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ameloblastic fibroma ( af ) is an extremely rare true mixed benign tumor that can occur either in the mandible or maxilla .
it is frequently found in the posterior region of the mandible , often associated with an unerupted tooth .
it usually occurs in the first two decades of life with a slight female predilection , causing delay in tooth eruption or altering the eruption sequence .
small tumors are asymptomatic , while larger ones produce significant swelling of the jaws . on radiographs ,
smaller lesions are well circumscribed and unilocular with a sclerotic border , while larger ones are multilocular .
histopathologically , af consists of odontogenic epithelium in the dental papilla - like background without dental hard tissue formation .
similar lesions with hard tissue deposits are categorized as ameloblastic fibro - odontoma or odontoma depending on the degree of calcification .
recently , there are few reports of this condition with high recurrence rates and malignant transformation .
a 19-year - old female patient came to the department of oral medicine and radiology with a chief complaint of slowly progressive swelling on the right side of her lower posterior jaw .
on intraoral examination , the right lower buccal vestibule was obliterated due to the expansion of the buccal cortical plate .
the mucosa over the swelling was normal , except for slight blanching due to expansion of the buccal cortical plate .
the lesion is seen extending from posterior aspect of first molar to the retromolar area , measuring 3 cm in size .
on palpation , the swelling was slightly tender and no abnormality was detected in the adjacent teeth .
panoramic radiograph showed a unilocular radiolucent area with well - defined borders , involving the posterior aspect of the right mandible .
the lesion was measuring approximately 3.5 cm in size and was extending posteriorly from the distal aspect of the second molar to the retromolar area .
microscopically , hematoxylin and eosin sections showed islands and strands of epithelial cells in a loose connective tissue stroma resembling primitive dental papilla [ figure 1 ] .
the peripheral epithelial cells lining the islands and strands were low columnar , similar to the cells found in the peripheral layer of the follicle in ameloblastoma .
the connective tissue resembled cellular fibroblastic tissue similar to the dental papilla in the developing tooth .
hyaline - like tissue is also seen adjacent to the epithelial strands and islands [ figure 2 ] .
it was interesting to note that both the epithelial islands and connective tissue stroma revealed high cellularity when compared with the conventional lesions of af . however , severe dysplastic features such as cellular and nuclear pleomorphism and increased number of abnormal mitotic figures were not observed . in this context ,
immunohistochemistry for ki-67 [ figure 3a ] proliferative marker along with proper positive [ figure 3b ] and negative [ figure 3c ] controls were performed .
this had ruled out the diagnosis of a malignant tumor , confirming the diagnosis of af .
photograph showing strands and islands of odontogenic epithelial cells in a loose connective tissue stroma resembling primitive dental papilla ( h and e 100 ) photograph showing the peripheral cells resembling ameloblasts with hyaline - like material surrounding the islands ( h and e 400 ) ( a ) photograph showing negative staining for ihc marker ki-67 antigen ( b ) photograph of positive control for ihc marker ki-67 antigen ( c ) photograph of negative control for ihc marker ki-67 antigen
the histopathological finding of high cellularity in this lesion is uncommon as conventional afs show mild to moderate cellularity in a loose myxomatous connective tissue background .
our case of af with high cellularity is unusual in the context of its histopathology . | ameloblastic fibroma is a rare odontogenic tumor comprising neoplastic epithelial and mesenchymal tissues .
this lesion was previously considered to be a benign lesion with very limited recurrence rate and malignant transformation .
however , recent reports have suggested that this lesion has the potential for recurrence and malignant transformation . in this brief report
, we report a case of af in the context of its high cellularity on histopathological examination . |
the burden of non - communicable disease in the developing countries is increasing and leading to high mortality rates . nowadays , type 2 diabetes ( t2 dm ) is pandemic and there are no signs of reduction in incidence rates .
diabetes is one of the most important risk factors for cardiovascular diseases and is the most prevalent cause of mortality in diabetic patients .
metabolic syndrome is highly prevalent in type 2 diabetic patients and is a strong risk factor for cardiovascular diseases in such patients .
it is estimated that 16% of people above 20 years of age globally have metabolic syndrome . in iran , this syndrome is dramatically prevalent and close to 34% of people above 20 years old have metabolic syndrome , according to the adult treatment panel ( atpiii ) .
differences in genetics , diet , physical activity , age and gender in different societies can affect the prevalence of this syndrome .
there is no globally accepted definition for the diagnosis of this syndrome and most studies use the atpiii criteria for defining metabolic syndrome . in 2005 , the international diabetes federation ( idf ) has suggested a definition for the diagnosis of people with metabolic syndrome , which is applicable to different ethnic groups . in the idf criteria ,
central obesity is a prerequisite and central obesity assessment by waist circumference for different genders and ethnic groups according to idf is necessary .
, the appropriate cutoff point for the diagnosis of metabolic syndrome in men and women in kerman has been determined .
currently , metabolic syndrome has become an important health problem and there is a need for investigation in this field .
considering the fact that diabetic patients with metabolic syndrome also have cardiovascular risk factors , the diagnosis of metabolic syndrome in such patients is very important , and leads to a better disease control and prevention of cardiovascular complications .
the present study was performed to determine the prevalence of metabolic syndrome and its related factors in type 2 diabetic patients of kerman , iran .
the data used in this study was part of the coronary artery disease risk factors ( kercadr ) study in kerman .
in kercadr , 5,900 people were recruited from which 950 people were diagnosed with diabetes and entered into our study in 2013 .
the variables evaluated in this study were age , gender , job , education , bmi , total cholesterol , hdl cholesterol , ldl cholesterol , fasting blood sugar ( fbs ) , systolic blood pressure , diastolic blood pressure , waist circumference , triglycerides , smoking , physical activity , the duration of diabetes , history of cardiovascular diseases , and the ratio of waist circumference to hip circumference .
data was analyzed by chi - square , t - test , and logistic regression using spss ( revision 20 ) software .
the prevalence of metabolic syndrome in people with diabetes was determined according to the below criteria .
the atpiii criteria that include three or more of the following items :
central obesity : waist circumference more than 102 centimeters in men and more than 88 cm in womentriglycerides 150 mg / dl or using drugs for hyper triglyceridemiacholesterol hdl < 40 mg / dl in men and less than 50 mgr / dl in women or under treatment for low hdl cholesterolhypertension systolic blood pressure more than 130 mmhg or diastolic blood pressure 85 mmhg or being under treatment for hypertensionfbs 100 mg / dl or being under treatment for diabetes or a diagnosis of type 2 diabetes.according to the idf 2005 criteria , central obesity ( waist circumference 94 cm in men and 80 cm in women ) and two or more of these criteria should be present : triglyceride 150 mg / dl or under treatment for hyper triglyceridemiacholesterol hdl < 40 mg / dl in men and less than 50 mgr / dl in women or under treatment for low hdl cholesterol levelshypertension systolic blood pressure more than 130 mmhg or diastolic blood pressure 85 mmhg or being under treatment for hypertensionfbs 100 mg / dl or being under treatment for diabetes or a diagnosis of type 2 diabetes .
central obesity : waist circumference more than 102 centimeters in men and more than 88 cm in women triglycerides 150 mg / dl or using drugs for hyper triglyceridemia cholesterol hdl < 40 mg / dl in men and less than 50 mgr / dl in women or under treatment for low hdl cholesterol hypertension systolic blood pressure more than 130 mmhg or diastolic blood pressure 85 mmhg or being under treatment for hypertension fbs 100 mg / dl or being under treatment for diabetes or a diagnosis of type 2 diabetes . according to the idf 2005 criteria , central obesity ( waist circumference 94 cm in men and 80 cm in women ) and two or more of these criteria should be present : triglyceride 150 mg / dl or under treatment for hyper triglyceridemia cholesterol hdl < 40 mg / dl in men and less than 50 mgr / dl in women or under treatment for low hdl cholesterol levels hypertension systolic blood pressure more than 130 mmhg or diastolic blood pressure 85 mmhg or being under treatment for hypertension fbs 100 mg / dl or being under treatment for diabetes or a diagnosis of type 2 diabetes .
study is the presence of central obesity ( waist circumference 86 cm in men and 83 cm in women ) plus two or more of these criteria :
triglyceride 150 mg / dl or under treatment for hyper triglyceridemiacholesterol hdl < 40 mg / dl in men and less than 50 mgr / dl in women or under treatment for low hdl cholesterol levelshypertension systolic blood pressure more than 130 mmhg or diastolic blood pressure 85 mmhg or being under treatment for hypertensionfbs 100 mg / dl or being under treatment for diabetes or a diagnosis of type 2 diabetes .
triglyceride 150 mg / dl or under treatment for hyper triglyceridemia cholesterol hdl < 40 mg / dl in men and less than 50 mgr / dl in women or under treatment for low hdl cholesterol levels hypertension systolic blood pressure more than 130 mmhg or diastolic blood pressure 85 mmhg or being under treatment for hypertension fbs 100 mg / dl or being under treatment for diabetes or a diagnosis of type 2 diabetes .
the present study was carried out on 950 patients with diabetes type 2 in which 392 ( 41.3% ) were male and 558 ( 58.7% ) were female .
the mean age of the patients was 56.011.6 ( females 55.111.1 and males 57.212.2 ) .
the prevalence of metabolic syndrome according to atpiii was 73.4% ( men 64.8% and women 79.4% ) . according to the idf criteria
, it was 64.9% ( men 46.7% and women 77.8% ) and according to the new criteria , it was 70.4% ( men 69% and women 71.3% ) .
figure 1 compares the prevalence of metabolic syndrome according to the three criteria . shows the prevalence of metabolic syndrome according to the three criteria among type 2 diabetics in different age groups .
based on the result of the three criteria , among the females low hdl , high bmi , weight , blood pressure , ldl , total cholesterol , and triglyceride were associated with increased metabolic syndrome prevalence .
the compatibility score of the atpiii and idf according to the kappa statistic was 0.49 , for idf and the new kerman criteria was 0.66 , and for atpiii and the new kerman criteria was 0.50 .
multivariate logistic regression adjusted for confounders , in people who had metabolic syndrome according to both idf and atpiii criteria , showed that gender , fasting blood sugar , triglycerides , hdl , waist circumference and systolic blood pressure were predictive factors for metabolic syndrome in the population .
also in people who had metabolic syndrome according to the three criteria , the same variables were able to predict the incidence of metabolic syndrome .
the adjusted or with 95% confidence intervals for factors predicting metabolic syndrome according to both atpiii and idf
in the present study , the prevalence of metabolic syndrome in patients with diabetes was 64% to 73% . in studies carried out in ahvaz ,
the prevalence of metabolic syndrome in diabetic patients was 73.1% ( atpiii ) and 64.9% ( idf ) , in esfahan 65% ( atpiii ) , and in england 94% ( both atpiii & idf ) .
in other studies , the prevalence of metabolic syndrome was estimated to be 62.3% and 56.8% ( idf and atpiii ) in india , and 71.1% and 60.4% ( idf and atpiii ) in sub - saharan africa . in the present study , according to the three criteria , the prevalence of metabolic syndrome was higher in women than men . in all other studies ,
the reason might be less activity , more weight and higher prevalence of dyslipidemia in women .
the female gender , fasting blood sugar , triglyceride , cholesterol , hdl , waist circumference and systolic blood pressure were able to predict the prevalence of metabolic syndrome in people with diabetes . in the ahvaz study , the female gender , physical activity , microalbuminuria , serum triglyceride and cholesterol , and in the isfahan study , age , gender , fasting blood sugar , hypertension , bmi , serum triglyceride and serum cholesterol were the predictors of metabolic syndrome . the female gender , cholesterol , and triglyceride in all these studies were a predicting factor .
the difference among other variables could be due to differences in data collection , sampling methods and using different criteria in diagnosing people with metabolic syndrome . among the limitations of the present study
is that the population was from kerman province and the results may therefore not be generalisable to other populations .
the present study shows that the prevalence of metabolic syndrome in people with type 2 diabetes is very high in kerman . | metabolic syndrome is highly prevalent in type 2 diabetics and is a strong risk factor for cardiovascular diseases in such patients .
the aim of this study was to determine the prevalence of metabolic syndrome according to the three criteria of atpiii , idf and the new criteria for metabolic syndrome diagnosis in kerman , iran .
this cross - sectional study was performed on 950 diabetic type 2 patients .
data was analyzed by independent t - test , chi - square and logistic regression using the spss ( revision 20 ) software .
the prevalence of metabolic syndrome in kerman was 73.4 , 64.9 , and 70.4% , according to the above criteria .
fasting blood sugar , gender , triglyceride , hdl , waist circumference , and systolic blood pressure were related to the prevalence of metabolic syndrome according to the above - mentioned criteria.the prevalence of metabolic syndrome is high in type 2 diabetic patients and the above - mentioned factors exacerbate the situation . |
insulin resistance is a classic risk factor for the development of type 2 diabetes mellitus ( t2 dm ) . a significant predisposition to insulin resistance
obesity and its associated comorbidities are reaching never - seen - before levels worldwide . with sufficient increases in basal and nutrient - stimulated insulin levels ,
systemic insulin levels can be increased by one of two general mechanisms : increased insulin secretion from individual beta cells ( that is , beta cell function ) and increased total numbers of beta cells ( that is , beta cell mass ) .
reduced beta cell function in t2 dm is clearly an early and important aspect underlying the pathophysiology of t2 dm ; hence , addressing this dysfunction is a valid therapeutic strategy for t2 dm .
( little is known of the effects of the therapeutics discussed in this review on alpha cell function . as dysregulated glucagon secretion has also been noted as a key issue in t2 dm pathophysiology , these are clearly important questions to address , but are outside of the scope of this review . )
the first t2 dm therapeutics to target the beta cells were the sulfonylurea compounds , which act directly on the atp - sensitive potassium channels of the beta cell to enhance membrane depolarization , stimulating exocytosis of insulin granules . while still used clinically and excellent drugs in some circumstances , sulfonylureas act in a glucose - independent manner , increasing the potential risk of hypoglycemia .
furthermore , sulfonylureas and other long - established therapeutics , including the insulin - sensitizer metformin , do not prevent the continuing loss of beta cell function observed in t2 dm . however , weight loss from metformin may indirectly benefit beta cell function and limit beta cell loss in t2 dm . a different aspect of beta cell dysfunction
is being addressed by recent t2 dm therapeutics : the lack of potentiation of insulin secretion in response to an oral glucose / nutrient challenge .
this potentiation occurs because of peptide hormones termed incretins that are secreted from the digestive tract and act on the pancreas and other peripheral tissues to elicit biological effects , including augmenting nutrient - stimulated insulin secretion .
the incretin receptors belong to a superfamily of transmembrane proteins termed as g protein - coupled receptors ( gpcrs ) that promote intracellular signal transduction pathways mediated by guanine nucleotide - binding proteins ( g proteins ) .
there are four major classes of g proteins : gs , gi , gq , and g12 .
these proteins are classified based on the sequence identity of their alpha subunit , which confers similar downstream signaling mechanisms among subfamily members .
gs is the sole member of its subfamily not restricted to olfactory / taste sensory cells , while similar members of the gi subfamily are gi1 , gi2 , gi3 , go1 , go2 and gz
. the gs and gi subfamilies also include specialized members : golf is a gs subfamily member restricted to olfactory cells , while gt and ggust are gi subfamily members restricted to the retina and taste cells , respectively . while taste and olfactory cell signaling is an interesting focus of current obesity and diabetes research ( for example , see work from dotson et al . ) , only the more ubiquitously expressed g proteins will be discussed in this review .
the gs and gi subfamily members all signal by modulating the levels of the intracellular second - messenger cyclic amp ( camp ) , either stimulating ( gs ) or inhibiting ( gi ) the production of camp by adenylate cyclase .
camp has also been shown to regulate beta cell proliferation and survival , potentially linking these receptors to regulation of beta cell mass .
the gq class of g proteins is also stimulatory toward insulin secretion , but acts through separate second - messenger pathways : inositol trisphosphate and diacylglycerol , which are formed when gq - activated phospholipase c- cleaves phosphatidylinositol bisphosphate
not as much is known regarding the role of g12 on beta cell function , but its actions through the small g protein , rho , appear to be important for promoting the exocytosis process itself .
drugs targeting the glucagon - like peptide 1 ( glp-1 ) receptor , a gs - coupled incretin receptor , are some of the newer t2 dm therapeutics , having been used clinically for nearly a decade .
the stable glp-1 analogs , such as exenatide ( also known as byetta , amylin pharmaceuticals , san diego , ca , usa ) , are resistant to inactivating cleavage by dipeptidyl peptidase 4 , which rapidly degrades endogenous glp-1 .
in addition , endogenous glp-1 can be stabilized by treatment with dipeptidyl peptidase inhibitors , such as sitagliptin ( also known as januvia , merck , whitehouse station , nj , usa ) .
several other gq- and gs - coupled incretin receptors are being explored as t2 dm therapeutic targets and have been reviewed elsewhere .
even with these successes , incretin - based therapeutics do not work in all individuals and safety concerns , including pancreatitis and gastrointestinal distress , in addition to possible side effects such as weight changes lead to non - compliance .
these concerns raise the possibility of targeting potential dysfunction in other components of beta cell signaling pathways .
endogenous inhibitors of beta cell function have long been known , even before the identity of their receptors and associated g proteins were established .
for example , alpha adrenergic receptor ( -ar ) agonists such as epinephrine and norepinephrine are released from the sympathetic nervous system as part of the fight - or - flight response , one aspect of which is to maintain higher glucose levels in the bloodstream .
-ars are expressed in beta cells , and agonists of the 2-ar subfamily , which are gi - coupled receptors , rapidly reduce glucose - stimulated insulin secretion .
another long - established inhibitor of beta cell function , prostaglandin e2 ( pge2 ) , is produced by cyclooxygenase ( cox)-catalyzed metabolism of arachidonic acid and acts in an autocrine and/or paracrine fashion to modulate beta cell growth and insulin secretion .
interestingly , receptors for adrenergic agonists and pge2 , as well as their associated g proteins , have all been linked with diabetic beta cell pathophysiology .
insulin - responsive tissues besides the pancreas also play critical roles in the pathophysiology of t2 dm . the pancreatic circulation empties
directly into the portal vein , meaning that the liver is the first stop for hormones secreted from the pancreas , including insulin and glucagon .
insulin acts on the liver to signal the fed state of the body , promoting glucose storage into glycogen and blocking gluconeogenesis and lipolysis , blocking hepatic glucose and free fatty acid production .
it is well accepted that hepatic fuel production is dysfunctional in t2 dm , as insulin - resistant alpha cells secrete glucagon in an unregulated manner , further exacerbating the phenotype of liver insulin resistance . in addition , muscle and adipose tissue are stimulated by insulin to increase glucose uptake for use as fuel and storage as glycogen and triglycerides , respectively .
inefficient muscle and adipose glucose uptake is also observed in t2 dm and contributes to the pathology of hyperglycemia .
there is much debate surrounding the impact of enhanced beta cell function and mass to the development of insulin resistance in t2 dm .
this discussion is beyond the scope of this review , but has been well described elsewhere .
specific gpcrs , including melatonin receptor isoform 2 ( mt2 ) , have been shown to have important roles in regulating insulin sensitivity .
metabolic syndrome is often linked to obesity and visceral fat accumulation known to perturb blood glucose regulation and increase insulin resistance ; however , obesity by itself is not a definitive predictor of metabolic syndrome as some obese individuals do not present with typical etiologies .
a diagnosis of metabolic syndrome is a significant risk factor , though , for the development of t2 dm , cardiovascular disease , neurodegeneration , polycystic ovarian syndrome , cancer and non - alcoholic fatty liver disease , among others .
therefore , developing anti - obesity therapeutics as a preventative for these significant , chronic diseases is a major focus of current research .
several effective medical treatments for obesity have been removed from the market or are strictly regulated due to unacceptable side effects .
fenfluramine / phentermine ( also known as phen - fen ) was pulled from the us market in 1997 .
sibutramine ( also known as reductil , meridia and sibutrex , abbott labs , abbott park , il , usa ) is a schedule iv - controlled substance and was pulled from the us market in 2010 .
dextromethamphetamine ( also known as desoxyn , abbot labs ) is a methamphetamine derivative and a schedule ii - controlled substance .
furthermore , the few widely available medical treatments such as orlistat ( also known as xenical , roche , basel , switzerland ; and alli , glaxosmithkline , research triangle park , nc , usa ) have only modest effects at best .
surgical techniques such as gastric bypass and sleeve gastrectomy are often quite effective , but are also major procedures that have continuously decreasing , yet still not insignificant , peri- and post - operative morbidity and mortality risks . a safe , effective medical treatment to augment healthy lifestyle changes in the efforts to combat obesity and metabolic syndrome is sorely needed .
one inhibitory gpcr in particular , cannabinoid receptor type 1 ( cb1 ) has been demonstrated as a potentially effective anti - obesity pharmacological target . in this review , we will discuss gi subfamily members that have been linked to beta cell dysfunction , insulin resistance or both .
next , we will discuss a select number of inhibitory gpcrs that have been shown to have significant pre - clinical or clinical evidence of efficacy as anti - t2 dm and/or anti - obesity targets .
these include gpcrs whose major effect is in the beta cell , as potential targets to maintain beta cell function and/or mass ; and gpcrs with primary effects in other insulin - responsive tissues , as targets to improve insulin resistance and/or obesity .
it is our hope that this review will stimulate further research into these relatively under - studied proteins as potentially useful therapeutic targets for the obesity and t2 dm that is becoming a worldwide public health problem .
beta cell dysfunction and insulin resistance are early and necessary events in the development of t2 dm , and gi signaling has been shown to be an important regulator of both these events . the gi family inhibitor , pertussis toxin ( ptx ) , was originally named islet - activating protein due to its ability to promote glucose- and hormone - stimulated insulin secretion long before its mechanism of action was known .
even so , no insulin secretion phenotypes had been reported in mouse lines deficient in individual gi subfamily members , until those lacking only the ptx - insensitive gi subfamily alpha subunit , gz , were studied ( since then , other gi subfamily members that are sensitive to ptx have been linked with regulating beta cell function in vivo , explaining the effect of ptx on insulin secretion : these will be discussed in more detail below ) . by sequence and functional similarity , gz is clearly a member of the gi subfamily , but it is unique in that its alpha subunit ( gz ) lacks the c - terminal cysteine residue that is the substrate for ptx - mediated adp ribosylation and inactivation .
gz - null mice exhibit improved insulin secretion response and glucose clearance after glucose challenge , correlating with constitutively increased camp levels and improved glucose - stimulated insulin secretion from their isolated islets . these effects
the slow gz gtp hydrolysis and deactivation rate were posited as a potential explanation for constitutive effects , although endogenous beta cell gpcrs have been linked specifically with gz .
agonists activating the prostaglandin e receptor 3 ( ep3 ) isoform of the pge2 receptor block glucose - stimulated insulin secretion from insulinoma cells and isolated mouse islets in a ptx - insensitive manner , suggesting coupling of the ep3 receptor specifically to gz and not other gi proteins .
in addition , the modulation of endocytosis following exocytosis ( a required event in the maintenance of insulin secretion ) by norepinephrine is also ptx - insensitive . a synthetic peptide mimicking the c - terminus of the gz blocked this norepinephrine effect .
this is not unexpected , as gz has little - to - no expression in the liver , adipose tissue or skeletal muscle , and although expressed in the brain , does not seem to regulate appetite or energy metabolism .
even so , gz - null mice are completely protected from the development of glucose intolerance upon high - fat diet feeding , primarily through a significant augmentation of beta cell mass and maintenance of beta cell function .
these results suggest gz as an emerging target to improve both defects of the diabetic islet : reduced beta cell mass and decreased beta cell function .
other gi subfamily members , including both gi and go isoforms , have been implicated in directly inhibiting exocytosis from beta cells , which , together with their subcellular localization , indicates a role for gi proteins in the regulation of a distal step in the stimulated secretion pathway . in studies in an insulinoma cell line ,
norepinephrine inhibited the refilling of the readily releasable pool of secretory granules , an effect that was abolished by a dual gi1/2-blocking peptide .
perplexingly , mice deficient in gi2 expression did not have noticeably improved insulin secretion , but instead had reduced insulin sensitivity .
furthermore , expression of constitutively active gi2 in mouse liver and adipose tissue enhanced glucose tolerance . among other effects , a direct effect of gi2 signaling on glut4 membrane translocation through a phosphoinositide 3-kinase - dependent mechanism may be responsible for these phenotypes .
early studies with go - null mice indicated changes in the nervous system structure and aberrant heart calcium channel signaling , but no effect on blood glucose levels . in more recent work , one of the two splice variants of go , go2 ,
mice lacking go2 , but not those lacking go1 or any gi isoforms have augmented glucose - stimulated insulin secretion and improved glucose tolerance , just as observed with gz - null mice . moreover , these results help support earlier findings that targeting ptx - sensitive proteins , such as go2 , can potentiate insulin secretion but in a different manner than gz .
these results are important , as they suggest that the gi subfamily members in the beta cell are not redundant , as has been suggested in other systems and that specific isoforms and/or their associated receptors might be useful t2 dm therapeutic targets . of note ,
no small - molecule pharmaceutical agents in use clinically target a gpcr / g protein pathway directly at the g protein level ( although some have been proposed hypothetically and/or tested in vitro ) .
in addition , biased gpcr ligands can preferentially effect one versus another g protein - dependent or -independent downstream signaling pathway , but these are still receptor - based drugs and not g protein - based .
thus , understanding more about the endogenous gpcrs that these inhibitory g proteins couple to will likely be very important in pharmaceutically targeting their activation .
many metabolites of the -6 polyunsaturated fatty acid ( pufa ) arachidonic acid have been linked directly with inflammation .
e - series prostaglandins , comprising a major family of arachidonate metabolites , were posited as playing a role in t2d over 30 years ago .
pge2 in particular has long been known as a physiological inhibitor of insulin secretion , acting via autocrine or paracrine mechanisms .
this means that islet cells themselves , including beta cells , express the enzymes required for synthesis of pge2 , including phospholipase a2 , which cleaves arachidonate from plasma membrane phospholipids ; cox , which convert the fatty acid into the intermediate pgh2 ; and pge synthases ( ptges ) , which convert pgh2 to pge2 .
many groups ( including the american diabetes association ) have recommended a high -3 pufa diet as a means to prevent disease .
yet , there still exists some controversy as to the benefits of -3 pufas on chronic disease as well as the impact of high -3 pufas in the context of a diet that remains high in -6 pufas .
notable exceptions include two groups that generated transgenic mice expressing a caenorhabditis elegans desaturase that converts -6 pufas to the -3 form .
these mice robustly increased -3 pufa concentrations ( and reduced -6 pufas to near zero ) in all tissues , with improved insulin secretion from isolated islets .
inhibition of cox ( which , in beta cells , is primarily cox-2 ) can completely block pge2 production along with that of numerous other metabolites downstream of pgh2 and the other cox intermediates .
the upregulation of cox-2 expression and/or activity has been clearly linked with the diabetic state .
the potential utility of cox-2 inhibitors in t2 dm is overshadowed by concerns of significant cardiovascular risks , especially in a population at greater intrinsic risk of cardiovascular disease .
interestingly , after nearly a half century , there has been a recent resurgence in studies on the utility of salicylate , which acts directly at the site of inflammation to reduce cox-1 and cox-2 expression , as a t2d therapeutic .
recently , a specific pge2 receptor isoform , ep3 , has been suggested as an emerging target for t2 dm therapeutics .
ep3 receptor expression and activity was significantly upregulated in diabetic mouse islets , as was the production of pge2 . targeting the ep3 receptor with a specific antagonist , l798,106 ,
interestingly , ep3 signaling was found to oppose the action of the glp-1 receptor , discussed above as a significant potentiator of glucose - stimulated insulin secretion .
specifically , the maximal effect of glp-1 on insulin secretion was blunted by ep3 activation , and a combination of an ep3 antagonist and glp-1 agonist additively promoted insulin secretion from diabetic mouse islets .
the mechanisms mediating dysfunctional ep3 signaling in diabetic beta cells and potential ways to target ep3 in t2 dm therapy are shown on the left half of figures 1 .
although the ep3 receptor plays critical roles in islet biology , extrapancreatic function may be equally important in t2 dm pathophysiology ; in particular , in mediating t2 dm complications . the migratory response of vascular smooth muscle cells is required for the dysfunctional vascular remodeling observed with cardiovascular disease .
genetic deletion or pharmaceutical inhibition of ep3 results altered polarity and directional migration of vascular smooth muscle cells , suggesting that blockade of ep3 might protect from dysfunctional vascular remodeling .
in addition , mice lacking ep3 have reduced baseline and stimulated mean arterial pressures , and pharmacological inhibition of ep3 blocks angiotensin 2-mediated vasoconstriction .
thus , it appears that pge2 signaling through ep3 may play a significant role in the progression of t2 dm and its associated comorbidities .
mice lacking ep3 develop obesity , insulin resistance , glucose intolerance and eat considerably more than wild - type controls .
part of this phenotype can be explained by increased light cycle eating , most likely due to unstable sleep patterns , as pge2 ( acting through hypothalamic ep3 ) may act as a somnogen .
furthermore , high levels of pge2 and increased signaling through ep3 both augment nitric oxide synthase expression , an enzyme critical in the brain development of newborns .
this signaling cascade is suggested to play a significant role in connecting brain circulation and synaptic activity in perinatal development .
if ep3 antagonists are pursued as therapeutics for t2 dm and/or metabolic disease , a peripherally restricted ep3 antagonist might be necessary .
interestingly , an ep3 antagonist , dg-041 , completed a european phase ii clinical trial by decode genetics testing its effect in treating peripheral vascular disease , which is typically caused and/or complicated by hypertension .
the results of this trial were never published , so questions remain as to whether this drug was effective as an anti - hypertensive agent . recently
, dg-041 treatment in mice was found to limit atherothrombosis without altering hemostasis , suggesting that dg-041 can decrease clot formation without increasing bleeding risk , critical in the prevention of cardiovascular disease .
dysfunctional -ar signaling was noted in t2 dm at least 40 years ago . in an early clinical study of six diabetic individuals , -ar blockade with
-ar antagonists were never much pursued as anti - diabetic agents , though , perhaps due to the systemic effects of -ar signaling , including smooth muscle contraction , inhibition of neurotransmitter release , transient hypertension followed by sustained hypotension , and constriction of certain veins and arteries , including those leading to and from the heart , among others .
this possible thinking that -ar signaling was too pleiotropic to be targeted therapeutically in t2 dm changed in 2010 , when a single nucleotide polymorphism in the 2a - ar gene , adra2a , was discovered as a susceptibility factor for t2d : rs553668 . in two different studies , including over 5000 individuals , rosengren et al
. showed that individuals with a specific rs553668 genotype trended toward elevated fasting plasma glucose levels and reduced serum insulin levels .
furthermore , these individuals were significantly more prone to a hypoinsulinemic response during glucose tolerance testing , indicating a defect in the insulin secretion process itself .
when human pancreatic islet preparations were genotyped , those with the specific rs553668 variant had reduced insulin granule docking and secreted less insulin in response to glucose due to increased expression of 2a - ar at the membrane .
treatment of isolated islets with an 2a - ar antagonist , yohimbine , corrected the insulin secretory dysfunction in these islets . a clinical trial initiated in
the us is currently enrolling patients with this specific genotypic risk variant in a randomized , controlled trial of yohimbine as a t2d therapeutic .
a more recent meta - analysis of four prospective studies confirmed the linkage of rs553668 with fasting glucose and t2 dm risk , and found another variant of adra2a , rs17186196 , associated with fasting glucose levels , fasting insulin levels and whole - body insulin sensitivity .
furthermore , rs491589 was associated with systolic and diastolic blood pressure , while rs36022820 correlated with body mass index .
additional adra2a variants have been linked with hypertension , a significant risk factor for cardiovascular disease , a common complication of t2 dm . in a study of 70 hypertensive patients , 35 with and 35 without t2 dm ,
individuals with a genotype encoding for a small deletion in adra2a were significantly more likely to have hypertension , regardless of t2 dm status .
a potential physiological mechanism for this association was the significantly lower high - density lipoprotein and higher low - density lipoprotein in individuals homozygous for the deletion phenotype .
overall , these results confirm the utility of targeting the 2a - ar to improve beta cell function in t2 dm in a specific population , and also indicate its possible utility as a target for insulin resistance , obesity and/or t2 dm complications .
the mechanisms mediating dysfunctional 2a - ar activity in a specific subset of diabetic beta cells and potential ways to target 2a - ar in t2 dm therapy are shown on the right half of figure 1 .
it has been demonstrated in humans that disruption of the circadian rhythm promotes obesity and diabetes , and leads to perturbations in plasma glucose and insulin concentrations .
for instance , melatonin ( n - acetyl-5-methoxytryptamine ) is secreted from the pineal gland and has a major role in the regulation of circadian rhythms .
melatonin acts through two receptors , mt1 ( gene name : mtnr1a ) and mt2 ( gene name : mtnr1b ) .
activation of mt1 or mt2 receptors in the beta cell inhibit insulin secretion . however , it is not melatonin antagonists that have been proposed as obesity / t2 dm therapeutics , but rather melatonin agonists .
this is because mt1/mt2 receptor signaling also reduces dyslipidemia , reduces hepatic glucose output , decreases adiposity and improves metabolic function .
thus , stimulating melatonin signaling has been proposed as a treatment for metabolic syndrome . altered melatonin receptor expression or loss - of - function mutations and altered melatonin secretion itself
melatonin was shown to function by reducing hepatic gluconeogenesis and phosphorylation and activation of akt in the hypothalamus .
most of the recent research into the links between the melatonin receptors and diabetes is focused on mt2 .
independent genome - wide association studies in european populations identified single nucleotide polymorphisms near mtnr1b that were associated with fasting plasma glucose , reduced beta cell function , and significantly increased t2 dm risk : rs1387153 and rs10830963 .
expression of these two single nucleotide polymorphisms is also significantly associated with the development of gestational diabetes , as well as elevated fasting glucose and reduced insulin secretion after parturition .
a diagnosis of gestational diabetes is one of the highest predictors for the later development of t2 dm .
a clinical trial was registered in late 2012 to test the effects of 3 months of melatonin treatment on glucose tolerance in individuals with different mtnr1b variants .
cb1 is a gi - coupled receptor located in the central and peripheral nervous system as well as other insulin - responsive tissues .
endogenous cb1 ligands include anandamide ( which is responsible for pleasurable effects , such as that from eating chocolate ) and 2-arachidonoyl glyceride .
natural and synthetic cannabinoids such as tetrahydrocannabinol , the main active compound of the psychoactive drug cannabis , and its synthetic analog , dronabinol , are potent cb1 agonists . in the brain ,
cb1 is highly expressed in the hypothalamus , which regulates appetite , and the medulla , which contains the chemoreceptor trigger zone , regulating nausea and vomiting , and where it is involved in the regulation of normal brain aging .
this explains why tetrahydrocannabinol is such an effective drug at combatting the nausea and loss of appetite associated with chemotherapy .
despite potential side effects such as nausea , the appetite - suppressive effects of central cb1 stimulated research into antagonists of the receptor as potential anti - obesity therapeutics .
several trials of the systemic cb1 antagonist , rimonabant , as a weight - loss agent were initiated in both europe and north america .
overall , rimonabant was superior to both sibutramine and orlistat , with a nearly 5-kg average weight loss over a 1-year period ( vs 4.5 kg for sibutramine and 2.5 kg for orlistat ) . also , t2 dm patients treated with rimonabant in addition to metformin or sulfonylurea had significantly lower hba1c levels than those treated with metformin or sulfonylurea alone .
not surprisingly , nausea was a common adverse effect in the rimonabant group ( 13% , as compared with 4% with placebo ) , as well as were a number of severe psychological alterations .
specifically , 15% percent of rimonabant - treated patients withdrew from the trials , and nearly half of these dropouts were attributed to mood alterations such as depression and increased suicide ideation .
these outcomes halted any ongoing rimonabant trials , and the cb1 antagonist field made little progress over the next few years . perhaps due to the importance of cb1 in mediating appetite in the central nervous system ,
these include the neutral cb1 antagonists , which have a weaker affinity for receptor , but also peripherally restricted cb1 antagonists , which can not cross the blood - brain barrier and thus have no direct central nervous system effects .
insulin - responsive tissues such as the liver , pancreas , adipose and muscle also express cb1 , and antagonism of these receptors promotes insulin signaling , blocks lipogenesis , promotes fatty acid oxidation and stimulates energy - requiring processes such as mitochondrial biogenesis and cell growth and differentiation .
cb1 receptors are also located on afferent and efferent nerve fibers ; thus , the insulin - responsive state of the body is transmitted to the brain , where it can send signals to decrease food intake .
overall , a requirement for direct central nervous system activation of cb1 receptors does not seem to exist in order to elicit positive metabolic changes , and perhaps , weight loss .
several recent publications show strong pre - clinical efficacy of these peripherally restricted cb1 antagonists .
in recent years , we have learned much about the role of inhibitory g proteins and gpcrs in mediating dysfunctional signaling pathways in metabolic diseases such as t2 dm and confirmed their efficacy in pre - clinical models and select human studies .
a number of other inhibitory gpcrs not discussed above may also have therapeutic potential ( for example , serotonin 2a , ghrelin , npy and gpr41 , among others ; reviewed by ahren ) .
yet , we still have a long way to go before demonstrating that any of these proteins are suitable targets for t2 dm and obesity in the general population . | the worldwide prevalence of obesity is steadily increasing , nearly doubling between 1980 and 2008 .
obesity is often associated with insulin resistance , a major risk factor for type 2 diabetes mellitus ( t2 dm ) : a costly chronic disease and serious public health problem .
the underlying cause of t2 dm is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance .
most current t2 dm therapeutics do not prevent continued loss of insulin secretion capacity , and those that do have the potential to preserve beta cell mass and function are not effective in all patients .
therefore , developing new methods for preventing and treating obesity and t2 dm is very timely and of great significance .
there is now considerable literature demonstrating a link between inhibitory guanine nucleotide - binding protein ( g protein ) and g protein - coupled receptor ( gpcr ) signaling in insulin - responsive tissues and the pathogenesis of obesity and t2 dm .
these studies are suggesting new and emerging therapeutic targets for these conditions . in this review
, we will discuss inhibitory g proteins and gpcrs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and t2 dm , improving satiety , insulin resistance and/or beta cell biology . |
overactivation of post - synaptic receptors for glutamate places a metabolic stress upon neurons which can contribute to neuronal injury in a wide range of conditions .
the evidence for a contribution of glutamate receptor overactivation ( termed excitotoxicity ) is strongest in conditions like ischemia , prolonged seizures , and trauma , in which there is rapid release and extracellular accumulation of glutamate .
however , there is also compelling evidence for important excitotoxic contributions to injury in neurodegenerative conditions including amyotrophic lateral sclerosis ( als ) .
glutamate acts at a number of receptors comprising both ligand gated ion channels ( ionotropic receptors ) and g - protein linked ( metabotropic ) receptors .
three families of ionotropic glutamate receptors ( nmda , ampa , and kainate receptors ) have been characterized both pharmacologically and by the identification and cloning of their subunit genes .
early studies of excitotoxic mechanisms focused on possible contributions of nmda receptors , which unlike most ampa , and kainate receptors ( collectively referred to as ampa / kainate receptors ) , are highly permeable to ca ions , and brief activation of which could potently trigger delayed injury to cultured neurons . however , despite clear contributions of these channels in some models of acute neurodegeneration , nmda blockers have to date failed to show impressive efficacy in therapeutic trials .
furthermore , in some studies ampa / kainate receptor blockade has shown greater efficacy against ischemic injury than nmda receptor antagonism ( buchan et al . , 1993 ) .
another early clue to contributions of ampa / kainite in neurodegeneration derives from studies of syndromes in humans or primates linked to the consumption of environmental toxins , beta - n - methylamino - l - alanine ( bmaa ) , beta - n - oxalylamino - l - alanine ( boaa ) , and domoic acid ( spencer et al . , 1986 , 1987 ; teitelbaum et al . ,
boaa and domoic acid , the respective causes of lathyrism and a form of shellfish poisoning , are both well established ampa / kainate receptor agonists .
bmaa , an unusual cyanobacterial toxin suspected of contributing to forms of human neurodegenerative disease , can induce selective injury to vulnerable subpopulations of neurons , including motor neurons ( mn , which degenerate in als ) , via activation of ampa / kainate receptors ( weiss et al .
, 1989 ; weiss and choi , 1991 ; rao et al . , 2006 ) .
whereas most ampa / kainate type glutamate channels are ca impermeable , subsets of these channels , which are expressed in characteristic temporal and spatial patterns on discrete populations of neurons , are ca - permeable , and it is the activation of these channels that likely mediates most of the neurodegeneration attributable to ampa / kainate receptor activation .
this review focuses specifically on ca - permeable ampa ( ca - ampa ) channels and clues that have accumulated over the past two decades to their roles in both acute and chronic neurodegenerative conditions .
ampa channels are the most prevalent glutamate channels in the mammalian central nervous system , and mediate most routine excitatory neurotransmission .
they are tetramers comprised of combinations of four subunits glua ( previously referred to as glur ) 1 , 2 , 3 , or 4 .
the presence of one or more glua2 subunits in a heteromeric channel prevents ca permeability .
the ability of glua2 subunits to block ca permeability is dependent upon the presence of a positively charged arginine residue at a critical site in the channel lining region of the peptide ( the q / r site ) ; the other ampa subunits ( glur1 , 3 , and 4 ) all have a neutral glutamine at the homologous site .
interestingly , the genomic dna encodes a neutral glutamine residue at this site in all four subunits , and the arginine codon is post - transcriptionally inserted into the glua2 mrna transcript via rna editing by the enzyme adar2 ( higuchi et al . , 2000 ) . after translation ,
the subunits are assembled in the er . likely indicative of the critical importance of these channels to normal physiological function , channel assembly , and membrane insertion are both sensitive to the editing state of glua2 ( greger et al .
it has further become apparent that pathological upregulation of these channels in disease states can occur via several mechanisms , including downregulation of glua2 mrna transcription , defective editing of the glua2 pre - mrna , and aberrant internalization of glua2 containing ( and ca impermeable ) channels and insertion of glua2 lacking channels .
the presence of ca - ampa channels is neither uniform nor constant , but varies with cell type and developmental stage . whereas most adult neurons possess relatively few of these channels , discrete populations of neurons , most prominently many gabaergic inhibitory interneurons , possess large numbers of ca - ampa channels ( jonas et al . , 1994
; yin et al . , 1994a ; leranth et al . , 1996 ) .
however , numbers of ca - ampa channels on pyramidal neurons change with developmental stage , and are present in markedly increased numbers in both rats and humans in early postnatal periods ( kumar et al .
, 2002 ; talos et al . , 2006a , b ; brill and huguenard , 2008 ) .
as mentioned above , relatively brief but strong activation of highly ca permeable nmda channels is sufficient to trigger degeneration of cultured neurons .
a large number of studies have highlighted mechanisms through which such neuronal ca loading can induce injury , which may vary depending upon the intensity of the ca load . with strong loading
, ca is taken up into mitochondria and can disrupt their function , causing release of damaging reactive oxygen species ( ros ; dugan et al . , 1995 ; reynolds and hastings , 1995 ) .
in addition , ca loading can induce ros generation through other mechanisms , including the activation of nitric oxide synthetase ( nos ) , with resultant nitric oxide production , and the activation of nadph - oxidase , with resultant superoxide production ( dawson et al .
these ros may induce dna damage , causing activation of the enzyme , parp , which can injure neurons via nad+ depletion , resulting in glycolytic block , depletion of atp , and induction of apoptotic signaling via release of aif from mitochondria ( hong et al .
it appears likely that many of these same downstream mechanisms may apply to injury caused by strong activation of ca - ampa channels ( carriedo et al . , 1998 ) .
a key question thus concerns differences between nmda and ca - ampa dependent injury , possibly accounting for the apparent greater contributions of ca - ampa channels in some conditions .
one difference is that nmda channels are subject to a voltage dependent block of their pores by extracellular mg ions , such that there is relatively little ion flux through these channels in the absence of significant post - synaptic depolarization .
this voltage dependence allows nmda channels to detect temporally and spatially convergent post - synaptic inputs and is thus important to their roles in activity dependent synaptic plasticity .
in contrast , ca - ampa channels are not blocked by extracellular cations ( but are subject to voltage dependent block by intracellular polyamines , resulting in inwardly rectifying currents ) , and thus freely permit ca entry with any level of receptor activation . secondly , whereas the expression of nmda channels is relatively static , ampa channels are strongly regulated in response to physiological and pathological signals , and numbers of ca - ampa channels may increase sharply in certain stress conditions and disease states ( as discussed further below ) .
thus , the increase in numbers of these channels in already stressed neurons adds an additional metabolic burden that may impair their ability to restore homeostasis and survive . a likely third factor in their propensity to contribute to injury concerns their permeability to other divalent cations .
zn is present in pre - synaptic vesicles of many ( but not all ) forebrain excitatory pathways , and appears to be co - released with glutamate upon pre - synaptic activation ( assaf and chung , 1984 ; howell et al . , 1984 ; qian and noebels , 2005 ) .
although the physiological significance of pre - synaptic zn release is uncertain , it can effectively block nmda channels , limiting their ion flux in response to intense pre - synaptic activation ( paoletti et al . , 2009 ) .
in contrast , zn readily permeates ca - ampa channels ( jia et al . , 2002 ) , and its entry through these channels can clearly injure cultured neurons and likely contributes to injury in conditions like ischemia ( yin and weiss , 1995 ; sensi et al . , 1999 ; yin et al . , 2002 ;
intracellular zn can trigger neurodegeneration via a number of mechanisms , including some previously attributed to ca , including the induction of mitochondrial dysfunction , which it appears to do with considerably greater potency than ca ( sensi et al .
as mentioned above , some early studies found ampa / kainate blockers to provide greater protection than nmda blockers against ischemic neurodegeneration , and it is likely that much of this protection reflects blockade of ca - ampa channels .
studies to date have suggested that ca - ampa channels may contribute to different forms of ischemic injury in a number of ways .
the immature brain is particularly susceptible to ischemia , and pre - term , or perinatal hypoxia ischemia of both white and gray matter is a major cause of early life morbidity , for instance as an antecedent of cerebral palsy .
a number of studies have indicated that periods of high ischemic susceptibility correspond to developmental periods of increased ca - ampa channel numbers , suggesting that the presence of these channels contributes to the ischemic susceptibility ( talos et al .
transient global ischemia ( tgi ) , as occurs in cardiac arrest , causes a variable degree of neuronal damage and loss , depending upon the duration of the ischemia . in hippocampus , pyramidal neurons ( hpns ) in the ca1 subfield
although adult hpns were previously felt to lack ca - ampa channels , especially in contrast to strongly ca - ampa channel expressing gabaergic interneurons , a number of studies have suggested that they may often be present under basal conditions in relatively low numbers , and preferentially on dendrites at a distance from the soma where they are not readily detected electrophysiologically ( lerma et al . , 1994 ; ikonomovic et al . , 1995
; yin et al . , 1999 ; ogoshi and weiss , 2003 ) . in specific support of a role for ca - ampa channels in ischemic injury , maneuvers to increase numbers of these channels increased susceptibility of ca1 pyramidal neurons to ischemic injury , whereas decreasing channel numbers decreased injury ( anzai et al .
a very intriguing clue to contributions of ca - ampa was provided by the observation that transient ischemia could induce a selective downregulation of glua2 mrna in ca1 pyramidal neurons , resulting in a delayed ( after 23 days ) incorporation of increased numbers ca - ampa channels into synapses ( gorter et al . , 1997 ;
; opitz et al . , 2000 ) . furthermore , indicating a direct role of these channels in the delayed neurodegeneration of ca1 neurons , the injury was attenuated by the delayed administration of a ca - ampa channel blocker ( noh et al . , 2005 ) .
first , highlighting contributions of zn to ischemic neurodegeneration , zn accumulates in degenerating neurons and injury was attenuated by an extracellular zn chelator ( tonder et al . , 1990 ; koh et al . , 1996 ) .
supporting a contribution of zn entry through ca - ampa channels , either an extracellular zn chelator or a ca - ampa channel blocker attenuated neuronal zn accumulation and subsequent degeneration in a hippocampal slice model of ischemia ( yin et al . , 2002 ) .
interestingly , zn may act in distinct ways during different temporal phases of the ischemic process .
addition of an extracellular zn chelator before the ischemic episode attenuated the glua2 downregulation , preventing the delayed increase in ca - ampa channels ( calderone et al . , 2004 ) .
a number of recent studies suggest that mitochondria might be an important target of these early zn rises ( calderone et al . , 2004 ;
these acute events appear to result in upregulation of the gene silencing transcription factor neuronal repressor element-1 silencing transcription factor ( rest)/neuron - restrictive silencer factor ( nrsf ) , which has been linked to the delayed downregulation of glua2 ( calderone et al . , 2003 ) .
in contrast , late addition of either the chelator or of a ca - ampa channel blocker , after the channel upregulation was already underway , attenuated the occurrence of a delayed intracellular zn rise and associated cell death ( calderone et al . , 2004 ; noh et al . , 2005 ) , consistent with the idea that zn entry through the newly inserted channels might contribute to the delayed injury .
of note , however , it is clear that not all ischemic zn accumulation reflects entry through ca - ampa or other channels , and that there are stores of zn already present in the ca1 neurons which can be mobilized in response to acidosis and oxidative stress associated with ischemia , contributing to the rises ( aizenman et al . , 2000 ;
whereas downregulation of glua2 results in the incorporation of new ca - ampa channels after a significant delay , other recent studies have suggested that ischemia can also induce a more rapid endocytosis of ca impermeable ampa channels and exocytosis of ca - ampa channels into synapses via mechanisms involving receptor trafficking events that may be involved in synaptic plasticity ( liu et al . , 2006 ; dixon et al . ,
another study suggested that defects in glua2 editing may occur after ischemia , resulting in the incorporation of unedited glua2 into channels , rendering them ca permeable ( peng et al . , 2006 ) .
interestingly , ampa receptors containing unedited glua2 may be particularly toxic to neurons , in part because of their constitutive trafficking to the plasma membrane ( mahajan and ziff , 2007 ) .
amyotrophic lateral sclerosis is a neurodegenerative disease characterized by progressive weakness due to the relatively selective degeneration of upper and lower mns , usually leading to death within 25 years of diagnosis . whereas most cases are sporadic , 10% are familial , and of those a fraction
observations of loss of the ability of astrocytes to take up extracellular glutamate provided an early clue that excitotoxicity might contribute to the mn injury .
consistent with this idea , glutamate uptake blockers caused preferential loss of mns both in organotypic slice and dissociated culture models ( rothstein et al .
furthermore , supporting a role of ampa / kainate receptors in the disease , this mn degeneration was prevented by ampa / kainate blockers , but not by nmda blockers .
first , they possess substantial numbers of ca - ampa channels ( carriedo et al .
, 1996 ; van den bosch et al . , 2000 ; vandenberghe et al . , 2000 ) .
in addition , populations of mns that degenerate in als are characterized by having low levels of cytosolic ca buffering proteins ( alexianu et al . , 1994 ; elliott and snider , 1995 ) , and buffer cytosolic ca loads poorly ( vanselow and keller , 2000 ) , such that excitotoxically induced ca loads are rapidly taken up into the mitochondria , with consequent strong mitochondrial ros release ( carriedo et al . , 2000 ) . in further studies ,
we have found evidence that this mitochondrial ros might not only damage the mns , but also exit the mns and contribute to the loss or dysfunction of glutamate transporters on surrounding astrocytes ( rao et al . , 2003 ) .
such a mechanism might contribute to the propagation of a feed forward cascade , with loss of glutamate transport resulting in increased extracellular glutamate levels , more activation of ca - ampa channels , and more mitochondrial ca loading resulting in more ros generation ( rao and weiss , 2004 ; yin et al . , 2007 ) .
transgenic mice overexpressing familial als associated sod1 mutations have provided by far the most studied animal models of the disease . providing strong evidence that ca - ampa channels play a role in this form of the disease , generation of sod1 mutant mice with increased numbers of ca - ampa channels in their mns accelerates disease progression , while decreasing numbers of the channels in these mice slows the disease ( tateno et al . , 2004 ; kuner et al . , 2005 ; van damme et al . , 2005 ) .
in addition to the presence of some ca - ampa channels on mns under basal conditions , an intriguing series of studies suggests that there may be a loss of glua2 editing in als mns , such that numbers of ca - ampa channels may increase , further predisposing them to injury ( kawahara et al .
, ampa receptors containing unedited glua2 may be particularly effective at inducing neurotoxicity ( mahajan and ziff , 2007 ) .
another recent study suggests that the loss of glua2 editing may be triggered as a consequence of excitotoxic exposures , via proteolytic cleavage of the editing enzyme , rna - dependent adenosine deaminase , adar2 ( mahajan et al . , 2011 ) .
although roles of ca - ampa channels have been most studied in the cases of ischemia and als , there are compelling clues to contributions in other conditions as well .
first , ca - ampa channels may well contribute to neurodegeneration in conditions of prolonged seizures , or brain , or spinal cord trauma , both of which , like ischemia are associated with prolonged elevations of extracellular glutamate .
much as in ischemia , glua2 mrna , and protein levels have been observed to decrease following prolonged seizures in ca1 and ca3 hippocampal pyramidal neurons prior to the death of these cells ( friedman et al . , 1994 ; grooms et al . , 2000 ) . in the case of traumatic injury ,
ventral root avulsion induced loss of glua2 in spinal mns ( nagano et al . , 2003 ) and
spinal cord contusion caused preferential loss of glua2 in mns that persisted for at least a month ( grossman et al .
in addition , recent studies in models of cerebral trauma provide evidence for endocytosis of glua2 containing receptors and insertion of ca - ampa receptors that contributed to the induction of injury ( spaethling et al . , 2008 ; bell et al . , 2009 )
other studies suggest possible contributions of ca - ampa channels to neurodegeneration in alzheimer s disease .
cholinergic neurons of the basal forebrain conspicuously degenerate in alzheimer s disease ( whitehouse et al . , 1982 ) , and there is evidence for loss of other subpopulations of interneurons including those containing parvalbumin or somatostatin ( davies et al . , 1980 ; rossor et al .
interestingly , in vitro studies indicate that these populations are all selectively sensitive to ampa / kainate receptor mediated excitotoxicity , probably in large part due to their possession of substantial numbers of ca - ampa channels ( weiss et al . , 1990 , 1994 ; yin et al .
other studies have found glua2 on basal forebrain cholinergic neurons to be markedly decreased in aged humans ( ikonomovic et al . , 2000 ) , and that levels of glua2 on pyramidal neurons in the entorhinal cortex and hippocampus of alzheimer brains decrease prior to neurofibrillary tangle formation ( ikonomovic et al . , 1997 ) , suggesting that increased numbers of ca - ampa channels may predispose to the degeneration of these neuronal populations .
further intriguing studies suggest a link between inflammation and increases in ca - ampa channel numbers .
the inflammatory cytokine , tissue necrosis factor - alpha ( tnf- ) can lead to rapid exocytosis of ca - ampa channels on pyramidal neurons ( beattie et al .
2005 ) , and recent studies strongly suggest that tnf- dependent trafficking of ca - ampa channels contributes to neurodegeneration after spinal cord trauma ( ferguson et al . , 2008 ) .
this mechanism could prove relevant to neurodegeneration in other immune or neurodegenerative conditions in which acute or chronic inflammation occurs .
the past two decades have seen the accumulation of a host of clues to contributions of ca - ampa channels in a wide range of diseases of the nervous system .
the presence of ca - ampa channels is not felt to be causative of the diseases .
but the preferential expression of ca - ampa channels on subpopulations of neurons may be a factor in the selective patterns of neurodegeneration seen in certain conditions .
in addition , the propensity for the upregulation of ca - ampa channel numbers , likely via a range of mechanisms , in response to disease associated signals may well subject affected neurons to new stresses that may tip the balance toward degeneration . despite the accumulation of numerous clues to roles of ca - ampa channels in disease , many questions , and obstacles exist .
there are as yet no proven therapeutics based on the modulation or block of these channels .
this likely reflects the absence to date of any highly effective , selective , and bioavailable drugs for blocking ca - ampa channels in whole animals , as would be necessary to fully test their contributions to disease .
such blockers , if available , might be remarkably useful neuroprotectants in a wide range of conditions , as they may effectively antagonize toxic ca and zn entry through these channels without overly suppressing overall glutamatergic neurotransmission .
the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | since the discovery and molecular characterization of ca2 + -permeable ampa channels just over two decades ago , a large body of evidence has accumulated implicating contributions of these unusual glutamate activated channels to selective neurodegeneration in certain conditions , including ischemia and amyotrophic lateral sclerosis .
factors likely involved in their contributions to disease include their distinct patterns of expression in certain neuronal populations , their upregulation via various mechanisms in response to disease associated stresses , and their high permeability to zn2 + as well as to ca2 + .
however , full characterization of their contributions to certain diseases as well as development of therapeutics has been limited by the lack of selective and bioavailable blockers of these channels that can be employed in animals or humans .
this review summarizes some of the clues that have emerged over recent years to the contributions of these channels in disease . |
transdermal delivery of nonsteroidal anti - inflammatory drugs is a topical treatment routinely used in physiotherapy to reduce pain and inflammation in musculoskeletal disorders.14 in order to weaken the skin barrier and to achieve a clinically effective drug concentration in the target tissues , various transdermal enhancement techniques , such as sonophoresis , iontophoresis , and occlusion , are used.511 however , little information is available concerning the efficacy of the treatment protocol and the penetration profiles of the substances used in the physiotherapy setting . indeed , most clinical studies have complex treatment protocols , and are unable to relate clinical outcome with drug penetration . a recent meta - analysis ( 2012 )
suggested a better clinical outcome for iontophoretically delivered substances.12 despite methodological flaws ( eg , combined therapies , lack of control for passive diffusion and occlusion ) , definite conclusions regarding the possible penetration enhancement of electrically assisted delivery can not be inferred.12 the building up of a stratum corneum ( sc ) reservoir is an important component of the pharmacodynamics of topically applied substances .
rougier et al used the reservoir effect of the sc after 30 minutes of application to predict the total amount absorbed.13 moreover , the human sc has the capacity to store topically applied substances through a prolonged time.14 the existence of a reservoir within the sc has been documented for several topically applied solutes such as corticosteroids,1418 caffeine,19,20 nicotine,21 diclofenac ( df),22 and chemical ultraviolet filters.23 roberts et al emphasized that reservoir properties can also be attributed to the viable epidermis and dermis .
specifically , df - binding capacities in dermal tissue have been reported.24 lambrecht et al estimated df skin bioavailability by quantifying the reduction of methyl nicotinate ( mn)-induced vasodilatation using chromametry and laser doppler flowmetry .
they detected a significant reduction in mn response 90 minutes after different application modalities of df .
open application resulted in a 32% reduction in mn response , application under occlusion accounted for a reduction of 66% , and the iontophoretic application resulted in a 65% reduction.11 at that time , one could not discriminate between the occlusive and electrically assisted delivered df . to obtain more insight into the pharmacodynamics and reservoir properties of df ,
the aim of the present study was to evaluate the bioavailability in the skin of df at longer time intervals after df application using different application modalities .
therefore , three different forms of application ( passive , occlusive , iontophoretic ) were examined in order to detect the anti - inflammatory effect and thereby get a better understanding of the skin - penetrating and reservoir - building behavior of df as a function of time .
mn responses were quantified by skin colorimetry at different time points ( 1.5 , 6 , 24 , 32 , 48 , 72 , 96 , and 120 hours ) after initial df application .
five different groups , ( group 1 , n=13 [ 1.5 hours ] ; group 2 , n=12 [ 6 hours ] ; group 3 , n=12 [ 24 , 32 hours ] ; group 4 , n=14 [ 48 , 72 hours ] and group 5 , n=9 [ 96 , 120 hours ] ) of healthy volunteers ( n=60 , 23 male and 37 female ) not treated with any drugs participated in this study .
approval by the ethical committee of the vrije universiteit brussel was obtained for this study ( 2002/136 ) . before testing , all subjects were informed of the research protocol and signed an informed consent . during the duration of the experiments , the volunteers were asked to maintain their daily activities , but to abstain from swimming and extensive showering .
the measurements were performed under standardized temperature ( 20c2c ) and relative humidity ( 45%5% ) conditions . before every df application and each measurement session
, the volunteers participated in a 30-minute acclimatization period . for each time interval tested , five randomized circular skin areas ( 7 cm ) were demarcated on the subjects volar forearms , for the following treatment and/or control modes : 1 ) open application ( without occlusion ) , 2 ) passive diffusion under a semiocclusive humid sponge , 3 ) iontophoretic application , 4 ) standard mn response , and 5 ) untreated skin side .
this meant that either five skin areas for groups 1 and 2 or ten skin areas for groups 3 , 4 , and 5 ( testing two time intervals ) were randomly demarcated on both arms .
this procedure enabled a single application of mn on every skin site at different time intervals , avoiding the occurrence of tachyphylaxis .
df skin bioavailability was assessed by quantifying the reduction of the mn response , as proposed by lambrecht et al.11 topical application of mn provokes an increase of local cutaneous blood flow ( erythema).25 when df is present in the skin , the nicotinate response is depressed in a concentration - dependent way.26 treffel and gabard demonstrated a good correlation between the in vivo inhibition of the mn - induced inflammation and the in vitro determination ( chromatographic analysis ) of nonsteroidal anti - inflammatory drug - concentration levels in the sc.27 lambrecht et al evaluated the inhibition of mn - induced erythema with both laser doppler flowmetry and the colorimetric method , which resulted in equivalent conclusions .
therefore it was decided that the chromametric evaluation be used.11,28 in the present study , the inhibition of the mn response with sum of the a * parameter ( a * ) was evaluated up to 50 minutes post - mn application , making the protocol more feasible and bearable for the subjects .
skin - surface colorimetric measurements were performed with a minolta cr 200 chromameter operating in the international commission on illumination l*a*b * color space . for the quantification of skin - surface color and erythema , the a
* parameter is especially useful , as it represents the chromaticity between red / magenta and green ( negative values indicate green , while positive values indicate magenta).29,30 chromameter measurements were carried out before df application , prior to mn application , and every 5 minutes until 50 minutes post - mn application for the different reservoir - estimation time points .
since different groups of subjects were involved , an untreated skin area was included in order to be able to correct for blank values . for treatment modes 1 , 2 , and 3 , a commercially available 1% df ( voltaren emulgel ; novartis pharma , bern , switzerland ) formulation ( 12 mg )
the mn test was applied using paper - filter disks that were saturated in a 0.005 m aqueous mn solution and applied on the circular demarcated skin areas for 30 seconds .
after removal of the filter disk , excess solution was gently wiped away using a tissue paper .
df was applied for 20 minutes under three different conditions : 1 ) open application ( without occlusion ) , 2 ) passive diffusion under a semiocclusive humid sponge , and 3 ) iontophoretic application ( direct current , 0.2 ma / cm , cathode ) . to reproduce physiotherapeutic conditions ,
two marked skin areas were not treated with df : the application side for the standard mn - induced response and the blank untreated skin side .
the bioavailability of df in the sc under the three conditions was assessed by quantification of mn induced erythema at 1.5 , 6 , 24 , 48 , 72 , 96 , and 120 hours post - df application . to estimate the presence of df in the skin , mn responses at the different time periods following initial df application ( 1.5 , 6 , 24 , 32 , 48 , 72 , 96 , and 120 hours ) were compared .
the a * corrected for blank values ( untreated skin ) up to 50 minutes post - mn application was used as an indicator for the magnitude of the mn response .
smirnov goodness - of - fit test . at the different time periods following initial df application , the skin response to the different application modalities
statistical significance between any application mode and the standard mn response was used as an indication for the presence of df in the skin at that particular time point following initial df application .
five different groups , ( group 1 , n=13 [ 1.5 hours ] ; group 2 , n=12 [ 6 hours ] ; group 3 , n=12 [ 24 , 32 hours ] ; group 4 , n=14 [ 48 , 72 hours ] and group 5 , n=9 [ 96 , 120 hours ] ) of healthy volunteers ( n=60 , 23 male and 37 female ) not treated with any drugs participated in this study .
approval by the ethical committee of the vrije universiteit brussel was obtained for this study ( 2002/136 ) . before testing , all subjects were informed of the research protocol and signed an informed consent . during the duration of the experiments , the volunteers were asked to maintain their daily activities , but to abstain from swimming and extensive showering .
the measurements were performed under standardized temperature ( 20c2c ) and relative humidity ( 45%5% ) conditions . before every df application and each measurement session
, the volunteers participated in a 30-minute acclimatization period . for each time interval tested , five randomized circular skin areas ( 7 cm ) were demarcated on the subjects volar forearms , for the following treatment and/or control modes : 1 ) open application ( without occlusion ) , 2 ) passive diffusion under a semiocclusive humid sponge , 3 ) iontophoretic application , 4 ) standard mn response , and 5 ) untreated skin side .
this meant that either five skin areas for groups 1 and 2 or ten skin areas for groups 3 , 4 , and 5 ( testing two time intervals ) were randomly demarcated on both arms .
this procedure enabled a single application of mn on every skin site at different time intervals , avoiding the occurrence of tachyphylaxis .
df skin bioavailability was assessed by quantifying the reduction of the mn response , as proposed by lambrecht et al.11 topical application of mn provokes an increase of local cutaneous blood flow ( erythema).25 when df is present in the skin , the nicotinate response is depressed in a concentration - dependent way.26 treffel and gabard demonstrated a good correlation between the in vivo inhibition of the mn - induced inflammation and the in vitro determination ( chromatographic analysis ) of nonsteroidal anti - inflammatory drug - concentration levels in the sc.27 lambrecht et al evaluated the inhibition of mn - induced erythema with both laser doppler flowmetry and the colorimetric method , which resulted in equivalent conclusions .
therefore it was decided that the chromametric evaluation be used.11,28 in the present study , the inhibition of the mn response with sum of the a * parameter ( a * ) was evaluated up to 50 minutes post - mn application , making the protocol more feasible and bearable for the subjects .
skin - surface colorimetric measurements were performed with a minolta cr 200 chromameter operating in the international commission on illumination l*a*b * color space . for the quantification of skin - surface color and erythema ,
the a * parameter is especially useful , as it represents the chromaticity between red / magenta and green ( negative values indicate green , while positive values indicate magenta).29,30 chromameter measurements were carried out before df application , prior to mn application , and every 5 minutes until 50 minutes post - mn application for the different reservoir - estimation time points .
since different groups of subjects were involved , an untreated skin area was included in order to be able to correct for blank values .
for treatment modes 1 , 2 , and 3 , a commercially available 1% df ( voltaren emulgel ; novartis pharma , bern , switzerland ) formulation ( 12 mg ) was applied .
the mn test was applied using paper - filter disks that were saturated in a 0.005 m aqueous mn solution and applied on the circular demarcated skin areas for 30 seconds .
after removal of the filter disk , excess solution was gently wiped away using a tissue paper .
df was applied for 20 minutes under three different conditions : 1 ) open application ( without occlusion ) , 2 ) passive diffusion under a semiocclusive humid sponge , and 3 ) iontophoretic application ( direct current , 0.2 ma / cm , cathode ) . to reproduce physiotherapeutic conditions ,
two marked skin areas were not treated with df : the application side for the standard mn - induced response and the blank untreated skin side .
the bioavailability of df in the sc under the three conditions was assessed by quantification of mn induced erythema at 1.5 , 6 , 24 , 48 , 72 , 96 , and 120 hours post - df application .
to estimate the presence of df in the skin , mn responses at the different time periods following initial df application ( 1.5 , 6 , 24 , 32 , 48 , 72 , 96 , and 120 hours ) were compared .
the a * corrected for blank values ( untreated skin ) up to 50 minutes post - mn application was used as an indicator for the magnitude of the mn response .
smirnov goodness - of - fit test . at the different time periods following initial df application , the skin response to the different application modalities
statistical significance between any application mode and the standard mn response was used as an indication for the presence of df in the skin at that particular time point following initial df application .
analysis of the skin color ( a * parameter ) after mn application for the different application modes ( open application , occlusion , and iontophoresis ) and at the different reservoir - estimation times ( 1.5 , 6 , 24 , 32 , 48 , 72 , 96 , and 120 hours ) clearly showed changes in mn response as a function of the application mode and reservoir - estimation time . as an example
, kinetics of the skin - color parameters are given in figures 1 and 2 . at 1.5 hours post - df application ,
a clear inhibition was visible for application under occlusion and under iontophoresis , while open df application did not provoke an inhibition in mn response ( figure 1 ) . at 96 hours
post - df application , inhibition of the mn response was no longer perceived ( figure 2 ) .
differences between application modes at the different reservoir - estimation times were assessed using the a * values calculated from the different kinetics ( table 1 ) . at 1.5 hours after the initial df application , a significantly decreased response was detected for the occluded and iontophoretically delivered df only . from 6 hours up to
32 hours post - df application , a significantly reduced mn response was detected at all df - pretreated skin sites . at 48 hours post - df application
, only the passive and occluded applications resulted in a significant reduction . at 72 hours post - df application ,
only the response after the passively delivered df remained significant . from 96 hours on ,
the applied method inhibition of a physiological reaction ( mn ) due to the presence of the inhibitor ( df ) in the skin reservoir enabled us to estimate penetration kinetics , df skin - reservoir building , and emptying properties of three different application conditions up to 120 hours after initial df application .
in contrast with other reservoir protocols , an active substance - liberating action , such as occlusion or increased hydration , was not required.24 this may be a confirmation of the presence of df in the viable tissue ( dermal compound ) , since mn - induced vasodilatation is inhibited immediately.24 the application modality influenced the formation and the emptying of the reservoir .
penetration - enhancing factors , such as occlusion and current , induced faster formation and emptying of the reservoir compared to an open ( passive ) df application .
the presence of the reservoir for the passive df application after 6 hours only is in contrast with earlier findings from our laboratory.11 lambrecht et al11 presented the data of the mn response as calculated areas under the curve , relating the inhibition of mn response throughout a 65-minute time period .
they found a 32% reduction of the mn response 1.5 hours after application , reaching borderline significance ( p=0.04 ) .
consequently , the weaker inhibition potential at 1.5 hours post - df application noticed in both studies indicates lower bioavailability at that moment , due to a slower percutaneous penetration compared to the other application modes . according to the literature
, the formation of a skin reservoir for topically applied substances is determined by a variety of factors ( eg , lipid / water - solubility , protein - binding capacity , percutaneous absorption , compound concentration , clearance , application time , and application mode).11,1416,23,24 our results support the previous findings of vickers , showing that increased drug diffusivity in the sc provoked by penetration enhancers results in faster formation of the reservoir.14 more specifically , takahashi et al suggested increased diffusivity of df into the sc from vehicles containing urea compared to vehicles without urea .
the latter was explained by the hydration - enhancement effect of urea on the sc.31 the results concerning the penetration - enhancement effect of iontophoresis are in line with the results of curdy et al on the in vivo uptake of piroxicam after passive , occlusive , and iontophoretic administration
. only after iontophoresis were enhanced drug uptakes found at 30 , 60 , and 125 minutes following the initial application at different depths in the sc .
in contrast with our results , curdy et al found no significant difference between passive delivery and the application under occlusion .
a possible explanation could be the low lipophilicity of piroxicam resulting in a low passive uptake into the sc.32 fang et al postulated that the route and mechanism for the iontophoretic delivery of df through the skin might be different compared to passive delivery .
therefore , the importance of the sc as a rate - limiting barrier is reduced for iontophoretic delivery of df.33 our results indicate that the emptying of the reservoir is influenced by the application mode .
after iontophoretic delivery , the df reservoir was present up to 32 hours following initial application .
however , after occlusive and passive delivery of df , the reservoir was present up to 48 and 72 hours , respectively .
it is well established that diffusivity influences clearance from the reservoir , with faster emptying as a function of increasing diffusivity . increasing sc hydration as well as iontophoresis have been shown to be effective methods for enhancing the percutaneous penetration of df.33 the present data fit in the model proposed by roberts et al with a shorter lag time resulting in a faster reservoir emptying.24 equally , based on the results of rougier et al13 one can assume that the application with the fastest reservoir - buildup conditions may result in a greater delivery of active substances to the viable tissues , which may have an effect on the clinical outcomes of the physiotherapeutic treatment .
within the limitations of our experimental design , we can hypothesize that the faster emptying of the reservoir after iontophoretic delivery compared to application under occlusion may be an indication for a higher diffusivity during the current - assisted application , leading to a superior delivery under iontophoretic circumstances .
limitations of the present study are the lack of information on absolute df quantities entering the viable skin and the fact that the methods used did not allow differentiation between an epidermal reservoir and a dermal reservoir .
further research estimating in vivo tissue concentrations after different modes of application are required for further elaboration of the pharmacodynamics of topical applied substances in physiotherapeutic practice .
this study measured the penetration kinetics and reservoir properties of df after a single topical passive , occlusive , and electrical assisted application in a realistic physiotherapeutic setting .
the results indicate that the contribution of occlusive and passive penetration in the iontophoretic delivery can be substantial .
the prompt inhibition of the vasoactive reaction may be an indication for a dermal df reservoir .
the formation and emptying of the reservoir was found to be dependent on the application mode . | aimthere is scarce information concerning the pharmacodynamic behavior of topical substances used in the physiotherapy setting .
the aim of the present study was to estimate the formation and emptying of the diclofenac ( df ) skin reservoir after passive , semiocclusive , and electrically assisted applications of df.subjects and methodsfive different groups of healthy volunteers ( ntotal=60 , 23 male and 37 female ) , participated in this study .
a 1% df ( voltaren emulgel ) formulation ( 12 mg ) was applied on the volar forearms on randomized defined circular skin areas of 7 cm2 .
df was applied for 20 minutes under three different conditions at the same time .
the presence of df in the skin results in a reduction of the methyl nicotinate ( mn ) response . to estimate the bioavailability of df in the skin ,
mn responses at different times following initial df application ( 1.5 , 6 , 24 , 32 , 48 , 72 , 96 , and 120 hours ) were analyzed.resultsat 1.5 hours after the initial df application , a significant decrease in mn response was detected for the occluded and iontophoretic delivery .
passive application resulted in a decrease of the mn response from 6 hours post - df application .
the inhibition remained up to 32 hours post - df application for the iontophoretic delivery , 48 hours for the occluded application , and 72 hours for the passive delivery . at 96 and 120 hours post - df application none of the mn responses was inhibited.conclusionthe formation and emptying of a df skin reservoir was found to be dependent on the df - application mode .
penetration - enhanced delivery resulted in a faster emptying of the reservoir . |
the primary hosts of this parasite are birds such as herons or ducks , and its second intermediate hosts are freshwater fish such as carp , crucian carp , or loach .
the parasite becomes a metacercaria inside the second intermediate host by forming a globular film around its body and waits for an opportunity to infect the final host .
although infections in humans are extremely rare , if a person consumes a raw freshwater fish infected by the larvae of this parasite , the larvae will excyst in the stomach , migrate through the esophagus , and attach to the throat and mature , causing parasitic pharyngitis or laryngitis . human laryngitis caused by
c. complanatum is a rare disease ; however , in japan , korea , and other asian countries where eating raw fish is a tradition , it is possible for individuals to become infected by this intestinal parasite . in this study , we describe a patient with laryngitis caused by c. complanatum that occurred after the patient ate freshwater fish sashimi .
a 64-year - old japanese man visited the outpatient s department of otolaryngology , yamaguchi university hospital , ube , japan .
his symptoms occurred two days after he had eaten raw carp sashimi for dinner at a japanese - inn situated at a hot spring .
furthermore , a blood test showed no increase in white blood cell count or c - reactive protein level , and a differential white blood cell count showed no eosinophilia .
a fiberoptic examination of the larynx revealed no clear signs of inflammation , such as redness or swelling of the pharynx or larynx .
however , there was a foreign object attached to the surface of the right aryepiglottic fold that moved slowly during observation ( figure 1 ) .
considering that the patient had consumed raw carp and that the shape of the object resembled a fluke when more closely observed , the probable diagnosis was infection by a parasitic fluke . after explaining the procedure to the patient and obtaining his consent , an endoscopic removal of the fluke
was performed under local anesthesia because he did not have a strong gag reflex . to stop the parasite moving and to make the removal easier , 1 ml of 4% lidocaine via inhalation
was administered to the patient ; with the patient in the right lateral decubitus position , a parasite of approximately 6-mm length was removed using biopsy forceps with a laryngeal endoscopy .
the parasite was almost completely motionless during removal because of the effects of the lidocaine .
removal was easy because the parasite was not strongly attached to the mucosa , too . the patient s throat discomfort disappeared almost immediately after the parasite was removed .
the patient was consulted and visited the gastroenterology department of our hospital for an upper gastrointestinal endoscopy to confirm that there were no residual parasites in the esophagus or stomach , and his treatment was completed .
microscopic findings showed that the frontal end consisted of an oral sucker with a ventral sucker slightly caudal to it .
there were black ovaries on both sides of the body , which was identified as the digenetic trematode c. complanatum .
endoscopic findings of clinostomum complanatum infection
a slow - moving worm attached to the surface of the larynx ( arrow ) . gross image of clinostomum complanatum ( 6.0 mm in length )
the frequency of parasitic infections in humans is decreasing because of changes in lifestyle and environments where food is cultivated .
for example , roundworm infections spread because of the agricultural use of human waste , but their incidence has been greatly reduced with the widespread use of chemical fertilizers . similarly , in japan , improvements in sanitation have decreased the incidence of parasitic diseases , such as tapeworms and pinworms .
sometimes overseas travelers carry parasitic diseases such as malaria . however , infections from parasites in food are still observed in areas where people consume raw meat or fish .
people in japan , korea , and other asian countries have consumed raw fish for long .
one traditional dish is sashimi and slices of raw fish that are now consumed all over the world as typical japanese food such as sushi .
sashimi prepared from fresh marine fish can be safely consumed , but not completely safe , because parasite such as anisakis can infect human , and other bacteria or virus also can cause gastroenteritis . however ,
wild fish that live in brackish water or fresh water may be infected with parasites ; thus , they are inappropriate to be consumed as sashimi .
sashimi made from carp , a freshwater fish , is a traditional dinner item that is sometimes served at japanese - style hot spring inns near lakes or streams .
currently , only carp farmed in spring water that can not be infected by parasites are used to make sashimi because cooking wild carp is an issue . to consume the wild fresh - water fish without heating it , it would normally have to be frozen for 24 h at 20c ( 4f ) in order to kill the parasites .
as far as we searched previous reports extensively , this case constitutes the 19th documented case of clinostomum infection in japan ( table 1 ) .
there are many reports from western japan , and the infection is prevalent in areas along the sea of japan . interestingly , there have also been two recent reports from korea .
sites of parasitic infection include the pharynx , arytenoid region , posterior oropharyngeal wall , and lateral lymphatic band .
however , some case reports also mention a sore throat or swallowing pain , while others report bloody phlegm and fever .
if the patient has consumed raw freshwater fish , the physician can search for a parasite while carefully inspecting the symptomatic area .
reported methods of removal include endoscopic removal under local anesthesia and removal using a direct laryngoscope under general anesthesia .
the alleged reason for choosing to remove the parasite under general anesthesia is to ensure a proper grip on the fluke , but the fluke can almost be completely removed under local anesthesia . in our case , the patient s gag reflex was not strong ; therefore , we had performed the endoscopic removal of the fluke under local anesthesia . if a direct laryngoscope was required to remove the fluke , the position of the laryngoscope should be changed in case the fluke moved , which would probably make the procedure more complicated .
in contrast , with an endoscope , even if the fluke moves the endoscope can easily bend and follow it .
kitagawa et al .
reported that when they were removing c. complanatum , the fluke was quickly moving ; therefore , they had to apply directly spraying it with 8% lidocaine solution to decrease its movements .
in our case , the patient inhaled 1 ml of 4% lidocaine to stop the parasite s movements before safely removing the parasite using biopsy forceps . in the report described above , a minimal amount of lidocaine
was used to effectively stop the parasite s movements , and we found that inhaled only 1 ml of 4% lidocaine was also effective . in the life cycle of c. complanatum , the metacercariae enter a patient s body , excyst in the stomach , migrate through the esophagus , and then attach to the throat and mature , causing parasitic laryngitis .
therefore , there may be a delay of several days between oral ingestion and emergence of symptoms .
even when a fluke is removed from the throat , the metacercariae may remain in the digestive tract or there may be more than one parasite , making it necessary to not only examine the throat but also perform an upper gastrointestinal endoscopy .
we also performed an upper gastrointestinal endoscopy in this case the day after removing the parasite and confirmed that there were no residual parasites .
we described a rare case of laryngitis caused by c. complanatum . in cases where patients develop throat discomfort after consuming raw freshwater fish ,
this disease should be considered as a possible diagnosis , and a fiberoptic examination of the laryngopharynx should be performed .
| abstracta 64-year - old japanese man visited our outpatient department complaining of an irritable sensation in the throat , occurring two days after eating raw freshwater fish ( carp sashimi ) at a japanese - style inn . during laryngeal endoscopy , a slow - moving worm ( fluke )
was found attached to the surface of the right aryepiglottic fold .
after inhalation of 4% lidocaine , the fluke was removed using endoscopic forceps .
patient s throat symptoms immediately improved .
the worm was microscopically identified as clinostomum complanatum . c. complanatum is a digenetic trematode that usually infects fish - eating water birds .
clinostomum infections in humans are rare , and only 21 cases have been described in japan and korea .
c. complanatum infection is known to occur after eating raw freshwater fish , which is a secondary intermediate host . in humans ,
the metacercariae are released into the stomach and migrate through the esophagus before lodging in the throat .
primary therapy involves endoscopic removal of the worm . |
it is common in countries with poor socio - sanitary conditions.1 humans act as intermediate hosts and get infected either through contact with definitive host ( dogs , wolves ) or its feces or by ingesting the infected food.2 due to its subtle presentation and absence of characteristic findings in plain radiographs , diagnosis is commonly missed .
differential diagnoses include potts disease , mycosis , abscesses and neoplasms.13 multiple recurrences are common .
mortality from this disease ranges from 3% to 50%1 and mean life expectancy has been reported to be 5 years.134 we present 13 year followup of spinal hydatid disease with multiple recurrences and intradural dissemination of the disease at the last followup .
intradural extension of the disease in our case was supposedly through the dural rent which has not been reported in english literature to the best of our knowledge .
a 40 year old female presented with history of progressive left leg pain and difficulty in walking since 2 months .
her walking capacity was restricted to 10 m. on examination , she had grade four power of ankle and digit dorsiflexors ( l4 and l5 myotomes ) on left side ( medical research council grade ) .
plain radiographs showed consolidation at d10-d11 ( old operated levels ) with stable anterior column and there were no implant related problems [ figure 1a ] .
magnetic resonance imaging showed a cystic lesion at l3-l4 , signal intensity same as of cerebrospinal fluid ( csf ) in t2 and t1 , displacing the cauda equina roots .
the proximal extent of the lesion could not be identified to certainty because of the artifacts from previous stainless steel instrumentation which was extending up to l3 level .
computed tomography ( ct ) myelogram showed complete block at l3-l4 intervertebral disc level [ figure 1b ] which pointed toward possibility of intradural lesion . again the proximal extent
ultrasonography abdomen - pelvis and ct scan chest and brain ruled out any other primary focus . in the year 1997 , patient had progressive onset of weakness in lower limbs , for which she underwent posterior decompression at d10-d11 level in peripheral hospital . she was started with anti tuberculosis treatment . at 1 year postsurgery ( 1998 ) when symptoms did nt improve ,
we then did excision biopsy of lesion through transpedicular approach along with posterior stabilization with hartshill rectangle and sublaminar wires .
intraoperatively we found pearly white daughter cysts and histopathology proved it to be hydatid cyst .
medical management in the form of tablet albendazole was then started in consultation with infectious disease physician .
postsurgery patient became ambulatory but again had a recurrence at the same level ( d10-d11 ) after 2 years and had to be reoperated for paraplegia .
it could not be repaired because dura was adherent to cyst wall and could not be separated .
she had further two recurrences at the same level ( d10-d11 ) which presented with paraparesis and right transthoracic resection of lesion ( 2002 ) and anterior corpectomy of d10-d11 with screw fixation with cement augmentation polymethyl methacrylate ( pmma ) ( 2004 ) was done .
