any of the tested microorganisms .
the association of ciprofloxacin with metronidazole resulted in a reduction of the mic .
the vehicle polyethylene glycol inhibited the growth of 100% of the tested strains , while natrosol inhibited 18% of the strains .
ciprofloxacin formulations with polyethylene glycol presented better effects than those of formulations to which metronidazole was added .
it was possible to conclude that ciprofloxacin presented antimicrobial action against all tested bacterial strains , and its association with metronidazole was synergic .
the vehicle polyethylene glycol showed antimicrobial effect and the ciprofloxacin / polyethylene glycol association was the most effective combination for reducing the tested bacteria and yeasts . |
the study protocol was approved by the ethics committee of the ministry of health and the school boards .
a total number of 30 clusters including 7,493 girls ( aged 620 years ) were selected .
students with a history of acute or chronic diseases or any chronic medication use were excluded . in all subjects , height , weight , and pubertal stages
general physicians determined pubertal stages under the supervision of specialists by visual inspection and palpation , using the criteria and definitions described by marshall and tanner.10 accordingly , breast stages 15 were determined by both inspection and palpation .
breast stage b2 corresponded to the breast bud stage with palpable glandular breast tissue and elevation of the papilla , stage b3 with further enlargement of the breast and areola with no separation of the contours , stage b4 where the areola and papilla form a secondary mound above the level of the breast , and stage b5 was assigned in case of the mature breast with projection of the papilla only .
the onset of puberty was measured as the age at breast development at tanner stage 2 ( b2 ) .
stage ph2 was assigned with the presence of long , slightly pigmented hair ( straight or curled ) .
participants were grouped by 1-year age intervals , which extended from the day of their birthday to the day before the next birthday . for comparison of different areas
, we used the national classification of the provinces according to their climate , ethnicity of the population , geographic locations , and socioeconomic variables .
classification of iranian provinces to regions according to climate , geographic location , ethnicity and socioeconomic status we estimated the mean of the time of men - arche , b2-b5 and ph2-ph5 stages .
statistical analysis was done using spss version 15 ( spss , chicago , il ) .
analysis of variance ( anova ) and post - hoc tests were used to compare the mean ages of menarche , pubarche , and telarche in different regions .
we estimated the mean of the time of men - arche , b2-b5 and ph2-ph5 stages .
statistical analysis was done using spss version 15 ( spss , chicago , il ) .
analysis of variance ( anova ) and post - hoc tests were used to compare the mean ages of menarche , pubarche , and telarche in different regions .
we estimated the mean of the time of men - arche , b2-b5 and ph2-ph5 stages .
statistical analysis was done using spss version 15 ( spss , chicago , il ) .
analysis of variance ( anova ) and post - hoc tests were used to compare the mean ages of menarche , pubarche , and telarche in different regions .
tehran had the lowest mean age of menarche , being 11.99 1.35 years , which was significantly lower than other 10 regions .
the second region with lowest age at menarche was fars ( 12.40 1.27 years ) .
the mean age at b2 stage of breast development was significantly lower in ghazvin - zanjan region ( 8.97 1.45 years ) followed by the region consisting of khorasan jonobi and sistan balochestan ( 10.11 1.61 years ; p = 0.003 ) , boshehr - khouzestan region ( 10.05 1.21 years ; p = 0.01 ) , tehran - relatd cities ( 10.46 1.67 years ; p < 0.0001 ) , fars ( 11.01 1.88 years ; p < 0.0001 ) and kordestan - lorestan - ilam region ( 9.94 1.25 years ; p = 0.02 ) . in fars region , the mean age at b2 stage of breast development ( 11.01 1.88 years ) was higher than other regions except for mazendran - guilan and tehran - related cities regions .
figure 2 shows the mean age at b1-b5 of breast development in different regions in iran .
mean age ( years ) at menarche in different regions mean age ( years ) at various stages of telarche in different regions in iran the mean age at ph2 stage of pubarche in kordestan - lorestan - ilam region ( 10.70 1.23 years ) was significantly higher than khorasan jonobi - sistan balochestan region ( p < 0.0001 ) , boshehr - khozestan region ( p < 0.0001 ) , tehran - relatd cities ( p < 0.0001 ) , chaharmahal - kerman - isfahan region ( p < 0.0001 ) , ghazvin - zanjan region ( p < 0.0001 ) , and boshehr - khozestan region ( p < 0.0001 ) .
the mean age at ph2-ph5 of pubic hair development in different geographic locations of iran is presented in figure 3 .
to the best of our knowledge , this is the first study of its kind to compare the pubertal stages in different geographic locations in a country .
it should be acknowledged that such comparison is not feasible in many countries with small population and no significant diversity in climate and other geographic characteristics .
we found that the mean age of menarche was significantly lower in tehran than all other regions .
tehran province , including the metropolitan city of tehran , is located in central part of the country with multiethnic population .
not only multi - ethnicity but also higher socioeconomic status and more industrialized lifestyle habits in tehran might be associated with earlier menarche age . in another study
the mean age of menarche was 12.68 years in tehran in 2004.11 in our study conducted 2 years later , this age decreased to 11.99 1.35 years . decreasing trend of menarche age
is observed in many developed and developing countries.1213 it might be attributed to changes in socioeconomic status of people14 and increasing rate of obesity.12 ethnicity is documented as a factor that describes some international varieties of puberty age.1516 in the current study , the mean age at menarche and ph2 was higher in girls with kurd ethnicity than in other ethnicities .
kurd ethnicity is the third most prevalent ethnicity in iran after persian and azeri ethnicities .
it can be suggested that the higher age of puberty in girls of this region might be attributed to their ethnicity as well as the cold climate and their high altitude mountainous region of living .
in addition to ethnicity , the geographic characteristics may influence the age of puberty . in our study , the lowest mean age at b2
was documented in zanjan - ghazvin region , with mediterranean weather and spring rains , and population of azeri ethnicity .
the earlier telarche in this region in comparison to other regions with azeri population might be attributed to environmental factors .
premature telarche is suggested to be associated with endocrine disrupting chemicals found in pesticides and plastics , with possible estrogenic and androgenic activities.1718 further investigations on environmental pollution might be necessary in this regard .
the considerably high prevalence of overweight among young iranian children19 might decrease the age of puberty in the near future .
the main limitation of this study was its cross sectional nature , and as a result , casualty can not be ascertained .
in addition , we considered the prominent ethnicity in each region ; but some participants might have other ethnicities .
we found significant differences in the age of pubertal stages of girls living in various regions with different ethnicity and geographic characteristics .
considering the impact of pubertal age on general health , more studies should be done about the lifestyle and environmental factors affecting the onset of puberty .
mem participated in the design and conducting the study ; ar participated in the design and conducting the study and helped to draft the manuscript ; rk drafted and edited the manuscript ; pm conducted the statistical analyses and helped to draft the manuscript ; ss participated in conducting the study ; ga participated in the design and conducting the study ; ha participated in conducting the study ; np participated in conducting the study ; sk in conducting the study ; pp helped to draft and edit the manuscript ; as participated in conducting the study . | background : this study aimed to compare the timing of puberty between various geographic locations and different ethnicities.methods:this national survey was conducted in 20 provinces in iran .
healthy iranian girls were selected from public schools using cluster random sampling .
a total number of 30 clusters including 7493 girls , aged 6.020 years , were selected . in order to compare different areas , the national classification of the provinces based on climate , ethnicity , geographic locations , and socioeconomic variables were used .
accordingly , there are 11 regions in iran .
analysis of variance was used to compare the mean ages of menarche , pubarche , and thelarche in different regions.results:tehranian girls , with 11.99 1.35 years ( mean sd ) , had the lowest age of menarche which was statistically significantly .
the second region with lowest age at menarche was fars ( 12.40 1.27 years ) .
the mean age at breast bud stage ( b2 ) was significantly lower in ghazvin - zanjan region ( 8.97 1.45 years ) . in fars region ,
the mean age at b2 stage of breast development ( 11.01 1.88 years ) was higher than other regions except for mazendran - guilan and tehran - related cities .
the mean age at public hair development at tanner stage 2 ( ph2 ) in kordestan - lorestan - ilam region ( 10.70 1.23 years ) was significantly higher than other regions.conclusions:we found significant differences in the age of pubertal stages of girls living in various regions with different ethnicity and geographic characteristics .
considering the impact of pubertal age on general health , more studies should be done about the lifestyle and environmental factors affecting the onset of puberty . |
ubiquitination is a process in which one or multiple ubiquitin moieties are covalently attached to a substrate through an enzymatic cascade involving ubiquitin - activating enzyme ( e1 ) , ubiquitin - carrier protein ( e2 ) , and ubiquitin - protein ligase ( e3 ) .
formation of a ubiquitin lys48 chain on the -nh2 group of a substrate 's internal lys residue ( polyubiquitination ) can target the substrate for degradation by the 26s proteasome .
ubiquitin can also be attached to the free -nh2 group in a substrate 's n - terminus to promote proteasomal degradation .
for example , polyubiquitination at lys63 is involved in inhibitor of nf-b ( ib ) kinase ( ikk ) activation .
in addition , a linear polyubiquitin chain can be achieved by conjugating the c - terminal glycine of ubiquitin and the a - nh2 group of the n - terminal methionine of its neighbor ubiquitin .
substrates can also undergo monoubiquitination or multi - monoubiquitination adding one ubiquitin to one or multiple lys residues , respectively .
recent evidence suggests that ubiquitin can be linked to cys , ser , or thr residues in a substrate through thio- or oxy - ester bonds ( i.e. , esterification ) , though the physiological relevance of these modifications remains to be defined. ubiquitin moieties can be released from a substrate by deubiquitinating enzymes .
for an organism to function properly , proteins must be degraded after they undergo specific functions .
moreover , proteins that are misfolded or damaged during translation , folding , or translocation must be degraded and eliminated in time .
either blocked degradation of oncogenic proteins / growth - enhancing factors or accelerated degradation of growth - suppressing proteins may disrupt the pathways controlling cell cycle progression , cell death , or survival , leading to cancer development , ( table 1 ) .
for example , the tumor suppressor cyld is mutated in several cancers , including cylindromatosis .
the deubiquitinating activity of cyld for ikk is critical for its cylindromatosis - suppressive function .
the ubiquitin ligase itch promotes the polyubiquitination and degradation of large tumor suppressor 1 ( ltsa1 ) , which is closely related to enhanced cell growth and epithelial - to - mesenchymal transition .
malt , mucosa - associated lymphoid tissue ; aml , acute myeloid leukemia . due to the critical roles of ubiquitination and the ubiquitin - mediated proteolysis in tumorigenesis and cell growth ,
it is a dipeptide boronate that specifically and reversibly blocks chymotrypsin - like activity of the proteasome in a variety of cancer cells .
although bortezomib inhibits nf-b activation and results in autophagy , this lethal effect of proteasome inhibition is probably due to loss of amino acid homeostasis .
notably , bortezomib has been used successfully as an anticancer drug for multiple myeloma and mantle cell lymphoma in the clinic,. because ubiquitination is generally substrate - specific , the components of the ubiquitination pathway might be more specific drug targets for cancer therapy than the proteasome .
cullin - ring ubiquitin ligases ( crls ) are involved in cellular processes such as cell cycle progression , cell death signaling , dna damage , and stress responses .
nedd8 is a ubiquitin - like protein that modifies cullin and is required for the activity of crls .
because nedd8-activating enzyme ( nae ) catalyzes the first step in the nedd8 pathway , targeting crls via inhibition of nae may be a promising anticancer strategy .
indeed , mln4924 , a selective inhibitor of nae , has potent tumor - suppressing activity in a wide range of tumors , including acute myeloid leukemia and diffuse large b cell lymphomas , .
apoptosis ( i.e. , programmed cell death ) is a cellular suicide process that is important for embryonic development and maintaining the size of cell populations .
the extrinsic pathway involves members of the tumor necrosis factor ( tnf ) receptor gene superfamily , which bind extracellular ligands and transduce intracellular signals during cell destruction .
the intrinsic pathway does not involve receptor - mediated intracellular signaling , but induces signaling in mitochondria . in mammals ,
the intrinsic pathway is regulated by the bcl-2 family of proteins , the adaptor protein apoptotic protease - activating factor-1 ( apaf-1 ) , and the caspases .
bcl-2 family members include both anti - apoptotic ( bcl-2 , bcl - xl , bcl - w , and mcl-1 ) and pro - apoptotic proteins ( bax , bak , bad , bid , and bim ) .
the release of cytochrome c from mitochondria causes the formation of the apoptosome ( apaf-1/caspase-9 complex ) , activates the downstream effector caspases , and finally results in cleavage of crucial substrates .
degradation of anti - apoptotic members is necessary for apoptotic progression , whereas degradation of pro - apoptotic members is required for the suppression of apoptosis , .
the levels of anti- and pro - apoptotic molecules can be regulated by ubiquitination and proteasomal degradation .
for example , trim17-mediated ubiquitination and subsequent degradation of mcl-1 , an anti - apoptotic bcl-2 family member , triggers neuronal apoptosis .
the pro - apoptotic bcl-2 member bax can be regulated by ubiquitination indirectly ; the ubiquitin ligase trim39 inhibits apc / c cdh1-mediated ubiquitination and degradation of the bax activator moap-1 , thus enhancing bax activation and apoptosis .
moreover , the levels of bcl2l10/bclb , an anti - apoptotic bcl2-like protein , are inversely correlated with survival in patients with several cancer types , including lung adenocarcinomas .
the inhibitors of apoptosis proteins ( iaps ) have one to three baculovirus iap repeat ( bir ) domains and can block apoptosis by directly binding and inhibiting caspases , .
furthermore , almost all iaps have ubiquitin ligase activity , which is required for the ubiquitination of certain substrates involved in apoptosis .
x - linked inhibitor of apoptosis protein ( xiap ) catalyzes the ubiquitination and degradation of caspase-3 , .
apoptosis inducing factor ( aif ) is also a substrate of xiap , and ubiquitination at k255 of aif shows a non - degradable role of ubiquitination in caspase - independent cell death . on the other hand ,
for example , ubiquitin - specific protease 19 ( usp19 ) is responsible for the inhibition of tnf--induced caspase activation and apoptosis in a ciap - dependent manner .
the activity of iaps can be suppressed by pro - apoptotic factors , such as second mitochondria - derived activator of caspase ( smac ) .
bruce / apollon is a large ( 528 kda ) , membrane - associated , essential iap in mammals .
furthermore , bruce / apollon can be degraded in a ubiquitin - dependent manner by the ubiquitin ligase nrdp1 during apoptosis .
the tumor suppressor p53 maintains the integrity of the genome and regulates cell cycle , dna repair , and apoptosis .
p53 promotes the activation of the pro - apoptotic bcl-2 family proteins and the release of cytochrome c. dysregulation of p53 is reported in numerous types of cancer .
several ubiquitin ligases , including mdm2 , have been reported to promote ubiquitination and degradation of p53 , while p53 is deubiquitinated and stabilized by ubiquitin - specific proteases ( usps ) .
thus , inhibiting the activity of p53 ubiquitin ligases or activating p53 usps can be a strategy for cancer therapy .
hausp regulates the activities of mdm2 and p53 by deubiquitination , while vif1 and vif2 antagonize hausp and promote p53-dependent apoptosis .
translationally controlled tumor protein ( tctp ) , which is down - regulated in tumor progression , inhibits mdm2 autoubiquitination and promotes mdm2-mediated ubiquitination and degradation of p53 .
in addition , fanconi anemia complementation group f ( fancf ) monoubiquitinates fancd2 , which is involved in the fa / brca dna damage response pathway .
silencing fancf elevates p53 activation in mitoxantrone - treated breast cancer cells . as a transcription factor involved in the extrinsic apoptosis pathway
, nf-b activates the expression of genes that contribute to cell proliferation , metastasis , and suppression of apoptosis .
sharpin , a ubiquitin - binding and ubiquitin - like - domain - containing protein , promotes linear ubiquitination of nemo / ikbkg , an adaptor of ikks , and subsequent activation of nf-b signaling .
ib , which inactivates nf-b under normal physiological conditions , can be phosphorylated by activated ikk , ubiquitinated by scf , and finally degraded by the proteasome in response to dna damage , .
nrdp1 promotes ubiquitination and degradation of the epidermal growth factor receptor family member erbb3 , which is upstream of nf-b activation , . in a word
, ubiquitination plays an important role in the regulation of apoptosis , and the components involved in the ubiquitination of key substrates can be potential targets for cancer therapy ( figure 1 ) .
autophagy , once categorized as programmed cell death type ii , is a cellular process by which intracellular proteins , lipids , and organelles are degraded in the lysosomal compartment after delivery from other cellular compartments .
there are three types of autophagy : macroautophagy , microautophagy , and chaperone - mediated autophagy .
although autophagy is generally thought to be non - selective , certain ubiquitinated proteins ( e.g. , catalase ) , organelles ( e.g. , peroxisomes and mitochondria ) , and invading bacteria have been shown to be selectively targeted for autophagic degradation .
, 18 autophagy - related proteins ( atgs ) in yeast , namely atg110 , atg1214 , atg1618 , atg29 , and atg31 , have been found to play a role in autophagosome formation .
atg8 , called lc3 in mammals , is a ubiquitin - like protein present on autophagic membranes as a phosphatidylethanolamine ( pe)conjugate .
p62/sqstm1 or nbr1 binds both ubiquitin and lc3 , probably providing a selective link between ubiquitinated substrates and autophagy .
nuclear dot protein 52 ( ndp52 ) , an autophagy receptor , targets intracellular ubiquitinated bacterial proteins for autophagic degradation .
misfolded polypeptides are usually recognized by molecular chaperones and degraded by the proteasome following polyubiquitination by ubiquitin ligases , such as chip and parkin .
however , when misfolded proteins can not be sufficiently removed by chaperone - mediated proteasomal degradation , protein aggregation occurs and may in turn inactivate the proteasome , resulting in cytotoxicity .
thus , p62/nbr1-mediated autophagic degradation may serve as an important compensatory mechanism for degradation of these ubiquitinated protein aggregates .
the crosstalk between autophagy and apoptosis is necessary for controlling the balance between cell survival and death .
beclin 1 , a mammalian atg6 ortholog , is a subunit of the class iii pi3-kinase complex .
beclin 1 interacts with bcl-2 via the bh3 domain in beclin 1 but can be released in starvation conditions to activate autophagy . this interaction can be terminated through c - jun n - terminal kinase ( jnk)mediated phosphorylation of bcl-2 and tnf receptor - associated factor 6 ( traf6)mediated ubiquitination of beclin 1 , .
under extreme conditions that can not be rescued by autophagy , jnk promotes hyperphosphorylation of bcl-2 , resulting in the release of bax to execute apoptosis .
caspase - mediated cleavage of beclin 1 inhibits beclin 1induced autophagy , and the cleavage product , the c - terminal region ( ct ) , enhances apoptosis by promoting the release of pro - apoptotic factors from mitochondria .
beclin 1 can also indirectly affect the crosstalk between apoptosis and autophagy by controlling the levels of p53 , a tumor suppressor that promotes apoptosis under genotoxic stress , .
furthermore , p53 can down - regulate lc3 levels in starved cells , preventing the autophagy burst that may be dangerous for cells . under normal conditions ,
p53 is kept at low levels by the ubiquitin ligase mdm2 . however , p53 levels can also be controlled by beclin 1 via regulating the deubiquitinating activity of usp10 and usp13 .
autophagy can target ubiquitinated misfolded proteins , caspases , and other cargo ( such as damaged mitochondria and invading bacteria ) for degradation , probably through p62 and nbr1 .
apoptosis and autophagy are counter - regulated in multiple steps , such as at p53 , the beclin 1/bcl-2 interaction , the cleavage of beclin 1 into the c - terminal region by caspases , and the autophagic degradation of caspases .
ubiquitination can promote degradation of both p53 and beclin 1 and thus , controls the mutual regulation of apoptosis and autophagy .
ubiquitination can occur on not only the -nh2 group of an internal lys residue , but also the -nh2 group of the n - terminal residue of a substrate .
moreover , recent evidence suggests that ubiquitin can be attached to cys , ser , or thr residue on a substrate by esterification .
these non - lys ubiquitinations might provide another layer of the regulation of protein functions , and further studies should focus on the identification of relevant substrates and physiological roles of these modifications .
future studies should also further explore how the ubiquitination of the critical proteins is involved in tumorigenesis and cancer therapy .
recent research efforts on cancer stem cells and personalized cancer genome sequencing are expected to help in this regard .
because the cytoprotection of autophagy and the evasion of apoptosis contribute to resistance to cancer therapy , it is important to unravel how these two pathways are mutually regulated .
the investigation on this issue has just begun and deserves more attention , especially with regard to how ubiquitination is involved in the counter - regulation of these critical processes . | ubiquitination is crucial for cellular processes , such as protein degradation , apoptosis , autophagy , and cell cycle progression .
dysregulation of the ubiquitination network accounts for the development of numerous diseases , including cancer .
thus , targeting ubiquitination is a promising strategy in cancer therapy .
both apoptosis and autophagy are involved in tumorigenesis and response to cancer therapy .
although both are categorized as types of cell death , autophagy is generally considered to have protective functions , including protecting cells from apoptosis under certain cellular stress conditions .
this review highlights recent advances in understanding the regulation of apoptosis and autophagy by ubiquitination . |
the south american sea lions ( otaria flavescens , carnivora : otariidae ) are common pinnipeds living along the eastern and western coasts of south america and are generally found in peru , chile , argentina , and south brazil [ 16 ] . along the chilean coastal shores , more than 200 colonies of free - ranging sea lions
a vast amount of data on feeding ecology , reproduction , life history parameters , and population dynamics of these otariid species was published [ 716 ] .
several studies have also addressed aspects of the helminth fauna of south american sea lions , comprising single species records , taxonomy , and population studies of some parasitoses [ 5 , 1422 ] . however , very little is known on protozoan parasite infections of these free - ranging marine mammals .
although their natural habitat is the marine environment , several pinniped and cetacean species are found in rivers containing fresh water . as such , a stable population of south american sea lions has been established within the city of valdivia , chile , resulting in permanent colonization for 20 years now .
these animals have adapted extremely well not only to the fresh water of the river but also to human activities in the river calle - calle , such as regular ship- and boat - trafficking , rowing , kayaking , and sealing activities .
sea lion colony is allocated approximately 7 km upstream from the ocean shore and animals mainly feed on fish captured by themselves in the river ( mainly carps , trouts , and salmons ) or on remains of the local fish market .
this unusual urban colony of south american sea lions consists of more than 72 individuals and is exclusively composed of males .
the age of these animals varies from 2 to 15 years but some animals might be even older . given that o. flavescens need to rest after swimming and diving activities , the sea lions in valdivia utilize river floats , riverside piers , and footways along the river promenade as recreation areas with all of them being allocated in close proximity to inhabitants , tourists , domestic pets , or the local fish market . since some of these animals behave rather aggressive towards humans , animals , or even vehicles and additionally tend to expand their territory into the city center , the local city authorities have established a
sea lion task force which should prevent the animals from harming humans and withhold them to premises alongside the river shore .
however , given that these animals nowadays represent a tourist attraction , direct contacts of humans with these animals or their faeces as well as faecal contamination of the river water or the terrestrial environment can not be avoided .
the present study therefore aimed to identify the actual gastrointestinal fauna in these free - ranging sea lions within their natural habitat in the river of the city of valdivia , chile , and to gain some insights in their potential zoonotic impact on public health issues .
south american sea lions ( o. flavescens ) were studied along the shores of the river calle - calle of the city of valdivia , chile .
the study area encompassed 3 km and comprised river floats , riverside piers , footways along the river promenade , and the local fish market with all of them being allocated in close proximity of humans and domestic pets ( figures 1(a ) , 1(b ) , 1(c ) , and 1(d ) ) .
whenever defecation occurred , scat samples were immediately collected and transferred into 2 ml plastic tubes ( eppendorf ) .
parasitological analyses were conducted at the institute of parasitology , justus liebig university giessen , germany .
coproscopical analyses were performed by using the standard sodium acetate acetic acid formalin ( saf ) technique .
the saf technique was used for the detection of parasite eggs , cysts , sporocysts , and oocysts within faecal material in marine mammals as described elsewhere .
additionally , a carbol fuchsin - stained faecal smear ( cfs ) was carried out for the detection of cryptosporidium oocysts .
moreover , coproantigen elisas ( prospect , oxoid ) were performed for the detection of cryptosporidium and giardia antigens in faecal samples .
the parasitological identification of eggs and cysts was based on morphological criteria referring to previous reports [ 24 , 26 , 27 ] .
all sampling procedures were conducted in accordance with institutional ethic commission of university austral of chile and the current chilean animal law .
giardia - positive sea lion samples were further analyzed for the presence of g. intestinalis dna by conventional and nested pcr detecting the beta - giardin gene ( assemblage c ) .
genomic dna was extracted from sea lion faecal material using the dna extraction stool kit ( qiagen ) according to the mammalian faecal protocol .
briefly , 1 ml of ethanol - fixed faeces was lysed in asl buffer containing 30 glass beads ( 4 mm diameter ) , under permanent stirring conditions .
the dna was thereafter purified using an anion exchange column ( qiagen ) and eluted in 100 l of distilled water .
for the conventional g. intestinalis - pcr , the following specific oligonucleotide sequences were used : the forward oligonucleotide -giardin g7f : 5-aagcccgacgacctcacccgcagtcg-3 and the reverse oligonucleotide -giardin g759r : 5-gaggccgccctggatcttcgagacgac-3 .
the pcr was performed in a total volume of 25 l containing 5 l faecal dna sample , 5 l faecal dna ( 1 : 100 ) , 1 l g7f oligonucleotides ( 10 m ) , 1 l g759r oligonucleotides ( 10 m ) , dntps 0.5 l ( 10 m ) , 0.5 l taq - polymerase ( 1 u/l ; peqlab ) , and 14.5 l h2o . the following thermocycle profiles were used : 95c for 5 min , 35 cycles at 95c for 30 s , 65c for 45 s , and 72c for 1 min and 30 s followed by a final extension step at 72c for 5 min and a final hold at 20c .
pcr amplificates were visualized in gelred - stained 2% agarose gels ( biotium incorporation ) .
in addition , a g. intestinalis - nested pcr was performed . for the nested pcr
the following forward oligonucleotide sequences of giarf were used : 5-gaacgagatcgaggtccg-3 and reverse oligonucleotide sequence of giarr : 5-ctcgacgagcttcgttgtt-3 .
the following thermocycle profiles for the nested pcr were used : 95c for 5 min , 35 cycles at 95c for 30 s , 50c for 40 s , and 72c for 1 min and 30 s followed by a final extension step at 72c for 5 min .
pcr amplificates were visualized using gelred - stained 2% agarose gels as described above ( biotium incorporation ) .
parasitological analyses of faecal samples of south american sea lions revealed 8 different protozoan ( 4 ) and metazoan ( 4 ) taxa .
the metazoan parasites consisted of trematodes ( one species ) , cestodes ( one species ) , and nematodes ( two species ) .
a complete list of the parasite stages and their prevalence is given in table 1 .
the most prevalent metazoan parasites found in this urban sea lion colony were anisakidae gen .
the most prevalent protozoan parasites were cryptosporidium ( 10% ) , giardia , and isospora showing the same prevalence ( 5.3% ) .
balantidium infections were detected at lower prevalence ( 2.5% , table 1 ) . within the metazoan endoparasites ,
the nematodes were the most rich in species ( two species ) followed by cestodes and trematodes with one species each . referring to parasite genus level
these parasitological findings included four new host records ( cryptosporidium , giardia , balantidium , and otostrongylus ) for o. flavescens .
to our best knowledge , the genus balantidium had only been described in fin whales ( balaenoptera physalus ) from the north atlantic .
all other protozoan parasites have already been reported for other marine mammals [ 4 , 5 , 17 , 19 , 20 , 24 , 30 ] .
some of the protozoan ( 3 ) and metazoan ( 2 ) endoparasite genera detected in sea lions bear an anthropozoonotic potential , such as cryptosporidium , giardia , balantidium , diphyllobothriidae gen .
although sea lion faecal samples were immediately fixed in 70% ethanol after collection in the field in order to avoid dna degradation , this goal was not successfully achieved .
thus no adequate giardia dna was possible to be extracted for further detailed molecular identification .
common collection methods for analyses of gastrointestinal parasites of wild sea lions generally rely on sections of accidentally stranded animals or on dead animals obtained from marine zoos [ 5 , 14 ] .
several studies are restricted to the helminth parasite fauna of south american sea lions and include several single species records [ 4 , 5 , 15 , 16 , 1822 , 31 ] .
conversely , by obtaining fresh faecal samples from resting sea lions , this record reveals unique insights into the actual gastrointestinal parasite fauna of free - ranging sea lions within their natural habitat . in the present survey ,
8 different parasite taxa were detected in individual sea lion faecal samples covering a respectable range of parasitic taxa .
the parasitological diagnosis based on morphological criteria revealed as quite a challenge since very little data on parasitic eggs , larvae , cysts and oocysts for sea lions are available in literature .
thus , the photo galleries provided here might supply a supportive tool for future parasitological research activities on sea lions and other marine mammals since many of the parasites described here infect a wide range of marine mammals , such as sea otters , seals , sea elephants , baleen , and toothed cetaceans .
the most prevalent parasitic stages found in the current study were eggs of anisakidae gen .
sp . however , owing to undistinguishable egg morphologies , characterization on species level was not possible .
based on parasite frequencies , these eggs most probably belong to the genera contracaecum , pseudoterranova , or anisakis since these parasite species appear to be quite common in south american sea lions [ 5 , 31 ] .
thus , south american sea lions are known as definitive hosts for the zoonotic nematodes anisakis spp . , contracaecum ogmorhini , and pseudoterranova cattani [ 5 , 31 ] .
ascarids parasitize either freely in the stomach or firmly attached , often as clusters , to the gastric mucosa .
mucosal penetration via larvae and adults can cause severe ulcers , gastritis , and perforation .
moreover , allergic reactions against epitopes of anisakis simplex major allergen ( ani s1 ) have been reported to occur in humans after the reexposure to these parasites [ 35 , 36 ] .
consistent to our findings , at least three different diphyllobothriid cestodes have previously been recorded in chile , two freshwater species ( diphyllobothrium latum and d. dendriticum ) and one marine species ( i.e. , adenocephalus pacificus ( diphyllobothrium pacificum ) ) [ 37 , 38 ] and also in the intestine of stranded sea lions from patagonia , but at a higher prevalence ( 26.8% ) .
diphyllobothriasis in sea lions generally is innocuous , but debilitation or even death of parasitized hosts might result in cases of high parasitic burdens .
in addition , diphyllobothriasis also represents an important fish - borne zoonosis worldwide [ 3943 ] .
trematode infections in the pancreas and liver occur in almost all marine mammals by members of the genera campula , zalophotrema , oschmarinella , and orthosplanchus .
additionally , also the genera apophallus , ascocotyle , ogmogaster , and pocitrema have been reported as intestinal parasites of otariids [ 5 , 44 ] .
these may induce necrosis of the parenchymal tissue , chronic fibrotic hepatitis , enteritis , and even meningoencephalitis by aberrant trematode migrations .
furthermore , the trematode genera pricetrema and nanophyetes have been reported to parasitize sea lions in the northern hemisphere . whilst pricetrema resides in the liver , nanophyetes infects the small intestine .
infections with the crenosomatid nematode otostrongylus in pinnipeds are generally associated with respiratory clinical manifestations , primarily in young animals .
interestingly , some sea lions of the current study showed strong coughing episodes , thereby expelling vast amount of mucus . based on the current findings this might indicate the presence of clinical otostrongylosis within the valdivian colony .
the parasitological findings of the study also included four new parasite host reports for o. flavescens , namely , cryptosporidium , giardia , balantidium , and otostrongylus , thereby providing a broader insight into the spectrum of parasitoses of this marine species .
in addition , the protozoan parasites ( i.e. , cryptosporidium , giardia , and balantidium ) clearly bear zoonotic potential and are considered as typical water - borne / food - borne diseases . in consequence ,
the urban sea lion colony in valdivia may function as relevant reservoir for these protozoans since the animals reside at the shore of the river and in close proximity to humans , pet animals , and especially in direct contact to the products of local fish market . within the genus balantidium ,
b. coli is the only species of trichostome ciliates nowadays considered as pathogenic for mammals [ 48 , 49 ] .
consistent with these findings , balatidium infections in free - ranging fin whales ( balaenoptera physalus ) from the north atlantic ocean were recently diagnosed , indicating that this terrestrial disease is circulating in the marine environment .
more importantly , b. coli infections have been demonstrated in humans and pigs in chile [ 50 , 51 ] . as seen for balantidiasis , giardiasis and cryptosporidiosis have an almost cosmopolitan distribution .
cryptosporidium and giardia are two common aethiological parasites of infectious enteritis in humans and animals [ 52 , 53 ] .
these enteric protozoans are usually transmitted by the faecal - oral route , following the ingestion of infective stages ( oocysts or cysts ) . moreover , marine mammals are well known as final hosts of giardia and cryptosporidium [ 24 , 5458 ] . keeping in mind that the urban sea lion colony is close to populated riversides and that some touristic activities , such as photos with sea lions or kayaking with sea lions , are becoming more popular
, sea lions may become infected by human excretions or vice versa . as seen for giardiasis
, cryptosporidium infections can cause severe diarrhoea in terrestrial mammals ; nonetheless , very little is known on the pathogenesis of cryptosporidiosis within the marine ecosystems [ 24 , 55 ] .
an essential component of the control of these diseases , from a public health perspective , is a better understanding of the sources and routes of transmission in different geographical regions .
unfortunately , the current knowledge and understanding of the ecto- and endoparasite fauna ( especially protozoan species ) in free - ranging sea lions are still very scarce .
although some parasitoses , such as hookworm ( e.g. , uncinaria hamiltoni ) or ascarid ( contracaecum ogmorhini , pseudoterranova cattani , and anisakis spp . ) , infections are discussed as pathogenic species for sea lions , the total parasite fauna of marine vertebrates has unfortunately not obtained sufficient attention , so far [ 24 , 59 ] . in conclusion , this study adds some new anthropozoonotic parasite records to free - ranging sea lions and calls for more integrated research to avoid the exposure of humans and domestic animals with these parasites .
in particular , regular monitoring programs should be established by local authorities for public health issues and for sea lion populations living in close proximity to human beings . | the present study represents the first report on the gastrointestinal endoparasite fauna of a free - ranging urban
colony of south american sea lions ( otaria flavescens ) living within the city of valdivia , chile .
a total of 40 individual faecal samples of south american sea lions were collected during the year 2012 within their natural habitat along the river calle - calle and in the local fish market of valdivia .
coprological analyses applying sodium acetate acetic formalin methanol ( saf ) technique , carbol fuchsin - stained faecal smears and giardia / cryptosporidium coproantigen elisas , revealed infections with 8 different parasites belonging to protozoan and metazoan taxa with some of them bearing anthropozoonotic potential . thus , five of these parasites were zoonotic ( diphyllobothriidae gen .
sp .
, anisakidae gen .
sp .
, giardia , cryptosporidium , and balantidium ) .
overall , these parasitological findings included four new parasite records for otaria flavescens , that is , giardia , cryptosporidium , balantidium , and otostrongylus .
the current data serve as a baseline for future monitoring studies on anthropozoonotic parasites circulating in these marine mammals and their potential impact on public health . |
in 2014 , the szent - gyrgyi prize for progress in cancer research was awarded to james allison , phd , chairman of the department of immunology at the university of texas md anderson cancer center ( figure 3 ) .
allison 's pioneering cancer research in the area of immunotherapy led to the successful development of immune checkpoint therapy and the first drug approved by the us food and drug administration for the treatment of metastatic melanoma . in bestowing the award
, nfcr 's selection committee recognized allison 's momentous achievement in the fight against cancer and his extraordinary leadership in the modern era of oncology . while mainstream cancer research and treatment focused for decades on radiation therapy and chemotherapy , dr .
allison 's trailblazing work in immunotherapy using the body 's own immune system to fight off cancer never ceased .
dr . allison has revolutionized the way science approaches cancer treatments , said sujuan ba , phd , co - chair of the 2014 szent - gyrgyi prize selection committee and chief operating officer of nfcr ( figure 4 ) .
he is on the front line in the war against cancer and could not be more deserving of this award .
this year 's szent - gyrgyi prize celebrates the world - renowned immunologist who opened the doors for an understanding of fundamental immune mechanisms and who used this newly found knowledge to advance cancer immunotherapy in a spectacular way , said dr .
alex matter , chief executive officer of the experimental therapeutics centre & d3 , a*star , singapore , winner of the 2013 szent - gyrgyi prize , and chair of this year 's prize selection committee .
he has validated the immunotherapy approach and turned previously widely held beliefs on their heads with his discoveries .
his work is extremely significant and constitutes a turning point in the history of progress in cancer treatments .
dr . allison earned his bachelor of science in microbiology in 1969 and his phd in biological science from the university of texas , austin , the us , in 1973 .
he was appointed a professor of immunology and director of the cancer research laboratory at the university of california , berkeley in berkeley , california , the us , in 1985 . in 2004
, he moved to the memorial sloan - kettering cancer center in new york , new york , the us , where he was director of the ludwig center for cancer immunotherapy , chair of the immunology program , david h. koch chair in immunologic studies , and attending immunologist . in 2012 , he joined md anderson cancer center , where he currently holds the position of professor and chair of immunology and executive director of the center 's immunotherapy platform .
dr . allison is a member of the national academy of sciences and the institute of medicine , a howard hughes medical institute ( hhmi ) alumnus , and a fellow of the american academy of microbiology and the american association for the advancement of science .
he has received numerous awards , including a recent aacr / su2c / cri cancer immunotherapy dream team grant , for which he will serve as the team leader . in december 2013 , he was awarded the breakthrough prize in life sciences for his work .
the cancer research community has been brewing the idea of harnessing the immune system to fight cancer for decades , but early attempts all failed . at least in part that was because we did not understand how complicated the immune system is .
there are also a lot of signals that are required to stop an immune response , said dr .
you have to stop an immune response or it will destroy you you'll just get collateral damage .
, it was understood that recognition of antigens by the t - cell antigen receptor was not sufficient to activate an immune response ; there had to be another co - stimulatory signal at the same time , and that kind of signal could only be provided by highly specialized cells . making an analogy to driving a car , dr .
every t cell has a different one , and unless you activate that , nothing is going to happen .
but unless you get the gas pedal pushed at the same time , it 's going to be an ineffective immune response .
the gas pedal in this analogy was a co - stimulatory molecule called cd28 . in the mid-1990s , dr .
cytotoxic t - lymphocyte - associated antigen 4 ( ctla-4 ) , which was known to compete with cd28was not an analogous co - stimulatory molecule , but actually quite the opposite .
if cd28 is the gas pedal , ctla-4 is the brakes , and ctla-4 was only induced after the t cells were activated .
it immediately occurred to me and to others in my lab that this might be the reason that attempts to vaccinate therapeutically against cancer would n't work because every time you gave vaccination gave the go signal that resulted in inducing the off signal as well , said dr .
if we could just block that signal , take it out of the pathway by something as simple as an antibody we could just let t - cell receptor and the gas pedal go to the floor and the t cells keep going , and hopefully eradicate the tumor cells . this was the paradigm shift : we 're not treating the tumor cells , we 're treating the immune system .
we 're treating the patient , we 're treating his or her t cells , said dr .
therefore , this has the possibility of becoming a treatment for all types of cancer .
this is a gross simplification , obviously , but the cancer cell 's irrelevant .
you 're just making the immune system attack something that ought n't to be there , whether it be melanoma , lymphoma , renal cancer , or colon cancer .
anything that kills tumor cells under inflammatory conditions will present tumor antigens to t cells , priming them for response .
so an anti - ctla-4 approach could also increase the effect of certain chemotherapies , radiotherapy , hormone therapy , or even new targeted therapies that kill tumor cells .
it could give memory to other therapies that kill tumor cells and give you that long - lived response that lasts for the rest of your life .
the experiments started in mice , and the results were stunning . in our early experiments we cured between 90% and 100% of the mice in most of these experiments , and the mice were permanently immuned when re - challenged with the tumor .
allison . all we were doing was covering up this one molecule out of all the things that are going on when the host immune system is detecting the tumor and turning tumor growth and inevitable death of the mice to a cure and long - lived immunity to re - challenge . in the late 1990s , dr .
allison 's lab teamed up with medarex to make completely human antibodies to ctla-4 . the resulting drug , ipilimumab ,
it 's been incredibly successful , having at least anecdotal responses in many kinds of cancer , including melanoma , renal cancer , prostate cancer , glioblastoma , pancreatic cancer , ovarian cancer , and lung cancer and the adverse effects are a lot milder than what you see with conventional chemotherapy . during his acceptance speech at the award ceremony , dr .
allison presented the story of a woman named sharon , who developed metastatic melanoma in her late 40s and subsequently failed every therapy .
allison , she just said she wanted to live long enough to see her sons graduate from high school .
sharon was placed on the phase i trial of ipilimumab and got a single dose at 3 mg / kg . six months later , she was completely clear of tumor . and she 's still completely tumor free , with no need for subsequent therapy at all , and now she 's 14 years out .
there are similar anecdotes for patients with metastatic melanoma to the brain , completely cured , and patients with prostate cancer and bone metastases , completely gone after anti - ctla-4 treatment .
the real proof was in an 800-patient , randomized , placebo - controlled trial of ipilimumab in metastatic melanoma .
median survival increased four months for patients in this trial , a result that had never been achieved with any therapy for metastatic melanoma .
even more remarkably , the survival curve flattens out at about 23% at two and a half years and goes out to three years , three and a half , four years , four and a half , and is absolutely flat .
recently there was actually a 5,000 patient retrospective study [ that ] showed that people who make it three years after this single round of therapy do not die of melanoma ; they die of something else .
ctla-4 is not the only molecule involved in down - regulating the immune response , and not all patients respond to anti - ctla-4 treatment .
work with another signaling molecule , programmed cell death 1 ( pd-1 ; which competes with the ignition switch and not the gas pedal , as the ctla-4 molecule does ) , demonstrated not just similar outcomes , but in fact , additive effects when combined with ipilimumab . we 're at a point where we know that immunotherapy by checkpoint blockade works
can we predict who 's going to respond and who wo n't respond ? how do we identify the best combinations to use , to try to raise the efficacy ? in addition , the concentration of translational research was on moving the median survival a few months to the right [ of the curve] what ipilimumab showed is that you can do that , but you can also get a tail on the curve where you get durable survivals that last for years and decades even of those patients . our goal now in this field is really to raise that tail not to move the median , but to raise the tail of the response to as high as we can get it in as many cancer types as we can get it , said dr .
and i 'm really optimistic that we can do that pretty soon , with continuing support for the basic science from the [ national cancer institute , national institutes of health ] , as well as from the national foundation for cancer research .
but also for support of the more translational aspects of this that you do n't get from the [ national cancer institute]they do n't provide [ this support ] ; you can get [ it ] only from organizations like the national foundation for cancer research that enable us to make that jump from basic demonstration that ctla-4 is an important regulatory molecule to letting us show that it works in preclinical and clinical models .
the 2014 szent - gyrgyi prize selection committee was chaired by alex matter , md , and co - chaired by sujuan ba , phd .
other selection committee members included leaders in cancer research and drug development from academic institutes and biotech and pharmaceutical industries : steven d. averbuch , md , bristol - myers squibb company ; webster k. cavenee , phd , ludwig institute for cancer research ; zhu chen , md , phd , shanghai jiao tong university ; sara courtneidge , phd , sanford - burnham medical research institute ; carlo m. croce , md , the ohio state university ; stan kaye , md , royal marsden nhs foundation trust ; marsha a. moses , phd , boston children 's hospital ; richard o'reilly , md , memorial sloan kettering cancer center ; scott d. patterson , phd , amgen , inc . ; peter k. vogt , phd , the scripps research institute ; and yi michael wang , md , phd , general secretary of nfcr .
2013 - alex matter , md chief executive officer , experimental therapeutics centre , agency for science , technology and research ( a*star ) , singapore .
2012 - zhu chen , md , phd professor , school of medicine of the shanghai jiao tong university ; chairman , chinese medical association ; former minister of health of china , shanghai , p. r. china .
2012 - zhen - yi wang , md professor , school of medicine of shanghai jiao tong university ; honorary director , shanghai institute of hematology , shanghai , p. r. china .
2011 - beatrice mintz , phd professor and jack schultz chair in basic science , fox chase cancer center , philadelphia , pennsylvania , us .
2010 - peter k. vogt , phd professor , department of molecular and experimental medicine , the scripps research institute , la jolla , california , us .
2009 - ronald a. depinho , md president , md anderson cancer center , houston , texas , us .
2008 - carlo m. croce , md director , human cancer genetics program ; director , institute of genetics , the ohio state university , columbus , ohio , us .
2007 - webster k. cavenee , phd director , ludwig institute for cancer research , san diego branch ; distinguished professor , university of california , san diego , california , us . 2006 -
harold f. dvorak , md mallinckrodt professor emeritus of pathology , harvard medical school ; chief , department of pathology , beth israel deaconess medical center , boston , massachusetts , us . | the szent - gyrgyi prize for progress in cancer research is a prestigious scientific award established by the national foundation for cancer research ( nfcr)a leading cancer research charitable organization in the united states that is committed to supporting innovative cancer research on the global scale that aims to cure cancer .
each year , the szent - gyrgyi prize honors an outstanding researcher whose original discoveries have expanded our understanding of cancer and resulted in notable advances in cancer prevention , diagnosis , or treatment .
the prize also promotes public awareness of the importance of basic cancer research and encourages the sustained investment needed to accelerate the translation of these research discoveries into new cancer treatments .
this report highlights the history and mission of the szent - gyrgyi prize , its role in promoting discovery - oriented cancer research , and the pioneering work led by the 2014 prize winner , dr .
james allison .
dr . allison 's work in the area of cancer immunotherapy led to the successful development of immune checkpoint therapy , and the first drug approved by the united states food and drug administration for the treatment of metastatic melanoma . |
the anterior choroidal artery ( achoa ) supplies several important structures including the optic tract , geniculate body , posterior limb of internal capsule , globus pallidus , cerebral peduncle , uncus , amygdala , ventrolateral nucleus of thalamus , and choroid plexus .
the vascular injury of achoa may result in severe neurological deficits such as hemiplegia , hemianesthesia , hemianopsia , decreased consciousness , and extrapyramidal symptoms .
therefore , complete knowledge about vascular anatomy of achoa is very important before surgical or endovascular manipulations .
although several detailed reports about anatomical variations of achoa have been reported , transposition of achoa as to posterior communicating artery ( pcoma ) origin was demonstrated only in 3 reports2 - 4 ) .
we report a case with ruptured cerebral aneurysm arising from pcoma , which originated from the internal carotid artery ( ica ) distal to the origin of achoa .
a 40-year - old woman presented with sudden onset of headache . computed tomography ( ct ) revealed subarachnoid hemorrhage ( sah ) which was slightly predominant in the left basal and sylvian cisterns .
two small aneurysms arose from both distal supraclinoid ica , these were observed on ct angiograms ( fig .
cerebral angiography demonstrated a small aneurysm arising from left fetal type of pcoma with suspicion of rupture and the other small aneurysm originating from right pcoma or perforating branch of ica . in this study , left achoa originated from ica proximal to the origin of fetal type of pcoma ( fig .
achoa usually arises from the posterolateral wall of ica , a few mm distal to the origin of pcoma , and it provides blood supply to the important neural structures around crural and ambient cisterns .
the achoa can have several variations in the course , size , number , branching pattern , and brain region supplied by this artery1 ) . among them
, there have been several anomalies related to the origin of achoa including transposition of achoa and pcoma , origin from pcoma , origin from junction of ica and pcoma , and origin from middle cerebral artery4,5 ) .
the clinical findings of transposed achoa cases including present one are summarized in table 1 .
all of the transpositions of achoa occurred in the left side . among all the cases ,
our case showed sah due to ruptured aneurysm arising from fetal type of pcoma among multiple cerebral aneurysms founded and achoa originating from ica proximal to pcoma .
typically , achoa runs posteromedially within the crural cistern and continues ambient cistern to enter the choroidal fissure .
the proximal portion of this artery is cisternal segment and following portion becomes intraventricular segment1 ) .
the transposed achoa in the previous cases including ours did not seem to affect the vascular territory or course despite of unusual vascular arrangements .
the achoa can be exposed or manipulated in surgical approaches into the pathological lesions around temporal lobe or involving supraclinoid ica .
although the achoa aneurysms accounting for 4% of all cerebral aneurysms are not so common , vascular injury of achoa with small caliber supplying the eloquent areas may be critical because of the possibility of significant morbidity and mortality . and
, another caution is that achoa do not have significant collateral flows despite a potential anastomosis with posterior choroidal artery3 ) .
we tried to preserve pcoma itself and all perforating branches arising from it because of the possibility to compromise the collateral flows of fetal type pcoma .
it is notable that careful review of the preoperative angiograms helps to find unexpected vascular anomalies which may impose an additional surgical risk . | the anterior choroidal artery has several kinds of variations . among them ,
the transposition of anterior choroidal artery and posterior communicating artery origins has been extremely rare .
we report a case with cerebral aneurysm arising from posterior communicating artery which origin was distal to the anterior choroidal artery and review the relevant literature . |
transposases are enzymes that catalyze the movement of mobile genetic elements in and between genomes and are the most abundant and ubiquitous genes in nature ( 1 )
. most often , transposases are part of transposable elements ( tes ) , which encode only the transposase gene and some short flanking sequences necessary for transposition .
classically , tes are considered to be selfish genetic elements or parasitic dna with no purpose other than reproducing themselves ( 24 ) . however , in more recent years , it has become clear that tes are not always parasites , but can also have beneficial effects that increase the fitness of their bacterial host ( for reviews of the ongoing debate , see references 35 ) .
tes ( especially is elements ) are involved in gene deletions , gene duplications , genome rearrangements , gene regulation , and horizontal gene transfer ( for a review , see references 6 and 7 ) , all of which can have beneficial effects on the host population by generating genomic diversity and thus enabling adaptation to environmental changes ( 810 ) .
therefore , transposase expression and , thus , transpositional activity are usually very low ( 11 ) , because the mutagenic effects of transposases would drive their hosts into extinction , thereby also eradicating their own existence ( 12 ) . accordingly , a large variety of mechanisms for the tight regulation of transposase expression exists at both the transcriptional and translational levels ( 11 ) . tes and , thus , transposase genes are particularly enriched in the genomes of some mutualistic symbionts ( here called
symbionts ) and some pathogens that have recently transitioned or are transitioning to an obligate , host - associated lifestyle ( 5 , 13 ) .
in contrast , tes are absent in the reduced genomes of most obligate , host - restricted bacteria that have been associated with their host over long evolutionary time periods ( tes absent in 65% of the genomes from symbionts classified as obligately intracellular in newton and bordenstein ) .
there are , however , some ancient obligate intracellular bacteria , such as wolbachia pipientis wmel and candidatus amoebophilus asiaticus , that have high numbers of tes in their genomes ( 14 , 15 ) ( tes present in 35% of the genomes from symbionts classified as obligately intracellular in newton and bordenstein ) . it has been shown that te expansion in bacteria in transition to an obligate , host - associated lifestyle plays a crucial role in the emergence and early evolution of pathogens ( 8 , 1618 ) and symbionts ( 19 , 20 ) .
currently , there is much uncertainty about the factors that lead to high te loads in host - restricted bacteria , and several hypotheses have been put forth ( reviewed in reference 3 ) .
the two main hypotheses , which represent partially opposing views , are ( i ) the selective advantage hypothesis and ( ii ) the relaxed natural selection hypothesis . in ( i ) , te expansion is selected for because it is beneficial for symbionts and pathogens transitioning to an obligate lifestyle , for example , by providing enhanced genomic plasticity for faster adaptation to the host environment ( 8 , 9 , 21 ) . in ( ii ) , temporary te expansion in the genomes of host - restricted bacteria is due to a reduced effectiveness of natural selection against deleterious transpositions ( 5 ) .
the relaxed natural selection , according to this second hypothesis , is caused by genetic drift due to small population sizes during transmission of symbionts from one host generation to the next , and the fact that when symbionts reside within a host , many of their genes become superfluous and can thus act as neutral integration sites for tes .
these two hypotheses can also explain the evolutionary processes involved in te expansion in ancient obligate intracellular bacteria that have a reduced genome , yet still have high te numbers in their genomes .
for these ancient symbionts , the intracellular arena hypothesis explains how tes first reenter their genomes : host switching events bring these symbionts into contact with other bacteria during coinfection within the same host and thus enable the uptake of foreign tes ( 13 , 14 ) .
these newly acquired tes then multiply in the genomes of the ancient symbionts either ( i ) because they confer a selective advantage or ( ii ) due to reduced effectiveness of natural selection in the intracellular environment ( 5 , 22 ) .
a recent study that tested hypothesis ( ii ) by subjecting escherichia coli for 4,000 generations to simulated conditions of relaxed natural selection raised doubts as to whether relaxed natural selection alone can account for te expansion ( 21 ) , because no te expansion occurred under the tested conditions .
although plague et al . ( 21 ) noted that there are several possible reasons why [ their ] experiment may not have adequately tested the [ relaxed natural selection ] hypothesis , they hypothesized that other factors , including increased transposase activity , might have enabled the massive te expansion observed in host - restricted bacteria .
common to both hypotheses ( i ) and ( ii ) is that they explain te expansion in host - restricted bacteria within the confines of the paradigm that transposase expression and , thus , transpositional activity are always low . high numbers of transposase genes were recently described in endosymbionts of the gutless marine oligochaete olavius algarvensis ( 23 ) .
o. algarvensis inhabits shallow water sediments in the mediterranean and lacks both a digestive and an excretory system , relying instead for nutrition and waste recycling on a symbiotic community of two gammaproteobacterial sulfur oxidizers ( 1 and 3 symbionts ) , two deltaproteobacterial sulfate reducers ( 1 and 4 symbionts ) , and a spirochete ( 23 , 24 ) .
the symbiotic bacteria cooccur in an extracellular space just below the worm s cuticle and above the host s epidermal cells where they are in direct contact with each other and have access to solutes < 70 kda from the environment that can easily diffuse through the worm s cuticle ( 25 ) .
metagenomic analyses of o. algarvensis showed that the 1 symbiont has a remarkably high percentage of transposases in its genome , at nearly 21% of all genes , followed by the 3 symbiont with 7.5% and the 1 symbiont with 2.3% ( 23 , 26 ) .
nothing is currently known about the factors that have led to such high te numbers in some of the o. algarvensis symbionts .
we assume that the association with the 1 symbiont is ancient because these symbionts occur in many gutless oligochaete species from around the world , indicating that the common ancestor of all gutless oligochaetes may have already had the 1 symbiont ( 25 ) .
however , the genome of the 1 symbiont shows no signs of reduction ( 23 ) .
the associations with the 3 and deltaproteobacterial symbionts may be more recent , as these symbionts are found in some but not all gutless oligochaetes ( 27 ) ; the genomes of these symbionts are also not reduced ( 23 ) .
the transmission mode of symbionts in gutless oligochaetes has been examined only in two host species from bermuda and appears to be vertical ( 28 ) .
however , occasional horizontal uptake of bacteria from the environment during egg deposition in the surrounding sediment can not be excluded .
thus , the o. algarvensis symbionts could acquire new tes from both environmental sources and cooccurring symbionts .
the aim of this study was to examine how the high percentage of tes in the o. algarvensis symbiont genomes affects their expression .
interestingly , the relationship between high te loads and transposase expression and activity has , to our knowledge , so far not been explored .
our metaproteomic analyses revealed that the 1 and 1 symbionts express a surprisingly high number of their transposase proteins , many of which are intact and possibly active , and we hypothesize that abundant transposase expression plays a key role in te expansion .
worms were collected as described previously ( 24 ) and either frozen immediately or symbionts were enriched via isopycnic centrifugation by layering the worm homogenate on top of a histodenz ( sigma ) multistep density gradient .
symbionts were separated from each other and from host tissue by 1 h of centrifugation at 4c as described by kleiner et al .
high proteome coverage was achieved , i.e. , 2,265 symbiont proteins were detected , via automated 24-h two - dimensional liquid chromatography followed by tandem mass spectrometry ( ms / ms ) with a hybrid linear ion trap - orbitrap ( thermo fischer scientific ) as described in detail by kleiner et al .
protein databases , peptide and protein identifications , and all ms / ms spectra are available from http://compbio.ornl.gov/olavius_algarvensis_symbiont_metaproteome/ , and detailed data on expressed transposases are available from http://compbio.ornl.gov / olavius_algarvensis_symbiont_transposases/. we detected the highest number of transposase proteins ever reported in bacteria in the 1 symbiont of o. algarvensis ( 22 proteins ) ( table 1 ) .
additionally , we detected two transposase proteins in the 1 symbiont and two transposase proteins that could not be unambiguously assigned to a given symbiont ( table 1 ) .
we did not obtain good proteome coverage for the 3 symbiont due to its low abundance in o. algarvensis and were therefore unable to determine if any of its numerous transposase genes were expressed . to compare our data to other studies , we searched for reports of transposase expression at the protein level and found that the highest numbers were found in free - living bacteria and not in host - associated bacteria .
the highest numbers of expressed transposases were seven in a proteome of the cultured bacterium deinococcus radiodurans ( 29 ) and eight in a metaproteome of the uncultured leptospirillum group ii bacterium from acid mine drainage ( 30 ) .
to our knowledge , transposase expression has so far not been examined at the protein level in symbionts or pathogens with high te numbers in their genomes .
overview of all expressed transposases grouped according to shared peptide matches accession numbers refer to the jgi img / m database ( http://img.jgi.doe.gov/cgi-bin/m/main.cgi ) .
some of the transposases that were identified as not intact may actually be intact and only appear to be fragmented due to the incomplete nature of the symbiont metagenome . identified with an unassigned symbiont metagenome fragment .
this number is bigger than the actual number , 134 , because one transposase fell into two groups .
transposases comprised up to 1.95% of the total 1 symbiont protein ( see table s1 in the supplemental material ) and up to 0.084% of the total 1 symbiont protein ( table s2 ) .
the abundance of transposases ( as estimated from label - free proteomic quantitation ) in the 1 symbiont was greater than that of some of its most abundant housekeeping proteins , such as the atpase b subunit ( 1.15% ) , malate dehydrogenase ( 0.38% ) , and 6-phosphofructokinase ( 0.35% ) ( 24 ) . within the context of natural microbial communities ,
highly abundant transposase protein expression has , so far , only been reported from a microbial biofilm in acid mine drainage ; however , relative abundances were estimated at less than half of the amounts observed here ( 30 ) .
since many transposase genes are present in multiple , nearly identical copies in the symbiont metagenomes ( 23 , 26 ) , the 26 groups of transposase genes ( table 1 ) could be encoded by as many as 134 transposase gene sequences in the metagenome ( see table s3 in the supplemental material ) . given that proteins encoded by almost - identical sequences with identical tryptic peptides can not be distinguished from each other by mass spectrometry - based proteomic analyses , we were not able to identify which of the 134 transposase genes were expressed and if multiple copies of identical genes were expressed
. therefore , we assigned the transposases that were identified with similar sets of peptides to 12 different groups ( table 1 ; see also table s4 in the supplemental material ) and used this grouping to identify the minimal set of transposases that must be expressed to explain all transposase - related peptides .
we classified the 26 nonredundant transposases into is element families using blastp against the curated is finder database ( 31 ) ( http://www-is.biotoul.fr/ ) and found that they belong to at least 10 different is element families in the 1 symbiont and 2 families in the 1 symbiont ( table 1 ) .
previous studies have reported increased transposase expression in response to stressful conditions ( 29 , 32 ) . however , relative abundances were much lower than those detected in our study . to exclude the possibility that transposase expression in the o. algarvensis symbionts was caused by stressful conditions during the 1 hour long symbiont enrichment procedure , we did a qualitative comparison of their proteomes with those of symbionts that were frozen in whole worms immediately following removal from the sediment
. we also measured high levels of transposase expression in these immediately frozen symbionts ( see tables s1 and s3 in the supplemental material ) .
thus , we conclude that the observed transposase expression is not due to stressful sampling and enrichment conditions but rather reflects expression under natural environmental conditions .
we inferred that some of the expressed transposases are active by excluding the two main reasons for potential inactivity : ( i ) transposase genes could be in the process of gene degradation so that their expression would lead to incomplete and potentially inactive transposases and ( ii ) the expression of some transposase genes is regulated through programmed translational frameshifting , which would lead to the translation of a truncated , nonfunctional version of the transposase protein ( 6 , 11 , 33 ) .
first , we checked for indications that the expressed transposase genes were intact rather than in a state of gene degradation .
we compared the gene sizes and protein sequences to those of closely related transposases in the is finder database and compared their protein domain structures with the domain structure of similar transposases using the domain organization feature available on pfam ( http://pfam.sanger.ac.uk/search ) .
we found that around half of the expressed transposase genes were intact , whereas for the other half , we were not able to exclude the possibility that they were in some stage of degeneration ( table 1 ) .
second , we checked the literature and found that only the is families of four out of the 26 detected transposases are known to be regulated by programmed translational frameshifting ( 11 ) ( table 1 ) .
therefore , we assume that the majority of the expressed transposases are translated to full - length proteins .
additional evidence for the expression of full - length transposase proteins comes from our proteomic data . for some of the identified transposases , we detected peptides not only in the beginning part of the protein but also in the middle or at the end of the protein , a finding which indicates that the protein was translated from beginning to end ( http://compbio.ornl.gov/olavius_algarvensis_symbiont_transposases/ )
our results show that the paradigm that transposase expression in bacteria must be tightly regulated and generally low to prevent the host population from going extinct does not always hold true .
we present evidence at the protein level that transposases are abundantly expressed in beneficial symbionts with high te numbers in their genomes .
this high expression of transposases indicates that the mechanisms typically found in bacteria for their tight regulation have been disabled or no longer exist , for example , possibly through mutations in proteins that are involved in transposase regulation ( 20 , 34 , 35 ) .
the fixation of such mutations may be enabled by the relaxed purifying selection suggested by moran et al . for symbionts and pathogens that recently transitioned to an obligate , host - associated lifestyle ( 20 ) .
currently , it is not possible to determine if abundant transposase expression is present in other symbionts and pathogens with high te numbers , because no comparable proteomic datasets exist for these bacteria .
however , many recent studies have shown high transcription of transposase genes in symbionts and pathogens ( 15 , 3640 ) , which may indicate that abundant transposase expression is common in these bacteria
. however , the presence of these transcripts does not represent conclusive evidence for transposase expression because it is possible that they are not translated into proteins due to regulatory mechanisms ( 11 , 15 , 37 ) .
based on the high numbers of transposase proteins in the o. algarvensis 1 symbiont and abundant transposase transcription in other symbionts and pathogens , we speculate that high transposase expression is one of the key factors in te expansion in host - restricted bacteria . as discussed above , an experiment that simulated conditions of relaxed natural selection failed to cause te expansion after 4,000 generations in e. coli . if , as we speculate , high transposase expression is the major catalyst for te expansion , the mutations that lead to increased transposase expression might have simply not occurred yet in the plague et al .
additional studies that investigate transposase expression in pathogens and symbionts are needed because high transposase expression may be an important factor in the sometimes very rapid emergence and evolution of new obligate symbionts and pathogens from facultative ones .
the remarkably high expression of transposases in the o. algarvensis 1 symbiont raises the question of how the symbiont can function over evolutionary time periods given the likelihood that high transposase expression leads to an increase in deleterious mutations .
in other organisms that are confronted with frequent transpositions , genome rearrangements and disruptions , it has been suggested that polyploidy buffers against the detrimental effects of the factors that lead to these genome disruptions such as transposable elements , introns , heat and ionizing radiation ( 4144 ) .
polyploidy has recently been shown for several symbionts , including sulfur - oxidizing symbionts of clams ( 45 ) , and it is possible that it also plays a role in the o. algarvensis 1 symbiont . to gain a better understanding of how the o. algarvensis 1 symbiont deals with high transposase expression , it would be crucial to know specifically how high the transpositional activity of the abundant transposases is .
do the symbionts have multiple genome copies that buffer against the deleterious effects of transposase activity ? to answer these questions , we are currently sequencing the genomes of single symbiont cells to examine how common transposition events are within the same individual host , within the host population , and between host populations .
worms were collected as described previously ( 24 ) and either frozen immediately or symbionts were enriched via isopycnic centrifugation by layering the worm homogenate on top of a histodenz ( sigma ) multistep density gradient .
symbionts were separated from each other and from host tissue by 1 h of centrifugation at 4c as described by kleiner et al .
high proteome coverage was achieved , i.e. , 2,265 symbiont proteins were detected , via automated 24-h two - dimensional liquid chromatography followed by tandem mass spectrometry ( ms / ms ) with a hybrid linear ion trap - orbitrap ( thermo fischer scientific ) as described in detail by kleiner et al .
protein databases , peptide and protein identifications , and all ms / ms spectra are available from http://compbio.ornl.gov/olavius_algarvensis_symbiont_metaproteome/ , and detailed data on expressed transposases are available from http://compbio.ornl.gov / olavius_algarvensis_symbiont_transposases/.
we detected the highest number of transposase proteins ever reported in bacteria in the 1 symbiont of o. algarvensis ( 22 proteins ) ( table 1 ) .
additionally , we detected two transposase proteins in the 1 symbiont and two transposase proteins that could not be unambiguously assigned to a given symbiont ( table 1 ) .
we did not obtain good proteome coverage for the 3 symbiont due to its low abundance in o. algarvensis and were therefore unable to determine if any of its numerous transposase genes were expressed . to compare our data to other studies , we searched for reports of transposase expression at the protein level and found that the highest numbers were found in free - living bacteria and not in host - associated bacteria .
the highest numbers of expressed transposases were seven in a proteome of the cultured bacterium deinococcus radiodurans ( 29 ) and eight in a metaproteome of the uncultured leptospirillum group ii bacterium from acid mine drainage ( 30 ) .
to our knowledge , transposase expression has so far not been examined at the protein level in symbionts or pathogens with high te numbers in their genomes .
overview of all expressed transposases grouped according to shared peptide matches accession numbers refer to the jgi img / m database ( http://img.jgi.doe.gov/cgi-bin/m/main.cgi ) .
some of the transposases that were identified as not intact may actually be intact and only appear to be fragmented due to the incomplete nature of the symbiont metagenome . identified with an unassigned symbiont metagenome fragment .
this number is bigger than the actual number , 134 , because one transposase fell into two groups .
transposases comprised up to 1.95% of the total 1 symbiont protein ( see table s1 in the supplemental material ) and up to 0.084% of the total 1 symbiont protein ( table s2 ) .
the abundance of transposases ( as estimated from label - free proteomic quantitation ) in the 1 symbiont was greater than that of some of its most abundant housekeeping proteins , such as the atpase b subunit ( 1.15% ) , malate dehydrogenase ( 0.38% ) , and 6-phosphofructokinase ( 0.35% ) ( 24 ) . within the context of natural microbial communities ,
highly abundant transposase protein expression has , so far , only been reported from a microbial biofilm in acid mine drainage ; however , relative abundances were estimated at less than half of the amounts observed here ( 30 ) . since
many transposase genes are present in multiple , nearly identical copies in the symbiont metagenomes ( 23 , 26 ) , the 26 groups of transposase genes ( table 1 ) could be encoded by as many as 134 transposase gene sequences in the metagenome ( see table s3 in the supplemental material ) . given that proteins encoded by almost - identical sequences with identical tryptic peptides can not be distinguished from each other by mass spectrometry - based proteomic analyses , we were not able to identify which of the 134 transposase genes were expressed and if multiple copies of identical genes were expressed . therefore , we assigned the transposases that were identified with similar sets of peptides to 12 different groups ( table 1 ; see also table s4 in the supplemental material ) and used this grouping to identify the minimal set of transposases that must be expressed to explain all transposase - related peptides .
we classified the 26 nonredundant transposases into is element families using blastp against the curated is finder database ( 31 ) ( http://www-is.biotoul.fr/ ) and found that they belong to at least 10 different is element families in the 1 symbiont and 2 families in the 1 symbiont ( table 1 ) .
previous studies have reported increased transposase expression in response to stressful conditions ( 29 , 32 ) . however , relative abundances were much lower than those detected in our study . to exclude the possibility that transposase expression in the o. algarvensis symbionts was caused by stressful conditions during the 1 hour long symbiont enrichment procedure , we did a qualitative comparison of their proteomes with those of symbionts that were frozen in whole worms immediately following removal from the sediment
. we also measured high levels of transposase expression in these immediately frozen symbionts ( see tables s1 and s3 in the supplemental material ) .
thus , we conclude that the observed transposase expression is not due to stressful sampling and enrichment conditions but rather reflects expression under natural environmental conditions .
we inferred that some of the expressed transposases are active by excluding the two main reasons for potential inactivity : ( i ) transposase genes could be in the process of gene degradation so that their expression would lead to incomplete and potentially inactive transposases and ( ii ) the expression of some transposase genes is regulated through programmed translational frameshifting , which would lead to the translation of a truncated , nonfunctional version of the transposase protein ( 6 , 11 , 33 ) .
first , we checked for indications that the expressed transposase genes were intact rather than in a state of gene degradation .
we compared the gene sizes and protein sequences to those of closely related transposases in the is finder database and compared their protein domain structures with the domain structure of similar transposases using the domain organization feature available on pfam ( http://pfam.sanger.ac.uk/search ) .
we found that around half of the expressed transposase genes were intact , whereas for the other half , we were not able to exclude the possibility that they were in some stage of degeneration ( table 1 ) .
second , we checked the literature and found that only the is families of four out of the 26 detected transposases are known to be regulated by programmed translational frameshifting ( 11 ) ( table 1 ) .
therefore , we assume that the majority of the expressed transposases are translated to full - length proteins .
additional evidence for the expression of full - length transposase proteins comes from our proteomic data . for some of the identified transposases , we detected peptides not only in the beginning part of the protein but also in the middle or at the end of the protein , a finding which indicates that the protein was translated from beginning to end ( http://compbio.ornl.gov/olavius_algarvensis_symbiont_transposases/ ) .
our results show that the paradigm that transposase expression in bacteria must be tightly regulated and generally low to prevent the host population from going extinct does not always hold true .
we present evidence at the protein level that transposases are abundantly expressed in beneficial symbionts with high te numbers in their genomes .
this high expression of transposases indicates that the mechanisms typically found in bacteria for their tight regulation have been disabled or no longer exist , for example , possibly through mutations in proteins that are involved in transposase regulation ( 20 , 34 , 35 ) .
the fixation of such mutations may be enabled by the relaxed purifying selection suggested by moran et al . for symbionts and pathogens that recently transitioned to an
currently , it is not possible to determine if abundant transposase expression is present in other symbionts and pathogens with high te numbers , because no comparable proteomic datasets exist for these bacteria .
however , many recent studies have shown high transcription of transposase genes in symbionts and pathogens ( 15 , 3640 ) , which may indicate that abundant transposase expression is common in these bacteria
. however , the presence of these transcripts does not represent conclusive evidence for transposase expression because it is possible that they are not translated into proteins due to regulatory mechanisms ( 11 , 15 , 37 ) .
based on the high numbers of transposase proteins in the o. algarvensis 1 symbiont and abundant transposase transcription in other symbionts and pathogens , we speculate that high transposase expression is one of the key factors in te expansion in host - restricted bacteria . as discussed above , an experiment that simulated conditions of relaxed natural selection failed to cause te expansion after 4,000 generations in e. coli . if , as we speculate , high transposase expression is the major catalyst for te expansion , the mutations that lead to increased transposase expression might have simply not occurred yet in the plague et al .
additional studies that investigate transposase expression in pathogens and symbionts are needed because high transposase expression may be an important factor in the sometimes very rapid emergence and evolution of new obligate symbionts and pathogens from facultative ones .
the remarkably high expression of transposases in the o. algarvensis 1 symbiont raises the question of how the symbiont can function over evolutionary time periods given the likelihood that high transposase expression leads to an increase in deleterious mutations .
in other organisms that are confronted with frequent transpositions , genome rearrangements and disruptions , it has been suggested that polyploidy buffers against the detrimental effects of the factors that lead to these genome disruptions such as transposable elements , introns , heat and ionizing radiation ( 4144 ) .
polyploidy has recently been shown for several symbionts , including sulfur - oxidizing symbionts of clams ( 45 ) , and it is possible that it also plays a role in the o. algarvensis 1 symbiont . to gain a better understanding of how the o. algarvensis 1 symbiont deals with high transposase expression , it would be crucial to know specifically how high the transpositional activity of the abundant transposases is .
do the symbionts have multiple genome copies that buffer against the deleterious effects of transposase activity ? to answer these questions , we are currently sequencing the genomes of single symbiont cells to examine how common transposition events are within the same individual host , within the host population , and between host populations .
organism normalized spectral abundance factor ( nsaf ) values ( relative abundance ) for the 1 symbiont transposases
. minimum number of 1 symbiont transposase proteins needed to explain all detected peptides ( according to the grouping in table s4 in the supplemental material ) .
the values in the table are nsaf , which indicate the relative abundance of the respective protein in relation to all identified 1 symbiont proteins .
minimum number of 1 symbiont transposase proteins needed to explain all detected peptides ( according to the grouping in table s4 in the supplemental material ) .
the values in the table are nsaf , which indicate the relative abundance of the respective protein in relation to all identified 1 symbiont proteins .
table s3 , xls file , 0.1 mb . identified transposases grouped according to the peptides with which they were detected . | abstracttransposases , enzymes that catalyze the movement of mobile genetic elements , are the most abundant genes in nature .
while many bacteria encode an abundance of transposases in their genomes , the current paradigm is that the expression of transposase genes is tightly regulated and generally low due to its severe mutagenic effects . in the current study
, we detected the highest number of transposase proteins ever reported in bacteria , in symbionts of the gutless marine worm olavius algarvensis with metaproteomics .
at least 26 different transposases from 12 different families were detected , and genomic and proteomic analyses suggest that many of these are active .
this high expression of transposases indicates that the mechanisms for their tight regulation have been disabled or no longer exist . |
joubert syndrome ( js ) was initially described in 1968 in 4 siblings with agenesis of the cerebellar vermis presented with episodic hyperpnea , abnormal eye movements , ataxia , and intellectual disability1 ) .
several years later , a pathognomonic midbrain - hindbrain malformation the molar tooth sign ( mts)was detected first in js2 ) , and then in several other conditions previously considered as distinct entities .
this neuroradiological sign is now represented as a mandatory criterion for the diagnose of js .
js can be assumed in all infants presented with hypotonia , altered respiratory pattern , abnormal eye movement , and developmental delay3 ) . in the newborn period , episodic hyperpnea , a subtle facial appearance , and abnormal eye movements
nystagmus , colobomas , strabismus , ptosis , and retinal pigmentation are associated with eye abnormalities in neonates3 ) .
concomitant symptoms of ptosis , anisocoria with unilateral miosis , and unilateral enophthalmos , which are compatible with horner syndrome ( hs ) , have not been documented in the children of js children thus far .
we now describe a neonate of js presented with hypotonia and unique eye abnormalities of nystagmus , ptosis , anisocoria , and unilateral enophthalmos . to the best of our knowledge ,
a 2-month - old boy was referred to the pediatric neurology clinic due to bilateral nystagmus , bilateral ptosis with left - dominant , left - sided enophthalmos , and anisocoria since the second week after birth .
the patient was the first baby of a healthy 28-year - old mother and a healthy 32-year - old father .
the baby was a 3,050 g ( 10th25th percentile ) infant born at gestational age of 38 weeks by normal vaginal delivery after an uneventful pregnancy .
his height was 48.5 cm ( 25th50th percentile ) , with a head circumference of 33 cm ( 10th25th percentile ) .
after birth , the baby had laryngomalacia , hypotonia , and a systolic murmur best heard at the upper left sternal border .
an electrocardiogram and echocardiogram revealed normal sinus rhythm , and a small sized secundum atrial septal defect .
on physical examination , both pupils promptly reacted to light , but unilateral miosis and enophthalmos of the left eye and left - dominant bilateral ptosis were observed , which could be indicative of hs ( fig .
other physical examination findings were unremarkable . in order to evaluate the symptoms of hs and horizontal nystagmus , magnetic resonance imaging ( mri ) of the orbit ,
orbit and brain mri showed hypoplastic cerebellar vermis and a narrowed anteroposterior diameter of the midbrain with a characteristic of molar tooth appearance ( fig .
2 ) , which were compatible with typical findings of js . neck mri showed no definite lesion or mass around the cervical sympathetic nerve chain , thymus , and mediastinum as the underlying cause of hs .
laboratory examinations including renal and hepatic function tests and an abdominal ultrasonography showed normal findings .
left - sided visual loss was found in ophthalmological examinations , and neither coloboma nor pigmentary retinal changes were observed .
no abnormality was noted in an audiological test and follow - up electrocardiogram and echocardiogram . molecular genetic test was not performed .
clinical development at the age of 10 months was global delayed , as explained by the following : he could not babble , follow with or turn his eyes on objects , control his head , sit independently , or hold his bottle .
he underwent ophthalmologic surgery ( bilateral frontalis operation ) due to persistent visual disturbance by bilateral ptosis , and had distinct improvement of ptosis ; however , unilateral enophthalmos , and anisocoria remained ( fig .
he is currently actively undergoing rehabilitation therapy , and will be regularly monitored through comprehensive developmental examination and follow - up imaging tests , including hepatorenal , cardiac , and ophthalmologic test .
the gross photos of the figure were permitted by his parents through the informed consent .
js is a rare autosomal - recessive disorder , which is characterized by midbrain - hindbrain malformation mainly in the form of agenesis or dysgenesis of cerebellar vermis3 ) .
the incidence rate is 1 out of 800,000 to 100,000 per a year in the newborn5 ) .
describe several diagnostic criteria ; ( 1 ) cranial mri findings demonstrating the mts on axial imaging with these three components : midline cerebellar vermis hypoplasia , deepened interpeduncular fossa , and thick , elongated superior cerebellar peduncles , ( 2 ) hypotonia in infancy , ( 3 ) global developmental delay / mental retardation , and ( 4 ) one or both of the following ( not absolutely required but supportive of the diagnosis ) : ( i ) irregular breathing pattern in infancy ( episodic tachypnea and/or apnea ) , and ( ii ) abnormal eye movement ( nystagmus , jerky eye movements , and difficulty with smooth visual pursuits ) .
pathological findings of mts correspond to an image of severe hypo - dysplasia of the cerebellar vermis with midline clefting , fragmentation of cerebellar nuclei and heterotopias of purkinje - like neurons , along with dysplasia of the pontine and medullary structures such as the basis pontis , reticular formation , inferior olivary , dorsal column , and solitary tract nuclei .
moreover , typical findings are expressed by the lack of decussation both of the superior cerebellar peduncles and of the corticospinal tracts at the medullary pyramids6 ) .
a diagnosis of js should be assumed in all infants presented with hypotonia , abnormal eye movement and developmental delay .
especially , a neonate of js shows the characteristic physical signs and symptoms ( table 1)3 ) . the infant may lie in a froglike position , and moderate to severe hypotonia can be observed3 ) .
the most common presented feature in the newborn period is an abnormal breathing pattern , characterized by episodic hyperpnea23 ) .
the distinctive short alternate episodes of apnea and hyperpnea or episodic hyperpnea lone , sometimes described as panting like a dog, may intensify with stress and progressively improve with age , usually disappearing around sixth months23 ) .
the 7 children were diagnosed with js at younger than 1 year old of age78910 ) , and the case of neonatal onset was only one7 ) .
the cardinal symptoms of them were unexplainable episodic tachypnea alternating with apnea during neonatal period , abnormal ocular movement , hypotonia , ataxia , and developmental delay . in rare cases
most of the patients showed nystagmus , saccadic palsy , or congenital ocular motor apraxia as the abnormal ocular movement , however , one 7-month - old boy had a severe vision loss as leber 's congenital amaurosis8 ) .
ocular and oculomotor involvement is common in js2411 ) , however , it has variable phenotypes among patients with js .
colobomas , nystagmus , strabismus , ptosis , and abnormal retinal pigmentation are noted in neonates3 ) .
genetype - phenytype correlations have stressed the predictive value for specific mutations of retinal involvement .
retinal involvement is present in about 80% of patients with ahi1 and cep290 mutations12 ) .
ocular misalignment is common in js411 ) , because the lesions of cerebellum lead to dysmetric and slow saccades .
this patient showed unique eye abnormalities with transient nystagmus , anisocoria and enophthalmos , and eyelid ptosis .
when both anisocoria and ptosis are coexistent , one must consider 2 main possible causes : a third nerve palsy ( parasympathetic pathway ) or a hs ( sympathetic pathway)13 ) .
in most cases , a third nerve palsy can usually have disconjugate gaze with limitations of eye movements .
hs is a problem of the disturbance of the oculosympathetic pathway13 ) , which is bilaterally originating in the hypothalamus , descending ipsilaterally in the reticular formation of the brainstem , eventually synapsing with the second - order neuron in the lower cervical or upper thoracic spinal cord .
the second - order neuron enters the sympathetic chain , which then ascends within the soft tissues of the neck , outside of the spinal cord , where it is vulnerable to disruption at various locations .
it finally synapses within the superior cervical ganglion to form the third - order neuron .
this third - order neuron forms a plexus surrounding the carotid artery and into the cavernous sinus , finally innervating the dilator muscle of the iris and the mller muscle in the upper and lower eyelids .
hs is characterized by a combination of ptosis due to superior tarsal muscle palsy , enophthalmos due to mller muscle palsy , rising of inferior eyelid due to inferior tarsal muscle palsy , miosis , and anhidrosis of the involved face when there is a defect of the sympathetic nerve route from hypothalamus to the orbit14 ) .
typical causes include birth trauma , neuroblastoma , vascular anomalies of the large arteries , and chest surgery .
if there is no history of birth trauma , an acquired hs in children should prompt evaluation for a tumor , particularly paraspinal neuroblastoma .
imaging studies , such as mri of the head , neck , chest , and abdomen , as well as the measurement of urine catecholamines should be evaluated .
these variable ocular phenotypes might be supported by a third nerve palsy and multiplex neuropathological findings of the cerebellar vermis and of several pontine and medullary structures .
it may arise from the genetic heterogeneity of js and related disorders15 ) . in conclusion
, we describe an infant of js with unique eye abnormalities , likely as symptoms of hs .
it may suggest that a neonate or young infant with diverse eye abnormalities should be considered for early neuroimaging to evaluate the underlying etiology , although there may be no focal neurologic deficit or significant developmental delay .
furthermore , once a diagnosis of js has been made , a further evaluation to assess the possibility of multiorgan involvement should be considered in children . | joubert syndrome ( js ) is characterized by the molar tooth sign
( mts ) with cerebellar vermis agenesis , episodic hyperpnea , abnormal eye movements , and hypotonia .
ocular and oculomotor abnormalities have been observed ; however , horner syndrome ( hs ) has not been documented in children with js .
we present the case of a 2-month - old boy having ocular abnormalities with bilateral nystagmus , left - dominant bilateral ptosis , and unilateral miosis and enophthalmos of the left eye , which were compatible with hs .
brain magnetic resonance imaging ( mri ) revealed the presence of the mts .
neck mri showed no definite lesion or mass around the cervical sympathetic chain .
his global development was delayed .
he underwent ophthalmologic surgery , and showed some improvement in his ptosis .
to the best of our knowledge , the association of hs with js has not yet been described .
we suggest that early neuroimaging should be considered for neonates or young infants with diverse eye abnormalities to evaluate the underlying etiology . |
the patient was a 77-year - old female patient admitted for surgery for the chief complaint of an intraductal carcinoma in the left breast that was diagnosed at another hospital .
the plain chest radiographs taken 3 years earlier showed " no detection of abnormal findings " , and dyspnea occurred during exercise from 2 years earlier . during a physical examination performed in march 2009 , she was diagnosed with intraductal carcinoma in the left breast , 3.8 cm in size , with the possibility of invasion to the lower areola . in the presurgical tests ,
1 ) . the bronchial endoscopic examination revealed the deposition of dark pigmentation ( anthracotic patches ) in the distal area of the right main bronchus , right middle lobe , right lower lobe and area from the left upper lobe to the left lower lobe , as well as a narrowing of the bronchial lumen of the right middle lobe due to fibrosis ( fig .
chest computed tomography revealed atelectasis in the right middle lobe associated with a calcified and ruptured lymph node and anthracofibrosis in the entire airway ( fig .
the presurgical pulmonary function test showed a fvc , fev1 and fev1/fvc of 1.59 l ( 73% ) , 1.04 l ( 71% ) and 66% , respectively . with the exception of a mild increase in liver enzymes in the blood test performed before surgery ,
no special findings were detected in the electrocardiogram , cardiac ultrasonography , and other tests .
the patient tested positive to the m. tuberculosis polymerase chain reaction test performed after the bronchial endoscopic examination , and she was placed on antituberculosis medicines from 3 weeks before surgery . for the induction of anesthesia ,
200 mg thiopental , 50 mg rocuronium , and 50 g fentanyl were injected , and the anesthesia was maintained with 2.0 - 2.5 vol% sevoflurane , 1 l / min oxygen , and 1 l / min air . because of the characteristics of the planned modified radical mastectomy , a large change in position from the supine position was not required , and a laryngeal mask airway ( proseal # 4 ) was used instead of endotracheal intubation to minimize airway stimulation . to reduce respiratory secretion , 0.2 mg glycopyrrolate
was injected intramuscularly during the induction of anesthesia . in the preparation for airway hemorrhage that might occur accidentally during the perioperative period
after inducing anesthesia , the arterial blood pressure and central venous pressure were monitored by inserting a 20 g and 7 fr , 3 lumen vascular catheter into the right radial artery and right internal jugular vein respectively . in arterial blood gas analysis
performed at the initiation of surgery under the conditions of 0.5 fio2 , 37 mmhg etco2 , and 100% spo2 , the ph was 7.480 , pco2 was 37.1 mmhg , po2 was 286.0 mmhg , hco3 was 27.0
mmol / l , be was 3.4 mmol / l , hb was 10.7 g / dl , hct was 31% , na was 136.0
the respiratory tidal volume , respiratory rate , peak pressure and pulse oximetry saturation level was 360 ml per respiration , 10 - 14 times , 15 mmhg and 100% , respectively .
the operation time was 1 hour 20 minutes , anesthesia time was 1 hour and 50 minutes , and surgery was completed without special events .
arterial blood gas analysis was performed at the time of the induction of mild hypercapnia by manual ventilation to recover self respiration and awaken the patient from anesthesia under the conditions of 0.5 fio2 , 45 mmhg etco2 , and 100% spo2 .
the results revealed a ph of 7.365 , pco2 of 49.5 mmhg , po2 of 233.6 mmhg , hco3 of 27.7 mmol / l , be of 1.7 mmol / l , hb of 11.1 g / dl , hct of 33% , na of 136.3 mmol / l , k of 3.45 mmol / l , ca of 1.04 mmol / l , and glu of 108 mg / dl .
after the recovery of sufficient self respiration , the laryngeal mask airway was removed under the inflation state , and endotracheal suction was not performed .
she recovered smoothly from anesthesia , and was transported to the ward via a recovery room .
, she did not live in a mining area , and farmed in yeongcheon city , gyeongsangbuk - do , which is believed to be less polluted than the larger cities .
she did not have prior history of tuberculosis or treatments for tuberculosis , and was a non - smoker .
therefore , the cause of the anthracofibrosis in this patient was speculated to be an undiagnosed tuberculosis infection .
she was confirmed to have bronchial tuberculosis by the positivity for m tuberculosis polymerase chain reaction performed prior to surgery .
as the mechanism for the deposition of pigment within the bronchus and fibrosis associated with tuberculosis , there is a theory that inflammation in the tubercular lymph nodes induces tuberculosis in the endobronchus through the bronchial wall , resulting in pigment deposition and stenosis .
consequently , endobronchial anthracofibrosis is the state of the development of the most undesirable sequelae of endobronchial tuberculosis .
chung and lee suggested that endobronchial tuberculosis progresses in 6 stages . among these stages , the infiltration of submucosal lymphocytes into the bronchus forms granulation tissues , and endobronchial stenosis occurs inevitably in cases in whom the disease progresses more than this decisive stage .
therefore , the most important point in the treatment of endobronchial tuberculosis is not to miss the critical time . for this , a bronchial endoscopic examination is required for both diagnosis and treatment .
anthracofibrosis associated with tuberculosis and tubercular endobronchial diseases occur more often in females than males ( 2.4 times and 5.4 times , respectively ) .
the typical characteristics of patients with bronchial stenosis due to anthracofibrosis are elderly women , without a prior history of pneumoconiosis or smoking , a chief complaint of cough without systemic symptoms or dyspnea , and findings of segmental or lobar consolidation on the plain chest radiographs .
in addition , chest computed tomography reveals diverse bronchial abnormalities surrounded by soft tissue or lymph nodes .
the most common invaded area is the right middle lobe bronchus , and an active tuberculosis infection is demonstrated in more than 60% patients , which concurs with the present findings .
huh and kang reported a case of an 84-year - old male patient with undiagnosed endobronchial anthracofibrosis before surgery , who developed severe endobronchial hemorrhage after endotracheal suction by a routine method performed in the recovery room . in the recovery room ,
the hemorrhage began while inserting a suction catheter through an endotracheal tube and aspirating airway secretion .
although the blood was aspirated continuously , the volume did not decrease , and the patient was transported to the intensive care unit .
bronchial endoscopy was performed to stop the hemorrhage , where at that time , together with a large hematoma , bronchial fibrosis and the deposition of dark pigment in a wide area of the right main bronchus adjacent to the carina were detected .
the patient showed repeated deterioration and improvement , and acute respiratory distress syndrome developed on the day 5 after surgery .
the patient died of multiple organ failure on day 63 after surgery . in our case , to reduce the risk of endobronchial hemorrhage , the amount of airway secretion was reduced using anticholinergic agents , and endotracheal suction using a suction catheter was not performed .
the entire length of the endotracheal tube ( mallincrodt medical inc . , ireland ) with a 7.0 mm
inner diameter , which is generally used for the general anesthesia in adult women , is approximately 34 cm when it is firmly fitted with force to prevent the separation of the connector from a catheter .
when a 12 fr suction catheter ( pvc suction catheter , qingdao sewoon medical co. ltd . , china ) was pushed to the end through this endotracheal tube , even the suction catheter completely passed the end of the endotracheal tube , and descends to the lumen of the bronchus by approximately 13 cm .
if a 14 fr suction catheter with a slightly larger inner diameter is used , it would enter the lumen of the bronchus by approximately 12 cm .
considering that the length of korean women from the upper incisors to the carina is approximately 18.5 - 25 cm , a suction catheter could enter the lumen of the lobar bronchus readily . in our case , pigment deposition plaque due to anthracofibrosis were formed at the distal area of right main broncus , and presented in the right middle and lower lobe and the left upper and lower lobe , too .
therefore , if endotracheal suction was performed without paying particular attention , a suction catheter possibly irritates the anthracofibrotic lesion at the distal area of the right main bronchus , and may induce the development of mass hemorrhage . the standard guidelines for the airway management for the cases with anthracofibrosis have not been established . in cases placing endotracheal tubes , particularly during the recovery of anesthesia , severe cough may develop due to bronchial stimulation and more hemodynamic changes may occur during the insertion and removal of the endotracheal tube .
it is possible that in cases using the laryngeal mask airway , as in our case , direct airway stimulation on the vocal cord and the proximal area of trachea could be decreased relatively . nevertheless , if it is not installed accurately , saliva , etc .
in addition , in cases with obstructive lung diseases due to endobronchial anthracofibrosis , adequate ventilation may be impossible with the laryngeal mask airway only . in our case ,
sufficient ventilation was achieved only by the placement of a laryngeal mask airway , and the maintenance of anesthesia and recovery were achieved without a large amount of airway secretion . similar to surgery that must use a laparoscope , for cases in whom an endotracheal tube must be inserted because of the characteristics of surgery itself requiring a high airway pressure , it would be desirable to assess the accurate position of anthracofibrosis lesions by performing a bronchoscopic examination in advance , to use agents that could reduce the level of airway secretion , and to restrain the use of a suction catheter as much as possible . in conclusion , for anthracofibrosis patients , anesthesia plans should be made by considering the possibility of the development of severe hemorrhage within the bronchus if airway suction is performed without paying attention .
in korea , where the incidence of tuberculosis is high , there is a strong likelihood of undiagnosed endobronchail anthracofibrosis in elderly women .
therefore , it is important to minimize airway stimulation . if continuous abnormal hemorrhage is detected after endotracheal suction , it is important to consider the possibility of massive hemorrhage within the bronchus caused by underlying anthracofibrosis .
in addition , in cases with anthracofibrosis findings detected accidentally during the endobronchial manipulation using a bronchoscope , it is highly probable that undiagnosed tuberculosis would be the cause . | bronchial anthracosis was recently defined in the english radiology literature as a luminal narrowing associated with anthracotic pigmentation on bronchoscopy without a relevant history of pneumoconiosis or smoking .
anthracosis refers to the presence of carbon particles in the lungs , not to a disorder per se .
anthracofibrotic lesions carry the potential risk of massive hemorrhage during endobronchial procedures .
this report describes a case of general anesthesia for a left modified radical mastectomy due to an intraductal carcinoma in a patient with known bronchial anthracofibrosis . |
a patient with severe papulopustular rosacea ( ppr ) and severe background erythema responded well to a treatment regimen consisting of a short course of antibiotics in combination with a corticosteroid , followed by monotherapy with isotretinoin.in patients with very severe , inflamed disease , it is necessary to achieve a rapid response with a fast - acting combinatorial treatment regimen to reduce the inflammation.patients with severe ppr and an inflammatory component can respond rapidly when treated using a multimodal , tailored approach .
because rosacea presents as a spectrum of overlapping symptoms of variable severity , it is important to adapt treatment decisions to individual clinical presentations .
papulopustular rosacea ( ppr ) is a commonly encountered subtype of rosacea characterized by the presence of persistent central facial erythema ( background erythema ) , accompanied by central facial inflammatory lesions ( papules and/or pustules ) .
patients with ppr often present with lesional and perilesional erythema , which is found around and immediately contiguous to a papule or pustule and contributes to the overall appearance of facial redness .
agents currently approved for the treatment of the inflammatory lesions of rosacea are known in clinical practice for their additional effect of decreasing the overall severity of facial erythema , as a result of the reduction of the lesional and perilesional erythema .
however , these agents are not indicated in the treatment of background erythema , which typically persists after clearance of the inflammatory lesions .
alternative treatments for the symptoms present in patients with ppr include short courses of second - generation macrolides such as clarithromycin and azithromycin , which have demonstrated a fast onset of action and a good safety profile in the treatment of patients with ppr [ 3 , 4 ] .
these agents may therefore prove useful as an off - label treatment in patients with severe disease who do not respond to tetracycline therapy , or when isotretinoin is contraindicated .
in addition , although corticosteroids are not indicated for use in rosacea , their short - term use may be considered in patients with severe disease who present with signs of rosacea fulminans .
there is an increasing awareness of the need to repair and protect the skin barrier as part of the treatment strategy when prescribing treatments aimed at strongly targeting the inflammatory component .
it is known that an impaired skin barrier leads to a heightened and potentially chronic inflammatory response .
repairing and protecting the skin barrier will decrease the risk of this process occurring , while also reducing the incidence of dermatological adverse events [ 7 , 8 ] . here
, we report a case of severe ppr with an important inflammatory component . a strategy aimed at strongly targeting the inflammation upfront
in may 2014 , a 30-year - old caucasian woman presented to our department ( fig .
for the past 4 years , the patient had suffered from severe ppr ( principally on the forehead , cheeks , and chin ) with signs of rosacea fulminans , as well as severe background erythema in the central facial and forehead regions .
she had previously been treated with metronidazole 7.5 mg / g cream and permethrin 50 mg / g cream , which had not provided a reduction in the number of inflammatory lesions .
she had also been prescribed doxycycline modified - release 40 mg for 18 months , which had also proved unsuccessful in reducing the inflammation.fig .