( a ) plain radiographs anteroposterior and lateral views showing instrumentation with hartshill and sublaminar wires showing fusion at d10-d11 and no implant breakage or cut - out ( b ) computed tomography myelogram showing complete cut off of dye flow at l3-l4 level a working diagnosis of recurrent hydatid cyst was established .
plan of surgery was to expose previous operated thoracic ( d10-d11 ) and lumbar level ( l3-l4 ) . through a posterior approach ,
site of previous surgery ( d10-d11 ) revealed a large cyst with multiple daughter cysts [ figure 2 ] .
there was no extra dural extension of the disease from the primary thoracic area ( d10-d11 ) to the lumbar region ( l3-l4 ) .
multiple small cysts were found from l4 to d12 largest being 1 1 0.5 cm .
morphology of cysts was typical of a hydatid cyst with translucent wall and clear fluid content .
few cysts were adherent to the cauda equina roots and were not excised completely to prevent root injury .
combination therapy of albendazole ( 10 mg / kg ) and praziquantel ( 50 mg / kg ) daily was started .
postoperative course was uneventful and at the latest followup of 1 year , patient was independently ambulatory with no complaints and showing complete neurological recovery .
hydatid involvement of the liver and lungs has been most commonly described ( 90%).23 bone affection is rare ( 0.5 - 4% ) of which spine involvement is seen in half of the cases.2 thoracic spine is most commonly affected ( 52% ) , followed by lumbar ( 37% ) sacral ( 5.5% ) and cervical ( 5.5% ) levels.67 involvement of the spine is through portovenous anastomoses or direct extension from pulmonary focus .
daughter cysts infiltrate the cancellous trabeculae of vertebral body , breach the bone and extend to the paravertebral soft tissue or extra dural space causing neurologic compression.278 intervertebral disc involvement is unusual .
braithwaite9 has described five pathologies for vertebral hydatidosis : ( 1 ) primary intramedullary cyst ( 2 ) intradural extra medullary cyst ( 3 ) extra dural intraspinal cyst ( 4 ) vertebral body disease ( 5 ) paravertebral cyst .
the first three groups are very rare.14710 our case had an initial primary thoracic vertebral involvement ( t7-t8 ) ( braithwaite type 4 ) with spread to paravertebral tissue and extra dural space ( braithwaite type 3 and 5 ) .
the patient had multiple recurrences with neurological involvement which required surgeries to decompress the cord . on each occasion ,
complete neural recovery was noted and was maintained at long term followup ( minimum 2 years to maximum 7 years ) . at last followup , there was intradural involvement of the disease with a block at the lumbar level ( braithwaite type 2 ) .
we believe that the possible mechanism of spread to the intradural space was through the rent in the dura mater that incurred during the second surgery . at surgery
there was no continuity of cyst at thoracolumbar region with the parent mass at the thoracic level ; either intradural or extra dural , thus indicating free fall of the daughter cyst in the csf column and settling under gravity at the lumbar level .
this is similar to drop metastasis described in literature.6 magnetic resonance imaging is the gold standard for the diagnosis of hydatid disease.1211 cyst has thin single wall , no septation and same intensity as of csf.211 in our case , the role of magnetic resonance imaging ( mri ) was limited because of stainless steel implant .
mri suggested recurrence of the disease ; however the presence of steel implant hindered with the determination of the actual extent and location of the disease .
diagnostic aspiration of the cyst has been described for primary hydatid disease though it should be avoided in view of possible risk of anaphylaxis and further spillage of the daughter cyst.2 dural injury , though unfortunate , are highly likely due to the peridural fibrosis both due to repeated surgery and the pathology itself .
furthermore the hydatid cyst wall may become continuous with the dural sac as was seen in our case at the thoracic level making complete excision of the sac impossible .
this type of spread has not been reported previously for a parasitic infestation.4 kaen et al.4 have reported a similar case of primary hydatid disease of lungs and liver with thoracic spine involvement .
primary hematic dissemination was regarded as most plausible mechanism , as there was no dural injury .
gnes et al.12 have described a case of primary pulmonary hydatid disease with multiple intradural dissemination .
the mode of spread in their case was postulated to be through local transforaminal invasion . in our case ,
dural tear at the time of previous surgery can be suggested as the most plausible mechanism of spread .
overall recurrence in spinal hydatid disease has been reported to be as high as 30 - 100%.1 incomplete primary removal of cyst due to extensive bony involvement , spillage during excision , improper chemotherapy and undetected local spread are few of the factors toward high recurrence rate .
adhesions between the cyst wall and the roots in the dural sac further complicate the situation for the fear of root injury if aggressive debridement is contemplated . in most of the cases
however this time , intradural involvement prohibited radical removal of the cyst for the fear of neurological injury.4 we agree that the patient may have further intradural recurrences ; therefore regular imaging during asymptomatic period may be justified .
pmma has been reported to have anthelmintic effect.3 medical management of the disease has been a matter of concern and debate .
albendazole is definitely preferred over mebendazole considering its better oral absorption and higher intracystic penetration.1 combination of albendazole with praziquantel has better scolicidal effect.10 this is one of the longest followup of hydatid disease of spine that has ever been reported .
this case highlights upon mode of progression , diagnosis , recurrence and modalities of management of hydatid disease .
hydatid disease of spine should be kept in mind in the differential diagnosis of destructive or infectious lesions of spine .
recurrence is known to occur even after successful evacuation of lesion and prolonged medical management .
therefore , high index of suspicion is the key to early diagnosis and long followup with critical clinical evaluation is necessary . | we present a long term followup ( 13 years ) of spinal hydatid disease with multiple recurrences and intradural dissemination of the disease at the last followup .
intradural extension of the disease in our case was supposedly through the dural rent which has not been reported in english literature .
an early followup of the same case has been reported previously by the authors .
a 53 year - old female came with progressive left leg pain and difficulty in walking since 2 months . on examination
, she had grade four power of ankle and digit dorsiflexors ( l4 and l5 myotomes ) on the left side ( medical research council grade ) .
there was no sensory loss , no myelopathy and sphincters were intact .
plain radiographs showed consolidation at d10-d11 ( old operated levels ) with stable anterior column and there were no implant related problems .
magnetic resonance imaging showed a cystic lesion at l3-l4 , signal intensity same as of cerebrospinal fluid in t2 and t1 , displacing the cauda equina roots .
the proximal extent of the lesion could not be identified because of artifacts from previous stainless steel instrumentation .
computed tomography myelogram showed complete block at l3-l4 junction with meniscus sign .
this is the longest followup of hydatid disease of the spine that has ever been reported .
hydatid disease should always be included in the differential diagnosis of destructive or infectious lesions of the spine .
aggressive radical resection whenever possible and chemotherapy is the key to good results .
recurrence is known to occur even after that .
disease can have long remission periods .
possibility of intradural dissemination through dural injury is highly likely .
hence , it should always be repaired whenever possible . |
acute myeloid leukemia ( aml ) is a clonal hematopoietic disorder characterized by uncontrolled self - renewal of hematopoietic stem cells , maturation arrest at myeloblast level , peripheral blood and bone marrow infiltration of blast cells .
it is demonstrated that pathogenesis of aml is associated with some disorders including genetic changes and chromosomal translocations .
investigation of molecular genetic alterations affecting npm1 ( nucleophosmin1 ) and flt3 genes as well as wt1 ( wilms ' tumor ) assay are known as important prognostic factors in aml , in addition to age , white blood cell counts and cytogenetic aberrations . in recent years
, epigenetic disorders including methylation of tumor suppressor genes like sfrps ( secreted frizzled related proteins ) family genes have also been shown to play a role in aml pathogenesis .
these alterations may lead to differentiation and apoptosis arrest in leukemic blasts as well as increase in proliferation and self - renewal .
secreted frizzled - related proteins ( sfrps ) are wnt antagonists that suppress this signaling pathway in healthy individuals .
wnt signaling pathway contributes to regulation of cell proliferation and differentiation . in some malignancies like colorectal cancers , head and neck tumors and gastric cancer , aberrant wnt signaling pathway has been shown to cause uncontrolled cell proliferation .
chronic myeloid leukemia was the first malignancy in which the important role of wnt signaling pathway has been described .
wnt controls the cytoplasmic level and stability of -catenin . in absence of wnt ligand and its protectional role , -catenin level decreases due to destruction by casein kinase 1 and glycogen synthase kinase 3b enzymes .
but when the ligand adheres to its receptor ( frizzled receptor ) , activates dv1 ( dishevelled ) proteins . having accumulated in cytoplasm
, -catenin migrates to nucleus where it causes expression of some genes involved in cell proliferation and differentiation .
it has recently been demonstrated that both chromosomal alterations and flt-3 mutations associated with aml pathogenesis affect wnt signaling pathway .
methylation of sfrp genes lose their inhibitory effect on wnt singnaling pathway leading to elevated cytoplasmic and nuclear levels of -cathenin and subsequently -cathenin as a transcription factor promotes expression of myc and cyclin d genes that are involved in cell cycle regulation . since the methylation of these genes may play a role in initiation and leukemogenesis of aml , in present study we investigated the methylation status of sfrp1 and sfrp2 genes in newly diagnosed aml patients who were admitted to the hematology , oncology and bone marrow transplantation research center in shariati hospital , tehran .
peripheral blood samples were drawn from 25 healthy individuals ( negative control group ) and 43 patients with newly diagnosed aml who were admitted to the hematology , oncology and bone marrow transplantation research center in shariati hospital , tehran .
the clinical parameters consisted of white blood cell count ; platelet , age , hemoglobin and rate of recovery following induction chemotherapy were extracted from patients ' medical records .
mononuclear cells of drawn samples including leukemic blast cells were isolated by concentration gradient sedimentation using ficoll - hypaque followed by dna extraction by saturated salt standard method . in the next step , extracted dna underwent bisulfite conversion with the epi tect bisulfite kit ( qiagen ) using producer instructions . by this treatment , unmethylated cytosine converted to uracil where methylated cytosine stayed intact .
then , the methylation status of sfrp1 and sfrp2 genes was investigated using msp ( methylation - specific pcr ) technique .
msp is a type of pcr used to investigate the methylation of cpg islands . in this method
, we used 2 pairs of primers specified for checking the methylated or unmethylated residue .
the sequences of these primers are designed in previous studies . sfrp-1 and sfrp-2 gene primers sequences , annealing temperature and product size for msp assays .
m : methylated , u : unmethylated , f : forward , r : reverse four msp reactions using methylated and unmethylated primers related to sfrp1 and sfrp2 were administered for each patient . in methylation testing , we used 2 l of bisulfite - treated dna , 4.5 l of dh20 , 12.5 l of master mix , 0.5 l of forward primer and 0.5 l of reverse primer , while we used 2 l of dna , 8.5 l of dh20 , 12.5 l of master mix , 0.5 l of forward primer , 0.5 l of reverse primer and 1 l of mgcl2 to investigate the unmethylated status .
in the first step of msp , reaction components put in pre - thermal condition including 98 c for 1 minute and 96 c for 3 minutes followed by 40 cycles including 99 c for 10 seconds , 97 c for 20 seconds , 54 c for 30 seconds ( sfrp1- um primer ) , 64 c for 30 seconds ( sfrp1 , 2-m primer ) and 72 c for 7 minutes ( extension ) . in this study
59695 ) containing unmethylated and completely methylated dnas as negative and positive controls , respectively .
whitney u - tests and spss analytic software version 21(version 21 , spss inc chicago , il ) were used for statistical analysis of data .
a total of 43 patients , 31 ( 72.1% ) males and 12 ( 27.9% ) females , were included in this study .
white blood cells and platelet counts were 600145000 and 2000280000 cells per microliter and their mean values were 27818.5 and 98633.3 cells per microliter , respectively .
sfrp1 gene was hemi - methylated in 13 of patients ( 30.2 ) , completely methylated in 13 patients ( 30.2 ) and completely unmethylated in 17 patients ( 39.5 ) , while sfrp2 gene was hemi - methylated in 16 of patients ( 37.2 ) , completely methylated in 9 patients ( 20.9 ) and completely unmethylated in 18 patients ( 41.8 ) ( figure 1 ) .
correlation between hypermethylation of sfrp1and sfrp2 genes and laboratory and clinical symptoms of patients are indicated in table 2 .
correlation between hypermethylation of sfrp-1 & -2 genes and laboratory and clinical symptoms of aml patients aml : acute myeloblastic leukemia , hb : hemoglobin , wbc : white blood cell , fab : french - american - british , m : methylated , u : unmethylated . in aml patients , hypermethylation frequency of sfrp1 and sfrp2 genes were 30.23 ( 13 out of 43 patients ) and 20.9 ( 9 out of 43 patients ) , respectively .
aberrant methylation of these genes was found in all fab classifications of aml , except subtype m6 .
hypermethylation of sfrp1 ( p = 0.028 ) and sfrp2 ( p = 0.004 ) genes were associated with fab - m0 subtype of aml ( table 2 ) .
there was no significant relationship between hypermethylation of genes ( sfrp1 and sfrp2 ) and clinical parameters of patients including sex , age , white cells and platelet counts ( table 2 ) .
the causes of relapse developed in 6 of 43 subjects were attributed to hypermethylated sfrp1 and sfrp2 genes in 2 ( 4.6% ) and 1 ( 2.3% ) patient , respectively .
there was no significant relationship between hypermehylation of both sfrp 1 and sfrp 2 genes and relapse of patients .
also , 30 ( 69.76 % ) out of 38 ( 88.37% ) patients had complete remission
wnt/-catenin signaling pathway has been implicated in many cellular procedures including proliferation , morphology , motions , destiny determination of cells and organ development . understanding the roles of wnt/-catenin signaling in survival , proliferation and differentiation of hematopoietic stem cells resulted in developing the hypothesis that this signaling pathway may be involved in leukemogenesis .
this protein binds to wnt protein and thus inhibits its binding to wnt - frizzled receptor .
hence , there may be an association between methylation of wnt signaling antagonist genes and the activation of this pathway in solid tumors and leukemia .
aberrant methylation of tumor suppressor genes is a more specific and common genetic events in human cancers . in present study , we investigated the methylation status of sfrp1 and sfrp2 genes in newly diagnosed aml patients .
the results of this study showed that hypermethylation of sfrp1 & sfrp2 genes occurs with a frequency of 30.23 % ( 13 out of 43 subjects ) and 20.9 % ( 9 out of 43 patients ) in aml patients at the time of diagnosis , respectively , while none of the normal samples showed methylation .
also , 13 out of 43 ( 32.2% ) patients showed simultaneous methylation of both sfrp1 and sfrp2 genes at the time of diagnosis .
veeck et al demonstrated that epigenetic changes of sfrp1 through methylation were even linked to poor prognosis in patients with breast cancer .
simon et al . suggested that recombinant sfrp1 might be a novel therapeutic strategy for cancers with suppressed sfrp1 expression .
moreover , sfrp2 methylation as one of the epigenetic targets occurs in cancers such as colon cancer , esophagus cancer ( 13 ) , bladder cancer , gastric cancer , liver cancer and lung cancer . like previous studies
, our study also showed that sfrp1 and -2 genes are epigenetic modulation targets in aml patient .
the percentage of patients with aberrant methylation of at least one sfrp gene in this study was 26 patients ( 60.5 % ) for sfrp1 and 25 patients ( 58.2 % ) for sfrp2 . therefore ; methylation of these genes may be involved in the onset of aml . in this study , the hypermethylation frequencies of sfrp1 and sfrp2 ( 30.2 % and 20.9 , respectively ; total 51.1 % ) were lower than those ( 41% & 31% , respectively ; total : 72% ) reported by valencia et al and higher than those ( 29 % and 19% , respectively ; total : 48 % ) reported by jost et al , but were similar to those ( 31.6 % and 19.3 % , respectively ; total : 50.9 % ) reported by h - a hou et al .
methylation of sfrp genes has also been shown in hematologic malignancies , so as pehlivan et al , showed the activation of wnt signaling pathway through methylation of sfrp1 leads to drug resistance in patients with chronic myeloid leukemia treated with imatinib msylate by suppression of imatinib mesylate effect over bcr - abl signaling pathway .
hong et al . , showed that methylation of sprp genes in patients with mds is associated with poor prognosis and less survivability . to investigate the prognostic relevance of aberrant methylation of sfrp1 and sfrp2 genes in aml patients , we concentrated on the clinical outcome of patients , including relapse , complete remission , failure and death . among 43 patients , complete remission after induction chemotherapy
however , we did not observe any significant association between hypermethylation of these genes and clinical outcome of patients .
jost et al and h - a hou et al also did not find any prognostic factor for aberrant methylation in sfrp promoters whereas griffiths et al .
, showed that sfrp2 and sfrp5 methylations were associated with poorer disease - free survival and os in cytogenetically normal aml valencia et al reported that methylation of sfrps and dkks was associated with a poorer prognosis only in young adult patients with intermediate - risk cytogenetics .
furthermore we could not find significant differences between aml patients with and without sfrp hypermetylation in conventional prognostic factors such as age , sex , wbc & platelet count , and hemoglobin concentration , though significant differences may be observed when increased sample size .
cheng et al pointed out that sfrp promoter methylation was significantly correlated with increased age ( median age : 12 years vs. 3.5 years ; p= 0.0001 ) and male sex in pediatric patients with aml .
moreover , h - a hou et al showed that male patients had a higher incidence of hypermethylation of wnt inhibitors than females .
our present study also showed that male patients had a higher incidence of hypermethylation of sfrp1 and sfrp2 than females ( 76.9% vs. 23% for sfrp1 and 66.6% vs. 33.3% for sfrp2 ) .
h - a hou et al . , showed that patients with fab m0 subtype of aml had the highest incidence of hypermethylation of wnt inhibitors , whereas those with m4/m5 subtype had the lowest incidence ( 28 ) .
our results also showed that aberrant methylation of these genes took place in all fab - aml subgroups including m0 , m1 , m2 , m4 and m5 , except subgroup m6 .
the highest hypermethylation rate of sfrp1 ( 100% , p=0.028 ) & sfrp2 ( 100% , p=0.004 ) were observed in m4 and m1 subgroups respectively , compared with m0 subgroup of aml that has the lowest rate of hypermethylation of sfrp1 ( 25% , p= 0.433 ) and sfrp2 ( 0% , p=0.936 ) , respectively .
the present study has showed that the methylation of sfrp1 and sfrp2 genes contingently takes place in all fab subgroups of aml except subgroup m6 in patients with aml at the time of diagnosis . in this study
, we did not observe an association between methylation of relevant genes and clinical findings in aml patients such as age , sex , wbc , platelet counts and complete remission after induction therapy .
however , it is recommended to conduct more studies to determine the role of hypermethylation of these genes in the pathogenesis of aml and other hematologic malignancies . | abstractintoduction : acute myeloid leukemia ( aml ) is a heterogeneous group of hematologic malignancies with abundant changeability in the pathogenesis .
dna methylation of cpg islands within the promoters of specific genes may play roles in tumor initiation and progression.secreted frizzled - related proteins ( sfrps ) are negative regulator of the wnt signaling pathway . in the present study
, we examined the methylation status of sfrp1 and sfrp2 genes in patients with aml.methods:isolated dna from peripheral blood of 43 aml patients and 25 healthy subjects as a control group was treated with sodium bisulfite was treated with sodium bisulfite and analyzed by methylation - specific polymerase chain reaction ( msp ) with primers specific for methylated and unmethylated promoter sequences of the sfrp1 & -2 genes .
we used mann - whitney u - tests to investigate the correlation between sfrp1 and sfrp2 genes hypermethylation and clinical parameters.results:the frequency of aberrant hypermethylation of sfrp1 and sfrp2 genes in patients with aml was determined 30.2% ( 13/43 ) and 20.9% ( 9/43 ) , respectively .
in addition , for all subjects in control group , methylation of sfrp1 and sfrp2 genes were negative .
patients with m0 subtype of fab - aml had the highest incidence of hypermethylation of sfrp1 ( p = 0.028 ) and sfrp2 ( p = 0.004).conclusion : the present study showed that , like many solid tumors , methylation of sfrp genes also occurs in aml .
therefore , the methylation of these genes may play a role in the initiation of leukmogenesis |
this prospective study was conducted among 488 patients admitted to oncology ward of our hospital , over a period of 1 year , after obtaining the approval of the institutional ethics committee ( iec 176/2012 ) .
it was an observational , focused pharmacovigilance study carried out as a part of the pharmacovigilance program of the hospital from january 1 , 2013 , to december 31 , 2013 . after obtaining informed consent ,
data on adrs were collected from the cancer patients belonging to both gender and of all ages , who were receiving either taxane based or vinca - based cancer chemotherapy under any standard regimen .
those patients who did not receive drugs belonging to either of these groups as part of drug regimen were excluded from the study .
no change in the treatment decision , schedule , or duration of cancer chemotherapy was made as a part of the study .
the patients who were admitted following the infusion were monitored for adverse effects till their discharge from hospital .
the drug effects which were described by the patients and effects which were diagnosed and documented by the physician during the infusion visit and the follow - up period were recorded .
patients demography , drug , dose , type of adr , severity , causality , and outcome of the treatment of adr were noted and reported in the indian pharmacopoeia commission ( ipc ) form .
the who causality assessment scale is the one recommended by the who uppsala monitoring center and classifies adr as certain , probable , possible , unlikely , conditional , and unassessable .
the naranjo 's algorithm is a questionnaire based scale consisting of ten objective questions with three types of answers , yes , no , or do not know .
scores are given accordingly , and the drug reaction can be classified as definite , probable , possible , and doubtful based on the total score . in naranjo 's algorithm , if there were alternate causes particularly other drugs in case of multi - drug regimen then the score for that question would be negative .
preventability and severity of adrs were assessed by modified schumock and thornton scale and modified hartwig et al .
the modified schumock and thornton scale classifies adrs as definitely preventable , probably preventable , and not preventable based on a set of questions for each level . the modified hartwig and siegel scale
classify the severity of adr as mild , moderate , or severe with various levels according to factors such as requirements for change in treatment , duration of hospital stay , and disability produced by the adr .
this was uploaded weekly on the vigiflow database for assessment by national coordinating centre , ipc which was reported to uppsala monitoring center a who program for international drug monitoring .
a total of 488 patients on microtubule - damaging anticancer drugs ( taxanes and vinca alkaloids ) were admitted to oncology ward during the above period . among them , 97 adrs were reported .
majority of adrs occurred in the age group of 4160 years ( n = 45 , 46.4% ) with a female preponderance ( n = 67 , 69.1% ) .
of the 180 male patients , 19 ( 10.56% ) reported with adrs and similarly of the 308 female patients , 39 ( 12.66% ) reported with adrs .
the observations for sex distribution of adrs were analyzed statistically using chi - square test .
the sex wise difference in the occurrence of adrs was statistically not significant ( p = 0.481 ) .
of the adrs reported , 73 ( 75.3% ) were from those being treated for an early stage of the tumor , whereas 24 ( 24.7% ) were from patients being treated for an advanced malignancy . of the total reactions , 34 ( 35.1% ) were from those who received anticancer drugs as the primary modality of treatment , 42 ( 43.3% ) were from those who received the drugs as adjuvant therapy , 16 ( 16.5% ) from those who received as neoadjuvant therapy , and 5 ( 5.2% ) of the reactions were from those who received as palliative care .
only 21 ( 21.6% ) reactions were from those who received these drugs as monotherapy , while 75 ( 77.3% ) reactions were from those who received these drugs as a part of a regimen .
the median cycle number at which adrs were reported is 3 ( interquartile range [ iqr ] 15 ) .
results revealed that gastrointestinal system 39 ( 40.2% ) was the most affected organ system followed by bone marrow 32 ( 33% ) , skin 8 ( 8.2% ) , kidney , central nervous system and others 6 ( 6.2% ) [ figure 1 ] .
frequency of adverse drug reactions categorized based on organ involved the various indications for the use of taxanes were tumors of head and neck , lung , peritoneal , and gynecological tumors .
the frequently reported adrs among taxanes were anemia 11 ( 18.6% ) , diarrhea 10 ( 16.9% ) , candidiasis 7 ( 11.9% ) followed by peripheral neuropathy 4 ( 6.8% ) [ figure 2 ] .
* others = respiratory tract infection thrombocytopenia , vomiting , * * others = cellulitis , elevated liver enzymes , angular stomatitis , flushing of face , photodermatitis , symptomatic hyponatremia , tinea corporis , alopecia , fever , glossitis , numbness , skin discoloration the various indications for the use of vinca alkaloids were tumors of head and neck and childhood tumors .
the frequently reported adrs among vinca alkaloids were febrile neutropenia 9 ( 23.7% ) , candidiasis 8 ( 21.1% ) , vomiting 6 ( 15.8% ) , followed by anemia 3 ( 7.9% ) , and obstipation 3 ( 7.9% ) [ figure 3 ] .
adverse drug reaction types with vinca alkaloids . * others = peripheral neuropathy , abdominal pain , diarrhea , neutropenia , thrombocytopenia , urinary tract infection , weakness most of the reported adrs were type a reactions as per the adr classification by rawlin and thomson .
predictability analysis showed that 43 ( 44.3% ) of the reactions were not predictable while 54 ( 55.7% ) were predictable . according to modified shumock and thornton scale
( n = 50 , 51.5% ) , followed by 27 ( 27.8% ) of the reactions not preventable while reactions like vomiting and neutropenia belonged to the category definitely preventable
. based on modified hartwig and siegal scale of severity assessment , most of the reactions were of mild level 1 severity , except for candidiasis and febrile neutropenia , which were of moderate level 3 severity .
the who causality assessment scale indicated 28 ( 28.9% ) probable and 69 ( 71.1% ) possible but no certain reactions .
naranjo 's algorithm indicated 48 ( 49.5% ) probable with score ranging from 5 to 8 and 49 ( 50.5% ) possible reactions with a score ranging from 1 to 4 [ table 1 ] .
however , the grade of causality for each adr remained low due to the presence of co - administered drugs .
of the reported cases , 69 ( 71.1% ) received either specific or symptomatic treatment while 28 ( 28.9% ) required no treatment .
the median duration of hospital stay of those who required admission was 7.50 ( iqr 020 ) .
from the study , it was found that 85 ( 87.5% ) recovered from the reaction , although none had fatal adrs . the remaining 12 ( 12.4% ) cases either continued to have the reaction during the study or else their reaction treatment outcome was unknown .
an adr , as defined by the who , is a response to a medicine which is noxious and unintended , and which occurs at doses normally used in man .
according to lazarou et al . in 1998 , adrs are considered fourth to sixth leading cause of death in the united states with an incidence of 0.13% fatal adrs requiring hospital admissions .
the plant alkaloids ( taxanes and vinca alkaloids ) , exert their antineoplastic effects by interfering with microtubule function , leading to inhibition of mitosis and apoptosis .
vinca alkaloids cause destabilization of the mitotic spindle , whereas taxanes cause disorganized stabilization of microtubule ultimately resulting in cell cycle arrest .
taxanes are the leading agents in the treatment of various malignancies including breast cancer , ovarian cancer , prostate cancer , kaposi 's sarcoma , squamous cell carcinoma of the head and neck , esophageal cancer , lung cancer , and bladder cancer .
vinca alkaloids are used in various treatment protocols of hematological malignancies and solid tumors including germ cell cancer , lung cancer , neuroblastoma , ewing sarcoma , and multiple myeloma .
taxanes and vinca alkaloids being natural plant products , though apparently considered as safer drugs , like all other drugs they too show some adrs in various patient conditions .
the incidence rate of adverse reactions in this study was found to be comparatively low when compared to other studies .
this lower incidence rate could be due to delivering periodic adr awareness classes to treating physicians and nurses , active involvement of clinical pharmacy and postgraduate students of pharmacology in clinical activities and coordination of quality control unit in the hospital . in this study
, we found that majority of the patients were females ( 69.1% ) which is consistent with findings in other studies . in india , most common cancer among males is oropharyngeal cancer followed by bronchogenic carcinoma with oropharyngeal cancer having surgical resection and irradiation as the modality of treatment .
thus , there was a proportionately higher ratio of females who were prescribed micro - tubule damaging drugs which account for the predominance of adverse reactions in them .
analysis of the age - wise distribution showed the predominance of patients in the age group of 4160 years ( 46.4% ) which is again similar to reports of previous studies .
the primary indications of the microtubule - damaging drugs were for breast , ovarian , cervical , endometrial carcinomas , and other solid tumors which were more prevalent among geriatric patients .
the presence of comorbid illnesses and usage of multiple drugs in them also could attribute to the adverse reactions in them .
this could also be because , in elderly patients , the metabolizing capacity and the excretory functions are reduced , leading to accumulation of drugs in the body and thus increasing the risk of adrs . in this study ,
the different treatment plans adopted depend on a variety of factors such as the staging of cancer , cost of the treatment plan , patient and physician - related factors .
the causality attributed to the drugs received as a part of a regimen received a lower score on the naranjo 's algorithm .
the documented adrs are mainly affecting the gastrointestinal tract , bone marrow and skin and this study also pointed out the same .
the study of shamna et al . and astolfi et al . also found the predominance of the gastrointestinal system followed by the skin in adr occurrence .
cancer chemotherapy damages rapidly dividing cells of bone marrow resulting in myelosuppression thus affecting white blood cells , platelets , and red blood cells .
this myelosuppression leads to a lowering of immunity and thus patients on cancer chemotherapy are at a high risk for developing various infections .
nausea and vomiting are prominent with most cytotoxic agents and is caused mainly due to direct stimulation of chemoreceptor trigger zone . the documented taxanes adrs are mainly alopecia , anemia , peripheral neuropathy , arthralgia / myalgia , nausea , diarrhea , neutropenia , flushing , rash , thrombocytopenia , and severe hypersensitivity reactions and this study also pointed out the same .
since patients in this study received the standard dose , there was no report of these adverse effects .
the frequency of candidiasis reported is more in this study as the majority of the patients were receiving repeated cycles of chemotherapy infusion .
hyperuricemia and symptomatic hyponatremia were not predictable ; though there are some reports of it , which can not be considered conclusive .
the documented adrs among vinca alkaloids are alopecia , peripheral neuropathy , constipation , obstipation , abdominal cramps , vomiting , diarrhea , paralytic ileus , urinary retention , polyuria , dysuria , syndrome of inappropriate antidiuretic hormone secretion ( siadh ) , weight loss , fever , stomatitis , headache and optic atrophy with blindness , and this study pointed out the same .
hyperuricemia and siadh occur during active cell lysis of highly proliferative tumors of massive burden ( e.g. , leukemia and lymphoma ) by cytotoxic chemotherapy .
obstipation is a severe form of constipation associated with fecal impaction and intense abdominal pain .
however , as per a study by astolfi et al . lung cancer , cervical and breast cancer were found to be the most common .
vincas by binding to intracellular microtubules causes intracellular edema of sensory axons and dose - dependent peripheral neuropathy . whereas taxanes by creating leaky axonal membranes are ultimately neurotoxic to schwann cells , clinically pictured as peripheral neuropathy .
paclitaxel was the most commonly used drug among taxanes and vincristine among vinca alkaloids in the inpatient settings and hence the higher incidence of adrs reported with this drug .
nausea , vomiting , and hypersensitivity reactions were not observed in this study due to the use of adequate premedication with parenteral dexamethasone , ranitidine and ondansetron and diphenhydramine , respectively .
analysis of the type of reported adrs according to rawlin and thompson revealed type a predominance .
this result is in line with the study conducted by bates et al . and lazarou et al . ,
but in another study by suthar and desai , all the reported reactions were type b reactions .
type a reactions are dose related and thus were preventable from their known pharmacology and therefore all of them were potentially avoidable .
type b reactions comprise approximately 1015% of all adrs and include hypersensitivity drug reactions . with adequate premedication , common adrs such as nausea , vomiting , glossitis , and candidiasis can be effectively controlled .
the incidence of definitely preventable reactions reveals the failure of rational drug use in the hospital .
this brings out the need for the treating physician to anticipate and counsel the patient adequately before starting of therapy .
most of the reactions were of mild to moderate severity and did not warrant stoppage or changing of drug considering the risk - benefit ratio in specific patients .
even though most of them were mild reactions , they resulted in an increased health care cost due to an increased length of stay and need of some medical interventions .
while severe reactions were more followed by moderate in a study by oshikoya et al .
similar studies may be used to identify iatrogenic adverse effects and may help in preventing such occurrences in the future . in this study ,
most of the reactions showed a similar causality assessment by both the who causality assessment scale and naranjo 's algorithm except for diarrhea and anemia , which were assessed as
possible with lower level of causality by the who scale , were judged as probable with higher level of causality by naranjo 's algorithm .
certain reactions as re - challenge was not attempted in any of the patients although some events like neutropenia recurred in the successive cycles of cancer chemotherapy .
contradict the study by oshikoya et al . which reported more number of definite reactions .
this study provided basic information regarding the safety profile of microtubule - damaging anticancer drugs in a variety of cancers .
similar studies have been done which focused on either multiple drugs used in one field of medicine or only on the causality aspect . to the best of our knowledge ,
a major limitation of the study is that only one case of alopecia and no case of thrombophlebitis reported though being a very common adverse effect with anticancer medications , there are chances of under - reporting and incomplete documentation of data regarding adrs in the case records .
the fact that all patients were not admitted following the infusion may have led to missing some adrs , particularly mild ones .
nevertheless , early detection of these adrs may help in minimizing the damage by either modifying the dose or changing the offending agent .
there is a great need to set up an effective adr monitoring and reporting system in all hospitals and also create awareness among healthcare professionals regarding the importance of this system .
| objectives : the aim of the study was to analyze the adverse drug reaction ( adr ) profile of microtubule - damaging antineoplastic drugs ( taxanes and vinca alkaloids ) and to look for unexpected adrs among the local population .
focused study on these drugs , rampantly used in oncology department for a wide variety of tumors including early and advanced malignancies , would enable better treatment care by physicians.materials and methods : data on adrs were collected from the cancer patients belonging to both gender and of all ages , on taxanes- or vinca - based cancer chemotherapy and reported in the indian pharmacopoeia commission form .
causality was assessed using the who criteria and naranjo 's algorithm .
preventability and severity of adrs were also assessed.results:a total of 97 adrs were reported among 488 patients on microtubule - damaging anticancer drugs admitted over a period of 1 year .
the incidence rate was 19.87% . gastrointestinal system ( 40.2% ) was the most affected followed by bone marrow ( 33% ) and skin ( 8.2% ) .
the highest incidence of adrs was reported among paclitaxel ( 54.6% ) , and vincristine ( 39.2% ) .
most of the reported adrs were of milder nature and preventable .
the who causality assessment scale indicated 71.1% possible reactions.conclusions:this study showed that most adrs are preventable with effective adr monitoring .
there is a great need to create awareness among healthcare professionals regarding the importance of the pharmacovigilance system .
judicious use of the preventive measures will lead to a reduction in the incidence of adrs due to the drug armamentarium , thereby enabling additional economic benefit to the patient and society . |
ultrasound is increasingly being applied in practical medicine for both clinical diagnosis and therapeutic purposes.1 , 2 for diagnostic applications , a better resolution of the morphological features of organs is desirable .
one of the most important factors in such diagnostic applications is the avoidance of biological damage.3 , 4 , 5 , 6 for therapeutic purposes , various applications have been developed including the healing of wounds in skin , muscle and other tissues , hemostasis , transdermal drug delivery , targeted drug delivery , in vivo oxygen delivery , and clot lysis.7 , 8 , 9 , 10 , 11 , 12 with regard to these applications , it is important to evaluate the effect of ultrasound sonication on the living body and cells.13 , 14 , 15 ultrasound has both thermal and nonthermal effects .
ultrasound on a mhz scale is considered to have thermal and/or nonthermal effects , whereas ultrasound on the order of several tens of khz generates mostly nonthermal effects .
although acoustic cavitation has both effects , it has been argued that the mechanical effect is dominant in the case of lowfrequency ultrasound .
in addition , therapeutic ultrasound often uses pulse wave ( pw ) rather than continuous wave ( cw).12 in the present study we focused on the effect of the frequency of pulsing ultrasound on doublestrand breaks ( dsbs ) in genomic dna molecules .
we used relatively lowfrequency , that is , several tens of khz , ultrasound , partly so that we can set the experimental conditions in a wellcontrolled manner to evaluate nonthermal effects .
lowfrequency ultrasound has been applied to lithotripsy , phacoemulsification , ultrasoundassisted liposuction , tissue cutting and vessel sealing , and skin permeabilization.4 , 15 , 16 , 17 , 18 in these applications , the nonthermal effect , that is , cavitation effect , plays the major role .
although several previous papers have discussed the cavitation effect on dna molecules , there have been few studies on the quantitative evaluation of damage to
it is important to unveil the effects of ultrasound from the standpoints of both safety and basic science .
dna damage is currently categorized into four types : base changes , crosslinking , and single and doublestrand breaks ( dsbs).25 among these , dsbs are considered to most strongly affect the function of genomic dna molecules.1 , 2 , 3 , 4 , 5 , 6 many studies have been performed to detect dsbs both in vivo and in vitro .
the polymerase chain reaction can be used to detect dna damage through observation of the termination of amplification.7 , 8 immunological assays are also commonly used to detect oxidative dna damage through the use of an antibody or immunoglobulin.7 , 9 in situ hybridization provides information on specific changes in certain dna sequences.7 , 10 although several methodologies have been proposed to detect dsbs in dna molecules , including the comet assay and singlecell gel electrophoresis assay,7 , 8 , 12 , 26 it has been rather difficult to evaluate the number of dsbs per unit length of dna in a quantitative manner , especially for long , genomicsized dna molecules . recently , it has been demonstrated that the direct visualization of single giant dna molecules above the size of 100 kbp by the use of fluorescence microscopy can provide useful information on the structure and function of genomic dna molecules.26 , 27 , 28 , 29 , 30 it is becoming increasingly clear that giant dna molecules undergo large discrete changes in their higherorder structure accompanied by a change in density on the order of 1010 , whereas shorter oligomeric dna molecules do not exhibit this property.31 , 32 recently , we studied the effect of ultrasound irradiation in cw mode on dsbs in dna by singlemolecule observation , and evaluated cavitation power acoustically through the use of a hydrophone.28 we found that dsbs are generated above a threshold ultrasound power , corresponding to the critical power to generate cavitation . in the present study , we measured dsbs in genomesized dna through singlemolecule observation with highresolution fluorescence microscopy to clarify the effect of pulse irradiation in comparison to cw irradiation .
figure 1 shows the results of the measurements of dsbs with different duty ratios for ultrasound sonication up to 60 s. as shown in the schematic in figure 1 a , we measured dsbs by changing the duty ratio of pw sonication : d=onon+off
, where on
is the sonication period and off
is the off period , that is , when sonication is not applied . a duty ratio of 1.0 corresponds to cw .
the total ultrasound energy applied to the dna solution is linearly proportional to the duty ratio d. figure 1 b shows examples of fluorescence images of dna molecules stretched on a glass slide , indicating marked fragmentation for pulse sonication with a duty ratio of d=0.8 .
figure 1 c shows the change in the average length of stretched dna molecules after ultrasound sonication ,
l
, depending on the duty ratio d ; there is a gradual decrease in l
with an increase in d. in this measurement , the average control length for more than 100 dna specimens under each experimental condition , l0
, was determined to be 29.5 m for samples without radiation ( d=0 ) .
this is smaller than the natural contour length ( 57 m ) and can be attributed to the procedure used to extract and purify t4 gt7 dna molecules from the phage and to the process of sample preparation , which includes pipetting and mixing.29 by denoting the average number of breaks per average length before sonication as n
, we obtain n
= l0
/l
1 .
to determine the frequency of dsbs per unit length n
, i.e. , per 10 kbp , we rescaled as n
= n
/l0
10 kbp , where l0
is in units of kbp.29 thus , figure 1 d shows the dependence of n
on the duty ratio d , where n
is almost linearly proportional to d , for d between 0 and 0.9 .
interestingly , for d above 0.9 , there is a large deviation from a linear relationship , including at d=1.0 , that is , for cw sonication . although it may be worthwhile to clarify the degree of deviation from the linear relationship , under the experimental conditions in figure 1 , it is difficult for obtain the value of n
in a precise manner , since the experimental error for n
increases with greater fragmentation .
under this situation , we performed additional experiments as described below .
a ) schematic representation of the periodic application , or pulsing , of ultrasound with different duty ratios , d. b ) fluorescence microscopy images of stretched dna molecules after exposure at various duty ratios ( see the procedure in figure 5 ) .
l
, versus the duty ratio , d , under the same sonication period , ttotal=60s
.
d ) number of dsbs versus duty ratio , d , under the same sonication period , ttotal=60s
. figure 2 a shows the experimental scheme that was designed to gain deeper insight into the effect of the duty ratio d , or pulse repetition period t
, on the degree of dsbs . in this scheme ,
the total input energy of ultrasound over 60 s is exactly the same for all of the experiments with different pulse repetition periods t
or different numbers of pulses n
p , where t=on+off
.
a ) schematic representation of the application of pulsed ultrasound with different pulse repetition periods , t
, under a constant ultrasound energy input .
b ) average length of dna , l
, versus number of pulses , n
p , ( or pulse repetition period , t
) under d=0.4 .
c ) number of dsbs versus number of pulses , n
p , ( or pulse repetition period , t
) under d=0.4 .
figure 2 b shows the change in the average length l
with respect to the number of pulses n
p at an ultrasound pressure of 90 kpa and with a duty ratio of 0.4 , where the horizontal axis is on a logarithmic scale . in these experiments , the average length , l0
, was 22.9 m for about 100 dna molecules without sonication .
figure 2 c indicates that , with a pulse repetition period t
above the order of second(s ) , there are fewer dsbs than with cw sonication . in contrast , the number of dsbs with t
below 1 second(s ) , becomes greater than that for cw , and tends to increase with a decrease in t
.
figure 3 shows the results of a similar series of experiments as in figure 2 under the condition d=0.8 .
the average length l
after sonication is somewhat smaller than that in figure 2 b. if we consider that the total input energy of ultrasound in figure 3 is twice that in figure 2 , it is natural to observe that dsbs are more frequent in figure 3 .
interestingly , a closer inspection of figure 2 and figure 3 indicates a similar dependence of dsbs on the change in n
p ; i.e. , compared to dsbs under cw , dsbs tend to decrease with fewer pulses and tend to increase with a larger number of pulses , which reflect longer and shorter pulse repetition periods t
, respectively .
a ) average length of dna , l
, versus number of pulses , n
p , ( or pulse repetition period , t
) under d=0.8 .
b ) number of dsbs versus number of pulses , n
p , ( or pulse repetition period , t
) under d=0.8 .
next , we consider the timedependent change in dsbs under ultrasound sonication . the experimental trend that a larger number of pulses causes greater damage to dna implies that the probability of dsbs per unit time tends to decrease gradually during the period of individual pulses
thus , we assume that the probability of dsbs for a single pulse , x , is simply dependent on its duration or period , on
, as in [ eq .
( 1)]:(1)xon
where we regard 0<<1 , by considering the weaker effect on the probability of dsbs per unit time with a longer period for a pulse .
the period is given as on = dttotalnp
, where d : duty ratio , ttotal
: total sonication period , and n
p : number of pulses .
thus , the number of dsbs per unit length n
is given under the approximation that the dsbs caused by individual pulses are mutually independent [ eq . ( 2)]:(2)nnpx = npdttotalnp
now , we may expect the following relationship between < n > and n
p.[eq .
log relationship between the number of pulses , n
p , and the number of dsbs ,
< n>. from the linear portions of the lines for both d=0.4 ( the blue line ) and 0.8 ( the red line ) , we obtain =0.8 .
( 4)]:(4)log10n=0.2log10np+c
scaling relationship between the number of pulses , n
p , and the number of dsbs , < n > , shown as the log log plot of the experimental data in figures 2 and 3 .
( 4 ) ] , whereas the curves are given by [ eq . ( 6 ) ] .
in other words , the difference in c is given as c 0.2 . from eq .
thus , it has become clear that the parallel linear relationship depicted as blue and red solid lines in figure 4 can be well interpreted based on the theoretical expectation as in [ eq .
the deviation of the plots for larger values of n
p with d=0.8 is attributable to the breakdown of the assumption that the timedependent change in the probability of dsbs is not influenced by prior pulses . in other words ,
when the resting time between neighboring pulses becomes on the order of 0.1 s , the aftereffect of the previous pulse becomes significant.33 from the experimental finding that the parameter ( = 0.8 ) is smaller than unity , it becomes clear that the kinetics of dsbs exhibit a scaling behavior .
this fact indicates the existence of a longer timetail compared to that under usual exponential decay .
next , we can discuss the deviation from a linear relationship at small values of n
p , especially at n
p=1 .
we can check the degree of deviation from a linear relationship by simply adding an additional term to [ eq .
( 6)]:(6)log10n=0.2log10np+c+0.3np.
this additional term results in the curved lines in figure 4 , suggesting that the fitting becomes rather good when we choose =2 .
this parameter in the scaling equation implies rather rapid decay of the aftereffect of pulsed sonication .
the detailed mechanism to account for why the probability of dsbs decays on a time scale of second(s ) during pulse sonication is not yet clear .
however , the kinetic data together with their analysis reported here may contain important information on the timedependent change in the physicochemical effect of pulsed sonication , including the strength of the shockwave generated through cavitation .
recently , we studied the effect of ascorbic acid against dsbs in dna and found that ascorbic acid has a marked protective effect against the damage induced by reactive oxygen species . in contract ,
ascorbic acid offers almost no protection against the damage caused by ultrasound.29 thus , dsbs caused by ultrasound are most likely caused by the shockwave under the generation of cavitation.28
the results reported here are expected to stimulate studies on the effects of periodic perturbations on the time evolution of nonlinear systems , including on many chemical and biochemical kinetics.34 it has been suggested that periodic perturbations significantly change the essential features of nonlinear dynamical systems , including the appearance of mode bifurcation , breakdown of additivity , etc.35 , 36 , 37
we studied dsbs in giant dna caused by ultrasound sonication by using singledna observation with fluorescence microscopy .
the results showed that the probability of dsbs under pulse sonication is lower than that under cw when the repetition of pulses is above the order of second(s ) .
in contrast , the probability of dsbs under pulse sonication is higher than that under cw when the pulse repetition is below the order of second(s ) .
the discovery of this biphasic nature of the effect of ultrasound is expected to attract considerable interest from scientists in a wide range of disciplines , including not only the field of ultrasonics , but also biophysics , physical chemistry , and macromolecular science , to shed light on the biphasic effect of pulse sonication , we proposed a simple phenomenological model by considering a characteristic decay in the probability of causing dsbs during singlepulse sonication .
the data analysis shows scaling behavior between the number of pulses and the number of dsbs , as in figure 4
. it may be interesting to clarify the actual physicochemical mechanism of the timedependent change in the probability of dsbs during pulse sonication in a future study . additionally , in the practical medical application of ultrasound
, these results suggest that through the selection of a suitable pulsing time profile together with a consideration of the effect of ultrasound power28 , 29 , 38 , 39 it may be possible to minimize any harmful effects on the human body .
similarly , we may be able to deduce the most suitable pulsing conditions for the treatment of malignant tumors .
t4 gt7 dna ( 166 kbp , contour length 57 m ) and trishcl solution ( ph 7.5 ) were purchased from nippon gene ( toyama , japan ) .
the fluorescent cyanine dye yoyo1 ( 1,1(4,4,7,7tetramethyl4,7diazaundecamethylene)bis4(3methyl2,3dihydro(benzo1,3oxazole)2methylidene)quinolinium tetraiodide ) was obtained from molecular probes , inc .
the antioxidant 2me ( 2mercaptoethanol ) and poly(llysine ) were purchased from wako pure chemical industries ( osaka , japan ) and from sigma
a pair of facing langevin transducers ( fbl28452hs ; fuji ceramics , fujinomiya , japan ) was attached to a cell so that they faced one another .
the cell measured 808050 mm ( depth ) and was filled with distilled water .
the experimental system was essentially the same as in a previous study.28 thus , we will only describe it briefly . a sinusoidal signal at a frequency of 30 khz was input to the transducers to form a standing acoustic wave .
we measured the threedimensional distribution of the sound pressure amplitude and confirmed that , under the condition with the fixation of a test tube , the standing wave showed maximum amplitude in the center of the cell , as shown in ref . . throughout the present study
inside the cell , a polypropylene tube containing a dna sample was located at the central antinode . t4 gt7 dna ( final concentration : 0.1 m )
we examined the relationship between the current entering the transducers ( i
0p ) and the sound pressure from zero to the peak value ( p
0p ) .
the sound pressure was measured using a calibrated hydrophone ( model 8103 ; brel & kjr , nrum , denmark ) with a frequency bandwidth ranging from 200 hz to 60 khz after the tube was removed from the cell . if we assume a linear relationship between the current and sound pressure , the pressure in the presence of the tube at the antinode was extrapolated from the i
0pp
0p curve . to visualize individual dna molecules by fluorescence microscopy , the fluorescent dye yoyo1 ( final concentration : 0.1 m ) and 2me ( 3.8 % ( v / v ) )
fluorescence images of dna molecules were captured by the use of an axiovert 135 tv microscope ( carl zeiss , oberkochen , germany ) equipped with an oilimmersed 100 objective lens , and recorded on a dvd through an ebccd camera ( hamamatsu photonics , japan ) .
the recorded videos were analyzed by virtualdub , a free and opensource videocapture and videoprocessing utility for microsoft windows written by avery lee .
the dna solution was subjected to ultrasound sonication for 60 s under different pulse conditions .
after ultrasound exposure , for the measurement of dna length , dna molecules were fixed on a glass surface .
glass slides were pretreated with poly(llysine ) ( concentration : 510 % ( v / v ) ) solution , and washed with distilled water .
a droplet ( 5 l ) of a sample was adsorbed on a modified glass slide to obtain images of the elongated dna conformation .
a droplet of the sample solution was situated on a glass slide and covered with a slip glass under weak shearing to observe elongated dna .
the captured fluorescent images were analysed by imagej software ( national institutes of health , md , usa ) .
the experimental error in the dna length is mainly due to the blurring effect associated with the fluorescence microscopic observation and is estimated to be on the order of 0.30.5 m .
experimental scheme to evaluate dsbs caused by exposure to ultrasound . | abstractdna doublestrand breaks ( dsbs ) caused by ultrasound were evaluated in a quantitative manner by singlemolecule fluorescence microscopy .
we compared the effect of timeinterval ( or pulse ) sonication to that of continuous wave ( cw ) sonication at a fixed frequency of 30 khz .
pulses caused fewer dsbs than cw sonication under the same total input ultrasound energy when the pulse repetition period was above the order of a second .
in contrast , pulses caused more dsbs than cw sonication for pulse widths shorter than a second .
these effect of ultrasound on dna were interpreted in terms of the timedependent decay in the probability of breakage during the duration of a pulse .
we propose a simple phenomenological model by considering a characteristic decay in the probability of dsbs during singlepulse sonication , which reproduces the essence of the experimental trend .
in addition , a data analysis revealed a characteristic scaling behavior between the number of pulses and the number of dsbs . |
as a result of increased access to dental care , people are retaining their natural dentition to a greater age . nevertheless , the prevalence of edentulism remains significant , especially among the elderly.1 the population continues to age worldwide , especially in developing countries and among groups with high socioeconomic status .
a world health organization statement from 2005 reported that 25% of polish people aged 6574 years were completely edentulous a very similar situation to that in the usa , where 26% of inhabitants aged 6569 years were completely edentulous.2 prosthodontic treatment with removable acrylic dentures is still frequent among older people , even in the developed world .
denture - related stomatitis is a disorder that often affects denture wearers.35 a number of studies suggest that two - thirds or more of the patients who wear removable complete dentures may suffer from denture - related stomatitis.1,6,7 despite being so common , the etiology of denture - related stomatitis is not completely understood .
the current thinking is that the etiology is multifactorial but , in many cases , it includes a pathogenic response to candida infection.810 denture plaque can be a reservoir of bacteria and fungi and may be responsible for respiratory tract diseases.11 our previous study showed that 58.5% of individuals wearing partial or full removable denture were affected by candida species.12 the authors of various studies have isolated candida species other than candida albicans that are perhaps involved in the pathogenesis of patients with oropharyngeal candidiasis.1315 a number of studies have compared the prevalence of denture - related stomatitis in men and women.1,1618 some authors have suggested that the development of denture - related stomatitis in women is higher due to hormonal changes.19 the aim of the retrospective study described here was to evaluate the intensity , genera , and frequency of yeast in the oral cavity of complete dentures wearers in terms of the gender and age of the subjects .
the null hypothesis is that there are no differences in candida species infection between male and female patients wearing complete dentures .
the patient data ( age , gender , general health status , length of time wearing denture , and type of denture ) were collected on individual electronic medical charts using ks - somed , an integrated software system produced by kamsoft ( katowice , poland ) .
the data of complete upper denture wearers who attended the department of prosthodontics at the university dental clinic in krakw were examined with a view to their possible inclusion in the study .
all of the described diagnostic and therapeutic methods were standard procedures . on the basis of our earlier study ,
a mycological examination was carried out on all patients with acrylic dentures in the maxilla , on the assumption that these patients were at risk of infection by candida species.20 we excluded patients with a postoperative prosthesis in the maxilla , or who had been using the denture for < 1 year , or who were within 1 year of oncology therapy , or who were within 3 weeks of antibiotic treatment , or who had extremely bad oral hygiene , or for whom the necessary data were lacking .
the data were analyzed in terms of four age groups : 50 , 5160 , 6170 , and > 70 years .
the examination procedures and methods of data collection were as described in our previous reports.12,21 the research was done with the accordance of the bioethics committee of jagiellonian university kbet/103/b/2004 and conformed to the criteria of the helsinki declaration and the international council on harmonisation good clinical practice guidelines .
the methodology of sample taking consisted of instructing the patients on what to do with their dentures in the evening and morning before attending the examination .
eating , drinking , smoking , and removing the denture from the mouth prior to the examination was not permitted .
a sterile cotton swab was moistened with saline and rubbed on the palate between the second and third palatal folds , immediately after the removal of the upper denture .
the swab was then inserted into a tube containing steward transport medium and sent the same day to the mycological laboratory at krakw university hospital .
the data from the mycological examination were examined for number of yeast colonies , their growth , and the candida species identified .
the cases were described as follows : not infected for 010 colonies , scarce growth for 1120 colonies , intermediate growth for 2150 colonies , intense growth for 51100 colonies , and abundant growth for over 100 colonies .
scarce or higher growth rates resulted in a diagnosis of infection . in the analysis of the growth intensity data ,
samples showing scarce growth then were identified by colony color using chromagar candida ( chromagar , paris , france ) medium as either c. albicans or non - albicans species . in all other cases , preliminary identification of the species
was made by colony color on chromagar candida medium , and a commercially available validation system the vitek 2 compact automated identification method with yst cards ( biomerieux sa , lyon , france ) was used from june 2009 onward to identify the non - albicans species . from january 2007 to may 2009 , individual candida species were identified using i d 32 c testing strips , following the manufacturer s instructions ( biomerieux sa , lyon , france).12 the number of candida species colonies for each patient was evaluated . the distribution of the genders by age groups , the intensity of yeast growth , the distribution of the number of yeast colonies , and the distribution of candida species morphotypes were then determined .
the data were analyzed using a statistical program ( spss statistics 17.0 for windows 2008 ) .
wallis test were used to examine the differences between the groups ; p - values < 0.05 were considered statistically significant .
the research was done with the accordance of the bioethics committee of jagiellonian university kbet/103/b/2004 and conformed to the criteria of the helsinki declaration and the international council on harmonisation good clinical practice guidelines .
the methodology of sample taking consisted of instructing the patients on what to do with their dentures in the evening and morning before attending the examination .
eating , drinking , smoking , and removing the denture from the mouth prior to the examination was not permitted .
a sterile cotton swab was moistened with saline and rubbed on the palate between the second and third palatal folds , immediately after the removal of the upper denture .
the swab was then inserted into a tube containing steward transport medium and sent the same day to the mycological laboratory at krakw university hospital .
the data from the mycological examination were examined for number of yeast colonies , their growth , and the candida species identified .
the cases were described as follows : not infected for 010 colonies , scarce growth for 1120 colonies , intermediate growth for 2150 colonies , intense growth for 51100 colonies , and abundant growth for over 100 colonies .
scarce or higher growth rates resulted in a diagnosis of infection . in the analysis of the growth intensity data ,
samples showing scarce growth then were identified by colony color using chromagar candida ( chromagar , paris , france ) medium as either c. albicans or non - albicans species . in all other cases ,
preliminary identification of the species was made by colony color on chromagar candida medium , and a commercially available validation system the vitek 2 compact automated identification method with yst cards ( biomerieux sa , lyon , france ) was used from june 2009 onward to identify the non - albicans species . from january 2007 to may 2009 , individual candida species were identified using i d 32 c testing strips , following the manufacturer s instructions ( biomerieux sa , lyon , france).12 the number of candida species colonies for each patient was evaluated . the distribution of the genders by age groups , the intensity of yeast growth , the distribution of the number of yeast colonies , and the distribution of candida species morphotypes were then determined .
the data were analyzed using a statistical program ( spss statistics 17.0 for windows 2008 ) .
the pearson chi - square test , the mann whitney u - test , and the kruskal
wallis test were used to examine the differences between the groups ; p - values < 0.05 were considered statistically significant .
over the 5-year period , 920 complete denture wearers ( 307 males and 613 females ) who did not match the exclusion criteria were incorporated in the analysis .
the results of the kolmogorov smirnov and shapiro wilk tests for the normality of the age distribution were statistically significant ( p<0.001 ) , meaning that the data were not from a normally distributed population and that nonparametric tests should be used .
patient ages ranged from 25 to 88 years ( males from 44 to 88 and females from 25 to 88 , respectively ) , with a mean age of 65.7 years ( males 67.7 and females 64.7 ) .
the difference in ages between sexes was statistically significant ( p<0.001 ) according to the mann
the number of females was greater by a factor of three in the two youngest groups .
diagnoses of yeast infection were made for 542 of 920 patients ( 58.8% ) , of which 161 were males ( 52.4% ) and 381 were females ( 62.2% ) , respectively .
the intensity of yeast growth was assessed by patient age , with statistical analysis showing significant differences ( p=0.002 ) . in the youngest group of patients ( those 50 years of age ) ,
the number of patients without a yeast infection was much smaller than in the other groups .
the greatest percentage of intermediate , intense , and abundant growths of yeast are seen in this age group .
data on the intensity of yeast growth with respect to age groups in females and males were analyzed .
males did not differ significantly in terms of intensity of yeast growth across the age groups ( p=0.178 ) . in the female group ,
the smallest percentage of uninfected females was found in the youngest group and the greatest percentage was seen in the oldest group .
intermediate , intense , and abundant growth of yeast occurred most frequently in the youngest group of females .
the results are shown in figure 4 . a statistically significant difference between males and females in the intensity of yeast growth
was found in the 61- to 70-year - old group ( p=0.029 ) . in this group ,
the percentage of males with scarce yeast growth was greater than that of females , and the percentage of females with intermediate , intense , or abundant growth of yeast was greater than in males .
one candida species colony was found in 421 patients ( 45.8% ) , two colonies in 116 patients ( 12.6% ) , and three colonies in five patients ( 0.5% ) .
for further analysis , we combined the patients with two or three candida colonies into one group .
the distribution of the number of yeast colonies by gender is presented in figure 5 .
the difference was statistically significant ( p=0.017 ) . in both categories of infection ( one colony and two or more colonies ) , the percentage of females was higher than the percentage of males .
the next evaluation concerned the distribution of the number of candida species colonies based on age groups .
an assessment was then made of the relative numbers of yeast colonies in males and females with respect to age .
males did not differ significantly in terms of the number of candida colonies in either age group ( p=0.313 ) .
however , in the female group , the differences were statistically significant ( p=0.021 ) . among those 50 years of age , 28% of females had two or more yeast colonies ( the highest number ) . in
the next two age groups examined , one colony was detected in > 50% of females . in the oldest age group ,
the results are shown in figure 7 . finally , correlations between the number of colonies , gender , and age were calculated . in all age groups ,
the males and females did not differ significantly in the distribution of the number of candida colonies .
the significance values were p=0.759 in the youngest group , p=0.263 in the next age range , p=0.149 in the second oldest group , and p=0.39 in the oldest group .
in the course of this survey , 22 morphotypes of candida species were collected from the participants palates .
the difference in the distribution of candida species across patient sex and age was not statistically significant ( pearson s chi - square test , p=0.590 and p=0.109 , respectively ) . c. albicans was the most common species found in both sexes .
the distribution of the types of yeasts most frequently detected in males and females is shown in figure 8 .
the most frequently detected yeasts were c. albicans , candida glabrata , and candida tropicalis .
these species occurred in respectively , 60.8% , 20.5% , and 4.8% of all affected patients .
the statistical analysis did not disclose significant differences in relation to gender in the distribution of these three most frequently occurring yeasts in the different age groups ( p=0.912 for males and p=0.223 for females ) . in each age group ,
the distribution of candida species with respect to gender was examined , but was not statistically significant for the age groups .
the only interesting finding was that females below and equal to 50 showed the greatest frequency of candida species other than c. albicans , c. glabrata , and c. tropicalis ( 23.1% versus 7.1% in males ) .
over the 5-year period , 920 complete denture wearers ( 307 males and 613 females ) who did not match the exclusion criteria were incorporated in the analysis .
the results of the kolmogorov smirnov and shapiro wilk tests for the normality of the age distribution were statistically significant ( p<0.001 ) , meaning that the data were not from a normally distributed population and that nonparametric tests should be used .
patient ages ranged from 25 to 88 years ( males from 44 to 88 and females from 25 to 88 , respectively ) , with a mean age of 65.7 years ( males 67.7 and females 64.7 ) .
the difference in ages between sexes was statistically significant ( p<0.001 ) according to the mann
the number of females was greater by a factor of three in the two youngest groups .
diagnoses of yeast infection were made for 542 of 920 patients ( 58.8% ) , of which 161 were males ( 52.4% ) and 381 were females ( 62.2% ) , respectively .
the intensity of yeast growth was assessed by patient age , with statistical analysis showing significant differences ( p=0.002 ) . in the youngest group of patients ( those 50 years of age ) ,
the number of patients without a yeast infection was much smaller than in the other groups .
the greatest percentage of intermediate , intense , and abundant growths of yeast are seen in this age group .
data on the intensity of yeast growth with respect to age groups in females and males were analyzed .
males did not differ significantly in terms of intensity of yeast growth across the age groups ( p=0.178 ) . in the female group ,
the smallest percentage of uninfected females was found in the youngest group and the greatest percentage was seen in the oldest group .
intermediate , intense , and abundant growth of yeast occurred most frequently in the youngest group of females .
the results are shown in figure 4 . a statistically significant difference between males and females in the intensity of yeast growth
was found in the 61- to 70-year - old group ( p=0.029 ) . in this group ,
the percentage of males with scarce yeast growth was greater than that of females , and the percentage of females with intermediate , intense , or abundant growth of yeast was greater than in males .
one candida species colony was found in 421 patients ( 45.8% ) , two colonies in 116 patients ( 12.6% ) , and three colonies in five patients ( 0.5% ) .
for further analysis , we combined the patients with two or three candida colonies into one group .
the distribution of the number of yeast colonies by gender is presented in figure 5 .
the difference was statistically significant ( p=0.017 ) . in both categories of infection ( one colony and two or more colonies ) ,
the next evaluation concerned the distribution of the number of candida species colonies based on age groups .
an assessment was then made of the relative numbers of yeast colonies in males and females with respect to age .
males did not differ significantly in terms of the number of candida colonies in either age group ( p=0.313 ) .
however , in the female group , the differences were statistically significant ( p=0.021 ) . among those 50 years of age , 28% of females had two or more yeast colonies ( the highest number ) . in
the next two age groups examined , one colony was detected in > 50% of females . in the oldest age group
the results are shown in figure 7 . finally , correlations between the number of colonies , gender , and age were calculated . in all age groups ,
the males and females did not differ significantly in the distribution of the number of candida colonies .
the significance values were p=0.759 in the youngest group , p=0.263 in the next age range , p=0.149 in the second oldest group , and p=0.39 in the oldest group .
in the course of this survey , 22 morphotypes of candida species were collected from the participants palates . the difference in the distribution of candida species across patient sex and age was not statistically significant ( pearson s chi - square test , p=0.590 and p=0.109 , respectively ) . c. albicans was the most common species found in both sexes .
the distribution of the types of yeasts most frequently detected in males and females is shown in figure 8 .
the most frequently detected yeasts were c. albicans , candida glabrata , and candida tropicalis .
these species occurred in respectively , 60.8% , 20.5% , and 4.8% of all affected patients .
the statistical analysis did not disclose significant differences in relation to gender in the distribution of these three most frequently occurring yeasts in the different age groups ( p=0.912 for males and p=0.223 for females ) . in each age group ,
the distribution of candida species with respect to gender was examined , but was not statistically significant for the age groups .
the only interesting finding was that females below and equal to 50 showed the greatest frequency of candida species other than c. albicans , c. glabrata , and c. tropicalis ( 23.1% versus 7.1% in males ) .
the use of removable prosthetic appliances is commonly associated with denture - related stomatitis , particularly in the upper arch.9,22 there are many factors that influence the severity of this disease , including continuous denture wearing , denture plaque , bad fitting and occlusal instability , general health problems like diabetes or hypertension , and the age of the denture .
regardless of these problems , candida species are the most frequently diagnosed.1,10,16,17,19 in our examination , a smaller percentage of the females were not infected by yeast , and the intensity of growth was greater in the female group than among the males , presenting microbiologically as intermediate , intense , and abundant growths of yeast .
we also noticed that , in the youngest group of patients ( those 50 years of age ) , the number of uninfected patients was smallest , and was even smaller than the number of subjects with two or more yeast colonies in the oral cavity .
there are some studies that report a greater prevalence of denture - related stomatitis in men than in women .
the literature review by gendreau and loewy shows that , in a study conducted in slovenia , finland , and chile , the number of female denture users was greater than the number of males.1 in our report , the number of women wearing an upper full denture was also greater than the number of men in each of the age groups analyzed .
a similar relation was reported by jainkittivong et al,5 abaci et al,16 and figueiral et al,17 but in other studies , the results were the opposite.3 more detailed studies should be carried out to determine the reason for this difference in gender prevalence , possibly in psychosocial and dental terms . in our study ,
when the number of yeast colonies in the whole group was examined , significant gender differences were seen .
this means that a difference between males and females does occur , but that various levels of yeast infection also occurs at different ages .
it is especially noticeable that , in the female group , the number of yeast colonies changes with age group .
no statistical differences were found between males and females when the number of yeast colonies present in the different age groups was considered .
similar results were obtained for calculating the intensity of the growth of yeast . the only gender difference between age groups
there were significantly more females with intermediate , intense , and abundant growth of yeast than males .
this shows that drawing conclusions from groups of patients with a broad spread of ages can lead to misinterpretation of results .
this could apply , for example , to the work of figueiral et al where the groups of individuals involved were < 35 years of age , between 35 and 75 years , and > 75 years.17 our research has concluded that , in each age group , the number of uninfected females was smaller than the number of males .
patients wearing complete dentures under and equal to the age of 50 suffered from more infection and had a greater diversity of infecting species .
dentists should therefore pay special attention to the condition of the palatal mucosa of all complete denture wearers , especially if the denture has been used for many years .
our investigation of candida species from the oral cavity of full denture wearers showed that c. albicans , c. glabrata , and c. tropicalis were most common . c. albicans was found in 60.8% of our complete denture users , and there were no statistical differences between genders ; these results are similar to those of other researchers,4,15 though some reports have described a greater frequency of c. albicans.3,16,17 in our earlier article , which involved patients with complete and partial acrylic dentures in the upper jaw , the percentage with c. albicans was 64.9%.12 when we examined separate age groups in the current study , we found that the distribution between gender and age of the percentage of c. albicans in complete denture wearers varied from 42.3% in the youngest group of females to 72.4% in males aged 5160 years .
we noticed a statistically insignificant , but nevertheless important finding , in the youngest group of females in relation to the yeast species present : this group had the greatest number of candida species , other than c. albicans , c. glabrata , and c. tropicalis .
however , it should be noted that the methods used to examine the yeast varied in different studies .
some results come from saliva specimens , while others are from swabbing the dorsum of the tongue , the denture , or the palate .
we conducted swab examinations of the mucosa of palate , this being the location of the infection connected with dentures .
also , different amounts of yeast have been obtained from different locations in the same patient.16 the comparison of results may therefore be unreliable . on the basis of our research , it has been possible to reject the null hypothesis that there were no differences in candida species infection between the sexes in patients wearing complete dentures . however , if the group of patients examined is congruous and includes those of similar ages ( within a 10-year span ) , it is not possible to reject the hypothesis .
yeast infections in the oral cavity of full denture wearers are common , but the genera of yeasts and their frequencies appear to be influenced by both age and gender . with regard to the patients in our study population ,
complete denture wearers of 50 years of age appeared to have the greatest proclivity for oral candidiasis infections .
researchers should pay special attention to age when selecting patients with complete dentures for investigation . | aimdenture - related stomatitis is a disorder that often affects denture wearers .
the purpose of this study was to evaluate the intensity , genera , and frequency of yeasts in the oral cavity of complete denture wearers in terms of subject gender and age.materials and methodsnine hundred twenty patients ( 307 males and 613 females ) with complete upper dentures were selected for the study and divided into four age groups : 50 years , 5160 , 6170 , and > 70 years .
yeast samples were taken as a smear from the palate .
the data were collected from january 15 , 2007 to january 15 , 2012.resultsthe distribution of the number of yeast colonies by gender was statistically significant ( p=0.02 ) . across all subjects , there was a statistically significant relationship between the intensity of yeast growth and the gender ( p=0.01 ) . in every age group ,
the number of infection - free individuals was greater among males than females .
intermediate , intense , and abundant growth of yeast occurred most frequently in the youngest group of females.conclusionthe genera of candida species and the frequency of yeast infection in denture wearers appear to be influenced by both age and gender .
the complete denture wearers 50 years of age appeared to have the greatest proclivity to oral candida infections . |
the study subjects were type 2 diabetic patients enrolled in the diabetes management through an integrated delivery system ( dmids ) project ( nct00288678 , clinicaltrials.gov ) ( 6 ) .
the patient recruitment period was between august 2003 and december 2005 , with the project carried out through december 2009 .
all type 2 diabetic subjects were eligible to be recruited unless they met any of the following exclusion criteria : 1 ) those aged < 30 or > 70 years ; 2 ) type 1 diabetic patients ; 3 ) pregnant women ; and 4 ) those who had major diabetes complications including leg amputation , uremia , or hospitalization in the previous year as the result of acute myocardial infarction , heart failure , or stroke .
of these enrollees , 22 subjects receiving insulin injection , 323 with a urine albumin - to - creatinine ratio ( acr ) 30 mg / g in at least one of the first two urine tests , and 126 subjects with missing fasting insulin data at baseline were excluded .
the remaining 738 subjects , who had normoalbuminuria ( acr < 30 mg / g in the first two consecutive urine tests ) , were selected for further investigation . written informed consent was obtained from all enrollees .
the institutional review board at the national health research institutes reviewed and approved this study . fasting ( overnight for at least 8 h ) venous blood and spot urine specimens were collected every 6 months .
glycated hemoglobin ( hba1c ) was measured by high - performance liquid chromatography ( variant ii ; bio - rad laboratories , hercules , ca ) .
fasting glucose , triglycerides , and hdl cholesterol were measured by an automatic analyzer ( hitachi 7060 ; hitachi high technologies , tokyo , japan ) .
insulin was measured by a chemiluminescent immunometric assay ( immulite 1000 ; diagnostic products , los angeles , ca ) .
urinary albumin was measured by the immunoturbidimetric method ( hitachi 7060 ; hitachi high technologies , tokyo , japan ) .
dipstick urinalysis was performed by an automated chemical analyzer ( clinitek 500 ; bayer , elkhart , in ) .
all blood and urine samples were kept in temperature - proof containers at 2c to 8c , transported by express delivery to a central laboratory , and measured within 8 h. waist circumference was measured at the level of the midpoint between the lowest rib and the iliac crest .
a mercury sphygmomanometer was used to measure blood pressure after subjects sat for 5 min .
blood pressure was measured three times separated by 1 min ; the mean of these three measurements was recorded .
we used the homa of insulin resistance ( homa - ir ) to assess insulin resistance ( homa - ir = { insulin [ mu / l ] fasting glucose [ mmol / l]}/22.5 ) ( 7 ) .
those who had acr 30 mg / g in two consecutive urine tests were defined as having microalbuminuria .
the urine samples were excluded from analysis if microscopic urinalysis showed erythrocytes > 5/high - power field ( hpf ) , white blood cells > 5/hpf , epithelial cells > 5/hpf , and appearance of casts or bacteria .
those who had smoked fewer than 100 cigarettes in their lifetime were defined as nonsmokers . for those who had smoked > 100 cigarettes ,
ex - smokers were defined as having stopped smoking completely at least 1 month prior to recruitment and current smokers as having a daily or occasional smoking habit at the time of recruitment .
data were expressed as mean sd for continuous variables , or as counts and proportions for categorical variables .
student t tests and analyses were used for continuous and categorical variables , respectively .
the incidence rate of microalbuminuria in the follow - up period was estimated by the number of observed new microalbuminuria cases per 1,000 person - years .
the person - years were calculated as the time elapsed from the date of recruitment until the date of death , microalbuminuria development , or the end of follow - up , whichever came first .
the calculation of a 95% ci for the incidence rate was based on the assumption that the observed incident cases followed a poisson distribution .
we estimated the incidence rate of microalbuminuria in different homa - ir quartiles not only for all study subjects but also for different subgroups according to their glycemic control ( hba1c 8 vs. >
kaplan - meier analyses and univariate cox proportional hazards models were used to explore the association between homa - ir and microalbuminuria development .
the predictors used in cox proportional hazards models including baseline demographic and metabolic profiles ( age at diabetes onset , sex , education , diabetes duration , smoking status , bmi , waist circumference , triglycerides and hdl cholesterol , urine acr , hba1c , and hypertension status ) .
multivariable cox proportional hazards modeling was used to determine the independent effects of homa - ir on microalbuminuria development .
observations were censored at the end of the study or the date that patients died or dropped out of the study , whichever occurred first .
results were expressed as hazard ratios ( hrs ) and 95% cis compared with the reference group . to eliminate residual confounding effects , for the survival analysis
they were those who met the following criteria at baseline : no acei ( angiotensin - converting enzyme inhibitor ) and arb ( angiotensin ii receptor blocker ) use , bmi < 24 kg / m , triglycerides < 150 mg / dl , waist circumference < 80 cm ( female ) or < 90 cm ( male ) , hdl cholesterol > 40 mg / dl ( male ) or > 50 mg / dl ( female ) , and blood pressure < 130/80 mmhg .
the proportional hazards assumption , the constant hr over time , was evaluated by comparing estimated log - log survival curves for all covariates .
all assessed log - log survival plots graphically showed two parallel lines , indicating no violation of the assumption . a test for trend
analyses were performed with sas software , version 9.1 ( sas institute , cary , nc ) .
fasting ( overnight for at least 8 h ) venous blood and spot urine specimens were collected every 6 months .
glycated hemoglobin ( hba1c ) was measured by high - performance liquid chromatography ( variant ii ; bio - rad laboratories , hercules , ca ) .
fasting glucose , triglycerides , and hdl cholesterol were measured by an automatic analyzer ( hitachi 7060 ; hitachi high technologies , tokyo , japan ) .
insulin was measured by a chemiluminescent immunometric assay ( immulite 1000 ; diagnostic products , los angeles , ca ) .
urinary albumin was measured by the immunoturbidimetric method ( hitachi 7060 ; hitachi high technologies , tokyo , japan ) .
dipstick urinalysis was performed by an automated chemical analyzer ( clinitek 500 ; bayer , elkhart , in ) .
all blood and urine samples were kept in temperature - proof containers at 2c to 8c , transported by express delivery to a central laboratory , and measured within 8 h.
waist circumference was measured at the level of the midpoint between the lowest rib and the iliac crest .
a mercury sphygmomanometer was used to measure blood pressure after subjects sat for 5 min .
blood pressure was measured three times separated by 1 min ; the mean of these three measurements was recorded .
we used the homa of insulin resistance ( homa - ir ) to assess insulin resistance ( homa - ir = { insulin [ mu / l ] fasting glucose [ mmol / l]}/22.5 ) ( 7 ) .
those who had acr 30 mg / g in two consecutive urine tests were defined as having microalbuminuria .
the urine samples were excluded from analysis if microscopic urinalysis showed erythrocytes > 5/high - power field ( hpf ) , white blood cells > 5/hpf , epithelial cells > 5/hpf , and appearance of casts or bacteria .
those who had smoked fewer than 100 cigarettes in their lifetime were defined as nonsmokers . for those who had smoked > 100 cigarettes ,
ex - smokers were defined as having stopped smoking completely at least 1 month prior to recruitment and current smokers as having a daily or occasional smoking habit at the time of recruitment .
data were expressed as mean sd for continuous variables , or as counts and proportions for categorical variables .
student t tests and analyses were used for continuous and categorical variables , respectively .
the incidence rate of microalbuminuria in the follow - up period was estimated by the number of observed new microalbuminuria cases per 1,000 person - years .
the person - years were calculated as the time elapsed from the date of recruitment until the date of death , microalbuminuria development , or the end of follow - up , whichever came first .
the calculation of a 95% ci for the incidence rate was based on the assumption that the observed incident cases followed a poisson distribution .
we estimated the incidence rate of microalbuminuria in different homa - ir quartiles not only for all study subjects but also for different subgroups according to their glycemic control ( hba1c 8 vs. >
kaplan - meier analyses and univariate cox proportional hazards models were used to explore the association between homa - ir and microalbuminuria development .
the predictors used in cox proportional hazards models including baseline demographic and metabolic profiles ( age at diabetes onset , sex , education , diabetes duration , smoking status , bmi , waist circumference , triglycerides and hdl cholesterol , urine acr , hba1c , and hypertension status ) .
multivariable cox proportional hazards modeling was used to determine the independent effects of homa - ir on microalbuminuria development .
observations were censored at the end of the study or the date that patients died or dropped out of the study , whichever occurred first .
results were expressed as hazard ratios ( hrs ) and 95% cis compared with the reference group . to eliminate residual confounding effects , for the survival analysis
they were those who met the following criteria at baseline : no acei ( angiotensin - converting enzyme inhibitor ) and arb ( angiotensin ii receptor blocker ) use , bmi < 24 kg / m , triglycerides < 150 mg / dl , waist circumference < 80 cm ( female ) or < 90 cm ( male ) , hdl cholesterol > 40 mg / dl ( male ) or > 50 mg / dl ( female ) , and blood pressure < 130/80 mmhg .
the proportional hazards assumption , the constant hr over time , was evaluated by comparing estimated log - log survival curves for all covariates .
all assessed log - log survival plots graphically showed two parallel lines , indicating no violation of the assumption . a test for trend
analyses were performed with sas software , version 9.1 ( sas institute , cary , nc ) .
table 1 displays demographic and biochemical characteristics of study subjects . compared with those who retained a status of normoalbuminuria , patients who developed microalbuminuria were more likely to have lower education , higher homa - ir , and higher baseline acr .
baseline demographic and biochemical characteristics of type 2 diabetic patients with and without progression to microalbuminuria data are n ( % ) or mean sd unless otherwise indicated .
sbp , systolic blood pressure ; dbp , diastolic blood pressure ; ccb , calcium channel blocker . the kaplan - meier survival curves in fig .