1patient with rosacea presenting with overlapping symptoms of severe inflammatory lesions and severe background erythema , receiving sequential treatment with azithromycin 500 mg plus prednisolone , isotretinoin 10 mg , and topical brimonidine 3 mg / g gel ; topical metronidazole 7.5 mg / g was administered throughout the entire period . a may 2014 ( baseline ) : patient with severe papulopustular rosacea , with signs of rosacea fulminans , and severe background erythema at initial presentation .
b june 2014 ( 4 weeks after baseline ) : 4 weeks after initiation of treatment with azithromycin 500 mg three times weekly plus once - daily prednisolone ( 30 mg for 1 week ; 10 mg for 3 weeks ) , in combination with metronidazole 7.5 mg / g cream twice daily .
c july 2014 ( 8 weeks after baseline ) : following a further 4 weeks of azithromycin 500 mg three times weekly , in combination with once - daily prednisolone 5 mg treatment ;
the patient continued to apply metronidazole 7.5 mg / g cream twice daily . isotretinoin
d october 2014 ( 25 weeks after baseline ) : following 4 months of treatment with isotretinoin 10 mg once daily .
e december 2014 ( 32 weeks after baseline ) : following a further 2 months of treatment with isotretinoin 10 mg once daily in combination with brimonidine 3 mg / g gel applied to the left side of the face 3 h before presentation to our outpatient department .
f march 2015 ( 43 weeks after baseline ) : following an additional 3 months .
treatment with isotretinoin 10 mg once daily was stopped after 9 months ; treatment with metronidazole 7.5 mg / g cream twice daily was continued .
the patient had applied brimonidine 3 mg / g gel to the left side of her face on the morning of the visit to the clinic patient with rosacea presenting with overlapping symptoms of severe inflammatory lesions and severe background erythema , receiving sequential treatment with azithromycin 500 mg plus prednisolone , isotretinoin 10 mg , and topical brimonidine 3 mg / g gel ; topical metronidazole 7.5 mg / g was administered throughout the entire period . a may 2014 ( baseline ) : patient with severe papulopustular rosacea , with signs of rosacea fulminans , and severe background erythema at initial presentation .
b june 2014 ( 4 weeks after baseline ) : 4 weeks after initiation of treatment with azithromycin 500 mg three times weekly plus once - daily prednisolone ( 30 mg for 1 week ; 10 mg for 3 weeks ) , in combination with metronidazole 7.5 mg / g cream twice daily .
c july 2014 ( 8 weeks after baseline ) : following a further 4 weeks of azithromycin 500 mg three times weekly , in combination with once - daily prednisolone 5 mg treatment ;
the patient continued to apply metronidazole 7.5 mg / g cream twice daily . isotretinoin
d october 2014 ( 25 weeks after baseline ) : following 4 months of treatment with isotretinoin 10 mg once daily .
e december 2014 ( 32 weeks after baseline ) : following a further 2 months of treatment with isotretinoin 10 mg once daily in combination with brimonidine 3 mg / g gel applied to the left side of the face 3 h before presentation to our outpatient department . f march 2015 ( 43 weeks after baseline ) : following an additional 3 months .
treatment with isotretinoin 10 mg once daily was stopped after 9 months ; treatment with metronidazole 7.5 mg / g cream twice daily was continued .
the patient had applied brimonidine 3 mg / g gel to the left side of her face on the morning of the visit to the clinic at presentation , the patient reported feelings of hopelessness , indicating a profound impact of the disease on her quality of life . following diagnosis of ppr with severe background erythema , different treatment options were evaluated .
in such a severe case , a specific strategy tailored to the patient was essential , particularly given the lack of success with prior regimens , which seemed to indicate that a more potent treatment regimen was needed in this patient to combat the inflammation upfront .
isotretinoin was initially considered ; however , because the patient was not taking hormonal contraception at the time of her visit to our department , this therapy was contraindicated .
the patient was offered a gynecology appointment to start hormonal contraception , so that isotretinoin could be an option in the future .
the patient was initially prescribed an 8-week regimen of azithromycin 500 mg three times weekly ( taken in the morning ) in combination with prednisolone 30 mg once daily ( reduced to 10 mg once daily after 1 week ) .
the choice of this regimen was based on results from the literature showing that a rapid response can be obtained with a multimodal , tailored approach with a short course of macrolide antibacterials .
furthermore , the use of corticosteroids over the short term is not strictly contraindicated in the treatment of rosacea and can be considered as an option to reduce inflammation in patients who present with signs of rosacea fulminans .
metronidazole 7.5 mg / g cream twice daily was continued with this combination therapy .
after 4 weeks of treatment , a reduction in the inflammatory lesion count was observed ( fig .
a slight decrease in the severity of facial redness was also seen , especially on the forehead , most likely resulting from a reduction in lesional and perilesional erythema .
the patient continued to take azithromycin 500 mg three times weekly and the prednisolone dose was reduced to 5 mg once daily . in addition , the patient continued to apply metronidazole 7.5 mg / g cream twice daily . during examination of the patient s face 1 month later , only a small number of inflammatory lesions were present ( fig .
the severity of erythema remained similar to that observed during the previous visit , indicating a reduction in the severity of the inflammation .
therapy with azithromycin 500 mg three times weekly and prednisolone 5 mg once daily was stopped ; treatment with isotretinoin 10 mg once daily ( taken with the main meal ) was initiated , and application of metronidazole 7.5 mg / g cream twice daily was continued .
when the patient returned to our department 4 months later , no inflammatory lesions were present ; however , the erythema had worsened on the forehead and cheeks ( fig .
the patient continued to apply metronidazole 7.5 mg / g cream twice daily , as well as isotretinoin 10 mg once daily for further improvement and as maintenance therapy to prevent relapse of inflammatory lesions .
treatment with brimonidine 3 mg / g gel , used as needed and applied in the morning , was initially started to the left side of her face in order to assess the impact of this agent on the background erythema ( split - face assessment ) .
brimonidine , a highly selective 2-adrenergic receptor agonist , is approved for the symptomatic treatment of facial erythema in adults with rosacea , in a formulation of brimonidine 3 mg / g gel ( mirvaso ; galderma international , la dfense , france ) .
this agent has previously been shown to be an effective option in the treatment of moderate to severe erythema , with a good safety profile . in december 2014 , 2
months after commencing treatment with brimonidine 3 mg / g gel on the left side of her face and while continuing treatment with isotretinoin 10 mg once daily to maintain remission of inflammatory lesions , the patient returned to our department , showing mild erythema on the right side of the face , with background erythema cleared on the left side of the face ( fig .
1e ) . of note , during the treatment period with brimonidine 3 mg / g gel , the patient did not experience any dermatological adverse events that required her to stop treatment temporarily .
at the most recent visit to our department in march 2015 , the patient showed an increase in erythema severity on the right side of her face ( not treated with brimonidine 3 mg / g gel ) compared with the previous visit , reflecting the dynamic and changing nature of this symptom .
the left side of the face , treated with brimonidine 3 mg / g gel on the morning of the visit to the clinic , remained clear of erythema ( fig .
isotretinoin treatment was stopped and the patient was advised to continue maintenance therapy with metronidazole 7.5 mg / g cream twice daily and brimonidine 3 mg / g gel .
here , we have reported the case of a patient with severe , inflamed ppr with a high inflammatory lesion count and severe erythema .
although corticosteroids are not generally recommended for the treatment of rosacea , because the level of inflammation in this patient was high , and similar to levels seen in patients with rosacea fulminans , it was decided that the initial course of treatment should consist of azithromycin 500 mg three times weekly in combination with prednisolone ( initially 30 mg once daily and then gradually reduced and stopped ) . following this short course of treatment , the next step was to continue decreasing the inflammation with isotretinoin 10 mg once daily monotherapy , and finally isotretinoin 10 mg once daily with brimonidine 3 mg / g gel on one side of the face to address the background erythema component of the disease . in cases of recalcitrant ppr , oral isotretinoin can be used over a 3- to 4-month treatment period to achieve a complete clearance of inflammatory lesions . in selected cases , a longer duration of therapy may be required in order to achieve an adequate level of improvement . in this case , following 4 months treatment with isotretinoin , all inflammatory lesions had disappeared .
treatment was continued for another 5 months to ensure maintained remission of inflammatory lesions . as the skin permeability barrier
is often impaired in rosacea , causing additional skin sensitivity and irritation , metronidazole 7.5 mg / g cream twice daily was applied throughout the courses of treatment , based on its proven effects in reducing skin irritation , transepidermal water loss , and erythema .
based on the positive outcome seen overall , the patient is currently on maintenance therapy with metronidazole 7.5 mg / g cream twice daily and brimonidine 3 mg / g gel , with no relapse of disease .
in patients with very severe , inflamed rosacea , a multimodal , tailored approach with a short course of macrolide antibiotics in combination with corticosteroids is needed to strongly address the inflammatory component upfront and improve patient outcomes ; corticosteroids are not strictly contraindicated in the treatment of rosacea if used for a short period to reduce inflammation showing signs of rosacea fulminans . in these patients , long - term therapy is needed to improve outcomes , with isotretinoin being effective in further reducing inflammatory lesions count and in maintaining remission .
the patient experienced a continuous reduction in the severity of her erythema , which was further improved with the use of brimonidine 3 mg / g .
the lack of reported dermatological adverse events with this topical treatment could in part be due to the continued application of metronidazole ; we believe metronidazole contributed to repairing and protecting the skin barrier , helping to restore skin barrier function and consequently lessening inflammation . for complex cases such as this one , it has been acknowledged that careful selection and use of a combination of clinical treatments is essential to treat the individual .
we believe that the publication of additional multifaceted cases such as the one presented here will help to facilitate the development of such guidelines .
| rosacea is a chronic inflammatory disease that can manifest as a spectrum of symptoms including erythema , inflammatory lesions , edema , and telangiectasia .
treatment decisions need to be adapted to reflect the nature and severity of the different symptoms present . in this report
, we discuss the case of a female patient diagnosed with severe , inflamed papulopustular rosacea ( ppr ) presenting with a large number of inflammatory lesions and severe background erythema .
this patient responded well to a treatment regimen consisting of a short course of antibiotics in combination with a corticosteroid , followed by monotherapy with isotretinoin to reduce the inflammation .
brimonidine gel , used as needed , was then added to isotretinoin to target the remaining background erythema .
this case of severe ppr required a combinatorial treatment regimen to effectively target all symptoms present .
the patient continued to apply topical metronidazole throughout the different treatment regimens prescribed over the course of almost 1 year .
use of topical metronidazole helped to repair and protect the skin barrier , which minimized the occurrence of dermatological adverse events when topical treatments were used .
we conclude that in patients with severe disease and an important inflammatory component , a rapid response can be obtained with a multimodal , tailored approach that also includes treatment to repair and protect the skin barrier . |
the typical adult individual seen by a primary care physician most likely believes that cancer screening is a good idea , but without comprehending the trade - off of harms to benefit or the uncertainties surrounding their magnitude .
the simplistic but effective mantra that early detection saves lives is one cause of the general enthusiasm for cancer screening . in the united states ( u.s . ) , a double standard exists : women are encouraged to participate in screening mammography , while men are advised to make informed decisions regarding prostate screening , although the fundamental issues to consider are very similar .
while professional society screening guidelines generally promote breast screening , they may be influenced by intellectual and financial conflicts of interest and generally have a poor evidence base compared with , for example , those from the u.s .
for informed choice to occur , the estimates of harms and benefit from screening mammography , along with their uncertainty , must be effectively communicated to women .
one challenge is that important screening harms are often not mentioned or misrepresented in promotional campaigns for screening and in the medical literature .
primary care physicians are a trusted source of information about cancer screening , but many are not trained in interpreting and presenting cancer screening statistics , which are often complex or even counterintuitive .
the goal of this review is to inform primary care physicians about four central screening principles applicable to mammography , so that they can help women make an informed decision on whether or not to participate .
breast cancer screening is a type of secondary prevention that is justified if the screening technology can identify and advance the diagnosis of otherwise lethal tumors and earlier treatment for this asymptomatic disease is more effective than usual treatment . in addition
, this benefit must outweigh any harms from the screening intervention . in order for screening to be beneficial and reduce mortality and mastectomy rates
, screening must reduce the incidence rate of advanced disease . in measuring advanced disease , stage is a less robust measure than size , as detection methods and stage definitions change over time .
have shown that there has been no decrease in the incidence rate of tumors over 2 cm in six western countries with population screening through many years , including the united states .
found identical decreases in advanced stage incidence in screened and unscreened areas of norway where breast screening was introduced in a staggered fashion , creating a control group . in the u.s .
there has been a slight reduction in the incidence rate of regional metastatic cancers , but none in the rate of distal ( stage 4 ) metastatic disease , even though screening mammography has existed for 30 years ( fig .
1 ) . the findings from norway suggest caution rather than uncritically attributing the reduction in regional metastatic cancer in the u.s . to screening , as such a reduction can happen unrelated to this .
surveillance , epidemiology , and end results data for invasive breast cancers without metastases , with regional metastases , and those with distant metastases involving other organs at diagnosis , as well as ductal carcinoma in situ lesions ( dcis ) are included , for women aged 4084 years at diagnosis , apart from dcis ( 50 + years ) .
there was no decline in the occurrence of cancers with distant metastases over the observation period , a slight decline for cancers with regional metastases , and vast increases for both localized and in situ cancers . since screening likely has little or no impact on the incidence rate of advanced disease , the most plausible explanation for the large and persistent increase in both in situ lesions and localized breast cancers wherever screening has been implemented is overdiagnosis .
overdiagnosed tumors are those screen - detected cases that would never present clinically or cause any problems in a woman 's lifetime .
overdiagnosis means that many healthy women will receive unnecessary surgery , chemotherapy , endocrine therapy , and radiation , and this overtreatment can only cause harm .
tumors , but this claim ignores extra treatment caused by overdiagnosis and is based on what seems intuitively correct rather than on evidence . even if some individual women with earlier detected disease receive less invasive therapy , overall the number of women with surgeries increases .
the randomized trials showed that screening participation increases lumpectomy and mastectomy rates by 30% and 20% , respectively .
in situ lesions
were rare before screening , and now most cases are detected as microcalcifications with mammography ( fig .
1 ) . in both the u.s . and the united kingdom , the proportion of in situ cases treated by mastectomy are 29% , and these new cases likely account for much of the increase in mastectomy rates due to screening . in the u.s . , about 20% of all diagnosed breast cancers and one - third of screen - detected cancers are in situ cases .
newer screening technology such as digital mammography , computer - aided detection , and magnetic resonance imaging likely increase overdiagnosis .
earlier detection does not necessarily improve prognosis either , because adjuvant therapy and chemotherapy are effective in all prognostic groups , even for metastatic disease .
in fact , improved treatment has caused the majority of the impressive declines in breast cancer mortality over the past 20 years .
countries that introduced screening late have experienced declines in breast cancer mortality that are equal to , and sometimes larger than , countries that introduced breast screening early . this conclusion is supported by the fact that reductions in breast cancer mortality across europe have been about 50% in women who are below the screening age ( < 50 years ) , which is considerably more than in age groups most often invited ( about 30% in women aged 5069 years ) . with better medical treatment , the absolute benefit of screening declines as there are fewer breast cancer deaths to prevent , a development that will continue with further advances in treatment . the u.s .
breast cancer death risk without screening for women age 50 is now 9/1000 over 15 years , a decrease from 11/1000 in 19781980 due to better treatment .
breast cancer awareness means women are more likely to notice a smaller symptomatic tumor than in the past , adding to this development .
screening statistics must be interpreted with caution , since they are subject to several biases .
many have difficulty distinguishing between the most reliable effect estimate for a screening intervention , reduced all - cause mortality rates in randomized trials , from two misleading but often cited screening statistics : increased survival - time and stage distribution percentages . instead of all - cause mortality , disease - specific mortality in the breast cancer screening trials was chosen because many fewer trial participants were required , but it is biased ( e.g. , due to uncertain attribution of cause of death and may be misleading because of increased mortality from other causes due to overdiagnosis ) . overdiagnosis leads to overtreatment with radiotherapy and chemotherapy and thus increased mortality from heart disease and other malignancies that may entirely outweigh the benefit in terms of reduced breast cancer mortality . only all - cause mortality avoids these biases and measures both the major benefit and harm , but the benefit of breast screening on all - cause mortality is so small that hundreds of thousands of participants are required to test if the effect is there .
the randomized trials , including > 600,000 women , did not show an effect on total mortality , which tells us that the absolute benefit of the intervention must be quite small , if it is there . the relative risk reduction ( rrr ) for disease - specific mortality in the screening mammography trials was 10%20% , but this number can be misleading and is not informative on its own . for informed choice , a woman needs to know the absolute benefit or absolute risk reduction ( arr ) , which is the rrr times the base rate for the breast cancer death risk without screening . for u.s .
women at age 50 years , this base rate is about 5/1000 over 10 years .
the base rate over 10 years for women in the swedish trials , which excluded women with a preexisting breast cancer diagnosis , was 3.4/1000 .
the cochrane review of all the screening trials calculated an arr of 0.5 percentage points , which means that 2000 women need an invitation to screening to avoid one breast cancer death over a 10 year period .
a different perspective is obtained by presenting the mortality benefit in relation to the higher base rate of all - cause death risk .
routine screening starting at age 50 with an arr of ( 0.15 * 5/1000 ) or 0.8/1000 is equivalent to a rrr of ( 0.8/37 ) or 2.2% using all - cause mortality ( fig .
routine screening increases a nonsmoking woman 's overall survival chance from 96.3% ( 1003.7 ) to 97.1% over 10 years .
women 's inaccurate and exaggerated perceptions of the benefit of screening are a challenge in promoting informed choice . in a 2003 survey , more than half of u.s .
women thought that screening mammography can prevent or reduce the risk of contracting breast cancer .
most believed that the rrr was over 50% , and the arr was greater than 80/1000 .
this is 100 times the actual benefit of 0.8/1000 , and twice the all - cause death risk .
women by smoking status ( age 40 and 50 years ) compared with the breast cancer death risk and lives extended through screening .
starting at age 50 , 713 women die from something else for every breast cancer death . the absolute risk reduction
( arr ) or benefit from routine screening mammography is the assumed relative risk reduction ( rrr ) times the breast cancer death risk without screening .
the arr in absolute numbers is the same as lives extended through inviting 1000 women to routine screening .
instead of using the arr , some patient organizations like the susan g. komen foundation argue that screening improves survival time .
lead - time bias means that earlier diagnosis through screening will increase the measured survival - time from diagnosis as one will live longer knowing about the cancer , regardless of whether screening makes people live to an older age .
because there is more time available to detect slow - growing rather than fast - growing tumors , routine screening will preferentially detect slow - growing and more indolent tumors , and is less likely to catch fast - growing , more lethal tumors .
these biases mean that women with screen - detected tumors will inevitably have a longer survival time and a more favorable prognosis than clinically detected ones , even if screening has no real benefit .
overdiagnosed tumors therefore all have a very favorable prognosis and survival time but contribute to making survival time noninformative .
the second misleading statistic is changes in cancer stages observed after screening due to lead - time and length biases .
there is an increase in percentage of localized cancers as a fraction of total cancers ( accounting for size , affected lymph nodes , and metastases ) with screen - detected versus symptomatic cancers ( fig .
the correct measure is the rate ( say , per 100,000 ) of advanced tumors in a population . as esserman
et al . have shown , the best - case screening scenario has a declining advanced cancer rate with a stable total cancer rate , because every case of early detection of an invasive cancer or in situ lesion causes one less case of advanced cancer ( true stage - shift ) .
the worst - case screening scenario has a stable advanced cancer rate with a growing total cancer rate , because all early detection is overdiagnosis ( no stage shift ) . in summary ,
an increase in the percentage of localized cancers always occurs , regardless if screening is effective , just like improved survival time .
the increased percentage of localized breast cancers ( stages 0 and 1 , < 2 cm ) after screen - detection does not prove screening is beneficial .
this result is due to lead - time bias and length bias ( overdiagnosis ) .
likewise , the increased percentage of smaller invasive tumors after screen - detection does not prove a morbidity or mortality benefit .
finally , case - control and cohort studies that measure exposure or screening participation regularly estimate double or triple the benefit , with rrrs of 30%40% , or more .
however , we know from recalculations of results from the randomized trials that the case - control design can yield effect estimates of more than a 50% reduction in breast cancer mortality when in fact there is none . the apparent , but spurious , effect is caused by selection or volunteer bias ; those who choose to go for screening are generally healthier and less likely to die from breast cancer , as well as from many other causes .
some researchers claim to avoid this bias when they use statistics that count only women who attend screening , but reliably adjusting for selection bias , the size of which varies and is unknown in a given setting , is likely not possible .
a systematic review of seventeen relevant population studies concluded that the benefit of screening mammography today is probably smaller than in the randomized trials .
3 ) . a 10-mm diameter tumor has 1 billion cells after 29 doublings , and assuming a median volume doubling time of 260 days , is about 20.7 years old . in the united states ,
the median symptomatic invasive tumor is about 21 mm ( 20 mm = 32 doublings , 22.8 years ) , and the median screen - detected invasive tumor is 14 mm ( 13 mm = 30 doublings or 1 cm , 21.4 years ) .
the difference in average size would therefore seem to be about 7 mm or 1.4 years , but in reality it is smaller because the many small , overdiagnosed tumors exaggerate the difference . in the randomized trials ,
the mean diameter of tumors in the screened groups was 16 mm ( 16 mm = 31 doublings ) , and in the control groups it was 21 mm .
the 5 mm size difference , including the overdiagnosed tumors , represents just over one volume doubling , or 340 days , which is 4% of a tumor 's lifetime . again disregarding overdiagnosis , the time difference between u.s .
screen - detected ( 21.4 years old ) and clinically detected ( 22.8 years old ) tumors ( or two doublings ) would be at most 520 days .
screening mammography therefore likely advances the time of diagnosis ( lead - time ) by far less than the 27 years that is usually assumed in studies of overdiagnosis that attempt to compensate for this lead - time effect .
women with breast cancer do not die from the primary tumor , but from metastatic disease .
only tumors that metastasize during the part of the tumor 's lifetime when it can be detected by mammography but is not yet symptomatic may have their prognosis improved . we know that some very small tumors send out micrometastases below the mammogram threshold , while some large symptomatic tumors have late occurring or no metastases .
the window narrows even more because in order to be successful , the screen - detection must occur before the tumor metastasizes . for every 5 mm of tumor growth , node - positivity increases by about 5% .
based on the mean 16-mm screened group tumor size from the trials , 35% of tumors would have already metastasized before being detectable . given the mean size in the control group of 21 mm , which is also the median size of u.s .
symptomatic tumors today , 40% of tumors would have metastasized . therefore , 5% would metastasize during the screening window , yielding a plausible rrr of breast cancer mortality of 12% due to screening . in summary ,
a 1- to 2-year window is too narrow for screening mammography to have a large effect .
each concentric circle depicts one volume doubling of an invasive breast tumor ( time scale ) .
the difference in median tumor size between screen - detected and clinically detected tumors ( 57 mm ) is one to two of 32 tumor - doubling times .
we provide a decision model with a summary of possible outcomes for women considering screening ( fig . 5 ) .
about 55% of breast cancers diagnosed in the u.s . are screen - detected , with symptomatic tumors diagnosed in women who did not screen , or as interval cancers between screening rounds . a u.s .
woman with a screen - detected in situ lesion or invasive cancer will have a true positive mammogram .
using optimistic and pessimistic screening scenarios as limits , she may be helped , hurt , or her prognosis unchanged ( table 1 ) .
for the baseline scenario single screening round for women ages 5059 years , for every 100 women with screen - detected cancer , over 15 years , 8 will have their lives extended .
this estimate of lives saved is optimistic , and is probably fewer than 5 lives saved .
since 21 women die despite screen - detection , of the remaining 79 survivors , 10% ( 8/79 ) are cured by earlier treatment , but over half of the survivors ( 42/79 ) are overdiagnosed and harmed .
these findings are contrary to the popular belief that screen - detection is synonymous with cure , reflecting the lack of balanced messaging regarding screening mammography . simplified screening mammography decision model with benefit and harms .
the top branch , or no screening decision , shows that some early cancers do not progress to cause symptoms . with screening ( bottom branch ) , many of these lesions are detected earlier , resulting in the harm of overdiagnosis .
although some otherwise lethal cases can be cured from earlier treatment , most cases that are not overdiagnosis remain curable or remain lethal despite screen - detection .
since mammography is imperfect , some cancers are not detected ( false negatives ) , and some healthy women are recalled for additional testing and biopsy ( false positives ) .
women with screen - detected cancer equivalent to true positive mammograms in figure 5 . includes invasive cancer and in situ lesions .
the baseline scenario is 15% relative risk reduction from screening and 30% overdiagnosis ( 1.30/1.0 or 23% ( 0.3/1.30 ) of diagnosed cancers ) .
the ranges correspond to optimistic ( 20%/10% ) and pessimistic ( 10%/50% ) screening scenarios .
the ranges correspond to optimistic ( 35%/10% ) and pessimistic ( 25%/50% ) screening scenarios .
besides overdiagnosis of screen - detected cancers , false - positive radiologist interpretations are another important screening harm in healthy women ( fig .
the associated diagnostic imaging recalls can lead to negative ( benign ) core - needle and open surgical biopsies in up to 19% of women after 10 mammograms .
these recalls are much more common than overdiagnosis and happen at least once to 40%60% of u.s .
we estimated single round screening events associated with 100 screen - detected cases by age ( table 2 ) .
recent insurance data reveal that recall and biopsy rates are much higher compared with data utilized in 2009 by the u.s .
preventive services task force besides direct patient costs , there are other indirect and intangible harms from false - positive recalls that require further investigation . estimated
single - round screening events and performance measures for 100 women with screen - detected cancer performance measures are breast cancer surveillance consortium 20002005 data from nelson et al . , 2009 unless specified .
insurance claims data based on 20102013 from alcusky et al . , 2014 , second row ages 5064 , third row ages 6575 years .
women showed that less than 10% have been informed by their physicians of the possibility of overdiagnosis and overtreatment after cancer screening .
breast cancer patients may have been overtreated because of screening mammography over the last 30 years , or 31% of diagnosed cancers in the screened age group ( 70,000 women annually ) .
extensive overdiagnosis of invasive breast cancer and in situ lesions has been confirmed by the 25-year follow - up of the randomized canadian screening trial .
as the physical , psychological and economic consequences of unnecessary cancer diagnoses and treatment are substantial , primary care physicians have an ethical duty to discuss overdiagnosis before ordering or recommending a screening mammogram .
once a cancer is detected , it is currently not possible to distinguish life - threatening from indolent cases .
the nordic cochrane centre has produced an evidence - based leaflet on screening mammography , available in 17 different languages , to facilitate discussion with patients .
we provide a decision model with a summary of possible outcomes for women considering screening ( fig . 5 ) . about 55% of breast cancers diagnosed in the u.s .
are screen - detected , with symptomatic tumors diagnosed in women who did not screen , or as interval cancers between screening rounds . a u.s .
woman with a screen - detected in situ lesion or invasive cancer will have a true positive mammogram .
using optimistic and pessimistic screening scenarios as limits , she may be helped , hurt , or her prognosis unchanged ( table 1 ) . for the baseline scenario single screening round for women ages 5059 years
, for every 100 women with screen - detected cancer , over 15 years , 8 will have their lives extended .
this estimate of lives saved is optimistic , and is probably fewer than 5 lives saved .
since 21 women die despite screen - detection , of the remaining 79 survivors , 10% ( 8/79 ) are cured by earlier treatment , but over half of the survivors ( 42/79 ) are overdiagnosed and harmed .
these findings are contrary to the popular belief that screen - detection is synonymous with cure , reflecting the lack of balanced messaging regarding screening mammography . simplified screening mammography decision model with benefit and harms .
the top branch , or no screening decision , shows that some early cancers do not progress to cause symptoms . with screening ( bottom branch ) , many of these lesions are detected earlier , resulting in the harm of overdiagnosis .
although some otherwise lethal cases can be cured from earlier treatment , most cases that are not overdiagnosis remain curable or remain lethal despite screen - detection .
since mammography is imperfect , some cancers are not detected ( false negatives ) , and some healthy women are recalled for additional testing and biopsy ( false positives ) .
women with screen - detected cancer equivalent to true positive mammograms in figure 5 . includes invasive cancer and in situ lesions .
the baseline scenario is 15% relative risk reduction from screening and 30% overdiagnosis ( 1.30/1.0 or 23% ( 0.3/1.30 ) of diagnosed cancers ) .
the ranges correspond to optimistic ( 20%/10% ) and pessimistic ( 10%/50% ) screening scenarios .
the ranges correspond to optimistic ( 35%/10% ) and pessimistic ( 25%/50% ) screening scenarios .
besides overdiagnosis of screen - detected cancers , false - positive radiologist interpretations are another important screening harm in healthy women ( fig .
the associated diagnostic imaging recalls can lead to negative ( benign ) core - needle and open surgical biopsies in up to 19% of women after 10 mammograms .
these recalls are much more common than overdiagnosis and happen at least once to 40%60% of u.s .
we estimated single round screening events associated with 100 screen - detected cases by age ( table 2 ) .
recent insurance data reveal that recall and biopsy rates are much higher compared with data utilized in 2009 by the u.s .
preventive services task force besides direct patient costs , there are other indirect and intangible harms from false - positive recalls that require further investigation .
estimated single - round screening events and performance measures for 100 women with screen - detected cancer performance measures are breast cancer surveillance consortium 20002005 data from nelson et al . , 2009 unless specified .
insurance claims data based on 20102013 from alcusky et al . , 2014 , second row ages 5064 , third row ages 6575 years .
women showed that less than 10% have been informed by their physicians of the possibility of overdiagnosis and overtreatment after cancer screening .
breast cancer patients may have been overtreated because of screening mammography over the last 30 years , or 31% of diagnosed cancers in the screened age group ( 70,000 women annually ) .
extensive overdiagnosis of invasive breast cancer and in situ lesions has been confirmed by the 25-year follow - up of the randomized canadian screening trial .
as the physical , psychological and economic consequences of unnecessary cancer diagnoses and treatment are substantial , primary care physicians have an ethical duty to discuss overdiagnosis before ordering or recommending a screening mammogram .
once a cancer is detected , it is currently not possible to distinguish life - threatening from indolent cases .
the nordic cochrane centre has produced an evidence - based leaflet on screening mammography , available in 17 different languages , to facilitate discussion with patients .
the principle of informed choice and the harm of overdiagnosis are well recognized in prostate cancer screening , and both are applicable to screening mammography . screening mammography is a complex topic , and many seemingly plausible screening statistics are misleading .
overdiagnosis distorts screening statistics and can change the delicate balance between benefit and harms from positive to negative .
primary care physicians can use the principles explained here to help women make their own informed decisions .
| abstractthis article reviews four important screening principles applicable to screening mammography in order to facilitate informed choice .
the first principle is that screening may help , hurt , or have no effect . in order to reduce mortality and mastectomy rates
, screening must reduce the rate of advanced disease , which likely has not happened . through overdiagnosis , screening produces substantial harm by increasing both lumpectomy and mastectomy rates , which offsets the often - promised benefit of less invasive therapy .
next , all - cause mortality is the most reliable way to measure the efficacy of a screening intervention .
disease - specific mortality is biased due to difficulties in attribution of cause of death and to increased mortality due to overdiagnosis and the resulting overtreatment with radiotherapy and chemotherapy . to enhance participation ,
the benefit from screening is often presented in relative instead of absolute terms .
third , some screening statistics must be interpreted with caution .
increased survival time and the percentage of early - stage tumors at detection sound plausible , but are affected by lead - time and length biases .
in addition , analyses that only include women who attend screening can not reliably correct for selection bias .
the final principle is that accounting for tumor biology is important for accurate estimates of lead time , and the potential benefit from screening . since
early detection
is actually late in a tumor 's lifetime , the time window when screen detection might extend a woman 's life is narrow , as many tumors that can form metastases will already have done so . instead of encouraging screening mammography , physicians should help women make an informed decision as with any medical intervention . |
h bonds in complex molecules
represents a most useful
but also one of the most challenging transformations in organic chemistry . over the past two decades , notable advances in the area of chemical
c
h oxidation involving the use of oxidizing reagents , biomimetic supramolecular complexes , or transition metal catalysts have
disclosed opportunities to afford the oxidation of electronically
activated c
h
sites could be targeted for oxidation by taking advantage of proximal
directing groups pre - existing or preinstalled in the
target compound . despite this progress ,
the vast majority of c
h bonds in molecules of high complexity ,
in particular isolated , ( stereo)electronically unbiased c
the use of cytochrome p450 enzymes constitutes
an attractive alternative
strategy toward the oxyfunctionalization of unactivated c
these biological
catalysts can provide a valuable complement to chemical oxidation
strategies as their site - selectivity could be , in principle , steered
toward a remote c h site in a given compound by influencing
their molecular recognition properties via protein engineering . in practice , however , fine - tuning the regio- and stereoselectivity
of p450s toward a predefined position in a non - native substrate of
interest has represented a fundamental problem .
various factors contribute to
complicate these efforts including , among others , the difficulties
inherent to controlling the site - selectivity of the p450-catalyzed
oxidation reaction via rational design , the notoriously rare occurrence
of variants with the desired selectivity properties within engineered
p450 libraries , and the requirement for laborious and time - consuming
analyses ( i.e. , via hplc or gc ) for regio / stereoselectivity determination . as a result
, this goal has been historically
met with only limited or partial success also when ingenious ad hoc
high - throughput assays reporting on the p450 regio- or stereoselectivity
on a given substrate had been applied .
alternatively ,
considerable screening efforts need to be invested for any single
substrate , as exemplified by a recent study focused on improving the
regioselectivity of a testosterone - hydroxylating p450 ( 50%
94% ) , an effort that required the analysis of several thousand engineered
variants ( > 9,000 ) by hplc . clearly ,
more
general ( i.e. , substrate - independent ) and streamlined strategies to
gain access to p450 catalysts with refined regio- and stereoselectivity
are needed in order to make p450 catalysis accessible for chemical
synthesis .
artemisinin ( 1 , art ; figure 1a ) , a naturally occurring sesquiterpene
lactone of prominent value
in the pharmacological treatment of human malaria , exemplifies the scope and limitations of currently available
tools for late - stage functionalization of a complex molecule . over
the past two decades ,
extensive synthetic efforts directed at elaborating
this molecule in search for improved antimalarial agents have been
largely confined to the lactone ring , and primarily c10 and c9 , which
can be readily accessed through chemical methods . whereas substitutions at c10 have yielded derivatives of clinical
relevance such as artesunate , artemether , and artemisone ( figure 1a ) , methods to functionalize sp c
h
sites in the upper hemisphere of the molecule , and in particular positions
c7 and c6a , would be highly desirable , as these positions constitute
major target sites during phase i metabolism of this molecule in humans ,
a process that contributes to the rapid clearance and undesirably
short in vivo half - life of artemisinin - based antimalarials .
these sites have also remained
inaccessible to chemical oxidation via iron - based catalysts , which
have preference toward the more ( stereo)electronically activated tertiary
c h bond in c6 . on the other hand ,
attempts to hydroxylate artemisinin by means of oxidizing microbial
strains have resulted in mixtures of multiple ( over)oxidation products
and/or a large extent of art deoxygenation to give deoxoartemisinin ,
which lacks the endoperoxide bridge essential for biological activity .
( a ) chemical structure of artemisinin ( 1 ,
art ) and
artemisinin - based antimalarial drugs .
using artemisinin as a model substrate , we developed
and report
here an efficient , new strategy for developing highly regio- and stereoselective
p450 catalysts for the oxyfunctionalization of a set of predefined ,
isolated c
the described approach
hinges upon three key elements , namely ( 1 ) the generation of p450
catalyst diversity via mutagenesis of first - sphere active site residues
in a parent p450 ; ( 2 ) mapping of the active site configurations of
the resulting p450 variants via high - throughput fingerprinting to rapidly identify the library members with
potentially unique regio / stereoselectivity features ; and ( 3 ) prioritization
of these variants via a general method of fingerprint - based prediction
of p450 reactivity toward the target substrate ( i.e. , artemisinin ) .
we demonstrate how this rationally driven , systematic strategy enabled
the rapid optimization of an unselective artemisinin - hydroxylating
p450 variant ( figure 1c ) into a set of p450
oxidation catalysts with highly refined regio- and stereoselectivity
for the hydroxylation of each of the three unactivated sp c h bonds in positions c7 and c6a of artemisinin .
importantly ,
the entire procedure required the empirical testing of only a handful
of p450 variants via conventional hplc - based analyses and yielded
a set of synthetically useful p450 catalysts for the selective , late - stage
oxyfunctionalization of a relevant yet previously inaccessible region
of artemisinin scaffold at both preparative scales and in high yields .
the starting point of this work
was fl#62 , an engineered variant of the
catalytically self - sufficient fatty acid monooxygenase p450bm3 from bacillus megaterium . in previous studies
, fl#62 was found
to exhibit a broad substrate profile that comprises large decaline-
and steroid - based compounds .
accordingly , we envisioned this variant
could also accept the bulky tricyclic terpene artemisinin ( figure 1b ) as a substrate for oxidation .
upon testing , fl#62
was indeed found to be capable of hydroxylating art with high activity ,
i.e. , supporting about 340 total turnovers ( ttn ) in the presence of
a nadph cofactor regeneration system . in comparison , wild - type p450bm3 showed no art - oxidation activity . the site - selectivity
of fl#62 was poor , however , producing a mixture of 7(s)-hydroxy - art ( 2 ) , 7(r)-hydroxy - art
( 3 ) , and 6a - hydroxy - art ( 4 ) in a 83:10:7
ratio ( figure 1c ) . this notwithstanding , the
aliphatic c
h bonds targeted by this enzyme resided in the
relevant , upper hemisphere of the art scaffold , overlapping in two
cases ( 3 , 4 ) with those targeted by human
liver p450s , making fl#62 a promising starting point toward evolving
selective p450 catalysts for the oxidative activation of each of these
sites . to this end
, our first step involved altering the
fl#62 active site via mutagenesis . in p450bm3 , more than
a dozen amino acids extend their side chains into the heme cavity
and the long hydrophobic substrate channel connecting the latter to
the protein surface . among these ,
mutation of those
that are most proximal to the heme ( referred to here as first - sphere
residues ) was expected to have the largest impact on the orientation
of the enzyme - bound artemisinin above the reactive iron center and ,
thus , on the regio- and stereoselectivity of fl#62 .
accordingly , a
series of active site libraries were constructed via site - saturation
mutagenesis ( nnk ) of first - sphere residues 74 , 78 , 81 , 82 , 87 , 181 ,
and 184 , which lie within 12 from the heme iron and within
5 from the terminal carbons of n - palmitoylglycine
in the substrate - bound structure of p450bm3 heme domain ( figure 2 ) .
first - sphere
residues along the i helix ( e.g. , a264 , t268 ) were not taken into
consideration because of their structural role or involvement in catalysis , while a328 was excluded because most substitutions at this site
would likely disfavor access of the bulky terpene to the heme .
overall ,
the size of the resulting libraries included more than 10 members , many of which , in all likelihood , would be misfolded or
functionally redundant as a result of deleterious or neutral mutations ,
respectively .
crystal structure of p450bm3 ( heme domain )
in complex
with n - palmitoylglycine ( pdb code 1jpz(24 ) ) .
the bound substrate is displayed as sphere models ( orange ) ,
and the heme is displayed as stick model ( red ) .
the dotted circle
highlights the first - sphere active site residues targeted for mutagenesis
in this study . to rapidly identify the most relevant fraction of
these libraries , namely , the variants that not only are functional
but also have the highest probability to exhibit altered regio- and
stereoselectivity in art oxidation
this method provides a means to quickly map the active
site geometry of these enzymes via a set of structurally diverse and
conformationally rigid chromogenic probes ( compounds p1p5 , figure s1 in the supporting
information ) .
accordingly , a total of 12,500 recombinants from
the mutagenesis libraries were fingerprinted in 96-well plates using
probes p1p5 and a purpald - based
colorimetric assay for quantification of probe activity ( figure 3a ) .
the acquired fingerprints were then compared
against each other in order to identify the p450 variants with a unique
profile , this feature indicating that the corresponding enzyme possesses
a unique active site configuration . using
this approach , about 1,950 out of the 12,500 screened variants were
determined to be catalytically competent ( criterion : > 10% of parent
enzyme activity on at least one of the fingerprint probes ) .
among
these , 522 variants were established to be functionally unique ( criterion :
larger than 20% variation on at least one of the five fingerprint
components compared to the parent enzyme and any other member of the
library ) and thus worthy of further consideration .
notably , this fraction
represented only the 4.1% of the original library , highlighting the
efficiency of this tier-2 procedure in drastically reducing the number
of candidate enzymes to consider in the subsequent steps .
fingerprint - based
strategy to guide the discovery of regio- and
stereoselective artemisinin - hydroxylating p450 catalysts .
( a ) schematic
summary of the process of identification of the functionally diverse
p450 variants from the active site libraries via high - throughput fingerprinting
followed by fingerprint comparative analysis ( tier 2 ) .
( c ) outline
of the method applied to predict art reactivity via multivariate fingerprint
analysis ( tier 3 ) .
, we sought to develop a tool
to further guide our search for art - hydroxylating variants within
this collection of 522 functionally diverse p450 catalysts .
since
art shares little or no structural similarity with any of the fingerprint
probes , a p450 reactivity prediction approach alternative to the one
previously implemented in the context of probe - related substrates was required . to this end
, we envisioned that
a more general strategy based on fingerprint multivariate analysis
could prove useful in the context of a probe - unrelated target substrate
like art .
as outlined in figure 3b , this method
relies on generating a fingerprint - based model predictive of art reactivity
by correlating p450 fingerprints with experimental art oxidation activities
across a small subset ( training set ) of the enzyme
collection .
since the fingerprints relay information on the p450 active
site shape and geometry , this process translates into finding a fingerprint
signature that defines an active site configuration compatible with
art binding and , thus , its oxidation .
the model is then applied to
rank the entire collection of fingerprinted p450s according to their
predicted art hydroxylation activity . according to this design ,
a training set of 20 p450s was assembled using fl#62 and 19 randomly
chosen variants from the 522-member p450 collection ( training set
a , table s2 in the supporting information ) .
the art - hydroxylating activity for each of these enzymes was measured
in ttn from small - scale reactions followed by derivatization of the
art hydroxylation products with benzoyl chloride and hplc analysis .
the derivatization procedure was implemented to enable sensitive and
precise quantification of the reaction products as art contains no
strong chromophores . out of the 19 variants , four exhibited art hydroxylation
activity , supporting up to about 110 total turnovers and showing varying
regio- and stereoselectivity ( table s2 in the supporting information ) .
these experiments also indicated
that only a small fraction ( 20% ) of this p450 catalyst collection
would be expected to be active on art . to generate the fingerprint - based
model predictive of art reactivity ,
the experimental art activities
were then correlated with the fingerprints
of the corresponding p450s across the whole training set via multiple
linear regression analysis ( mlr ) ( figure 4a ) ,
which provides a statistical tool to analyze the correlation between
a dependent variable y and multiple independent variables
potentially correlated with y ( equation : y = b0 + b1x1 + b2x2 + ... + bnxn ) .
in the applied mathematical function ,
y corresponded
to relative art activity and the five fingerprint components were
the five independent variables x1 ... x5 . from the resulting best - fit regression coefficients ,
a fingerprint - based model was obtained , which was then applied to
rank the remaining p450s in the collection according to their predicted
art reactivity ( high - scoring = predicted art active , low - scoring =
predicted art inactive ) ( figure 4b ) .
( a ) plot
of experimental versus calculated art activity from multiple linear
regression analysis of art reactivity / fingerprint correlation across
p450 training set a ( table s1 in the supporting
information ) .
( b )
ranking of the 522 p450 variants according to their predicted artemisinin
activity calculated based on the fingerprint - based model ( training
set a ) .
( c ) total turnovers in art hydroxylation for the 50 top - scoring
p450 variants ( solid box in panel b ) arranged from the most to the
least active variant .
guided by these predictions , we focused our attention on the 50
best - scoring p450 variants from the ranking list ( figure 4b , solid box ) , which were characterized for art hydroxylation
activity .
gratifyingly , we found that 78% ( 39/50 ) of these variants
showed art - hydroxylation activity at synthetically useful levels ( > 100
ttn ; average = 323 ttn , figure 4c ) .
furthermore ,
nearly half of the correctly identified variants ( 22/50 ) were found
to possess higher art - hydroxylating activity than the parent enzyme ,
with one ( ii - c3 ) supporting almost twice as many total turnovers ( 602
vs 339 ) ( figure 4c ) .
notably , the hit
rate achieved using this method ( 78% ) greatly exceeded that expected
from random sampling of the collection ( 20% ) . in order to
further assess the quality of these predictions
, we then tested the
twenty lowest - scoring variants from the ranking list ( figure 4b , dotted box ) , which were expected to have minimal
or no activity on art .
90% of these p450s ( 18/20 ) show no detectable
oxidation activity on the compound , with the two active variants supporting
only up to 130 ttn .
the fraction of false negatives ( 10% ) within this
group was thus lower than the expected occurrence of art - active p450s
from random sampling ( 20% ) , further supporting the reliability of
the predictions and functionality of the method to generate them .
since the p450
catalysts in the collection were preselected to possess unique active
site geometries ( figure 3a ) , the 42 art - reactive
variants identified using the strategy described above were expected
to exhibit markedly different regio- and stereoselectivity in art
oxidation , ideally displaying the sought - after high selectivity toward
c7- , with both s and r stereoselectivity ,
and c6a - hydroxylation .
inspection of the product distribution of these
variants confirmed our expectations ( table s3 in the supporting information ) .
importantly , as many as five variants
( 12% of the pool ) were found to exhibit 95% selectivity for
c7 hydroxylation with s - stereoselectivity . among
these
, the triple mutant iv - h4 displayed the desired absolute regio-
and stereoselectivity for this transformation , in addition to supporting
higher ttn than the parent enzyme ( table 1 ) .
on the other hand , three variants showed much improved ( > 70% ) selectivity
for 7(r)-hydroxylation ( table s3 in the supporting information ) , which is produced in
only 10% relative ratio with the parent enzyme . among these
, variant
ii - h10 displayed absolute regio- and r - stereoselectivity
for hydroxylation of this site , thereby providing a second , exquisitely
selective p450 catalyst for c7-oxyfunctionalization with complementary
stereoselectivity compared to iv - h4 ( figure 5 ) .
finally , p450 catalysts with improved 6a - hydroxylation activity
were also captured through the strategy , with the best variant ( ii - e2 )
hydroxylating this position with considerably higher ( 48% ) , albeit
still suboptimal , regioselectivity compared to fl#62 ( 7% ) ( table s3
in the supporting information ) .
the comparatively
lower occurrence of 6a - hydroxylating p450s within these first - generation
variants could be explained on the basis of the low level of 6a - hydroxylation
activity in the parent enzyme combined with the higher strength of
this primary c h bond compared to the secondary ones in c7 .
importantly , the improvement in 6a - selectivity in ii - e2 was accompanied
by an increase in catalytic activity ( 338 393 ttn , table 1 ) , making this variant a promising intermediate
toward further refining 6a - selectivity via another round of directed
evolution .
mutations in fl#62 vs p450bm3 are v78a ,
f81s , a82 v , f87a , p142s , t175i , a180 t , a184v , a197v ,
f205c , s226r , h236q , e252 g , r255s , a290v , l353v .
rates are measured over initial 30 s. ratio between product formation
rate and nadph oxidation rate in the presence of artemisinin .
overview of the divergent evolution process leading to the selective
art - hydroxylating p450 variants . by sequencing ,
ii - e2 was determined to carry three active site mutations ( a78n / s81f / v82a ) ,
which left additional first - sphere positions available for mutagenesis
( figure 2 ) .
accordingly , a triple site - saturation
library ( 74/181/184 ) was constructed based on this enzyme , from which
3,000 recombinants were subjected to high - throughput fingerprinting
followed by fingerprint comparative analysis according to the tier-2
procedure of figure 3a .
this step revealed
the occurrence of just 50 unique - fingerprint p450s out of a total
of 120 catalytically active variants ( 1.6% of the library ) .
after
expanding the training set to include ii - e2 (= training set b ) , these
50 variants were scored based on their predicted art reactivity according
to the tier-3 method of figure 3b as described
above for the fl#62-derived variants ( figures s2 and s3 in the supporting information ) . upon evaluation of the
25 top - scoring variants , we found that about half ( 52% ) were capable
of hydroxylating art , supporting up to 474 ttn ( average : 146 ttn )
( table s4 in the supporting information ) . on the other hand , 90% ( 9/10 ) of the predicted inactive ones ( bottom - ranking )
furthermore , nearly two - thirds
( 8/13 ) of the correctly identified art - active variants exhibited significantly
improved 6a - selectivity ( > 80% ) compared to ii - e2 ( table s4 in the supporting information ) .
most importantly , this
approach enabled the identification of two highly regioselective p450
catalysts for c6a hydroxylation , namely , x - e12 ( 94% selectivity ) and
x - f11 ( 92% selectivity ) , thereby completing our desired divergent
evolution process ( figure 5 ) .
for x - e12 , the
improvement in 6a - selectivity was accompanied by a reduction in catalytic
activity ( 113 vs 393 ttn in ii - e2 ) .
in contrast , x - f11 was found to
support nearly as many turnovers as ii - e2 and higher ttn than the
initial enzyme fl#62 ( table 1 ) . to determine
whether potentially interesting catalysts could have been missed in
this process ,
these studies revealed the occurrence of only
two additional art - oxidizing variants , none of which showed superior
performance in terms of either regioselectivity or ttn compared to
those already identified ( table s4 in the supporting
information ) .
based on these results , we concluded that the
method allowed efficient capturing , within the top one - fifth ( 10/50 )
and half ( 25/50 ) of the ranked variants , of 37% ( 6/16 ) and 81% ( 13/16 ) ,
respectively , of the art - active p450 catalysts in the pool , with the
ten top - ranking p450s comprising those with the highest 6a - regioselectivity .
consistent with their divergent reactivity ,
the best variants for 7(s)-hydroxylation ( iv - h4 ,
v - h2 ) , 7(r)-hydroxylation ( ii - h10 , iii - b1 ) , and 6a - hydroxylation
( x - e12 , x - f11 ) show distinct fingerprints compared to each other ,
the parent fl#62 , and the evolutionary intermediate ii - e2 ( figure 6 ) .
these variants were further characterized to
gain insights into the effect of the mutations on their catalytic
and substrate binding properties .
interestingly , the selective 7(s)-hydroxylating variants were found to share a similar
mutational pattern ( table 1 ) , both carrying
three active site mutations with two identical substitutions at position
78 and 82 and a similar solution
( val vs ile ) at the
neighboring site 81 ( figure 2 ) . in comparison ,
refinement of 7(r)- and c6a - selectivity required
a more extensive remodeling of the enzyme active site as judged by
the five to six mutations occurring in ii - h10 , x - e12 , and x - f11 ( table 1 ) .
overall , this trend well reflected the functional
distance of these variants , in terms of site - selectivity , from the
parent enzyme .
ii - h10 mutations were found to comprise a triad of
phenylalanine substitutions within the heme pocket ( s81f , a87f , l181f ) .
interestingly , iii - b1 , the second best ( r)7-hydroxylating
variant found in this study , also carries a pair of phenylalanine
mutations in proximity of the heme .
these common features suggest
a distinctive requirement for bulky substitutions at the level of
the heme pocket to favor art hydroxylation at c7 with r stereoselectivity , when compared to iv - h4 and v - h2 .
another interesting
observation emerged from comparing the mutations in the 6a - hydroxylating
variants with those of ii - h10 , which evidenced the importance of substitutions
at positions 74 and 181 in steering the enzyme selectivity toward
c6a .
probe activities correspond to mean values from
triplicate experiments ( se within 10% ) after normalization to those
of the reference enzyme p450bm3(f87a ) . upon incubation with art
, all the p450 variants
were found to exhibit
a 5 to 20% shift of the heme iron spin - state equilibrium ( figure 7 and figure s4 in the supporting
information ) , which is indicative of the ability of this substrate
to displace the heme - bound water ligand upon complex formation .
this
property also enabled estimation of the binding affinity ( kd ) of these variants for art via titration experiments .
for all the p450 variants ( figure 7 and figure
s5 in the supporting information ) , hyperbolic
curves were observed , suggesting a noncooperative binding interaction .
interestingly , the 7(s)- and the 7(r)-selective variants showed kd values
similar to that of fl#62 ( 3060 m ; table 1 ) , indicating that the acquired mutations had little impact
on art binding affinity compared to the parent enzyme . 5- to 10-fold
higher kd values were observed instead
for variants x - e12 and x - f11 as well as for ii - e2 , indicating that
the mutations responsible for improving 6a - hydroxylation selectivity
resulted in a somewhat weaker interaction with art .
these equilibrium
dissociation constants fall however in the range of that observed
for wild - type p450bm3 and the fatty acid laurate ( 270 m ) .
artemisinin binding experiments with ( a ) variant iv - h4 ,
( b ) variant
ii - h10 , and ( c ) variant x - e12 .
left panel : overlay of the enzyme absorbance
spectrum before ( gray line ) and after ( red line ) addition of artemisinin
( 1 mm ) , illustrating the substrate - induced shift of the heme spin
state equilibrium .
right panel : representative plot of the artemisinin - induced
heme spin shift versus artemisinin concentration . the dissociation
constant ( kd ) for the enzyme
artemisinin
complex was calculated via nonlinear fitting of the experimental data
( dots ) to a noncooperative 1:1 binding model equation ( solid line ) .
data relative to the other variants of table 1 are provided in the supporting information .
further experiments were carried out to analyze
the impact of the
mutations on the catalytic rates of the enzymes .
these analyses revealed
that all the selective 7(s)- , 7(r)- , and 6a - hydroxylating variants exhibited a 3- to 10-fold lower
initial product formation rate compared to fl#62 and the less selective
ii - e2 ( table 1 ) .
this trade - off between oxidation
rate and site - selectivity has been observed in other studies as well , and it can be rationalized here considering
that the improved site - selectivity in the former enzymes must inherently
involve a more specific enzyme substrate interaction and thus
a restriction of the ( productive ) binding orientations available to
art during oxidation . in the case of
ii - h10 and v - h2 , the observed
reduction in product formation rate is likely to result also from
a reduction in their coupling efficiency (= ratio of product formation
rate / nadph oxidation rate ) as compared to the parent enzyme .
for all
the other selective p450 variants ( iv - h4 , x - e12 , x - f11 ) , however ,
the improvement in regio / stereoselectivity came at no cost of the
coupling efficiency , as evidenced by the values reported in table 1 .
notably , for both the 6a - hydroxylating variant
x - f11 and the 7(s)-hydroxylating variant iv - h4 the
coupling efficiency significantly improved compared to fl#62 , exceeding
values of 55% and 70% , respectively .
the catalytic turnovers achieved
in analytical - scale settings indicated that the evolved p450 variants
could be valuable for synthetic purposes .
prior attempts toward enzymatic
hydroxylation of art have involved the use of oxidizing strains . besides requiring long reaction times ( 314
days ) ,
these transformations have however resulted in the unselective
oxidation of the natural product and/or its degradation to the biologically
inactive deoxoartemisinin via deoxygenation of the endoperoxide group . to assess the synthetic utility of the engineered
p450 catalysts developed here , large - scale reactions involving up
to 0.4 g of art
were carried out for 12 h using iv - h4 ( 0.19 mol % ) ,
ii - h10 ( 0.25 mol % ) , and x - f11 ( 0.28 mol % ) in buffer ( 50 mm kpi ,
ph 8.0 ) in the presence of a cost - effective nadph regeneration system
consisting of a thermostable phosphite dehydrogenase and sodium phosphite as sacrificial reductant . from these
reactions , 0.26 to 0.41
g of the three desired products ( 7(s)- , 7(r)- , and 6a - hydroxy - art , respectively )
could be isolated in over 90% yields .
interestingly , both iv - h4 and
ii - h10 were found to support higher total turnovers under these settings
( 485 and 350 ttn , respectively ) , likely due to more favorable oxygen
transfer conditions .
importantly , access to preparative amounts
of 3 and 4 could now allow for the direct
modification of two metabolically labile sites in artemisinin and
derivatives thereof via stereoconservative hydroxyl group functionalizations ( e.g. ,
alkylation or acylation ) .
at the same time , the complementary stereoselectivity
of iv - h4 for c7 hydroxylation can be leveraged upon to chemically
protect , in a two - step sequence , the pro - r c h
bond in c7 via deoxofluorination , a transformation that typically
proceeds through inversion of configuration at secondary carbon centers . to illustrate this point , the enzymatically
produced 7(s)-hydroxy - artemisinin 2
was
reacted with dast to afford enantiopure 7(r)-fluoro - artemisinin 5 in 82% yield ( scheme 1 ) .
5 was then converted in two steps into 6 and 7 , which correspond , respectively , to the clinical antimalarial drugs
artemether and artesunate bearing a c h to c f substitution ,
with the ideal configuration , at the major site of metabolic attack
of these drugs by human hepatic p450s .
altogether , these results demonstrated the viability
of these p450 catalysts for oxyfunctionalization of art at preparative
scales as well as their utility toward enabling relevant , late - stage
elaborations of art scaffold via p450-mediated c h functionalization .
in summary , we have demonstrated how a
systematic approach based
on first - sphere active site mutagenesis , high - throughput p450 fingerprinting ,
and fingerprint - based p450 reactivity predictions can provide an efficient
solution to the problem of developing p450 catalysts with fine - tuned
site - selectivity for the oxidative activation of multiple , isolated
sp c h bonds in a complex molecule . by guiding
and
focusing our search for the desired catalysts across a large engineered
p450 library , this method allowed us to accomplish our goal of refining
the site - selectivity of a p450 enzyme in a time- and cost - effective
manner . to our knowledge , the complete inversion in stereoselectivity
achieved with ii - h10 ( 100% ee ( r ) from 78% ee ( s ) in fl#62 ) , the absolute regioselectivity and stereocomplementarity
obtained with the iv - h4/ii - h10 pair , and the regioselectivity shift
achieved with x - e12 to afford the selective hydroxylation of a primary ,
nonallylic / nonbenzylic c h site ( 7% 94% ) remain unprecedented
in previous p450 engineering efforts .
it is also
worth noting that the overall process required the empirical testing
of only 75 p450 variants via laborious hplc - based methods , thereby
reducing by orders of magnitude the screening efforts currently associated
with the search for selective p450 catalysts . from a methodological standpoint , another important aspect is that
the tier-2 component of this approach is independent of the nature
of the target substrate whereas the tier-3 method could be readily
extended to any probe - unrelated compound of interest , making this
strategy potentially very general in terms of substrate scope . from
the characterization of the evolved artemisinin - hydroxylating variants ,
intriguing trends
have emerged with respect to the active site positions
and mutations responsible for steering the selectivity of these enzymes
toward each one of the three , spatially close sp c h
bonds in c7 and c6a of artemisinin ( figure 1b ) .
these findings call for future studies to explore in more detail
the molecular mechanisms that permit such a subtle control on the
c h oxidation reaction catalyzed by this group of related variants .
finally , the p450-based oxidation catalysts developed in this work
disclose now the opportunity to selectively functionalize relevant
aliphatic positions within the artemisinin scaffold that have so far
remained inaccessible to chemical methods / catalysts . as demonstrated with the preparation
of 7(r)-fluoroartemether and 7(r)-fluoroartesunate , the suitability of this set of engineered p450s
for late - stage c h functionalization of artemisinin at practically
useful scales is expected to render them of particularly high value
toward the synthesis and investigation of new classes of c7- and c6a - modified
artemisinin - based antimalarial agents .
multiple
site - saturation ( nnk ) mutagenesis libraries were prepared using pcwori_fl#62
as template , primers bamhi_2_fwd ( 5-ggaaacaggatccatcgatgc-3 )
and saci_2_rev ( 5-aatatcgagctcgtagtttgtatgatc-3 )
as megaprimers , and the oligonucleotides of table s1 in the supporting information as mutagenizing primers .
construction of libraries 78/87 , 78/81/87 , 78/87/181 , 78/87/184 , 78/81/82/87 ,
and 81/82/87/184 was described earlier .
additional libraries ( 78/81/82/87/181/184 and 74/81/82/87/181/184 )
were constructed in a similar manner by pcr overlap extension mutagenesis .
the soe products ( 1.5 kbp ) were digested with bamhi and saci restriction enzymes and ligated to bamhi / saci double - digested pcwori_fl#62
vector .
the ligation mixtures were transformed in chemically competent
dh5 cells and plated on lb agar plates containing ampicillin
( 100 mg l ) followed by overnight incubation at
37 c .
96-deep - well plates containing 400 l of lb medium ( 100 mg
of ampicillin l ) per well were inoculated with
single colonies from the p450 libraries and shaken overnight at 37
c and 200 rpm . a row ( 8 wells ) in each plate was inoculated
with dh cells expressing fl#62 ( or ii - e2 in the case of ii - e2-based
libraries ) as a reference .
after overnight growth , the lb plates were
used to inoculate a second set of 96-deep - well plates containing 900
l of terrific broth ( tb ) medium ( 100 mg of ampicillin l ) . at od600 = 1.0
, the tb cultures were
induced with 0.25 mm -d-1-thiogalactopyranoside ( iptg )
and 0.3 mm -aminolevulinic acid ( ala ) .
after incubation at
30 c and 200 rpm for 18 h , the plates were centrifuged at 3500
rpm and the pellets were separated from the supernatant and frozen
at 80 c .
cell lysates were prepared by adding 400 l
of lysis solution ( 4 u of deoxyribonuclease i , 0.8 mg / ml lysozyme ,
10 mm mgcl2 , 50 mm phosphate buffer , ph 7.5 ) to each well .
after incubation at 37 c for 70 min , clarified lysates for the
screening were obtained by centrifuging the plates at 4,000 rpm .
p450
demethylation activity on probe p1p5 ( figure s2 in the supporting information ) was measured in parallel reactions with the aid of a beckman coulter
multimek 96 automated pipettor and a tecan infinity plate reader .
reactions were carried out by mixing 50 l of clarified cell
lysate with 150 l of 50 mm phosphate buffer ( ph 7.5 ) containing
the probe ( final concentration : 1 mm ) and a phosphite dehydrogenase
( ptdh)-based cofactor regeneration system ( final concentrations : 1.8
m ptdh , 50 mm sodium phosphite , 150 m nadp ) .
after incubation for 1 h at room temperature , to each well was
added 50 l of 2 m naoh containing 150 mm purpald following
by reading of the absorbance at 550 nm with the plate reader .
the
measured demethylation activity of each member of the library on the
probes p1p5 was then normalized
to the activity of the parent enzyme from the same plate .
the p450 enzymes
were expressed from pcwori - based vectors and purified by ion - exchange
chromatography as described previously .
p450 concentration was determined from co binding difference spectra
( 450500 = 91,000 m cm ) .
the vector encoding for the thermostable phosphite
dehydrogenase ( ptdh ) was kindly provided by the zhao group .
ptdh was expressed and purified via ni - affinity
chromatography according to the published procedure .
a 200 ml scale reaction was set up adding fl#62
( 1 m ) to a buffered solution ( 50 mm potassium phosphate , ph
8.0 ) containing 32 mg of artemisinin ( 0.53 mm ) , ptdh ( 2 m ) ,
nadp ( 150 m ) , and sodium phosphite ( 50 mm ) .
the
reaction mixture was stirred overnight at room temperature and then
extracted with dichloromethane ( 3 30 ml ) .
the collected organic
layers were dried with na2so4 , concentrated
under reduced pressure , and purified by flash chromatography ( dichloromethane / hexanes / ethyl
acetate : 1/1/1 ) to afford 2 ( 18 mg ) , 3 ( 2
mg ) , 4 ( 1 mg ) , and recovered artemisinin ( 10 mg ) .
h nmr ( 500 mhz , cdcl3 ) : = 1.18 ( 3
h , d , j = 7.2 hz ) , 1.23 ( 1 h , m ) , 1.27 ( 3 h , d , j = 7.2 hz ) , 1.44 ( 1 h , m ) , 1.50 ( 3 h , s ) , 1.57 ( 2 h , m ) ,
1.79 ( oh , br ) , 1.95 ( 1 h , m ) , 2.022.20 ( 3 h , m ) , 2.48 ( 1 h ,
m ) , 3.32 ( 1 h , ddd , j = 10.6 hz , j = 10.6 hz , j = 4.5 hz ) , 3.42 ( 1 h , m ) , 5.98 ( 1
h , s ) . c nmr ( 100 mhz , cdcl3 ) : = 12.6 ,
15.5 , 24.8 , 25.1 , 32.1 , 32.6 , 35.8 , 42.3 , 44.5 , 47.9 , 73.5 , 78.8 ,
93.5 , 105.5 , 171.6 . ms ( esi ) calcd for c15h23o6 [ m + h]m / z : 299.15 , found 299.47 .
the 7(s ) configuration of 2 was confirmed based on the j coupling constant between the 7(h ) proton and 6(h ) and 8(h ) protons
and the known chair conformation of the cyclohexyl ring in artemisinin .
the observed coupling constants ( j = 10.6 hz ; j = 10.6 hz ; j = 4.5 hz ) are consistent with 7(h ) being in trans to 6(h ) and thus with c7 atom having s configuration .
h nmr ( 500 mhz , cdcl3 ) : = 1.12 ( 3
h , d , j = 6.7 hz ) , 1.23 ( 3 h , d , j = 7.3 hz ) , 1.34 ( 1 h , dd , j = 13.7 hz , j = 2.1 hz ) , 1.461.51 ( 4 h , m ) , 1.56 ( 1 h , m ) , 1.81
( oh , br ) , 1.95 ( 2 h , m ) , 2.10 ( 2 h , m ) , 2.402.53 ( 2 h , m ) ,
3.45 ( 1 h , m ) , 3 .
89 ( 1 h , ddd , j = 2.8 hz , j = 2.8 hz , j = 2.5 hz ) , 5.90 ( 1 h , s ) .
c nmr ( 100 mhz , cdcl3 ) : = 12.5 , 15.8 ,
24.6 , 25.2 , 30.7 , 32.4 , 36.0 , 37.6 , 41.3 , 43.4 , 69.1 , 79.3 , 93.4 ,
105.5 , 172.4 . ms ( esi ) calcd for c15h23o6 [ m + h]m / z : 299.15 , found 299.41 .
the 7(r ) configuration of 3 was confirmed based on the j coupling constant between the 7(h ) proton and 6(h ) and 8(h ) protons
and the known chair conformation of the cyclohexyl ring in artemisinin . the observed coupling constants ( j = 2.8 hz ; j = 2.8 hz ; j = 2.5 hz ) are consistent with 7(h ) being in cis to 6(h ) and thus with c7 atom having r configuration .
h nmr ( 500 mhz , cdcl3 ) : = 1.15 ( 1 h , m ) , 1.26
( 3 h , d , j = 6.6 hz ) , 1.361.49 ( 4 h , m ) ,
1 .
1.541.61 ( 3 h , m ) , 1.65 ( oh , br ) , 1.761.85 ( 2 h ,
m ) , 1.952.14 ( 3 h , m ) , 2.50 ( 1 h , ddd , j =
17.3 hz , j = 13.3 hz , j = 3.9 hz ) ,
3.45 ( 1 h , m ) , 3 .
68 ( 1 h , dd , j = 10.6 hz , j = 5.6 hz ) , 3.79 ( 1 h , dd , j = 10.6 hz , j = 3.0 hz ) , 5.93 ( 1 h , s ) .
c nmr ( 100 mhz ,
cdcl3 ) : = 12.6 , 23.0 , 24.5 , 25.2 , 27.9 , 32.9 , 35.9 ,
44.2 , 44.5 , 44.8 , 64.1 , 79.5 , 93.6 , 105.4 , 172.0 . ms ( esi ) calcd for
c15h23o6 [ m + h]m / z : 299.15 , found 299.39 .
analytical - scale reactions ( 1 ml ) were
carried using 0.51 m p450 , 0.5 mm artemisinin , 2 m
ptdh , 100 m nadp , and 50 mm sodium phosphite in
potassium phosphate buffer ( 50 mm , ph 8.0 ) .
the p450 variants described
in table 1 were characterized in purified form ,
while those described in tables s2s4 in the supporting information were characterized directly from cell
lysates .
the hydroxylation products were functionalized with benzoyl
chloride followed by hplc analysis as described in more detail in
the supporting information .
ttn values
were calculated based on the total amount of hydroxylated products
in the reactions as quantified based on the calibration curves generated
using purified 24 .
the enzyme regio-
and stereoselectivity was determined based on the peak areas corresponding
to the hydroxylation products 2 , 3 , and 4 .
mean and standard deviation values reported for p450 variants
in table 1 were calculated from experiments
performed at least in triplicate .
initial product formation rates were measured from 1 ml scale reactions
containing 250 m artemisinin , 0.10.5 m purified
p450 , and 200 m nadph in potassium phosphate buffer ( 50 mm ,
ph 8.0 ) at room temperature .
after 30 s , the samples were extracted
with dichloromethane and subjected to benzoyl chloride derivatization
followed by hplc analysis as described above
. cofactor oxidation rate
in the presence of artemisinin was measured by monitoring nadph depletion
at 340 nm ( = 6.22 mm cm ) using 0.1 m purified p450 , 250 m artemisinin , and
200 m nadph .
coupling efficiency was calculated from the ratio
between the initial product formation rate and the initial nadph oxidation
rate . reported mean and standard deviation values were calculated
from experiments performed at least in triplicate .
binding experiments were performed using 3 m
purified p450 in potassium phosphate buffer ( 50 mm , ph 8.0 ) by titrating
increasing amounts of artemisinin ( 5 m to 1 mm ) from an ethanol
stock solution ( 50 mm ) . at each concentration ,
a difference spectrum
from 350 to 500 nm was recorded and binding curves were generated
by plotting the change in absorbance at 390 and 420 nm corresponding
to the high - spin and low - spin state of the enzyme , respectively , against
the artemisinin concentration .
kd values
were calculated using sigma plot via nonlinear fitting of the experimental
binding curves to an equation describing a standard 1:1 binding interaction .
reported mean and standard deviation values were calculated from experiments
performed at least in triplicate .
after high - throughput fingerprinting , p450 variants displaying a
unique fingerprint were identified by selecting the parent - normalized
fingerprints according to the following criteria : ( a ) > 10% of parent
activity on at least one probe ; and ( b ) larger than 20% variation
in activity on at least one probe compared to the parent or any other
member of the library .
the p450 variants with a unique fingerprint
were transferred from the library plates to new 96-well plates containing
p450bm3(f87a ) as reference enzyme .
the p450 variants of
this collection were then fingerprinted in triplicate using the probe
activity of p450bm3(f87a ) for fingerprint normalization .
the standard error in the values of the fingerprints provided in figure 3b is within 10% . to generate the fingerprint - based
model predictive of artemisinin reactivity , 19 randomly chosen p450
variants from the collection were characterized for artemisinin hydroxylation
activity measuring their ttn values ( table s2 in the supporting information ) .
after normalization against the activity
of the parent enzyme ( ttn(p450 variant)/ttn(fl#62 ) ) , the normalized
activities were then correlated with the corresponding fingerprints
using multiple linear regression ( mlr ) analysis using the equation y = b0 + b1x1 + b2x2 + b4x4 + b4x4 + b5x5 , where y corresponds to relative artemisinin
reactivity , x1 to x5 correspond to the five fingerprint components ( activity on
probes 1 to 5 , respectively ) , and b1 to b5 correspond
to the regression coefficients for the five independent variables x1 to x5 .
this process
yielded the plot of calculated versus experimental artemisinin activity
presented in figure 2a , with the calculated
regression coefficients being b1 = 0.0109 , b2 = 0.0016 , b3 =
0.0551 , b4 = 0.0338 , and b5 = 0.01271 .
the low rmsd value ( 0.0859 ) indicated
an excellent fit between the calculated and experimental values .
this
model was then used to rank the p450 variants of the collection according
to their predicted artemisinin reactivity .
the same method was applied
for generating a fingerprint - based predictive model for ranking the
second - generation variants derived from ii - e2 . in this case , the training
set used for calculations was expanded to include ii - e2 ( training
set b , table s2 in the supporting information ) as this variant exhibited the highest selectivity toward 6a - hydroxylation .
mlr analysis of the correlation between fingerprints and artemisinin
hydroxylation activities across this training data set yielded a best - fitting
model with regression coefficients b1 =
0.0037 , b2 = 0.0278 , b3 = 0.0839 , b4 = 0.1158 , and b5 = 0.1202 and a rmsd
of 0.1789 ( figure s2 in the supporting information ) .
based on this model , the 50 ii - e2 derived variants were scored
according to their predicted artemisinin reactivity ( figure s3 in
the supporting information ) and subsequently
prioritized according to their score . to prepare 2 , purified p450 variant iv - h4 ( final concentration : 1 m ;
0.19 mol % )
was dissolved in 1 l of 50 mm phosphate buffer ( ph 8.0 )
in the presence of artemisinin ( 150 mg , final concentration : 0.53
mm ) , ptdh ( 2 m ) , nadp ( 150 m ) , and sodium
phosphite ( 50 mm ) .
the collected organic layers were dried with na2so4 , concentrated , and purified by flash chromatography
( dichloromethane / hexanes / ethyl acetate : 1/1/1.5 ) to afford 2 ( 138 mg , 92% ) .
the same procedure was repeated two more times to
yield a total of 410 mg of 2 . to prepare 3 , purified p450 variant ii - h10 ( final concentration 1 m ; 0.25
mol % )
was dissolved in 1 l of 50 mm phosphate buffer ( ph 8.0 ) in
the presence of artemisinin ( 110 mg , final concentration : 0.39 mm ) ,
ptdh ( 2 m ) , nadp ( 150 m ) , and sodium phosphite
( 50 mm ) .
the collected organic layers were dried with na2so4 , concentrated , and purified by flash chromatography ( dichloromethane / hexanes / ethyl
acetate : 1/1/1.5 ) to afford 2 ( 100 mg , 91% , ttn : 350 ) .
the same procedure was repeated two more times to yield a total of
305 mg of 2 . to prepare 4 ,
purified p450
variant x - f11 ( final concentration : 1 m ; 0.28 mol % ) was dissolved
in 1 l of 50 mm phosphate buffer ( ph 8.0 ) in the presence of artemisinin
( 100 mg , final concentration : 0.35 mm ) , ptdh ( 2 m ) , nadp ( 150 m ) , and sodium phosphite ( 50 mm ) .
the collected
organic layers were dried with na2so4 , concentrated ,
and purified by flash chromatography ( dichloromethane / hexanes / ethyl
acetate : 1/1/2 ) to afford 4 ( 90 mg , 90% ) .
the same procedure
was repeated two more times to yield a total of 260 mg of 4 . to a solution of 3 ( 50 mg , 0.17 mmol )
in anhydrous ch2cl2 ( 5 ml ) was added ( diethylamino)sulfur
trifluoride ( 55 mg , 0.34 mmol ) dropwise at 78 c . the
reaction mixture was warmed up to 0 c and stirred until the
starting material disappeared as determined by tlc .
the reaction mixture
was quenched with 60 l of triethylamine and extracted with
ch2cl2 ( 2 5 ml ) .
the collected organic
layers were dried with na2so4 and concentrated ,
and the residue was purified by flash chromatography ( hexanes / ethyl
acetate : 3/1 ) to afford 5 ( 42 mg , 82% ) .
h
nmr ( 500 mhz , cdcl3 ) : = 1.20 ( 3 h , d , j = 7.0 hz ) , 1.27 ( 3 h , d , j = 7.4 hz ) , 1.281.50
( 6 h , m ) , 1.85 ( 1 h , m ) , 1.94 ( 1 h , m ) , 2.03 ( 1 h , m ) , 2.292.36
( 2 h , m ) , 2.53 ( 1 h , m ) , 3.49 ( 1 h , m ) , 4.67 ( 1 h , m ) , 5.87 ( 1 h ,
s ) .
c nmr ( 100 mhz , cdcl3 ) : = 10.12 ,
12.4 , 24.7 , 25.2 , 28.7 ( d , j = 21.8 hz ) , 32.3 , 35.9 ,
37.9 , 40.7 ( d , j = 20.6 hz ) , 43.9 , 78.7 , 90.6 ( d , j = 166.0 hz ) , 93.0 , 105.5 , 171.9 .
f nmr ( 376.5
mhz , cdcl3 ) : = 133.9 . ms ( esi ) calcd for
c15h22fo5 [ m + h]m / z : 301.15 , found 301.42 . to a solution of 5 ( 20 mg , 0.07 mmol ) in
2 ml of anhydrous methanol was added sodium borohydride ( 8 mg , 0.21
mmol ) at 0 c .
the reaction mixture was stirred at 0 c
until the starting material disappeared as determined by tlc .
the
reaction mixture was quenched with 5 ml of ice - cold water , and methanol
was removed by evaporation in vacuo .
the aqueous layer was then extracted
with ch2cl2 ( 3 5 ml ) , and the collected
organic layers were dried with na2so4 and concentrated .
the crude product was dissolved in 2 ml of anhydrous meoh followed
by the addition of sulfuric acid ( 11 l , 0.21 mmol ) .
the mixture
was stirred overnight at room temperature and then quenched with 50
l of triethylamine .
the mixture was concentrated under reduced
pressure and purified by flash chromatography ( hexanes / ethyl acetate :
3/1 ) to afford 6 ( 11 mg , 55% ) .
h nmr ( 500
mhz , cdcl3 ) : = 0.97 ( 3 h , d , j = 7.5 hz ) , 1.15 ( 3 h , d , j = 6.8 hz ) , 1.50 ( 5 h ,
m ) , 1.821.92 ( 2 h , m ) , 1.98 ( 1 h , m ) , 2.052.20 ( 3
h , m ) , 2.47 ( 1 h , m ) , 2.72 ( 1 h , m ) , 3.48 ( 3 h , s ) , 4.57 ( 1 h , d , j = 49.5 hz ) 4.76 ( 1 h , d , j = 3.3 hz ) ,
5.43 ( 1 h , s ) .
c nmr ( 100 mhz , cdcl3 ) :
= 12.7 , 16.1 , 24.6 , 26.1 , 29.7 ( d , j = 20.7 hz ) ,
36.4 , 37.3 , 40.6 ( d , j = 19.9 hz ) , 45.1 , 56.1 , 80.2 ,
86.9 , 91.9 ( d , j = 172.5 hz ) , 92.3 , 103.6 , 104.2 .
ms ( esi ) calcd for c16h25fnao5 [ m
+ na]m / z : 339.16 , found
339.25 . to a solution of 5
( 20 mg , 0.07 mmol ) in
2 ml of anhydrous methanol was added sodium borohydride ( 8 mg , 0.21
mmol ) at 0 c .
the reaction mixture was stirred until the starting
material disappeared as determined by tlc , followed by quenching with
5 ml of ice - cold water .
the mixture was evaporated in vacuo to remove
methanol , and the remaining aqueous layer was extracted with ch2cl2 ( 3 5 ml ) .
the
crude product was dissolved in 2 ml of 1,4-dioxane followed by the
addition of succinic anhydride ( 8 mg , 0.077 mmol ) and triethylamine
( 50 l , 0.35 mmol ) .
the mixture was then concentrated
under reduced pressure and purified by flash chromatography
( dichloromethane / methanol : 20/1 ) to afford 7 ( 24 mg ,
90% ) .
h nmr ( 500 mhz , cdcl3 ) : = 0.90
( 3 h , d , j = 7.1 hz ) , 1.051.11 ( 4 h , m),1.321.60
( 6 h , m ) , 1.90 ( 2 h , m ) , 2.102.22 ( 3 h , m ) , 2.47 ( 1 h , m ) ,
2.662.79 ( 4 h , m ) , 4.65 ( 1 h , d , j = 50.1
hz ) , 5.46 ( 1 h , s ) , 5.82 ( 1 h , d , j = 10.1 hz ) .
c nmr ( 100 mhz , cdcl3 ) : = 11.7 , 15.9 ,
24.4 , 25.9 , 27.2 ( d , j = 20.6 hz ) , 28.5 , 28.7 , 31.0 ,
36.1 , 38.3 , 40.1 ( d , j = 19.2 hz ) , 44.3 , 52.1 , 79.2 ,
91.5 ( d , j = 146.5 hz ) , 93.2 , 104.6 , 171.1 , 172.6 . f nmr ( 376.5 mhz , cdcl3 ) : = 135.2 .
ms ( esi ) calcd for c19h27fnao8 [ m
+ na]m / z : 402.17 , found
402.33 . | the selective oxyfunctionalization of isolated sp3 c h
bonds in complex molecules represents a formidable challenge in organic
chemistry . here , we describe a rational , systematic strategy to expedite
the development of p450 oxidation catalysts with refined regio- and
stereoselectivity for the hydroxylation of remote , unactivated c
h
sites in a complex scaffold . using artemisinin as model substrate
,
we demonstrate how a three - tier strategy involving first - sphere active
site mutagenesis , high - throughput p450 fingerprinting , and fingerprint - driven
p450 reactivity predictions enabled the rapid evolution of three efficient
biocatalysts for the selective hydroxylation of a primary and a secondary
c h site ( with both s and r stereoselectivity ) in a relevant yet previously inaccessible region
of this complex natural product .
the evolved p450 variants could be
applied to provide direct access to the desired hydroxylated derivatives
at preparative scales ( 0.4 g ) and in high isolated yields ( > 90% ) ,
thereby enabling further elaboration of this molecule . as an example
,
enantiopure c7-fluorinated derivatives of the clinical antimalarial
drugs artesunate and artemether , in which a major metabolically sensitive
site is protected by means of a c h to c f substitution ,
were afforded via p450-mediated chemoenzymatic synthesis . |
the loss of teeth , very common in this population , is related to the reduction of bone tissue , receptors ( proprioceptors and periodontal ligaments ) , and muscle atrophy .
consequently , orofacial functions are impaired in toothless individuals.1 in addition , prostheses are functionally less efficient when compared to individuals with natural dentition.1,2 the stability of the jaw occlusion of the posterior teeth or prostheses is important for the swallowing function.2 the dental condition and the use of poorly fitting prostheses may contribute to the difficulties arising from old age , and the oral diet and nutritional status of the elderly may be affected by changes related to aging3 and changes in skill and desire to feel , bite , chew4 and swallow food,3,5 significantly compromising and further aggravating the quality of life of these individuals.6 the literature has shown the influence of oral health condition on swallowing and nutrition , indirectly influencing the daily life activities of the elderly.7 furthermore , it is clear that maintenance of oral health is essential for the general health , quality of life , chewing ability8 and , mainly , the reduction of pneumonia risk in fragile elderly people,8,9 for it is already known that bacterial colonies in oropharyngeal tissues and in dental plaque are the greatest precursors for the development of aspiration respiratory infections.10 besides the influence of the loss of dentition , the functional impairments resulting from aging may be potentiated by neurological changes resulting in dysphagia in patients , an important cause of morbidity and mortality in this population.11 in the adult and elderly population , dysphagia is most commonly associated with stroke.12 more than half of patients who have suffered stroke present between six and ten types of disability , muscle weakness being the most prevalent one , followed by communication , speech , and swallowing disorders.13 specifically regarding swallowing , the stroke entails increased oropharyngeal transit time,14,15 changes in motor control of tongue,16 as well as the presence of laryngotracheal aspiration of food.16,17 once the physiological changes resulting from aging in swallowing may be aggravated by loss of teeth and the use of poorly fitting prostheses , the severity of dysphagia could be influenced by the oral health condition and use of dental prostheses in elderly patients with neurological diseases .
however , according to the literature studied , no research has made the correlation between the findings of the swallowing performance with oral health in elderly patients after stroke
. therefore , the aim of this study was to relate the condition of oral health to the level of oral intake and the degree of swallowing dysfunction in elderly patients with stroke in chronic phase .
thirty poststroke individuals , aged 61 to 90 years , whose injury happened 6 months to 9 years previously to their participation in this prospective study .
the inclusion criteria considered the following cases : affected by stroke as attested by a medical report , through clinical diagnosis and imaging , with a minimum time of 6 months ; aged over 60 years , being in regular clinical neurological monitoring , not having undergone dysphagia rehabilitation ; presenting general stable health that would enable the realization of the proposed tests . in order to achieve a greater delineation of the group , in addition to considering the group of 30 individuals ( heterogeneous group [ hg ] ) ,
the total dentate , the total edentulous without dental prostheses , and individuals with more than one episode of stroke were excluded and the participants were divided into two groups , according to the affected body side : right hemiplegic group ( rhg , n=8 ) and left hemiplegic group ( lhg , n=11 ) . a distribution and recruitment fluxogram
the participants underwent assessment of various components of oral health through traditional health indicators , based on the presence or absence of disease .
the data were collected from the clinical examination by a dentist as instructed by the who oral health surveys : basic methods,18 including the assessment of dental status by the number of decayed , missing , and filled teeth ( dmft ) and evaluation of dental prostheses .
the prostheses were evaluated for retention , functional stability , smile aesthetics , degree of bone resorption , and quality of the mucosa.19 from the evaluation , the dental arches were classified as without prosthesis or with the presence of partial or complete dental prosthesis ; later , the use or need for replacement of dental prostheses was indicated .
the need for prostheses replacement was as follows : 0 , no need to change ; 1 , partial dental prosthesis was necessary in one of the dental arches ; 2 , need for partial dental prosthesis in both dental arches ; 3 , need for complete dental prosthesis in one of the dental arches ; 4 , need for dental prosthesis in both dental arches .
research of oral ingestion was performed by reviewing the usual food consumption patterns referred to in the 24-hour dietary recall . from the data obtained by the research of oral ingestion
, patients were classified according to the levels of the functional oral intake scale ( fois),20 on a scale from i to vii , considering the diet characteristics based on the properties and texture of the food.21 the fiberoptic endoscopic evaluation of swallowing ( fees ) was performed by an otorhinolaryngologist physician collaboratively with the speech therapist .
subjects were asked to remain seated with their heads arranged in the direction of the body axis , without bending or rotation , and the examination was performed using a flexible endoscopic equipment of fiberoptic bronchoscopic type ( model olympus clv - u20 ) and an olympus otv sc nasopharyngoscope ( olympus corporation , tokyo , japan ) .
three consistent standardized foods were evaluated : liquid ( 10 ml of filtered water ) , thick pudding ( 10 ml of thickened dietary grape juice , reaching a final consistency similar to that of pudding ) , and solid ( half a slice of bread , 1 cm thick ) . from the data obtained by the fees ,
the severity rating of the swallowing disorder was determined in accordance with the scale of functional impairment of swallowing of macedo filho,22,23 which subdivides the dysphagia severity levels ( normal , mild , moderate , and severe ) .
spearman s test was used to verify the correlation between the oral health condition ( dmft , number of teeth present , and the need for prostheses replacement ) and the performance in the swallowing function ( classification in the fois scale and dysphagia rating in each consistency ) . for this analysis to be performed
, the fois scale and the degree of dysphagia were numerically classified from 1 to 7 and from 1 to 4 , respectively .
for comparison of fois results and dysphagia classification , among the different types of prostheses used in the upper and lower dental arches ( complete dental prosthesis , partial dental prosthesis , denture or none ) , the kruskal
thirty poststroke individuals , aged 61 to 90 years , whose injury happened 6 months to 9 years previously to their participation in this prospective study .
the inclusion criteria considered the following cases : affected by stroke as attested by a medical report , through clinical diagnosis and imaging , with a minimum time of 6 months ; aged over 60 years , being in regular clinical neurological monitoring , not having undergone dysphagia rehabilitation ; presenting general stable health that would enable the realization of the proposed tests . in order to achieve a greater delineation of the group , in addition to considering the group of 30 individuals ( heterogeneous group [ hg ] ) ,
the total dentate , the total edentulous without dental prostheses , and individuals with more than one episode of stroke were excluded and the participants were divided into two groups , according to the affected body side : right hemiplegic group ( rhg , n=8 ) and left hemiplegic group ( lhg , n=11 ) . a distribution and recruitment fluxogram
the participants underwent assessment of various components of oral health through traditional health indicators , based on the presence or absence of disease .
the data were collected from the clinical examination by a dentist as instructed by the who oral health surveys : basic methods,18 including the assessment of dental status by the number of decayed , missing , and filled teeth ( dmft ) and evaluation of dental prostheses .
the prostheses were evaluated for retention , functional stability , smile aesthetics , degree of bone resorption , and quality of the mucosa.19 from the evaluation , the dental arches were classified as without prosthesis or with the presence of partial or complete dental prosthesis ; later , the use or need for replacement of dental prostheses was indicated .
the need for prostheses replacement was as follows : 0 , no need to change ; 1 , partial dental prosthesis was necessary in one of the dental arches ; 2 , need for partial dental prosthesis in both dental arches ; 3 , need for complete dental prosthesis in one of the dental arches ; 4 , need for dental prosthesis in both dental arches .
research of oral ingestion was performed by reviewing the usual food consumption patterns referred to in the 24-hour dietary recall . from the data obtained by the research of oral ingestion
, patients were classified according to the levels of the functional oral intake scale ( fois),20 on a scale from i to vii , considering the diet characteristics based on the properties and texture of the food.21 the fiberoptic endoscopic evaluation of swallowing ( fees ) was performed by an otorhinolaryngologist physician collaboratively with the speech therapist .
subjects were asked to remain seated with their heads arranged in the direction of the body axis , without bending or rotation , and the examination was performed using a flexible endoscopic equipment of fiberoptic bronchoscopic type ( model olympus clv - u20 ) and an olympus otv sc nasopharyngoscope ( olympus corporation , tokyo , japan ) .
three consistent standardized foods were evaluated : liquid ( 10 ml of filtered water ) , thick pudding ( 10 ml of thickened dietary grape juice , reaching a final consistency similar to that of pudding ) , and solid ( half a slice of bread , 1 cm thick ) . from the data obtained by the fees ,
the severity rating of the swallowing disorder was determined in accordance with the scale of functional impairment of swallowing of macedo filho,22,23 which subdivides the dysphagia severity levels ( normal , mild , moderate , and severe ) .
spearman s test was used to verify the correlation between the oral health condition ( dmft , number of teeth present , and the need for prostheses replacement ) and the performance in the swallowing function ( classification in the fois scale and dysphagia rating in each consistency ) . for this analysis to be performed
, the fois scale and the degree of dysphagia were numerically classified from 1 to 7 and from 1 to 4 , respectively .
for comparison of fois results and dysphagia classification , among the different types of prostheses used in the upper and lower dental arches ( complete dental prosthesis , partial dental prosthesis , denture or none ) , the kruskal
the participants underwent assessment of various components of oral health through traditional health indicators , based on the presence or absence of disease .
the data were collected from the clinical examination by a dentist as instructed by the who oral health surveys : basic methods,18 including the assessment of dental status by the number of decayed , missing , and filled teeth ( dmft ) and evaluation of dental prostheses .
the prostheses were evaluated for retention , functional stability , smile aesthetics , degree of bone resorption , and quality of the mucosa.19 from the evaluation , the dental arches were classified as without prosthesis or with the presence of partial or complete dental prosthesis ; later , the use or need for replacement of dental prostheses was indicated .
the need for prostheses replacement was as follows : 0 , no need to change ; 1 , partial dental prosthesis was necessary in one of the dental arches ; 2 , need for partial dental prosthesis in both dental arches ; 3 , need for complete dental prosthesis in one of the dental arches ; 4 , need for dental prosthesis in both dental arches .
research of oral ingestion was performed by reviewing the usual food consumption patterns referred to in the 24-hour dietary recall . from the data obtained by the research of oral ingestion
, patients were classified according to the levels of the functional oral intake scale ( fois),20 on a scale from i to vii , considering the diet characteristics based on the properties and texture of the food.21
the fiberoptic endoscopic evaluation of swallowing ( fees ) was performed by an otorhinolaryngologist physician collaboratively with the speech therapist .
subjects were asked to remain seated with their heads arranged in the direction of the body axis , without bending or rotation , and the examination was performed using a flexible endoscopic equipment of fiberoptic bronchoscopic type ( model olympus clv - u20 ) and an olympus otv sc nasopharyngoscope ( olympus corporation , tokyo , japan ) .
three consistent standardized foods were evaluated : liquid ( 10 ml of filtered water ) , thick pudding ( 10 ml of thickened dietary grape juice , reaching a final consistency similar to that of pudding ) , and solid ( half a slice of bread , 1 cm thick ) . from the data obtained by the fees ,
the severity rating of the swallowing disorder was determined in accordance with the scale of functional impairment of swallowing of macedo filho,22,23 which subdivides the dysphagia severity levels ( normal , mild , moderate , and severe ) .
spearman s test was used to verify the correlation between the oral health condition ( dmft , number of teeth present , and the need for prostheses replacement ) and the performance in the swallowing function ( classification in the fois scale and dysphagia rating in each consistency ) . for this analysis to be performed
, the fois scale and the degree of dysphagia were numerically classified from 1 to 7 and from 1 to 4 , respectively .
for comparison of fois results and dysphagia classification , among the different types of prostheses used in the upper and lower dental arches ( complete dental prosthesis , partial dental prosthesis , denture or none ) , the kruskal
according to the evaluation of oral condition , the mean of dmft of elderly individuals affected by stroke in this study was 28.7 , while the mean of remaining teeth was 5.6 , showing that most individuals were edentulous .
table 2 shows the types of prostheses used by the elderly individuals affected by stroke in each dental arch and the need for prostheses replacement . according to the results of the functional oral intake scale ,
most individuals ( 53% ) presented as level v on the fois , followed by level iv ( 34% ) and level vii ( 13% ) . with the severity rating of the swallowing disorder , no occurrence of severe
the distribution of the elderly affected by stroke according to severity rating of the swallowing disorder is shown in table 3 , as well . according to the results of the statistical analysis ,
correlations between the oral health condition and the results of the fois scale in elderly individuals affected by stroke in this study were found . there was a statistically significant negative correlation between the need for replacement of prostheses and the rating scale , and also in the hg and in rhg , showing that the greater the need for replacement of the prosthesis , the worse the rating of the fois scale .
there were differences in the classification of the fois scale , for the lhg , in the different types of prostheses used in the upper dental arch , whereas for the rhg the difference in the classification of fois occurred between groups of individuals with different types of prostheses used in the lower dental arch .
after completion of the dunn s post - test , the difference was confirmed only in the lhg , demonstrating that individuals of this group with partial prostheses in the upper arch ( ie , that still had natural elements and whose missed teeth had been rehabilitated ) had better ratings in the fois scale compared to individuals with total prostheses .
the results of the correlation between the fois scale and the oral health condition are shown in table 4 . in the statistical analysis between the degree of swallowing dysfunction and oral health condition , a significant negative correlation between the number of teeth and the swallowing performance of liquid in the lhg
was found , demonstrating that the greater the number of teeth , the lower the degree of swallowing dysfunction . according to the kruskal
wallis test , there was no difference in the performance of swallowing solid boluses depending on the type of prosthesis used in the lower dental arch ; in the hg , however , when applying the post - dunn s test , this difference was not confirmed ( p>0.05 ) .
the comparison of the fois scale between the types of dental prostheses is shown in table 5 .
the results of the correlation between the degree of dysphagia and the oral health condition are shown in table 6 , and the comparison of the degree of swallowing dysfunction between types of dental prostheses is described in table 7 .
according to the literature , individuals affected by stroke often exhibit decreased level of consciousness , paralysis of the muscles involved in swallowing , sensory deficits of the pharynx and oral cavity , as well as loss of appetite.24 with these disorders related to swallowing , dysphagia becomes an important symptom in this population , and may be aggravated by teeth loss and the use of poorly fitting prosthesis , common during aging .
furthermore , the hypothesis that oropharyngeal colonization is primarily responsible for the subsequent respiratory infection , owing to aspiration , is strongly supported by the literature.10 no literature studies have made the correlation between the findings of the swallowing performance and the condition of the oral health in elderly patients after stroke .
the results of this study showed some correlations between the oral condition and the data of the swallowing performance even when elderly individuals affected by stroke were part of the heterogeneous group . besides the statistical analysis for the study group , in an attempt to reduce its heterogeneity
, the subjects were divided according to the side of the motor impairment after stroke .
former studies have suggested that dysphagia maybe associated with diffuse lesions in one or both hemispheres , anterior fossa , or brainstem , as swallowing has a bilateral cortical representation .
it has been well established that the protection of the airways during swallowing requires a precise coordination with accurate movements of the structures involved and adequate propulsion of the bolus to the oropharynx and esophagus;25 thus , the motor ability for swallowing seems to be essential for this protection . in the hg and rhg
, it was found that the higher the need for replacement of the prosthesis , the worse the rating in the fois scale ; ie , individuals with greater need for replacement of the prostheses had a greater perception in making more compensation and changes in their diet , which reflected the classification level of oral intake .
the fois scale was developed to describe the functional level of the daily oral intake of food and liquid of a patient , considering the changes in diet and need for compensation during swallowing.20 these limitations and changes in oral intake result from patient perception and self - perception of swallowing ability that is impaired .
therefore , from the results , it can be inferred that when there was a greater need for replacement of the prosthesis , the patient s perception of doing more compensation and dietary modifications is reflected in the classification of the fois scale .
significant changes in oral condition can have a major effect on the functions of eating and drinking and on the nutritional status of elderly individuals and those with neurological diseases.26 thus , the selection of food is determined by the difficulty in chewing and swallowing of the individuals .
missing teeth and the use of prosthesis cause changes in the chewing ability of the elderly according to the type of food , and influences the swallowing function;27 thus , soft or mashed foods are preferentially ingested by the elderly , due to the difficulty in mastication,28 showing that there is an influence of oral health condition on the selection of foods .
besides the influence of the oral condition on swallowing , there was a difference between the types of prostheses and classification of the fois scale when the results of the evaluation of individuals with impaired right and left hemiplegia were analyzed separately .
the difference between the types of prostheses could be confirmed by post - dunn s test in the lhg , and demonstrated that individuals with partial dentures had better ratings in the fois scale than individuals with complete prostheses .
thus , we can infer that when there was partial prosthesis rehabilitation along with the presence of some dental elements , subjects needed less compensation and changes in the oral diet . in this sense , studies show that the use of prostheses in the edentulous contributes to the maintenance of the physiological process of swallowing in the elderly,29 the fixation of the prosthesis in the mandible,30 and the presence of natural elements enables greater preservation of functional swallowing .
when comparing the swallowing performance according to the type of prosthesis used , there was no difference between the types of prostheses fitted in the lower dental arch in swallowing solids in the hg .
studies in healthy elderly subjects demonstrate the importance of the oral condition in the performance of the oral phase of swallowing .
changes in masticatory function related to the oral condition , such as difficulties biting and chewing food27 demonstrated an influence on the pharyngeal phase of swallowing , such as premature escape of food , waste retention in the vallecula and pyriform sinuses , and the presence of coughing and gagging.27 moreover , the absence of dental prosthesis can result in a longer duration of swallowing,29 and the chewing and swallowing difficulties in healthy older adults decreased after the replacement of removable total prostheses by mandibular implant - supported prostheses.30 the literature has shown positive effects in the swallowing function with the use of prostheses and the differences in swallowing performance with prostheses in the upper or inferior arches.8 a study with healthy elderly individuals showed the differences between the total duration of swallowing , the latency period before pharynx elevation , duration of preparatory and oral phase , when using or not using the dental prostheses.29 finally , in the lhg , it was observed that the smaller the number of teeth present , the worse the rating of dysphagia for liquid . although this study aimed at finding an influence of the presence and number of teeth on chewing and consequently on swallowing solids,1,27,28 it also demonstrates the influence of the oral condition on liquid swallowing for this population .
according to tamura et al,2 especially for liquid food , which demands more coordination during swallowing , stability in the jaw is also required , so that there is greater stability of the hyolaryngeal complex . to start the pharyngeal phase ,
the rise of the hyoid bone by the suprahyoid muscles and laryngeal elevation should occur . to complement these events ,
the jaw must be stabilized by natural or rehabilitated teeth into the proper position , because when the elevation of the hyoid bone and larynx is insufficient , there is an increased risk of aspiration during swallowing .
the findings of this study indicate that the oral health condition of elderly individuals after stroke in chronic phase showed an association with the level of oral intake and the degree of oropharyngeal dysphagia . the limitations of the study should be considered , one being the lack of access to the imaging examinations to characterize the individuals according to the type of stroke , damaged areas , and extent of injury , and heterogeneity of the time of brain involvement , in addition to the effects of medications and other comorbidities , which were not taken into account .
also , owing to the small sample , strong correlations could not be observed , and a cause / effect relationship can not be determined . despite the limitations of this study , and the fact that hemiplegia does not necessarily represent a direct relationship with the injured brain hemisphere contralaterally , since cerebellum lesions may cause ipsilateral motor damages,31 the findings indicated that the level of oral intake was influenced by the condition or type of dental prostheses , regardless of the side of the body affected . on the other hand ,
the number of teeth were demonstrated to be related to the level of dysphagia in individuals with right hemiplegia , which shows that the oral health condition differentially affects the physiological responses of swallowing , influenced by the brain damage .
considering the present sample , further research is necessary pertaining to the oral condition and the neurological involvement of individuals .
in addition , these issues demonstrate the need for an interdisciplinary research team comprising dentists , physicians , speech pathologists , and other health professionals , so as to study and define the treatment and guidelines to patients and caretakers , in order to maintain the best oral health condition in the elderly presented with oropharyngeal dysphagia . | backgroundaccording to the literature , the occurrence of dysphagia is high in cases of stroke , and its severity can be enhanced by loss of teeth and the use of poorly fitting prostheses.objectiveto verify that the status of oral health influences the level of oral intake and the degree of swallowing dysfunction in elderly patients with stroke in chronic phase.methodsthirty elderly individuals affected by stroke in chronic phase participated .
all subjects underwent assessment of their oral condition , with classification from the functional oral intake scale ( fois ) and nasoendoscopic swallowing assessment to classify the degree of dysphagia .
the statistical analysis examined a heterogeneous group ( hg , n=30 ) and two groups designated by the affected body part , right ( rhg , n=8 ) and left ( lhg , n=11 ) , excluding totally dentate or edentulous individuals without rehabilitation with more than one episode of stroke.resultsthere was a negative correlation between the need for replacement prostheses and the fois scale for the hg ( p=0.02 ) and rhg ( p=0.01 ) .
differences in fois between types of prostheses of the upper dental arch in the lhg ( p=0.01 ) and lower dental arch in the rhg ( p=0.04 ) . a negative correlation was found between the number of teeth present and the degree of dysfunction in swallowing liquid in the lhg ( p=0.05 ) .
there were differences in the performance in swallowing solids between individuals without prosthesis and those with partial prosthesis in the inferior dental arch ( p=0.04 ) for the hg.conclusionthe need for replacement prostheses , type of prostheses , and the number of teeth of elderly patients poststroke in chronic phase showed an association with the level of oral intake and the degree of oropharyngeal dysphagia . |
in recent years , video - assisted intubation devices have flooded theaters , and , to a lesser extent , are also being used in icus . but can such new technology really contribute to making tracheal intubation in the icu less life - threatening , or even more life - saving ? noppens and colleagues , in a study just published in critical care , evaluated the efficacy of the c - mac video laryngoscope for endotracheal intubation in an icu managed by anesthesiologists and compared the results to those obtained during a previous period when conventional direct laryngoscopy was used . in patients with at least one predictor for difficult intubation , poor glottic views ( cormack and lehane grades iii and iv )
were reduced from 38% to 19% , and intubation success on the first attempt increased from 56% with direct laryngoscopy to 79% when the c - mac video laryngoscope was used . even in all patients , the incidence of at least two intubation attempts
though this was not statistically significant , halving this incidence may have substantial clinical impact .
although the overall intubation success was 100% in either group , successful tube placement in the trachea on the first attempt is crucial in icu patients with poor tolerance to apnea .
after implementation of a video laryngoscope , however , intubations were more often performed by less experienced physicians .
even if one reason may be that younger physicians are more open to the newer technique , it must be guaranteed that physicians ' experience is not substituted solely by technical equipment . similarly , the use of endoscopic intubation was reduced substantially in the intervention phase ( 4% versus 13% ) , so experience with such devices may decrease , causing problems if video laryngoscopy is not possible or is contraindicated .
video laryngoscopy for every patient ? sparing such devices for the really difficult cases ? to answer these questions it needs to be emphasized that all video - assisted and optical intubation devices have different view angles , thus producing different images with particular distortions , and even experts in ' old - fashioned ' airway management need a substantial level of training with a certain device before using it safely and successfully in critical situations and patients .
video laryngoscopes , regardless of a particular brand or device , can not be used intuitively - they require expert skills and routines to be tamed and turned into life - saving tools .
tube placement can turn out to be difficult and even impossible , despite the excellent glottic exposure video laryngoscopes most often provide .
the reason is that the operator 's eye is no longer in a straight - optical and anatomical - line with the larynx , but at the tip of the laryngoscope blade .
optical and anatomical axes divert , and tube placement is sometimes difficult or fails . becoming alert to these potential problems associated with video laryngoscopy and constantly improving one 's technical skills to overcome such pitfalls can only be achieved when such devices are used on a daily basis and in routine patients .
apart from the primary aim of the study - evaluating video laryngoscopy - it needs to be highlighted that noppens and colleagues routinely used capnography to verify correct tube placement after intubation . capnography has become a mandatory requirement in any operating room , but its use in the icu seems still to be somewhat dowdy . as of 2010 , only 25% of icus in the uk and the republic of ireland used capnography to confirm correct tube placement after intubation . such refusal may produce catastrophic outcomes : data collected from all uk national health service hospitals over a period of one year in 2008/2009 revealed that 61% of adverse events during airway management in the icu resulted in death or permanent brain damage . in 74% of such fatal cases no capnography was used !
doubtless , video laryngoscopy is a helpful tool in the hands of experienced physicians , but should not be considered a bailout procedure for beginners in airway management . and
as attractive and simple video laryngoscopy appears , we must always remind ourselves to maintain our skills in endoscopic intubation .
both authors receive continuous material support for clinical studies from karl storz gmbh & co. kg , the manufacturer of the c - mac video laryngoscope . | the c - mac video laryngoscope substantially reduced poor glottic views and increased intubation success in icu patients with at least one predictor for difficult intubation . however , all video - assisted and optical intubation devices have different view angles , thus producing different images with particular distortion , and even experts in ' old - fashioned ' airway management need a substantial level of training with a certain device before using it safely and successfully in critical situations and patients .
video laryngoscopes , regardless of a particular brand or device , can not be used intuitively - they require expert skills and routines to be turned into a life - saving airway management tool . |
in gipuzkoa , 27 human listeriosis episodes were reported during january 2013february 2014 . all cases produced sepsis in the patients , except 1 case that produced diarrheal disease in a 34-year - old parturient woman who had undergone a splenectomy .
eleven episodes ( 40.7% ) occurred in pregnant or parturient women , and 8 of the children of these patients were affected : 5 newborns ( 4 of them premature infants ) became ill , 2 pregnancies ended in miscarriage , and 1 infant was stillborn .