1 indicate that the probability of remaining normoalbuminuric for those in the 2nd to 4th homa - ir quartiles was significantly lower than for subjects in the lowest quartile .
the significant adjusted hrs for those in 3rd and 4th quartiles in the multivariable cox proportional hazards model , shown in table 2 , confirm the independent association between insulin resistance and microalbuminuria development .
figure 2 shows the adjusted hr and trend test for those with good metabolic profiles were similar to what was observed for overall subjects .
kaplan - meier estimates of probability of normoalbuminuria according to 4 quartiles of homa - ir at baseline .
cox proportional hazards models for progression to microalbuminuria * data are hr ( 95% ci ) . *
adjusted hr of microalbuminuria development in type 2 diabetic patients for overall subjects and those with good metabolic profiles at baseline .
type 2 diabetic patients with good metabolic profiles were those who had no acei / arb use , bmi < 24 kg / m , triglycerides < 150 mg / dl , waist circumference < 80 cm ( female ) or < 90 cm ( male ) , hdl cholesterol > 40 mg / dl ( male ) or > 50 mg / dl ( female ) , and blood pressure < 130/80 mmhg .
the controlled covariates in the survival analyses included demographics ( baseline age , sex , education , smoking status , and diabetes duration ) and baseline biomedical profiles ( waist circumference , bmi , triglycerides , acr , hdl cholesterol , hba1c , and mean arterial pressure ) .
the reference group for each model was those who were in the lowest quartile of the corresponding homa - ir index .
the mean arterial pressure ( map ) was calculated by the formula : mean arterial pressure = diastolic blood pressure + 1/3 ( systolic blood pressure diastolic blood pressure ) .
in addition to overall subjects , this dose - response relationship remained true for type 2 diabetic patients with poor glycemic or blood pressure control . furthermore , we classified the homa - ir score based on its tertile instead of quartile
. the incidence rate ratios of tertile 3 , in comparison with tertile 1 , were 1.60 ( 95% ci 1.002.55 ) , 2.71 ( 1.804.15 ) , 1.92 ( 1.163.27 ) , and 1.70 ( 1.142.54 ) for subgroups with hba1c 8% , hba1c > 8% , blood pressure 130/80 mmhg and blood pressure > 130/80 mmhg , respectively .
their p values ( 0.041 , 0.022 , 0.013 , and 0.007 , respectively ) for dose - response trend tests were also significant ( data not shown ) .
incidence of microalbuminuria in subjects in different homa - ir quartiles , according to their glycemic and blood pressure control bp , blood pressure ; rr , rate ratio . * test for trend for incidence rate ratio from homa - ir q1 to q4 .
insulin resistance is shown in this longitudinal study to be a strong predictor in determining the development of microalbuminuria within 5 years for the type 2 diabetic patients with acr < 30 mg / g at enrollment .
compared with those with a homa - ir score in the lowest quartile , those in the highest quartile had increased incidences of microalbuminuria by about 17% ( for those with poor blood pressure control ) to 92% ( for those with good blood pressure control ) .
furthermore , the adjusted dose - response effect of insulin resistance on development of microalbuminuria was significant in overall subjects and the subgroup with good metabolic profiles .
overall , this prospective study provides new epidemiological evidence linking elevated risk of diabetic nephropathy with insulin resistance .
a growing body of studies has demonstrated that insulin resistance is associated with hemodynamic alteration in the kidney .
hyperinsulinemia has been reported to be able to raise glomerular hydrostatic pressure , increase renal vascular permeability , aggravate glomerular hyperfiltration , and enhance renal sodium reabsorption ( 810 ) .
altogether , the steady state of renal endothelial functions and hemodynamic harmonization may be changed because of cascading reactions caused by insulin resistance .
this could explain underlying mechanisms for the significant association between the homa - ir score and microalbuminuria development observed in this study .
similar to previous studies ( 3,11,12 ) , our study found that those with a higher homa - ir were more likely to have metabolic syndrome ( data not shown ) and higher urine acr at baseline .
metabolic syndrome and urine acr have been well recognized as risk factors associated with microalbuminuria ( 2,4,5,1315 ) .
because of this intertwining feature in the cluster of insulin resistance , metabolic syndrome , and microalbuminuria , we could not completely exclude the possibility that insulin resistance is a biochemical marker rather than a causal factor in the process of microalbuminuria development . in order to ensure the effects of insulin resistance
, we calculated the incidence rate ratio and performed multivariable survival analyses for subjects stratified by different baseline characteristics .
the causal association is considered to be truly independent because the significant dose - response effect of homa - ir on microalbuminuria development was demonstrated ( fig .
2 ) not only in overall subjects but also in type 2 diabetic subjects with no acei / arb use and good metabolic profiles .
we found that the less educated were also more likely to have higher hba1c and to be current smokers ( data not shown ) , both of which are risk factors for the development of microalbuminuria .
these findings are in accordance with previous studies in both western and oriental countries ( 16,17 ) .
this universal evidence implies that those with lower educational attainment are at higher risk of developing chronic kidney disease .
first , we used the homa - ir index instead of the hyperinsulinemic euglycemic glucose clamp ( 18 ) to measure insulin resistance .
although some previous papers argued that the homa - ir index might have limitations to assess insulin resistance in certain circumstances ( 19 ) , it is the most frequently used index to evaluate insulin resistance in the general population and diabetic patients ( 20 ) .
the uk prospective diabetes study also used the homa - ir index to monitor insulin sensitivity in its type 2 diabetic cohort ( 21 ) . in line with other epidemiologic studies
, we believe the classification of insulin resistance by quartiles in this study is reliable .
second , we did nt measure fat deposition by using standardized dual - energy x - ray absorptiometry or computed tomography to evaluate association between abdominal adiposity and nephropathy ( 22 ) . instead
, abdominal adiposity was estimated by waist circumference , which is suggested as a predictor of insulin resistance and albuminuria ( 23,24 ) .
although we adjusted for waist circumference in all of our multivariable survival analyses , the homa - ir score remained a significant predictor of microalbuminuria development , either in the entire cohort ( table 2 ) or in a subgroup of individuals with good metabolic profiles ( fig .
2 ) . moreover , small sample size in subgroups in quartiles 14 of glycemic control or blood pressure control in table 3 could affect statistical significance . if we classified homa - ir based on its tertile , the incidence rate ratios of tertile 3 for subgroups with hba1c 8% , hba1c > 8% , blood pressure 130/80 mmhg , and blood pressure > 130/80 mmhg were all significant as compared with those in tertile 1 .
these results indicate the dose - response effects of homa - ir on microalbuminuria were robust . in conclusion
, this is the first prospective study investigating an association between insulin resistance and development of microalbuminuria in type 2 diabetic patients .
we reconfirm that type 2 diabetic patients predisposed to higher insulin resistance are more likely to develop microalbuminuria .
future research should aim at stopping renal function deterioration by improving insulin sensitivity for type 2 diabetes with high insulin resistance . | objectivean association between insulin resistance and microalbuminuria in type 2 diabetes has often been found in cross - sectional studies .
we aimed to reassess this relationship in a prospective taiwanese cohort of type 2 diabetic subjects.research design and methodswe enrolled 738 normoalbuminuric type 2 diabetic subjects , aged 56.6 9.0 years , between 2003 and 2005 and followed them through the end of 2009 .
average follow - up time was 5.2 0.8 years .
we used urine albumin - to - creatinine ratio to define microalbuminuria and the homeostasis model assessment of insulin resistance ( homa - ir ) to assess insulin resistance .
the incidence rate ratio and cox proportional hazards model were used to evaluate the association between homa - ir and development of microalbuminuria.resultswe found incidences of microalbuminuria of 64.8 , 83.5 , 93.3 , and 99.0 per 1,000 person - years for the lowest to highest quartiles of homa - ir .
compared with those in the lowest quartile of homa - ir , the incidence rate ratios for those in the 2nd , 3rd , and highest quartiles were 1.28 ( 95% ci 0.881.87 ) , 1.44 ( 0.992.08 ) , and 1.52 ( 1.062.20 ) , respectively ( trend test : p < 0.001 ) . by comparison with those in the lowest quartile , the adjusted hazard ratios were 1.37 ( 0.932.02 ) , 1.66 ( 1.122.47 ) , and 1.76 ( 1.202.59 ) for those in the 2nd , 3rd , and highest homa - ir quartiles , respectively.conclusionsaccording to the dose - response effects of homa - ir shown in this prospective study ,
we conclude that insulin resistance could significantly predict development of microalbuminuria in type 2 diabetic patients . |
schizophrenia and related psychoses are chronic disorders with an onset usually in early adulthood that affect patients all over their life and generally have moderate prognosis .
several previous [ 1 , 2 ] and more recent [ 3 , 4 ] studies have suggested that early intervention is crucial for the improvement of the prognosis and outcome of these disabling disorders both medium- and long - term .
however , the results of such studies have been criticized by others , and it is supported that the effectiveness of current protocols of early intervention is not superior to standard care [ 5 , 6 ] .
moreover , it has been recently proposed that adequate funding and good clinical governance are critical in ensuring service quality and maintaining continuity of care , whether the early intervention in psychosis service is specialized or integrated within a generic mental health team . despite the ongoing debate about the effectiveness of specialized early intervention services , at recent years several such services have been developed worldwide for the early detection , intervention and comprehensive care of people who experience a first episode of psychosis ( fep ) .
the first step for the establishment of new services is the estimation of the needs of a defined area .
this study aimed to explore the needs and provide epidemiological data on fep patients in the prefectures of ioannina and thesprotia in greece .
this is the first greek study on the rates of first episode of psychosis in a defined catchment area .
the epidemiologic survey was performed in the context of the recently established , early intervention service ( eis ) of the university hospital of ioannina .
the catchment area of the prefectures of ioannina and thesprotia belongs to the epirus region , north - western greece , and has a population of about 220000 inhabitants .
mental health services of the area comprise the inpatient and outpatient psychiatric department of the university hospital of ioannina , the outpatient psychiatric department of the general state hospital , the outpatient department of the social insurance organization ( ika ) , and the mobile mental health unit ( mmhu i - t ) which delivers services in rural and remote areas .
although in greece care is public , a large proportion of patients prefer to be examined by private practice physicians .
there is no registration system for the first diagnosed psychotic patients , so for the performance of a reliable epidemiological survey data should be obtained from all those who may have examined and treated such patients .
data for first episode psychotic patients during a two - year period ( 2008 and 2009 ) were obtained by reviewing the medical records of the patients examined in the two hospitals , ika and the mmhu i - t .
for patients treated in the private sector , data was obtained by personal communication with the 12 treating psychiatrists of the area for the aforementioned period .
all diagnoses were made by treating clinicians according to icd-10 criteria , as this classification is officially used in the country .
however , only clinicians of the eis regularly use a standardized assessment interview , while their private sector colleagues rely only on usual clinical assessment .
however , there is evidence that stability of icd-10 psychotic - disorder diagnoses over time is high .
duration of untreated psychosis ( dup ) was calculated by the first author ( vp ) with the retrospective application of the principles of the symptom onset in schizophrenia ( sos ) inventory to the patients information as recorded at their charts ( public sector ) , or as provided by treating psychiatrists ( private sector ) . for the determination of patients socio - economic status we used an adapted form of the uk classification system , which has been used in previous research .
more specifically , three social classes were defined , namely upper ( corresponding to the class i ) , middle ( comprising classes ii and iii ) and lower ( comprising classes iv and v ) , respectively .
this adaptation was based on economic criteria of the patients families rather , than on their working status , since a large proportion of patients were very young , were still students and may have never worked .
the statistical package of the social sciences ( spss 19.0 ) was used t perform all analyses .
analysis was made with the use of the student t - tests and the statistical level for significance was chosen at p<0.05 .
a total of 132 fep patients were examined in the 2-year period in the catchment area .
most of the patients ( 81 or 61.4% ) were diagnosed and treated by private practicing psychiatrists .
the majority of patients examined in the public sector ( 45 out of 51 , 88.2% ) were referred to the eis .
data regarding base - line symptom severity were not available for private sector patients , because private practice psychiatrists do not regularly use assessment tools .
statistical analysis showed no differences between the two sectors in terms of patients age , gender , family and social status , profession and dup .
education was associated with the treatment setting selection , and college education was predictive of the use of the public sector ( p<.001 ) .
first episode patients who had a history of alcohol / substance abuse were more likely been treated in the private sector ( p=.021 ) .
a positive family history of a psychiatric disorder was associated with treatment in the public sector ( or 0.43 , 95% ci 0.20 - 0.92 , p=0.028 ) .
there was an interaction of the variables substance abuse and family history .
logistic regression showed that treatment setting was determined by the combination of these two variables , and that patients who were abusing alcohol or substances and had no family psychiatric history were less likely been treated in the public sector ( or 0.19 , 95% ci 0.04 - 0.90 , p=0.036 ) .
the acquisition of data on the rates on fep in a defined area is essential for planning and providing mental health services . we were able to identify 132 fep cases who received treatment in every available setting in our catchment area in a 2-year - period
this makes an annual incidence rate of 30 new cases per 100000 which is within the range reported in previous research in different countries . in our study
this may reflect patients attitudes toward hospital psychiatric treatment ; or it may be that the eis unit , where most public sector cases were referred had just been established at the time of the study .
moreover , at that time financial crisis in greece was in the beginning and more patients could afford treatment in the private sector .
however , this is a relevant finding , because most patients did not receive the comprehensive multidisciplinary care delivered by the eis unit of the university hospital of ioannina .
moreover , it has been suggested that specialized first - episode psychosis services may effectively address the issues of involving and educating families about psychosis as well as stigma .
it is not known whether this is the case in the private sector in our country . on the other hand , preference for the psychiatric private sector
is widespread in western countries , and there is evidence that , with the exception of some university centers , the quality of treatment in the public sector is poor [ 14 , 15 ] .
notably , there was not a single case of first episode patient treated by the mmhu , but patients from rural areas would be rather examined by outpatient hospital services or private practicing psychiatrists .
the mmhu delivers services since 2007 and has contributed significantly to the reduction of hospitalizations of chronic psychotic patients living in rural and remote areas of the prefectures of ioannina and thesprotia .
we assume that perceived stigmatization of fep patients and their families in these rural areas prevents them from seeking help by a local mental health service .
immigrants comprised only a small proportion of the patients ( 8 cases , 6% ) .
the immigrant population in our catchment area is estimated at 4.4% , mostly at working age .
it has been demonstrated , that migration is associated with high incidence rates of psychosis . in a recent study at a socioeconomically deprived area of inner london immigrants
were over - represented among fep cases . from a total of 484 fep patients ,
only 23.1%% were british , while the proportion of british in the population at risk was as high as 41.6% .
recent evidence in our country suggests that immigrants experienced higher degrees of inequity in primary health care that is possibly caused by their restricted access to social insurance health care
. this may be the case of some fep cases which go unrecognized in this population .
we assume that fep immigrant patients may have problems of access to the mental health system , resulting from socioeconomic reasons , insurance issues and barriers within the system , i.e. difficulties in language and little appreciation of the culture and adversities of this population by mental health staff and primary care physicians .
conceivably , efforts should be made for the identification of such cases by the mental health system .
duration of untreated psychosis was not significantly different for patients been treated in the private or the public sector ( mean duration 18.2 and 22.5 months , respectively ) .
the interval between the onset of psychotic symptoms and treatment initiation has been shown to be a predictor of outcome , and shorter dup has been associated with better chance for recovery [ 21 , 22 ] .
it is unknown whether private sector care providers can make efforts to reduce dup , but public health services should arrange initiations for facilitating access to mental health care and educating the public regarding psychotic illness in young persons . according to statistical analysis , education was associated with treatment setting selection , and having or being studied in college predicted treatment in the public sector .
this finding is not easy to be interpreted and it is not known whether this level of education contributes to the development of a positive attitude toward the public health system .
another finding of statistical and probably clinical significance was that patients with a history of alcohol or substance abuse were more likely to be treated in the private sector .
it may be possible that the public sector physicians tend to underestimate the role of alcohol or substance abuse in psychopathology .
this is supported by the relatively low rates of 11.8% of alcohol / substance abuse recorded for public sector patients , compared to previously reported , much higher rates . or
, that dual diagnosis patients would prefer the perceived less restricted context of the private sector . no matter the explanation , dual diagnosis patients , who are a particular challenging subgroup , would not receive a more intensive and comprehensive care provided by the multidisciplinary eis team .
the selection of the private sector as treatment setting was found merely to be determined by the combination of having a history of alcohol or substance abuse and a negative family psychiatric history .
this means that patients with a positive family history for mental disorder would prefer to be treated in the public health system .
perhaps the families of those fep patients , who were familiar with mental illness , were aware of the early intervention service of the university hospital , where most public sector cases were treated .
however , there is evidence that families with a past history of psychiatric hospitalization of a family member were less likely to recommend other family members to mental health services , as indicated by the long delay between onset of psychotic symptoms and first admission which was found to be independently predicted by a family history of psychiatric hospitalization [ 24 , 25 ] . regarding illness severity at baseline
, we have to note that panss scores were not available for patients treated in the private sector .
our data are supposedly complete because we obtained data from every resource in the area patients could seek mental health care .
a small number of cases may have been missed if they have been examined in other cities , such as the capital of greece , athens or the second large city , thessaloniki .
other cases may have been missed if they were hospitalized in private hospitals in other cities , as in our region there is no private inpatient facility . a possible explanation for such a preference may be the perception that in those large cities with well - organized private facilities , fep patients would receive better care ; or it was the perceived stigmatization that led patients and families to seek care in other cities .
we believe that the number of such cases is small , and we also assume that these patients were difficult to receive optimal long term care due to distance from their treating service which would render regular follow - up difficult . it should be mentioned that the structure of the health system in greece which is largely based on private practice physicians makes any epidemiologic survey performance a difficult task .
for example , in the large population cities of athens and thessaloniki where there are many psychiatrists working in private practice , an effort to collect epidemiological data would be extremely difficult .
patients diagnoses at the private sector were made according to usual clinical examination and history of the patients , without the use of a diagnostic interview , or other instrument .
moreover , dup was calculated retrospectively , by applying the principles of sos to the information at patients charts , without interviewing the patients and other informants .
in our catchment area most first episode patients are been treated by private sector clinicians .
the selection of the private sector as treatment setting was found merely to be determined by the combination of having a history of alcohol or substance abuse and a negative family psychiatric history .
immigrants comprised a small proportion of the patients , despite evidence of higher rates of psychotic disorders in this population .
the results of this first greek study on the rates of first episode of psychosis in a defined area are relevant and may inform clinical practice and mental health policy .
| this is the first greek study presenting epidemiologic data on first - episode psychosis ( fep ) patients in a defined catchment area .
data for first episode psychotic patients during a two - year period ( 2008 and 2009 ) were obtained by all the mental health providers in the area , public or private .
a total of 132 fep patients were examined in the 2-year period in the catchment area .
most of the patients ( 61.4% ) were diagnosed and treated by private practicing psychiatrists .
statistical analysis showed no differences between the two sectors in terms of patients age , gender , family and social status , profession and duration of untreated psychosis ( median duration 6 months ) .
patients who were abusing substances and had no family psychiatric history were less likely been treated in the public sector .
immigrants comprised only a small proportion of the patients , probably because they have difficulties in accessing the mental health system . |
a new thermodynamic model for calculating the dissociation constants of complexes formed between the aryl hydrocarbon receptor ( ahr ) and polychlorinated biphenyls ( pcbs ) is reported .
the free energies of binding of pcbs to ahr are controlled by their lipophilicities , electron affinities , and entropies .
the corresponding physicochemical properties of polychlorinated dibenzo - p - dioxins and dibenzofurans also control their interactions with ahr .
we present evidence supporting the hypothesis that the majority of pcbs are likely to interact with ahr in their nonplanar conformations .
in addition , we demonstrate that the affinities of pcbs for ahr relative to 2,3,7,8-tetrachlorodibenzo - p - dioxin correlate with corresponding toxic equivalency factors in animals .
the reported methodology is likely to be applicable to other polyhalogenated and mixed polyhalogenated bi- and terphenyls and related xenobiotics ; thus , it could minimize the number of in vivo studies in laboratory animals and facilitate the identification of potentially hazardous aromatic xenobiotics.imagesp422-afigure 2 . | |
if i wish to explain it to him who asks me , i do not know .
an initial issue regarding time is that , in our western mode of thinking , we have retained heracleitus ' metaphor of time being a river which we never can enter twice , because it never remains the same .
this is one the main assertions or principles attributed to heracleitus ( ~510 ~450 bc ) : all things flow and nothing stands .
marcus aurelius ( 121 - 180 ) completed the metaphor when he wrote :
time is like a river made up of the events which happen , and a violent stream . and salvador dali ( 1904 - 1989 ) more recently expressed the same idea in his famous painting of soft melting pocket watches .
the message of heracleitus is that everything passes , therefore not only time , but we ourselves , also pass .
everything changes constantly , and it is the world that flows . the term the past in itself contains the idea of flow . in the case of the river
, we know ( and have done since galileo 's time ) that gravity makes it flow .
the use of this river metaphor to describe time leads to the illogical idea that the past might be be located at a higher position than the future .
a second issue regarding time is its unicity , ie , whether there is only one time or a variety of times
. e. t. hall ( 1914 - 2009 ) , studying various cultures , created the concepts of monochrony versus polychrony , which he illustrated with examples of waiting in line : in northern countries , everybody patiently waits in line , while in more mediterranean areas one sees several people being served simultaneously in the markets .
for thinkers and researchers in general physics , from galileo to einstein , monochrony rather than polychrony is the accepted principle .
they critize polychrony , seeing it as a metaphor , because it includes versions of time that can not be measured .
however , as of the last few years , scientists in particle physics imagine several simultaneous times .
all of them , except time as we know it , would be wound or rolled up on themselves , ie , they would be cyclical .
a third issue is the question of causality , as defined in philosophy and physics : if the principle is to be respected , there is no possibility of any beginning , either with linear nor with cyclical time .
when one turns toward dictionaries , with their usual charming circularity , one reads that time is a duration , that it is a succession , or that it is represented in its essence . in the first
meaning , time is a duration ; it can then be indeterminate and continuous ; it can also be a fragment of a given duration , itself limited by the activity of a person , or by the nature of a biological process .
in many cases , duration can be objectively measured , as finite phenomena within complex ensembles .
for example , part of a step in dancing , a beat in music , time - sharing in data processing , etc . in the second
meaning , time is a succession : it is a moment in a series of states , of single events ; one speaks of the time of ancient culture , of a person 's period in life , etc .
time is associated , depending on historical period and literary genre , with precariousness , with the fleeting nature of life , with the end of all human achievements . through personification , time is described as the allegory of an old man holding a scythe . in religion , for example christianity , the coming of jesus inserts human time into the eternity of god . in philosophy , time is a recurrent theme .
it is a daily observation that there is an opposition between the time of physics and the psychological time , between chronos and tempus .
philosophical distinctions are defined in box 1 . guide to philosophical concepts cited in this review materialism : matter constructs reality ; all things are composed of material and all phenomena ( including consciousness ) are the result of material interactions ; opposed to any kind of transcendence ( superstition , mythology , supernatural , spiritualism , theology , religion , deism , idealism ) .
enipirisim : knowledge comes only or primarily from sensory experience ; authors : aristotle , okham , bacon , hobbes , locke , hume , einstein . opposed to innatism ( innate ideas ) or knowledge a priori .
authors : plato , descartes , leibniz . idealism : the nature of reality rests on the mind , on abstract forms or mental representations .
authors : for idealism : plato , plotinus , descartes , leibniz , kant , poincar .
opposed to realism , which asserts that the external world has an independent existence of human consciousness or human knowledge ; it may consist in refusing all reality to phenomens , it may assert that thought is the only certain reality , it may derive reality from a spiritual principle .
chronos , time according to physics , is objective ( does not depend on us ) , is uniform ( shows no acceleration ) , is linear , and we know how to measure it . as of october 13 , 1967 ,
a second is the duration of about 9 billion periods of the electromagnetic wave emitted by a cesium 133 atom , when it changes from one level of energy to another .
astronomic clocks were the first to be used by man . before clepsydra and hourglasses ( egg timers )
, we counted the days , taking advantage of the colossal clock that is the sun .
there have been methodological difficulties in calculating the mean solar day , because one has to assume that the sun moves at a constant speed , or to calculate the mean sidereal day , because one has to assume that stars keep the same relative position with respect to each other . in precision clocks ,
the regularity of movement comes from a pendulum that oscillates under the influence of gravity : the duration of a half - cycle depends on two elements , the length of the pendulum and the intensity of gravity ( which is not uniform at different earth locations ) .
the unit of time is thus defined using a measure of length . according to aristotle ( 384 - 322 bc )
, time is just this a degree of motion with respect to before and after . and
hence time is not movement , but only movement in so far as it admits of enumeration .
what aristotle includes in the term movement concerns the place ( moving , shifting ) as well as the quality ( change in shape or state ) , the quantity ( increment , decrement ) , and the essence ( appearance / disappearance , birth / death ) .
there are ties between time and movement , but time is not movement : movement varies , and is polymorphous , while time does not change . but time , which is a number , does not exist without the soul ( nowadays we would say the mind ) , outside of the soul .
the instant , the now , that separates between before and after , is an abstraction that exists only in the mind .
the measure , of time determines what separates the beginning and the end of a movement .
plotinus ( 205 - 270 ) , a neo - platonician philosopher , raised the issue of self - reference in a definition of time based on time , and said that this was a confusion between what is numbered ( measured ) and what numbers ( measures ) .
in contrast to aristotle , he argued that time existed before number was applied to it .
one should remember that , at the time of plotinus , christianity strongly influenced philosophy and science : adam had committed a sin , and since then the soul of man was separated from god and thrown out of eternity into temporality .
saint augustine ( 354 - 430 ) says that there is no time outside of the soul .
here is the quotation that follows the epigraph : but , then , how is it that there are the two times , past and future , when even the past is now no longer and the future is now not yet ?
but if the present were always present , and did not pass into past time , it obviously would not be time but eternity .
if , then , time present if it be time comes into existence only because it passes into time past , how can we say that even this is , since the cause of its being is that it will cease to be ?
thus , can we not truly say that , time is only as it tends toward nonbeing ? this quotation follows the idealistic philosophy that , from parmenides ( ~520~455 bc ) to plato ( ~428~348 bc ) , stands in opposition to the empiricism of aristotle . as another illustration
if time only exists by and for the soul , then what does one measure when speaking of time ?
he was the first to relate time to memory ; much later , dali did the same when he entitled his 1931 painting of soft watches , mentioned above , the persistence of memory ; currently , the role of memory functions in relation to time is the theme of much research in neuroscience .
the opposition between the phenomenological description of time using memory by saint augustine and the mechanistic explanation of aristotle of time being the number of movement has never fully been solved in western philosophy .
the materialist tradition also favors eternity in relation to time : atoms and the emptiness of the universe are infinite , uncreated , and imperishable .
time represents an illusion due to the appearance in consciousness of events that , in themselves , are accidental , according to lucretius ( ~98~55 bc ) . until the 17th century
, one did not make the distinctions between three versions of time : time as an abstract number , time as it is measured in physics , and duration ( a version of time as felt in our consciousness ) .
galileo ( 1564 - 1642 ) , the founder of modern physics , considered the universe to be written in the language of mathematics
an idealistic idea opposite to aristotle 's empirism and saw the world as expressing an eternal order of things , that we can conceive , although we can not feel them .
galileo 's idea of an eternal and undifferentiated time replaced the concept of a time consecutive to movement .
from galileo to einstein , separating rest from uniform movement became a matter of frame of reference , or , put differently , a matter of position of the observer .
a popular example of the role of the observer 's position is when we are seated in an immobile train , and the departure of another train gives us the impression that our train is moving .
galileo also invented thought experiments : if one makes the hypothesis that a theory is true and one demonstrates that reasoning based on this hypothesis leads to dead ends , then the theory is false .
performing such a thought experiment , he concluded that the speed of fall of an object is proportional to the duration of the fall and independent of the mass of the object .
this was the first historical occurrence of a physical law being expressed using the parameter of time .
later on , newton ( 1643 - 1727 ) asserted the reality of an absolute space and of an absolute time : absolute , true , and mathematical time , of itself , and from its own nature , flows equably without relation to anything external , and by another name is called duration . he defined time as a succession of mathematical instants ( an entity with no length ) .
thus , with respect to his own definition , using the name of duration is inadequate .
leibniz ( 1646 - 1716 ) was as idealistic as plato , when he stated :
i hold space to be something merely relative , as time is , taking space to be an order of coexistences , as time is an order of successions .
conversely , wrote kant ( 1724 - 1804 ) , one can neglect all information coming from our senses ( sensitive data ) , but never can one leave out time and space , which are indispensable for any representation .
the representation of space can not , therefore , be empirically obtained from the relations of outer appearance . on the contrary , this outer experience is itself possible at all only through that representation .
appearances may , one and all , vanish ; but , time ( as the universal condition of their possibility ) can not itself be removed .
physicists chose the linear version of time on the basis of the principle of causality , which was first introduced by leibniz .
there are several descriptions of this principle , ie , the relationships between causes and effects .
first , a cause necessarily precedes its effects ( this precludes a cyclical time ) .
second , the same causes induce the same effects ( and the repetition of a cause leads to the repetition of the effects , sometimes leading to cycles .
third , there is a mandatory chronology between the effect and the cause , and the effect of a cause can not act retrospectively on the cause . in cyclical time
, a cause a leads to an effect b , and in the future of b one could come back to the past of a : while growing older , one could enter one 's parents ' past and prevent them from meeting one another !
cyclical time is not time , since there are then no distinctions between past and future . and fourth , the past can not be modified ( it will always be that what occurred in the past did occur ) . as of the 19th century
, the definition of what is a cause became more complex as the science of thermodynamics developed : mathematical distribution functions were used to describe gas molecules in terms of probabilities , a rather new concept .
more recently , at the beginning of the 20th century , a form of causality principle without cause arose from quantum physics : with certain types of phenomena , which are causally related , a chronology is mandatory , but one can not establish that the first phenomenon causes the second . despite these new ideas about the nature of causes , physicists kept the principle of causality as a valid axiom for their work . in conclusion , in the terms of physics , the principle of causality makes it such that time can not be cyclical ( repeating itself indefinitely ) , but that cycles in time can exist ( phenomena can repeat themselves ) .
the other time , tempus , is time as we experience it subjectively . it does not flow uniformly , it depends on our emotional status , in the broad sense of activation , vigilance , and mood : time is elastic , and the phenomenology of this characteristic has been the theme of many studies .
tempus , the subjective or psychological time can not be measured with chronos , but nevertheless can be compared with it .
tempus rarely flows as we would wish it to : a minute , being bored or an hour of passion can not be measured using the tools of physics .
this is why we carry watches , so that we do not lose track of time .
aristotle had already observed the subjective nature of time : but neither does time exist without change ; for when the state of our own minds does not change at all , or we have not noticed its changing , we do not realize that time has elapsed .
many myths speak of a world before time , a world in which time had yet to come into existence .
amusingly , astrophysicists have a similar argument when they describe the origin of the universe .
recall that in the bible , after adam ate the apple , god said : behold , the man has become like one of us , knowing good and evil ; and now , lest he put forth his hand and take also of the tree of life , and eat , and live for ever .
adam was punished for wanting to archieve eternity and he was thrown into temporality .
etienne klein ( 1958- ) reinterpreted a greek myth about the birth of time as follows : at the beginning of all things , gaia arose from chaos and gave birth to her son ouranos while she was sleeping out of time .
gaia was in the center , ouranos all around , and there was no space between them .
gaia became pregnant and the titans , children of ouranos , could not be born because of the lack of space .
one of the titans , kronos , made it possible to chase away ouranos , and this enabled space to be created .
according to klein , a confusion we make between time and future dates back to that myth . a major contribution from this author
many other temporal physiological functions are distributed in other brain areas , independent of the suprachiasmatic nucleus .
they are the temporal coordination of spatially distributed neuronal networks , as well as brain structures that relate to tempus , ie , the prediction and evaluation of durations .
the measurement of short durations , of less than a second , are probably less dependent on attention or emotion , although these can modulate even the measurement of short durations .
the evaluation of longer durations , from seconds to hours or weeks , involves cognitive functions such as attention , emotion , and memory .
more than 100 years ago , the swiss psychiatrist auguste forel ( 1848 - 1931 ) , known for his work in psychiatry , neurohistology and for his extensive studies on ants , noticed that bees came to his breakfast table regularly at the same time , even when no breakfast was being served .
indeed , bees can be conditioned to go at a given moment of the day to a given location where food was previously provided .
a theoretical model on how biological systems could measure duration , ie , how a brain system might generate a continuous metric , was proposed by treisman in 1963 .
the model proposed by treisman provides the organism with a centralized system of time measurement .
this has been criticized on the following basis : it could be that each biological function has an inbuilt duration measuring system , for example , a different one for motor action , for vision or for audition .
moreover , there might even be independent duration measuring systems for subfunctions in each motor or perceptive system .
david m. eagleman summarized issues raised by the handling of time by the brain : a challenge for the brain is that afferent signals from the different , sensory modalities are processed at different , speeds .
when receiving signals from several modalities , how does the brain determine the timing correspondence ?
for example , with short durations , when the event occurs in the immediate proximity or during an eye saccade , there is compression of time .
an experiment by stetson illustrates this recalibration of duration , in the case of the visual modality .
when a delay of 100 ms is artificially introduced between the moment of pressing a button and the occurrence of a flash of light , the subject rapidly adapts to this delay , which seems progressively shorter .
when the presence of this delay is abruptly interrupted , the subject can have the impression that the flash occurred before he or she pressed the button .
these adaptations of duration judgments are independent of one another , in the sense that if duration compression or dilation occurs in one perceptive aspect or system , eg , vision , it generally does not occur in other modalities , eg , audition .
these observations speak in favor of more than one neuronal networks that judges duration , since the temporal outputs of these networks can become desynchronized .
imprecision , ambivalence , and contradiction are often how we speak of time , and this influences how we think about it .
a major imprecision is that we do not set apart time and temporal phenomena : without noticing it , we attribute to time properties that are those of the temporal phenomena that we observe . for example , succession of days and nights , or the repetitive obligation to fill out tax declarations , will lead us to say that time repeats itself , and thus that is is cyclical . defining time is a challenge . indeed , defining a concept is feasible when the concept is referred to something more fundamental . and nothing has been found that could be considered to be more fundamental than time .
thus , definitions of time are circular ; they are tautological , as found by aristotle , who saw time as the amount of movement with respect to before and after .
when thinking about time , we remain under the influence of old analogies and metaphors .
we hesitate between two pillars of greek philosophy , parmenides and the concept of immobility , and heracleitus and the concept of a future .
heracleitus has shaped our discourse about time for the last two millennia , and still today , we can not consider other metaphors than that of time analogous to a river and the flow of water .
space remains , while time passes : could this be a manner to differentiate them ?
but if we define time as a machine that produces instants , then we have to conclude that it is not time that passes , but that all these instants are fabricated by time .
we should learn not to confuse time with duration , time with future , or time with temporal phenomena .
a stimulating but radical view about time was proposed by wittgenstein ( 1889 - 1951 ) : ... there is no such thing and it is just a form of objects . the solution of the riddle of life in space and time lies outside space and time .
we stand inexorably in time and space , and our challenge is to live with time as an object that we remain unable , to define .
and i confess to thee , o lord , that i am still ignorant as to what time is .
and again i confess to thee , o lord , that i know that i am speaking all these things in time , and that i have already spoken of time a long time , and that very long is not long except when measured by the duration of time .
how , then , do i know this , when i do not know what time is ? or , is it possible that i do not know how i can express what i do know ? | we are unable , using our five senses , to feel time , nor , using our intelligence , to define it , because we stand inexorably within time .
we achieve a representation of time through evaluation of changes in ourselves and in our environment .
this is made possible by memory functions .
what if time only existed as a construct in our minds , and what if the absence of this construct made our mode of thinking uncomfortable to us ?
if our two major tools for constructing our world , feeling and reasoning , are of little help , then the study of time , ie , chronology , might exist as a list of scientific hypotheses , and remain , to some extent , a philosophical question an enigma that has been approached by thinkers for more than two millenia . in this review ,
various fields of knowledge are discussed in relation to time , from philosophy and physics to psychology and biology .
we discuss the differences between chronos and tempus , respectively the time of physicists and that of psychologists . |
current comprehensive cancer care is centered on reducing the bulk of disease through surgery , chemotherapy , and radiation . despite the increasing effectiveness of these cornerstones of treatment and high cure rates of multiple cancer forms
, cancer remains a leading cause of death.1 recent breakthroughs in cancer immunotherapy have added several promising new therapies to the traditional armamentarium of oncology treatment regimens .
the strategy of utilizing the immune system in the treatment of cancer dates back to the 1890s and the work of william coley.2 coley observed that some tumors regress in the setting of acute bacterial infection .
he attempted to recapitulate this phenomenon by studying the injection of heat - inactivated streptococcus erysipelas and serratia marcescens ( coley s toxins ) in cancer patients .
the field of cancer immunology and immunotherapy has greatly advanced since coley s initial studies , a time when little was known about the mechanisms underlying the antitumor effects of bacterial toxins .
there is now a growing understanding of how the immune system identifies tumor cells and targets them for elimination . just as important is the growing understanding of how tumors can undermine the immune system s ability to recognize and eliminate cancer cells . briefly ,
an adaptive immune response against tumor cells is classically believed to be initiated when tissue - resident antigen - presenting cells , such as dendritic cells , take up and process tumor - specific or tumor - associated antigens , and present these antigens in the context of major histocompatibility complex ( mhc ) complexes to nave t - cells in secondary lymphoid organs .
nave t - cells can differentiate and expand into different classes of antigen - specific t - cells , including cluster of differentiation ( cd)4 t helper cells and cd8 effector cytotoxic t lymphocytes ( ctls ) . at each step of this process ,
various signals shape whether an antitumor t - cell response will be produced , or conversely , an immunosuppressive and/or tolerogenic response will be made by such mediators as regulatory t - cells and myeloid - derived suppressor cells ( reviewed by palucka and banchereau,3 chen and mellman,4 and blattman and greenberg5 ) .
immunotherapies for cancer can target each or many of these steps to skew toward an antitumor response and away from an immunosuppressive response .
cancer immunotherapies can be categorized as non - antigen - specific or antigen - specific therapies .
non - antigen - specific immunotherapies aim to either enhance the immune response in a general fashion or to decrease the immunosuppression present in the tumor environment .
non - antigen - specific therapies include cytokines and immune growth factors ( eg , interferon ( ifn]- , interleukin [ il]-2 , or granulocyte macrophage colony - stimulating factor ) , immunologic adjuvants
( eg , bacille calmette - gurin ) ; toll - like receptor ( tlr)-3 agonists , such as poly - i : c ( rintatolimod , ampligen ; hemispherx biopharma , inc . , philadelphia , pa , usa ) and poly - iclc ( hiltonol ; oncovir , washington , dc , usa ) ; tlr-4 agonists , such as monophosphoryl lipid a ; the tlr-7 agonist , imiquimod ; immune checkpoint blockers , eg , anticytotoxic t - lymphocyte antigen-4 ( ctla-4 ) antibody;6,7 and the programmed death-1 ( pd-1 ) pathway agents , nivolumab and lambrolizumab.811 compared with non - specific immunotherapies , antigen - specific therapies , such as therapeutic vaccines against cancer , aim to induce immune cells to target cancer cells that express a particular set of antigens .
different classes of cancer vaccines include peptide - based or protein - based vaccines , cancer cell - based vaccines , viral vector vaccines , dna vaccines , messenger rna vaccines , and carbohydrate vaccines.1219 in all cases , these vaccines involve two components , an antigen and an adjuvant , aimed at promoting local inflammation and the resulting immunization .
additionally , all of the above types of cancer vaccines rely on the patients endogenous dendritic cells ( dcs ) for their uptake and effective antigen presentation to tumor - specific cd8 and cd4 t - cells .
another category of cell - based cancer vaccines is use of patients ex vivo - generated and tumor antigen - loaded dcs ( or more precisely , autologous cellular therapeutics ) .
this strategy limits the dependence of the immune system on patients resident dcs , which have been shown to be defective in the advanced stages of cancer3,20,21 or even redirected to differentiate toward myeloid - derived suppressor cells.22,23 regardless of whether endogenous or ex vivo - generated dcs are utilized for immunization , therapeutic cancer vaccines need to overcome several common challenges to induce immunity in the presence of established tumors and can benefit from recent developments in the area of dc biology .
for a therapeutic cancer vaccine to be effective , it must be capable of inducing a high number of antigen - specific t - cells against an established tumor , which can migrate to the tumor and perform their effector functions at the tumor site ( figure 1 ) .
the first challenge is achieving high numbers of antitumor t - cells when the vaccine is being administered in the presence of an ongoing , although dysfunctional , immune response . due to the ongoing antitumor immune response
, the vaccine - carrying antigen - presenting cells ( using either endogenous dcs that have taken up vaccine - introduced antigens or ex vivo - generated tumor antigen - loaded dcs ) , may be recognized by the cd8 t - cells as
tumor.24,25 since this encounter occurs in the periphery , away from the immunosuppressive tumor microenvironment , the cd8 t - cells may be capable of eliminating the vaccine , and thus limiting the vaccine s effectiveness before it can induce an immune response.3 additionally , there is a lack of the proinflammatory signals required to promote effective immune responses .
these signals are replaced by tumor - induced immunosuppressive / anti - inflammatory signals predominating in cancer patients .
therefore , to achieve the goal of inducing high numbers of tumor - specific t - cells , the vaccine - carrying antigen - presenting cells must not only survive long enough to present antigen , but must also provide the inflammatory signals to drive effector cell functions.3,2628 unfortunately , the presence of high numbers of antigen - specific t - cells does not ensure an effective antitumor response if these t - cells are unable to home to the tumor . in a normal infection scenario , where the immune response is targeting invading pathogens
, the microorganisms and local tissue damage induce chemokines that recruit effector cells such as ctls , type 1 helper cd4 t - cells , or natural killer cells to the site of pathogen entry.27,29 however , one of the immune evasion mechanisms evoked by tumors to support tumor growth and metastatic spread is downregulation of the chemokines that attract immune effector cells28,29 and upregulation of chemokines that attract suppressor cells , such as regulatory t - cells,3032 suppressive plasmacytoid dcs,33 and myeloid - derived suppressor cells.34,35 thus , a therapeutic vaccine needs to either induce t - cells that can respond to the spontaneously expressed tumor - associated chemokines or be administered as part of a combinatorial therapy with additional factors to alter the chemokine profile in the tumor microenvironment.32,34 once high numbers of vaccine - induced tumor - specific t - cells have been generated and arrive at the tumor site , the t - cells must be capable of killing the tumor cells in order for the vaccine to be effective .
most types of cancer ( including melanoma , ovarian , breast , renal , prostate , lung , and head and neck cancer ) produce many factors , including il-10 , transforming growth factor beta , vascular endothelial growth factor , il-6 , and cyclooxygenase-2 products like prostaglandin e2 , that contribute to immune dysfunction by suppressing the functions of endogenous or adoptively transferred dcs and t - cells.21,3638 these factors not only act to directly suppress dc and t - cell functions , but they can also promote cell - mediated immune suppression by enhancing the recruitment , expansion , and activation of regulatory t - cells and myeloid - derived suppressor cells.30,31,39,40 while in some patients the high numbers of tumor - specific t - cells induced by the vaccine may be able to overcome the immunosuppressive tumor microenvironment , most therapeutic cancer vaccine strategies would greatly benefit from a combinatorial approach that alters the tumor to reduce immunosuppressive factors .
currently , the only therapeutic cancer vaccine approved by the us food and drug administration is sipuleucel - t , a treatment for metastatic androgen - independent prostate cancer that was approved in 2010.41,42 sipuleucel - t consists of antigen - presenting cells that are activated ex vivo from autologous peripheral blood mononuclear cells by a fusion protein , pa2024 , which is comprised of granulocyte macrophage colony - stimulating factor and prostatic acid phosphatase , a prostate adenocarcinoma - associated antigen.41,43 in two randomized , double - blind , placebo - controlled multicenter phase iii trials , sipuleucel - t increased median survival by 4 months when compared with placebo.43,44 sipuleucel - t was administered in three doses at weeks 0 , 2 , and 4 , each at 2 days following leukapheresis . in the d9901/d9902a trials of 225 patients with asymptomatic metastatic hormone - refractory prostate cancer randomized in a 2:1 ratio to treatment with sipuleucel - t or a control infusion , the primary objective was time to disease progression . while there was no statistically significant difference in time to progression ( median 11.1 weeks with sipuleucel - t versus 9.7 weeks with control )
, there was a 33% reduction in risk of death with sipuleucel - t compared with control and a statistically significant difference in survival ( median 23.2 months for sipuleucel - t versus 18.9 months for control , p=0.011).44 in the impact ( immunotherapy for prostate adenocarcinoma treatment ) study of 127 metastatic castration - resistant prostate cancer patients with the primary endpoint of overall survival , there was a 22% adjusted relative reduction in risk of death and a statistically significant increase in median survival of 4.1 months ( median 25.8 months for sipuleucel - t versus 21.7 months for placebo , p=0.03 ) , although there was no difference in disease progression.43 patients in the treatment group who had antibody titers of more than 400 against pa2024 had an increased survival compared with those who had titers of less than 400 ( p<0.001).43 cumulative antigen - presenting cell activation measured by cd54 upregulation , antigen - presenting cell number , total nucleated cell number , and antigen - specific immune responses to pa2024 and/or prostatic acid phosphatase in the treatment group correlated with overall survival ( p<0.05).41 the clinicaltrials.gov registry gives an insight into upcoming cancer vaccines in development that show promise in improving outcomes.45 a query of this website in november 2013 with a targeted search of phase iii and iv clinical trials with known statuses and cancer listed as the condition , vaccine as the intervention , and survival as the outcome measure , resulted in 42 studies .
a summary of selected cancer - specific vaccines from this query is listed in table 1 with additional information from publications and abstracts.43,4655 in addition to the sipuleucel - t trials , a phase iii trial of a glycoprotein 100 peptide vaccine also posted positive results . in a randomized , multicenter trial ,
patients with advanced melanoma received il-2 and glycoprotein 100:209217 ( 210 m ) peptide vaccination or il-2 alone.47 for the primary endpoint of clinical response , the il-2 with vaccination group had a significantly higher response rate of 20% ( complete response 11% , partial response 9% ) versus a response rate of 10% in the il-2 only group ( complete response 2% , partial response 8% ; p=0.05 ) .
median progression - free survival was also significantly longer in the il-2 with vaccination group ( 2.2 months ) than in the il-2 alone group ( 1.6 months ; p=0.008 ) .
there was a trend of increased overall survival with the addition of vaccination to il-2 ( 17.8 months ) compared with il-2 alone ( 11.1 months ; p=0.06 ) .
it is important to note that this study was not powered to detect a difference in overall survival .
another vaccine in phase iii trials is tg4010 , a poxvirus vector vaccine encoding for the tumor - associated antigen mucin-1 ( muc1 ) and il-2 , which is being investigated in non - small cell lung cancer ( nsclc).49 ima901 , a multiple peptide vaccine for renal cell carcinoma , has also completed accrual for its phase iii study and its results are pending . the ten peptides for ima901 were uniquely chosen using an antigen discovery platform that analyzed renal cell carcinoma tissue.56 the hyperacute vaccines ( newlink genetics , ames , ia , usa ) for pancreatic and nsclc consist of allogeneic cancer cells that have been genetically modified to express murine (1,3)galactosyl.50,51 prostatak ( advantagene inc . , auburndale , ma , usa ) and prostvac - v / f - tricom ( bavarian nordic ; washington , dc , usa ) are viral - based vaccines for prostate cancer .
prostatak involves intratumoral injection of an adenovirus containing a herpes virus thymidine kinase gene followed by valaciclovir .
prostvac - v / f - tricom is composed of recombinant vaccinia and fowlpox viral vectors that encode for prostate - specific antigen and tricom , a combination of three costimulatory molecules , lfa-3 , b7.1 , and intercellular adhesion molecule-1.52 a few phase iii trials have failed to meet their primary endpoints , and highlight the difficulties of cancer vaccine development .
one of the largest studies in nsclc , the magrit ( mage - a3 as adjuvant nsclc immunotherapy ) trial , which utilized a melanoma - associated antigen 3 ( mage - a3 ) protein vaccine , was stopped in early 2014 after failing to increase the primary endpoint of disease - free survival in mage - a3-positive patients overall or mage - a3-positive patients without chemotherapy treatment , compared with control.5759 this was following a double - blind , randomized , placebo - controlled phase ii study that showed clinical activity , with all treated patients developing anti - mage - a3 antibodies and with a pretreatment 84-gene expression signature being associated with increased disease - free response.60,61 however , the subsequent phase iii trial was not able to determine a subpopulation of gene signature - positive patients who would benefit from treatment since there was an insufficient treatment
effect.59 belagenpumatucel - l , an allogeneic genetically modified nsclc tumor cell vaccine , showed a trend toward increased median survival but this did not reach statistical significance.53 however , the subgroup of patients who received vaccination within 12 weeks of chemotherapy had a statistically significant improvement , and the study is continuing in this subgroup of patients .
similarly , tecemotide , a muc1 peptide vaccine for nsclc , failed to demonstrate a statistically significant difference in overall survival compared with placebo , but a significant increase in median overall survival in the subgroup of patients who had concurrent chemoradiation has led to plans for a randomized trial of tecemotide with concurrent chemoradiation in stage iii nsclc patients.54 one of the largest studies in metastatic melanoma was mmait - iv ( malignant melanoma active immunotherapy trial for stage iv disease ) , an international , multicenter , randomized , double - blind phase iii trial in 1,656 stage iii and iv patients of an allogeneic whole melanoma cell vaccine , canvaxin ( cancervax corporation , carlsbad , ca , usa ) with a bacille calmette - gurin adjuvant , compared with placebo plus bacille calmette - gurin , that was closed early after interim analysis showed a low probability of demonstrating a significant increase in survival in the canvaxin with bacille calmette - gurin arm.62 although the trial had negative results , an ancillary study of pretreatment and post - treatment circulating tumor cell biomarkers for melanoma antigen recognized by t - cells 1 ( mart1 ) , mage - a3 , and paired box 3 ( pax3 ) from patients in the mmait - iv trial was able to demonstrate that pretreatment and serial circulating tumor cell levels were significantly associated with decreased disease - free survival and overall survival.63 another large melanoma vaccine study , the randomized phase iii trial of adjuvant ganglioside ( gm2 ) conjugated to keyhole limpet hemocyanin ( klh ) admixed with adjuvant qs-21 ( gm2-klh / qs-21 ) vaccine versus observation in 1,314 stage ii melanoma patients , was terminated after the second interim analysis due to failure to increase recurrence - free survival and a trend toward increased overall survival in the observation arm , which was also confirmed on final analysis after a median follow - up of 4 years.64 a challenge in evaluating therapeutic cancer vaccines is appropriate patient selection .
while clinical trials of new oncologic therapies are traditionally first tested in patients with advanced cancers who have failed multiple treatment regimens , vaccines may be more effective when the disease burden is low.65 another challenge in trial design and evaluation is that the kinetics of tumor growth rates for vaccine therapy differ from those of traditional chemotherapy and radiotherapy.66 compared with these directly cytotoxic therapies in which the treatment response occurs immediately following their administration and the tumor growth rate often returns to pretreatment levels following termination of treatment , positive responses to vaccine therapy may begin months after treatment , with a potentially prolonged treatment effect persisting long after administration.67 therefore , the intermediate endpoint of progression - free survival based on the commonly used response evaluation criteria in solid tumors or world health organization criteria has very limited value in vaccine therapies , and more relevant immunologic endpoints are needed.6668 a common phenomenon with immunotherapy trials is that overall survival may improve without a change in progression - free survival.43,46,69 in fact , there may even be a treatment response after initial progression or tumor growth.67 in result , the recently formulated immune - related response criteria67 are better predictors of prolonged overall survival of patients treated with immunotherapy than the classical response criteria used to evaluate the effectiveness of chemotherapeutic agents ( response evaluation criteria in solid tumors [ recist ] and world health organization).67 finally , another important trial design consideration is immunologic selection and response monitoring of patients .
pretreatment markers would help to determine which patients would benefit the most from vaccine treatment but this work is still in its infancy.70 the discovery of markers to monitor immune responses that correlate with clinical outcomes is still in development .
current biomarkers to evaluate the immune response focus on ctl antigen recognition and the humoral response .
markers shown to correlate with clinical outcome include antigen - specific t - cell response based on ifn- enzyme - linked immunospot ( elispot ) assays , cytokine expression levels , and reduction in regulatory t - cells.41,7173 furthermore , two clinical trials involving dc vaccines indicated a role of dc - produced il-12p70 as a predictive marker of the clinical benefit of vaccination.74,75
the primary aim of cancer vaccines is to generate a ctl response against cancer cells.76 an important advantage of therapeutic immunizations , compared with traditional cancer treatments , is that the treatment effect is typically durable due to the induction of long - lived effector memory and central memory t - cells , which can persist for prolonged periods after administration of the vaccine .
the second advantage is the very high selectivity of the immune response in targeting tumor cells , while not damaging healthy tissue .
as mentioned before , several strategies , such as protein or dna vaccines , utilize a patient s endogenous dcs at the injection site for uptake and presentation of tumor antigens , but the observed dysfunction of dcs in cancer patients due to tumor - related suppressive factors may limit the effectiveness of these vaccines , which rely on endogenous dcs for antigen uptake.37,7779 therefore , the use of ex vivo - generated dc vaccines is an attractive option for circumventing this issue , enabling dcs to mature in the absence of tumor - related immunosuppression and allowing more control of the dc maturation process to direct the nature of the immune response .
effective induction of an antigen - specific t - cell response requires delivery of at least four types of signals ( figure 2 ) by dcs , each of which can be optimized to improve the cancer vaccine.80 the first signal ( signal 1 ) is the presentation of processed antigen in the context of mhc molecules by dcs to nave t - cells via the t - cell receptor.81 one of the key characteristics of dcs that makes these cells a unique tool for cancer vaccination is their ability to take up different forms of antigens , process them , and then cross - present these antigens to nave cd4 and cd8 t - cells .
this broadens the source of antigens that can be used in vaccines to include not only peptides ( which are mhc - restricted and limited to known , well characterized tumor antigens and thus only applicable to patients who express the appropriate mhc haplotype and have tumors that express the specific antigen ) , but also recombinant proteins , tumor lysates , or whole tumor cells from either autologous or allogeneic sources .
the use of proteins or whole cell sources of antigen increases the ability to prime immune responses to undefined patient - specific tumor antigens .
various methods of processing tumor cells for loading have been studied , such as freeze - thaw lysates , irradiation , and oxidation of tumor cells , to enhance the uptake and cross - presentation of whole tumor cells by dcs.79,8284 of note , loading dcs with apoptotic cells was shown to be more effective in stimulating ctls compared with loading with necrotic cells.85 signal 2 involves costimulatory signals that amplify the t - cell receptor signal and prolong the mhc : t - cell receptor interaction to ensure t - cell activation .
this amplification signal is provided by b7 family molecules , such as cd80 and cd86 , that bind to cd28 on the t - cell.86,87 the mhc : t - cell receptor and cd28:cd80/cd86 interactions are stabilized by integrins , notably leukocyte function - associated antigen-1 ( lfa-1 ) : intercellular adhesion molecule-1 interactions , so that the cell - cell interactions are not prematurely terminated , resulting in incomplete activation.88 the absence of costimulation during antigen presentation by dcs can induce cd8 t - cell tolerance to the antigen.89 the molecules involved in costimulation are upregulated upon dc maturation , when the dc also gains the ability to respond to the lymph node - homing chemokines ccl ( chemokine [ cc motif ligand]-19 and ccl22 ) by upregulating cc chemokine receptor type 7 ( ccr7).90,91 the first generation of dc vaccines utilized immature or partially matured dcs capable of cross - presentation of antigens but deficient in costimulatory and lymph node - homing abilities.86 this led to protocols for dc maturation producing the second generation of dc vaccines that are able to provide both signals 1 and 2 .
in these protocols , the dcs are matured using either monocyte - conditioned medium92 or a cytokine cocktail consisting of il-6 , il1 , tumor necrosis factor alpha , and prostaglandin e2.93 while these maturation strategies induce upregulation of costimulatory molecules and ccr7 , and have enhanced immunogenicity in vitro and in vivo in healthy volunteers , their initial promise diminished in a randomized multicenter phase iii trial for advanced melanoma when less than 5% of patients receiving the vaccine demonstrated a clinical response and there was no impact on overall survival.94 signal 3 is the dc - produced cytokine profile that skews the type of immune response generated ( ie , type 1 cell- mediated versus type 2 humoral responses ) , and provides survival and differentiation signals to nave t - cells .
a prototypical example of a signal 3 cytokine that promotes cell - mediated immunity is il-12p70,95 which is produced by dcs when they are matured in the presence of ifn- , a cytokine produced by activated natural killer cells at the site of infection , and in the absence of the chronic inflammatory cytokine , prostaglandin e2 .
one possible factor in the negative results of the clinical trials using second generation dcs is the use of prostaglandin e2-containing maturation cocktails , since prostaglandin e2 has subsequently been shown to have a deleterious effect on il-12p70.9597 in order to generate mature dcs with high costimulatory molecules and lymph node - homing ability , as well as high il-12-producing capacity to promote the desirable cell - mediated immunity , a third generation of dcs was generated.73,74 the third generation dcs are generally matured in conditions mimicking viral infection , which predominantly drives cell - mediated immunity .
some of the strategies to mature dcs are : to coculture immature dcs with other immune cells , such as il-18 activated natural killer cells98 or memory cd8 t - cells;25,99 to mature with conditioned medium from activated ctls;100,101 or to use cytokine cocktails that include viral - mimicking tlr
( dc1 ) , possessing not only high antigen cross - presentation and costimulatory abilities , but also a superior ability to secrete il12 for up to 48 hours after interaction with cd40l - expressing cd4 t - cells.102,103,108110 additional inclusion of ifn- to a type 1 polarizing cytokine cocktail consisting of ifn- , il1 , tumor necrosis factor alpha , and poly - i : c enhanced the expression of the lymph node homing chemokine receptor ccr7.111113 these dc1s also preferentially produce chemokines that promote migration of nave , memory , and effector t - cells , but show reduced expression of chemokines that promote immunosuppressive cell recruitment , further enhancing the ability of dc1s to interact and prime strong antitumor immune responses.111113 a recent clinical trial utilizing dc1 vaccines and an alternative type of type 1 polarized dcs
induced by the combination of cd40l and ifn- demonstrated that the ability of dc1 vaccines to produce high il-12p70 levels was the strongest predictor of prolonged progression - free survival in vaccinated patients.74,75 the last type of signal ( signal 4 ) delivered to t - cells during priming interactions with dcs results in programming of specific chemokine receptor expression on activated t - cells that directs them to specific tissues.86 in vitro and ex vivo studies have demonstrated that different dc subsets isolated from various tissues can modulate the chemokine expression profile on activated t - cells , thereby directing t - cells back to the tissues of dc origin.114,115 this differential chemokine expression programming is not limited to dcs developed in various tissues in vivo , but also extends to ex vivo - generated , cytokine - matured dcs .
a comparison of cd8 t - cells from melanoma patients sensitized ex vivo by either prostaglandin e2-matured dcs ( second generation ) or type 1 polarized dc1s ( third generation ) demonstrated different chemokine expression on the activated cd8 t - cells.97 specifically , t - cells sensitized by dc1s had higher expression of ccr5 and cxc chemokine receptor 3 ( cxcr3 ) , two chemokine receptors involved in peripheral homing to the skin and entry into melanoma and other tumors , compared with t - cells sensitized by prostaglandin e2-matured dcs.111,112,114,116
future developments in cancer immunotherapy research will likely focus on the challenges that vaccine - induced ctls encounter in reaching the tumor microenvironment and performing their antitumor cytotoxic functions .
areas of current investigation in changing the tumor milieu include promoting ctl entry via chemokine modulation , inhibiting immune checkpoints that block ctl effector function , and decreasing immunosuppressive cells , such as regulatory t - cells and myeloid - derived suppressor cells .
chemokine modulation aims to shift the balance of the tumor environment toward expression of effector t - cell attracting chemokines , and away from regulatory t - cell attracting chemokines.32 tumor infiltration of certain immune cells such as ctls , type 1 helper cd4 t - cells , dcs , and m1 macrophages has positive prognostic value , while infiltration by regulatory t - cells , type 2 helper cd4 t - cells , myeloid - derived suppressor cells , and m2 macrophages is associated with poor outcomes.117120 there are currently several monoclonal antibodies and small molecule inhibitors targeting various chemokine receptors in clinical trials.121 our group has also shown that ex vivo treatment of tumor tissue with type 1 ifns , a tlr-3 ligand , and a cyclo - oxygenase-2 inhibitor increased the production of the effector t - cell attracting chemokines , ccl5 and cxcl10 , while decreasing the production of regulatory t - cells attracting chemokine ccl22.32 combining vaccines with agents that reduce the levels of immunosuppressive cells ( such as myeloid - derived suppressor cells and regulatory t - cells ) has also been an attractive strategy .
low - dose cyclophosphamide has been used extensively for its ability to suppress regulatory t - cells since it is inexpensive and easily obtained.122 a randomized phase ii study of the renal cell cancer peptide vaccine , ima901 , demonstrated that a single cyclophosphamide dose was effective in reducing the number of regulatory t - cells , and that among patients who were immune responders , those treated with cyclophosphamide had increased survival.56 another phase i / ii trial of a multipeptide - loaded dc vaccine in advanced ovarian cancer showed a trend toward increased survival with the addition of cyclophosphamide treatment.123 other combination strategies to reduce regulatory t - cells in vaccine trials have included anti - cd25 monoclonal antibodies and a cd25 targeting immunotoxin.124,125 preliminary data from an ongoing randomized dc vaccine trial targeting myeloid - derived suppressor cells using all - trans - retinoic acid show that the treatment arm receiving all - trans - retinoic acid and vaccination had an improved immune response compared with vaccination alone.126 other inhibitors of immunosuppressive targets shown to correlate with decreased survival , such as prostaglandin e2 , indoleamine 2,3-dioxygenase , and nitric oxide synthase , are also potential targets for combinatorial therapy with cancer vaccines.127130 in contrast with combinatorial therapies that reverse immunosuppressive cells , cancer vaccines may be combined with cytokine treatments that promote effector t - cell activity and prolong t - cell memory ( see figure 1c ) .
il-7 , il-15 , il-21 , and il-27 are similar to il-2 as part of the common gamma chain cytokine receptor family.131 il-7 has a role in development , homeostasis , and survival of t - cells and b - cells.132,133 administration of recombinant il-7 to cancer patients has been shown to be safe and to rapidly expand circulating cd4 and cd8 cells that express cd127 , but not regulatory t - cells.134 il-15 has a role in t - cell and natural killer cell activation and proliferation and maintenance of memory t - cell responses.135,136 early phase clinical trials utilizing il-15 for cancer treatment are ongoing or recently completed , but without published results as yet.136 il-21 is produced by activated cd4 t - cells and natural killer t - cells , and contributes to antitumor immunity by its induction and activation of cd8 t - cells , natural killer cells , and natural killer t - cells.131,137,138 early phase i and ii studies have shown encouraging results in metastatic melanoma and metastatic renal cell carcinoma.139142 il-27 is produced by antigen - presenting cells and can enhance cd8 t - cell and natural killer cell activation , but development of il-27 as a therapeutic is still in preclinical stages.143,144 the approval by the us food and drug administration of ipilimumab for metastatic melanoma in 2010 signaled a change in the landscape of cancer therapies .
ipilimumab ( mdx-010 , yervoy ; bristol - myers squibb , new york , ny , usa ) is a fully human monoclonal antibody against ctla-4 , and a homologue of cd28 with greater affinity to b7 molecules which outcompetes cd28 binding , effectually preventing the costimulatory signal 2.11 anti - ctla-4 antibodies block this inhibitory interaction or immune checkpoint and restore signal 2 for t - cell activation . in a randomized , double - blind , three - arm phase iii trial comparing ipilimumab with and without a glycoprotein 100 vaccine ( mdx-1379 ) with vaccination alone in patients with metastatic melanoma , subjects in the ipilimumab treatment groups were found to have a significantly higher median survival compared with those receiving vaccination alone ( 10 months versus 6.4 months).46 the failure of the vaccination arms in the phase iii study to improve overall survival was unexpected , but it is possible that this resulted from the application of a single - epitope glycoprotein 100 peptide vaccine .
a similar glycoprotein 100 vaccine did not show an improvement in survival , although that study was only powered to detect a difference in progression - free survival and not overall survival.47 furthermore , the original phase iii study had ipilimumab and vaccination administration occurring concurrently , whereas there is more recent evidence from a murine model that sequential therapy of vaccination followed by anti - ctla-4 antibody was superior to the anti - ctla-4 antibody when administered first.145 some of the early preclinical studies of ipilimumab indeed focused on using it in combination with cell - based cancer vaccines , and other anti - ctla-4/vaccine combinations are in clinical trials.11,146148 a recent phase ii study comparing ipilimumab alone or in combination with gm - csf - secreting whole cell vaccine showed a higher survival rate when ipilimumab was combined with vaccine.146 another actively studied immune checkpoint receptor is pd-1 ( cd279).149 pd-1 and its ligands , pd-1 ligand 1 and 2 ( pdl1 and pdl2 ) , are expressed on more cell types than ctla-4 .
pd-1 expression can be induced not only on activated t - cells , but also on b - cells and natural killer cells , while pdl1 and pdl2 can be upregulated on tumor cells , antigen - presenting cells , and other cells in inflammatory conditions .
several clinical trials of anti - pd-1 and anti - pdl1 antibodies have shown durable response rates.6,150152 while studies using combinatorial pd pathway agents and vaccine therapy are not as advanced as those with anti - ctla-4 agents , there is promising preclinical and early clinical trial data suggesting that the dual combination or even the triple combination with anti - ctla-4/pd pathway blockade / vaccination therapy will have increased clinical benefit by further enhancing the antigen - specific t - cell response from vaccination and decreasing regulatory t - cells.153157 an effective combinatorial vaccine therapy will likely need to address three goals : building a robust antigen - specific ctl response ; altering the tumor microenvironment to allow ctl infiltration and reduce migration of regulatory t - cells and myeloid - derived suppressor cells ; and counteracting ctl inhibitory mechanisms such as immune checkpoints that lead to immunosuppression ( figure 1 ) .
an encouraging study using a combination of a peptide vaccine , anti - pd-1 antibody , and low - dose cyclophosphamide in a murine tumor model demonstrated that this combination of drugs synergized in increasing survival and reducing tumor burden.158 one of the concerns about optimal application of complex immunotherapies is determination of the optimal sequence and duration of application of each of the components .
it also needs to be determined how to optimally incorporate immunotherapy , different forms of which can either suppress or enhance both the induction of immune responses and the susceptibility of cancer tissues to immune attack .
the next era of vaccine development is likely to involve both continued improvement of the vaccines themselves as well as combinatorial application of vaccines with agents that target the tumor microenvironment to promote entry of vaccination - induced cells , while eliminating local predominance of suppressive cells , and amplifying and prolonging the duration of the effector phase of antitumor immunity at tumor sites .
the development of optimized immunotherapies for advanced cancer will also benefit from identification of the most relevant laboratory correlates of clinical effectiveness and integration of immunotherapy with other elements of comprehensive cancer care . | recent us food and drug administration approvals of provenge ( sipuleucel - t ) as the first cell - based cancer therapeutic factor and ipilimumab ( yervoy/anticytotoxic t - lymphocyte antigen-4 ) as the first checkpoint blocker highlight recent advances in cancer immunotherapy .
positive results of the clinical trials evaluating additional checkpoint blocking agents ( blockade of programmed death [ pd]-1 , and its ligands , pd-1 ligand 1 and 2 ) and of several types of cancer vaccines suggest that cancer immunotherapy may soon enter the center stage of comprehensive cancer care , supplementing surgery , radiation , and chemotherapy .
this review discusses the current status of the clinical evaluation of different classes of therapeutic cancer vaccines and possible avenues for future development , focusing on enhancing the magnitude and quality of cancer - specific immunity by either the functional reprogramming of patients endogenous dendritic cells or the use of ex vivo - manipulated dendritic cells as autologous cellular transplants .
this review further discusses the available strategies aimed at promoting the entry of vaccination - induced t - cells into tumor tissues and prolonging their local antitumor activity .
finally , the recent improvements to the above three modalities for cancer immunotherapy ( inducing tumor - specific t - cells , prolonging their persistence and functionality , and enhancing tumor homing of effector t - cells ) and rationale for their combined application in order to achieve clinically effective anticancer responses are addressed . |
the morphologically similar appearing clear cells are ambiguous in nature and believed to be originated from different cell lineages such as epithelial , mesenchymal , melanocytic , or hematopoietic .
clear cell odontogenic carcinoma ( ccoc ) is one among the rare neoplasm with only 74 reported cases in the literarure .
it was first described by hansen et al . and later by waldron et al . , in 1985 , as clear cell odontogenic tumor . in 2005 , the world health organization ( who ) had reclassified as ccoc owing to its high rate of recurrence , local and distant metastasis , and tumor - related deaths .
ccoc usually present with a variable degree of pain and mobility of regional teeth with or without the involvement of bone .
approximately 60% of patients show evidence of soft tissue involvement in the anterior portion of the mandible as lesion perforates bone .
radiographically , a poorly delineated , unilocular or multilocular radiolucent lesion with prominent bone destruction is observed .
histopathologically , ccoc may show one or more architectural patterns such as biphasic , monophasic , and ameloblastomatous .
the undetermined source of clear cells makes the diagnosis based on conventional histology nearly impossible .
they require special attention to be differentiated from other clear cell lesions either by means of special stains such as mucicarmine , congo red and periodic acid schiff 's ( pas ) or immunohistochemical staining such as cytokeratin ( ck ) , epithelial membrane antigen ( ema ) , s-100 protein , and vimentin .
expression of ck-19 and ema is a consistent finding in ccoc whereas the tumor shows negativity for vimentin , s-100 protein , desmin , and smooth muscle actin .
this article presents another unique case of ccoc with varied clinicopathological , radiological , and histopathological findings affecting the symphyseal region of the mandible in a middle - aged patient .
a male patient aged 46 years reported to outpatient department with the chief complain of swelling in the lower front tooth region since 45 months .
intraorally , firm , nontendered , and exophytic lesion measuring 2.5 cm 4 cm was observed involving mandibular anterior jaw region with exfoliation of 31 , 32 and 41 , 42 .
orthopantomogram revealed the presence of an ill - defined radiolucent destructive lesion in the mandibular anterior region [ figure 2 ] .
computed tomography view showed well - defined hypodense area with the complete loss of buccal cortical plate and severe thinning of the lingual cortical plate in mandibular symphysis region [ figure 3 ] .
intraoral view showing lesion in lower alveolar ridge orthopantomogram and computed tomography scan showing bone destruction in the symphysis region h and e stained section showing ( a ) islands of clear cells in fibrous connective tissue stroma ( 40 ) , ( b ) clear cells mixed with columnar cells with eosinophilic cytoplasm ( 100 ) keeping the clinical and radiological features in view , the provisional diagnosis of squamous cell carcinoma was made .
the incisional biopsy was performed after obtaining informed consent from the patient , and the tissue sections were stained with hematoxylin and eosin ( h / e ) for microscopic evaluation .
h / e staining of tissue sections revealed the presence of islands and sheets of round or polyhedral shaped hyperchromatic clear cells with the distinct cytoplasmic membrane .
the tumor cells adjacent to the fibrovascular septa were cuboidal to columnar with reversed polarity of the nucleus and eosinophilic cytoplasm whereas central cells were large , round to polygonal with clear cytoplasm .
mitotic figures were insignificant without any fibrous capsule at the periphery of the tumor [ figure 4 ] .
photomicrograph showing ( a ) congo red stain , ( b ) mucicarmine stain and ( c ) periodic acid - schiff stain .
congo red and mucicarmine are negative while periodic acid - schiff stain has taken up by cytoplasmic granules special stains such as mucicarmine , congo red , and pas were done to check the content of clear cells for mucin , amyloid , and glycogen deposition , respectively .
mucicarmine and congo red were negative while pas was strongly positive suggesting the glycogen content of clear cells [ figure 5 ] .
ck-19 and ema showed strong immunoreation to the tumor cells whereas s-100 protein and vimentin were nonreactive .
photomicrograph showing ( a ) cytokeratin , ( b ) epithelial membrane antigen , ( c ) vimentin , ( d ) s-100 .
the tumor shows strong positivity for keratin ( cytokeratin ) and epithelial islands ( epithelial membrane antigen ) .
the vimentin immunoreactivity is positive for fibrous tissue stroma only while s-100 was negative the histopathological features confirmed the odontogenic origin of tumor and diagnosis of ccoc of the biphasic pattern was made .
further , the patient was referred to oral surgery department for surgical excision of the lesion .
the postoperative recovery was uneventful , and no recurrence was observed in 3 years follow - up period .
initially , it was considered to be a benign neoplasm , but later due to its aggressive nature , spread , and recurrence , it is redesignated by the who as ccoc in 2005 .
clear cells appear to be clear due to the presence of an intracellular accumulation of nonstaining compounds , such as glycogen , mucopolysaccharides , lipids , and mucin .
the derivatives of the dental lamina or cell rests of malassez have been suggested to be the origin of clear cells in jaws .
it is believed that during late bell stage , cells of inner enamel epithelium undergo histodifferentiation to presecretory ameloblast . electron microscope shows the presence of lysosomes , mitochondria , tonofilaments , and desmosomes .
it is now known that bone morphogenetic protein ( bmp-2 ) plays a crucial role in regulating ms 1 and ms 2 along with dl 2 .
the combined effect of ms 1 , ms 2 , and dl 2 influences dl 3 , which normally help ameloblast cell to differentiate .
absence of these two homobox genes may be due to dysregulation of bmp-2 expression affecting the terminal differentiation of ameloblast cell .
in addition to this , the genomic analysis also revealed a polypoid population of cells with dna index of 1.93 and overall s - phase of 10.2% suggesting chromosomal alterations in cases of ccoc .
a wide age range from 14 to 89 years has been described with the peak incidence noted in the sixth decade of life .
females out numbers males with the ratio of 2:1 . more than 80% of the lesions developed in the anterior portion of the mandible .
the conventional radiological findings usually show poorly delineated , unilocular or multilocular radiolucent lesion that occurs with prominent bone destruction .
the light microscopic features of ccoc reveal three histological patterns : biphasic , monophasic , and ameloblastomatous .
the most common biphasic pattern shows two sets of cellular population arranged in sheets and islands .
center cells are clear , round to polygonal in shape mixed with another population of cuboidal to columnar cells with eosinophilic cytoplasm at the periphery .
the ameloblastomatous pattern is least common , characterized by the presence of clear cells inside the follicular network .
it has a higher tendency for the invasion to the medullary bone , muscle , and the neural tissue .
differential diagnoses include calcifying epithelial odontogenic tumor , mucoepidermoid carcinoma , myoepithelial carcinoma , hyalinising clear cell carcinoma , epithelial - myoepithelial carcinoma , amelanotic melanoma , and metastatic renal cell carcinoma [ table 1 ] .
differential diagnosis of ccoc we could demonstrate intracellular glycogen by means of pas - positive , diastase - sensitive granule accumulation within the tumor cells whereas mucin and amyloid were excluded by negative mucicarmine and congo red staining .
ccoc is differentiated from calcifying epithelial odontogenic tumor due to the absence of calcifications and amyloid deposition ( congo red negative ) .
lack of intermediate cells , squamous differentiation , and mucin ( mucicarmine negative ) production excluded mucoepidermoid carcinoma .
metastatic lesion is differentiated from ccoc microscopically as later lacks the prominent sinusoidal vascularity and intramural hemorrhage that characterize metastatic renal carcinoma .
immunohistochemically , the tumor cells showed positive staining for wide spectrum ck and ema , but negative staining for vimentin and s-100 protein .
the histochemical staining profile clearly spells the odontogenic nature of neoplasm excluding the possibility of salivary gland origin .
the treatment should aim at wide surgical resection with tumor - free margins adjunct to radiotherapy .
a long - term follow - up is necessary to look for any loco - regional recurrence and distant metastasis .
conspicuous nature of clear cells in ccoc poses a great challenge to a pathologist to diagnose and differentiate from other clear cell lesions .
these type of lesions demands diagnosis based on conventional histopathology adjuvant to special staining and immunohistochemistry for optimal management considering the risk of recurrence and metastases .
| clear cell odontogenic carcinoma is a rare , aggressive neoplasm of the jaw with only 74 reported cases .
it occurs predominantly in the mandibular anterior region during fifth to seventh decades of life .
clinically it manifests as intra - bony swelling with a variable degree of pain .
microscopically , it reveals nests of cells with clear cytoplasm in connective tissue stroma arranged in different patterns .
it is often misdiagnosed due to the rarity of lesion and confusing histopathology .
immunohistochemical staining plays an intricate role to uncertain the native of the clear cell to reach a confirmative diagnosis .
the article aims to highlight the clinicopathologic features of clear cell odontogenic carcinoma in a middle - aged man with special emphasis on its differential diagnosis . |
whole blood viscosity ( wbv ) is one of virchow 's triad , which is an established concept of three phenomena including stasis , endothelial dysfunction and atherothrombosis that ultimately lead to , and/or result from cardiovascular complications[14 ] .
each phenomenon represents a subclinical vascular process , which in turn is indicated by a clinical pathology index .
specifically , wbv is an intrinsic resistance of blood flow in the vascular system , and index for stasis .
increase in wbv is a potential risk factor for future cardiovascular disease . in current clinical practice
considering the implication of stasis in metabolic diseases , its clinical management with antiplatelet and the controversies regarding antiplatelet and its bleeding complications[79 ] , such usage is under - utility .
international normalized ratio ( inr ) is used to determine the clotting tendency of blood and the need for anticoagulant ( especially warfarin ) therapy .
normal range is about 1.0 for a healthy person , and 2.0 - 3.5 for people who are on anticoagulant therapy .
a higher inr level indicates a high tendency of bleeding complications , whereas lower inr level is indication of high risk of having a thrombotic event in patients who are on anticoagulant therapy .
thus , while anticoagulant and antiplatelet are used interchangeably , there is an established laboratory index ( inr ) being employed for routine monitoring to assess contraindication to and/or need for anticoagulant ; but none for antiplatelet .
the objective of this work is to establish whether difference in wbv level is associated with different levels of inr and platelet count .
it would be expected , and thus hypothesized that a patient with low inr requiring anticoagulant therapy should present with higher wbv .
the findings from this study would provide evidence to suggest wbv as a potential laboratory index to assess for contraindication to antiplatelet therapy synonymous to how inr is being used for anticoagulants .
it is supported materially by the albury south west pathology a unit of western pathology cluster of nsw health australia .
one year of de - identified archived clinical pathology data for the period of january to december 2008 was audited .