( for this study , a pregnancy - associated episode was counted only once , whether the causative strain was isolated from mother , child , or both . ) ten cases ( 37.0% ) occurred among the elderly ( > 70 years of age ) and 6 ( 22.2% ) in adults 4560 years of age . of the 6 patients in the 45- to 60-year age group ,
a total of 29 human l. monocytogenes isolates were available for microbiological characterization : 22 from blood , 3 from placental membranes , 2 from cerebrospinal fluid , and 1 each from stool and dermal exudate .
serotypes were established by agglutination ( listeria - o - antisera , difco ; bd diagnostics , sparks , md , usa ) and by multiplex - pcr ( 4 ) .
the predominant serotypes identified were 1/2b ( n = 17 , 58.6% ) and 4b ( n = 8 , 27.6% ) ; 4 isolates were serotype 1/2a ( 13.8% ) .
pulsed - field gel electrophoresis ( pfge ) was performed by using the restriction endonucleases smai and asci ( 5 ) .
the sts of l. monocytogenes isolates were determined by using the multilocus sequence typing ( mlst ) primers and conditions described by the pasteur institute ( 6 ) .
mlst showed that 16 of 17 serotype 1/2b isolates we tested were st87 ; the remaining serotype 1/2b isolate was st3 .
only 2 human l. monocytogenes st87 serotype 1/2b isolates , both from japan , were listed in the pasteur institute mlst database ( 6 ) .
further , pfge showed 2 large clusters within serotype 1/2b , and epidemiologic research detected 2 main outbreaks .
all listeriosis episodes during the study period that were caused by isolate types other than serotype 1/2b and st87 were sporadic .
in the first case cluster ( first outbreak ) , 5 episodes were detected during august
all isolates from 1 woman at 28 weeks gestation , 1 parturient woman and her newborn child , and 2 newborn twins ( no samples from the mother were available ) showed the same pfge pattern with the restriction enzyme asci ( arbitrarily named as pattern a ) and were st87 ( figure ) .
another 2 l. monocytogenes isolates from cases apparently not related to this first outbreak , isolated in january 2012 and in april 2013 from a 53-year - old man with meningitis and an 84-year - old woman , respectively , showed the same pfge pattern .
pulsed - field gel electrophoresis patterns of 6 listeria monocytogenes serotype 1/2b isolates collected from listeriosis patients in gipuzkoa , northern spain , during january 2013february 2014 .
lanes 1 and 2 , isolates from first outbreak ( pattern a ) ; lanes 3 , 4 , and 5 , isolates from second outbreak ( pattern b ) , including ( lane 5 ) 1 isolate from a food product of foie gras ; lane 6 , l. monocytogenes sequence type 3 serotype 1/2b isolate , unrelated to outbreaks .
for this first outbreak , epidemiologic interviews were conducted starting with the cases observed during september 2013 .
patients were asked about their alimentary habits , focusing on consumption of raw or semicooked animal or animal derivate products .
although no specific food was identified as the possible source of the outbreak , all but 1 of the patients remembered eating cooked ham bought in the butcher s department of a certain supermarket chain located in different villages of the province .
were investigated by pcr and culture after selective enrichment ( 24 listeria enrichment broth ; oxoid , basingstoke , uk ) ) . in the microbiological investigation ( 7 )
, 1 of 6 brands of cooked ham studied grew l. monocytogenes at a low level , but the sample strain was not the serotype 1/2b identified in the outbreak .
no listeria spp . were identified in the remaining 5 brands of cooked ham studied . in the second outbreak ,
10 episodes were reported during early november 2013 through late february 2014 ; patients were 5 elderly persons , 4 previously healthy parturient women , and 1 parturient women who had undergone a splenectomy .
no isolates could be obtained from the mother of an infected newborn and from a miscarriage .
the 10 isolates available for study ( 1 per episode ) were of the same serotype and sequence type , 1/2b and st87 , as isolates from the first outbreak , but these isolates differed in pfge asci pattern ( this pattern arbitrarily named pattern b ) .
isolates from these 2 outbreaks had the same pfge pattern when the smai enzyme was used ( figure ) .
after the epidemiologic survey for this outbreak , we conducted microbiological analysis of several foods .
a foie gras product kept by a patient in his refrigerator yielded a positive culture of l. monocytogenes that had the same phenotype and molecular profile as isolates from the second outbreak .
the presence of the outbreak strain was confirmed in high amounts ( 5.2 10 cfu / g ) by several other cultures from 3 unopened samples from the same brand of foie gras .
after this food source was identified as the source of this second outbreak , all patients from the first outbreak were specifically interviewed again about the consumption of this product , but none remembered having eaten it .
l. monocytogenes infection is serious and has high fatality rates . among the 35 persons infected in this region of spain since 2013 , a total of 6 deaths have occurred : 3 adults , 2 fetuses ( miscarriages ) , and 1 child ( stillbirth ) . without rapid case detection and early treatment ,
l. monocytogenes infections mainly affect elderly persons , pregnant women , newborns , and immunocompromised adults ( 810 ) .
humans are usually infected after eating contaminated food , although the source of the infection is infrequently detected in sporadic cases . for outbreaks , after the epidemiologic survey , a food is usually implicated as the source of infection , but it is not always possible to obtain microbiological confirmation ( 11 ) . in this study , the strain ( same phenotype and genotype ) that caused the second outbreak was obtained from a recently consumed food in the home and in several unopened samples from the same batch of food . however , this source could not be identified as related to the first outbreak .
pregnant women were frequently infected during these outbreaks ; epidemic clones infected 8 pregnant women and their offspring . however , during 2012 and 2013 , serotype 4b isolates of different genotypes also infected 5 pregnant women , resulting in 1 miscarriage , 3 premature newborns , and 1 stillborn infant and 1 premature newborn in a twin pregnancy . in summary , we defined 2 epidemiologically unrelated outbreaks of listeriosis caused by a rare type of l. monocytogenes that occurred at the same time in a small region of spain .
management of this frequently fatal disease requires careful investigation of the source of infection to stop its spread and prompt treatment of infected persons to prevent severe illness and death . | in the province of gipuzkoa , spain ( 700,000 inhabitants ) , 712 episodes of human listeriosis were recorded annually during 20092012 . however , during january 2013february 2014 , 27 episodes were detected , including 11 pregnancy - associated cases .
fifteen cases in 2 epidemiologically unrelated outbreaks were caused by a rare type of listeria monocytogenes , sequence type 87 serotype 1/2b . |
toothbrushes play an essential role in oral hygiene and are commonly found in both community and hospital settings .
toothbrushes may play a significant role in disease transmission and increase the risk of infection since they can serve as a reservoir for microorganisms in healthy , oral - diseased and medically ill adults .
contamination is the retention and survival of infectious organisms that occur on animate or inanimate objects . in healthy adults ,
contamination of toothbrushes occurs early after initial use and increases with repeated use [ 2 , 3 ] .
toothbrushes can become contaminated from the oral cavity , environment , hands , aerosol contamination , and storage containers .
bacteria which attach to , accumulate , and survive on toothbrushes may be transmitted to the individual causing disease [ 4 , 5 ] . in the hospital
there is a need for standardized nursing guidelines to prevent toothbrush contamination , which may increase the risk of infections from potentially pathogenic microorganisms and is clinically relevant for assessing the risks and benefits of oral care and informing nursing practice .
this review of peer - reviewed literature was conducted to evaluate the cumulative state of knowledge related to toothbrush contamination , its possible role in disease transmission , and in preparation for a research study related to toothbrush contamination in critically ill adults .
articles published from 1977 to 2011 , on human subjects and using the english language were obtained .
the review included studies that evaluated toothbrush contamination in healthy and oral - diseased adults , guidelines for toothbrush and oral care in both healthy and medically ill persons , hospitalized and nonhospitalized patients , and interventions for reducing contamination of toothbrushes .
the following databases were searched : pub med ( clinical inquiries and mesh ) , cinahl , cochrane library , national guidelines clearinghouse , web of science , and google scholar .
key search terms used in the review were toothbrush , tooth brushing , colonization , bacterial contamination , contamination , oral hygiene , oral health , nursing practice , microbial contamination , and adults .
a total of three separate searches were conducted in a systematic fashion using the inclusion and exclusion criteria and search terms . the first search ( search 1 ) identified articles in the selected databases and complete copies of articles that were considered to have met the inclusion criteria were obtained for further review ( table 1 ) .
articles were excluded if they did not meet the inclusion criteria listed above , were conducted on a pediatric population , were duplicates from other databases , or only explored antibacterial methods . the second search ( search two ) included articles identified through cited articles and were reviewed following the same criteria . there were a total of 23 new articles identified through the second search . a third search ( search three )
was conducted one year after the first search in order to capture any recently published articles .
after a review of the abstracts for the articles obtained through the three searches , a total of 88 relevant articles were identified for further evaluation .
after inclusion criteria were applied , 38 articles were selected ; after exclusion criteria were applied , ten articles were retrieved to be read in their entirety and included in this review ( figure 1 ) .
the selected studies are grouped by setting in vivo , in vitro , and studies that combined both types of settings .
the sample sizes ranged from 3 to 103 with the majority of studies having a sample size under 30 .
overall , the studies evaluated several perspectives related to toothbrush contamination to include : contamination , methods for decontamination , storage , design , and environmental factors .
all of the studies examined toothbrush contamination and found significant bacterial retention and survival on toothbrushes after use [ 6 , 7 ] .
glass found that toothbrushes from both healthy patients and patients with oral disease contained potentially pathogenic bacteria and viruses such as staphylococcus aureus , e. coli , pseudomonas , and herpes simplex virus .
glass also found toothbrushes contaminated with herpes simplex virus 1 in numbers sufficient to cause an infection in the patient .
bunetel et al . found that toothbrushes used by patients with existing oral disease quickly became contaminated .
this study also found a significant relationship between repeated use and bacterial retention on toothbrushes and that the oral cavity can be inoculated from a contaminated toothbrush .
several of the studies found that toothbrushes were contaminated before use [ 5 , 9 ] .
mehta et al . found that 70% of the toothbrushes in their study became heavily contaminated with pathogenic microorganisms after use .
studies by both taji and rogers and glass found extensive toothbrush contamination after use except in cases where an oral antiseptic , such as mouthwash , was used immediately prior to brushing .
verran and leahy - gilmartin found that toothbrushes supported many different bacteria and the amount of growth was varied .
bunetel et al . found that toothpaste , mouthwash , and oral antiseptics all decrease microbial load on toothbrushes .
their study found that the toothbrushes became heavily contaminated after use . soaking the toothbrush in listerine for 20 minutes prior to and after brushing decreased the microbial load .
the use of antimicrobial coated toothbrushes in adults with oral disease was explored by efstratiou et al . as a means to prevent toothbrush contamination .
this study , however , found that coating the bristles with triclosan did not change bacterial growth but the use of toothpaste did .
glass and jensen explored ultraviolet light as a means of decontamination and found this method to be effective at reducing the bacterial load on toothbrushes .
the use of coated tufts and toothpaste was investigated in adult patients with oral disease .
found that coated tuffs did not inhibit contamination but use of toothpaste did reduce contamination .
mehta et al . found that an overnight immersion in chlorhexidine gluconate was highly effective in decreasing toothbrush contamination and chlorhexidine was more effective than listerine in reducing the microbial load of bacteria .
sato et al . found that rinsing toothbrushes with tap water resulted in continued high levels of contamination and biofilm .
. found that the use of regular and triclosan - containing toothpaste resulted in lower toothbrush contamination than no toothpaste use .
toothbrushes can become contaminated through contact with the environment , and bacterial survival is affected by toothbrush storage containers .
found that toothbrushes placed in closed containers and exposure to contaminated surfaces yielded higher bacterial counts than those left open to air .
mehta et al . found that the use of a cap for toothbrush storage increased bacteria survival .
in addition , glass found that bacteria survived more than 24 hours when moisture is present .
toothbrush bristles range from soft to hard with different cluster patterns and plastic shapes while toothbrush handles included different plastic shapes and decorative moldings .
bunetel et al . found that bacteria become trapped inside the bristles of the toothbrush and bacterial survival is dependent upon the bacteria ( aerobic versus anaerobic ) and toothbrush design .
in addition , the researchers found that solid handles had less bacteria retention and that as the surface area increased , so did the microbial load .
toothbrushes with bristles that are frayed and arranged closely together trapped and retained more bacteria .
this finding was also echoed in a study by glass that explored the level of bacterial retention based on toothbrush brand , color and bristle pattern .
contamination was the lowest in soft and round , clear , two bristle row toothbrushes .
mehta et al . found that the retention of moisture and oral debris in the bristles increased bacterial survival .
due to the limited number of publications specifically related to toothbrush contamination , it was necessary to conduct a preliminary evaluation of the majority of identified articles for this review .
for example , several of the articles combined an in vivo examination of bacterial survival on actual patient 's toothbrushes and then conducted an in vitro autoinoculation experiment to examine decontamination methods on sterile toothbrushes in the laboratory .
the selected studies all found that toothbrushes of healthy and oral diseased adults become contaminated with potentially pathogenic bacteria from the dental plaque , design , environment , or a combination of factors .
the trend identified in the literature is to evaluate methods to reduce toothbrush contamination or toothbrush design rather than evaluating the process related to how the toothbrush initially becomes contaminated , is stored , or is disinfected . in a vulnerable population such as critically ill adults , pathogenic contamination may increase the risk of infection and mortality . although some interventions such as chlorhexidine , toothpaste , mouthwash , and ultraviolet sanitizers reduce bacterial survival , oral hygiene practices in the hospital setting by nurses vary . currently , there are no nursing guidelines related to toothbrush frequency of use , storage , and decontamination . in the hospital
setting , the environment as a source of pathogenic bacteria is now a hot topic and the focus of many current infectious disease research studies .
surfaces in close contact with the patient such as bed frames , countertops , sinks , bedside tables , linens , and mattresses may act as fomites .
toothbrushes may come into contact with these surfaces prior to or after use thus increasing risk . while there is significant literature available on environmental contamination and risk for infection ,
no studies have specifically examined the toothbrush on more vulnerable hospital populations such as critically ill adults .
toothbrush storage is inconsistent in both community and hospital environments and may increase exposure to pathogenic organisms .
the storage conditions of toothbrushes play an important role in bacterial survival : toothbrushes stored in aerated conditions had a lower number of bacteria than those stored in plastic and bacterial growth on the toothbrush increased 70% in a moist , covered environment . in clinical practice , the author has observed that there is no standardized nursing protocol for the storage or replacement of toothbrushes and that some commonly observed nursing practices include storing the toothbrush in the bath basin with other bathing / personal supplies and linens , in a paper towel , in a plastic wrapper , on the bedside table , next to the sink , and in an oral rinse cup at the bedside .
these practices may impact the contamination of toothbrushes . in this review , the majority of studies identified had small sample sizes .
importantly , despite multiple studies supporting toothbrush contamination and the likely relationship between contamination and disease transmission , there are no studies that specifically examine toothbrush contamination and the role of environmental factors , toothbrush contamination and vulnerable populations in the hospital setting ( e.g. , critically ill adults ) , and toothbrush use in nursing clinical practice
. additional descriptive studies to evaluate these relationships would be beneficial and informative for future research .
the relationship between environmental factors , toothbrush contamination , and patient oral colonization would inform development of nursing oral care guidelines for adults that minimize risks related to toothbrush contamination . | toothbrushes are commonly used in hospital settings and may harbor potentially harmful microorganisms .
a peer - reviewed literature review was conducted to evaluate the cumulative state of knowledge related to toothbrush contamination and its possible role in disease transmission .
a systematic review was conducted on adult human subjects through three distinct searches .
the review resulted in seven experimental and three descriptive studies which identified multiple concepts related to toothbrush contamination to include contamination , methods for decontamination , storage , design , and environmental factors .
the selected studies found that toothbrushes of healthy and oral diseased adults become contaminated with pathogenic bacteria from the dental plaque , design , environment , or a combination of factors .
there are no studies that specifically examine toothbrush contamination and the role of environmental factors , toothbrush contamination , and vulnerable populations in the hospital setting ( e.g. , critically ill adults ) and toothbrush use in nursing clinical practice . |
this article briefly reviews the need for emergency care in africa and barriers to its integration into health systems in the region .
we describe an example of an emergency care initiative at muhimbili national hospital ( mnh ) in dar es salaam that has integrated a multi - cadre training program with development of a dedicated emergency department in the largest public hospital in tanzania .
this article should be of interest to those concerned with prevention and treatment of acute illness in the sub - saharan region , and those with a particular interest in global emergency care and physician training initiatives in resource - limited settings .
low - income countries suffer the highest rates of every category of injury from road traffic to drowning ; the highest rates of maternal death from acute complications of pregnancy ; and the highest rates of acute complications of communicable diseases including tuberculosis , malaria , and hiv .
the rapidly growing prevalence of cardiovascular and diabetic disease has only increased the burden of acute illness , as patients with chronic disease in low - income countries also have the highest rates of mortality from acute complications .
the disease control priorities in developing countries project estimates that 45 per cent of deaths and 36 per cent of disability - adjusted life years ( dalys ) in low- and middle - income countries could be addressed by the implementation of emergency care systems .
while definitive specialty care will never be available at all times in all places , several studies suggest that emergency care is an effective and cost - effective means of secondary prevention : prioritizing an integrated approach to early resuscitation and stabilization of acutely ill patients greatly reduces the morbidity and mortality associated with a range of medical , surgical , pediatric , and obstetric conditions .
in addition , many acute conditions can be mitigated by primary prevention ; disease surveillance at acute and emergency care facilities has been shown to increase preparedness and disease control capabilities .
in general , analysis of acute disease patterns and
the distribution of mortality across the acute care continuum is a crucial part of identifying the preventive and training initiatives most likely to improve outcomes .
facility - based and pre - facility emergency care are essential components of a continuum ranging from prevention , through primary and chronic care , to inpatient critical care and surgery , and with resolution 60.22 the world health assembly has called for all its member states to consider formal , integrated emergency care systems ' .
there are four foundational challenges to integrating emergency care into health systems in sub - saharan africa :
the burden of acute disease in sub - saharan africa is severely under - documented .
some data exist on the distribution of inpatient diagnoses in the region , but the actual range of acute presentations to health facilities is largely unknown .
studies that compare police , hospital , and insurance records with other sources in low - income countries , for example , suggest that as few as 1 in 10 injuries may be documented in official counts .
health - care facilities often lack an integrated approach to triage , resuscitation , and stabilization of acutely ill patients . even at a single small district hospital , acutely ill patients
may be cared for by several different departments , depending on age , pregnancy status , and specific disease states .
this vertical approach means that there is rarely a dedicated acute intake area staffed with nonrotating personnel who can be trained in resuscitation and stabilization.essential components of acute and emergency care have not been determined , and there is no consensus on how to define success . to date , there has been no systematic analysis of the acute care delivery systems most appropriate to these varied contexts . while there are scattered examples of successful interventions , little
is known about what makes these programs effective or how others might replicate their success .
impact is often quantified by the number of providers trained , rather than by any measure that incorporates quality or performance assessment.there is no current advocacy plan for placing emergency care on the global health agenda . despite the essential role of early resuscitation and stabilization in averting morbidity and mortality ,
emergency care is conspicuously absent from discussions of global health priority initiatives ( such as the millennium development goals and un consensus statements ) and large - scale global health funding strategies .
the burden of acute disease in sub - saharan africa is severely under - documented .
some data exist on the distribution of inpatient diagnoses in the region , but the actual range of acute presentations to health facilities is largely unknown .
studies that compare police , hospital , and insurance records with other sources in low - income countries , for example , suggest that as few as 1 in 10 injuries may be documented in official counts .
health - care facilities often lack an integrated approach to triage , resuscitation , and stabilization of acutely ill patients . even at a single small district hospital , acutely ill patients
may be cared for by several different departments , depending on age , pregnancy status , and specific disease states .
this vertical approach means that there is rarely a dedicated acute intake area staffed with nonrotating personnel who can be trained in resuscitation and stabilization .
essential components of acute and emergency care have not been determined , and there is no consensus on how to define success . to date , there has been no systematic analysis of the acute care delivery systems most appropriate to these varied contexts . while there are scattered examples of successful interventions , little is known about what makes these programs effective or how others might replicate their success .
impact is often quantified by the number of providers trained , rather than by any measure that incorporates quality or performance assessment .
there is no current advocacy plan for placing emergency care on the global health agenda . despite the essential role of early resuscitation and stabilization in averting morbidity and mortality ,
emergency care is conspicuously absent from discussions of global health priority initiatives ( such as the millennium development goals and un consensus statements ) and large - scale global health funding strategies . despite these challenges ,
interest in emergency care training is rapidly expanding in africa , and there are planned or established emergency training programs for specialists in botswana , ethiopia , ghana , kenya , liberia , rwanda , south africa , sudan , tanzania , and uganda ; and for other provider cadres in ghana , tanzania , south africa , and uganda .
we review one such initiative at mnh in dar es salaam , with a focus on the development of the emergency medicine residency program .
the united republic of tanzania is designated as a low - income country with an estimated population of 46 million , a per capita income of around us$500 , and an average life expectancy of 51 .
more than three quarters of the population lives in rural areas and a third lives below the poverty line .
the infant mortality rate is around 51 per 1000 population , and there are approximately 3 doctors and 39 nurses and midwives per 100 000 population . in january 2010 , mnh opened tanzania 's first dedicated public emergency department via a public private partnership between tanzania 's ministry of health and social welfare ( mohsw ) , and abbott fund tanzania .
the mnh emd is fully staffed with physicians ( registrars have completed medical school and internship , whereas residents have worked as registrars and are now pursuing specialty training ) and nurses 24 hours per day and currently sees 100130 patients daily , or approximately 40 000 patients per year .
the department has full resuscitation capacity , and to add to the main hospital - operating suite a single - room operating theater dedicated to emergency cases has recently been opened within the emd . a business plan to ensure the department 's financial sustainability
has just been finalized through an agreement between the hospital , the ministry , and abbott fund tanzania .
nearly 40 per cent of mnh emd patients are female , 25 per cent are under 18 years of age , and 13 per cent are under 5 years of age ( table 1 ) .
approximately , one quarter of all patients and 20 per cent of pediatric patients present with injuries . in adults , intracranial injuries
are very common , and road traffic is a very common cause of injury in adults and children . other common diagnoses in adults and children include anemia and infections , particularly pneumonia in children .
mental illness is also a top diagnosis in adults , with the majority of these patients presenting with psychosis .
while the muhimbili emd project began as an infrastructure - development initiative , the necessity for a coordinated professional development strategy emerged early at the time the emd opened , there were no emergency physicians in tanzania and no dedicated emergency care nurse training programs .
abbott fund tanzania initiated and supports an academic collaboration among united states ( us ) , south african , and canadian institutions to provide clinical training for nurses and physicians , with the goal of ensuring long - term sustainability of the muhimbili emd and training leaders to promote , disseminate , and sustain emergency care throughout tanzania . via this program , the emd providers are supported by international emergency nursing and emergency physician faculty year round .
visiting faculty serve minimum 1-month stints , and there is an emergency medicine residency program director from the university of california san francisco onsite for the majority of the year ( author tr ) .
the collaboration has developed site - specific training materials for a 10-module nursing curriculum , a 1-year registrar program , and a 3-year residency program .
ninety per cent of nursing staff have completed the program ; 15 registrars have filed to complete the 1-year emergency care credentialing process ; and the first class of eight residents and second class of six residents will be joined by a third class this fall .
the first residency cohort will graduate in july 2013 , and leadership of the program will transition to local faculty by the end of 2014 .
the program at muhimbili is the nation 's only emergency medicine residency and is fully integrated into the muhimbili university of health and allied sciences ( muhas ) master of medicine ( mmed ) track .
the initial curriculum was developed and implemented in a collaboration between the mnh head of department and the us emergency medicine faculty serving as abbott fund tanzania consultants .
the curriculum was subsequently revised as part of a university - wide initiative to introduce a competency - based format . at the time of the curriculum development
, the international federation for emergency medicine model curriculum had not been released , but we have since conducted a curricular review to ensure that our curriculum meets its guidelines .
residents currently spend approximately half their time in the emd itself , and half on outside rotations in other clinical departments relevant to the practice of emergency medicine , such as surgery , obstetrics gynecology , cardiology , orthopedics , and pediatrics .
the competency - based curriculum guides evaluation and allows resident performance to be appraised with reference to context - oriented proficiency , rather than traditional , discipline - based evaluation .
this mode of targeted learning is particularly appropriate to the broad , setting specific practice of emergency medicine especially as a newly established field where the transmission of a culture of practice is as essential as the transmission of a body of scientific knowledge .
the program is monitored by a combination of resident examinations and other provider performance metrics .
each semester residents undergo a multidimensional evaluation including a written multiple - choice exam , a written essay exam , an oral case - based exam , and an observed clinical exam ( with case presentation ) on volunteer hospitalized patients .
exams are proctored by local faculty and international outside examiners approved by the university academic senate .
performance gaps are recorded and addressed in subsequent review sessions , and any gaps shared by multiple residents trigger review of the relevant component of the didactic and off - service curriculum .
in addition , all doctors , nurses , and health attendants undergo regular professional performance audits that evaluate on - time attendance , room - response times , and documentation quality .
the emd operations team is currently specifying metrics to evaluate throughput times , triage performance , and resuscitation process .
emergency residents have also agreed to participate in a 10-year tracking study , which currently surveys their experiences on off - service rotations , and will continue to track their practice sites and the disease burden they encounter over the next decade .
in general , medical trainees in tanzania face an extraordinary burden of disease and take on a degree of individual responsibility that rarely if ever falls on their us counterparts .
the ratio of patients to providers is daunting , and the ratio of faculty to residents in most specialties is simply not adequate to the supervision and training needs .
whereas this latter condition is mitigated to some extent by the visiting faculty in the muhimbili emergency program , other challenges result from the acuity of the specialty and its nascent status .
managing dynamically evolving , unstable patients is new to most trainees as their previous clinical training was often in settings with limited resources for intervening with critically ill patients ( who usually died ) . for now , emergency residents also face the challenge of being taught and supervised by faculty who have not been trained within the same system .
although they are guided by faculty in clinical decision making , ultimately the residents themselves are charged with developing the practice of regionally specific emergency care as they build their individual funds of knowledge .
they frequently serve as advisors and teachers for each other , and are often in the position of researching a condition as they treat it in a patient . on rotations in other departments , besides mastering the clinical content of that respective specialty , the emergency residents must serve as ambassadors and educate others as to the nature of their own specialty and their learning goals . as the first and only emergency physicians in the country ,
they are called into institutional and political service to a degree that only a handful of their international colleagues will experience much later in their careers . even in terms of the scientific advancement of the specialty ,
because the burden of acute disease is so poorly documented in the region , many of these dissertations will be the first , and for a time the definitive literature on the topic .
although the practice of emergency medicine has indeed evolved during the past 40 years into a coherent global discipline with a unique set of cognitive , technical , and administrative skills for managing acute illness or injuries , ' there remain notable differences in the scope of practice required of providers in low - income countries .
simple translation of curricular content from other regions is unlikely to prepare trainees for what they will face during training and after graduation .
acute care facilities serve to fill many gaps in the health systems of low - income countries , and providers at facilities distant from referral centers may be required to perform tasks considered beyond the scope of emergency specialists in other countries , such as basic obstetric , abdominal , and orthopedic surgeries . in this context ,
off - service rotations serve a different role than they might in areas where consultants are expected to be accessible within minutes to hours . as much as possible , informed by interviews with local providers who have worked in district and sub - district facilities , we have adapted the content of our curriculum to accommodate this reality ( either by emd - based or off - service training ) .
in addition , to ensure that our curriculum is appropriate to the local disease burden , we are currently analyzing diagnostic and demographic data from the first 2 years of the mnh emd , and the 2013 curriculum will be adjusted based on these results and other emerging regional data .
eventually , we hope that the results of our physician tracking study will also serve to inform future emergency care curricula at muhimbili and elsewhere .
in an effort to advocate for the dissemination of high - quality emergency care in tanzania , muhimbili providers formed the emergency medicine association of tanzania ( emat ) , the first emergency medicine professional organization in the region .
emat was ratified by the ministry in may of 2011 with the mandate to :
promote and improve emergency care through teaching , research , and education.advocate for and provide emergency care across tanzania.collaborate with other emergency care research and educational organizations , regardless of location , to the benefit of emergency care in tanzania .
advocate for and provide emergency care across tanzania . collaborate with other emergency care research and educational organizations , regardless of location , to the benefit of emergency care in tanzania . with the approval of the mohsw , a research team including the authors ( tr , hs , jm ) and other emat members
has just completed site visits to every district hospital in tanzania to assess emergency care capacity .
the results will serve to guide future emergency care planning and development , as well as prevention initiatives .
other ongoing emat projects include expanding the registrar training program to accommodate visiting stints by district hospital providers ; onsite training of district hospital providers in regionally appropriate basic and advanced resuscitation and trauma management skills ; the development of a transport medicine skills course ; and a series of needs analysis studies that will facilitate the development of coordinated pre - hospital emergency services .
in addition , emat aims to expand collaboration with other emergency provider training efforts in arusha , bugando , moshi , and zanzibar and has begun an exchange of site visits and planned collaborative training projects . to amplify and support its national efforts ,
emat has affiliated with the african federation for emergency medicine and the international federation for emergency medicine , and plans are underway to establish an east african emergency medicine federation that will allow resource - sharing and coordination of research and training efforts . the first east african regional meeting on emergency care
sub - saharan africa faces a disproportionate burden of acute disease and many challenges to meeting the call of world health assembly resolution 60.22 for formal integrated emergency care systems . the abbott fund tanzania - supported program at mnh in dar es salaam represents one effort to integrate a multi - cadre professional development strategy with the establishment of a high - volume emergency care facility .
the muhas - based residency will soon enroll its third class and will produce its first graduates in 2013 . their and others ' future efforts will benefit from greater national and regional coordination to allow multi - site research and surveillance and sharing of educational resources .
emat has been formed to help expand regional and global advocacy efforts in collaboration with the african and international federations for emergency medicine . | even though sub - saharan africa faces a disproportionate burden of acute injury and illness , few clinical facilities are configured to take an integrated approach to resuscitation and stabilization .
emergency care is a high - impact and cost - effective form of secondary prevention ; disease surveillance at facilities delivering acute and emergency care is essential to guide primary prevention .
barriers to emergency care implementation in the region include limited documentation of the acute disease burden , a lack of consensus on regionally appropriate metrics to facilitate impact evaluation , and the lack of coordinated advocacy for acute disease prevention and emergency care . despite these challenges ,
interest in creating dedicated acute care facilities and emergency training programs is rapidly expanding in africa .
we describe one such initiative at muhimbili national hospital in dar es salaam , with a focus on the development of the emergency medicine residency program . |
the notion of peripheral immune regulation by t cells that shut off other immune cells has been around for many decades ( gershon and kondo , 1971 ) .
, 1995 ; takahashi et al . , 1998 ) and a lineage - specific transcription factor ( hori et al . , 2003 ; walker et al
. , 2003 ) confirmed the existence of a distinct regulatory t cell ( treg ) population .
originally identified by their aptitude to hold off autoimmune reactions ( sakaguchi et al . , 1995 ; takahashi et al . , 1998 ;
treg own far - ranging suppressive activity affecting the function , maturation , and survival of all types of immune cells ( thornton and shevach , 1998 ; jonuleit et al . , 2001 ; grossman et al . , 2004 ; kojima et al . ,
2005 ) in response to self and non - self antigens , including pathogens ( belkaid et al . , 2002 ;
additionally , treg have been shown to confer regulatory properties upon suppressed t cells implementing a second layer of regulation ( jonuleit et al . , 2002 ; stassen et al . , 2004 ; andersson et al . , 2008 ) .
attempts to define the molecular basis of treg suppression have lead to the description of numerous putative pathways and molecules ( tang and bluestone , 2008 ; shevach , 2009 ) .
a majority of studies agreed on cell contact - dependent suppression by treg in vitro ( thornton and shevach , 1998 ; jonuleit et al . , 2001 ) and
the observation of persistent contacts between treg and dendritic cells ( dcs ) during active suppression by intravital microscopy suggests that cell contact - dependent suppression might also play a role in vivo ( tang et al . , 2006 ) . analyzing the molecular mechanism of contact - dependent treg suppression by comparison of gene expression in treg and non - regulatory t cells
, we found that treg up - regulate camp in their cytosol upon activation and consign camp to conventional cd4 t cells and dcs ( bopp et al .
2010 ) by gap junction intercellular communication ( gjic ; oviedo - orta et al . , 2000 ) . upon transfer camp
inhibits the proliferation and differentiation of responder cells , most probably through the induction of inducible camp early repressor ( icer ) expression ( foulkes et al . , 1991 ; bodor et al .
continuative work revealed that camp transmission is an essential component of treg - mediated suppression in vivo ( bopp et al .
dc interaction ( tang and bluestone , 2006 ) , transfer of treg - derived camp into conventional t cells in vivo was inevitably dependent on the presence of antigen presenting cells ( apc ) and restricted to the draining lymph node ( bopp et al . , 2007 ) .
correspondingly , repression of camp accumulation in treg either by adenylyl cyclase inhibition , application of a camp - specific antagonist or phosphodiesterase ( pde ) overexpression abrogated treg suppression ( bopp et al .
, 2007 ; oberle et al . , 2007 ; becker et al . , 2009 ; klein et al . , 2012 ; martin et al . ,
inversely , blockade of camp degradation by pde inhibition improved treg - mediated suppression in a murine asthma model ( bopp et al . , 2009 ) .
regarding camp regulation in treg , foxp3 has been shown to repress pde3b expression ( gavin et al . , 2006 ) thereby preventing camp degradation .
( 2009 ) showed that the high camp content in treg and their suppressive property depend on foxp3-mediated repression of the adenylyl cyclase 9 ( ac9 ) regulating mirna 142 - 3p . in line with these observations ,
2009 ) demonstrated that non - functional treg in foxp3 mutant scurfy mice harbor significantly reduced levels of cytosolic camp .
hence , the transcription factor foxp3 participates in camp buildup by concomitantly regulating the expression of camp - generating and degrading enzymes .
it is noteworthy that the transmission of camp is actually involved both in the suppression of other t cells ( bopp et al .
huang et al . , 2009 ; klein et al . , 2012 ) and in suppression of dcs ( fassbender et al . , 2010 ) .
together these findings classify camp as a key component of treg suppressive mechanism in vitro and in vivo and disclose camp - regulating enzymes as molecular targets for therapeutic intervention with treg activity in pathological processes like allergy and autoimmunity .
next to the transfer of camp through gap junctions , production of extracellular adenosine has been suggested as an alternative mechanism in camp - dependent suppression by treg ( deaglio et al . , 2007 ) .
extracellular nucleotides are anti - inflammatory mediators produced by a variety of cell types including treg ( deaglio et al . , 2007 ;
physiologically , extracellular nucleotide production represents a protective mechanism in response to tissue injury ( fredholm , 2007 ) . in treg suppression adenosine formation through the ectoenzymes cd39 and cd73 , expressed by murine treg and a subpopulation of human treg ( mandapathil et al . , 2009 ) , has been assumed to induce camp production in conventional t cells or dcs upon binding to the a2a receptor ( deaglio et al .
however , the role of adenosine as a major suppressive mechanism employed specifically by treg is questionable .
blockade of camp production in responder t cells by inhibition of adenylyl cyclases does not alter their susceptibility to treg - mediated suppression ( klein et al . , 2012 ) .
in addition , a2a receptor expression is detectable on t cells 4 days after stimulation ( deaglio et al . , 2007 ) while t cells are susceptible to treg suppression exclusively within the first 24 h after stimulation ( hagness et al . , 2012 ) . finally , blockage of ectonucleotidase activity only slightly abrogates suppression of human t cells by cd39 expressing treg ( mandapathil et al . , 2010 ) .
thus , while nucleotides certainly affect numerous cellular functions including de novo camp generation in treg their role in treg suppression is most likely of an indirect nature .
interestingly , camp up - regulation in treg coincides with another cell contact - dependent mechanism of suppression : treg constitutively express the two co - inhibitory membrane - bound molecules ctla-4 and tigit ( read et al . , 2000 ; takahashi et al . , 2000 ) which are believed to provide inhibitory signals . in mice ctla-4 deficiency ( bachmann et al .
, 1999 ) , ctla-4 blockade ( takahashi et al . , 2000 ) , and treg - specific ablation of ctla-4 ( wing et al
, ctla-4 deficient treg remain suppressive in vitro and in vivo ( tang et al .
, 2004 ; read et al . , 2006 ) suggesting additional mechanisms to be involved . studies on human treg in vitro revealed only a minor role of ctla-4 in treg suppression ( birebent et al . , 2004 ) or
firmly excluded ctla-4 as a suppressive mechanism ( baecher - allan et al . , 2001 ;
however , discrepancies regarding the importance of ctla-4 in treg suppression might in part be due to the use of different target cells . while the role of ctla-4 in suppression of t cells remains uncertain , it is unequivocally required in the suppression of apc .
suppression of dcs by treg via ctla-4 has been shown to induce the downregulation of cd80 and cd86 ( cederbom et al . , 2000 ) preventing effector t cell activation by the apc in vitro ( oderup et al . , 2006 ) and in vivo ( wing et al .
notably , elevated camp levels in t cells have been shown to increase ctla-4 expression ( vendetti et al . , 2006 ) and
camp and ctla-4 expression are simultaneously up - regulated in treg upon activation ( becker et al . , 2009 ) . while a majority of studies firmly excluded soluble factors in treg suppression in vitro ,
there is growing evidence that cytokines substantially add to the immune regulatory function of treg in vivo .
in particular , transforming growth factor- ( tgf- ) and il-10 seem to be indispensable for sustained tolerance induction by treg . a role for tgf- in maintenance of peripheral tolerance
was initially suggested by its importance in infectious tolerance ( chen et al . , 2003 )
particularly its long - lasting production by cd4 t cells from tolerant mice in long - term acceptance of allografts ( daley et al . , 2007 ) .
however , in order to exert its biological functions , tgf- needs to be converted from its latent ( bound to latency associated peptide , lap ) into its active conformation by proteolytic cleavage ( khalil , 1999 ) .
yet , there are multiple mechanisms of activating tgf- from its latency ( lawrence , 2001 ; annes et al . , 2003 ) and it is unclear how tgf- is activated in vivo . although repeatedly observed in disease models ( nakamura et al . , 2001 )
a direct contribution of tgf- in treg suppression remained controversial because anti - tgf- antibodies and soluble tgf - rii failed to affect the suppressive function of treg ( andersson et al . , 2008 ) .
glycoprotein a repetitions predominant ( garp ) expressed on the surface of treg upon activation ( wang et al .
2011 ) has been shown to act as a receptor for the tgf-/lap complex ( stockis et al . , 2009 ) .
stassen et al . , 2004 ) latent tgf- bound to garp on the surface of activated treg has been demonstrated to convert responder t cells into induced treg ( andersson et al . , 2008 ) .
thus , apart from acting as a soluble modulator of immune cells , tgf- supposedly helps treg to execute their contact - dependent suppressive activity by binding to garp ( battaglia and roncarolo , 2009 ) .
il-10 has been unequivocally shown to form another important mediator in treg suppression in vivo ( kearley et al . , 2005 ; collison et al . , 2007 )
particularly in suppression of pathogenic th17 cells ( chaudhry et al . , 2011 ; huber et al . ,
correspondingly , treg - specific ablation of il-10 leads to inflammation ( rubtsov et al . ,
in contrast to general treg deficiency , however , treg - specific il-10 paucity leads to mucosal but not systemic autoimmunity , suggesting mucosal restriction of il-10-mediated treg tolerance induction .
this view is supported by our previous observation that human treg expressing gut - homing 7 integrin preferentially induce il-10 production in converted secondary t helper suppressor cells ( stassen et al . , 2004 ) .
due to their far - ranging tolerizing capability treg have become key targets in the development of tolerance - inducing therapies ( wing and sakaguchi , 2010 ) . like other t cells ,
attempts to exploit treg for therapeutic purposes therefore depend on treg activation , either by antigen or polyclonal stimulation ( jordan et al . , 2001 ) .
current efforts to increase the frequency and potency of treg in vivo include the use of cytokines ( tawara et al . , 2010 ) ,
antigen targeting to immature dc ( mahnke et al . , 2003 ) , and monoclonal antibodies ( mab ) against surface molecules ( belghith et al . ,
are biased toward recognition of self - antigens ( hsieh et al . , 2004 ) , however , because antigenic specificities of treg in diseases have not been elucidated , potential clinical applications have mainly focused on polyclonal treg activation methods ( horwitz et al . ,
t cell surface molecules that participate in t cell receptor - mediated stimulation have a significant influence on t cell function .
mab against coreceptors have been successfully shown to allow intentional tolerance induction in rodent and non - human primate models ( krieger et al . , 1996 ) .
one particularly well - established regimen of tolerance induction is the administration of anti - cd4 mab ( waldmann and cobbold , 1998 ) .
although the mechanisms underlying tolerization by anti - cd4 mab are not yet fully understood , the activation of treg has been recognized as the entering wedge to successful tolerance induction ( becker et al . , 2009 ;
cd4 , a 55-kda glycoprotein with four extracellular domains ( littman , 1987 ) , recruits the protein kinase p56 ( rudd et al .
1988 ) to the tcr complex ( holdorf et al . , 2002 ; kim et al . , 2003
2010 ) and strengthens the contact between t cells and apcs through its interaction with non - polymorphic regions of mhc class ii molecules ( greenstein et al . , 1984 ; doyle and strominger , 1987 ; konig et al . , 1992 , 1995 ) .
cd4 molecules on t cell surface have been shown to preferentially form disulfide - linked dimers and tetramers ( 110 and 220 kda ; li et al . , 1998 ; moldovan et al . , 2002 ) and mutations disabling dimerization
cd4 expression on mature t cells is uniform with the exception of polarized t helper 2 cells ( itoh et al . , 2005 ) and treg ( bryl et al . , 2001 ) which both show decreased cd4 expression supposedly entailing altered proximal tcr signaling ( hannier et al . , 2002 ; itoh et al . , 2005
through its interaction with tyrosine kinase p56 , cd4 engagement alone can induce tcr - independent signaling events in t cells ( zhou and konig , 2003 ) .
selective engagement of the cd4 coreceptor by certain mab raises intracellular calcium and il-2 production ( carrel et al . , 1991 ) , whereas other anti - cd4 mab prime t helper cells to activation - dependent cell death triggered by subsequent tcr / cd3-mediated signals ( newell et al .
colleagues suggested that the gene - activating and proapoptotic potential of different anti - cd4 mab may be associated with different epitopes ( baldari et al .
, 1995 ; di somma et al . , 1995 ; milia et al . , 1997 )
however , a similar range of divergent responses can be induced through a single cd4 epitope as demonstrated for the cd4-binding ( lasky et al . , 1987 )
human immunodeficiency virus-1 ( hiv-1 ) envelope protein gp120 ( liegler and stites , 1994 ; westendorp et al .
it is therefore tempting to speculate that the functional outcome of cd4-stimulation might mainly depend on the functional state of the t cell addressed rather than on a specific cd4 epitope .
however , the functional state is believed to affect the formation of cd4 oligomers , which , in turn , regulate the activation of the cd4 cytoplasmic tail - associated tyrosine kinase p56 , by trans - phosphorylation ( veillette et al . , 1989 ) . even before the role of the cd4 molecule in t cell activation had been fully recognized , three groups reported that short courses of anti - cd4 mab application induce long - term tolerance to foreign proteins ( benjamin and waldmann , 1986 ; benjamin et al .
subsequent studies revealed that anti - cd4-mediated tolerance induction was not based on t cell depletion but rather an activation of regulatory mechanisms ( benjamin et al . , 1988 ; carteron et al . , 1988 , 1989 ; qin et al . , 1990 ) .
further , tolerance could not only be induced to foreign proteins but also to various transplanted allografts ( shizuru et al . , 1987 ;
qin et al . , 1989 ; davies et al . , 1996 ) , demonstrating that the tolerizing potential of anti - cd4 mab is not restricted to a particular type of antigen .
immunoregulatory mechanisms initially suggested to operate in anti - cd4 induced tolerance include a predisposure of developing t cells to selective deletion , or anergy in the thymus ( arima et al . , 1997 ) ;
immune deviation ( scully et al . , 1997 ) ; receptor blockade ( fehervari et al . , 2002 ; harding et al . , 2002 ) ; modulation of cd4 expression ( portoles et al . , 1999 ) ; and transmission of negative signals ( chirmule et al . , 1999 ) .
however , none of these not mutually exclusive processes could reasonably explain the infectious tolerance
rather than being submissive , anti - cd4 induced tolerance relied on dominant immune suppression by t cells activated in presence of the antibody . in regard to the dominant suppressive t cell type in charge
several functionally and phenotypically different anti - cd4 mab - induced tolerogenic cd4 t cell populations have been proposed ( bushell et al . , 2003 ; chen et al . , 2003 ;
however , whether these had been directly or indirectly induced by anti - cd4 treatment remained undefined at first .
the impressive capacity of treg and their ability to confer regulatory properties upon suppressed t cells ( jonuleit et al .
, 2002 ; stassen et al . , 2004 ; andersson et al . , 2008 ) in particular , strongly suggested a role of treg in anti - cd4-mediated infectious tolerance induction .
in support of this assumption administration of non - depleting anti - cd4 mab into mice had been shown to result in pre - activation of treg in vivo ( karim et al . , 2005 ; yang et al . , 2007 )
foxp3(hcd2 ) mice to ablate treg with an anti - hcd2 mab kendal et al .
( 2011 ) formally demonstrated that treg are crucial for infectious tolerance induced by non - ablative anti - t cell mab .
motivated by the description of activated treg in murine anti - cd4 tolerance models we previously analyzed the effect of cd4 binding agents on human treg .
comparing numerous anti - cd4 mab we found that certain anti - cd4 mab have the potential to induce the suppressive function of isolated human treg in a supposedly t cell receptor - independent manner ( becker et al .
in addition , we and others observed that the cd4-binding hiv-1 surface protein gp120 activates the suppressive function of treg ( nilsson et al .
, 2006 ; kinter et al . , 2007 ) in vitro and in two humanized mouse models in vivo ( becker et al . , 2009 ;
ji and cloyd , 2009 ) signifying that stimulation via the cd4 receptor represents an efficient treg activating pathway with potential to induce immunological tolerance in humans .
difference between anti - cd4 mab to trigger treg suppressive activity could not be related to a particular cd4 epitopes . however , comparing the treg activating potential of different anti - cd4 mab and cd4 binding virus envelopes we observed that one crucial event that separates treg activating and non - activating cd4 ligands consists in up - regulation of the second messenger camp ( becker et al . , 2009 and unpublished results ) . moreover
, the binding affinity of cd4 ligands seems to play a role as suggested by the fact that weak cd4 binding viral envelopes from hiv-2 ( gp105 ) and siv ( gp130 ) did not activate human treg in vitro and in vivo .
however , apart from these general observations the signaling events initiated by separate ligation of cd4 on treg so far remain unexplored . in particular , it is unclear whether cd4 stimulation of treg is truly independent of tcr signals , whether and how both pathways resemble or differ from another , and , most important , whether cd4-mediated signals are differently or similarly handled in treg and conventional cd4 t effector cells
. the latter question is of particular interest since treg are believed to maintain an activated phenotype through constant stimulation by self antigens , yet , require additional stimulation to become suppressive .
future insights into how tcr and cd4 signaling pathways drive the suppressive activity of treg will undoubtedly help to understand treg biology and discover alternative intervention points for functional manipulation of treg suppressive activity .
as summarized in figure 1 at least three different immune mechanisms can be distinguished that help to explain the tolerizing effect of cd4-specific agents : first , a general treg - independent mechanism that consist in interference with proper cd4 coreceptor function resulting in induction of t cell anergy or t cell depletion ( figure 1a ) .
this effect seems to depend either on cd4/mhc class ii binding blockade or additional tcr - independent signaling .
second , by modulating antigenic stimulation , individual cd4 mab induce differentiation of naive t cells into adaptive tregs ( oliveira et al . , 2008 ) , which are suggested to control pathogenic effectors through tgf- ( oliveira et al . , 2011 ) or il-10 release ( figure 1b )
finally , and crucially important for tolerance induction , cd4-specific mab activate the suppressive function of treg ( becker et al .
2011 ) , which , upon activation , exert control on pathogenic t cells by direct and linked suppression ( figure 1c ) .
these different effects of cd4 stimulation are intrinsic functions of individual anti - cd4 mab .
potential modes of tolerance induction by cd4-specific monoclonal antibodies.a short - term treatment with non - depleting cd4-specific mab induces dominant tolerance to foreign proteins and transplanted allografts .
this figure represents the different immune mechanisms that have been proposed to explain the tolerizing effect of cd4-specific mab .
three intervening points can be distinguished : ( a ) cd4 binding by non - depleting cd4-specific mab modulates antigenic stimulation through the t - cell receptor complex resulting in induction of t cell anergy .
( b ) by modulating antigenic stimulation , cd4-specific mab induce differentiation of naive t cells into adaptive regulatory t cells , which control pathogenic effectors through transforming growth factor- ( tgf- ) and il-10 release .
( c ) crucially important for tolerance induction , cd4-specific mab activate the ( camp - dependent ) suppressive function of treg , which , upon activation , exert control on pathogenic t cells by direct and linked suppression .
current immunosuppressive therapies are efficient in preventing acute transplant rejection and dampening inflammation in autoimmune diseases such as rheumatoid arthritis or lupus .
nevertheless , immune suppression remains inadequate , as it comprises significant side effects such as organ toxicity and hypersuppression disabling protective immune responses against pathogens and enhancing the risk of chronic infections .
hence , there is a clinical need for novel immunotherapeutic drugs with the ability to rebalance the immunologic tolerance network without persistently affecting immune function .
in contrast to pharmacological immune suppression , re - induction of tolerance through the exploitation of evolutionarily established tolerance mechanisms is expected to offer a parentally operative cure . among mechanisms
operative in self - tolerance , the immune - suppressive activity of treg appears to be exceptionally well suited for therapeutic exploitation for several reasons : first , activated treg dampen the function of a wide range of immune cells including t cells ( pandiyan et al . , 2007 ) , b cells ( lim et al . , 2005 ) ,
dc ( misra et al . , 2004 ; larmonier et al . , 2007 ) , and monocytes ( taams et al . , 2005 ) and affect a broad range of immune contexts including cardiovascular disease ( ait - oufella et al . , 2006 ) and obesity - induced insulin resistance ( feuerer et al . , 2009 ) .
second , the activation of treg is antigen - specific defined by the selected t cell receptor repertoire in the thymus .
however , once activated the suppressive mechanisms of treg operate in an antigen - non - specific manner , sidestepping the need to identify disease - specific antigens to affect a particular treg population .
prime examples of the treg immune dampening potential are experiments demonstrating that treg can be expanded and re - infused to limit immune responses ( hoffmann et al . , 2002 ) preventing gvhd induction without causing toxicity .
while persistent polyclonal treg activation would lead to general immune hyporesponsiveness , a short - term treg activation as established for tolerance induction with non - depleting anti - cd4 mab in mice is expected to induce ( or re - induce ) antigen - specific regulatory networks that maintain antigen - specific tolerance when treg activity has returned to normal levels .
based on the evidence for treg activation by cd4 ligands as outlined above , anti - cd4 mab seem to represent ideal compounds for treg - mediated tolerance induction . however , although animal studies have provided a compelling basis for clinical application of anti - cd4-mediated tolerance induction , this approach has been remarkably unsuccessful when transferred to the clinic .
although short interventions with particular mab have been shown to offer quick symptomatic relief , improvements supposedly caused by inactivation and depletion of cd4 t cells ( kon et al . , 2001 ; choy et al . ,
failure to establish an anti - cd4-based tolerogenic therapy in humans is most likely due to difficulties in translating the timing and dosage used in animal models for human application .
importantly , in contrast to animal models , mab are administrated at late disease stages in clinical studies .
whereas the immature immune system seems to dependably allow tolerance induction with anti - cd4 mab , it seems more difficult to tolerize the experienced immune system in patients , in part due to the presence of effector and memory t cells resistant to the suppressive action of treg ( yang et al . , 2007 ) .
in fact , treg - based therapies have been found to be generally less effective in models of autoimmune diseases .
( 2011 ) for example observed that functionally active treg failed to control hyperactivated t effector cells in rheumatoid arthritis patients with ongoing inflammation but prevented autoaggressive immune responses in non - inflammatory arthritis .
impaired treg suppression under inflammatory conditions has been mainly ascribed to the influence of tnf- , il-1 , and il-6 , which turn effector t cells resistant to treg - mediated suppression ( walker , 2009 ; goodman et al . , 2011 ) .
certainly , resistance to treg - mediated suppression can be overcome by blockade of il-6 ( chen et al . ,
2009 ) and supposedly , the beneficial effects of anti - tnf- treatment include a similar effect too ( ehrenstein et al . , 2004 ; valencia et al . , 2006 ) .
thus , provided treg can be sufficiently activated in the host , their suppressive efficiency might depend on the disease stage , which strongly argues for a combination of treg enhancing strategies with biologicals that reverse treg resistance in autoaggressive t effector cells . as exemplified with anti - cd3 mab already in the clinic evacuation of t effectors cells and concomitant enhancement of treg activity can form a very effective treatment ( chatenoud and bluestone , 2007 ) . with regard to anti - cd4-mediated tolerance induction in humans , it is important to emphasize again that anti - cd4 mab vary in their capacity to activate treg ( becker et al .
, 2007 ) and antibodies used in clinical trials so far have not been analyzed with regard to their treg activating potential .
however , clinical trials with treg enhancing agents such as the anti - cd4 mab tregalizumab in rheumatoid arthritis have been initiated to investigate the efficacy of treg - based anti - cd4-mediated tolerance induction in patients with autoimmune diseases .
in summary , polyclonal activation of treg through their surface molecules by biologicals that enhance their intracellular camp level are effective to induce the suppressive function of treg for re - induction of tolerance in small animal models and in humanized mice .
it is therefore expected that polyclonal treg activation forms a rational for tolerance induction in humans . however , both the exact conditions , efficiency in different stages of disease and cooperation with additional treatment regiments to diminish t effector cells need to be thoroughly explored
. moreover differential signals in treg versus t effector cells are far from being clear .
in addition to deepening our understanding of treg biology investigation of the latter holds the key to define alternative entry points for therapeutic manipulation of treg function .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | understanding tolerance mechanisms at the cellular and molecular level holds the promise to establish novel immune intervention therapies in patients with allergy or autoimmunity and to prevent transplant rejection .
administration of mab against the cd4 molecule has been found to be exceptionally well suited for intentional tolerance induction in rodent and non - human primate models as well as in humanized mouse models .
recent evidence demonstrated that regulatory t cells ( treg ) are directly activated by non - depleting cd4 ligands and suggests treg activation as a central mechanism in anti - cd4-mediated tolerance induction .
this review summarizes the current knowledge on the role of treg in peripheral tolerance , addresses the putative mechanisms of treg - mediated suppression and discusses the clinical potential of harnessing treg suppressive activity through cd4 stimulation . |
hepatitis c virus ( hcv ) infection is responsible for considerable morbidity and mortality worldwide .
moreover the hcv genotype has been shown to have an important role in clinical and histological features as well as the response to antiviral treatment .
most studies suggest that subtype 1b isolate possibly represent more aggressive variants of the virus and better responses to ifn are found in patients infected with either hcv genotypes 2 or 3 in comparison with those infected with genotypes 1 and 4 .
considering that approximately 1% of iran s population is infected
, chronic hepatitis c remains a serious medical problem with considerable burden on iran s health care system .
these observations have led us to determine hcv genotypes , particularly in the region of khuzestan province , southwest iran , which has no available data on hcv genotypes .
herein , we report the results of our cross - sectional analyses of hcv genotype distribution and its association with certain clinical features in patients with chronic hcv infection in khuzestan province conducted from september 2006 to september 2009 .
during a three year period ( between september 2006 and september 2009 ) , we conducted a cross - sectional study on 223 consecutive hcv positive individuals who referred to the ahwaz jundishapour university hospitals ( ajsuh ) and hepatitis clinic .
the presence of anti - hcv antibody was determined by an elisa test ( ortho hcv 3.0 diagnostics , raritan , nj , usa ) .
the presence of hcv rna in anti - hcv positive and indeterminate samples was detected by rt - pcr with the qualitative amplicor hcv test v.2.0 ( roche molecular systems , branchburg , nj , usa ) .
hcv genotyping / sub - typing was performed by two rt - pcr assays , restriction length polymorphism analysis ( rflp ) of the 5noncoding region ( 5ncr ) and nested pcr with type specific primers following primary rt - pcr with consensus primers designed for the core region of the hcv genome .
hcv genotyping by rflp was carried out using the restriction enzymes avaii and rsai on pcr - amplified from 5ncr as previously described .
type - specific pcr assay was accomplished by means of the nested pcr assay originally developed by okamoto et al . for sub typing genotypes 1 and 2 , and
sequences of the hcv core region were detected after synthesis of cdna and nested pcr with conserved primers covering positions 439 to 751 of the hcv genome .
rank - sum and kruskal - wallis tests compared continuous variables such as age between the groups . fisher exact test assessed associations .
, 1989 - 2002 , chicago , il , usa ) for windows in addition to univariate analysis and multivariate logistic regression .
all 223 patients had documented positive anti - hcv antibodies for at least six months .
there were 170 males ( mean age : 34.6 years ) and 53 females ( mean age : 49.1 years ) with an age range of 13 - 70 years .
samples comprised 42 patients with unknown risk factors and 179 with parenteral risk of infection [ 11 dialyzed , 10 hemophiliacs , 95 intravenous drug users ( ivdu ) , with tattoos , 54 polytransfused , and needle sticks in 2 cases ] .
possible routes of infection were recognized in 181 ( 81.2% ) patients . among these , patients with a history of ivdu , blood transfusion recipients , undergoing chronic hemodialysis , history of tattooing , accidental inoculation and non - marital sexual contact comprised 42.6% , 23.3 % , 4.93% , 3.15% , 0.9% and 0.9% of samples , respectively ( table 1 ) .
rflp identified hcv genotypes in 181 of 223 samples , whereas 42 cases could not be sub - typed with this method .
patients ages were analyzed as a categorical variable ( < 40 and > 40 years ) .
genotypic distribution was as follows : 1a ( 41.7% ) ; 1b ( 2.7% ) ; 2 ( 4.1% ) ; 3a ( 31.4% ) and 4 ( 1.8% ) .
there were 42 samples ( 18.84% ) not classified into any of the known hcv subtypes .
univariate analysis demonstrated an independent association between ivdu and genotypes 1 ( 60.7% 1a ) and 3a ( 30% ) .
statistical analysis did not show a difference in the frequency distribution of genotypes according to gender and age ( p > 0.56 ) .
in this study , 64 ( 28.7% ) of 223 patients received blood transfusions before 1995 , whereas 5 ( 2.25% ) of 64 patients were positive for hcv rna who received transfusions after 1995 .
hepatitis c infection is currently the most common cause of end - stage liver disease in many countries , however the whole population , prevalence is less than 1% in iran .
a comparison of hcv genome sequences from various geographical regions of the world has shown substantial heterogeneity of nucleotide sequences within several regions of the viral genome .
based on these genomic differences ,
six major genotypes with several subtypes have been identified and a nomenclature for these was given following a consensus proposal .
in the first report on iranian patients , zali et al . in tehran studied the prevalence of specific genotypes in 15 samples with the following results : type i /1a ( 7 ) , type ii/1b ( 3 ) , type v/3a ( 4 ) and type 4 ( 1 ) . a recently published article by samimi - rad et al . in iranian patients with anti - hcv ab positive from tehran and
five cities from different locations of iran showed a predominance of genotype 1a ( 47% ) , followed by 3a ( 36% ) , 1b ( 8% ) and 4 ( 7% ) , respectively .
the current study has shown that in khuzestan province , southwest iran , genotype 1a accounted for the majority of hcv infections ( 41.7% ) .
this is the first study of the genotype distribution in this area which suggests a pattern similar to that reported in other parts of iran , where most cases studied thus far belonged to genotype 1 .
however the results of our study differed from other middle - east countries such as the republic of yemen , kuwait , iraq and saudi arabia where genotype 4 has been identified as the most prevalent .
a study of saudi patients detected hcv genotype 4 in 50% of cases and genotype 1b in nearly 40% .
from other parts of the world , studies have revealed that genotype 3 is prevalent in south east asia whereas genotype 1 is common in the usa and western europe .
patients with risk factors for hcv infection enrolled in the present study were very few and therefore we were unable to reach any conclusion of genotype distribution within this group .
the distribution of the two most common hcv viral types ( 1a and 3a , ) were not statistically different in terms of mean age or gender .
the relative influence of hcv genotype , risk factors for infection , duration of infection , and host factors on progression of liver disease seemed to be important .
our study indicated that ivdu and blood transfusions were the leading risk factors for hcv acquisition ( 71.3% ) , of which ivdu was the most frequent risk factor ( 42.6 % , ) .
all hcv infected ivdu patients were male , which was consistent with other reports .
the route of hcv transmission mainly causes this epidemiological pattern in genotype distribution such that ivdu associated with genotype 1a and 3a is currently the primary risk factor of hcv infection .
screening for hcv is routine for all blood donors since 1995 in iran but 5 ( 2.25% ) out of 64 patients who received transfusions after 1995 were positive for hcv rna .
therefore , more careful pre transfusion screening of blood for anti - hcv must be introduced in our blood banks .
there were 42 cases ( 18.8% ) unable to be classified for hcv genotype by rflp technique .
this may be due to multiple infecting genotypes in these patients , difficulties in sample handling or unusual hcv genotypes in this patient group which could not be classified by this method .
we can conclude that genotype 1 , particularly the subtype 1a and genotype 3 , subtype 3a are predominate in this geographic part of iran .
knowledge on the distribution of various genotypes in our area is essential for its prognostic implications in chronic hepatitis c infection .
further investigations with larger sample numbers are necessary to determine the major genotypes that cause hcv infection in various clinical conditions and potential associations with disease severity .
this work was supported by the tropical and infection research center , ahwaz jundishapour university of medical sciences , ahwaz , iran .
we are grateful to the research deputy of ahwaz jundishapour university of medical sciences for his encouragement and constructive advice during the preparation of the manuscript .
we thank the subjects for their compliance in participating in this study , and danesh lab center for genetic studies , sequence alignment and immunoassays .
| background
hepatitis c virus ( hcv ) infection is responsible for considerable morbidity and mortality worldwide .
the hcv genotype has a geographic distribution and an important role in clinical and histological outcomes .
this study determined hcv genotypes and their related risk factors among patients from khuzestan province , southwest iran .
methods
in a cross - sectional study , 223 patients infected with hcv who referred to ahwaz jundishapour university hospitals ( ajsuh ) and hepatitis clinic were enrolled .
specific and nested polymerase chain reaction ( pcr ) and restriction fragment length polymorphisms ( rflps ) were performed to determine viral infection and genotype analysis .
liver enzymes including alt and ast and the correlated risk factors were also determined .
results
the hcv genotype distribution was as follows : genotype 1a ( 41.7% ) ; genotype 1b ( 2.7% ) ; genotype 2 ( 4.1% ) ; genotype 3a ( 31.4% ) ; and genotype 4 ( 1.8% ) .
there were 42 samples ( 18.84% ) not classified into any of the known hcv subtypes .
no patient was infected with more than one genotype .
hiv was found in four ( 1.8% ) cases , of which all were intravenous drug users .
univariate analysis demonstrated an independent association of intravenous drug use ( ivdu ) and genotypes 1a ( 60.7% ) and 3a ( 30% ) .
conclusion
our findings suggest that genotypes 1a and 3a are the most common ones among iranian patients with chronic hepatitis c infection in khuzestan province , southwest , iran . |
in the past few decades , there has been a tremendous growth in information and communication technology leading to easier access to any form of digital data including medical data .
medical data are those that are generated from medical information system ( mis ) , hospital information system ( his ) , radiology information system ( ris ) , and electronic patient records ( epr ) . most of the medical diagnosis is based on images from ct scans , x - rays , mri scans , mammography , and other forms of image modalities .
the time factor is very important to discover the abnormality issues in target images , especially in various cancer tumors such as the breast cancer .
researchers at the january 1997 consensus development conference presented data discussing the facts related to breast cancers detected by mammography .
their data showed that mammograms have more prognoses than cancers detected by other imaging modalities .
though the survival rate of a cancer patient is less , it makes a difference for a breast cancer patient . to increase the survival rate of breast cancer patients , mammography is used for earlier detection and treatment stages with reduced risk .
doctors ' advice and treatment are based on the data the doctor analyses from the mammogram .
nowadays , most of the patients prefer a second / third opinion on the medical analyses received from their doctor
. the second / third opinion may be from a doctor located at a different place through telemedicine .
telemedicine applications are those that provide for diagnostics , prescription , consulting , and sometimes conferences to telesurgery .
telemedicine plays an important role in today 's world , where the patient 's medical data is transmitted over the internet .
the medical data considered may be medical images ( mammogram ) which has to be shared or transmitted over internet .
this medical data is crucial data and is at the risk of unauthorized access or manipulation .
authenticating as well as protecting it is essential since critical judgment is done on medical images especially mammograms during breast cancer detection .
cryptography , steganography , and watermarking are such schemes that are used to protect digital data .
digital image watermarking is a technique of embedding a watermark ( say logo ) into the host image for dealing with security issues .
medical image watermarking deals with medical data authentication , ownership , security , source identification , and patient identification .
when dealing with epr , it is mandatory to consider confidentiality to access the information , availability of the data to be used when required , and reliability of the data .
these are maintained through security issues , namely , integrity , availability , authentication , and confidentiality nonrepudiation .
medical image watermarking provides for security at the origin reducing piracy of crucial medical data and also protects medical documents or images that can be illegally modified .
part of the medical information , the region of interest ( roi ) , is crucial and must not be altered .
several watermarking schemes exist , and these schemes adopt both additive and multiplicative approaches in time domain .
these schemes adopted ss ( spread spectrum ) , dct ( discrete cosine transform ) , dft ( discrete fourier transform ) , dwt ( discrete wavelet transform ) , svd ( singular value decomposition ) , ridgelets , and contourlets in frequency domain .
proposed a watermarking scheme to verify the integrity and authenticity of ct scan images . here
have identified three different schemes to watermark a medical image : embedding the information within the non - region of interest ( nroi ) : this method generally places the watermark in the gray portions of the image .
, this method may seem too annoying to the physician.these schemes deal with reversible watermarking and were better suited for integrity control and data hiding .these methods normally minimize distortion .
have proposed a method where the physician selects the maximum power of the watermark under the level of interference with diagnosis .
wakatani proposed a scheme wherein the watermark was embedded in the nroi ( non - region of interest ) by adopting dwt .
medical image watermarking schemes must consider computational complexities which may lead to time delays for the physician and are still in the early stages of development .
it is difficult to evaluate watermark interferences with diagnosis . embedding the information within the non - region of interest ( nroi ) : this method generally places the watermark in the gray portions of the image .
during application of salt and pepper noise , this method may seem too annoying to the physician .
these schemes deal with reversible watermarking and were better suited for integrity control and data hiding .
this was achieved by replacing some of the image details . in this paper , a robust medical image watermarking scheme using fractional wavelets is proposed .
the proposed scheme is divided into two stages , namely , the embedding stage and the extraction stage .
frft ( fractional fourier transform ) has good reconstruction capability compared to fft ( fast fourier transform ) . when frft is combined with wpt ( wave packet transform ) , it has the capability to retain the coefficients after attacks .
firstly , arnold transform is applied to the host image and the host image is scrambled .
secondly , the value of the transform order is used as the key . according to this key
thirdly , the position of all frequency subbands is changed at each level using some secret rule known only to the user or creator .
quality of the extracted watermark to the original watermark is directly proportional to degradation of the image quality .
sections 2 and 3 describe fractional wave packet transform and arnold transform , respectively .
we conclude the work presenting the future enhancements to obtain more robust medical image watermarking schemes .
based on the idea of fractional fourier transform ( frft ) and wave packet transform ( wpt ) , fractional wave packet transform ( frwpt ) was introduced by huang and suter .
mathematically , for an input signal x(t ) represented along the time axis and its fourier transform represented along the frequency axis , frft , f(t , u ) , of the input signal , x(t ) , is given by(1)ft , u=+ct , uxtdt , where c(t , u ) is the fourier transformation kernel .
the wave packet transform ( wpt ) is a combination of short time fourier transform ( stft ) and continuous wavelet transform ( cwt ) .
wpt ( w ) of an input signal , x(t ) , is given by(2)w=12+expjuttxtdt.wpt can also be defined as the ft of a signal windowed by a wavelet , dilated by and translated by . frwpt , w(u , a , b ) , for a given input signal x(t ) can be defined as(3)wu , a , b=1a+ctbaxtdt.the frwpt is a function of time , frequency , and scale .
the computation of frwpt corresponds to the following steps as explained by huang and suter : a product by a wavelet.a product by a chirp.a fourier transform.another product by a chirp.a product by a complex amplitude factor.for computational purposes , a simple implementation of frwpt is used as shown in figure 1 . a product by a wavelet . a product by a chirp .
the computational algorithm can be described as follows:(1)the transform order of is used for the frwpt .
if frwpt is applied to the original signal , and the transformed signal is reconstructed from inverse frwpt , then the transform order is that value which results in minimum mean square error between the original and the reconstructed signal .
the process may sometimes be long and cumbersome due to its trial and error approach , but it is limited only to once per signal.(2)frft is performed on the input signal , with user defined transform order , followed by wavelet packet transform .
this phase is the decomposition phase.(3)inverse wavelet packet transform and inverse frft are performed on the transformed signal with transform order . this phase is the reconstruction phase .
if frwpt is applied to the original signal , and the transformed signal is reconstructed from inverse frwpt , then the transform order is that value which results in minimum mean square error between the original and the reconstructed signal .
the process may sometimes be long and cumbersome due to its trial and error approach , but it is limited only to once per signal .
frft is performed on the input signal , with user defined transform order , followed by wavelet packet transform .
inverse wavelet packet transform and inverse frft are performed on the transformed signal with transform order . this phase is the reconstruction phase .
to further improve the security and improve the proposed watermarking scheme , the host image is preprocessed ( scrambled ) by applying arnold transform .
consider an image of size n n ; the arnold transform is defined by ( 4)xy=11121112xymodn , where x , y are the coordinates of the image and x , y are the coordinates after scrambling .
the proposed scheme can be divided into two phases : the embedding phase and the extraction phase . in the embedding phase , the host image is converted to a reference image and the watermark is embedded on the reference image . during the extraction phase ,
the watermark is first extracted and then the host image is obtained from the reference image .
the embedded watermark is smaller than the host image by a factor raised to the power of 2 along both the directions .
the proposed embedding and extraction scheme is shown in figures 2 and 3 , respectively .
assuming that i(x , y ) represents the original image of size p q , w(x , y ) represents the watermark of size p q ; the embedding and the extraction phase can be detailed as follows .
the proposed watermarking embedding process can be outlined as follows:(1)scramble the host image by applying arnold transform.(2)apply l - level fractional wave packet transform to the arnold transformed image .
the subbands obtained are hh , hl , lh , and ll.(3)change the position of the subbands .
the pattern to swap / change the image subband is defined by the creator.(4)apply l - level inverse fractional wavelet transform to obtain the reference image iref(x , y ) and then divide the reference image into subblocks.(5)apply watermark to the subblocks to obtain the modified reference watermarked image .
the modified subblocks are represented by(5)wref = irefx , y+wp , q , where denotes the strength of the watermark .
integrate the subblocks to obtain the reference watermarked image , wref(x , y).(6)perform l - level fractional wavelet transform.(7)change the position of subbands .
perform l - level inverse fractional wavelet transform.(8)apply arnold transform to scramble back the image to obtain the watermarked image , w(x , y ) .
apply l - level inverse fractional wavelet transform to obtain the reference image iref(x , y ) and then divide the reference image into subblocks .
the modified subblocks are represented by(5)wref = irefx , y+wp , q , where denotes the strength of the watermark .
apply arnold transform to scramble back the image to obtain the watermarked image , w(x , y ) .
the watermark extraction process can be outlined as follows:(1)apply arnold transform on the watermarked image w(x , y).(2)perform l - level fractional wavelet transform on the arnold transformed image.(3)change the positions of all the subbands.(4)perform l - level inverse fractional wavelet transform to obtain the watermarked reference image , wref(x , y).(5)extract the watermark , w(x , y).(6)perform l - level fractional wavelet transform on the watermarked image , w(x , y).(7)change the positions of all the subbands.(8)perform l - level inverse fractional wavelet transform to obtain the host image , i(x , y ) .
perform l - level inverse fractional wavelet transform to obtain the watermarked reference image , wref(x , y ) .
perform l - level fractional wavelet transform on the watermarked image , w(x , y ) .
the watermark used is the bmw logo ( any grey scale image can be used ) ; reference image is obtained using 2-level decomposition of frwpt . since 2-level decomposition gives a block size of 64 64 , the watermark is embedded 16 times into the modified reference image to get the watermarked reference image .
table 1 shows the values of psnr and ssim values of the watermarked image with reference to the original image without any attack .
the watermarked image is subjected to common attacks to investigate the robustness of the proposed scheme .
the attacks considered are average and median filtering , gaussian noise , salt and pepper noise , cropping , resizing , rotation , and sharpening attacks .
the performance of the watermarking algorithm under these attacks is tabulated in tables 2 and 3 .
the extracted watermark quality is also degraded . when the image is subjected to weiner filtering the image quality
this is due to addition of noise which degrades the quality of the image and hence the watermark extracted is degraded .
similar effects are also seen when gaussian noise and salt and pepper noise are added .
cropping is done by deleting certain number of rows and columns . since a large part of the image
was cropped ( 200 200 pixels towards upper left corner of the image ) , most of the medical information was removed . but this can be further modified to crop only the non - region of interest , preserving important medical data .
the image was rotated by an angle of 5 degrees towards the left and right .
the proposed algorithm was also tested for sharpening and contrast adjustments and results show that the proposed scheme could withstand these results .
graphical analysis of the proposed scheme under various common attacks discussed above is depicted in figures 6 and 7 .
the graph shows the relation between psnr and correlation coefficient for different values of , where denotes the strength of the watermark .
it is observed that the proposed watermarking scheme is not image variant and maintains a linear relationship for most of the mammogram images used .
in this work , a robust nonblind medical image watermarking scheme for mammograms was proposed applying frwpt .
experimental results show that the scheme is robust to common attacks like the compression attacks , interference attacks , and signal processing attacks .
it is vital to adopt imperceptible watermarks to ensure security issues of integrity and authenticity . | exchanging of medical data requires efficient authentication and protection of medical data that can be illegally modified .
watermarking plays an important role in protecting , sharing , and securing medical data . in this work
, a robust nonblind medical image watermarking scheme is proposed .
the process involves two steps : the embedding and the extraction phase . during the embedding phase
, l - level frwpt is performed on the host image and the watermark is embedded into the modified reference image . in the second phase , inverse frwpt is performed on the watermarked image to extract the watermark from the watermarked image .
the proposed scheme is tested on mammograms images and is subjected to common attacks like gaussian filtering , median filtering , compression , sharpening , and contrast adjustments .
experimental results show that the proposed scheme is robust . |
septic shock is consistently the most common causative factor identified for acute kidney injury ( aki ) in critical illness , and has been associated with nearly 50% of cases internationally . despite advances in our understanding of the pathophysiology of septic aki ,
treatment aimed at reversing or preventing septic aki remains primarily based on supportive haemodynamic management . in the previous issue of critical care , legrand and colleagues examine the association between haemodynamic targets of resuscitation ( cardiac output , mean or diastolic blood pressure , central venous pressure ( cvp ) and central venous oxygen saturation ) and development or persistence of aki in a single centre study of patients with septic shock admitted to a surgical icu .
the authors found that only higher cvp and low diastolic blood pressure were associated with increased risk of development of new aki , or persistence of renal dysfunction present at icu admission . in these patients
importantly , the association between cvp and aki remained when potentially confounding effects of positive fluid balance and higher positive end - expiratory pressure were accounted for in a multivariable analysis .
so , a 5 mmhg increase in cvp predicted 2.7-fold odds of new or persistent aki .
furthermore , when the association between different levels of mean cvp and aki was examined in a non - parametric logistic regression , there was a trend for higher cvp to be associated with worse renal outcome for all levels of cvp from 4 mmhg upward , so that a cvp of 15 mmhg was associated with an approximately 80% risk of new or persistent aki , compared to approximately 30% at a cvp of 6 mmhg .
these findings are important because current surviving sepsis campaign guidelines recommend fluid resuscitation of patients with sepsis - induced tissue hypoperfusion to target a cvp of 8 to 12 mmhg ( or 12 to 15 mmhg in mechanically ventilated patients ) within 6 hours of presentation . in patients with sepsis - induced hypotension
the rationale for cvp targeted fluid resuscitation is to ensure ' adequate cardiac preload and hence maintain cardiac output and organ perfusion .
however , absolute levels or changes in cvp poorly predict cardiovascular response as sepsis - induced hypotension is multi - factorial , related to changes in myocardial performance , vascular tone , regional blood flow distribution , venous reservoir capacity and capillary permeability . in contrast
, elevated cvp will cause a direct and predictable increase of renal venous pressure that , experimentally , has been associated with elevated renal interstitial and intra - tubular pressure .
resultant renal venous congestion can reduce renal blood flow ( lower trans - renal pressure gradient ) and decrease the pressure gradient for ultrafiltration ( higher intra - tubular pressure ) . in septic shock ,
. aggressive fluid resuscitation beyond reversal of hypovolaemia to arbitrary cvp targets could result in increased venous congestion without substantial benefit to forward renal perfusion , thus predisposing to renal dysfunction .
the association between elevated cvp and renal dysfunction has been made previously in the setting of chronic cardiac failure .
now legrand and colleagues have provided evidence that such an association is observed in septic shock and its treatment .
their findings are also in accord with secondary analysis of the vasopressin in septic shock trial , which reported that a more positive fluid balance and elevated cvp were associated with increased mortality in patients with septic shock even when accounting for differences in baseline illness severity and demographics .
although these observational findings are hypothesis - generating rather than confirmatory , they add weight to the literature suggesting the adverse effects of fluid overload in critical illness and aki in particular , effects that may be mediated in part by venous congestion causing impaired organ function .
it is now important to determine whether uncritical pursuit of cvp targets such as those advocated by the surviving sepsis campaign might , in some patients , add insult to sepsis - induced organ injury .
prospective data are required to evaluate the true contribution of raised venous pressure to renal and other organ dysfunction and to determine better endpoints for fluid resuscitation in sepsis .
rajkumar rajendram bsc mbbs mrcp frca fficm , specialty registrar in anaesthesia , royal free hospital , london .
john r prowle ma msc md mrcp fficm , consultant in renal medicine and intensive care , the royal london hospital , london . | in critical illness , septic shock is a contributing factor in nearly half of all cases of acute kidney injury ( aki ) .
traditional approaches to prevention of organ dysfunction in early sepsis have focused on prevention of hypoperfusion by optimisation of systemic haemodynamics , primarily by fluid resuscitation .
fluid administration to a target central venous pressure ( cvp ) of 8 to 12 mmhg ( 12 to 15 mmhg in mechanically ventilated patients ) is currently recommended for the early resuscitation of septic shock .
however , in the previous issue of critical care , legrand and colleagues report that higher cvp in the first 24 hours of icu admission with septic shock was associated with increased risk for development or persistence of aki over the next 5 days .
this study highlights a potential association between venous congestion and the development of septic aki , suggesting that cvp - targeted fluid resuscitation in septic shock might not be beneficial for renal function . |
in modern endodontic practice , the number of indications for endodontic periapical surgery is decreasing . still periapical surgery accounts for 3% to 10% of typical endodontic practice .
this case report has been presented to emphasize that all nonvital teeth with periapical lesion should be first treated conservatively , and the treatment mode is to be changed to surgical therapy only when conventional treatment proves ineffective .
oztan and kalaskar et al . have confirmed that large periapical lesions can respond favorably to nonsurgical treatment using calcium hydroxide paste .
cohn proposed periapical surgery as a predictable option when root canal treatment is either not possible or fails .
thus surgical endodontic treatment is indicated in limited number of cases of persistent periradicular pathosis .
selection between alternative treatments should be based on assessment of individual case , as done in the present case report .
a meticulous removal of periapical granulation tissue is an important aspect of surgical endodontic treatment .
removal of very large anterior cysts in maxilla may produce oro - nasal communication in cases wherein the cyst has destroyed the bony floor of the nasal cavity , with only soft tissue lining left in between .
an innovative method of surgical removal of the periapical lesion to save teeth with periapical pathosis refractory to conventional treatment and to preserve the adjacent vital structures has been introduced .
the large through and through periapical pathosis with both buccal and palatal bone loss is managed by periapical surgery adopting a novel method of separation of lesion after nonresponsiveness of the pathosis to intracanal medicament and of the smaller periapical pathosis to long - term intracanal medicament . to the best of our knowledge ,
a 19-year - old female patient was referred to the department of conservative dentistry and endodontics with the chief complaint of pain and recurrent swelling in the upper front teeth region since 10 to 12 weeks .
medical history and investigations did not reveal anything that could oppose or influence the proposed treatment plan .
on clinical examination , labial and palatal swelling was seen in association with maxillary right central incisor ( tooth no .
maxillary right and left central incisors ( tooth no 11 and 21 ) were discolored and fractured .
11 , 12 , 13 and 21 gave a negative response with the electrical pulp tester ( digitest , farmingdale , ny ) .
the maxillary occlusal radiograph showed a large radiolucent periapical lesion in association with maxillary right anterior teeth and a relatively small radiolucent periapical lesion in association with maxillary left anterior tooth [ figure 1 ] .
preoperative radiograph based on the history , clinical examination , sensitivity test and radiographic examination , a diagnosis of pulp necrosis with symptomatic periapical pathosis was made for the right maxillary central incisor , lateral incisor and canine .
a decision of conventional root canal treatment with the placement of intracanal medicament was made after discussion with the patient .
the operative field was isolated , and access preparation was done in 11 , 12 , 13 and 21 without local anesthesia . after the radiographic determination of the working length , biomechanical preparation following step back method was done using 2.6% sodium hypochlorite and normal saline as an irrigating agent .
iodine containing calcium hydroxide ( vitapex , neo dental chemical products , tokyo , japan ) was placed as intracanal medicament , and the root canal access was sealed with zinc oxide eugenol cement at the end of the first appointment . after 21 days , the patient reported with no relief from her symptoms .
there was no resolution of the buccal - palatal swelling , and tenderness to percussion continued to be present in 11 , 12 and 13 ; but tooth 21 became asymptomatic .
teeth 11 , 12 and 13 were obturated with zinc oxide eugenol sealer and gutta - percha using the lateral condensation technique .
assessment of the surgical site showed adequate mouth opening , little attached gingiva and adequate depth of the vestibule .
local anesthesia of 2% lignocaine with 1:80,000 adrenaline was infiltrated in the alveolar buccal mucosa superficial to the periosteum at the level of root apices from 13 to 21 to get added advantage of less surgical bleeding .
the buccal full - thickness triangular mucoperiosteal flap was elevated to expose the area of periapical lesion . already existing pathological cortical bone window
was expanded until underlying pathology was adequately exposed and sufficient space was available for thorough curettage .
sterilized gauze was placed between the pathological soft tissue mass and the lateral wall of the crypt , and lateral pressure was applied on the gauze with the concave surface of the curette facing the bone .
this helped in separation of the periapical lesion from the surrounding bone and the nasal mucosal and palatal linings [ figure 2 ] .
the dental assistant supported the palatal lining from inside the oral cavity with his finger while the cystic lining was being separated from the palatal lining .
the periodontal curette was used to remove the tissue firmly attached to the root surface [ figure 3 ] .
caution was exercised in separating the lesion from the roof because no intervening bone was present between the nasal lining and the lesion .
however , the lesion was curetted in toto , leaving the nasal and the palatal linings intact .
sterilized gauze placed between the soft tissue mass and the lateral wall of the crypt from the distal aspect with the curette after separation of the periapical lesion from the surrounding bone and the nasal and palatal linings but attached to the root surface the enucleated periapical lesion was stored in 10% buffered formalin solution and sent for histopathological examination .
the root resection was not done to maintain maximum cemental covering on the root surface and to maintain the original root length to help tooth stability .
the bone cavity was irrigated with sterile normal saline and gently dried with moist gauze .
careful clinical and radiographic inspection of the area was done to ensure no residual lesion tissue was left behind .
the flap was repositioned and the flap margins were ensured to rest on sound bone .
on the 4 postoperative day , healing of covering mucosa with primary intention was observed and the sutures were removed .
the patient was free from all symptoms and signs with optimum tissue healing at the 6-month follow - up visit .
tooth 21 was obturated with zinc oxide eugenol sealer and gutta - percha using lateral condensation technique .
a radiographic review at the 1-year follow - up showed good evidence of bone healing bilaterally [ figure 4 ] .
conservative approach of treatment of any lesion is always preferable over surgery as most periapical lesions are the result of an inflammatory response to bacterial infection within the root canal , i.e. , intracanal infection .
nonsurgical root canal treatment involves cleaning and disinfecting the root canal system , thereby reducing the bacterial load and creating an environment in which periapical healing can occur .
this has limited periapical surgery in contemporary dental practice to very selective cases those in which the causative factors are located outside the root canal , such as microorganisms colonizing the periapical tissues , cysts and foreign body reactions . to prevent tooth loss , surgical solution for the inflammatory periapical lesions
was considered in the present case as right maxillary anterior region did not respond favorably to conservative treatment .
nonsurgical root canal treatment using calcium hydroxide as intracanal medication was attempted on both sides , emphasizing conservative treatment , expecting that the long - term intracanal medicament will be effective in resolving the periapical lesions .
follow - up after 21 days revealed , left tooth was asymptomatic but the teeth in the right region were symptomatic .
treatment for the right side was changed to surgical intervention according to the clinical need .
intracanal medicaments are advocated to eliminate remaining bacteria after chemomechanical instrumentation , even in the inaccessible areas of the root canal system , reduces inflammation of the periapical tissues , dissolves remaining organic material and counteracts coronal microleakage . in the case
presented here , calcium hydroxide intracanal medicament created a more favorable environment leading to healing of periapical lesion in the left central incisor .
the periapical lesion on the right side , however , did not respond to the intracanal medicament because of the causative factor being located beyond the root canal system , viz .
. conventional method of removing soft tissue from the bony crypt consists of using curettes .
curettes are initially used with the concave surface facing the internal wall of the osseous crypt to separate the soft tissue lesion from the lateral borders of the crypt and subsequently used in a scraping manner to remove the remainder of the lesion from the osseous defects .
curette being sharp margined , there always remains a risk of penetration of the vital anatomic structures or pathological soft tissue mass , which will make the removal of the tissue mass difficult and time consuming .
the surgical technique is a fundamental consideration , since it largely conditions the prognosis of periapical surgery .
a novel method of separation of lesion was adopted in this case with large periapical lesion to prevent injury to other surrounding vital anatomic structures .
caution was required in separating the lesion from the roof because no intervening bone was present between the nasal lining and the lesion .
application of gentle lateral pressure on the sterilized gauze placed between the pathological soft tissue mass and the lateral wall of the crypt with the concave surface of the curette facing the bone to free the soft tissue in the present case helped in the removal of the entire tissue mass without injuring the nasal mucosa and palatal lining .
the present method has the advantage of more predictable removal of the soft tissue lesion in toto because of the blunt technique used , compared to using sharp curettes with higher chances of penetration of the soft tissue lesion making the removal of the remaining tissue tags difficult and more time consuming . at the same time , the present method results in greater safety of the approximating vital and vulnerable anatomical tissues , resulting in better postoperative comfort and outcome for the patient . at the 6-month and 1-year postoperative recall examination ,
the teeth were asymptomatic and radiographically showed signs of healing of periapical lesions . evaluating success of the periapical surgery based on clinical and radiographic criteria of von arx and kurt
it can be concluded that endodontic treatment with calcium hydroxide as intracanal medicament might be a viable approach for promoting periapical healing and success in root canal treatment in nonvital teeth associated with periapical lesion .
however , periapical surgery may be the only alternative when the tooth with periapical lesion fails to respond to calcium hydroxide as intracanal medicament . to prevent injury to other vital anatomic structures ,
a novel method of surgically treating large periapical lesions has also been introduced in the present case report . | conventional nonsurgical endodontic treatment has a high degree of clinical success , but in certain cases surgical intervention becomes necessary .
this is the case report of a patient presenting with bilaterally separate periapical lesions in the maxillary anterior region .
one lesion was managed by periapical surgery using a novel and safe method of separating the soft tissue mass of the periapical lesion which was in proximity to vital and vulnerable anatomical tissues , and the other was managed by long - term intracanal medicament , emphasizing the need of application of treatment in the best interest of the patient . |
it has been well established that patients with end - stage renal disease ( esrd ) undergoing hemodialysis ( hd ) had a higher association of peripheral arterial disease ( pad ) , lower extremity amputation ( lea ) , and foot ulcerations ( fu ) [ 13 ] . such an association could be attributed to a common atherosclerotic cause . moreover ,
diabetic patients with esrd had a 10-fold increased risk of lea in comparison to diabetes patients without renal insufficiency .
several studies have reported increased rates of lea among esrd patients , irrespective of the concomitant presence of diabetes mellitus ( dm ) [ 46 ] .
the mechanism of fu development is multifactorial which depends upon various physiological , mechanical , and treatment factors .
reported significantly higher incidence of foot complications in patients with concomitant esrd and dm in comparison to those who had only dm suggesting a possible relationship between esrd and fu .
in addition , lea and fu are well established complication of diabetic nephropathy [ 8 , 9 ] .
margolis et al . observed a strong correlation between progressions of chronic kidney disease ( ckd ) with the development of diabetic fu .
game et al . also demonstrated a close association of fu and amputations in diabetic patients started to undergo dialysis .
performed a meta - analysis to look for the relationship of diabetic fu with cardiovascular disease ( cvd ) and all - cause mortality .
the authors found an increased association of cvd and mortality in diabetic patients with fu in comparison to those without fu .
interestingly , a recent study advocated a higher incidence of risk factors ( pad and peripheral neuropathy ) for fu among hd patients . in qatar , an earlier report from our center identified a high incidence of pad in hd patients .
however , there is a lack of information that describes the risk factors and prognostic implications of fu among hd patients in the arab middle east .
we aim to evaluate this association and its impact on the outcomes over a 5-year period .
we conducted a retrospective analysis of all hemodialysis patients enrolled at the hd unit at hamad general hospital ( hgh ) , qatar , over five years ( 20072012 ) duration .
patients surviving for at least 3 months on the initial hd were included in the study .
hd patients were categorized into two groups according to whether they had foot ulcer ( fu ) or not ( no - fu ) based on physical examination .
the attending physician in the hd unit collected the data regarding the demographics characteristics , clinical evaluation , medical history , and comorbidities . during the follow - up period over 5 years , hd - related procedures such as vascular access ( arteriovenous fistula , arteriovenous grafting , and tunneled catheter ) , renal transplantation , and vascular complication were also reported .
the need for 3 vascular accesses ( due to mechanical obstruction , poor flow , or infection ) had been analyzed .
we speculated that hd patients with fu had poor outcomes compared to no - fu patients .
the study end - points ( major amputation , vascular complications , and mortality ) were also subanalyzed according to the presence or absence of dm and pad .
fu was defined according to the clinical findings as a full thickness skin break below the level of malleoli .
patients were considered to have pad if they had one of the following criteria : abi < 0.9 , history of intermittent claudication , vascular bypass or endovascular intervention for occlusive vascular disease , or amputation due to occlusive vascular disease .
major amputations were defined as amputations proximal to the ankle joint , and minor amputations were defined as those through or distal to the ankle joint .
diabetic nephropathy was diagnosed during patient follow - up in the nephrology out - patient clinic according to the patient chart .
it was defined as presence of macroalbuminuria that is a urinary albumin excretion of more than 300 mg in a 24-hour collection or macroalbuminuria and abnormal renal function as represented by an abnormality in serum creatinine , calculated creatinine clearance , or glomerular filtration rate .
analyses were conducted using the student 's t - test for continuous variables and pearson chi - square ( ) test for categorical variables .
multivariate logistic regression analysis was performed for predictors of development of fu and mortality . adjusted odds ratios ( or ) , 95% ci , and p values
a significant difference was considered when the two - tailed p value was < 0.05 .
data analysis was carried out using the statistical package for social sciences version 18 ( spss inc . ,
the study has been approved by the medical research center , hamad medical corporation , qatar ( irb # 12007/12 ) .
a cohort of 252 hd patients was included in the study , of which 42 had fu ( 17% ) and 210 were without fu .
demographic , clinical characteristics , and risk factors for hd patients with and without fu were shown in table 1 and figure 1 .
patients with fu were 6 years older and had higher incidence of retinopathy ( 67% versus 40.5% ; p = 0.002 ) , polyvascular disease ( 26.2% versus 6.7% ; p = 0.001 ) , angina ( 24% versus 11% ; p = 0.02 ) , pad ( 71% versus 32% ; p = 0.001 ) , and nephropathy ( 79% versus 42% ; p = 0.001 ) compared to no - fu patients .
also , a high percent of patients needed tunneled catheters ( 88% versus 82% ; p = 0.03 ) and aspirin ( 62% versus 45% ; p = 0.04 ) in the fu group , whereas , history of renal transplant ( 14% versus 2.4% ; p = 0.03 ) was observed more in no - fu group .
patients in fu group also had higher percentage of dm ( 83.3% versus 56% ; p = 0.001 ) , major amputation ( 26% versus 1% ; p = 0.001 ) , need for 3 vascular accesses ( 69% versus 47% ; p = 0.01 ) , and coronary artery disease
the two groups were comparable regarding the mean baseline laboratory investigations ( table 2 ) .
similarly , the percentage of dyslipidemia , hypertension , smoking , and cerebral vascular accident ( cva ) was comparable between the two groups ( table 1 ) .
the types of hd vascular accesses such as arteriovenous fistulas , graft , or catheter were comparable in the 2 groups .
overall mortality rate within 3 years was 24.2% among the study cohort and was comparable between the two groups .
development of pad ( 71% versus 32% ; p = 0.001 ) , new foot ulcers ( 47.6% versus 2.4% ; p = 0.001 ) , and need for major amputation ( 16.7% versus 1% ; p = 0.001 ) were significantly higher among fu group as compared to no - fu group . in the following two years ,
significantly more number of patients in fu group developed new pad ( 22% versus 1.4% ; p = 0.001 ) , underwent major amputation ( 22% versus 0% ; p = 0.001 ) , and died ( 70.4% versus 17.5% ; p = 0.001 ) in comparison to no - fu group ( table 1 ) .
the frequency of renal transplant and angina was comparable among both the groups , while cerebral vascular accident ( 3.5% ) was only observed in no - fu group .
figure 2 shows that dm patients had significantly higher incidence of fu ( 23% versus 7% ; p = 0.001 ) , need for major amputation ( 8.6% versus 0% ; p = 0.003 ) , and mortality ( 65% versus 23% ; p = 0.001 ) than non - dm patients . in dm group ,
figure 3 demonstrates the outcomes in hd patients based on the presence / absence of dm and/or pad .
patients who had pad showed increased incidence of fu ( 41% versus 4% ; p = 0.001 ) , major amputation ( 14% versus 2% ; p = 0.02 ) , and mortality ( 80.5% versus 29% ; p = 0.001 ) than non - pad . on the other hand , among non - dm patients
of these patients , mortality rate was significantly higher among pad group ( 39% versus 13% ; p = 0.007 ) in comparison to non - pad .
univariate analysis ( figure 1 ) shows the mortality rate in patients with fu in both diabetic and nondiabetic patients . on multivariate analysis , after adjusting for dm , hba1c , age , and gender , the presence of pad was the major independent predictor of development of fu in hd patients with an adjusted odd ratio ( aor ) of 16.0 ( 95% confidence interval ( ci ) : 4.4162.18 , p = 0.001 ) followed by duration of hd with aor 1.14 ( 95% ci : 1.0051.299 , p = 0.042 ) .
furthermore , after adjusting for age , sex , and cad , predictors of mortality in hd patients were pad ( aor 4.1 ; 95% ci : 1.948.59 , p = 0.001 ) , fu ( aor 3.6 ; 95% ci : 1.2810.002 , p = 0.01 ) , and dm ( aor 2.7 , 95% ci : 1.235.89 , p = 0.01 ) ( figures 4 and 5 ) .
the present study highlights the frequency and implications of fu in patients undergoing maintenance hd over a 5-year duration .
the mortality rates were higher in patients with fu in both diabetic and nondiabetic patients ; however it was relatively higher in diabetic patients .
moreover , fu was associated with 4-fold increased risk of mortality after adjusting for age , sex , and cad .
presence of pad was associated with 16-fold increased risk of fu in hd after adjustment for age , sex , dm , and duration of hd .
patients in the fu group underwent higher number of repeated hd vascular accesses in comparison to non - fu group .
recent studies have identified an increased risk of fu and lea in ckd patients who did not receive renal replacement therapy [ 10 , 17 ] .
other studies investigated patients of combined dm with esrd and found a higher risk of fu in patients undergoing hd [ 11 , 15 ] .
a swedish study demonstrated a 2.45 times increased risk of lea in esrd patients compared to those without esrd .
similarly , prompers et al . found the risk of nonhealing of fu to be 2.3-fold higher in esrd than in non - esrd patients .
the correlation between dialysis and foot complications among patients with dm and ckd has been initially described by mcgrath and curran .
they observed 50% mortality rate at one - year follow - up after lea . in our study , the rate of major amputation was significantly higher in fu patients which corroborates with an earlier study showing increased rate of amputation in ckd patients undergoing dialysis ( 57% ) as compared to those without dialysis ( 25% ) .
the relevance of selecting fu in our report is that it is potentially preventable , and its progression generally leads to serious foot complications , major amputation , and mortality . in their long - term follow - up study
( 10 years ) , morbach and coworkers concluded that patients with diabetic foot had high mortality particularly in the presence of pad or renal failure . in comparison to that study
, our 5-year study showed that fu patients had higher mortality ( 81% versus 70.4% ) although our patients were 6 years younger and less likely to have dm ( 83% versus 100% ) and pad ( 34% versus 55.5% ) .
moreover , the entire cohort of the present study was undergoing hd ( 100% versus 4% ) .
the association of severe complications in hd patients might be attributed to the cardiovascular risk factors .
several contributing factors have been proposed for the development of fu in patients with esrd and dm .
the important risk factors for the development of diabetic fu involve distal polyneuropathy , microangiopathy , and macroangiopathy . also , cardiovascular autonomic neuropathy is another coexisting complication of dm .
the authors found that pad is independently associated with esrd . according to one of our recent studies ,
pad patients had 4- to 5-fold increased incidence of fu and lea in comparison to non - pad patients . in our study ,
a higher incidence of retinopathy , polyvascular disease , angina , pad , and nephropathy was associated with fu .
studied the risk factors associated with prevalent fu in patients with dm and ckd ( predialysis versus on - dialysis ) .
the authors reported that dialysis therapy and previous fu were the only independent predictors of the development of new fu .
kaminski et al . reported a high prevalence of risk factors for fu present in patients with esrd either with or without the coexistence of dm .
the authors did not indicate the severity of esrf or whether the patients were on dialysis or not .
. found that dm , preexisting comorbidities , cvd , hd inadequacy , and lower serum albumin level are the major factors for lea .
consistent with our findings , a recent meta - analysis reported higher association of cvd , dm , and fu . ischemic in comparison to neuropathic ulcers are associated with higher mortality rate . moreover ,
the marked inflammatory response during the process of ulceration has a significant role in the initiation and worsening of the atherosclerosis . in our study , among hd patients without diabetes , only 7% developed fu , despite a high prevalence of pad and cad .
wolf et al . reported that the presence of dm in esrf patients increases the risk of lea 10 times in comparison to those who are diabetic without esrf .
moreover , during hd , around 4% of patients require an amputation each year [ 17 , 25 ] .
our subanalysis showed significantly higher incidence of fu , amputation , and mortality in diabetic patients than in non - dm patients .
also , patients with combined dm and pad revealed increased association of fu , amputation , and mortality .
our findings are supported by a large meta - analysis which showed that pad is independently associated with cvd and all - cause mortality .
further , the authors reported an increased risk of all - cause mortality in diabetic patients who developed fu than in diabetics without fu .
it is not possible to specify the extent of infection , neuropathy , ischemia , depth , or extent of tissue loss grade of fu .
another limitation is the additive effect of diabetes on hd patients who developed foot ulcers .
we did not know how many hd patients developed dm during the follow - up . in order to confirm our findings
, large sample - sized studies are needed to establish the implications of fu in hd patients .
abi < 9 was used as a part of the diagnosis of pad which may lead to underestimation of the disease .
previous data showed that as with low abi , high rates of mortality , vascular events , and amputation were reported in patients with high abi or noncompressible vessels . in a large study of patients with dm and cad , singh et al . reported a high prevalence of arterial stiffness , similar to that seen in older individuals and dialysis patients .
( 2014 ) defined pad as an abi < 0.9 or > 1.4 , these high abi values were observed in patients with diabetes , particularly for those with concomitant ckd . in conclusion , hemodialysis is a significant risk factor for fu which needs special attention .
further , pad is significantly associated with fu , amputation , and mortality in diabetic hd patients .
the increased risk of mortality could be explained by the greater burden of cvd in these patients .
therefore , hd patient needs intensive foot care to avoid complications of the lower limb and warrant progressive modification of cvd risk factors . | foot ulceration ( fu ) remains a serious concern for patients worldwide .
we analyzed the incidence , risk factors , and outcome of fu in hemodialysis ( hd ) patients .
a retrospective cohort study was conducted for 252 hd patients who were followed up for 5 years .
patients were categorized according to whether they developed fu or not .
the fu group ( 17% ) was older and had significantly higher incidence of nephropathy , retinopathy , peripheral ( pad ) , coronary artery disease ( cad ) , and diabetes mellitus ( dm ) as compared to no - fu group .
fu group had higher frequency of major amputation ( p = 0.001 ) and hd vascular access ( p = 0.01 ) .
patients with combined dm and pad had a 10-fold increased risk of fu in comparison to those who had dm alone .
presence of pad was the main independent predictor for development of fu in hd with an adjusted odd ratio ( aor ) of 16.0 ( 95% ci : 4.4162.18 ; p = 0.001 ) .
after adjusting for age , sex , and cad , predictors for mortality were pad ( aor 4.3 ) , fu ( aor 3.6 ) , and dm ( aor 2.6 ) .
fu is common in hd patients regardless of dm .
however , the presence of pad is significantly associated with more fu and mortality in hd .
hd patients need intensive foot care and warrant progressive modification of vascular risk factors . |
the neuronavigation had proven to be a great device for the surgical treatment of variable neurosurgical problems such as tumor resection or biopsy , the surgery for epilepsy , vascular neurosurgery , the localization of functional and eloquent area , spinal neurosurgery and the operation for hydrocephalus9,13,16,17 ) .
because the device helps the identification of pathologic condition and intraoperative anatomy distorted by pathologic anatomic conditions , the application of this device makes the operations more exact , safe , fast and less invasive due to finding the way and avoiding collision16,17 ) .
the endoscopic surgery in pediatric patients can also be applied to the some operations such as shunt for the hydrocephalus , the fenestration for the cystic lesion and the biopsy or resection of intraventricular and periventricular lesions .
but the pathologic condition distorts the normal anatomy , so the operator confuses normal anatomical structures in small endoscopic surgical view . for these difficulties
the pinless electromagnetic ( em ) image - guided neuronavigation has expanded the application of neuronavigation in intracranial neurosurgery , because the other neuronavigation systems needed the rigid pinning cranial fixation , these techniques were not always suitable for very young patients13 ) .
in addition , some reported that this device has proven to be successful in pediatric patients17,18 ) . in this study ,
the usefulness of em neuronavigation is described with the review for the neuroendoscopic surgery in 11 pediatric surgical cases in one institute .
between jan . 2007 and jul . 2011 , nine pediatric patients ( 8 boys and 1 girl ) were managed with endoscopic surgery using pinless em neuronavigation ( axiem , medtronics , usa ) .
the age of the patients ranged from 4 months to 12 years ( mean age : 4.0 years old ) .
a total of 11 neuroendoscopic procedures on 9 patients with cystic lesions ( 7 patients ) and hydrocephalus ( 2 patients ) were performed .
the cystic lesions consisted of five arachnid cysts and two ependymal cysts . in two hydrocephalus , one patient had an obstructive hydrocephalus due to an aqueductal stenosis by a tectal glioma and the other had the multi - loculated hydrocephalus caused by ventriculitis and ventricular abscess .
the clinical signs and symptoms were increased head circumference , headache or the prematurity ( table 1 ) .
then the registration with fiducial markers and em stylets was performed . with the guidance of neuronavigation
the clinical outcome of the procedure was evaluated by improvements of symptoms and sign and the postoperative radiologic study . on classical optical neuronavigation with rigid pinning cranial fixation , preoperative
1a ) which were attached on patient 's head ( scalp and/or face ) . in the operation room ( or )
, the patient 's head was fixed with rigid pinning fixation such as mayfield frame ( fig .
the preoperative navigation - image was transported to the navigation workstation in or and reconstructed to 3-dimensional image .
and then the position of the infra - red camera was controlled to recognize the reference frame and navigation pointer .
at the end of the registration , the fiducial markers on patient 's scalp were removed and the surgery started .
on pinless and frameless neuronavigation , adhesive tape replaces rigid pinning fixation as the fixation of head .
the patient 's head was put on the gelly head ring and electromagnetic coil was installed around head ( fig .
on classical optical neuronavigation with rigid pinning cranial fixation , preoperative mri or ct was performed with several fiducial markers ( fig .
1a ) which were attached on patient 's head ( scalp and/or face ) . in the operation room ( or )
, the patient 's head was fixed with rigid pinning fixation such as mayfield frame ( fig .
the preoperative navigation - image was transported to the navigation workstation in or and reconstructed to 3-dimensional image .
and then the position of the infra - red camera was controlled to recognize the reference frame and navigation pointer .
at the end of the registration , the fiducial markers on patient 's scalp were removed and the surgery started .
on pinless and frameless neuronavigation , adhesive tape replaces rigid pinning fixation as the fixation of head .
the patient 's head was put on the gelly head ring and electromagnetic coil was installed around head ( fig .
all procedures were performed with using of em neuronavigation and not interfered by any magnetic devices during navigation .
the fenestration was performed for the cystic lesion and made on the cystic wall for communication to cistern and/or ventricle .
the csf diversion was performed for the patients suffered with hydrocephalus and included shunt operation , third ventriculostomy and septostomy .
the combined procedures were performed at the same time in some cases . in author 's study
, the fenestration was used in 8 cases and the csf diversion in 6 cases . in all cases , the goal of surgery was accomplished successfully at the time of surgery , as confirmed immediately by postoperative imaging with the check of multiple pneumocephalus as the evidence of communication between the lesion and other space such as cistern or ventricle .
surgery related intraoperative problems such as error of the surgical trajectory , the csf leakage , infection and the injury of neurovascular structures were not occurred in all patients . and
one patient ( case 6 ) had mild third nerve palsy due to manipulation intraoperatively and that was transient .
the imaging study for postoperative status and follow - up showed the improvement and the stabilization of the lesion in 7 cases . in 2 cases ,
all patients have the improvement of preoperative symptom and normal development except one baby who was transferred to the other hospital during follow up on out - patient department . a 4-month - old male infant visited to the hospital due to incidentally detected intracranial cystic lesion .
the baby was born with 2820 gram body weight at 38 weeks 5 days of gestation .
the cystic lesion was detected on prenatal ultrasonography and the size enlarged on follow up examination .
brain mri demonstrated about 8 cm sized interhemispheric multiloculated cystic lesion without enhancement and associated hypogenesis of corpus callosum , right lateral ventricular dilatation ( fig .
the baby underwent endoscopic cystic wall fenestration at multiple sites and insertion external ventricular drainage ( evd ) catheter in the cyst .
postoperative brain ct showed decreased size of the cyst and subdural hygroma on bilateral frontoparietal region ( fig .
but the cyst was enlarged again with the aggravation of ventriculomegaly on postoperative imaging study of 3 months ( fig .
therefore , the endoscopic cyst fenestration and ventriculoperitoneal shunt was performed because the function of csf circulation might be impaired .
the size of cyst and ventricle decreased and the brain mantle has gotten thicker ( fig .
a 2-year - old boy visited with the complaint of prolonged headache after slipping down . on neurological examination , there was no specific finding .
brain ct showed about 6.5 cm sized cystic lesion on the right middle cranial fossa with midline shifting to the left side by 7 mm ( fig .
the neuroendoscopic cyst fenestration with the guidance of em neuronavigation and the insertion of evd catheter were performed .
the boy discharged with the removal of evd catheter and mild ptosis and medial gaze limitation on right eye . on the follow - up on out - patient department ,
4b ) . a 12-year - old boy visited to the hospital due to intermittent syncope and headache . there was no abnormal finding on neurological examination , on past history and on other examination such as electrocardiography . however , brain mri demonstrated about 8 cm sized cystic lesion on antrum of left lateral ventricle without hydrocephalus ( fig .
he underwent pinless em image - guided neuroendoscopic septostomy and ventriculo - cystostomy through a right postcoronal burr hole on prone position .
postoperative brain ct demonstrated pneumocephalus on frontal horn of both lateral ventricles without any acute hemorrhagic or ischemic lesion ( fig .
postoperative neurologic examination was non - specific . on follow - up , the size of cyst had decreased ( fig .
a 4-month - old male infant visited to the hospital due to incidentally detected intracranial cystic lesion .
the baby was born with 2820 gram body weight at 38 weeks 5 days of gestation .
the cystic lesion was detected on prenatal ultrasonography and the size enlarged on follow up examination .
brain mri demonstrated about 8 cm sized interhemispheric multiloculated cystic lesion without enhancement and associated hypogenesis of corpus callosum , right lateral ventricular dilatation ( fig .
the baby underwent endoscopic cystic wall fenestration at multiple sites and insertion external ventricular drainage ( evd ) catheter in the cyst .
postoperative brain ct showed decreased size of the cyst and subdural hygroma on bilateral frontoparietal region ( fig .
but the cyst was enlarged again with the aggravation of ventriculomegaly on postoperative imaging study of 3 months ( fig .
therefore , the endoscopic cyst fenestration and ventriculoperitoneal shunt was performed because the function of csf circulation might be impaired .
the size of cyst and ventricle decreased and the brain mantle has gotten thicker ( fig .
a 2-year - old boy visited with the complaint of prolonged headache after slipping down . on neurological examination , there was no specific finding .
brain ct showed about 6.5 cm sized cystic lesion on the right middle cranial fossa with midline shifting to the left side by 7 mm ( fig .
the neuroendoscopic cyst fenestration with the guidance of em neuronavigation and the insertion of evd catheter were performed .
the boy discharged with the removal of evd catheter and mild ptosis and medial gaze limitation on right eye .
on the follow - up on out - patient department , the symptom improved and the size of the cyst decreased ( fig .
4b ) . a 12-year - old boy visited to the hospital due to intermittent syncope and headache . there was no abnormal finding on neurological examination , on past history and on other examination such as electrocardiography .
however , brain mri demonstrated about 8 cm sized cystic lesion on antrum of left lateral ventricle without hydrocephalus ( fig .
he underwent pinless em image - guided neuroendoscopic septostomy and ventriculo - cystostomy through a right postcoronal burr hole on prone position .
postoperative brain ct demonstrated pneumocephalus on frontal horn of both lateral ventricles without any acute hemorrhagic or ischemic lesion ( fig .
postoperative neurologic examination was non - specific . on follow - up , the size of cyst had decreased ( fig .
a 4-month - old male infant visited to the hospital due to incidentally detected intracranial cystic lesion .
the baby was born with 2820 gram body weight at 38 weeks 5 days of gestation .
the cystic lesion was detected on prenatal ultrasonography and the size enlarged on follow up examination .
brain mri demonstrated about 8 cm sized interhemispheric multiloculated cystic lesion without enhancement and associated hypogenesis of corpus callosum , right lateral ventricular dilatation ( fig .
the baby underwent endoscopic cystic wall fenestration at multiple sites and insertion external ventricular drainage ( evd ) catheter in the cyst .
postoperative brain ct showed decreased size of the cyst and subdural hygroma on bilateral frontoparietal region ( fig .
but the cyst was enlarged again with the aggravation of ventriculomegaly on postoperative imaging study of 3 months ( fig .
therefore , the endoscopic cyst fenestration and ventriculoperitoneal shunt was performed because the function of csf circulation might be impaired .
the size of cyst and ventricle decreased and the brain mantle has gotten thicker ( fig .
a 2-year - old boy visited with the complaint of prolonged headache after slipping down . on neurological examination , there was no specific finding .
brain ct showed about 6.5 cm sized cystic lesion on the right middle cranial fossa with midline shifting to the left side by 7 mm ( fig .
the neuroendoscopic cyst fenestration with the guidance of em neuronavigation and the insertion of evd catheter were performed .
the boy discharged with the removal of evd catheter and mild ptosis and medial gaze limitation on right eye . on the follow - up on out - patient department ,
a 12-year - old boy visited to the hospital due to intermittent syncope and headache .
there was no abnormal finding on neurological examination , on past history and on other examination such as electrocardiography . however , brain mri demonstrated about 8 cm sized cystic lesion on antrum of left lateral ventricle without hydrocephalus ( fig .
he underwent pinless em image - guided neuroendoscopic septostomy and ventriculo - cystostomy through a right postcoronal burr hole on prone position .
postoperative brain ct demonstrated pneumocephalus on frontal horn of both lateral ventricles without any acute hemorrhagic or ischemic lesion ( fig .
postoperative neurologic examination was non - specific . on follow - up , the size of cyst had decreased ( fig .
the neuroendoscopy has used since 1922 by dandy1 ) and the appliance of endoscopy are variable in neurosurgical area22 ) : the biopsy or resection of intraventricular or periventricular lesion , third ventriculostomy , cyst fenestration for arachnoid or ependymal cyst , aqueductal stenosis , multilobulated hydrocephalus and the insertion of intraventricular catheter such as shunt proximal tip and ommaya reservoir etc .
this procedure requires exact anatomic orientation for every step of the operation due to narrow surgical view .
however , this procedure had relative high complication rate that reported up to 20%6,14,17,19,22 ) : the neurovascular injury , csf leakage and infection , seizure and subdural fluid collection ( table 2 ) . and abtin et al.1 ) and navarro et al.14 ) reported the discontinuation of the procedure due to the lack of anatomical landmark with distorted intracranial anatomy in several cases .
after the appliance of the neuronavigation makes the neurosurgical procedures more safe and easy , so it can help to reduce the complication and morbidity and improve outcome in neurosurgical procedures13,17 ) ( table 2 ) .
and the appliance of the neuronavigator helps the operator understand the orientation of distorted anatomy and blind areas over the septum and the cystic wall20 ) .
but the classical optical neuronavigation with rigid cranial fixation has some limitations for the application .
the fixation has a risk of scalp laceration , skull fracture , extradural hemorrhage and intracranial injury13,17 ) .
in addition , the pain involved in using the fixation cause the increased use of anesthetic agents and the increased operation time7 ) .
the rigid pinning makes that the appliance to children with thin and immature skull is especially difficult .
therefore , the advance of pinless neuronavigation can lead to an increase in its range of uses in several neurosurgical conditions including pediatrics17 ) .
but this system does not solve the limitation of the operator 's motion and that of the head fixation with adhesive tape .
em neuronavigation leads to be non - invasive due to the use without rigid pinning cranial fixation and with free head motion on the operation .
due to non - invasiveness , the system can apply to very young pediatric patients who have very thin and immature skull .
this advantage makes lower limitation of the application neuronavigation in age . as shown table 2 ,
the age in which neuroendoscopic surgery was performed is lower in em neuronavigation than in classical neuronavigation .
the system needs shorter time for set - up and registration compared with previous neuronavigation system .
in addition , it allows head movement at any time after registration with no restriction on the freedom of movement of instruments within the surgical field and does not limit the operator motion due to no need of optical line between the pointer , reference frame , and the infrared camera .
also , the operation using neuronavigation can be performed under local anesthesia due to no fixation with pinning frame9,11,13,15 - 17,21 ) . in an author 's case , which was 5 months baby with congenital hydrocephalus , this device was very useful to decide the proper trajectory for proximal shunt tip with the change of head position after registration .
several operators reported the appilication of em neuronavigation on neuroendoscopic surgery in variable conditions3,7,9,13,17 ) .
mcmillen et al.13 ) applied em neuronavigation in 13 months children who had obstructive hydrocephalus caused by cystic hypothalamic glioma . in the case ,
neuroendoscopic surgery for shunt and cyst fenestration was performed in two different surgical points with head reposition without accuracy changes . in this study ,
the patient ( case 1 ) had post - infectious multiloculated hydrocephalus and needed multiple fenestrations for multiple cystic walls .
the decision for the site to fenestrate was difficult , because the loculated walls were not defined exactly and not discriminated clearly from normal ventricular walls , in addition , normal anatomy was distorted .
em neuronavigation without rigid pinning fixation was able to been applied for endoscopic surgery usefully and successfully .
hayhurst et al.9 ) suggested some indications for appliance of em neuronavigation in neurosurgical procedures ( table 3 ) . among them , the em neuronavigation had additional several usefulness in neuroendoscopy with the advantage of previous system as follows2,8,9,13,16,17,23 ) : 1 ) the control of probe 's depth due to the flexibility of the probe is feasible .
2 ) this system allows to be passed through the endoscope working channel for use in fenestration of both arachnoid and floor of the third ventricle on the tip of the endoscope .
4 ) the handling of endoscope containing em probe is not difficult due to a character of weightless stylet .
5 ) if the endoscope needed to be repositioned , the navigation stylets could easily be replaced in the endoscope 's working channel to confirm the location of the tip of the instrument .
6 ) the additional procedure with head reposition can be performed without distortion of preoperative registration .
the navigation devices such as probe and tracer were disposable and expensive , and some authors reported that the accuracy for application is lower than other system .
in addition , it has the risk of loss of sterility and unsuspected injury in awaken procedures due to not rigidity fixation9,17,21 ) .
some authors reported that the em neuronavigation in the insertion of the shunt proximal catheter insertion can be worthier than the cost of stylets because the costs for revision and/or complications which caused by not using em neuronavigation might be higher than the cost for usage of em neuronavigation7,9 ) .
in addition , rosenow and sootsman15 ) and mascott12 ) reported that application accuracy of em neuronavigation is not lower than optical navigation .
the usefulness of em neuronavigation in neuroendoscopy has been reported and proven by several authors and cases .
this technique in 9 pediatric patients who were younger than 12 year - old showed successful surgical management in this study , however , there were too small cases to prove usefulness of em neuronavigation compared with other authors . but
this result of operations is similar with others and can be supports for usage of em neuronavigation and help prove usefulness of em neuronavigation in neuroendoscopic surgery .
the appliance of the neuronavigation reduces the complication rate and enhances for the indication of the neuroendoscopic surgery . but the use of classical optical neuronavigation which needs rigid pinning fixations has been limited in several situations such as pediatrics with immature skull .
em neuronavigation , in this aspect , solved that problem without using rigid pinning fixation . in conclusion
, it can be a useful device and alternative to a classical optical neuronavigation in the pediatric neuroendoscopic surgery . | objectiveneuroendoscopy is applied to various intracranial pathologic conditions . but this technique needs informations for the anatomy , critically .
neuronavigation makes the operation more safe , exact and lesser invasive procedures .
but classical neuronavigation systems with rigid pinning fixations were difficult to apply to pediatric populations because of their thin and immature skull .
electromagnetic neuronavigation has used in the very young patients because it does not need rigid pinning fixations .
the usefulness of electromagnetic neuronavigation is described through our experiences of neuroendoscopy for pediatric groups and reviews for several literatures.methodsbetween january 2007 and july 2011 , nine pediatric patients were managed with endoscopic surgery using electromagnetic neuronavigation ( axiem , medtronics , usa ) .
the patients were 4.0 years of mean age ( 4 months-12 years ) and consisted of 8 boys and 1 girl .
totally , 11 endoscopic procedures were performed .
the cases involving surgical outcomes were reviewed.resultsthe goal of surgery was achieved successfully at the time of surgery , as confirmed by postoperative imaging . in 2 patients ,
each patient underwent re - operations due to the aggravation of the previous lesion . and
one had transient mild third nerve palsy due to intraoperative manipulation and the others had no surgery related complication.conclusionby using electromagnetic neuronavigation , neuroendoscopy was found to be a safe and effective technique . in conclusion , electromagnetic neuronavigation is a useful adjunct to neuroendoscopy in very young pediatric patients and an alternative to classical optical neuronavigation . |
one hundred forty - seven clinical isolates of m. tuberculosis were obtained from the molecular mycobacteriology laboratory , department of microbiology , faculty of medicine , siriraj hospital , mahidol university , thailand , and the t-2 project from 1997 to 2001 ( table ) .
these strains were isolated from 74 cerebrospinal fluid ( csf ) samples and 73 sputum samples from 147 different patients .
dna from these isolates was isolated by an enzymatic method and submitted for genotyping by performing the is6110 restriction fragment length polymorphism with the standard method ( 12 ) and for sequencing the pks15/1 region ( 8) .
* csf , cerebrospinal fluid . using the genotyping results , we categorized m. tuberculosis isolates into beijing , single - banded , few - banded ( 25 bands ) , nonthaburi , and heterogeneous with > 5 bands ( table and figure 1 ) , as recently reported ( 13,14 ) .
all m. tuberculosis genotypes were sequenced around the junction of pks15 and pks1 ( corresponding to the m. tuberculosis h37rv sequence ) to determine whether they contained an intact pks15/1 or separated pks15 and pks1 .
unexpectedly , the results showed that the 7-bp insertion of pks15 that causes a frameshift mutation resulting in an intact pks15/1 was found in most strains of all genotypes , except the heterogeneous group with > 5 bands ( table and figure 2 ) .
r indicates the m. tuberculosis mt 14323 strain used as the positive control for is6110 typing .
sequence alignment of region corresponding to the 3 portion of pks15 and 5 portion of pks1 in various mycobacterium tuberculosis genotypes .
b ) m. tuberculosis strains isolated from sputum . letters in brackets refer to is6110 restriction fragment length polymorphism patterns : bj , beijing ; sb , single banded ; fb , 25 bands ; nb , nonthaburi ; h , heterogeneous .
the 7-bp insertion is shown in boldface , and the start codon of the pks1 gene is underlined .
the intact pks15/1 has been shown to be responsible for the production of phenolic glycolipids and is seemingly found in m. tuberculosis w - beijing family , but it was not found in m. tuberculosis cdc1551 and h37rv ( 8) .
previous studies suggested that pgls produced by the m. tuberculosis w - beijing family were associated with the hypervirulent phenotype by inhibiting the innate immune response ( 6,7 ) .
the intact pks15/1 has also been shown to be nonpolymorphic in the w - beijing family ; it was found in all 102 w - beijing strains tested ( 15 ) . from this observation
, we hypothesized that if the ability to produce pgls is among the factors that make this family more virulent than others , the intact pks15/1 should be absent in strains other than the w - beijing family .
our results showed that the 7-bp insertion of the pks15/1 was not only present in the w - beijing family but also in other m. tuberculosis genotypes .
although almost all beijing strains contain the intact pks15/1 ( 97% ) , 38.5%100% of strains of other genotypes also contain it .
these strains could , therefore , produce pgls and cause both pulmonary and disseminated diseases as the w - beijing strains do .
our results showed no significant difference in the percentage of m. tuberculosis isolates with an intact pks15/1 gene between csf isolates ( 65 [ 87.8% ] of 74 ) and sputum isolates ( 62 [ 84.9% ] of 73 ) .
the hypothesis that the hypervirulence of the w - beijing family is solely attributable to pks15/1 is still inconclusive .
this family may have only recently been transmitted globally and may have had more chances to cause infections and disease than other families .
although pgls are involved in the hypervirulence of the pgl - producing strains , they are not a unique characteristic of the w - beijing family
. if w - beijing strains are more virulent than others , other virulence determinants besides pgls must be responsible for the hypervirulent phenotype . | to determine whether intact pks15/1 is unique to the w - beijing family , we investigated 147 mycobacterium tuberculosis strains with different is6110 genotypes .
intact pks15/1 was found in 87.8% of cerebrospinal fluid and 84.9% of sputum isolates .
it was found not only in w - beijing strains ( 97% ) but also in other genotypes ( 38.5%100% ) . |
although , the incidence of multicentricglioma accounts nearly 2.5% of all gliomas , mostcases are glioblastoma or anaplastic gliomas .
oligodendroglioma cells are detectablein the cerebrospinal fluid in up to 14% of patients , and we recently treated a case of multiple anaplastic oligodendrogliomas , describe the case in detail and discuss the distinction between multicentric and dissemination via cerebrospinal pathways and the treatment .
a 72-year - oldman revealed aphasia , abnormal behavior , and rapidly progressivedementia since october 13 , 2009 , and hospitalized in our department on oct .
17 , 2009.magnetic resonance imaging ( mri ) enhanced by contrast media demonstrated multiplebrain tumors in left parietal lobe and left paraventricular region [ figure 1a ] . computed tomography ( ct ) scan showed high density of microbleeding in the left paraventricularregion [ figure 1b ] .
biopsy for the tumor in the left parietal tumor was performed . in the permanent section ,
the hematoxylin and eosin - stained specimen showed that thetissue was composed of cells with clear cytoplasm and with round or irregular nucleushaving dense chromatin ( so called honey comb appearance ) that existed in between highly proliferative vasculature [ figure 2 ] .
the nuclei had pleomorphism and a variety ofsize , a lot of mitosis were observed .
microvascular proliferation and necrotic findingswere also seen . occasionally , calcification deposit and microbleeding were observed.immunocy to chemical study showed positive for glial fibrillary acidic protein , stronglypositive for the olig-2 ( oligodendrocyte lineage genes ) stain .
these findings were compatible for anaplastic oligodendroglioma , so geneticalevaluation was performed , showing loss of heterozygosity ( co - deletions ) in chromosome1p19q .
local brain for the left parietal regionradiotherapy was given to a total dose of 50gy , in34 fractions of 1.5gy daily on weekdays from november to december , 2009 .
at the sametime , temozolomide of 120 mg daily was orally administrated for 42 days .
mri was performed just after these combined chemo- and radiotherapy showed lessening of themass effect in the left parietal tumor , but enlarging the size of the tumor in the left paraventricular region [ figure 3 ] .
we considered that enlargement of the paraventriculartumor was pseudoprogression , as consolidating therapy , temozolomide of 240 mg daily ( 150mg / m2/day ) for 5 days was orally administered for 5 times from february to october 2010 at outpatient clinic . despite of the high levels of promoterhypermethyltransferase ( mgmt ) activity , temozolomide is the first linechemotherapeutic agent against anaplastic oligodendroglioma , so we tried temozolomide . but during those period , cerebellar tumor appeared and graduallyenlarged and hydrocephalus was observed [ figure 4 ] . in october 2010 ,
one cycle of temozolomidevia intravenous administration was performed and lessening of the masseffect was seen , so the patient was transferred to rehabilitation hospital . in the end
ofnovember 2010 , the patient 's consciousness deteriorated because of the enlargement ofthe 4th ventricle tumor , hydrocephalus and abnormality of serum electrolyte .
theconsciousness of the patient recovered for a while by correcting the electrolyte balance , but consciousness level worsened again , and respiration and clinical state wasdeteriorated , and died on january , 2011 .
( a ) magnetic resonance imaging ( mri ) enhanced by contrast media obtained atthe first hospitalization.multiple brain tumors in the left parietal and leftparaventricular region were observed .
the tissue was composed ofcells with clear cytoplasm and with round or irregular nucleus having dense chromatin ( so called honey comb appearance ) .
hematoxylin and eosinstained specimen , 400 mri after radiotherapy 50gy and temozolomide of 120 mg daily for 42 days .
although , lessening of the mass effect of left parietal tumor was observed , enlargementof the paraventricular tumor was seen , which seemed to be pseudoprogression mri performed about 1 year after initial hospitalization .
salvati m. et al.reported 25 cases with multicentricgliomas , 11 cases aremulticentricglioblastomas , 6 cases are multicentric anaplastic astrocytomas , and othercases are combinations of glioblastomas , anaplastic astrocytomas , and low gradegliomas , and no oligodendrogliomas were found .
solomon etal . stated a clear distinction must be made , in cases of multiple tumors , amongdissemination via cerebrospinal pathways , direct extension across commissures , satellitosis , and true multicentrism .
multicentric tumors are widely separated , arisingin different lobes or hemispheres , which is not readily explained by one of the pathwaysmentioned above.in our case , the 4th ventricle tumor which arose about 1 year afterinitial treatment might have been a metastasis via cerebrospinal fluid , although one ofthe multiple sites can not be dismissed entirely . however , left parietal tumor and lefttrigonal area tumor were found at the same time , and those two tumors were separated , arising in the distinct sites , and were not explained either by the dissemination viacerebrospinal pathways or direct extensions , so we considered those tumors weremulticentric.the pathogenesis of multicentricgliomas is unknown.the most quoted hypothesis is the two - step process reported by willis . in the firststage
a large area of the brain undergoes neoplastic transformation , while in the secondstage one or more foci of tumor proliferation develop within the affected area , givingorigin to separate lesions .
the pathogenesis of multicentricglioma include theembryonal rest theory and the two - stage theory , but zlch pointed out the metastasisvia an unknown pathway .
multicentricgliomas are characterized by rapid progression , which may be related tothe fact that they tend to exhibit malignant features on histology , this fact is also validfor the oligodendrogliomas .
the course of our case from initial diagnosis to death was 1year and 3 months , despite of the combined chemoradiotherapy.in our case , the 4thventricle portion tumor which appeared about one year after the initial treatment andwas not detected in the mri taken at initial hospitalization , might be the metastasis viacerebrospinal fluid , as histological type of malignant oligodendroglioma is a risk factor of cerebrospinal fluid dissemination .
finally , the tumor in the left paraventricular region showed progression even just after the temozolomide treatment , and we presumed it was pseudoprogression , because in the first 6 months of post - chemoradiotherapy period , showed a high incidence of pathologically confirmed pseudoprogression associated with treatment - related necrosiswithout viable tumor cells .
various imaging techniques including mr spectroscopy havebeen studied but neither has been found to be powerful to identify pseudoprogression , and unfortunately , the diagnosis of pseudoprogression still depends on pathologicexamination.the disruption of blood - brain barrier with the resultant observation of an earlyradiologic increase in contrast enhancement at mri , which may falsely mimic trueprogression , and this fact made the treatment of this case more complicated . | a 72 year - old male hospitalized with aphasia , abnormal behavior , and rapidlyprogressive dementia.magnetic resonance imaging ( mri ) enhanced by contrast media demonstrated multiplebrain tumors in left parietal lobe and left paraventricular region .
biopsy was performed , andhistopathological examination and genetical evaluation revealed anaplasticoligodendroglioma .
local radiation 50gy was given , and temozolomide via orallyadministered for 42 days .
after the chemoradiotherapy , even though the parietal tumorshowed lessening of the size , enlargement of the tumor in the left paraventricularregion was observed , and we considered that phenomenon was pseudoprogression .
5courses of temozolomide therapy was added , but cerebellar tumor appeared andenlarged with hydrocephalus , and died 1 year and 3 months after the firsthospitalization . |
metformin is widely used to improve insulin resistance ( ir ) , but the mechanism is not fully understood .
although liver is the primary target , metformin acts on a variety of tissues , including adipose tissue , skeletal muscles , endothelium , and ovary [ 1 , 2 ] .
previous studies have demonstrated metformin exerts the metabolic actions , especially the glucoregulatory action , mainly through amp activated protein kinase ( ampk ) [ 3 , 4 ] . in recent years , metformin has been reported to have an impact on the production of adipokines ( e.g. , adiponectin , leptin ) , which might be associated with the insulin - sensitizing effect of this agent [ 5 , 6 ] .
pigment epithelium - derived factor ( pedf ) , an adipokine discovered in recent years , plays an important role in obesity - induced ir .
it is one of the most abundant proteins secreted by human adipocytes with the property to induce ir and inflammatory signaling in adipocytes and skeletal cells .
serum pedf levels elevate in obese mice and reduce upon weight loss ; prolonged pedf administration stimulates adipose tissue lipolysis , results in ectopic lipid deposition , and reduces insulin sensitivity , whereas neutralizing pedf with pedf antibody in obese mice enhances insulin sensitivity [ 7 , 9 ] .
clinical researches have revealed that serum pedf level is an independent determinant of ir in patients with essential hypertension . in our previous work ,
increased serum pedf levels , which are closely associated with ir and chronic inflammation , are observed in patients with polycystic ovary syndrome .
additionally , pedf is also found to be an important determinant of oxidative stress , inflammation , and angiogenesis [ 12 , 13 ] .
it is presently unclear whether metformin could influence pedf production in adipose tissue and liver .
a study showed that the improvement of ir , following diet and/or surgery - induced weight loss , was associated with the magnitude of decrease in serum pedf levels and pedf expression of adipose tissue . in this context , the present study was undertaken to get insights into the effect of metformin on the expression and secretion of pedf in adipocyte and hepatocyte and whether this effect is associated with the ir - improving action of the agent , which may help to clarify pharmacological action of metformin .
our data showed metformin inhibits pedf production and release , and ampk plays a key role in this process , which is closely associated with the improvement of ir .
animal care and experimental procedures were performed with the approval from the animal care committees of chongqing medical university . after two weeks of adaptive feeding ,
the seven - week - old sd rats were randomly assigned to receive a normal chow diet ( 15% fat ) ( nc group , n = 6 ) or a high fat diet ( 45% fat ) ( n = 12 ) for 15 weeks ; then the high - fat - diet rats were gavaged saline ( hf group , n = 6 ) or metformin ( glucophage , sanofi , france ) 400 mg / kg / d ( met group ,
n = 6 ) for 4 weeks . at the end of the 19th week ,
insulin sensitivity was assessed by the hyperinsulinemic - euglycemic clamp , and then the rats were culled with cervical dislocation . when sacrificed , blood and tissue samples were collected for further assessments .
mouse embryo fibroblast 3t3-l1 cells were obtained from american type culture collection ( manassas , va ) and grown in dulbecco 's modified eagle 's medium ( dmem ) containing 1% antibiotics ( life technologies , maryland , us ) as described previously . human hepatoma cells ( hepg2 cells )
( obtained from american type culture collection ) were grown in 1640 medium supplemented with 10% calf serum and 1% antibiotics .
hepg2 cells and differentiated 3t3-l1 cells seeded on 96-well plates were treated with 0 , 0.05 , 0.5 , 5 , and 50 nm insulin for 24 h , and the insulin ( 100 nm ) mediated glucose uptake was examined to validate the establishment of the cell models .
the ir cell models were treated with or without 0.1 mm metformin ( sigma , us ) for 24 h , and the 40 m compound c ( sigma , us ) was added 1 h before metformin addition .
the tracer used to monitor glucose uptake in cells was 6-[n-(7-nitrobenz-2-oxa-1 , 3-diazol-4-yl ) amino]-6-deoxyglucose ( 2-nbdg ; sigma , us ) , with the method described previously , with some modification [ 19 , 20 ] .
after treatment , the cell medium was removed , and the cells were washed twice with krebs'-ringer's - hepes ( krh ) buffer ( 136 mm nacl , 20 mm hepes , 4.7 mm kcl , 1.25 mm mgso4 , and 1.25 mm cacl2 , ph 7.4 ) and the plate was measured by a fluorescence microplate reader ( spectramaxm2 multimode plate readers , molecular devices , usa ) set at an excitation wavelength of 488 nm and an emission wavelength of 520 nm , and the results were expressed as fa . treated with 2-nbdg ( 50
m ) for 15 min and washed twice with krh buffer , the cells were measured by the fluorescence microplate reader again and the results were expressed as fb . later on , treated with 2-nbdg combined with insulin ( 100 nm ) for 15 min and washed twice with krh buffer , the cells were measured by the fluorescence microplate reader for the third time and the results were expressed as fc .
finally , the cell viability was measured by 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and the results were expressed as od .
pedf concentration in the serum and the supernatants of 3t3-l1 and hepg2 cells was analyzed by the elisa kits ( uscn life science inc , china ) according to the manufacturer 's instruction .
serum insulin was determined by the elisa kits ( uscn life science inc , china ) .
serum lipids were measured as follows : total cholesterol ( tc ) using the cholesterol oxidase - hdaos method ( wako , osaka , japan ) ; triglycerides ( tg ) using the gpo - hdaos glycerol blanking method ( wako ) ; high - density lipoprotein cholesterol ( hdl - c ) using the immunoinhibition ( direct ) method ( wako ) ; and low - density lipoprotein cholesterol ( ldl - c ) using the selective protection enzymatic ( direct ) method ( wako ) .
clamp was performed as described previously , with some modifications [ 21 , 22 ] . in brief , after rats were anesthetized with 10% chloral hydrate ( 0.4 ml/100 g body weight ) , two polyethylene catheters were inserted into the right jugular vein and left carotid artery .
patency was maintained by 0.5% heparin ( 1 ml / kg body weight ) administration , and the vein catheter was connected with two digital syringe pumps ( medtronic , inc , usa ) by a y connector .
blood samples of 600 l were obtained for the measurement of serum insulin , lipids , and pedf before the infusion of insulin .
the infusion rate of insulin ( humulin regular insulin , eli lilly , usa ) , mixed with a variable volume of 0.1% bsa , was set at ( 10 mu / kg / min ) , and when the plasma glucose was lower than 5.0
mmol / l ) was maintained by a variable infusion rate that was adjusted according to the plasma glucose level at 5 min intervals . during the last 60 min of the clamp when the plasma glucose level became steady ,
the glucose infusion rate was calculated as the average results of the last thirteen times of plasma glucose levels .
total rna was extracted using the trizol reagent ( life technologies , maryland , usa ) according to the manufacturer 's instruction .
the rna purity was assessed by measuring od at 260 and 280 nm with the standard of a260/a280 1.8 .
rna was treated with 1 unit of dnase i ( life technologies , maryland , usa ) for 15 min at room temperature . cdna synthesis was performed with 0.5 mg total rna treated with dnase i in a 10 l reaction system using the first strand synthesis for rt - pcr kit ( takara ) .
real - time reverse transcription polymerase chain reaction ( rt - pcr ) was performed in the applied biosystems 7500 real - time pcr system , using sybr green dye ( qiagen , german ) according to the manufacturer 's protocol .
all the primers were designed by beacon designer software and synthesized by life technologies corporation .
proteins were extracted from tissues and cells using cell lysis buffer for western blotting ( beyotime , jiangsu , china ) , and protein concentration was determined using the bca protein assay kit ( beyotime ) .
sample proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in a bio - rad mini - protean apparatus .
membranes were blocked with blocking buffer and incubated with primary antibodies ( goat polyclonal anti - pedf , rabbit polyclonal anti - pampk , rabbit polyclonal anti - beta actin , santa cruz biotechnology , usa ) overnight at 4c , followed by horseradish peroxidase - labeled secondary antibodies ( santa cruz biotechnology , usa ) for one hour at room temperature .
immunoreactive proteins were visualized using enhanced chemiluminescence ( ecl , beyotime , china ) , and the band intensities were quantified using quantity one software ( bio - rad , usa ) .
data involving more than two groups were assessed by one - way anova ( with games howell test for post hoc analysis ) .
compared with the nc group , rats in the hf group showed higher levels of body weight , fasting plasma glucose , fasting serum insulin , ldl - c , and a lower level of glucose infusion rate ( table 2 ) .
in addition , the mrna expression levels of phosphoenolpyruvate carboxykinase ( pepck ) and glucose 6-phosphatase ( g-6-pase ) , in the liver were also significantly upregulated in the high - fat diet induced obese rats ( figure 1 ) . with metformin treatment for 4 weeks , the ir of the high - fat diet induced obese rats was obviously improved , manifested as enhanced glucose infusion rate , reduced serum tg and serum insulin levels , and down - regulated mrna expression of pepck , g-6-pase ( table 2 , figure 1 ) .
the high - fat diet induced obese rats showed higher levels of serum pedf as compared with the nc group ( 2.16 0.09 versus 1.77 0.14 g / ml , p < 0.05 , figure 2(a ) ) , and a negative association was found between the serum pedf concentration and glucose infusion rate ( r = 0.67 , p < 0.05 , figure 2(b ) ) .
the mrna and protein expression of pedf in the epididymal adipose tissue and liver were significantly up - regulated in the hf group as compared to the nc group .
however , the metformin group exhibited decreased serum pedf levels ( 2.16 0.09 versus 1.69 0.24 g / ml , p < 0.05 , figure 2(a ) ) and down - regulated pedf expression when compared with the hf group ( figures 2(c ) and 2(d ) ) . to be in accordance with the ir condition in vivo , ir models of adipocytes and hepatocytes
were established by the hyperinsulinemic method [ 1618 ] . with a low or moderate concentration of insulin ( 0.05 nm5 nm ) treatment for 24 h ,
the glucose uptake in the adipocytes and hepatocytes showed no significant changes ; however , when the insulin concentration was as high as 50 nm , an obvious decreased glucose uptake was observed , indicating an ir status in the cell models ( figure 3 ) .
similar to the results in vivo , increased pedf expression and secretion were found in the ir adipocytes and hepatocytes .
while with 0.1 mm metformin treatment , the pedf expression was down - regulated and the pedf secretion was lowered , accompanied with increased glucose uptake in the ir adipocytes and hepatocytes ( figure 4 ) . since metformin exerts its metabolic action mainly through ampk [ 3 , 4 ] , the phosphorylation at threonine 172 of the subunit of ampk was examined , and the results showed metformin treatment stimulated ampk phosphorylation in adipose tissue and liver of the obese rats , which was accompanied with the profound reduction in pedf protein content ( figure 2 ) . in vitro , when the ir adipocytes and hepatocytes were treated with metformin combined with an ampk inhibitor compound c , the inhibitive effects of metformin on pedf expression or secretion were abrogated ( figure 4 ) .
first identified in retinal pigment epithelium cells , pedf is expressed in various tissues throughout the body such as the eye , adipose tissue , liver , and muscle [ 79 , 13 ] .
it is reported as a multifunctional , pleiotropic protein with antiangiogenic , antioxidant , neurotrophic , and both pro- and anti - inflammatory properties [ 12 , 13 ] . in the previous work , we found the serum pedf level is elevated in women with polycystic ovary syndrome and is associated with ir , indicating pedf may play a role in the pathogenesis of ir in patients with polycystic ovary syndrome .
metformin , a widely prescribed agent to improve ir , has been reported to have an impact on the production of adipokines ( e.g , adiponectin , leptin ) , which might be associated with the ir - improving effect of the agent [ 5 , 6 ] . while pedf has been reported to play an important role in the development of ir [ 79 ]
for the first time , our study reports that metformin inhibits the expression and secretion of pedf in adipocyte and hepatocyte via stimulating ampk phosphorylation , and this effect is associated with the ir - improving action of metformin . to get insights into the effect of metformin on pedf when metformin exerts its ir - improving action ,
high - fat diet induced obese rats were used for the in vivo experiment and validated as being insulin resistant by the hyperinsulinemic - euglycemic clamp . in vitro
, the ir models of 3t3-l1 and hepg2 cells were established by the hyperinsulinemic method and validated through glucose uptake test .
it is generally accepted that ir is the most common cause of hyperinsulinemia ; however , recent studies have revealed that ir might also be a result of prolonged exposure of high concentrations of insulin [ 2426 ] .
liu et al . found that exposure of cultured hepatocytes to 10100 nm insulin for 24 h induces significant ir , which might be associated with disturbed insulin signal transduction and oxidative stress .
another study reported that exposure to 1 m insulin for 6 h , 3t3-l1 cell showed down - regulated mrna expression of glucose transporter 4 and lost their capacity for acute stimulation of glucose uptake by insulin .
similar to these studies , our data revealed that , incubated with 50 nm insulin for 24 h , the adipocytes and hepatocytes were markedly insensitive to insulin as shown by the decreased glucose uptake when stimulated by 100 nm insulin .
it is noteworthy that although the high concentration of 50 nm insulin induced ir in vitro , this might not be duplicated in vivo since the serum insulin level as high as 50 nm has rarely been reported in human . both in vivo and in vitro , increased pedf secretion as well as expression were found in tissues and cells with ir .
furthermore , we found the serum pedf levels were negatively correlated with the insulin sensitivity . in the study of crowe et al .
, an elevated serum pedf and expression in adipose tissue were also observed in high - fat diet induced mice , which are in line with our in vivo findings . with metformin intervention for 4 weeks , high - fat diet induced obese rats exhibited obvious improvement of ir , manifested as enhanced glucose infusion rate , reduced serum tg and fasting serum insulin levels , and down - regulated mrna expression of key enzymes which promote gluconeogenesis and fatty acid synthesis .
in addition , serum pedf levels as well as expression of pedf in the adipose tissue and liver decreased significantly during ir improvement caused by metformin . in accordance with the in vivo findings , insulin resistant adipocytes and
hepatocytes also showed decreased secretion and expression of pedf with metformin treatment , accompanied with enhanced glucose uptake . to further investigate the role played by the target molecule of metformin , the phosphorylation at threonine 172 of the subunit of ampk was measured in the rats , and we found the impact of metformin on pedf expression in adipose tissue and liver was associated with stimulation of ampk phosphorylation . when combined with compound c , the suppressive effect of metformin on pedf
those results suggest that metformin exerts the effect on pedf via stimulation of ampk phosphorylation .
additionally , our study indicates ampk might be a potential regulator of pedf production , on transcriptional or posttranscriptional level , although the detailed mechanism needs further study .
the study conducted by akin et al . on 36 turkish type 2 diabetic patients showed that , after metformin treatment for 6 months , the serum pedf levels increased .
the different subjects in akin 's study ( type 2 diabetic patients ) and ours ( high - fat diet induced obese rats ) might partially explain the discrepancy in the results of the two studies .
besides , metformin exerts complex effect in vivo , whether the increase of serum pedf in type 2 diabetic patients is a direct effect of metformin is still unclear .
although different from metformin in mechanism , troglitazone was an insulin sensitizer which was once used to improve ir .
, our study reveals that metformin inhibits expression and secretion of pedf in adipocytes and hepatocytes via the stimulation of ampk phosphorylation .
in addition , our study indicates ampk might be a potential regulator of pedf . although we found the effect of metformin on pedf
is closely associated with the ir - improving action of the agent , whether the action is mainly mediated through an inhibition in pedf still needs further study . | objective .
pigment epithelium - derived factor ( pedf ) plays an important role in obesity - induced insulin resistance ( ir ) .
the study aims to investigate the effect of metformin , a widely used agent to improve ir , on pedf production both in vivo and in vitro
. methods .
sd rats were divided into normal control group , high fat group ( hf group ) , and metformin group ( met group ) .
hyperinsulinemic euglycemic clamp was performed to evaluate insulin sensitivity .
ir models of 3t3-l1 and hepg2 cells were established and then treated with metformin and inhibitor of amp activated protein kinase ( ampk ) .
results . in vivo ,
the hf group showed increased serum pedf which is negatively correlated with insulin sensitivity , while the met group revealed decreased serum pedf and downregulated pedf expression in fat and liver , concomitant with significantly improved ir . in vitro , the ir cells showed enhanced pedf secretion and expression , whereas metformin lowered pedf secretion and expression , accompanied with increased glucose uptake .
metformin stimulated ampk phosphorylation in fat and liver of the obese rats , while in vitro , when combined with ampk inhibitor , the effect of metformin on pedf was abrogated .
conclusions .
metformin inhibits the expression and secretion of pedf in fat and liver via promoting ampk phosphorylation , which is closely associated with ir improvement . |
efforts in the field of biomarker research have pointed to the existence of circulating immune complexes as a novel class of tumor markers with diagnostic potential comparable with or greater than that of the corresponding free biomarker .
they include biomarker - immunoglobulin m ( igm ) complexes , which have been found in several neoplastic diseases , such as colorectal , liver , and prostate cancer [ 13 ] .
although they are supposed to be a valuable adjunct in differential diagnostics , the biological meaning of this kind of complexes has not been elucidated ; that is , it is not known whether they have any physiological role .
in addition , from the biochemical point of view , there is no insight into their composition in terms of the structural properties of the molecules recognized by igm .
prostate - specific antigen ( psa ) , a well - known tumor marker for prostate cancer ( pca ) , has been found complexed with igm in both benign prostatic hyperplasia ( bph ) and cancer . in general , psa comprises heterogeneous molecules differing in primary structure and in glycan composition .
structural variability exists among psa forms in serum , seminal plasma , and hyperplastic or cancerous tissues [ 46 ] .
thus , using different experimental approaches , more than 30 immunoreactive glycoisoforms ranging in molecular mass from 6 to 35 kd have been separated from serum , seminal plasma , or prostate tissue [ 712 ] .
investigation of the structural heterogeneity of psa revealed that some distinct forms are more frequently associated with pca than with bph [ 1315 ] .
for example , some isoforms of precursor or pro - psa are cancer specific , whereas other internally cleaved or nicked ( multichain ) psa isoforms are characteristic for bph [ 16 , 17 ] .
this study was aimed at complementing existing data on psa - igm by defining molecular species of psa in circulating immune complexes .
having in mind the oligoreactivity and multivalency of igm and its preference for carbohydrate antigens , there is the possibility that it can selectively recognize known psa glycoisoforms [ 18 , 19 ] . beyond the clinical relevance for cancer detection
, this might be of basic importance , since tumor - associated antigens are known to be targets of immune surveillance [ 20 , 21 ] .
circulating psa - igm complexes separated by gel filtration from subjects with bph and pca were subjected to on - chip immunoaffinity profiling using monoclonal anti - psa antibodies .
the detected molecular species were subsequently analyzed for protein and glycan composition and compared to established psa forms .
monoclonal anti - free psa antibody , clone 8a6 ( recognizing epitope i ) , was purchased from hy test ( turku , finland ) .
monoclonal anti - psa antibody , clone 8311 ( recognizing free and complexed psa forms ) , was from medix biochemica ( kauniainen , finland ) .
alkaline phosphatase - conjugated anti - human igm and p - nitrophenyl phosphate ( pnpp ) were from institut virion\serion gmbh ( wrzburg , germany ) .
broad range sds - page molecular mass standards and silver stain kit were purchased from bio - rad ( hercules , ca , usa ) .
proteinchip : ps20 ( preactivated surface ) , q10 ( strong anion exchanger ) , cm10 ( weak cation exchanger ) , sinapinic acid , and proteinchip all - in - one protein standards ii were from bio - rad ( hercules , ca , usa ) .
microwell plates were from nunc ( roskilde , denmark ) and star - bottom tubes were from spektar ( aak , serbia ) .
total psa ( tpsa ) and free psa ( fpsa ) were quantitated using the appropriate commercially available irma psa assays ( inep , serbia ) according to the manufacturer 's instructions .
serum samples from subjects with benign prostatic hyperplasia ( bph ) and prostate cancer ( pca ) seen at inep , zemun , for psa determination as a part of followup , were obtained according to local ethical standards ( document gsp/05 and pr030/09 , approved by the institutional committee of inep ) .
diagnoses were confirmed using clinically established protocols based on psa level , physical examination , ultrasound , and biopsy .
individual serum samples from 18 patients with pca and bph were randomly used ( to reflect intrinsic heterogeneity ) to form three pools ( each n = 6 ) .
psa concentration ranges of the pca pools were 15.6776 g / l ; 35.4987.2 g / l and 20.73063.3 g / l .
psa concentration ranges of the bph pools were 0.85.1 g / l ; 0.42.5 g / l and 0.35.1 g
serum samples ( 400 l ) were applied to a sephacryl s-300 column ( 1.3 25 cm ) equilibrated with 0.05 m pbs , ph 7.2 , and eluted with the same buffer and 1 ml fractions were collected .
psa - igm or psa was detected in the expected fractions , using solid phase assays with immobilized monoclonal anti - psa antibody .
antibody - coated tubes ( 1.2 g per tube ) were prepared by physical adsorption , overnight at 4c . after washing with 0.05 m pbs , ph 7.2 ,
tubes were blocked with 0.5% casein for 3 h at 37c and rinsed again .
( a ) gel filtration fractions ( fractions 120 ) were added ( 100 l ) to coated tubes and incubated for 4 h at room temperature ( rt ) .
after washing thrice with 0.05 m pbs , ph 7.2 , alkaline phosphatase - conjugated anti - human igm ( 100 l ) was added and incubated for 2 h at rt .
unbound conjugate was washed , and 100 l of pnpp substrate solution was added .
the reaction was quenched with 100 l 1 m naoh after 30 min .
the absorption was measured at 405 nm using a wallac 1420 multilabel counter ( perkin elmer , monza , italy ) .
fractions containing psa - igm were concentrated and used for further analysis . in parallel ,
the total igm was detected in void volume gel filtration fractions after direct immobilization on polystyrene microwell plates , using alkaline phosphatase - conjugated anti - human igm , as described above .
( b ) psa was probed by the addition of corresponding gel filtration fractions ( 100 l of fractions 130 ) to anti - psa coated tubes following simultaneous incubation with iodinated anti - psa antibody ( 300000 cpm / tube ) , overnight at rt . after washing steps
, bound radioactivity was detected on a wallac wizard 1470 automatic -counter ( perkin elmer , waltham , usa ) .
samples of igm or psa - igm preparations ( 10 l ) were subjected to immunoelectrophoresis on 1% agar gel for 1.5 h at 4c .
purified sheep anti - human igm ( inep , zemun , serbia ) was used for detection .
psa fractions ( psa - igm or fpsa ) separated by gel filtration were profiled using on - chip immobilized monoclonal anti - psa antibodies , as described .
the corresponding antibody ( 5 l ) was applied to each spot of the preactivated surface protein chip array and incubated in a humid chamber overnight at 4c .
the spots were washed thrice with 5 l of 0.05 m pbs ,
ph 7.2 for 1 min at rt and then blocked with 0.05 m tris - hcl , ph 8.0 buffer for 1 h in a humid chamber at rt . after washing three times using the same procedure , 5 l of examined sample was added to each spot and incubated for 2 h in a humid chamber at rt .
the spots were rinsed again thrice with 0.05 m pbs , ph 7.2 , and twice with deionized water .
all procedures included shaking ( 150 rpm ) . after drying , 1 l of 50% sinapinic acid in acetonitrile / dh2o / trifluoroacetic acid ( 50%/49.9%/0.1% ) was added to each spot , dried , and then reapplied .
nonspecific binding was monitored using igm ( figure 1 , fraction 10 ) as control sample .
protein chips were analyzed by surface - enhanced laser desorption / ionization time of flight mass spectrometry ( seldi - tof / ms ) using the proteinchip reader , series 4000 , personal edition ( bio - rad ) .
all spectra were acquired in 25 kv positive ion acquisition mode , in mass range 2.5300 kda , and with 8815 laser shots per spot .
all spectra were analyzed using ciphergen express software 3.0 ( bio - rad , hercules , ca , usa ) .
the spots with incorporated quaternized ammonium groups ( q10 , strong anion exchanger ) or carboxymethyl groups ( cm 10 , weak cation exchanger ) were prewetted twice with 5 l of 0.05 m tris - hcl , ph 8 , for 5 min at rt . after removing the prewetting buffer , 5 l of the examined sample ( bph fpsa or pca fpsa ) was added to each spot and incubated for 1.5 h in a humid chamber at rt .
the spots were rinsed thrice with 0.05 m tris - hcl , ph 8 , and twice with deionized water .
after drying , 1 l of 50% sinapinic acid in acetonitrile / dh2o / trifluoroacetic acid ( 50%/49.9%/0.1% ) was added to each spot , dried , and then reapplied .
a representative psa - immunogram , revealing typical differential elution positions of the main serum fractions , is shown in figure 1 .
psa - igm ( b ) is eluted at the trailing edge of the total igm peak ( a ) .
as deduced from its elution position and immunoelectrophoretic appearance ( data not shown ) , psa - immunoreactivity of igm complexes seemed to be partly associated with low molecular mass igm .
gel filtration profiles of several individual runs of pooled pca and bph sera gave no striking difference in psa - igm peaks , but psa - act ( c ) and fpsa ( d ) peaks were considerably lower in bph , as expected .
psa - igm preparations from pooled pca and bph sera were further subjected to on - chip immunoaffinity chromatography , to determine psa forms in the immune complexes .
typical mass spectra of psa - igm preparations from bph and pca sera , profiled using anti - fpsa antibody , are shown in figure 2 and table 1 .
the general pattern was similar , comprising dominant peaks at 17 kda and 28 kda .
the main difference was the presence of a 26889.27 da species in pca psa - igm absent from bph psa - igm .
this appeared as doubly charged ion , which might be related to its structural properties . in comparison to the species at 26.8 kda ,
the observation of minor peaks at 15.8 kda or at 16.0 kda which have close molecular masses was not considered as relevant difference .
in addition to anti - fpsa antibody , anti - psa antibody , recognizing both free and complexed forms , was also used for on - chip chromatography .
comparable spectra were obtained but the intensity and frequency of particular peaks differed slightly . however
, this did not additionally contribute to separation and individual characterization of psa ( data not shown ) .
psa - immunoreactive forms complexed with igm , in both bph and pca , overlapped distinct molecular species observed in mass spectra of fpsa fraction ( figure 3 ) .
cluster at 28 kda was common for both pca psa - igm ( figure 2(a ) ) and pca fpsa ( figure 3(a ) ) , whereas the 15.8 kda species was more abundant in pca fpsa compared to pca psa - igm .
as for bph , fpsa fraction displayed a more restricted pattern with the main forms at 27595.81 da and 28244.95 da ( figure 3(b ) ) in contrast to bph psa - igm ( figure 2(b ) ) . taking the mass spectra of psa - igm preparations obtained from different pools of sera together , subtle differences ( some peaks were occasionally present ) were noticeable but they all revealed psa separation into several peaks ; that is , they suggested the presence of different psa glycoisoforms in immune complexes with igm .
taking into account the theoretical mass of psa peptides and their carbohydrate moiety , the observed psa - igm species were matched to amino acid sequence and glycan composition of established psa isoforms ( table 2 ) .
the deduced glycan masses of dominant peaks at 17 kda in bph psa - igm and pca psa - igm ranged between 1121 and 1259 da .
the distinct pca psa species at 26889.27 is supposed to contain trimannosyl core / paucimannosidic structures ( mm ) , based on the deduced glycans mass of 800.16 da .
psa species at 28 kda are assessed to contain fucosylated or nonfucosylated monosialylated biantennary chain with terminal n - acetylglucosamine ( gnnaf / nagnf ) or galactose ( naa / ana ; anaf / naaf ) .
in addition , the modification through bisecting glucosaminylation ( nagnfbi / gnnafbi ) is also suggested .
28.9 kda peaks in both bph and pca have estimated glycan mass of 2817 da .
this is higher than mass of complete psa glycan ( 2352 da ) , and it might be assigned to triantennary oligosaccharide chains . due to the limited amounts of particular psa species for direct analysis , the predictions were validated using ion - exchange chromatography of uncomplexed psa as reference . taking into account ,
known data on isoelectric point of psa glycoisoforms ( pi 6.97.7 ) , corresponding samples were separated on anion and cation exchangers at ph 8.0 .
the results obtained indicated that incompletely glycosylated psa ( 17 kda ) showed weak binding to both ionic exchangers in contrast to sialylated forms ( 28 kda ) which bound strongly to anionic exchanger ( figure 4 ) .
thus , the observed charge heterogeneity of psa molecular species was in agreement with their predicted various degrees of glycosylation .
according to the observed molecular masses , psa has been classified into two more or less heterogeneous groups of glycosylated ( gp28 , gp22 , gp18 , and gp12 ) or nonglycosylated ( p26-full length nonglycosylated psa , p20 , p16 , p10 , and p6 ) peptides .
lower molecular mass psa results from protein cleavage at several different sites , among which major forms comprise those cuts at either lys182/ser183 or lys145/ser146 or at both residues [ 4 , 16 ] .
our results indicate that the major molecular species of pca psa and bph psa found complexed with igm corresponded to gp18 , which is glycosylated psa amino acid residues 1145 .
in addition , minor forms corresponding to the entire mature glycosylated psa species , that is , gp28 , were also observed .
psa is defined as a 28.430 da glycoprotein containing 8% of carbohydrates mainly as a biantennary n - linked oligosaccharide of n - acetyllactosamine type with sialic acid group at the end of each of the two branches . however
, available data indicate that there is pronounced heterogeneity under both normal and pathological conditions .
psa isoforms differ , generally , in sialic acid content as well as outer chain features [ 2325 ] .
besides biantennary chains , monoantennary sugar chains and peripheral structures composed only of n - acetylhexosamine residues also occur .
the results obtained in this study indicated that psa species complexed with igm mostly contained incomplete glycan chain lacking sialic acid .
thus , the main psa species complexed with igm are assessed to have oligomannosidic structure in both bph and pca . in addition ,
alterations in psa glycosylation were reported in both bph and pca [ 2630 ] . specifically , psa forms with reduced sialylation , fucosylation changes , and increased pi were found in pca . as for unusual mannosidic structures found in psa - igm ,
it represented a minor fraction , that is , only 0.6% of the total psa isoforms , and comprised four psa species ranging from 26808.9 to 27295.8 da .
it seems that newly found igm - bound isoforms could have diagnostic potential to distinguish pca and bph , but with no advantage in comparison to other established parameters ( total psa - igm or fpsa / psa ) . due to intrinsic heterogeneity of total psa - igm and complexity of the method applied ,
their possible relevance should be confirmed by testing different types of samples , using different experimental designs suitable for clinical use .
however , the finding of distinct igm - bound psa forms may be of interest in light of recent data indicating paucimannose as marker of human cancers [ 31 , 32 ] .
in contrast to these poorly glycosylated psa species found in complex with igm , the main fpsa species contained predominantly mono- or disialylated biantennary chains .
the fpsa fraction was used as the reference , because it is supposed to comprise an abundant species that can be recognized by igm .
psa complexed with act was not considered relevant for this interaction , since it predominantly contains covalently bound enzymatically active psa .
psa glycosylation is very important for its biomarker potential and this was investigated in a number of studies under diverse physiological and pathological conditions .
generally , an examination of psa glycosylation , especially numerous psa species / glycoforms , is faced with the problem of small amounts , preventing isolation in sufficient quantity for glycan analysis .
in addition , the limitation is related to the known influence of both glycan and peptide on psa behavior during electrophoretic and chromatographic separations . using data on the psa standard preparation obtained by mass spectrometry , assessment of psa glycosylation by calculation of glycan mass
has already been proved relevant . in this study , this was combined with on - chip immunoaffinity chromatography and ion - exchange chromatography , which enables simultaneous sensitive detection of various psa glycoforms .
thus , despite the differences in methods and detection systems used for this analysis and those reported in the reference literature , generalizations about the composition of psa isoforms , based on congruence in patterns of protein and glycan moieties , can be made .
differences between calculated and theoretical masses extended to 0.1 kda , which may be due to common ion adducts observed in mass spectrometry .
however , for the psa species at 28.9 da , the difference was significant .
psa species having masses higher than 28.4 kda are also reported in seminal plasma and serum , but they are not characterized in detail [ 6 , 8 , 11 , 12 ] .
their relation to psa forms containing multiantennary chains or to pro - psa , as suggested by some authors , can not be confirmed or denied .
it is known that the structural integrity of psa significantly influences its biological activity and ability to interact with different molecules . as the first line of defense
, igm has a special position in the immune system and the discovery of psa - igm complexes opens an interesting point beyond its biomarker potential . is the binding of modified carbohydrates , as in the case of psa , a general principle , not only of basic , but also of clinical interest , for instance , in relation to cancer immunotherapy ?
different active and passive vaccines , including carbohydrate - based vaccines to aberrant glycostructures , are under clinical trials but have had limited success at proof of principle and efficacy stages .
characteristic psa glycoforms bound to igm , as emerged from this study , direct further investigations to structural comparison of different tumor markers found in immune complexes and their free forms and hold promise for advancement of their use as targets for cancer detection and therapy .
the results obtained indicated distinct differences in immuno - complexed pca psa compared to bph psa , although both were predominantly composed of nonsialylated incompletely glycosylated molecular species .
it poses the question of the possible role of glycosylation as a framework for immunoreactivity and immunoediting , representing a mechanism for early elimination or inactivation of aberrant cancer molecules and cells . | this study was aimed at defining molecular species of prostate - specific antigen ( psa ) in immune complexes with immunoglobulin m ( igm ) . having in mind the oligoreactivity of igm and its preference for carbohydrate antigens
, there is the possibility that it can selectively recognize known psa glycoisoforms .
psa - igm complexes and free psa fractions were separated from the sera of subjects with prostate cancer ( pca ) and benign prostatic hyperplasia ( bph ) by gel filtration and subjected to on - chip immunoaffinity and ion - exchange chromatography .
psa - immunoreactive species were detected using surface - enhanced laser desorption / ionization time of flight mass spectrometry .
the obtained spectra were analyzed for protein and glycan composition .
the general pattern of the molecular species of pca psa and bph psa found in complexes with igm was similar .
it comprised major peaks at 17 kda and minor peaks at 28 kda , corresponding to the entire mature glycosylated psa .
the main difference was the presence of incompletely glycosylated 26.8 kda species , having putative paucimannosidic structures , observed in pca psa - igm , but not in bph psa - igm .
characteristic pca psa - igm glycoforms pose the question of the possible role of glycosylation as a framework for immune surveillance and may be of interest in light of recent data indicating mannose - containing glycans as cancer biomarker . |
the design and baseline characteristics of the whi have been described in detail elsewhere ( 8) . in brief , the whi enrolled postmenopausal women 5079 years of age who provided written informed consent and had an expected survival and local residency of 3 years .
exclusion criteria included current alcoholism , drug dependency , dementia , or other conditions that would limit full participation in the study .
a total of 161,808 women ( 68,132 in clinical trials and 93,676 in an observational study ) were enrolled in the whi between 1993 and 1998 .
data used in this report were obtained from women at baseline and then at periodic follow - up points over an average of 10.4 years . the protocol and consent forms were approved by the institutional review boards of all participating institutions .
prevalent diabetes was defined as a self - report at baseline of ever having received a physician diagnosis of diabetes when not pregnant .
incident diabetes was based on data collected at annual follow - up visits at which participants were asked , since the date given on the front of this form , has a doctor prescribed any of the following pills or treatments ? choices included
cases of incident treated diabetes reported as of 31 august 2009 were included in the analysis .
a study of the accuracy of self - reported diabetes conducted in a subset of whi participants indicated that self - reported disease status was a reasonably valid indicator of diagnosed diabetes when compared with medication and laboratory criteria ( 9 ) .
however , self - report fails to identify undiagnosed diabetes and tends to underestimate diabetes prevalence and incidence in our study group ( 9 ) . at baseline , whi participants
self - reported their race / ethnicity , choosing from non - hispanic white ( referred to hereafter as white ) , non - hispanic black ( referred to hereafter as black ) , hispanic , american indian / alaska native , asian ( ancestry was chinese , indo - chinese , korean , japanese , pacific islander , or vietnamese ) , and other .
we excluded 1,849 women who reported race / ethnicity as other and 413 women without race / ethnicity information .
the sample size was limited among american indians ( n = 714 ) ; therefore , analyses were restricted to whites , blacks , hispanics , and asians .
body weight , height , and waist circumference were measured at baseline and year 3 .
demographic and health history data were self - reported at baseline and included age , years of education , cigarette smoking status , family history of diabetes , and hormone therapy use .
the baseline physical activity questionnaire asked about usual frequency and duration of several types of recreational and household activities using a standardized classification of physical activity intensity ( 10 ) .
total energy expenditure was summarized in metabolic equivalent ( met ) hours per week ( met - h / week ) , computed as the summed product of frequency , duration , and intensity for reported activities .
participants also completed a standardized food frequency questionnaire ( ffq ) developed for the whi to estimate average daily nutrient intake over the 3-month period prior to baseline visit ( 11 ) .
dietary quality , assessed by the alternate healthy eating index ( ahei ) ( 12,13 ) , was computed based on food items and nutrients derived from the ffq , including 1 ) fruit , 2 ) vegetables , 3 ) nuts and legumes , 4 ) ratio of white to red meat , 5 ) total dietary fiber , 6 ) trans - fat , 7 ) ratio of polyunsaturated fat to saturated fat , 8) alcohol , and 9 ) multivitamin use .
the reliability and validity of physical activity measurements were assessed among a random sample of 536 participants by a second measure of physical activity 10 weeks after the first measure . the test - retest reliability ( weighted ) for the physical activity variables ranged from 0.53 to 0.72 , and the intraclass correlation for the total physical activity variable was 0.77 ( 8) . for the ffq ,
we assessed bias and precision of the ffq by comparing the intake of 30 nutrients estimated from the ffq with means from four 24-h dietary recalls and a 4-day food record from 113 women who participated in the whi ( 11 ) . for most nutrients , means estimated by the ffq were within 10% of the records or recalls .
energy - adjusted correlation coefficients ranged from 0.2 ( vitamin b12 ) to 0.7 ( magnesium ) , with a mean of 0.5 .
we concluded that the ffq produced nutrient estimates , which were similar to those obtained in other studies comparing short - term dietary recall and recording methods .
we acknowledge that the reliability and validity are not perfect ; however , they provided reasonable measures in a large clinical trial , as published by several of our authors in high - impact articles ( 1416 ) .
racial / ethnic differences in the prevalence of diabetes were assessed using logistic regression and are expressed as odds ratios ( ors ) and associated 95% cis , with whites used as the reference group .
we present four logistic regression models : 1 ) unadjusted , including only race / ethnicity as a covariate ; 2 ) age - adjusted ; 3 ) adjusted for multiple potential confounding factors , including study arm , baseline age , bmi , waist circumference , physical activity , dietary quality , and smoking status ; and 4 ) adjusted for the same covariates as in model 3 plus educational attainment . among women who were free of diabetes at baseline ,
incidence rate was calculated as the number of newly reported postbaseline diabetes cases divided by total follow - up time in person - years .
the time to diabetes ( i.e. , time to event ) was calculated as the interval between enrollment date and the earliest of the following : 1 ) date of annual medical history update when new diagnosis of diabetes and initiation of treatment for diabetes were ascertained ( observed event ) ; 2 ) date of last annual medical update during which participants were identified to be without diabetes ( censored event ) ; or 3 ) date of death from any cause ( censored event ) .
cox proportional hazards models were used to estimate the hazard ratios ( hrs ) and associated 95% ci of incident diabetes , with whites used as the reference group .
four cox regression models that parallel those described for the analysis of diabetes prevalence are presented . to identify determinants of diabetes incidence that might vary by racial / ethnic group ,
the impact of several covariates was evaluated by assessing the extent to which the hr estimate for a specific race / ethnicity group changed when each covariate was added individually to the unadjusted model . the percentage change in hr between the model with and without the covariate was used to describe the contribution of each covariate , considered singly , to diabetes risk estimated by race / ethnic group . to identify the most parsimonious prediction model ,
a stepwise cox proportional hazards regression analysis was conducted ( p values for entry = 0.25 and for retaining in the model = 0.05 ) .
subgroup analyses also were conducted by race / ethnicity . weight change from baseline to 3 years was evaluated in relation to risk of incident disease by race / ethnicity among women who reported being free of diabetes at the 3-year measurement point .
a similar analysis was conducted for waist circumference . to assess how the combined role of healthy lifestyle habits in diabetes prevention may vary by race / ethnicity ( 6,7 ) ,
categories of four modifiable lifestyle factors were defined as follows : physical activity ( upper vs. lower two tertiles ) , dietary quality score ( upper vs. lower two tertiles ) , smoking ( nonsmokers vs. past and former smokers combined ) , and bmi ( < 25 vs. 25
kg / m ) . in subgroup analyses by race / ethnicity , hrs and 95% cis were calculated for diabetes incidence for each lifestyle risk factor individually and several lifestyle factors in aggregate , adjusting for age , family history of diabetes , hormone therapy use , study arm , and lifestyle risk factors not already included in the model .
the design and baseline characteristics of the whi have been described in detail elsewhere ( 8) . in brief , the whi enrolled postmenopausal women 5079 years of age who provided written informed consent and had an expected survival and local residency of 3 years .
exclusion criteria included current alcoholism , drug dependency , dementia , or other conditions that would limit full participation in the study .
a total of 161,808 women ( 68,132 in clinical trials and 93,676 in an observational study ) were enrolled in the whi between 1993 and 1998 .
data used in this report were obtained from women at baseline and then at periodic follow - up points over an average of 10.4 years . the protocol and consent forms were approved by the institutional review boards of all participating institutions .
prevalent diabetes was defined as a self - report at baseline of ever having received a physician diagnosis of diabetes when not pregnant .
incident diabetes was based on data collected at annual follow - up visits at which participants were asked , since the date given on the front of this form , has a doctor prescribed any of the following pills or treatments ? choices included
cases of incident treated diabetes reported as of 31 august 2009 were included in the analysis .
a study of the accuracy of self - reported diabetes conducted in a subset of whi participants indicated that self - reported disease status was a reasonably valid indicator of diagnosed diabetes when compared with medication and laboratory criteria ( 9 ) .
however , self - report fails to identify undiagnosed diabetes and tends to underestimate diabetes prevalence and incidence in our study group ( 9 ) .
at baseline , whi participants self - reported their race / ethnicity , choosing from non - hispanic white ( referred to hereafter as white ) , non - hispanic black ( referred to hereafter as black ) , hispanic , american indian / alaska native , asian ( ancestry was chinese , indo - chinese , korean , japanese , pacific islander , or vietnamese ) , and other .
we excluded 1,849 women who reported race / ethnicity as other and 413 women without race / ethnicity information .
the sample size was limited among american indians ( n = 714 ) ; therefore , analyses were restricted to whites , blacks , hispanics , and asians .
body weight , height , and waist circumference were measured at baseline and year 3 .
demographic and health history data were self - reported at baseline and included age , years of education , cigarette smoking status , family history of diabetes , and hormone therapy use .
the baseline physical activity questionnaire asked about usual frequency and duration of several types of recreational and household activities using a standardized classification of physical activity intensity ( 10 ) .
total energy expenditure was summarized in metabolic equivalent ( met ) hours per week ( met - h / week ) , computed as the summed product of frequency , duration , and intensity for reported activities .
participants also completed a standardized food frequency questionnaire ( ffq ) developed for the whi to estimate average daily nutrient intake over the 3-month period prior to baseline visit ( 11 ) .
dietary quality , assessed by the alternate healthy eating index ( ahei ) ( 12,13 ) , was computed based on food items and nutrients derived from the ffq , including 1 ) fruit , 2 ) vegetables , 3 ) nuts and legumes , 4 ) ratio of white to red meat , 5 ) total dietary fiber , 6 ) trans - fat , 7 ) ratio of polyunsaturated fat to saturated fat , 8) alcohol , and 9 ) multivitamin use .
the reliability and validity of physical activity measurements were assessed among a random sample of 536 participants by a second measure of physical activity 10 weeks after the first measure . the test - retest reliability ( weighted ) for the physical activity variables ranged from 0.53 to 0.72 , and the intraclass correlation for the total physical activity variable was 0.77 ( 8) . for the ffq ,
we assessed bias and precision of the ffq by comparing the intake of 30 nutrients estimated from the ffq with means from four 24-h dietary recalls and a 4-day food record from 113 women who participated in the whi ( 11 ) . for most nutrients , means estimated by the ffq were within 10% of the records or recalls .
energy - adjusted correlation coefficients ranged from 0.2 ( vitamin b12 ) to 0.7 ( magnesium ) , with a mean of 0.5 .
we concluded that the ffq produced nutrient estimates , which were similar to those obtained in other studies comparing short - term dietary recall and recording methods .
we acknowledge that the reliability and validity are not perfect ; however , they provided reasonable measures in a large clinical trial , as published by several of our authors in high - impact articles ( 1416 ) .
racial / ethnic differences in the prevalence of diabetes were assessed using logistic regression and are expressed as odds ratios ( ors ) and associated 95% cis , with whites used as the reference group .
we present four logistic regression models : 1 ) unadjusted , including only race / ethnicity as a covariate ; 2 ) age - adjusted ; 3 ) adjusted for multiple potential confounding factors , including study arm , baseline age , bmi , waist circumference , physical activity , dietary quality , and smoking status ; and 4 ) adjusted for the same covariates as in model 3 plus educational attainment . among women who were free of diabetes at baseline ,
incidence rate was calculated as the number of newly reported postbaseline diabetes cases divided by total follow - up time in person - years .
the time to diabetes ( i.e. , time to event ) was calculated as the interval between enrollment date and the earliest of the following : 1 ) date of annual medical history update when new diagnosis of diabetes and initiation of treatment for diabetes were ascertained ( observed event ) ; 2 ) date of last annual medical update during which participants were identified to be without diabetes ( censored event ) ; or 3 ) date of death from any cause ( censored event ) .
cox proportional hazards models were used to estimate the hazard ratios ( hrs ) and associated 95% ci of incident diabetes , with whites used as the reference group .
four cox regression models that parallel those described for the analysis of diabetes prevalence are presented . to identify determinants of diabetes incidence that might vary by racial / ethnic group ,
the impact of several covariates was evaluated by assessing the extent to which the hr estimate for a specific race / ethnicity group changed when each covariate was added individually to the unadjusted model . the percentage change in hr between the model with and without the covariate was used to describe the contribution of each covariate , considered singly , to diabetes risk estimated by race / ethnic group . to identify the most parsimonious prediction model ,
a stepwise cox proportional hazards regression analysis was conducted ( p values for entry = 0.25 and for retaining in the model = 0.05 ) .
subgroup analyses also were conducted by race / ethnicity . weight change from baseline to 3 years was evaluated in relation to risk of incident disease by race / ethnicity among women who reported being free of diabetes at the 3-year measurement point .
a similar analysis was conducted for waist circumference . to assess how the combined role of healthy lifestyle habits in diabetes prevention may vary by race / ethnicity ( 6,7 )
, categories of four modifiable lifestyle factors were defined as follows : physical activity ( upper vs. lower two tertiles ) , dietary quality score ( upper vs. lower two tertiles ) , smoking ( nonsmokers vs. past and former smokers combined ) , and bmi ( < 25 vs. 25
kg / m ) . in subgroup analyses by race / ethnicity , hrs and 95% cis
were calculated for diabetes incidence for each lifestyle risk factor individually and several lifestyle factors in aggregate , adjusting for age , family history of diabetes , hormone therapy use , study arm , and lifestyle risk factors not already included in the model .
at baseline , the average age of the 158,833 women with evaluable data was 63 years .
the racial / ethnic distribution was 84.1% white , 9.2% black , 4.1% hispanic , and 2.6% asian .
blacks and hispanics tended to have more risk factors , whereas asians tended to have fewer ( table 1 ) .
baseline characteristics of participants in the whi 19932009 ( n = 158,833 ) at enrollment , diabetes prevalence was highest among blacks ( 12.2% ) , followed by hispanics ( 7.2% ) , asians ( 5.9% ) , and whites ( 3.3% ) ( table 2 ) .
compared with the unadjusted ors , which were significantly higher in all three race / ethnic groups when compared with whites , the age - adjusted and multivariable - adjusted ors were attenuated for blacks and hispanics but strengthened for asians . additional adjustment for educational attainment further attenuated the ors for blacks and hispanics ; however , ors for all three racial / ethnic minority groups remained significantly higher than that of whites .
prevalence and incidence of diabetes in the whi 19932009 during an average of 10.4 years ( sd = 3.2 ) of follow - up , 14,604 new cases of diabetes were reported ( 11,127 whites , 2,181 blacks , 879 hispanics , and 417 asians ) , with incidence being highest in blacks and lowest in whites . compared with whites ,
unadjusted analyses showed a significantly higher diabetes incidence of 136% in blacks , 114% in hispanics , and 43% in asians . after adjusting for age , study arm , bmi , physical activity , smoking status , and educational attainment ,
1 ) , the highest cumulative incidence of diabetes was seen among women who had both high bmi and low levels of physical activity .
the cumulative incidence of diabetes was 23.4% among black women who were both obese and in the lowest tertile of physical activity , but decreased to 8.8% in those who had a healthy weight and exercised .
white women who were overweight ( bmi = 2529.9 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 8.7% , whereas white women who were obese ( bmi 30 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 18.6% . across all racial / ethnic groups , women who were normal weight ( bmi < 25 kg / m ) and in the highest tertile of physical activity had less than one - third to one - sixth the incidence of diabetes compared with women with bmi 30 kg / m and in the lowest tertile of physical activity .
cumulative incidence of diabetes by race / ethnicity , physical activity level , and bmi category .
analyses conducted to estimate the influence of individual factors that may account for the observed racial / ethnic variation in diabetes incidence by comparing hrs obtained from unadjusted versus fully adjusted models ( results not shown ) revealed that if asian women had the same waist circumference , bmi , and dietary quality intake as whites , their hr for diabetes would be increased by 44 , 29 , and 3.5% , respectively . among blacks ,
if they had the same bmi , waist circumference , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 25 , 19 , 11 , 7 , and 6% , respectively . among hispanics ,
if they had the same educational attainment , bmi , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 14 , 10 , 9 , 7 , and 6% , respectively .
results from the stepwise cox proportional hazards regression analysis conducted to identify the most parsimonious predictive model for diabetes incidence , fitting candidate predictors plus race / ethnicity , showed that race / ethnicity , age , bmi , waist circumference , education , smoking status , physical activity , family history of diabetes , and dietary quality score entered the model .
hrs of incident diabetes corresponding to single and specific combinations of lifestyle risk factors in race / ethnicity subgroups in the whi 19932009 subgroup analyses by race / ethnicity ( results not shown ) conducted to assess the effect of interval ( i.e. , baseline to 3 years ) changes in factors found to be significant in the cox proportional hazards models revealed a significant effect of 5% increased risk of diabetes for each 5-cm increase in waist circumference ( hr 1.05 [ 95% ci 1.041.07 ] ) . the observed effect was consistent across all race / ethnicity groups .
an alternative model fit to assess if weight gain could explain some of the observed racial / ethnic variation in diabetes incidence revealed an 3% increase in subsequent risk for each 1-kg / m increment in bmi ( 1.03 [ 1.021.04 ] ) .
higher levels of physical activity , better diet , and having a healthy weight tended to be associated with a significantly lower risk of diabetes in each racial / ethnic group , with some exceptions .
whites , blacks , and hispanics with all factors in the low - risk category ( 4.0 , 1.1 , and 2.2% ) had 60 , 69 , and 63% lower risk for incident diabetes ( table 3 ) .
healthy weight ( bmi < 25 kg / m ) demonstrated the greatest role in reducing risk of diabetes in each racial / ethnic group : 66% in whites , 55% in blacks , 64% in hispanics , and 66% in asians .
at baseline , the average age of the 158,833 women with evaluable data was 63 years .
the racial / ethnic distribution was 84.1% white , 9.2% black , 4.1% hispanic , and 2.6% asian .
blacks and hispanics tended to have more risk factors , whereas asians tended to have fewer ( table 1 ) .
at enrollment , diabetes prevalence was highest among blacks ( 12.2% ) , followed by hispanics ( 7.2% ) , asians ( 5.9% ) , and whites ( 3.3% ) ( table 2 ) . compared with the unadjusted ors , which were significantly higher in all three race / ethnic groups when compared with whites ,
the age - adjusted and multivariable - adjusted ors were attenuated for blacks and hispanics but strengthened for asians .
additional adjustment for educational attainment further attenuated the ors for blacks and hispanics ; however , ors for all three racial / ethnic minority groups remained significantly higher than that of whites .
prevalence and incidence of diabetes in the whi 19932009 during an average of 10.4 years ( sd = 3.2 ) of follow - up , 14,604 new cases of diabetes were reported ( 11,127 whites , 2,181 blacks , 879 hispanics , and 417 asians ) , with incidence being highest in blacks and lowest in whites . compared with whites ,
unadjusted analyses showed a significantly higher diabetes incidence of 136% in blacks , 114% in hispanics , and 43% in asians . after adjusting for age , study arm , bmi , physical activity , smoking status , and educational attainment ,
1 ) , the highest cumulative incidence of diabetes was seen among women who had both high bmi and low levels of physical activity .
the cumulative incidence of diabetes was 23.4% among black women who were both obese and in the lowest tertile of physical activity , but decreased to 8.8% in those who had a healthy weight and exercised .
white women who were overweight ( bmi = 2529.9 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 8.7% , whereas white women who were obese ( bmi 30 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 18.6% . across all racial / ethnic groups ,
women who were normal weight ( bmi < 25 kg / m ) and in the highest tertile of physical activity had less than one - third to one - sixth the incidence of diabetes compared with women with bmi 30 kg / m and in the lowest tertile of physical activity .
cumulative incidence of diabetes by race / ethnicity , physical activity level , and bmi category .
analyses conducted to estimate the influence of individual factors that may account for the observed racial / ethnic variation in diabetes incidence by comparing hrs obtained from unadjusted versus fully adjusted models ( results not shown ) revealed that if asian women had the same waist circumference , bmi , and dietary quality intake as whites , their hr for diabetes would be increased by 44 , 29 , and 3.5% , respectively . among blacks , if they had the same bmi , waist circumference , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 25 , 19 , 11 , 7 , and 6% , respectively . among hispanics ,
if they had the same educational attainment , bmi , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 14 , 10 , 9 , 7 , and 6% , respectively .
results from the stepwise cox proportional hazards regression analysis conducted to identify the most parsimonious predictive model for diabetes incidence , fitting candidate predictors plus race / ethnicity , showed that race / ethnicity , age , bmi , waist circumference , education , smoking status , physical activity , family history of diabetes , and dietary quality score entered the model .
hrs of incident diabetes corresponding to single and specific combinations of lifestyle risk factors in race / ethnicity subgroups in the whi 19932009 subgroup analyses by race / ethnicity ( results not shown ) conducted to assess the effect of interval ( i.e. , baseline to 3 years ) changes in factors found to be significant in the cox proportional hazards models revealed a significant effect of 5% increased risk of diabetes for each 5-cm increase in waist circumference ( hr 1.05 [ 95% ci 1.041.07 ] ) . the observed effect was consistent across all race / ethnicity groups .
an alternative model fit to assess if weight gain could explain some of the observed racial / ethnic variation in diabetes incidence revealed an 3% increase in subsequent risk for each 1-kg / m increment in bmi ( 1.03 [ 1.021.04 ] ) .
higher levels of physical activity , better diet , and having a healthy weight tended to be associated with a significantly lower risk of diabetes in each racial / ethnic group , with some exceptions .
whites , blacks , and hispanics with all factors in the low - risk category ( 4.0 , 1.1 , and 2.2% ) had 60 , 69 , and 63% lower risk for incident diabetes ( table 3 ) .
kg / m ) demonstrated the greatest role in reducing risk of diabetes in each racial / ethnic group : 66% in whites , 55% in blacks , 64% in hispanics , and 66% in asians .
previous reports , mainly focused on younger men and women , have indicated significant racial / ethnic disparities in diabetes in the u.s . ( 1,4,5 ) .
the 20072009 national health interview survey found that 7.1% of whites , 8.4% of asians , 11.8% of hispanics , and 12.6% of blacks reported having a diagnosis of diabetes ( 1 ) .
the whi allows a unique opportunity to deepen understanding of the patterns and determinants of diabetes in older women , including racial / ethnic minorities that represent growing segments of the u.s .
we found that the prevalence and incidence of diabetes ranged from approximately two to three times higher in blacks and approximately two times higher in hispanics and in asians , compared with whites . observed racial / ethnic differences in diabetes incidence
were explained , in large part , by modifiable lifestyle factors that included diet quality , physical activity , and smoking status or factors resulting from lifestyle behaviors including bmi and waist circumference .
adjustment for differences in these variables indicates that asians have the highest inherent risk , though women of all four racial / ethnic groups would experience a large reduction in diabetes risk by maintaining a healthy body weight , healthy diet , and a physically active lifestyle .
maintaining a bmi < 25 kg / m appears to be particularly important , and interval changes in both bmi and waist circumference predicted newly incident disease . in this study ,
both bmi and waist circumference were found to be related to prevalent diabetes , and interval changes were associated with risk of incident disease . because waist circumference reflects centralized obesity , and the propensity toward large waist circumference varies according to race / ethnicity , its effect may differ from that of bmi ( 17,18 ) . although bmi and waist circumference among asian , postmenopausal women in the whi were relatively lower than whites , asians were at higher risk of diabetes at lower levels of bmi and with smaller waist circumferences ( i.e. , by 44 and 29% , respectively ) compared with whites , a result consistent with that seen in the multi - ethnic study of atherosclerosis ( 19 ) .
our results on asians suggest that additional risk factors , which might be biological , social , or a combination , drive the higher prevalence and incidence of diabetes in this population .
diabetes risk in blacks with the same bmi and waist circumference as whites was lower by 25 and 17% , respectively .
similarly , diabetes risk in hispanics with the same bmi and waist circumference as whites was lower by 10 and 2% , respectively .
we noted that within each group , bmi was the most important determinant for diabetes incidence .
physical inactivity also was found to be associated with increased risk of diabetes in all groups . in a previous publication using whi data , we reported the association between physical inactivity and development of diabetes ( 20 ) .
our present study showed that , compared with whites , given the same level of physical activity , the risk for diabetes for blacks and hispanics is 6% lower .
several studies have found differences in dietary intake by race / ethnicity ( 26,27 ) .
although their diet quality was better than that of whites , we found , for example , if their overall dietary quality were to decrease to that of whites , their risk of diabetes would increase by 4% relative to whites .
in fact , if blacks and hispanics improved their diet to a quality similar to that of whites , their risk of diabetes would be 7% lower .
although previous studies demonstrated that diabetes is preventable by relatively simple lifestyle modifications ( 6,7 ) , our data suggest that some minority groups might obtain greater benefit from improving lifestyle factors ( i.e. , blacks and hispanics ) than others ( i.e. , asians ) due to differences in the amount of change possible or the vulnerability to these factors .
the divergent findings in asian women are of interest , suggesting that additional approaches to prevention deserve attention .
for example , asians may need to achieve even greater weight loss to have the same low risk of diabetes as nonoverweight whites , and the world health organization already uses a lower bmi cut point for asians .
the fact that asians in the whi had much healthier body weights at baseline set the stage for much lower overall diabetes risk .
however , further research with this population is warranted because our statistical models , with additional covariates , may be unstable and less reliable due to small numbers .
we found that blacks and hispanics are more sensitive to lifestyle modifications and weight loss than whites , and this is corroborated by our previous lifestyle intervention study results from a hispanic population ( 28 ) .
hispanic sensitivity to the development of an insulin - resistant state or diabetes with lower weight gain is well described ( 29 ) .
if confirmed in weight - loss studies , the sensitivity to modest weight loss that we observed in high - risk groups in the whi will have important clinical and public health implications .
it will also be important to explore possible social and genetic underpinnings for such population sensitivity .
two recent studies indicate that social economic status ( ses ) may account for some of the observed race / ethnicity disparities in diabetes prevalence ( 30,31 ) .
similarly , in our study , adjustment for educational attainment resulted in the largest decrease in diabetes incidence in hispanics ; if hispanics had achieved the same education levels as whites , their risk of diabetes would be 14% lower . however , education is only one component of ses , and thus consideration of an adjustment for other ses parameters ( e.g. , income and occupation ) would likely account for a greater proportion of the disparities observed in black and hispanic women .
first , observed prevalence of diabetes in the whi was lower than expected , as relatively healthy postmenopausal women were enrolled in the study .
second , reported education level among black women in the whi was considerably higher than education level among black women in the u.s .
the education differential for blacks and hispanics is much more extreme than that observed in whites , and this may underlie some of the other observations in the data ( 32,33 ) .
however , observed patterns are consistent with the population - based literature in terms of race / ethnic patterns of diabetes prevalence and incidence of diabetes .
third , asian women appear to have the greatest inherent risk in that their risk factor profile was in fact better than whites , and large increases in diabetes risk would occur if the risk factors deteriorate .