7387 cases ( female - male ratio = 3528 - 3859 ) for international normalizing ratio , which were concomitantly tested from one phlebotomy collection point for full blood count ( fbc ) haematocrit and total proteins , were extracted .
haematocrit results from the fbc were used in conjunction with total protein to extrapolate wbv values based on previously published algorithm . whether whole blood viscosity differs between normal vs. high international normalizing ratio and thrombocytopenia vs. thrombocytosis were evaluated .
inr level was commonly determined arithmetically from the result of prothrombin time using the formula inr = ptpatient / ptnormal inr data for this study were determined with this formula included in a standard operational procedure ( sop ) .
the sop included measurement of prothrombin time ( pt ) by quantitative analysis using the sysmex ca540 method and inr calibration curve , which is plotted for every new lot of pt reagent .
results of inr were reported with the following interpretative consideration to risk of bleeding
normal patients no therapy = 1.0anitcoagulated patient = 2.0 3.5 including
prophylaxis for prosthetic heart valves = 2.5 3.5all other indications = 2.0 3.0
critical results for anticoagulated patients = < 1.5 or > 4.5
normal patients no therapy = 1.0 anitcoagulated patient = 2.0 3.5 including
prophylaxis for prosthetic heart valves = 2.5 3.5all other indications = 2.0 3.0
prophylaxis for prosthetic heart valves = 2.5 3.5 all other indications = 2.0 3.0 critical results for anticoagulated patients = < 1.5 or > 4.5 in this study , numerical levels of inr results have been used as reported . it was assumed on the premise of the hypothesis that a pathology that impacts on inr should also impact on platelet count and wbv .
the data were sorted by wbv results and categorized into three groups of low , normal or high wbv levels based on the continuum of 15.00 , 15.01 - 19.01 and 19.02 respectively .
high , normal and low wbv were named groups i , 2 and 3 respectively . in the statistical analysis , it was considered to avoid errors due to unequal sample size such as exaggerating the effects of inequality of variance . the high wbv ( group 1 ) had the least number ( n = 173 ) and was used as the base sample size for the sub - groups .
the discretional criterion for selection of [ n = 173 ] from the other two groups was to make the comparison be between the lowest - median - highest intervals in ranking ( fig .
1 ) . therefore , the lowest [ n = 173 ] in the low wbv rank and the median [ n = 173 ] in the wbv groups were selected . to determine association of high , normal or low wbv with inr and platelet count , multivariable analysis of variance ( manova )
indication of selections from the ranks of wbv - groups . 1 - 173 , 2102 - 2274 , 7215 - 7387 , 173 , g1 , g2 & g3 : hyperviscosity , normoviscosity and hypoviscosity groups respectively , r : ranking , s : selection for statistical analysis .
the complete data set were further re - analyzed thrice to observe for consistency in result .
first , data were sorted by , and selection of ( n = 173 ) from groups 2 and 3 were repeated to match for age and gender .
second , data were sorted by inr . the top ( n = 120 ) vs. the bottom ( n = 120 ) were selected to represent high vs. low inr sub - groups respectively and wbv level was compared between the sub - groups .
the top ( n = 120 ) vs. the bottom ( n = 120 ) were selected to represent thrombocytosis vs. thrombocytopenia sub - groups respectively and wbv level was compared between the sub - groups .
the statistics of the groups for the complete data set are presented in table 1 .
the result shows that platelet count increases with whole blood viscosity or vise versa , whereas low viscosity is associated with high inr ( table 1).manova show that levels of international normalizing ratio and platelet counts statistically significantly differs between different ranges of whole blood viscosity levels ( p<0.001 ) .
platelet count is statistically significantly lower in low whole blood viscosity compared to hyperviscosity and normoviscosity ( p<0.001 ) .
conversely , international normalized ratio is statistically significantly higher in low whole blood viscosity compared to hyperviscosity ( p<0.001 ) and normoviscosity ( p<0.002 ) .
no difference was observed between hyperviscosity and normoviscosity in platelet count or international normalized ratio ( fig .
2 ) . comparison of inr & plt between wbv - groups . 1 - 3 : hyperviscosity , normoviscosity and hypoviscosity groups respectively , inr : international normalized ratio , plt : platelet count x10/l , wbv : whole blood viscosity . in the repeat analyses for observation of consistency ,
manova on the age and gender matched data set also showed statistical significance ( p<0.005 ) .
wbv was observed to statistically significantly differ between high vs. low inr ( p<0.001 ) , as well as between thrombocytopenia vs. thrombocytosis ( p<0.005 ) ( fig .
inr : international normalized ratio , plt : platelet count x10/l , wbv : whole blood viscosity .
this study reports observation of significantly higher level of inr associated with lower level of wbv .
this observation is in consonance with the study 's hypothesis and has relevant applicability in clinical practice . on one hand
, it affirms that the use of anticoagulant therapy is in tandem with relative high wbv level .
more importantly on the other hand , low wbv being associated with relative high inr , whereby the latter indicates risk of bleeding , suggests that blood viscosity can be utilized as a laboratory index of contraindication / indication to anticoagulant and/or antiplatelet therapies .
the issue is that inr is mainly used to assess the status of extrinsic ( clotting ) pathway in order to determine indication / contraindication for anticoagulant therapy .
neither is wbv assessed on the chronic patients , nor is the risk of bleeding complication of less concern .
therefore , if the latter is assessed to determine possible risk of bleeding complication anticoagulant , chronic disease patients who are not qualified for inr assessment , but who are to be treated with antiplatelet would benefit from assessment of wbv .
a recommended guideline is not to prescribe antiplatelet if contraindicated[1720 ] . yet , lack of laboratory assessment of compliance is an acknowledged major issue .
the report provides insight to assessment of hypoviscosity as part of antiplatelet drug monitoring and in compliance to guidelines and good evidence - based clinical practice .
further , this study reports observation of significantly higher and lower level of platelet count associated with high and low wbv respectively .
it is known that antiplatelet therapy is employed in the management of hyperviscosity / stasis through modulation of platelet hyperreactivity .
therefore , it is not out of place that relative high platelet count is associated with high blood viscosity .
this logic is not in line with the fact that several studies have reported an association between platelet hyperreactivity after antiplatelet therapy .
nevertheless , platelet function can be measured by different methods and platelet counting has been in use , except that the usage to monitor antiplatelet drugs is not common .
factors that increase wbv include haematocrit , total plasma protein , erythrocyte aggregation , erythrocyte deformability and oxidative stress . in current clinical practice ,
a low platelet count is a factor that determines temporary stopping chemotherapy , but not antiplatelet therapy .
this observation of thrombocytopenia associated with low blood viscosity provides evidence to suggest that low platelet count is also occasion to consider stopping any antiplatelet therapy .
a review has reported gender - specific differences in platelet function and response to antiplatelet therapy .
the report further acknowledged role for laboratory monitoring of antiplatelet medications in predicting individual responsiveness .
the results from this study show that age and gender differences may not impact on the association between wbv and inr or platelet count . in corroboration with previous observation of infrequent prevalence of hyperviscosity in thromboembolic state ( whole blood viscosity issues
ii on this series ) , it surmises that platelet count should necessarily be used as adjunct laboratory index to determine indication or contraindication for antiplatelet therapy .
it is known that thrombocytopenia is associated with bleeding complications[2628 ] . what this article contributes is that inr
, platelet count and wbv are laboratory indices to consider in constituting antiplatelet monitoring panel .
the observations also corroborate with previous report to suggest putting into perspective the specificity of wbv relative to stasis .
in addition , the result indicates that wbv identify individuals in whom anticoagulant and antiplatelet therapies may be contraindicated that is , from the association and clinical utility of inr .
perhaps , one question is : will hypoviscosity be associated with gastrointestinal bleeding to prove evidence of being a possible contraindication index ?
this report presents that higher level of inr is associated with lower level of wbv and vise versa , whereas thrombocytopenia is associated with hypoviscosity .
the results indicate that wbv identifies individuals in whom anticoagulant and antiplatelet therapies are indicated or contraindicated .
the findings suggest that inr , platelet count and wbv are laboratory indices to consider in constituting antiplatelet monitoring panel . | background : anticoagulant and antiplatelet therapies are being used interchangeably or in combination .
while international normalized ratio is assessed to determine anticoagulant 's contraindication / need , whole blood viscosity is not assessed to determine the need for antiplatelet.aims:the objective of this study is to investigate whether whole blood viscosity value is associated with levels of international normalized ratio and platelet count.materials and methods : de - identified archived clinical pathology data for the year 2008 were audited .
all cases of international normalized ratio , which were concomitantly tested for haematocrit and total proteins , were extracted ( n=7,387 ) .
whole blood viscosity levels were extrapolated . whether differences are associated with normal vs. high international normalized ratio and thrombocytopenia vs. thrombocytosis were evaluated.results:multivariate analysis show
that whole blood viscosity levels statistically significantly differs between international normalized ratio and platelet counts ( p<0.001 ) .
platelet count is statistically significantly lower in low blood viscosity when compared with hyperviscosity and normoviscosity ( p<0.001 ) .
conversely , international normalized ratio is statistically significantly higher in low blood viscosity relative to hyperviscosity ( p<0.001 ) and normoviscosity ( p<0.002 ) .
no difference was observed between hyperviscosity and normoviscosity in platelet count or international normalized ratio.conclusion:the observation corroborates with previous reports to suggest putting into perspective the specificity of whole blood viscosity relative to stasis , against which antiplatelet is employed .
it indicates that low whole blood viscosity is synonymous to high international normalized ratio whereby anticoagulant and antiplatelet therapies are contraindicated .
international normalized ratio , platelet count and blood viscosity are laboratory indices to consider in constituting antiplatelet monitoring panel . |
the most frequent tumors and simulating lesions in ocular adnexa are lymphoproliferative disorders ( lpds ) , including malignant lymphomas and orbital inflammation with lymphoid hyperplasia or infiltration , some of which are historically called orbital pseudotumors . in japanese studies , goto reported that the rate of lpd among 409 cases with orbital tumors and simulating lesions was 43% , while ohtsuka et al .
described that the rate was 49% out of 213 cases . in the united states , lpd is a common orbital disease although the rate seems to be somewhat lower : 24% of lpd out of 703 cases with orbital lesions was reported by shields et al . , and 26% of lpd out of 268 cases was reported by shinder et al . . when patients with suspected orbital lpd are encountered , tissue biopsy is preferred since image examinations alone can not distinguish inflammatory lesions from malignant lymphomas .
igg4-related disease ( igg4-rd ) often involves lacrimal glands , which is now known as igg4-related mikulicz 's disease [ 5 , 6 ] or igg4-related dacryoadenitis [ 7 , 8 ] by many recent reports over several years .
recently , it was also elucidated that igg4-related orbital inflammatory lesions include other ocular adnexal tissues such as extraocular muscles and periorbital membrane .
therefore , igg4-rd is a differential diagnosis in orbital lpd . the question is then raised as to what percentage of orbital lpd is related to igg4 . in this study ,
an orbital lpd case series was investigated and clinical aspects of igg4-related orbital lymphoproliferative disorders were evaluated .
in kanazawa university hospital in japan , a 47-year - old woman with igg4-immunopositive histopathology and an elevated serum igg4 level of 1000 mg / dl was diagnosed as a first case of igg4-related dacryoadenitis in november , 2004 . from that time through march , 2011 , sixty - two cases ( 27 men and 35 women ; mean age , 66 14 yrs ; range 3289 yrs ) were pathologically diagnosed with orbital lymphoproliferative diseases ( lpd ) from surgical samples of ocular adnexal tissue .
the two main categories of orbital lpd were malignant lymphomas and orbital inflammations : the latter included reactive lymphoid hyperplasia , lymphoid infiltrated lesions , and inflammatory pseudotumor .
conjunctival lesions were not enrolled in this study because conjunctival involvement in igg4-rd has never been experienced in previous reports [ 9 , 11 ] or in the author 's institution .
intraocular lymphoma belongs to cns lymphoma and thus was also excluded in this study . in most cases , immunoglobulin heavy chain gene rearrangement in surgical samples was also examined to support the differential diagnosis of malignant lymphoma .
diagnostic criterion for positive igg4-immunostaining in orbital tissue ( igg4-related orbital disease ) was either ( 1 ) the ratio of igg4-positive cells to igg - positive cells ( igg4+/igg+ cells ) was more than 40% , or ( 2 ) the number of igg4-positive cells was more than 30 per high power microscopy field .
mouse monoclonal antibody anti - human igg4 ( 05 - 3800 , zymed , usa ) and rabbit polyclonal antibody anti - human igg ( a0423 , dako , usa ) were used for immunostaining .
serum igg and igg4 were measured in all of the cases with an igg4-positive pathological diagnosis .
igg4-related orbital lesions including lacrimal gland swelling , extraocular muscle enlargement , and other mass lesions were evaluated using computed tomography ( ct ) and/or magnetic resonance imaging ( mri ) .
histological types of the 62 orbital lpd were 22 cases ( 35% ) with extranodal marginal zone lymphoma of mucosa - associated lymphoid - tissue type ( malt lymphoma ) , 11 cases ( 18% ) with diffuse large b - cell lymphoma ( dlbcl ) , 3 cases ( 5% ) with other malignant lymphomas ( 1 mantle cell lymphoma , 1 nk / t cell lymphoma , 1 small lymphocytic lymphoma ) , 16 cases ( 26% ) with igg4-related orbital inflammation ( igg4-roi ) , and 10 cases ( 16% ) with non - igg4-related orbital inflammation ( non - igg4-roi ) ( figure 1 ) .
none of the malignant lymphomas showed a relationship with igg4 in this case series .
figure 2 depicts the ages of the four groups of dlbcl , malt lymphoma , igg4-roi , and non - igg4-roi .
ages of the igg4-roi group averaged 56 10 yrs , which was significantly lower than those of the malt lymphoma ( 71 12 yrs , p = 0.00027 in a student 's t - test ) and dlbcl ( 75 14 yrs , p = 0.00107 ) groups .
clinical data of sixteen cases of igg4-roi are summarized in table 1 , which were sorted in ascending order of serum igg4 level .
the age ranged between 41 to 76 yrs ( mean sd ; 56 10 yrs , median ; 58 yrs ) , and there was no sex difference ( 8 men and 8 women ) . these cases except one ( number 1 , detailed in the discussion ) were accompanied by elevated serum igg4 concentration ( 549 293 mg / dl , n = 15 , normal range < 135 mg / dl ) and an increased ratio of serum igg4/igg ( 28 12% , n = 15 , normal range < 7% ) . in 9 patients ,
these were characterized by lymphoplasmacytic infiltration , forming lymphoid follicles ( germinal center ) and sclerosing fibrosis .
orbital lesions other than lacrimal glands were detected by mri in six cases ( cases numbers 4 , 7 , 8 , 11 , 14 , and 16 in table 1 ) as shown representatively in figure 3 .
swollen extraocular muscles were seen in four cases ( numbers 4 , 7 , 8 and 14 ) .
supraorbital nerve ( frontal nerve ) , and/or infraorbital nerve enlargements were observed in 4 cases ( numbers 7 , 8 , 11 , and 14 ) .
a mass lesion surrounding the optic nerve was detected in two patients ( numbers 8 and 11 ) .
steroid therapy was performed for all the igg4-roi cases except three patients because their eyelid swellings decreased in size after biopsy surgery ( case numbers 1 , 2 , and 5 ) and because of normal serum igg4 ( case number 1 ) and diabetes mellitus ( case number 2 ) .
eleven cases underwent oral prednisolone tapering therapy with an initial dose of 20 , 30 , or 40 mg per day . in two cases with diabetes
mellitus , lower doses of oral prednisolone ( 10 mg daily in case number 10 and 8 mg daily in case number 15 ) were administered initially and reduced to a maintenance dose .
all of the 11 cases essentially responded well to initial doses of oral prednisolone , but in 5 cases ( numbers 4 , 8 , 10 , 11 , and 14 ) ocular symptoms such as eyelid swelling deteriorated during tapering , and thus increase in the dosage was required . in case number 11 with retrobulbar mass ( figure 3(c ) ) , oral prednisolone therapy alone ( 30 mg daily initially ) failed to diminish his exophthalmos , and then intravenous methylprednisolone pulse ( 500 mg for 3 days ) was additionally performed three times .
however , some previous reports suggested that orbital malignant lymphomas can be related to igg4 .
cheuk reported three cases of ocular adnexal lymphoma ( 2 malt lymphoma and 1 follicular lymphoma ) arising in igg4-related dacryoadenitis , and that the rate of transformation of malignant lymphoma in the background of igg4-roi was approximately 10% .
first detected igh gene rearrangement in two cases of ocular adnexal igg4-related disease and later reported seven patients with ocular adnexal malt lymphoma arising from igg4-related orbital disease .
on the other hand , he described a case of igg4-producing malt lymphoma of the lymph node .
also in the orbital region , oyama reported a case of igg4-expressing malt lymphoma in the lacrimal gland .
based on these findings , there could be two possibilities of orbital malt lymphoma arising from preexisting igg4-roi and de novo igg4-positive orbital malt lymphoma . in any case
, orbital biopsy should be mandatory to evaluate malignancy before treatment even if serological examination already detected elevated serum igg4 .
there is no doubt that igg4-roi most frequently involves the lacrimal gland , which is reported as igg4-related dacryoadenitis and mikulicz 's disease [ 58 ] .
sato et al . reported that orbital masses other than the lacrimal gland were detected in 7 out of 21 cases with igg4-roi .
mehta et al . presented a case of igg4-roi with enlargement of the infraorbital canal and periorbital membrane involvement .
similar lesions were observed in the present case series of igg4-roi ( figure 3 ) .
in addition , mass lesions around the optic nerve were seen in two cases , indicating that involvement of the optic nerve sheath may not be rare in igg4-roi . among 16 cases of igg4-roi , one patient ( case number 1 ) presented normal levels of serum igg4 . in this case , the ratio of igg4+/igg+ cells in immunohistochemistry was around 30% , somewhat lower than values in diagnostic criteria [ 6 , 12 ] , but igg4-positive cells were more than 30 per high power microscopy field .
the pathological findings that lymphoplasmacytic infiltration with germinal centers and dense sclerosing fibrosis was characteristic for igg4-roi .
so far , we can not resolve the reason for this discrepancy between pathological and serological findings .
however , kubota previously reported a patient with an inverse discrepancy who had negative findings on the igg4 immunostaining despite presumably typical igg4-roi with elevated serum igg4 .
the rate of igg4-rd in 62 cases with orbital lpd was 26% in this study and was reported to be 33% out of 58 orbital lpd cases from another japanese institute .
thus , it is likely that nearly a quarter of orbital lymphoproliferative disorders are estimated to be related to igg4 .
further multicenter studies will be required to confirm this rate and to evaluate the frequency and location of igg4-roi other than the lacrimal glands . | the most frequent ocular adnexal tumors and simulating lesions are lymphoproliferative disorders ( lpds ) , including malignant lymphomas and orbital inflammation with lymphoid hyperplasia or infiltration .
igg4-related orbital inflammation ( igg4-roi ) often involves lacrimal glands and other orbital tissues and is an important differential diagnosis .
the present study evaluated clinical aspects of igg4-roi in a case series of orbital lpd .
sixty - two consecutive cases of orbital lpd , pathologically diagnosed from november , 2004 , through march , 2011 , were investigated .
histological types were 22 cases with malt lymphoma , 11 cases with diffuse large b - cell lymphoma ( dlbcl ) , 3 cases with other malignant lymphomas , 16 cases with igg4-roi , and 10 cases with non - igg4-roi .
ages of the igg4-roi group ( 56 10 yrs ) were significantly lower than the malt lymphoma ( 71 12 yrs ) and dlbcl ( 75 14 yrs ) groups .
orbital lesions other than lacrimal glands were present in six cases including extraocular muscle swelling , mass lesions surrounding the optic nerve , and supraorbital and infraorbital nerves enlargements .
although none of the malignant lymphomas were related to igg4 , previous evidence suggested that malignant lymphomas can arise from igg4-roi .
based on this study ( 26% ) and another report ( 33% ) , it is likely that nearly a quarter of orbital lpd are igg4-roi . |
bone metastases affect up to two - thirds of patients with advanced solid tumours such as breast , prostate or lung cancer , and osteolytic bone lesions are typical of multiple myeloma .
individuals with bone metastases or lesions are at a high risk of experiencing skeletal - related events ( sres ) , including pathologic fractures ( pfs ) , spinal cord compression and radiation or surgery to bone , , , .
pfs have commonly been reported in the placebo arms of clinical trials that evaluated the effect of bisphosphonates in patients with bone metastases secondary to advanced cancers , , and have been shown to be detrimental to patients quality of life .
statistically significant declines in the physical and emotional well - being of patients have been reported after experiencing pfs .
patients with pfs often require substantial orthopaedic treatment , including rehabilitation and supportive care , such as pain relief , and therefore utilise considerable healthcare resources .
furthermore , the occurrence of pfs has been correlated with reduced survival rates in patients with bone metastases .
several studies worldwide have revealed that hru and costs associated with sres , including pfs , in patients with bone metastases or lesions are substantial , , , .
however , these studies have focused either on single countries or on small numbers of european countries .
increasing healthcare providers knowledge of the hru associated with pfs in patients with bone metastases or lesions in europe would highlight the potential economic value of using treatments that prevent or delay sres .
such treatments may also reduce hru and maintain patients quality of life . this study aimed to estimate the hru associated with pfs in patients with bone metastases secondary to solid tumours or lesions
this was a multinational , retrospective study to assess hospital - related hru associated with pfs in patients with bone metastases or lesions from austria , the czech republic , finland , greece , poland , portugal , sweden and switzerland .
patients eligible to participate in the study were aged 20 years or older , had bone metastases secondary to breast , lung or prostate cancer , or bone lesions due to multiple myeloma .
patients also had to have experienced an index pf ( defined as a pf preceded by a sre - free period of at least 6.5 months ) during the study period ( from 1 july 2004 to 1 july 2009 ) to be included .
exclusion criteria included current enrolment or previous participation in a denosumab clinical trial , death less than 2 weeks after the index pf and patient chart data of insufficient quality .
relevant patient charts were identified at each site from electronic or paper patient lists using the international classification of diseases ( icd ) ninth revision ( icd-9 ) and tenth revision ( icd-10 ) codes .
data from consecutive patient charts for those fulfilling the inclusion criteria and not meeting the exclusion criteria were captured during the study period . the patient chart with
the most recent pf was analysed first ( even if this is not the most recent index sre ) , then the second most recent was analysed ( i.e. systematically in reverse consecutive order ) until the planned number of pfs were documented on a country level . according to european legislation for this type of retrospective research , informed consent is generally not required .
pfs were grouped into those affecting long bones ( i.e. bones that are longer than they are wide , such as the femur ) or those involving other bones .
long bone fractures are usually major clinical events , whereas fractures of some other bones may be asymptomatic and may be discovered only by routine bone scanning . in this study ,
all pfs were symptomatic but the hru for these fracture types may differ . based on epidemiology and feasibility studies , the target number was 10 patients with at least one pf of a long bone and 30 patients with at least one pf of other bones for each participating country .
retrospective hru data were collected from patient charts during the study period . for patients who experienced only the index pf during the study period , data were extracted from hospital charts for a period beginning 3.5 months before and ending 3 months after the index pf ( fig .
in order to attribute hru in patients with multiple sres , it was necessary to set a diagnostic window . in line with a previous study , a period of 3 months starting 3.5 months before the index pf was used to establish baseline hru , and a 14-day ( 2 week ) period immediately before the index pf was used to estimate any diagnostic hru . for patients with multiple sres ,
the data - extraction period was extended until 3 months after the last sre that the patient experienced during the study period ( fig .
there was no limit to the number of sres included in the period following the index pf . to ensure lack of carry - over of hru from a previous sre that occurred before the 3.5-month period immediately preceding the index pf , a clean window of an additional 3 months without a sre
if multiple sres were present at the same anatomical site and occurred within a 21-day window , they were considered to be linked and the total hru was attributed to the index pf . in cases in which sres occurred at the same anatomical site but outside the 21-day window , or at different anatomical sites , the sres were considered to be unlinked and hru was attributed to the appropriate sre type following chart review by an expert panel .
primary hru outcome measures were : the number and duration of inpatient stays ( overall and by type of hospital unit ) ; the number of outpatient visits ( overall and by healthcare provider type ) ; the number of day - care hospital visits ( visits to day - care centres were made by patients who required more prolonged treatment or investigations than outpatients , but who did not require an overnight stay ) ; the number of emergency room visits ; and the number and types of procedures provided .
the proportion of patients receiving bisphosphonate medications at baseline and post - sre ( and the dose frequency ) was recorded . to estimate hru associated with an index pf ,
statistical analyses were descriptive in nature ; data are presented as mean ( standard deviation [ sd ] ) , because this better describes the total hru for the study population .
pfs were grouped into those affecting long bones ( i.e. bones that are longer than they are wide , such as the femur ) or those involving other bones .
long bone fractures are usually major clinical events , whereas fractures of some other bones may be asymptomatic and may be discovered only by routine bone scanning . in this study ,
all pfs were symptomatic but the hru for these fracture types may differ . based on epidemiology and feasibility studies , the target number was 10 patients with at least one pf of a long bone and 30 patients with at least one pf of other bones for each participating country .
retrospective hru data were collected from patient charts during the study period . for patients who experienced only the index pf during the study period , data were extracted from hospital charts for a period beginning 3.5 months before and ending 3 months after the index pf ( fig .
in order to attribute hru in patients with multiple sres , it was necessary to set a diagnostic window . in line with a previous study , a period of 3 months starting 3.5 months before the index pf was used to establish baseline hru , and a 14-day ( 2 week ) period immediately before the index pf was used to estimate any diagnostic hru . for patients with multiple sres ,
the data - extraction period was extended until 3 months after the last sre that the patient experienced during the study period ( fig .
there was no limit to the number of sres included in the period following the index pf . to ensure lack of carry - over of hru from a previous sre that occurred before the 3.5-month period immediately preceding the index pf , a clean window of an additional 3 months without a sre was required .
if multiple sres were present at the same anatomical site and occurred within a 21-day window , they were considered to be linked and the total hru was attributed to the index pf . in cases in which sres occurred at the same anatomical site but outside the 21-day window , or at different anatomical sites , the sres were considered to be unlinked and
hru was attributed to the appropriate sre type following chart review by an expert panel .
primary hru outcome measures were : the number and duration of inpatient stays ( overall and by type of hospital unit ) ; the number of outpatient visits ( overall and by healthcare provider type ) ; the number of day - care hospital visits ( visits to day - care centres were made by patients who required more prolonged treatment or investigations than outpatients , but who did not require an overnight stay ) ; the number of emergency room visits ; and the number and types of procedures provided .
the proportion of patients receiving bisphosphonate medications at baseline and post - sre ( and the dose frequency ) was recorded .
to estimate hru associated with an index pf , the following calculation was used.estimateofhruassociatedwithpf=hrurecordedduringpost-pfperiod+hruduringdiagnosisperiodhrurecordedduringthebaselineperiodaadjusted to allow for the different lengths of the periods .
statistical analyses were descriptive in nature ; data are presented as mean ( standard deviation [ sd ] ) , because this better describes the total hru for the study population .
in total , 118 patients with long bone pfs and 241 patients with pfs of other bones were included .
the baseline demographics of participants were generally consistent across all countries ; however , the mean age of patients with pf of long bones was higher in finland ( 74.5 [ sd 4.2 ] years ; n=8 ) and sweden ( 75.8 [ sd 7.4 ] years ; n=9 ) compared with the other countries ( range 61.568.9 years ) ( table 1 ) .
overall , the most common cancer types were breast cancer ( long bones 37.3% ; other bones 32.4% ) and multiple myeloma ( long bones 23.7% ; other bones 40.7% ) ( table 1 , table 2 ) .
the mean time since initial diagnosis of bone metastases or lesions was approximately 1 year in both patients with long bone pfs and those with pfs of other bones .
the mean duration of follow - up was similar for patients with long bone pfs ( 3.2 months ; sd 1.2 months ) and other bone pfs ( 3.3 months ; sd 1.2 months ) .
overall , the most common fractures affecting long bones were those of the femur ( 58.5% ) and humerus ( 32.2% ) ( table 3 ) .
fractures of the vertebrae were the most common fracture type in those with pfs affecting other bones ( 66.7% overall ; thoracic vertebrae 40.2% ; lumbar vertebrae 23.2% ; cervical vertebrae 3.3% ) ( table 4 ) .
retrospective review of those patients who had a long bone or other bone pf , 28.0% ( n=33 ) and 25.3% ( n=61 ) , respectively were receiving a bisphosphonate at baseline .
after a sre , the number of patients receiving a bisphosphonate was 61.9% ( n=73 ) in those patients with a long bone pf and 69.3% ( n=167 ) in those patients with a pf of other bones .
the most commonly used bisphosphonate was zoledronic acid ( table 5 , table 6 ) . in the long bone pf group , more patients had multiple sres than had a single sre ( i.e. only the index pf ) ( 79.7% [ n=94 ] and 20.3% [ n=24 ] , respectively ; table 1 ) .
however , in patients with pfs of other bones , the proportions with multiple or single sres were similar ( 51.0% [ n=123 ] and 49.0% [ n=118 ] , respectively ; table 2 ) .
sres could be classified as being linked or unlinked ; subsequent linked sres were more frequent in patients with long bone pfs ( 73.4% of multiple sres ; n=69 ) than in those with other bone pfs ( 47.2% ; n=58 ) .
the most common linked sres were surgery to bone ( long bones 42.4% [ n=50 ] ; other bones 6.6% [ n=16 ] ) and radiation to bone ( long bones 18.6% [ n=22 ] ; other bones 17.4% [ n=42 ] ) .
the mean number of inpatient stays per pf increased from baseline for both pf of long bones ( 1.2 [ sd 1.2 ] ) and other bones ( 0.8 [ sd 1.2 ] ( fig .
the mean duration of inpatient stays also increased in both groups ( long bones 20.9 [ sd 22.1 ] days ; other bones 12.3 [ sd 19.5 ] days ) ( fig .
the greatest increases in duration of stay per pf were reported in portugal ( 32.1 [ sd 19.8 ] days ; n=14 ) and finland ( 29.4 [ sd 34.6 ] days ; n=8 ) .
the smallest increase was reported in greece ( 5.8 [ sd 8.3 ] days ; n=8 ) ( fig .
. the types of hospital units that patients stayed in differed according to the type of fracture they experienced .
the largest mean change from baseline in the number of stays per pf of long bones occurred in orthopaedic units ( 0.5 [ sd 0.6 ] ) , but stays in oncology units and other units ( including trauma surgery , casualty units , cardiology units and nursing units ) also increased slightly ( 0.1 [ sd 0.3 ] ) and 0.1 [ sd 0.8 ] , respectively ) . for individuals with pfs of other bones , the largest mean increases from baseline in
the number of inpatient stays per pf were observed in internal medicine units ( 0.2 [ sd 0.6 ] ) and oncology units ( 0.2 [ sd 0.7 ] ) .
there was a larger increase in the number of visits per event for patients with pfs of other bones ( 4.0 [ sd 5.8 ] ) than for those with pfs of long bones ( 2.6 [ sd 4.7 ] ) ( fig .
3a ) . compared with the other countries in this study , finland recorded the largest increase in the mean number of outpatient visits per event ( long bones 8.6 [ sd 6.1 ] ; n=8 ; other bones 6.8 [ sd 6.1 ] ; n=30 ) .
the smallest changes were reported in greece ( 0.9 [ sd 3.3 ] ; n=8 ) and poland ( 0.9 [ sd 3.2 ] ; n=28 ) for those with pfs of long bones . for individuals with pfs of other bones ,
poland had the smallest change in the mean number of outpatient visits ( 0.8 [ sd 1.7 ] ; n=30 ) .
the largest changes in the mean number of outpatient visits per pf from baseline were in visits to radiation oncologists / radiotherapists ( long bones 1.0 [ sd 2.5 ] ; other bones 1.5 [ sd 3.8 ] ) and oncologists ( long bones 0.5 [ sd 1.9 ] ; other bones 0.8 [ sd 2.3 ] ) .
the increase in the number of visits to orthopaedic surgeons was greater in patients with pfs of long bones ( 0.3 [ sd 0.7 ] ) than in those with pfs of other bones ( 0.1 [ sd 0.3 ] ) .
the number of visits to radiologists also increased , and this change was greater in patients with pfs of other bones ( 0.6 [ sd 1.7 ] ) than in those with pfs of long bones ( 0.2 [ sd 1.1 ] ) .
the increase in the mean number of day - care visits was smaller for individuals with long bone pfs ( 0.8 [ sd 2.7 ] than for those with other bone pfs ( 1.4 [ sd 3.6 ] ) .
the mean number of emergency room visits increased from baseline per pf of long bones ( 0.3 [ sd 0.7 ] ) and other bones ( 0.2 [ sd 0.9 ] ) .
overall , the number of procedures provided per event increased in both pf groups ( long bones 6.1 [ sd 7.1 ] ; other bones 5.9 [ sd 6.6 ] ) ( fig .
finland reported the greatest increase in the mean number of procedures in patients with pfs of long bones ( 12.9 [ sd 9.2 ] ; n=8 ) . in individuals with pfs of other bones ,
the greatest increase was observed in austria ( 10.3 [ sd 7.5 ] ; n=22 ) . for both fracture groups ,
the smallest changes in the number of procedures were reported in poland ( long bones 4.1 [ sd 3.9 ] ; n=28 ; other bones 2.7 [ sd 2.7 ] ; n=30 ) .
the largest overall increase was seen in the use of external beam radiation ( long bones 2.1 [ sd 3.9 ] ; other bones 2.6 [ sd 4.9 ] ) . in patients with pfs of long bones , surgery to bone
other procedures increased in those with pfs of long bones ( 2.3 [ sd 3.9 ] ) ; however , these encompassed a wide range of diagnostic procedures , none of which showed a large change when considered alone . in patients with pfs of other bones , the number of
other procedures also increased from baseline ( 1.7 [ sd 3.3 ] ) , and use of computerised tomography also increased ( 0.6 [ sd 1.1 ] ) .
in total , 118 patients with long bone pfs and 241 patients with pfs of other bones were included .
the baseline demographics of participants were generally consistent across all countries ; however , the mean age of patients with pf of long bones was higher in finland ( 74.5 [ sd 4.2 ] years ; n=8 ) and sweden ( 75.8 [ sd 7.4 ] years ; n=9 ) compared with the other countries ( range 61.568.9 years ) ( table 1 ) .
overall , the most common cancer types were breast cancer ( long bones 37.3% ; other bones 32.4% ) and multiple myeloma ( long bones 23.7% ; other bones 40.7% ) ( table 1 , table 2 ) .
the mean time since initial diagnosis of bone metastases or lesions was approximately 1 year in both patients with long bone pfs and those with pfs of other bones .
the mean duration of follow - up was similar for patients with long bone pfs ( 3.2 months ; sd 1.2 months ) and other bone pfs ( 3.3 months ; sd 1.2 months ) .
overall , the most common fractures affecting long bones were those of the femur ( 58.5% ) and humerus ( 32.2% ) ( table 3 ) .
fractures of the vertebrae were the most common fracture type in those with pfs affecting other bones ( 66.7% overall ; thoracic vertebrae 40.2% ; lumbar vertebrae 23.2% ; cervical vertebrae 3.3% ) ( table 4 ) .
retrospective review of those patients who had a long bone or other bone pf , 28.0% ( n=33 ) and 25.3% ( n=61 ) , respectively were receiving a bisphosphonate at baseline .
after a sre , the number of patients receiving a bisphosphonate was 61.9% ( n=73 ) in those patients with a long bone pf and 69.3% ( n=167 ) in those patients with a pf of other bones .
the most commonly used bisphosphonate was zoledronic acid ( table 5 , table 6 ) .
in the long bone pf group , more patients had multiple sres than had a single sre ( i.e. only the index pf ) ( 79.7% [ n=94 ] and 20.3% [ n=24 ] , respectively ; table 1 ) .
however , in patients with pfs of other bones , the proportions with multiple or single sres were similar ( 51.0% [ n=123 ] and 49.0% [ n=118 ] , respectively ; table 2 ) .
sres could be classified as being linked or unlinked ; subsequent linked sres were more frequent in patients with long bone pfs ( 73.4% of multiple sres ; n=69 ) than in those with other bone pfs ( 47.2% ; n=58 ) .
the most common linked sres were surgery to bone ( long bones 42.4% [ n=50 ] ; other bones 6.6% [ n=16 ] ) and radiation to bone ( long bones 18.6% [ n=22 ] ; other bones 17.4% [ n=42 ] ) .
the mean number of inpatient stays per pf increased from baseline for both pf of long bones ( 1.2 [ sd 1.2 ] ) and other bones ( 0.8 [ sd 1.2 ] ( fig .
the mean duration of inpatient stays also increased in both groups ( long bones 20.9 [ sd 22.1 ] days ; other bones 12.3 [ sd 19.5 ] days ) ( fig . 2b ) . for individuals with long bone pfs ,
the greatest increases in duration of stay per pf were reported in portugal ( 32.1 [ sd 19.8 ] days ; n=14 ) and finland ( 29.4 [ sd 34.6 ] days ; n=8 ) .
the smallest increase was reported in greece ( 5.8 [ sd 8.3 ] days ; n=8 ) ( fig .
the types of hospital units that patients stayed in differed according to the type of fracture they experienced .
the largest mean change from baseline in the number of stays per pf of long bones occurred in orthopaedic units ( 0.5 [ sd 0.6 ] ) , but stays in oncology units and other units ( including trauma surgery , casualty units , cardiology units and nursing units ) also increased slightly ( 0.1 [ sd 0.3 ] ) and 0.1 [ sd 0.8 ] , respectively ) . for individuals with pfs of other bones ,
the largest mean increases from baseline in the number of inpatient stays per pf were observed in internal medicine units ( 0.2 [ sd 0.6 ] ) and oncology units ( 0.2 [ sd 0.7 ] ) .
there was a larger increase in the number of visits per event for patients with pfs of other bones ( 4.0 [ sd 5.8 ] ) than for those with pfs of long bones ( 2.6 [ sd 4.7 ] ) ( fig .
3a ) . compared with the other countries in this study , finland recorded the largest increase in the mean number of outpatient visits per event ( long bones 8.6 [ sd 6.1 ] ; n=8 ; other bones 6.8 [ sd 6.1 ] ; n=30 ) .
the smallest changes were reported in greece ( 0.9 [ sd 3.3 ] ; n=8 ) and poland ( 0.9 [ sd 3.2 ] ; n=28 ) for those with pfs of long bones . for individuals with pfs of other bones , poland had the smallest change in the mean number of outpatient visits ( 0.8 [ sd 1.7 ] ; n=30 ) .
the largest changes in the mean number of outpatient visits per pf from baseline were in visits to radiation oncologists / radiotherapists ( long bones 1.0 [ sd 2.5 ] ; other bones 1.5 [ sd 3.8 ] ) and oncologists ( long bones 0.5 [ sd 1.9 ] ; other bones 0.8 [ sd 2.3 ] ) .
the increase in the number of visits to orthopaedic surgeons was greater in patients with pfs of long bones ( 0.3 [ sd 0.7 ] ) than in those with pfs of other bones ( 0.1 [ sd 0.3 ] ) .
the number of visits to radiologists also increased , and this change was greater in patients with pfs of other bones ( 0.6 [ sd 1.7 ] ) than in those with pfs of long bones ( 0.2 [ sd 1.1 ] ) .
the increase in the mean number of day - care visits was smaller for individuals with long bone pfs ( 0.8 [ sd 2.7 ] than for those with other bone pfs ( 1.4 [ sd 3.6 ] ) .
the mean number of emergency room visits increased from baseline per pf of long bones ( 0.3 [ sd 0.7 ] ) and other bones ( 0.2 [ sd 0.9 ] ) .
overall , the number of procedures provided per event increased in both pf groups ( long bones 6.1 [ sd 7.1 ] ; other bones 5.9 [ sd 6.6 ] ) ( fig .
finland reported the greatest increase in the mean number of procedures in patients with pfs of long bones ( 12.9 [ sd 9.2 ] ; n=8 ) . in individuals with pfs of other bones ,
the greatest increase was observed in austria ( 10.3 [ sd 7.5 ] ; n=22 ) . for both fracture groups ,
the smallest changes in the number of procedures were reported in poland ( long bones 4.1 [ sd 3.9 ] ; n=28 ; other bones 2.7 [ sd 2.7 ] ; n=30 ) .
the largest overall increase was seen in the use of external beam radiation ( long bones 2.1 [ sd 3.9 ] ; other bones 2.6 [ sd 4.9 ] ) . in patients with pfs of long bones , surgery to bone
other procedures increased in those with pfs of long bones ( 2.3 [ sd 3.9 ] ) ; however , these encompassed a wide range of diagnostic procedures , none of which showed a large change when considered alone . in patients with pfs of other bones , the number of
other procedures also increased from baseline ( 1.7 [ sd 3.3 ] ) , and use of computerised tomography also increased ( 0.6 [ sd 1.1 ] ) .
this is the first study to capture real - world changes in hru associated with pfs of long bones and , separately , also pfs of other bones .
overall , pfs were associated with considerable hru owing to increases in the number of inpatient and outpatient visits , duration of inpatient stays , and number of procedures required . over the follow - up period of approximately 3 months ,
the mean number of inpatient stays and outpatient visits per pf increased in both groups .
a multicentre , observational european study conducted over an 18-month period also found that the mean number of inpatient stays and outpatient visits increased in patients with pfs ( range 0.40.5 increase in inpatient stays per pf ; range 1.93.1 increase in outpatient visits per pf ) .
an observational study in spain revealed that a large proportion of patients with pfs secondary to solid tumours required hospital stays ( 4060% ) and outpatient visits ( 6770% ) . in our study , the mean duration of inpatient stays also increased as a result of pf in both the long bone and other bone groups . in a previous retrospective - prospective study of hru associated with sres in four european countries ,
pfs were found to be associated with a considerable number of inpatient stays , with a duration ranging from 19 to 22 days , similar to the durations of inpatient stays that were observed in our study .
inpatient stays appear to be a substantial driver of overall hru and therefore are likely to contribute considerably to the costs associated with pfs . in addition , a large study in spain demonstrated that the mean length of hospital stay following the first admission for pf was substantial for those with breast , lung or prostate cancer ( 1220 days ) .
the results of our study indicate for the first time that the increase in the number and duration of inpatient stays is generally much higher for pfs of long bones than for pfs of other bones
. this may be due in part to patients with long bone pfs requiring complicated and lengthy rehabilitation , including surgery and physiotherapy .
owing to limited resources for the care of patients with cancer and fractures in general hospitals , these individuals may have to receive inpatient care at specialised units , which will further increase the hru costs associated with pfs of the long bones .
it has previously been shown that patients who experience one sre are more likely to experience subsequent events , .
a retrospective analysis of patients with prostate cancer found that skeletal morbidity ( including pfs and bone pain ) was higher in patients who had experienced a sre than in those with no history of sres .
our study found that the majority of patients with pfs of long bones experienced multiple sres . in some pivotal clinical trials ,
multiple sres were not recorded because all sres that occurred within a 21-day window were counted as a single event , .
our study used retrospective real - world data , and may therefore reflect clinical practice more closely than clinical trials .
the treatment of multiple sres is likely to require more outpatient hospital visits and inpatient stays than the treatment of a single sre , and this may also contribute to the greater increases in hru observed for long bone pfs compared with those affecting other bones .
the proportion of patients receiving bisphosphonates increased from baseline at similar levels during the duration of this study for patients with pfs of long bones and those with pfs of other bones .
the fact that some patients were already receiving bisphosphonate treatment yet still experienced a pf indicates that further treatment optimisation with bone - targeted agents ( btas ) and new antineoplastic agents may be required to minimise the frequency of pfs , and thereby hru .
pfs affect a large proportion of patients with advanced cancers and require a considerable amount of healthcare resources for their treatment ; this means that pfs result in substantial costs , .
another multinational european study of patients with solid tumours and multiple myeloma estimated that the mean costs associated with each individual pf ranged from 1000 to 7000 for vertebral fractures and from 1700 to 3200 for non - vertebral fractures . in spain and belgium ,
the mean hru cost per pf has also been reported to be high ( 3209 and 7087 for non - vertebral fractures and
5015 and 6968 for vertebral fractures in spain and belgium , respectively ) , . in portugal ,
one of the countries included in our analysis , the estimated annual cost of pfs per patient was 8730 .
the costs associated with surgery to bone and radiation to bone are also substantial and were the linked sres with largest increases from baseline in our study .
a strength of this study is the ability to distinguish between the different pfs because hru is different for each fracture type ; long bone fractures are rarely asymptomatic and will have hru requirements that are different from those of other bone fractures .
the main limitation of our study was the low number of patients with pf of long bones identified in each country , reflecting a low incidence of this fracture type in patients with cancer .
one study of patients with advanced breast cancer found that the incidence of long bone fracture was half that of other fracture types .
furthermore , the use of bone - targeted agents in routine clinical practice may have reduced the incidence of sres overall , as seen in clinical trials , , , .
however , the baseline demographics of patients from countries with low recruitment were generally consistent with those of the other countries in the study .
this is the first study to differentiate between the hru attributed to pfs of long bones and to those affecting other bones .
both fracture types were associated with increases from baseline in the mean number of inpatient and outpatient stays , and visits to day care centres and emergency rooms .
the number of sres can be reduced by using bone - targeted agents such as bisphosphonates , , , radiopharmaceuticals or denosumab , , , or new antineoplastic agents for prostate cancer such as enzalutamide and abiraterone acetate .
combining these new antineoplastic agents with bone - targeted agents may lead to greater reductions in hru in patients with advanced cancer . | backgroundskeletal - related events ( sres ; pathologic fracture [ pf ] , spinal cord compression and radiation or surgery to bone ) are common complications of bone metastases or bone lesions and can impose a considerable burden on patients and healthcare systems . in this study ,
the healthcare resource utilisation ( hru ) associated with pfs in patients with bone metastases or lesions secondary to solid tumours or multiple myeloma was estimated in eight european countries.methodseligible patients were identified in austria , the czech republic , finland , greece , poland , portugal , sweden and switzerland .
hru data were extracted from hospital charts from 3.5 months before the index pf ( defined as a pf preceded by a 6.5-month period without a sre ) until 3 months after the last sre during the study period .
changes from baseline in the number and duration of inpatient stays , number of outpatient visits and number of procedures provided were recorded.resultsoverall , 118 patients with pfs of long bones ( those longer than they are wide , e.g. the femur ) and 241 patients with pfs of other bones were included .
overall , hru was greater in patients with long bone pfs than in those with pfs of other bones . a higher proportion of patients with long bone pfs had multiple sres ( 79.7% ) , and more of their sres were considered to be linked ( 73.4% ) compared with patients with pfs of other bones ( 51.0% and 47.2% , respectively).conclusionthe increased number and duration of inpatient stays for pfs of long bones compared with those for pfs of other bones may be due in part to the requirement for complicated and lengthy rehabilitation in patients with long bone pfs .
implementing strategies to delay or reduce the number of pfs experienced by patients with bone metastases or lesions may therefore reduce the associated hru and patient burden . |
hemangiopericytomas ( hpcs ) were first categorized by stout as malignant tumors arising from the blood vessels and described as a sarcoma .
the cell of origin is thought to be the pericyte which interacts with endothelial cells and is critical for blood vessel maintenance .
hpcs are extremely rare and occur usually outside the central nervous system ( cns ) with the most common sites being the lower extremities or retroperitoneum . in rare instances ,
hpcs can arise from the meninges in the cns and can be confused with solitary fibrous tumors . in the case series by guthrie and et al .
the primary mode of treatment included surgical resection , followed by radiotherapy , but , unfortunately , hpcs can both recur and metastasize .
paraneoplastic syndromes have been reported as the initial presentation or part of the diagnosis of hpc .
the paraneoplastic syndrome of oncogenic osteomalacia , defined by hypophosphatemia , hyperphosphaturia , and osteomalacia , has been seen in soft tissue sarcomas , including hpcs .
while the roles of surgical resection and radiotherapy are well understood , the role of chemotherapy in the treatment of multiple recurrences of hpc has not been well established .
we present a case of a male with a right sphenoid wing hpc who presented initially with the paraneoplastic syndrome of oncogenic osteomalacia 9 years before his hpc diagnosis .
after multiple recurrences , he has been stabilized with molecularly targeted monotherapy utilizing dasatinib , a small molecule that inhibits src - related tyrosine kinases .
a 22-year - old healthy male presented with diffuse bone pain . on clinical laboratory testing , serum phosphorus was extremely low at 0.8 mg / dl .
the patient was later found to have hyperphosphaturia and hypercalcemia and was diagnosed with hypophosphatemic osteomalacia .
etiology of the hypophosphatemic osteomalacia was unclear at that time , but the patient was treated with neutral sodium phosphate and dihydrotachysterol , which improved his pain .
he also began to experience sharp pain at the right retro - orbital region and right - sided hearing loss .
later , placement of a right tympanostomy was attempted because of presumed chronic otitis media and a vascular tumor was visualized in his right middle ear .
initial biopsy was only remarkable for granular tissue . at 30 years of age , he developed numbness at the right temple and a head ct revealed a mass arising from the right sphenoid wing that infiltrated into the floor of the middle cranial fossa .
embolization was performed which was followed by a partial resection of the mass via right temporal craniotomy .
the previous diagnosis of hypophosphatemic osteomalacia was attributed to a paraneoplastic presentation of oncogenic osteomalacia due to hpc . within 4 years from initial tumor diagnosis , the tumor recurred , and the patient was found to have a right seventh nerve palsy .
a second embolization was performed for this recurrent tumor , and the patient was followed with serial scans . approximately 3 years after this resection , the tumor increased in size . despite partial resection ,
he was also noted to have brown tumors associated with hyperparathyroidism involving the mandible and the right hand .
octreoscan ( indium - labeled pentetreotide scan ) showed active uptake by the remaining tumor .
after identification of the brown tumors , the patient underwent a total parathyroidectomy with reimplantation of one parathyroid into the left arm to address the tertiary hyperparathyroidism .
after 3 years of octreotide treatment , he developed dj vu episodes ( consistent with possible seizure activity ) and magnetic resonance imaging ( mri ) revealed progression of his tumor .
when the mass recurred approximately 2 years later , he had fractionated stereotactic radiotherapy to the right temporal mass .
eleven months after fractionated radiotherapy , he participated in a clinical trial for 5 months using somatostatin receptor radiopharmaceutical called ( 90 ) y - dodecane - tetrateaacetic acid - phe1-tyr3-octreotide .
after completion of 3 six - week cycles of this therapy , he was followed with serial mri scans .
approximately 1 year later , he developed worsening right facial weakness and was found to have recurrent tumor extending into the middle ear cavity , external auditory canal , intratemporal fossa , inferior orbital fissure , and the anterior aspect of the cavernous sinus .
there was no change in his treatment course and he continued to be followed with serial scans until 4 years later . at that time
( age 56 years ) , he underwent a right temporal craniotomy with partial resection for recurrent tumor ( fig .
testing of his tumor from the second resection showed high immunoreactivity to platelet - derived growth factor receptor ( pdgfr ) , therefore , dasatinib was considered for monotherapy . after the third resection
, he was placed on dasatinib at 70 mg orally once a day and has been followed with serial scans .
currently , he has been on monotherapy with dasatinib for 2 years and continues to be both clinically and radiographically stable by mri an dfdg - pet ( fig . 2c ) .
hpc is a rare malignancy that is believed to arise from pericytes , a support cell of the vasculature that interacts with endothelial cells .
usually , hpcs present in the lower extremities or retroperitoneum , but in some circumstances , they can occur within the cns .
when they involved the cns , hpcs were previously described as angioblastic meningiomas , as the tumors were uniquely vascular and arose from the meninges .
. two case series of meningeal hpcs have described the presentation , course , and treatment of these rare tumors . in both case series ,
patients present in adulthood , with a median occurrence of hpc in the fifth decade .
the initial presentation was usually focal neurological signs , and headache was also a prominent symptom that led to the diagnosis of meningeal hpcs . a majority of patients experienced recurrences of their tumors within a range of 60.6 - 76% .
in rare circumstances , clinicians have documented that hpcs can metastasize with sites including the bone , liver , lung , and peritoneum , with a 5-year metastasis rate at 33% .
treatment of meningeal hpcs is similar to meningiomas in that surgical resection is utilized at the initial presentation if amenable to surgery .
total resection is often hindered by the extensive vasculature of the tumor , and pre - embolization of the tumor before resection can prevent blood loss .
radiotherapy is used post - operatively to prevent recurrence , but in a study by dufour et al .
doxorubin - based regimens have been used , but have yet to establish a clear role in the treatment of hpc . with the recurrent nature of hpcs as seen in our current case report , there is a vital need for better treatments of persistently recurrent hpcs .
one possible therapy could be the use of molecularly targeted therapies based on the pattern of expression of growth factors in hpcs .
a recent report showed that angiomatous meningiomas and hpcs often overexpress pdgfr as detected by immunohistochemistry .
therefore , it is provocative that this overexpression of pdgfr could be utilized to treat recurrent angiomatous meningiomas and hpcs .
dasatinib , at nanomolar concentrations , inhibits the following kinases : bcr - abl , src family ( src , lck , yes , fyn ) , c - kit , epha2 , and pdgfr .
dasatinib is approved by the federal drug administration for the treatment of chronic myelogenous leukemia or philadelphia - chromosome positive acute lymphoblastic leukemia . in our case presentation ,
the patient 's tumor tissue showed overexpression of pdgfr based on immunohistochemical analysis . because of dasatinib 's ability to inhibit pdgfr , we initiated therapy with this agent . since initiation of therapy
, the patient has tolerated that therapy well with little toxicity and has continued on monotherapy for over 2 years .
mri and fdg - pet imaging reveal no evidence of recurrent disease at this time .
thus , this suggests that there may be a role for targeted therapy in the treatment of hpcs . while this tumor is extremely rare and it is thus difficult to evaluate the drug 's effectiveness in clinical trials , the further use of dasatinib in the treatment of recurrent hpc may be warranted . in conclusion ,
our case report provides a first example of treatment of recurrent hpc with molecular targeted monotherapy . | hemangiopericytoma ( hpc ) is a rare sarcomatous tumor arising from pericytes , a support cell found in blood vessels .
these tumors can occur throughout the body , particularly in the lower extremities and retroperitoneum . in rare circumstances ,
hpcs can arise from the meninges . in these cases ,
they behave similar to meningiomas , in particular angiomatous meningiomas , but tend to be more aggressive and are likely to recur .
treatment usually focuses on surgical resection and radiotherapy with possible inclusion of chemotherapy for control of recurrent disease .
we describe a case of recurrent right temporal hpc that first manifested as a paraneoplastic syndrome of oncogenic osteomalacia . despite maximum therapy , this patient experienced multiple recurrences of the tumor , and immunohistochemical analysis revealed overexpression of platelet - derived growth factor receptor , a member of the src - related tyrosine kinases .
after multiple recurrences , the patient 's tumor has been stable with treatment with monotherapy utilizing molecularly targeted therapy to src - related tyrosine kinases .
this is the first case report of the treatment of recurrent meningeal hpc with molecularly targeted therapy to src - related tyrosine kinases . |
due to the nature of the job , physical therapists provide medical services in close
relationships with patients1 , 2 .
the turnover of physical therapists is a major loss in
quality improvement of health services and rehabilitation related to human resources3 .
currently , medical services are moving from healthcare provider - centered to
patient - centered and hospitals are also converting into patient - centered healthcare
systems4 .
in addition , hospital
organizations are recognizing recruitment , training , and retraining for healthcare providers
in each specialized field5 . to provide good quality health services , healthcare providers beliefs in significantly
contributing to patient care and their own work satisfaction
emotional labor refers to the idea of
suppressing and limiting emotions to satisfy hospitals changing patient - centered goals and
patients feelings6 , 7 .
stress caused by excessive emotional labor
has a negative effect on workers commitment to the organization and satisfaction toward the
job9 , 10 , resulting in work - related stress ( ws ) .
the work environment ( we )
of human service workers has also been presented as a major factor in turnovers11 . if an organization member leaves the organization due to turnover , ensuring good quality
staffing is threatened ; large amounts of money are spent in the selection and training of
new staff , finally resulting in loss to the organization12 .
additionally , new employees are highly likely to make mistakes or
cause accidents compared to employees with experience .
employee replacement costs are also
much higher than the costs of actually recruiting , hiring , and training new staff12 ; therefore , this has negative effects on
the overall health and economic sectors .
byun13 said in her previous research that promoting efficient task
execution and reducing turnovers by reducing ws and increasing job satisfaction are very
important .
for the efficient management of internal employees , it is necessary to accurately
identify job satisfaction and ws and analyze their correlations with turnovers . examining similar research on the correlation between a variety of factors in the workplace
and turnovers within medical institutions , radiological technologists showed higher job
satisfaction with higher annual income and lower working years and showed higher turnovers
depending on the number of working years , wages , and night watch duty14 .
age , characteristics of medical institutions in which
they are working , and job satisfaction are related to turnover of occupational
therapists15 . for nurses , excellent
nursing is dependent on job satisfaction enabling them to handle work positively and
efficiently ; higher job satisfaction reduces turnovers16 .
the reality is that studies related to physical therapists include
studies on ws17 , research on working
conditions and job satisfaction18 ,
empowerment and job satisfaction , research on organizational commitment19 , etc .
however , the number is remarkably small compared to
studies targeting other health service workers and the data on turnover intention ( ti )
itself as well as studies that analyze the relationship between ti and we and ws of physical
therapists are very rare .
therefore , by investigating the effect of ws and we on physical therapists and turnover ,
this study can provide basic data for reducing the turnover of physical therapists and
seeking effective human resource and organizational management measures in the future .
the study participants were 241 physical therapists currently working in medical
institutions who were directly visited during the three months from january to march 2014
for questionnaire distribution and collection .
five questionnaires were miswritten due to
error or insufficient survey answer contents ; 236 questionnaires were examined
statistically .
those who fully comprehended the study procedure and voluntarily agreed to
participate were selected as participants .
the ti measurement tool used seven questions ; for turnover factors , four questions modified
and used by gang20 were modified and
reconstructed for this study .
, this
study used the questionnaire consisting of 15 questions after modifying and complementing
the method used by byun13 .
the we
measurement tool was reconstructed for this study based on the survey tool reconstructed by
lim21 .
each question on the measurement tool was based on a likert
scale from 1 ( not at all ) to 5 ( strongly agree ) points and higher scores meant better we .
cronbach s alpha values in a previous and the present study were more than 0.700 .
spss 18.0 was used for the collected data and the analysis method is as follows .
ti , ws , and we
of the participants were obtained as means and standard deviations .
the data were analyzed
using cronbach s alpha coefficient to verify the reliability of questionnaire internal
consistencies .
the correlation between ti , ws , and we used correlation analysis through
pearson s correlation coefficient and linear regression analysis to determine the impact of
ws and we on ti2 .
there were 106 males ( 44.9% ) and
130 females ( 55.1% ) . for age ,
155 were less than 30 years old ( 65.7% ) and 81 were older than
30 years ( 34.3% ) .
most were single ( n=190 , 80.5% ) ; 46 were married ( 19.5% ) .
there were 104
college graduates ( 44.1% ) , 88 university graduates ( 37.3% ) , and 44 graduate students or
higher ( 18.6% ) .
most were hopeful ( n=198 , 83.9%).this was the first job for 113 ( 47.9% ) . in
career tenure , 56 had less than one year ( 23.7% ) , 99 had 14 years ( 41.9% ) , 42 had 59 years
( 17.8% ) , and 39 had more than 10 years ( 16.5% ) . in current hospital tenure ,
79 had less than
1 year ( 33.5% ) , 125 had 13 years ( 53.0% ) , and 32 had more than four years ( 13.6% ) .
sixty
people had less than 1.5 million monthly income ( 25.4% ) , 98 had 1.52 million ( 41.5% ) , 57
had 22.5 million ( 24.2% ) , and 21 had more than 2.5 million ( 8.9% ) .
the total ti mean of participants was 3.09 0.78 ; for ws , 3.31 0.56 ; and for we , 3.25
0.46 .
ti and ws ( r=0.531 , p<0.01 ) had a statistically significant positive correlation
and ti and we ( r=0.511 , p<0.01 ) had a statistically significant negative correlation .
ws
( =0.415 ) had a significant positive impact and we ( =0.387 ) had a significant negative
impact on ti ( p<0.01 ) ( table 1table 1.impact of work environment and work related stress of study subjects on the
turnover intentionvariablestwork related stress0.4157.929work environment0.3877.395r=0.419 , adjust r=0.414 , f=83.848 ( * p<0.001 ) ) .
the effects of physical therapy appear through emotional and cognitive interactions in the
relationship with patients . in this process
, emotional labor of a physical therapist affects
both the therapist and the patient .
however , therapists who have previously adapted to the
healthcare system have emphasized only ability and have neglected the emotional aspect22 .
accordingly , psychological factors may
burden poor we and reduced job involvement and job satisfaction levels9 , 10 . to provide the
best care services to patients
, physical therapists should continue to have a smooth
interactive relationship with patients , constantly watch the patient condition , and find the
optimum treatment in this process .
thus , physical therapists satisfaction with their job
and working with responsibility are essential elements in increasing treatment efficiency as
well as cementing the relationship with patients8 . in this study , the correlations between ti , ws , and we were analyzed ; there was a
statistically significant positive correlation between ti and ws .
ti
and we had a statistically significant negative correlation ; results of a study21 targeting 290 nurses support the results
of this paper .
the results of regression analysis on physical therapists ti by ws showed a positive
explanatory power of 41.5% ( r=0.415 ) ; the size of the influence ( =0.415 ) shows
that higher ws leads to relatively higher ti .
lim s study targeting 248 nursing teachers
showed 53.6% explanatory power ( r=0.536 ) and influence of =0.540 ; ws also
affected ti in other occupations , highly supporting the results of this study19 . however , heo s study , targeting 353
nurses obtained only 12% explanatory power ( influence =0.36 ) showing relatively low support
for the results of this study24 . on the
other hand , satisfaction with we showed a negative explanatory power of 41.5% of ti
( r=0.415 ) and
the size of the influence was =0.387 , indicating that low
satisfaction with we leads to relatively high ti .
other research results include explanatory power of 52.1% ( r=0.521 ) , and
influence of =0.229 ( working environment ) , =0.382 ( self - development ) , and =0.263
( working facilities ) in lee s study targeting 236 golf course employees25 ; explanatory power of 53.6% ( r=0.536 ) and
influence of =0.141(relationship with superintendent ) and =0.118 ( compensation adequacy )
in lim s study targeting nursing teachers21 ; and explanatory power of 36% ( r=0.360 ) and influence
of =0.228 in sin s study targeting local hospital nurses23 .
thus , we affected ti in other occupations , showing high support
for the results of this study .
according to the results of this study , the higher the ws , the higher the ti , and the
better the we , the lower the ti . in particular , ws had a greater impact on ti than we .
this
is similar to the results of lim21 and
jeongs26 studies in which ws had the
most significant impact on ti .
therefore , ws becomes the direct cause of turnovers and if
not solved , leads to turnovers .
thus , it is essential to reduce the ws of physical
therapists and improve the we ; ways to reduce turnovers should be sought through this .
the limitation of this study is that it can not be generalized to all physical therapists
due to its potential selection bias ; physical therapists in daejeon ( chungcheong ) , physical
therapists working in rehabilitation hospitals , and young physical therapists were surveyed .
further , the correlations between various stress factors and we affecting ti could not be
clearly identified because survey questions were not segmented . in conclusion ,
future
research including national physical therapists , all medical institutions , and participants
of various ages needs to be conducted . the root cause can be found and solved when
correlations of each factor are identified more clearly through the segmentation of the
survey questions . | [ purpose ] this study was conducted to provide basic data for solutions to reduce the
turnover rate of physical therapists .
it should help create efficient personnel and
organization management by exploring the impact of the work environment and work - related
stress on turnover intention and analyzing the correlation between them .
[ subjects and
methods ] a survey was conducted with 236 physical therapists working at medical
institutions in the daejeon and chungcheong areas . for the analysis on the collected data ,
correlational and linear regression analyses
were conducted using the spss 18.0 program
and cronbach s alpha coefficient .
[ results ] the results showed a statistically significant
positive correlation between turnover intention and work - related stress but a
statistically significant negative correlation respectively between turnover intention and
work environment .
work - related stress ( =0.415 ) had a significant positive impact on
turnover intention and work environment ( =0.387 ) had a significant negative impact on
turnover intention .
[ conclusion ] to increase satisfaction level with the profession as
well as the workplace for physical therapists , improvement of the work environment was the
most necessary primary improvement . |
one of the important risks is the existence of high levels of gasoline vapors including benzene that during work shifts employees are exposed to .
employees in functional units are more vulnerable population because of their continuous contact with harmful substances such as benzene .
based on usepa ( united state environmental protection agency ) and iarc ( international agency for research cancer ) reports , benzene has been classified to be a group a and class 1 human ( 1 ) .
adverse health effects of benzene appear in two different type ; short - term and long - term .
short - term effects are associated with high concentration and may involve headaches , dizziness , distraction and defects temporary memory and tremors . whereas , exposure to benzene in a long - term is connected to intricate adverse health effects such as immunological , hemato - toxicity , geno - toxicity , adverse effects on reproductive organs , and as well as various cancers ( 1 , 2 ) .
therefore , a powerful method is required to predict existent exposure to harmful substances besides to evaluate probable adverse effects ( 3 ) .
although several mathematical models have been carried out so far , there is high complexity related to chemical exposure in terms of human health . due to the lack of information about chemical impacts on human particularly in long - term impacts ,
some unpredictable factors that called uncertainties can affect health risk assessment ( 4 , 5 ) .
health risk assessment through inhalation concerning benzene emission from equipment at the unit of benzene production was accomplished .
this project is an example of using a hybrid approach that can incorporate uncertainties to assessment s process .
major variables affecting the absorption of chemicals moreover key parameters in the dispersion of chemicals can be reflected in a hybrid system which combining fuzzy logic and neural networks ( 5 , 6 ) . in this research
however , breathing rates are affected by many individual characteristics , including age , sex , weight , health , and level of physical activity ( running , jogging , etc . )
( 5 , 9 ) . perhaps the first and distinguished use of fuzzy sets in health risk assessment was on the application of fuzzy logic in the environmental risk assessment ( 10 ) .
one more example which connected to human health risk assessment is the application of fuzzy sets in human health risk assessment .
fuzzy sets were employed to estimate carcinogenic risk caused by air pollution in ten russian cities ( 11 ) .
the study area was in the northern part of the persian gulf with a distance of 10 miles from the coast .
the plant mainly produced about one million tons annually of benzene , paraxylene , orto - xylene used to produce ethylbenzene , styrene , cyclohexane , and nitrobenzene .
benzene from refinery streams was typically produced from catalytic reformats pyrolysis , gasoline , and toluene de - alkylation .
this study focused on tasks and activities those are exposed to potential contact with benzene during refinery .
main workplaces related to workers exposed to benzene such as loading of tankers were listed in table 1 .
each workplace maybe had a different group of job and activities . in order to increase the information about jobs , activities , tasks , and
main work places for workers the sampling procedure was based on absorption of benzene in an active charcoal tube ( active sampling ) ( 12 ) .
skc model 222 pumps have been utilized for gas sampling . the glass tubes with a 6 mm external diameter , 4 mm internal diameter and 70 mm height , containing activated charcoal holder with a restrictive orifice ( separated by a 2-mm part of urethane foam ) were installed .
the pump was adjusted to work for 30 min at a flow rate of 100 ml / min ( 12 ) .
four measuring procedures were performed lasting one week in the middle of each season ( 12 , 13 ) .
consequently , by the end of the week 70 samples were taken and during 15/2/2012 to 21/09/2013 totally 280 samples were collected . for detailed information stickers attached on each tube to tag sampling number , the time of end duration of sampling , the pump number , the humidity , the wet and dry temperature , and the date of sampling ( 13 ) . the human lifetime average daily dose ( ladd )
equation for a single chemical exposure proposed by u.s.epa ( 2011 ) that related to cancer risk model ( presuming that the inhalation is the only route of intake ) is , as shown in
eq .
( 14 ) :
ladd = ca.ir.ef.ed / bw.at
the cancer risk ( cr ) is calculated for exposure to benzene using eq .
( 15 ) :
cancer risk = ladd ( g / kg / day)sf ( g / kg / day)1 for equation 1 , ca is the concentration of a chemical in an exposure medium ( g / m ) , ir represents the inhalation rate ( m / h ) , ef is the exposure frequency ( number of working days per year ) , ed is the exposure duration ( working years ) , bw is the body weight ( kg ) , at is averaging time ( at=70 yr 365 d / year for carcinogens ) , ladd shows lifetime average daily dose ( g / kg / day ) and sf in equation 2 , is the cancer slope factor of benzene ( linear low - dose cancer potency factor ) ( 15 ) .
the quality of data in equation 1 is uncertain even if definite exposure - related measurements are available for variables ( 16 ) . to explain more , some unpredictable parameters can change the results . as an illustration , movement of contaminants among environmental media changes
remarkably the quality of data , therefore , uncertainty plays a crucial role involved in each variable as well as affected by physical and chemical factors ( 11 , 16 ) .
these variables ; wind speed , ambient temperature , humidity and rainfall cause fluctuation in concentration of air pollutants ( 17 ) .
furthermore , the inhalation rate is also influenced by multiple individual aspects including age , body weight , and amounts of physical activity ( 5 , 11 ) .
owing to these numerous variables , the trustworthy approach is required to make a better risk evaluation . therefore , to overcome the problems of uncertainties the hybrid method was prepared ( 5 , 18 ) .
in this figure , each tablet represents a sub - system combined ; they determine the chemical absorption rate through inhalation .
the receptor description ( inhalation rate ) and dose estimation sections use a set of fuzzy rules and obtain the average daily inhalation dose , based on fuzzy inference .
the exposure prediction section consists of a designed neural network using a new back - propagation algorithm .
this section created in order to calculate of real ambient concentration . in the following sections , details about the subdivisions will be explained ( 5,18 ) .
integrated system of risk assessment ( 5 ) a. artificial neural network b. life lifetime average daily inhalation dose standard quantities technique in health issues has been often vague and unclear adjectives such as a little , too much ; so at the start it is necessary to quantify these adjectives and change them into fuzzy sets ( 19 , 20 ) . in this section
age , body weight , and activity tend to be the significant reasons of daily energy expenditure in healthy people for as long as is also in energy balance ( 20 , 21 ) .
variables must be delineated with linguistic values rather than numerical values ( 5 , 21 ) .
2 shows one of the prepared fuzzy sets ( fuzzy numbers ) that related to age with three linguistic values .
the horizontal vector indicates the size of the parameter and the vertical vector represents the degree of membership ( degree of dependence ) of each value ( 5 ) .
membership functions of the variable age ( 5 ) the membership function for age was set to range from 18 to 80 yr ( 22 ) .
using census and demographic details , age ( in years ) was divided into three fuzzy linguistic sets : low , ranging from 18 to 30 ; medium , ranging from 25 to 64 ; and high , ranging from 59 to 80 ( 5 , 22 ) . according to brainard and burmaster ( 23 ) , body weight ( 45110 kg )
it has been divided into three linguistic sets : low ( from 45 to 68 kg ) , medium ( from 56 to102 kg ) , and high ( from 90 to110 kg ) .
fuzzy set was made for physical activity by means of four linguistic values and follows skewed ( normal ) distribution ( 5 ) . as for long - term physical activity levels ( pals ) , it is ranging from 1.2 to 2.5 times the bmr ( basal metabolic rate ) , where 1.2 represents the minimal intensity of activity and 2.5 shows a very physically active lifestyle . aging is a parameter that can remarkably decline high - intensity activity ( 21 ) .
moreover , seven linguistic values ranging from 525 m / day with normal distribution have been provided to show inhalation rate , which is compatible with the human capability ( 14 ) .
anns are working based on specified transfer function and made by artificial neurons ( 17 ) .
using particular function anns can adjust the value of different weights properly . in order to neural network learning
hence , the data set for this project were randomly divided into a ratio of 3:1 between training and testing sets , respectively ( 7 , 10 , 17 ) . as a result , available data ( 280 samples ) arbitrarily fragmented into two subsets .
one of those was training groups and included 75% of data . by developing the model this group predicted the concentration of benzene for the second group that contained the 25% of data ( 10 , 17 ) .
parameters ; wind speed , temperature , relative humidity , rainfall and measured concentration was chosen as model input data . the predicted benzene concentration was the output parameter .
the third layer involved just one neuron which is output parameter and shows actual benzene concentration .
a typical three - layer neural network is shown in fig . 3 ( 17 ) .
selected three layer neural network ( 17 ) details of the meteorological monitoring devices before conducting the network preparation procedure , a training group ( 75% of the data ) consisting of 210 cases had to be prepared from the environmental data done by the back propagation paradigm .
training group itself became divided into training set and testing set that this process conducted randomly .
first one is the training set consisted of 90% of the data or 189 cases used to train the model .
the second or final 10% contains 21 cases were left out as a test set ( 7 , 17 , 24 ) .
output variables from the artificial neural network stage and fuzzy logic section contributed to the last part of the hybrid model and produced a chronic daily intake dose ( 18 ) .
the predicted benzene concentrations lie between 0 and 67 ( g / m ) considered very high .
( 25 ) , the peak concentrations in the study area are high and the membership function for output and some fuzzy rules must be adjusted to range 0 and 67 ( g / m ) .
the process of adjustment was easily done that indicates a powerful feature of the dose assessment modeling system as well as the adaptability to different scenarios . in different circumstances ,
the user can change fuzzy rules according to the different situations ( 6 , 9 ) .
the outputs from the benzene prediction divided into six linguistic variables based on the frequency distribution of the data .
the membership function of variable concentration is shown in fig . 4 ( 18 ) .
the output from the receptor block was divided by the body weight to derive m / d / kg then contributed as input to the dose estimation section ( 5 , 18 ) .
the daily intake rate for the dose estimation is expressed by four linguistic fuzzy sets and is expressed in m / d / kg ( 18 ) . finally , the life average daily intake dose defined as seven fuzzy linguistic values and ranging from 0 to 6 g / kg / d .
input variable predicted concentration ( g / m ) a sample fuzzy inference calculation for the dose estimation block is shown in fig .
5 . where ir denotes inhalation rate normalized for body weight ( m / day / kg )
; cc indicates chemical concentration ( g / m ) ; ladid = lifetime average daily inhalation dose ( g / kg / d ) . as an illustration , if the inhalation rate were considered 0.35
m / kg / d , the ambient benzene concentration is 15 g / m , lifetime average daily inhalation dose would be 1.63 g / kg / day .
fuzzy inference for estimation of ladid ( matlab software ; fuzzy toolbox ) comparison of observed and predicted concentration ( source : author 's calculations ) the output from this last module gives the overall dose through inhalation for each individual around the site , taking into account both the exposure factors and the receptor factors in a quantitative way , based on neural network and fuzzy inference .
when the exposure is quantified , combined with integrated risk information system ( iris ) toxicity factors , then as a final point the related risk can be estimated .
the study area was in the northern part of the persian gulf with a distance of 10 miles from the coast .
the plant mainly produced about one million tons annually of benzene , paraxylene , orto - xylene used to produce ethylbenzene , styrene , cyclohexane , and nitrobenzene .
benzene from refinery streams was typically produced from catalytic reformats pyrolysis , gasoline , and toluene de - alkylation .
this study focused on tasks and activities those are exposed to potential contact with benzene during refinery .
main workplaces related to workers exposed to benzene such as loading of tankers were listed in table 1 .
each workplace maybe had a different group of job and activities . in order to increase the information about jobs , activities , tasks , and
the sampling procedure was based on absorption of benzene in an active charcoal tube ( active sampling ) ( 12 ) .
skc model 222 pumps have been utilized for gas sampling . the glass tubes with a 6 mm external diameter , 4 mm internal diameter and 70 mm height , containing activated charcoal holder with a restrictive orifice ( separated by a 2-mm part of urethane foam ) were installed .
the pump was adjusted to work for 30 min at a flow rate of 100 ml / min ( 12 ) .
four measuring procedures were performed lasting one week in the middle of each season ( 12 , 13 ) .
consequently , by the end of the week 70 samples were taken and during 15/2/2012 to 21/09/2013 totally 280 samples were collected . for detailed information stickers attached on each tube to tag sampling number , the time of end duration of sampling , the pump number , the humidity , the wet and dry temperature , and the date of sampling ( 13 ) .
the human lifetime average daily dose ( ladd ) equation for a single chemical exposure proposed by u.s.epa ( 2011 ) that related to cancer risk model ( presuming that the inhalation is the only route of intake ) is , as shown in
eq .
( 14 ) :
ladd = ca.ir.ef.ed / bw.at
the cancer risk ( cr ) is calculated for exposure to benzene using eq .
( 15 ) :
cancer risk = ladd ( g / kg / day)sf ( g / kg / day)1 for equation 1 , ca is the concentration of a chemical in an exposure medium ( g / m ) , ir represents the inhalation rate ( m / h ) , ef is the exposure frequency ( number of working days per year ) , ed is the exposure duration ( working years ) , bw is the body weight ( kg ) , at is averaging time ( at=70 yr 365 d / year for carcinogens ) , ladd shows lifetime average daily dose ( g / kg / day ) and sf in equation 2 , is the cancer slope factor of benzene ( linear low - dose cancer potency factor ) ( 15 ) . the quality of data in equation 1 is uncertain even if definite exposure - related measurements are available for variables ( 16 ) . to explain more , some unpredictable parameters can change the results . as an illustration , movement of contaminants among environmental media changes
remarkably the quality of data , therefore , uncertainty plays a crucial role involved in each variable as well as affected by physical and chemical factors ( 11 , 16 ) .
these variables ; wind speed , ambient temperature , humidity and rainfall cause fluctuation in concentration of air pollutants ( 17 ) .
furthermore , the inhalation rate is also influenced by multiple individual aspects including age , body weight , and amounts of physical activity ( 5 , 11 ) .
owing to these numerous variables , the trustworthy approach is required to make a better risk evaluation .
therefore , to overcome the problems of uncertainties the hybrid method was prepared ( 5 , 18 ) . the construction of the system is displayed in fig . 1 ( 5 ) .
in this figure , each tablet represents a sub - system combined ; they determine the chemical absorption rate through inhalation .
the receptor description ( inhalation rate ) and dose estimation sections use a set of fuzzy rules and obtain the average daily inhalation dose , based on fuzzy inference .
the exposure prediction section consists of a designed neural network using a new back - propagation algorithm .
this section created in order to calculate of real ambient concentration . in the following sections , details about the subdivisions will be explained ( 5,18 ) .
integrated system of risk assessment ( 5 ) a. artificial neural network b. life lifetime average daily inhalation dose
standard quantities technique in health issues has been often vague and unclear adjectives such as a little , too much ; so at the start it is necessary to quantify these adjectives and change them into fuzzy sets ( 19 , 20 ) . in this section
age , body weight , and activity tend to be the significant reasons of daily energy expenditure in healthy people for as long as is also in energy balance ( 20 , 21 ) .
variables must be delineated with linguistic values rather than numerical values ( 5 , 21 ) .
2 shows one of the prepared fuzzy sets ( fuzzy numbers ) that related to age with three linguistic values .
the horizontal vector indicates the size of the parameter and the vertical vector represents the degree of membership ( degree of dependence ) of each value ( 5 ) .
membership functions of the variable age ( 5 ) the membership function for age was set to range from 18 to 80 yr ( 22 ) . using census and demographic details , age ( in years )
was divided into three fuzzy linguistic sets : low , ranging from 18 to 30 ; medium , ranging from 25 to 64 ; and high , ranging from 59 to 80 ( 5 , 22 ) . according to brainard and burmaster ( 23 ) , body weight ( 45110 kg )
it has been divided into three linguistic sets : low ( from 45 to 68 kg ) , medium ( from 56 to102 kg ) , and high ( from 90 to110 kg ) .
fuzzy set was made for physical activity by means of four linguistic values and follows skewed ( normal ) distribution ( 5 ) .
as for long - term physical activity levels ( pals ) , it is ranging from 1.2 to 2.5 times the bmr ( basal metabolic rate ) , where 1.2 represents the minimal intensity of activity and 2.5 shows a very physically active lifestyle . aging is a parameter that can remarkably decline high - intensity activity ( 21 ) .
moreover , seven linguistic values ranging from 525 m / day with normal distribution have been provided to show inhalation rate , which is compatible with the human capability ( 14 ) .
anns are working based on specified transfer function and made by artificial neurons ( 17 ) .
using particular function anns can adjust the value of different weights properly . in order to neural network learning
hence , the data set for this project were randomly divided into a ratio of 3:1 between training and testing sets , respectively ( 7 , 10 , 17 ) . as a result , available data ( 280 samples ) arbitrarily fragmented into two subsets .
one of those was training groups and included 75% of data . by developing the model this group predicted the concentration of benzene for the second group that contained the 25% of data ( 10 , 17 ) . parameters
; wind speed , temperature , relative humidity , rainfall and measured concentration was chosen as model input data .
the third layer involved just one neuron which is output parameter and shows actual benzene concentration .
a typical three - layer neural network is shown in fig . 3 ( 17 ) .
selected three layer neural network ( 17 ) details of the meteorological monitoring devices before conducting the network preparation procedure , a training group ( 75% of the data ) consisting of 210 cases had to be prepared from the environmental data done by the back propagation paradigm .
training group itself became divided into training set and testing set that this process conducted randomly .
first one is the training set consisted of 90% of the data or 189 cases used to train the model .
the second or final 10% contains 21 cases were left out as a test set ( 7 , 17 , 24 ) .
output variables from the artificial neural network stage and fuzzy logic section contributed to the last part of the hybrid model and produced a chronic daily intake dose ( 18 ) .
the predicted benzene concentrations lie between 0 and 67 ( g / m ) considered very high .