( 19 ) , which showed that asians had a higher diabetes risk per unit increase in bmi and waist circumference . recognizing that each of the racial / ethnic groups is heterogeneous due to differences by national origin and other relevant parameters
fourth , it is encouraging to note the effect of interval changes in weight amounting to a 3% reduction in risk of diabetes for each unit decrease in bmi .
individual efforts to reduce weight and increase physical activity are difficult to sustain ; however , current efforts are underway to tackle weight regulation through a variety of approaches that go beyond the individual .
lessons from the tobacco literature indicate that behavior change is influenced by our social and political structure ; thus , multilevel approaches are likely needed . in the u.s . , the race / ethnicity categories used most often in medical and public health research are from self - report , the same as the u.s . census categories . genetic race / ethnicity information known as admixture data ( i.e. , ancestry - informative markers ) from another study indicates great diversity within all four of the groups that we examined ( 34 ) .
yet , few studies obtain such data due to issues concerning cost , feasibility , practicality , and comparability . thus , the differences observed and reported here reflect any inherent biological differences across the groups studied as well as differences in life experiences ( e.g. , exposure to specific environments and stressors ) , which may also contribute to acquired physiological differences in reactivity , and in turn diabetes risk ( 35 ) .
the challenges for interpreting the results associated with studying self - reported racial / ethnic groups will likely increase in future studies , as more people identify themselves as belonging to multiple racial / ethnic groups .
although geographically diverse , racial / ethnic groups vary in their representation of the general population .
data for whites show that many characteristics of the whi participants are similar to white women participating in the national health and nutrition examination survey ( 11 ) ; however , ethnic groups are underrepresented in the whi .
women from parts of the country where we see large disparities in certain minorities ( e.g. , rural southern blacks ) were not represented .
thus , both prevalence and racial differentials are smaller in the whi than what we might expect to see in the u.s . as a whole .
second , only self - reported prevalence of diabetes and treated incident diabetes were ascertained ; thus , prevalence and incidence of diabetes may be underestimated .
we acknowledge that this is a limitation , and we did not account for nontreated diabetes .
however , self - reported diabetes in the whi was found to be reliable and sufficiently accurate to allow its use in epidemiologic studies ( 9 ) .
third , there could be other factors for which we did not control that further contribute to racial / ethnic disparities , such as health care access ( 36 ) . however , >
90% of the whi participants had insurance coverage , and diabetes prevalence and incidence were assessed at regular study visits .
fourth , although incident diabetes in older women is likely to be type 2 diabetes , the whi question did not specify type of diabetes .
other limitations include missing data ; however , the rates of retention in the whi were > 95% during an average of 7 years of follow - up ( 37 ) . balancing the limitations , there are several major strengths .
in addition , the whi collected detailed information on a comprehensive range of diabetes risk factors relevant to this investigation , with a 10-year follow - up for diabetes outcome . in conclusion ,
significant disparities exist between the major ethnic groups in diabetes prevalence and incidence in postmenopausal women ; these differences withstand adjustment for a very comprehensive group of physiological and behavioral risk factors .
although these results highlight the potential benefits of tailored diabetes prevention strategies directed at those specific factors that are most likely to increase the risk of diabetes among each racial / ethnic group , it is prudent to recommend avoidance of weight gain , weight loss , a healthy diet , and adequate levels of physical activity to all postmenopausal women for the purpose of diabetes risk reduction . | objectiveto examine determinants of racial / ethnic differences in diabetes incidence among postmenopausal women participating in the women s health initiative.research design and methodsdata on race / ethnicity , baseline diabetes prevalence , and incident diabetes were obtained from 158,833 women recruited from 19931998 and followed through august 2009 .
the relationship between race / ethnicity , other potential risk factors , and the risk of incident diabetes was estimated using cox proportional hazards models from which hazard ratios ( hrs ) and 95% cis were computed.resultsparticipants were aged 63 years on average at baseline .
the racial / ethnic distribution was 84.1% non - hispanic white , 9.2% non - hispanic black , 4.1% hispanic , and 2.6% asian .
after an average of 10.4 years of follow - up , compared with whites and adjusting for potential confounders , the hrs for incident diabetes were 1.55 for blacks ( 95% ci 1.471.63 ) , 1.67 for hispanics ( 1.541.81 ) , and 1.86 for asians ( 1.682.06 ) .
whites , blacks , and hispanics with all factors ( i.e. , weight , physical activity , dietary quality , and smoking ) in the low - risk category had 60 , 69 , and 63% lower risk for incident diabetes .
although contributions of different risk factors varied slightly by race / ethnicity , most findings were similar across groups , and women who had both a healthy weight and were in the highest tertile of physical activity had less than one - third the risk of diabetes compared with obese and inactive women.conclusionsdespite large racial / ethnic differences in diabetes incidence , most variability could be attributed to lifestyle factors .
our findings show that the majority of diabetes cases are preventable , and risk reduction strategies can be effectively applied to all racial / ethnic groups . |
cholelithiasis presented as chronic right back pain in the case discussed in this report . according to the clinical practice guidelines , gallbladder lithiasis is a common condition . around two - thirds of the cases
are asymptomatic , with the most frequent clinical presentation being the presence of attacks of acute abdominal pain . however , chronic right back pain persisting for months as a main symptom is uncommon .
this report presents a patient with right back and subscapular pain for 9 months , demonstrating both biliary lithiasis and chronic cholecystitis .
it is important to take into account the atypical manifestations of a prevalent illness as well as the limitations of the diagnostic tests , since the presence of gallstones can easily be missed or misinterpreted and delay in diagnosis may lead to complications which could worsen the prognosis of the patient .
a 47-year - old male presented with a history of chronic neck pain that had started 10 years back .
he underwent a magnetic resonance ( mr ) which showed cervical canal stenosis of degenerative etiology and also had an episode to self - limited abdominal spasmodic pain that appeared 10 years ago , considered as being functional by the gastroenterologist .
abdominal ultrasonography was performed twice , and both times it was normal . one and a half years ago , the patient had right subscapular and paralumbar back pain at the t10l2 level for 9 months .
he described it as a permanent pain , which persisted throughout the day , with very intense exacerbations , especially in the evening and at night .
these exacerbations usually lasted for 4 - 6 h , worsening on inhalation and sometimes radiating to subcostal and right hemithorax .
the only feature of note on physical examination was the palpation of painful spinal processes from t10 to l2 .
blood analysis showed the level of gamma - glutamyl traspeptidase to be 79 u / l , while all the other laboratory parameters were normal .
abdominal computed tomography ( ct ) showed an extensive hepatic steatosis and a moderately relaxed gallbladder , with no stones observed within thin walls [ figure 1 ] .
abdominal ct : gallbladder without gallstones despite the regular use of analgesics and the rehabilitation treatment that followed , the patient presented a torpid evolution . due to the pain in the right paralumbar area ,
an abdominal ultrasonography was performed , which showed moderate hepatic steatosis and a gallbladder with multiple inner calculi [ figure 2 ] .
on diagnosis of cholelithiasis , a cholecystectomy was carried out , which revealed a gallbladder with a large number of yellow lithiasic structures , thereby confirming an anatomopathological diagnosis of chronic cholecystitis [ figure 3 ] .
the previous pain completely disappeared after surgery and the patient remained asymptomatic 7 months later .
back pain affects 80% of adults at some point of their life and occurs at all ages .
the first objective is to rule out a visceral disorder or a serious potentially life - threatening condition , which occurs in 2.7% of the cases
. back pain may be the manifestation of thoracic involvement as in esophageal disease , pleurisy , aortic aneurysm , or coronary heart disease , or an abdominal process , such as gastrointestinal ulcer , gastric cancer , pancreatic cancer , pancreatitis , or biliary pathology .
only one - third of the cases of gallstones are symptomatic and give an indication for surgery .
the most frequent symptomatic manifestation is episodic upper abdominal pain called biliary colic , with complications such as cholecystitis , acute pancreatitis , cholangitis , or choledocholithiasis being less frequent .
characteristically , this pain is severe and located in the epigastrium and/or the right upper quadrant .
the pain may radiate to the upper back or the right scapula in 60% of the cases and might be associated with nausea or vomiting , usually occurring in the late evening or at night , in 80% of the cases .
one study carried out to characterize the presentation in cholelithiasis reported that all patients described pain in the right upper quadrant of the abdomen , including the epigastrium .
the pain was located in the right subcostal area ( 20% ) and epigastrium ( 14% ) , which radiated to the back in 63% .
in fact , chronic cholecystitis is thought to be a result of a delay in diagnosis .
the test of choice to diagnose cholelithiasis is transabdominal ultrasound , with a sensitivity and specificity greater than 95% for detecting gallstones larger than 4 mm .
abdominal ct scan has a very low sensitivity and gallstones may be visible due to most being isodense .
microlithiasis ( stones <3 mm in diameter ) is not detectable by transabdominal ultrasonography ; however , it may cause complications such as acute pancreatitis . in some patients
the diagnosis can be confirmed by the detection of biliary crystals by microscopic examination of bile collected from the duodenum . in patients with a clinical history of biliary colic with negative transabdominal ultrasonography ,
endoscopic ultrasonography is an important diagnostic tool , since it can detect microlithiasis in a proportion of patients ranging from 41 to 78% .
patients with pain and a negative abdominal ultrasound show an endoscopic ultrasound and/or a positive analysis of the bile .
most of these patients show positive results on cholecystectomy , with resolution of abdominal pain and improvement in their quality of life .
non - vertebral causes such as the presence of gallstones should be considered in patients with back pain .
physicians should be aware of atypical manifestations of common diseases , so as to avoid diagnostic delays .
gallbladder disorders should be taken into account in patients with dull , undefined right back pain , despite a normal physical examination .
abdominal ultrasound should be chosen as the first diagnostic procedure when considering gallstones , since ct scans have a very low sensitivity for their detection . in a patient with a typical clinical report of biliary colic and a negative abdominal ultrasound ,
this was the case of our patient in whom the abdominal ultrasonography was normal 5 years earlier , thereby making correct differential diagnosis crucial . | chronic right back pain is a symptom in both biliary lithiasis and chronic cholecystitis
. ten percent of the population in the world suffers from biliary lithiasis .
only 20% are symptomatic .
the first diagnostic test of choice is an abdominal ultrasound .
when a suggestive clinical sign of biliary colic with negative abdominal ultrasound is identified , we should consider the option of carrying out an endoscopic ultrasound in order to rule out microlithiasis .
the case discussed in the report presented with chronic right back pain , which is an atypical manifestation of biliary lithiasis and chronic cholecystitis .
it is important to know about the atypical manifestations of the prevalent illnesses as well as the limits of the diagnostic tests , in order to avoid diagnostic delays which may cause complications that could worsen a patient 's prognosis .
this case should contribute to the medical knowledge and must have educational value or highlight the need for a change in clinical practice , especially in primary care . |
the process of odontogenesis is under the control of homeobox ( hox ) genes ; a number of different mesenchymal regulatory molecules and their receptors .
hox genes are classified as muscle segment ( msx1 and msx2 ) , distal - less ( dlx ) , orthodontical , goosecoid , paired box gene 9 ( pax9 ) and sonic hedgehog ( shh ) .
msx1 and msx2 genes are responsible for the developmental position and further development of tooth buds , respectively .
pax9 is a transcription factor required for tooth morphogenesis and plays a role in the establishment of the inductive capacity of the tooth mesenchyme as it is necessary for the mesenchymal expression of bone morphogenetic protein ( bmp4 ) , msx1 and lef1 genes .
tumor necrosis factor , fibroblast growth factor , bmp , shh and wnt pathways are involved in signaling pathways of organogenesis on the 9 to 11 embryonic days to initiate tooth epithelium . any mutation in these genes and any disruption of regulatory molecules may result in the anomaly of dental characteristics .
hypodontia , oligodontia and anodontia are the terms used to describe the numerical values of tooth agenesis .
hypodontia is the absence of 1 - 6 teeth ( excluding third molars ) , whereas oligodontia refers to the absence of more than 6 teeth ( excluding third molars ) and anodontia is the complete absence of teeth .
hypodontia is frequently accompanied with cleft - lip or palate , reduction in tooth size , short root anomaly , malformation of other teeth , impaction , maxillary canine and first premolar transposition , delayed formation or eruption of other teeth , microdontia , taurodontism , enamel hypoplasia and altered craniofacial growth .
sometimes , it is caused by environmental factors such as ; infection , different kinds of trauma in the apical area of dentoalveolar process , chemical substances or drugs , radiation therapy or disturbances in the jaw innervations , but in the majority of cases , hypodontia is impacted by genetics .
autosomal dominant inheritance with incomplete penetrance and variable expressivity was first proposed by burzynski and escobar . for a genetic linkage study on hypodontia , arte et al .
, evaluated 214 family members in three generations and concluded that incisor - premolar agenesis is transmitted by autosomal dominant genes with small upper incisors , ectopic canines , taurodontism and rotated premolars as accompanying anomalies .
a familial autosomal dominant hypodontia was demonstrated to be caused by a point mutation in the msx1 gene .
vastardis et al . , using a linkage analysis in a family with second premolar and third molar agenesis , defined a locus on the chromosome 4p16 as the site of the msx1 .
sequence analysis revealed arg31-to - pro missense mutation in the msx1 homeodomain for all of the affected subjects .
arg31-to - pro mutation is known to influence the msx1 interactions , which are critical in the normal development of human teeth .
this finding supported the result of an animal study , which showed a knockout mutation of msx1 gene leading to the inhibition of dental development ; however , when the premolar agenesis was accompanied with a lateral incisor , the influence of msx1 and msx2 genes could not be proved .
another gene , causing tooth agenesis is pax9 in chromosome 14 ( 14q21-q13 ) . in hypodontia cases ,
a frameshift mutation and a nonsense termination mutation of the same gene have been observed in oligodontia . according to das et al .
, this situation suggests that hypodontia and oligodontia are not fundamentally different or at least can be caused by different mutations in the same gene .
the frameshift mutation of pax9 gene located on chromosome 14 was identified as responsible for autosomal dominant oligodontia in a large family for four generations . in some of the affected members , maxillary and mandibular second premolars and mandibular central incisors
were absent in addition to the lack of permanent molars ; although , a normal primary dentition was present . in 2001 , nieminen et al . , identified an a - to - t transversion of the pax9 gene in a family with autosomal dominant oligodontia .
they reported that all the second and third permanent molars , maxillary lateral incisors and in some cases all of the first permanent molars and several second premolars were missing , in addition to deciduous second molar agenesis .
a year later , frazier - bower et al . concluded that the molar oligodontia is due to allelic heterogeneity in pax9 , which is an important regulator of molar teeth development . in 2003 , lamni showed a distinct phenotype with canine agenesis , which is quite rare , in a family segregating autosomal dominant oligodontia .
in recent studies , different transition , insertion and transversion mutations in pax9 gene were reported , providing more comprehensive explanations for the pathogenic mechanisms of the interactions between mutations and tooth agenesis .
in addition to pax9 and msx1 genes , contribution of a regulatory molecule in the mesenchyme , called transforming growth factor alpha , might play a role in isolated dental agenesis .
furthermore , axin2 is a negative regulator of canonical wnt signaling and mutations of this gene can cause tooth agenesis associated with colorectal cancer .
another mode of inheritance for hypodontia associated with other dental anomalies such as dental malformations , enamel hypoplasia and eruption failure is the autosomal recessive inheritance , with polygenic inheritance also suggested .
studies showed that tooth agenesis could be manifested as an isolated feature or as a part of a syndrome .
tooth agenesis was associated with a large number of syndromes , which indicating that the development of teeth and certain organs are under the control of the same molecular mechanisms .
hypodontia is a major component of ectodermal dysplasia , oral - facial digital syndromes with oral - facial clefting .
ectodermal dysplasia is characterized by small , misshapen and missing teeth , delayed eruption , prominent lip , maxillary hypoplasia , sparse hair and hypohidrotic skin .
this disorder is inherited as an x - linked trait , though the autosomal recessive form has also been reported .
oral - facial digital syndrome type 1 has symptoms such as hypodontia of lower incisors , facial asymmetry , hypertelorism , micrognathia , supernumerary frenulum and thickened alveolar ridges .
oral - facial clefting is affected by chromosome 6p24 , 2p13 , 19q13 and 4q and the prevalence of hypodontia increases with cleft severity .
the upper lateral incisor is the most frequently affected tooth in the cleft area both in primary and in permanent dentitions . in syndromic cleft lip and palate ( clp ) like pierre - robin sequence 50% hypodontia and van der woude syndrome 70% hypodontia prevalence is associated with other disorders .
the genetic basis of hyperdontia has been extensively explained in the literature . in 1932 ,
brook reported a greater occurrence of supernumerary teeth in the close relatives of effected individuals , compared with the general population .
it was also reported that the supernumerary and impacted teeth occupied the same location in the identical twins and in their parents .
although there is no difference in the sex distribution for the primary dentition , supernumeraries occur more frequently in the permanent dentition of males .
supernumerary teeth may occur in isolation or as part of a syndrome , such as in clp , cleidocranial dysplasia and gardner 's syndrome and less commonly in fabry anderson 's syndrome , chondroectodermal dysplasia , ehlers - danlos syndrome and tricho - rhino - phalangeal syndrome .
supernumerary and impacted teeth , retarded eruption of primary and permanent dentition , aplasia or hypoplasia of clavicles and other skeletal anomalies are the features of this disorder exhibiting autosomal dominant inheritance .
hypodontia , oligodontia and anodontia are the terms used to describe the numerical values of tooth agenesis .
hypodontia is the absence of 1 - 6 teeth ( excluding third molars ) , whereas oligodontia refers to the absence of more than 6 teeth ( excluding third molars ) and anodontia is the complete absence of teeth .
hypodontia is frequently accompanied with cleft - lip or palate , reduction in tooth size , short root anomaly , malformation of other teeth , impaction , maxillary canine and first premolar transposition , delayed formation or eruption of other teeth , microdontia , taurodontism , enamel hypoplasia and altered craniofacial growth .
sometimes , it is caused by environmental factors such as ; infection , different kinds of trauma in the apical area of dentoalveolar process , chemical substances or drugs , radiation therapy or disturbances in the jaw innervations , but in the majority of cases , hypodontia is impacted by genetics .
autosomal dominant inheritance with incomplete penetrance and variable expressivity was first proposed by burzynski and escobar . for a genetic linkage study on hypodontia , arte et al .
, evaluated 214 family members in three generations and concluded that incisor - premolar agenesis is transmitted by autosomal dominant genes with small upper incisors , ectopic canines , taurodontism and rotated premolars as accompanying anomalies .
a familial autosomal dominant hypodontia was demonstrated to be caused by a point mutation in the msx1 gene .
vastardis et al . , using a linkage analysis in a family with second premolar and third molar agenesis , defined a locus on the chromosome 4p16 as the site of the msx1 .
sequence analysis revealed arg31-to - pro missense mutation in the msx1 homeodomain for all of the affected subjects .
arg31-to - pro mutation is known to influence the msx1 interactions , which are critical in the normal development of human teeth .
this finding supported the result of an animal study , which showed a knockout mutation of msx1 gene leading to the inhibition of dental development ; however , when the premolar agenesis was accompanied with a lateral incisor , the influence of msx1 and msx2 genes could not be proved .
another gene , causing tooth agenesis is pax9 in chromosome 14 ( 14q21-q13 ) . in hypodontia cases ,
a frameshift mutation and a nonsense termination mutation of the same gene have been observed in oligodontia . according to das et al .
, this situation suggests that hypodontia and oligodontia are not fundamentally different or at least can be caused by different mutations in the same gene .
the frameshift mutation of pax9 gene located on chromosome 14 was identified as responsible for autosomal dominant oligodontia in a large family for four generations . in some of the affected members , maxillary and mandibular second premolars and mandibular central incisors
were absent in addition to the lack of permanent molars ; although , a normal primary dentition was present . in 2001 , nieminen et al . , identified an a - to - t transversion of the pax9 gene in a family with autosomal dominant oligodontia .
they reported that all the second and third permanent molars , maxillary lateral incisors and in some cases all of the first permanent molars and several second premolars were missing , in addition to deciduous second molar agenesis .
a year later , frazier - bower et al . concluded that the molar oligodontia is due to allelic heterogeneity in pax9 , which is an important regulator of molar teeth development . in 2003 , lamni showed a distinct phenotype with canine agenesis , which is quite rare , in a family segregating autosomal dominant oligodontia .
in recent studies , different transition , insertion and transversion mutations in pax9 gene were reported , providing more comprehensive explanations for the pathogenic mechanisms of the interactions between mutations and tooth agenesis .
in addition to pax9 and msx1 genes , contribution of a regulatory molecule in the mesenchyme , called transforming growth factor alpha , might play a role in isolated dental agenesis .
furthermore , axin2 is a negative regulator of canonical wnt signaling and mutations of this gene can cause tooth agenesis associated with colorectal cancer .
another mode of inheritance for hypodontia associated with other dental anomalies such as dental malformations , enamel hypoplasia and eruption failure is the autosomal recessive inheritance , with polygenic inheritance also suggested .
studies showed that tooth agenesis could be manifested as an isolated feature or as a part of a syndrome .
tooth agenesis was associated with a large number of syndromes , which indicating that the development of teeth and certain organs are under the control of the same molecular mechanisms .
hypodontia is a major component of ectodermal dysplasia , oral - facial digital syndromes with oral - facial clefting .
ectodermal dysplasia is characterized by small , misshapen and missing teeth , delayed eruption , prominent lip , maxillary hypoplasia , sparse hair and hypohidrotic skin .
this disorder is inherited as an x - linked trait , though the autosomal recessive form has also been reported .
oral - facial digital syndrome type 1 has symptoms such as hypodontia of lower incisors , facial asymmetry , hypertelorism , micrognathia , supernumerary frenulum and thickened alveolar ridges .
oral - facial clefting is affected by chromosome 6p24 , 2p13 , 19q13 and 4q and the prevalence of hypodontia increases with cleft severity .
the upper lateral incisor is the most frequently affected tooth in the cleft area both in primary and in permanent dentitions . in syndromic cleft lip and palate ( clp ) like pierre - robin sequence 50% hypodontia and van der woude syndrome 70% hypodontia prevalence is associated with other disorders .
the genetic basis of hyperdontia has been extensively explained in the literature . in 1932 ,
brook reported a greater occurrence of supernumerary teeth in the close relatives of effected individuals , compared with the general population .
it was also reported that the supernumerary and impacted teeth occupied the same location in the identical twins and in their parents .
although there is no difference in the sex distribution for the primary dentition , supernumeraries occur more frequently in the permanent dentition of males .
supernumerary teeth may occur in isolation or as part of a syndrome , such as in clp , cleidocranial dysplasia and gardner 's syndrome and less commonly in fabry anderson 's syndrome , chondroectodermal dysplasia , ehlers - danlos syndrome and tricho - rhino - phalangeal syndrome .
supernumerary and impacted teeth , retarded eruption of primary and permanent dentition , aplasia or hypoplasia of clavicles and other skeletal anomalies are the features of this disorder exhibiting autosomal dominant inheritance .
the structural anomalies of teeth are caused by the disturbances at the level of enamel and dentin during tooth development .
amelogenesis imperfecta as one of these disorders , is characterized by discolored teeth and anterior open bite and exhibits x - linked , autosomal dominant and recessive inheritance caused by mutation of five different genes ; amelogenin amel , enamelin enam , matrix metalloproteinase- 20 mmp20 , kallikrein - related peptidase 4 klk4 and family with sequence similarity 83 , member h fam83h .
there are three types of amelogenesis imperfecta ; as hypoplastic ; with thin , normally calcified enamel , hypocalcified ; with less mineralized , but normal thickness enamel and hipomaturation with enamel structure that has the same radiodensity of dentin and which is easily dislodged from dentin . in a study of 50 patients with hypoplastic amelogenesis imperfecta ,
anterior openbite occurred in 24% of the cases and severe vertical discrepancy was observed in almost all of them .
furthermore , it was shown that this specific malocclusion with autosomal recessive inheritance was caused by the mutation in the enamelin gene , which is the largest protein in the enamel matrix of developing teeth .
when hypoplastic teeth coexist with an anterior openbite , the orthodontists should investigate the genetic background for any existing condition .
type i is syndromic form of dgi which is inherited with osteogenesis imperfecta and the genes encoding collagen , type i , alpha 1 , ( col1a1 ) and col1a2 .
radiographically , the teeth have short , constricted roots and dentin hypertrophy leading to pulpal obliteration either before or just after eruption .
expressivity is variable even within an individual , with some teeth showing total pulpal obliteration while in others the dentin appears normal .
the other two forms seem to result from mutations in the gene dentin sialophosphoprotein , encoding dentin phosphoprotein and dentin sialoprotein .
bulbous crown are typical features of dgi type ii , with hypotrophy in dentine structure .
dentin dysplasia ( dd ) , which has radicular and coronal subtypes is another structural anomaly and has the same genetic disorder with dgi , except dd type i ( radicular type ) .
the importance of the genetic factors controlling tooth size and morphology has been shown by twin studies .
some authors reported that tooth crown dimensions , especially buccolingually and mesiodistally were genetically determined .
it has been suggested that there is an association between oversized teeth and supernumerary teeth .
similarly , the existence of peg shaped or strongly mesiodistally reduced lateral incisors may be the result of a variation in the expression of hypodontia .
the displacement of canines in a palatinal direction is a positional dental anomaly and can generally happen even if there is adequate place in the dental arch .
there is an association between the malpositions of certain teeth , such as palatally displaced canines , mandibular lateral incisor - canine transposition and maxillary canine - first premolar transpositions and tooth agenesis .
similar for tooth agenesis , the positional anomalies of canines have been shown to affect some family members and thought to be under strong genetic control .
some authors suggested that the ectopic canines exhibit a multifactorial inheritance pattern with high phenotypic variance and low penetrance .
reported that palatally displaced and frequently impacted canines and mandibular lateral incisor - canine transposition were related to congenitally missing third molars in the posterior orofacial area . similarly , an association between the maxillary canine - first premolar transposition and maxillary lateral incisor agenesis in the anterior orofacial area may exist .
furthermore , the agenesis of the lower second premolar , which is located in an intermediate zone is related to all the positional anomalies of canines .
the anteroposterior morphogenetic field concept , proposed by butler in 1939 , supports the current molecular investigations such as the determination of the interaction between a single gene and site specific orofacial expressivity .
hox genes , which play a role in oral and dental development are known to show site specific anteroposterior expression patterns .
msx1 , regulator gene in the third molar and lower second premolar agenesis , may be responsible for posterior site development .
in addition to the other posterior area genes , which are dlx-1 , dlx-2 and barx-1 , pax9 also control the development of all of the molars .
furthermore , neubser et al . , reported that there is an association between pax9 transcription factor and repositioning of tooth buds on the mesenchymal level .
this theory might give a clue to researchers about the genetic mechanisms of dental positional anomalies such as palatally displaced canines or different kind of transpositions .
it appears that tooth agenesis , tooth size and position anomalies , which are often seen together , are the components of a complex , genetically controlled dental condition .
dental malpositions such as rotations , eruption failures and ankylosis are among other anomalies complicating this dental condition .
the etiology of dental anomalies is partly environmental and partly genetic . because of the polygenic nature of dental characteristics
, it is very challenging to identify one single defective gene responsible for a specific dental anomaly .
further studies are required and the rapid progress in the field of genetics may help the clinicians to more accurately discern the environmental and genetic factors contributing to the development of dental anomalies . currently , the orthodontist , probably the first to diagnose hereditary dental anomalies and malocclusion of an individual , will remain responsible for the detection of any additional defects in the same patient in order to provide the best treatment .
the clinician should always keep in mind that some of those dental anomalies can coexist with certain syndromes and other family members might also have been affected .
whenever it seems necessary , a genetic consultation should be added as part of the orthodontic treatment .
finally , this interdisciplinary approach may help to reveal any risk of recurrence in subsequent generations . | the interruption of odontogenesis by any etiological factor may result in dental anomalies .
apart from the environmental factors , the impact of genetics in dental anomalies was found to be a factor in different levels .
many authors had questioned a common genetic defect resulting in different phenotypic conditions such as absent , malformed , malposed or ectopic teeth . because the multidisciplinary treatment of these dental anomalies such as hypodontia , impaction etc .
, involves orthodontic intervention , orthodontists must be aware of the etiology and possible correlative conditions with dental anomalies . |
homeopathy claims a curative reaction from a small dose of a drug of which high doses cause symptoms similar to those from which the patient is suffering .
homeopathy originated in 19 century , prior to the acceptance of the germ and gene bases of disease ; it has never been based on scientific evidence .
results of randomized trials do not provide acceptable evidence that homeopathic treatments are more effective than placebo ; although there are also contradicting statements .
it is however possible that some empirical knowledge is successfully used in homeopathy unrelated to its axioms like can be cured with like , less is more or the memory of water .
hormesis has been defined as a biphasic dose - response relationship in which the response at low doses is opposite to the effect at high doses . according to this concept ,
however , claims that homeopathy is based on hormesis create an illusion that it employs a scientific method .
the difference between hormesis and homeopathy is that hormesis can be observed at low but measurable concentrations ; while homeopathy claims effects of infinite dilutions , whereas the concept of memory of water is used as an explanation .
there is an opinion , shared by the author that the term hormesis should not be linked with homeopathy .
if homeopaths have valuable empirical knowledge , it should be verified by the methods of evidence - based medicine .
potentially useful empirical knowledge gathered in homeopathy , alternative or complementary medicine , should be scientifically tested and discussed in the professional literature .
among the known hormetic agents are pro - oxidants , heavy metals , heat , exercise , food restriction , and different kinds of stress .
living organisms come in contact with all these factors in the natural environment , so that the hormetic effects can be explained from an evolutionary standpoint .
the term hormetins has been used in the literature for hormesis - inducing compounds . for antibiotics ,
hormetic effects develop secondarily along with the adaptation of microorganisms and development of antibiotic resistance .
another example : thousands years adaptation of certain human populations to ethanol resulted in detectable hormesis also for this toxic agent : moderate alcohol consumption was reported to be associated with a reduced risk of coronary heart disease and other health benefits .
there has been no plausible explanation of hormesis as a default principle in the pharmacological theory .
scientific foundations of some hormetic mechanisms were discussed within the framework of stress response pathways . however , different kinds of stress are an integral part of the environmental impact on living organisms , who have been accordingly adapted to it .
hormesis as a general principle is conceivable only for the agents that have induced adjustment of living organisms , so that a deviation in either direction from an optimum would be harmful .
this is obviously the case for visible light , ultraviolet and ionizing radiation , atmospheric pressure , as well as for many chemical substances and microelements present in the environment .
it is not surprising that potentially toxic heavy metals , which are present in the natural environment , act hermetically in plants .
there are no general reasons to expect hormetic responses for the factors absent in the natural environment . among explanations for the hormetic effects , discussed in the literature ,
are an excess of repair mechanisms in response to mild damage and a proposed existence of two receptor types ( small quantity of high - affinity receptors and large numbers of low - affinity ones ) .
both hypotheses have not been sufficiently proven , in particular , as umbrella mechanisms for different types of agents . moreover , some reported hormetic effects can be doubtful because of the difficulties of differentiation between low - level hormetic and placebo effects questionable reliability of some data , poor study designs , etc . it should be stressed that a response to an agent usually increases with increasing concentration ; in contrast , a placebo effect does not depend on concentrations , while homeopathic remedies can be extremely diluted so that the agent can be absent in the solution . for research purposes ,
placebo effects can be excluded in animal experiments without conditioning and especially in plants , where hormetic effects can be studied .
hormesis phenomenon was discussed in the context of homeopathy ; it was sometimes generalized and treated as a matter - of - course .
for example , the question : is hormesis likely to occur for all types of drugs ? was answered : there are sufficient data to conclude that the hormetic dose response is common , reproducible , and a biological expectation in the vast majority of biological systems , end points measured , and chemical classes tested
. the question may drugs be acting hormetically even though the experimental data appear inconsistent with this interpretation ?
was responded : the hormesis concept establishes a biological context for some of the key
hormetic - like biphasic dose responses may represent a general biological dose - response pattern or strategy have never been substantiated .
moreover , hormesis , usually , relates to a single response , while toxic impacts can have different responses .
some noxious stimuli can act synergistically with other factors , for example , on the cells with a limited or no capacity for cellular regeneration such as cardiomyocytes or neurons .
it can be of particular importance in conditions when such cells are pre - damaged by ischemia so that even a mild additional damage would act according to a no - threshold dose - response pattern without hormesis . in conditions close to a functional decompensation of an organ
, even minimal additional damage can be detrimental . in such conditions , which are not uncommon especially in elderly patients
, the concept of hormesis can be dangerous if used in the clinical decision - making .
for example , it would hardly be indicated to apply mild asphyxia in angina pectoris or small doses of ethanol in end - stage liver disease with a hope for a hormetic effect as a general biological dose - response pattern .
considering the above , the statement : the hormesis concept is a fundamental dose response , highly conserved , and set in an evolutionary framework is true a priori only for the factors that have induced evolutionary adjustment .
if even hormesis was observed in studies of the substances that are absent in the environment such as antineoplastic , anxiolytic or anti - seizure drugs , or resveratrol ( the latter was extensively discussed in the volume 29 of human and experimental toxicology , while relevance of the hormetic effects was questioned ) , there is still no reason to conclude that
hormetic dose responses are broadly generalizable , being independent of biological model , endpoint measured , and stressor agent , and represent a basic feature of biological responsiveness to chemical and physical stressors .
the publications containing generalizations of this kind can be cited in support of homeopathy and placebo , in gerontology and other fields of medicine , also to endorse official registration of drugs without specific effects or efficacy not exceeding that of placebo .
it can pave the way for homeopathy and placebos instead of evidence - based treatments , as inexpensive substitutes , especially for elderly patients .
there are many examples of marketed compounds without scientifically demonstrated efficacy , in russia often in the guise of evidence - based medications ; while artificial theoretic concepts are created to promote them .
promotion of unproven health schemes can be harmful especially for elderly people . in the medical practice ,
deception is normally objectionable on the grounds that it limits autonomy and breaches trust ; these grounds possibly do not apply to placebos when they are prescribed within appropriate ethical limits ; although there is an opinion that clinical placebo interventions are unethical and unnecessary .
if even placebo therapy with misinformation of a patient might be ethically acceptable in certain cases , it is still not a reason to publish biased information .
remarkably , it seems that some patients are influenced not only by medical advertizing , which is sometimes misleading in russia , but also directly or indirectly by professional publications . in conditions when commercial considerations tend to replace medical ethics , some patients try to come clear with their ailments with the help of professional literature , which is their right .
moreover , persistence and development of spurious theoretic concepts can sooner or later result in the application of invasive procedures with questionable clinical indications .
for example , in the preceding article a series of studies was commented that has become internationally known in 1986 after a publication in the lancet with participation as coauthor of the health minister of that time .
there followed numerous publications in russian and foreign journals continued until today [ 40 - 47 ] ( more references are in )
. cultures of smooth muscle cells or macrophages were used for testing of blood atherogenicity , anti- or pro - atherogenic action of various substances .
in addition to the drugs , many natural substances were shown by the same researchers using the cell culture method to be effective against serum atherogenicity : black elder berries , calendula and violet flowers , grape seeds and stems etc .
extracts from 13 different mushrooms were shown to significantly lower serum atherogenicity . however , as discussed in , the relationship between serum atherogenicity in a cell culture and atherogenesis in vivo must be inverse rather than direct .
for example , in familial hypercholesterolemia , caused by abnormality of lipoprotein receptors , ineffective clearance of low density lipoprotein ( ldl)-cholesterol from serum causes hypercholesterolemia and predisposes to atherosclerosis .
up - regulation of ldl - receptors ( and , correspondingly , of the ldl - cholesterol uptake by cells ) is one of the paradigms to the atherosclerosis therapy .
accordingly , if an agent reduces cholesterol uptake by cells in - vitro , it can be expected to cause serum cholesterol elevation in - vivo .
nevertheless , following their concept , the same researchers started applying extracorporeal apheresis through a column with immobilized ldl aimed at the removal of non - lipid atherogenicity factor(s ) twice monthly for the period of 7 - 9 months .
the patients were men 46 - 59 years old with functional class ii - iii angina pectoris , an angiographically documented stenosis of 2 - 3 coronary arteries and a normal cholesterol level . during this trial ,
the patients were reported to feel better , endure higher physical loads , and have heightened sexual activity , which could have been caused by a placebo effect .
it is reasonable to assume that invasive procedures are associated with a placebo effect , which might be stronger than that of non - invasive procedures .
blood apheresis is associated with certain risks , although severe side - effects such as shock or allergic reactions were reported to be very rare .
efficiency of the apheresis in the study can not be excluded , although apheresis is usually aimed at removal of lipids and lipoproteins e.g. , in patients with severe drug - resistant ldl - hypercholesterolemia or lipoprotein elevation and premature atherosclerosis . considering the above , indications to apheresis in
hormesis as a general principle has never been proven as an umbrella theoretic basis for factors that are absent in the environment .
if an agent is present in the natural environment , existence of its optimal level can be assumed , which would correspond to the current environmental level or , considering that the natural selection is a slow process , to some average from the past .
low - dose impacts may be associated with a higher risk in a state of organ sub - compensation or failure especially in elderly patients .
accordingly , practical recommendations should be based neither on the hormesis as a default approach nor on the like cures like , less is more or other postulates of homeopathy .
all clinically relevant effects , including hormetic ones , should be tested by the methods of evidence - based medicine . | homeopathy claims a curative reaction from small doses of a substance , high doses of which cause symptoms similar to those the patient is suffering from .
hormesis is a concept of biphasic dose - response to different pharmacological and toxicological agents . according to this concept ,
a small dose of a noxious agent can exert a beneficial action .
a hypothesis is defended here that hormesis as a general principle can be assumed only for the factors present in the natural environment thus having induced adaptation of living organisms .
generalizations of the hormesis phenomenon used in support of homeopathy are unfounded .
low - dose impacts may be associated with a higher risk in a state of organ sub - compensation or failure especially in the elderly patients .
practical recommendations should be based neither on the hormesis as a default approach nor on the postulates of homeopathy .
all clinically relevant effects , hormetic or not , should be tested by the methods of evidence - based medicine . |
testicular adrenal rest tumors ( tarts ) are benign intratesticular masses that occur in male patients with congenital adrenal hyperplasia ( cah ) , with more than 90% of cases caused by a deficiency of 21--hydroxylase .
tarts originate from aberrant adrenal cells in the testes and can impair both spermatogenesis and endocrine testicular function .
studies have indicated that tarts may be present in childhood , with an increasing prevalence after the onset of puberty .
therefore , the early detection of tarts is important to preserve testicular function , and scrotal baseline ultrasound ( bus ) screening is recommended beginning in early childhood .
although several reports of tart sonographic features have been described previously , the reports are limited by a small numbers of cases or the old age of the patients .
this situation prompted us to investigate the sonographic features of tarts during puberty , evaluating patients no older than 18 years .
from june 2009 to january 2015 , 79 male patients had been diagnosed as cah and screened for testicular evaluation in our institute , and only 15 patients had tarts in this study .
the prevalence of tarts in our patient population was 19% ( 15/79 ) , which was all bilateral , without unilateral .
written informed consent was obtained from all patients , and the study was approved by the first affiliated hospital of sun yat - sen university institutional review board .
the first machine was an high density interconnector 5000 us machine ( phillips healthcare , tokyo , japan ) equipped with a transducer with a frequency range of 7.0 to12.0 mhz .
the other was an aplio xv machine ( toshiba medical systems , tokyo , japan ) equipped with a transducer with a frequency range of 7.5 to 10.0 mhz .
all bus examinations were performed by 1 of 2 experienced radiologists who had more than 3 years of experience in diagnosing andrological diseases .
the entire testicle was scanned thoroughly using bus , and the target lesions were identified . the location , size , shape , boundary , and echogenicity of the lesion , as well as the testicular size and the presence or absence of the testicular mediastinum were recorded .
afterwards , the transducer was maintained in a stable position and shifted to the color doppler imaging mode , and the blood supply of the lesions was recorded .
from june 2009 to january 2015 , 79 male patients had been diagnosed as cah and screened for testicular evaluation in our institute , and only 15 patients had tarts in this study .
the prevalence of tarts in our patient population was 19% ( 15/79 ) , which was all bilateral , without unilateral .
written informed consent was obtained from all patients , and the study was approved by the first affiliated hospital of sun yat - sen university institutional review board .
the first machine was an high density interconnector 5000 us machine ( phillips healthcare , tokyo , japan ) equipped with a transducer with a frequency range of 7.0 to12.0 mhz .
the other was an aplio xv machine ( toshiba medical systems , tokyo , japan ) equipped with a transducer with a frequency range of 7.5 to 10.0 mhz .
all bus examinations were performed by 1 of 2 experienced radiologists who had more than 3 years of experience in diagnosing andrological diseases .
the entire testicle was scanned thoroughly using bus , and the target lesions were identified . the location , size , shape , boundary , and echogenicity of the lesion , as well as the testicular size and the presence or absence of the testicular mediastinum were recorded .
afterwards , the transducer was maintained in a stable position and shifted to the color doppler imaging mode , and the blood supply of the lesions was recorded .
fifteen male patients involved in this study were aged 4 to 17 years ( mean age , 8.8 3.8 years ) , height 104.6 to 172.5 cm ( mean height , 138.7 22.5 years ) , and weighted 15 to 57 kg ( mean weight , 36.5 14.0 kg ) .
all of the patients had been diagnosed with cah for at least 1 year ; the blood pressure and blood glucose of the cases were all at normal levels ; 33.3% ( 5/15 ) of the cases exhibited normal bone age , and nearly 66.7% ( 10/15 ) of the cases exhibited advanced bone age ; and the andrological evaluation of these cases were listed in table 1 . in addition , 80% ( 12/15 ) of the cases were confirmed by pathological examination using surgical specimens , and immunohistochemistry showed that cd56 , inhibin , and vimentin were all positive , which were the pathological characteristics of adrenal gland ( figure 1 ) .
the remaining 3 cases were clinically confirmed ( diagnostic criteria : the testicular lesions near the mediastinum were gradually diminishing with an increasing amount of hormone therapy in patients who had been diagnosed with cah ) .
the hormone levels of these 15 patients with tarts before and after operation and hormone therapy were showed in table 2 .
basic characteristics of pubescent patients with congenital adrenal hyperplasia and testicular adrenal rest tumors testicular adrenal rest tumor was growing into testis ( a ) .
and immunohistochemistry showed that cd56 ( b ) , inhibin ( c ) , and vimentin ( d ) were all positive , which was the pathological characteristics of adrenal gland .
the basic hormone levels of pubescent patients with congenital adrenal hyperplasia and testicular adrenal rest tumors before and after operation and hormone therapy all 15 cases were bilateral , and there were 30 lesions in total .
the lesions were 1.4 0.8 cm ( range : 0.23.8 cm ) in diameter on average .
in 93.3% of the patients ( 14/15 ) , the testicular mediastinum were clearly visualized , and only 1 mediastinum exhibited a fuzzy appearance .
all of the lesions had clear boundaries and were located near the testicular mediastinum or the testicular hilum ; 83.3% ( 25/30 ) of the lesions had an irregular shape , whereas 5 ( 5/30 , 16.7% ) lesions were round .
furthermore , 56.7% ( 17/30 ) of the tart lesions exhibited homogeneous hypoechogenicity ( figure 2 ) , 36.7% ( 11/30 ) of the tart lesions exhibited heterogeneous hypoechogenicity ( figure 3 ) , and 6.6% ( 2/30 ) of the tart lesions exhibited heterogeneous isoechogenicity ( figure 4a ) .
color doppler revealed that 76.7% ( 23/30 ) of the lesions had a rich blood supply and that 23.3% ( 7/30 ) of the lesions had a scarce blood supply .
testicular adrenal rest tumor in a 4-year - old patient with congenital adrenal hyperplasia ( patient 13 ) .
sonographic examination of the left testis revealed a homogeneous hypoechoic lesion ( arrow ) with a round clear boundary , 4 mm in diameter , adjacent to the mediastinum of the testis ( a , b ) .
testicular adrenal rest tumor in a 10-year - old patient with congenital adrenal hyperplasia ( patient 5 ) .
( a ) sonographic examination of the right testis revealed a clearly defined heterogeneous hypoechoic lesion ( arrow ) , 14 mm in diameter , adjacent to the mediastinum of testis .
( b ) color doppler demonstrated marked vascularization compared with the normal testicular parenchyma ( arrow ) .
testicular adrenal rest tumor in a 15-year - old patient with congenital adrenal hyperplasia ( patient 4 ) .
( a ) sonographic examination of the left testis revealed a heterogeneous isoechoic lesion with a lobulated boundary ( arrow ) , 31 mm in diameter , adjacent to the mediastinum of testis .
( b ) sonographic examination of the left testis half a year later after operation revealed no tart lesion in the heterogeneous echoic testis caused by operation ( arrow ) .
sonographic features of pubescent patients with congenital adrenal hyperplasia and testicular adrenal rest tumors the follow - up period was from 3 to 12 months , and the tart lesions were almost disappeared after operation and hormone therapy .
the testis exhibited heterogeneous echogenicity , which might be caused by operation ( figure 4b ) .
fifteen male patients involved in this study were aged 4 to 17 years ( mean age , 8.8 3.8 years ) , height 104.6 to 172.5 cm ( mean height , 138.7 22.5 years ) , and weighted 15 to 57 kg ( mean weight , 36.5 14.0 kg ) .
all of the patients had been diagnosed with cah for at least 1 year ; the blood pressure and blood glucose of the cases were all at normal levels ; 33.3% ( 5/15 ) of the cases exhibited normal bone age , and nearly 66.7% ( 10/15 ) of the cases exhibited advanced bone age ; and the andrological evaluation of these cases were listed in table 1 . in addition , 80% ( 12/15 ) of the cases were confirmed by pathological examination using surgical specimens , and immunohistochemistry showed that cd56 , inhibin , and vimentin were all positive , which were the pathological characteristics of adrenal gland ( figure 1 ) .
the remaining 3 cases were clinically confirmed ( diagnostic criteria : the testicular lesions near the mediastinum were gradually diminishing with an increasing amount of hormone therapy in patients who had been diagnosed with cah ) .
the hormone levels of these 15 patients with tarts before and after operation and hormone therapy were showed in table 2 .
basic characteristics of pubescent patients with congenital adrenal hyperplasia and testicular adrenal rest tumors testicular adrenal rest tumor was growing into testis ( a ) .
and immunohistochemistry showed that cd56 ( b ) , inhibin ( c ) , and vimentin ( d ) were all positive , which was the pathological characteristics of adrenal gland .
the basic hormone levels of pubescent patients with congenital adrenal hyperplasia and testicular adrenal rest tumors before and after operation and hormone therapy
the lesions were 1.4 0.8 cm ( range : 0.23.8 cm ) in diameter on average . in 93.3% of the patients ( 14/15 )
, the testicular mediastinum were clearly visualized , and only 1 mediastinum exhibited a fuzzy appearance .
all of the lesions had clear boundaries and were located near the testicular mediastinum or the testicular hilum ; 83.3% ( 25/30 ) of the lesions had an irregular shape , whereas 5 ( 5/30 , 16.7% ) lesions were round .
furthermore , 56.7% ( 17/30 ) of the tart lesions exhibited homogeneous hypoechogenicity ( figure 2 ) , 36.7% ( 11/30 ) of the tart lesions exhibited heterogeneous hypoechogenicity ( figure 3 ) , and 6.6% ( 2/30 ) of the tart lesions exhibited heterogeneous isoechogenicity ( figure 4a ) .
color doppler revealed that 76.7% ( 23/30 ) of the lesions had a rich blood supply and that 23.3% ( 7/30 ) of the lesions had a scarce blood supply .
testicular adrenal rest tumor in a 4-year - old patient with congenital adrenal hyperplasia ( patient 13 ) .
sonographic examination of the left testis revealed a homogeneous hypoechoic lesion ( arrow ) with a round clear boundary , 4 mm in diameter , adjacent to the mediastinum of the testis ( a , b ) .
testicular adrenal rest tumor in a 10-year - old patient with congenital adrenal hyperplasia ( patient 5 ) .
( a ) sonographic examination of the right testis revealed a clearly defined heterogeneous hypoechoic lesion ( arrow ) , 14 mm in diameter , adjacent to the mediastinum of testis .
( b ) color doppler demonstrated marked vascularization compared with the normal testicular parenchyma ( arrow ) .
testicular adrenal rest tumor in a 15-year - old patient with congenital adrenal hyperplasia ( patient 4 ) .
( a ) sonographic examination of the left testis revealed a heterogeneous isoechoic lesion with a lobulated boundary ( arrow ) , 31 mm in diameter , adjacent to the mediastinum of testis .
( b ) sonographic examination of the left testis half a year later after operation revealed no tart lesion in the heterogeneous echoic testis caused by operation ( arrow ) .
sonographic features of pubescent patients with congenital adrenal hyperplasia and testicular adrenal rest tumors the follow - up period was from 3 to 12 months , and the tart lesions were almost disappeared after operation and hormone therapy .
the testis exhibited heterogeneous echogenicity , which might be caused by operation ( figure 4b ) .
in male patients with cah , tarts are relatively common , especially in adult males , with a prevalence of nearly 94% . in recent years
, it has become evident that some of these lesions can be detected in childhood , with a prevalence ranging from 18.3% to 29% in studies performed on children .
a similar prevalence of 19% ( 15/79 ) was found in our patient population . to the best of our knowledge
, few reports have described the imaging characteristics of tarts in puberty and adolescent ; the present study is the first to evaluate the sonographic features of tarts in a large pediatric cah population from 0 to 18 years of age .
we retrospectively analyzed the imaging features of 30 tart lesions that had been pathologically or clinically confirmed within the past 5 years .
our results revealed that all 15 cases were bilateral in onset , similar to previous reports .
although the results of delfino et al indicated that 2 of 11 cases were unilateral , we believe that this result might be influenced by lesions too small to detect ; in our series , the size of the smallest lesion was 2 mm in diameter , and it is sometimes difficult to detect lesions smaller than 2 mm .
ultrasonography revealed that all 30 lesions in our series were located adjacent to the testicular mediastinum and had clear boundaries .
the location in the mediastinum has previously been described and is considered a typical feature of tarts in cah .
the results of stikkelbroeck et al revealed blurring of the margins in 36% of lesions , but all of our lesions had clearly defined margins , which may be due to the advanced ultrasonic technology compared with that used a decade ago . in our study , 83.3% ( 25/30 ) of the lesions had an irregular , lobulated shape , and ranged from 0.7 to 3.8 cm in size .
five ( 5/30 , 16.7% ) lesions were round , ranging from 0.2 to 0.6 cm in size .
we believe the tarts begin as small , distinct lesions that could grow and coalesce to form 1 large , lobulated lesion .
the histopathological hypothesis of the development of these lesions was that they originated from aberrant adrenal rest cells ; when stimulated by adrenocorticotropic hormone , these adrenal rest cells in the testicular tissue were activated and became hyperplastic , subsequently appearing as confluent lesions .
the tarts in cah in our group exhibited hypoechogenicity compared with normal testicular tissue except in 6.6% ( 2/30 ) of the lesions , which exhibited isoechogenicity .
by contrast , with respect to lesion size , stikkelbroeck et al reported that 17 of 20 lesions < 2 cm were hypoechoic , whereas all lesions > 2 cm were hypoechoic with hyperechoic reflections .
the significant difference in the echogenicity of tarts was likely due to the selection of patients , who were all adults in stikkelbroeck 's report and might have had a longer disease course compared with the pubescent patients ; furthermore , hyperechogenicity might indicate fibrotic changes or calcifications in the lesions .
color doppler revealed that 76.7% ( 23/30 ) of the lesions exhibited a rich blood supply , whereas 23.3% ( 7/30 ) of the lesions exhibited a scarce blood supply .
in addition , the vessels coursing through the lesions did not deviate or change in caliber .
the latter observation has also been made by avila et al and stikkelbroeck et al , who suggested that this appearance may be useful in distinguishing tarts because it has not been described with other testicular tumors .
therefore , tart screening by scrotal bus beginning from early childhood is important in male patients with cah to prevent the development of future complications , especially infertility .
notably , the high tart prevalence in adult patients is due to failure of detection during childhood .
our study still has a limitation , which is the uncertain relationship between tarts size and abnormal hormone levels .
so , a well designed clinical study is needed to get the relationship between them . in conclusion ,
the sonographic characteristics of tarts are bilateral growth , location adjacency to the testicular mediastinum , hypoechogenicity , and a rich blood supply .
we recommend that ultrasound be performed from the onset of puberty in all boys with classic cah to avoid the risk of infertility . | abstractthe aim of this study was to evaluate the imaging features of testicular adrenal rest tumors ( tarts ) on baseline ultrasound ( bus).the imaging features of 30 tart lesions pathologically or clinically confirmed in 15 patients who had undergone bus were evaluated , and the sonographic characteristics of the lesions were analyzed.all 15 cases were bilateral and located near the testicular mediastinum .
approximately 56.7% ( 17/30 ) of the tart lesions exhibited homogeneous hypoechogenicity , 36.7% ( 11/30 ) of the lesions exhibited heterogeneous hypoechogenicity , and 6.6% ( 2/30 ) of the lesions exhibited heterogeneous isoechogenicity .
in addition , 76.7% ( 23/30 ) of the lesions exhibited a rich blood supply , whereas 23.3% ( 7/30 ) of the lesions exhibited a scarce blood supply.the sonographic characteristics of the tarts were bilateral growth , location adjacent to the testicular mediastinum , hypoechogenicity , and rich blood supply , which may play important roles in early clinical diagnosis . |
hip fractures are a major problem for the elderly , and especially for those confined to long - term care , both nursing homes and residential homes .
those in institutions constitute about 6%-7% of the population of those over 65 years , but this population contributes almost one third of the hip fracture numbers [ 1 , 2 ] .
the residents of long - term care establishments are , however predominantly female and tend to be particularly old , both risk factors for hip fracture .
they are also frailer , which is what usually necessitates their institutionalization and makes them more likely to fall .
in addition , they may have fragile bones , though whether more fragile than their community - dwelling peers is not known . in a later report
, however , zimmerman and colleagues demonstrated that the predictors of bone density in nursing home patients were the same as for those living in the community , namely , age , gender , race , and weight .
we have compared hip fracture rate in long - term care ( ltc ) and community - dwelling men and women at different age strata .
subsequently we corrected for age and gender distribution in the ltc setting to see what proportion of the excess rate of hip fracturing the different age and gender balance of the ltc population might account for . any remaining excess risk would point to an added risk factor , be it endogenous or exogenous to the patient , as important in increasing the hip fracture rate in these people .
the study was conducted in london , ontario , canada , a city of approximately 350000 residents , using secondary deidentified data . hospital discharge abstract databases ( dads )
are reported to the canadian institute for health information from all acute care hospitals in canada ( except those in the province of quebec ) to form a national database .
dads were obtained from the two local acute care hospitals which handle all hip fractures from the city .
data for 5 calendar years , 20022006 inclusive , were obtained . to distinguish between hip fractures occurring in institutionalized versus community - dwelling populations
, a prefracture residence classification was created using information from the residential postal code , place of injury and institution from data fields contained in the dads .
a hip fracture was classified as having occurred in an institution if one of the following three criteria was met : ( i ) the patient 's residential postal code matched a specific postal code for an institution ( most of the institutions are large enough to have their own specific postal code ) , ( ii ) the patient 's residential postal code was consistent with , but not specific to , an institution , and the place of injury on the dad was coded for an institution , or ( iii ) the patient 's residential postal code was consistent with but not specific to an institution , and the patient 's institution from designation in the dad was unique to a known institution .
residents of both nursing homes and residential homes are included as we could not distinguish between them in the databases .
all subjects aged 65 years or over , who were residents of london , were included in the data set .
patients with subcapital ( s72.0-s72.091 ) or intertrochanteric ( s72.1-s72.191 ) fractures were identified from the data field containing the most responsible diagnosis for the length of stay indicator .
patients coded as having a fracture of the femur other than at the subcapital or intertrochanteric sites , those with identified malignant neoplasms ( c00-d09 , n = 71 ) or motor vehicle accident - related fractures ( v01-v99 , n = 48 ) were excluded .
those with more than one admission within a calendar year for hip fracture were only counted once to exclude those with more than one admission for the same fracture or transfer between hospitals . a few ( n = 12 ) with missing postal codes were excluded . the final number for analysis was 1209 ( 902 women and 307 men ) .
the number of london residents for the city of london was obtained from statistics canada , census of canada data for the years 2001 and 2006 categorized according to census tract . given that intercensal estimates are not generated at this level of aggregation , population estimates for each year
this assumes a linear change in population numbers and distribution between the two census times .
the population count for the middle of the study period ( 2004 ) was used in all analyses that amalgamated the study years .
the number of institutionalized people ( n = 3199 ) was available from the local agency responsible for admissions to ltc .
the age and gender distribution was calculated by a sample survey of 6 ltc homes housing 1448 residents , representing a 45% sample of all ltc residents in the city . for the institutionalized population
the numbers in three age strata 6574 , 7584 , and 85 years and over were calculated for each gender . from the statistics canada data the community dwelling population in each age stratum
was derived and the rate of hip fracture for each age stratum calculated from this and the dads .
standard rate of fracture for each age stratum which was used to calculate the expected or theoretical rate of hip fracture for the institutionalized subjects that would be expected if they had the same rate as those of that age stratum and gender living in the community . a total for each of the actual fracture counts and the theoretical counts could then be summed to provide a comparison of the two counts for the total institutionalized population .
although this is similar to the methods used in other studies , for example , that employed by guilley et al . , there is a difference between the two populations that should be noted .
beds which are vacated , usually in the canadian system by the demise of the resident , are filled by a new admission which may or may not be of the same gender and in the same age stratum .
it is likely , however , that overall , the population is reasonably stable regarding gender and age distribution , in the absence of any major extraneous influences . in the community ,
but once again , in the absence of major demographic trends , such as the arrival of the baby boomers , the population will remain reasonably stable in terms of age and gender distribution .
there will be some immigration into and emigration from the region of interest somewhat similar to the discharge and admission processes of the institution and which can not be accounted for .
furthermore in the method used whereby secondary data analysis of 5 consecutive years of administrative data are studied , the institution dwellers are not a true cohort as the turnover is higher , and the composition of the cohorts , in terms of the individual members of the study group year by year , will change more than the community dwellers .
this turnover may actually be part of the cause of an excess rate of hip fractures as people newly admitted are known to be at higher risk of hip fracture .
accordingly the definition of the rate of fracture presents some difficulty but for simplicity sake we have chosen to call it the rate per person - year but bearing in mind the above provisos .
data analysis was performed using spss 18 , and confidence intervals for ratios followed the method of altman et al . .
figures 1 and 2 compare the crude rates of fracture in community and institutional dwelling subjects for the three age strata and the two genders . in the community ,
the expected rapid rise with age is seen , with women higher than men for all age strata . for men in institutions the fracture rate increases with age and
is much higher than that seen in the community men at all age strata . for women
the pattern is somewhat different , with the rate tending to plateau and the rate in the oldest band being very similar for women living in the community and those in the institutions .
the men in institutions at the two younger age bands have a fracture rate similar to those of women of the same ages whereas the oldest men in institutions actually have a higher rate than the oldest women in institutions ( p < .05 ) .
table 1 shows that the mean age of the patients in the various age bands was very similar for community and ltc dwelling patients .
table 2 shows the actual numbers of hip fractures for men , women , and both together for those living in institutions and the theoretical , or expected , number that would be seen if their rate was the same as those of similar age and same gender living in the community , that is , standardized to the community population .
the theoretical number of fractures is calculated by multiplying the total institutional population of that age and gender by the rate of fracturing shown by the community dwellers of same age stratum and gender .
for the total of all institutional subjects the rate of fracturing is 1.8 ( 95% ci 1.62.0 ) times higher than expected , but it is only 1.5 ( 95% ci 1.31.7 ) times higher for women while being 4.3 ( 95% ci 3.75.6 ) times higher for men ( p < .05 for men versus women ) .
overall , 207 of the 373 institution - based hip fractures or 55% ( + / 2.6 se ) can be explained by the age and gender distribution of the institutional population , but the corresponding percentages explained for the women and men are 66% ( + / 2.8 ) and 23% ( + / 4.5 ) , respectively .
our study suggests that the excess number of hip fractures that are seen in the ltc population is only partly accounted for by the age and gender distribution in this population .
furthermore , a much lower proportion of the fractures in men appears to be attributable to the age distribution than is the case in women .
hip fractures are a complicated phenomenon produced in part by increasing fragility of the bones with age , an increasing tendency to fall , and a change in the manner of falling in older life , where the fall is more likely to be sideways or backwards [ 10 , 11].the studies of zimmerman et al . and
chen et al . provided several predictors of hip fracture in the ltc population , namely , age , cognition , gender , race , and weight .
the population studied here is almost exclusively of white caucasian origin and race is not an issue .
we did not have access to weight , which may be an important factor as residents of ltc may lose weight .
this has been well described in the context of private for - profit nursing homes in the united states , but evidence from a canadian context is less available [ 1214 ] .
undernutrition , as opposed to actual weight loss , is well described , especially in cognitively impaired residents and , indeed , low body weight is a risk factor for admission to an institution . where the hip fracture phenomenon fits into this scenario clearly requires more study . within certain populations
cumming was able to explain the difference in hip fracture rate between institution and community on the basis of various potential confounders , such as weight , dementia , diet , activity , and so on , but whether the final common pathway was through thinning of the bones or risk of falling , was not explored .
dementia , for example , is associated with an increased rate of hip fracture , but it is associated especially with an increased rate of falling [ 17 , 18 ] .
. demonstrated that for a given bone density there were more fractures with aging , suggesting other factors operate , the obvious one being a tendency to fall more often .
interestingly , none of these studies has corrected for the rate of falling nor may this be the whole answer , as the way of falling may be as important as the frequency .
the possible consequence of the higher turnover in the ltc group needs to be considered .
our hip fracture rates , especially in the older women , are lower than some of the figures reported in the literature .
for example , rapp et al . found a markedly higher rate but their study was a prospective cohort following subjects from the time of admission .
thus this included the early stages of the admission to the home when risk of fracture is highest , due perhaps to disorientation and unfamiliarity with the surroundings , deconditioning from recent illness , and so on , but even allowing for that our rates are low . for example , chen et al
. found an overall rate of 40 per 1000 person years in a prospective study of subjects already residing in ltc . whether the difference is due to patient factors such as different levels of mobility , or system characteristics , such as admission criteria or
one might have predicted that the need to be admitted to long - term care would have had a leveling effect on the rate of fracturing as individuals need to have reached a threshold level of dysfunction , and perhaps frailty , in order to be admitted . to some degree
this does happen as the relative increase in risk is much higher for the younger strata compared to those in the community ( table 2 ) .
however , the observation that for the most part absolute risk is higher for those who are in an institution and are in the most advanced age category suggests that even in older patients , for whom institutionalization seems necessary , increasing frailty is seen with increasing age .
the markedly higher hip fracture rate in institutionalized men of all ages and institutionalized women in the two younger age strata , compared to those in the community , is notable .
the anomaly seems to be the similar rate of fracturing in the oldest women whether in the community or ltc .
possibly , the reasons the oldest women are admitted to ltc are not related to the risk of falling , this therefore remaining the same for both community and ltc dwellers from that stratum .
it is known that falling in the ltc population is much more prevalent than in the community but whether this is correct for the oldest female group is unknown .
as more very old men have surviving spouses than do old women , this may enable them to be cared for in the community until they are particularly frail and/or ill , and institutionalization is required . thus the oldest men may be frailer than their female counterparts . however , this is speculative and the explanation is by no means clear .
the situation in the men , who are presumably admitted to the ltc sector for reasons other than osteoporosis , suggests that the high rate in ltc is not just bone - related and , compared to the oldest men , the lower rate in the oldest institutionalized women , who might be expected to have the most osteoporosis , supports a different etiology .
our study has some obvious shortcomings , the main one being an absence of information regarding some of the risk factors , such as weight and cognitive status , in the subjects in the two settings .
our study does , however , include all hip fractures over a 5-year period so should it be free of selection biases .
there is some urgency to sorting out the cause of the excess fracture rate in the ltc setting , where it may be easy to assume that the underlying problem is osteoporosis , leading to the use of osteoporosis medications in this setting despite absence of efficacy for this population . as van den kroonenberg
et al . have shown , a fall onto the greater trochanter will generate enough force to break most if not all hip bones , and the real problem of hip fractures in ltc may well be the much more difficult one of falling and the way of falling , with osteoporosis simply determining where the hip will break , with those with thin bones more likely to suffer an intertrochanteric fracture than a subcapital fracture . | introduction .
this study compares hip fracture rates in long term care ( ltc ) residents with those in the community to determine if their high rate of fracturing reflects the extreme age and predominantly female nature of that population .
methods .
hospital discharge data in london ontario ( population 350,000 ) and statistics canada data were used to correct the hip fracture rate in the ltc setting for age and gender .
results .
the risk of hip fracture is 1.8 times greater in ltc than in the community for people of similar age and gender .
the rate in women is 1.5 times higher whereas in men it is 4.3 times higher . in the oldest residents , the risk in men exceeds that of women in ltc .
conclusion .
the high hip fracture rate in ltc is not just a reflection of the age and predominantly female nature of this population .
the oldest men in ltc are a particularly high risk group , deserving more attention . |
angiogenesis is a constant hallmark of multiple myeloma ( mm ) progression and has prognostic potential .
we have previously isolated endothelial cells ( ec ) from bone marrow of patients with mm ( mmec ) .
they show intrinsic angiogenic ability , because they rapidly form a capillary network in vitro , and extrinsic ability , because they generate numerous new vessels in vivo in the chick embryo chorioallantoic membrane ( cam ) .
moreover , mmec secrete angiogenic cytokines , such as fibroblast growth factor-2 ( fgf-2 ) and vascular endothelial growth factor ( vegf ) , as well as matrix metalloproteinase-2 and 9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells .
studies by rajkumar et al . , dominici et al . , and laroche et al .
the bone marrow microenviromnent plays a crucial role in inducing the angiogenic response in mm [ 6 , 7 ] .
reciprocal positive and negative interactions between plasma cells and bone marrow stromal cells , namely haematopoietic stem cells , fibroblasts , ostoblasts / osteoclasts , ec , ec progenitor cell , t lymphocytes , macrophages and mast cells are mediated by an array of cytokines , receptors and adhesion molecules .
these cells secrete angiogenic factors , such as interleukin-6 ( il-6 ) , vegf , fgf-2 , hepato - cyte growth factor / scatter factor ( hgf / sf ) , transforming growth factor- ( tgf- ) and tumour necrosis factor ( tnf- ) [ 6 , 7 ] . here , we have attempted a fine characterization of the angiogenic response induced by mmec , as compared with monoclonal gammopathy of undetermined significance ec ( mgusec ) in the cam assay .
we investigated the angiogenic response induced by gelatin sponges soaked with the cell suspensions and implantedonthecamsurfacefrom8today12ofincu - bation and we studied the effects of mmec and mgusec on the expression of endogenous levels in the cam of vegf , fgf-2 , angiopoietin-1(ang-1 ) , hypoxia - inducible factor-1 ( hif-1 ) and endostatin by reverse transcriptase - polymerase chain reaction ( rt - pcr ) .
mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described .
fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell .
the window was sealed with a glass and the eggs were returned to the incubator . gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions .
the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively .
the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) .
blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x .
means 1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data .
total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna
were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining .
the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) .
level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin .
pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) .
preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) .
intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) .
six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] .
after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide .
the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst .
peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film .
control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load .
stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) .
mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described .
fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell .
the window was sealed with a glass and the eggs were returned to the incubator .
gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions .
the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively .
the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) .
blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x .
means 1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data .
total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna
were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining .
the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) .
level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin .
pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) .
preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) .
intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) .
six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] .
after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide .
the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst .
peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film .
control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load .
stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) .
the ability of mmec and mgusec to induce an angiogenic response in vivo was assessed with the cam - gelatin sponge assay .
1 ) . cam implanted with the medium containing fgf-2 or mmec gave significantly higher vessel counts and numerous allantoic vessels converging like spokes toward the sponges were recognizable ( table 1 and fig .
when the sponges were loaded with the medium alone and with mgusec , physiologic angiogenesis was observed in the form of few allantoic vessels partly around and partly converging toward the sponge ( table 1 and fig .
angiogenic response assessed by the chick embryo chorioallantoic membrane ( cam)-gelatin sponge assay p<0.001 versus rpmi-1640 and mgusec angiogenic activity in the chorioallantoic membrane ( cam ) assay . gelatin sponges loaded with fgf-2 ( a ) , mmec ( b ) , rpmi-1640 ( c ) and mgusec ( d ) were implanted on top of the cam on day 8 .
macroscopic view of the cam on day 12 shows numerous allantoic vessels converging like spokes toward the sponge in a and b , while few allantoic vessels are recognizable in c and d. original magnification : a d , x 50 . in order to evaluate the angiogenic effects of mmec and mgusec , mrna expression of pro - angiogenic factors including vegf , fgf2 , ang-1 , hif-1 and mrna expression of anti - angiogenic molecule endostatin
the expression level of each transcript was compared to those detected in control cam . as shown in fig .
2 , treatment of cam with mmec and mgusec did not produce significant difference in the expression of pro - angiogenic factors mrna in comparison with control cam treated with medium alone .
moreover , no statistical difference was found in vegf , fgf-2 , ang-1 , hif-1 and endostatin mrna content between mm and mgus treated cam ( p<0.05 ) .
in contrast , the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam ( mgus od:120.113 and mm od:48.319.6 versus ctrl od:205.84 19.6 ; p<0.05 ) .
expression levels of mrna coding for vegf , fgf-2 , ang-1 , hif1 and endostatin evaluated by semi - quantitative rt - pcr .
transcript levels from the cam assay are referred to ec obtained from 6 mm and 6 mmgus patients and the error band represents the standard deviation of six experiments .
western blot analyses were also performed to study whether the expression of endostatin changes in mmec and mgusec treated cam respect to control . as shown in fig .
3 , according to rt - pcr quantification , endostatin was significantly lower also at the protein level in both mgusec and mmec treated cam respect to control cam ( mgus od : 97.1410 and mm od : 52.313.6 versus ctrl od : 158.715.52 ;
p<0.05 ) . immunoblotting analysis and quantification after western blot analysis of endostatin expression in cam treated with ec obtained from 3 mm and 3 mgus patients .
mm plasma cells secrete vegf , whereas both vegfr-1 and vegfr-2 are markedly elevated in the bone marrow stromal cells , suggesting that a paracrine growth pathway mediated by vegf - activated stromal cells , secretion of il-6 on their part , and subsequent activation of plasma cells may occur . in active mm , we found secretion of the vegf - a isoform by plasma cells , overexpression of vegfr-2 by bone marrow microvessels and isolated ec and of vegfr-1 by the other stromal cells , secretion of vegf - c and vegf - d by stromal cells , and expression of their cognate receptor vegfr-3 by plasma cells , which suggests another paracrine loop of angiogenesis and tumour growth in mm .
parallel studies by kumar et al . found secretion of vegf by several mm cell lines and fresh patients ' bone marrow plasma cells , as well as expression of vegfr-1 and vegfr-2 , which suggests multiple vegf - mediated autocrine pathways of tumour growth and paracrine stimulation of angiogenesis .
when we studied the angiogenic potential of plasma cell suspension obtained from patients with active mm and implanted them onto the cam , we demonstrated a vasoproliferative response , significantly higher than that induced by cell suspension obtained from patients either with non - active mm or mgus .
hgf / sf is yet another angiogenic factor identified in the culture medium of both human mm cell lines and freshly isolated plasma cells .
still other factors may be responsible for bone marrow angiogenesis in mm : the plasma cell secretion products , such as tgf- , il-1 , il-6 and il-8 ; granulocyte - colony stimulating factor ( g - csf ) , granulocyte macrophage - colony stimulating factor ( gm - csf ) and tnf- , secreted by bone marrow microenvironment cells recruited and activated by plasma cells .
since human mm cell lines produce ang-1 that up - regulates the tie-2 cognate receptor in bone marrow ec , involvement of the ang-1/tie-2 loop in mm neovascularization has been demonstrated .
conversely , mmec secrete angiogenic cytokines , such as fgf-2 , vegf and ang-1 as well as matrix metalloproteinase-2 and -9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells .
other studies , however , showed that the expression levels of vegf , fgf-2 , and their receptors were similar among plasma cells from mgus and mm , suggesting that increasing angiogenesis from mgus to mm is , at least in part , explained by increased tumour burden rather than increased expression of vegf / vegfr-2 by plasma cells .
another possibility is that the inhibition of angio - genesis in mgus is lost with progression , hence that the switch from mgus to mm may involve a loss of an anti - angiogenic activity .
our data are in accord with this statement , because the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam .
the phenotypic switch to angiogenesis in tumour growth involves more than simple up - regulation of angiogenic activity and is thought to be the result of a net balance of positive and negative regulators , and our study suggests that angiogenic switch in mm , investigated by using an in vivo experimental model such as the chick cam , may involve loss of an endoge - nous angiogenesis inhibitor , namely endostatin . | abstractwe have attempted a fine characterization of the angiogenic response induced by multiple myeloma endothelial cells ( mmec ) by using the chick embryo chorioallantoic membrane ( cam ) assay and by reverse transcriptase - polymerase chain reaction ( rt - pcr ) .
results showed that in the cam assay mmec induced an angiogenic response comparable to that of a well - known angiogenic cytokine , namely fibroblast growth factor-2 ( fgf-2 ) , while rt - pcr demonstrated that the expression of endostatin mrna detected in mm treated cam was significantly lower respect to control cam .
these data suggest that angiogenic switch in mm may involve loss of an endogenous angiogenesis inhibitor , such as endostatin . |
preeclampsia , a pregnancy - complicated syndrome , is one of the leading causes of maternal and fetal morbidity and mortality .
this potentially dangerous condition affects approximately 27% of all pregnancies in the western world , but the incidence in developing countries can be greater [ 1 , 2 ] .
preeclampsia is a maternal systemic symptom of late second or third trimester of pregnancy , characterized by hypertension , proteinuria , fetal intrauterine growth restriction , and other maternal abnormity . in the worst cases
now , except for induced delivery , there is no effective treatment to cure it .
risk factors involved in preeclampsia have been increasingly identified ( table 1 ) , and emergence of any risk factor may cause a woman to develop preeclampsia . however , exact pathogenesis of this disorder remains uncertain since numerous research into the etiology of preeclampsia did not progress significantly .
understanding the detailed mechanism underlying preeclampsia will help to develop novel prophylactic and therapeutic interventions for this condition .
the current understanding about preeclampsia is that it appears to develop in a two - stage progress , with a preclinical initial stage which has an abnormal placenta , followed by a second stage during which release of soluble factors from hypoxic placenta leads to various observed maternal conditions , including hypertension , proteinuria , and generalized damage to the maternal organ .
preeclampsia is commonly considered to result from the presence of a poor placenta since the only effective cure of the clinical syndrome is delivery of the placenta .
placentation occurs from weeks 6 to 18 of gestation , during which extravillous cytotrophoblast invasion is required for extensive remodeling of maternal spiral arteries . in preeclampsia , placenta is extremely shallow because of inadequate trophoblast invasion and deficient remodeling of spiral arteries [ 5 , 6 ] .
implantation failure of placenta predisposes it to hypoxia and oxidative stress , thus resulting in a maternal systemic inflammatory response characterized by enhanced expression of inflammatory mediators .
it was found that dysfunctional placenta can release adverse factors into the maternal circulation , causing the clinical features of this condition .
these factors include some hormones or chemical substances , such as sflt1 ( soluble fms - like tyrosine kinase 1 ) , at1-aa ( angiotensin ii at1 receptor auto - antibody ) , and cardiac glycosides ( marinobufagenin , mbg ) . among these
, sflt1 can cause endothelial dysfunction in the maternal blood vessels by binding vascular endothelial growth factors and placental growth factor ; at1-aa can react with the at1 receptor in a stimulatory fashion similar to angiotensin ii signalling , which could contribute to maternal hypertension ; mbg , involved in the pathogenesis of preeclampsia , has been demonstrated to cause hypertension , proteinuria , and intrauterine growth restriction in the rat model of preeclampsia .
augmented release of these factors secondary to the original poor placenta will eventually lead to damage of the endothelium , metabolism dysfunction , and inflammation as well as other typical symptoms .
then , what is the exact mechanism leading to deficient trophoblast invasion and further poor placenta ?
immune responses and maternal endocrine system can be considered to be two main elements involved in the development of placenta . there is evidence suggesting that shallow placenta is thought to stem from the lack of immune tolerance to pater - derived fetal antigens .
initial maternal rejection to the fetoplacenta may be the main reason for inadequate spiral arteries remodeling in preeclampsia . during the normal pregnancy , maternal innate immune response
th1/th2 activity balance is strongly shifted toward th2 activity to maintain the normal fetomaternal interaction .
many have found that maternal t cell can not recognize fetal human lymphocyte antigens ( hlas ) rightly and decidua nk cell has an unusual phenotype in preeclampsia .
moreover , another important difference found between normal pregnancy and preeclampsia is the shift of th1/th2 balance toward th1 responses .
in addition to immune maladaptation , it is also found that maternal endocrine system is perturbed in preeclampsia . in normal pregnancy ,
components of renin - angiotensin - aldosterone system , including renin , angiotensin , and aldosterone , are increased , while these hormones are suppressed in preeclampsia .
a recent report found that activation of mineralocorticoid receptor in trophoblast by maternal aldosterone appears to be required for trophoblast proliferation and placenta development .
additionally , other pregnancy - related hormones also make great contribution to favourable pregnancy outcome . for example , progesterone plays a dominant role in maintaining uterine quiescence and modulating fetomaternal immune response locally , while estrogen appears also to be a potent immunomodulator in pregnancy - associated immune function .
aberration in these hormones level may disrupt homoeostasis of placenta development , thus causing the onset of preeclampsia eventually .
overall , abnormal change in immune responses and maternal endocrine system throughout pregnancy may be one explanation for preeclampsia ( figure 1 ) .
following extensive research into placentation , recent advances have identified emerging key players relevant to placenta establishment , including renin - angiotensin system ( ras ) , 1 , 25-dihydroxyvitamin d ( 1,25(oh)2d3 ) , and lipoxin a4 .
evidence indicates that aberration in them may disrupt placenta development , contributing to adverse pregnancy outcome , such as preeclampsia ( described subsequently ) .
v - atpase , another key molecule in placentation , is closely associated with all of them . during normal pregnancy ,
ras , 1,25(oh)2d3 , and lipoxin a4 may work independently , in parallel or jointly on the central molecule , v - atpase , while v - atpase also affects them at the same time .
therefore , the intriguing molecule v - atpase may represent a key link between various causes of preeclampsia . in the following
the present review will focus on some new findings about interaction between v - atpase and ras , 1,25(oh)2d3 and lipoxin a4 in pregnancy , which will provide new insights into the molecular mechanisms underlying preeclampsia .
the acidity regulation of intracellular and extracellular environment is crucial for a variety of biological processes , including acidity homeostasis , membrane trafficking , protein degradation , and sperm maturation .
central to this regulation is the vacuolar atpase ( v - atpase ) , which is a large multisubunit complex functioning as an atp - driven proton pump .
v - atpase is of significant importance in basic physiological processes , and it has been implicated in a number of human diseases , such as renal disease , tumour metastasis , and bone disease .
v - atpase is composed of two large domains , v0 and v1 . the v0 domain is a 260 kda membrane - embedded complex that contains six different subunits ( a , d , e , c , c , and c ) and functions as a proton - translocation domain , whereas the v1 domain is a 650 kda cytoplasmic complex that contains eight different subunits ( a , b , c , d , e , f , g , and h ) and functions as an atp - hydrolytic domain .
it is the structural complexity of v - atpase that makes it an important player in various physiological functions .
many of v - atpase subunits have multiple isoforms , and so specific v - atpase can be composed of different combinations of these subunit isoforms .
v - atpase , mediating various physiological processes and cellular homeostasis , may be closely involved in different stages of reproduction .
in fact , there are many reports demonstrating that v - atpase has an important role in all aspects of mammalian reproduction , such as spermatogenesis , fertilization , embryonic implantation , and embryo development . on one hand ,
the acidic microenvironment regulated by v - atpase is essential for embryonic development following embryonic implantation . on the other hand ,
different v - atpase has specific tissue distribution in reproduction system and exerts differential crucial function . among all of the v - atpase subunits , subunit a
is involved in acrosomal reaction of sperm and embryonic implantation , both of which provide vital foundation for successful pregnancy . subunit
a is a large membrane - spanning protein with a hydrophilic cytoplasmic n - terminal domain and a nine - transmembrane c - terminal domain , and it contains the information necessary to target the v - atpase to different cellular destinations and is responsible for proton transport into the lumen of intracellular vesicles or across membranes .
( a1 , a2 , a3 , and a4 ) , which exhibit diverse tissue distribution and subcellular location .
( atp6v0a2 , referred to as a2v - atpase ) can be detected in the epididymis , kidney , lung , thymus , spleen , and placenta .
recently , increasing evidence indicates the crucial role of a2v - atpase in placental immune modulation associated with pregnancy . in early pregnancy
, many important cytokines secreted by placenta trophoblast cells and immune cells in decidua play a key role in embryonic implantation and fetomaternal immune tolerance .
interestingly , it is reported that a2v - atpase is important in controlling the expression of these essential cytokines .
thus , the normal expression and localization of a2v - atpase in fetoplacental unit is essential for pregnancy .
have reported that placental a2v - atpase expression is a link between multiple causes of spontaneous abortion in mice , and dysfunction of a2v - atpase in early pregnancy has been associated not only with spontaneous abortion but also with preeclampsia and fetal growth restriction .
another interesting study found that nox2/nadph oxidase regulates antigen crosspresentation in adaptive immunity through changing phagosome lumen ph , whereas the effect of nox2/nadph oxidase on phagosomal ph is antagonized by v - atpase , which may represent a novel mechanism responsible for the maintenance of immune tolerance .
taken together , these indicate the importance of v - atpase in maintaining reproduction process and placentation and modulating fetomaternal immune tolerance in pregnancy , whereas defective v - atpase may increase the risk of causing a poor placenta and inducing preeclampsia ( figure 2 ) .
the circulating renin - angiotensin system ( ras ) is an endocrine hormone system that plays a key role in regulating blood pressure and systemic electrolyte balance .
apart from systemic circulating ras , local ras is present in many tissues or organs , such as brain , heart , ovary , and placenta , and one of the major extrarenal ras during pregnancy is the placental ras . in pregnancy ,
the circulating ras maintains maternal electrolyte balance and blood pressure homoeostasis , facilitating expanding blood supply for fetus , while the placental ras has indispensable role in the uteroplacental unit .
components of placental ras have been confirmed to be involved in many molecular events of placental establishment .
a recent review reported that placental cytotrophoblast is rich in at1 receptor , and ang ii - at1 receptor signaling regulates multiple genes associated with normal trophoblast invasion , such as plasminogen activator inhibitor-1 ( pai-1 ) , such as soluble fms - like tyrosine receptor-1 ( sflt-1 ) and soluble endoglin ( s - eng ) .
additionally , ang ii signaling also activates nf - kappa b and stimulates the synthesis of reduced nicotinamide - adenine dinucleotide phosphate ( nadph ) oxidase by trophoblast .
moreover , it has been demonstrated by a recent study that activation of mineralocorticoid receptor in placental trophoblast by maternal aldosterone appears to be required for trophoblast growth and normal fetoplacental function .
therefore , in response to pregnancy , both systemic ras and placental ras need to be adapted to sustain a healthy pregnancy .
, there is an increase in almost all the components of maternal ras , including renin , angiotensin , and aldosterone , but these hormones are suppressed in preeclampsia .
however , the changes happening in the components of uteroplacental ras are different from those observed in circulating ras .
recent studies demonstrated that upregulation of the at1 receptor expression , increase in renin level , and enhancement of ang ii production occur in uteroplacental unit of preeclamptic women .
so , the expression profile of both systemic ras and placental ras in preeclampsia differs greatly from that of healthy uncomplicated pregnancy . additionally , there is a growing body of reports indicating the presence of the angiotensin ii type i receptor agonistic autoantibody ( at1-aa ) in preeclamptic women , and this autoantibody through at1 receptor signaling can result in dysregulation of the ras , then leading to clinical features of preeclampsia .
all abnormalities in both systemic ras and placental ras can cause poor placentation and preeclampsia .
there is accumulating evidence suggesting that components of ras are interrelated to v - atpase and are essential for regulating v - atpase activity .
the ( pro)renin receptor ( ( p)rr ) , encoded in atp6ap2 , plays a key role in activation of the local ras , while a truncated form of ( p)rr , termed m8.9 , was found to be associated with the v - atpase assembly and function , implicating a novel role of ( p)rr in v - atpase activity .
recently , another interesting report indicated that ( p)rr functions as an adaptor between wnt receptor and v - atpase , which is required for mediating wnt signaling .
it was found that angiotensin ii has profound effects on h transport and urinary acidification in kidney , mainly through its regulation on v - atpase activity , and it has also been confirmed to stimulate vacuolar h - atpase activity in renal acid - secretory intercalated cells from the outer medullary collecting duct .
not long before , a report showed that aldosterone may participate in the regulation of v - atpase activity in acid - secretory intercalated cells through rapid nongenomic stimulatory action and contribute to urinary acidification . meanwhile , another study , prior to this review , described that aldosterone has both genomic and nongenomic stimulatory effects on v - atpase in proximal s3 segments .
considering the content described earlier , it is reasonable that uteroplacental v - atpase function is a key link between ras and placentation .
therefore , any aberration in interaction between ras and v - atpase may induce preeclampsia by interfering various processes in placent development .
1,25-dihydroxyvitamin d ( 1,25(oh)2d3 ) , the active form of vitamin d , has long been considered to be a leading molecule in calcium and bone homeostasis . however , in the past years , it was found that vitamin d receptor ( vdr ) is present in most tissues and many tissues contain the 1,25(oh)2d3 synthase , 25(oh)d3 - 1-hydroxylase [ 31 , 32 ] , which makes us begin to be aware of multiple nonclassic actions of 1,25(oh)2d3 .
in fact , advances have shown that 1,25(oh)2d3 deficiency may increase the risk of developing a wide range of chronic diseases , including cancer , autoimmune disease , infectious disease , and cardiovascular disease . through binding to the vdr
, 1,25(oh)2d3 can generate a wide array of favorable nonclassic biological responses that are different from its classic actions in maintaining bone and calcium homeostasis , whereas these nonclassic actions are mainly linked to its regulation of cell proliferation , cell differentiation , and immune responses .
it is worth emphasizing that some of nonclassic biological effects of 1,25(oh)2d3 have been suggested to participate in modulation of embryo implantation , fetomaternal immune tolerance , and placental antimicrobial and anti - inflammatory responses .
therefore , 1,25(oh)2d3 deficiency in pregnancy may not only impair maternal and fetal bone health , but also cause various adverse pregnancy outcomes such as preterm birth , fetal intrauterine growth restriction , and preeclampsia .
al demonstrated that the level of 1,25(oh)2d3 in both maternal circulation and umbilical cord compartment is low in preeclampsia .
furthermore , expression and activity of 25-hydroxyvitamin d-1-hydroxylase has been confirmed to be significantly restricted in cultures of placental syncytiotrophoblast cells from preeclamptic pregnancy .
typical feature of preeclampsia is maternal systemic inflammatory response , characterized by upregulation of proinflammatory mediators in both uteroplacental unit and peripheral circulation .
in fact , as described previously , the systemic inflammatory response in preeclampsia is thought to stem from initial disorder of fetomaternal immune tolerance .
vitamin d , particularly 1,25(oh)2d3 , has received particular attention in recent years as it has been shown to have important effect on both innate and adaptive immune systems .
moreover , 1,25(oh)2d3 has been demonstrated to be essential for modulating tissue - specific immune responses and preventing chronic inflammation and autoimmunity .
increasingly , the immunomodulation of 1,25(oh)2d3 has been proposed as a key link between vitamin d and pregnancy .
in addition to be mainly synthesized in the kidney , 1,25(oh)2d3 can also be locally produced by immune cells , such as macrophage , dendritic cell , activated t cell , and probably b cell . in autocrine or paracrine manner ,
the local produced 1,25(oh)2d3 can exert a marked inhibitory effect on adaptive immune responses , promoting humoral th2 responses whilst suppressing cellular th1 responses [ 33 , 36 ] .
for example , 1,25(oh)2d3 is able to inhibit the differentiation , maturation , and immunostimulatory capacity of dcs by decreasing the expression of mhc class ii molecule , cd40 , cd80 , and cd86 .
a recent report by saito speculated that imbalance between regulatory t cell and th17 cell differentiation may explain pathogenesis of preeclampsia , which provides new light on pathophysiology of preeclampsia .
interestingly , 1,25(oh)2d3 has been confirmed to enhance tolerogenic immunity by inducing immunosuppressive treg , while proinflammatory th17 cell is suppressed by 1,25(oh)2d3 . in summary , maternal vitamin d level may influence pregnancy outcome through a variety of potential mechanisms , and impaired vitamin d status may lead to preeclampsia .
indeed , a recent study demonstrated that in the presence of vitamin d , human osteoclasts express elevated v - atpase levels .
meanwhile , this work also described that 1,25(oh)2d3 can upregulate the activity of v - atpase complex .
although the study did not study the case in pregnancy , it will imply us that 1,25(oh)2d3 may have an important effect on uteroplacental v - atpase activity .
moreover , the ability of 1,25(oh)2d3 to regulate secretion of pregnancy - related hormones may explain its role in mediating v - atpase activity .
1,25(oh)2d3 has been confirmed to be an important player in the upregulation of estrogen biosynthesis , and the previous description has also revealed that v - atpase expression is estrogen dependent , which makes it reasonable to speculate that 1,25(oh)2d3 can act on v - atpase indirectly by influencing hormones level .
recently , a significative review suggested that autophagy is a general basis for the multiple health - promoting effects of vitamin d .
autophagy , depending on lysosomal self - digestion machinery , is an evolutionarily conserved self - catabolism process that is essential for cell growth , development , and homeostasis . to a great extent ,
v - atpase is vital for autophagy since v - atpase is of great importance for lysosomal function .
thus , coupled with v - atpase , 1,25(oh)2d3 could induce and activate autophagy , protecting human against many diseases .
autophagy is induced by several forms of cell stress including hypoxia , infection , and starvation .
if autophagy could not be initiated properly , disturbance in autophagy will result in a wide range of disorder . in the case of preeclampsia , abnormal placentation in early pregnancy predisposes placenta to ischemia , hypoxia , and oxidative stress , which accumulates large numbers of apoptotic trophoblast cells at uteroplacental interface .
if not treated through cell autophagy , the apoptotic trophoblast cells may impair further placenta development seriously and induce onset of preeclampsia .
thus , maternal vitamin d deficiency may trigger preeclampsia through affecting v - atpase activity , which seems to represent a general and basic pathogenesis of preeclampsia .
lipoxin , the stop signal for inflammation , is the first mediator recognized to have dual anti - inflammatory and proresolution activities . in mammals ,
lipoxin a4 , mainly depending on transcellular biosynthesis in the inflammation microenvironment , is derived from arachidonic acid by the sequential action of lipoxygenase ( lox ) .
this pattern of biosynthesis involves the initiation of arachidonic acid oxygenation by 5-lox in leukocytes , and then the intermediate leukotriene a4 is converted into lipoxin a4 by 12-lox in platelets .
interestingly , our previous results indicated that platelet - derived microparticles containing 12-lox can be transferred to leukotriene - producing mast cells , which represents a new pathway for lipoxin a4 biosynthesis in vivo .
lipoxin a4 can act on many cell types including blood cell , neural cell , and stromal cell .
lipoxin a4 displays selective regulation for leukocyte responses and trafficking in vivo through activating its specific receptor , alxr .
for example , in neutrophils , lipoxin a4-alxr interaction blocks neutrophil migration and infiltration into sites of inflammation .
by contrast , in monocytes , lxa4-alxr interaction stimulates monocyte recruitment and the phagocytosis of apoptotic neutrophils .
recently , we found that lipoxin a4 can suppress hepatocellular carcinoma via remodeling tumor microenvironment .
moreover , we found that alxr ( also termed fpr2 ) has an appreciable pleiotropic regulator role in macrophage polarization , and lxa4-induced m2a+m2c - like macrophage phenotype showed antitumorigenesis activities . taken together ,
all previous results show us the key role of lipoxin a4 as an essential modulator of immune response and homoeostasis .
inflammatory processes are central to a series of pregnancy events , while inflammation dysregulation is a typical feature of pregnant complications , such as preeclampsia and preterm .
normal pregnancy is characterised by a state of mild maternal systemic inflammation , which develops when pregnancy is progressing .
although preeclampsia is also associated with inflammatory response , it is an exaggerated maternal systemic inflammatory response .
in fact , this disorder is an extreme condition of a range of uncontrolled inflammatory responses induced by abnormal pregnancy .
lipoxin a4 , as an anti - inflammatory and proresolution mediator , will play an important role in human pregnancy .
in fact , level of lipoxin a4 in nonpregnant women is significantly lower than that in pregnant women , and circulating level of lipoxin a4 in pregnant women displayed a modest but significant increase throughout pregnancy .
thus , it is tempting to speculate that inadequate production of lipoxin a4 can lead to abnormality of placental immunomicroenviroment , increasing the risk of preeclampsia .
this has been confirmed by our results from clinical study that level of lipoxin a4 in preeclampsia is significantly lower than that in normal pregnancy .
additionally , our results from animal study demonstrated that lipoxin a4 could suppress the increased production of proinflammatory cytokines and alleviate the symptoms of preeclampsia in lps - induced rat model for human preeclampsia .
moreover , results from our previous study verified that lipoxin a4 can inhibit the enhanced il-1 production of monocytes from severe preeclampsia women .
recently , preliminary results from animal study indicated that lipoxin a4 may participate in the regulation of rat fetomaternal immune tolerance in early pregnancy
. therefore , lipoxin a4 , an endogenous anti - inflammatory and proresolution mediator , is a key player in terminating exaggerated inflammatory responses and restoring maternal systemic homoeostasis in pregnancy .
the recent findings by russell et al . found that lipoxin a4 is a novel estrogen receptor modulator and exhibits estrogenic activity in vivo , revealing a previously unappreciated facet of various bioactions of this anti - inflammatory and pro - resolution mediator .
thus , lipoxin a4 may exert an important role in regulating the v - atpase expression , as v - atpase expression level is estrogen dependent , which has been mentioned earlier in this review .
in addition , v - atpase may also participate in the modulation of lipoxin a4 bioactions .
there is evidence implicating that v - atpase may get involved in the complex interaction between lipoxin a4 and its receptor in lipoxin a4-stimulated signal pathways .
reported that internalization and trafficking is critical for lxa4-stimulated phagocytosis in the resolution of inflammation .
transmembrane receptors internalization is of physiological importance , which is required for right signal transduction , proper dissociation of ligand from receptor , and receptor - mediated endocytosis .
although receptor internalization may occur in different ways , v - atpase - mediated intraendosomal pathway is the main endocytic trafficking machinery .
therefore , internalization and trafficking of lipoxin a4 receptor is likely to require the right assembly and activity of v - atpase in the endocytic complex of alxr internalization . if there is some defect in v - atpase , biological actions of lxa4 through alxr could be severely hindered . in summary
, lipoxin a4 may play an important role in modulating v - atpase activity during pregnancy while the key role of lipoxin a4 in fetomaternal immunoregulation also depends on normal v - atpase activity extensively .
based on the previous novel findings , a model of how placental v - atpase function can be a key link between multiple causes of preeclampsia is proposed ( figure 3 ) . on one hand ,
v - atpase is an essential modulator in regulating bioaction of ras , 1,25(oh)2d3 , and lipoxin a4 during pregnancy . on the other hand ,
key roles of ras , 1,25(oh)2d3 , and lipoxin a4 can be integrated into the v - atpase function in pregnancy .
right cross - talk between v - atpase and ras , 1,25(oh)2d3 , and lipoxin a4 can facilitate normal placentation and ensure successful pregnancy .
once disorder of v - atpase activity takes place at the fetomaternal interface , various bioactivity of ras , 1,25(oh)2d3 , and lipoxin a4 will come into great trouble , and vice versa .
any abnormality occurring at the interaction between v - atpase and ras , 1,25(oh)2d3 and lipoxin a4 could cause a defective placenta , which will be eventually involved in the progression of preeclampsia .
overall , the previous proposal not only suggests the importance of v - atpase in modulating essential components of maternal endocrine system ( ras ) and key molecules of fetomaternal immune interaction ( 1,25(oh)2d3 and lipoxin a4 ) , but also shows the key role of ras , 1,25(oh)2d3 , and lipoxin a4 in regulating v - atpase function .
maternal endocrine system and immune responses have been long considered to be involved in normal placenta development .
the renin - angiotensin system is a typical endocrine hormone system that plays a key role in regulating placentation as well as maintaining normal pregnancy .
recently , the immunomodulation of vitamin d in pregnancy has been proposed as a key aspect of placental homoeostasis .
additionally , lipoxin a4 , as an anti - inflammatory and proresolution mediator , has been demonstrated to be vital in modulating fetomaternal immune tolerance during pregnancy . nevertheless , the complexity of various molecular events that occur at the fetomaternal interface in placenta development is far from being completely elucidated .
a series of molecular mechanisms needs to be coordinated for establishing a perfect placenta , while any deviation from this ordered course of molecular events may result in pregnancy complications , such as preeclampsia .
v - atpase , a key molecule in placentation , is beginning to be appreciated .
it is closely linked to renin - angiotensin system , 1,25(oh)2d3 , and lipoxin a4 .
further studies regarding v - atpase - related molecular mechanisms necessary for placentation are required , since clinical intervention to these potential dysregulated molecular events may eventually improve pregnancy outcome .
clarify the potential role of placental v - atpase in preeclampsia.investigate the nature of interaction between v - atpase and ras , 1,25(oh)2d3 , and lipoxin a4 in normal pregnancy.investigate the causes of abnormal interaction between v - atpase and ras , 1,25(oh)2d3 , and lipoxin a4 in preeclampsia.understand the mechanisms of placental damage associated with defective v - atpase , ras , 1,25(oh)2d3 and lipoxin a4 in preeclampsia.study the effect of potential therapeutic agents on placental damage and maternal clinical signs of preeclampsia .
investigate the nature of interaction between v - atpase and ras , 1,25(oh)2d3 , and lipoxin a4 in normal pregnancy .
investigate the causes of abnormal interaction between v - atpase and ras , 1,25(oh)2d3 , and lipoxin a4 in preeclampsia .
understand the mechanisms of placental damage associated with defective v - atpase , ras , 1,25(oh)2d3 and lipoxin a4 in preeclampsia .
study the effect of potential therapeutic agents on placental damage and maternal clinical signs of preeclampsia . | preeclampsia , a relatively common pregnancy disorder , is one of the major causes of maternal and fetal morbidity and mortality . despite numerous research ,
the etiology of this syndrome remains not well understood as the pathogenesis of preeclampsia is complex , involving interaction between genetic , immunologic , and environmental factors .
preeclampsia , originating in placenta abnormalities , is induced by the circulating factors derived from the abnormal placenta .
recent work has identified various molecular mechanisms related to placenta development , including renin - angiotensin system , 1 , 25-dihydroxyvitamin d , and lipoxin a4 .
interestingly , advances suggest that vacuolar atpase , a key molecule in placentation , is closely associated with them . therefore , this intriguing molecule may represent an important link between various causes of preeclampsia . here , we review that vacuolar atpase works as a key link between multiple causes of preeclampsia and discuss the potential molecular mechanisms .
the novel findings outlined in this review may provide promising explanations for the causation of preeclampsia and a rationale for future therapeutic interventions for this condition . |
spending hours in plastic surgery opd in a government medical college and dealing with the kind of queries posed to us by patients and often having to see delayed and neglected patients made us question and wonder on various views that people have of plastic surgery .
most of the patients had no idea of the range of our services and had reached us after having visited a series of generalists and eventually being referred to us .
hence , we undertook this study with an aim to ascertain current awareness among the medical students and residents ( the future practitioners ) about the surgical procedures that a plastic surgeon performs .
this study was conducted in four medical colleges chosen from four zones of india : king george medical college ( kgmu ) , lucknow ; all india institute of medical sciences ( aiims ) , new delhi ; king edward memorial ( kem ) hospital , mumbai ; and stanley medical college ( smc ) , chennai .
the questionnaire had 35 surgical situations , whereby the respondents were to refer a patient to any department or departments where a particular problem can be managed .
a total of 2000 questionnaires were distributed and 1552 responses were received . there were 440 respondents from kgmu , 312 from aiims , 418 from kem , and 382 from smc .
the respondents were categorized into four groups : mbbs students , interns , junior residents , and senior residents .
the y - axis reveals percentage of referral to plastic surgeons and x - axis shows the group studied .
facial fractures , craniofacial surgery , temporomandibular joint dysfunction cleft lip palate , burns liposuction , rhinoplasty , hair grafting wound cover , pressure ulcer , scalp avulsion peripheral nerve injury , hand anomalies and trauma
it was observed [ table 1 ] that there was a good deal of awareness ( > 60% ) regarding the role of plastic surgeons in dealing with burn injuries , rhinoplasty , liposuction , hair grafting and cleft lip and palate .
there was a moderate degree of awareness ( 4060% ) regarding wound management and pressure ulcer , being managed by plastic surgeons .
only 1530% respondents were aware that craniofacial anomalies , congenital hand anomalies , facial fractures , scalp avulsion , hand injuries and peripheral nerve injuries are reconstructed by us .
there was a poor knowledge ( < 15% ) about brachial plexus surgery and temporomandibular joint ( tmj ) ankylosis being managed by plastic surgeons .
table 1 is an ascending order depiction of the awareness regarding treatment of various conditions by plastic surgeons .
the findings of our study are similar to those of previous studies on different population and socioeconomic groups which show the public to be poorly informed about plastic surgery . in his paper
perception of plastic surgery in the society , the author concludes that indian public associates plastic surgeons with cosmetic and burn surgeries . the knowledge is also limited among medical students and nurses and the source of their knowledge is magazines and newspapers . in their study
perception and reality a study of public and professional perceptions of plastic surgery conducted in the british public population , medical students and general practitioners , plastic surgery was associated with reconstruction for trauma and cancer and procedures with a strong aesthetic element by all the three groups and hand was associated with orthopaedic surgeons . in a study conducted in philadelphia in the usa , among consecutive patients visiting a primary care facility , medical students , and primary care physicians , the authors demonstrate that public perception of the plastic and reconstructive surgeon is limited and grossly underestimates this specialty .
although the plastic surgeons are associated with reconstructive surgery , they are not necessarily identified as primary surgeons for procedures fundamental to this specialty .
many people who do not come into contact with plastic surgery in their hospitals are under the popular misconception that plastic surgeons spend their time fixing noses and altering the size of breasts .
it is not really surgery since it can be done in the office and is not covered by insurance .
the media is also little informed about the versatility of plastic surgeons and ends up building false expectation in public and wrong images of plastic surgeons as scavengers for money . in their paper plastic surgery in the cinema
, the authors concluded that films usually incorporate cosmetic surgery and burns in the screenplay .
also , plastic surgeons are depicted to be living in affluent localities , making a lot of money .
patients visiting plastic surgeons are also shown to be ultra rich.[710 ] this often deters many patients from primarily consulting us .
patients visiting the department late in the course of disease process have often revealed that they had feared to visit our department fearing the expenditure they would incur . although the findings of this paper may not surprise practicing plastic surgeons , it does quantify a reality we all share that various works of our specialty are not easily identified by public and medical professionals .
we have made an attempt to reason out the cause for the same in our country and suggest remedial measures .
also , the commonly followed textbook of general surgery in the undergraduate curriculum discusses mostly burns and cleft lip and palate under plastic surgery section . to add to these ,
the lectures devoted to plastic surgical topics are few and often delivered by general surgeons who are less equipped to spread awareness among medicos .
hence , plastic surgery department must be developed in all colleges , and medical students and surgical residents must be rotated for at least a week or two as part of their training programme .
this is a long - term goal , and till it is in place , a few committed and reverent teachers in the society may come forward to deliver planned lectures to mbbs students in colleges where the department is lacking .
this can be put forward by the association of plastic surgeons of india ( apsi ) as part of expansion of plastic surgery .
we should also develop methods to educate the general practitioners because they are the people who reach out to public at large .
our primary aim then should be to educate and sensitise the media regarding the role of plastic surgeon even in day - to - day life of a common man
. the contribution of prominent members of plastic surgery society will be more than sufficient to open the eyes of media regarding various subspecialities .
apsi may invite media personnel on an awareness trip to well - established centres to see a variety of procedures and the necessity of plastic surgeon in trauma centres , burn care , as reconstructive surgeons in trauma , congenital and oncological surgeries besides their role as an aesthetic surgeon .
we should encourage media personnel to project a real picture to the general public and publicise the various conditions in which plastic surgeons play a role . at the village level , this may be done by plays and distributing pamphlets and posters showing preoperative and postoperative photos .
regular monitoring of progress in the endeavour to spread awareness can be conducted yearly , and necessary intervention may be added as and when required welcoming newer ideas for the same .
our study clearly shows that knowledge about the field of plastic surgery is highly lacking and lot needs to be done to spread awareness .
the best way to describe plastic surgeons is that we are a problem solving specialty .
the amazing developments made in our field must be made available and accessible to the mass and a collective part played by plastic surgeons can go a long way in making our specialty really known to even layman as to it being a necessity rather than a privilege . | the field of plastic surgery , while being famous for aesthetic surgery , also includes craniofacial surgery , hand surgery , burn surgery , microsurgery , reconstructive plastic surgery and paediatric plastic surgery
. the magnanimous progress in these areas , though a hot topic in conferences , remains cryptic to the layman and also to generalists who are and will remain to be the most important referral source of these patients.[1 ] hence , it becomes the duty of plastic surgeons themselves to spread awareness regarding their chosen field of endeavour . |
this mixed - methods study ( yin , 2003 ) seeks to more accurately identify perceptions of acceptance for various age- , race- , and sexual identity - based segments of the population within the gay and mainstream communities at a pivotal time in the evolution of both urban gay communities and mainstream societal attitudes . while the use of survey data aligns with extant studies that measure gay community attachment ( barrett & pollack , 2005 ; frost & meyer , 2012 ) , the addition of interviews with self - identified gay and bisexual men and service providers allows for the creation of new , grounded theory ( strauss & corbin , 1990 ) about gay community inclusion and its relationship to the changing social contexts of north america .
we employ the concept of perceived acceptance to gauge how the gay and mainstream communities as imagined by individuals are perceived to be oriented toward various groups ( e.g. , men of color , trans men ) , rather than measuring individuals levels of attachment to a pre - given gay community .
first , measuring perceived acceptance is a more meaningful indicator of the inclusivity of gay and mainstream communities than simple attachment ( i.e. , participation ) .
second , asking respondents to identify levels of acceptance toward multiple groups ( e.g. , men of color ) and not just the one being surveyed ( i.e. , gay and bisexual men ) allows individuals to reflect on personal exclusion or discrimination that might be associated with their other intersecting identities ( moghaddam & studer , 1997 ) . finally , our mixed - methods approach allowed for interview narratives to inform our survey design and , through triangulation , corroborate and explain the results it produced .
this study is part of a larger project called health in middlesex men matters ( himmm ) , which was initiated following a 2006 forum organized by the regional hiv / aids connection ( rhac , formerly aids committee of london ) to discuss health needs and challenges for london - middlesex s lgbt , two - spirit , and queer communities .
the himmm project was formed to examine how the forum - identified themes of community , communication , and homophobia influence the lives and health of local gay and bisexual men , specifically . as a community - based collaborative research project ,
partners on himmm include rhac , the university of western ontario , middlesex - london health unit , options clinic for hiv testing at the london intercommunity health centre , st .
joseph s infectious diseases care program , and gay men s sexual health alliance of ontario .
london - middlesex , ontario , the study site , is located halfway between toronto , ontario , and detroit , michigan .
london is the seat of middlesex county and the eleventh largest city in canada , comprising approximately 366,000 residents and another 70,000 in the surrounding county ( statistics canada , 2011 ) .
statistics canada estimates that 34% of the canadian population lives in areas considered to be london - middlesex s peer group : mixed urban - rural areas with average numbers of both aboriginal residents and immigrants ( statistics canada , 2009 ) . as of 2006 , 11.7% of the population of middlesex county was a visible minority , with the largest populations being black , latin american , and arab ( about 2% of the population each ) and chinese and south asian residents representing slightly smaller segments ( statistics canada , 2006 ) . the study sample , which generally mirrors these characteristics
, therefore offers an opportunity to assess the dynamics of social acceptance outside of canada s largest cities .
himmm project data were collected in two phases : an initial phase of qualitative semistructured interviews and a second phase of quantitative information gathered using an online questionnaire . during 2009 ,
semistructured interviews were conducted with 15 local gay and bisexual community members and five service providers .
gay and bisexual men , 16 years or older and residing in middlesex county , were sampled purposively based on age , ethnicity , hiv status , location , and sexual orientation .
service providers , which comprised a physician , an hiv testing provider , and counselors and coordinators from community organizations , were also sampled purposively based on their experiences working with local gay and bisexual men .
community members were asked about gay and bisexual community and identity ; access to health and wellness services ; and spiritual , emotional , and sexual health .
service providers were asked about their experiences serving gay and bisexual populations , including coming out counseling and working with other local organizations to meet service user needs . since gay- and bi - identified trans men ( female - to - male )
are increasingly viewed as part of the gay community ( bockting , benner , & coleman , 2009 ) , and since some trans women ( male - to - female ) have histories or attachments within communities of gay and bisexual men , results relevant to these populations are also considered .
no remuneration was offered for participation . after reviewing the transcripts , the himmm team designed the survey , which was pretested and pilot - tested by local gay and bisexual volunteers .
eligibility criteria allowing access to the online survey included age 18 + , having an address in middlesex county ( though not necessarily a permanent address , as in the case of students ) , and identifying as gay , bisexual , or as a man who has had sexual experience with or strong , continual sexual attractions to another man .
questionnaire participants were recruited through online web sites , smartphone apps , and informal referrals between gay and bisexual men .
participants received a $ 10 gift card as a token gift for completing the survey , with chances to win additional prizes if they referred other gay and bisexual men who also completed the survey .
sociodemographic variables in the survey included age , ethno - racial identity , birth country , educational attainment , household income , employment status , area of residence , marital status , relationship status , and sexual orientation identity , adapted from the canadian community health survey ( statistics canada , 2008 ) and other community surveys .
the project team developed 10 questions assessing perceptions of acceptance of gay men , bisexual men , men of color ( sexuality not specified ) , transgender men ( sexuality not specified ) , and transgender women ( sexuality not specified ) from within the gay community and the broader middlesex - london community .
responses to each question asking how accepting the broader and ( separately ) gay communities were toward each group were expressed in likert scales ranging from 1 ( not at all accepting ) to 7 ( completely accepting ) .
using nvivo 10 , qualitative interview texts were open - coded and grouped into categories and themes based on team discussions of themes emerging from both the initial forum and the interview data .
the team then used coded data reports to outline perceived processes of community development and evolution .
first , frequencies for sociodemographic variables were calculated for both the interview and survey samples .
next , paired t - tests were used to test for differences ( = 0.05 ) between respondents perception scores for acceptance of different sexual orientation , race , and gender identity groups within the gay community .
similarly , tests were used to compare acceptance for those same groups within the broader
table 1 demographic attributes of survey respondents and interview participants from the health in middlesex men matters ( himmm ) study survey sample ( n = 202 ) n ( % ) interview sample ( n = 20)age group 182448 ( 23.8)3 253462 ( 30.7)7 354430 ( 14.9)2 455439 ( 19.3)6 55 + 23 ( 11.4)2ethno - racial group non - aboriginal white176 ( 87.1)16 non - aboriginal racialized19 ( 9.4)3 aboriginal7 ( 3.5)1ethnic or cultural identity indicated * white canadian / american / european180 ( 89.1)16 aboriginal7 ( 3.5)0 east / south / southeast asian7 ( 3.5)0 latin american5 ( 2.5)1 black canadian / american / african / caribbean4 ( 2.0)2 middle eastern3 ( 1.5)0 indo - caribbean3 ( 1.5)0education high school not completed12 ( 6.0)3 high school completed20 ( 10.0)5 some postsecondary57 ( 28.4)8 postsecondary graduate112 ( 55.7)4area of residence non - rural194 ( 97.0)18 rural6 ( 3.0)2marital status married or living common - law with a man55 ( 27.4)8 married or living common - law with a woman6 ( 3.0)0 separated / divorced / widowed / never married140 ( 69.7)12sexual orientation identity homosexual153 ( 89.5)13 bisexual17 ( 9.9)1 do nt know / would rather not say1 ( 0.6)1 heterosexual0 ( 0.0)5trans - identified yes5 ( 2.5)1 no195 ( 97.5)18country of birth canada185 ( 91.6)n / a other17 ( 8.4)n / ahousehold income per person < $ 15,00030 ( 15.6)3 $ 15,000$29,99963 ( 32.8)3 $ 30,000$49,99948 ( 25.0)3 $ 50,000$79,99928 ( 14.6)5 $ 80,000 + 23 ( 12.0)3employment status employed163 ( 81.2)11 non - employed38 ( 18.9)8student status not attending school146 ( 72.6)n / a attending school full - time39 ( 19.4)n / a attending school part - time16 ( 8.0)n / arelationship status single95 ( 47.3)n / a in a monogamous relationship71 ( 35.3)n / a in a non - monogamous or polyamorous relationship35 ( 17.4)n / a
demographic attributes of survey respondents and interview participants from the health in middlesex men matters ( himmm ) study community . paired t - tests were also used to assess whether participants viewed the gay community and the broader community as different in their acceptance of each group .
finally , linear regression was used to model the association between age and the perception of acceptance of each group by the gay community , and again separately by the broader community .
following these analyses , the team used an iterative process to triangulate and establish complementarity of information across qualitative and quantitative data sources ( yin , 2003 ) .
qualitative and quantitative phases of the himmm project were approved by the non - medical research ethics board at the university of western ontario .
within the broader community , survey respondents perceived distinct hierarchies of acceptance for men of various sexual orientation , genders , and racial identities , with men of color ( sexuality not specified ) being seen as most accepted and trans persons as least accepted ( table 1 ) . as shown in table 2 , respondents reported significantly different levels of perceived broader community acceptance of each of the selected identity subgroups ( p < 0.01 ) , excepting perceived broader community acceptance of gay versus bisexual men .
racialization thus appeared to be less of a perceived detriment to acceptance than sexual minority or transgender status .
we questioned whether this may be the result of optimistic perceptions of racial tolerance within our majority - white sample , but as shown in table 3perceptions of broader community acceptance for all men of color did not differ significantly between aboriginal participants ( 4.71 on a scale of 17 ) , non - aboriginal participants of color ( 4.68 ) , and white participants ( 4.61 ) .
since we queried perceived acceptance for single - characteristic groups , we can not comment on acceptance for different intersections of sexual orientation , gender identity , and race .
table 2 differences in gay and bisexual men s mean perceived levels of broader community acceptance and gay community acceptance , comparisons of subgroups differencetpbroader community acceptance of
gay men compared to bisexual men0.111.520.1305 gay men compared to trans men1.3113.51<0.0001 gay men compared to trans women1.1711.83<0.0001 gay men compared to men of color0.736.74<0.0001 bisexual men compared to trans men1.1913.22<0.0001 bisexual men compared to trans women1.0611.41<0.0001 bisexual men compared to men of color0.847.43<0.0001 trans men compared to trans women0.142.680.0081 trans men compared to men of color2.0417.95<0.0001 trans women compared to men of color1.9016.01<0.0001gay community acceptance of
gay men compared to bisexual men0.919.630.1305 gay men compared to trans men1.4313.4<0.0001 gay men compared to trans women1.4413.18<0.0001 gay men compared to men of color0.171.710.088 bisexual men compared to trans men0.535.37<0.0001 bisexual men compared to trans women0.565.31<0.0001 bisexual men compared to men of color0.766.79<0.0001 trans men compared to trans women0.030.650.51 trans men compared to men of color1.2711.64<0.0001 trans women compared to men of color1.3111.76<0.0001
differences based on a 7-point likert scale measure .
paired t - test .
as an example ,
gay / bi men perceived that gay men s acceptance within the broader london community was , on average , 0.11 points higher than bisexual men s acceptance , using a 7-point likert scale to measure acceptance .
table 3 differences in gay and bisexual men s mean perceived levels of broader community acceptance and gay community acceptance of men of color , comparisons between respondent ethno - racial groups differencetpbroader community acceptance of men of color aboriginal compared to white0.100.180.86 aboriginal compared to men of color0.030.040.96 white compared to men of color0.070.200.84gay community acceptance of men of color aboriginal compared to white0.300.530.60 aboriginal compared to men of color0.921.100.28 white compared to men of color0.611.690.09
differences based on a 7-point likert scale measure .
as an example , gay / bi aboriginal men s perception of the broader community s acceptance of men of color was on average 0.10 points more than white gay / bi men s perception of the broader community s acceptance of men of color ( p = 0.0.86 ) .
differences in gay and bisexual men s mean perceived levels of broader community acceptance and gay community acceptance , comparisons of subgroups
differences based on a 7-point likert scale measure .
as an example , gay / bi men perceived that gay men s acceptance within the broader london community was , on average , 0.11 points higher than bisexual men s acceptance , using a 7-point likert scale to measure acceptance .
differences in gay and bisexual men s mean perceived levels of broader community acceptance and gay community acceptance of men of color , comparisons between respondent ethno - racial groups
differences based on a 7-point likert scale measure .
as an example ,
gay / bi aboriginal men s perception of the broader community s acceptance of men of color was on average 0.10 points more than white gay / bi men s perception of the broader community s acceptance of men of color ( p = 0.0.86 ) .
the high levels of perceived acceptance for gay men in the broader mainstream community align with an emerging narrative of equal rights and greater social acceptance for gay identities in western countries ( weeks , 2007 ; smith , 2008 ) .
one interview participant reflected on an adoption support group meeting where he saw widespread support for a middle - class gay couple :
i went to these meetings by myself and there were maybe primarily five or six [ male / female ] couples but there was two men there they presented themselves as a couple awaiting an adoption and i was very pleased to see that the other couples interacted with these two men as they would all of the other couples like , you know ,
i hope you get a child soon and that kind of thing so that was very affirming .
( peter , 51 , gay - identified )
i went to these meetings by myself and there were maybe primarily five or six [ male / female ] couples but there was two men there they presented themselves as a couple awaiting an adoption and i was very pleased to see that the other couples interacted with these two men as they would all of the other couples like , you know ,
i hope you get a child soon and that kind of thing so that was very affirming .
( peter , 51 , gay - identified ) others felt that gay identities had become widely acceptable in the broader london community :
um , i think [ visibility is ] a good thing and i m not one of those people who is really out there or i need to put myself out there . um ,
if i m at an event so , the visibility for [ my partner and me ] has been very good and it hasnt been very negative because of our positions and our reputations in the [ london ] community .
( mark , 31 , gay - identified)a very mainstream , you know , social organization [ united church of canada ] linking themselves with gay pride , i mean that s a very positive thing .
( peter , 51 , gay - identified )
um , i think [ visibility is ] a good thing and i m not one of those people who is really out there or i need to put myself out there
. um , if it comes up in conversation then we ll talk about it or
if i m at an event so , the visibility for [ my partner and me ] has been very good and it hasnt been very negative because of our positions and our reputations in the [ london ] community .
( mark , 31 , gay - identified ) a very mainstream , you know , social organization [ united church of canada ] linking themselves with gay pride , i mean that s a very positive thing .
( peter , 51 , gay - identified ) at the same time , historical events in london suggest a more mixed attitude toward gay and bisexual men within the broader community . in 1993 , two local men were arrested on child pornography charges following the discovery of bags of videotapes in a nearby river . over the next two years ,
a widely publicized police investigation called project guardian linked the gay community with the possible existence of a large child pornography ring .
while only the initial two men arrested were convicted on child pornography charges , at least 2,296 interviews were conducted by police , 64 men were charged for various offenses , some were arrested , and several were also outed ( sas & hurley , 1997 ) . during the same period , the london police arrested large numbers of men for cruising in bathrooms and parks ( janoff , 2005 , pp .
in 1995 , the year that project guardian concluded , former mayor diane haskett refused the homophile association of london ontario s ( halo s ) request for the city to issue a proclamation for the london gay pride celebration .
haskett insisted that she would not issue controversial proclamations , and the city council voted 135 against it .
halo filed a complaint with the human rights commission of ontario , which decided in 1997 that both haskett and the london city council had discriminated against halo .
haskett subsequently posted an ad in the london free press claiming the decision was incorrect and violated constitutional freedoms . in this historically hostile social environment , it is feasible that the overall high reported acceptance for gay and bisexual identities masks some variation in men s experiences based on their socioeconomic and professional positions .
those who do not fit easily into the mainstream or require services tend to suffer from the lack of visibility and resources in a smaller community where the closet may be bigger than people on the street
mainstream public health services in london - middlesex , for example , have built only limited capacity to address lgbt health needs .
[ w]e live in a conservative government who does nt care about gay issues , msm issues , the health aspects of a small minute population and there are no services , one participant said .
there is a variety of mental health services , um , but again , they re not directed to the gay population and oftentimes there you may have to go to through two or three counsellors before you find one that s appropriate ( steve , 45 , gay - identified ) .
another compared the availability of health information in london and toronto : in toronto , you know , you have a community that s so well connected and so well informed that information is just , you know , jumping off the trees really .
but in london you really have to go searching for it to find it
concerns about anonymity of services in a mid - sized city may discourage some from using the limited services available .
one provider recounted ,
when i first started [ at a satellite clinic ] and i could nt draw blood very well , i would say you can come by tomorrow to our main site and they would say no , no i ca nt go down there because so and so works there .
i do nt want them to know that i m getting blood work for hiv cause i m not out to her in the community .
( jason , 53 , service provider )
when i first started [ at a satellite clinic ] and i could nt draw blood very well , i would say you can come by tomorrow to our main site and they would say no , no i ca nt go down there because so and so works there .
i do nt want them to know that i m getting blood work for hiv cause i m not out to her in the community .
( jason , 53 , service provider ) another participant reflected that the continued marginalization of gay and bisexual men in mainstream health care settings had made the local hiv clinic and counseling center an important support structure for local gay and bisexual men .
there s a fundamental belief that even within the structured systems that [ gay people are ] not right , you know , and i m talking about the aids committee focusing on gay issues as a necessity rather than simply focusing on hiv issues even though all the psychologists and psychiatrists say , you know , this is natural .
( steve , 45 , gay - identified )
there s a fundamental belief that even within the structured systems that [ gay people are ] not right , you know , and i m talking about the aids committee focusing on gay issues as a necessity rather than simply focusing on hiv issues even though all the psychologists and psychiatrists say , you know , this is natural .
( steve , 45 , gay - identified ) in other institutions , such as schools and media outlets , the subtle , ongoing erasure of lgbt issues and identities persists despite official proclamations of equality , diversity , and inclusion ( see young & meyer , 2005 ; bauer et al .
, 2009 ) . while the london region is associated increasingly with a creative cities
discourse that equates certain local industries ( e.g. , education , biosciences ) with cosmopolitanism and tolerance ( bradford , 2010 ) , other dominant sectors such as finance ( e.g. , london life insurance ) , manufacturing ( general motors , toyota , mccormick foods ) , and military - industrial ( general dynamics land systems tank manufacturing ) , have been associated with heteronormative and sometimes homophobic work cultures and environments ( mcdowell , 2001 ; embrick , walther , & wickens , 2007 ) . according to one participant ,
even the higher education sector in london - middlesex suppressed gay visibilities intermittently :
at kings [ college ] there was a group of students last year who wanted to do something in the cafeteria in conjunction with pride activities on main campus at [ university of ] western [ ontario ] and they were initially refused to do that they could have a booth and they could educate about sexual orientation but not celebrate use words that were like too condoning .
( peter , 51 , gay - identified , white )
at kings [ college ] there was a group of students last year who wanted to do something in the cafeteria in conjunction with pride activities on main campus at [ university of ] western [ ontario ] and they were initially refused to do that they could have a booth and they could educate about sexual orientation but not celebrate use words that were like too condoning .
( peter , 51 , gay - identified , white ) in local high schools , suppression of gay identities has been often more overt : we have the safe schools project here and since that s been instigated , numerous teachers have been made to take down their safe space signage because of objections from parents , so it was a good idea but the follow through is lousy ( steve , 45 , gay - identified ) .
others felt that the inaccessibility of the local media limited the possibility of a more connected , supportive gay community .
one participant said ,
small groups like [ rhac ] ca nt afford to take out full page ads each september or the high schools wo nt print them in their newsletters that there is a safe space to come talk about your issues or concerns so the problem is nt necessarily the resource being available , it s the awareness of the availability of the resources and either we do nt have the financial resource to make that happen or we live in a culture that does nt want it to happen .
( steve , 45 , gay - identified )
small groups like [ rhac ] ca nt afford to take out full page ads each september or the high schools wo nt print them in their newsletters that there is a safe space to come talk about your issues or concerns so the problem is nt necessarily the resource being available , it s the awareness of the availability of the resources and either we do nt have the financial resource to make that happen or we live in a culture that does nt want it to happen .
( steve , 45 , gay - identified ) he added that the london - middlesex mainstream media also seemed to ignore ongoing homophobia and hate crime in the region :
i saw on [ gay hook - up site ] squirt today there that somebody was assaulted a couple days ago here and someone in kitchener got the crap beat out of them too so gay violence is on the rise again , but you know , you do nt ever hear it . another agreed , in london
although both national - level advances in rights and ongoing , more localized marginalization might be therefore described as issues affecting the canadian lgbt community at large , they have been experienced differently by the various subgroups of gay and bisexual men , potentially leading to the diverging perceptions of inclusion in both the gay community and broader society .
acceptance of gay and bisexual men in the mainstream community , then , may be reserved largely for middle - class community members ( e.g. , business owners ) who , in turn , mask or minimize their sexual expression to continue being accepted .
there s a number of high profile gay [ -owned ] businesses opened up and they really revitalized the core , but for the most part their sexuality has been downplayed [ in the media ] .
they re young , exciting , interesting entrepreneurs bringing diversity to our core many of the senior gay businesses , the people who i knew were gay had visible , high traffic locations , had the resources to make an impact and change , they did nt want to be associated with gay pride or gay business , exclusively because the comment was 90% or more of their business comes from a heterosexual population who enjoy the sass , that classing up and london is an upper middle - class community and so they appreciate that sense of decadence not necessarily going to buy into it but they appreciate it .
( steve , 45 , white )
there s a number of high profile gay [ -owned ] businesses opened up and they really revitalized the core , but for the most part their sexuality has been downplayed [ in the media ] .
many of the senior gay businesses , the people who i knew were gay had visible , high traffic locations , had the resources to make an impact and change , they did nt want to be associated with gay pride or gay business , exclusively because the comment was 90% or more of their business comes from a heterosexual population who enjoy the sass , that classing up and london is an upper middle - class community and so they appreciate that sense of decadence not necessarily going to buy into it but they appreciate it .
( steve , 45 , white ) another added , there s more publications geared to a gay audience now than ever , but if you witness the way media is going over the last number of years
it s not about developing a community , it s using the community to profit from ( steve , 45 , gay - identified ) .
middle - class participants also tended to deal with perceived exclusion or marginalization in the broader community by seeking services elsewhere or relocating altogether .
one talked about his past unwillingness to disclose his sexual identity to local doctors and , eventually , the choice to have a doctor in toronto :
i ve had doctors in the past where i havent disclosed my sexual orientation out of fear of being rejected and , you know , i m pretty sure that it would have happened before because not all doctors are as good as my gp [ general practitioner ] in toronto ( dean , 27 , gay - identified ) .
another found acceptance primarily in white - collar institutions he worked in :
i think [ acceptance in london is ] pretty good to the extent that people feel comfortable sort of being out and you know public in their sexuality in a sense they do nt hide it people will support that and i d certainly in my experience here at the firm they ve been extremely supportive at the law school and in undergrad that s sort of when i was coming out .
i mean , you do nt have to make an apology for it because , you know , you re fortunate where you work that it s just not an issue .
( will , 29 , gay - identified )
i think [ acceptance in london is ] pretty good to the extent that people feel comfortable sort of being out and you know public in their sexuality in a sense they do nt hide it people will support that and i d certainly in my experience here at the firm they ve been extremely supportive at the law school and in undergrad that s sort of when i was coming out .
i mean , you do nt have to make an apology for it because , you know , you re fortunate where you work that it s just not an issue .
( will , 29 , gay - identified ) in contrast , he downplays persistent harassment among gay men in london s social spaces as part of the human condition :
i mean , sure yeah , sometimes you ll see people in bars or something who are jerks , but that s just you know the human condition
i think london is a fairly progressive - minded town and um , you know , it s got a fairly educated population between the university and the hospital system and the health system so you know it s really not a bad place to be gay .
i mean , sure yeah , sometimes you ll see people in bars or something who are jerks , but that s just you know the human condition
i think london is a fairly progressive - minded town and um , you know , it s got a fairly educated population between the university and the hospital system and the health system so you know it s really not a bad place to be gay .
respondents also perceived high acceptance for men of color ( sexual orientation not specified ) in the broader london - middlesex community , even more than for gay and bisexual men . as noted ,
the mean perceived acceptance for all men of color did not differ statistically for participants from different ethno - racial groups . however , research participants may be more likely to report discrimination toward the identity group that is the study s focus ( gay and bisexual men , in this case ) than they are to report personal discrimination , which may be connected to intersecting statuses such as race ( moghaddam & studer , 1997 ) .
in contrast , participants observed sustained exclusion of transgender individuals in london - middlesex , mirroring research reporting profound transphobia in ontario s health care institutions and communities more generally ( bauer et al . ,
i knew there was a large trans population but it s not a very visible one i mean those boys and girls they keep themselves well hidden
i believe they just allowed gender reassignment surgeries to be covered by [ universal public health insurance ] again but for years that was taken out ( steve , 45 , gay - identified ) .
measuring perceived acceptance of various groups within the london gay community elicited responses markedly different from those regarding the broader london community ( see table 2 ) . here , being a man of color or gay was perceived as most acceptable , with bisexuality less acceptable , and being a transgender man or woman even less so . as shown in table 2 ,
perceived acceptance levels for each group differed significantly from one another at p < 0.0001 , excepting gay men versus men of color , and transgender men versus transgender women ; neither of these comparisons were statistically significant ( p > 0.05 ) . finally , as shown in table 4 , the broader london community was perceived as being less accepting than the gay community for each group ( p < .0001 for each comparison ) .
table 4 differences in gay and bisexual men s mean perceived levels of acceptance of subgroups , broader community compared to the gay community differencetpbroader community acceptance compared to gay community acceptance of gay men1.2111.43<0.0001bisexual men0.424.45<0.0001trans men1.0910.15<0.0001trans women0.928.68<0.0001men of color0.323.96<0.0001
differences based on a 7-point likert scale measure .
paired t - test .
as an example ,
gay / bi men perceived that the level of acceptance for gay men within broader community was on average 1.21 points less than their acceptance within the gay community ( p < 0.0001 ) .
differences in gay and bisexual men s mean perceived levels of acceptance of subgroups , broader community compared to the gay community
differences based on a 7-point likert scale measure .
as an example , gay / bi men perceived that the level of acceptance for gay men within broader community was on average 1.21 points less than their acceptance within the gay community ( p < 0.0001 ) .
the high perceived acceptance for gay men within the gay community is perhaps an unsurprising finding . located within a rural , regional bible belt , london acts as a magnet for younger , rurally situated lgbt people moving from homophobic environments ( bruce & harper , 2011 ) . at the same time , because it is also located less than 200 miles from toronto , groups such as gay students and white - collar workers might travel to toronto for nightlife and services , or eventually move there ( rowe & dowsett , 2008 ; lewis , 2012 ) .
the london - middlesex gay community is also a transient entity whose shifting dynamics affect the durability of community infrastructures . in 2005 ,
halo , the city s central gay community nonprofit organization , bar , and referral center , closed permanently .
halo s closure left a large physical and psychological gap in the community , leaving a rotating circuit of 12 bars , a bathhouse , and the local aids service organization ( aso ) as the only visible community entry points . for gay men who might not fit easily into the remaining gay commercial scene
[ volunteering at rhac is ] an activity that just makes me feel so good inside and you know , it s a reason to get out of bed in the morning . if it was nt for [ rhac ] , you know , i would not want to see where i would be at just because , you know , coming to [ rhac ] and having that social interaction and knowing that i m not being judged for being gay , i m not being judged for being hiv positive it s really important that i come here ( dean , 27 , gay - identified )
[ volunteering at rhac is ] an activity that just makes me feel so good inside and you know , it s a reason to get out of bed in the morning . if it was nt for [ rhac ] , you know , i would not want to see where i would be at just because , you know , coming to [ rhac ] and having that social interaction and knowing that i m not being judged for being gay , i m not being judged for being hiv positive it s really important that i come here ( dean , 27 , gay - identified ) some felt , however , that rhac was still perceived primarily as a place for the economically disadvantaged or hiv - positive despite its broadened service umbrella . [ rhac ] primarily supports more marginalized members of society ,
another added , [ london s gay men s community is ] either for people who like the bar scene or who are [ hiv- ] infected , but nothing nothing for anybody else . that s just my take on what i m hearing ( jason , 53 , service provider ) . as in the analysis of the broader community ,
the high reported perceived acceptance for men of color may mask important differences in how white men and men of color perceive the local gay community .
one participant recounted :
i saw one person of colour in the [ pride london ] parade and i do nt remember seeing any persons of color observing the parade people of colour , um , generally do nt advertise their lives but if i m one who was interested in [ the parade ]
i m going to , you know , enthusiastically provide that information to other black people so they can be there too .
( ronald , did not wish to share age or sexual identity )
i saw one person of colour in the [ pride london ] parade and i do nt remember seeing any persons of color observing the parade people of colour , um , generally do nt advertise their lives but if i m one who was interested in [ the parade ]
i m going to , you know , enthusiastically provide that information to other black people so they can be there too .
( ronald , did not wish to share age or sexual identity ) he explained further that gay and bisexual men of color may also feel marginalized where few health or social resources are geared to their specific needs and experiences :
you wo nt get a black person or a person of colour coming into the group that has 30 white people , one indian and one chinese people and expecting them to share and be open but if they re in a group with people that are in the same health circumstances that they are in and they re all the same colour , then there s gon na be more openness if there were 10 support groups right now and none of them were multicultural i would nt go to none of them even if i felt that i needed them .
you wo nt get a black person or a person of colour coming into the group that has 30 white people , one indian and one chinese people and expecting them to share and be open but if they re in a group with people that are in the same health circumstances that they are in and they re all the same colour , then there s gon na be more openness if there were 10 support groups right now and none of them were multicultural i would nt go to none of them even if i felt that i needed them .
ronald s narrative indicates that men of color may feel disconnected from the gay community unless providers make efforts to create communal experiences , avoid tokenization , and allay fears of being outed .
trans participants also delineated themselves as separate from a mainstream gay community that they equated with socializing and nightlife at gay venues .
i would say the community that i have run into that s more of the mainstream community , you know , downtown at the clubs and things like that that is a community that i am uncomfortable around
you know there is like the club - hopping community and the not - club hopping community i guess that is what i d call it
this echoes browne and lim s ( 2010 ) study of brighton ( england s gay capital ) , which found that trans residents connected more closely geographically and psychologically with a regional gender clinic than the local gay community defined by tourism and nightlife .
middle - aged respondents perceived the lowest levels of acceptance for the various identity subgroups in the broader london - middlesex community ( see figure 1 ) . when asked to evaluate acceptance for the same subgroups within the gay community ,
middle - aged respondents again reported lower levels of acceptance than younger or older respondents ( see figure 2).we found a significant age effect ( as indicated by a statistically significant quadratic term ) on the assessment of acceptance of all groups other than bisexual men .
figure 1 regression results of gay and bisexual men s mean perceived levels of broader community acceptance of different subgroups , by respondent age
figure 2 regression results of gay and bisexual men s mean perceived levels of gay community acceptance of different subgroups , by respondent age
regression results of gay and bisexual men s mean perceived levels of broader community acceptance of different subgroups , by respondent age regression results of gay and bisexual men s mean perceived levels of gay community acceptance of different subgroups , by respondent age younger gay men , who have come of age in a milieu of greater social acceptance and gay rights ( hammack & cohler , 2011 ) , typically demonstrated the most optimism about the inclusion of sexual minorities within mainstream society : i have a lot of straight friends too that are fine with me being gay and one of my straight friends is really adamant about gay rights
i think it s come a long way in both communities because people in the straight community are more accepting now and we are more willing to answer that acceptance and speak out for ourselves
bisexuality was deemed equally acceptable as we observed no statistically significant difference in gay and bisexual men s perceived acceptance of bisexual versus gay men in the broader london community , though some suggested that the acceptance was due to perceptions of bisexuality as a stepping stone to being gay rather than a distinct identity . at the same time , the levels of perceived acceptance for gay men compared with other sexual / gender minority groups echoes recent work claiming that developments in gay rights have cultivated a group of inoffensive , normal gay male subjects that are acceptable to mainstream society , while other groups ( e.g. , trans people ) are made marginal ( duggan , 2002 ) .
middle - aged men s more pessimistic perceptions of community acceptance appeared based , in part , on experiences from earlier in life ( see also kertzner , 2001 ; hammack & cohler , 2011 ) .
one spoke about experiences of school - based , family - based , and sometimes internalized homophobia among middle - aged men who had grown up in the surrounding rural areas .
we re a feeder community drawing from a lot of smaller communities i grew up in a place called [ name withheld ] , when i came out with my father he said now why do you want to do that you know how much i hate fags .
there s this instantaneous revulsion and yet when he sold his house they found a bag of porn and in there was two very , very hard - core gay porns .
( steve , 45 , gay - identified )
we re a feeder community drawing from a lot of smaller communities i grew up in a place called [ name withheld ] , when i came out with my father he said now why do you want to do that you know how much i hate fags .
there s this instantaneous revulsion and yet when he sold his house they found a bag of porn and in there was two very , very hard - core gay porns .
( steve , 45 , gay - identified ) he continued , our school system where you get the majority of this very few parents who would even consider that [ their ] child may be homosexual for the most part , no , and it s a sense of failure .
other middle - aged participants perceptions were influenced by religious upbringings in the london - middlesex region .
one noted that experiences of self - devaluation relating to religion were common among his peers , but also among younger men : you know , so many guys .
you re a gay man , well , the devil was punishing you and stuff like
i m still hearing that , you know from young guys that come out and say i was abused like whoa there s still a lot of discrimination , there s still even from within
internalized guilt and shame may therefore keep some men from coming out or participating in gay community until later in life :
i havent heard a lot of people coming through saying that um , they ve been shunned , except you know , unless they ve been shunned by their family or by their community um , i think the lack of acceptance is often from within a lot of people who are reassessing their sexuality or 40 years into a marriage to a woman where the intimacy is long gone and everything is great except that they wind up down at the bathhouse and they re feeling terrible about it .
( jason , 53 , service provider )
i havent heard a lot of people coming through saying that um , they ve been shunned , except you know , unless they ve been shunned by their family or by their community um , i think the lack of acceptance is often from within a lot of people who are reassessing their sexuality or 40 years into a marriage to a woman where the intimacy is long gone and everything is great except that they wind up down at the bathhouse and they re feeling terrible about it .
( jason , 53 , service provider ) men in midlife also appeared most likely to carry forward trauma from events in the community s past :
i ve heard like historically you know going back to days where , you know , the mayor would nt declare gay pride day and right - wing groups demonstrating at some pride activities and how much of that is still happening ( peter , 51 , gay - identified ) .
younger men , in contrast , were more likely to dismiss discrimination from the mainstream community as a thing of the past .
it s something that sort of is like the , you know , diane haskett thing from years ago , but i mean , you d think surely after she gets trounced in the subsequent election [ in 2006 , despite being re - elected immediately after the legal decision in 1997 ] that people would say oh , maybe it s not so bad .
( will , 29 , gay - identified )
i think people perceive london as being more conservative than it actually is
it s something that sort of is like the , you know , diane haskett thing from years ago , but i mean , you d think surely after she gets trounced in the subsequent election [ in 2006 , despite being re - elected immediately after the legal decision in 1997 ] that people would say oh , maybe it s not so bad .
( will , 29 , gay - identified ) mirroring their perceptions of low acceptance for gay and bisexual groups in london , men in or approaching midlife felt especially disenfranchised by a funny split like young people as in university students or college students and then everyone else ( will , 29 , gay - identified ) .
while younger men tend to join university and college lgbt groups and continue to patronize the city s main commercial gay bar , men in midlife have been left with fewer social supports and opportunities to connect with other men their age .
there s been inklings in attempts to have a support group for older adults , [ young adult groups are ] basically up to 25 , so if you re coming out at 30 or in a married relationship and that falls apart because you really discover that you re not just bi , you re not just interested in sex with men there really is nt many supports out there .
( steve , 45 , gay - identified).like maybe it would be useful periodically to chat with some other middle - aged gay men about if there are some issues that come up sexual activity or sort of moving into middle age
i realize that all of my close friends in london are heterosexual and i was thinking this is odd because anywhere else at least a number of my close friends were gay men .
( peter , 51 , gay - identified )
there s been inklings in attempts to have a support group for older adults , [ young adult groups are ] basically up to 25 , so if you re coming out at 30 or in a married relationship and that falls apart because you really discover that you re not just bi , you re not just interested in sex with men there really is nt many supports out there .
like maybe it would be useful periodically to chat with some other middle - aged gay men about if there are some issues that come up sexual activity or sort of moving into middle age
i realize that all of my close friends in london are heterosexual and i was thinking this is odd because anywhere else at least a number of my close friends were gay men .
( peter , 51 , gay - identified ) middle - aged participants also commented on the limited number of gay - associated activities and venues that they felt were age - appropriate or interesting .
they re not all into bingo i m sure there s a lot of gay men that love to go boating maybe i m at an age where i do my own thing .
( carl , 54 , msm - identified)with the demise of halo a few years ago there is no social network or body as such , and typically i ll hear this at the bathhouse , the folks are there , and you know they re not especially sexual but , they do nt like the bar scene , they do nt like the music , they do nt like the atmosphere or what the bathhouse is , but they ll go but saying why is there nothing for folks for whom sex is not the be all and the end all ?
( jason , 53 , service provider )
not all gays / bisexual or men having sex with men are into drag
they re not all into bingo i m sure there s a lot of gay men that love to go boating maybe i m at an age where i do my own thing .
( carl , 54 , msm - identified ) with the demise of halo a few years ago there is no social network or body as such , and typically i ll hear this at the bathhouse , the folks are there , and you know they re not especially sexual but , they do nt like the bar scene , they do nt like the music , they do nt like the atmosphere or what the bathhouse is , but they ll go but saying why is there nothing for folks for whom sex is not the be all and the end all ?
( jason , 53 , service provider ) the comments indicate that while some gay community infrastructure exists in the london region , it is seen as supporting primarily a younger , often transient population . while there was insufficient qualitative evidence to examine why older men demonstrated less pessimism about acceptance than middle - aged men , it is possible that this generation sees the current state of the community as more positive because they have survived the aids epidemic , a long - term lack of human rights protections , and the pathologization and criminalization of sexuality .
within the broader community , survey respondents perceived distinct hierarchies of acceptance for men of various sexual orientation , genders , and racial identities , with men of color ( sexuality not specified ) being seen as most accepted and trans persons as least accepted ( table 1 ) . as shown in table 2 , respondents reported significantly different levels of perceived broader community acceptance of each of the selected identity subgroups ( p < 0.01 ) , excepting perceived broader community acceptance of gay versus bisexual men .
racialization thus appeared to be less of a perceived detriment to acceptance than sexual minority or transgender status .
we questioned whether this may be the result of optimistic perceptions of racial tolerance within our majority - white sample , but as shown in table 3perceptions of broader community acceptance for all men of color did not differ significantly between aboriginal participants ( 4.71 on a scale of 17 ) , non - aboriginal participants of color ( 4.68 ) , and white participants ( 4.61 ) .
since we queried perceived acceptance for single - characteristic groups , we can not comment on acceptance for different intersections of sexual orientation , gender identity , and race .
table 2 differences in gay and bisexual men s mean perceived levels of broader community acceptance and gay community acceptance , comparisons of subgroups differencetpbroader community acceptance of
gay men compared to bisexual men0.111.520.1305 gay men compared to trans men1.3113.51<0.0001 gay men compared to trans women1.1711.83<0.0001 gay men compared to men of color0.736.74<0.0001 bisexual men compared to trans men1.1913.22<0.0001 bisexual men compared to trans women1.0611.41<0.0001 bisexual men compared to men of color0.847.43<0.0001 trans men compared to trans women0.142.680.0081 trans men compared to men of color2.0417.95<0.0001 trans women compared to men of color1.9016.01<0.0001gay community acceptance of
gay men compared to bisexual men0.919.630.1305 gay men compared to trans men1.4313.4<0.0001 gay men compared to trans women1.4413.18<0.0001 gay men compared to men of color0.171.710.088 bisexual men compared to trans men0.535.37<0.0001 bisexual men compared to trans women0.565.31<0.0001 bisexual men compared to men of color0.766.79<0.0001 trans men compared to trans women0.030.650.51 trans men compared to men of color1.2711.64<0.0001 trans women compared to men of color1.3111.76<0.0001
differences based on a 7-point likert scale measure .
paired t - test .
as an example ,
gay / bi men perceived that gay men s acceptance within the broader london community was , on average , 0.11 points higher than bisexual men s acceptance , using a 7-point likert scale to measure acceptance .
table 3 differences in gay and bisexual men s mean perceived levels of broader community acceptance and gay community acceptance of men of color , comparisons between respondent ethno - racial groups differencetpbroader community acceptance of men of color aboriginal compared to white0.100.180.86 aboriginal compared to men of color0.030.040.96 white compared to men of color0.070.200.84gay community acceptance of men of color aboriginal compared to white0.300.530.60 aboriginal compared to men of color0.921.100.28 white compared to men of color0.611.690.09
differences based on a 7-point likert scale measure .
as an example , gay / bi aboriginal men s perception of the broader community s acceptance of men of color was on average 0.10 points more than white gay / bi men s perception of the broader community s acceptance of men of color ( p = 0.0.86 ) .
differences in gay and bisexual men s mean perceived levels of broader community acceptance and gay community acceptance , comparisons of subgroups
differences based on a 7-point likert scale measure .
as an example , gay / bi men perceived that gay men s acceptance within the broader london community was , on average , 0.11 points higher than bisexual men s acceptance , using a 7-point likert scale to measure acceptance .
differences in gay and bisexual men s mean perceived levels of broader community acceptance and gay community acceptance of men of color , comparisons between respondent ethno - racial groups
differences based on a 7-point likert scale measure .
as an example ,
gay / bi aboriginal men s perception of the broader community s acceptance of men of color was on average 0.10 points more than white gay / bi men s perception of the broader community s acceptance of men of color ( p = 0.0.86 ) .
the high levels of perceived acceptance for gay men in the broader mainstream community align with an emerging narrative of equal rights and greater social acceptance for gay identities in western countries ( weeks , 2007 ; smith , 2008 ) .
one interview participant reflected on an adoption support group meeting where he saw widespread support for a middle - class gay couple :
i went to these meetings by myself and there were maybe primarily five or six [ male / female ] couples but there was two men there they presented themselves as a couple awaiting an adoption and i was very pleased to see that the other couples interacted with these two men as they would all of the other couples like , you know ,
i hope you get a child soon and that kind of thing so that was very affirming .
( peter , 51 , gay - identified )
i went to these meetings by myself and there were maybe primarily five or six [ male / female ] couples but there was two men there they presented themselves as a couple awaiting an adoption and i was very pleased to see that the other couples interacted with these two men as they would all of the other couples like , you know ,
i hope you get a child soon and that kind of thing so that was very affirming .
( peter , 51 , gay - identified ) others felt that gay identities had become widely acceptable in the broader london community :
um , i think [ visibility is ] a good thing and i m not one of those people who is really out there or i need to put myself out there . um ,
if i m at an event so , the visibility for [ my partner and me ] has been very good and it hasnt been very negative because of our positions and our reputations in the [ london ] community .
( mark , 31 , gay - identified)a very mainstream , you know , social organization [ united church of canada ] linking themselves with gay pride , i mean that s a very positive thing .
( peter , 51 , gay - identified )
um , i think [ visibility is ] a good thing and i m not one of those people who is really out there or i need to put myself out there
. um , if it comes up in conversation then we ll talk about it or
if i m at an event so , the visibility for [ my partner and me ] has been very good and it hasnt been very negative because of our positions and our reputations in the [ london ] community .
( mark , 31 , gay - identified ) a very mainstream , you know , social organization [ united church of canada ] linking themselves with gay pride , i mean that s a very positive thing .
( peter , 51 , gay - identified ) at the same time , historical events in london suggest a more mixed attitude toward gay and bisexual men within the broader community . in 1993 , two local men were arrested on child pornography charges following the discovery of bags of videotapes in a nearby river . over the next two years ,
a widely publicized police investigation called project guardian linked the gay community with the possible existence of a large child pornography ring .
while only the initial two men arrested were convicted on child pornography charges , at least 2,296 interviews were conducted by police , 64 men were charged for various offenses , some were arrested , and several were also outed ( sas & hurley , 1997 ) . during the same period , the london police arrested large numbers of men for cruising in bathrooms and parks ( janoff , 2005 , pp .
in 1995 , the year that project guardian concluded , former mayor diane haskett refused the homophile association of london ontario s ( halo s ) request for the city to issue a proclamation for the london gay pride celebration .
haskett insisted that she would not issue controversial proclamations , and the city council voted 135 against it .
halo filed a complaint with the human rights commission of ontario , which decided in 1997 that both haskett and the london city council had discriminated against halo .
haskett subsequently posted an ad in the london free press claiming the decision was incorrect and violated constitutional freedoms . in this historically hostile social environment , it is feasible that the overall high reported acceptance for gay and bisexual identities masks some variation in men s experiences based on their socioeconomic and professional positions .
those who do not fit easily into the mainstream or require services tend to suffer from the lack of visibility and resources in a smaller community where the closet may be bigger than people on the street
mainstream public health services in london - middlesex , for example , have built only limited capacity to address lgbt health needs .
[ w]e live in a conservative government who does nt care about gay issues , msm issues , the health aspects of a small minute population and there are no services , one participant said .
there is a variety of mental health services , um , but again , they re not directed to the gay population and oftentimes there you may have to go to through two or three counsellors before you find one that s appropriate ( steve , 45 , gay - identified ) .
another compared the availability of health information in london and toronto : in toronto , you know , you have a community that s so well connected and so well informed that information is just , you know , jumping off the trees really .
but in london you really have to go searching for it to find it
concerns about anonymity of services in a mid - sized city may discourage some from using the limited services available .
one provider recounted ,
when i first started [ at a satellite clinic ] and i could nt draw blood very well , i would say you can come by tomorrow to our main site and they would say no , no i ca nt go down there because so and so works there .
i do nt want them to know that i m getting blood work for hiv cause i m not out to her in the community .
( jason , 53 , service provider )
when i first started [ at a satellite clinic ] and i could nt draw blood very well , i would say you can come by tomorrow to our main site and they would say no , no i ca nt go down there because so and so works there .
i do nt want them to know that i m getting blood work for hiv cause i m not out to her in the community .
( jason , 53 , service provider ) another participant reflected that the continued marginalization of gay and bisexual men in mainstream health care settings had made the local hiv clinic and counseling center an important support structure for local gay and bisexual men .
there s a fundamental belief that even within the structured systems that [ gay people are ] not right , you know , and i m talking about the aids committee focusing on gay issues as a necessity rather than simply focusing on hiv issues even though all the psychologists and psychiatrists say , you know , this is natural .
( steve , 45 , gay - identified )
there s a fundamental belief that even within the structured systems that [ gay people are ] not right , you know , and i m talking about the aids committee focusing on gay issues as a necessity rather than simply focusing on hiv issues even though all the psychologists and psychiatrists say , you know , this is natural .
( steve , 45 , gay - identified ) in other institutions , such as schools and media outlets , the subtle , ongoing erasure of lgbt issues and identities persists despite official proclamations of equality , diversity , and inclusion ( see young & meyer , 2005 ; bauer et al .
, 2009 ) . while the london region is associated increasingly with a creative cities
discourse that equates certain local industries ( e.g. , education , biosciences ) with cosmopolitanism and tolerance ( bradford , 2010 ) , other dominant sectors such as finance ( e.g. , london life insurance ) , manufacturing ( general motors , toyota , mccormick foods ) , and military - industrial ( general dynamics land systems tank manufacturing ) , have been associated with heteronormative and sometimes homophobic work cultures and environments ( mcdowell , 2001 ; embrick , walther , & wickens , 2007 ) . according to one participant ,
even the higher education sector in london - middlesex suppressed gay visibilities intermittently :
at kings [ college ] there was a group of students last year who wanted to do something in the cafeteria in conjunction with pride activities on main campus at [ university of ] western [ ontario ] and they were initially refused to do that they could have a booth and they could educate about sexual orientation but not celebrate use words that were like too condoning .
( peter , 51 , gay - identified , white )
at kings [ college ] there was a group of students last year who wanted to do something in the cafeteria in conjunction with pride activities on main campus at [ university of ] western [ ontario ] and they were initially refused to do that they could have a booth and they could educate about sexual orientation but not celebrate use words that were like too condoning .
( peter , 51 , gay - identified , white ) in local high schools , suppression of gay identities has been often more overt : we have the safe schools project here and since that s been instigated , numerous teachers have been made to take down their safe space signage because of objections from parents , so it was a good idea but the follow through is lousy ( steve , 45 , gay - identified ) .
others felt that the inaccessibility of the local media limited the possibility of a more connected , supportive gay community .
one participant said ,
small groups like [ rhac ] ca nt afford to take out full page ads each september or the high schools wo nt print them in their newsletters that there is a safe space to come talk about your issues or concerns so the problem is nt necessarily the resource being available , it s the awareness of the availability of the resources and either we do nt have the financial resource to make that happen or we live in a culture that does nt want it to happen .
( steve , 45 , gay - identified )
small groups like [ rhac ] ca nt afford to take out full page ads each september or the high schools wo nt print them in their newsletters that there is a safe space to come talk about your issues or concerns so the problem is nt necessarily the resource being available , it s the awareness of the availability of the resources and either we do nt have the financial resource to make that happen or we live in a culture that does nt want it to happen .
( steve , 45 , gay - identified ) he added that the london - middlesex mainstream media also seemed to ignore ongoing homophobia and hate crime in the region :
i saw on [ gay hook - up site ] squirt today there that somebody was assaulted a couple days ago here and someone in kitchener got the crap beat out of them too so gay violence is on the rise again , but you know , you do nt ever hear it . another agreed , in london
although both national - level advances in rights and ongoing , more localized marginalization might be therefore described as issues affecting the canadian lgbt community at large , they have been experienced differently by the various subgroups of gay and bisexual men , potentially leading to the diverging perceptions of inclusion in both the gay community and broader society .
acceptance of gay and bisexual men in the mainstream community , then , may be reserved largely for middle - class community members ( e.g. , business owners ) who , in turn , mask or minimize their sexual expression to continue being accepted .
there s a number of high profile gay [ -owned ] businesses opened up and they really revitalized the core , but for the most part their sexuality has been downplayed [ in the media ] .
they re young , exciting , interesting entrepreneurs bringing diversity to our core many of the senior gay businesses , the people who i knew were gay had visible , high traffic locations , had the resources to make an impact and change , they did nt want to be associated with gay pride or gay business , exclusively because the comment was 90% or more of their business comes from a heterosexual population who enjoy the sass , that classing up and london is an upper middle - class community and so they appreciate that sense of decadence not necessarily going to buy into it but they appreciate it .
( steve , 45 , white )
there s a number of high profile gay [ -owned ] businesses opened up and they really revitalized the core , but for the most part their sexuality has been downplayed [ in the media ] .
many of the senior gay businesses , the people who i knew were gay had visible , high traffic locations , had the resources to make an impact and change , they did nt want to be associated with gay pride or gay business , exclusively because the comment was 90% or more of their business comes from a heterosexual population who enjoy the sass , that classing up and london is an upper middle - class community and so they appreciate that sense of decadence not necessarily going to buy into it but they appreciate it .
( steve , 45 , white ) another added , there s more publications geared to a gay audience now than ever , but if you witness the way media is going over the last number of years
it s not about developing a community , it s using the community to profit from ( steve , 45 , gay - identified ) .
middle - class participants also tended to deal with perceived exclusion or marginalization in the broader community by seeking services elsewhere or relocating altogether .
one talked about his past unwillingness to disclose his sexual identity to local doctors and , eventually , the choice to have a doctor in toronto :
i ve had doctors in the past where i havent disclosed my sexual orientation out of fear of being rejected and , you know , i m pretty sure that it would have happened before because not all doctors are as good as my gp [ general practitioner ] in toronto ( dean , 27 , gay - identified ) .
another found acceptance primarily in white - collar institutions he worked in :
i think [ acceptance in london is ] pretty good to the extent that people feel comfortable sort of being out and you know public in their sexuality in a sense they do nt hide it people will support that and i d certainly in my experience here at the firm they ve been extremely supportive at the law school and in undergrad that s sort of when i was coming out .
i mean , you do nt have to make an apology for it because , you know , you re fortunate where you work that it s just not an issue .
( will , 29 , gay - identified )
i think [ acceptance in london is ] pretty good to the extent that people feel comfortable sort of being out and you know public in their sexuality in a sense they do nt hide it people will support that and i d certainly in my experience here at the firm they ve been extremely supportive at the law school and in undergrad that s sort of when i was coming out .
i mean , you do nt have to make an apology for it because , you know , you re fortunate where you work that it s just not an issue .
( will , 29 , gay - identified ) in contrast , he downplays persistent harassment among gay men in london s social spaces as part of the human condition :
i mean , sure yeah , sometimes you ll see people in bars or something who are jerks , but that s just you know the human condition
i think london is a fairly progressive - minded town and um , you know , it s got a fairly educated population between the university and the hospital system and the health system so you know it s really not a bad place to be gay .
i mean , sure yeah , sometimes you ll see people in bars or something who are jerks , but that s just you know the human condition
i think london is a fairly progressive - minded town and um , you know , it s got a fairly educated population between the university and the hospital system and the health system so you know it s really not a bad place to be gay .
respondents also perceived high acceptance for men of color ( sexual orientation not specified ) in the broader london - middlesex community , even more than for gay and bisexual men . as noted ,
the mean perceived acceptance for all men of color did not differ statistically for participants from different ethno - racial groups . however , research participants may be more likely to report discrimination toward the identity group that is the study s focus ( gay and bisexual men , in this case ) than they are to report personal discrimination , which may be connected to intersecting statuses such as race ( moghaddam & studer , 1997 ) .
in contrast , participants observed sustained exclusion of transgender individuals in london - middlesex , mirroring research reporting profound transphobia in ontario s health care institutions and communities more generally ( bauer et al . ,
i knew there was a large trans population but it s not a very visible one i mean those boys and girls they keep themselves well hidden
i believe they just allowed gender reassignment surgeries to be covered by [ universal public health insurance ] again but for years that was taken out ( steve , 45 , gay - identified ) .
measuring perceived acceptance of various groups within the london gay community elicited responses markedly different from those regarding the broader london community ( see table 2 ) . here , being a man of color or gay was perceived as most acceptable , with bisexuality less acceptable , and being a transgender man or woman even less so . as shown in table 2 ,
perceived acceptance levels for each group differed significantly from one another at p < 0.0001 , excepting gay men versus men of color , and transgender men versus transgender women ; neither of these comparisons were statistically significant ( p > 0.05 ) .
finally , as shown in table 4 , the broader london community was perceived as being less accepting than the gay community for each group ( p < .0001 for each comparison ) .
table 4 differences in gay and bisexual men s mean perceived levels of acceptance of subgroups , broader community compared to the gay community differencetpbroader community acceptance compared to gay community acceptance of gay men1.2111.43<0.0001bisexual men0.424.45<0.0001trans men1.0910.15<0.0001trans women0.928.68<0.0001men of color0.323.96<0.0001
differences based on a 7-point likert scale measure .
paired t - test .
as an example ,
gay / bi men perceived that the level of acceptance for gay men within broader community was on average 1.21 points less than their acceptance within the gay community ( p < 0.0001 ) .
differences in gay and bisexual men s mean perceived levels of acceptance of subgroups , broader community compared to the gay community
differences based on a 7-point likert scale measure .
as an example ,
gay / bi men perceived that the level of acceptance for gay men within broader community was on average 1.21 points less than their acceptance within the gay community ( p < 0.0001 ) .
the high perceived acceptance for gay men within the gay community is perhaps an unsurprising finding .
located within a rural , regional bible belt , london acts as a magnet for younger , rurally situated lgbt people moving from homophobic environments ( bruce & harper , 2011 ) . at the same time , because it is also located less than 200 miles from toronto , groups such as gay students and white - collar workers might travel to toronto for nightlife and services , or eventually move there ( rowe & dowsett , 2008 ; lewis , 2012 ) .
the london - middlesex gay community is also a transient entity whose shifting dynamics affect the durability of community infrastructures . in 2005 ,
halo , the city s central gay community nonprofit organization , bar , and referral center , closed permanently .
halo s closure left a large physical and psychological gap in the community , leaving a rotating circuit of 12 bars , a bathhouse , and the local aids service organization ( aso ) as the only visible community entry points . for gay men who might not fit easily into the remaining gay commercial scene
[ volunteering at rhac is ] an activity that just makes me feel so good inside and you know , it s a reason to get out of bed in the morning . if it was nt for [ rhac ] , you know , i would not want to see where i would be at just because , you know , coming to [ rhac ] and having that social interaction and knowing that i m not being judged for being gay , i m not being judged for being hiv positive it s really important that i come here ( dean , 27 , gay - identified )
[ volunteering at rhac is ] an activity that just makes me feel so good inside and you know , it s a reason to get out of bed in the morning . if it was nt for [ rhac ] , you know , i would not want to see where i would be at just because , you know , coming to [ rhac ] and having that social interaction and knowing that i m not being judged for being gay , i m not being judged for being hiv positive it s really important that i come here
( dean , 27 , gay - identified ) some felt , however , that rhac was still perceived primarily as a place for the economically disadvantaged or hiv - positive despite its broadened service umbrella .
another added , [ london s gay men s community is ] either for people who like the bar scene or who are [ hiv- ] infected , but nothing nothing for anybody else .
( jason , 53 , service provider ) . as in the analysis of the broader community ,
the high reported perceived acceptance for men of color may mask important differences in how white men and men of color perceive the local gay community .
one participant recounted :
i saw one person of colour in the [ pride london ] parade and i do nt remember seeing any persons of color observing the parade people of colour , um , generally do nt advertise their lives but if i m one who was interested in [ the parade ]
i m going to , you know , enthusiastically provide that information to other black people so they can be there too .
i saw one person of colour in the [ pride london ] parade and i do nt remember seeing any persons of color observing the parade people of colour , um , generally do nt advertise their lives but if i m one who was interested in [ the parade ] i m going to , you know , enthusiastically provide that information to other black people so they can be there too .
( ronald , did not wish to share age or sexual identity ) he explained further that gay and bisexual men of color may also feel marginalized where few health or social resources are geared to their specific needs and experiences :
you wo nt get a black person or a person of colour coming into the group that has 30 white people , one indian and one chinese people and expecting them to share and be open but if they re in a group with people that are in the same health circumstances that they are in and they re all the same colour , then there s gon na be more openness if there were 10 support groups right now and none of them were multicultural i would nt go to none of them even if i felt that i needed them .
you wo nt get a black person or a person of colour coming into the group that has 30 white people , one indian and one chinese people and expecting them to share and be open but if they re in a group with people that are in the same health circumstances that they are in and they re all the same colour , then there s gon na be more openness if there were 10 support groups right now and none of them were multicultural i would nt go to none of them even if i felt that i needed them .
ronald s narrative indicates that men of color may feel disconnected from the gay community unless providers make efforts to create communal experiences , avoid tokenization , and allay fears of being outed .
trans participants also delineated themselves as separate from a mainstream gay community that they equated with socializing and nightlife at gay venues .
i would say the community that i have run into that s more of the mainstream community , you know , downtown at the clubs and things like that that is a community that i am uncomfortable around
you know there is like the club - hopping community and the not - club hopping community i guess that is what i d call it
this echoes browne and lim s ( 2010 ) study of brighton ( england s gay capital ) , which found that trans residents connected more closely geographically and psychologically with a regional gender clinic than the local gay community defined by tourism and nightlife .
middle - aged respondents perceived the lowest levels of acceptance for the various identity subgroups in the broader london - middlesex community ( see figure 1 ) . when asked to evaluate acceptance for the same subgroups within the gay community ,
middle - aged respondents again reported lower levels of acceptance than younger or older respondents ( see figure 2).we found a significant age effect ( as indicated by a statistically significant quadratic term ) on the assessment of acceptance of all groups other than bisexual men .
figure 1 regression results of gay and bisexual men s mean perceived levels of broader community acceptance of different subgroups , by respondent age
figure 2 regression results of gay and bisexual men s mean perceived levels of gay community acceptance of different subgroups , by respondent age
regression results of gay and bisexual men s mean perceived levels of broader community acceptance of different subgroups , by respondent age regression results of gay and bisexual men s mean perceived levels of gay community acceptance of different subgroups , by respondent age younger gay men , who have come of age in a milieu of greater social acceptance and gay rights ( hammack & cohler , 2011 ) , typically demonstrated the most optimism about the inclusion of sexual minorities within mainstream society : i have a lot of straight friends too that are fine with me being gay and one of my straight friends is really adamant about gay rights
i think it s come a long way in both communities because people in the straight community are more accepting now and we are more willing to answer that acceptance and speak out for ourselves
bisexuality was deemed equally acceptable as we observed no statistically significant difference in gay and bisexual men s perceived acceptance of bisexual versus gay men in the broader london community , though some suggested that the acceptance was due to perceptions of bisexuality as a stepping stone to being gay rather than a distinct identity . at the same time , the levels of perceived acceptance for gay men compared with other sexual / gender minority groups echoes recent work claiming that developments in gay rights have cultivated a group of inoffensive , normal gay male subjects that are acceptable to mainstream society , while other groups ( e.g. , trans people ) are made marginal ( duggan , 2002 ) .
middle - aged men s more pessimistic perceptions of community acceptance appeared based , in part , on experiences from earlier in life ( see also kertzner , 2001 ; hammack & cohler , 2011 ) .
one spoke about experiences of school - based , family - based , and sometimes internalized homophobia among middle - aged men who had grown up in the surrounding rural areas .
we re a feeder community drawing from a lot of smaller communities i grew up in a place called [ name withheld ] , when i came out with my father he said now why do you want to do that you know how much i hate fags .
there s this instantaneous revulsion and yet when he sold his house they found a bag of porn and in there was two very , very hard - core gay porns .
( steve , 45 , gay - identified )
we re a feeder community drawing from a lot of smaller communities i grew up in a place called [ name withheld ] , when i came out with my father he said now why do you want to do that you know how much i hate fags .
there s this instantaneous revulsion and yet when he sold his house they found a bag of porn and in there was two very , very hard - core gay porns .
( steve , 45 , gay - identified ) he continued , our school system where you get the majority of this very few parents who would even consider that [ their ] child may be homosexual for the most part , no , and it s a sense of failure .
other middle - aged participants perceptions were influenced by religious upbringings in the london - middlesex region .
one noted that experiences of self - devaluation relating to religion were common among his peers , but also among younger men : you know , so many guys .
you re a gay man , well , the devil was punishing you and stuff like i m still hearing that , you know from young guys that come out and say i was abused like whoa
there s still a lot of discrimination , there s still even from within
internalized guilt and shame may therefore keep some men from coming out or participating in gay community until later in life :
i havent heard a lot of people coming through saying that um , they ve been shunned , except you know , unless they ve been shunned by their family or by their community um , i think the lack of acceptance is often from within a lot of people who are reassessing their sexuality or 40 years into a marriage to a woman where the intimacy is long gone and everything is great except that they wind up down at the bathhouse and they re feeling terrible about it .
( jason , 53 , service provider )
i havent heard a lot of people coming through saying that um , they ve been shunned , except you know , unless they ve been shunned by their family or by their community um , i think the lack of acceptance is often from within a lot of people who are reassessing their sexuality or 40 years into a marriage to a woman where the intimacy is long gone and everything is great except that they wind up down at the bathhouse and they re feeling terrible about it .
( jason , 53 , service provider ) men in midlife also appeared most likely to carry forward trauma from events in the community s past :
i ve heard like historically you know going back to days where , you know , the mayor would nt declare gay pride day and right - wing groups demonstrating at some pride activities and how much of that is still happening ( peter , 51 , gay - identified ) .
younger men , in contrast , were more likely to dismiss discrimination from the mainstream community as a thing of the past .
it s something that sort of is like the , you know , diane haskett thing from years ago , but i mean , you d think surely after she gets trounced in the subsequent election [ in 2006 , despite being re - elected immediately after the legal decision in 1997 ] that people would say oh , maybe it s not so bad .
( will , 29 , gay - identified )
i think people perceive london as being more conservative than it actually is
it s something that sort of is like the , you know , diane haskett thing from years ago , but i mean , you d think surely after she gets trounced in the subsequent election [ in 2006 , despite being re - elected immediately after the legal decision in 1997 ] that people would say oh , maybe it s not so bad .
( will , 29 , gay - identified ) mirroring their perceptions of low acceptance for gay and bisexual groups in london , men in or approaching midlife felt especially disenfranchised by a funny split like young people as in university students or college students and then everyone else ( will , 29 , gay - identified ) .
while younger men tend to join university and college lgbt groups and continue to patronize the city s main commercial gay bar , men in midlife have been left with fewer social supports and opportunities to connect with other men their age .
there s been inklings in attempts to have a support group for older adults , [ young adult groups are ] basically up to 25 , so if you re coming out at 30 or in a married relationship and that falls apart because you really discover that you re not just bi , you re not just interested in sex with men there really is nt many supports out there .
( steve , 45 , gay - identified).like maybe it would be useful periodically to chat with some other middle - aged gay men about if there are some issues that come up sexual activity or sort of moving into middle age
i realize that all of my close friends in london are heterosexual and i was thinking this is odd because anywhere else at least a number of my close friends were gay men .
( peter , 51 , gay - identified )
there s been inklings in attempts to have a support group for older adults , [ young adult groups are ] basically up to 25 , so if you re coming out at 30 or in a married relationship and that falls apart because you really discover that you re not just bi , you re not just interested in sex with men there really is nt many supports out there .
like maybe it would be useful periodically to chat with some other middle - aged gay men about if there are some issues that come up sexual activity or sort of moving into middle age
i realize that all of my close friends in london are heterosexual and i was thinking this is odd because anywhere else at least a number of my close friends were gay men .
( peter , 51 , gay - identified ) middle - aged participants also commented on the limited number of gay - associated activities and venues that they felt were age - appropriate or interesting .
they re not all into bingo i m sure there s a lot of gay men that love to go boating maybe i m at an age where i do my own thing .
( carl , 54 , msm - identified)with the demise of halo a few years ago there is no social network or body as such , and typically i ll hear this at the bathhouse , the folks are there , and you know they re not especially sexual but , they do nt like the bar scene , they do nt like the music , they do nt like the atmosphere or what the bathhouse is , but they ll go but saying why is there nothing for folks for whom sex is not the be all and the end all ?
( jason , 53 , service provider )
not all gays / bisexual or men having sex with men are into drag
they re not all into bingo i m sure there s a lot of gay men that love to go boating maybe i m at an age where i do my own thing .
( carl , 54 , msm - identified ) with the demise of halo a few years ago there is no social network or body as such , and typically i ll hear this at the bathhouse , the folks are there , and you know they re not especially sexual but , they do nt like the bar scene , they do nt like the music , they do nt like the atmosphere or what the bathhouse is , but they ll go but saying why is there nothing for folks for whom sex is not the be all and the end all ?