( 25 ) , the peak concentrations in the study area are high and the membership function for output and some fuzzy rules must be adjusted to range 0 and 67 ( g / m ) .
the process of adjustment was easily done that indicates a powerful feature of the dose assessment modeling system as well as the adaptability to different scenarios . in different circumstances ,
the user can change fuzzy rules according to the different situations ( 6 , 9 ) .
the outputs from the benzene prediction divided into six linguistic variables based on the frequency distribution of the data .
the membership function of variable concentration is shown in fig . 4 ( 18 ) .
the output from the receptor block was divided by the body weight to derive m / d / kg then contributed as input to the dose estimation section ( 5 , 18 ) .
the daily intake rate for the dose estimation is expressed by four linguistic fuzzy sets and is expressed in m / d / kg ( 18 ) . finally , the life average daily intake dose defined as seven fuzzy linguistic values and ranging from 0 to 6 g / kg / d .
input variable predicted concentration ( g / m ) a sample fuzzy inference calculation for the dose estimation block is shown in fig .
5 . where ir denotes inhalation rate normalized for body weight ( m / day / kg ) ; cc indicates chemical concentration ( g / m ) ; ladid = lifetime average daily inhalation dose ( g / kg / d ) . as an illustration ,
m / kg / d , the ambient benzene concentration is 15 g / m , lifetime average daily inhalation dose would be 1.63 g / kg / day .
fuzzy inference for estimation of ladid ( matlab software ; fuzzy toolbox ) comparison of observed and predicted concentration ( source : author 's calculations ) the output from this last module gives the overall dose through inhalation for each individual around the site , taking into account both the exposure factors and the receptor factors in a quantitative way , based on neural network and fuzzy inference .
when the exposure is quantified , combined with integrated risk information system ( iris ) toxicity factors , then as a final point the related risk can be estimated .
in this section , leave - one - out cross - validation ( loocv ) was followed with the aim of training and testing the ann model . frequently , one sample is kept for testing while the rest is used for training up to all samples are finally tested ( 26 ) .
before the proposed model is applied to the particular application , it must be trained using all available samples ( 27 ) .
the training of network continued until maximum correlation within the measured and predicted output was achieved ( table 3 ) .
correlation expressed by r - squared that r is coefficient of multiple determinations and relative root mean square error ( rmse ) ( 26 ) .
correlation results are perfect when an r - squared value of 1 , a very good fit is next to 1 and a very poor fit less than 0 . on the other side ,
how much the value of rrmse is smaller ; the performance of the model is better .
ambient air temperature affects significantly the exposure levels , as a result , the high temperatures in the summer and spring explains the increased benzene concentrations in exposure values .
when temperatures are very low ( winter - time ) , the exposure levels are less than usual for an equivalent quantity of close benzene level ( fig . 8) .
the presence of wind reduces exposure levels , especially to employees who are performing outdoor activities .
no strong correlation found between wind speed and exposure levels of employees working in laboratory and control room .
annually average concentration ( source : author 's calculation ) seasonal comparison of ambient concentration of benzene ( source : author 's calculations ) the measured benzene concentrations and the related exposure levels are not considered as an acute health risk ; meanwhile , this is the possibility of leukemia existence after chronic occupational contact with benzene due to measured concentration .
long - term individual risk will be calculated by combining usepa integrated risk information system ( iris ) toxicity features with results from exposure assessment section ( 18 ) .
the individual excess cr was calculated for exposure to benzene through inhalation is presented in table 4 .
the best estimate for the variable distribution of risk in a population implied 33% of people exposed less than 110 .
based on epa clean air act risk range , 110 risk range is considered as the most health protective end of the range ( 27 , 28 ) . whereas 110 is the midpoint of risk range and 67% of the population is ranging from 110 to 9.810 cancer risk probability ( 27 , 28 ) .
individual health risk assessment ( based on average exposure obtained by passive sampling ) the average estimated risk for all work areas considering exposure to benzene is equal to 2.410 , ranging from 1.510 to 6.910 .
the results counsel potential cancer risk for period exposure to benzene within the numerous areas but at different levels ( fig .
these differences result from differences in the employee 's type of activity , age , weight , and breathing rate that in common methods of risk assessment they did not pay attention to them
in this section , leave - one - out cross - validation ( loocv ) was followed with the aim of training and testing the ann model . frequently , one sample is kept for testing while the rest is used for training up to all samples are finally tested ( 26 ) .
before the proposed model is applied to the particular application , it must be trained using all available samples ( 27 ) .
the training of network continued until maximum correlation within the measured and predicted output was achieved ( table 3 ) .
correlation expressed by r - squared that r is coefficient of multiple determinations and relative root mean square error ( rmse ) ( 26 ) .
correlation results are perfect when an r - squared value of 1 , a very good fit is next to 1 and a very poor fit less than 0 . on the other side ,
how much the value of rrmse is smaller ; the performance of the model is better .
ambient air temperature affects significantly the exposure levels , as a result , the high temperatures in the summer and spring explains the increased benzene concentrations in exposure values .
when temperatures are very low ( winter - time ) , the exposure levels are less than usual for an equivalent quantity of close benzene level ( fig . 8) .
the presence of wind reduces exposure levels , especially to employees who are performing outdoor activities .
no strong correlation found between wind speed and exposure levels of employees working in laboratory and control room .
annually average concentration ( source : author 's calculation ) seasonal comparison of ambient concentration of benzene ( source : author 's calculations )
the measured benzene concentrations and the related exposure levels are not considered as an acute health risk ; meanwhile , this is the possibility of leukemia existence after chronic occupational contact with benzene due to measured concentration .
long - term individual risk will be calculated by combining usepa integrated risk information system ( iris ) toxicity features with results from exposure assessment section ( 18 ) .
the individual excess cr was calculated for exposure to benzene through inhalation is presented in table 4 .
the best estimate for the variable distribution of risk in a population implied 33% of people exposed less than 110 .
based on epa clean air act risk range , 110 risk range is considered as the most health protective end of the range ( 27 , 28 ) . whereas 110 is the midpoint of risk range and 67% of the population is ranging from 110 to 9.810 cancer risk probability ( 27 , 28 ) .
individual health risk assessment ( based on average exposure obtained by passive sampling ) the average estimated risk for all work areas considering exposure to benzene is equal to 2.410 , ranging from 1.510 to 6.910 .
the results counsel potential cancer risk for period exposure to benzene within the numerous areas but at different levels ( fig .
these differences result from differences in the employee 's type of activity , age , weight , and breathing rate that in common methods of risk assessment they did not pay attention to them . potential cancer risk
the complete conception in establishing an integrated product is a dynamic model for risk estimation , able to evaluate probable interactions among the levels of benzene exposure through different task designs , those lead to different levels of benzene metabolic rate and subsequently to discount risk estimations ( 29 , 30 ) . in this model ,
risk factors that can influence inhalation rate were age , body weight , and physical activity of persons .
these parameters considered as the input variable of fuzzy logic in order to estimate exposure level for the various situation of employees .
the hybrid system also used artificial neural network approach to predict actual atmospheric benzene concentration .
major variables affecting pollutant concentration were wind speed , ambient temperature , humidity , and rainfall .
expert viewpoint is used as fuzzy rules and the rules can be changed according to the needs of the user .
the produced composite model shows promise as a new tool for chemical exposure and health risk assessment , which it allows multiple uncertainties incorporate into health risk assessment .
in this project , the new hybrid dose model has been able to replicate a measured exposure prospering believed to be more representative of actual intake dose than data from previously accepted methods .
overall , according to aforementioned reasons this system can produce risk assessment data that appear realistic .
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 author . | background : reliable methods are crucial to cope with uncertainties in the risk analysis process .
the aim of this study is to develop an integrated approach to assessing risks of benzene in the petrochemical plant that produces benzene .
we offer an integrated system to contribute imprecise variables into the health risk calculation.methods:the project was conducted in asaluyeh , southern iran during the years from 2013 to 2014 .
integrated method includes fuzzy logic and artificial neural networks .
each technique had specific computational properties .
fuzzy logic was used for estimation of absorption rate .
artificial neural networks can decrease the noise of the data so applied for prediction of benzene concentration .
first , the actual exposure was calculated then it combined with integrated risk information system ( iris ) toxicity factors to assess real health risks.results:high correlation between the measured and predicted benzene concentration was achieved ( r2= 0.941 ) . as for variable distribution , the best estimation of risk in a population implied
33% of workers exposed less than 1105 and 67% inserted between 1.0105 to 9.8105 risk levels .
the average estimated risk of exposure to benzene for entire work zones is equal to 2.4105 , ranging from 1.5106 to 6.9105.conclusion:the integrated model is highly flexible as well as the rules possibly will be changed according to the necessities of the user in a different circumstance .
the measured exposures can be duplicated well through proposed model and realistic risk assessment data will be produced . |
type i ifns can be produced by almost any cell type in the body in response to stimulation of an array of transmembrane and cytosolic receptors .
in most cell types , the prevalent pathway of type i ifn induction is the activation of cytosolic receptors that recognize viral or other xenogeneic or autologous nucleic acid , particularly double - stranded rna ( dsrna ; kawai and akira , 2010 ; fig .
inducible gene i ( rig - i ) and melanoma differentiation - associated gene 5 are the prevalent cytoplasmic receptors responsible for triggering type i ifn secretion . these receptors ( rig - i like receptors ) transmit signals through the mitochondria - localized adaptor molecule ips-1 or mavs and the kinases tbk1 and ikki to activate irf3 and induce transcription of the type i ifn genes ( kawai and akira , 2010 ) . a cytosolic dna sensor , dna - dependent activator of irfs ( dai ) or z - dna binding protein 1 , was shown to induce type i ifn in response to the right - handed dsdna conformation ( b - dna ) in a tbk1- and irf3-mediated mechanism ( kawai and akira , 2010 ) .
however , dai may not be a unique dna sensor and may not be active on all cell types .
the existence of other cytoplasmic dna sensors is also suggested by the ability of the bacterial second messenger cyclic - di - gmp to potently induce type i ifn via a mechanism that is independent of dai or other known cytoplasmic receptors but requires tbk1 and irf3 ( mcwhirter et al . , 2009 )
. an alternative mechanism of dsdna sensing depends on the ability of cytosolic dna - dependent rna - polymerase iii to transcribe b - dna into 5-ppp rna , which then activates type i ifn transcription through rig - i ( chiu et al . , 2009 )
type i ifn production can be triggered by recognition of dsrna by the cytosolic receptors mda5 and rig - i or of dsdna ( b - dna ) by dai or as yet unknown cytosolic dna receptors ( dna - rx ) , leading to activation of the irf3 via the kinase tbk-1 ( or ikki ) and culminating in production of type i ifn ( ifn/4 ) .
dsdna can also be transcribed by rna - polymerase iii into 5-ppprna , which triggers rig - i .
the ability of the bacterial product cyclic - di - gmp to induce type i ifn suggests the existence of additional dna sensors .
the production of type i ifn can be amplified by a positive feedback loop in which the early produced ifn- and ifn-4 trigger the transcription of irf7 and , at least in dcs , irf8 . in macrophages and dcs ,
ligation of tlr3 , tlr4 , tlr7 , and tlr9 triggers type i ifn production via signaling through adaptor molecules , including myd88 , tirap , tram , and trif .
trif associates with tbk1 to activate irf3 , whereas tlr7 and tlr9 signal through myd88 and irak4 to activate irf7 .
the cytoplasmic nod receptors can also induce type i ifn production in response to bacterial cell wall components via activation of the kinase rick and downstream signaling molecules including nf-b and tbk-1 .
the production of type i ifn is further regulated by a positive feedback loop that is based on the ability of ifn- and ifn-4 to induce the transcription of irf7 and , at least in dcs , irf8 ( mari et al . , 1998 ;
1 ) . once phosphorylated , these transcription factors help drive the expression of all genes of the type i ifn family , thereby amplifying type i ifn production . in some cell types ( e.g. , macrophages and dcs ) ,
type i ifn is produced in response to triggering of the transmembrane receptors toll - like receptor 3 ( tlr3 ) and tlr4 by dsrna and lipopolysaccharide , respectively ( fig .
1 ) . tlr3 and tlr4 signal through the adaptor molecule trif , which associates with tbk1 and activates irf3 ( kawai and akira , 2010 ) .
tnf receptor - associated factor 3 ( traf3 ) associates with trif , as well as tbk1 and ikki , and is required for production of type i ifn and il-10 , but not other cytokines , in the response to tlr signaling .
thus , traf3 links tlr adaptors and downstream regulatory kinases important for irf activation ( hcker et al . , 2006 ; oganesyan et al . , 2006
although all or most cell types can produce type i ifn , the existence of a cell type specialized for the production of large amounts of type i ifn and required for nk cell mediated killing of virus - infected cells ( natural ifn - producing cell ) was identified in humans over 30 yr ago ( trinchieri et al . , 1978 ) , and more recently was discovered in the mouse ( asselin - paturel et al . , 2001 ) .
natural ifn - producing cells are now usually referred to as plasmacytoid dcs ( pdcs ) because of their round morphology , eccentric nucleus , and abundant endoplasmic reticulum ( colonna et al .
pdcs preferentially express the intracellular endosomal receptors tlr7 and tlr9 , allowing them to respond to single - stranded rna and dna viruses , respectively , by triggering signal transduction through the adaptor protein myd88 ( colonna et al .
these receptors are efficient in inducing type i ifn only in pdcs because these cells constitutively express irf7 ( and irf8 ) , and the myd88irf7 complex undergoes a spatiotemporal regulation upon tlr ligation such that it is retained in the endosomal compartment , where it induces type i ifn production ( colonna et al . , 2004 ) .
bacterial dna can activate tlr9 in these cells , and it has recently been shown that phagosomal bacteria such as group b streptococcus , but not cytosolic bacteria , also induce type i ifn in conventional dcs via tlr7 , myd88 , and irf1 ( mancuso et al . , 2009 ) .
in many ( but not all ) viral infections , pdcs are important for the production of most early circulating type i ifn , but are not essential for antiviral innate resistance , as many other cell types are able to produce type i ifn during the infection ( colonna et al . , 2004 ) . a novel mechanism of type i ifn production by cytoplasmic ligands was shown to involve the nucleotide - binding oligomerization domain containing protein ( nod ) 1 and nod2 receptors ( pandey et al . , 2009
nods are card - containing cytoplasmic molecules in epithelial and hematopoietic cells that contain leucine - rich repeats recognizing related muropeptide subunits of the bacterial wall .
nods signal through the kinase rip2 ( also called rick ) and activate nf-b and other transcription factors , resulting in the production of proinflammatory cytokines .
it has recently been observed , however , that rick can interact with traf3 and mavs , thus inducing the activation of tbk1 and ikki , phosphorylation of irf5 or irf7 , and transcription of the ifn- gene ( fig .
1 ) . type i ifn can be induced by mycobacterium tuberculosis through the activation of nod2 in macrophages ( pandey et al . , 2009 ) and
by helicobacter pylori through nod1 in epithelial cells ( watanabe et al . , 2010 ) .
exposure of cells to ifn before infection induces an antiviral state that prevents productive viral infection .
ifn receptor signaling induces and activates intrinsic antiviral factors such as rna - activated protein kinase , the 2 - 5a system , and the mx proteins .
in addition , ifns potently regulate both innate and adaptive immune responses , acting directly or indirectly on nk cells , t cells , b cells , dcs , and phagocytic cells ( prchal et al . , 2009 ) . in
most or all pathogenic virus infections , early production of type i ifn is required to limit initial viral replication before effective humoral or cellular adaptive immune mechanisms become operational .
not surprisingly , many viruses have evolved molecular mechanisms to either prevent type i ifn induction or block the signaling through its receptors ( bowie and unterholzner , 2008 ) . however , in addition to their cell intrinsic or extrinsic antiviral effects , ifns mediate many anticellular effects by modulating cell viability and function ( e.g. , induction of apoptosis , autophagy , differentiation , cell migration , and inhibition of cell proliferation and angiogenesis ; bekisz et al .
thus , ifn produced during viral infection , other pathological conditions , or in the presence of dna released by dying cells may mediate unwanted toxicity or induce pathological damage and inflammatory or autoimmune syndromes ( pascual et al . , 2010 ) .
hiv induces type i ifn , which together with tryptophan catabolism , may drive t cell dysfunction , and also contribute to other aspects of the disease pathogenesis ( boasso et al . , 2008 ) .
in bacterial infections , type i ifn is often produced , but its effects are complicated and do not always favor the immune response of the host against the infection . as in viral infections ,
the first observation of the ability of type i ifn to prevent the intracellular replication of bacteria was for pathogens of the genus chlamydia replicating in nonhematopoietic human cells ( de la maza et al . , 1985 ) .
in this case , rather than inducing intracellular killing of the bacteria , type i ifn inhibits the chlamydia growth cycle at the point of transformation from elementary to reticulate body , resulting in chronic infection and failure to produce infectious organisms ( ishihara et al . , 2005 ) .
the anti - chlamydia effect of type i ifn is reversed by exogenous l - tryptophan and iron , which are essential for chlamydial growth , possibly secondary to induction of indoleamine 2,3-dioxygenase ( ishihara et al . , 2005 ; puccetti , 2007 ) .
in human endothelial cells , mavs , irf3 , irf7 , and endogenous ifn- are necessary for the control of c. pneumoniae replication ( buss et al . , 2010 ) .
similarly , the limited ability of legionella pneumophila to replicate in murine macrophages is caused by an effective innate response involving autocrine type i ifn signaling . in these cells
, ifn suppresses intracellular replication of l. pneumophila via a stat1- , 2- , and 3-independent pathway that induces the differentiation of classically activated macrophages as well as the production of nitric oxide , which is required for the antibacterial effect ( plumlee et al . , 2009 ) .
type i ifn also protects mice against salmonella typhimurium infection by activating ifn- production via stat4 activation in an il-12-independent way ( freudenberg et al . , 2002 ) .
phagosomal bacteria such as group b streptococci can induce type i ifn via tlr7 , and this is important for production of proinflammatory cytokines by macrophages and resistance to the infection ( mancuso et al .
other possible antibacterial effects of type i ifn have been postulated based on its ability to modulate innate and adaptive response against bacteria , e.g. , nk cell activation , antibody production , and dc activation ( decker et al . , 2005 ) .
often , however , the production of type i ifn is associated with suppression of the innate response to the bacterial infections .
indeed , type i ifn receptor deficient mice ( ifnar ) are more resistant to infection with francisella tularensis and listeria monocytogenes than are wt mice ( fehr et al . , 1997 ; auerbuch et al .
, 2004 ; carrero et al . , 2004 ; stockinger and decker , 2008 ; oconnor et al .
furthermore , the ability of mycobacterium tuberculosis strains to induce type i ifn production has been proposed to correlate with bacterial virulence ( manca et al .
m. tuberculosis infected ifnar mice have decreased and late mortality compared with wt animals ( manca et al . , 2005 ) ; however , at least in the first 23 mo after infection , their bacterial load was similar in the lungs and only slightly lower in the spleen ( cooper et al .
, 2000 ; manca et al . , 2005 ; stanley et al . , 2007 ) .
modular and pathway analysis of the transcriptomes from the blood of patients with tuberculosis revealed that the signature was dominated by a neutrophil - driven ifn - inducible gene profile , consistent with both type i and type ii ifn signaling , suggesting both changes in cellular composition and altered gene expression ( berry et al . , 2010 ) .
thus , type i ifn has a hitherto underappreciated role in tuberculosis pathogenesis , which has implications for vaccine , diagnostic , and therapeutic development .
several mechanisms may account for the deleterious effects of type i ifn in bacterial infections .
type i ifn increases the susceptibility of different cell types to apoptosis - inducing stimuli , in particular macrophages and lymphocytes .
this is well described in infection with listeria , mycobacteria , brucella , and chlamydia spp ( carrero et al . , 2004 ; gold et al . , 2004 ;
oconnell et al . , 2004 ; decker et al . , 2005 ; qiu et al . ,
2008 ) . in l. monocytogenes infection , the pore - forming virulence factor listeriolysin o is required for ifn induction in response to infection , and ifn in turn facilitates the listeriolysin o induced lysis of lymphocytes and macrophages ( carrero et al . , 2004 , 2006 ) .
indeed , more virulent strains induce a higher level of type i ifn than do less virulent strains , and the difference in virulence disappears in ifnar mice ( reutterer et al . , 2008 ) .
type i ifn alone is not sufficient to induce apoptosis in most cell types ; rather , its effects likely involve up - regulation of caspases and pro - apoptotic innate sensors , such as tlrs and the inflammasomes .
for example , production of type i ifn is required for francisella spp to induce inflammasome activation , possibly caused by the ifn - driven up - regulation of the cytosolic aim2 inflammasome ( choubey et al . , 2010 ; fernandes - alnemri et al . , 2010 )
activation of aim2 then results in caspase1 activation and in the production of il-1 and il-18 as well as inflammasome - mediated cell death in infected cells .
type i ifn is also important to induce the up - regulation of rig - i and melanoma differentiation - associated gene 5 in epithelial and other cells and this may represent a type i ifn amplifying loop in several types of infections ( haller et al . , 2006 ; jaitin and schreiber , 2007 ) .
these receptors respond to foreign nucleic acids and initiate a type i ifn - dependent proapoptotic signaling pathway that requires the mitochondrial adapter ips-1 , induces the production of the proapoptotic bh3-only proteins puma and noxa , and culminates in the efficient activation of caspase-9- and apaf-1-dependent mitochondrial apoptosis ( besch et al . , 2009 ) .
type i ifn is also required for the apoptosis resulting from infection - induced activation of the trif - associated receptors tlr3 and tlr4 , in part by suppressing akt signaling and degradation of the cdk inhibitor p27 ( hasan et al .
for example , type i ifn , may prevent the ability of ifn- to activate antibacterial function of macrophages either by directly interfering with ifn- signaling , as discussed or by lowering ifn- production .
type i ifn also exerts direct effects on phagocytic cells thus decreasing the activation of anti - microbial products and favoring bacterial replication and release ( rayamajhi et al . , 2010 ) .
thus , the evolutionary pressure for a rapid and strong innate resistance in the early stages of aggressive viral infections may have been stronger than that exerted by the relative decrease in resistance to bacterial infections .
it constrains the secretion of il-17 by t cells , as observed in f. tularensis and l. monocytogenes infections ( henry et al .
, 2010 ) and limits th1 immune responses to m. tuberculosis in part by inducing negative regulator molecules such as suppressor of cytokine signaling proteins ( manca et al . , 2005 ) .
type i ifn can also suppress th17 responses by inducing the expression of il-27 , which then suppresses il-17 production ( guo et al . , 2008 ) .
type i ifn also triggers the generation of il-10producing regulatory t ( tr1 ) cells that dampen both innate and adaptive resistance to infection ( dikopoulos et al . , 2005 ; levings et al . , 2001 ) .
type 1 ifn might also affect antibacterial resistance by modifying the recruitment and activation of myeloid cells at the site of infection .
resistance to l. monocytogenes infection requires the ccl2- and ccl7-dependent recruitment of a class of activated monocytes / dcs that produces tnf and inducible nitric oxide synthase ( tip - dcs ; fig .
2 ) and mediates innate immune defense ( jia et al . , 2009 ) .
signals through myd88 are involved in the recruitment and activation of tip - dcs , but these signals can be replaced in part by type i ifn .
thus , the deletion of ifnar in myd88-deficient mice results in increased rather than decreased susceptibility to infection ( jia et al .
, 2009 ) , indicating that type i ifn has both positive and negative effects on the resistance to l. monocytogenes infection .
type i ifn drives the production of chemokines , such as ccl2 and ccl7 , which recruits ccr2-expressing tnf and inducible nitric oxide synthase producing dcs ( tip - dcs ) that are required for defense against listeria infection .
ccr2-expressing macrophages also contribute to lung pathology during influenza infection and are highly permissive to infection with m. tuberculosis . during influenza infection ,
type i ifn inhibits the production of the cxcr2 ligands cxcl1 and cxcl2 , thus decreasing neutrophil recruitment and dampening host defense against secondary bacterial infections .
however , type i ifn also drives the production of cxcl10 , which recruits activated cxcr3-expressing neutrophils , thus protecting against sepsis in response to clp .
the opposing roles of type i ifn are illustrated by influenza virus and secondary bacterial infections ( fig .
however , ifn also induces chemokines such as ccl2 that recruit ccr2 monocyte - derived dcs and macrophages to the site of infection , resulting in infection - associated pulmonary pathology and mortality ( lin et al . , 2008 ) .
type i ifn inhibits the production of cxcr2 ligands , thus reducing neutrophil recruitment and facilitating the development of secondary bacterial pneumonia ( shahangian et al . , 2009 ) .
a similar type i ifn mediated decrease in neutrophil numbers was observed in mice infected with lymphocytic choriomeningitis virus and was associated with susceptibility to bacterial infection ( navarini et al . , 2006 ) .
in addition , ifn induced by intranasal poly - ic treatment exacerbates tuberculosis in mice by recruiting a population of ccr2-expressing macrophages that is highly permissive for m. tuberculosis proliferation ( antonelli et al . , 2010 ) .
type i ifn also has complex and often opposing effects in controlling susceptibility and lethality in various models of sepsis or endotoxic shock . during endotoxic shock in mice , type
i ifn deficiency reduces lps - induced lethality but not the production of other proinflammatory cytokines or nitric oxide ( karaghiosoff et al . , 2003 ) .
similar results were reported in a model of high lethality septic peritonitis induced by colon ascendens stent , as well as in a model of liver injury after ischemia and reperfusion ( weighardt et al . , 2006 ; zhai et al . , 2008 ) . in the septic peritonitis model
, ifnar mice displayed a significantly increased peritoneal neutrophil recruitment and activation , which is perplexing given that ifnar mice had increased local production of ccl2 , which primarily recruits macrophages , decreased cxcl10 , which recruits nk cells and activated th1 cells , and unaltered cxcl1 , which recruits neutrophils ( weighardt et al . , 2006 ) .
however , despite their increased endotoxin resistance , ifnar mice have increased late mortality in a low lethality model of cecal ligation and puncture ( clp)induced sepsis ( fig .
2 ) . increased mortality in the clp model was attributed to decreased production of cxcl10 and failure to recruit the phagocytic neutrophils that are necessary for resistance in this model ( kelly - scumpia et al . , 2010 ) .
the differential effect of type i ifn on neutrophil recruitment in the two models of polymicrobial sepsis is difficult to explain and might relate to differences in bacterial load and lethality . in influenza virus infected mice , decreased neutrophil recruitment to the lungs was attributed to the ability of type i ifn to decrease the production of cxcr2 ligands , such as cxcl1 and 2 .
this was not observed in either sepsis model , whereas cxcl10 production was decreased in both sepsis models , albeit with opposite effects on neutrophil recruitment and activation ( weighardt et al . , 2006 ; shahangian et al . , 2009
the role of type i ifn in chemokine - induced neutrophil recruitment falls short of explaining the full range of type i ifn s regulatory effects on toxicity and infection in these complex models . a quite different mechanism
should also be invoked to explain how type i ifn drives tnf - induced lethal shock ( huys et al . , 2009 ) .
tnf induces ifn- production in vitro , and this may establish an autocrine loop that promotes the sustained production of chemokines and other stat1-dependent genes ( yarilina et al . , 2008 ) .
although type i and type ii ifn share some common mechanisms in the activation of the immune response , ifn- is generally a more potent activator of phagocytic cell and antigen - presenting cell function than type i ifn .
the interaction between type i and type ii ifn signals is complex and has received relatively little attention .
both types of ifn are often produced during the course of the same immune response , and evidence suggests that cross - regulation exerts physiologically meaningful effects ( fig .
, exposure of macrophages to type i ifn in vitro inhibits ifn- binding to the cells and prevents ifn- from enhancing class ii mhc expression , fc receptor expression , and oxidative burst ( ling et al . , 1985 ; yoshida et al . , 1988 ) .
l. monocytogenes infection induces type i ifn production , which suppresses macrophage activation by ifn- by decreasing ifngr1 mrna and ifn- receptor surface expression ( rayamajhi et al . , 2010 ) .
cross - talk between type i ifn and ifn-. high levels of type i ifn ( solid arrows ) decrease the expression of ifngr1 and inhibit ifn--induced activation of mhc class ii expression , oxidative burst , and bactericidal activity in macrophages .
however , low constitutive levels of type i ifn ( dashed arrows ) prime cells for secondary responses to type i and ii ifns and il-6 by favoring expression and activation of stat1 ( shown in fibroblasts ) . in dcs , low concentrations of type i ifn are essential for the optimal production of the ifn-inducing cytokine il-12p70 , whereas higher levels of type i ifn suppress tlr- and ifn-induced il-12p40 expression , thus dampening il-12p70 production .
type i ifn can also directly induce the production of ifn- in nk cell and t cells via the activation of stat4 .
in addition to signaling through the isgf3 complex , type i ifn can activate stat1 homodimers , and thus mimic the gene induction pattern of ifn- ( van boxel - dezaire et al . , 2006 ) .
in addition , cells are continuously exposed to low levels of type i ifn , and the resulting weak signal is required for the cells to produce high levels of ifn in response to inducing stimuli ( taniguchi and takaoka , 2001 ) .
for example , the priming of cells by constitutive sub - threshold type i ifn signaling enhances their response to stimulation with other cytokines , including an at least 10-fold enhancement of the response to ifn- , by favoring stat1 activation .
this sensitization has been attributed to the association of the two nonligand binding receptors components , ifnar1 and ifngr2 , in the caveolar membrane domains ( takaoka et al . , 2000 ) .
more recently , constitutive expression of c - jun was shown to induce endocrine or paracrine production of low levels of ifn- that maintains expression of stat1 required for optimal cellular responses to type i ifn , as well as to ifn- and il-6 ( gough et al . , 2010 ) .
furthermore , the induction of low levels of type i ifn during stimulation of dcs through innate receptors is essential for optimal production of the il-12 p70 heterodimer ( gautier et al . , 2005 ) , thus indirectly contributing to ifn- production . in the presence of high concentrations of type
i ifn , however , the production of il-12p40 is suppressed , limiting the amount of il-12 heterodimer ( byrnes et al . , 2001 ) .
by signaling through stat4 , type i ifn has also been reported to directly induce the production of ifn- in nk and t cells , particularly in the absence of stat1 , which normally suppresses ifn- production ( brinkmann et al .
, 1993 ; freudenberg et al . , 2002 ; nguyen et al . , 2000
because of the transient nature of stat4 activation by type i ifn and the regulatory effect of stat1 , the physiological role of type i ifn in controlling ifn- production , as compared with classical ifn-inducing cytokines such as il-2 and il-12 , remains to be evaluated .
the connection between viral or other infections and autoimmunity has been known for a long time , although the precise mechanisms and the possible role of type i ifn in pathogenesis has remained difficult to determine ( mnz et al . , 2009 ; rose , 2010 ) .
the development of lyme arthritis after borrelia burgdorferi infection is associated with an ifn - induced gene expression signature that is independent of t and b cells ( miller et al . , 2008 ) .
blocking type i , but not type ii , ifn signaling reduced both the ifn signature and the development of arthritis ( miller et al . , 2008 ) . therapeutic treatment with type
i ifn for cancer , chronic viral infections , and multiple sclerosis has been associated with significant collateral toxic effects characterized by chronic fatigue because of transient or chronic autoimmune pathology ( malik et al .
. the mechanisms of type i ifn - induced autoimmunity has been attributed in part to increased dc activation and antigen uptake and presentation from apoptotic cells , as well as increased production of autoantibodies ( jego et al .
a prominent feature of systemic lupus erythematosus ( sle ) is the increased expression of type i ifn regulated genes in blood cells , which is often associated with the presence of ifn in the plasma ( bennett et al .
the cells producing type i ifn in sle appear to be predominantly pdc , which are reduced in number in the blood but are abundant in skin and lymph nodes ( rnnblom et al . , 2009 ) .
in sle patients , immune complexes consisting of autoantibodies bound to self - dna and rna can stimulate production of type i ifn through tlr7 and tlr9 after being internalized following binding to fc receptors ( rnnblom et al . , 2009 ) .
stimulation of pdcs through tlr7 and tlr9 also renders these cells resistant to the apoptotic effect of glucocorticosteroids , thus decreasing the ability of these drugs to reduce type i ifn production and effectively treat the patients ( guiducci et al . , 2010 ) .
several genes that have been associated with susceptibility to sle are involved either in the regulation of the ifn response or in the ability to clear immune complexes through fc receptors , and thus to degrade nucleic acid released from dying cells ( morel , 2010 ) .
the pathogenic role of type i ifn in sle is mediated in part by its ability to induce immune activation , including a positive feedback loop that induces plasma cell maturation and increases autoantibody formation ( jego et al . ,
pathology in sle could also be caused by the ability of type i ifn to directly induce pathogenic effects at the tissue level . for example , type i ifn directly induces vasculogenesis and may be responsible for the premature atherosclerosis found in sle patients ( denny et al . , 2007 ) .
the role of type i ifn in this disease has now been confirmed in lupus - prone mouse strains ( agrawal et al .
the presence of pdcs in autoimmune target tissues is associated with an ifn - induced gene signature , suggesting the possible involvement of type i ifn in several other autoimmune disorders , including diabetes mellitus , dermatomyositis , and sjogren s syndrome ( guiducci et al . , 2009 ) .
a clear association also exists between type i ifn and cutaneous skin diseases characterized by interface dermatitis , such as lichen planus , dermatomyositis , lichen sclerosus , cutaneous lupus , and cutaneous graft - versus - host disease , in which pdc infiltrates and deposition of nucleic acid containing immune complexes is observed ( wenzel and tting , 2008 ) .
a pdc infiltrate linked to an ifn signature is also observed in psoriasis . although tissue deposition of immune complexes is not observed in psoriasis , the antimicrobial cationic peptide ll-37 accumulates in lesions where it binds to and transports self - dna and rna to the early endosomal compartment of pdc , thereby stimulating type i ifn production through tlr9 and tlr7 , respectively ( ganguly et al . , 2009 ) .
the involvement of type i ifn in the pathogenesis of several autoimmune diseases indicates the possibility to therapeutically target either type i ifn or the mechanisms leading to ifn production in those diseases ( guiducci et al . , 2009 ; pascual et al . , 2010 )
paradoxically , however , in several experimental and clinical autoimmune conditions , treatment with type i ifn or its induction has proven beneficial . in most cases , these conditions are tissue - specific autoimmune or inflammatory syndromes characterized by activation of effector th1 and th17 responses , including arthritis , inflammatory bowel diseases , and multiple sclerosis ( lee et al . , 2006 ; yarilina et al . , 2007 ;
ifn- is a common and effective treatment for reducing disease recurrence in multiple sclerosis ( noseworthy et al . , 2000 ;
the mechanisms by which type i ifn affects these pathological conditions are most likely linked to its immunoregulatory effects , including its ability to affect il-12 production , to activate regulatory t cells , and to constrain th17 cell mediated autoimmune inflammation ( byrnes et al . , 2001 ; levings et al .
several of the biological functions of type i ifn , including its regulation of innate and adaptive immunity and its antiangiogenic and proapoptotic effects , make it an obvious candidate for anti - cancer therapy .
indeed , type i ifn has been used with some success for the treatment of several types of cancer , including hematological malignancies ( hairy cell leukemia , chronic myeloid leukemia , and some b and t cell lymphomas ) , and solid tumors ( melanoma , renal carcinoma , and kaposi s sarcoma ; ferrantini et al .
, 2007 ; moschos and kirkwood , 2007 ) . in some of those indications ,
type i ifn therapy is still in use ; in others it has been superseded by more effective targeted therapies .
the antitumor effect of type i ifn therapy is accompanied by severe side effects , including autoimmune and inflammatory symptoms , as well as direct tissue toxicity , that are probably responsible for the hematological and neurological side effects .
it was recently shown that endogenous type i ifn , like ifn- , prevents the growth of primary carcinogen - induced and transplantable tumors ( dunn et al . , 2005 ) .
unexpectedly , unlike ifn- , type i ifn does not act directly on tumor cells .
hematopoietic cells , in particular nk cells , are critical ifn-/ targets during development of protective antitumor responses ( dunn et al . , 2005 ; swann et al . , 2007 )
thus , one of the earliest described immunoregulatory functions of type i ifn its ability to regulate nk cell functions ( trinchieri and santoli , 1978)has now been proven to be one of the major mechanisms of the regulation of tumor growth by endogenous type i ifn . however , a possible intrinsic role of type i ifn in regulating tumor cell proliferation , apoptosis , and autophagy , as discussed above for infectious diseases , should not be discounted and has only recently begun to be investigated in depth ( goldszmid et al . , 2009 ) .
the ability of oncogenes such as ras and hpv16 e6e7 to down - regulate ifn - inducing innate receptors , rig - i and tlr9 , respectively , is suggestive of a role of type i ifn in modulating not only the infectiveness of oncogenic viruses but also their cell transforming ability ( hasan et al . , 2007 ; shmulevitz et al . , 2010 ) .
in these last few years , our understanding of the mechanisms controlling the production and the function of type i ifn in infection , immunity , autoimmunity , and oncology has been progressing at a very fast pace , and the role of this family of cytokines in physiology and pathology is finally starting to be fully appreciated .
type i ifn are essential for organisms to survive acute viral infection , even if many viruses have developed mechanisms to escape their effects . however , the organism pays a price for this effective innate resistance , as the toxicity of ifn causes acute and chronic morbidity and alters innate and adaptive defenses against other opportunistic infections .
louis cruveilhier wrote during the spanish flu epidemic of 19181919 : si la grippe tue , cest parce quelle frappe au thorax or ( to paraphrase ) , flu condemns , and additional infection executes
however , as the mechanisms underlying the actions of type i ifn are becoming better understood , there is hope of a more rationale therapeutic targeting . | although the role of type i interferon ( ifn ) in the protection against viral infections has been known and studied for decades , its role in other immunologically relevant scenarios , including bacterial infections , shock , autoimmunity , and cancer , is less well defined and potentially much more complicated . |
in gynecology endosalpingiosis is a benign condition in which fallopian tube - like epithelium is found outside of the fallopian tube .
endosalpingiosis is associated with the formation of psammoma bodies , which may lead to the misdiagnosis of malignancy such as serous carcinoma.1 the significance of endosalpingiosis is not settled ; medical experts differ on whether it causes pelvic pain , or is an incidental ( asymptomatic ) finding discovered in the course of investigating pelvic pain , menstrual irregularities or infertility .
endosalpingiosis is occasionally found in lymph nodes , and may be misinterpreted as an adenocarcinoma metastasis . in one study , they found that 34.5% of endosalpingiosis cases had concurrent endometriosis ; 40% of the endosalpingiosis group were postmenopausal .
in contrast , there was no significant link between endosalpingiosis and infertility nor chronic pelvic pain .
a 75-year - old woman , gravida 2 , postmenopausal women , presented with vaginal bleeding which occurred a week ago .
she had hyperlipidemia and took medical therapy . under the impression of pelvic mass , cancer antigen 125 ( ca-125 ) ,
ca 19 - 9 , complete blood cell count , serum chemistry , contrast enhanced - abdomen - computer tomography were done .
ca 19 - 9 is 9.8 u / ml , ca-125 is 13.5 u / ml and glucose is 118 mg / dl .
in contrast enhanced chest computer tomography , image showed multiseptated cystic mass in left adnexa which adhesion with left psoas muscle , adjacent small and large bowel loops and there is possibility of underlying chronic inflammatory process with abscess ( fig .
vaginal bleeding is caused by the atrophic vaginitis considering that the patient 's age is 75 years old .
endosalpingiosis is a non - neoplastic process and is generally considered to be derived from the secondary mllerian system , which consists of structures covering the peritoneal mesothelium , the adjacent mesenchyme in the small pelvis and the lower part of the female abdominal cavity.3 proliferation of these structures can result in the creation of three different types of lesions : endometriosis , which occurs most frequently , and the less common endosalpingiosis and endocervicosis.4 the symptom of endosalpingiosis is not settled ; medical experts differ on whether it causes pelvic pain,5 or is an incidental ( asymptomatic ) finding discovered in the course of investigating pelvic pain , menstrual irregularities or infertility.6 the causes of the endosalpingiosis are unknown . in this case
however , it is almost always an incidental finding , either at the time of operation or , more commonly , on microscopic examination.7 it is most commonly encountered on the pelvic peritoneum covering the uterus , fallopian tubes , ovaries and culde - sac .
less frequent sites include the pelvic parietal peritoneum , omentum , bladder and bowel serosa , paraaortic area and skin.8 only a few examples of tumor - like cystic endosalpingiosis localized to the female pelvis have been described , and most of these cases are reported to be derived from the serosal surface of the uterus and the ovary 6 - 10 or from the paraovarianregion.8 for diagnosis of endosalpingiosis , pathology on excision ( e.g. biopsy ) , is needed .
it is characterized by cysts with tubal - type epithelium ( e.g. ciliated epithelium ) surrounded by a fibrous stroma . unlike endometriosis , it is not associated with hemorrhage .
a tubal - type epithelial surrounded by endometrial - type stroma is a variant of endometriosis , not endosalpingiosis .
endosalpingiosis is occasionally found in lymph nodes , and may be misinterpreted as an adenocarcinoma metastasis.9 the pathological differential diagnosis of endosalpingiosis is discussed in detail in previously reported cases.7,10,11,12,13,14 however , the clinical differential diagnosis of an intramural mass , whether it causes any symptoms or not , is more important for clinicians when discussing further surgery with the patient .
therefore the symptoms and the imaging findings of this rare entity are very important . as endosalpingiosis ,
endosalpingiosis appears to affect an older age group , with 40% of endosalpingiosis cases occurring in postmenopausal women.2 premenopausal women with endosalpingiosis were more likely to have a gynecologic malignancy.2 endosalpingiosis appears to affect postmenopausal women at a much higher rate than previously thought , and in consequence the average age of women presenting with endosalpingiosis was higher than expected.2 in this case , 75-year - old woman , gravida 2 , postmenopausal women , presented with vaginal bleeding was reported .
however , it must be kept in mind that vaginal bleeding can be caused by endosalpingiosis , especially in the postmenopausal elderly women .
possibility of endosalpingiosis must be considered in the differential diagnostic spectrum of vaginal bleeding , thus avoiding the problem of misdiagnosis of endosalpingiosis . | in gynecology , endosalpingiosis is a benign condition in which the fallopian tube - like epithelium is found outside the fallopian tube .
the thirty - four point five percent of endosalpingiosis cases have concurrent endometriosis and 40% of the endosalpingiosis group are in postmenopausal states .
in contrast with endometriosis , there are no significant links between infertility , chronic pelvic pain and endosalpingiosis .
the symptoms of endosalpingiosis are not yet settled .
endosalpingiosis is almost always an incidental finding ; it is commonly found through microscopic examinations , and is then confirmed by pathologists for excision and biopsy .
therefore , the clinical differential diagnosis of an intramural mass is more important for clinicians when discussing further surgery with the patients .
we report case of woman who has endosalpingiosis and is presented with vaginal bleeding .
we first suspect the disease during physical examination . under the impression of pelvic mass , laboratory tests and radiological images of contrast
enhanced chest computer tomography are taken .
images show multisepted cystic masses in left adnexa . to rule out the pelvic mass , we executed exploratory laparotomy .
pathologic results show endosalpingiosis near the ovary section .
but the endosalpingiosis , is not generally considered a pathology , and thus , no treatment is necessary . |
breast cancer is the most common cancer diagnosed in women worldwide . in the united states the estimates for 2010 were 209,060 new cases of invasive breast cancer and 40,230 deaths .
the overall 5-year relative survival of patients with metastatic breast cancer is 23% , while the relative survival of breast cancer patients with nonmetastatic tumors is 98% .
patients who have no evidence of tumor dissemination at the time of diagnosis are still at risk of metastatic disease .
approximately one - third of women who are sentinel lymph node negative at the time of surgical resection of the breast primary tumor will eventually develop clinically detectable secondary tumors .
therefore , understanding the mechanisms governing tumor dissemination and developing new strategies to control or effectively treat patients with or at risk of metastatic disease would significantly improve the overall outcome of the disease .
metastasis is a multistep complex process that involves the detachment of tumor cells from the primary tumor , migration and invasion through the surrounding tissues and basement membranes , intravasation and survival in the small blood vessels or lymphatic channels , and colonization in a distant target organ .
these steps are usually followed by extravasation into the surrounding tissue , survival in the foreign microenvironment , proliferation , and induction of angiogenesis ( figure 1 ) .
it has become apparent that the vast majority of tumor cells within the primary tumor and also the disseminated tumor cells will not form distant metastases , either because they die or remain dormant .
the dormancy phenomenon probably explains what is seen in the clinic in which some cancer patients remain free of clinical evidence of metastatic disease for years or even decades after primary tumor resection , and after this prolonged period of time these patients show signs of tumor relapse .
the development of the primary tumor microenvironment is also an important determinant of tumor dissemination ; this tumor microenvironment may influence the release of cancer cells into the blood and the lymphatic systems and subsequently promote continued survival and proliferation at the secondary site .
it has been well known that the interaction between tumor cells and their microenvironment is important for establishing metastatic colonies and for defining the balance between dormancy and malignant growth [ 5 , 6 ] .
furthermore , the microenvironment of metastatic tumor cells has recently been thought to play a major role in tumor progression .
although tumor cells may be continually released from the primary site , relatively few of them are able to efficiently form macrometastasis .
what are the factors controlling this important step ? in this paper we will discuss a more global view of host - tumor interactions in which the metastatic potential of tumors is an inherent component of cells forming the primary tumor mass at an early time in tumor progression .
we will also discuss the association of two genes , brd4 and sipa1 ( signal - induced proliferation - associated 1 ) , with mammary tumor progression in both the mouse and the human . here
, we will focus on the role that the metastasis susceptibility gene brd4 plays in the regulation of extracellular matrix ( ecm ) gene expression and subsequently metastatic progression .
studies from our laboratory have demonstrated that the inherited polymorphism , or the genetic background on which a tumor arises , plays an important role in determining the probability that the given tumor will progress to metastatic disease .
these findings are based on a series of genetic mapping studies using the highly metastatic polyoma middle - t ( pymt ) transgenic mammary mouse model .
this mouse expresses the mouse polyoma virus middle - t antigen in the mammary epithelium of fvb / n inbred mice from an early age , which results in the development of highly aggressive mammary tumors that metastasize to the lung with high frequency . specifically , when the male pymt transgenic mouse was bred to different inbred strains , the f1 progeny showed significant variation in tumor characteristics , such as tumor latency , growth kinetics , and metastatic capacity .
it is important to point out that these tumors were all induced by the same oncogenic event , the activation of the pymt transgene .
subsequent analysis identified several interacting quantitative trait loci ( qtl ) , some of which were found to map to homologous regions associated with loss of heterozygosity in human breast cancer [ 8 , 10 , 11 ] .
together , these findings suggest that inherited germline polymorphisms may contribute to the age of onset in human breast cancer and also the ability of tumor cells to metastasize
. further investigation of these earlier observations identified the first known polymorphic metastasis susceptibility gene , the rap - gtpase activating protein ( gap ) sipa1 .
experimental manipulation of cellular sipa1 mrna levels in a highly metastatic mouse mammary tumor cell line showed that subtle differences in sipa1 levels significantly affected the ability of the cells to colonize to the lungs , while not impacting primary tumor kinetics .
studies of human breast cancer have suggested that sipa1 germline polymorphisms are associated with aggressive disease behavior and with indicators of poor prognosis [ 13 , 14 ] , suggesting that sipa1 may play an important role in establishing metastatic susceptibility in humans as well as in mice .
we have shown so far that hereditary polymorphisms modulate metastatic potential . to further study whether genetic polymorphisms could be an important factor in the induction of prognostic signature profiles ,
previously published metastasis - predictive gene expression signatures were examined both in the mouse and in humans .
ecm genes were found to be common components of the metastasis - predictive gene signature in both human breast tumors [ 1517 ] and in pymt - induced mouse mammary tumors [ 18 , 19 ] , suggesting an important association of these genes with breast cancer progression .
briefly , the ecm components constitute a structure that is not only essential for the maintenance of tissue integrity but is also important for regulating cell migration .
historically , tumor interaction with the basement membrane was defined as the critical event in tumor invasion that signals the initiation of the metastatic cascade .
many steps in metastasis formation require specific interactions with the ecm . the nature and degree of this interaction will change from step to step during the metastatic process .
however , the type of specific interactions between tumor cells and the ecm might be influenced by the type of tumor cells and the type of matrix in which they reside .
for example , tumor cells may respond differently to various extracellular matrices and stromal cells that are encountered during metastasis formation , and this might result in the emphasis of some steps over others at particular points in the metastatic cascade ( reviewed in ) .
a study by bergamaschi and colleagues has portrayed the tumor - stroma composition of invasive breast carcinomas by characterizing the ecm components .
the ecm classification was recapitulated in a set of early - stage primary breast carcinomas [ 22 , 23 ] .
survival analysis on the early - stage breast carcinoma dataset showed significant differences in clinical outcome among the various ecm subclasses .
several studies that explored gene expression differences of primary breast and metastatic lymph node tumors have shown that genes involved in changes in extracellular matrix stability are critical for the early stages of the metastatic process [ 2427 ] .
furthermore , ecm gene dysregulation has been shown to be a very prominent feature of metastatic progression and may well explain why highly metastatic mouse mammary tumor cell lines are typically more adhesive , invasive , and migratory than the less metastatic lines . to determine whether the ecm dysregulation is under germline control ,
the akxd recombinant inbred mice ( ri ) were used to define ecm expression quantitative trait loci ( eqtl ) .
an eqtl is a genetically defined genomic locus associated with variation of gene expression , in this case ecm gene expression .
we chose the akxd ri mice because they are considered a useful tool for the study of germline - encoded metastatic propensity since they are derived from a highly metastatic strain , akr / j , and a weakly metastatic strain , dba/2j .
we found that the most significant eqtl in these mice is located on proximal mouse chromosome 17 .
both of these eqtl and metastasis loci colocalize and reside in a genomic region that contains the gene brd4 , suggesting that brd4 modulates ecm gene expression .
brd4 is the mammalian member of the bet ( bromodomain and extra - terminal ) family [ 32 , 33 ] , whose members carry two tandem bromodomains [ 34 , 35 ] . brd4 has been shown to regulate cell growth by acting at different stages of the cell cycle and also to interact with acetylated chromatin through its two bromodomains [ 32 , 33 ] .
given the apparent modulation of ecm gene expression , we further investigated the possibility that brd4 might be a metastasis susceptibility gene .
indeed , we found that ectopic expression of brd4 in a highly metastatic mouse mammary tumor cell line reduces both primary tumor growth and metastatic capacity in our mouse model . in vitro analyses showed that brd4 ectopic expression reduces both cell invasion and cell migration and also reduces cellular growth in three - dimensional cultures .
microarray gene expression analysis of the cell lines ectopically expressing brd4 , further confirmed that brd4 is a regulator of at least some of the ecm gene family members .
some of the ecm genes that were altered by ectopic expression of brd4 are the collagen genes col1a1 , col5a3 , col6a2 , the fibrillin gene fbn1 and serping1 , indicating that brd4 is a causative factor in the transcriptional regulation of these genes .
brd4 has been previously found to interact in vitro and in vivo with the metastasis modifier sipa1 .
this interaction modulates the enzymatic activity of sipa1 by increasing its rap - gap activity .
the n - terminus bromodomain ii of brd4 was shown to be the domain where brd4 and sipa1 interact .
deletion of bromodomain ii resulted in further suppression of primary tumor growth and lung metastasis mediated by brd4 and also induced a conversion to a more epithelial state .
these results are consistent with our previous findings that sipa1 is associated with greater malignancy .
it is important to mention here that brd4 and sipa1 were shown to regulate each other 's subcellular localization , with brd4 being redirected from the nucleus to the cytoplasm .
it is possible that the interaction between these two proteins contributes to tumor progression , and also the activity of brd4 might be modulated by compartmentalization ; however , the mechanism by which this occurs has yet to be explored .
one possibility could be that there is a balance between brd4 and sipa1 within the cell . under normal conditions brd4 and sipa1 interact in the nucleus while the cytoplasmic sipa1 does not take part in this interaction .
upon brd4 overexpression , sipa1 accumulates in the perinuclear region and in some cases in the nucleus near the nuclear membrane .
however , when sipa1 is overexpressed , a large fraction of brd4 gets moved to the cytoplasm leading to a more malignant phenotype .
our results suggest that the loss of the ability of sipa1 to relocalize or sequester the bromodomain ii mutant to the cytoplasm would increase the nuclear concentration of brd4 , leading to a more differentiated state and a less malignant phenotype . at this point
it is not known whether the brd4-sipa1 interaction influences the small gtpase rap1 levels within the tumor cell .
rap1 activity has been shown to play an important role in tumor formation and progression to malignancy [ 39 , 40 ] .
further investigations of the brd4-sipa1 relationship and the influence that it could have on rap1 levels might reveal a novel mechanism associated with malignant progression .
a recent report has shown that the extraterminal ( et ) domain of brd4 is an important transcriptional regulatory domain .
the c - terminal domain contains a single defined domain that binds the transcriptional elongation factor p - tefb .
brd4 also contains regions of high serine , proline , and glutamine content of unknown function .
indeed , microarray gene expression analysis of the cell lines that ectopically express brd4 has revealed that brd4 modulates the expression of genes involved in processes such as cellular proliferation , cell cycle progression , and chromatin remodeling .
other processes that are critical for metastasis , such as cytoskeletal remodeling , cell adhesion , and as mentioned earlier ecm expression regulation , were also regulated by brd4 .
furthermore , microarray gene expression analysis of cell lines that express a c - terminal deletion of brd4 show modulation of other classes of genes involved in emt and stem cell conversion processes .
emt is a multigstep process in which the cells acquire molecular changes that lead to a loss of cell - cell junctions , dysfunctional cell - cell adhesion , and rearrangement of the cytoskeleton , leading to a loss of polarity and the acquisition of a more spindle - shape morphology [ 4348 ] .
these alterations might eventually promote cancer cell progression and invasion through the basement membrane and into the surrounding tissues .
indeed , several studies have associated emt with cancer progression and metastasis [ 4952 ] .
for example , emt markers have been found to be present in invasive breast cancer especially in the invasion - metastasis cascade [ 47 , 53 ] .
recently , a concept of the migratory cancer stem cell has been described , in which a tumor cell possesses both stemness and motility properties .
it is suggested that cancer stem cells that have undergone emt can disseminate , and those that retain stem - cell functionality can form metastatic colonies .
more recently the emt process has also been linked to the ability of self - renewal .
current thinking suggests that disseminated cancer cells may need to acquire self - renewal properties similar to those exhibited by the stem cells , in order to achieve formation of macroscopic metastases .
the role of emt in tumor - initiating cells has also been described in human specimens .
breast cancer tumor - initiating cells and mesenchymal claudin - low - subtype cells show an association based on gene expression pattern
. furthermore , higher expression of mesenchymal genes was detected in breast cancer tumors before and after treatment with letrozole , indicating that the epithelial cancer cells have undergone emt .
ectopic expression of the c - terminal deletion mutant of brd4 ( c ) in a highly metastatic cell line induced significant morphological and physiological changes reminiscent of emt - like and cancer stem cell - like properties .
microarray gene expression analysis of these cell lines demonstrated that ectopic expression of the c mutant modulated the expression of some previously described emt markers and stem cell markers .
it is important to point out here that this mutant still contains the p - tefb - binding domain suggesting that emt - like and stem cell - like changes appear to be mediated by this p - tefb - binding region .
brd4 has two alternatively spliced variants that differ in the coding region and have a distinct 3 utr .
both isoforms have the same n - terminal region containing the chromatin - binding bromodomains and the serine - rich domain ; however , the c - terminal proline - rich and p - tefb - binding domains are absent in the shorter isoform . we have found that ectopic expression of the short isoform enhances metastatic colonization , as opposed to that seen by ectopic expression of the longer isoform .
this would suggest that the brd4 short isoform might be a competitive inhibitor of the longer isoform and that this inhibition would increase the ability of tumors to progress to metastatic disease .
this also suggests that metastatic susceptibility might be encoded by a ratio between the two isoforms .
the above data also suggest that the carboxy terminal half of the full - length isoform mediates the ability of brd4 to suppress progression and metastasis .
this was confirmed by the finding that expression of the c - terminal c mutant of brd4 increased lung colonization .
this increased malignancy is consistent with the in vitro data that cells expressing this mutant possess emt- and stem cell - like properties .
it is not known at this point whether the ratio between the two brd4 isoforms influences the expression of sipa1 or vice versa .
it would also be highly interesting to determine whether the brd4 short isoform and sipa1 could change each other 's subcellular localization as seen with the longer isoform .
the ratio between these three proteins and their cellular localization could be critical for malignant progression .
it is important to mention here that , in rare midline carcinomas , a highly malignant form of human squamous carcinoma , the brd4 short isoform is frequently fused to the nut ( nuclear protein in testis ) oncogene via an intronic translocation [ 5760 ] .
the major oncogenic effect of brd4-nut fusion protein appears to lie in its ability to arrest the differentiation of the so - called nut - midline carcinoma cells .
this is consistent with our findings that the shorter brd4 isoform promotes metastatic capacity and also that the competitive inhibition of the longer brd4 isoform would increase the ability of tumors to progress to metastatic disease .
several studies have demonstrated that primary tumors with a higher propensity to metastasize exhibit gene expression patterns that predict the likelihood of metastatic potential [ 1517 ] . as mentioned earlier
, brd4 is responsible , at least partially , for the presence of ecm components in the metastatic - predictive gene signatures , suggesting that brd4 itself might be a predictive of survival .
we have found that the brd4 long isoform induces a gene expression signature that predicts good outcome in human breast cancer datasets .
this suggests that brd4 activation is an important determinant in the overall likelihood of relapse and/or survival .
the brd4 gene expression signature was also able to stratify breast cancer patients with lymph - node - negative and estrogen - receptor - positive at presentation into high- and low - risk patients .
the gene expression signature induced by the brd4 short isoform , however , predicted poor outcome in these human breast cancer datasets , confirming that the shorter isoform might be a competitive inhibitor of the longer isoform .
additionally , the brd4 long- and short - isoform gene expression signatures were compared to a 19-gene signature that was defined by correlating tumor growth expression , histological grade , and survival .
we found that the brd4 longer isoform signature matches low - grade g1 breast cancer tumors while the shorter isoform matches high - grade g3 tumors [ 36 , 38 ] .
the outcome prediction and the signature convergence might be of potential importance in the clinic where it could improve the stratification of patients into different subtypes and in turn enable clinicians to tailor treatments for individual patients .
selective inhibitors of the bet family members have been recently developed [ 6265 ] .
a competitive binding of the small molecule inhibitor jq1 , for example , was shown to displace the brd4 fusion oncoprotein from chromatin , promoting squamous differentiation and specific anti - proliferative effects .
these effects were seen in brd4-dependent cell lines and patient - derived xenograft models . in another study , zuber and
colleagues studied acute myeloid leukemia ( aml ) , which is an aggressive hematopoietic malignancy that is often associated with aberrant chromatin states .
suppression of brd4 by shrna or by jq1 compound led to robust antileukemic effects both in vivo and in vitro .
brd4 inhibition also led to myeloid differentiation and leukemia stem - cell depletion . at this point
it is not known whether the small - molecule inhibition of brd4 would have any effect on breast cancer and metastatic progression .
the genetic background impacts not only the primary tumor but all of the tissues , which play a role in the establishment of the microenvironment in both primary and metastatic tumor cells .
this would suggest that an earlier prognosis in nontumor tissues should be possible even before cancer develops .
this is only possible if a sufficient fraction of metastatic risk is encoded by germline polymorphisms , rather than autonomous somatic events within the tumor .
our recent data suggest that the metastasis susceptibility gene brd4 appears to play a significant role in establishing transcriptional programs that predict breast cancer outcome via a balance between the tumor- and metastasis - suppressive long isoform and the metastasis - promoting short isoform .
given the fact that brd4 regulates important intermediates and processes within the metastatic cascade suggests that brd4 , and possibly other metastatic susceptibility genes , may be altering the risk of developing distant metastases by predisposing the tumors of high - risk patients to undergo conversion to a more dedifferentiated or primitive state .
finally , the brd4 gene expression signature identified could be applied as a useful predictive tool by identifying those patients with low risk of relapse at presentation .
this combined with the traditional clinical variables such as lymph node - negative and er - positive patients would facilitate the identification and the initiation of new treatment protocols that could be applied for individual patients .
invasiona process that initiates metastasis and consists of changes in tumor cell adherence to the extracellular matrix , proteolysis of the extracellular matrix and the surrounding tissues and migration through these tissues .
a process that initiates metastasis and consists of changes in tumor cell adherence to the extracellular matrix , proteolysis of the extracellular matrix and the surrounding tissues and migration through these tissues .
extravasationthe escape of tumor cells from the circulation into the parenchyma of an organ .
the escape of tumor cells from the circulation into the parenchyma of an organ .
colonizationa process by which disseminated tumor cells grow to form clinically detectable metastatic lesions .
a process by which disseminated tumor cells grow to form clinically detectable metastatic lesions .
angiogenesisthe formation of new blood vessels that are needed for the growth of the primary tumor and metastases .
the formation of new blood vessels that are needed for the growth of the primary tumor and metastases .
dormancya period in which the cells are in a non - dividing state .
a period in which the cells are in a non - dividing state .
polymorphisma variation within a gene where two or more alleles exist at a frequency of at least 1% in the general population .
a variation within a gene where two or more alleles exist at a frequency of at least 1% in the general population .
extracellular matrixthe matrix that is laid down by cells in which they adhere and move .
the matrix that is laid down by cells in which they adhere and move .
expression quantitative trait locus ( eqtl)a genetically defined genomic locus associated with variation of expression of the genes that underlie the trait in question .
a genetically defined genomic locus associated with variation of expression of the genes that underlie the trait in question .
epithelial - to - mesenchymal transition ( emt)a potential mechanism in tumor progression by which some cancer cells acquire the ability to convert from polarized epithelial cells to mesenchymal motile cells facilitating metastasis at distant sites .
a potential mechanism in tumor progression by which some cancer cells acquire the ability to convert from polarized epithelial cells to mesenchymal motile cells facilitating metastasis at distant sites . | metastasis is an extremely complex process that accounts for most cancer - related deaths .
malignant primary tumors can be removed surgically , but the cells that migrate , invade , and proliferate at distant organs are often the cells that prove most difficult to target therapeutically . there is growing evidence that host factors outside of the primary tumors are of major importance in the development of metastasis .
recently , we have shown that the bromodomain - containing protein 4 or bromodomain 4 ( brd4 ) functions as an inherited susceptibility gene for breast cancer progression and metastasis . in this paper
, we will discuss that host genetic background on which a tumor arises can significantly alter the biology of the subsequent metastatic disease , and we will focus on the role of brd4 in regulating metastasis susceptibility . |
lumbar spondylolysis , a defect in the pars interarticularis , is a common cause of low back pain in adolescents and young adults .
it occurs in 6% of the general population and has been reported more commonly in men .
spondylolysis commonly occurs at the fourth and fifth lumbar vertebrae , accounting for more than 95% of total cases of spondylolysis .
symptomatic pars lesions appear to be particularly a clinical problem in adolescents , especially adolescent athletes .
conservative treatment is commonly considered first in most patients , but those who remain symptomatic may benefit from operative treatment . when the source of back pain is the pars defect itself , direct repair of spondylolysis seems to be most effective and appears to be more logical than posterior spinal fusion in the patients without disc degeneration or spondylolisthesis .
multilevel spondylolyses are rare , varying between 1.2% and 5.6% of lumbar spondylolysis cases , and majority involve 2 levels , l4 and l5 .
so for , to our knowledge , only 11 cases of 3-level lumbar spondylolyses have been reported in the existing english literature . in this paper
, we reported 3 cases of 3-level lumbar spondylolyses in young adults and review the management choices made in these cases .
this study obtained the approval of the medical ethics committee of our hospital ( general hospital of armed police force , beijing , china ) .
case 1 , a 22-year - old male , presented with low back pain for 2 years .
he did not complain of radicular pain , numbness , and concomitant claudication in his lower extremities .
he had a history of lumbar injury dropped down from 2 meters high 2 years ago .
motor , sensory , and tendon reflex were all normal in his 2 lower limbs .
lumbar radiography and computed tomography ( ct ) scan showed left l3 and bilateral l4 and l5 spondylolyses without any slippage .
before admission to our hospital , the patient had underwent a 3-month course of conservative treatment with physiotherapy and nonsteroidal anti - inflammatory drugs in a local clinic , but these therapies did not relieve his low back pain . because ct scans have showed terminal - stage findings in the isthmic lysis areas , no abnormality of the posterior arches such as dysplasia and spina bfida was found , surgical indication for left l3 and bilateral l4 and l5 direct isthmic repair via a pedicle screw
case 2 , a 22-year - old male , was admitted with low back pain for 14 months .
pain was originated from an acute lumbar heavy blow 14 months ago , and increased gradually .
one month ago , he felt numbness in his right hip and right lateral thigh .
motor , sensory , and tendon reflex were all normal in his 2 lower limbs .
radiographs and ct scans showed bilateral l3l5 spondylolyses without any slippage ( figures 1 and 2 ) .
after his lumbar injury , he was sent to a local hospital , and was diagnosed with l3l5 bilateral spondylolyses by plain radiography . since then , he has undergone conservative treatment with bracing to protect lumbar spine , physiotherapy , and oral nonsteroidal anti - inflammatory drugs in a local clinic , but these therapies did not relieve his low back pain .
after he underwent a 4-month course of conservative treatment , further ct scan showed terminal - stage findings in the l3l5 bilateral isthmic lysis areas .
surgical indication for bilateral l3l5 direct isthmic repair via a screw hook technique was recommended .
case 3 , a 23-year - old man , was admitted with low back pain for 6 months . in the beginning
a month ago , he sprained his waist carelessly , which aggravated his back pain .
since then , he has undergone conservative treatment in a local hospital , but conservative treatment did not relieve his back pain
motor , sensory , and tendon reflex were all normal in his 2 lower limbs .
because mri showed l5/s1 disc degeneration , we needed to identify whether this disc was a pain - generating disc .
the result of discography showed l5/s1 disc disruption with pain reproduction , which indicated the l5/s1 disc as the one of the back pain sources . because ct scan had shown terminal - stage findings in the l3l5 bilateral isthmic lysis areas , accordingly ,
surgical indication for bilateral l3 and l4 direct isthmic repair and l5/s1 interbody fusion was recommended .
three cases of 3-level lumbar spondylolyses were diagnosed and subsequently direct isthmic repair in 2 cases and a combined surgery of isthmic repair and interbody fusion in 1 case were recommended for the 3 patients preoperatively based on clinical findings consistent with imaging study .
case 1 underwent a surgery of direct isthmic repair in 5 lytic defects . under general anesthesia , conventional posterior midline approach to lumbar spine was used to exposure l3l5 spinous processes , vertebral laminae , and facet joints .
first , a 6.0 45 mm pedicle screw was inserted to involved vertebra under fluoroscopic guidance .
second , the involved lysis area was curetted , freshened , and grafted with autologous iliac bone .
finally , sublaminar hook was placed , and connected through a short rod under compression with corresponding pedicle screw .
the total surgical time was 3 hours with 300 ml of blood loss .
a lumbar - sacral brace was used for 3 months after surgery . surgical procedure in case 2 was the same as case 1 .
the total surgical time was 3 hours with 500 ml of blood loss .
postoperatively , the symptoms of low back pain and numbness in right hip and right lateral thigh disappeared .
four pedicle screws were placed in l5 and s1 vertebrae bilaterally , then left laminectomy was performed to allow a cage placement ( 10 26 mm ) filled with autogenous local bone derived from laminectomy between the decorticated endplates of l5 and s1 , and finally 2 rods were connected with 4 pedicle screws under compression .
the total surgical time was 3.5 hours with 400 ml of blood loss .
after surgery , a brace was placed for 3 months , and symptom of low back pain was almost completely disappeared .
case 1 has undergone a follow - up of 2 years , and his low back pain was almost completely disappeared . at 6 months after surgery
, bone healing was found in all 5 lytic defects . at 8 months after surgery in case 2 ,
bone healing was found in all 6 lytic defects . during a 1-year follow - up period
, he was pain - free . at 2-year follow - up in case 3 ,
his low back pain had not recurred , and bone fusion was found between l5 and s1 vertebral bodies and in all 4 isthmi of l3 and l4 according to the evaluation of lumbar radiographs and ct scans .
the surgery for the 3 patients was successfully performed and their symptoms were obviously relieved and lumbar function was markedly improved .
the alleviation of back pain and improvement of lumbar function was assessed by the change in the degree of pain with a self - assessment of pain by an 11-point visual analog scale ( vas , 010 ) pain scale and the oswestry disabiligy index ( odi , version 1.0 , 0100 ) .
lumbar spondylolysis is a common disease , in more than 90% of cases located in l5 .
a pars defect develops into a chronic nonunion , and becomes bridged by tissue composed of a combination of fibrous , cartilaginous , or osseous material . in this circumstance
, the origin of chronic low back pain could be in the scar and connective tissue rich in nociceptive nerve endings that bridges the gap of a pars defect .
extra load exerted on the motion segment may cause disc degeneration , which is therefore an accompanying disorder of spondylolysis .
the prevalence of progressive disc degeneration in the involved level is markedly increased in the adult spondylolytic patients who often appear low back pain later in life , after an asymptomatic childhood .
the pathology of lumbar spondylosis was considered being congenital failure of fusion of 2 ossification centers or a hyperflexion injury at birth , but spondylolysis had never been founded in embryos , fetus and at birth .
following wiltse 's proposal in 1957 , the pathology of spondylolysis was shifted to a consensus of fatigue fracture of the pars interarticularis with a strong hereditary basis . for now , 2 factors can explain isthmic lysis , both genetic and mechanical .
no specific genetic variation was identified , but it would seem that a genetic predisposition to this pathology may exist .
yamada et al reported 3 cases of lumbar spondylolysis in juveniles from the same family .
the highest incidence was reported in native residents in greenland up to 54% . from a mechanical point of view , heavy work and repeated injuries seem to favor the appearance of lumbar spondylolysis .
a number of studies have described the association of sports activities with spondylolysis , and all of them support the stress fracture theory .
traumatic spondylolysis can be diagnosed early with the use of isotope bone scan or single photon emission computed tomography scan .
preoperative ct scan is very important to evaluate the degree of sclerosis in the bony margins of the isthmic defect .
the obvious osteosclerosis was shown in all the isthmic defect margins in the 3 patients by ct scan , which indicated that direct isthmic repair was necessary .
preoperative lumbar mri is imperative to evaluate the state of intervertebral disc . in our case 3 , due to l5/s1 disc degeneration
, an additional lumbar discography was used to indicate this disc as a painful disc .
this result finally determined the indication for fusion surgery at the l5/s1 segment instead of a direct isthmic repair in l5 .
there are many types of direct repair methods , including scott 's wiring , buck 's screwing , louis butterfly plate , and screw hook technique ( pedicle screw and hook rod system ) . from clinical aspects ,
screw hook technique allows for fixation of the defects in the pars interarticularis with rigid implants and exerts force along the lamina to effectively suppress the defect motion , which is critical for better bone healing .
the precondition of direct defect repair is normal signal intensity in the involved disc shown in t2-weighted mri . if not
, lumbar fusion should be considered . in the present cases 1 and 2 , the patients were young with normal discs and normal posterior arches ,
so l3l5 direct isthmic pair was reasonable . in the case 3 , because mri showed both the l3/4 and l4/5 discs normal and the l5/s1 disc degeneration with disruption and pain reproduction in discography , accordingly a combined surgery of bilateral l3 and l4 direct isthmic repair and l5/s1 interbody fusion was performed for the young patient .
surgical treatment varies between fusion , direct isthmic repair , and combined management associating 2 procedures at different levels .
success of management of multilevel spondylolyses depends on the choice of appropriate treatment for every patient .
we reported here 3 cases of 3-level lumbar spondylolyses with good clinical and radiological results .
hook technique is a simple and safe procedure for the motion segment with normal disc .
although our clinical results are satisfactory , all 3 patients are young and the duration of their follow - up periods has not been long .
therefore , further observation of these patients clinical course and radiological results at the segments treated as well as adjacent segments is needed . | abstractthree - level lumbar spondylolyses are extremely rare .
so far , only 11 cases were reported in the literature .
treatment of multilevel spondylolyses has not been consistent .
conservative treatment is commonly considered first in most patients , but those who remain symptomatic may benefit from operative treatment .
we report here 3 cases of 3-level lumbar spondylolyses that were treated successfully with direct isthmic repair in 2 cases and a combined surgery of isthmic repair and interbody fusion in 1 case .
our clinical results indicated that direct defect repair using the screw
hook technique is a simple and safe procedure for the motion segment with normal disc .
if the involved disc shows degenerative change , fusion surgery should be consideredsurgical treatment of multilevel spondylolyses varies between fusion , direct isthmic repair , and combined management associating 2 procedures at different levels .
the success of management of the 3 patients with 3-level spondylolyses depends on the choice of appropriate treatment for every patient . |
angina bullosa hemorrhagica is a rare benign subepithelial oral mucosal blisters filled with blood that are not attributable to a systemic disorder or haemostatic defect .
angina bullosa hemorrhagica ( abh ) is a sub epithelial oral mucosal hemorrhagic blister of unknown aetiology .
this condition was first described in 1933 by argentina as traumatic oral haemophlyctenosis and badham in 1967 first used the currently accepted term abh .
clinically , it presents as acute , spontaneous , asymptomatic hemorrhagic vesicle / bullae , commonly on the soft palate in middle aged individuals without any sex predilection .
though exact aetiology is not known , many local and systemic factors are incriminated in the causation of abh .
we report two cases of abh which was idiopathic in one case and other was associated with chronic renal failure on hemodialysis .
a 48-year - old woman presented with asymptomatic recurrent blood filled blister in the oral cavity with a history of irregular and intermittent appearance since 25 years . in most instances , lesion appeared singly on the lateral aspect of tongue but also involved soft palate occasionally .
lesions healed spontaneously without any sequel but lesion recurred every year once or twice without any seasonal predilection .
no history of intake of any other drugs , no history suggestive of precipitating factors like trauma , steroid inhalation , blood dyscrasias . not a known diabetic , hypertensive or asthamatic .
, there was solitary oval tense hemorrhagic bulla measuring about 2 1.5 cm over the lateral margin of tongue on the right side [ figure 1 ] .
hemorrhagic blister over lateral tongue in case 1 a 50 years old male patient with chronic renal failure since two years , on haemodialysis since 8months with blood pressure , developed hemorrhagic blister at the junction of buccal mucosa and alveolar ridge on right side . over a period of 12 hrs it ruptured discharging thin clear blood mixed fluid and healed within four days without any scarring or other squeal . on examination
, there was solitary oval tense hemorrhagic bulla measuring about 2.5 2 cm present at the junction of buccal mucosa and alveolar ridge on right side .
hemorrhagic blister at the junction of buccal mucosa and alveolar ridge on right side which broke on manipulation tzanck smear in both cases showed numerous rbc 's and there was no acantholytic or multi - nucleated giant cell .
biopsy from case -1 of the ruptured blister revealed sub - epithelial split with minimal perivascular infiltrate .
biopsy could not be done in case -2 , since the patient did not consent .
routine blood and urine tests including bleeding and clotting time , liver function test , renal function test , thyroid function test , lipid profile , ultra sound examination of abdomen and pelvis , chest
in case-2 , all the investigations were normal except for high blood pressure ( 130/90 mm hg ) and elevated serum urea and creatinine levels ( 50 and 2.5 mg / dl respectively ) .
spontaneous onset , isolated nature , constant presence of blood in the blister fluid and rapid healing in the absence of an identifiable local cause or systemic disorder differentiates abh from mucous membrane pemphigoid , bullous pemphigoid , bullous lichen planus , epidermolysis bullosa , dermatitis herpetiformis , linear iga disease and oral amyloidosis .
the classical history and morphology of lesions helped us to arrive at the clinical diagnosis of angina bullosa hemorrhagica in both the presenting cases .
abh is a benign phenomenon that is characterized by the sudden appearance of a blood filled blister on the oral mucosa in the absence of an identifiable cause or systemic disorder , commonly seen in middle and elderly people with no sex predilection .
bulla is typically seen on soft palate and occasionally on lateral border of tongue , gingiva and buccal mucosa .
onset is rapid and blistering starts over few minutes which breaks down in minutes to hours to form shallow ulcer and heals spontaneously over 3 - 10 days .
recurrences can be seen at the same or different oral site in 30% of people .
local factors implicated are trauma from hot foods , dental procedures and endoscopy , local anaesthesia , and chronic steroid inhalation and systemic causes are hypertension , asthma , diabetes , rheumatoid arthritis , gastrointestinal disturbances and hyperuracemia .
yamamoto et al . in his report of 11 cases , described a case of abh associated with renal failure .
it has been known that abh presents with recurrences at regular intervals , usually associated with some internal disease or with some medications . in our cases reported here , case-1 had a classical presentation with pronged history of irregular presentation for 25 years without any other associated disease or drug intake .
case 2 presented with a classical presentation of first episode associated with blood pressure and renal failure on dialysis .
these cases are reported for the relative rarity of this entity and the diversity of presentation with reference to site within the oral cavity ( in case -1 , presenting at an atypical site ) and diverse aetiology .
this report though does not convey significant new information it would certainly help to create more awareness among dermatologists and oral physicians to be able to differentiate from a host of other blistering disorders of varying aetiologies in the oral cavity .
angina bullosa hemorrhagica may be seen in renal failure patients on dialysis and may have recurrence for long periods .
this reprt helps to create awareness among dermatologists and oral physicians to be able to differentiate from a host of other blistering disorders of varying aetiologies in the oral cavity .
a 48-year - old woman presented with asymptomatic recurrent blood filled blister in the oral cavity with a history of irregular and intermittent appearance since 25 years . in most instances , lesion appeared singly on the lateral aspect of tongue but also involved soft palate occasionally .
lesions healed spontaneously without any sequel but lesion recurred every year once or twice without any seasonal predilection .
no history of intake of any other drugs , no history suggestive of precipitating factors like trauma , steroid inhalation , blood dyscrasias . not a known diabetic , hypertensive or asthamatic .