( jason , 53 , service provider ) the comments indicate that while some gay community infrastructure exists in the london region , it is seen as supporting primarily a younger , often transient population . while there was insufficient qualitative evidence to examine why older men demonstrated less pessimism about acceptance than middle - aged men
, it is possible that this generation sees the current state of the community as more positive because they have survived the aids epidemic , a long - term lack of human rights protections , and the pathologization and criminalization of sexuality .
with regard to both the broader london community and the local gay community , respondents perceived a hierarchy of acceptance , with men of color and gay men perceived as most accepted , bisexual men as somewhat less accepted , and trans men and women as least accepted . at the same time , notable variations emerged .
respondents perceived that in the broader community , men of color were more accepted than gay men and bisexual men ( who were deemed to be roughly equally accepted ) . with regard to the gay community , it was men of color and gay men who were deemed equally accepted , while bisexual men were seen as less accepted .
respondents also perceived overall greater acceptance in the gay community than in the broader london - middlesex community .
in addition , middle - aged respondents perceived less acceptance for all groups than did younger and older respondents . the high level of perceived acceptance for gay men may signify the growing normalization of gay identities and advances in gay rights .
at the same time , comments suggested that acceptance might be directed specifically toward upwardly mobile , professional gay men , and that these segments of the community are also those most able to overcome the lack of social supports in a mid - sized city with a long history of intolerance .
the figures for men of color suggested that respondents felt that racial diversity was as accepted as sexual diversity in the gay community , and even more accepted than sexual diversity within the broader community . although the lack of differentiation for acceptance of gay men and men of color in the gay community could be due simply to higher perceived acceptance for gay men in the gay community ( and not lower acceptance for men of color ) , the qualitative comments suggested that there was only limited integration of those with non - white identities into london s gay community .
at the same time , the high perceived acceptance for men of color in the broader community might be inflated due to predominance of white respondents in the sample .
the high overall perceived acceptance for all men of color in both gay and broader communities and limited quantifiable differences in perceptions of acceptance across race groups mirrors frost and meyer s ( 2012 ) finding that there are few differences in gay community attachment by race group .
however , the qualitative comments regarding perceived invisibility of men of color in the gay community echo other recent qualitative work noting racial hierarchies within gay communities ( e.g. , han , 2007 ) . while the difference in perceived acceptance for men of color and gay men in the gay community was diminished ( compared with the broader community ) , the difference for bisexual men and gay men was amplified ( though not significantly so ) .
this might be due to the perception of bisexuality as a conflicted or incomplete sexual identity within the gay community , but as roughly analogous to homosexuality ( in terms of acceptability ) within the broader community .
trans persons were perceived as least accepted from both the mainstream community and the gay community .
trans persons have not necessarily enjoyed the greater mainstream social acceptance recently achieved for gay men and other sexual minorities , and they may also lack safe , inclusive spaces within urban gay communities characterized more by nightlife and social scenes dominated by gay men than by mutually supportive interaction between multiple sexual and gender minority groups . finally , the age - based segmentation of perceived acceptance suggests differing experiences of the london region and its gay community among men of different generations .
while younger men emphasized recent advances in rights and potential for the future , men at middle age frequently mentioned both individual traumas from growing up in homophobic environments as well as more recent traumas ( e.g. , project guardian , the pride controversy ) affecting their perceived acceptance of sexual and gender diversity in broader london and its gay community .
in addition , these feelings may be amplified among older , long - term residents of the region compared with a younger , transient population who fits more easily into the commercial gay scene and may be living in london only temporarily .
these trends mirror barrett and pollack s ( 2005 ) study noting less gay community attachment among older and working - class men , as well as qualitative interventions that observe age , gender , and generational differences in broader experiences of gay community ( browne & lim , 2010 ; zablotska , holt , & prestage , 2011 ; wilkinson , 2011 ; hammack & cohler , 2011 ) .
the differences also reflect social , political , and institutional changes that have occurred across canada but that affect different geographic regions and community subgroups differently . in canada , the advent of legal rights such as same - sex marriage , adoption , and pension benefits have been treated as roughly universal goods for gay communities ( smith , 2008 ) .
even so , their lived impacts across the sub - communities and sociodemographic groups within them may be more mixed . in the new milieu of rights - based activism , greater social inclusion in the form of same - sex marriage , pension , and adoption
rights was pursued in the federal courts , while conservative - led regional governments ( e.g. , ontario s harris administration during the 1990s and early 2000s ) and federal government ( the harper administration since 2006 ) disinvested in health and social services infrastructure geared toward vulnerable and minority groups ( hackworth , 2008 ; whiteside , 2009 ) . sustained , grassroots lgbt movements in large cities ( e.g. , toronto ) have effectively resisted the trend of rollback neoliberal governance in canada , running services through critical masses of volunteers , in - kind donations , and petitions for public funding .
however , smaller cities have not been able to rely on these types of infrastructures ( smith , 2005 ) .
a lack of gay community connectedness among some groups in london - middlesex , then , may reflect declining infrastructures and services more than it does assimilation into mainstream society ( weeks , 2007 ) or an individualist gravitation toward personal communities ( holt , 2011 ) .
the qualitative evidence suggests that class also mediates the observed hierarchies of perceived acceptance among respondents . for younger gay and bisexual men who are also middle - class , optimistic perceptions of acceptance may stem from coming of age during the promotion of human rights protections for sexual orientation in canada , insulation within a university environment , ability to participate in the limited nightlife scene , or assuredness of their ability to move ( e.g. , to toronto ) . for middle - aged gay and bisexual respondents , class status in the form of white - collar jobs , dual incomes , or ability to engage in more normative practices ( e.g. , raising children ) may to counteract the overall less optimistic perceptions of acceptance .
middle - class respondents also seem most able to dismiss , bypass , or circumvent the ( under- ) development of more inclusive , sustained community infrastructures by socializing in private and traveling elsewhere , while other groups ( e.g. , trans men ) may remain marginalized .
while our study did not compare the survey and interview responses with those from a big - city referent group ( e.g. , gay and bisexual men in toronto ) , our preliminary results suggests that recent advances in gay rights and mainstream social acceptance may have a mixed effect in smaller and mid - sized cities .
as recent interventions have shown ( knopp & brown , 2003 ; lewis , 2013 ) , cultural hierarchies that position large metropolitan , commercial gay scenes as the birthplaces and anchors of gay community never truly applied to smaller cities such as london . in the 1980s and 1990s , a single venue , halo , fostered an active , self - sustaining gay community .
it is only in the new milieu of advanced rights , reduced funding for health and social services , and the closure of organizations such as halo , that toronto a city with established lgbt services , networks , and neighborhoods emerges as the region s dominant urban gay community while smaller - city communities experience continued disinvestment and social fragmentation .
incidentally , the relative security of community identity and visibility in toronto has spawned both a post - mo media discourse that suggests a declining need for village - based gay venues and the emergence of alternative , non - bar queer spaces as well as trans and people of color spaces ( nash , 2013 ) .
london - middlesex , in contrast , seems to have suffered many of the drawbacks of the new right - based milieu ( e.g. , homonormativity and gay community disinvestment ) without seeing the supposed benefits , such as visibilities for more diverse gay and bisexual groups or the development of a rich array of alternative gay and queer spaces .
london and other mid - size or small cities might therefore benefit from community centers that offer opportunities for volunteering and networking across identity - based groups .
such centers even in an online capacity would offer new health promotion opportunities amid persistent perceptions of asos as spaces solely for the hiv - infected ( zablotska , holt , & prestage , 2011 ) .
creating such spaces , however , may require public endorsements , funding mechanisms , or public
meanwhile , groups who are perceived as most marginalized within the gay community ( e.g. , trans people , racialized immigrants ) might be included through greater network - building with extant organizations such as student groups or the local newcomer settlement center , and through explicitly multicultural and gender - fluid events .
thus , for london and potentially other mid - sized cities , narrowing the social cleavages among gay and bisexual men may require reframing the form and function of gay community rather than merely celebrating ascendance to a
. more work , therefore , needs to be done to assess whether gay communities are beginning to evolve differently in smaller cities than they are in large metropolitan areas .
the conclusions drawn in this study , however , must also be considered in light of several limitations .
first , as a small - scale , convenience - sample study , the results can not easily be extrapolated to gay and bisexual men and communities more generally , though it may suggest possibilities for this larger group .
second , while the inferential statistics used in this study highlight gradients in perceived social acceptance among gay and bisexual men , they can not be interpreted in the same way as in a larger , random - sample study .
this is primarily a descriptive analysis , and the quantitative analysis does not provide information as to why differences in perceived acceptance exist .
however , the study s mixed - methods design overcomes some of this limitation , in allowing more detailed explanation .
third , since we queried perceived acceptance for men of color without sexual identity being specified , we can not comment on the acceptance of gay or bisexual men of color .
fourth , the higher perceived acceptance for all groups within the gay community may be due to positive bias stemming from surveying respondents who are members of the community they are being asked to describe . despite these limitations
, the study provides an important point of departure for monitoring and assessing emerging cleavages in both gay communities and gay equalities , particularly in mid - sized cities that do not follow dominant metropolitan narratives of gay advancement . | changes in gay and bisexual men s connectedness to the gay community are related to the declining public visibility of hiv / aids and greater acceptance for homosexuality and bisexuality in mainstream society
. little work , however , has focused on perceived acceptance for subgroups within the gay community or broader society . using interviews ( n = 20 ) and a survey ( n = 202 ) of gay and bisexual men in a mid - sized canadian city
, we find perceived hierarchies of acceptance for the various subgroups as well as an age effect wherein middle - aged men perceive the least acceptance for all groups .
these differences are linked with the uneven impact of social , political , and institutional changes relevant to gay and bisexual men in canada . |
according to the international diabetes federation , metabolic syndrome ( ms ) is clinically characterized by central obesity ( waist circumference 94 cm for men and 80 cm for women ) , and at least two of these risk factors : high triglyceride levels ( 150 mg / dl ) ; low hdl cholesterol ( 40mg / dl ) ; high blood pressure levels ( systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg ) ; and fasting plasma glucose levels 100 mg / dl .
this cluster of cardiovascular risk factors is intrinsically related to increased incidence of diabetes mellitus and cardiovascular mortality .
studies have demonstrated several ms - induced abnormalities of cardiac geometry and function . both increased left ventricular mass and relative wall thicknesses , as well as rapid deceleration time , have been found in hypertensive subjects with ms when compared with a hypertensive cohort without the syndrome . on the other hand , patients with ms
in addition , it is well known that insulin resistance plays a key role in ms and as such contributes to the development of premature cardiovascular atherosclerosis , independent of the association with diabetes and obesity [ 68 ] .
it seems reasonable to assume that the association of several risk factors , as in the ms , favors an increased incidence of cardiovascular diseases and death risks in humans .
thus , a better understanding of the pathophysiological mechanisms of this syndrome becomes of paramount importance in clinical practice .
studies on animal models can be relevant as they mimic the aspects of the human disease as the development and maintenance of ms characteristics , particularly obesity , type 2 diabetes , dyslipidemia , and hypertension .
thus , the aim of this paper is to both describe and discuss the animal models used for the study of ms .
a special focus was given to cardiovascular changes which can be seen in tables 1 , 2 , and 3 .
the db / db mouse ( bks.cgm+/+leprdb/j ) is a genetic model widely used as ms animal model , as it presents a leptin receptor mutation which causes hyperglycemia and insulin resistance .
however , the evaluation of these animals with 8 - 9 weeks of age did not lead to changes in blood pressure as compared to their wild controls . as the animals age ( 14 - 15 weeks ) ,
noradrenergic responsiveness of the heart is reduced and indications of sympathetic denervation are observed .
the increase in blood pressure of these animals is associated with an increase in plasma angiotensin converting enzyme activity and angiotensin ii levels .
using spectral methods for autonomic function evaluation , no changes in heart rate variability , blood pressure variability , or baroreflex function are observed [ 11 , 13 ] .
in addition , db / db mice show impaired cardiac functional reserve capacity during maximal beta - adrenergic stimulation ( with dobutamine ) , which is associated with unfavorable changes in cardiac energy metabolism .
kkay is a congenital strain established by the transduction of the yellow obese gene ( ay ) into the moderate hyperglycemic kk strain .
kkay mice are obese and have high blood pressure levels , increased urinary excretion of catecholamines , and exacerbated responses to sympathetic blockade , suggesting a sympathetic role in the genesis of their hypertension [ 16 , 17 ] .
the ob / ob mice have a mutation in the ob gene resulting in leptin deficiency .
short - term direct blood pressure measurements suggested that the ob / ob mice are hypotensive with low sympathetic nerve activity .
however , when blood pressure is measured chronically , that is , data are collected for 5 s every 2 min and are averaged for the light cycle ( 7 am4 pm ) and the dark cycle ( 7 pm5 am ) with radiotelemetry ( 24 h full - time ) , ob / ob mice remain hypertensive during the light period . interestingly , during the dark cycle , ob / ob mice show no difference in blood pressure . on examination of the data over 24 h ( the dark and light cycles combined ) , ob / ob mice
, blood pressure was measured 24 h / day , which obviously includes every activity performed by the animals .
thus , behavior and feeding may be especially relevant since the ob / ob animals have leptin deficiency and eat throughout the day and at night , leading to the hypertension observed during the day .
furthermore , these animals develop left ventricular hypertrophy with decreased cardiac function at 24 weeks of age and cardiac fibrosis after 20 weeks of age .
finally , the db / db ( c57bl / ksj - db / db ) mice have inherited an autosomal recessive mutation in the leptin receptor gene present on chromosome 4 .
the metabolic alterations most frequently observed in this strain are hyperglycemia , hyperinsulinemia , hypertriglyceridemia , hypercholesterolemia , high levels of nonesterified fatty acids , and reduced hdl cholesterol .
in addition to these changes , both infiltration with inflammatory cells and fibrosis were observed in the heart after 12 weeks of age .
these mice also show vascular endothelial dysfunction , although no blood pressure changes are observed .
we studied the cardiovascular and autonomic phenotype of male db / db mice and evaluated the role of angiotensin ii at(1 ) receptors .
radiotelemetry was used to monitor 24 h blood pressure in mice for 8 weeks .
although there were no changes in heart rate variability and spontaneous baroreflex sensitivity between control and db / db mice , the results indicate an age - related increase in mean arterial pressure in db / db mice , which can be reduced by the antagonism of angiotensin ii at(1 ) receptors .
wistar ottawa karlsburg w ( wokw ) rats were developed from a wistar rat outbred strain of the biobreeding laboratories . as observed in humans , the features of ms in this model
in addition to obesity , this animal model shows other metabolic alterations , such as dyslipidemia , hyperinsulinemia , and impaired glucose tolerance . specifically , insulin resistance in this animal model
genomic scan studies have revealed a linkage of the ms and/or diabetes to a region on chromosome 3 ( 3q26 - 27 ) , where the gene encoding adiponectin , apm1 , is located .
one important clinical characteristic displayed by these animals was the impaired coronary function , due to increased alpha(1)-adrenoceptor - mediated coronary constriction ( at 3 and 10 months of age ) , and to a seriously blunted beta - adrenoceptor - mediated coronary relaxation ( at 16 months of age ) .
zucker obese ( zo ) rat model develops ms characterized by obesity since they are polyphagic due to a mutation in the leptin receptor [ 30 , 31 ] , insulin resistance , hypertriglyceridemia , and hypertension .
obesity leads to an inflammatory state which is linked to reduced insulin sensitivity and expression of glut4 in adipose tissue , skeletal muscle , and heart [ 33 , 34 ] .
this is likely to be related to increased circulating free fatty acids which compete with glucose as energy substrate and also inhibit the translocation of glut4 to the cell membrane .
myocardial fatty acid uptake and utilization lend support to the hypothesis that myocardial insulin resistance is associated with cardiac dysfunction , characterized by increased left ventricle mass , reduction systolic function , and survival .
increasing fatty acid as fuel energy can lead to increased reactive oxygen species , thus contributing to structural and functional damage in the myocardium . accordingly ,
these abnormalities occur prior to the onset of hypertension , which becomes elevated above control levels around 12 weeks of age .
furthermore , cardiovascular complications similar to human obesity also include higher resting sympathetic nerve activity and reduced heart rate variability [ 35 , 36 ] which were observed in zo rats attenuated baroreflex - mediated changes in sympathetic nerve activity to vascular targets .
these are due to impairments in sympathetic and parasympathetic control of the heart [ 37 , 38 ] .
zucker diabetic fatty rats ( zdf ) phenotype originated from selective breeding of zucker rats with high glucose levels , which developed diabetes after 10 weeks of age .
zdf presents hyperphagia , as a result of a nonfunctioning leptin receptor , which in turn leads to obesity similar to the prediabetic state in humans .
furthermore , hyperglycemia in the zdf model is different from that observed in the zo ; zdf rats do not have sufficient pancreatic -cell function .
it has been observed that changes in left ventricular chamber morphology occurred in the untreated zdf animals as early as 16 weeks of age .
( 2009 ) found an increase in myocardial fatty acid oxidation , a reduction in insulin - mediated myocardial glucose utilization , associated with impairment of myocardial function in zdf rats with 14 weeks of age .
in addition , vascular and neural dysfunction observed in zdf rats with 12 weeks of age has been improved with vasopeptidase inhibitors .
in addition , this animal model shows an increase in plasma angiotensin converting enzyme activity and angiotensin ii levels that , associated with high glucose levels , can lead to advanced nephropathy .
this may be the reason by which zdf rats started to die at 50 weeks of age ( 63 weeks for the control group ) , in association with an abrupt increase in blood urea nitrogen , suggesting that the cause of death was renal insufficiency .
one of the most recently created ms models is the dahls.z - lepr / lepr rat ( ds / obese ) .
this ms model was established by crossing dahl salt rats and zucker rats with the missense mutation in the leptin receptor gene ( lepr ) .
the animals , once fed with a normal diet , developed obesity , as well as hypertension , dyslipidemia , insulin resistance , and type 2 diabetes .
in addition , these animals developed cardiac hypertrophy , as well as renal and liver damage , which may account for their premature death .
hattori et al . ( 2011 ) showed that in an experimental period of 18 weeks , 13 ( 65% ) of 20 ds / obese rats died ( seven from renal failure , two from cerebrovascular events , and four from sudden cardiac death ) ; there were no deaths in the control group .
( 2012 ) have recently demonstrated that body weight , as well as visceral and subcutaneous fat mass , was significantly increased in ds / obese female rats , which was associated with diastolic dysfunction and marked left ventricle hypertrophy and fibrosis .
myocardial oxidative stress and inflammation , serum insulin , and triglyceride were also increased in ds / obese rats compared with ds / lean rats .
otsuka long - evans tokushima fatty ( oletf ) rats are a cholecystokinin 1 receptor knockout model which become obese secondarily to hyperphagia . the increased food intake is characterized by a large increase in meal size with a decrease in meal frequency which is not sufficient to compensate for the meal size increase . as a result ,
these animals usually present hyperglycemia after 18 weeks of age , mild obesity , and diabetes mellitus , more frequently observed in males
. however , insulin resistance in oletf rats emerges at 12 weeks of age , before the impairment of pancreatic -cell function .
( 2000 ) have previously demonstrated that oletf rats present diastolic dysfunction in their prediabetic state ( 15 weeks ) , observed by a prolongation in deceleration time and a decrease in amplitude of peak velocity of the early diastolic filling wave , without changes in the blood pressure and heart rate .
in addition , the researchers observed extracellular fibrosis and abundant transforming growth factor-1 receptor ii in the left ventricle of these rats . while still in the prediabetic stage , oletf rats exhibited a lower coronary flow reserve and increased coronary vascular resistance during hyperemia , which was associated with increased wall - to - lumen ratio and perivascular fibrosis . in the late stage of diabetes ( 22 and 62 weeks of age ) , it was demonstrated that these animals displayed an impairment in diastolic function and changes in the geometry of conductance and resistance arteries .
the goto - kakizaki ( gk ) model was created by selective breeding of an outbred colony of wistar rats , selected for high glucose levels in an oral glucose tolerance test .
these animals develop hyperglycemia after 4 weeks of age and increased liver and plasma lipid concentration after 8 weeks of age . after 16 weeks of age
, at prediabetic state , gk rats displayed left ventricle remodeling with marked hypertrophy of cardiomyocytes and increased extracellular matrix deposition , culminating in increased heart size . despite the progressively worsening of glucose metabolism derangement , at 18 months of age
the contractile function of the heart appears to be well preserved , as observed by maintenance of amplitude of shortening in electrically stimulated myocytes .
in addition , untreated gk rats presented mild hypertension and a blunted vascular relaxation by acetylcholine and sodium nitroprusside when compared to control animals .
we studied in our laboratory the cardiovascular autonomic function of obesity induced by monosodium glutamate ( msg ) in a normotensive model .
male wistar rats receiving msg neonatal treatment showed metabolic abnormalities , as well as increased body weight , lee index , epididymal white adipose tissue , and insulin resistance .
in addition , these animals exhibited reduced glucose / insulin index ( 62.5% ) , and increased insulin secretion during glucose overload ( 39.3% ) , and hyperinsulinemia .
these rats showed a slight increase in mean arterial pressure with no difference in the heart rate from their controls .
however , they showed cardiovascular autonomic dysfunction , as shown by reduced baroreflex sensitivity and vagal and sympathetic effects when compared to their controls .
we observed a reduced sympathetic effect which was not followed by changes in the basal heart rate or tachycardic responses to arterial pressure changes .
recently , we carried out studies on cardiovascular abnormalities of the spontaneously hypertensive rat ( shr ) [ 59 , 60 ] treated with msg in the neonatal period .
although this animal model is not new [ 62 , 63 ] , no research , to our knowledge , has been undertaken using metabolic and cardiovascular parameters over time ( 3 , 6 , and 9 months of age ) .
the use of msg in genetically hypertensive rats led these animals to progressively increase body adiposity and triglyceride levels .
besides developing and maintaining insulin resistance , they presented low hdl cholesterol and increased inflammation state ( high c - reactive protein , interleukin 6 , tumor necrosis factor- levels , and low adiponectin levels ) , which reached highest levels at 6 and 9 months of age . in ms ,
therefore , the use of a genetically hypertensive rats associated with msg treatment , which leads to metabolic changes including obesity , seems to work well as an ms model resembling the human disease .
however , early studies showed that shr treated with msg did not maintain hypertension , but it should be noted that indirect tail - cuff methods were used .
thus , we evaluated blood pressure by direct method ( analyzed on a beat - to - beat basis ) at 3 , 6 , and 9 months in msg - treated shr compared to shr and normotensive rats .
it was observed that mean blood pressure was similarly higher in shr and msg - treated shr at all ages , when compared to normotensive rats .
furthermore , there were no changes in blood pressure over time in the groups studied .
the increased cardiovascular mortality which characterizes ms may be partially attributed to cardiac sympatho - vagal imbalance [ 64 , 65 ] .
thus , we evaluated temporal cardiovascular autonomic dysfunction in this model by spectral analysis and observed that the changes in cardiovascular autonomic control evaluated up to 9 months are similar to those observed in hypertensive rats . at 9 months
the animals maintained the hypertensive state , a feature of shr , and presented reduced heart rate variability , decreased spontaneous baroreflex sensibility , and increased cardiac sympathovagal balance .
in addition to these changes , it was observed that shr and shr with msg - induced obesity showed increments of 6- to 8-fold of systolic arterial pressure variability at 6 and 9 months , as compared to normotensive rats .
interestingly , at 6 months , msg - induced shr had increased systolic arterial pressure variability when compared to only shr . taken together ,
sympathetic activation plays an important role in the pathogenesis of insulin resistance [ 6668 ] and in the activation of the renin - angiotensin system , which are related to cardiovascular autonomic control dysfunction of ms .
the injection of streptozotocin ( stz ) to mice , rats , and rabbits has been widely used as a model of type 1 diabetes .
streptozotocin destroys pancreatic cells , resulting in a diabetic syndrome in animals , characterized by hyperglycemia , hypoinsulinemia , glycosuria , and body weight loss [ 70 , 71 ] .
these rats have systolic and diastolic dysfunction at rest , as evaluated by left ventricular catheterization and echocardiography , as well as reduced capacity for cardiac adjustment to volume overload and maximal oxygen consumption [ 72 , 73 ] .
furthermore , stz induces reduction in baroreflex - mediated bradycardia and tachycardia , as well as impairment in cardiac vagal tone in the face of unaltered sympathetic tone [ 71 , 74 , 75 ] . although well accepted in the literature as a model for type 1 diabetes ,
the injection of stz alone does not allow it to mimic the characteristics of ms in humans , since these animals show hypoinsulinemia , loss or no change in body weight , and hypotension [ 71 , 74 , 75 ] .
an alternative approach , previously used , is the administration of low doses of stz ( 25 mg / kg ) associated with high fructose diet in rats .
this combination induced mild hyperglycemia and hypertriglyceridemia associated with mild fasting hyperinsulinemia and whole body insulin resistance , without significant increase in body weight . in addition , the authors also observed decreased left ventricular contractile function and reduced myocardial metabolic efficiency . since 1978
the mean daily intake of added fructose and total fructose has increased in both sexes and all age groups , having as the main sources of consumption soft drinks and other sweetened beverages [ 77 , 78 ] .
fructose overload in drinking water or chow has been used to promote metabolic , hemodynamic , structural , and functional derangements in rodents .
fructose overload in experimental animals has been associated with high triglyceride levels , adiposity , insulin resistance , and glucose intolerance [ 7986 ] .
these animals have been used by our group in order to understand the various aspects of obesity , dyslipidemia , and insulin resistance - associated cardiovascular changes [ 8188 ] .
we observed that an increased sympathetic modulation to the vessels and heart preceded metabolic dysfunction in fructose - consuming mice , thus suggesting that changes in autonomic modulation may be a triggering mechanism underlying the cluster of symptoms associated with cardiometabolic disease [ 8386 , 88 ] .
animal studies have shown strong associations between high fructose intake and the onset of arterial hypertension . according to farah et al . , mice which received a high fructose diet showed higher blood pressure and heart rate in the dark ( active ) period when compared with the light ( resting ) period .
this augment in blood pressure was related to an increase in the blood pressure variability and , consequently , to increased vascular sympathetic modulation .
this is a relevant finding since during the dark period the mice are active ( grooming , eating , and drinking ) and the sympathetic activity should be high . on the other hand , it was displayed that female fructose - fed rats presented reduced vagal tonus , unaltered sympathetic tonus , and intrinsic heart rate .
however , this study also showed that although the sympathetic tonus remained unchanged , the autonomic balance ( sympathetic / parasympathetic ) was altered , leading to a sympathetic predominance . a positive correlation between reduced cardiac vagal tonus and insulin resistance
was also found , thus reinforcing the relationship between autonomic and metabolic dysfunction . since arterial
baroreflex influences both sympathetic and parasympathetic outflow , disorders in autonomic neuronal pathways ( efferent or afferent ) may affect cardiovascular health and be related to high blood pressure .
high fructose consumption induces an impairment of baroreflex sensitivity , as evaluated by heart rate responses associated with arterial pressure changes induced by vasoactive drugs [ 81 , 86 ] , as well as by linear regression and alpha - index .
furthermore , unpublished data from our laboratory demonstrated that impairment of baroreflex sensitivity was positively correlated with the number of elastic lamellae in the ascendant aorta .
this data suggest that the possible loss of distensibility of the aorta may be associated with changes in baroreflex sensitivity , since the mechanical stress of the arterial wall can not be effective to properly trigger the mechanoreceptors .
regarding the rennin - angiotensin system , it has been widely acknowledged that high - fructose diet induces increased angiotensin ii plasma levels , which contributes to hypertension , insulin resistance , and dyslipidemia , and may account for cardiac remodeling [ 80 , 90 ] and vascular oxidative stress in this ms model .
in fact , it appears that angiotensin ii promotes fibroblast proliferation due to activation of the angiotensin ii type 1 receptor , which results in increased collagen type iii expression and accumulation in the heart . also , there is evidence that the insulin - resistant state is associated with angiotensin ii type 1 receptor upregulation and increased endothelial ( aorta ) superoxide anion levels , which is likely to be caused by an increase in nad(p)h oxidase expression in fructose - fed rats .
furthermore , it should be stressed that angiotensin 1a receptors are critical in mediating the response to a high - fructose diet and the resultant state of glucose intolerance , because in the absence of these receptors , a fructose diet decreases the blood pressure , as observed in angiotensin 1a knockout mice . as an important consequence of these metabolic , hemodynamic , autonomic , and structural changes displayed in this ms model , our group recently demonstrated that fructose overload promoted changes in left ventricular morphometry , diastolic dysfunction , and increased cardiac effort , as evidenced by the increase in the myocardial performance index .
other researchers have found similar results for wistar rats receiving a 10% fructose overload for 8 weeks .
sucrose is a disaccharide composed by one molecule of glucose linked to one molecule of fructose through an 14 glycoside bond .
similar to fructose , sucrose induces ms in animals , as increased plasma concentrations of insulin , leptin , triglycerides , glucose , and free fatty acids and impaired glucose tolerance were shown .
sucrose - treated rats ( 32% in drinking water ) revealed early abnormalities in diastolic function ( 2.5 weeks of treatment ) followed by late systolic dysfunction and concurrent alterations in myocardial structure ( 10 weeks of treatment ) .
furthermore , the authors demonstrated that after 10 weeks of sucrose treatment the animals presented reduced calcium uptake in sarcoplasmic reticulum of cardiomyocytes .
in addition , animals treated with an 8% sucrose solution developed hypertension and tachycardia after 2 weeks of treatment , and this was not related to weight gain .
the authors concluded that sucrose ingestion may stimulate the ventromedial hypothalamus to increase sympathetic activity and elevate blood pressure in rats .
high fat diets have been used with fat fractions between 20% and 60% energy as fat , and the basic fat component varies between animal - derived fats and plant oils , for example , corn , coconut , or safflower oil .
after 10 weeks of a high fat diet , rats displayed high fat mass , insulin resistance , and hyperleptinemia , typically associated with obesity .
rabbits receiving a high fat diet during 3 weeks presented an impairment in leptin sensibility , along with increased mean arterial pressure , heart rate , and plasma norepinephrine concentration .
renal sympathetic nerve activity was also higher in high fat diet rabbits when compared to control diet rabbits and was correlated to plasma leptin .
2012 ) have demonstrated that the baroreceptor dysfunction which controls renal sympathetic nerve activity is an initial change in the obesity - induced high fat - fed rats , which might be a predictor of sympathoexcitation and hypertension associated with obesity .
the renin - angiotensin system has been involved in the hypertension genesis linked to obesity .
( 2004 ) observed that both angiotensinogen gene expressions in retroperitoneal fat mass and plasma angiotensinogen concentration were increased in rats receiving high fat diet , thus showing increased activity of the adipose and systemic rennin - angiotensin system in obesity - related hypertension .
proponents of chemically induced ms models have been challenged by researchers who argue for diet - induced models .
the latter contend that these reflect more accurately the condition of human obesity when compared to chemically induced or genetic modifications .
thus , several diet - induced experimental models have been proposed , suggesting that there is an aggregation of factors in ms .
the cafeteria diet model is a fine example of a diet - induced experimental model . in this model ,
animals are allowed free access to standard chow and water while concurrently given highly palatable , energy dense , unhealthy human foods ad libitum , which promotes voluntary hyperphagia .
this diet results in weight gain , increased fat mass , glucose intolerance , and insulin resistance [ 106 , 107 ] .
wistar rats fed a cafeteria diet showed , in addition to the classical metabolic disorder , cardiovascular alterations , such as increased heart rate and blood pressure .
cafeteria diet has been shown to lead to impairment of the endothelium - derived hyperpolarization mechanism , in particular , potassium channel signaling mechanisms .
thus , this finding may explain , at least in part , the link between cafeteria diet and increased blood pressure in rats ; this link is further reinforced by understanding that vascular tone refers to the balance between constrictor and dilator actions and influences the control of blood flow and pressure , and this phenomenon is influenced by endothelium - derived hyperpolarization mechanism .
although normotensive rats fed a cafeteria diet showed an increase in blood pressure , this diet may have a better outcome if used in shr , since this animal model has cardiac disorders , for example , increased sympathetic activity , hypertension , and cardiac hypertrophy [ 59 , 60 ] which may be potentiated by the cafeteria diet .
spontaneously hypertensive rats fed with cafeteria diet for 12 weeks showed metabolic changes similar to those of the ms , for example , high plasma levels of glycemia , insulin , triglyceride , leptin , and obesity .
many studies have dealt with the autonomic dysfunction of shr by spectral analysis [ 111 , 112 ] .
there is considerable evidence to support the hypothesis that the cluster of complications from metabolic syndrome status converges to a derangement of the cardiovascular system . in fact , cardiovascular diseases are the leading cause of morbidity and mortality in this condition .
the use of different experimental models that mimic the metabolic derangements seen in this syndrome is extremely important in order to better understand the mechanisms involved in cardiovascular changes caused by metabolic syndrome .
new approaches , such as genetically modified models , can provide mechanistic information on the genetic and environmental factors involved in the development of cardiovascular dysfunction in the metabolic syndrome . | metabolic syndrome has been defined as a group of risk factors that directly contribute to the development of cardiovascular disease and/or type 2 diabetes . insulin resistance seems to have a fundamental role in the genesis of this syndrome . over the past years to the present day , basic and translational research has used small animal models to explore the pathophysiology of metabolic syndrome and to develop novel therapies that might slow the progression of this prevalent condition . in this paper
we discuss the animal models used for the study of metabolic syndrome , with particular focus on cardiovascular changes , since they are the main cause of death associated with the condition in humans . |
the prostate is the site of two common diseases in aging men , benign prostatic hyperplasia and prostate cancer ( pca ) .
epidemiological studies show that , in the united states , pca accounts for 28.7% of new non - skin cancers and 12.7% of cancer - related deaths.1 androgen deprivation therapy ( adt ) retards pca progression in 60%80% of patients but may cause cardiovascular complications and alterations in body composition , lipoprotein metabolism , and sexual function . in general , adt may result in a decreased quality of life .
furthermore , studies in men demonstrate that low serum testosterone ( t ) levels are associated with several cardiovascular risk factors,2,3 such as lower high density lipoprotein cholesterol , higher triglyceride concentrations , hyperinsulinemia , and increased abdominal adiposity .
these factors are all characteristic of the metabolic insulin resistance syndrome . in addition , many patients treated with adt experience rapid bone loss that increases the risk of debilitating osteoporotic fractures.46 reports indicate that total and free levels of serum t decline in aging men and that abdominal visceral fat is inversely associated with t levels . resting energy expenditure ( ree ) , which accounts for 60%70% of total daily energy expenditure ,
decreases with age , physical inactivity , sympathetic nervous system and endocrine status.7,8 to our knowledge , the effects of adt on ree have not been fully explored in controlled trials .
the goal of this prospective study was to determine the effects of adt on body composition and resting metabolic rate in patients with metastatic pca .
sixteen men , aged 63 to 96 years ( mean age 71 ) , with untreated metastatic pca were enrolled in this study . prior to orchiectomy , all patients were evaluated clinically and by laboratory analysis to establish a baseline . after a careful medical history had been taken from each patient , we assessed several exclusion criteria , namely , cognitive alterations , depression and emotional liability .
laboratory examinations included tests of fsh - lh serum concentrations , and an evaluation of body mass index , fat / lean body mass , bone mineral density , carbohydrate / lipid oxidation , and resting energy expenditure .
following orchiectomy , the same evaluation was carried out monthly and after 12 months , the period chosen for final analysis . to evaluate body composition
, all patients underwent densitometry testing ( dual - energy x ray absorptiometry - dexa).9 the ree , fat and carbohydrate oxidation levels were measured using indirect calorimetry , with a delta - trac metabolic monitor.10 body composition analyses and indirect calorimetry were carried out at baseline and after 12 months following the orchiectomy procedure . written consent was obtained from all patients , and this protocol was approved by the ethics committee at our institution .
we used the non - parametric two - sided wilcoxon t - test , and p<0.05 was considered significant to appropriately compare time differences for each variable.11
we used the non - parametric two - sided wilcoxon t - test , and p<0.05 was considered significant to appropriately compare time differences for each variable.11
statistically significant differences in body composition pre and post orchiectomy were seen in terms of weight , body mass index ( bmi ) and lean body mass ( lbm ) .
total fat body mass ( fbm ) exhibited a tendency to increase but failed to reach significance .
no significant changes were observed in bone mineral density ( bmd ) and ree , but we identified a statistically significant change in terms of increased carbohydrate oxidation ( choox ) and a decrease in lipid oxidation ( lipidox ) .
aging is associated with sarcopenia ( loss of muscle mass ) , with an average 12 kg loss of lbm between the ages of 25 and 70 years and an increase in fbm of 18%36% during the same period.2 testosterone levels exhibit a positive correlation with lbm and a negative correlation with fbm .
this has led to the hypothesis that adt may help mitigate age - related alterations in body composition .
our study reports two major findings : 1 ) adt was associated with decreased body weight and with decreased lbm ; 2 ) adt led to increased choox and decreased lipidox .
androgens modulate body composition , including lipid and protein metabolism.12 smith et al,13 galvo et al,14 levy et al,15 and van londen et al16 observed significant changes in metabolic and body composition parameters after 6 months of induced hypogonadism in all patients .
the researchers observed a significant increase in fbm ( 20.29.4 to 21.99.6 kg,13 13.82.3%,14 1.172.3 % , 15 and 2167.15 g,16 respectively ) and a significant decrease in lbm ( 63.28.0 to 62.35.4 kg,13 2.4 0.4%,14 1.152.20%;15 and 1785.81 g,16 respectively ) with no significant changes in total body weight , lipid readings or glucose metabolism .
they also reported a positive correlation between the changes in fbm and insulin concentrations ( r=0.56 ; p=0.013 ) .
these results are in agreement with other studies that demonstrated a relationship between t and abnormalities in carbohydrate and lipid metabolism.17 in healthy male populations , low t concentrations are negatively correlated with the degree of central abdominal obesity . in hypogonadal males , there is a tendency toward increased visceral adiposity and reduced muscle mass that can be reversed by androgen replacement.18 the underlying mechanisms for these observations are not well defined , but androgen receptors are known to be present on visceral adipocytes .
it is likely that t is directly involved in the mobilization of free fatty acids.19 we observed significant changes in metabolic and body composition in our patients after adt .
we identified significant decreases in total body weight , bmi and lbm ( 2.5 kg , p = 0.01 ; 0.8 kg / cm , p = 0.02 , 11.7 kg , p = 0.002 , respectively ) .
fbm was found to increase ( 7.15 kg , p = 0.06 ) with no significant change in bone mineral density ( bmg ) .
there were no changes in ree , but we did identify a significant increase in choox and a decrease in lipidox ( 52.62% , 4.10% respectively ) . in one study , suppression of t levels after 10 weeks of gnrh agonist administration was found to reduce ree in young men .
in this study , researchers were unable to determine whether the effect was directly mediated by a reduction in t levels or if it was due to a subsequent reduction in t aromatization to estrogen.20 we were unable to show alterations in ree in our study , possibly due to the size of our study sample .
the altered concentrations of gonadotropins following orchiectomy may be linked with the effects of this procedure on the pituitary gland .
it is important to consider that adt not only acts on pca but also affects metabolism , including hematogenesis , bone , fat , protein , and sarcho - metabolism . despite pca being one of the leading causes of death , a substantial proportion of aging patients die of unrelated causes that are due to comorbidities , often in the context of metabolic syndrome .
to our knowledge , this is the first prospective study to examine changes in body composition , resting energy expenditure , and consequences involving the oxidation of carbohydrates and lipids in the context of adt .
a study on a larger group of patients would be of great interest once a limited number of patients included from only one centre may be considered a limitation in this study .
after 12 months of androgen deprivation therapy , elderly males with metastatic prostate cancer exhibit declines in lean body mass and lipid oxidation , together with an increase in carbohydrate oxidation . | introductiontestosterone is needed for normal male development , muscle strength , bone mineralization , hematopoietic function , and sexual and reproductive functions .
the main purpose of androgen deprivation therapy in prostate cancer is to reduce tumor progression , but therapy is often accompanied by significant adverse effects.objectivethis study aimed to determine the effects of androgen deprivation therapy on body composition and resting metabolic rate in patients with prostate cancer.patients and methodsa prospective study was performed to evaluate the body composition of 16 elderly males ( aged 6396 ; median age 71 ) with prostate cancer scheduled for orchiectomy , one year before and after surgery .
body composition was measured by dexa , and energy expenditure , fat and carbohydrate oxidation were measured by indirect calorimetry.resultsbody weight ( p=0.01 ) , lean mass ( p=0.004 ) , and lipid oxidation ( p=0.001 ) decreased significantly .
carbohydrate oxidation ( p=0.02 ) , fsh ( p=0.0001 ) and lh ( p=0.0001 ) levels increased significantly .
changes in fat mass ( p=0.06 ) and bone mineral density ( p=0.48 ) were not significant.conclusionsafter 12 months of androgen deprivation therapy , elderly men with metastatic prostate cancer exhibit a decline in lean body mass and lipid oxidation , together with increased carbohydrate oxidation . |
5xfad tg mouse model ( b6sjl - tg(appswfllon , psen1*m146l*l286v)6799vas ) ( jackson laboratories ) is bred on a non - transgenic background ( b6sjlf1 ) .
hela is a human cervical cancer cell line ; nih/3t3s is a mouse embryonic fibroblast line ; hepg3 is from hepatocellular carcinoma ; raw264.7 is a mouse leukemic monocyte macrophage cell line .
16 nm magnetite nanoparticles were synthesized by decomposition of iron - oleate at 320c as described in an earlier report .
10.8 g of iron ( iii ) chloride hexahydrate and 36.5 g sodium oleate were dissolved in a mixture of 60 ml distilled water , 80 ml ethanol and 140 ml hexane and heated at 60c for 4 hr . the organic layer of the biphasic mixture becomes dark , indicating phase transfer of iron ( iii ) ions and formation of iron oleate complex .
18 g of iron oleate complex and 2.58 g of oleic acid were dissolved in 100 g of octadecene at room temperature and heated to 320c at a rate of 3.3c per minute .
resulting nanoparticles are separated from the solution by addition of ethanol and ethyl acetate followed by centrifugation . to make the organic phase synthesized mns suitable for biological application we stabilized the mns using carboxylate terminated ligand with ndopa as an anchor .
synthesis of carboxylate terminated ndopa ligand and functionalization of the mns was carried out according to the following protocol .
tetraethylene diacide , n - hydroxysuccinimide ( nhs ) , n , n-dicyclohexylcarbodiimide ( dcc ) , ndopa hydrochloride and anhydrous sodium bicarbonate was dissolved in chloroform under argon atmosphere and stirred for 4 hr .
hexane stabilized mns were added and stirred for another 24 hr . the precipitate formed was separated by magnet , dispersed in water and purified by dialysis .
the conjugation of buffer stabilized mns with antibody was done using a conventional carboxyl - amine crosslinking method .
we first activated the carboxyl terminated mns by sulfo - n - hydroxy succinimide ( snhs ) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ( edc ) followed by incubation with corresponding antibody ( nu4 or igg1 , with or without fluorescent label ) overnight .
conjugated mns were separated by magnet to remove excess reagent and antibody then re - dispersed in working media .
conjugation efficiency was estimated using uv spectroscopy ( absorbance at 280 nm ) of the magnetically separated supernatant . of note , because both fluorophore and mns conjugation use carboxyl - amine crosslinking , fluorescent nu4 that has more than 2.5 mol fluorophore / mol antibody does not conjugate well to the mns .
optimal concentration of fluorophore / nu4 is ~1.02.5 mol fluorophore / mol antibody for fluorescent - nu4mns preparation .
mice were anesthetized with isoflurane and then placed on their backs with their heads positioned to maximize the residency time for the delivered material to remain on the olfactory surface .
each naris was administered with 568-nu4 , nu4mns or non - immune iggmns ( 5 l / naris ) , using a sterile micropipette , slowly over a period of 1 min , keeping the opposite naris and mouth closed to allow complete aspiration of delivered material .
a falcon tube containing the mns treated tissue samples was placed in a bruker pharmascan 7 t animal mri scanner .
sample were imaged using a t2 * -weighted gradient echo ( gre ) flash pulse sequence 3-dimensional acquisition .
the image resolution was 0.07 mm isotropic , with field - of - view ( fov ) 4.62cm/2.52cm/0.65 cm and image matrix ( mtx ) 700 pixels/385 pixels/100 pixels .
the number of averages ( nex ) was 20 to improve the signal - to - noise ratio .
following intranasal inoculation , the probe was allowed to distribute for 4 hours before mr imaging was performed according to imaging methodology described in mundt et al .
t1 , t2 , and t2 * weighted mr images were acquired on a bruker biospec 7 t magnet , using a 25 mm rf quadrature coil .
t1- and t2-weighted images provide anatomical guidance as well as some localization of the nu4mns and were acquired with a fat suppressed spin echo sequence ( rapid acquisition with relaxation enhancement , rare ) with the following parameters for t1-weighted ( tr=1000 ms , teeff=13.2 ms , rare factor 2 , number of excitations , nex=4 ) and for t2-weighted ( tr=3500 ms , teeff=58.5 ms , rare factor 4 , nex=4 ) . t2
* -weighted imaging provides more of the localization of the nu4mns as the iron causes local changes in magnetic susceptibility which t2 * weighted images can be sensitive to .
a gradient echo sequence was used with the following parameters ( gradient echo fast imaging , gefi ; tr=1200 ms , te=5.6 ms , flip angle 35 and nex=4 ) .
5xfad tg mouse model ( b6sjl - tg(appswfllon , psen1*m146l*l286v)6799vas ) ( jackson laboratories ) is bred on a non - transgenic background ( b6sjlf1 ) .
hela is a human cervical cancer cell line ; nih/3t3s is a mouse embryonic fibroblast line ; hepg3 is from hepatocellular carcinoma ; raw264.7 is a mouse leukemic monocyte macrophage cell line .
16 nm magnetite nanoparticles were synthesized by decomposition of iron - oleate at 320c as described in an earlier report .
10.8 g of iron ( iii ) chloride hexahydrate and 36.5 g sodium oleate were dissolved in a mixture of 60 ml distilled water , 80 ml ethanol and 140 ml hexane and heated at 60c for 4 hr . the organic layer of the biphasic mixture becomes dark , indicating phase transfer of iron ( iii ) ions and formation of iron oleate complex .
18 g of iron oleate complex and 2.58 g of oleic acid were dissolved in 100 g of octadecene at room temperature and heated to 320c at a rate of 3.3c per minute .
resulting nanoparticles are separated from the solution by addition of ethanol and ethyl acetate followed by centrifugation .
10.8 g of iron ( iii ) chloride hexahydrate and 36.5 g sodium oleate were dissolved in a mixture of 60 ml distilled water , 80 ml ethanol and 140 ml hexane and heated at 60c for 4 hr . the organic layer of the biphasic mixture becomes dark , indicating phase transfer of iron ( iii ) ions and formation of iron oleate complex .
18 g of iron oleate complex and 2.58 g of oleic acid were dissolved in 100 g of octadecene at room temperature and heated to 320c at a rate of 3.3c per minute .
resulting nanoparticles are separated from the solution by addition of ethanol and ethyl acetate followed by centrifugation .
to make the organic phase synthesized mns suitable for biological application we stabilized the mns using carboxylate terminated ligand with ndopa as an anchor .
synthesis of carboxylate terminated ndopa ligand and functionalization of the mns was carried out according to the following protocol .
tetraethylene diacide , n - hydroxysuccinimide ( nhs ) , n , n-dicyclohexylcarbodiimide ( dcc ) , ndopa hydrochloride and anhydrous sodium bicarbonate was dissolved in chloroform under argon atmosphere and stirred for 4 hr .
hexane stabilized mns were added and stirred for another 24 hr . the precipitate formed was separated by magnet , dispersed in water and purified by dialysis .
the conjugation of buffer stabilized mns with antibody was done using a conventional carboxyl - amine crosslinking method .
we first activated the carboxyl terminated mns by sulfo - n - hydroxy succinimide ( snhs ) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ( edc ) followed by incubation with corresponding antibody ( nu4 or igg1 , with or without fluorescent label ) overnight .
conjugated mns were separated by magnet to remove excess reagent and antibody then re - dispersed in working media .
conjugation efficiency was estimated using uv spectroscopy ( absorbance at 280 nm ) of the magnetically separated supernatant . of note , because both fluorophore and mns conjugation use carboxyl - amine crosslinking , fluorescent nu4 that has more than 2.5 mol fluorophore / mol antibody does not conjugate well to the mns .
optimal concentration of fluorophore / nu4 is ~1.02.5 mol fluorophore / mol antibody for fluorescent - nu4mns preparation .
mice were anesthetized with isoflurane and then placed on their backs with their heads positioned to maximize the residency time for the delivered material to remain on the olfactory surface .
each naris was administered with 568-nu4 , nu4mns or non - immune iggmns ( 5 l / naris ) , using a sterile micropipette , slowly over a period of 1 min , keeping the opposite naris and mouth closed to allow complete aspiration of delivered material .
a falcon tube containing the mns treated tissue samples was placed in a bruker pharmascan 7 t animal mri scanner .
sample were imaged using a t2 * -weighted gradient echo ( gre ) flash pulse sequence 3-dimensional acquisition .
the image resolution was 0.07 mm isotropic , with field - of - view ( fov ) 4.62cm/2.52cm/0.65 cm and image matrix ( mtx ) 700 pixels/385 pixels/100 pixels .
the number of averages ( nex ) was 20 to improve the signal - to - noise ratio .
following intranasal inoculation , the probe was allowed to distribute for 4 hours before mr imaging was performed according to imaging methodology described in mundt et al .
t1 , t2 , and t2 * weighted mr images were acquired on a bruker biospec 7 t magnet , using a 25 mm rf quadrature coil .
t1- and t2-weighted images provide anatomical guidance as well as some localization of the nu4mns and were acquired with a fat suppressed spin echo sequence ( rapid acquisition with relaxation enhancement , rare ) with the following parameters for t1-weighted ( tr=1000 ms , teeff=13.2 ms , rare factor 2 , number of excitations , nex=4 ) and for t2-weighted ( tr=3500 ms , teeff=58.5 ms , rare factor 4 , nex=4 ) . t2
* -weighted imaging provides more of the localization of the nu4mns as the iron causes local changes in magnetic susceptibility which t2 * weighted images can be sensitive to .
a gradient echo sequence was used with the following parameters ( gradient echo fast imaging , gefi ; tr=1200 ms , te=5.6 ms , flip angle 35 and nex=4 ) .
| one way to image the molecular pathology in alzheimer s disease ( ad ) is by positron emission tomography using probes that target amyloid fibrils .
however , these fibrils are not closely linked to the development of the disease .
it is now thought that early stage biomarkers that instigate memory loss comprise of a oligomers ( aos ) .
here we report a sensitive molecular magnetic resonance imaging ( mri ) contrast probe that is specific for aos .
we attach oligomer - specific antibodies onto magnetic nanostructures and show the complex is stable and it binds to aos on cells and brain tissues to give a mri signal .
when intranasally administered to an ad mouse model , the probe readily reached hippocampal aos . in isolated samples of human brain tissue
, we observed an mri signal that distinguished ad from controls .
such nanostructures that target neurotoxic aos are potentially useful for evaluating the efficacy of new drugs and ultimately for early - stage ad diagnosis and disease management . |
cataract extraction requires analgesia , ocular akinesia , and is performed in the outpatient surgery .
topical anesthesia is indicated in procedures involving the anterior part of the eye , lasting less than two hours .
sedation is performed so that the patients remain quiet and do not interfere with the procedure .
clonidine is one of the drugs used in this context because it causes sedation without inducing hypnosis and reduces the blood pressure ( bp ) and heart rate ( hr ) [ 3 , 4 ] .
the reduction of the intraocular pressure ( iop ) represents an additional advantage of clonidine because it makes the eye surgery easier and reduces the frequency of complications , such as expulsion of the vitreous humor when the lens posterior capsule is broken during phacoemulsification .
following the development of phacoemulsification , topical anesthesia became a well - accepted minimally invasive technique , although it does not provide an analgesia that is as efficacious as blocks .
this led us to conduct the study with the aim of assessing the effect of clonidine on the intraoperative analgesia , sedation , and intraocular pressure in patients subjected to cataract extraction under topical anesthesia .
the secondary outcomes were better sedation and a reduction of the intraocular pressure ( iop ) and arrhythmia .
the study was prospective , randomized , and double - blind and registered at clinicaltrials.gov under code nct01677351 .
before the onset of anesthesia , a nurse or anesthetist who was not involved in the participant 's later follow - up opened the envelope and prepared the syringes with clonidine or 0.9% saline solution as indicated .
no other investigator who participated in the study or data collection was aware of the group to which each participant was allocated .
following the approval by the research ethics committee of federal university of so paulo ( number 0609/01 ) and signature on the informed consent form , 40 patients who were 40 to 80 years of age , of both genders , with an asa ( american society of anesthesia ) i , ii , or iii physical status , and subjected to cataract extraction by means of phacoemulsification under topical anesthesia at a tertiary university hospital were assessed .
the following were excluded : patients with coronary heart disease , heart arrhythmias , or chronic pain ; patients using analgesics 2 weeks before surgery ; patients with tremors or cough ; patients who had previous surgery ; patients with claustrophobia , glaucoma , psychiatric disorders , or difficulty in communicating ; and pregnant patients .
monitoring included the following : electrocardiogram using a holter device , arterial pressure ( ap ) , heart rate ( hr ) , oxygen saturation , and placement of a venous catheter followed by pupil dilation using 10% phenylephrine ( two drops ) and 1% tropicamide ( two to eight drops ) .
thirty minutes later , the patients in clonidine group were given a clonidine solution ( 4 mcgkg ) , while the patients in placebo group were given 0.9% saline solution .
twenty minutes later , topical anesthesia was performed with 2% lidocaine gel that was placed on the inferior conjunctival sac , and the patient was released for surgery .
whenever it was needed , anesthesia was supplemented by instillation of 2% lidocaine ( three to five drops ) or an intracameral injection of 2% lidocaine ( 0.5 ml ) .
intraoperative analgesia was assessed on a numerical scale ranging from 0 to 10 , before injection of the solution , after 5 and 15 minutes , blepharostat placement , incision of the cornea , capsulorhexis , phacoemulsification , irrigation and aspiration , and lens implantation , and at the end of surgery .
sedation was assessed by means of the ramsay sedation scale ( ( 1 ) patient is anxious ; ( 2 ) patient is calm and awake ; ( 3 ) patient is sleepy but opens eyes on command ; ( 4 ) patient is sleeping and only responds to vigorous verbal stimuli ; ( 5 ) patient is sleeping and responds to pain stimulation by glabellar tap ; ( 6 ) patient does not respond to pain stimuli ) . the iop was measured before and 15 minutes after administration of the solutions to both eyes and after the surgical procedure in only the eye not subjected to the surgery .
the continuous electrocardiogram , holter series 8500 , was applied after the pupil dilation and was discontinued immediately at the end of surgery .
the instat graph statistical software was used for the sample calculations and analysis of the results . a difference of 30% in pain intensity according to a numerical scale was considered clinically significant for 80% power and a 95% confidence interval ( 95% ci ) . based on a preliminary evaluation , a standard deviation ( sd ) of 2.2 was estimated for the pain intensity score within the group of patients ; the calculated sample size was 20 patients per group .
the statistical tests used were as follows : student 's t - test for age , body weight , height , iop , hr , ap , and length of surgery and the mann - whitney test for hr , ap , pain intensity , sedation , iop , and arrhythmia .
measures of central tendency , mean , and dispersion ( standard deviation ) were used .
there was no difference between the groups regarding the demographic data ( table 1 ) .
the physical status was class asa i for six patients in clonidine and eight patients in placebo group , and it was asa ii for eight patients in clonidine and 12 patients in placebo , without a significant difference between the groups ( p = 0.7411 ; mann - whitney test ) .
the surgery lasted 22 9.9 min for clonidine and 24 12 min for placebo ( p = 0.05692 ; student 's t - test ) .
the intensity of pain exhibited a significant difference between the groups at the assessed time points ( table 2 ) .
sedation was greater in g1 at almost all the assessed time points ( p < 0.05 ; mann - whitney test ) ( table 3 ) .
sedation increased in g1 between the onset ( 2.0 0.0 ) and end ( 2.5 0.6 ) of the surgery , which did not occur in g2 ( 2.0 0.0 and 2.0 0.0 , respectively ; kruskal - wallis analysis of variance ( anova test ) ) .
the greatest hr measured was 69.4 8.6 in g1 and 76.0 18.5 in g2 , while the lowest hr was 63.2 11.1 in g1 and 69.3 17.9 in g2 . at some time points ,
the systolic arterial pressure ( sap ) was lower when clonidine was used ( p < 0.05 ; mann - whitney test ) . at some time points ,
the iop of the nonmydriatic eye exhibited a significant difference between the groups 15 minutes after injection of the solution ( clonidine = 12.9 3.1 and placebo = 15.5 3.2 ; p = 0.0135 ; mann - whitney test ) and at the end of surgery ( clonidine = 12.5 3.4 and placebo = 15.3 2.7 ; p = 0.0065 ; mann - whitney test ) .
the incidence of arrhythmia was higher in placebo only at the end of surgery , and there was no difference in the incidence of myocardial ischemia ( table 4 ) .
it also promoted sedation and was associated with reductions in the bp , hr , and iop .
the intensity of pain decreased during the surgical steps that are considered to be uncomfortable .
although supplementary analgesia was not required in either group , the intensity of pain was lower when clonidine was used , thus showing that this drug is appropriate for the intended procedure . in spite of its disadvantages , many patients prefer a topical anesthesia to a block due to the greater discomfort caused by the use of needles .
the analgesic effect is not as good as after blocks but there are fewer complications .
complementary analgesia might be achieved with intracameral 1% lidocaine , which is effective as a supplement to topical anesthesia in cataract surgery via phacoemulsification . in the present study ,
the sedative and anxiolytic effects of clonidine , which are mediated through its binding to alpha-2 receptors , might avoid complications related to sympathetic stimulation . in this study
, no patients required supplementary sedation . in spite of the sedation induced by clonidine ,
the patients who received it were as awake at the end of surgery as the ones who were given a placebo , although the length of the procedure was shorter .
reduced iop during cataract extraction facilitates the surgery performance and reduces the risk of complications . in the present study ,
the iop of the patients given clonidine decreased at the end of the surgery ( approximately 30 min ) , which was similar to another study .
clonidine might reduce the hr and bp mediated by a decreased release of peripheral norepinephrine and a central sympatholytic effect . in the present study ,
the hr was lower when clonidine was used ; however , it remained within the normal range .
there are no reports on significant alterations of the ap or hr when these mydriatics are used at their usual doses .
the bp was lower when clonidine was used , but neither hyper- nor hypotension occurred with the dose used in the present study .
clonidine might also reduce the incidence of myocardial ischemia in patients with coronary heart disease [ 1113 ] . in the present study , the incidence of arrhythmia exhibited a difference only at the end of surgery , which suggests the antiarrhythmic action of clonidine .
arrhythmia before anesthesia might be related to the use of mydriatics and to anxiety . by inhibiting the sympathetic activity
, clonidine might act as an anxiolytic and antiarrhythmogenic agent [ 14 , 15 ] .
the results of the present study suggest that clonidine at a dose of 4 /kg via an intravenous route promotes an analgesic effect and sedation appropriate for cataract extraction by means of phacoemulsification under topical anesthesia without altering the heart rate or the systemic arterial pressure .
the results of the recent study further suggest that the use of clonidine reduces the incidence of arrhythmia .
although no cardiovascular adverse effects were observed , the sample size of this study is too small to make any clinically relevant conclusions . in fact
, the cataract population is usually old , has several comorbidities , and thus is at a very high risk of cardiovascular complications . | objectives . the aim of this study was to assess the effects of clonidine on intraoperative analgesia , sedation , intraocular and blood pressure , arrhythmia , and ischemia .
methods .
forty patients undergoing cataract surgery were allocated into two groups .
they were monitored with holter machine , the pupil was dilated , and 30 minutes later , 20 patients received clonidine ( 4 g / kg ) , while the other 20 patients were given a 0.9% saline intravenously .
twenty minutes later , 2% lidocaine gel was applied . there were assessed intraoperative analgesia , intraocular pressure , blood pressure , heart rate , and the occurrence of arrhythmias and myocardial ischemia .
results .
pain intensity was lower in g1 during the phacoemulsification , irrigation , aspiration , and intraocular lens implantation .
the hr and bp were lower with clonidine .
the iop was lower with clonidine after 15 minutes and at the end of the surgery .
sedation was higher with clonidine .
the incidence of arrhythmia was lower at the end of surgery with clonidine .
the incidence of myocardial ischemia did not differ between the groups .
conclusions .
clonidine ( 4 g / kg ) before a phacoemulsification reduced the intensity of pain during cataract surgery .
it also induced sedation , reduction of bp , hr , and incidence of arrhythmia at the end of the surgery , and did not alter myocardial ischemia .
this trial is registered with clinicaltrials.gov nct01677351 . |
the prevalence of childhood obesity has been increasing at unsettling rates across the globe .
in addition to striking the developed world , this pattern has also been noted in developing countries undergoing rapid epidemiological transitions , including those in east africa . in sudan , a study of children in secondary school in the capital khartoum found that rates of overweight and obesity were 28.5% and 5.6% , respectively .
rates of obesity for younger schoolchildren in east africa remain unclear , though obesity at younger ages may carry greater importance because younger children possess improved potential for early intervention .
hypertension , a notable sequela of obesity , was already common in sub - saharan africa but has been reported to be worsening in prevalence in recent years .
hypertension often goes underdiagnosed in children , in part because its accurate diagnosis requires the use of standardized growth charts specific for age , gender , and height , with hypertension defined as a systolic and/or diastolic blood pressure > 95th percentile and pre - hypertension defined as systolic and/or diastolic blood pressure 9095th percentile .
underdiagnosis of hypertension may be even more common in developing countries , where medical care is limited to symptomatic diseases , and childhood hypertension has been overlooked in lieu of more urgent disease . given the importance of childhood hypertension and pre - hypertension in determining adult cardiovascular disease outcomes , our goal was to determine the prevalence of hypertension and pre - hypertension among urban school children in sudan and to determine whether hypertension was associated with obesity in this population .
these data serve to alert providers in developing and developed countries to the extent of the current obesity epidemic .
sudan is in east africa , bordering egypt on the north , ethiopia on the east , kenya , uganda , and congo on the south , and central african republic , chad , and libya on the west .
sudan has 2.5 million square kilometers and 37 million inhabitants , of whom 57 million live in the capital of khartoum .
traditionally an impoverished country , sudan has experienced an economy that has expanded rapidly with oil exportation over the past 10 years , with khartoum being the epicenter of economic activity .
this was a cross - sectional study using a sample of 304 children aging six through twelve years from two schools randomly selected in khartoum , the capital of sudan .
all students were given a questionnaire to be filled out by their parents , which asked for signed consent for their child to participate in the study , in addition to other demographic and health information .
ethics approval was obtained from the university of medical sciences and technology ( khartoum , sudan ) research ethics board .
questionnaires sent to parents inquired regarding the child 's age , gender , and health conditions , both previous and current .
the questionnaire also asked whether there was a medical diagnosis of hypertension , diabetes , or heart disease among blood - related family members and which family members were affected .
the child was classified as having a positive family history only if disease was present in a first - degree relative .
body weight was measured to the nearest tenth of a kilogram on a calibrated digital scale , with the child 's shoes removed .
height was measured with the child standing with shoes removed , measured in centimeters to the nearest millimeter .
blood pressure was measured twice , once manually and once digitally , using an appropriate cuff size , based on arm circumference , and the mean was taken and used for analysis . body mass index ( bmi ) , defined as body weight in kilograms divided by the square of height in meters ( kg / m ) , was used as the measure of obesity in this study .
bmi is an accepted measure of obesity in clinical practice , and its use in children has been supported internationally by the international obesity task force ( iotf ) , which agreed that it provides a reasonable index of adiposity and in that it is a simple and inexpensive measure .
it provides reliable estimations , with the exceptions of extremes of age , height , and musculature .
bmi was categorized according to the centers for disease control and prevention ( cdc ) age- and sex - specific growth charts .
the following categories were used : underweight < 5th percentile ; normal weight , 5th to 85th percentile ; overweight , 85th to 95th percentile ; obese , > 95th percentile .
blood pressure was categorized according to bp tables from the fourth report on the diagnosis , evaluation , and treatment of high blood pressure in children and adolescents , using age and height percentiles , with normotension defined as a bp under the 90th percentile , prehypertension 90th to 95th percentile , and hypertension greater than 95th percentile or an absolute systolic bp ( sbp ) of 120 mm hg or diastolic bp ( dbp ) of 80 mm hg .
children were also classified as hypertensive if they were taking antihypertensive medication or had been diagnosed with hypertension previously .
descriptive statistics were calculated for gender , family history , and bmi and bp categories .
analyses were performed using the chi - squared test for association , and multinomial logistic regression to investigate the odds of systolic and diastolic prehypertension and hypertension with overweight or obesity , controlling for family history of hypertension .
analyses were conducted using stata 10.0 ( 2008 statacorp lp , college station , tx ) .
complete data on height , weight , bp , and family history were available for the entire sample ( n = 304 ) .
the average age of participants was 7.8 years , with a median age of 9 years ( range 612 years ) .
all were of sudanese nationality , and the gender distribution was 236 female ( 77.6% ) and 68 male ( 22.4% ) .
the number of children who were overweight was 45 ( 14.8% ) , and 32 were obese ( 10.5% ) .
females had a nonsignificant trend toward higher rates than males of overweight ( 14.0% versus 11.8% ) and obesity ( 11.0% versus 5.9% , p > .05 ) .
prehypertension was detected in 15 ( 4.9% ) participants and additional 15 ( 4.9% ) had hypertension , all on the basis of bp measurement , as none were taking antihypertensives or had a previous diagnosis of hypertension .
the rate of elevated bp ( prehypertension and hypertension combined ) in males and females was 13.2% versus 8.9% .
a family history of hypertension was reported for 64 ( 21.1% ) of the participants overall , including 20.1% of children with a normal blood pressure , 20.0% of those with pre - hypertension , and 40.0% of hypertensive children ( table 2 ) . though there is a higher rate of family history with hypertension compared to normal bp or prehypertension , this difference was not significant by chi - square test ( p > .05 ) . regarding the association between bmi and bp , 31.2% of obese children had hypertension , versus 17.8% of overweight children and 5.3% of normal bmi children .
these results were found to be highly statistically significant by pearson chi - squared test ( p < .001 ) .
further classification of prehypertension and hypertension into systolic and diastolic is stratified by bp category in table 3 for descriptive purposes .
the results are given as the relative risk of being in a certain bp category by weight category compared to children with a normal bmi , adjusting for gender and family history .
after adjustment for these factors , sudanese children who were obese had a relative risk of systolic hypertension of 14.7 compared to their normal - weight counterparts ( p < .01 ) .
we found a high rate of overweight and obesity among 612-year - old primary schoolchildren in urban sudan , with 14.8% of the children overweight and 10.5% obese . despite moderate rates of hypertension reported previously among children in sub - saharan africa up to 11% in rural children , suggesting an underlying genetic predisposition we found that obesity and not family history was the factor most strongly associated with hypertension in our sample .
obese children carried a relative risk of 14.7 for systolic hypertension after adjustment for family history , while family history of hypertension was not significantly associated .
that obesity was so highly correlated with hypertension in a part of the world more commonly linked to undernutrition underscores the pervasiveness of the obesity epidemic and its sequelae throughout the world .
rates of hypertension in sub - saharan africa have been noted to be increasing among adults [ 5 , 6 , 13 ] concurrent with rising rates of obesity in urban areas .
prior studies to demonstrate these effects among children in east africa have been lacking , however .
the appearance of early cardiovascular sequelae of obesity at these young ages suggests that urban areas in the developing world may begin to face increasing health concerns in children related to the obesity epidemic . as in other sub - saharan african nations ,
these high rates of obesity in sudan are felt to be due to the epidemiological transition that has come with westernization .
while food availability has improved , dietary habits have shifted away from traditional agricultural choices to an increase in processed foods as seen in western countries .
the appearance of obesity - associated hypertension in children may signal that developing countries are likely to face similar difficulties as developed countries in overcoming lifestyle choices .
similar reports of obesity have been reported from other less - developed nations outside of africa .
a study of schoolchildren in beijing , china , reported that approximately 20% of children were overweight or obese . in karachi ,
pakistan , an almost identical prevalence of 25% of children had a high bmi , with 6% of children overweight and 19% obese . in iran ,
rates of hypertension in other populations are less clear , although increasing bmi has been consistently associated with increased blood pressure throughout childhood [ 1719 ] .
the total number of children involved was relatively small ( 304 subjects ) , particularly with respect to boys ( 68 subjects ) .
in contrast to many studies of blood pressure in african children that did not determine rates of hypertension based on normal ranges for height , age , and gender , we determined blood pressure status for each of our participants , dividing children into normotensive , pre - hypertensive , and hypertensive categories . in common with other studies [ 20 , 21 ]
, we used the average of blood pressure measurements at a single time point for each subject , acknowledging that measuring blood pressure on separate occasions is necessary for the diagnosis of hypertension on a clinical basis .
it is possible that the prevalence of hypertension in our sample would be lower were three measurements used to determine hypertension .
other measurements estimating adiposity ( waist circumference , bioelectrical impedance , and mri ) are more specific to the amount of fat mass .
nevertheless , for population - based studies bmi correlates well with these other measures and remains widely used as a definition of obesity in hypertension research [ 2325 ] .
in conclusion , primary schoolchildren in urban sudan exhibited a high degree of overweight and obesity , and hypertension among these children was more closely linked to obesity than to family history . that obesity - associated cardiovascular sequelae exist in the developing world at such young ages
is a harbinger of worsened cardiovascular outcomes in sub - saharan africa in the future . to overcome these trends , children in urban settings in africa are likely to require similar dietary and activity lifestyle adjustments as needed by their counterparts in developed nations
| cardiovascular disease ( cvd ) frequently has roots in childhood , including following childhood - onset hypertension .
incidence of cvd has increased in developing countries in east africa during recent urbanization .
effects of these shifts on childhood hypertension are unclear .
our objectives were to ( 1 ) determine the prevalence of hypertension among primary schoolchildren in khartoum , sudan ; ( 2 ) determine whether hypertension in this setting is associated with obesity .
we performed a cross sectional study of 6 - 12y children from two schools randomly selected in khartoum , sudan .
height , weight , bmi , bp and family history of hypertension were assessed .
age- , height- and gender - specific bp curves were used to determine pre - hypertension ( 9095% ) and hypertension ( > 95% ) . of 304 children , 45 ( 14.8% )
were overweight ; 32 ( 10.5% ) were obese ; 15 ( 4.9% ) were pre - hypertensive and 15 ( 4.9% ) were hypertensive .
obesity but not family history of hypertension was associated with current hypertension . in multiple logistic regression , adjusting for family history , children who were obese had a relative - risk of 14.7 ( ci 2.45 - 88.2 ) for systolic hypertension compared to normal - weight children . we conclude that overweight and obesity are highly prevalent among primary schoolchildren in urban sudan and are strongly associated with hypertension . that obesity - associated cardiovascular sequelae exist in the developing world at young ages
may be a harbinger of future cvd in sub - saharan africa . |
incisional hernia is a common long - term complication of abdominal surgery and is estimated to occur in 3% to 13% of laparotomy incisions .
however , its incidence is greater than 23% in patients who have developed an infection in the laparotomy wound .
approximately 50% of incisional hernias develop within the first 2 years after the primary operation , and 74% develop after 3 years .
approximately more than 100,000 men , women , and children in the usa undergo surgery for ventral hernia repair each year .
the recurrence rate of incisional hernia , after primary closure is high , ranging between 10% and 50% , and has been reduced to 3% to 18% after the introduction of prosthetic materials ( meshes ) in hernia repair .
nevertheless , open hernia repair can be a major operation with considerable morbidity due to mesh - related infections .
an increasing interest in laparoscopic surgery and the availability of new materials have encouraged the adoption of laparoscopic techniques in ventral hernia repair .
laparoscopic ventral hernia repair ( lvhr ) was introduced into surgical practice by leblanc and booth in 1991 .
it is based on the same physical and surgical principles as the open underlay procedure described by stoppa , rives , and wantz .
surgeons and patients have rapidly accepted laparoscopic repair for incisional and primary ventral hernia over the past decade .
lvhr is now being used with increasing frequency even for the management of patients with complex incisional hernias . with the use of the laparoscopic approach , large incisions and drain placement
can be avoided , which leads to a reduction in postoperative wound - related problems .
recent reports on this topic have supported minimal postoperative morbidity , a shorter convalescence period , and an acceptable recurrence rate . limiting factors in most studies include technical variations , limited sample size , and restricted follow - up . to date , more than 100 studies on lvhr have been published , although most of them are case series lacking control groups . recently , several studies based on large numbers of patients have been accumulated due to increasing experience .
herein , we analyze the existing literature on lvhr , in an effort to examine the optimal surgical technique , complications , and long - term results in comparison with the open ventral hernia repair ( ovhr ) .
early postoperative development of incisional hernia implies an important role of perioperative factors , such as type of incision , suture technique , wound infection , or wound dehiscence .
it is hypothesized that incisional hernias are caused by acute subclinical fascial separations in the early postoperative period .
several studies have compared different suture material , and it has been recommended that an absorbable suture be used for fascial closure , as it decreases the likelihood of infection and eventually dissolves , preventing a saw - like effect on the fascia , which may predispose to fascial disruption . however , more recently , 2 meta - analyses identified more incisional hernias after closure with absorbable sutures than with nonabsorbable sutures . another meta - analysis performed by van't
riet et al found no difference in incisional hernia development between slowly absorbable materials , such as pds , and nonabsorbable sutures , such as nylon .
in addition , nonabsorbable sutures were found to be associated with an increased incidence of prolonged wound pain and suture sinus formation ; therefore , the use of slowly absorbable sutures should be considered in abdominal wound closure .
late development of an incisional hernia implicates other mechanisms , such as advanced age , diabetes , malignancy , and connective tissue disorders .
however , there is evidence that the abdominal wound can weaken over the course of years and give rise to an incisional hernia . wound healing depends on a variety of cellular and molecular factors capable of inducing hemostasis , angiogenesis , inflammation , fibroplasias , and wound remodeling .
one of the major extracellular proteins involved in wound healing is collagen , with 2 main types : type i and type iii collagen .
type i collagen is found in fascia , bone , and skin , and has a high tensile strength .
type iii collagen has a lower tensile strength but increased flexibility and is found in blood vessels , smooth muscle , and organ parenchyma .
early aponeurotic wound healing is based mainly on type iii collagen , which provides the wound with little tensile strength .
therefore in the early phase , closure and healing of the aponeurosis depends largely on technical closure .
however , fascial strength increases later as type i collagen increases and forms tight intermolecular links .
it is believed that abnormalities in collagen synthesis may predispose to weak healing of the aponeurosis and progression to development of an incisional hernia . in an experimental study by dubay et al , , a fibrablast growth factor (
bfgf)-releasing rod was placed into the fascial wound and as a result a significant increase in type i collagen staining around the bfgf treated fascia was noted .
this contributed to a considerable increase in the fascia - breaking strength , and as a consequence a significant reduction in primary and recurrent incisional hernia formation rate was observed .
advances in analyzing the cellular and molecular mechanisms of the pathogenesis of incisional hernia will help in applying therapeutic agents to strengthen wound healing and prevent incisional hernia formation .
traditional primary open repair is based on suture approximation of aponeurosis on each side of the hernia defect . however , recurrence rates after this procedure range from 41% to 52% in the long term .
the introduction of prosthetic meshes in hernia repair has substantially helped in the reduction of recurrence rates .
indeed , luijendijk et al demonstrated a significant reduction in recurrence rates between primary suture repair compared with mesh repair , 43% to 24% , respectively , for first - time incisional hernia repairs .
however , the mesh repairs still require wide dissection of soft tissue , which contributes to an increased incidence of wound - related complications ( more than 12% ) . among the open repairs ,
this technique is easy , convenient , but it has a considerable morbidity rate and recurrence rate ( 8% to 27% ) . another repair option is the inlay technique , such that the mesh is sutured to the aponeurotic edges .
this technique has been used to cover large aponeurotic gaps , and it carries extremely high recurrence rates . in the extra - peritoneal underlay technique , widely known as the rives - stoppa repair , the mesh is placed retromuscularly and anterior to the posterior rectus sheath , followed by primary closure of the anterior fascia .
this technique requires limited soft tissue dissection ; therefore , it carries acceptable morbidity and recurrence rates . because there is no direct contact of mesh with the abdominal viscera ,
moreover , problems resulting from placement of the prosthesis in the subcutaneous space , such as seroma formation and wound infection , are also avoided .
the intraperitoneal underlay technique was first described by mccarthy et al in 1981 . at the beginning , they used polypropylene mesh , which was sutured to the peritoneal edge of the hernia sac .
polypropylene was gradually abandoned for intraperitoneal mesh fixation due to its trend to cause adhesions to bowel loops and was replaced by the polytetraflouoroethylene ( eptfe ) mesh or bilayer polytetrafluoroethylene and polypropylene mesh in the more recent series .
a recent study by millikan et al with this technique using full - thickness transfascial sutures in 102 patients showed a 0% recurrence rate in a median follow - up of 28 months .
nevertheless , in the majority of studies analyzing the intraperitoneal underlay technique , the recurrence rate did not surpass 10% .
although the onlay technique seems to be the most popular due to its simplicity , it carries a higher morbidity rate than the other 3 techniques .
in addition , rudmik et al performed an extensive review of the articles referring to the main techniques used in open ventral hernia repair and calculated an overall 4.5% , 8% , 14% , and 48% recurrence rate for the intraperitoneal underlay repair , the extraperitoneal underlay repair , onlay repair , and inlay repair , respectively .
the relatively high morbidity and recurrence rates of the open repair techniques prompted the development of the laparoscopic approach in an effort to improve the clinical outcome .
since the introduction of the laparoscopic approach in ventral hernia repair by leblanc and booth , the laparoscopic technique has gradually become increasingly popular worldwide , as it offers early recovery , decreased hospital stay , minimal morbidity , and low recurrence rates .
the laparoscopic technique has numerous variations of the methodology used by surgeons , although several common steps are followed by all .
the procedure starts with entering the peritoneal cavity by using a veress needle , an open hasson method , or an optical trocar allowing view of the abdominal wall layers during penetration .
three trocars are used , one 10-mm trocar and two 5-mm , which are placed as laterally as possible on the abdominal wall , so they are at an adequate distance from the hernia orifice ( figure 1 ) .
perforation of the intestine is the most serious injury associated with laparoscopic ventral hernia repair .
schematic presentation of a midline hernia defect , trocar sites , suture fixation of the mesh , and tack application points .
after adhesiolysis , the sac contents are gently reduced into the peritoneal cavity , while the peritoneal sac is left in situ .
closure of large hernia defects is recommended by franklin et al with nonabsorbable sutures , even if only a limited closure is possible .
the periphery of the hernia defect is evaluated by direct vision and palpation and is marked on the abdominal wall skin with a marker .
the carbon dioxide should be released prior to measurement , revealing the true size of the hernia defect .
the craniocaudal and lateral measurements are taken to define the size of the prosthetic mesh .
the surgeon should add 6 cm to these measurements in both directions , which provides a 3-cm overlap of the aponeurotic edges of the hernia by the mesh .
some surgeons suggest a 4-cm to 5-cm overlap , especially if the patient is morbidly obese , or if the hernia is recurrent or of large size .
the size of mesh that most closely approaches this measurement is selected for the repair .
four main types of mesh have been used : polypropylene ( prolene , ethicon , sommerville , nj , usa ) , expanded polytetrafluoroethylene ( dual mesh , gore - tex , gore medical , flagstaff , az , usa ) , composite polypropylene+polytetrafluoroethylene ( composix , cr bard , cranston , nj , usa ) , or composite polypropylene+collagen ( parietene , sofradim , trevoux , france ) . polypropylene prosthesis has been abandoned in the laparoscopic approach , because it may create adhesions with bowel loops .
it has been replaced by proceed ( ethicon ) , which is composed of polypropylene covered with oxidized regenerated cellulose ( orc ) ( table 1 ) .
the most popular prosthetic biomaterials available for incisional hernia repair eptfe = expanded polytetrafluoroethylene , ppm = polypropylene , pol = polyester , ha = hyalouronic acid , pds = polydioxanone , orc = oxidized regenerated cellulose , cmc = carboxymethyl cellulose .
gore - tex ( eptfe ) mesh has been widely used worldwide , but composix has become more popular in the last few years .
all collagen meshes have been treated to eliminate all cells and proteins other than collagen , which may evoke adverse reactions to the host .
the most popular of these meshes is surgisis ( surgisis gold , cook surgical , bloomington , il , usa ) , made of porcine small intestinal mucosa .
these meshes are mainly used to reconstruct the abdominal wall in an infected field , but they are of limited use in laparoscopic ventral hernia repair .
after selection of the appropriate - sized mesh , 4 to 6 sutures are placed on the edges of the prosthetic mesh .
the suture sites are numbered with a marker to allow easier orientation of the prosthetic mesh in the abdominal cavity .
the tailored prosthetic mesh is rolled tightly and inserted in the peritoneal cavity through the 10-mm to 11-mm trocar .
two - mm skin incisions are made in the marked points on the abdominal wall . with the help of a berci fascial closure instrument
( karl storz gmbh & co. kg , tuttlingen , germany ) or the endoclose ( autosuture ) inserted through each skin incision into the peritoneal cavity , the 2 ends of each suture are grasped and drawn outside through the skin incisions by separate passages and at different angles .
the mesh is further secured with 5-mm titanium tacks using protack ( autosuture , norwalk , ct , usa ) , the ems stapler ( ethicon ) , or the reusable salute ( cr bard ) .
lvhr achieves adequate closure of the hernia defect by using intraperitoneal mesh fixation with minimal soft - tissue dissection .
the technique has all the advantages of the laparoscopic approach , such as less postoperative pain , earlier recovery , and a shorter convalescence period than the ovhr .
moreover , the patients feel more comfortable and tolerate oral intake earlier than after the open procedure . for patients undergoing laparoscopic repair of a primary ventral hernia
however , for patients undergoing laparoscopic repair of an incisional hernia , the benefit in terms of body image is limited .
lvhr also has significant technical advantages . with this approach , minimal fascial defects , known as
swiss cheese defects , which may be missed during the open repair , can be identified and closed with one mesh .
in addition , by placing the mesh intraperitoneally , the intraabdominal pressure pushes upwards and holds the mesh into position .
the major debate for this type of repair is which mesh fixation technique should be used , tacks plus transfascial sutures or tacks alone .
a variety of reports are available on this type of mesh fixation , which represents the traditional technique in lvhr .
heniford et al , in a large series of 850 consecutive hernias , used an eptfe mesh that was fixed on the abdominal wall with tacks and tranfascial eptfe stitches in 97% of cases .
tacks alone were used to secure the mesh in several cases at the beginning of the study , but later this practice was discontinued . during a mean follow - up of 20.2 months ,
leblanc et al have also suggested the importance of suture anchorage at 4-cm to 5-cm anchorage intervals around the perimeter of the mesh to minimize the risk for mesh migration . in another study ,
leblanc 's group retrospectively compared 2 patient groups , one early and one late lvhr group , of 100 patients each . in a median follow - up of 36 months ,
the recurrence rate droped from 9% to 4% , which was attributed to the use of transfascial sutures and the increased mesh overlap in the more recent group .
franklin et al published their 11-year experience in 384 patients who underwent lvhr using transfascial sutures . in a median follow - up of 47.1 months ,
perrone et al recently reported their results in a group of 116 patients who had lvhr . in a median follow - up of 22 months ,
the majority of cases were performed with transfascial sutures ; however , 35% of cases were performed with tacks alone .
the 2 groups , ie , the transfascial suture group and the tack alone group , had similar recurrence rates , 9.2% and 8.5% , respectively .
in a recent large review in lvhr by cobb et al , a 3.8% overall recurrence rate was found , when transfascial sutures and tacks were used , compared with a 5.6% recurrence rate , when tacks only were used .
frantzides et al followed up 208 patients for a median period of 24 months and reported a very low 1.4% recurrence rate .
bageacu et al studied a similar group of 159 patients for a median follow - up of 49 months .
the recurrence rate was very high , 15.7% , and this was attributed by the authors to the learning curve effect : their recurrence rate decreased from 20% to 10% between the early ( 1993 to 1995 ) and the late period ( 1996 to 1998 ) of the study .
the largest study was performed by carbajo et al who followed up 270 patients for a median period of 44 months . in this study ,
et al review of the related literature , the calculated recurrence rates after the suture and tack fixation technique and the tack fixation technique were approximately equal , 4.5% and 4.4% .
therefore , it seems that the fixation technique does not really affect the final outcome in lvhr , and that the laparoscopic repair yields very low recurrence rates .
however , there is a general idea that tacks do not have the same holding strength as full - thickness abdominal sutures , and this is further supported by experimental evidence .
up to the present , no randomized prospective trial has compared the 2 fixation techniques , and therefore we can not draw any safe conclusion in this matter .
the laparoscopic approach in ventral hernia repair carries the risk of intestinal or bladder injury intraoperatively .
adhesions to the abdominal scar represent a significant problem during lvhr , with the risk of bowel injury around the neck of the hernia during dissection .
rudmik et al , in their review , calculated an overall risk of enterotomy of 2.1% .
injury of a hollow organ is a very serious event and should be recognized and treated immediately .
this should be managed with endoscopic suturing if it is limited , whereas the procedure should be converted to open in case a more extensive laceration occurs of the small bowel or the colon , or if spillage of bowel contents occurs in the abdominal cavity . in the latter case ,
if the enterotomy remains unnoticed , it may result in an acute abdominal condition and sepsis within a few hours after surgery .
the main disadvantage of the laparoscopic approach is that the hernia sac is usually retained in place , which predisposes to postoperative seroma formation .
the latter is a fluid collection inside the hernia sac , which develops in most cases . actually , in a series , systematic ultrasound examinations detected seromas in 93% of patients postoperatively .
many of these cases are not noticed by the patient , and the majority resolve without intervention .
however , some seromas persist for more than 8 weeks or cause symptoms requiring intervention , which is usually a sterile aspiration .
the incidence of symptomatic seromas according to various reports ranges from 1% to 24% . according to the carolina medical center 's experience
, only 2.6% of a large group of 860 patients with lvhr had seromas that persisted for more than 8 weeks or caused symptoms requiring intervention .
long - term complications , such as infection , do not usually develop from these fluid collections , regardless of whether they are aspirated early or allowed to persist for 8 weeks or longer .
seroma formation is a common problem after lvhr ; however , its incidence is similar to that after ovhr . despite the fact that no specific measures are available to prevent this complication , leblanc et al showed that applying a postoperative compressive bandage or wearing an abdominal binder for a couple of months after surgery may decrease clinically significant seroma formation . in lvhr ,
the mesh comes into minimal contact with the patient 's dermal flora while it is introduced intraperitone - ally .
minimal tissue dissection in the laparoscopic approach further reduces the risk of infection . despite the potential benefit of a reduction in infectious complications from lvhr , still
a percentage of patients , ranging from 0.7% to 5% in different series , may acquire infection related to the hernia repair .
the overall calculated incidence of mesh infection is 0.6% , whereas the incidence of fistula formation is 0.1% .
any associated abscesses should be drained percutaneously under computed tomography guidance , and the patient should receive systemic antimicrobial therapy .
prophylactic measures to reduce the risk of infection include efforts to minimize skin contamination at surgery , elimination of any potential sources of infection before surgery , limit the contact of mesh with the skin , and the use of antibiotic prophylaxis perioperatively . protracted pain ( more than 12 weeks ) at the sites of the transfixing sutures on the abdominal wall is not uncommon ( 1.3% to 3.3% ) .
patients with prolonged pain are initially treated with anti - iflammatory medications , or injections of local anesthetics . in case of persisitent pain , removal of the anchoring suture
however , there may be neuroma formation , which does not resolve after suture removal .
prolonged ileus may develop in a minority of patients ( 3% ) and should be treated expectantly , in - hospital , until patients can tolerate oral intake . in some patients , adhesions may form between the omentum or the intestines and the mesh at the tack sites , and this may predispose to intestinal obstruction .
exacerbation of chronic obstructive pulmonary disease may occur after the laparoscopic procedure , as a result of pneumoperitoneum .
cardiac complications may also occur in patients with preexisting cardiac disease , such as congestive cardiac failure , coronary artery disease , and other such things .
heniford et al found significant associations between recurrence and larger hernias , longer operative times , previous hernia repairs , morbid obesity , and higher complication rates .
sains et al performed an excellent meta - analysis of recent high - quality studies comparing lvhr with ovhr and they noted a trend towards lower hernia recurrence rates following the laparoscopic approach , though not achieving statistical significance . pierce et al have presented a pooled data analysis of 45 published series in lvhr , representing 5340 patients ( 4582 lvhr , 758 ovhr ) .
they demonstrated a significantly lower recurrence rate with lvhr for both the pooled ( 4.3% ) and the paired ( 3.1% ) study cohorts ( studies comparing patients who had undergone lvhr or ovhr within a given institution ) , compared with ovhr series ( 12.1% , p<0.0001 ) .
although recurrence still remains an important problem after lvhr , it does not surpass 5% to 10% in most series .
the laparoscopic technique has numerous variations of the methodology used by surgeons , although several common steps are followed by all .
the procedure starts with entering the peritoneal cavity by using a veress needle , an open hasson method , or an optical trocar allowing view of the abdominal wall layers during penetration .
three trocars are used , one 10-mm trocar and two 5-mm , which are placed as laterally as possible on the abdominal wall , so they are at an adequate distance from the hernia orifice ( figure 1 ) .
perforation of the intestine is the most serious injury associated with laparoscopic ventral hernia repair .
schematic presentation of a midline hernia defect , trocar sites , suture fixation of the mesh , and tack application points .
after adhesiolysis , the sac contents are gently reduced into the peritoneal cavity , while the peritoneal sac is left in situ .
closure of large hernia defects is recommended by franklin et al with nonabsorbable sutures , even if only a limited closure is possible .
the periphery of the hernia defect is evaluated by direct vision and palpation and is marked on the abdominal wall skin with a marker .
the carbon dioxide should be released prior to measurement , revealing the true size of the hernia defect .
the craniocaudal and lateral measurements are taken to define the size of the prosthetic mesh .
the surgeon should add 6 cm to these measurements in both directions , which provides a 3-cm overlap of the aponeurotic edges of the hernia by the mesh .
some surgeons suggest a 4-cm to 5-cm overlap , especially if the patient is morbidly obese , or if the hernia is recurrent or of large size .
the size of mesh that most closely approaches this measurement is selected for the repair .
four main types of mesh have been used : polypropylene ( prolene , ethicon , sommerville , nj , usa ) , expanded polytetrafluoroethylene ( dual mesh , gore - tex , gore medical , flagstaff , az , usa ) , composite polypropylene+polytetrafluoroethylene ( composix , cr bard , cranston , nj , usa ) , or composite polypropylene+collagen ( parietene , sofradim , trevoux , france ) . polypropylene prosthesis has been abandoned in the laparoscopic approach , because it may create adhesions with bowel loops .
it has been replaced by proceed ( ethicon ) , which is composed of polypropylene covered with oxidized regenerated cellulose ( orc ) ( table 1 ) .
the most popular prosthetic biomaterials available for incisional hernia repair eptfe = expanded polytetrafluoroethylene , ppm = polypropylene , pol = polyester , ha = hyalouronic acid , pds = polydioxanone , orc = oxidized regenerated cellulose , cmc = carboxymethyl cellulose . the clinical experience with all these types of mesh varies from country to country .
gore - tex ( eptfe ) mesh has been widely used worldwide , but composix has become more popular in the last few years .
all collagen meshes have been treated to eliminate all cells and proteins other than collagen , which may evoke adverse reactions to the host .
the most popular of these meshes is surgisis ( surgisis gold , cook surgical , bloomington , il , usa ) , made of porcine small intestinal mucosa .
these meshes are mainly used to reconstruct the abdominal wall in an infected field , but they are of limited use in laparoscopic ventral hernia repair .
after selection of the appropriate - sized mesh , 4 to 6 sutures are placed on the edges of the prosthetic mesh .
the suture sites are numbered with a marker to allow easier orientation of the prosthetic mesh in the abdominal cavity .
the tailored prosthetic mesh is rolled tightly and inserted in the peritoneal cavity through the 10-mm to 11-mm trocar .
two - mm skin incisions are made in the marked points on the abdominal wall . with the help of a berci fascial closure instrument
( karl storz gmbh & co. kg , tuttlingen , germany ) or the endoclose ( autosuture ) inserted through each skin incision into the peritoneal cavity , the 2 ends of each suture are grasped and drawn outside through the skin incisions by separate passages and at different angles .
the mesh is further secured with 5-mm titanium tacks using protack ( autosuture , norwalk , ct , usa ) , the ems stapler ( ethicon ) , or the reusable salute ( cr bard ) .
lvhr achieves adequate closure of the hernia defect by using intraperitoneal mesh fixation with minimal soft - tissue dissection .
the technique has all the advantages of the laparoscopic approach , such as less postoperative pain , earlier recovery , and a shorter convalescence period than the ovhr .
moreover , the patients feel more comfortable and tolerate oral intake earlier than after the open procedure . for patients undergoing laparoscopic repair of a primary ventral hernia
however , for patients undergoing laparoscopic repair of an incisional hernia , the benefit in terms of body image is limited .
lvhr also has significant technical advantages . with this approach , minimal fascial defects , known as
defects , which may be missed during the open repair , can be identified and closed with one mesh .
in addition , by placing the mesh intraperitoneally , the intraabdominal pressure pushes upwards and holds the mesh into position .
the major debate for this type of repair is which mesh fixation technique should be used , tacks plus transfascial sutures or tacks alone .
a variety of reports are available on this type of mesh fixation , which represents the traditional technique in lvhr .
heniford et al , in a large series of 850 consecutive hernias , used an eptfe mesh that was fixed on the abdominal wall with tacks and tranfascial eptfe stitches in 97% of cases .
tacks alone were used to secure the mesh in several cases at the beginning of the study , but later this practice was discontinued . during a mean follow - up of 20.2 months ,
leblanc et al have also suggested the importance of suture anchorage at 4-cm to 5-cm anchorage intervals around the perimeter of the mesh to minimize the risk for mesh migration . in another study ,
leblanc 's group retrospectively compared 2 patient groups , one early and one late lvhr group , of 100 patients each . in a median follow - up of 36 months ,
the recurrence rate droped from 9% to 4% , which was attributed to the use of transfascial sutures and the increased mesh overlap in the more recent group .
franklin et al published their 11-year experience in 384 patients who underwent lvhr using transfascial sutures . in a median follow - up of 47.1 months ,
perrone et al recently reported their results in a group of 116 patients who had lvhr . in a median follow - up of 22 months , the recurrence rate was 9.3% .
the majority of cases were performed with transfascial sutures ; however , 35% of cases were performed with tacks alone .
the 2 groups , ie , the transfascial suture group and the tack alone group , had similar recurrence rates , 9.2% and 8.5% , respectively .
in a recent large review in lvhr by cobb et al , a 3.8% overall recurrence rate was found , when transfascial sutures and tacks were used , compared with a 5.6% recurrence rate , when tacks only were used .
frantzides et al followed up 208 patients for a median period of 24 months and reported a very low 1.4% recurrence rate .
bageacu et al studied a similar group of 159 patients for a median follow - up of 49 months .
the recurrence rate was very high , 15.7% , and this was attributed by the authors to the learning curve effect : their recurrence rate decreased from 20% to 10% between the early ( 1993 to 1995 ) and the late period ( 1996 to 1998 ) of the study .
the largest study was performed by carbajo et al who followed up 270 patients for a median period of 44 months . in this study ,
et al review of the related literature , the calculated recurrence rates after the suture and tack fixation technique and the tack fixation technique were approximately equal , 4.5% and 4.4% .
therefore , it seems that the fixation technique does not really affect the final outcome in lvhr , and that the laparoscopic repair yields very low recurrence rates .
however , there is a general idea that tacks do not have the same holding strength as full - thickness abdominal sutures , and this is further supported by experimental evidence . up to the present , no randomized prospective trial has compared the 2 fixation techniques , and therefore we can not draw any safe conclusion in this matter .
the laparoscopic approach in ventral hernia repair carries the risk of intestinal or bladder injury intraoperatively .
adhesions to the abdominal scar represent a significant problem during lvhr , with the risk of bowel injury around the neck of the hernia during dissection .
rudmik et al , in their review , calculated an overall risk of enterotomy of 2.1% .
injury of a hollow organ is a very serious event and should be recognized and treated immediately .
this should be managed with endoscopic suturing if it is limited , whereas the procedure should be converted to open in case a more extensive laceration occurs of the small bowel or the colon , or if spillage of bowel contents occurs in the abdominal cavity . in the latter case , mesh repair of the ventral hernia should be postponed for a second stage .
if the enterotomy remains unnoticed , it may result in an acute abdominal condition and sepsis within a few hours after surgery .
the main disadvantage of the laparoscopic approach is that the hernia sac is usually retained in place , which predisposes to postoperative seroma formation .
the latter is a fluid collection inside the hernia sac , which develops in most cases . actually , in a series , systematic ultrasound examinations detected seromas in 93% of patients postoperatively .
many of these cases are not noticed by the patient , and the majority resolve without intervention .
however , some seromas persist for more than 8 weeks or cause symptoms requiring intervention , which is usually a sterile aspiration .
the incidence of symptomatic seromas according to various reports ranges from 1% to 24% . according to the carolina medical center 's experience ,
only 2.6% of a large group of 860 patients with lvhr had seromas that persisted for more than 8 weeks or caused symptoms requiring intervention .
long - term complications , such as infection , do not usually develop from these fluid collections , regardless of whether they are aspirated early or allowed to persist for 8 weeks or longer .
seroma formation is a common problem after lvhr ; however , its incidence is similar to that after ovhr .
despite the fact that no specific measures are available to prevent this complication , leblanc et al showed that applying a postoperative compressive bandage or wearing an abdominal binder for a couple of months after surgery may decrease clinically significant seroma formation . in lvhr ,
the mesh comes into minimal contact with the patient 's dermal flora while it is introduced intraperitone - ally .
minimal tissue dissection in the laparoscopic approach further reduces the risk of infection . despite the potential benefit of a reduction in infectious complications from lvhr
, still a percentage of patients , ranging from 0.7% to 5% in different series , may acquire infection related to the hernia repair .
the overall calculated incidence of mesh infection is 0.6% , whereas the incidence of fistula formation is 0.1% .
any associated abscesses should be drained percutaneously under computed tomography guidance , and the patient should receive systemic antimicrobial therapy .
prophylactic measures to reduce the risk of infection include efforts to minimize skin contamination at surgery , elimination of any potential sources of infection before surgery , limit the contact of mesh with the skin , and the use of antibiotic prophylaxis perioperatively . protracted pain ( more than 12 weeks ) at the sites of the transfixing sutures on the abdominal wall is not uncommon ( 1.3% to 3.3% ) .
patients with prolonged pain are initially treated with anti - iflammatory medications , or injections of local anesthetics . in case of persisitent pain , removal of the anchoring suture
, however , there may be neuroma formation , which does not resolve after suture removal .
prolonged ileus may develop in a minority of patients ( 3% ) and should be treated expectantly , in - hospital , until patients can tolerate oral intake . in some patients , adhesions may form between the omentum or the intestines and the mesh at the tack sites , and this may predispose to intestinal obstruction .
exacerbation of chronic obstructive pulmonary disease may occur after the laparoscopic procedure , as a result of pneumoperitoneum .
cardiac complications may also occur in patients with preexisting cardiac disease , such as congestive cardiac failure , coronary artery disease , and other such things .
heniford et al found significant associations between recurrence and larger hernias , longer operative times , previous hernia repairs , morbid obesity , and higher complication rates .
sains et al performed an excellent meta - analysis of recent high - quality studies comparing lvhr with ovhr and they noted a trend towards lower hernia recurrence rates following the laparoscopic approach , though not achieving statistical significance .
pierce et al have presented a pooled data analysis of 45 published series in lvhr , representing 5340 patients ( 4582 lvhr , 758 ovhr ) . they demonstrated a significantly lower recurrence rate with lvhr for both the pooled ( 4.3% ) and the paired ( 3.1% ) study cohorts ( studies comparing patients who had undergone lvhr or ovhr within a given institution ) , compared with ovhr series ( 12.1% , p<0.0001 ) .
although recurrence still remains an important problem after lvhr , it does not surpass 5% to 10% in most series .
our review indicates that lvhr is a safe and effective approach to abdominal wall hernias .
the technique offers the advantages of the laparoscopic approach , ie , a short hospital stay , less postoperative pain , and early convalescence .
the procedure carries an acceptable risk of complications , a low risk of recurrence , and an excellent cosmetic result .
however , larger , long - term multicenter studies comparing the laparoscopic with the open technique are needed to establish its efficacy . for the time being , it is considered a good alternative to its open counterpart , at least in experienced hands . | background : the purpose of this study was to analyse the surgical techniques , perioperative complications , and recurrence rate of laparocopic ventral hernia repair ( lvhr ) , in comparison with the open ventral hernia repair ( ovhr ) , based on the international literature.methods:a medline search of the english literature was performed using the term laparoscopic ventral hernia repair .
further articles were found by cross - referencing the references of each main article.results:current literature on the topic suggests that lvhr is a safe alternative to the open method with the main advantages being minimal postoperative pain , a shorter convalescence period , and better cosmetic results .
main complications after the laparoscopic approach , such as incidental enterotomy , protracted pain , postoperative seroma , or mesh infection occur at an acceptable rate .
furthermore , most articles favor lvhr versus ovhr in terms of recurrence rate.conclusions:although further randomized studies are needed to draw safe conclusions in terms of complications and recurrence , lvhr is fast becoming the standard approach in the repair of abdominal wall hernias . |
5-fluoruracil ( 5-fu ) , pyrimidine antimetabolite , is a chemotherapeutic agent widely used for various tumors .
neurotoxicites of 5-fu with manifestations of somnolence , upper motor neuronal signs , cerebellar ataxia and a cluster of symptoms and signs of organic brain syndrome have rarely been reported .
we report a case of serious neurotoxicity following 5-fu infusion for malignant gastrointestinal stromal tumor ( gist ) and also briefly review 5-fu related neurotoxicity .
a 44-year - old male patient received his first cycle of palliative 5-day chemotherapy containing 5-fu ( 800 mg / m ) , adriamycin ( 40 mg / m ) and mitomycin- c ( 8 mg / m ) for a recurrent , massive , malignant gastrointestinal stromal tumor ( gist ) which developed at 3 years after surgery .
his tumor was about 20 cm in diameter and invaded the urinary bladder and sigmoid colon ( figure 1 ) .
about 1 week after the completion of his first cycle of chemotherapy , he displayed slurred speech and inappropriate behavior for 35 days at home . such abnormal symptoms improved spontaneously .
on the beginning of the second cycle of chemotherapy , he appeared well and no remarkable abnormality was found on physical examination and laboratory test .
he received the second cycle of chemotherapy with the same regimen as the first and was discharged without any complaint . on the 7 day after the completion of his second chemotherapy ,
his mental status deteriorated with slurred speech , inappropriate language , hyperventilation , insomnia , disorientation and incontinence of stool and urine .
the er , his vital signs were as follows : blood pressure 120/70 mmhg , pulse rate 92/minute , respiration rate 24 /minute and body temperature 36.8c . on physical examination
neurological examination revealed confused mental status , bizarre behavior , lethargy , slurred speech and somnolence .
light reflex was prompt with symmetric , isocoric pupils . orientation to time , person and place
however , he showed neither focal neurological sign nor pathological reflex . on the laboratory findings , white blood cell count was 3,020/ul ,
bun and creatinine were 24.0 mg / dl and 1.4 mg / dl , respectively , serum natrium 135.0 meq / l , serum pottasium 4.3 meq / l , ionized calcium 4.25 mg / dl and serum albumin 2.9 g / dl . on arterial blood gas analysis ( abga ) , respiratory alkalosis pattern was found with ph 7.546 , pco2 19.3 mmhg , po2 133.3 mmhg , bicarbonate 16.8 meq / l and o2 saturation 99.4 % at room air .
his serum ammonia level was at 326 ug / dl , higher than the upper limit of normal range ( 0150 ug / dl ) .
chest x - ray was normal , abdominal simple x - ray showed mild paralytic ileus pattern .
brain computed tomography at the er was normal without evidence of metastasis or cerebral vascular accident .
after a few sessions of lactulose enema , his mental status recovered somewhat with intact orientation to persons . during the admission period for 15 days , his mental status and general condition gradually recovered .
he complained of intermittent abdominal pain and gastric distension due to partial obstruction of the proximal small bowel by the mass .
cerebellar function tests , such as finger to nose , rapid alternative test , heel to shin test and romberg test , were all negative .
brain magnetic resonance imaging revealed no abnormal lesion in brain parenchyma . his mental status and general condition recovered ,
so that we changed the chemotheapeutic regimen to adriamycin ( 60 mg / m ) and cisplatin ( 60 mg / m ) for 1 day , omitting 5-fu .
on the 5 day of postchemotherapy , he was in stuporous mentality and lethargic condition . at er ,
he was found to have neutropenic fever and sepsis syndrome with pancytopenia , respiratory alkalosis and hypokalemia .
cerebrospinal fluid ( csf ) study was normal in pressure , cell count and chemistry . on blood culture ,
after treatment with intravenous fluids and broad - spectrum antibiotics , his mental status and general condition rapidly improved for 23 days .
on the 17 day after the 3 chemotherapy , he was discharged since he completely recovered from sepsis syndrome .
after discharge , he intermittently experienced altered mentality which developed after constipation and abdominal distention .
his neurological symptoms were relieved after repeated lactulose enema at a local hospital . on the last visit after 2 months of 5-fu administration , he still displayed slightly slurred speech and slowly progressive cachexia .
brain magnetic resonance imaging revealed newly developed hyperintense lesions in the bilateral cerebellar hemisphere , thalami and midbrain on t2wi without mass effect .
on t1wi , hypertensive lesion was developed in bilateral basal ganglia ( figure 3 ) .
there are many reports on serious neurotoxicity of 5-fu in patients with dihydropyrimidine dehydrogenase ( dpd ) deficiency .
dpd is the rate - limiting enzyme of 5-fu catabolism and is encoded by the human dihydropyrimidine dehydrogenase gene ( dpyd ) .
so far , about 20 different mutations of dpyd have been reported in dpd deficient subjects .
g to a point mutation within the 5-splicing site ( gt to at ) , also known as dpyd2a ( intron 14 g1a ) , is one of the common mechanisms for dpd deficiency171 .
therefore we examined the presence of dpyd2a ( intron 14 g1a ) by polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) .
when a pcr - rflp based genotyping was performed , fragments of 278 and 131 bp were produced by the restriction endonuclease mae ii in the wild type allele .
however , the mutant allele had no restriction site and remained undigested ( 409 bp ) .
therefore , as shown in figure 4 , this patient had a wild - type pattern .
although both acute and delayed forms have been reported , fluorouracil - induced neurotoxicity is rare .
the acute form consists of cerebellar syndrome and encephalopathy , whereas the delayed variety takes the form of subacute multifocal ieukoencephalopathy .
about 5% ( 0.67% ) of the patients who received 5-fu have been reported to experience neurotoxic symptoms .
the clinical manifestations of acute cerebellar syndrome , such as ataxia , slurred speech and nystagmus , were first described by riehl and brown in 1964 .
encephalopathy or organic brain syndrome following 5-fu treatment is a less commonly observed neurotoxicity with confusion , disorientation and other cognitive disorders .
subacute multifocal leukoencephalopathy with typical brain magnetic resonance imaging findings of multifocal , enhancing white matter lesions and biopsy findings of demyelination has been reported as a 5-fu sequela .
we reported a case of delayed form of 5-fu neurotoxicity characterized by subacute multifocal leukoencephalopathy .
our case initially showed moderate diffuse cerebral dysfunction on electroencephalography ( figure 2 ) without abnormality in brain magnetic resonance imaging . during the 2 months after the last administration of 5-fu , the neurological symptoms waxed and waned depending on the partial obstruction of the bowel and did not fully recover .
however , we could not find any relevant papers on the neurotoxicities of adriamycin and mitomycin .