, there was solitary oval tense hemorrhagic bulla measuring about 2 1.5 cm over the lateral margin of tongue on the right side [ figure 1 ] .
a 50 years old male patient with chronic renal failure since two years , on haemodialysis since 8months with blood pressure , developed hemorrhagic blister at the junction of buccal mucosa and alveolar ridge on right side . over a period of 12 hrs it ruptured discharging thin clear blood mixed fluid and healed within four days without any scarring or other squeal . on examination
, there was solitary oval tense hemorrhagic bulla measuring about 2.5 2 cm present at the junction of buccal mucosa and alveolar ridge on right side .
hemorrhagic blister at the junction of buccal mucosa and alveolar ridge on right side which broke on manipulation tzanck smear in both cases showed numerous rbc 's and there was no acantholytic or multi - nucleated giant cell .
biopsy from case -1 of the ruptured blister revealed sub - epithelial split with minimal perivascular infiltrate .
biopsy could not be done in case -2 , since the patient did not consent .
routine blood and urine tests including bleeding and clotting time , liver function test , renal function test , thyroid function test , lipid profile , ultra sound examination of abdomen and pelvis , chest
in case-2 , all the investigations were normal except for high blood pressure ( 130/90 mm hg ) and elevated serum urea and creatinine levels ( 50 and 2.5 mg / dl respectively ) .
spontaneous onset , isolated nature , constant presence of blood in the blister fluid and rapid healing in the absence of an identifiable local cause or systemic disorder differentiates abh from mucous membrane pemphigoid , bullous pemphigoid , bullous lichen planus , epidermolysis bullosa , dermatitis herpetiformis , linear iga disease and oral amyloidosis .
the classical history and morphology of lesions helped us to arrive at the clinical diagnosis of angina bullosa hemorrhagica in both the presenting cases .
abh is a benign phenomenon that is characterized by the sudden appearance of a blood filled blister on the oral mucosa in the absence of an identifiable cause or systemic disorder , commonly seen in middle and elderly people with no sex predilection .
bulla is typically seen on soft palate and occasionally on lateral border of tongue , gingiva and buccal mucosa .
onset is rapid and blistering starts over few minutes which breaks down in minutes to hours to form shallow ulcer and heals spontaneously over 3 - 10 days .
recurrences can be seen at the same or different oral site in 30% of people .
local factors implicated are trauma from hot foods , dental procedures and endoscopy , local anaesthesia , and chronic steroid inhalation and systemic causes are hypertension , asthma , diabetes , rheumatoid arthritis , gastrointestinal disturbances and hyperuracemia .
yamamoto et al . in his report of 11 cases , described a case of abh associated with renal failure .
it has been known that abh presents with recurrences at regular intervals , usually associated with some internal disease or with some medications . in our cases
reported here , case-1 had a classical presentation with pronged history of irregular presentation for 25 years without any other associated disease or drug intake .
case 2 presented with a classical presentation of first episode associated with blood pressure and renal failure on dialysis .
these cases are reported for the relative rarity of this entity and the diversity of presentation with reference to site within the oral cavity ( in case -1 , presenting at an atypical site ) and diverse aetiology .
this report though does not convey significant new information it would certainly help to create more awareness among dermatologists and oral physicians to be able to differentiate from a host of other blistering disorders of varying aetiologies in the oral cavity .
angina bullosa hemorrhagica may be seen in renal failure patients on dialysis and may have recurrence for long periods .
this reprt helps to create awareness among dermatologists and oral physicians to be able to differentiate from a host of other blistering disorders of varying aetiologies in the oral cavity . | angina bullosa hemorrhagica ( abh ) is a disorder of unclear aetiology characterized by abrupt and unprovoked presentation of blood - filled blister over the oral mucosa .
histopathology reveals sub epithelial blister containing erythrocytes with superficial perivascular lymphocytic infiltrate .
we report two cases of abh of which one was idiopathic and other was associated with chronic renal failure on hemodialysis . |
congenital epulis , the rare benign soft - tissue tumors , derived from greek word meaning on the gum present with an aggressive appearance with large size , causing swelling , and difficulties during the breastfeeding . any exophytic , asymptomatic soft - tissue tumor in infants , unifocal / multifocal of the mucogingival zone of the anterior labial gingiva , should be considered in the differential diagnosis of the following nosologic entities : granular cell tumor , gingival cyst of the newborn , hamartoma , choristoma , vascular malformations , melanotic neuroectodermal tumor of infancy , oral teratoma , and rhabdomyosarcoma .
this paper reports a rare case of congenital oral midline subcutaneous smooth muscle ( leiomyomatous ) hamartoma of incisive papilla in a rare presentation as solid tubular overgrowth of incisive papilla .
a 2-year - old female child accompanied by her mother referred to the department of pediatric dentistry with chief complaint of a solid tubular overgrowth of incisive papillary region which leads to difficulty in feeding and respiration .
medical history was normal , and the mother reported normal , uncomplicated full - term pregnancy .
intraoral examination revealed growth on incisive papilla , i.e. , palatal aspects of primary maxillary central incisors .
this growth was present as a small nodule at birth which progressively increased in size extending to 22.5 cm below the incisal edge of maxillary central incisors [ figure 1 ] .
the soft - tissue examination of the lesion revealed a solitary ( 22.5 cm 0.5 cm 0.5 cm ) firm , painless , pedunculated growth on the incisive papilla .
soft - tissue examination revealed normal oral mucosa , gingiva , tongue , and floor of the mouth .
total excisional biopsy was done under local anesthesia , which was sent for histopathological examination in 10% formalin solution .
( a and b ) growth attached to incisive papilla between deciduous maxillary central incisors .
( c ) tissue received after excisional biopsy hematoxylin- and eosin - stained paraffin - based sections ( 5 m thick ) showed a parakeratinized stratified squamous epithelium with long and thick rete ridges . underlining connective tissue stroma showed irregular and interlacing bundles of collagen fibers and numerous muscle fiber bundles .
plump- and spindle - shaped fibroblasts along with proliferating nerve bundles and endothelial cell - lined blood vessels underneath the epithelial surface with extravasated red blood corpuscles [ figure 2 ] .
reticulin - stained slide which showed the presence of immature muscle fibers whereas masson 's trichrome stain revealed collagen fibers and smooth muscles .
histopathological findings confirmed the diagnosis of oral midline subcutaneous smooth muscle ( leiomyomatous ) hamartoma of incisive papilla .
( a ) h and e stained section showing parakeratinized stratified squamous epithelium with irregular and interlacing bundles of collagen fibers along with numerous muscle fiber bundles in connective tissue stroma .
( c ) masson 's trichrome - stained section showing collagen fibers ( blue ) and smooth muscles ( red )
hematoxylin- and eosin - stained paraffin - based sections ( 5 m thick ) showed a parakeratinized stratified squamous epithelium with long and thick rete ridges . underlining connective tissue stroma showed irregular and interlacing bundles of collagen fibers and numerous muscle fiber bundles .
plump- and spindle - shaped fibroblasts along with proliferating nerve bundles and endothelial cell - lined blood vessels underneath the epithelial surface with extravasated red blood corpuscles [ figure 2 ] .
reticulin - stained slide which showed the presence of immature muscle fibers whereas masson 's trichrome stain revealed collagen fibers and smooth muscles .
histopathological findings confirmed the diagnosis of oral midline subcutaneous smooth muscle ( leiomyomatous ) hamartoma of incisive papilla .
( a ) h and e stained section showing parakeratinized stratified squamous epithelium with irregular and interlacing bundles of collagen fibers along with numerous muscle fiber bundles in connective tissue stroma .
( c ) masson 's trichrome - stained section showing collagen fibers ( blue ) and smooth muscles ( red )
hamartoma is an abnormal proliferation of mature tissues , composed of elements that are normally found in the normal location for the tissue in which it develops , often with one predominating element .
similar lesion like choristoma is a histologically normal tissue proliferation of a type that is normally not found in the anatomic site .
the present case appeared clinically as a simple soft - tissue mass present at birth ; therefore , diagnosis of congenital epulis was made .
excisional biopsy was done under local anesthesia as feeding and respiration problems were encountered . due to rare site of the involvement of incisive papilla ,
reticulin - stained slide showed the presence of immature muscle fibers whereas masson 's trichrome stain revealed immature collagen fibers and smooth muscles confirmed the diagnosis of oral leiomyomatous hamartoma mimicking hypertrophy of incisive papilla .
intraoral smooth muscle hamartomas of midline in palate are very rare and tend to occur at sites where fusion of processes and prominences occurs in developing embryo [ table 1 ] .
summary of reported cases of leiomyomatous hamartomas of the midline maxillary gingiva differential diagnosis for congenital epulis includes hemangioma , fibroma , rhabdomyoma , rhabdomyosarcoma , lymphangioma , sarcomas , hamartomas , teratoma , and granular cell tumor .
this is defined as a cohesive tumor - like mass consisting of normal cells in abnormal location .
many a times , they are found in the form of rests , without forming a mass lesion . although neuromuscular hamartoma , composed of neural and skeletal muscle differentiation , has been referred to as
benign triton tumor , it is technically incorrect . this has also been proved by karyotyping .
some clinical details may aid in distinguishing the oral leiomyomatous hamartoma from other gingival lesions in newborns .
vascular malformations often have reddish surface and tend to decrease in size after compression . the melanotic neuroectodermal tumor of infancy is presented as a pigmented lesion , usually located in the anterior alveolar ridge of the maxilla , characterized by a rapid growth , different from the features showed by congenital epulis .
oral teratoma and rhabdomyosarcoma are rare tumors that arise as a solid mass with progressive and invasive growth .
although very rare , peripheral odontogenic tumors in newborns were also reported and should be included in the differential diagnosis .
the congenital epulis cells are negative for neural , fibroblastic , myofibroblastic , myogenous , vascular , and histiocytic markers , and there are not evidences that congenital epulis grows after the birth .
the majority of benign tumors in the young are probably developmental rather than true neoplasms .
differences found in various population group children and adolescents and those from other countries may be attributable to genetic and geographic differences .
treatment consists of simple conservative excision under general or local anesthesia ; no recurrence has been reported .
surgery should not be radical as it minimizes danger of damaging underlying alveolar bone and developing tooth buds .
oral smooth muscle ( leiomyomatous ) hamartomas should be considered in the clinical differential diagnosis of congenital epulis . early and prompt surgical excision is the widely preferred approach in this condition .
it is important for dental professionals to be aware of this congenital tumor and its presentation , differential diagnosis , histopathology , and management .
a multidisciplinary approach is required for managing such patients by considering a significant reduction in parent 's anxiety and patient 's morbidity .
| congenital soft - tissue tumors of oral cavity are mostly hyperplastic and benign in nature .
this article presents an unusual case of congenital subcutaneous hamartoma of incisive papilla in a 2-year - old female child causing feeding and breathing difficulty .
total excisional biopsy was done under local anesthesia .
histopathology of tissue in reticulin - stained slide showed the presence of immature muscle fibers whereas masson 's trichrome stain revealed collagen fibers and smooth muscles confirming the diagnosis of oral midline subcutaneous smooth muscle ( leiomyomatous ) hamartoma of incisive papilla .
it is important for dental professionals to be aware of this oral lesion present from birth mimicking overgrowth of incisive papilla , by its presentation , differential diagnosis , histopathology , and management . |
systemic lupus erythematosus ( sle ) is a prototypic autoimmune disease affecting various parts of the body including skin , kidneys , lungs , joints , heart , nervous system , and hematopoietic organs .
it is a disease whereby a diverse array of autoantibody production , complement activation , immune complex deposition , and inflammation cause damages in those organs .
although the exact aetiology of sle still remains unclear , a combination of genetic risk factors and environmental events is believed to contribute to an irreversible break in immunological self - tolerance . with the introduction of genome - wide association studies ,
a huge breakthrough has been made in the discovery of sle associated susceptibility genes that in turn advances our understanding of pathogenesis of sle .
recently , several reviews have categorised the susceptible genes according to their immunological pathways and cell types .
: ( i ) innate immune response including toll - like receptor ( tlr)/interferon ( ifn ) signalling pathways ; ( ii ) adaptive immune response including b , t , and antigen - presenting cells immune signal transduction ; and ( iii ) immune complex clearance mechanism .
defects in tlr / ifn signalling pathways cause immune complexes containing self - nucleic acids to interact with tlr7 and tlr9 inside plasmacytoid dendritic cells and b cells endosomes , resulting in the secretion of type i ifn and interleukin ( il)-6 . the combined triggering of both b cell receptors and tlr leads to autoreactive b - cell proliferation .
their further differentiation into plasmablasts and autoantibody - secreting plasma cells is induced by type i ifn and il-6 , respectively .
tnfaip3 , stat4 , irf5 , trex , and irak1 are the genes involved in upstream and downstream pathways of type i ifn production that have been recently identified .
the stat4 gene consists of 24 exons that spread over a 120 kb region on chromosome 2q32.3 .
it encodes a transcription factor that mediates signals induced by il-12 , il-23 , and type i ifn and activates the production of ifn- and il-17 .
it also directs the differentiation of helper t cells toward the proinflammatory t - helper type 1 and t - helper type 17 lineages that have been shown to play a critical role in the pathogenesis of sle .
the stat4 null allele in lupus - prone mouse model confers reduced autoantibody production and glomerulonephritis , indicating that stat4 may be involved in multiple sle - associated phenotypes .
there are a few studies involving stat4-deficient lupus - prone mice which demonstrate the role of stat4 in autoantibody production only [ 4 , 5 ] .
polymorphisms in the stat4 gene have been found to be strongly associated with sle susceptibility , in particular rs7574865 [ 6 , 7 ] .
the simultaneous association of the risk allele t of stat4 rs7574865 with both lower serum ifn- activity and increased ifn--induced gene expression has been reported , confirming that this polymorphism was associated with increased ifn- sensitivity [ 8 , 9 ] .
tnfaip3 , or tumour necrosis factor alpha - induced protein 3 gene , encodes the a20 protein which is a negative regulator of the nf-b signalling pathway , an essential pathway in the pathogenesis of sle .
a20 is an ubiquitin - editing enzyme required for effective termination of nf-b - mediated proinflammatory responses induced by tlrs , tnf receptor , il-1 receptor , and nod2 .
a meta - analysis and imputation study identified a 109 kb risk haplotype spanning tnfaip3 region with lupus nephritis and hematologic manifestation . a nonsynonymous mutation ( c.380 t > g ) , rs223092 , in tnfaip3 gene which causes phenylalanine - to - cysteine change at position 127 of a20 protein has been consistently linked with sle various ethnic groups .
the final candidate gene , irf5 , which is ifn regulatory factor 5 , is a transcription factor that mediates inflammatory and immune responses .
this factor stimulates the production of the proinflammatory cytokines tnf- , il-12 , and il-6 following tlr signalling as well as transactivation of type i ifn and ifn - induced genes [ 13 , 14 ] .
polymorphisms in irf5 cause functional changes in messenger rna , which in turn alter ifr5-mediated transcription resulting in elevated sle risk .
it was also suggested that sle patients who carry irf5 risk haplotype and are positive for either anti - rbp or anti - dsdna potentially have higher serum ifn- activity . in this study , we aimed to investigate the association between seven single nucleotide polymorphisms ( snps ) in stat4 , tnfaip3 , irf5 genes , and sle in the south east asian scenario , particularly in the malaysian participants .
we also attempted to compare and pool the ors of snps which were significant in the malaysian sle with the other studies through meta - analysis .
a total of 790 malaysians were included in this study , which is comprised of 360 sle patients and 430 healthy controls .
blood samples were collected from patients diagnosed with sle according to 4 out of acr criteria and healthy volunteers recruited at the university of malaya medical centre ( ummc ) , kuala lumpur , in compliance with requirements as stipulated by the ummc medical ethics committee ( ummc ethics approval code : 733.19 ) .
the distribution of samples from malays , chinese , and indians , as well as the ratio of females to males , is shown in table 1 .
genomic dna was isolated from the peripheral blood samples by using the standard dna extraction method as described previously .
the concentration and purity of the extracted dna were further quantified by measuring the absorbance values at 260 nm and 280 nm via a spectrophotometer .
tetraprimer arms - pcr was performed in the genotyping of rs10168266 and rs7601754 in stat4 region , rs2230926 and rs3757173 in tnfaip3 region , and rs4728142 in irf5 region .
primers were designed using computer software accessible through the internet at http://cedar.genetics.soton.ac.uk/public_html/primer1.html , developed by ye and team . in silico pcr as described previously was further carried out to ensure the self - designed primers were targeted to the gene regions of interest [ 1921 ] .
each pcr reaction was carried out in a total of 10 l , containing 50 ng of template dna , appropriate concentration of inner and outer primers and mgcl2 ( table 3 ) , 200 m dntp , 20 mm tris - hcl ph8.4 , 50 mm kcl , and 0.15 u taq polymerase ( fermentas , vilnius , lithuania . ) .
the pcr mixture was then subjected to touchdown pcr , whereby it was incubated for 5 min at 95c , followed by 30 cycles of 45 s denaturation at 95c , 45 s of annealing ( started at temperature 10c higher than annealing temperature , decreasing by 1c per cycle , maintained at annealing temperature for the remaining 20 cycles ) and 45 s of extension at 72c , and a final extension at 72c for 10 min at the end of the cycles .
five microlitres of pcr amplicons was electrophoresed on a 2% ( w / v ) agarose gel .
the agarose gel was viewed under uv illumination and image was recorded using a gel documentation system .
predesigned taqman snp genotyping assays were used to genotype snps where tetraprimers could not be designed for arms - pcr ( probe i d : rs7574865 in stat4 region , c_29882391_10 ; rs729302 in irf5 region , c_2691216_10 ; applied biosystems , ny , usa ) .
fifty nanograms of template dna was mixed with 2x taqman gtxpress master mix ( applied biosystems ) and 20x taqman genotyping assay ( applied biosystems ) to make up to a total volume of 10 l .
real - time pcr reaction was initiated with pre - pcr read step at 60c for 1 min , followed by dna polymerase activation at 95c for 20 s , 40 cycles of denaturation ( 95c for 3 s ) and annealing / extension ( 60c for 30 s ) , and ended with a final extension step at 60c for 1 min .
allele and genotype frequencies were calculated , followed by performing a goodness - of - fit test to evaluate whether or not the observed genotype frequencies of each polymorphisms were departures from hardy - weinberg equilibrium ( hwe ) in control subjects ( p values > 0.05 ) .
fisher 's exact test was conducted on 2 2 contingency table using spss software to assess the association of each snp with sle susceptibility in malays , chinese , and indians .
p values were adjusted according to bonferroni correction and p < 0.05 was regarded as significant .
adjusted ors were computed using logistic regression , whereby major allele and major homozygous genotype of each snp were set as reference group and their ors were adjusted to 1 .
the ors of the three ethnicities were combined using mantel - haenszel test to evaluate the overall association of each snp in the malaysian population .
the mantel - haenszel ors were calculated using comprehensive meta - analysis version 2.0 software ( biostat , nj , usa ) .
random effect model was used when heterogeneity was significant ( p < 0.10 ) ; otherwise , the fixed effect model was used .
meta - analysis was conducted using comprehensive meta - analysis version 2.0 software ( biostat ) for the snp(s ) which was / were significantly associated with sle in the malaysian population by including data from other studies as well as the current study . due to insufficient number of indian subjects ,
we examined the association between stat4 polymorphisms and sle fully and rigorously , with the use of the key words
systemic lupus erythematosus , and sle . electronic databases including pubmed , embase , and web of science
only fully published articles were included and the eligible studies were identified based on the following criteria : ( a ) the study was original , ( b ) the patients were sporadic cases , ( c ) having available allele and genotype frequency data , and ( d ) having sufficient published data to determine or with 95% ci .
data extraction was performed by collecting the following information from each study : the first author 's name , year of publication , ethnicity , the number of cases and controls , and the frequency of minor allele ( maf ) of each polymorphism in both cases and controls . for studies including several independent case - control populations ,
malay and chinese populations from this study were also included in the meta - analysis .
random effect model was used for meta - analysis when heterogeneity was significant ( p < 0.10 ) ; otherwise , fixed effect model was used . by inputting the study name , total number of cases and controls , and maf of cases and controls , comprehensive meta - analysis version 2.0 software calculated the ors , 95% ci , and p values for each study , as well as the pooled or , 95% ci , and p values for the meta - analysis .
the goodness - of - fit test demonstrated that all polymorphisms investigated in this study fulfilled hwe in the control group .
the malay sle patients were significantly associated with minor alleles of stat4 rs7574865 , rs10168266 , and tnfaip3 rs2230926 , and heterozygous genotype tg of tnfaip3 rs2230926 ( table 4 ) . increased sle susceptibility in chinese population was significantly conferred by minor alleles and minor homozygous genotypes tt of stat4 rs7574865 and rs10168266 .
however , after bonferroni adjustment , significant associations were only observed between minor allele t of stat4 rs7574865 and chinese sle patients ( p = 0.028 , or = 1.42 , 95% ci : 1.121.82 ) and between minor allele t of stat4 rs10168266 and malay sle patients ( p = 0.035 , or = 1.80 , 95% ci : 1.202.71 ) .
stat4 rs7601754 , tnfaip3 rs3757173 , and irf5 rs4728142 and rs729302 did not show significant association with sle in any of the ethnicities .
combined analysis of the three ethnicities was carried out to represent the association of each snp with sle in the malaysian population as a whole .
the analysis revealed that only minor allele t of stat4 rs10168266 was significantly associated with the malaysian sle ( p = 0.014 , or = 1.435 , 95% ci : 1.1431.802 ) ( table 5 ) . since stat4 rs10168166 showed significant association with the malaysian sle , meta - analysis
was carried out to combine the data from other studies with the current one [ 6 , 2325 ] .
four relevant articles were identified eligible and a total of 5 subgroups were included for comparison .
data extracted from these articles is shown in table 6 . in the overall analysis , significant association of stat4 rs10168166 with sle
the fixed effect model was used as the heterogeneity test did not appear significant ( p > 0.10 ) .
the pooled or for the minor allele t was 1.559 , with 95% ci of 1.4591.665 and p value of < 0.001 ( figure 1 ) .
understanding the full molecular pathology of sle remains a great challenge , although many insights have been revealed .
recognition of self - nucleic acids by toll - like receptors tlr7 and tlr9 on plasmacytoid dendritic cells and b cells is believed to be an important step in the pathogenesis of this disease
. increased antinuclear antibodies and production of type i ifn are both correlated with the severity of disease .
stat4 , tnfaip3 , and irf5 are genes involved in regulating tlr / ifn signalling pathways .
snps investigated in this study have consistently shown associations with sle susceptibility in many populations , especially in asians [ 7 , 24 , 2729 ] .
when the various ethnic groups were considered , rs7574865 and rs10168266 of stat4 gene were significant in chinese and malays , respectively .
however , only rs10168266 of stat4 was observed to have correlations with sle in the malaysians generally .
none of the snps seemed to influence sle in indians . due to population demographics and lower sle risk predisposition ,
this suggests that the irf5 genetic variants tested for in this study are not linked to sle in our cohort and that there may be other variants that are more important in the indian ethnic group .
therefore , it may be concluded from the present study that stat4 gene polymorphisms feature more prominently as the genetic risk factors in the malaysian sle rather than those polymorphisms in tnfaip3 and irf5 .
rs10168266 which is located in intron 5 of stat4 gene has been frequently related to sle susceptibility in the asian population , particularly in korean population , and also in the european population [ 6 , 2325 , 30 ] .
nevertheless , not many studies were done on this snp and thus only four studies were included in the meta - analysis of this study .
after the analysis , the pooled or and p value once again showed that this snp was overall an important risk factor for sle and more attention should be taken .
rs7574865 , which was significantly associated with sle in chinese in this study , is located in the third intron of the stat4 gene .
the minor / risk allele t has reported associations with other immune - mediated diseases such as rheumatoid arthritis , primary sjgren 's syndrome , type-1 diabetes , crohn 's disease , and ulcerative colitis [ 3134 ] .
the association of this particular snp with sle susceptibility was observed in many populations , including both european and asian populations .
snp haplotype in the third intron of stat4 marked by rs7574865 was found to be associated with sle susceptibility and it could be responsible for splice variation or regulatory effects of stat4 [ 31 , 35 ] .
this coding snp is a nonsynonymous variant causing a phenylalanine - to - cysteine change at residue 127 of the a20 protein .
it has been already proven that minor cys127 is relatively stable compared to the phe127 protein , causing it to be less effective at inhibiting tnf - induced nf-b activity .
interestingly , as opposed to other findings suggesting that minor allele g was the risk factor of sle , the results of this study demonstrated that it conferred protection against sle in our cohort [ 11 , 24 , 27 , 3640 ] .
this study speculates that apart from rs223093 , other factors such as adjacent snps may possibly alter the structure of a20 protein .
we suggest that multiple ( at least four ) genes may collectively play critical roles in the development of this disease [ 4143 ] .
finally , both snps in irf5 gene investigated in our study were not significant in the malaysian patients although both are fairly established sle risk factors for europeans and some asians [ 4447 ] .
the present study was relatively small in contrast to larger studies of sle by other researchers . nevertheless , we present evidence to suggest that the genes involved in tlr / ifn signalling pathways , especially stat4 rs10168266 polymorphisms , contribute to the development of sle in malays and chinese . | polymorphisms in genes involved in toll - like receptor / interferon signalling pathways have been reported previously to be associated with sle in many populations .
this study aimed to investigate the role of seven single nucleotide polymorphisms within tnfaip3 , stat4 , and irf5 , which are involved in upstream and downstream pathways of type i interferon production , in sle in the south east asian populations .
genotyping of 360 malaysian sle patients and 430 normal healthy individuals revealed that minor alleles of stat4 rs7574865 and rs10168266 were associated with elevated risk of sle in the chinese and malay patients , respectively ( p = 0.028 , odds ratio ( or ) = 1.42 ; p = 0.035 , or = 1.80 , respectively ) .
polymorphisms in tnfaip3 and irf5 did not show significant associations with sle in any of the ethnicities .
combined analysis of the malays , chinese , and indians for each snp indicated that stat4 rs10168266 was significantly associated with the malaysian sle as a whole ( p = 0.014 ; or = 1.435 ) .
the meta - analysis of stat4 rs10168266 , which combined the data of other studies and this study , further confirmed its importance as the risk factor for sle by having pooled or of 1.559 and p value of < 0.001 . |
ultrasound is widely available in emergency departments and point - of - care ultrasound is increasingly used by emergency physicians .
detection of intraperitoneal free air ( ifa ) is of great value in identifying life - threatening conditions in patients with acute abdominal pain .
ifa usually occurs as a result of a perforated hollow viscous , intraperitoneal gas insufflations , penetrating abdominal injury , infection with gas forming organisms , or spontaneous pneumoperitoneum .
however , many patients with acute abdominal pain can not stand to have a chest radiograph .
if perforation is suspected , those patients are usually subjected to abdominal ct scan exposing them to high dose of radiation .
we have to highlight that point - of - care ultrasound examination is of great value in these circumstances .
point - of - care ultrasound is an extension of the clinical examination in patients having an acute abdomen .
it is a goal - directed study that can be used for rapidly diagnosing intraperitoneal fluid , aortic aneurysms , gallstones , appendicitis , and hydronephrosis .
failure of ultrasound to reveal ifa is considered by many as a weakness of abdominal sonographgy .
herein we review the sonographic findings of ifa and technical manoeuvres that can improve clinicians detection of ifa using point - of - care ultrasound .
it is claimed that , sonography is superior to erect chest x - ray in diagnosing ifa and as little as 2 ml of ifa can be detected by ultrasound . nevertheless , its detection is difficult even for an experienced sonographer .
the sonographic appearance of ifa results from scattering of the ultrasound waves at the interface of soft tissue and air which is accompanied by reverberation of the waves between the transducer and the air .
this , typically , results in a high - amplitude linear echo ( increased echogenicity of a peritoneal stripe ) accompanied by posterior artifactual reverberation echoes with characteristic comet - tail appearance [ figure 1 ] .
sagital sonographic section of the right hypochondrium using a curvilinear probe showing enhanced peritoneal stripe ( empty arrow ) and reverberation artefacts ( small arrows ) which partially obscure the right lobe of the liver ( l ) and right kidney ( k ) .
laparotomy confirmed that the patient had perforated diverticulitis reverberation may not be seen in small air collections .
when extensive pneumoperitoneum is present , then reverberation may even obscure the underlying abdominal organs . trapped air bubbles in ascitic fluid or in a localized fluid collection will give rise to echogenic foci due to air from perforated viscus [ figure 2 ] .
transverse sonographic section of the right hypochondrium using a linear probe showing a hyperdence echogenic small area ( arrow head ) moving within a fluid collection .
laparotomy confirmed that the patient had a perforated duodenal ulcer associated sonographic findings can help in the diagnosis of the etiology of the ifa [ figure 3 ] .
detection of interaperitoneal free or localized fluid collections can be seen in perforated peptic ulcer or perforated appendix . thickened bowel or gall bladder wall and decreased bowel motility or
ileus may point toward a peritoneal inflammatory process that can be associated with perforation of diverticulitis or small bowel perforation .
sagital sonographic section of the right hypochondrium using a curvilinear probe showing a hyperdence interrupted echogenic lines under the liver in morrison 's pouch ( arrow head ) , fluid collection ( white arrow ) , and a hyperdense echogenic line in the anterior wall of the duodenum representing the scar of a duodenal ulcer ( black arrow ) .
the presence of intraperitoneal air outside the intestinal lumen is unusual and can be mistaken for air within the bowel .
the linear - array transducer ( 10 - 12 mhz ) is more sensitive than standard curvilinear abdominal transducer ( 2 - 5 mhz ) for detecting ifa because it has higher resolution in the near field where air accumulates .
ifa is best detected when scanning the right upper quadrant between the anterior abdominal wall and liver where there is no intervening bowels except rarely with interposition of the bowel between the liver and the diaphragm .
furthermore , the intraluminal gas can be seen inside a bowel loop having visible peristalsis and a normal wall thickness .
the presence of air in abnormal sites such as along with the fissure of ligamentum teres and morrison 's pouch should raise the suspicion of ifa .
patients should be first scanned in the supine position concentrating on the midline and right upper quadrant [ figure 1 ] .
the presence of even small amount of ifa behind the anterior abdominal wall causes an enhancement of the peritoneal stripe that will move when changing the patients position . in morbidly obese patients , the identification of enhanced peritoneal stripe can be facilitated by asking the patient to take a deep breath .
this can help in the differentiation between the enhanced peritoneal stripe and the moving intraperitoneal fat and bowel .
when scanning the right upper quadrant , care should be taken to avoid artifacts resulting from the adjacent lung .
the shadows produced by the intervening ribs originate above the peritoneal line and move with respiration .
it is important to repeat the abdominal ultrasound in clinically suspected cases of perforated bowels with non conclusive evidence of ifa because more free air may accumulate and become more evident .
sonography is a valuable tool not only for the detection of ifa , but also for the diagnosis of the etiology of ifa . in comparison with ct scan ,
ultrasound is easily available , safe , portable , less expensive , and does not have radiation .
it is operator dependant and some ultrasound machines have low - quality images that may not be able to detect ifa .
furthermore , very ill and irritable patients may be less cooperative to allow for scanning of different regions .
ultrasound of ifa typically appears as an enhanced peritoneal stripe accompanied by posterior artifactual reverberation echoes with characteristic comet - tail appearance .
emergency physicians performing point - of - care ultrasound should be familiar with the sonographic findings of ifa which has a great value in diagnosing life - threatening conditions . repeated abdominal scanning of clinically suspected cases of bowel perforation is essential to detect accumulating free air overtime . | detection of intraperitoneal free air is important for the diagnosing of life - threatening conditions in patients with acute abdominal pain .
point - of - care ultrasound is an extension of the clinical examination in patients presenting with acute abdomen .
failure of sonographers to detect intraperitoneal free air was clinically considered as a limitation of abdominal ultrasound .
it is now increasingly appreciated that ultrasound may detect intraperitoneal free air .
emergency physicians should be familiar with the sonographic features of intraperitoneal free air which may be essential to recognize bowel perforation .
herein we review the literature on the sonographic findings of intraperitoneal free air and the technical manoeuvres that can improve clinicians detection of intraperitoneal free air using point - of - care ultrasound . |
there is still no evidence to base interventions on neuropsychological rehabilitation or cognitive stimulation related with learning development .
this is not derived at all from a lack of studies but to methodological difficulties finding trials suitable for meta - analysis .
this is a real hazard for translational research because of the difficulties of developers in testing new designs and showing efficacy , and also for the development of the body of knowledge , making difficult to find main theories and design appropriate experiments .
advances in communication technologies allow delivery of intensive , personalized , monitored neuropsychological intervention , through telerehabilitation platforms , and cognitive neuroscience based content , to guide plastic changes towards the recovery of more appropriate behaviour .
advances in information technologies have provide new possibilities to assess efficacy , opening new pathways to knowledge discovery from databases generated from clinical practice .
institute guttmann , from its initiative previrnec , is pioneering the application of this dual approach , bridging knowledge to the end user through communication technologies ( internet ) and extracting implicit knowledge ( efficacy on neuropsychological rehabilitation ) from data generated from each intervention . | introductionthere is still no evidence to base interventions on neuropsychological rehabilitation or cognitive stimulation related with learning development .
there are two main problems to solve .
the first is the sustainability of the clinical interventions .
the second one is the lack of evidence .
this is not derived at all from a lack of studies but to methodological difficulties finding trials suitable for meta - analysis .
this is a real hazard for translational research because of the difficulties of developers in testing new designs and showing efficacy , and also for the development of the body of knowledge , making difficult to find main theories and design appropriate experiments.description of careicts have profoundly modified this scenario .
advances in communication technologies allow delivery of intensive , personalized , monitored neuropsychological intervention , through telerehabilitation platforms , and cognitive neuroscience based content , to guide plastic changes towards the recovery of more appropriate behaviour .
advances in information technologies have provide new possibilities to assess efficacy , opening new pathways to knowledge discovery from databases generated from clinical practice.conclusioninstitute guttmann , from its initiative previrnec , is pioneering the application of this dual approach , bridging knowledge to the end user through communication technologies ( internet ) and extracting implicit knowledge ( efficacy on neuropsychological rehabilitation ) from data generated from each intervention . |
acute hospital settings do not provide the ideal care environment for older patients with heart failure ( hf ) .
a european integrated home - care model has been developed with the goals that patients will experience participation , safety and have knowledge about their disease and treatment .
to describe the meaning of participation in care of hf patients who receive home - care .
strategic sampling was used to reach a variation regarding severity of hf , different needs of home - care , age and gender .
the interviews were analyzed with qualitative content analysis , where categories were developed inductively . a preliminary analysis based on nine out of 18 interviews has been initiated .
five preliminary categories that describe patient participation have been identified : caring communication , accessibility , individual activity , trust and prerequisites for making decisions .
significance of the study : participation in care is regulated in health - care legalizations and is important from the individual s perspective .
therefore it is important to describe hf patient s experiences of participation within home - care . | introductionacute hospital settings do not provide the ideal care environment for older patients with heart failure ( hf ) .
an option is to provide advanced hf care in the patient s home .
a european integrated home - care model has been developed with the goals that patients will experience participation , safety and have knowledge about their disease and treatment .
participation in care has shown to have positive effects on self - care activities.aimto describe the meaning of participation in care of hf patients who receive home-care.methodspatients receiving home - care at four different settings were included in the study .
strategic sampling was used to reach a variation regarding severity of hf , different needs of home - care , age and gender .
a total of 18 open ended interviews have been conducted .
the interviews were analyzed with qualitative content analysis , where categories were developed inductively . a preliminary analysis based on nine out of 18 interviews has been initiated .
five preliminary categories that describe patient participation have been identified : caring communication , accessibility , individual activity , trust and prerequisites for making decisions .
each category consists of different subcategories.significance of the study : participation in care is regulated in health - care legalizations and is important from the individual s perspective .
home - care is becoming more common in patients with hf .
therefore it is important to describe hf patient s experiences of participation within home - care . |
brain and other central nervous system ( cns ) cancers include a variety of histopathologic subtypes , but the most common , by far , are gliomas . these tumours , which arise from the glial cells that surround and support neurons , include astrocytoma , glioblastoma , oligodendroglioma , oligoastrocytoma , and ependymoma .
medulloblastoma , another neuroepithelial cancer , is relatively common in children but rare in adults . brain cancers in children typically arise in the cerebellum , whereas brain cancers in adults are more likely to occur in the cerebral hemispheres . in adults , older age at diagnosis of brain cancer
is associated with higher tumour grade and poorer prognosis . indeed , glioblastoma is among the most lethal of all cancers .
brain and central nervous system ( cns ) tumours occur at each stage of life and are therefore classified as embryonic , paediatric , and adult cancers [ 2 , 3 ] . according to central brain tumour registry of the unites states ( cbtrus ) , the prevalence rate for all primary brain and central nervous system tumours
the five - year relative survival rate following diagnosis of a primary malignant brain and central nervous system tumour is 33.8% for males and 37.5% for females
brain and other cns cancers accounted for 3.1% of all cancers in egyptians , a large majority of cancers were located in the brain ( 85.2% ) ( middle east cancer consortium 19952001 ) . due to the lack of effective therapies for aggressive brain and cns tumours , the identification of new targets and prognostic indicators is required .
current studies in this area are focused on developing new therapies that target specific molecular events that lead to malignant transformation of cells .
the pi3k / akt pathway is one of the major cell survival pathways activated on stimulation of receptor tyrosine kinases such as epidermal growth factor receptors ( egfr ) that are over expressed in 4060% of gliomas [ 810 ] .
activation of pi3k / akt pathway has been associated with malignant transformation of cells and is frequently overexpressed in glioblastoma tumours when compared to nonglioblastoma tumours .
this activation is also associated with increased tumour grade that correlates positively with adverse clinical outcome in gliomas .
mammalian target of rapamycin ( mtor ) is a serine / threonine kinase that functions downstream of the pi3k / akt pathway .
mtor is known to regulate cell proliferation , growth , and survival by regulating translation initiation .
akt is shown to activate mtor through inhibition of tsc1/2 ( tuberous sclerosis complex 1 and 2 ) and activation of ras homologue - enriched in brain ( rheb ) .
upon activation by mtor , s6k1 phosphorylates s6 ribosomal protein , leading to increased translation of mrna with oligopyrimidine tract at the 5 terminal ( 5top ) .
s6k1 itself has no specific inhibitors that are available commercially but it responds to inhibitors that target its upstream regulators as mtor and pi3k .
rapamycin ( sirolimus ) , a macrolide antibiotic , blocks mtor kinase activity by forming a complex with fk506-binding protein ( fkbp-12 ) , thereby leading to the blockade of translation initiation through its action on s6k and 4ebp1 and cell cycle arrest at g1 phase [ 16 , 17 ] .
rapamycin 's growth inhibitory action has also been correlated with a decrease in glucose and amino acids uptake by rapamycin - sensitive glioblastoma cells .
several clinical trials of rapamycin and its derivatives are being conducted to evaluate their efficacy .
rapamycin and its derivatives have been shown to inhibit growth in several cancers , including breast cancer , pancreatic cancer , prostate cancer , melanoma , renal cell cancer , leukemia , and glioblastoma [ 2022 ] .
phase ii trial with temsirolimus , an ester analog of rapamycin , showed that this drug was well tolerated in patients with recurrent glioblastoma and this study has also shown that patients with high baseline levels of s6k1 responded to the drug treatment . using human glioma cell lines and
have found that suppression of mtor or raptor was sufficient to significantly reduce anchorage - independent growth in soft agar , an assay of transformation .
furthermore , s6k1 , but not eif4e , rescued glioma growth in soft agar from rapamycin - mediated suppression , and transient s6k1 inhibition was sufficient to significantly reduce glioma growth in soft agar . additionally , they found that in vivo s6k1 suppression in intracranially implanted glioma xenografts reduced levels of phosphorylated s6 and also resulted in reduced intracranial tumour growth .
their findings define a significant role for the mtor - raptor ( mtorc1)-s6k pathway in supporting gliomagenesis .
the ribosomal protein s6 kinase ( s6k ) family is a key regulator of cell growth and cell size and acts downstream of the pi3k / mtor pathway , and it has been identified to date to exert crucial functions in many other cellular processes such as metabolism and transcription regulation . the importance of the pi3k and mtor pathways in the regulation of s6k activity was revealed by the ability of inhibitors of those pathways to block the activity of s6 kinases . activating mutations in kinases involved in signalling via the pi3k / mtor / s6k pathway
on the other hand , inhibitory mechanisms were described to target s6k pathway through inhibiting its upstream signalling .
inhibitory mechanisms are exerted by a number of tumour suppressor genes such as pten , tsc1/2 , redd1 , and others .
deregulation of those proteins in many tumours has resulted in activation of pi3k and mtor pathways and in turn to s6k activation .
this has raised the possibility that s6 kinases are involved in tumour progression processes ( reviewed in ) .
the initial link between mtor / s6k regulation and the cellular response to hypoxia has come from several studies , which have demonstrated that mtor signalling upregulates expression of the hypoxia inducible factor ( hif-1 ) and vascular endothelial growth factor ( vegf ) , reviewed in .
recently , a tos motif has been found in hif-1 subunit , which also binds to raptor .
loss of the tos motif disrupts regulation of hypoxia via hif-1. immunofluorescent analysis of s6k1 and akt phosphorylation status has revealed positive correlation between a high p - akt and p - s6k expression , and a venous and capsular invasion of hepatocellular carcinoma ( hcc ) .
this suggests that activation of the akt - mtor - s6k pathway plays a significant role in hcc progression by promoting neoangiogenesis .
a proposed model for the inhibition of mtor / s6k pathway occurs under hypoxic conditions through coordination of different upstream tumour suppressors .
this model involves three different mechanisms : ( 1 ) redd1 upregulation , ( 2 ) ampk activation resulting in mtor inhibition through the activation of the tsc1/tsc2 tumour suppressor complex [ 33 , 34 ] , and ( 3 ) mtor accumulation in the nucleus through the action of the pml tumour suppressor .
all three regulatory events inhibit the interaction of mtor with rheb , which in turn reduces the phosphorylation / activation of s6k .
inhibition of the mtor / s6k pathway through these mechanisms results in decreased translation of hif-1 and expression of vegf [ 3335 ] .
deregulation of these tumour suppressors in cancer results in activation of the mtor / s6k pathway and subsequent elevation of hif-1 and vegf , which promote tumour angiogenesis .
the combination of mtor inhibitors with angiogenesis inhibitors could be a useful tool for cancer therapy . in this study
, we aimed to investigate the gene expression profile of s6 kinases in human brain tumours and its association with patients ' clinical outcomes as response to treatment , recurrence , and survival .
we used oncomine cancer microarray database ( http://www.oncomine.org/ ) to study the profile of s6k1 , s6k2 , and hypoxia - induced genes expression in human brain and cns tumour types versus their normal tissue counterparts . in order to compare the gene expression in a tumour type to its normal counterpart ,
gene expression data from a same study , performed with the same methodology , were used .
the gene expression data were log transformed , median centered per array , and the standard deviation was normalized to one per array . a gene was considered as overexpressed when its mean value in tumour samples was significantly higher to its mean value in the normal tissue counterpart using a t - test ( p 0.05 ) and the fold of induction was 1.5 .
brain and cns cancer data sets used in this study are summarised in table 1 .
the results were analysed using graphpad prism computer system ( graphpad software , san diego , usa ) .
statistical analysis comparisons were done with mann - whitney or student t - tests for gene expression analysis .
in the present study , we queried the oncomine database to systematically assess relative gene expression levels of s6k1 and s6k2 genes in brain and cns tumours .
gene expression data from embryonic , pediatric , and adult brain tumours were collected from oncomine database ( http://www.oncomine.com/ ) .
we compared gene expression in normal brain versus cancer tissues , and in different histological subtypes .
differential analysis of s6 kinases gene expression in brain and cns tumours versus normal tissue counterparts has retrieved 9 results in oncomine database .
we have analysed the studies that showed a significant difference value of gene expression ( p 0.05 ) in cancerous tissues compared with normal counterparts .
s6k1 gene ( rps6kb1 ) is significantly overexpressed in different brain tumours compared with normal brain tissue in 4 independent studies . on the other hand , s6k2 ( rps6kb2 ) gene expression profile did not change between cancerous and normal brain tissues ( data not shown ) .
study that analysed four different tumour types against normal cerebellum ; atypical teratoid / rhabdoid tumour ( fold = 20.023 , p = 0.015 ) , classic medulloblastoma ( fold = 13.827 ,
p = 0.02 ) , desmoplastic medulloblastoma ( fold = 6.658 , p = 0.05 ) , and malignant glioma ( fold = 5.389 , p = 0.06 ) .
study , s6k1 is overexpressed in glioblastoma compared to normal brain ( fold = 1.57 , p = 4.21e 11 ) , and in anaplastic astrocytoma ( fold = 1.5 , p = 4.72e 5 ) .
furthermore , we have found that s6k1 is overexpressed in glioblastoma compared to normal brain in a murat brain study , 2008 ( fold = 1.644 , p = 9.89e 4 ) .
figure 1(a ) shows some representative results of the analysis performed of s6k1 in brain tumours against normal brain .
furthermore , we have found that s6k1 is differentially overexpressed in specific histology types of brain tumours . in van den boom
study , s6k1 gene is differentially overexpressed in mixed glioma when compared to astrocytoma and glioblastoma ( p = 0.017 ) .
study , s6k1 is differentially overexpressed in glioblastoma against oligodendroglial tumour ( p = 0.002 ) .
these results indicate that s6k1 gene but not s6k2 is overexpressed in a number of brain and cns tumours and shows differential expression profile in specific histological types .
to assess the clinical significance of s6k1 overexpression in brain tumours , we investigated the association between its gene expression levels and the patient 's clinical outcome including response to treatment , recurrence , and survival status .
no significant association between s6k1 gene expression levels and patients response to therapy or sample recurrence status was identified , while a significant association between s6k1 overexpression and patients ' poor survival status was observed . in french
study , s6k1 gene is significantly overexpressed in anaplastic oligoastrocytoma compared to normal brain ( p = 5.20e 4 ) and in anaplastic oligodendroglioma compared to normal brain ( p = 0.0045 , figure 2(a ) ) . in the same study , as shown in figure 2(b )
, s6k1 is significantly overexpressed in patients who died later after 3 and 5 years of diagnosis ( p = 0.007 and p = 0.018 , resp . ) .
additionally , we found that s6k1 is overexpressed in patients who died after 5 years of surgical resection compared to alive patients ( p = 0.0136 ) .
furthermore , we have tested the association between s6k1 gene expression and patients ' survival by analysing the data available from pomeroy et al .
we have found that , in classic medulloblastoma , s6k1 overexpression is significantly associated with patients ' poor survival ( p = 0.04 ) ( supplementary figure 1(a ) available at doi:10.1155/2012/416927 ) .
taken together , we conclude that s6k1 is overexpressed in patients that showed poor survival status .
this indicates that s6k1 gene expression could be used as a useful marker to predict patients ' survival .
the high proliferation rate of solid tumours causes the continuous outstripping of the oxygen supply provided by the local vasculature , resulting in hypoxic regions within the tumour .
hypoxia inducible factor ( hif ) is the key mediator of cellular response to hypoxia , activating the expression of multiple genes that participate in angiogenesis , iron metabolism , glycolysis , glucose transport , and cell proliferation and survival .
to assess the gene expression profile of hypoxia - induced genes in brain and cns tumours , we queried the oncomine database for the following genes ; hif1a1 , vegfa , mmp2 , nedd9 , sox4 , and sox9 .
we found that hypoxia - induced genes are overexpressed in a number of brain tumours .
table 2 summarizes the analysis results of the selected genes indicating the induction fold and the significance difference of gene expression in a tumour type compare to normal brain .
only analysis that showed fold of induction > 2 and p < 0.01 was included .
the results we show here highlights the importance of hypoxic mechanism in brain tumours and the high level of overexpression of angiogenic factors as hif1a , vegfa , and mmp2 that are implicated in metastasis could be target molecules in those tumour types .
here , we aimed to investigate the coexpression profile of s6k1 and hypoxia - induced genes in brain tumours . to achieve
that , we queried the oncomine database using a literature defined concept concept : upregulated genes in response to hypoxia and in response to hif-1 expression .
this concept included a gene list of genes reported in literature to be upregulated by hypoxia and in response to hypoxia inducible factor 1 .
this gene list incorporated hif1a , vegfa , sox4 , sox9 , nedd9 , clo4a1 , col1a1 , and other genes .
the query included s6k1 has been done in brain and cns tumours versus normal brain .
we have observed that s6k1 is co - overexpressed with known genes that are induced by hypoxia .
figure 3(a ) shows a heat map of coexpression profile analysis of s6k1 and hypoxia - induced genes in pomeroy brain study .
the analysis showed a significant overexpression of s6k1 and hypoxia - induced genes in atypical teratoid / rhabdoid tumour compared to normal cerebellum ( figure 3(a ) ) . to extend the analysis to more studies we compared the expression profile of s6k1 and hypoxia - induced genes in 4 different brain tumours comprising 3 independent studies [ 2729 ] ( 298 patients ) .
figure 3(b ) shows the comparison of s6k1 and hypoxia - induced genes expression profile across 4 different analyses .
we noticed that vegfa , hif1a , sox4 , sox9 , mmp2 , tgfb1 genes are overexpressed together with s6k1 in all 4 tumour types investigated .
taken those results together , we indicate that s6k1 overexpression in brain tumours is associated with overexpression of hypoxia - induced genes and reflects a possible connection between s6k1 and hypoxia in that tumour type .
deregulation of this pathway is reported in various cancers highlighting it as a possible target in cancer .
region 17q23 is amplified in several breast cancer cell lines and in about 30% of primary tumours , whereas s6k1 is overexpressed in the majority of breast cell lines and breast primary tumours with this amplification [ 4548 ] . in brain and cns tumours
, there is no data regarding the s6 kinases gene expression profile and gene amplification . using a rich source of publically available cancer microarray data as the oncomine database , we investigated the gene expression of s6 kinases in different tumours .
the overexpression of s6k1 in brain tumours not s6k2 attracted our attention to a distinct role of this homologue in brain tumours .
we then surveyed the literature and found no gene expression analysis of s6 kinases in brain tumours has been done . in this study , we interrogated the oncomine database for s6k1 gene expression level and its association patient 's clinical outcomes . furthermore , we investigated the gene expression profile of hypoxia - induced genes and their association with s6k1 gene expression . here , we show that s6k1 but not s6k2 is overexpressed in different brain tumours as atypical teratoid tumour , classic medulloblastoma , glioblastoma , and anaplastic oligoastrocytoma compared to normal brain in cancer microarray database ; oncomine ( figure 1 ) .
no reports indicated the upregulation of s6k1 in brain tumours at the gene expression level .
s6k1 gene is known to be amplified in breast and cervical cancer patients but no reports regarding its amplification in brain tumours have been identified . to test the hypothesis if the overexpression of s6k1 mrna could be due to its gene amplification , we investigated the oncomine database and found only two records for s6k copy number in brain tumours .
no significant difference between s6k dna copy number in normal and brain tumour was detected in the investigated datasets ( data not shown ) .
this excludes the possibility of s6k1 gene amplification and explains that the gene expression upregulation involves upstream gene transcription regulators that bind to s6k1 gene promoter and enhance its expression .
we also observed that the overexpression of s6k1 in anaplastic oligoastrocytoma was significantly associated with patients ' poor survival status in 3 different analyses , which could attract the attention for s6k1 as a possible marker for prognosis in those brain tumour types .
furthermore , we have observed an association of s6k1 gene overexpression and patients ' poor survival in classic medulloblastoma when we analysed pomeroy et al .
study ( supplementary figure 1(a ) ) . due to limited number of studies available on brain tumours and cns , extending the analysis to other tumours types as glioblastoma retrieved only one study ; murat brain study 2008 .
no significant difference was observed between the s6k1 expression levels in the patients ' survived after 5 years and patients who died after the same period ( supplementary figure 2(a ) ) .
the tumours adapt themselves to hypoxic condition by upregulation of hif1 gene transcription factor which upregulates vascular endothelial growth factor vegf which is a crucial modulator of new blood vessels formation in tumour .
. the critical role of the hypoxia response network and hif in cancer has resulted in it being viewed as an ideal target for small molecule intervention ( reviewed in ) . in this study , we investigated the expression profile of hypoxia - induced genes in oncomine cancer microarray database using a literature defined concept .
we investigated a list of genes that are known from literature to respond to hypoxia ; for example : hif1 , vegfa , mmp2 , sox9 , sox4 , and nedd9 .
we have found that all those genes are significantly overexpressed in different brain tumour types compared to normal brain .
furthermore , we showed that the overexpression of s6k1 is associated with the upregulation of hypoxia - induced genes .
targeting hypoxia and s6k pathway could be a useful tool in brain tumours and better prognosis .
overall , we report here the induction of s6k1 gene expression in different brain tumours compared to normal brain .
this overexpression pattern is associated with patient 's poor survival and upregulation of hypoxic - induced genes .
these results highlight the possible role of s6k1 in brain tumour progression and as an upstream regulator of hypoxic response .
this attracts a further investigation of reported gene combination as a predictor for patients ' survival . | mtor / s6k pathway is a crucial regulator of cell growth and metabolism .
deregulated signalling via s6k has been linked to various human pathologies , including metabolic disorders and cancer .
many of the molecules signalling upstream of s6k have been shown to be either mutated or overexpressed in tumours , leading to s6k activation .
the role of s6k1 in brain tumours is not fully investigated . in this study , we investigated the gene expression profile of s6 kinases in brain and cns tumours using the publically available cancer microarray database .
we found that s6k1 but not s6k2 gene is overexpressed in brain tumours and this upregulation is associated with patients ' poor survival . furthermore , we interrogated oncomine database for the expression profile of hypoxia - induced genes using a literature - defined concept .
this gene list included hif1a , vegfa , sox4 , sox9 , mmp2 , and nedd9 .
we show that those genes are upregulated in all brain tumour studies investigated . additionally , we analysed the coexpression profile of s6k1 and hypoxia responsive genes .
the analysis was done across 4 different brain studies and showed that s6k1 is co - overexpressed with several hypoxia responsive genes .
this study highlights the possible role of s6k1 in brain tumour progression and prediction of patients ' survival .
however , new epidemiological studies should be conducted in order to confirm these associations and to refine the role of s6k1 in brain tumours as a useful marker for patients ' survival . |
since its discovery in the mid-1960s , the development of stable ,
well - defined , and functional - group - tolerant olefin metathesis catalysts
has greatly influenced the fields of organic synthesis and polymer
and materials science .
although alkene metathesis has
found a wide range of applications , alkyne metathesis has only recently
become the focus of attention .
moreover , living ring - opening olefin metathesis polymerization ( romp )
has had a great impact in the areas of biomimetic synthetic polymers ,
self - assembled nanomaterials , and monolithic supports . despite recent synthetic advances toward
highly functionalized
ring - strained alkynes , the application of ring - opening alkyne metathesis
polymerization ( roamp ) to the field of polymer synthesis has remained
limited due to the lack of commercially available well - behaved catalysts .
presently , poly(arylene ethynylene)s , used in applications
ranging
from molecular photonics , electronics , to sensing , can be accessed
through acyclic diyne metathesis ( adimet ) polymerization of diynes
using highly active molybdenum and tungsten catalysts .
however , this step - growth process provides only very limited control
over the polydispersity , length , and modality of the polymer product .
previous attempts at synthesizing polymers using ring - opening of strained
alkynes showed polydispersities ranging from 1.1 to 7.0 . while polymers with polydispersities
as low as 1.1 have been obtained ,
the active catalyst species is poorly
defined , and the reaction requires low temperatures and rigorous air - free
conditions .
polymers resulting from these
catalysts tend to have higher molecular weights than predicted on
the basis of the monomer to catalyst loading .
h nmr experiments
show that only a fraction of the catalyst is activated and contributes
to the linear chain growth , indicating that the rate of propagation
is larger than the rate of initiation ( kp / ki > 1 ) .
the poor selectivity of
alkyne
metathesis catalysts for strained over unstrained alkynes in the growing
polymer chain leads to significant broadening of the polydispersity
index ( pdi ) through chain - transfer processes and backbiting
to form cyclic structures . in this study
, we report the synthesis
and the detailed mechanistic
investigation of the first molecularly defined living
ring - opening alkyne metathesis catalyst [ tolcmo(ono)(or)]kor ( r = cch3(cf3)2 , ono = 6,6-(pyridine-2,6-diyl)bis(2,4-di - tert - butylphenolate ) ) 1 ( figure 1 ) . in solution ,
a rapid equilibrium between the -ate complex 1 and the pentacoordinate 14-electron complex 2 is observed ( electron count does not include potential -donation
of electron density from alkoxide lone pairs ) .
while the reversible
association of a free alkoxide prevents undesired side reactions ,
the dissociation of 1 does not represent a rate - limiting
step during the propagation .
kinetic studies reveal that the growing
polymer chain efficiently limits the rate of propagation with respect
to the rate of initiation ( kp / ki 10 ) .
we herein
demonstrate the outstanding control over molecular weight and polydispersity
achieved in living roamp with 1 and the first synthesis
of block copolymers through alkyne metathesis .
catalyst 1 was synthesized through ligand exchange
from the trisalkoxy molybdenum benzylidyne complex [ tolcmo(or)3(dme ) ] 3 . while structurally
related 12-electron molybdenum and tungsten complexes have been reported
as catalysts for alkyne cross - metathesis and ring - closing metathesis ,
these highly active complexes are unsuitable for controlled roamp .
extensive chain transfer reactions lead to undesired broad weight
distributions ( pdi > 2 ) . in an effort to increase the selectivity of our catalyst for the
activation of strained monomers over unstrained alkynes in the growing
polymer chain
, we incorporated a permanent electron donating , sterically
demanding ono pincer ligand 4 .
this tridentate ligand stabilizes the high oxidation state of the
molybdenum benzylidyne complex , prevents its dimerization in solution , and irreversibly blocks one of the catalyst s
active sites .
deprotonation of the ono pincer
ligand 4 with potassium benzyl followed by addition to
[ tolcmo(or)3(dme ) ] in toluene
quantitatively converted 3 to the desired
product 1 , by h and f nmr spectroscopy .
color coding : c ( gray ) , o
( red ) , n ( blue ) , f ( green ) , mo ( turquoise ) .
dark brown crystals of 1 were isolated
in 36% yield
after recrystallization from diisopropyl ether at 35 c .
x - ray crystallography
of 1 ( figure 1 ) confirms the presence
of a c(1)mo(1 ) triple bond with bond length of 1.760(2 )
and c(2)c(1)mo(1 ) angle of 176.91(19). the tridentate
ono pincer ligand adopts a skewed conformation featuring typical mo(1)o(1 )
and mo(1)o(3 ) distances of 1.9876(16 ) and 2.0010(16 ) ,
respectively .
the mo(1)n(1 ) distance of 2.2227(19 )
corresponds to a neutral l - type n
mo bond , indicating the presence
of an interaction between the lone pair of the pyridine ring and the
metal center .
the presence of two alkoxides and one potassium cation
in the crystal structure of 1 confirms that only one
alkoxide in 3 has been displaced by the ono pincer ligand .
the mo o distances are 2.0038(16 ) and 2.2475(16 ) for
the hexafluoro - tert - butoxide cis , mo(1)o(2 ) , and trans , mo(1)o(4 ) ,
to the carbyne , respectively .
the elongated mo(1)o(4 ) bond
for the alkoxide trans to the carbyne suggests a
weak interaction with an oxygen lone pair .
crystals of 1 are stable in air for hours and can
be stored for indefinite time under an atmosphere of nitrogen . in
the absence of moisture and air , a solution of 1 in
toluene - d8 shows less than 5% decomposition after one
month at 24 c . in toluene - d8 ,
the
pseudo - octahedral -ate complex 1 is in dynamic equilibrium
with the dissociated pentacoordinate complex [ tolcmo(ono)(or ) ]
( r = cch3(cf3)2 ) 2 ( supporting information figure s1 ) . in thf - d8
the alkoxide trans to the
carbyne is replaced by the solvent , and only a single species , corresponding
to a thf bound hexacoordinate complex , is observed by h and f nmr .
we studied the roamp of 3,8-dihexyloxy-5,6-dihydro-11,12-didehydrodibenzo[a , e]annulene ( 5a ) ( scheme 1 ) , a readily accessible highly solubilized ring - strained
alkyne , with 1 .
addition of 1 to a solution
of 5a in toluene ( [ 5a]/
= 10 ) at 24 c does not lead to the formation of polymeric species
within 24 h. h and f nmr indicate that the
roamp catalyst 1 quantitatively initiates with a half - life
of t1/2 < 5 min with 1 equiv of 5a to form the initiated complex 6 ( n = 1 ) ( scheme 1 ) . at 90 c , however ,
the initiation reaction is instantaneous , and the living roamp of
monomer 5a ( 10 equiv ) in toluene is completed in less
than 2 h , as determined by h nmr spectroscopy . in the
absence of monomer ,
the molybdenum catalyst attached to the propagating
polymer chain remains active and continues to incorporate equivalents
of monomer added sequentially to the reaction mixture ( supporting information figure s2 ) .
precipitation
of the resulting polymers in meoh affords poly-5a in greater than 90% isolated yield .
gpc analysis for various
monomer / catalyst loadings at 90 c in toluene shows a pdi of
1.02 , the lowest value ever reported for roamp ( figure 2 , table 1 ) . extended reaction
times
the molecular weights
of poly-5a determined by gpc , calibrated
to polystyrene standards , scale linearly with the conversion of monomer
( supporting information figure s2 ) , are
proportional to the initial [ 5a]/ loading ,
and show a unimodal distribution ( figure 2 ) .
no evidence for branching or the formation of cyclic polymers could
be observed by h nmr analysis and mass spectrometry ( supporting information figure s3 ) .
h nmr end - group analysis of the tolyl group reveals that gpc overestimates
the mn of poly-5a . a correction factor 0.71.0 correlates
well with the degree of polymerization determined by nmr analysis
and the expected molecular weight based on the [ 5a]/ loading .
gpc traces for poly-5a produced through
roamp of 5a with catalyst 1 at variable
loadings of [ 5a]/ = 100 ( red ) , 50 ( blue ) ,
20 ( green ) , 10 ( black ) ( t = 90 ) , calibrated
to polystyrene standards .
the proposed kinetic scheme for the polymerization of a ring - strained
monomer 5a with catalyst 1 is depicted in
scheme 2 . in a fast initiation reaction ,
1
equiv of 5a reacts with 2 to form the initiated
complex 7 ( n = 1 ) .
binding of kor to 7 stabilizes the initiated complex and reversibly blocks the
active site .
dissociation of kor from 6 regenerates the
active propagating species that undergoes linear chain - growth polymerization
with further equivalents of 5a to form extended living
polymer chains . calibrated to narrow polydispersity
polystyrene standards .
degree
of polymerization determined
by h nmr end - group analysis . to meet the stringent criteria
for a living polymerization
the
initiation of the catalyst must be fast and quantitative ( ki > kp ) , the
concentration
of propagating species has to remain constant throughout the reaction ,
all propagating chains have to grow at the same rate , and irreversible
termination and chain - transfer processes should be absent .
the rate laws for both the initiation and the propagation reaction
are derived employing the following assumptions : ( i ) the release of
ring - strain stored in the cyclic monomer 5a makes the
initiation and the propagation irreversible .
( ii ) the rate of propagation kp is comparable for all propagating species
irrespective of the degree of polymerization .
( iii ) the dissociation
equilibria are faster than the rate of initiation / propagation .
it is thus reasonable
to assume that , during the polymerization , the concentration of 7 reaches steady state .
the resulting rate law for the polymerization
is1where [ m ] is the concentration
of monomer 5a , [ c]0 is the starting concentration
of 1 , and kdiss , p is the
dissociation
constant of 6 . since the rate of initiation of complex 2 is very fast at the temperatures used throughout the polymerization ,
we herein rely on an approximation based on initial rates of reaction2where [ ci ] is
the concentration
of all initiated species 6/7 , and kdiss is the dissociation constant of 1 .
plots
of ln([m]/[m]0 ) over time ( figure 3 ) are linear throughout the entire polymerization and fit a rate
law first order in monomer .
the concentration of propagating species
is constant throughout the reaction , and irreversible termination
processes are absent
. the observed rate of propagation shows a linear
dependence on the catalyst loading ( supporting
information figure s4 ) . a plot of 1/([kor]/kdiss , p+1 ) versus kp , obs at
90 c shows a linear correlation between the rate of propagation
and the inverse of the concentration of kor ( supporting
information figure s5 ) .
similarly , the rate of initiation shows
a linear dependence on the concentration of monomer [ m ] and catalyst
[ c]0 .
the observed rate constants ki , obs and kp , obs at various
temperatures are summarized in table 2 . the
standard activation enthalpy for the initiation ( h = 20.7 1.2 kcal mol ) and for the propagation reaction ( h = 23.0 1.2 kcal mol ) can
be derived from eyring analysis ( supporting information figures s6s7 ) .
kinetic studies of the rate of polymerization
of 5a and 5b by 1 at various
temperatures . from eyring analysis
we conclude that , for the polymerization of 5a with catalyst 1 at 90 c , the rate of
initiation is 10 times faster than the rate of
propagation .
quantitative initiation of catalyst 1 is
practically instantaneous upon addition to a solution of the monomer .
all propagating species incorporate monomer 5a at comparable
rates ( kp ) to give polymers with exceptionally
narrow weight distributions . the rate of initiation at t >
40 c is too fast to be monitored by f nmr .
we studied the role of the weakly
coordinating alkoxide ligand
during the initiation and the polymerization reaction . at elevated
temperatures ( t > 60 c )
a rapid equilibrium
is established between the -ate complexes 1 and 6 , and the dissociated complexes 2 and 7 , respectively ( supporting information figure s1 ) .
the dissociation constants of 1 ( kdiss ) and 6 ( kdiss , p ) at selected temperatures are summarized in table 3 .
vant hoff analysis reveals that the changes
in standard free enthalpy ( h = 7.1
0.2 kcal mol ) and entropy ( s = 13.8 0.6 eu ) associated with the dissociation of
kor from 6 are smaller than the respective changes observed
for the dissociation of 1 ( h
= 9.5 0.5 kcal mol , s = 16.8 2.1 eu ) ( supporting information figures s8s9 ) .
the rates of dissociation ( k1 and k2 ) at various temperatures
were measured by selective inversion recovery ( sir ) f
nmr experiments ( table 3 , supporting information figures s10s12 ) .
the standard activation enthalpies for the dissociation of 1 ( h = 10.4
0.6 kcal mol ) and 6 ( h = 12.8 0.4 kcal mol ) were derived from eyring plots ( supporting
information figures s13s14 ) . to highlight the importance
of the kor dissociation equilibrium for the performance of roamp catalyst 1 , we polymerized 5a in the presence of varying
amounts of a lewis acid .
addition of 2 equiv of bph3 to
a solution of 1 in toluene efficiently shifts the dissociation
equilibrium toward the pentacoordiante complex 2 ( 2 equiv
of a lewis acid are required to trap the labile hexafluoro - tert - butoxide and the isopropyl ether found in the crystal
unit cell of 1 ) .
polymers formed in the absence of free
hexafluoro - tert - butoxide feature broad weight distributions
( pdi > 1.3 ) and mn values that do not
reflect the initial [ 5a]/ loading ( supporting information figure s15 ) .
the resonance signals
in the f nmr are broadened and could not be inverted
for sir experiments .
figure 4 summarizes the experimentally determined
kinetic and thermodynamic parameters for the initiation and the propagation
reaction at standard conditions .
the association of kor is a fast
pre - equilibrium to the rate - determining step .
the rate of initiation
is faster than the rate of propagation even though the equilibrium
concentration of 2 is lower than the concentration of
the propagating species 7 .
the rate - determining transition
state for the propagation is 3.6 kcal mol higher
than the transition state for the initiation reaction .
the observed
difference in metathesis activity between 2 and 7 can be rationalized by a combination of electronic and steric
effects imposed by the growing polymer chain . the steric bulk associated
with
the ortho - substituted polymer backbone increases
the barrier for the incorporation of the next ring - strained monomer .
the initiated catalyst 7 features an electron donating
hexyloxy substituent on the benzylidyne that further stabilizes the
mo(vi ) complex as compared to the ch3 group in 2 ( scheme 1 ) .
reaction coordinate diagram for the initiation
( black ) and the
propagation reaction ( red ) at 25 c . to expand the substrate scope of roamp with catalyst 1 we synthesized ring - strained monomer 5b ( scheme 1 ) featuring solubilizing triethylene glylcol chains .
even though the ether oxygen atoms in the side chains compete with
the free alkoxide and the ring strained monomer for binding to the
propagating molybdenum species 7 , the mn and the pdis for polymers obtained from the ring opening
of 5b are comparable to 5a and
the observed rate constant for
the roamp of 5b at 90 c is slower ( kp , obs = 0.144 m s ) than for 5a resulting in a t1/2 38 min ( figure 3 ) .
sample taken from the reaction mixture
after t = 30 min . with two chemically distinct monomers at hand we studied
the performance
of roamp catalyst 1 in the synthesis of amphiphilic block
copolymers . at 90 c , 10 ( 20 )
prior to the addition of 10 ( 20 ) equiv
of 5b , an aliquot was removed from the reaction mixture
and analyzed by gpc . after the consumption of all monomers , as judged
by h nmr spectroscopy , the reaction was quenched with
meoh .
unlike poly-5a , low molecular
weight ( mn = 8000 ) poly-5a - block - poly-5b is soluble in meoh and only precipitates from concentrated
solutions as a pale orange solid in > 90% yield .
gpc analysis reveals
an increase in mn upon addition of 5b to the living chains of poly-5a ( supporting information figure s16 ) .
the pdi of poly-5a - block - poly-5b is exceptionally low ( 1.08 )
and matches the catalyst performance achieved for the respective homopolymers .
end - group analysis reveals that the ratio of monomers in poly-5a - block - poly-5b scales linearly with the monomer loading .
in summary , we have described the synthesis of the first molecularly
well - defined 16-electron roamp catalyst based on a molybdenum benzylidyne
ono pincer complex [ tolcmo(ono)(or)]kor ( r = cch3(cf3)2 ) 1 .
the incorporation
of a permanent electron donating tridentate ligand irreversibly blocks
one of the catalyst s active sites , prevents undesired alkyne
polymerization reactions , and significantly increases its stability
toward air and moisture .
the catalyst is capable of selectively ring - opening
strained alkynes in a controlled polymerization to yield high molecular
weight polymers with exceptionally low pdis ( 1.02 ) .
mechanistic studies
reveal that the roamp catalyst 1 meets all the criteria
for a controlled living polymerization : the initiation reaction is
quantitative and 10 times faster than the propagation
( ki > kp ) ,
the concentration of catalytically active complex is constant throughout
the reaction , and all propagating chains grow at the same rate . the
reversible coordination of kor to the propagating catalyst prevents
undesired chain termination and bimolecular decomposition of the catalyst .
we demonstrate for the first time the synthesis of structurally well - defined
block copolymers through a controlled living roamp .
the catalyst developed
herein provides an unprecedented control and access to functionalized
homo- and block copolymers derived from ring - strained alkynes with
potential applications in advanced thin - film electronics / photonics ,
molecular sensing , and nanopatterning .
unless otherwise stated ,
all manipulations of air and/or moisture sensitive compounds were
performed in oven - dried glassware , under an atmosphere of ar or n2 .
solvents were dried by passing through a column of alumina
and were degassed by vigorous bubbling of n2 or ar through
the solvent for 20 min .
all h , { h}c , and f nmr spectra were recorded on bruker av-600 ,
drx-500 , av-500 , and av-900 mhz spectrometers , and are referenced
to residual solvent peaks ( cdcl3h nmr
= 7.26 ppm , c nmr = 77.16 ppm ; c6d6h nmr = 7.16 ppm , c nmr
= 128.06 ppm ; tol - d8h nmr = 2.08 ppm ; thf - d8h nmr = 1.78 ppm , c nmr = 67.21
ppm ) or trifluorotoluene ( f nmr = 63.72
ppm ) .
the concentrations of 1 , 2 , 6 , 7 , and kocch3(cf3)2 were determined by f nmr using the eretic method
against an external standard of 13.6 mm trifluorotoluene in tol - d8 .
the concentration
of monomer 5a , b was verified by h nmr applying the eretic method against an external standard of
19.4 mm of hexamethyldisiloxane in tol - d8 .
selective inversion recovery ( sir )
experiments were performed using topspin for data acquisition , and
fitted with cifit .
the temperature in all vt nmr
experiments is calibrated to ethylene glycol or meoh standards .
esi
mass spectrometry was performed on a finnigan ltqft ( thermo ) spectrometer
in positive ionization mode .
maldi mass spectrometry was performed
on a voyager - de pro ( applied biosystems voyager system 6322 ) in positive
mode using a matrix of dithranol .
elemental analysis ( chn ) was performed
on a perkinelmer 2400 series ii combustion analyzer ( values are given
in % ) .
gel permeation chromatography ( gpc ) was carried out on a lc / ms
agilent 1260 infinity set up with a guard and two agilent polypore
300 mm 7.5 mm columns at 35 c and calibrated to narrow
polydispersity polystyrene standards ranging from mw = 100 to 4 068 981 .
x - ray crystallography
was performed on apex ii quazar , using a microfocus sealed source
( incoatec is ; mo k radiation ) , kappa geometry with dx
( bruker - axs build ) goniostat , a bruker apex ii detector , quazar multilayer
mirrors as the radiation monochromator , and oxford cryostream 700
for 1 .
crystallographic data were refined with shelxl-97 ,
solved with sir-2007 , visualized with ortep-32 , and finalized with
wingx . 4 , and kbn were synthesized following literature procedures .
a 25 ml vial was charged with 4 ( 88
mg , 0.18 mmol , 1.0 equiv ) in dry toluene ( 3 ml ) .
a suspension of kbn
( 48 mg , 0.37 mmol 2.05 equiv ) in dry toluene ( 8 ml ) was added dropwise
and the reaction mixture stirred for 15 min at 24 c . the resulting
suspension
was added dropwise to a solution of 3 ( 164
mg , 0.2 mmol , 1.1 equiv ) in toluene ( 7 ml ) .
an immediate color change
to dark brown was observed , and the reaction mixture was stirred for
30 h at 24 c . the suspension was filtered , and the solvent was
removed under dynamic vacuum .
the precipitate was dissolved in cold
ch2cl2/pentane ( 3:2 , 4 ml ) and filtered through
a precooled frit .
pr2o ( 1
ml ) was added to the solution , and the solvent was removed under vacuum .
the residue was recrystallized from pr2o ( 2 ml ) ( 35 c ) , to yield pure 1 ( 78 mg , 36% ) as a dark brown crystalline solid .
crystals for x - ray
analysis were grown from saturated pr2o solutions at 35 c . in toluene , 1 is in equilibrium with the dissociated pentacoordinate complex 2 and free koc(cf3)2ch3 .
h nmr ( 500 mhz , tol - d8 , 22 c )
= 7.70 ( 2 ) , 7.63 ( s , 2h , ar - h ) , 7.42 ( s , 2h , ar - h ) , 7.27 ( 2 ) , 7.20
( d , j = 8.0 hz , 2h , 3,5-nc5h2h ) , 6.91 ( t , j = 8.0 hz , 1h , 4-nc5h2h ) , 6.58 ( d , j = 7.6 hz , 2h , c6h2h2ch3 ) , 6.44 ( 2 ) , 6.30 ( d , j = 7.6 hz , 2h , c6h2h2ch3 ) , 6.26 ( 2 ) , 2.01 ( s , 3h , c6h4ch3 ) , 1.93 ( s , 3h ,
oc(cf3)2ch3 ) , 1.71
( 2 ) , 1.64 ( s , 18h , bu - h ) , 1.46 ( s , 18h , bu - h ) , 1.37 ( 2 ) , 1.00 ( s , 3h , k - oc(cf3)2ch3 ) ppm .
f
nmr ( 470 mhz , tol - d8 , 22 c )
= 76.79 ( 2 ) , 77.80 , 78.26 , 81.18
( dissociated koc(cf3)2ch3 ) ppm . in thf , only the dissociated species 2thf is observed , resulting in the presence of free koc(cf3)2ch3 .
h nmr ( 500 mhz , thf - d8 , 22 c ) = 7.92 ( t , j = 8.0 hz , 1h , 4-nc5h2h ) ,
7.70 ( d , j = 8.0 hz , 2h , 3,5-nc5h2h ) , 7.52 ( d , j = 2.3 hz , 2h ,
ar - h ) , 7.46 ( d , j = 2.3 hz , 2h ,
ar - h ) , 6.74 ( d , j = 7.9 hz , 2h ,
c6h2h2ch3 ) , 6.12 ( d , j = 7.9 hz , 2h , c6h2h2ch3 ) , 2.20 ( s , 3h ,
c6h4ch3 ) , 1.78 ( s ,
3h , oc(cf3)2ch3 ) ,
1.52 ( s , 18h , bu - h ) ,
1.39 ( s , 18h , bu - h )
ppm .
{ h}c nmr ( 126 mhz , thf - d8 , 22 c ) = 307.5 , 166.2 , 155.6 , 141.5 , 140.5 ,
139.1 , 138.8 , 137.5 , 136.8 , 130.3 , 127.6 , 126.0 , 125.4 , 124.9 , 123.1 ,
84.2 , 36.0 , 34.8 , 32.4 , 30.8 , 23.5 , 21.6 ppm .
f nmr ( 470
mhz , thf - d8 , 22 c ) = 76.92
ppm .
ftms ( esi - tof ) ( m / z ) : [ [ tolcmo(ono)(occh3(cf3)2 ) ] + h ] calcd [ c45h54f6mono3 ] , 868.3056 ;
found 868.3076 .
calcd for [ [ tolcmo(ono)(occh3(cf3)2)2]kopr2]2pr2o : c , 56.21 ; h , 6.26 ; n , 1.13 . found : c , 56.04 ; h ,
6.40 ; n , 1.38 .
crystal data : ccdc no . , 998197 ; formula , c60.5h83f12kmono6.25 ; fw , 1297.32
g mol ; temp , 100(2 ) k ; cryst syst , monoclinic ;
space group , p21/n ; color ,
black ; a , 12.751(5 ) ; b , 29.140(5 )
; c , 17.008(5 ) ; , 90.000(5) ;
, 93.406(5) ; , 90.000(5) ; v , 6308(3 ) ; z , 4 ; r1 , 0.0367 ;
wr2 , 0.0818 ; gof , 1.051 .
a 10 ml resealable schlenk tube was charged with
a stock solution of 5a ( 220 mm ) in toluene .
if required ,
the solution was diluted with additional dry toluene to reach a total
of 0.5 ml . a stock solution of 1 ( 11 mm , 100 l )
in toluene was added , and the reaction mixture was heated in a bath
at 90 c for 2 h. the reaction mixture was cooled , and polymers
were precipitated with meoh ( 2 ml ) .
the precipitate was filtered ,
washed with meoh ( 2 ml ) , and dried in vacuum to yield poly-5a ( 92% isolated yield ) as a pale brown solid .
h nmr ( 600 mhz , cdcl3 , 22 c ) = 7.40
( d , j = 8.4 hz , 2h , ar - h ) , 6.776.52
( m , 4h , ar - h ) , 3.67 ( t , j = 6.5
hz , 4h , och2 ) , 3.19 ( s , 4h , ch2 ) , 1.691.58 ( m , 4h , o(ch2)5ch3 ) , 1.411.19 ( m , 12h , o(ch2)5ch3 ) , 0.87 ( t , j = 7.0 hz , 6h , ch3 ) ppm .
nmr ( 151 mhz , cdcl3 , 22 c )
= 159.2 , 145.3 , 133.5 , 115.3 , 114.6 , 113.0 , 90.5 , 67.9 , 36.6 ,
31.8 , 29.4 , 25.9 , 22.8 , 14.2 ppm .
a 10 ml resealable schlenk tube was charged with
a stock solution of 5b ( 220 mm ) in toluene .
if required ,
the solution was diluted with additional dry toluene to reach a total
of 0.5 ml . a stock solution of 1 ( 11 mm , 100 l )
in toluene was added , and the reaction mixture was heated in a bath
at 90 c for 7 h. the reaction mixture was concentrated and the
solid residue suspended in cold meoh ( 2 ml ) .
the precipitate was filtered ,
washed with cold meoh ( 2 ml ) , and dried in vacuum to yield poly-5b ( 53% isolated yield ) as a pale orange
solid .
h nmr ( 900 mhz , cdcl3 , 22 c )
= 7.38 ( s , 2h , ar
h ) , 3.913.24
( m , 30h ) , 3.17 ( s , 4h , ch2 ) ppm .
nmr ( 226 mhz , cdcl3 , 22 c )
= 158.8 , 145.1 , 133.6 , 115.7 , 114.8 , 113.1 , 90.6 , 72.0 , 70.9 ,
70.7 ( 2c ) , 69.7 , 67.4 , 59.2 , 36.2 ppm .
a 10 ml resealable schlenk
tube was charged with a stock solution of 5a ( 230 mm ,
200 l ) in toluene . a stock solution of 1 ( 7.7
mm , 300 l ) in toluene
was added , and the reaction mixture was
heated at 90 c for 30 min .
an aliquot ( 150 l ) was quickly
removed and precipitated with meoh ( 2 ml ) . a stock solution of 5b ( 46 mm , 700 l ) in toluene
was added , and the reaction
was heated for an additional 7 h. the reaction mixture was cooled ,
and polymers were precipitated with meoh ( 2 ml ) .
the precipitate was
filtered , washed with meoh ( 2 ml ) , and dried in vacuum to yield poly-5a - block - poly-5b ( 94% isolated yield ) as a pale orange solid .
h nmr ( 500
mhz , cdcl3 , 22 c ) = 7.40 ( d , j = 8.4 hz , 4h , ar - h ) , 6.796.42 ( m , 8h , ar - h ) , 4.193.42 ( m , 34h ) , 3.18 ( s , 8h , ch2 ) , 1.791.49 ( m , 4h , o(ch2)5ch3 ) , 1.401.16 ( m , 12h , o(ch2)5ch3 ) , 0.86 ( t , j = 6.9 hz , 6h , ch3 ) ppm .
{ h}c nmr ( 126 mhz , cdcl3 , 22 c )
= 159.2 , 145.2 , 133.4 , 115.2 , 114.5 , 113.0 , 90.4 , 72.0 , 70.9 ,
70.7 ( 2c ) , 69.7 ( 2c ) , 67.9 , 59.2 , 36.6 , 31.8 , 29.4 , 25.9 , 22.8 , 14.2
ppm . | the pseudo - octahedral
molybdenum benzylidyne complex [ tolcmo(ono)(or)]kor ( r = cch3(cf3)2 ) 1 , featuring a stabilizing ono pincer ligand , initiates
the controlled living polymerization of strained dibenzocyclooctynes
at t > 60 c to give high molecular weight
polymers
with exceptionally low polydispersities ( pdi 1.02 ) .
kinetic
analyses reveal that the growing polymer chain attached to the propagating
catalyst efficiently limits the rate of propagation with respect to
the rate of initiation ( kp / ki 103 ) .
the reversible coordination
of kocch3(cf3)2 to the propagating
catalyst prevents undesired chain - termination and -transfer processes .
the ring - opening alkyne metathesis polymerization with 1 has all the characteristics of a living polymerization and enables ,
for the first time , the controlled synthesis of amphiphilic block
copolymers via roamp . |
conjugated polymers
have been studied extensively because of their
semiconducting and optical properties , which offer great potential
for a diverse range of applications such
as organic light - emitting diodes , field - effect
transistors , and polymer solar cells .
recently , macrocycles have attracted increasing attention as quasi - infinite
-conjugated systems exhibiting unique electronic and optical
behavior .
several studies have investigated
electron or energy transfer , nonlinear optical phenomena , and topological
effects in these structures . a promising approach to synthesis of nanosized fully -conjugated
macrocycles based on porphyrin units
however , the difficulty of synthesizing
suitable templates has greatly limited the size of macrocycles accessible .
to this end
, vernier effects were exploited to direct synthesis of
large nanorings using multiple smaller templates .
vernier templating not only proves to be a powerful new
strategy to construct large nanorings , but also renders shape - persistent
structures with diverse geometries .
exciton
delocalization and emission depolarization processes are expected
to be strongly affected by the change of geometry in the structures .
conformational effects on photophysical
properties have been examined
extensively using both experimental and theoretical
methods on a variety of conjugated polymer
systems , such as ppv , p3ht , and dendrimers . in particular ,
effects
on polarization memory loss and energy transfer have attracted much attention . at the center of our study
,
we address the question of how deliberate
distortion of a molecular nanoring influences the emissive properties
of a molecule following excitation .
we examine to what extent out - of - plane
distortions of a particular nanoring topology affect the polarization
memory of the transition dipole moment and whether such effects can
be predicted by molecular mechanics simulations . in search for answers
to these questions , a range of conjugated zinc porphyrin nanorings
of similar size but different conformations are examined using ultrafast
time - resolved spectroscopy and the results are compared to molecular
dynamics simulations of ring structures .
the synthesis and characterization of the
nanoring templates and complexes are described in detail elsewhere .
figure 1 introduces the porphyrin nanorings under investigation ,
which comprise porphyrin units joined by butadiyne bridges . through
insertion of templates binding to the porphyrin units ,
a range of
different molecular shapes are created with spatial extent on the
nanometer scale .
untemplated nanorings c - p10 and c - p12 only differ slightly in
ring diameter and have almost identical conformation and absorption / emission
spectra ( further information in the supporting
information ( si ) ) .
both samples are prepared in toluene/1%
pyridine solution to prevent aggregation . templating changes the geometry
considerably by rigidifying the nanoring into two smaller porphyrin
loops and introducing significant out - of - plane distortions ,
such as bending and twisting .
molecular mechanics simulations ( vide
infra ) show that c - p10(t5)2 and c - p10(t6)2 have similar structures ,
with the main difference being in the shape of templates as shown
in figure 1b , c .
the angle between the mean
template planes in c - p10(t5)2 is calculated to be
= 72. the twist in c - p12(t6)2 is even more pronounced and
has the form of a figure - of - eight , with = 28 , as shown
from crystal structure data in a previous study . on the other hand ,
these conformations have been deduced
from molecular mechanics and dynamics simulation using hyperchem , which will be described later in detail .
samples with template
complexes were prepared in pure toluene solution , as the templates
would otherwise be displaced by competition with pyridine .
chemical structures
of the budadiyne - linked zinc porphyrin nanoring
assemblies under investigation : ( a ) c - p10 , ( b ) c - p10(t5)2 , ( c ) c - p10(t6)2 , ( d ) c - p12 , ( e ) c - p12(t6)2 , and ( f ) c - p12(t8)2 .
all compounds have octyloxy
side chains ( ar ) which are omitted in the graphic for clarity .
the photoluminescence
( pl ) upconversion technique was engaged to investigate pl dynamics
of sample solutions held in quartz cuvettes as described in detail
elsewhere .
an excitation pulse was generated
by a mode - locked ti : sapphire laser with pulse duration of 100 fs and
a repetition rate of 80 mhz .
pl is collected and optically gated in
a beta - barium - oxide ( bbo ) crystal by a vertically polarized time - delayed
gate beam .
the upconverted signal , which consists of sum - frequency
photons from the gate pulse and the vertical component of the pl ,
was collected , dispersed in a monochromator , and detected using a
nitrogen - cooled ccd . using a combination of a half - wave plate and
a glan - thompson polarizer , the polarization of the excitation pulse
was varied and pl intensity dynamics were recorded separately for
components polarized parallel ( i ) and perpendicular ( i ) to the
excitation pulse polarization .
the pl anisotropy is defined using
= ( i i ) /(i + 2i ) and calculated from the
measured components .
the full - width - half - maximum ( fwhm ) of the instrumental
response function ( irf ) was
measured to be 270 fs , which gives the time - resolution limit
of the system ( further details in the si ) . by recording the irf with both polarizations , a temporal shift
of 15 fs between i and iwas found , and
thus , the calculation
of (t ) was adjusted accordingly ( further information
in the si ) .
to investigate pl decay dynamics at longer delay time after
excitation ( > 1 ns ) , electronic gating through time - correlated single - photon
counting ( tcspc ) technique was explored , using a becker & hickl
module . here
, emission was detected with a silicon single - photon avalanche
diode , yielding a temporal resolution of around 40 ps . by fitting
the experimental data to a single exponential decay model ipl
steady - state absorption and time - integrated pl spectra at room
temperature were recorded using a perkinelmer lambda 1050 uv / vis / nir
spectrometer and a horiba fluorolog fluorimeter , respectively .
in order to gain basic
insight about the photophysical and electronic properties of the nanorings ,
steady state absorption and emission spectroscopy was performed .
the
resulting spectra for c - p12 and c - p12(t6)2 are compared in figure 2 ; spectra for
further structures are provided in the si .
zinc porphyrin monomers exhibit a strong s0
s2 transition at 400 nm ( soret band ) and a weaker
s0 s1 transition at 550 nm
( q - band ) . for the nanorings ,
-conjugation
extends through the butadiyne bridges , lifting the degeneracy in both
the lowest - energy q band and soret band of the porphyrin
monomers .
transitions polarized parallel ( qx ) and perpendicular ( qy ) to the acetylenic backbone can therefore be observed
( see figure 2 ) . when the nanorings bind to
the templates , qx is
observed to be red - shifted with respect to the soret band , while qy shows no considerable change .
in addition , a significant sharpening of features in both emission
and absorption spectra can be observed for the templated complexes .
templates bring rigidity to the system ; the molecules are less prone
to interporphyrin torsional motions and the molecular backbone may
be planarized , resulting in the observed
red shift of qx .
the
four templated nanorings hence exhibit similar spectral features ( see
also further spectra in the si ) .
normalized
steady - state absorption ( blue lines ) and time - integrated
photoluminescence ( green lines ) spectra at 295 k for ( a ) c - p12 in toluene/1% pyridine and ( b ) c - p12(t6)2 in toluene solution .
the emission spectra were recorded after excitation
at 520 nm ( into the soret band ) .
pl upconversion under ambient condition was performed to reveal
the extent of emission depolarization following photoexcitation with
a linearly polarized laser pulse .
the templated nanorings were excited
at 820 nm and the untemplated rings at 760 nm , with emission being
monitored at the peak wavelength for each nanoring . to avoid pl quenching
via exciton
figure 3 depicts the time - dependent pl
of c - p12 and c - p12(t6)2 over
the first 15 ps after excitation with a pulse polarized either parallel
( i ) or perpendicular ( i ) to the polarization of the detected
emission .
for c - p12 , the pl intensity remains constant over the experiment
time window after the onset of the signal at t =
0 , whereas c - p12(t6)2 shows a slight decaying trend , in agreement
with the shorter lifetime obtained using tcspc ( shown in table 1 ) .
time - resolved pl dynamics of ( a ) c - p12 in toluene/1% pyridine ( concentration 0.2 mm ) and ( b ) c - p12(t6)2 in toluene solution ( concentration 0.4 mm ) .
samples were excited
by excitation pulse polarized either parallel ( blue dots ) or perpendicular
( red dots ) to the detection polarization , as illustrated in the inset .
selective excitation near the qx band of a rigid butadiyne - bridged
porphyrin oligomer is expected
to result in an excited state with a transition dipole moment along
the molecular backbone . in the absence
of any depolarization mechanisms
, emission will occur in the same
polarization direction as the absorption , and for a randomly oriented
distribution of such molecules in solution , an initial anisotropy
value of 0.4 is expected .
however , for a complete
depolarization of the transition dipole moment within the 2d plane ,
as may be the case for the untemplated rings , a pl anisotropy value
of 0.1 should be found .
other effects , such
as out - of - plane distortion , could further lower the value toward zero .
figure 4 shows
the temporal dependence of
the anisotropy (t ) for a number of the investigated
nanoring topologies .
nanoring topologies
investigated , the anisotropy is found to be static over the first
20 ps after excitation , within the temporal resolution of 270 fs .
it is possible that
excitation near the band edge ( 900 nm ) may lead to creation
of localized states that have higher anisotropy and/or slower pl depolarization ; however , we are unable to probe such effects
here .
these transients therefore indicate that while an initial ultrafast
depolarization process may occur within the first hundred femtoseconds
after excitation , slower effects such as molecular reorientation are
absent over these time scales .
the values
extracted in this way for the initial pl anisotropy are listed
in table 1 for all molecules investigated .
these values have been independently determined multiple times at
5 ps after excitation , and an overall accuracy of 0.02 was achieved .
the untemplated rings c - p10 and c - p12 display anisotropy values around 0.1 ,
as expected for a complete memory loss in the 2d plane and in the
absence of significant out - of - plane distortions of the rings .
for all templated rings on the other hand , a
significant reduction of below the value of 0.1 was observed .
these results suggest that the excitation can access any segment
on the rings , both templated and untemplated , within the first few
hundred femtoseconds after excitation .
such dynamics may be accounted
for by either of two different mechanisms . in the first possible scenario
,
excitons are initially fully delocalized over the entire ring in the
absorbing state , but may subsequently self - localize
following geometric relaxation .
in the second scenario , the absorbing state is only delocalized
over a subset of monomers , but excitons are subject to ultrafast energy
migration along the ring within hundreds of femtoseconds and thus
beyond the time - resolution of the upconversion system , similar to
observations reported in lh2 antenna complexes .
it has recently been shown that as the size of untemplated
porphyrin nanorings increases , a shift from the former to the latter
scenario may gradually occur .
we find that
the measured anisotropy values below 0.1 for the templated
nanorings can be explained by the presence of significant out - of - plane
distortions .
molecular dynamics simulations discussed in detail below
show that the measured is closer to zero for nanoring topologies
with larger distortions . as illustrated in figure 4 , the rigid planar c - p12(t8)2 has an appreciably higher anisotropy
( = 0.07 ) than the other three templated nanorings which are
found to display significant out - of - plane distortions .
pl polarization anisotropy
as a function of time after excitation
at 820 nm for c - p12 ( blue circles , detection
at 872 nm ) , c - p12(t8)2 ( red triangles , detection at 904 nm ) , and c - p12(t6)2 ( green squares , detection at 914 nm ) .
the structures shown
on the right are energy minimized geometries calculated using a modified
form of mm+ force field in hyperchem .
side chains
are not shown in the diagram but were included in the simulations .
to quantitatively examine the
distortions present in the different
nanoring topologies , molecular dynamics simulations were conducted
for the templated nanorings with side chains using hyperchem .
the starting configurations were energy - minimized geometries ( at
0 k ) calculated using a modified form of hyperchem s mm+ force field . additional bond stretch , angle bend , and
torsion terms were added to describe metalloporphyrins , alkynes , and butadiynes .
molecular dynamics simulation were conducted
assuming ambient vacuum at two constant temperatures , 300 and 600
k. simulations ran in time steps of 1 fs for a time window of 500
ps , which gave a representative average . since the templates are fairly
rigid , it can be assumed that distortions mainly originate from motions
between two template planes where two fundamental motions can be distinguished :
twisting and bending , as illustrated in figure 5a , b for the case of 300 k. corresponding results for 600 k are shown
in the si .
the corresponding angles
and are introduced in the figure to quantitatively describe
the deviation of the structure from a planar conformation .
all starting
configurations hold (0 ) 0. the distortion angles
of the conformations in the 500 ps simulation window are plotted as
a histogram , and the results are summarized in figure 5c f , with the root - mean - square ( rms ) values listed
in table 1 . due to the nature of the simulations
and the ambiguity in defining the distortion angles
it is remarkable
that , compared to the bending angle , the spread in the twisting
angle is generally much narrower and the rms remains closer
to the energetically optimized values , which indicates that , in the
molecular dynamics , thermal energy has a greater influence on the
bending motion than the twisting motion .
c - p10(t5)2 and c - p10(t6)2 show large out - of - plane distortions in twisting ( around 65 )
as well as bending motion at 300 k , consistent with the experimentally
observed low pl anisotropy values of = 0.05 .
c - p12(t6)2 and c - p12(t8)2 both show similar distributions in around
25 , much lower than that for the templated c - p10 topologies in comparison and in good agreement
with structures obtained from crytallographic investigations .
these simulations explain the only slight decrease
in from 0.1 to 0.07 experimentally observed for c - p12(t8)2 . however , c - p12(t6)2 has a much lower pl anisotropy , which can
be accounted for by its large bending angle similar to c - p10(t5)2 and c - p10(t6)2 .
in contrast , c - p12(t8)2 remains rigid :
is narrowly distributed around the planar starting conformation . in summary
, the molecular dynamics simulations reveal that the
templates provide an effective means to control the nanoring structure ,
as can be seen in the narrow distribution in figure 5c f as well as in the close resemblance between rms
values at 300 k and the energetically optimized values at 0 k for
all nanorings .
furthermore , a good correspondence is evident between
simulated conformations and measured pl anisotropy : the more pronounced
the out - of - plane distortions in both twisting and bending motions ,
the further the anisotropy is found to be lowered from 0.1 . using a simplified model based
on the results from hyperchem as described in the
main text , simulated anisotropy values s are given . to link the value
experimentally
obtained for the pl anisotropy
with the calculated molecular structures , simple simulations were
performed using a distributed point - dipole model .
the porphyrin units in each nanoring are
assumed to be arranged in two smaller circles around each template
as a result of the template s rigidity , with their transition
dipole moments aligned along the molecular backbone , as shown in the
schematic in the si .
the angle between
the two template planes in the calculation is taken to be the rms
values simulated by hyperchem at 300 k. corresponding
values for 600 k are given in the si and
exhibit similar trends .
for simplicity , the emitting and absorbing
states are assumed to extend over one monomer unit .
this approximation
seems sensible as the pl depolarization dynamics are observed to be
sufficiently fast ( < 300 fs ) to suggest that all sites are visited
with similar probability .
therefore , the calculated average based
on contributions from individual monomers will yield representative
results , independent of the extent of exciton wave function delocalization .
the polarization anisotropy is then calculated using1with the only parameter being , the
angle between absorbing and emitting dipole moments .
excitation can
access any segment of a nanoring , and so each monomer can act as absorbing
or emitting site with equal probability .
therefore , angles between
dipoles of any two porphyrin monomers on the rings are considered
and the average of is calculated according to eq 1 . even though this model is rather simplified and primarily
focuses on the effect of bending and twisting distortions on the pl
anisotropy , the results agree well with the experimental findings .
the calculated anisotropy results s are listed in
table 1 and lie within the error range of the
experimental values .
this correspondence confirms that the out - of - plane
distortion is in fact the major contribution causing the lowered anisotropy
value from 0.1 .
the modeled values tend to be slightly lower than
the experimental values , but are surprisingly close , given the simplicity
of the model .
deviations between experimental and simulated values
may arise because simulations were carried out assuming vacuum conditions
( screening of intermolecular interactions arising from the solvent
was not taken into account ) and because of some variation with the
molecular mechanics potential chosen .
in addition , a more complete
description of exciton depolarization dynamics ought to include effects
such as exciton localization dynamics , for example , to sites that are preferred through specific local
geometries
. however , the good agreement between the experimental results
and the simple simulation performed here ultimately results from the
intimate link between the topology of the molecule to the polarization
directions its emitting state can explore .
( a ) the twisting angle
between two template planes is defined using c - p12(t6)2 as an example .
four atoms on the porphyrin ring ( red ) are chosen
to form three vectors a , b , and c , with a and c lying approximately parallel to each
other when the molecule does not exhibit any twist .
is the
torsional angle defined by the four atoms and is the angle between
the planes formed by a , b and b , c.
represents the twisting angle from a planar position .
( b ) the bending
angle is illustrated using c - p12(t8)2 as an example .
two angles and are
calculated , where = + describes the deviation
from a planar position .
simulations were carried out with octyloxy
side chains , which are omitted in the diagram for clarity .
( c f )
area normalized histogram of twisting angle ( blue ) and bending
angle ( red ) for ( c ) c - p10(t5)2 , ( d ) c - p10(t6)2 , ( e ) c - p12(t6)2 , ( f ) c - p12(t8)2 .
molecular dynamics simulation carried
out using hyperchem at 300 k for 500 ps , as described
in detail in the text .
-conjugated nanorings containing
10 or 12 porphyrin units
have been investigated to explore the influence of molecular topology
on exciton dynamics and polarization memory loss .
it is found that
excitations can access any part of the templated nanorings , despite
their twisted conformations , thus showing similar exciton dynamics
to their more planar untemplated counterparts .
time - dependent ultrafast
spectroscopy reveals that all nanorings exhibit static pl anisotropy
transients within the first 20 ps after excitation , but with an anisotropy
much lower than the value of 0.1 found for untemplated rings .
molecular
dynamics simulations show that this phenomenon can be fully explained
and well described by the out - of - plane distortions of the rings from
a planar conformation .
numerical simulations of the pl anisotropy
taking into account such distortions yield values that correlate well
with the experimentally obtained results . in summary ,
template - directed
synthesis not only plays a vital role in the synthesis of large porphyrin
rings , but it also rigidifies the nanorings and introduces a means
to control their geometric structures while maintaining excellent
-conjugation . these findings open exciting new possibilities
for fast and directional energy transfer in nanoscale molecular structures . | conjugated
polymers with cyclic structures are interesting because
their symmetry leads to unique electronic properties .
recent advances
in vernier templating now allow large shape - persistent fully conjugated
porphyrin nanorings to be synthesized , exhibiting unique electronic
properties .
we examine the impact of different conformations on exciton
delocalization and emission depolarization in a range of different
porphyrin nanoring topologies with comparable spatial extent .
low
photoluminescence anisotropy values are found to occur within the
first few hundred femtoseconds after pulsed excitation , suggesting
ultrafast delocalization of excitons across the nanoring structures .
molecular dynamics simulations show that further polarization memory
loss is caused by out - of - plane distortions associated with twisting
and bending of the templated nanoring topologies . |
with the evolution of radical prostatectomy from an open technique to a laparoscopic or robotic approach , several discussions have ensued regarding the anatomy around the posterior bladder neck ( bn ) , because the antegrade dissection proceeds differently in the new approaches from that used in the open approach [ 1 - 4 ] . since 2000 , the anterior layer of denonvilliers ' fascia ( dnf ) or a posterior layer of the detrusor apron have been reported as the structure at the posterior aspect of the bn , which needed to be incised horizontally during posterior bn dissection .
more recently , tewari et al reported this structure as the retrotrigonal layer and emphasized that this tissue may serve as a key surgical anatomical landmark to orient the location of the vasa and seminal vesicles , which are located just beneath this layer ( fig .
, we have used this structure to identify the vas deferens and seminal vesicles in the midline at the posterior aspect of the prostato - vesical junction .
however , aside from the presence and position of the layer , it seems that there have been no reports about the natural function of the layer .
the aim of our study , therefore , was to discover a possible role of this structure . considering this layer 's positioning around the bn
, we postulated that it might have some role in the control of urinary continence . to verify this
, we assessed whether there was difference in the recovery of postprostatectomy incontinence ( ppi ) according to whether the retrotrigonal layer ( which was incised during posterior dissection of ralp ) was reconstructed or not .
from may 2007 to september 2010 , a total of 181 consecutive patients underwent ralp by a single surgeon ( bhc ) at our institution .
all of the preoperative , perioperative , and postoperative data were collected through a retrospective review of the patients ' charts .
all men were continent for urine before surgery ( no involuntary urine loss of any kind ) and had neither undergone transurethral resection of the prostate nor had a history of neurological disease . to control the learning curve ,
we divided our patients into two groups depending on whether a retrotrigonal layer backup stitch ( rtbs , as described below ) was performed .
these stitches were not used in group 1 ( cases 31 to 124 ) but were used for the patients in group 2 ( cases 125 to 181 ) .
all cases were carried out by use of the transperitoneal , six - port technique .
after the endopelvic fascia was opened and the dorsal vein complex ( dvc ) ligated , the anterior bn was divided . the posterior bn was then divided at the midline of the prostate - vesical junction . at this stage ,
after division of the retrotrigonal layer , both the vasa and seminal vesicles were found just beneath the retrotrigonal layer ( fig .
the seminal vesicles were then completely dissected as the transected distal ends of the vasa were pulled anteriorly by using the fourth robotic arm .
subsequently , posterior dissection was continued as far distally as possible , and the posterior aspect of the prostate was nearly freed from the rectum . then the nerve sparing ( interfascial ) was performed from the apex to the base , if deemed indicated according to the patient 's preoperative potency and tumor characteristics ( table 2 ) . after the prostatic pedicles were ligated , apical dissection and urethral transection were performed .
posterior anastomosis was performed in a clockwise direction starting at the 5 o'clock position and ending at the 10 o'clock position .
anterior anastomosis was performed with the second arm of the suture in a counterclockwise direction , and both sutures were tied together . here , the only technical difference between our two groups was an incorporation of the retrotrigonal layer into the posterior aspect of the vesicourethral anastomosis in group 2 . in group 2 , when we made conventional , continuous running sutures on the posterior bladder wall ,
this stitch was performed at a minimum of four points on the posterior bladder wall . after completing the anastomosis ,
routine postoperative care was administered , and the urethral catheter was removed 5 to 7 days after surgery .
follow - up for all patients was conducted at 1 week , 1 month , 3 months , and then every 3 months for up to 2 years .
continence recovery was determined by direct interview with the patients at each visit to our outpatient clinic .
the two groups were statistically compared for patient age , body mass index ( bmi ) , prostate volume , pre - ralp prostate - specific antigen ( psa ) level and international prostate symptom score ( ipss ) , length of membranous urethra , number of nerve - sparing procedures , pathologic gleason score , pathologic stage , margin - positive rates , biochemical recurrence , rates of adjuvant radiation therapy , overall operative time , robotic console time , and postoperative continence rate .
the student 's t - test or the mann - whitney test was used to analyze numerical variables , and the chi - square test or the fisher 's exact test was used to analyze categorical variables . to compare the interval before the return of urinary continence between the two groups
, we used the kaplan - meier method with the log - rank test to analyze the differences between the curves . to minimize and control for selection bias
there were no significant differences between the groups with respect to patient age , bmi , prostate volume , preoperative psa level , or preoperative obstructive and irritative ipss ( p>0.05 ) ( table 1 ) .
there were also no significant differences between the groups in the number of nerve - sparing procedures , pathologic gleason score , pathologic stage , positive surgical margin , biochemical recurrence , or rates of adjuvant radiation therapy ( p>0.05 ) ( table 2 ) . for overall operative time and robotic console time
, times were considerably shorter in group 2 ( cases 125 to 181 ) than in group 1 ( p<0.0001 ) ( table 2 ) .
according to our definition , group 1 showed continence rates of 40.4% , 70.2% , and 90.4% at 3 , 6 , and 12 months , respectively ; in group 2 , the continence rates were 42.1% , 70.1% , and 89.7% , respectively . the median ( 95% confidence interval ) time to continence recovery was four months ( range , 1 to 12 months ) in group 1 and four months ( range , 1 to 9 months ) in group 2 .
kaplan - meier curves showed no significant difference in the recovery of continence between the two groups ( log rank test , p=0.629 ) ( fig .
prostate volume , serum psa , preoperative total ipss , pathologic gleason score , length of membranous urethra , and rtbs technique were not significant risk factors for the recovery of continence after ralp ( p>0.05 ) ( table 3 ) . in patients with no preoperative erectile dysfunction ( international index of erectile function-5 score > 21 ) ,
intercourse was reported in 72.5% and 76.2% of the patients undergoing bilateral nerve - sparing surgery at 12 and 24 months of follow - up , respectively .
various intraoperative techniques for improving ppi have been introduced for use in open radical prostatectomy [ 6 - 14 ] , lrp , and ralp series [ 16 - 18 ] . although many of these techniques showed continence outcomes that were superior to those of procedures performed without the techniques , some controversies regarding the efficacy or limitations of these techniques persist [ 19 - 24 ] .
besides , we had found that our initial experiences with ralp ( cases 1 to 124 ) showed acceptable continence outcomes without the use of such techniques .
for these reasons , we did not fully trust the efficacy of the current and past intraoperative techniques for improving ppi until now . for techniques focusing on the bn , which is regarded as an internal sphincter
a bn preservation technique was introduced to theoretically improve ppi on the basis of the idea that sparing as much bn as possible during rp might result in a sphincter mechanism more closely resembling that of the preoperative state .
however , srougi et al found that the technique did not improve ppi and in fact might compromise cancer control because of the marginal positivity of the bn .
intussusception of the bn , a technique introduced by walsh and marschke , was another modification intended to result in earlier return of urinary control .
concerning the anatomy around the bn , there are some newly visited aspects ( especially posterior to the bn ) in addition to the well - known anatomy that was described by myers .
such trends have resulted from the evolution of rp from an open technique to lrp or ralp .
that is to say , the anatomy around the posterior bn has been examined under the enhanced and magnified vision of laparoscopic surgery and , more importantly , as a result of the different ( antegrade ) dissection of lrp / ralp .
for example , in 2001 and 2002 , myers described the extension of the longitudinal detrusor muscle of the bladder anteriorly , in front of the anterior commissure of the prostate .
he termed this the detrusor apron and explained that this structure had generally been unappreciated during rp by many urologists . in another example , in 2006 , secin et al described some longitudinal muscle fibers extending from the bn to the base of the prostate during the dissection of the posterior bn in lrp .
they proposed that these fibers actually correspond to the posterior longitudinal fascia of the detrusor muscle .
an earlier report regarding this structure could be found . when describing the ' montsouri ' technique of lrp , guillonneau and vallancien described the presence of a fascial structure with cephalocaudal striations that needed to be incised horizontally during the posterior bn dissection .
however , this terminology was challenged by several authors , because , according to our current anatomical knowledge , the dnf does not extend anterior to the seminal vesicle . instead , the term posterior layer of the detrusor apron was suggested , in view of the similarity between this structure and the detrusor apron that myers had described .
more recently , tewari et al used cadaveric dissections and real - time videos from ralps to describe a tissue layer posterior to the bn that extended from the posterior aspect of the trigone to the base of the prostate , which is consistently encountered after division of the posterior bn .
, they emphasized that this tissue may serve as a key surgical anatomical landmark to locate the vasa and seminal vesicles , which are located just beneath this layer . aside from its presence or accurate termination , however , it seems that the natural role or function of the layer is not clearly known .
therefore , we postulated that this layer might have some role in the control of urinary continence , considering its position around the bn . to verify this
, we analyzed the outcomes in continence recovery after dividing our ralp cases into two groups ( with or without rtbs ) .
accordingly , we started to perform rtbs techniques from the 125th case of our ralp series .
we compared the data of the patients with rtbs ( cases 125 to 181 ) with those of our initial ralp cases ( without rtbs , cases 31 to 124 ) , in which the retrotrigonal layer had been used only as a landmark for posterior dissection of the bn .
our ralp technique in all 151 cases did not include any of the current or past techniques for improving ppi , including puboprostatic ligament ( ppl ) preservation , additional anterior support of the ppl / dvc , bn preservation , membranous urethral lengthening , or posterior reconstruction of denonvilliers ' musculofascial plate ( such as the rocco stitch ) .
moreover , there was no significant difference in the number of nerve - sparing procedures between our two groups .
accordingly , we think that our comparison of the two groups was quite reliable in terms of both surgical aspects and baseline patient characteristics . during posterior dissection of the bn
, the retrotrigonal layer was clearly identified in all but four of our patients , although the thickness varied from one case to another . from our experience , we fully agree with tewari et al that this tissue may be a key landmark for use in the dissection of the posterior bn .
although some authors have reported the use of an " ultradissection technique " or modified ultradissecction ( in asians with relatively small body sizes ) during bn dissection , we felt that the approach via the retrotrigonal layer was easier . also , incorporating the retrotrigonal layer into the posterior aspect of the vesicourethral anastomosis presented no difficulty .
however , upon analysis of our results , our rtbs technique did not show any role in the improvement of ppi .
we think that this conclusion is bolstered by the fact that the surgeries in group 2 ( with rtbs ) were performed when our procedures for ralp were far more stabilized in every other step that might affect the continence outcome ( e.g. , nerve sparing , etc ) , and yet there was no significant difference in continence recovery between the two groups .
the finding of significant differences in operative time and console time between the groups ( table 2 ) also supports this conclusion .
our study would have more power if verified knowledge existed of the innate , accurate point at which the retrotrigonal layer has its distal insertion .
such knowledge will be essential to put our conclusions in context , because the residual retrotrigonal layer was sutured to the membranous urethra ( fig .
the limitations of the present study include the retrospective comparison with a historical cohort and the lack of randomization .
as such , the results should be considered exploratory or observational , not definitive . to address this concern
, we are planning a prospective randomized trial with validated continence measures to more rigorously assess the effectiveness of rtbs .
. a verified description of the innate , accurate point at which the retrotrigonal layer has its distal insertion and prospective randomized studies with larger numbers of patients will be essential to confirm our results . | purposeto evaluate the impact of a retrotrigonal layer backup stitch ( rtbs ) during robot - assisted laparoscopic radical prostatectomy ( ralp ) on post - prostatectomy incontinence.materials and methodswe compared the difference in continence recovery between 94 patients ( group 1 , as historical controls ) and 57 patients ( group 2 ) .
the only technical difference between our two groups was the incorporation of the retrotrigonal layer into the posterior aspect of the vesicourethral anastomosis ( group 1 : without rtbs ; group 2 : with rtbs ) .
postoperative continence recovery was defined as the use of no absorbent pads.resultsin group 1 , the continence rate at 3 , 6 , and 12 months postoperatively was 40.4% , 70.2% , and 90.4% , respectively;in group 2 , the continence rate was 42.1% , 70.1% , and 89.7% , respectively . the median ( 95% confidence interval )
time to continence recovery was four months ( range , 1 to 12 months ) in group 1 and four months ( range , 1 to 9 months ) in group 2 .
kaplan - meier curves showed no significant difference in the recovery of continence between the two groups ( log rank test , p=0.629).conclusionsa rtbs does not appear to improve urinary incontinence after ralp .
further anatomical study and prospective randomized studies will be needed to confirm this . |
postoperative as are well described major and long - term complications of cervical laminoplasty for csm .
the overall incidence ranges from 6% to 60% 1,2 . the incidence and severity of as appears to vary among different types of laminoplasty procedures .
further , various procedural approaches to the cervical spine may affect alignment , influence quality of decompression , as well as impair stability of the pmlc postoperatively 2,3,4,5 .
to determine if there is a difference in ; ( 1 ) the rate and severity of postoperative as ; ( 2 ) the change of cervical spine sagittal alignment ; and ( 3 ) spinal cord decompression between patients treated with a preservation of the unilateral pmlc procedure and hsap for the treatment of csm .
inclusion criteria : we included all patients with csm and presence of the following clinical conditions : three or more segments of spinal - cord compression developmental and degenerative stenosis of the cervical spine + /-
ossified posterior longitudinal ligament presence of both preoperative and postoperative clinical and radiological examinations including x - ray and magnetic resonance imaging ( mri ) scans .
exclusion criteria : patients were excluded if they had : previous cervical spine surgery ankylosing spondylitis congenital deformities cerebrovascular disease sequela motor neuron disease , or neuromuscular junction disease required simultaneous instrumented fusion or combined anterior procedures patient population : nine hundred and forty - six patients underwent one of two cervical laminoplasty procedures between january 2006 and december 2009 .
there were 552 patients eligible for the study ; however , 203 ( 37% ) did not have either radiology or clinical details in the follow - up records and 213 ( 39% ) did not return for follow - up .
the remaining 136 of those eligible ( 24% ) were analyzed ( fig 1 ) .
interventions : seventy patients received preservation of unilateral pmlc procedures ( fig 2 ) and 66 received hsap ( figs 3a
there was no reason to believe that one procedure was selected over another based on disease severity .
outcomes : the existence and severity of as were measured and graded at final follow - up according to kawaguchi s description 6 and hosono grading system 7 by one of the investigators : no as : not any axial symptoms at all minor as : slight symptoms which do not affect activities of daily living major as : obvious symptoms and occasional analgesic pain medication may be required severe as : very obvious symptoms that affect patient s activities of daily living , regular analgesics are taken .
the change of sagittal cervical spinal alignment was measured using the ishihara 8 method by one of the investigators .
the expansive effect was evaluated by measuring the diameter of spinal canal on standard lateral view x - rays .
spinal cord decompression was measured using mri , when available , by evaluating the appearance of the dura sac and the paraspinal muscles .
continuous baseline variables , between group changes in preoperative to postoperative changes in cervical spine curvature and preoperative to postoperative changes in the expansive effect were compared using a 2-tailed independent t - test . within group
changes in preoperative to postoperative changes in cervical spine curvature and preoperative to postoperative changes in the expansive effect were compared using a 2-tailed matched pair t - test .
patient sampling and selection procedure a : preservation of muscular - ligament complex method procedure b : hinge side - anchoring method
seventy patients in the preservation of unilateral pmlc group and 66 in the hsap group were analyzed ( fig 1 ) . the mean time to follow - up in the preservation of unilateral pmlc group was 6.9 months ( range , 68 months ) and the hsap group was 6.4 months ( 68 months ) . there were no statistically or clinically significant differences between the two groups with respect to baseline demographic and preoperative japanese orthopaedic association ( joa ) scores ( table 1 ) .
there was no significant difference in intraoperative and immediate postoperative characteristics , such as operative time , blood loss between two groups , and postoperative joa scores and recovery rates ( table 1 ) .
the most common decompression levels were c3c7 for both groups , this difference was not statistically significant ( p = .23 ) .
differences in other decompression levels were not clinically significant . the overall rate of any as among all subjects was 35% .
no as was reported in 45.7% of the pmlc group and 23.8% of hsap ( p = .008 ) ( table 2 ) .
severe as was reported in 4.3% and 11.1% of patients per group , respectively ( p = .14 ) ( table 2 ) .
cervical lordosis increased in the preservation of unilateral pmlc group and decreased in the hsap group ( p = .04 ) ( table 3 ) .
the risk of losing lordosis was more than two times higher in the hsap cohort compared with the pmlc group ( 70% and 34% , respectively ; p <
there was a clear structure of muscular ligament complex without significant atrophy and fibrosis found in all preservation of unilateral pmlc cases .
there was derangement and atrophy / fibrosis of the posterior muscular structure on the other side in all unilateral pmlc cases and both sides in all hsap cases .
joa = japanese orthopaedic association fisher analysis p = .048 matched - paired t - test
the rate of postoperative as in this study is lower than most rates reported in the literature .
the hsap group presented higher rates and severity of postoperative as , deterioration of cervical - spine lordosis , and worse derangement of paraspinal muscles .
the preservation of unilateral pmlc procedure appears to have the advantage of less soft - tissue denervation that provides higher dynamic and static stability of the c - spine 9 , better muscular alignment and power , and the possibility and capacity of early mobilization .
strengths : this is the first comparative study of two laminoplasty procedures evaluating important clinical outcomes .
the patients appeared evenly matched with respect to the baseline measures that we collected except for the decompression levels between c27 .
limitations : the greatest limitation of this study is the significant loss to follow - up ( 76% ) due to missing radiological materials or clinical details or nonreturning patients .
loss to follow - up can lead to selection bias . although the losses were evenly distributed between the two groups , we can not be sure that those lost were similar to those remaining ; hence we can not rule out bias in this study .
important factors that may also influence the outcomes that we can not be sure were evenly distributed between the remaining subject groups include baseline symptoms severity , general health , smoking status , collar protection duration , and physiotherapy status .
differences were observed between the two groups ; however , a high loss to follow - up rate weakens our confidence in the findings .
laminoplasty with preservation of unilateral pmlc may have the advantage of a lower rate of postoperative as and a satisfactory maintenance of cervical - spine lordosis compared with hsap .
editorial staff perspective this is our first scientific article published through the aospine methodological support offered through ebsj in an effort to encourage a wider group of members from our global spine community to engage in regular clinical science undertakings .
this article underscores some of the potential of providing methodological support to the regions ; it also demonstrates the long road ahead in terms of overcoming infrastructural shortcomings in some of the regions .
the topic at hand was chosen for our ebsj enhanced methodological support program ( emsp ) after a fair bit of discussion among our editorial staff due to the relevance of the topic , the substantial number of patients studied , and the presence of two compelling comparison groups .
the authors deserve praise for having diligently worked with the ebsj methodological staff to optimize analysis of the available data points .
substantial shortcomings , which could not be overcome , remain ; however , the substance of the paper and aspects of the findings deserve publication and will provide substance for discussion .
hopefully a learning benefit will be gained for our scientifically minded aospine international community through increased methodological awareness , which not only apply to research but also to our clinical practices .
specifically the subject of loss to follow - up in retrospective studies is very common and of such relevance to the interpretation of data that we have dedicated a separate science in spine section to this topic in the next edition of ebsj . to be taken seriously as spine surgeons we need to try to optimize follow - up of our patients , not just for research but in our everyday practices .
of course there are tremendous financial and infrastructural hurdles to contend with . however , creative use of adjuvant strategies , such as structured telephone interviews , mailings of outcomes questionnaires , and use of regional providers notes with inclusion of the findings in a designated location of the available medical records system is a very acceptable fall - back option .
the occurrence of axial neck pain after laminoplasty is well known and is often taken into account when considering surgical options .
the figures provided also give the reader a clear understanding of the differences between the two procedures . here
are some noteworthy observations about this study : although the study is well presented within the parameters of the journal , its largest weakness is the loss of follow - up for 76% of their patients , which the authors acknowledge . despite demographic similarities ,
24% of the eligible patient population is not a sufficient representation . in examining postoperative axial neck pain symptoms
, it would be useful to examine the prevalence of preoperative axial neck pain symptoms . in the hinge group , if there were a disproportionate number of patients who had preoperative neck pain that did not change , or actually improved to mild pain
after surgery , this may alter the final conclusions of the authors . in the discussion of pain having fixed time points of observation ,
ie , 2 years after elective procedures , is recommended to allow for a more consistent comparison of this outcomes variable . in general , it is not appropriate to declare that
there was no statistically significant difference , unless a prestudy power analysis had been performed and adequate power had been achieved by the study .
it is more accurate to state , we did not observe a statistically significant difference while the primary outcome of measure was the postoperative axial symptoms , it would be beneficial for surgeons to also know rates of complications , revisions , and failures of these procedures to have a wider scope of comparison . since preservation of neurological status and , hopefully , improvement of myelopathy are main factors in performing laminoplasties , reporting of the neurological outcomes can be considered of prime outcomes parameters to study and report . in the end
we hope that this presented project was helpful for the authors in implementing principles of clinical research for the future and will provide positive insights for our readership as well as in terms of methodological principles .
we encourage your comments on the ebsj enhanced methodological support program ( emsp ) . | study design : retrospective cohortobjective : to determine if there are differences in important clinical outcomes between patients treated with a preservation of the unilateral posterior muscular - ligament complex ( pmlc ) procedure and a hinge side anchoring procedure ( hsap ) for the treatment of cervical spondylotic myelopathy ( csm).methods : nine hundred and forty - six patients underwent the above procedures for treatment of csm between january 2006 and december 2009 .
five hundred and fifty - two ( 58% ) subjects met study criteria and 136 were analyzed ( 76% unavailable for follow - up ) .
one group ( 70 patients ) received a preservation of unilateral pmlc procedure and another group ( 66 patients ) received an hsap .
the rate and severity of postoperative axial symptoms were assessed , and the changes of cervical - spine curvature and postoperative decompression were measured.results : the mean time to follow - up in the preservation of unilateral pmlc group was 6.9 months ( range , 68 months ) and the hsap group was 6.4 months ( 68 months ) .
the overall rate of any axial symptoms ( as ) among all subjects was 35% .
no as was reported in 45.7% of the preservation of unilateral pmlc group and 23.8% of the hsap group ( p = .008 ) .
severe as was reported in 4.3% and 11.1% of patients per group , respectively ( p = .14 ) .
cervical spinal lordosis was improved in the unilateral pmlc group and decreased in the hsap group .
the risk of losing lordosis was more than two times higher in the hsap group compared with the unilateral pmlc group ( 70% and 34% , respectively ; p < .001).conclusion : differences were observed between the two groups ; however , a high loss to follow - up rate weakens the findings .
cervical expansive laminoplasty with preservation of unilateral pmlc may have the advantage of a lower rate of postoperative as and maintenance of cervical spinal lordosis compared with the hsap group . |
caricom countries like many other developing nations are in varying stages of an epidemiologic and nutrition transition , such that the traditional public health problems of infectious disease have decreased in prevalence , only to be largely replaced by the diseases of life style that are associated with western - type diets and reduced activity .
a challenge facing the incoming millennium generation of the region is the easy availability of calories in the face of sometimes monotonous diets and reduced opportunity for exercise .
the who proclaims that childhood obesity is one of the most serious public health challenges of the 21st century , with close to 35 million overweight children under the age of five in developing countries .
undernutrition in early childhood is associated with developmental deficits of reduced cognitive and psychosocial functioning as well as later physical and work capacity .
in addition , many children who experience undernutrition will be at increased risk of developing chronic diseases .
nutritional status in children clearly has implications for the future of the individual as well as for these nation states as a whole .
tracking these patterns over time is important if policy tailored to the unique needs of the different countries is to be devised . the millennium declaration signed by 189 countries , including caricom in september 2000 , set out to create an environment which is conducive to development and the elimination of poverty .
the first five millennium development goals ( mdg ) have bearing on children 's nutritional status , health , and development .
these goals are to eradicate extreme poverty and hunger , achieve universal primary education , promote gender equality and empower women , reduce child mortality , and improve maternal health .
the mdgs included these gross health indicators for monitoring progress : infant mortality , the under - five mortality rate , and antenatal care coverage .
associated recommendations were to prevent low birth weight , improve early feeding practices , begin breastfeeding within one hour of birth , exclusive breastfeeding for the first six months of life , and timely and appropriate complementary feeding from six months of age - continued breastfeeding up to two years of age . in this paper , we describe the nutritional status of children under 5 years in caricom countries , reported since 2000 , and seek to assess the current situation in relation to the first five mdgs , which contain aspects that pertain to children 's nutritional status .
in addition , we reviewed available work on childhood obesity in the caribbean because of the who proclamation that childhood obesity is one of the most serious public health challenges of the 21st century , with close to 35 million overweight children under the age of five in developing countries .
reviews of this nature have been conducted that group the caribbean together with latin america .
however , this paper focuses specifically on the caribbean and should assist with planning and policy making that caters to the unique characteristics of these small states that form caricom .
a pubmed and google scholar search up to 2010 was made using the following keywords : food , childhood ,
caribbean , under - nutrition , overweight , obesity , low birth weight , infant mortality , breast feeding , maternal education , mothers , maternal , education ,
height for age , weight change , developing countries , west indian , millennium development goals , weight for height , and nutrition .
the reference lists of articles retrieved from pubmed and google scholar were also reviewed .
publicly available literature for original data on child development in caricom countries published since the year 2000 was used .
data from published reports from who , unicef , cia world fact book , caribbean food and nutrition institute , and unesco were used .
information on child growth and wellness indices was used as a basis on which to track early childhood development .
feeding practices were used to estimate the progress of interventions aimed at eliminating poor nutritional status [ 5 , 9 ] .
we specifically collated information on infant mortality , low birth weight , stunting , wasting , overweight , and infant feeding . where original data was not available we used estimates collated by international health and information agencies [ 4 , 10 ] . from the literature review , we found primary data from surveys for jamaica , trinidad and tobago , and guyana in the form of multiple indicator cluster surveys ( mics ) .
most of the other data used in calculations was taken from unicef , other international agencies , or from country reports that tabulate health and economic data [ 10 , 14 ] .
the three countries that had published surveys , jamaica , trinidad and tobago , and guyana , were used to represent more detailed descriptive comparisons for each indicator .
we added barbados and haiti to represent the range of gross national income ( gni ) across the region .
the gni for barbados was estimated from gross domestic product ( gdp ) , 2007 . as an initial step
, we examined the relationship of gni and infant mortality in the caricom states to determine whether the data for the region presented by unicef followed expected trends .
in addition to using individual country data , a summary statistic for developed countries was included .
as mentioned before , the mdgs that have bearing on child nutritional status , health , and development are as follows : goal 1 : eradicate extreme poverty and hunger , goal 2 : achieve universal primary education , goal 3 : promote gender equality and empower women , goal 4 : reduce child mortality , goal 5 : improve maternal health .
goal 1 : eradicate extreme poverty and hunger , goal 2 : achieve universal primary education , goal 3 : promote gender equality and empower women , goal 4 : reduce child mortality , goal 5 : improve maternal health .
for each goal ,
indicators for monitoring progress were identified for analysis in the results : goal 1 : eradicate extreme poverty and hunger indicator 1.8 : prevalence of underweight children under five years of age goal 2 : achieve universal primary education indicator 2.1 : net enrolment ratio in primary education goal 3 : promote gender equality and empower women indicator 3.1 : ratios of girls to boys in primary , secondary , and tertiary education goal 4 : reduce child mortality indicator 4.1 : under - five mortality rate , 4.2 : infant mortality rate goal 5 : improve maternal health indicator 5.5 : antenatal care coverage .
goal 1 : eradicate extreme poverty and hunger indicator 1.8 : prevalence of underweight children under five years of age indicator 1.8 : prevalence of underweight children under five years of age goal 2 : achieve universal primary education indicator 2.1 : net enrolment ratio in primary education indicator 2.1 : net enrolment ratio in primary education goal 3 : promote gender equality and empower women indicator 3.1 : ratios of girls to boys in primary , secondary , and tertiary education indicator 3.1 : ratios of girls to boys in primary , secondary , and tertiary education goal 4 : reduce child mortality indicator 4.1 : under - five mortality rate , 4.2 : infant mortality rate indicator 4.1 : under - five mortality rate , 4.2 : infant mortality rate goal 5 : improve maternal health indicator 5.5 : antenatal care coverage . indicator 5.5 : antenatal care coverage .
additional gross health indicators were employed to examine nutritional status in the separate states of caricom that were not specifically identified by the mdg targets or indicators .
these are interrelated ; for example , neither low birth weight nor infant feeding practices are specifically named as an mdg , but both relate to infant mortality and antenatal care which are clearly identified mdg indicators for monitoring progress . therefore , these common health indicators were noted under the specific mdg and indicators that they correlate with in the results .
the results are presented with respect to overweight and obesity in the caribbean region and in accordance with the aforementioned mdg targets and recommendations . for the purposes of this paper , overweight in children
is defined as a z - score value > 1 s.d . above the who / nchs mean weight - for - height while obesity in children is defined as weight - for - height z - scores > 2 s.d . .
using data compiled from various sources including some unpublished country surveys , the caribbean food and nutrition institute ( cfni ) compiled and published in 2001 regional estimates of overweight among the children ages of 05 years .
they report an overall prevalence of 36% and from clinic data a trend of increasing prevalence over a ten - year interval .
information on trinidad and haiti was available from other sources which used who definitions of overweight and obesity .
the estimate for jamaica for overweight prevalence among children of 05 years from a 1993 survey was 6% . a lower estimate of 4% was arrived from an unpublished survey conducted in 1998 by cfni .
data from a 1987 national nutrition survey recorded the prevalence of overweight of approximately 8.9% and obesity at 1.9% among children 1236 months old . however , there were significant rural urban and ses differences .
another author using slightly older children from this same sample ( 1259 months old ) estimated overweight prevalence at 3% . however , the definition of overweight used here was what was used as obesity by the previous author .
the overweight estimates quoted in the cfni 2001 report ( 3% ) in the 05-year age group are in keeping with the latter study but a source is not identified . for guyana , there was an estimated overweight prevalence children 15 years old of 2.3% from a 1981 survey .
the cfni estimate from an unpublished survey conducted in 1981 of 1% in the 04-year age group is considerably lower .
the prevalence of overweight in haiti for 1994 - 1995 defined in a national survey as > 1 sd above mean weight - for - height in children 1259 months of age was approximately 5.7% and obesity was 1.4% .
these data show no difference in prevalence by geographical setting , ses , sex , nor level of maternal education . among a slightly younger group from the same sample ( 359 months )
another author estimated the prevalence of overweight as 2.8% defined as > 2 sd above mean weight - for - height .
the incidence of overweight among children of 05 years of age at 1981 was an estimated 3.8% for barbados in a national nutrition survey .
obesity was defined as > 120% of the weight for length using the gomez classification .
notably , levels in the caribbean of wasting and stunting are below those for other developing countries . however , there appears to be no trend with gni .
half of the caricom countries report no undernutrition among children under five years at all .
the countries that report wasting also report stunting but stunting is most prevalent ( table 1 ) and is high in haiti where levels approach the mean for developing countries . levels in belize and guyana are also of concern .
indicator 2.1 : net enrolment ratio in primary education indicator 3.1 : ratios of girls to boys in primary , secondary , and tertiary education .
indicator 2.1 : net enrolment ratio in primary education indicator 3.1 : ratios of girls to boys in primary , secondary , and tertiary education .
net enrollment in primary school was high in guyana in 2006 with no significant gender bias ( boys 96.3% ; girls 96.0% ) .
there was a relatively sharp decline for entrance to secondary school ( boys 66.1% ; girls 72.6% ) . for jamaica ,
net enrollment in 2005 was 89% with no gender bias with high rates of transition to secondary school ( 97% both boys and girls ) .
for trinidad , 2006 , the net primary school enrollment rate was 82% with equal numbers of boys and girls .
for the period 20032008 , unicef reports barbados net primary enrollment at 97% and transition to secondary school ( boys 88% ; girls 93% ) .
no estimates were available for transition to secondary school but attendance rates were low ( boys 18% ; girls 21% ) .
maternal education appears to be of negligible importance for the region because it is only associated with mortality when the mother has not finished primary school .
indicator 4.1 : under - five mortality rate , 4.2 : infant mortality rate .
there was an inverse relationship of infant mortality to gni ( p = 0.051 ) for caricom countries ( figure 1 ) .
infant and under-5 mortality rates were lowest in the rural areas and highest in other towns ( excluding kingston and the metropolitan area ) .
the under - five mortality rate is estimated to be around 35 per one thousand live births .
there are regional differences between tobago at 48/1000 and the south west region at 16/1000 . in guyana
child mortality was lower among east indian children than among children of the other ethnicities .
infant mortality rate in barbados is estimated at 12 deaths per thousand live births for 2009 .
infant mortality rate for haiti was estimated for 2009 at 60 deaths per thousand live births .
a child is almost ten times more likely to die if they are severely underweight than if they are of average weight for their age .
there is a general increase in lbw with increasing gni with haiti having highest incidence of lbw , likely due to intrauterine growth retardation ( iugr ) .
for example , the prevalence in barbados does not fit the general caribbean pattern so that despite high gni and antenatal care the incidence of low birth weight is higher than for several lower income caribbean countries ( table 2 ; figure 2 ) .
results from the jamaica mics estimated low birth weight ( birth weigh less than 2500 grams ) at 12 percent of live births .
this is a reliable estimate since ninety - seven percent of infants are weighed at birth .
trinidad and tobago reported lbw from the mics at 18.8% as 89.8 percent of live births were weighed at birth .
the highest percentages of lbw infants occurred among the poorest ( 20.6 ) and upper middle quintile ( 21.5 ) groups while the other quintiles represented the lowest percentages of lbw ( 16.4 , 18.2 , and 17.1 , resp . ) . in guyana
there was geographic variation with higher incidence in the interior among amerindian women ( 24 percent ) .
interestingly , guyana recorded a 7 percent increase in low birth weight between 2000 and 2006 .
mothers ' level of education and household wealth did not appear to have much impact on low birth weight status among infants .
haiti occupies the top of the rank with respect to prevalence of lbw in the caribbean ; this is in keeping with its low gdp and uneven distribution of wealth .
there is some speculation that higher rates in barbados reflect teenage pregnancies ; this seems unlikely given that rates of teenage pregnancy are low in barbados .
an alternate explanation of higher rates of pregnancy among older women leading to greater iugr and lbw seems more credible , but the supporting data has not been collected . according to nation
95% and 24% of children are delivered by a skilled attendant in barbados and haiti , respectively , so this would reflect , though not exactly , the number of weighed births in these countries .
this suggests that the estimates of low birth weight at least for barbados are likely to be close to the true value .
underweight in children can be prevented through initiatives promoting breastfeeding , complementary feeding , micronutrient supplements , and social protection mechanisms .
only five of the caricom countries have not formally reported on breastfeeding in the recent past . in haiti , just under half of mothers
meet the recommendation to exclusively breastfeed for six months ( table 3 ) . in those countries that report breastfeeding with supplementation at 69 months , the percentage of children breastfeeding with supplementation is higher than of exclusive feeding ( table 4 ) .
haiti has the highest percentage of mothers ( 35 percent ) who meet the recommendation for breastfeeding up to 24 months ( table 4 ) .
when gni is controlled for antenatal care , the expected inverse relationship of lbw to gni is revealed ( p = 0.064 ) .
nutritional status is the best global indicator of well - being in children and is an indicator of the overall well - being of a society .
children are growing and are therefore more susceptible to environmental change , so growth is a good indicator of nutritional status among children . for this reason , child growth and wellness indices are frequently monitored and form a useful basis on which to track development [ 5 , 9 ] .
the detailed country descriptions suggest that the urban , rural , and geographic regional differences that presumably correspond to socioeconomic status ( ses ) were also loosely associated with income , but the patterns are not consistent from country to country with rural urban patterns moving in opposite directions in some cases . in the five countries selected for more detailed comparisons ,
this led to a trend of the higher estimates of child mortality to previous estimates for these countries .
it should be noted that data on specific causes of death has not been presented mostly because indirect means are used to estimate infant mortality .
evidence on ethnic differences in the epidemiology of infant mortality in the united states indicates that differences in social circumstances are likely to be associated with a different distribution of early mortality .
this may partially explain the high and regional variation of rates experienced in trinidad and tobago .
child mortality rates in both infants and those under five are critical indicators of well - being of children . in the caribbean , rates are very moderate to low .
haiti has the highest rates in the region and ranks the 48th in the world , whereas barbados ranks at 140 , with rates much closer to rates of 8 and 6 per thousand live births seen in the us and uk , respectively .
rates in trinidad and tobago are surprisingly high , given the standard of living and gross national income ( figure 1 ) .
information on the causes of infant mortality is often not readily available in the caribbean .
so , although there is an obvious macroscopic association of poverty with child mortality ( figure 1 ) , the patterns are not so obvious at the county level .
when gni increases to around $ 5000 usd per capita , its correlation with infant mortality is no longer evident .
this suggests that decreasing poverty as an intervention to reduce infant mortality has limited effect after this point at the country level . since the infant mortality rates in many of the caribbean countries approach those of the uk and usa
, we can assume that the broad interventions applied in the mid - twentieth century have worked to produce as much improvement in infant mortality as they are likely to .
clearly , different types of study must be conducted in the region if we are to understand and address the causes of infant mortality especially in the more developed of the territories where the broad indicators like poverty and maternal education have lost some of their sensitivity .
the prevalence patterns of low birth weight for most of the countries have remained virtually static since the mid - nineteen - eighties ( walker 1989 ) . this may be attributed to a lack of reliable estimates in some cases . in guyana and trinidad and tobago , ethnic groups may have an effect in that around half of the trinidad population and one - third of the guyana population are of indian extract , known to have smaller babies .
nevertheless , the regional differences appear to represent to some extent women with different access to education and antenatal care .
lucia , the obvious inverse associations of reduced antenatal care and of education when less than primary level is attained indicate that addressing vulnerable women remains an important strategy for regional development . in the developing world , estimates of incidence of low birth weight are often hampered by infants not being weighed at birth .
sometimes prevalence is estimated from biased nonrepresentative samples taken from health facilities or may be estimated from maternal recall . however , because there are skilled attendants at most births in caricom countries , this is less of a problem than in other developing countries . as a consequence of the lack of studies that elicit details on reasons for low birth weight , it is possible only to speculate about the reasons for the variations observed .
one might expect low birth weight to consistently decrease in the face of improved antenatal care ; however , figure 2 demonstrates that this indicator like infant mortality paradoxically can rise when conditions in a country become quite good such that better antenatal care prolongs pregnancies that formally would not have been viable .
a higher proportion of these infants succumb to the problems of being born early for gestational age , thereby increasing both infant mortality and incidence of low birth weight .
this effect is likely more pronounced in states like barbados where there are fewer resources to sustain premature infants than is ideal compared to the developed world .
linear growth retardation is known to affect about 30% of children in the developing world and to have long term effects on development [ 32 , 33 ] .
early childhood stunting is associated with poor school achievement and reasoning in young adults [ 33 , 34 ] .
children stunted before age 2 years have poorer emotional and behavioral outcomes in late adolescence than nonstunted children .
however , studies have demonstrated that some of the effects can be ameliorated with early intervention .
although , by comparison to other countries in the developing world , the rates of stunting in the caribbean are relatively low , in absolute terms , this represents quite a large burden on these vulnerable states . of the approximately 16.7 million children under five years in caricom countries ,
it highlights the fact that interventions such as those conducted in jamaica in the late twentieth century still have a high degree of relevance for many pockets of children in the region .
overall prevalence in the caribbean and gross numbers of stunted children are low compared to regions like eastern africa and asia , where as recently as 2005 prevalence ranged from 43.7% to 34.9% , respectively , of the 5 years and under population .
trinidad and tobago remains an anomaly with respect to under - five growth and development .
they record significant negative measures of every childhood growth and development indicator despite having concurrently one of the highest gnis in the region ( tables 1 - 2 ) .
the lack of a direct relationship between gni and measures of childhood undernutrition is possibly explained by differences in public health management of the problem and due to historical differences in economic and natural disaster misfortunes , in places like haiti .
these challenges need to be addressed , for the children currently living in these circumstances . while for most of the developing world obesity does not appear to be a public health problem among preschool children , in a number of countries in latin america and the caribbean , levels equal those of the usa . the who proclaims that childhood obesity is one of the most serious public health challenges of the 21st century , with close to 35 million overweight children under the age of five in developing countries . of concern in this region as elsewhere
is the coexistence of undernutrition and high levels of overweight . grappling with this paradox is a troubling prospect for a region with scarce resources .
it is nevertheless difficult to find credible data for caricom countries that confirms this especially among children under 5 years .
the data on overweight and obesity prevalence and incidence among caribbean children are even sparser than those on undernutrition .
the few measures that are available are not on exactly comparable groups and different definitions of overweight are used [ 16 , 20 ] .
the necessary measurements have not been routinely collected for most countries up to 2012 and few population surveys have been conducted .
nevertheless , there is some evidence , for example , from haiti that prevalence of overweight is increasing and anecdotal evidence from the other territories that this is a growing problem .
this is important because early growth is associated with the prevalence of obesity later in the life course .
breastfeeding is known to be associated with many positive childhood outcomes including protection against overweight [ 38 , 39 ] , although there is debate as to the reasons for the positive associations .
early introduction of solid foods on the other hand appears to potentiate negative outcomes including allergy and obesity .
mothers and grandmothers are sometimes known to hold beliefs that may promote development of overweight .
this is important in many caricom countries where the prevalence of obesity and associated cardiovascular disease has risen drastically in the last 20 years [ 2 , 43 ] ; importantly , rates among children are also rising [ 44 , 45 ] .
exclusive breastfeeding before six months is recommended as it is thought to have a protective role in conditions spanning asthma to overweight .
who recommendations for adequate feeding seek to have infants and toddlers exposed to optimal nutrition as a means of reducing the burden of disease among coming generations .
how well the caribbean has heeded these recommendations is an indicator of likely progress with helping children to exploit their developmental potential .
cultural norms , social pressure , and working mothers are some of the factors that impinge upon this important nutrition intervention .
there is scant information on early childhood feeding practices in the region ; as a whole , many of the higher income countries like barbados and bahamas do not even report basic statistics .
haiti reports that 87% of mothers breastfeed at some point ; 35% of them are still breastfeeding when the child is 2023 months , in keeping with recommendations .
so even where resources are scarce there is a trend of early introduction of complementary food .
this propensity for introduction of early solid foods is of concern as it appears to be most prevalent in those countries like barbados where adult obesity and chronic cardiovascular disease are major problems , ( unpublished data ; beverly stanford , national nutrition centre , barbados ) .
it is not currently known whether heavier breastfed babies are at higher risk of becoming overweight than slimmer breastfed babies ; however , it is known that breastfed babies have a lower risk of later becoming overweight than formula - fed babies .
most of the substantive interventions carried out in the caribbean region were conducted in jamaica .
one major intervention study in jamaica was initiated with a cohort of stunted children 924 months old from inner city kingston .
children received nutritional supplementation , stimulation ( mothers playing with homemade toys ) , or both .
initially , both interventions showed positive effects on development but by age 17 to 18 years only the effect of stimulation remained .
stimulation administered to stunted children in early childhood was associated with reduced cognitive and educational deficits compared to controls in late adolescence .
children stunted in early childhood had significant more anxiety than their nonstunted counterparts but those who received early stimulation were somewhat better than controls but remained at a disadvantage with respect to emotional and behavioural outcomes in late adolescence . this important work out of jamaica demonstrates that the effects of stunting can be alleviated with feasible interventions using materials available to poor working class parents .
we were unable to identify a regional study that successfully intervened in early childhood overweight or obesity .
some school based interventions in children and adolescents have shown promise whereby children have been shown to change habits associated with development of overweight [ 45 , 50 ] . however , in general in the world most school based interventions to reduce bmi at the population level have been unsuccessful .
the states of caricom except for haiti have in most instances achieved the mdgs of primary education goals and gender equality .
although enrolment in primary schooling is high , transition to secondary school is less consistent .
in addition because education is a major tool of empowerment the trend of equal opportunity for schooling both at the primary and secondary levels demonstrates commitment to the goal of promoting gender equality .
the nutritional status of caribbean children under 5 years is relatively good compared to children in other developing nations .
caricom countries by and large have reached the millennium development goals with respect to early childhood nutritional status , although there are lingering pockets of undernutrition in some caribbean states .
some of these differences may be due to the transition from low income to high income country lifestyles .
it appears that the large scale poverty alleviation interventions such as lowering of childhood mortality and low birth weight have reached their peak of efficiency in the region .
consequently , to improve the health status of children under 5 years , it is important to explore the drivers for disparities on an individual country basis .
research and interventions that deal more pointedly with country specific needs are required including those targeting obesity if the mdgs for caricom are to be attained by all member states . | previous reviews of nutritional status in children under 5 years describe the caribbean grouped with latin america .
this paper focuses specifically on the caribbean and the goals and targets of the millennium declaration that have bearing on childhood development .
the results indicate that caricom countries have made progress in terms of child health as assessed by gross health indicators . yet ,
the millennium generation experiences coexistence of undernutrition and overweight in early childhood . the associations of gni with markers such as poverty indices are somewhat inconsistent with traditional findings and highlight a need to reassess the causes of infant mortality and low birth weight .
however , a lack of systematic local data has hampered progress on an individual country basis .
interventions that deal more pointedly with country specific needs are required including those targeting obesity if the mdgs are to be attained by all member states . |
copd is a lifestyle - related disease of the lungs that is primarily caused by regular smoking.1 it is an inflammatory disease associated with damage to the alveolar septa and increased levels of inflammatory mediators , such as c - reactive protein ( crp ) , interleukin-6 , interleukin-1 , and tumor necrosis factor- ( tnf- ) in the lung periphery.2 this local inflammation in the lungs spills into the systemic circulation , where it causes systemic inflammation.3 research has suggested that the systemic effects of copd can also cause comorbidities , such as cardiovascular diseases.3,4 thus , copd has attracted attention as a systemic disease.3,5 the systemic inflammation of copd is suggested to be involved in the pathogenesis of ischemic heart diseases and arteriosclerosis.6 several reports demonstrated that athosclerotic plaques in copd patients show a low - grade inflammation similar to that in the peripheral lungs.3,7,8 actually , the cause of death for 20%30% of patients with copd is cardiovascular disease , and death due to cardiovascular disease is highly evident in patients with mild or moderate copd.9,10 however , the precise mechanism of arteriosclerotic cardiovascular diseases and their effective preventive strategy in patients with copd are still unknown .
thus , it is associated with the pathology of cardiovascular diseases and is a factor that governs the severity of cardiovascular disease .
long - acting inhaled anticholinergics , such as tiotropium , are used in curing copd , and the uplift trial examined the usefulness of these medications .
the results of this trial and a subanalysis indicated that patients using tiotropium had improved respiratory function and a decreased risk of cardiovascular complications , such as myocardial infarction.11,12 in other words , research has suggested that copd is a therapeutic target for the improvement of prognosis in patients with cardiovascular diseases .
various epidemiological studies have indicated that crp is a factor that predicts cardiovascular events , such as myocardial infarction.1315 in addition , a study that examined systemic inflammation in patients with copd reported that these patients had increased levels of inflammatory markers , such as circulating neutrophils , fibrinogen , tnf- , and crp.5 1-antitrypsin low - density lipoprotein ( at - ldl ) complex is a recently identified oxidized ldl . at - ldl
is found in human serum and at sites of atherosclerotic lesions , and the levels of at - ldl in the blood indicate the activity of foam cells in atherosclerotic lesions.16,17 serum amyloid a ( saa ) bonds to high - density lipoprotein . when reactive oxygen species from inflammatory cells are activated by inflammation in blood vessels , they bond saa to ldl to form a complex known as saa - ldl .
saa - ldl is a potential marker that can be used to predict and thus prevent the occurrence of coronary events.18 curcumin is a polyphenol naturally found in turmeric and has anti - inflammatory and antioxidant activities .
turmeric has been used as a remedy in traditional indian and chinese medicine for over 6,000 years .
it is also extensively used as a spice , flavoring agent , food preservative , and food color .
curcumin has a number of biological activities , such as anti - inflammatory and anticancer actions.19,20 over the past few years , some useful effects of curcumin in treating various disorders have been identified.1921 we previously reported the use of curcumin in two rat models of chronic heart failure following hypertensive heart disease and myocardial infarction.22 curcumin was effective at inhibiting the progression of heart failure . because of its various biological activities , curcumin may be used clinically .
however , the drawbacks of curcumin are a very limited absorbability when ingested and a poor bioavailability .
thus , we used a drug delivery system to develop a highly absorbable curcumin preparation ( theracurmin ; theravalues corporation , kioi , tokyo , japan ) to increase its absorption in the intestinal tract .
natural curcumin forms extremely large granules in an aqueous solution , but theracurmin is a preparation that features coated , extremely fine microparticles of curcumin .
with such a drug delivery system , curcumin is consistently dispersed in an aqueous solution , thus improving its absorption by the intestinal tract.23 curcumin is reported to have anti - inflammatory activity as it inhibits nuclear factor-b ( nf-b ) as well as antioxidant activity as it eliminates reactive oxygen species.24 thus , curcumin may inhibit inflammation and oxidative stress , both implicated in increasing the risk of cardiovascular diseases .
accordingly , our objective was to evaluate the efficacy of theracurmin in patients with mild copd by examining its effects on oxidative stress markers and inflammatory markers .
the purpose of this trial was fully explained to potential subjects verbally and in writing .
once potential subjects provided written informed consent , they were allowed to participate in this trial .
the subjects were men or women ( age , 2085 years ) who met the following criteria : 1 ) patients with copd at stage 0 , i , or ii according to the definition by the japanese respiratory society and 2 ) patients who have never smoked at least for the past 4 months .
we enrolled patients with copd ( including those being treated for the condition ) who were classified as having stage i ( mild ) or stage ii ( moderate ) copd based on the classification system of the japanese respiratory society . during observation , the quantity and frequency of the dose remained unchanged for the patients being treated for copd , and the patients had stable disease with no acute exacerbation .
this trial was reviewed and approved by the ethics committee of the kyoto medical center .
based on the philosophy of the ethical principles originating in the declaration of helsinki , this trial protected the rights and welfare of subjects . to ensure the scientific nature and reliability of this trial and to ensure safety ,
this trial was conducted in accordance with the principles of good clinical practice and strictly adhered to the ministry of health , labor , and welfare s ethical guidelines for clinical research .
we conducted this double - blind , parallel - group , randomized trial from october 2012 to september 2014 .
once the written informed consent was obtained , the subjects were randomly assigned to one of the two groups ( the theracurmin [ theravalues corporation , kioi , tokyo , japan ] group or the placebo group ) .
the subjects took three capsules of theracurmin ( theracurmin is sold as a health supplement ) or a placebo ( 90 mg in total , 30 mg per capsule ) twice a day ( after breakfast and dinner , total dose : 180 mg ) .
the primary end points were oxidative stress markers and inflammatory markers ( crp , saa - ldl , and at - ldl ) .
parametric data , ie , continuous data , were expressed as mean standard deviation . as part of statistical analysis , a mann whitney u - test was used to compare continuous data between the two groups . for analysis of three or more matched groups ,
friedman test was performed followed by the wilcoxon signed - rank test with bonferroni correction as a post hoc test .
all analyses were done using spss version 22.0 for windows ( ibm corporation , armonk , ny , usa ) , with p<0.05 indicating statistical significance .
the purpose of this trial was fully explained to potential subjects verbally and in writing .
once potential subjects provided written informed consent , they were allowed to participate in this trial .
the subjects were men or women ( age , 2085 years ) who met the following criteria : 1 ) patients with copd at stage 0 , i , or ii according to the definition by the japanese respiratory society and 2 ) patients who have never smoked at least for the past 4 months .
we enrolled patients with copd ( including those being treated for the condition ) who were classified as having stage i ( mild ) or stage ii ( moderate ) copd based on the classification system of the japanese respiratory society . during observation , the quantity and frequency of the dose remained unchanged for the patients being treated for copd , and the patients had stable disease with no acute exacerbation .
this trial was reviewed and approved by the ethics committee of the kyoto medical center .
based on the philosophy of the ethical principles originating in the declaration of helsinki , this trial protected the rights and welfare of subjects . to ensure the scientific nature and reliability of this trial and to ensure safety ,
this trial was conducted in accordance with the principles of good clinical practice and strictly adhered to the ministry of health , labor , and welfare s ethical guidelines for clinical research .
we conducted this double - blind , parallel - group , randomized trial from october 2012 to september 2014 .
once the written informed consent was obtained , the subjects were randomly assigned to one of the two groups ( the theracurmin [ theravalues corporation , kioi , tokyo , japan ] group or the placebo group ) .
the subjects took three capsules of theracurmin ( theracurmin is sold as a health supplement ) or a placebo ( 90 mg in total , 30 mg per capsule ) twice a day ( after breakfast and dinner , total dose : 180 mg ) .
the primary end points were oxidative stress markers and inflammatory markers ( crp , saa - ldl , and at - ldl ) .
parametric data , ie , continuous data , were expressed as mean standard deviation . as part of statistical analysis ,
a mann whitney u - test was used to compare continuous data between the two groups . for analysis of three or more matched groups ,
friedman test was performed followed by the wilcoxon signed - rank test with bonferroni correction as a post hoc test .
all analyses were done using spss version 22.0 for windows ( ibm corporation , armonk , ny , usa ) , with p<0.05 indicating statistical significance .
of the 48 subjects enrolled , four dropped out : one because of soft stool ( placebo group ) , one because of gastrointestinal bleeding ( theracurmin group ) , one because of muscle aches ( placebo group ) , and one because of a rash on the back ( placebo group ) .
five subjects withdrew their consent ( four in the placebo group and one in the theracurmin group ) because they would not like to take the trial medication of their own free will despite a lack of symptoms , leaving 39 subjects in the final analysis .
the placebo group comprised 17 subjects ( 15 men and two women ) , and the theracurmin group comprised 22 subjects ( 19 men and three women ) .
there were no significant differences between the two groups in terms of sex , age , systolic blood pressure , diastolic blood pressure , glycated hemoglobin , blood sugar , triglyceride , ldl - cholesterol , high - density lipoprotein - cholesterol , uric acid , -glutamyl transpeptidase , creatinine , crp , saa - ldl , and percent forced expiratory volume in 1 second .
the patients in the placebo group had a significantly higher body mass index than those in the theracurmin group ( p=0.046 ) .
the theracurmin group had significantly higher levels of at - ldl than did the placebo group ( p=0.037 ) .
the percent changes in parameters between the baseline and after 6 months of theracurmin or placebo administration are listed in table 2 .
there were no significant differences in the percent changes in crp and saa - ldl ( primary end points ) between the groups .
however , the percent change in all primary end points increased in the placebo group but decreased in the theracurmin group .
the levels of at - ldl , which is oxidatively modified ldl , were also a primary end point .
the percent change in at - ldl was significantly lower in the theracurmin group compared with that in the placebo group ( p=0.020 ) .
curcumin significantly decreased the blood levels of at - ldl in patients with mild copd .
systemic inflammation in copd is known to be associated with a risk of cardiovascular diseases.25 copd itself is a risk factor for arteriosclerosis , and the systemic effects of copd are known to cause various comorbidities , such as a 1.5- to 2-fold increase in the risk of ischemic heart disease.26 currently , because there is no definitive treatment for copd and no treatment to restore the lung function , bronchodilators and steroids are used to treat copd by improving respiratory function .
however , some of these medications , such as 2-agonists , worsen comorbidities such as myocardial infarction that are caused by the systemic effects of copd .
the 2-agonists are used as bronchodilators to treat patients with asthma or copd and are thought to cause fewer adverse reactions in the cardiovascular system than 1-agonists ; however , 2-agonists are reported to increase heart rate and lower potassium levels , potentially increasing the cardiovascular risk for patients with asthma or copd.27 a study examining steroids and the occurrence of cardiovascular events reported that steroids significantly increased the risk of a cardiovascular event.28 curcumin was used in the current trial , and no studies have reported serious adverse reactions to curcumin .
curcumin is a highly safe natural herbal product , and a study has suggested that curcumin may be effective in treating numerous disorders , such as heart disease , malignancies , lifestyle - related diseases , neurological disorders , inflammatory diseases , and infections.1922 a comparison of at - ldl levels in smokers and nonsmokers indicated that at - ldl levels are significantly elevated in smokers.29 at - ldl is an oxidatively modified ldl , and at - ldl levels quickly drop as a result of smoking cessation , which suggests that at - ldl promotes atherosclerosis and is closely related to smoking .
based on these findings , it may be a useful marker to assess cardiovascular risk . given that at - ldl levels decreased significantly as a result of taking theracurmin , curcumin may prevent the onset of arteriosclerosis in copd and consequently reduce the cardiovascular risk .
the baseline at - ldl levels were significantly higher in the theracurmin group , but levels of at - ldl and other inflammatory markers and oxidative stress markers , such as crp and saa - ldl , tended to decrease in the theracurmin group , suggesting that the inflammation in copd is likely to be inhibited by theracurmin .
many studies have reported that curcumin has anti - inflammatory and antioxidant activities.3032 tnf- , cyclooxygenase-2 ( cox-2 ) , stat , cyclin d1 , and nf-b are related to inflammation , and curcumin displays anti - inflammatory action by inhibiting tnf- , cox-2 , stat , cyclin d1 , and nf-b signaling .
curcumin displays antioxidant activity because the ch2 located in the center of the molecule and hydroxyl groups in the benzene rings eliminate reactive oxygen species.30,33 these actions of curcumin may be related to the mechanism by which curcumin inhibits oxidized ldl . a retrospective cohort study of 854 patients with copd examined differences in the mortality rate depending on whether patients were treated with statins.34 in this study , the hazard ratio for death of patients taking statins was 0.57 ( 95% confidence interval 0.380.87 ) compared with patients not taking statins .
the study reported that treatment with statins was associated with an improvement in the survival rate after the exacerbation of copd .
a cohort study of 11,212 patients reported that angiotensin - converting enzyme inhibitors and angiotensin ii receptor blockers improved the prognosis for patients with copd.35 cardiovascular events are complications of copd , and treatment must inhibit cardiovascular events to improve the prognosis for patients with copd .
we examined blood inflammatory markers and respiratory function in patients with mild copd over 6 months .
compared with the placebo , theracurmin significantly decreased the blood levels of the inflammatory marker at - ldl and tended to decrease ( albeit not significantly ) levels of crp and saa - ldl .
however , as a limitation , this trial had a small sample and was conducted over a short period of time ( 6 months ) . a long - term study with cardiovascular events as a primary end point and numerous patients with copd
if a natural product that is inexpensive and safe can be used to prevent and treat cardiovascular disease , then such a treatment could greatly benefit public health and welfare and reduce medical expenses in the process .
in patients with copd and inflammatory disease , the current trial indicated that theracurmin might reduce levels of at - ldl , a complex that promotes arteriosclerosis .
copd is an inflammatory disease , and curcumin may help to prevent ischemic disorders ( eg , myocardial infarction ) , which are a comorbidity of copd .
this study suggests that curcumin could help in preventing the development of vascular events in patients with copd . | purposecopd is mainly caused by tobacco smoking and is associated with a high frequency of coronary artery disease .
there is growing recognition that the inflammation in copd is not only confined to the lungs but also involves the systemic circulation and can impact nonpulmonary organs , including blood vessels .
1-antitrypsin low - density lipoprotein ( at - ldl ) complex is an oxidatively modified ldl that accelerates atherosclerosis .
curcumin , one of the best - investigated natural products , is a powerful antioxidant .
however , the effects of curcumin on at - ldl remain unknown . we hypothesized that theracurmin , a highly absorptive curcumin with improved bioavailability using a drug delivery system , ameliorates the inflammatory status in subjects with mild copd.patients and methodsthis is a randomized , double - blind , parallel - group study .
subjects with stages i ii copd according to the japanese respiratory society criteria were randomly assigned to receive 90 mg theracurmin or placebo twice a day for 24 weeks , and changes in inflammatory parameters were evaluated.resultsthere were no differences between the theracurmin and placebo groups in terms of age , male / female ratio , or body mass index in 39 evaluable subjects .
the percent changes in blood pressure and hemoglobin a1c and ldl - cholesterol , triglyceride , or high - density lipoprotein - cholesterol levels after treatment were similar for the two groups
. however , the percent change in the at - ldl level was significantly ( p=0.020 ) lower in the theracurmin group compared with the placebo group.conclusiontheracurmin reduced levels of atherosclerotic at - ldl , which may lead to the prevention of future cardiovascular events in mild copd subjects . |
craniopharyngioma is a type of benign tumor that arises from rathke s pouch , a derivative of the oral ectoderm , and accounts for approximately 33.5% of all intracranial tumors3 ) .
although regarded as a world health organization ( who ) grade i tumor , it often demonstrates aggressive local behavior after surgery and requires adjuvant radiotherapy .
craniopharyngiomas are known to recur even after presumed total resection , especially at the primary site or in its vicinity , suggesting microscopic residual disease .
it has been estimated that the recurrence rates after total and subtotal resection are 30% and 5070% , respectively .
craniopharyngioma rarely undergoes malignant transformation , a phenomenon that was most likely first described by salyer in 197310 ) .
the etiology and pathogenesis of malignant transformation is unclear , although previous literature has suggested a correlation with radiotherapy9 ) .
the prognosis of malignantly transformed craniopharyngioma is poor and because of its low incidence rate , there is insufficient evidence for various treatment modalities .
herein , we report a case of craniopharyngioma with subsequent malignant transformation 2 years after the first operation .
a 26-year - old male patient presented with a 1-week history of progressive headache and visual disturbance without any other neurological deficits . brain magnetic resonance imaging ( mri ) revealed a suprasellar mass lesion with enhanced solid and multiseptated cystic components measuring 3.02.8 cm ( fig .
preoperative investigations of pituitary function including prolactin and thyroid function did not reveal any abnormalities .
the patient underwent an excision via an interhemispheric transcallosal approach with near - total resection of the tumor .