5-fu is a pyrimidine antimetabolite which has to be metabolically activated within the cells and incorporated into both dna and rna , which results in dna strand breakage and abnormal processing and function of rna .
5-fu itself seems to be relatively nontoxic to the nervous system in laboratory animals when administered intrathecally .
koenig et al . proposed that fluorocitrate , the major catabolite of 5-fu , inhibited krebs cycle and a blockade of krebs cycle impaired the activity of the urea cycle .
reported that , in animal experiments , direct toxic effect on myelin with splitting of the intraperiodline and vacuole formation was not due to 5-fu itself , but to monofluoroacetic acid and -fluoro--alanine ( the major catabolites of 5-fu ) .
the other explanations for 5-fu neurotoxicity , such as induction of thiamine deficiency and inherited deficiency of the enzyme dihydropyrimidine dehydrogenase ( dpd ) , have also been presented .
the frequency of low and deficient dpd activity in the general population is estimated to be 35% and 0.1% , respectively .
recent efforts to explain the relationship between genotype and phenotype in patients with dpd deficiency have brought much progress .
dpd protein is encoded by dpyd , which is a large gene ( > 950 kb ) containing 23 exons with approximately 3 kb of coding region . to date , about 20 variant dpyd alleles , containing single amino acid substitutions , nucleotide deletions or a donor splice site mutation resulting in exon skipping , have been described . among them , g to a point mutation within the 5-splicing site ( intron 14 g1a ) is the most common ( 2.2% out of 90 alleles in finnish and 2.7% out of 72 alleles in taiwanese general population ) and analytical methods for this mutation are well established .
although this mutant allele was not found in our case , a possibility of decreased dpd activity by other dpyp mutations could not be excluded . as a chemotherapeutic agent ,
5-fu is active against a wide variety of solid tumor , including cancers of gastrointestinal tract , ovary and breast .
, there are many possible differential diagnoses , such as brain metastasis , infection of brain or meninges , sepsis syndrome , metabolic abnormality . based on our present case
, we emphasize that 5-fu - induced neurotoxicity should also be an important category of differential diagnosis in these patients .
the treatment of 5-fu neurotoxicity is recommended as supportive care . in the literature , several treatments , such as thiamine infusion , glucocorticosteroid , uridine infusion , charcoal hemoperfusion or hemodialysis , were recommended .
however , their efficiency has not been proven . to understand the mechanism of neurological manifestation of 5-fu , | 5-fluorouracil ( 5-fu ) is a commonly used chemotherapeutic agent . however , its neurotoxicity is rare and not well recognized .
we report a case of 5-fu neurotoxicity with organic brain syndrome and progression to multifocal leukoencephalopathy in a 44-year - old male patient having malignant gastrointestinal stromal tumor .
5-fu - induced neurotoxicity should , therefore , be considered as an important differential diagnosis in cancer patients with neurological abnormality and history of chemotherapy . |
celiac disease is an under diagnosed condition in which gluten ingestion in genetically susceptible individuals results in small - bowel mucosal inflammation and villous atrophy .
most patients are asymptomatic or suffer from mild symptoms only , ( 1 ) and many present with extraintestinal manifestations such as neurologic disorders ( 2 ) .
the anti - tissue transglutaminase antibody ( ttga ) test is a sensitive and specific tool in disclosing celiac disease with overt villous atrophy ( 3 ) with a specificity of approximately 95% , false positive tests are uncommon , and ttga may appear in serum at an early stage in the disease , in other words before the clinical manifestations and even before the development of villous atrophy ( 4 ) .
psychiatric disorders are also common in untreated celiac disease , especially depressive symptoms ( 58 ) .
hallert & derefeldt reported that nine out of 42 studied subjects had attended a psychiatric clinic because of neurotic problems and most of them are involved with depressive disorders ( 7 ) .
celiac disease was considered relatively uncommon in iran , until recently an estimated population prevalence of 1:166 was reported ( 9 ) .
greater awareness of its varying presentation and the availability of new serologic tests have shown celiac disease to be relatively common ( 10 ) .
these observations prompted us now to assess the association between celiac disease and severe chronic depression and schizophrenia .
this cross - sectional study was carried out in 2006 - 2007 . by random sampling
, 200 inpatient men comprising 100 with chronic depression and 100 with schizophrenia ( mean age 37 years , range 18 - 68 years ) were enrolled in razi hospital , tabriz , iran .
chronic depression was defined according to dsm - iv criteria , ( 11 ) diagnosed and treated by semi - structural clinical interview by two psychiatric experts .
the duration of the diseases was more than two years , and the diseases were unbearable without antipsychotic drugs .
patients with schizophrenia suffered from different types of the disease such as paranoid , phrenetic or undistinguished .
two hundred healthy males were selected as controls , matched for age and birthplace ( mean age 32 , range 4 - 77 years ) .
a written informed consent was obtained from patients ( or from next of kin if necessary ) and the study was approved by the institutional ethics committees of the research center for gastroenterology and liver disease , tabriz medical university .
iga class ttga antibody was measured by enzyme - linked immunosorbent assay using a commercially available kit ( eu - ttg iga , eurospital , trieste , italy ) .
a titer of > 7 u / ml was considered positive as recommended by the manufacturer .
a frequency of 0.6% celiac disease has been reported in iran ( 9 ) . assuming this frequency in 200 controls and a tenfold frequency in psychiatric patients ( 6% , as has been reported in many autoimmune conditions ) the statistical power of 0.80 at a significance level of 0.05
percentages were compared by rates and proportion ; 95% confidence intervals ( ci ) were reported .
a frequency of 0.6% celiac disease has been reported in iran ( 9 ) . assuming this frequency in 200 controls and a tenfold frequency in psychiatric patients ( 6% , as has been reported in many autoimmune conditions ) the statistical power of 0.80 at a significance level of 0.05
percentages were compared by rates and proportion ; 95% confidence intervals ( ci ) were reported .
none of the 200 patients had a history of chronic diarrhea and all were taking antipsychiatric drugs ( antipsychotic such as risperidone , haloperidol or perphenazine and anticholinegic for schizophrenic patients , and fluoxetine and tricyclic antidepressants for depressive patients ) .
three patients with chronic psychiatric disorders were ttga positive , in which one ( age 52 years ) with schizophrenia and two ( both 30 years of age ) with chronic depression . of these three patients ,
two refused duodenal biopsy and one died during the study period . in the control group ,
one ( age 25 years ) out of 200 individuals was positive , but duodenal histology proved normal .
the prevalence of positive celiac disease serology in patients with chronic psychiatric disorders was thus slightly but not significantly higher than in controls ; 1.5% , ( 95% ci : 0.38 - 4.03 ) and 0.5% ( 95% ci : 0.00025 - 2.44 ) , respectively ( p = 0.06 ) . clinical features and laboratory findings among the patients with schizophrenia and depression are shown in table 1 .
five in the study group and none in the control group had selective iga deficiency .
clinical and laboratory features of male patients with schizophrenia and depression
ttga iga - class anti - tissue transglutaminase antibody ; meanstandard deviation
in our reports , the frequency of positive celiac disease serology in iranian inpatients suffering from depression or schizophrenia was 1.5% . by comparison , pynnnen et al .
have shown the prevalence of celiac disease in patients with depression to be 0.7% ( 5 ) .
the same authors have reported that in adolescent celiac disease patients the frequency of depression and disruptive behavioral disorders was higher than in controls , 31% and 7% , respectively ( 6 ) .
the present observations support earlier findings that celiac disease is not increased in patients with schizophrenia .
( 12 ) studied 7754 schizophrenia patients in denmark and found a frequency of untreated celiac disease of 0.05% . in uk , west et al . showed that in subjects with celiac disease the prevalence of schizophrenia was 0.25% , the adjusted odds ratios showing no association between the two conditions ( celiac disease vs. controls 0.76 , 95% ci : 0.41 - 1.4 ) ( 13 ) .
by contrast , a study in the uk revealed that patients with celiac disease developed schizophrenia 3 times more frequently than non - celiac controls ( 14 ) .
some studies have suggested that schizophrenia and celiac disease may be associated with similar or adjacent genes ( 15 , 16 ) .
it has indeed been reported that genetic susceptibility in schizophrenia lies in human leukocyte antigen ( hla ) dq , similarly to autoimmune disorders such as celiac disease ( 17 ) .
by contrast , a recent study showed no such hla association in schizophrenia ( 18 ) . in a case report
, the symptoms of schizophrenia were improved in a celiac patient aged 33 years after the introduction of gluten free diet ( 19 ) .
here we had no opportunity to investigate the effect of gluten - free diet , since two patients refused and one died during the study . in this present study ,
the frequency of positive celiac disease serology in patients with chronic depressive ( 2% ) and schizophrenia ( 1% ) was in fact similar to that found in healthy blood donors in iran ( 0.6% ) ( 9 ) . in the latter study ,
the frequency of celiac disease in males ( 1.8% ) was higher than in females ( 0.5% ) , although usually 60%-70% of celiac disease patients are female . we could not investigate females with psychiatric disorders , which may be considered as a limitation to the current study . on the other hand ,
all our patients were inpatients , indicating that they suffered from severe manifestations of chronic psychiatric disorders .
patients with selective iga deficiency remain negative by ttga iga class screening , and we had no opportunity to test our 5 such subjects by igg class ttga . in blood donors
there may thus be additional celiac case in our study group , but we consider that this would not change our conclusions . to conclude , mass screening for celiac disease in patients with depression or schizophrenia is not advocated . despite this , alertness to celiac disease should be high , since early diagnosis and treatment by gluten - free diet may ameliorate the symptoms and quality of life of these patients . | aimthe aim of this study was to determine the prevalence of celiac disease in iranian patients suffering from chronic depression or schizophrenia.backgroundpsychiatric disorders are common in untreated celiac disease.patients and methodstwo hundred iranian inpatient men with in chronic phase of depressive disorders or schizophrenia , and 200 age - matched healthy male subjects were screened for celiac disease by anti - tissue transglutaminase iga antibodies .
the mean age of the study patients was 37 years.resultsone ( 1% ) schizophrenic and two ( 2% ) depressive patients were positive for anti - tissue transglutaminase iga antibodies ; duodenal biopsy was not possible in these subjects . in the control group one ( 0.5% )
individual was positive for anti - tissue transglutaminase iga antibodies , but had normal duodenal histology .
the difference between patients and controls was not statistically significant.conclusionthe frequency of celiac disease serology in schizophrenic and depressive inpatients was not significantly higher than that in the general population
. we would therefore not advocate systematic serologic screening in these patients , but alertness to celiac disease should be kept in mind . |
we carried out a retrospective study by testing blood and serum samples on 1,740 patients who consulted the emergency department of cayenne hospital seeking treatment for fever compatible with malaria and/or dengue during a 1-year period , july 2004june 2005 ( figure ) .
diagnosis of malaria has always been quick ; dengue diagnosis was initially conducted only in malaria - negative patients . in our study ,
dengue investigations were conducted retrospectively at the pasteur institute of french guiana for 99% of patients ( 1,723/1,740 ) by using serum samples obtained at admission and frozen at 80c .
comparison of confirmed cases of dengue fever and of symptomatic malaria in patients examined at the emergency department of cayenne hospital , cayenne , french guiana , january 2004march 2006 .
the black frame corresponds to the period of the retrospective study ( july 2004june 2005 ) .
* cases confirmed by positive test results from reverse transcription pcr or virus isolation ( pasteur institute , cayenne ) .
cases diagnosed based on recorded fever or history of fever in the previous 24 h associated with microscopic detection of asexual forms of plasmodium spp . by blood smear .
malaria diagnosis was based on the identification of hematozoa on a thin blood film and/or on a thick blood film stained with giemsa .
the asexual parasite load ( pl ) was quantified in percent parasitized erythrocytes for values > 0.1 % . for lower values
, classes were created using thick smears : class 1 , < 0.00125% but positive ; class 2 , > 0.00125% but < 0.0125% ; and class 3 , > 0.0125% but < 0.125% .
carriage was considered for classes 1 and 2 ( in the absence of prior antimalarial treatment and for case - patients residing > 1 year in an area of malaria transmission ) . virus isolation or reverse transcription
( 8) was performed on all serum samples obtained during the acute phase of infection , between day 0 and day 4 ( n = 264 ) . for malaria - positive samples ,
virus isolation was conducted on all samples without a date of onset of disease ( n = 163 ) .
serologic immunoglobulin ( ig ) m testing was performed on all serum samples ( n = 1,723 ) .
dengue was detected in 238 case - patients ( 13.8% ) ; among these , 73% ( 174/238 ) were confirmed by positive virologic diagnosis ( isolation or rt - pcr ) , whereas 27% were probable dengue cases ( positive igm serology only ) .
the first group was named early dengue cases ( edc ) and the second group late dengue cases ( ldc ) . of the 1,723 patients , 393 ( 22.8% ) had smear - positive malaria ; of those , 251 ( 63.9% ) were p. vivax , 133 ( 33.8% ) were p. falciparum , 2 were p. malariae , and 7 were mixed p. vivax and p. falciparum .
most malaria - positive case - patients had a parasite count above class 2 ( 371/393 [ 94.4 % ] ) , indicating acute malaria .
concurrent dengue ( edc and ldc ) and malaria were confirmed in 17 of the 1,723 patients ( 1% ) , corresponding to 7.1% ( 17/238 ) of dengue cases and 4.1% ( 16/393 ) of malaria cases , respectively ( table ) .
when considering acute malaria associated with edc , the percentages of confirmed associations were 3.4% for dengue ( 6/174 , 95% confidence interval [ ci ] 0.76.2 ) and 1.6% for malaria ( 6/371 , 95% ci 0.32.9 ) .
all 17 associations were considered clinically as malaria , including the 2 case - patients with low parasite counts .
antimalarial drugs were administered promptly in every case . dengue serology and virology reports were available after the initial episode ; however , these results did not influence patient management . among the 6 acute concurrent infections ,
three patients were hospitalized , all in the igm - seropositive group , i.e. , ldc ; only 1 was severely ill .
this patient , who had p. vivax malaria infection , was hospitalized for interstitial pneumonia with severe anemia .
intubation , blood transfusion , and antimicrobial drugs were required , but he was discharged from the intensive care unit after 11 days .
the second patient was hospitalized for diabetes , the third because treatment with riamet ( arthemether + lumefantrine ) was only available to inpatients . * igm , immunoglobulin m ; rt - pcr , reverse transcription
pcr ; iso , isolate ; pv , plasmodium vivax ; , negative ; + positive ; den , dengue ; nd , not done ; pf , p. falciparum ; tp , thrombocytopenia++ ( < 100,000 platelets/l ) ; hb , hemoglobin ( reference range < 10
g / l ) ; bp , arterial blood pressure ( systolic / diastolic ) ; sat , blood oxygen saturation ; hr , heart rate ; bpm , beats per minute .
for diabetes requiring insulin . for treatment with riamet ( arthemether + lumefantrine ) .
malaria and dengue must be suspected in febrile patients living in or returning from areas endemic for these infections .
although the usual places of contamination by malaria and dengue viruses are quite different in french guiana , considering that the incubation phase is longer for malaria than for dengue and that the population s mobility is high , a simultaneous clinical expression of the 2 diseases is plausible . moreover , in french guiana , dengue viruses have spread to malaria - endemic rural areas ( 9 ) .
the confirmation of malaria is rapid , and after malaria is confirmed , dengue is usually ruled out without screening for it .
two methods can confirm dengue : dengue - specific igm seroconversion or detection of dengue virus particles during the acute phase ( day 0 to day 4 after onset of fever ) by rt - pcr , which is faster and more specific . in published case reports ( 17 ) , the diagnosis of dengue infection is usually made based on positive dengue igm ; however , this can not confirm recent dengue , because igm can persist for months and cross - react with other arboviruses ( 10 ) .
if rt - pcr requires a specific laboratory and can not be performed on site , a new test , the platelia , is now easily included in any laboratory and is indicated particularly for early - acute phase samples ( 11 ) . to investigate the frequency of dengue and malaria co - infection ,
the platelia test should be used in all cases of dengue - like or malaria - like syndrome , even when malaria diagnosis was positive , in regions where both infections may overlap . of the 1,723 patients investigated in this study , 17 had concurrent dengue and malaria . in 10 of these patients , recent acute dengue fever could not be confirmed ( ldc ) .
two patients , 1 of whom was part of the edc group , could have been asymptomatic carriers of plasmodium spp .
( 1 patient with p. falciparum and 1 with p. vivax ) because of low parasitemia ( 12 ) . a true acute concurrent infection ( strictly defined diagnosis )
concurrent acute malaria and recent dengue fever had a lower frequency than predicted by the multiplication of both prevalences , but such reasoning implies the same overlapping contamination areas for malaria and dengue , which it is not the situation in french guiana
. the greater prevalence of ldc than edc associated with acute malaria infection illustrates the prolonged persistence of specific igm or igm cross - reaction , which increases the probability of a malaria case when comparing the short 45 day period corresponding to edc .
virologic investigations using isolation or rt - pcr techniques were not performed on samples taken after the 4th day because of the usual disappearance of viremia .
additional associations where fever was initially caused by malaria and followed by dengue after the 4th day of malaria fever could have been undiagnosed .
edc were diagnosed on average after 4 days of fever , never 5 .
thus , delayed complications of dengue or malaria may not be detected using this definition .
one of these patients had pneumonia , which has recently been described as a complication of p. vivax ( 13 ) .
although acute concurrent infections were benign in our study , special attention should be given to the possibility of co - infection with malaria and dengue , especially when p. falciparum is implicated .
the distinction between severe dengue and severe malaria must be made in an emergency department or hospital setting because in both situations , early diagnosis is essential for patient care . | dengue malaria co - infection reports are scarce . of 1,723 consecutive febrile patients in cayenne hospital ,
238 had dengue ( 174 early dengue fever cases ) and 393 had malaria ( 371 acute malaria ) ; 17 had both .
diagnosis of 1 of these 2 infections should not rule out testing for the other infection . |
to date , kaposi sarcoma has not been mentioned among the adverse effects of triptolide / tripdiolide , ethyl acetate extracts or polyglycosides of the chinese herbal remedy tripterygium wilfordii hook f.
she underwent treatment with corticosteroids , methotrexate and gold sodium thiosulfate , and was chronically taking ketoprofen . at the age of 59 years
she started to take a powder ( 2 g / day ) from a chinese physician for treatment of rheumatoid arthritis .
this powder was supplied to her regularly for 10 years . at the age of 69 years
, multiple soft , violaceous to dark - red patches , plaques , nodules and blisters of varying sizes appeared on a background of severely edematous skin on her legs , and later on her arms .
triptolide ( 235 g/1 g ) and tripdiolide were found in the chinese powder by the use of liquid chromatography electrospray ionization mass spectrometry .
this case indicates a possible association between triptolide / tripdiolide chronic intake and development of human herpesvirus 8-associated kaposi sarcoma .
triptolide / tripdiolide could contribute to development of kaposi sarcoma by reactivation of latent human herpesvirus 8 , permitted by immunosuppression induced by triptolide .
our aim is to show a case of disseminated cutaneous kaposi sarcoma ( ks ) , occurring during a long - term usage of a powder containing triptolide / tripdiolide , for treatment of rheumatoid arthritis ( ra ) .
triptolide and tripdiolide , compounds originally purified from tripterygium wilfordii hook f and used for centuries in traditional chinese medicine to treat ra [ 24 ] , could contribute to development of ks by reactivation of latent human herpes virus 8 ( hhv8 ) , permitted by triptolide - induced immunosuppression . to date , ks has not been mentioned among adverse effects of triptolide , ethyl acetate extracts or polyglycosides of the chinese herbal remedy tripterygium wilfordii hook f[4,69 ] , although dermal reactions including different kinds of rush with the tendency to develop erosion and scarring were described with incidence of 55.5% during 12 weeks of treatment with polyglycosides of tripterygium wilfordii hook f.
a patient was diagnosed to have ra when she was 29 years old . since 55 year of age
the patient was regularly controlled by a nephrologist due to infections of the urinary tract resulting in chronic tubulo - interstitial nephritis , and due to possible ra influence on kidneys .
echocardiography was normal , proteinuria was not present , and glomerular filtration rate was over 60 ml / min/1.73 m body surface area .
a cyst ( diameter 20 mm ) was shown in the left kidney , not confirmed in further evaluations .
high sensitivity c - reactive protein ( hscrp ) was 4.2 mg / l ( normal value < 5 mg / l ) .
medication with chloroquine diphosphate was added to ketoprofen ( table 1 ) , but did not influence ankle edema and joint pain .
serum perinuclear antineutrophil cytoplasmic antibodies were negative . since 69 years of age , leg edema was worse and she stopped working in her profession ( a pharmacist ) .
doppler ultrasonography of leg vessels again did not reveal abnormalities , proteinuria was not present , and renal function was normal .
six months later , multiple soft , violaceous to dark - red patches , plaques , nodules and blisters of varying sizes started to appear on a background of edematous skin on her legs .
ambulatory laboratory data of the blood revealed hemoglobin level of 5.87 mmol / l ( normal range 7.449.92 g / l for women ) , total protein of 55.2
g / l ( normal range 3550 g / l ) and creatinine of 144 mol / l .
proteinuria was not present . although there was no infection of the urinary tract and renal function
was deteriorated no more then before appearance of skin changes , she was admitted to the nephrologic ward to determine her health problem . at the admission ,
severe skin lesions on the edematous background were present on the patient s legs ( figure 1 ) and incipient skin changes on her arms .
rheumatoid factor ( rf ) was 2,310 iu / ml ( normal range < 14 iu / ml ) , but decreased to 566 iu after introduction of treatment with corticosteroids . functional class iii / iv of ra was classified according to steinbrocker et al . .
g / l ) and lymphocytes constituted 22% of all leukocytes ( normal range 2044% ) .
lymphocyte subset percentage was abnormal : cd3 84.0% ( normal range 6776% ) , cd3+dr+ 43.0% ( normal range 815% ) , cd4 32% ( normal range 3846% ) , cd8 52% ( normal range 3140% ) , cd19 2% ( normal range 1116% ) , nk 6% ( normal range 1019% ) .
the histological examination of 2 biopsy specimens of the leg lesions showed a mixed proliferation of irregular slit - like vascular channels associated with pleomorphic spindle cells , a low - grade lymphocytic inflammatory infiltrate and extravasated erythrocytes consistent with a diagnosis of ks ( figure 2 ) .
immunophenotypic analysis showed cell markers f viii+ , ck ae1/ae3 , cd34 + and cd31 + ( figure 3 ) , demonstrating the endothelial nature of the proliferating tumor cells .
expression of the proliferative antigen ki-67 was shown in 20% ( specimen 1 ) and in 30% ( specimen 2 ) of cell nuclei ; p53 immunoreactivity was found in less than 5% of cell nuclei .
hh8 latent nuclear antigen protein was detected in biopsy specimens by immunohistochemistry ( figure 4 ) .
knowing the diagnosis of hh8 ks , a patient s spouse confessed that at the age of 59 years she started to take a chinese powder of unknown composition and continued it until the current hospitalization .
she evaluated this powder as a good pain reliever and was convinced that it would help her to avoid severe ra signs and symptoms .
according to the patient s spouse , she used to take 1 dose of powder a day , and sporadically took 2 doses per day . for brief intervals she would stop taking the powder .
he calculated that his wife had taken approximately 300 doses per year . during hospitalization in the nephrologic ward , administration of the powder
the patient was transferred to the oncological ward and treatments with paclitaxel ( sindaxel , sindan , romania ) were initiated according to earlier promising reports .
sixteen weeks after withdrawal of the chinese powder and after 12 weeks of paclitaxel administration ( 6 doses of paclitaxel were given ) the patient s condition improved and the skin lesions diminished significantly , presenting postinflammatory hyperpigmentation and crust ( figure 5 ) . at the 10 month of treatment with paclitaxel , she died at the age of 71.3 years due to sepsis complicating perforation of the sigmoid diverticulum .
we obtained a sample of the powder from the patient s spouse and performed an analysis .
qualitative examination was done using liquid chromatography electrospray ionization mass spectrometry ( lc / esi - ms ) system .
the presence of triptolide m / z=359 ( m - h ) and tripdiolide m / z=375 ( m - h ) , 2 major active components of trypterigium wilfordii hook f , was confirmed .
the content of triptolide in 1 g of the analyzed sample , determined by hplc quantitative analysis , was 235 g .
we obtained a sample of the powder from the patient s spouse and performed an analysis .
qualitative examination was done using liquid chromatography electrospray ionization mass spectrometry ( lc / esi - ms ) system .
the presence of triptolide m / z=359 ( m - h ) and tripdiolide m / z=375 ( m - h ) , 2 major active components of trypterigium wilfordii hook f , was confirmed .
the content of triptolide in 1 g of the analyzed sample , determined by hplc quantitative analysis , was 235 g .
a question arises how triptolide / tripdiolide , taken in the high daily dose for years , could influence the patient s health status .
triptolide is a small molecule known to act as an anti - inflammatory and anti - cancer compound .
aa amyloidosis , present in 4.44% of early ra patients , was shown to be inhibited in mice by experimental treatment with triptolide .
thus , progression of ra seemed to be slower than usually observed , independently of triptolide / tripdiolide administration .
however , only while receiving triptolide / tripdiolide was she almost free from joint pain .
the most important point to determine is the possible association of ks with triptolide / tripdiolide medication .
ks is a multifocal angioproliferative neoplasm of the skin ( mainly affecting the skin of the limbs ) and mucosa , frequently seen in immunosuppressed patients , also due to ra .
regardless of their source or clinical subtype , have been found to be infected with hhv8 .
hhv8 latent transcripts , such as latency - associated nuclear antigen , viral cyclin , viral flip and viral - encoded micrornas , drive cell proliferation and prevent apoptosis , whereas hhv8 lytic proteins such as viral g protein - coupled receptor , k1 and virally encoded cytokines ( viral interleukin-6 and viral chemokines ) contribute to the angioproliferative and inflammatory ks lesions through a mechanism called paracrine neoplasia . in patients infected with hiv ,
expression of the proliferative antigen ki-67 , shown in our patient in 2030% of cell nuclei , does not correlate with skin and organ lesions or early- and late - stage ks lesions , but is valuable in ks diagnosis .
decreasing cd4 cells , triptolide could contribute to development of ks by reactivation of latent hhv8 , permitted by triptolide - induced immunosuppression .
on the other hand , triptolide is an anti - cancer compound and ks is not mentioned among the adverse effects of triptolide , ethyl acetate extracts or polyglycosides of the chinese herbal remedy tripterygium wilfordii hook f[4,69 ] .
moreover , ks lesions are attributed to the release of angiogenic molecules , most notably vascular endothelial growth factor ( vegf ) and angiopoietin - like 4 .
it was recently found that the antitumor action of triptolide is partly via inhibition of tumor angiogenesis by blocking 2 endothelial receptor - mediated signalling pathways , and triptolide can be a promising antiangiogenic agent .
thus , triptolide as an immunosuppressant could contribute to development of ks , but also could potentially be helpful by vegf inhibition
although we can not definitively answer the question of whether triptolide/ tripdiolide contributed to development of ks in our patient , we also can not exclude such a possibility .
the case of our patient indicates an association between triptolide / tripdiolide chronic intake and development of hhv8 ks .
triptolide/ tripdiolide could contribute to development of ks by reactivation of latent hhv8 , permitted by immunosuppression induced by triptolide .
we believe that this case will help other physicians to be vigilant for a possible association between ks and medication with chinese remedies containing triptolide . | summarybackgroundto date , kaposi sarcoma has not been mentioned among the adverse effects of triptolide / tripdiolide , ethyl acetate extracts or polyglycosides of the chinese herbal remedy tripterygium wilfordii hook f.case reporta patient was diagnosed with rheumatoid arthritis at the age of 29 years .
she underwent treatment with corticosteroids , methotrexate and gold sodium thiosulfate , and was chronically taking ketoprofen . at the age of 59 years
she started to take a powder ( 2 g / day ) from a chinese physician for treatment of rheumatoid arthritis .
this powder was supplied to her regularly for 10 years . at the age of 69 years
, multiple soft , violaceous to dark - red patches , plaques , nodules and blisters of varying sizes appeared on a background of severely edematous skin on her legs , and later on her arms .
biopsy specimens of the leg lesions were diagnostic for human herpesvirus 8-associated kaposi sarcoma .
triptolide ( 235 g/1 g ) and tripdiolide were found in the chinese powder by the use of liquid chromatography electrospray ionization mass spectrometry .
administration of the powder was stopped and medication with paclitaxel was introduced .
general condition of the patient improved and skin lesions diminished significantly.conclusionsthis case indicates a possible association between triptolide / tripdiolide chronic intake and development of human herpesvirus 8-associated kaposi sarcoma .
triptolide / tripdiolide could contribute to development of kaposi sarcoma by reactivation of latent human herpesvirus 8 , permitted by immunosuppression induced by triptolide . |
. one of the most common cardiac diseases is congestive heart failure , with the highest incidence , mortality , and hospitalization .
this is the only cardiac disease with a progressive incidence and mortality , in which almost 10% of the individuals over 75 years of age are involved .
the reported statistics reveal an increase in its mortality by six times in the last 40 years , indicating that about 300,000 people expired yearly directly or indirectly due to heart failure . on the other hand
, this disease is so disabling and costly that over 70% of the patients are readmitted in a hospital 3 months after their initial discharge , imposing a high financial burden to health care systems .
one of the important strategies to promote clinical outcomes in these patients is promotion of self - care behaviors .
self - care in heart failure contributes to issues such as medical and food diet , limitation of intake of sodium and fluids , daily weighing , level of permitted activities , and a decision for appropriate therapeutic interventions when the disease gets worse .
various researches show that self - care behavior can significantly diminish the number of hospitalizations , mortality , and care cost burdens . in patients with heart failure , administration of self - care
is difficult as a result of the complicated medical diet , the chronic nature of the disease , as well as the complications in various systems of the body which result in lower self - care .
therefore , provision of an efficient support for self - care is essential and important .
family as the most important source of social support is tightly related with self - care activities .
since most of the patients with heart failure live with other family members at their home , participation and support of family members can play a key role in self - care behaviors and efficiency of disease control .
therefore , family can influence a patient 's success and stability of their behavior change in self - care programs .
several studies revealed the association between family support and heart failure patients self - care .
earlier studies showed that the patients with more support had better compliance of self - care health behaviors .
showed the self - care behaviors such as restricting fluid intake , regular medication , sports , and referring to physicians in case of observed overweight among the patients who had received more support .
sayers et al . showed that the level of family support was directly associated with medical and food diets and other dimensions of self - care in heart failure patients .
meanwhile , most of the families , especially the caregivers , have low knowledge about the disease and its signs and treatment , and do not know what is beneficial for the patients or how they can support and encourage the patients to follow self - care behaviors .
nurses , with their unique position in family members , can play a key role in support , education , counseling , and taking care of heart failure patients and their caregivers .
they can familiarize the family members , especially caregivers , with these behaviors , which act as a knowledgeable and capable source of family - focused nursing interventions to give a better support to these patients . despite evidences proving the important role of family support in heart failure
patients self - care , family - focused supportive interventions with goal of their clinical outcomes promotion have been less conducted among these patients .
on the other hand , the researcher , despite searching thoroughly , did not find any studies on family - focused supportive care in heart failure patients in iran . therefore , the present study aimed to define the effect of family supportive intervention on heart failure self - care behaviors .
the present study was a pre- and post - test case and control clinical trial conducted in the selective university hospitals in isfahan , iran , in 2012 .
it aimed to study the effect of family support intervention on self - care behavior in heart failure patients .
inclusion criteria were : confirmation of heart failure diagnosis by a cardiologist , being in grade ii , iii , or iv of heart failure based on american heart association classification , having history of at least one time hospitalization due to cardiac failure , at least 1 year of experience of heart failure , being over 21 years of age , complete consciousness , no history of myocardial infarction ( mi ) or heart surgery in the past 6 months , no history of other chronic or disabling diseases except cardiovascular risk factors ( diabetes , hypertension , and hyperlipidemia ) , having a family , not living alone and the caregiver being from the family members , being over 18 years of age , and being literate .
the sample size was calculated to be 36 heart failure patients in each group of study and control .
a total of 62 patients with heart failure were initially selected by convenient sampling , and then randomly assigned to experimental and control groups equally . the data collection tool was a two - section questionnaire .
the first section of the questionnaire contained demographic characteristics , the existing data in patients medical profile ( ejection fraction , disease grade , and consumed drugs ) , as well as some information about patients caregiver ( age , sex , relation to the patient , length of care , and education level ) .
this questionnaire was designed by shoji fard et al . , and its face and content validity and reliability were confirmed by experts panel , and cronbach 's alpha was 0.8 . in this tool , 15 self - care activities were listed and investigated using a likert 's 5-point scale ( from zero = never to 4 = always ) . based on this scale , the obtained score can vary from 0 to 60 .
scores of 0 - 20 were assigned to poor , 21 - 40 to moderate , and 41 - 60 to good . after obtaining an informed written consent from the subjects and filling the self - care behaviors questionnaire , the subjects were assigned to study and control groups , and then family support intervention was conducted in the study group .
as the subjects in the study group were from three medical centers , the patients caregivers in each center were considered as a control group and three sessions with 8 - 12 attendees were held for each group .
these sessions were held weekly for 1 - 1.5 h in an appropriate classroom in the same medical educational center . in the first session ,
the caregivers were familiarized with the definition and heart failure disease process , its etiology and treatment , importance and manner of self - care behaviors , and related skills such as reading food labels and taking strategies to lower food salt intake . at the end of the first session ,
a booklet of heart failure self - care guidelines was distributed among the caregivers , not only to read but also to discuss the points with the other family members in order to be able to answer the questions related to the disease and administration of self - care indicated at the end of the booklet , with the cooperation of the patients .
they were also asked to write down their possible questions and deliver them to the researcher in the following session . in the second session ,
patients and caregivers responses to the questions in the booklet were collected and their learning and practical administration of the learned issues were evaluated , and the required guides were given to the caregivers .
then , the importance and role of family in disease control and patients care was explained .
the caregivers held group discussions about living with heart failure patients and the way of supporting them . in order to increase emotional and affective support toward these patients , efficient communication skills
case scenario , role play of suggested strategies , and supportive discussion were adopted to empower and practice learned skills . at the end of the second session ,
the caregivers were given a booklet about the importance and manner of patients practical and emotional support , and they were asked to pass it among the other family members . in the third session , caregivers gave examples of the patient supportive strategies taken , and communication as well as prohibiting and facilitating factors they faced during the latest week .
then , some indications about the manner of self - care as well as other related points were explained to finalize the subject and get a conclusion during the sessions . at the end of the third session ,
a contact number was given to the caregivers in order to get answers for their disease - related and self - care - related questions .
phone call follow - ups were carried out for 2 weeks to guide the subjects and answer their questions .
one month after the group sessions and 1 month after the introductory test in control group , self - care behavior questionnaire was again filled up for the subjects in both the groups .
the data were analyzed by descriptive and analytical statistics through spss version 12 . in analytical statistical tests ,
p < 0.05 was considered significant . in the present study , letter of introduction issued by isfahan nursing and midwifery school
the subjects were also assured that their personal information would be kept confidential by the researcher , and the subjects were free to leave the research any time they wanted to .
in the present study , 81 heart failure patients entered and filled the initial questionnaire . before the subjects random allocation
, nine subjects were excluded from the study ( three patients died and six were not interested to attend ) . during the study ,
four subjects dropped out of control group ( two died and two were not available ) . in the study group , four subjects dropped out ( two did not participate in all stages of the research and two died ) .
research findings showed that the subjects were identical in both groups concerning age ; sex ; marital status ; education level ; grade of disease ; hospitalization history ; sex , age , and education level of caregivers ; relationship of the caregiver with the patient ; and length of care .
most of the subjects in both groups were males ( 54.6% ) , married ( 87.15% ) , illiterate ( 51.55% ) , and in grade iii of the disease ( 59.35% ) .
most of the caregivers were females ( 81.2% ) , had education level of high school and lower ( 81.2% ) , and were the children of the patients ( 46.9% ) . demographic characteristics of the subjects in each group are presented and compared in table 1 .
demographic characteristics of patients and caregivers in table 2 , it is observed that the highest mean scores of each self - care behavior before intervention in both the study and control groups were for not smoking , drug consumption based on doctor 's prescription , time of preparation of the medications , regular visits to a physician , and avoiding canned foods , respectively . meanwhile , the lowest self - care behavior mean scores were for visiting a physician due to an observed overweight , daily weighing , and fluid restriction compliance and intake of low - salt food . the comparison between each self - care behavior 's mean score of the subjects in the intervention and control groups before and after the intervention in table 3 , it is seen that there was no significant difference between self - care behavior mean scores in the two groups of study and control before intervention ( p = 0.79 ) , but there was a significant difference between self - care behavior mean scores in the two groups of study and control after intervention ( p < 0.001 ) .
the comparison between self - care behavior mean scores of the subjects in the intervention and control groups before and after the intervention
the results of the present study show that administration of supportive intervention and promotion of family support led to promotion of self - care behavior in heart failure patients .
the results show that subjects had a moderate level of self - care behavior before intervention .
shoja fard et al . and gallager et al . reported subjects self - care behavior mean scores in the range of the present study , which is consistent with our findings .
the results obtained show that not smoking , taking the medications based on physician 's prescription , preparation of the medications in time , regular visits to physicians , and avoiding canned foods were among the self - care behaviors , and were in a moderate level .
meanwhile , subjects function in the other behaviors such as daily weighing , intake of less salt , referring to a physician when observing the signs , and fluid restriction compliance were found to be poor .
also reported that medication intake based on physician 's prescription was the most frequent self - care behavior practiced by the subjects , while daily weighing was the least . in sayers et al .
reported that lack of daily weighing in heart failure patients was as a result of the following factors : either the subjects did not find it helpful or they did not have an appropriate tool for weighing or they could not interpret the results . in the present study ,
the subjects mentioned being illiterate , not having scales available for daily weighing , and consideration of low importance as the reasons for lack of daily weighing .
the results of the present study showed that self - care behaviors in heart failure patients were promoted after supportive intervention and promotion of family support toward the patients .
showed that supportive intervention and conducting group sessions with heart failure patients caregivers and counseling regarding the manner of positive and supportive communication of the family with the patients led to better compliance concerning following a low - salt diet .
piette et al . showed that supportive strategies promoted self - care behaviors in the subjects .
the results of other researches also showed an association between social support from family side and self - care , as well as a positive association between family support and self - care , which is consistent with the present study .
argue that this finding may be due to the long time of follow - up and the inadequate interventions administered , and recommend further studies in this regard . in the present study , the process of skills administration and use of supportive strategies was followed up , evaluated , and checked through phone calls and filling checklists in sessions intervals . on other hand , lofvemark et al . showed that administration of a mere educational program for heart failure patients concerning care does not affect their level of re - hospitalization despite promotion of their caring knowledge .
lack of an educational effect on clinical outcomes and self - care behaviors in the aforementioned study can show that knowledge alone is not adequate for a behavior change and promotion of self - care .
in addition , clark et al . reported a poor relationship between heart failure knowledge and self - care . in the present study ,
the goal was not just promotion of caregivers knowledge , but their familiarization with strategies and supportive communication in the direction of patients emotional support promotion .
the researchers believe the observed patients self - care promotion may be as a result of caregivers participation in efficient communication skills education sessions , accompanied with the patients , and application of supportive communication and strategies by them leading to promotion of patients motivation toward conducting self - care . in the present study ,
the highest promotion of self - care behavior was observed after administration of family supportive intervention in dimensions such as compliance to a low salt - diet , fluid restriction , and referring to a physician after observing the signs of disease .
heart failure patient 's family plays a vital role in the control of signs and symptoms , and can help the patients to diagnose the symptoms of the disease getting worse earlier , encourage their compliance to a medical food diet , and support them through changes in their lifestyle .
showed that the patients who received more support by their spouses had better compliance to fluid restriction and referring to a physician .
dunbar et al . also observed a significant reduction in daily sodium intake after family supportive interventions .
one of the most important challenges in heart failure patients self - care is compliance to a low - salt diet .
since the self - care concerning compliance to a diet often occurs at home , family members can play a specific role in compliance to diet restrictions .
therefore , making the family members familiar with the need for compliance to this behavior and patients encouragement to do it is of great importance .
the findings of the present study reveal the major and important role of family members support in self - care among congestive heart failure patients .
nurses , as the professionals in the field of health and with regard to their important role in support , education , and care of these patients and their caregivers , can support , educate , and guide these patients through family members education and designing appropriate care programs in order to take steps toward promotion of self - care . designing ,
administration , and application of these supportive educational programs by family 's participation can reveal the role of nurses in promotion of patients and community health behavior and self - care .
based on the findings of the present study , it is suggested to design and conduct more family - focused interventions and studies for promotion of self - care in other chronic diseases . | background : heart failure is one of the chronic heart diseases and a debilitating condition of increased prevalence in the elderly .
one of the important and non - pharmacological strategies for improving clinical outcomes in these patients is promotion of the self - care .
background and social environment in which a patient is trying to control his disease is an important factor in the self - care .
the aim of this study was to evaluate the effects of family support intervention on the self - care behaviors in patients with heart failure.materials and methods : this study was a randomized clinical trial conducted in university hospitals in isfahan , iran , in 2012 .
a total of 62 patients with heart failure were randomly assigned to experimental ( n = 32 ) and control ( n = 32 ) groups .
supportive intervention including three educational sessions with the delivery of educational booklet and follow - up by telephone was performed for caregivers of patients in the experimental group .
data were collected using the questionnaire of self - care behaviors , which was completed before and 1 month after the intervention in both the groups , and the questionnaires were analyzed using descriptive statistics and independent and paired t-tests.results:the results indicate that after the intervention , self - care behavior scores in the experimental group and control group were 47.2 and 28.4 , respectively , and independent t - test revealed that the difference was statistically significant.conclusion:family-focused supportive interventions can be used as an effective method for improving the self - care behaviors in patients with heart failure . |
vaccinations are an effective prevention tool for maintaining the health of the individual and society as a whole and have significantly increased life expectancy and quality [ 1 , 2 ] .
smallpox was officially declared eradicated by the world health organization ( who ) in 1980 , all industrialized countries are considered " poliofree " , while other important infectious diseases such as diphtheria , tetanus and hepatitis b reached a significant reduction [ 3 - 5 ] , with important benefits for childhood , by the planning of vaccination campaigns and development of national pediatric immunization schedules in many european countries [ 6 , 7 ] .
strategies adopted by european union countries to achieve and maintain high immunization rates in target population include compulsory and recommended vaccinations , free of charge and co - payment offer .
some countries have preferred mandatory vaccinations such as france , greece , portugal and italy ( for some pediatric vaccines ) ; others , such as the united kingdom , voluntary decision supported by appropriate service offer , incentive for caregivers and health education .
others , like usa , canada and germany , opted for a middle way ( no sanctions for non - vaccination , but need for a certificate of admission to school ) [ 8 - 10 ] . according to latest edition of national immunization schedule ,
italian children are mandatory vaccinated against polio , diphtheria , tetanus and hepatitis b , while vaccinations against pertussis , measles , mumps , rubella , pneumococcus , meningococcus and invasive forms of h. influenzae type b are recommended .
this difference is merely theoretical since no coercion is actually performed for parents who express their dissent to compulsory vaccinations .
accomplishment of mandatory vaccinations and successful extensive immunization programs permitted prevention of many infectious diseases which resulted in lower concern about them while , nowadays , worsening attention to side effects and availability of new vaccines has progressively increased burden of commitment within vaccination policy .
current scenario is also complicated by strongly varied regional framework in immunization schedules ( determined by modification of title v of constitution ) , by inadequate information on low risk in terms of safety and significant benefits concerning protection that facilitate activity of antivaccinal groups .
all italian national vaccine plans ( nvp ) , from 2005 to present , notice the opportunity to begin a cultural and social awareness growth to overcome difference between compulsory and recommended vaccinations , inviting to same active offer for both vaccine groups , in favor of a modern approach , based on health education , health promotion and patient self - determination .
regions which possess following characteristics are encouraged to move towards abolition of mandatory vaccinations : an effective information system with well organized vaccines register ; adequate immunization coverage ; a sensitive and specific surveillance system of communicable diseases , are able to mix corporate / regional data flows ; a good system of monitoring vaccine adverse events , are able to ensure appropriate follow up of cases .
piedmont , for example , introduced in 2006 a method of simplification of immunization offer , defining priority vaccines ( proposed free and in active manner ) and all other vaccines ( available in health care organizations at cost ) obtaining very satisfactory results in terms of agreement .
tuscany , instead , eliminated distinction between mandatory and recommended vaccinations in 2007 and 2010 through tuscan vaccine schedules [ 13 , 14 ] .
only recently autonomous province of trento started a step - by - step overcoming of compulsory vaccination in children , depending on achievement of provincial immunization coverage percentage not less than that expected by the nvp and in absence of conflicting epidemiological evaluations . with regional law 7/2007 " suspension of mandatory vaccination for children and adolescents " , veneto region has suspended obligation of compulsory immunizations for all infants born from 1 january 2008 , confirming that they still are included in essential level of assistance and therefore free and actively offered by the local health units ( ulss ) and part of immunization schedule , periodically reviewed and approved by the regional council in accordance with nvp guidelines .
a monitoring plan of vaccination system , shared with ministry of health and superior institute of health , must ensure vaccination coverage achieves at least 95% of newborns , otherwise obligation suspension is stopped .
president of regional council may also come back to previous system , if significant epidemiological events occur or if the content of the document drafted by the technical scientific committee , expressly created , signals an alarm situation with regard to immunization coverage rates .
this paper aims to analyze whether innovations introduced by veneto regional law 7/2007 have modified immunization coverage rates and parents participation in vaccination acceptance .
levels of vaccination coverage achieved in local health unit of rovigo ( ulss18 ) at 24 months in children born in 2002 - 2008 regarding immunizations for which obligation is suspended ( including an estimate of non - vaccination against polio percentage and reasons for non - vaccination ) and immunization rates for some recommended vaccinations ( pertussis , h. influenzae b , measles , mumps and rubella ) are considered . for birth cohorts 2006 - 2011 , we assessed percentage of children who have received , within the first year of life , full or partial cycle of vaccination against diphtheria , tetanus , pertussis , hepatitis b to detect possible differences between before and after the appliance of regional law 7/2007
. similarly we considered noncompulsory vaccinations against pneumococcus ( partial and complete cycle ) , meningococcal c , measlesmumps- rubella ( mmr ) and chickenpox ( one dose ) to examine whether high levels of adhesion to recommended vaccinations were confirmed even after suspension of compulsory vaccination .
records were obtained from two regional forms adopted for monitoring vaccination activities : model 19 for coverage rates and enclosure c for immunization acceptance . in both cases
adjusted rates were calculated by dividing number of administered doses by number of children born in investigated cohort excluding unreachable ( e.g. domiciled abroad and untraceable ) .
data on immunization coverage were collected from annual survey form ( so called model 19 ) that each ulss annually transmit to veneto region in order to assess coverage rates .
years from 2004 to 2010 were considered , therefore , vaccination coverage refers to birth cohorts 2002 - 2008 ( 2008 birth cohort is the first affected by the suspension of immunization compulsoriness ) . according to regional directives , we examined immunization coverage at 24 months as the number of completed vaccinations in children born two years before the year of reference .
coverage rates at 24 months were assessed as well as for previously mandatory vaccines ( polio , diphtheria , tetanus and hepatitis b ) as for those against pertussis , h. influenzae b ( hib ) , measles , rubella and mumps .
trends in immunization offer acceptance were calculated from regional form , called enclosure c , that each ulss send to department prevention services - promotion and development of hygiene and public health of veneto region twice a year ( march 31 and september 30 ) in order to strictly monitor vaccination compliance .
number of doses administered to newborns divided in cohorts , according to three previous semesters are recorded , e.g. the detection of 30 september 2011 considered separately children born in the first half of 2010 , those born in the second half of 2010 and those born in the first half of 2011 in order to assess vaccines received within 6 , 12 and 18 months from birth respectively .
presented data relate to three doses of hexavalent and anti - pneumococcal vaccines and first dose of measles , chickenpox , rubella , mumps and meningococcal c vaccines in the period september 2007- september 2011 .
data on immunization coverage were collected from annual survey form ( so called model 19 ) that each ulss annually transmit to veneto region in order to assess coverage rates .
years from 2004 to 2010 were considered , therefore , vaccination coverage refers to birth cohorts 2002 - 2008 ( 2008 birth cohort is the first affected by the suspension of immunization compulsoriness ) . according to regional directives , we examined immunization coverage at 24 months as the number of completed vaccinations in children born two years before the year of reference .
coverage rates at 24 months were assessed as well as for previously mandatory vaccines ( polio , diphtheria , tetanus and hepatitis b ) as for those against pertussis , h. influenzae b ( hib ) , measles , rubella and mumps .
trends in immunization offer acceptance were calculated from regional form , called enclosure c , that each ulss send to department prevention services - promotion and development of hygiene and public health of veneto region twice a year ( march 31 and september 30 ) in order to strictly monitor vaccination compliance .
number of doses administered to newborns divided in cohorts , according to three previous semesters are recorded , e.g. the detection of 30 september 2011 considered separately children born in the first half of 2010 , those born in the second half of 2010 and those born in the first half of 2011 in order to assess vaccines received within 6 , 12 and 18 months from birth respectively .
presented data relate to three doses of hexavalent and anti - pneumococcal vaccines and first dose of measles , chickenpox , rubella , mumps and meningococcal c vaccines in the period september 2007- september 2011 .
coverage percentages at 24 months for the vaccines for which suspension of compulsory immunization was introduced are shown in figure 1 .
for each vaccination and for each birth cohort considered , 95% coverage , set by region as goal , was always exceeded . for birth cohort
2008 percentage of vaccinated children , that under mandatory tended to decrease , showed an increase compared to previous birth cohort , reaching 98.5% for polio and hepatitis b and 99% for diphtheria - tetanus .
rovigo ulss18 coverage rate at 24 months for formerly mandatory vaccinations ( birth cohorts 2002 - 2008 ) .
the percentage of children vaccinated against pertussis and hib at 24 months after birth was always fully satisfactory and , in the first cohort affected by the suspension of obligation , amounted to 96.7% .
measles , mumps and rubella vaccinations , that in cohort 2002 recorded a coverage of 96% , has shown , over the years , a marked reduction , however , in recent years percentage of vaccinated children has increased to 94.9% for measles and 94.5% for mumps and rubella .
rovigo ulss18 coverage rate at 24 months for formerly recommended vaccinations ( birth cohorts 2002 - 2008 ) .
the motivations of non - vaccination against polio at 24 months after birth are indicated in figure 3 .
the leading cause was the inability to contact parents and deliver invitation to vaccination because they moved abroad , do not have a fixed address or are untraceable .
percentage of children at 24 months not vaccinated against polio according to motivation that led to non - vaccination ( birth cohorts 2002 - 2008 ) . since 2007 ,
standard definitions to define several types of vaccination postponement have been broadened considering also vaccination delay .
proportion of children actually defaulting vaccination ( having expressed an informed dissent ) was a minority , but the percentage showed an increasing trend from 0.2% in 2002 cohort to 0.9% in 2008 cohort .
data collected every six months , before and after suspension of compulsory vaccination , are reported in table i. the monitoring do not have the purpose of determine vaccine coverage , that can be estimated on consolidated data at 24 months of life , but to evaluate performance of immunization activities on cohorts of newborns to make a comparison over time and between ulss ( benchmarking ) , therefore it should not concern the detection of percentages slightly lower than 95% even for previously mandatory vaccinations .
proportion of children receiving hexavalent vaccine has always been elevated and a steady increase , more pronounced for the third dose , has been recorded . in all measurements ,
the percentage of vaccinated children has reached levels ranging from acceptable ( 90 - 94% ) and desirable ( 95% ) .
vaccination against pneumococcal presented a comparable situation , with a more marked rise for third dose passing from 33% in september 2007 to 64% in september 2011 .
percentage of children vaccinated against meningococcal ( offered from the thirteenth month ) was stable around 90% .
measles - mumps - rubella and chickenpox vaccinations starting from very lower rates of acceptance ( respectively 72.3 and 67.3% ) showed an increase , reaching levels fairly above 80% , always remaining within a not satisfactory range .
hexavalent vaccine contains poliomyelitis , diphtheria , tetanus , pertussis , hepatitis b , h. influenzae b vaccines .
regional autonomy has allowed , in healthcare sector , the undertaking of an important initiative of public health such as that started by veneto region with the promulgation of regional law 7/2007 that , overcoming the dichotomy between mandatory and recommended vaccinations , meets the growing demand of citizens for self - determination in choices regarding their health by introducing a legislative action with a european outlook .
the debate on recommended or mandatory vaccinations has also collected conflicting opinions because it raises several ethical issues . the possibility for regions to independently determine vaccination schedule and introduce new vaccines and methods of promotion
has created a national framework strongly non homogeneous which could have a negative impact on immunization programs and , apparently , does not ensure equal access to immunization .
furthermore , at first sight , the comparison between countries that only recommend certain vaccinations and countries that force them does not seem to show remarkable differences [ 17 , 18 ] .
the experience of rovigo ulss18 showed that , in a context of already high levels of coverage for recommended vaccinations , rates for ex - mandatory immunizations remain unchanged .
results obtained in the 2008 birth cohort , the first involved in the suspension of the obligation , showed that no loss of coverage occurred for previously mandatory vaccines , in fact coverage rates moved from values ranging from 95 - 97% in previous birth cohorts to 98.5 for poliomyelitis and hepatitis b and to 99 for diphtheria and tetanus in 2008 birth cohort . obviously available data on a single birth cohort
are too limited to express a judgment of full validity of the regional law , therefore it will be necessary to continue to monitor levels of vaccination coverage and offer compliance in order to highlight situations of deviation from expected value before warning situation occurs .
opponents of this law consider that liberalization in the field of immunization poses a threat to consolidated practices of public health with the risk of decreasing vaccine coverage and increase the number of cases of infectious diseases easily preventable .
however , first monitoring data are fully encouraging and demonstrate that there were no changes with regard to coverage , while acceptance of immunization offer remained constant for ex - mandatory and was even growing for exrecommended vaccinations . in that regard , it should be noted that the legislative measure adopted by veneto provides for a constant monitoring of vaccination coverage and compliance to invitation , a rigorous epidemiological surveillance of infectious diseases for which the requirement of vaccination is suspended , and immediate suspension of the regional law 7/2007 when coverage levels fall below the established threshold or in case of significant epidemiological events related to the disease for which the vaccination requirement is suspended .
the developed delivery system of vaccinations appears able to allow the suspension of mandatory vaccinations without risk to coverage , continuing to ensure a strong adhesion to all available vaccinations , as it is characterized by some essential elements : a strong degree of integration of vaccination services with pediatricians and family physicians ; active and free offer of all vaccinations recognized as safe and effective ; computerized immunization database that allows rapid recovery of non - adhesion to first call ; the remarkable cultural maturity reached by population that is constantly affected by actions of counseling and health education .
these observations support the conclusion that similar legislative measures can be adopted with comparable success , as advocated by nvp 2012 - 14 , also in other italian regions , provided that they possess specific starting conditions : high levels of immunization coverage , ability to ensure an effective vaccination offer system , continuous monitoring of membership , a rapid recovery of any non - compliance based on dialogue and information .
the introduction of compulsory vaccination suspension law in veneto region has allowed experiencing a public health setting established on self - guided individual choice in making decisions regarding health , creating a modern and efficient vaccine service .
further research and reports are required to careful value whether the choice between compulsory and voluntary vaccination takes effect on coverage rates . in conclusion
a healthcare system should promote and actively offer all vaccinations approved as safe , effective and with a positive public health impact .
however decision about proposal for vaccine strategies should be elaborated in agreement with organizational features of vaccination services combined with traditional and cultural habits .
trust on health authorities will lead to more compliance with recommendations producing not only benefits for health of citizen but also enhancing their awareness on this issue and support the overall effectiveness of vaccination programs through the herd immunity effect . | summarythe burden of infectious diseases preventable by vaccination decreased considerably over last decades in all countries provided with effective immunization schedules .
implementation of these programs with new vaccines has started discussion on duality between mandatory and recommended vaccinations .
regional autonomy has allowed the veneto region , with introduction of law 7/2007 , to experience suspension of all mandatory childhood vaccinations , replaced by an active and free offer of all vaccines recognized as safe and effective .
coverage 's trends and acceptance of invitation are carefully monitored to avoid loss of compliance and reduction of immunized children .
the evaluation , performed on population of rovigo ulss18 for 2002 - 2008 birth cohorts ( 2008 is the first concerned by the change ) , revealed no fall for previously mandatory vaccinations , but rather a slight increase .
percentage of non - vaccinated children was negligible .
the leading cause of non - vaccination to polio was inability to deliver the letter of invitation due to wrong address .
an informed dissent was expressed only by a minority ( 0.9% in 2008 birth cohort).compliance to immunization offer was elevated both for previously compulsory and recommended vaccinations .
experience of rovigo ulss 18 showed that , in a context of already high levels of coverage for recommended vaccinations with optimal organization of immunization services , coverage rates for exmandatory vaccines remain unchanged .
further research and reports are required to carefully assess the effect on coverage rates in next birth cohorts , but available data are encouraging .
similar legislative measures can be successfully adopted by other regions with starting conditions comparable to veneto region . |
one of the leading causes of death are non - communicable diseases ( ncds ) , killing more people each year than all other causes combined .
it has been reported that nearly 80% of ncd deaths occur in low- and middle- income countries .
it has been estimated that about one fourth of global ncd - related deaths take place before the age of 60(1 - 4 ) .
it has been indicated that the prevalence of non - communicable diseases such as hypertension and diabetes including obesity has increased over the past few years in iran(1 , 4 ) .
the prevalence of hypertension , diabetes , obesity and overweight in big cities in iran was 14% , 13% , 27% and 20% respectively(1 ) .
hypertension is a risk factor for life threatening diseases such as cerebrovascular accidents ( cva ) , coronary artery diseases ( cvd ) , congestive heart failure ( chf ) and chronic renal failure ( crf ) ( 3 - 13 ) .
based on national health and nutrition examination survey ( nhanes ) database in united states , the prevalence of hypertension was 24.5% and 28.4% in 1988 - 1994 and 1999 - 2000 respectively ( 11 , 12 ) .
the prevalence of hypertension in the capital city of big provinces of iran was 11%.(1 ) also , in zabol city in south east of iran , the prevalence of hypertension was 13.9% among adults ( 4 ) . in semnan province ,
the prevalence of hypertension was 24.1% and 24.8% in urban and rural area respectively.(5 ) in tehran , the prevalence of hypertension among taxi drivers was 35.4% ( 7 ) .
based on phase 1 of isfahan healthy heart study , the prevalence of hypertension in female and male were 15.6% and 18.8% respectively .
the prevalence of hypertension was higher in women than in men , except in under 25-year age group .
overall 26.7% of hypertensive male and 47.7% of hypertensive female were on anti - hypertensive pharmacological treatment among which bp was under control in 6.4% of the male and 13.8% of the female ( 8) .
ncd risk factors can be categorized as " modifiable " and " non - modifiable " .
modifiable parameters include factors that can be altered such as individual and community influences , living and working conditions and socio - cultural factors .
non - modifiable parameters include those factors that are beyond the control of the individual , such as age , sex and genetic factors ( 1 , 4 ) . diabetes mellitus , cardiovascular diseases , cancer , and chronic obstructive pulmonary diseases are linked by common preventable risk factors related to lifestyle ( unhealthy diet , physical inactivity , obesity and overweight , and tobacco use ) ( 1 ) . also , four behavioral risk factors that are pervasive aspects of economic transition , rapid urbanization and 21st - century lifestyles are tobacco use , unhealthy diet , insufficient physical activity and the harmful use of alcohol .
these risk factors affect low- and middle- income countries , and on poorer people within all countries , mirroring the underlying socioeconomic determinants .
there is a fact that poverty exposes people to behavioral risk factors for ncds and , in turn , the resulting ncds may become an important driver to the downward spiral that leads families towards poverty ( 1 ) .
it has been shown that a major reduction in the burden of ncds will come from population - wide interventions , which are cost effective and may even be revenue - generating .
however , it has been reported effective interventions , such as tobacco control measures and salt reduction , are not implemented on a wide scale because of inadequate political commitment , insufficient engagement of non - health sectors , lack of resources , vested interests of critical constituencies , and limited engagement of key stakeholders ( 1 ) .
according to global status report on non - communicable diseases 2010
smoking is estimated to cause about 71% of lung cancer , 42% of chronic respiratory disease and nearly 10% of cardiovascular disease .
it has been demonstrated that adequate consumption of fruit and vegetables reduces the risk for cardiovascular diseases , stomach cancer and colorectal cancer .
moreover , most populations consume much higher levels of salt than recommended by world health organization ( who ) for disease prevention ; high salt consumption is an important determinant of high blood pressure and cardiovascular risk .
high consumption of saturated fats and trans - fatty acids is linked to heart disease .
in addition , it has been indicated that raised blood pressure is estimated to cause 7.5 million deaths , about 12.8% of all deaths .
the prevalence of raised blood pressure is similar across all income groups , though it is generally lowest in high - income populations ( 1 ) .
it has been illustrated that at least 2.8 million people die each year as a result of being overweight or obese . also , it is estimated that raised blood pressure cause about 7.5 million deaths , about 12.8% of all deaths.(2 , 6 ) risks of heart disease , strokes and diabetes increase steadily with increasing body mass index ( bmi ) .
prevalence of overweight is over 50% in the who european region , the eastern mediterranean region and the region of the americas ( 1 ) .
physical inactivity is a risk factor of ncds that causes 3.2 million people die each year .
it has been reported that people who are insufficiently physically active have a 20% to 30% increased risk of all - cause mortality . moreover
, regular physical activity reduces the risk of cardiovascular disease including high blood pressure and diabetes ( 1 ) .
according to the alma ata declaration , it is obvious that in effective management of diseases , three aspects should be considered .
the second aspect was preventive intervention which includes personal and behavioral actions and education to encourage behavioral modification .
the third aspect was curative and rehabilitative intervention that includes services focusing on individuals who are already diagnosed , to prevent complications ( 1 ) .
the first step of planning and policy making for intervention and modification of cardiovascular diseases in a defined population is to assess the prevalence of their risk factors .
therefore , this study performed to assess personnel blood pressure and its risk factors in one of the medical universities of tehran in te health day of 2013 .
this cross - sectional study was performed from may 19 , 2013 to may 24 , 2013 ( i.r . of iran s health weak ) in one of the medical universities of tehran .
participants were personnel of the university that completed a researcher - made questionnaire voluntarily ; the questionnaire composed of demographic variables such as sex , age and variables about risk factors and preventive factors of cardiovascular diseases such as smoking , history of diabetes , history of hypertension , physical exercise status and so on .
systolic and diastolic pressures were measured after the participants had rested in a quiet area for 5 minutes .
participants were advised to avoid exercise for at least 30 minutes before their blood pressure measurement .
the korotkoff phase i ( appearance ) and phase v ( disappearance ) were recorded for the systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) respectively ( least of 3 readings taken on 1 occasion ) .
one - sample kolmogorov - smirnov test used to check normal distribution of quantitative variables .
binary logistic regression used to analyze relationship between hypertension status and its risk factors .
one - sample kolmogorov - smirnov test used to check normal distribution of quantitative variables .
binary logistic regression used to analyze relationship between hypertension status and its risk factors .
of 195 persons participated in this study , 180 persons ( 92.3% ) were male .
table 1 shows age , duration of employment , weight , height , body mass index ( bmi ) and systolic and blood pressure . only 8 persons ( 5.6% ) were cigarette smokers and 26 persons ( 14.9% ) were hookah smokers .
table 3 shows the history of some diseases in participants and first - degree relatives . among hypertensive cases ( 18 persons ) ,
most of the patients ( 87.0% ) used 1 - 2 cups of tea per day . among participants , only 29 persons ( 15.8% ) attended healthy life style classes .
of participants , only 75 persons ( 40.5% ) had normal bmi ( table 4 ) .
figure 1 shows classification of blood pressure based on the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 7 category ) ( 14 ) .
hypertension status based on jnc7
table 5 shows relationship between demographic and other factors among normal , pre - hypertensive and hypertensive groups based on jnc7 categories .
according to binary logistic regression analysis , ( enter model )
, there was a significant relationship between pre - hypertension or hypertension status with first relatives cardiovascular history ( p= 0.029 ) .
however , forward conditional model showed no significant relationship between pre - hypertension or hypertension status and its risk factors .
the result of our study showed that only 40.5% of samples had a normal bmi .
although overweight and obesity are risk factors of hypertension , diabetes and other ncd , it could be modified by life style intervention such as physical activity promotion .
yarahmadi and his colleagues study showed that the prevalence of overweight and obesity was 27.0% and 20.0% respectively ( 1 ) .
based on global status report on non - communicable diseases 2010 , the age - adjusted prevalence of overweight in bahrain and egypt was 70.3% and 69.8% respectively .
in addition , the age - adjusted prevalence of obesity in bahrain and egypt was 32.6% and 34.6% respectively ( 2 ) .
godarzi and his colleagues study showed that the prevalence of obesity in male and female was 64% and 70% in male and female respectively ( 4 ) .
demirchi and mehrabani s study showed that the prevalence of overweight and obesity was 26.6% and 40.6% respectively ( 7 ) .
sadeqi and her colleagues study showed that the prevalence of bmi > 25 in male and female was 41.9% and 59.0% respectively.(8 ) ahmadi and his colleagues study showed that the prevalence of obesity in hypertensive and control group was 21.7% and 14.2% respectively ( 10 ) .
ong and his / her colleagues study showed that the mean of bmi ( se ) was 27.81 ( 0.22 ) , 27.87 ( 0.16 ) and 27.99 ( 0.13 ) in 1999 - 2000 , 2001 - 2002 and 2003 - 2004 respectively based on nhnes database ( 11 ) .
in 2001 , the prevalence of obesity ( bmi>= 30 ) was 20.9% versus 19.8% in 2000 .
saeedi study showed that the prevalence of obesity was 56.5% and 25.0% in diabetic female and male respectively ( 16 ) .
mcniece and his / her colleagues ' study showed that the prevalence of overweight was 18.1% in adolescent ( 17 ) .
tazi and his colleagues ' study indicated that the prevalence of obesity in non - hypertensive and hypertensive adult was 11.1% and 23.6% respectively . also , the prevalence of overweight in non - hypertensive and hypertensive adult was 24.7% and 32.8% respectively ( 18 ) .
fukuda and his / her colleagues ' study showed that the mean bmi ( sd ) of male and female employees was 22.6 ( 2.4 ) and 21.2 ( 2.9 ) respectively ( 19 ) . based on a report from a community - based cohort study in taiwan ,
mean bmi in normotensive , pre - hypertensive , stage 1 hypertensive and stage 2 hypertensive group were 22 , 23.2 , 24 and 24.3 respectively.(20 ) ( 20 ) steyn and his / her colleagues ' study showed that the prevalence of obesity in male and female adult was 11.1% and 23.6% respectively ( 21 ) .
singh and his / her colleagues ' study indicated that the prevalence of overweight and obesity ( bmi>23 kg / m2 ) in normotensive , pre - hypertensive and hypertensive female and male was 14.3% , 82.3% , 83.2% , 16.4% , 70.7% and 71.3% respectively.(22 ) ishikawa and his / her colleagues ' study showed that the mean bmi ( sd ) of normotensive , prehypertensive and hypertensive male and female was 22.0 ( 2.5 ) , 23.0 ( 2.8 ) , 23.8 ( 3.0 ) , 22.1 ( 2.7 ) , 23.3 ( 3.1 ) and 24.3 ( 3.4 ) respectively ( 23 ) .
egan and his / her colleagues ' study showed that the prevalence of obesity was 21.8% , 29.5% , 29.6% , 31.4% , 33.4% and 33.2% in 1988 - 1994 , 1999 - 2000 , 2001 - 2002 , 2003 - 2004 , 2005 - 2006 and 2007 - 2008 based on nhnes database respectively ( 24 ) .
a prevalence study in iranian military personnel showed that about 20% of participants were overweight ( 25 ) .
our results showed that the prevalence of norm tension , prehypertension and hypertension was 47.9% , 40.4% and 11.7% respectively .
also , there was a significant difference between cardiovascular diseases and diabetes history and hypertension status based on jnc7 categories .
yarahmadi and his colleagues study showed that the prevalence of hypertension was 14.0% in the capital city of 6 big provinces of iran ( 1 ) .
based on global status report on non - communicable diseases 2010 , the age - adjusted prevalence of hypertension in bahrain and egypt was 43.7% and 38.1% respectively ( 2 ) .
godarzi and his colleagues study showed that the prevalence of hypertension was 13.9% among zabol city adults ( 4 ) .
another study showed that the prevalence of hypertension was 24.1% and 24.8% in urban and rural area respectively ( 5 ) .
a study on taxi drivers blood pressure showed that the prevalence of hypertension was 35.4% .
this study concluded that hypertension prevalence are correlated positively with age increase , physical activity decrease , cardiovascular diseases history , obesity , low level of education , income increase and family member increase ( 7 ) .
sadeqi and her colleagues showed that the prevalence of hypertension was 15.6% and 18.8% in male and female respectively .
moreover , the prevalence of hypertension was higher in female than in men , except in under 25-year age group ( 8) .
gu and his colleagues indicated that the prevalence of hypertension was 27.2% in chinese adult population ( 13 ) .
in addition , tazi and his colleagues study showed that the prevalence of hypertension was 37.2% and 41.3% in male and female respectively . also , the prevalence of hypertension was 30.6% , 46.5% and 58.1 in normal , overweight and obese samples respectively . ( 18 ) singh and his / her colleagues study indicated that the prevalence of female and male prehypertension and hypertension in five indian cities was 27.2% , 27.2% , 30.0 and 30.6% respectively ( 22 ) .
ishikawa and his / her colleagues study showed that the prevalence of the prevalence of normotension , prehypertension and hypertension was 32.7% , 33.0% ( 34.8% in males and 31.8% in females ) , 34.3% ( 36.8% in males and 32.7% in females ) respectively ( 23 ) .
a prevalence study in iranian military personnel showed that 58% and 49.4% of participants had systolic and diastolic pre hypertension respectively .
additionally , the prevalence of systolic and diastolic hypertension was 9.4% and 22.2% respectively ( 25 ) .
based on mustafavi and zare study the prevalence of hypertension was 66.4% and 10.1% in elderly and young population respectively ( 26 ) .
angell et al showed that the prevalence of hypertension among nyc adults was 25.6% ( 95% ci 23.4% to 27.8% ) and was similar for men and women ( men : 25.5 ( 22.528.7 ) , female : 26.0 ( 23.528.8 ) ) ( 27 ) .
kearney and his / her colleagues study indicated that 26.4% of the adult population in 2000 had hypertension ( 26.6% of men and 26.1% of women ) , and 29.2% were projected to have this condition by 2025 ( 29.0% of men and 29.5% of women ) ( 28 )
based on nhanes database in united states , the prevalence of hypertension was 24.5% and 28.4% in 1988 - 1994 and 1999 - 2000 respectively.(11 , 13 , 24 , 29 , 30 ) also , the prevalence of hypertension was 26.0% and 29.3% in 2001 - 2002 and 2003 - 2004 respectively ( 11 , 24 , 29 ) .
based on jaipur heart watch in india , the prevalence of hypertension in jhw - r , jhw-1 , jhw-2 , jhw-3 and jhw-4 studies in men was 21.6% , 29.1% , 29.6% , 42.5% and 45.1% and in women 15.7% , 21.7% , 25.5% , 35.2% and 38.2% respectively ( 31 ) .
a survey by wolf - maier and colleagues showed that the age- and sex - adjusted prevalence of hypertension was 28.0% in north american countries and 44.0% in the european countries based on jnc7 categories ( 32 ) .
based on global status report on non - communicable diseases 2010 , the age - adjusted prevalence of current daily cigarette smoking in bahrain and egypt was 14.3% and 16.5% respectively ( 2 ) .
godarzi and his colleagues showed that the prevalence of tobacco and cigarette smoking in male and female was 36.0% and 15.0% respectively ( 4 ) .
sadeqi and her colleagues study showed that the prevalence of current daily cigarette smoking in male and female was 29.0% and 1.6% respectively ( 8) .
based on ahmadi and his colleagues study , the prevalence of current daily cigarette smoking in hypertensive and normal population was 9.4% and 14.2% respectively ( 10 ) .
tazi and his colleagues showed that the prevalence of current smoking in non - hypertensive and hypertensive samples was 16.4% and 9.1% respectively ( 18 ) .
based on a report from a community - based cohort study in taiwan , the prevalence of smoking in normal blood pressure , pre - hypertensive , stage 1 hypertensive and stage 2 hypertensive men was 72.1% , 69.9% , 70.1% and 75.4% respectively .
these percentages in women were 5.3% , 3.8% , 7.0% and 6.5% respectively ( 20 )
based on singh and his colleagues study , the prevalence of tobacco use in normotension , prehypertension and hypertension group of men and women was 35.7% , 30.5% , 30.8% , 9.2% , 10.2% and 10.7% respectively ( 22 ) .
based on the jichi medical school cohort study , the prevalence of current smoking in normotension , prehypertension and hypertension group was 57.2% , 51.1% and 44.4% respectively ( 23 ) .
based on jaipur heart watch in india , the prevalence of smoking in jhw - r , jhw-1 , jhw-2 , jhw-3 and jhw-4 studies in men were 50.0% , 38.7% , 35.7% , 27.1% and 59.9% respectively ( 31 ) .
despite the low prevalence of hypertension in our samples , the high prevalence of prehypertension and overweight needs great attention .
interventions like lifestyle education , life style modification such as increasing time of physical activity programs per week , dietary modification and low caloric diet and avoiding junk and fast foods could be effective . | background hypertension is a risk factor for life threatening diseases such as cerebrovascular accidents , coronary artery diseases , congestive heart failure and chronic renal failure .
the prevalence of non - communicable diseases such as hypertension and diabetes including obesity has increased over the past few years in iran .
the first step for modification of cardiovascular diseases in a defined population is to assess the prevalence of their risk factors .
this study was conduceted to assess personnel blood pressure and its risk factors in one of the medical universities of tehran in the health day of 2013 .
methods : this cross sectional study was performed from may 19 , 2013 to may 24 , 2013 ( i.r . of iran s health weak ) in one of the medical universities of tehran .
participants completed voluntarily a researcher - made questionnaire which composed of demographic characteristics and variables about risk factors and preventive factors of cardiovascular diseases such as smoking , history of diabetes , history of hypertension , physical exercise status and so on .
blood pressure was measured by mercury sphygmomanometer and weight and height were measured by a ground analogue scale .
results : of 195 persons participated in this study , 180 persons ( 92.3% ) were male .
the mean age of participants was 33.75 ( 9.87 ) yr .
the mean of systolic and diastolic blood pressure was 114.44 ( 8.67 ) mmhg and 73.06 ( 8.45 ) mmhg , respectively .
the prevalence of overweight , obesity , prehypertension and hypertension was 41.7% , 17.8% , 40.4% and 11.7% respectively . only 8 persons ( 5.6% ) were cigarette smokers .
conclusion : despite the low prevalence of hypertension in our samples , the high prevalence of prehypertension and overweight need great attention .
interventions like life style modification could be effective in prevention of hypertension . |
lysosomes are cellular organelles that play a pivotal role in the cell homeostasis through their involvement in degradation and recycling processes of extracellular material that has been internalized by endocytosis and intracellular components that have been sequestered by autophagy ( 1 ) .
lysosomes may also fuse with the plasma membrane , emptying their contents outside the cell .
this is important for processes such as cellular immune response and plasma membrane repair , both in normal and pathological conditions ( 2 , 3 ) .
mutations that cause lysosomal enzyme deficiencies result in different syndromes , known as lysosomal storage disorders ( lsds ) ( 4 ) .
in addition , they are characterized by intracellular deposition and protein aggregation , events also found in age - related neurodegenerative disorders , such as alzheimer s and parkinsons s diseases ( 57 ) .
these studies underline the importance of the lysosome as a central player in cell metabolism .
hence , the characterization of genes participating in lysosomal biogenesis and function is a critical step toward the understanding of basic processes in cell biology and pathogenic mechanisms in many human diseases .
recently , it was found that most lysosomal genes exhibit coordinated transcriptional behavior and are regulated by the transcription factor eb ( tfeb ) , which also links autophagy to lysosome biogenesis ( 8 , 9 ) . gene expression at the post - transcriptional level can be regulated by micrornas ( mirnas ) .
mirnas play important roles in diverse biological processes , including development , cell differentiation , proliferation and apoptosis , in which the lysosomal system also plays an important role .
notably , mirnas have been recently identified as involved in the regulation of autophagy ( 10 , 11 ) .
the human lysosome gene database ( hlgdb ) is the first searchable database focused on the census of genes belonging to the lysosomal system and on their regulation by mirnas .
no database resources entirely dedicated to the regulation of lysosomal genes by mirnas or other regulators are currently available .
several lists of lysosomal genes were collected from public gene databases , published proteomics articles and reviews edited by biochemists and cell biologists working in the lysosome field .
we paid special attention on balancing predictions , which were as follows : ( i ) more suitable to look for confirmatory evidence ( targetscans ) ( 15 ) ; ( ii ) more suitable to identify any possible target for a particular mirna , to form the basis for in vitro or in vivo experiments ( pictar four - way and five - way ) ( 16 ) ; ( iii ) more suitable to find in silico evidence for the interaction between a mirna and a gene of a certain family or function ( pita , miranda ) ( 17 ) . to increase mirna - target mrna information , experimentally verified mirna targets from mirtarbase were also reported .
hlgdb aims to providing a useful resource to anyone studying the lysosomes and a tool for identifying common regulatory features of lysosomal genes .
hlgdb provides a user - friendly interface through which information can be easily retrieved , including the union and intersection of different gene lists , searches for mirna predictions and visualization on the gene transcript sequence of the mirna target predictions .
the data reported in the current version 1.1 , derived from ncbi pubmed searches for review articles regarding human ( 8 , 1825 ) and murine ( 22 , 2629 ) proteome of the lysosome and from lists of lysosomal genes present in the gene ontology ( 30 ) , kegg ( 31 ) , reactome ( 32 ) and uniprot databases ( 33 ) [ uniprot : lysosome ( kw-0458) and organism : homo sapiens ( human ) ( 9606) ; kegg : lysosome ( ko04142) ; go : go:0005764 data stamp from the source 20120303 ] .
the references listed within each review article ( 34 , 35 ) were examined and lists of genes were extracted from either the full text or the supplementary information following a manual curation .
there are 16 sources of information divided in four main categories : proteomic studies , databases , reviews and system biology approaches .
each gene has been associated to its official hgnc gene symbol ( 36 ) and to its entrez gene i d ( mappings were based on data provided by entrez with a date stamp from the source of 7 march 2012 ) .
the gene transcripts associated to each gene are annotated accordingly to ncbi refseq or genbank ( release 57 ) .
mirna target predictions were extracted from the tables downloaded from the websites of the different algorithms used to predict the binding between mirna and gene transcripts .
coordinated lysosomal expression and regulation ( clear ) is a nucleotide motif ( gtcacgtgac ) found to be highly enriched in the promoter set of lysosomal genes ( 8) .
we mapped this motif on both strands on the human genome ( hg19 ) by means of fuzznuc utility of the emboss package allowing one single mismatch ( 37 ) .
the binding sites of the tfeb come from a chip - seq experiment carried out on hela cell lines ( 38 ) .
hlgdb is a mysql 5.0.95 database ( constructed in the fourth normal form , some redundancy being kept to increase retrieval performance ) , and the interface is built in php .
search for gene lists : hlgdb can be used to retrieve and combine lists of lysosomal genes from different sources . the any and all options allow the user to either merge or intersect lists .
search for a gene using the gene symbol is also allowed ( figure 1 ) .
search for mirna targets : once the user has selected or created a gene list , he / she can find mirna ( or families of mirna ) targets choosing different combinations of prediction softwares .
results are returned in a table showing information about the gene ( gene symbol and gene name ) and the mirna ( identifier and number of softwares predicting each binding ) .
gene symbols are hyperlinked to gene - centered page where additional gene information is provided ( figure2a ) . on each gene transcript associated to the selected gene ,
search for tfeb binding sites / clear motifs : once the user has selected or created a list , he / she can choose the filter and find the lists of genes with tfeb binding sites or clear motifs within a range around the transcription start site ( tss ) .
results are provided in the table showing gene - centered information , as the hit count and distance from tss of tfeb binding sites / clear motifs .
all data in hlgdb are freely available for download as tab - delimited text files without password protection for any user
. concerning other organisms , currently the database provides a parallel orthology annotation for mouse : the user can select the species of interest using the upper right botton .
it collects information about these genes and their transcriptional regulation such as tfeb binding sites and mirnas .
hlgdb was designed to become a lysosomal gene census . when new lysosomal genes will be discovered
lysosomal genes include lysosomal hydrolases , lysosomal membrane proteins , lysosomal proteins involved in acidification and non - lysosomal proteins fundamental for this organelle biogenesis .
in addition , there is increasing evidence that lysosome genes play a role in the pathogenesis of common neurodegenerative diseases such as alzheimer s , parkinson s and huntington s .
researchers may benefit from hlgdb because they have in a single reference to the broadest compendium of lysosomal gene lists .
results of mirna targets may be directly compared with other transcriptional regulation elements such as the distance from the tss of tfeb binding site or the distance to a clear sequence to identify common features of regulation .
hlgdb has been designed to integrate additional layers of biological information , such as experimental data and comparative genomics . currently the database present information for human and mouse species ; in the next versions , additional species , such as rat , will be integrated .
finally , hlgdb provides a powerful resource to system biology approaches and network analysis to dissect the map of interactions taking place in the lysosomal system .
| lysosomes are cytoplasmic organelles present in almost all eukaryotic cells , which play a fundamental role in key aspects of cellular homeostasis such as membrane repair , autophagy , endocitosis and protein metabolism .
the characterization of the genes and enzymes constituting the lysosome represents a central issue to be addressed toward a better understanding of the biology of this organelle . in humans , mutations that cause lysosomal enzyme deficiencies result in > 50 different disorders and severe pathologies .
so far , many experimental efforts using different methodologies have been carried out to identity lysosomal genes .
the human lysosome gene database ( hlgdb ) is the first resource that provides a comprehensive and accessible census of the human genes belonging to the lysosomal system .
this database was developed by collecting and annotating gene lists from many different sources .
references to the studies that have identified each gene are provided together with cross databases gene related information .
special attention has been given to the regulation of the genes through micrornas and the transcription factor eb .
the hlgdb can be easily queried to retrieve , combine and analyze information on different lists of lysosomal genes and their regulation by microrna ( binding sites predicted by five different algorithms ) .
the hlgdb is an open access dynamic project that will permit in the future to collapse in a unique publicly accessible resource all the available biological information about lysosome genes and their regulation.database url :
http://lysosome.unipg.it/ |
a malignant peripheral nerve sheath tumor ( mpnst ) is a sarcoma that arises from peripheral nerves or cells of the associated nerve sheath ( schwann cells , perineural cells , fibroblasts ) .
the term " mpnst " has substituted previously used terms such as malignant schwannoma , neurofibrosarcoma , and neurogenic sarcoma1,2 ) .
the mpnsts comprise approximately 5% to 10% of all soft tissue tumors and have an incidence of 0.001% in the general population and the etiology is unknown .
however , more than half of mpnst cases develop in patients with neurofibromatosis type-1 ( nf-1 ) and there is a higher incidence in patients that have undergone prior radiotherapy1,2,5,8,13 ) .
mpnsts are most likely to metastasize to the lungs , followed by the bone and finally the pleura , but , spinal cord and brain metastasis is very rare in non - nf-1 mpnst3,7 ) .
the authors report a very rare case of spinal cord and brain metastasis of mpnst in the lumbar spine that occurred in a 18-year - old man without nf .
an 18-year - old man presented initially with a 6-month history of low back pain that worsened over time .
one month prior to admission , the patient developed radiating pain to his anterior thigh .
neither caf au lait spots nor neurofibromas were seen and his family history was absent of nf-1 .
the neurologic examination showed normal motor power in his lower extremities . magnetic resonance imaging ( mri ) showed a paraspinal mass that extended from the central space of l2 to the right psoas muscle through right l2 - 3 foraminal space .
it was an asymmetrical dumbbell shaped mass and the thecal sac was displaced left by the mass .
the mass showed iso - signal intensity on t1-weighted images and mixed signal intensity on t2-weighted images .
a paramedian incision was made from the l1 to l4 and we exposed the right l2 and l3 lamina with transverse process .
right l2 hemilaminectomy was performed using a high speed drill and kerrison rongeur to expose the thecal sac .
the margin of mass was relatively distinct from neural tissue , thus gross total resection was possible under microscopic view . to remove the mass extended to the psoas muscle ,
the right transverse process of l2 and l3 was resected and the mass was visible .
after the operation , his symptom had improved . however , a few days later , his confirmed pathologic report was presented as mpnst ( fig .
the contrast - enhanced mri was followed at 1 month after the operation and revealed a total removal of tumor ( fig .
however , the patient declined the external beam radiation due to the fear of potential radiation risk .
spine and brain mri showed that the tumor recurrence with leptomeningeal , multiple spinal cord and brain metastases ( fig .
4 ) . complete surgical clearance of the tumor was not technically feasible , owing to the multiple metastases .
therefore , the patient was referred for adjuvant chemotherapy . in addition , intrathecal chemotherapy was performed .
after 4 months of chemotherapy , he was referred to the hospice hospital and died within a month .
an 18-year - old man presented initially with a 6-month history of low back pain that worsened over time .
one month prior to admission , the patient developed radiating pain to his anterior thigh .
neither caf au lait spots nor neurofibromas were seen and his family history was absent of nf-1 .
the neurologic examination showed normal motor power in his lower extremities . magnetic resonance imaging ( mri ) showed a paraspinal mass that extended from the central space of l2 to the right psoas muscle through right l2 - 3 foraminal space .
it was an asymmetrical dumbbell shaped mass and the thecal sac was displaced left by the mass .
the mass showed iso - signal intensity on t1-weighted images and mixed signal intensity on t2-weighted images .
a paramedian incision was made from the l1 to l4 and we exposed the right l2 and l3 lamina with transverse process .
right l2 hemilaminectomy was performed using a high speed drill and kerrison rongeur to expose the thecal sac . for removal of intradural portion
the margin of mass was relatively distinct from neural tissue , thus gross total resection was possible under microscopic view . to remove the mass extended to the psoas muscle ,
the right transverse process of l2 and l3 was resected and the mass was visible .
after the operation , his symptom had improved . however , a few days later , his confirmed pathologic report was presented as mpnst ( fig .
the contrast - enhanced mri was followed at 1 month after the operation and revealed a total removal of tumor ( fig .
however , the patient declined the external beam radiation due to the fear of potential radiation risk .
spine and brain mri showed that the tumor recurrence with leptomeningeal , multiple spinal cord and brain metastases ( fig .
4 ) . complete surgical clearance of the tumor was not technically feasible , owing to the multiple metastases .
in addition , intrathecal chemotherapy was performed . however , no significant effect was observed and the patient 's condition deteriorated gradually .
after 4 months of chemotherapy , he was referred to the hospice hospital and died within a month .
non - nf-1 mpnsts are very rare and the occurrence of mpnsts occurring in adolescents is very rare
. mpnsts generally occur in adulthood typically between the ages of 20 and 50 years of age .
approximately 10 - 20% of cases have been reported to occur in the first 2 decades of life , with occasional cases involving infants as young as 11 months of age5,6 ) .
hruban et al . described 43 patients with mpnst and of these patients , 28 ( 65% ) had distant metastasis3 ) .
sites of metastasis included the lungs ( 22 cases ) , bone ( 9 cases ) , pleura ( 6 cases ) , retroperitoneum ( 4 cases ) , diaphragm ( 3 cases ) , inguinal lymph nodes ( 2 cases ) , liver ( 2 cases ) , chest wall ( 2 cases ) , soft tissue ( 2 cases ) , pulmonary hilum , pericardium , thyroid gland , and adrenal gland ( 1 case each ) .
described 11 distant metastasises in 54 patients with mpnst that included metastases to lymph nodes , pleura , lung , liver , adrenal gland , leptomeninges and brain7 ) .
william et al.4 ) reported a non - nf-1 mpnst case of metastasis to the spinal cord4 ) .
our case showed that leptomeningeal , spinal cord and brain metastasis of non - nf-1 mpnst for relatively short period after tumor removal in young male patient , reflecting aggressive nature of this malignancy .
the outcome with respect to both local recurrence and distant metastasis largely depends on grade of surgical excision .
radiation therapy combined with wide surgical excision offers statistical significant reduction in the rates of local disease recurrence .
however , it has not had a meaningful reduction in either rate of distant metastasis or overall survival15 ) .
benefit of chemotherapy is unproven and its application is limited to high grade metastatic disease3 ) .
the italian and german soft tissue sarcoma cooperative group reported an overall pediatric response rate of 45% , with the highest response noted within the ifosfamide group9,11,12 ) .
the recurrence rate for mpnsts has been reported to range from 40 - 68%7,10,14 ) .
the 5-year survival rate has been reported at 16 - 52% and a favorable prognosis has been related with complete surgical excision of the tumors that are sized less than 5 cm7,10 ) .
historically , mpnsts have been difficult tumors to treat due to their inherently aggressive nature and dismal prognosis .
our patient underwent complete surgical excision , but the patient was not treated with radiotherapy .
follow - up images revealed multiple spinal cord and brain metastases . the patient was managed with chemotherapy , but expired several months later . despite complete surgical excision ,
we reported a case of mpnst that had metastasized to the brain and spinal cord .
because the mpnst showed both rapid and aggressive progression , patients should be followed carefully to identify local recurrence or metastasis and be mandatory an adjuvant radiotherapy . | a malignant peripheral nerve sheath tumor ( mpnst ) is a type of sarcoma that arises from peripheral nerves or cells of the associated nerve sheath .
this tumor most commonly metastasizes to the lung and metastases to the spinal cord and brain are very rare .
we describe a case of young patient with spinal cord and brain metastases resulting from mpnst .
an 18-year - old man presented with a 6-month history of low back pain and radiating pain to his anterior thigh . magnetic resonance imaging showed a paraspinal mass that extended from the central space of l2 to right psoas muscle through the right l2 - 3 foraminal space .
the patient underwent surgery and the result of the histopathologic study was diagnostic for mpnst .
six months after surgery , follow - up images revealed multiple spinal cord and brain metastases .
the patient was managed with chemotherapy , but died several months later . despite complete surgical excision ,
the mpnst progressed rapidly and aggressively .
thus , patients with mpnst should be followed carefully to identify local recurrence or metastasis as early as possible . |