2 ) . there was no cytologic atypia and mitoses . in the immediate postoperative period ,
after 6 months , mri showed a residual mass enhanced along the right anterior margin of the third ventricle floor ( fig .
the patient was treated with medication for endocrine dysfunction and refused radiation therapy for the residual tumor .
two years after his first operation , the patient presented to our department with confused mental status and progressive visual disturbance with bitemporal hemianopsia .
mri showed a recurrence of a multiseptated cystic mass measuring 4.75.0 cm in the suprasellar cistern , third ventricle , and parts of the lateral ventricles ( fig .
he was operated on via an interhemispheric transcallosal approach along the previous incision , and gross total resection of the tumor was performed .
macroscopically , the tumor was soft and calcified with relatively defined boundaries compared to the surrounding tissue .
immunological tests showed overexpression of p53 and ki67 ( ki67 labeling index 15% ) ( fig .
5c , d ) , and the tumor cells were positive for pancytokeratin and vimentin ( fig .
the patient s symptoms of headache and impaired consciousness improved but the endocrine dysfunction and visual disturbances persisted . after three months , mri showed no residual tumor ( fig .
6 ) . however , seven months after the second operation , he visited the emergency room ( er ) with a high fever , general weakness , and drowsy mentality .
while being evaluated in the er , he showed a sudden drop in blood pressure and expired shortly after despite immediate efforts to resuscitate him . no autopsy was performed .
malignant transformation of craniopharyngioma is extremely rare and is not included in the current who classification of tumors of the central nervous system published in 20077 ) .
malignant craniopharyngiomas are presumably derived from the remnants of rathke s pouch , which was first described by akachi in 19871 ) .
a total of 23 cases have been reported since then and only five cases exhibited malignant craniopharyngioma without radiotherapy .
four of these cases developed de novo ( primary type ) and only one case developed by malignant transformation of craniopharyngioma ( secondary type ) as in the present case ( table 1 ) .
according to the literature , the pathologic findings of malignant craniopharyngiomas are heterogeneous including squamous cell carcinoma , myoepithelial carcinoma , ameloblastoma , ameloblastic carcinoma , and malignant craniopharyngioma mimicking odontogenic ghost cell carcinoma . a case of malignant craniopharyngioma with spindle cell proliferation that presented with sarcomatous transformation
although diagnostic criteria for malignant craniopharyngioma have not yet been established , the following histology is suggestive of malignantly transformed craniopharyngioma : increased cellularity , nuclear pleomorphism , increased mitotic activity or ki67 proliferation indices , necrosis , or vascular proliferation .
our case is similar to the case reported by wang et al.11 ) in that , the malignantly transformed portion was derived from the stromal spindle cell portion of the preexisting craniopharyngioma .
there are various causes of malignant transformation in craniopharyngioma ; however , the exact mechanism is currently unknown because of the limited number of reported cases . according to the 13 case reports in the literature , radiotherapy might have an influence on malignant transformation .
similar to our case of malignant transformation without radiotherapy , gao et al.4 ) reported histopathological confirmation of papillary craniopharyngioma after the patient s first surgery , followed by histopathological confirmation of adamantinomatous craniopharyngioma with ameloblastic carcinoma due to regrowth of the mass in some areas after the second surgery .
after one year , the patient underwent a third surgery owing to recurrence and histopathological examination showed clear malignant features . in our case , the patient was diagnosed with a typical adamantinomatous craniopharyngioma during the first surgery ; however , after the second surgery , histopathological examination showed malignant features and confirmed that the craniopharyngioma was of the adamantinomatous type . therefore , other factors besides radiotherapy might contribute to malignant transformation of craniopharyngioma .
recurrence of craniopharyngioma within a short period , especially after radiation therapy , could result in its malignant transformation . | craniopharyngiomas exhibiting histologic malignancy are extremely rare .
herein , we report the case of a 26-year - old male patient who underwent suprasellar mass excision via an interhemispheric transcallosal approach .
histopathological examination indicated that the craniopharyngioma was of the adamantinomatous subtype .
the patient received postoperative medical treatment for endocrine dysfunction and diabetes mellitus without radiation treatment .
two years after the operation , he presented with progressive visual disturbance and altered mentality .
magnetic resonance imaging revealed a huge mass in the suprasellar cistern and third ventricle .
he underwent a second operation via the same approach .
the histopathological examination showed an adamantinomatous craniopharyngioma with sheets of solid proliferation in a spindled pattern , indicating malignant transformation .
malignant transformation of craniopharyngioma in the absence of radiation therapy has been reported in only five cases , including this one .
we present a case of malignant transformation of craniopharyngioma with a brief review of relevant literature . |
since the initial evidence for the existence of transposable elements ( tes ) reported by mcclintock , large - scale sequencing of the genome of a wide range of living organisms has highlighted the abundance of te - derived sequences relative to the coding portion of genomes .
parasitic dna , are mobile genetic elements that encode their own mobility enzymes and move from one genomic locus to another . based on their transposition mechanisms
, they can be classified into two main categories [ 2 , 3 ] : class i elements transpose via the reverse transcription of an rna molecule , while class ii elements transpose via a dna intermediate .
class ii transposons , also called dna transposons , are found in variable proportions among eukaryotic and prokaryotic genomes ; for instance , they constitute the major fraction of resident tes in bacteria ( reviewed in ) but are underrepresented relative to class i elements in the human genome and absent from the genome of the yeast saccharomyces cerevisiae . among dna transposons , cut - and - paste transposons move in two steps : ( i ) excision from the donor site , as a result of transposase - induced dna cleavages at their ends and ( ii ) integration into the target site through strand transfer of their free 3oh ends .
the most widespread cut - and - paste transposons , which are found in eukaryotes and in prokaryotes , encode a so - called dde transposase , an enzyme that bears a conserved triad of acidic residues ( dde or ddd ) that catalyzes the excision and integration steps [ 2 , 7 ] . upon integration , these particular cut - and - paste transposons duplicate a short ( 2 to 15 bp ) target sequence ( tsd or target site duplication ) on each side of the newly integrated copy ( reviewed in ) . upon excision ,
staggered double - strand cleavages at their ends generally leave the two copies of the tsd at the donor site and the resulting double - strand break can be repaired by end joining [ 911 ] .
this generates a footprint at the excision site , formed by two copies of the tsd flanking a few remaining bp from the transposon .
however , if transposition takes places during replication , homologous recombination with the sister chromatid may restore the initial transposon copy at the donor locus , which leads to a net increase in transposon copy number in the genome [ 1113 ] .
increase in transposon copy number may have detrimental effects on host fitness , since insertions can disrupt coding regions , modify the expression of adjacent cellular genes , or trigger ectopic recombination between distant transposon copies ( reviewed in ) .
several defense strategies have been developed by the host to inactivate transposition . in eukaryotes ,
posttranscriptional inactivation of tes is mediated by homologous small rnas , which may also induce histone and dna methylation to inactivate the transcription of transposon genes ( reviewed in ) . in some hosts , like filamentous fungi
, heavy mutagenesis of repeated sequences , a phenomenon named repeat - induced point mutation ( or rip ) , was also reported , but the involvement of small rnas in this process has not been clearly demonstrated . as a result of these defense responses ,
many extant genomes harbor large numbers of defective copies of transposable elements that have lost their ability to transpose .
transposons , however , may also provide novel and advantageous functions to the host , as illustrated by the growing list of entire or truncated transposase genes found in eukaryotic genomes , either isolated or fused to genes encoding unrelated protein domains , which still appear to be expressed and encode proteins , but are not embedded within a mobile element anymore . according to several criteria described in [ 8 , 17 ] , these genes have been domesticated to become cellular genes , but , in most cases , their function has not been elucidated . most often ,
only putative transposase domains involved in nucleic acid binding have been conserved and may play a role in cellular dna or rna metabolism .
intriguingly , most domesticated transposases have lost their characteristic dde ( or ddd ) signature and only for very few of them have evidence been obtained for in vivo or in vitro dna cleavage activity .
remarkable examples of catalytically active domesticated dde transposases were reported in different organisms : the rag1 endonuclease , related to transib transposases , catalyzes v(d)j recombination of immunoglobulin genes during the differentiation of lymphocytes in vertebrates ( reviewed in ) ; alpha3 , a domesticated mutator - like transposase , is involved in mating - type switching in the yeast kluyveromyces lactis ; finally , setmar , identified in the human genome , carries a histone methyltransferase domain fused to the partially active catalytic site of a mariner transposase and is thought to participate in the repair of dna double - strand breaks , in the restart of stalled dna replication forks and in chromosome decatenation ( , reviewed in ) .
evidence for a role of domesticated piggybac transposases in programmed genome rearrangements was reported recently in ciliates [ 24 , 25 ] .
thanks to their unique nuclear dimorphism and to their original mechanism of programmed dna elimination from their somatic nucleus , ciliates , and most specifically paramecium , have emerged as novel model organisms for the study of the impact of transposable elements on genomes ( for recent reviews , see [ 26 , 27 ] ) . here
, we will present an overview of the life cycle of these unicellular eukaryotes and of the massive and developmentally programmed genome rearrangements that take place at each sexual cycle .
we will discuss how the domestication of an ancient piggybac cut - and - paste transposase in paramecium might subsequently have allowed tc1/mariner elements to spread throughout the germline genome , without strong counterselection against insertion within genes .
these unicellular organisms are characterized by the coexistence , in their cytoplasm , of two functionally distinct types of nuclei ( figure 1 ) .
the diploid germline micronucleus ( mic ) is transcriptionally silent during vegetative growth , but harbors the genetic information that is transmitted to the next sexual generation .
according to ciliate species , the number of mics per cell may vary ( one in tetrahymena thermophila and two in paramecium tetraurelia , e.g. ) .
gene expression is carried out from the highly polyploid somatic macronucleus ( mac : ~800n in paramecium ) , which is therefore essential for cell survival at all stages . during sexual events ( conjugation between compatible mating types or , for some species ,
self - fertilization also called autogamy ) , mic meiosis leads to the formation of haploid nuclei , one of which divides once to yield two identical gametic nuclei .
the fusion of two gametic nuclei ( reciprocally exchanged between mating partners during conjugation or originating from the same cell during autogamy ) gives rise to the zygotic nucleus . in the meantime ,
new mics and macs differentiate from mitotic copies of the zygotic nucleus . throughout this developmental process
, the old mac ensures all gene transcription and is progressively replaced by the new mac .
therefore , the development of a functional new mac is essential for the survival of sexual progeny , once the old mac has disappeared from the cell .
mac chromosomes of ciliates are shorter than their mic chromosomes and apparently do not carry centromeres , which is consistent with the observation that the mac divides through an amitotic process , with no chromosome condensation ( reviewed in ) .
moreover , early studies of the complexity of mic and mac genomes pointed out that the two nuclei do not harbor the same dna content , although they derive from the same zygotic nucleus .
indeed , the mic genome contains additional sequences that are removed from the somatic genome during mac development ( reviewed in [ 31 , 32 ] ) .
pulse field electrophoresis analyses indicated that the size of paramecium mac chromosomes varies between 50 kb and 1 mb .
the study of particular mac chromosome ends in p. primaurelia and p. tetraurelia [ 35 , 36 ] revealed that they are capped by a mixture of g3t3 or g4t2 telomeric repeats added at heterogeneous positions by a single , error - prone telomerase [ 37 , 38 ] ; several telomere - addition regions distant of several kbp have been identified for some mac chromosomes , each one extending over ~1 kb . the mac genome sequence of paramecium tetraurelia was obtained in 2006 by a consortium of european labs .
this study provided a global view of the structure of some 150 acentromeric mac chromosomes and highlighted the fact that the somatic genome is streamlined for gene expression , with a very high gene density ( 78% coding ) and essentially no repeated sequences . even more striking , ~40,000 genes were annotated in the mac genome , as a consequence of at least three successive whole genome duplications ( wgd ) during evolution of the paramecium aurelia group of sibling species , to which p. tetraurelia belongs .
chromosomes , only limited knowledge of the number and structure of paramecium germline chromosomes is available ( figure 2 ) .
early microscopy studies proposed that 35 to 50 pairs of 1 to 7 mb chromosomes harbor the genetic content of the mic in p. tetraurelia .
molecular analyses of a couple of germline regions encompassing mac chromosome ends revealed no conserved nucleotide sequence motif for chromosome fragmentation in paramecium .
this situation is quite different from that observed in other ciliates , in which consensus chromosome breakage sequences ( cbs ) were found at fragmentation sites ( reviewed in ) .
instead , the fragmentation of paramecium mac chromosomes seems to be associated with heterogeneous elimination of repeated germline sequences ( minisatellites , germline transposons , etc . )
southern blot hybridization experiments confirmed that known germline transposons are eliminated from the somatic genome during mac development ( o. garnier , unpublished and [ 24 , 42 ] ) . on a genome - wide scale ,
more work is clearly needed to gain full insight into the dna content ( and more specifically their te landscape ) of large germline regions that are eliminated from the mac in association with chromosome fragmentation .
in contrast , along chromosomes , the availability of a phage library of p. tetraurelia mic dna constructed by preer et al . in 1992which has represented a technical tour de force made it possible to compare the nucleotide sequence of particular mac and collinear mic loci ; these studies led to the identification of short , noncoding sequences called iess ( internal eliminated sequences ) that interrupt both coding and noncoding regions in the germline genome and are excised precisely from mac chromosomes ( , reviewed in [ 45 , 46 ] ) .
recent genome - wide sequencing of a set of 45,000 iess confirmed the early description of these sequences .
paramecium iess are very short ( 93% are shorter than 150 bp long and one - third are within the 2630 bp size range ) and each one appears to be single copy in the genome .
their exquisitely precise excision is essential for the recovery of a functional new mac , since 47% of genes are interrupted by at least one of these intervening sequences in the germline genome .
their only absolutely conserved feature is the presence of one flanking 5-ta-3 at each end , while a single ta is retained at their excision site on mature mac chromosomes . because of this conservation
, paramecium iess have defined the family of the so - called ta - iess . in other ciliates ,
iess are also eliminated during mac development , but their structure varies from one species to the other . the existence of short ta - iess has been reported in euplotes crassus and oxytricha fallax , while iess in tetrahymena thermophila are larger and are generally not flanked by ta repeats ( reviewed in ) .
statistical analysis of the nucleotide sequence of the ends of ~20 iess from different paramecium aurelia species was performed by klobutcher and herrick , who identified a degenerate 8 bp consensus ( 5ta(c / t)ag(c
/ t)n(a / g)3 ) that defines a loosely conserved terminal inverted repeat ( tir ) at ies ends .
this consensus sequence , which includes the flanking ta , was confirmed by all the following analyses of increasing numbers of iess [ 41 , 46 , 49 ] .
interestingly , it also matches the ends of the short ta - iess found in euplotes and oxytricha and also of tc1/mariner - related transposons tec1 and tec2 present in high copy numbers in the germline genome of euplotes crassus . based on the observation that ta - iess and tec transposons coexist in euplotes , klobutcher and herrick proposed their ibaf model ( invasion / bloom / abdicate / fade ) , according to which
ta - iess within a given ciliate species have evolved from tc1/mariner transposons , which would have invaded the mic genome and accumulated internal substitutions / deletions during evolution .
therefore , transposon remnants would have lost their coding capacity while being kept under strong selection pressure for their elimination from the somatic genome .
interestingly , a common feature of tc1/mariner transposons is their preference for ta dinucleotides as integration targets , which they duplicate upon insertion ; thus , the conserved tas at the boundaries of ta - iess would simply be the tsds generated by integration of ancestral tc1/mariner - related tes .
the ibaf model for the evolutionary origin of iess has recently obtained further support in paramecium .
transposon - like sequences were indeed identified in the heterogeneously eliminated fraction of the germline genome of different p. aurelia strains [ 34 , 41 ] .
sequence alignment of these elements has led to the establishment of a consensus for each family ( tennessee in p. primaurelia , sardine and thon in p. tetraurelia ) and to the unambiguous identification of open reading frames encoding putative transposases harboring the characteristic dd(35)e triad of tc1/mariner / is630 transposons .
reminiscent of the situation described in e. crassus , the six outward terminal nucleotides ( including the flanking ta ) of the long ( 500 to 700 bp ) and complex tirs of the sardine and thon elements of p. tetraurelia match the consensus of ies ends ( figure 3(a ) ) .
analysis of the three wgds that took place during the evolutionary history of p. tetraurelia provided evidence that iess have appeared continuously in the germline genome and that their size tends to shorten over time . among the largest iess ( > 500 bp ) ,
some of them exhibit significant sequence similarities with the long tirs of known tes from the thon family and are excised from mac chromosomes just like any other ies .
the existence of these solo tirs provides further support to the notion that some iess at least have derived from recently mobile tes from the tc1/mariner family .
genetic evidence indicates that the conserved tas , which are supposed to represent the tsd created by integration of the ancestral tc1/mariner , are essential for the developmentally programmed excision of iess [ 5155 ] .
one of the assumptions of the ibaf model is that , at first , ancestral invading germline tc1/mariner transposons were eliminated from the somatic genome during mac development , thanks to the action of their own transposase ; thus , programmed genome rearrangements have allowed tes to proliferate in the mic , with little or no effect on the phenotype of the cell , as long as they are correctly excised from the mac .
then , at some point during evolution , a cellular gene ( possibly a domesticated or preexisting tc1/mariner transposase gene ) took over the catalysis of excision of all these elements , allowing them to accumulate internal mutations and give rise to current iess , while still being able to excise from the mac .
as already discussed , one caveat of this model is that tc1/mariner transposition leaves a characteristic footprint at the excision ( or donor ) site , while iess are excised precisely at the nucleotide level , leaving only one copy of the original duplicated ta at the excision junction ( figure 3(b ) ) .
molecular analyses of ies excision intermediates formed in vivo during sexual processes in p. tetraurelia provided important information about the mechanisms involved in this process .
ies excision starts after a few rounds of endoduplication of the germline genome have taken place , so that at least 16 copies of each ies need to be excised in each developing mac .
it is initiated by 4 bp staggered double - strand dna cleavages at both ends of each ies , centered on the conserved ta dinucleotides . as a result , transient double - strand breaks ( dsbs ) with characteristic 4-base 5 overhangs can be detected by ligation - mediated pcr during mac development , at the ends of linear excised ies molecules and at flanking mac - destined dna ends ( figure 3(b ) and [ 57 , 58 ] ) .
strikingly , these dsbs have the same geometry as those catalyzed in vitro by piggybac transposases . indeed
, piggybac cut - and - paste transposons duplicate a 5-ttaa-3 target site upon integration , and when they transpose to a new locus , their transposase cleaves dna on each side of each duplicated tsd to generate a 5 ttaa overhang . thus , piggybac excision is highly precise and reconstitutes the ttaa sequence at the donor site .
the discovery of piggymac ( pgm ) , a domesticated piggybac transposase gene in p. tetraurelia , represented a significant breakthrough towards the identification of protein partners involved in ies excision .
this gene is only expressed during sexual processes , with an induction peak during the development of new macs , which corresponds to the time when ies excision starts .
it encodes a large 1065 aa protein with a recognizable central domain homologous to the transposase of piggybac transposons , including a potentially active ddd catalytic triad ( figure 4(a ) ) . during sexual processes ,
pgm - gfp fusion proteins were found to localize specifically in the developing new macs , in which ies excision takes place ( figure 4(b ) and , dubois , unpublished ) . in cells silenced for expression of the pgm gene ,
ies excision is blocked as well as other known programmed genome rearrangements ( chromosome fragmentation and heterogeneous elimination of sardine transposons ) ; as a result , strong lethality is observed in the sexual progeny .
nuclei of pgm - silenced cells ( purified during the development of new macs , before the cells die ) provided the source of dna that was used for whole - genome sequencing and identification of the set of 45,000 iess described above .
indicative of a catalytic function of pgm , microinjection of a mutant transgene encoding a protein in which the ddd catalytic triad was switched to aaa induces a dominant negative effect on the survival of sexual progeny , while a normal phenotype is obtained with a wild - type transgene ( dubois , unpublished ) .
thus , even though paramecium iess are probably relics of tc1/mariner transposons , their precise excision from the mac genome appears to be carried out by a domesticated transposase related to a different family of transposable elements , the piggybac family .
dna transposons generally assemble a synaptic nucleoprotein complex called the transpososome , which includes both transposon ends and oligomers of the transposase ( see for a review ) .
assembly of this complex activates the successive hydrolysis and transesterification steps that ultimately lead to transposon excision .
likewise , genetic evidence has indicated that ies excision in paramecium involves an interaction between the two ends of each ies , before dna cleavage . for three iess of different sizes ( 28 , 66 , and 370 bp )
, it was indeed shown that a mutation within the ta at one end not only inhibits cleavage of the mutant end but also strongly impairs dna cleavage at the wild - type end of the same ies .
moreover , thorough analysis of the currently available set of 45,000 iess of p. tetraurelia provided support to the hypothesis that ies excision , similar to transposition , involves the formation of an intramolecular dna loop on a double - strand substrate .
indeed , the size distribution of iess exhibits a striking 10 bp periodicity , which coincides with the length covered by one turn of the dna double helix .
this suggests that interactions between pgm molecules bound at each end of an ies depend critically on helical phasing , especially for very short sequences ( 93% of paramecium iess are shorter than 150 bp , the persistence length of double - strand dna ) . as discussed for other systems ( site - specific recombination ,
transposition , or repression ) , dna looping between very closely spaced sites might also be favored by dna bending factors and/or local melting of the double helix .
the ends of cut - and - paste transposons are generally made of two parts : an internal sequence - specific binding site for their cognate transposase and a few nucleotides at their termini that constitute the dna cleavage site per se ( see , for example , ) .
for piggybac , the site cleaved by the transposase is the ttaa duplicated target sequence on each side of the integrated copy of the element .
a 13 bp terminal repeat ( tr ) and a 19 bp internal repeat ( ir ) separated by a spacer are present in inverted orientation at each end of the element and may be binding sites for the transposase [ 65 , 66 ] .
analysis of the nucleotide sequence of 45,000 iess from p. tetraurelia showed that the ttaa tetranucleotide is actually largely underrepresented at ies ends ( marmignon , unpublished ) , even though the sequence cleaved by piggymac at the termini of paramecium iess bears some similarity with the site cleaved by the piggybac transposase ( i.e. , a 4 bp sequence with a central ta ) .
furthermore , neither tr nor ir repeats are found at the ends of paramecium iess .
the situation is even more striking for tetrahymena iess , which depend on a close piggymac homolog , the piggybac - like transposase called tpb2 , for their elimination ; indeed , the sequence cleaved at their ends ( 5annnnt3 ) does not even carry a conserved central ta .
this suggests that ciliate domesticated piggybac transposases do not recognize a specific nucleotide motif at ies ends and raises the question of how germline sequences are targeted for elimination .
part of the answer may lie in the epigenetic mechanisms that control programmed genome rearrangements in paramecium and tetrahymena ( reviewed in [ 26 , 27 , 69 , 70 ] ) .
it was proposed for both ciliates that a comparison between the dna content of parental mic and mac genomes takes place during mic meiosis through the annealing of two kinds of noncoding rna molecules .
short rnas , also called scnrnas ( 25 nt in paramecium , 28 nt in tetrahymena ) , are generated by a specialized rna interference pathway from noncoding rna precursors transcribed specifically from the mic during meiosis . according to the scanning
model , these scnrnas would pair to larger transcripts that are produced constitutively by generalized transcription of the parental mac genome , which was rearranged during the previous sexual cycle .
those scnrnas that do not find homologous mac sequences , and therefore represent the fraction of the germline genome that was absent from the parental mac , are then imported into the new developing mac , in which they are thought to target the deletion of homologous sequences . in tetrahymena ,
the methylation of ies - associated histones is clearly one of the scnrna - dependent epigenetic modifications that trigger the elimination of heterochromatin regions [ 71 , 72 ] .
in contrast , the putative epigenetic marks that are deposited by scnrnas on paramecium germline eliminated sequences have not been identified yet , especially for iess , the vast majority of which are much shorter than the length of dna wrapped around a nucleosome ( ~150 bp ) .
whatever the exact mechanism may be , a strong implication of the scanning model is that ciliates tend to reproduce their pattern of developmentally programmed genome rearrangements from one sexual generation to the next .
thus , epigenetic control may have contributed to loosen the requirement for a specific nucleotide sequence to direct ciliate domesticated piggybac transposases towards regions that have to be eliminated from the developing mac .
quite interestingly , piggymac and tpb2 present variant domains relative to piggybac transposases ( within their catalytic site and a downstream cysteine - rich region ) and have acquired long c - terminal extensions ( figure 4(a ) ) ; the role of these domains in ies recognition still has to be elucidated . thanks to the particular cleavage properties of their transposase , the transposition of piggybac transposons leaves no footprint at the donor site ( figure 3(b ) ) , and excision junctions can be closed through direct annealing of the fully complementary 5-ttaa-3 overhangs generated on flanking dna ends , with no need for any additional processing step . for paramecium iess , however , the situation is quite different , since only the central ta is conserved on the 4-base overhangs created by pgm - dependent cleavage .
it was proposed that the closure of ies excision junctions on mac chromosomes involves partial pairing of the flanking ends through annealing of their conserved tas and limited additional processing ( removal of the unpaired 5-terminal nucleotides and gap - filling by addition of one nucleotide at each 3 recessive end ) , before the final ligation step ( figure 5 and ) .
iess are assumed to be excised as linear molecules and , at least for those larger than 200 bp , to be circularized in a second step using the same pathway ( partial pairing of overhangs , 5 and 3 processing , ligation ) .
however , recent work uncovered the essential role played by the ligaseiv and its partner xrcc4 in the closure of ies excision sites and the circularization of excised iess . ligaseiv and xrcc4
are core actors of the nonhomologous end joining ( nhej ) pathway , which repairs dsbs through the direct joining of broken ends , without requiring sequence homology .
two very closely related lig4 genes originating from the most recent wgd and a single xrcc4 gene were identified in the genome of p.tetraurelia .
their expression reaches a peak during mic meiosis , even before new macs have differentiated from mitotic copies of the zygotic nucleus ; this implies that induction of dsb repair genes is part of a developmental program in paramecium rather than a response to dna damage . in cells depleted either for ligaseiv or xrcc4 ,
pgm - dependent cleavages are introduced normally , but no detectable chromosomal nor circular junctions are formed ; unrepaired dsbs accumulate at ies excision sites as well as linear forms of excised iess .
noteworthy , dsbs are processed normally at their 5 end in ligase iv - depleted cells ( removal of the 5terminal nucleotide ) , but no nucleotide addition is observed at their 3 recessive end . as already inferred from in vitro studies of reconstituted eukaryotic nhej systems , this indicates that the ligaseiv participates in the recruitment or activation of a gap - filling dna polymerase , prior to end joining ( figure 5 ) .
the participation of actors of the nhej pathway in the final step of ies excision raises the question of how accurate end joining is achieved following the massive introduction of programmed dsbs throughout the genome .
indeed , given the number of iess per haploid genome , thousands of dsbs are introduced all along chromosomes within a restricted time window during mac development .
the formation of a pgm - containing synaptic excision complex prior to ies end cleavage might contribute to hold together adjacent fragments of mac - destined dna , ensuring , therefore , that somatic chromosomes are assembled in the right order during dsb repair ( figure 6 ) .
furthermore , the human xrcc4 protein was recently shown to form filaments with another nhej factor , cernunnos ( or xlf ) , independently of ligase iv [ 7577 ] .
two cernunnos homologs are encoded by paralogs of the recent wgd in p. tetraurelia , and their expression is induced during sexual processes . during ies excision , such filaments may provide an alignment scaffold and favor the correct assembly of broken mac ends . at the nucleotide level ,
an additional requirement for assembly of a functional mac genome is the highly precise joining of each ies excision site to reconstitute open reading frames .
increasing evidence that the classical nhej pathway is inherently precise ( , reviewed in ) has pointed to the key role played by the ku70/ku80 heterodimer in protecting broken dna ends against resection and inhibiting other dsb repair pathways , such as alternative end - joining ( which would create imprecise deletions ) or homologous recombination ( which would restore the non - rearranged molecule ) .
ku proteins have been conserved through evolution , from bacteria to humans , and several genes encoding putative ku70 and ku80 homologs were found in the genome of p. tetraurelia .
efficient recruitment of ku proteins at ies excision sites probably plays a determinant role in the precision of dna rearrangements .
p. tetraurelia also harbors a unique gene encoding a homolog of the dna - pkcs , a dna - dependent protein kinase ( malinsky et al .
, in preparation ) that interacts with the ku dimer , facilitates the synapsis of broken dna ends , and , after autophosphorylation , activates downstream nhej proteins .
the conservation of dna - pkcs in paramecium , even though this protein has been lost from other model organisms such as budding yeast or drosophila , suggests that this protein was present in the ancestral eukaryotic nhej core machinery .
functional inactivation of the ku and dna - pkcs genes by rna interference indicates that ku70/ku80 and the dna - pkcs homolog are required for ies excision ( marmignon , unpublished ; malinsky et al .
strikingly , among the three ku80 genes identified in the genome , only one is specifically expressed during mac development and appears to have acquired a specialized function in genome rearrangements ( marmignon , unpublished ) .
the availability of a genome - wide set of ~45,000 iess in paramecium tetraurelia has broadened our current view of the evolutionary history of the germline genome of ciliates .
all iess that have been identified so far in paramecium belong to the ta - ies family .
consistent with the ibaf model proposed by klobutcher and herrick , some of them at least seem to have evolved from ancestral tc1/mariner - related transposons , still recognizable in the fraction of the mic genome that is eliminated in an imprecise manner .
this putative evolutionary link between iess and tes is similar to that proposed for euplotes , a distant spirotrichous ciliate in which tec transposons and related ta - iess are excised precisely from the mac genome , although the details of the mechanism may be somewhat different from ies excision in paramecium . in particular , the enzyme responsible for ies excision in euplotes
at least three families of tbe transposons , also related to the tc1 family and initially designated as telomere - bearing elements , have been identified in the eliminated fraction of the mic genome . along the lines of the ibaf model , rna interference experiments have suggested that the tbe transposase itself mediates the elimination of tbe transposons from the somatic genome .
it appears to be also involved in other genome rearrangements reported in oxytricha , such as ies excision and the unscrambling of a subset of genes , for which macronuclear - destined sequences are not collinear in the mic and mac genomes .
this situation has provided a nice example of mutualism , rather than domestication , between resident transposons and their host ( discussed in ) . in paramecium ,
the discovery that elimination of iess and tc1/mariner - like transposons depends on a domesticated transposase related to the piggybac family has provided an unexpected extension of the ibaf hypothesis .
as discussed previously [ 24 , 41 ] , the existence of a catalytically active piggymac homolog , tpb2 , also required for programmed genome rearrangements in tetrahymena thermophila , indicates that domestication of a piggybac transposase occurred early during ciliate evolution , before the divergence between paramecium and tetrahymena ( figure 7(a ) ) .
the initial role of this ancestral piggybac transposase might have been to cope with a first invasion of piggybac elements , by removing them from ciliate genomes .
it may then have been recruited to carry out the elimination of other unrelated germline sequences from the mac genome .
intriguingly , except for a few ttaa - iess that may originate from piggybac transposons ( some of which add 3 exons to genes that would be expressed transiently during mac development ) , tetrahymena iess are generally not flanked by ta dinucleotides and differ significantly from those of paramecium ; they are larger and are usually multicopy elements , their excision generates microheterogeneity at chromosomal junctions , and they are very rarely found within coding sequences [ 26 , 86 ] .
this suggests , therefore , that invasion of the paramecium germline genome by tc1/mariner transposons took place after the separation of the two ciliate lineages ( figure 7(a ) ) . this idea has been supported by an analysis of ies evolution in paramecium , which led to the conclusion that the majority of ta - iess appeared between the intermediate and recent wgds , that is after divergence of paramecium and tetrahymena . in paramecium ,
the ability of piggymac and the nhej pathway to carry out the precise excision of tc1/mariner - related elements from the mac may have allowed these transposons to spread throughout the germline genome without harmful consequences on gene expression ( figure 7(b ) ) .
thus , thanks to nuclear dimorphism and to the existence of a precise mechanism for transposon elimination from the somatic genome , paramecium , in contrast to other organisms , may have tolerated insertions within genes .
this raises the question of whether currently known iess are the relatively harmless remnants of ancient tc1/mariner invaders or whether they have acquired some useful function for the cell . as suggested earlier
, some of them may contribute to the structuration of mic chromosomes , for example , by providing centromere - related functions or ensuring the condensation of chromosomes .
iess may also have a regulatory role , if they carry sequences that can control transient gene expression specifically before they are removed from genes during genome rearrangements .
a recent study of ~10 million genes annotated in sequenced genomes from individual bacteria , archaea , eukaryotes , and viruses as well as in metagenomes , has pointed to the remarkable evolutionary success of transposase genes , which appear to be the most abundant , the most ubiquitous genes in nature .
this brought further support to the idea that transposons should not simply be considered as selfish or parasitic elements , but also as a source of novel and sometimes essential functions for their host . in mammalian genomes for instance ,
numerous transposase genes seem to have been domesticated , but , for the most part , their function has remained elusive .
the best documented example is the rag1 nuclease involved in v(d)j recombination , a process that generates the highly diverse repertoire of immunoglobulin genes in differentiating b and t lymphocytes ( reviewed in ) .
rag1 is clearly a domesticated transposase from the transib family , and its target sites within immunoglobulin genes , also called the recombination signal sequences , present significant sequence similarities with the tirs of transib transposable elements . as in paramecium ,
the nhej double - strand break repair pathway has been recruited in this system to join the coding ( and signal ) ends and assemble functional immunoglobulin genes . in v(d)j recombination , however , additional factors ( such as nucleases and a template - free dna polymerase ) contribute to the observed variability of the coding junctions .
in addition to being yet another example of a catalytically active domesticated transposase involved in programmed dna elimination during differentiation , piggymac in paramecium represents a novel variation on the theme of how a genome can cope with invasion by transposable elements . here , a domesticated piggybac transposase , the nhej pathway and epigenetic control by noncoding rnas orchestrate a highly precise and accurate system for the programmed elimination of transposon - related sequences from somatic chromosomes . as discussed in , recent observations have indicated that some iess in paramecium may carry promoters or parts of coding sequences , the excision of which would be regulated during the development of a new mac and could also be submitted to homology - dependent epigenetic control of the old mac . whether ies excision may have provided an additional layer of variability for the control of gene expression at the genome - wide scale is an attractive hypothesis that will need to be investigated . | sequences related to transposons constitute a large fraction of extant genomes , but insertions within coding sequences have generally not been tolerated during evolution .
thanks to their unique nuclear dimorphism and to their original mechanism of programmed dna elimination from their somatic nucleus ( macronucleus ) , ciliates are emerging model organisms for the study of the impact of transposable elements on genomes . the germline genome of the ciliate paramecium , located in its micronucleus , contains thousands of short intervening sequences , the iess , which interrupt 47% of genes .
recent data provided support to the hypothesis that an evolutionary link exists between paramecium iess and tc1/mariner transposons . during development of the macronucleus ,
iess are excised precisely thanks to the coordinated action of piggymac , a domesticated piggybac transposase , and of the nhej double - strand break repair pathway .
a piggymac homolog is also required for developmentally programmed dna elimination in another ciliate , tetrahymena . here , we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle .
we discuss how ancient domestication of a piggybac transposase might have allowed tc1/mariner elements to spread throughout the germline genome of paramecium , without strong counterselection against insertion within genes . |
primary sclerosing cholangitis ( psc ) and primary biliary cirrhosis ( pbc ) are both immune - mediated chronic cholestatic liver conditions .
psc , which is frequently seen in association with inflammatory bowel disease , is a chronic biliary disease which may affect both the intra- and extrahepatic biliary tree while in pbc the main damage is noted in the small and medium size intrahepatic bile ducts .
many patients affected with psc develop progressive biliary strictures , leading to recurrent cholangitis . both psc and pbc lead to hepatic and systemic accumulation of toxic biliary compounds , resulting in progressive liver damage . in response , several defense mechanisms are induced to prevent from liver injury .
these comprise equilibrium changes in hepatobiliary transporters , downregulation of uptake systems , and induction of enzymes catalyzing detoxification processes [ 48 ] .
the end readout of the complex interplay of these systems is prevention of and compensation for the deleterious accumulation of toxic bile acids . at the transcriptional level , the pivotal fine - tuning
role for the homeostasis of these adaptive mechanisms is mainly played by members of the nuclear receptor ( nr ) family .
the pregnane x receptor ( pxr ; nr1i2 ) is a ligand - activated member of the nuclear receptor superfamily of transcription factors , which is highly expressed in human liver and gastrointestinal tract .
it serves as a xenobiotic sensor which induces phase i ( catalyzing hydroxylation ) and phase ii ( catalyzing glucuronidation and sulfation ) metabolism of many endogenous and exogenous compounds including bile acids ( ba ) . the primary bile acids in man are cholic and chenodeoxycholic acid .
they are metabolized by enteric bacteria to produce the secondary bile acids deoxycholic and lithocholic , respectively .
lithocholic acid ( lca ) is extremely lipophilic , rapidly partitions into membranes , and has a high potential for toxicity .
hydroxylation and sulphation of lca greatly reduce its intestinal reabsorption , thus minimising its enterohepatic circulation and promoting its excretion in faeces .
in contrast to the other bile acids which act as fxr ligands , lca is a preferred ligand of pxr .
sonoda et al . have shown that lca in micromolar amounts is a powerful inducer of the sulphotransferase ( sult2a1 ) responsible for its sulphation only in the presence of the rxr : pxr heterodimer .
sulphation , a phase 2 activity , converts lca to a less toxic and more water - soluble form which is readily excreted in faeces .
we had previously reported low levels of lithocholic acid sulphation in chronic cholestatic liver disease but could not distinguish between a causal and consequential linkage .
we postulated that failure of the liver 's coordinated defense against lithocholic acid toxicity could be critically involved in the pathogenesis of psc .
recently , we reported that concentrations of plasma lithocholic acid sulphate were significantly reduced in psc in comparison to patients with pbc and normal controls .
we postulated that an increased expression of sult 2a1 , in response to pxr activation , would be anticipated to occur as a hepatoprotective response to injurious cholestasis in conditions such as psc and possibly pbc . to test our hypothesis
, we analyzed the levels of pxr and its target gene sult2a1 in psc and pbc patients with comparable degrees of clinical cholestasis and sought out for potentially significant differences amongst psc and pbc in this regard .
liver tissue specimens were collected from explanted livers of patients with psc ( n = 11 ) and pbc ( n = 19 ) who underwent liver transplantation . control liver tissues ( n = 19 ) were obtained from large margin liver resections of colorectal metastases with no microscopic changes of liver disease identified by a pathologist . table 1 summarizes clinical and laboratory features of patients included in the analysis of liver expression of pxr and sult2a1 .
intestinal tissues were obtained from a group of 22 patients with psc who underwent their routine colonoscopies .
eleven patients ( 8 males , 3 females ; mean age 35 17 ) had macroscopic features of ulcerative colitis ( uc ) , called psc + uc group , and 11 ( 9 males , 2 females ; mean age 30 9 ) had never been diagnosed with inflammatory bowel disease ( called psc group ) .
all patients with psc were treated with ursodeoxycholic acid with an average dose of 15 mg / kg . b.w .
and patients with uc additionally received 5asa ( 2 - 3 g / daily ) .
specimens were collected from ileum and ascending and sigmoid colon . for this part of the study ,
the control group comprised 14 ( 8 males , 6 females ; mean age 50 16 ) subjects who underwent their colonoscopies for various indications and who were found to have no macroscopic changes in their colons .
then , biopsies were processed for future analyses , that is , either ( i ) stored in rnalater for analysis of mrna expression ( am7021 ; applied biosystems , carlsbad , ca , usa ) , ( ii ) fixed in neutral - buffered formalin for histological assessment , or ( iii ) immediately frozen in liquid nitrogen for proteomic analyses .
histology was assessed by a pathologist ( eu ) who was blinded to clinical diagnoses of analyzed patients , according to histological grading scale introduced by geboes et al . .
briefly , according to this score , 6 histological features are assessed ; these include ( i ) architectural changes ; ( ii ) chronic inflammatory infiltrate ; ( iii ) lamina propria neutrophils and eosinophils ; ( iv ) neutrophils in epithelium ; ( v ) crypt destruction ; ( vi ) erosion or ulceration .
the research protocol was approved by the ethics committee of pomeranian medical university and conformed to the ethical guidelines of the 1975 declaration of helsinki .
total rna were isolated using the rneasy mini kit ( qiagen , valencia , usa ) , according to the manufacturer 's protocol .
cdna synthesis was carried out using superscript ii rt kit ( invitrogen , carlsbad , ca , usa ) according to the protocol previously described and stored at 20c .
the expression of specific target genes was measured by quantitative real - time pcr using commercially available gene expression assays and 7500 fast real - time pcr system ( applied biosystems ) .
the following assays were used in the study : pxr ( hs01114267_m1 ) ; sult2a1 ( hs00234219_m1 ) ; and control human gapdh ( hs99999905_m1 ) .
a 20 l reaction mixture contained 10 l of taqman gene expression pcr master mix ( applied biosystems , foster city , ca , usa ) , 2 l diluted cdna template , and 1 l of the probe / primer assay mix .
( applied biosystems , carlsbad , ca , usa ) . the expression of target genes was calculated using the ct method of relative quantification .
proteins from frozen liver and intestine tissue were extracted through homogenization in an ice - cold ripa buffer ( 50 mm tris - hcl ph = 8 , 150 mm nacl , 1% np-40 , 0,5% nadoc , 0,1% sds , 1 mm edta , 100 mm pmsf , 100 mm naf ) containing protease inhibitor cocktail and phosstop ( roche diagnostics gmbh , mannheim , germany ) .
protein quantification was made using the bicinchoninic acid assay ( micro bca protein assay kit ; thermo scientific , waltham , ma , usa ) .
forty g of protein extracts from each liver sample was electrophoresed in sds polyacrylamide gels and subsequently blotted into pvdf membranes ( thermo scientific , rockford , il , usa ) under semidry transfer conditions .
membranes were blocked overnight at 4c with tbst containing 5% ( w / v ) milk ( merck ) and then probed using the following primary antibodies : pxr ( sc-48403 ; santa cruz , 1 : 500 ) , sult2a1 ( sc-8002 santa cruz , 1 : 200 ) , and anti-/-tubulin ( 2148 , cell signaling , 1 : 1000 ) . for the detection of antigen - antibody complexes
, peroxidase conjugated anti - rabbit secondary antibody ( na9340v , amersham , ge healthcare , uk ; 1 : 5000 dilution ) or anti - mouse secondary antibody ( na9310v , amersham ; 1 : 5000 dilution ) was used .
protein expression was detected using an enhanced chemiluminescence detection system ( chemiluminescent hrp substrate , millipore , billerica , ma , usa ) .
bands were visualized and quantified using microchemi 2.0 system and gelquant software ( israel ) .
[ a / g ] and rs112433193 [ c / g ] ) , located near / within the pxr binding site within promoter region of sult2a1 gene , were analyzed in genomic dna isolated from peripheral blood mononuclear cells of 151 psc patients ( 109 males , 42 females ; mean age 32 13 ) , ( dneasy blood & tissue kit , qiagen ) .
pcr reactions contained 20 ng dna , 900 nm of each primer , 12.5 l of taqman universal master mix , and 200 nm of vic - labelled and fam - labelled probes in 25 l - reactions .
amplification conditions were as follows : 95c for 10 min , 40 cycles of 92c for 15 s , and 60c for 1 min .
oligonucleotide primers and taqman probes for the sult2a1 polymorphisms were designed and synthesized by applied biosystems .
additionally , for the purpose of detecting a dna sequence of sult2a1 promoter region within the pxr binding sites represented by ir2 and dr4 motifs , the pcr reaction was performed in samples of genomic dna of 151 pscs patients with the use of the following primers : fw 5-gcacgattgcaggattatttag-3 ; rv 5agaaatcgtccgacatgatgat-3. the amplified dna ( 436 pb ) was purified with extractme dna gel out kit ( dna , gdansk , poland ) followed by sequencing in the laboratory of dna sequencing and oligonucleotide synthesis ( oligo.pl ; institute of biochemistry and biophysics , polish academy of sciences ) . the location of primers used in the sequencing and pxr binding site within the promoter region of sult2a1 gene is showed in figure 1 .
total rna from liver samples explanted from patients diagnosed with psc ( n = 4 ) , pbc ( n = 4 ) and aged and gender - matched control donors for each disease ( n = 4 per experimental group ) was isolated with the use of mirneasy mini kit ( qiagen ) .
microarray analysis comprised affymetrix genechip mirna 4.0 arrays and was performed by microarray core boston university ( http://www.bumc.bu.edu/microarray/ ) .
data were analyzed using stat - view-5 software ( sas institute , cary , nc , us ) and included fisher 's exact and anova analysis .
in cirrhotic liver tissues expression of pxr mrna was considerably enhanced in both psc ( 1.6-fold , p = 0,04 ) and pbc ( 3-fold increase , p < 0.0001 versus controls ; figure 2(a ) ) .
expression of pxr mrna did not correlate with biochemical features of cholestasis ( data not shown ) .
similarly , protein level of pxr was significantly augmented in psc and pbc compared to control tissues ( 2.7 0.3 versus 1.2 0.2 , p = 0.0003 and 3.1 0.5 versus 1.1 0.1 , p = 0.0005 resp . ;
the increase in pxr mrna and protein levels was significantly less pronounced in psc than pbc patients ( p = 0.002 and p = 0.0001 versus pbc , for pxr mrna and protein levels , resp . ; figures 2(a ) and 2(b ) ) . in pbc , the enhanced pxr expression was accompanied by the increased expression of sult2a1 mrna ( 4-fold , p = 0.0003 versus controls ; figure 2(c ) ) and sult2a1 protein level ( 1.9-fold , p = 0.0003 versus controls ; figure 2(d ) ) .
such changes were not observed in livers from patients with psc ( figures 2(c ) and 2(d ) ) .
data on histological findings in colons in patients with psc and controls are summarized in table 2 .
ileal tissues were not assessed by the pathologist as they are not included in the scoring system applied in this study .
expression of pxr mrna was similar in patients with psc and controls regardless of the examined part of colon .
the level of sult2a1 mrna was significantly lower in the ileum of patients : 0.37 0.1 in psc without uc and 0.41 0.1 in psc with uc versus 1.01 0.2 in controls , p = 0.02 and p = 0.03 , respectively .
the genotyping analysis has shown that the examined snps , that is , rs11569683 and rs112433193 , are not present among psc patients .
furthermore , since earlier studies established that ir2 and dr4 motifs within promoter region of human sult2a1 are involved in the pxr - induced activity of the this gene , the detailed analysis of the nucleotide sequence of the promoter region of sult2a1 gene containing the pxr binding sites was carried out ( figure 1 ) .
the genomic analysis did not identify any changes within the examined region ( data not shown ) .
a substantial increase in the level of microrna mir-378a-5p in liver tissue of psc patients ( 3,6-fold change ; p = 0.0047 versus pbc ) was seen .
in the present study , we looked at the expression of pregnane - x - receptor ( pxr ) and sulphotransferase 2a1 ( sult2a1 ) in the livers of patients with psc and pbc .
we documented , for the first time , in vivo evidence of increased pxr expression in these conditions .
we also found that , contrary to pbc , pxr activation is not accompanied by an enhanced expression of sult2a1 , suggesting a disease - specific impairment of sult2a1 expression in psc .
also , mirna analysis suggested that sult2a1 expression in psc is likely regulated by mir-378a-5p , further indicating a pathogenic role for this mir in psc .
pxr is a key member of the nrs family of ligand - modulated transcription factors .
it binds ( as a heterodimer with rxr ) to response elements in the promoter region of target genes involved in stimulation of the bile acid detoxification machinery .
although there is a wide evidence for nr interaction pathways in cholestatic conditions , most data derive from in vitro and animal studies in experimentally induced cholestasis .
sult2a1 is a target gene for pxr and plays an important role in pxr - mediated detoxification .
a comprehensive analyses of the ba profile in serum from cholestatic patients has shown significant reduction of the amount of sulfated lca present in patients with psc and reduced lca sulfotransferase activity was detected in human livers with psc [ 10 , 17 ] .
such data are in line with the diminished potential of sult2a1 expression following pxr activation noted in psc .
our study showed that pxr mrna and protein expression were significantly enhanced in psc and pbc livers . to our knowledge , in vivo evidence of increased expression of pxr
this finding is in agreement with data obtained in experimental cholestatic injury induced in animals , as well as in vitro observations .
these cumulative data confirm that in the course of cholestatic processes the expression of ba - activated pxr protein increases in order to protect against ba toxicity .
pxr was reported to be involved in pathogenesis of icp [ 18 , 19 ] and psc as well as in adaptation to cholestatic liver diseases , for example , in obstructive cholestasis . with regard to pbc
, who have shown a repression of cyp7a1 mrna and elevation of mrp4 protein level in patients with pbc compared to controls
. however , that study has found that expressions of nrs including pxr were not significantly changed .
although , in response to cholestatic insult , pxr expression was significantly increased in both conditions , this increase was markedly less effective in our patients with psc .
treatment of human intestinal caco-2 cells with activators of pxr leads to the induction of sult2a1 in a hepatocyte nuclear factor 4- ( hnf4- )
stimulation of pxr expression reduced liver injury triggered by lca administration in a sult2a1-dependent manner .
radominska et al . demonstrated that sult2a1 is the only enzyme responsible for bile - acid sulphation in the human liver [ 21 , 22 ] . in human hepatocytes
thus , in normal conditions , bile acid sulphation is a very efficient elimination process in humans , and lca , the most hydrophobic and toxic bile acid , is rapidly sulphated already on the first pass through the liver [ 25 , 26 ] . taking into account the biological importance of sult2a1 in protecting against the dysregulation of homeostasis caused by cholestasis , we considered that it is worthy to study its expression . in our study , the overexpression of pxr was accompanied by elevation of sult2a1 mrna and protein levels in patients with pbc , but not in psc . in the latter group of patients , mrna and protein levels were unchanged in comparison to controls samples , though an induction of pxr proteins was observed in this group .
this observation may suggest that , in psc , the positive regulation pathway between pxr and sult2a1 is disturbed , resulting in an impairment of sulphation capacity .
this finding is in line with our previous observations demonstrating a reduction of sulphation capacity in psc but not in pbc and controls .
lcms / ms analysis of the concentrations of lca and its sult2a1 metabolite - lca - s in patients with psc , pbc and controls clearly demonstrated a decrease of the sulphation potential of lca in psc with a metabolic lca - s / lca ratio reduction noted in psc when compared to both pbc patients and controls .
lca - s / lca ratio was 5.01 in psc as compared to 1.94 in pbc and 1.75 in controls .
analysis of human livers from various chronic liver diseases such as pbc , psc , autoimmune hepatitis , and alcoholic cirrhosis ( but not in cryptogenic cirrhosis ) has shown that sult2a1 activity and concentration are significantly reduced when compared to normal livers . however
as already mentioned , the interplay between pxr and hnf4alpha has been shown to be of importance in regulation of sult2a1 gene expression .
regardless of the enhanced expression of pxr , the transcript level of its target gene , that is , sult2a1 , was not changed in the livers of psc patients .
therefore , we decided to perform the genomic analysis of sult2a1 promoter in order to find out whether the alteration in nucleotide sequence may be accountable for the lack of adequate level of this detoxification enzyme .
we hypothesized that a possible change in dna sequence within sult2a1 promoter region , more precisely in the proximity of hnf4alpha and pxr binding sites , could explain the lack of the increased expression of sult2a1 gene in psc disease .
however , our hypothesis proved to be wrong , as neither snp genotyping assessments nor a detailed sequencing of sult2a1 promoter have provided evidence in support of our assumption . within the last decade , microrna ( mirna ) have emerged as a new class of small molecules that control intracellular gene expression at a posttranscriptional level .
increasing body of evidences confirms the fundamental role of mirna in the physiological and pathological processes in the liver .
our study provide a new insight into sult2a1-specific expression patterns that can be modulated by mir-378a-5p in psc patients .
the substantially increased expression of mir-378a-5p in psc liver may be responsible for the observed lower level of sult2a1 protein , as the identified microrna was predicted to be involved in the regulation of sult2a1 gene expression .
however further analysis is needed to understand the role of mir-378a-5p in cholestatic liver diseases like psc . in this study
we have also analyzed expression of both pxr and sult2a1 mrna in the intestine of patients with psc . to our knowledge ,
expression of pxr in the intestinal tissue has not been analyzed so far in psc .
the tendency of colitis to primarily involve the caecum and right hemicolon in psc patients is in contradistinction to non - psc related ulcerative colitis in which the disease is always distal and supports the hypothesis that elements of the enterohepatic circulation are implicated in the pathogenesis of psc - associated colitis .
we found a significantly decreased expression of sult2a1 mrna but not pxr in the ileum of patients with psc .
this finding further supports the notion of impaired sult2a1 function in psc . on the other hand
, we did not see any difference between analyzed groups in terms of expression of pxr and sult2a1 mrna in the colon .
as expected , patients with psc with concomitant uc had significantly more pronounced inflammatory features on their histology ; however , this did not appear to affect either pxr or sult2a1 mrna .
as already mentioned , there is no study in the literature dealing specifically with this issue .
thus , this finding requires further investigation and its interpretation is difficult at this point .
in conclusion , our results indicate disease - specificity of intrinsic pxr - coordinated hepatoprotective mechanism against ba toxicity .
in contrast to pbc , psc patients show an impaired signaling between pxr and sult2a1 .
the observed increase in liver mir-378a-5p level , a negative posttranscriptional modulator of sult2a1 gene , could contribute to the pathogenic processes seen in this condition . since most of the accessible research in this area was undertaken in a rodent model , which does not translate directly to humans , our data on pxr and sult2a1 expressions in humans are novel and may have a future translational clinical repercussion .
more research is needed to understand the enigmatic role of sult2a1 in the development of liver disease [ 29 , 30 ] . | background / aim .
sulphotransferase 2a1 ( sult2a1 ) exerts hepatoprotective effects .
transcription of sult2a1 gene is induced by pregnane - x - receptor ( pxr ) and can be repressed by mir-378a-5p .
we studied the pxr / sult2a1 axis in chronic cholestatic conditions : primary sclerosing cholangitis ( psc ) and primary biliary cirrhosis ( pbc ) .
materials / methods . western - blot / pcrs for sult2a1/pxr were performed in psc ( n = 11 ) , pbc ( n = 19 ) , and control liver tissues ( n = 19 ) .
pxr and sult2a1 mrna was analyzed in intestinal tissues from 22 psc patients .
genomic dna was isolated from blood of psc patients ( n = 120 ) and an equal number of healthy volunteers .
liver mirna expression was evaluated using affymetrix - gene - chip mirna4.0 .
results .
increased pxr protein was observed in both psc and pbc compared to controls and was accompanied by a significant increase of sult2a1 in pbc but not in psc . decreased expression of sult2a1 mrna
was also seen in ileum of patients with psc . unlike pbc
, mirna analysis in psc has shown a substantial increase in liver mir-378a-5p .
conclusions .
psc is characterized by disease - specific impairment of sult2a1 expression following pxr activation , a phenomenon which is not noted in pbc , and may account for the impaired hepatoprotection in psc .
mirna analysis suggests that sult2a1 expression in psc may be regulated by mir-378a-5p , connoting its pathogenic role . |
it is estimated that , globally , 466,000 new cases of cervical cancer are diagnosed
each year . as per estimate 231,000 women in a year
die of cervical cancer and 80% of them are from the developing world .
use of cytological screening technique , papanicolaou ( pap ) test , has significantly increased the detection of cervical
cancer .
cancer of cervix emerges from a defined series of preneoplastic
lesions with increasing cellular dysplasia , referred to as cervical
intraepithelial neoplasia ( cin ) grade i , ii , and iii
. however , cin lesions
frequently regress , and not all cin iii lesions , progress to invasive cancer
.
persistent infection with high - risk - type human papilloma viruses ( hpvs ) is the strongest independent
risk factor for cervical cancer .
cin lesions induced by hpv infection and
genomes of these viruses were detected in the vast majority of cervical cancer
specimens [ 57 ] . during the course of tumor initiation in an hpv - infected cervical epithelial cell , an aneuploid dna pattern develops ,
which along with its descendants shows genetic instability .
aneuploidy reflects
a situation of uncontrolled increase of dna and loss of essential information
and plays an important role in neoplastic transformation .
increased
aneuploid dna value with the increase in grades of cervical dysplasia has long
been considered to be a specific prognostic marker of malignancy .
however ,
at present , no well - established technique , which , qualitatively , predicts the
clinical outcome of cervical dysplasia except pap smear screening , is
available .
approximately 1530% of all women who developed low - grade squamous intraepithelial lesion ( lsil ) have a
probability of developing moderate - to - severe cervical intraepithelial neoplasia
( cin ii and iii ) identified on a subsequent cervical biopsy . to prevent
cervical malignancy and its precursors ,
several adjuvant diagnostic methods
have been proposed to strengthen the accuracy of cytological and histological
diagnosis . as chromosomal
aneuploidy has been used as an early key event
in tumorigenesis caused by genetic instability [ 13 , 14 ] , the cytometric
equivalence of chromosomal aneuploidy detected by dna flow - cytometry may serve
as a marker of neoplasia and provide valuable information for the diagnosis and
understanding of pathogenesis of cervical cancer . since aneuploid pattern of
dna is a useful parameter for the prediction of progression and persistence of atypical squamous cells of
unknown significance ( ascus ) ,
low - grade squamous intraepithelial
lesion ( lsil / mild dysplasia / cin i ) , and high - grade squamous intraepithelial
lesion ( hsil / moderate and severe
dysplasia / cin ii and iii ) in clinical
practice , the present study was designed to investigate whether
alteration in cellular / nuclear content predicts the occurrence of cancer in
cervical smears .
we have thus estimated their dna content using flow cytometry
and compared it with the cytological findings .
the cases were derived from the ongoing outpatient cytology screening at department
obstetrics and gynecology , king george 's medical university ( lucknow , india ) for
the present study .
cytologically diagnosed 127 cases of dysplasia ( 79 mild , 36
moderate , and 12 severe ) , 57 cases of
atypical
squamous cells of unknown significance ( ascus ) and 69 controls ,
which enrolled during the period of 20042006 , were recruited from a
prospective followup study to understand the biological behavior and the
natural history of cervical precancerous lesions .
none of the patients had any previous history of cin or had been treated
for cin .
the blunt end of the second spatula was
transferred into a cryo tube immediately , containing 3 ml chilled pbs ( 7.2 ph )
and quick frozen at 20c until further use for flow - cytometric
analysis .
precancerous lesions of the uterine cervix were classified as normal ,
ascus , lsil ( mild dysplasia / cin
grade i ) , and hsil ( moderate dysplasia / cin grade
ii , severe dysplasia / cin grade iii and carcinoma in situ / cis ) . to understand the biological behavior
of cervical precancerous lesions , the following criteria were followed.the cases initially diagnosed as lsil ( cin i ) and hsil
( cin ii and iii ) were made to undergo a second pap smear test
after 30 days.cytological confirmed dysplasia
cases were enrolled for the followup with the consent of the patients .
cases initially diagnosed as cin
iii were subjected to colposcopy directed biopsy on day 30 and were directed to
gynecologist for further management .
a cytological control examination was done
after every 6 and 3 months for lsil and hsil subjects , respectively , and were
categorized as returned to normal ( after the morphology normalized ) ;
progressed ( when presented with higher grade of dysplasia ) ; and with no change
( when remained at the same grade of dysplasia ) .
the samples were also tested
for the dna ploidy pattern.ascus and all aneuploid cases from control group were also followed for a
period of one year .
the cases initially diagnosed as lsil ( cin i ) and hsil
( cin ii and iii ) were made to undergo a second pap smear test after 30 days .
cytological confirmed dysplasia
cases were enrolled for the followup with the consent of the patients .
cases initially diagnosed as cin
iii were subjected to colposcopy directed biopsy on day 30 and were directed to
gynecologist for further management .
a cytological control examination was done
after every 6 and 3 months for lsil and hsil subjects , respectively , and were
categorized as returned to normal ( after the morphology normalized ) ;
progressed ( when presented with higher grade of dysplasia ) ; and with no change
( when remained at the same grade of dysplasia ) .
ascus and all aneuploid cases from control group were also followed for a
period of one year . to determine the cellular dna content , the protocol of nicoletti
et al .
was followed . in brief , cells were fixed with chilled ethanol for
30 minutes at 4c temperature . after adding propidium
iodide ( sigma - aldrich , miss , usa ) , the mixture was stored in dark for 1 hour at
room temperature .
dna histogram cell - cycle analysis was performed as
described by rabinovitch , using modfit lt for mac v2.0
software
after excluding debris by using electronic gate .
the events in g0-g1 , s , and g2-m
phases of the cell cycle were counted .
cases were regarded as acceptable for
analysis if the cv of the g0-g1 peak was 7.0 .
if two distinct g0-g1 peaks were present
with a dna index of 1.15 ( each containing 10% of
total cell population ) , the histogram was considered as aneuploid ( dna
content ) . by convention ,
a histogram was
classified as tetraploid only if a large peak observed in g2-m ( dna index of
1.802.2 ) , containing 20% of
the total curve and was also associated with a corresponding 8 n peak .
epi - info software was used for
calculating the odd ratio and ( chi - square ) values .
a p - value
of .05 or less was
considered statistically significant ( 95% ci ) .
the third and fourth groups comprised of lsil
( 79 cases ) and hsil ( 48 cases ; 36 cases in moderate dysplasia and 12 cases in
severe dysplasia ) .
a comparison of dna content analysis
with behavior of lsil or hsil in
patients having cytologically diagnosed various grades of dysplasia along with
the control and ascus groups were made ( table 1 ) .
127 cases of different grades
of dysplasia were employed for analysis of dna ploidy out of which 78 ( 62.99% )
cases were aneuploid ( 39 mild , 28 moderate , and 11 cases of severe dysplasia )
and remaining 49 ( 37.01% ) cases ( 40 mild , 8 moderate , and 1 severe ) were nonaneuploid ,
that is , diploid ( see figure 2 ) . in the control and ascus groups ,
6 ( 8.70% ) and
8 ( 14.04% ) cases were found to be aneuploid , respectively ( see figure 2 ) .
the
association of aneuploidy with increasing grades of dysplasia was found to be
significant ( p .001 ;
table 1 , see figure 3 ) .
all these cases with aneuploid dna
content and cytologically diagnosed ascus and various grades of dysplasia were
followed up for 1 year at aforementioned intervals , in order to study the
behavior of aneuploid dna in progression / regression / persistence of disease .
among 57 patients with ascus , 49 ( 85.96% ) diploid cases had smears that returned
to normal cytology and remaining 8 ( 14.04% ) aneuploid cases showed signs of
progression / regression .
one patient presented with hsil and three with lsil
after one year period of followup .
remaining four aneuploid cases of ascus
group regressed to normal , as evident from diploid dna .
out of six aneuploid
cases with normal cytological diagnosis , 4 cases were followed , where only one
case represented progression of disease from normal to mild dysplasia after a
year .
a total of 79 patients who had diagnosis
of lsil , 75 cases were followed up .
out of 39 aneuploid cases , 7 developed
moderate dysplasia ( cin ii ) and 30 cases presented with no change in cytology
( cin i ) , after a period of six months from the date of registration .
35 cases who had lsil with nonaneuploid
dna returned to normal and one patient presented with no change in cytology , that
is , remained at cin i. the sample for this particular subject showed aneuploid
dna content of cell in followup study while 35 normal cases were diploid ( table 2 ) . out of 36 cases of moderate dysplasia ,
20 patients out of 25 who were
diagnosed with moderate dysplasia ( hsil / cin ii ) and with aneuploid dna
remained cin ii ( 71.79% ) with an aneuploid dna in their followup study after
three months of registration .
in addition , 2 other patients were diagnosed
later with cis , who were positive for aneuploid dna in their followup .
1 patient
with diploid dna remained at cin ii in cytological followup and other 4 diploid
showed a regressive pattern . out of 11 only 3 aneuploid cases of severe
dysplasia ( cin iii ) were followed , rest underwent prescribed treatment .
of the 3
cases , which were aneuploid , 1 case progressed to cis and 2 remained at cin
iii .
the association of aneuploidy with progression of disease was found to be
significant ( table 2 ) .
test value for lsil
( p .001 ) and hsil ( p .001 ) was significant .
there is evidence that cervical
carcinoma evolves on the basis of persistence of aneuploidy .
studies have
shown that aneuploid conditions indicate either invasive carcinoma or
prospective neoplastic development in cervical dysplasia [ 18 , 19 ] and play a
causal role in neoplastic development [ 20 , 21 ] .
genetic abnormalities , such as numerical chromosomal aberrations ( aneuploidy and
tetraploidy ) , have been reported in women diagnosed with precancerous and
cancerous cervical lesions and are observed frequently in the presence of hpv
infections [ 10 , 22 , 23 ] . according to melsheimer et al .
, deregulated viral
oncogene expression appears to result first in chromosomal instability and
aneuploidization and is subsequently followed by integration of hpv genomes in
the affected cell clones . in cin and
invasive cervical cancer , dna ploidy assessment by flow cytometric technique has
been established as a prognostic factor that allows the estimation of the
relative progressive risk into more advanced lesions [ 8 , 2427 ] .
dysplastic
epithelium represents potentially precancerous conditions in which the risk of
progression to cancer is usually unknown .
bocking and motherby suggested
that the identification of dna aneuploidy in dysplastic squamous epithelia
could increase the predictive value for malignant transformation to over 90% .
similarly , the results of our study have also confirmed the predictive value of
aneuploidy with 92% aneuploid cases in all grades of dysplasia showing
progressive pattern of disease ( table 2 ) .
evidences indicate aberrant dna content
in cells as a cause rather than as a consequence of malignant transformation [ 29 , 30 ] .
duesberg et al . pointed out that aneuploid cancers
are generally considered to be more dangerous and aggressive than the diploid
.
. carried out a prospective study to determine the
progression to malignancy of cervical smears with ascus .
researchers concluded that the
aneuploid lesions should immediately be controlled histologically or be removed . in
accordance with the above findings
, the results of the present study also
demonstrate the progressive pattern of disease among aneuploid cases from ascus
group .
suggested that ascus cases , those with high - risk hpv and
with abnormally high dna content , represent similar biologic features as hsil
with elevated risk to develop cancer .
thus , dna content may reflect
genomic instability of the cells very early in the malignant process , at a
point where morphological changes in malignancies or premalignancies are not
yet evident .
conceivably , lesions that are defined as nondysplastic , and
therefore considered to represent minimal or no risk for developing cancer
cervix , may contain readily detectable large - scale genomic alteration viz dna
aneuploidy which could signal a malignant transformation [ 34 , 35 ] .
the
identification of a molecular biomarker of persistent hpv infection in women
who had been diagnosed as normal / ascus in pap smear could be a valuable tool in
the proper management of this disease .
we have also found 6 aneuploid cases
from control group and among them , after one - year duration , one case presented
the progression of disease . in this study , we have shown that
aneuploid dna content could be used as a biomarker of progressive cervical
changes towards malignancy . progressive
increase in aneuploidy increases the severity of the lesions ( 49.36% for mild
dysplasia , 77.77% for moderate dysplasia versus 91.66% for severe dysplasia ) .
our followup results clearly demonstrate that more and more progressive
pattern of disease was noted in aneuploid cases and that dna ploidy could be
used as a prognostic marker for cervical malignancies .
comparison of results obtained with pap screening and dna ploidy
measurement provide a positive correlation between aneuploid dna and
progression of disease ( table 2 ) .
furthermore , the percentage of aneuploid cases
towards the progression of disease was higher in followup data that supported
the prognostic value of aneuploidy as a marker to identify the progressive
behavior of lesion .
occurrence of aneuploidy was exceptional in patients
who had samples with morphology that returned to normal , whereas , patients who
had cytological signs of disease progression were found with a significantly
greater frequency ( p < .01 , p < .001 ; table 2 ) .
high - grade lesions ( according
to bethesda classification ) can be detected using cytodiagnosis method with
generally good accuracy , but the diagnosis occasionally relies on subjective
parameters .
moreover , inadequate sampling may result in a mild morphologic
change despite early progressive behavior .
thus , the measurement of dna content by flow cytometry provides qualitative
information and presence of aberrant aneuploid cells in cytological samples ,
which could serve as a useful parameter along with cytology with good
specificity and sensitivity [ 37 , 38 ] .
if patients at risk could be identified earlier
by analysis of parameter that reflects the true biological properties of
lesion , treatment regimes could be given selectively to improve the current
poor survival rate of cancer cervix cases .
flow cytometric analysis of dna
ploidy may provide a strategic diagnostic tool for early detection of carcinoma
cervix [ 8 , 25 ] .
therefore ,
we propose the concept of an hpv screening with reflex cytology in combination
with dna flow cytometry to detect progressive lesions with the greatest
possible sensitivity and specificity . | the majority of squamous cell carcinomas of cervix are preceded by visible changes in the cervix , most often detected by cervical smear . as cervical cancer is preceded by long precancerous stages , identification of the high - risk population through detection of dna ploidy may be of importance in effective management of this disease . here
we attempted to correlate aneuploid dna patterns and their influence on biological behavior of flow - cytometry analysis of dna ploidy which was carried out in cytologically diagnosed cases of mild ( 79 ) , moderate ( 36 ) , and severe ( 12 ) dysplasia , as well as
atypical squamous cells of unknown significance ( ascus ) ( 57 ) along with controls ( 69 ) , in order to understand its importance in malignant progression of disease .
cytologically diagnosed dysplasias , which were employed for dna ploidy studies , 39 mild , 28 moderate , and 11 severe dysplasia cases were found to be aneuploid .
out of the 69 control subjects , 6 cases showed aneuploidy pattern and the rest 63 subjects were diploid .
an aneuploidy pattern was observed in 8 out of 57 cases of cytologically evaluated ascus .
the results of the followup studies showed that aberrant dna content reliably predicts the occurrence of squamous cell carcinoma in cervical smear .
flow cytometric analysis of dna ploidy may provide a strategic diagnostic tool for early detection of carcinoma cervix .
therefore , it is a concept of an hpv screening with reflex cytology in combination with dna flow cytometry to detect progressive lesions with the greatest possible sensitivity and specificity . |
early life events contribute substantially to the likelihood of an individual becoming obese , although underlying mechanisms are not well understood .
obesity in women of reproductive age ( 15 to 44 years ) is increasing rapidly , and up to 50% of women in this age range in the usa are now either overweight or obese .
this has translated to an exponential increase in the prevalence of obesity during pregnancy with up to 20% of women entering pregnancy with a bmi which would define them as obese .
obesity in pregnancy increases the risks for complications of pregnancy including miscarriage , hypertension , and gestational diabetes [ 35 ] .
furthermore , it is now well established that maternal obesity leads to an increased risk of obesity and metabolic and cardiovascular disorders in offspring [ 69 ] . in view of the rising prevalence of obesity in pregnancy and its association with adverse maternal and offspring outcomes , there is a great deal of interest in understanding the mechanistic pathways that link maternal obesity and excess maternal nutrition to increased risk of disease in childhood and beyond [ 1013 ] .
clinical and experimental studies have consistently shown that maternal obesity predicts obesity , associated metabolic comorbidities , and reproductive disorders in adult offspring [ 2 , 1416 ] , often underpinned by a proinflammatory status .
however , there is a need to understand the relative contributions and interactions between maternal prepregnancy obesity and a gestational obesogenic environment on subsequent health and well - being of offspring .
we have previously shown that obesity in adult rat offspring arising as a consequence of maternal high fat ( hf ) nutrition was independent of the pre - conceptional maternal diet .
we have shown that offspring of mothers fed an hf diet restricted to pregnancy and lactation compared to those born to mothers fed a hf diet prior to conception and throughout pregnancy and lactation both exhibit a remarkably similar obesogenic phenotype , characterised by increased fat mass , hyperinsulinemia , and hyperleptinemia .
these data suggested that despite a common obesity phenotype , differential mechanisms based on the timing of maternal exposure to hf nutrition may underpin pancreatic adipoinsular axis dysfunction in these offspring [ 1719 ] .
we therefore investigated key components of the leptin - insulin ( adipoinsular ) and inflammatory signaling pathways in pancreatic tissue of offspring born to mothers fed a high fat diet during pregnancy in the presence or absence of pre - conceptional hf diet - induced obesity .
the rat model of maternal high fat nutrition has been described by our group previously [ 14 , 15 , 2024 ] . in brief ,
male and female wistar rats were acquired at a weaning age ( 22 days , vernon jansen unit , university of auckland ) and housed two per cage under standard conditions with a 12:12 light dark cycle and free access to water .
females were weight - matched and assigned to receive either standard rat chow ( n = 16 , 5% fat , diet 2018 , harlan teklad , oxon ) or a high fat ( hf ) diet ( n = 8 , 24% fat , d12451 , research diets , nj , usa ) to be fed ad libitum for the duration of the trial .
body weights were recorded every 3 days until postnatal day 120 . at postnatal day 110 , body composition in females was quantified using dual energy x - ray absorptiometry ( dexa , lunar prodigy , ge medical systems , madison , wi , usa ) . at postnatal day 120 , females were time - mated using an estrus cycle monitor ( model ec40 , fine science tools , ca , usa ) . upon confirmation of mating ,
three maternal dietary groups were established : ( 1 ) controls ( cont ) : females fed a standard chow diet throughout their life and maintained on a standard chow diet throughout pregnancy and lactation ; ( 2 ) maternal high fat diet ( mhf ) : females fed an hf diet throughout their life and maintained on the hf diet throughout pregnancy and lactation ; and ( 3 ) pregnancy + lactation hf diet ( plhf ) : females fed a standard chow diet until conception and then an hf diet throughout pregnancy and lactation .
all pregnant dams were weighed and had food intakes measured daily throughout pregnancy . following birth , pup weights and body lengths were recorded and litter size was randomly adjusted to 8 pups per litter to ensure standardized nutrition until weaning .
body weights and food intakes of dams were measured throughout the lactation period , and offspring were weighed every three days until weaning . at weaning ( d22 ) , offspring were housed 2 per cage ( 2 per litter / maternal background ) and fed a chow diet until the end of the trial . at postnatal day 150 , body composition was measured in male offspring ( n = 1018 per group ) by dexa . at postnatal day 160 , animals were fasted overnight and killed by decapitation following anaesthesia with sodium pentobarbitone ( 60 mg / kg , ip ) .
blood was collected into heparinized tubes , centrifuged and plasma supernatant stored for future analysis .
whole pancreata were removed and immediately snap - frozen in liquid nitrogen for later molecular analysis .
all animal experiments were approved under guidelines of the animal ethics committee at the university of auckland ( approval r652 ) .
fasting plasma leptin and insulin concentrations were analysed using commercial rat - specific elisas ( crystalchem 90040 and 90060 , resp . , downers grove , il , usa ) .
fasting whole blood glucose concentrations were measured using a glucose meter at the time of tissue collection ( d160 ) ( roche accuchek ) .
plasma tumor necrosis factor ( tnf- ) alpha , interleukin ( il- ) 1 and il-6 were measured using commercially available elisas ( quantikine kits , rta00 , rlb00 , and r6000b , resp . ,
the homeostatic model assessment ( homa ) method was used as a measure of insulin resistance ( ir ) and beta - cell function ( homa - ir = plasma glucose insulin/22.5 ) [ 25 , 26 ] .
briefly , whole pancreas tissue was disrupted using a homogenizer , and total rna was extracted using commercially available kits ( qiagen mini - prep ; catalogue number 80204 ) according to the manufacturer 's instructions . rna integrity and quality
were assessed using a nanodrop spectrophotometer ( nd-1000 ; biolab ltd . ) using nanodrop software ( version 3.1.2 ) where sample ratio a260/280 and a260/230 were ~2.0 , and intact bands were visualised by gel electrophoresis .
complimentary ( c ) dna was synthesised from rna ( 5 g ) by reverse transcription pcr using mmlv - rt ( invitrogen , ca , usa ) .
quantitative pcr assays were performed in 15 l reaction volumes using express sybr greener ( invitrogen ) , and fluorescence was measured and quantified using an abi-7900ht ver.2.3 sequence detection system ( applied bio systems , ca , usa ) .
the qpcr thermal profile for amplification of all genes was as follows : melting at 95c for 15 secs , followed by annealing / extending at 60c for 1 min , for 40 cycles .
primers were obtained either commercially or designed de novo ( table 1 ) using ncbi primer - blast .
a standard curve was generated using 6-fold serial 1 : 5 dilutions of pooled stock cdna .
all samples were assayed in triplicate and only those assays displaying a single pcr peak in the melt - curve analysis were used .
all qpcr results were normalized against the geometric mean of housekeeper genes cyclophilin and hprt , according to the genorm method of vandesompele et al . .
tnf , il-1r1 , and cd68 ( and respective controls hprt and cyclophilin ) were analysed using taqman gene expression assays . to control for between - sample variability , mrna levels were normalized to the geometric mean of the controls .
the ct for each treatment sample was compared to the mean ct for control samples using the relative quantification 2-(ct ) method to determine fold - change .
all data were analysed by analysis of variance ( anova ) with maternal diet as factor with post hoc testing to compare groups of interest as required ( holm - sidak ) .
a pre - conceptional hf diet ( mhf ) resulted in a significant increase in female body weight ( chow 270 5 g , mhf 310 8 , p < 0.05 ) and total fat mass compared to chow fed animals ( chow 17.8 0.7% , mhf 27.7 1.7 , p < 0.0001 ) . at the start of pregnancy , there were no differences in body weights between females randomly assigned to cont and plhf groups ( cont 270 8 g , plhf 269 7 ) .
total caloric intake was similar between cont and mhf groups but was significantly increased in plhf dams during the first 10 days of pregnancy ( average caloric intake : cont 0.23 0.004 kcal / gbw / day , mhf 0.23 0.004 , plhf 0.32 0.01 , p < 0.05 for plhf versus cont and mhf ) ; thereafter , caloric intake was similar to that of cont and mhf dams .
as reported previously , mhf and plhf pups had slightly but statistically significantly reduced body weights and were hypoinsulinemic and hypoleptinemic compared to cont offspring at birth .
mhf and plhf neonates displayed rapid catch - up growth and were heavier than cont neonates at the time of weaning ( day 22 : cont 61.6 0.7 g , mhf 70.5 0.9 , plhf 64.2 1.2 ) .
final adult body weights were increased in mhf and plhf male offspring compared to cont ( cont 562 9 g , mhf 635 18 , plhf 628 17 ) .
mhf and plhf adult male offspring had significantly increased total body fat mass at day 150 compared to cont ( figure 1 ) .
there were no differences in total fat mass between mhf and plhf adult offspring .
mhf and plhf adult male offspring were hyperleptinemic and hyperinsulinemic compared to cont ( figures 2(a ) and 2(b ) ) .
fasting blood glucose levels were similar between groups ( cont 5.6 0.2 mmol / l , mhf 5.7 0.3 , plhf 5.7 0.2 ) .
glucose : insulin ratios were significantly ( p < 0.001 ) reduced in mhf and plhf male offspring compared to control ( cont 2.5 0.2 , mhf 1.3 0.1 , plhf 1.2 0.1 ) and offspring exhibited increased insulin resistance using homa analysis ( figure 2(c ) ) .
plasma tnf- levels were not significantly different between the groups ( cont 3.6 0.9 pg / ml , mhf 6.7 1.9 , plhf 5.1 1.9 ) .
plasma il-1 levels were significantly increased in mhf ( p < 0.05 ) offspring compared to control ( cont 31.9 0.7 pg / ml , mhf 38.3 2.8 , plhf 36.1 2.2 ) but were not different between cont and plhf offspring .
mhf offspring showed significantly higher levels of pancreatic mrna levels of socs3 , obrb , and irs1 compared to cont and plhf groups ( figure 3 ) .
mrna levels of these genes were similar between cont and plhf offspring . despite these similarities in expression levels between cont and plhf offspring ,
significant positive correlations that existed between these and other measured genes were primarily found in cont and mhf offspring only and were not present in plhf offspring ( table 2 ) .
levels of mrna of irs2 , pdx-1 , and kir6.2 were significantly reduced in plhf offspring compared to mhf offspring but were not different between any other groups .
pancreatic ins1 and ins2 expression was significantly increased in mhf and plhf offspring compared to cont ( figure 3 ) .
stat3 expression was significantly increased in plhf offspring compared to cont and mhf offspring ( figure 4(a ) ) .
pi3k expression was significantly decreased in plhf compared to cont and mhf ( figure 4(b ) ) . of note , pi3k was positively correlated with irs1 and irs2 expression in plhf offspring ( r = 0.44 , p < 0.05 and r = 0.72 , p < 0.0005 , resp . ) but not in cont or mhf offspring . insulin receptor , leptin , obra , and phosphodiesterase 3b ( pde3b ) were similar between groups ( data not shown ) .
a highly significant relationship was observed in all offspring groups between pdx-1 and atp - sensitive potassium ( kir6.2 ) channel ( figure 5 ) . given the potential link between socs3 and inflammation , mrna levels of key proinflammatory cytokines
there was an increase in tnf- in plhf offspring compared to cont and mhf offspring ( figure 6(a ) ) .
the macrophage marker cd68 was increased in mhf and plhf offspring compared to cont ( figure 6(b ) ) .
there was an increase in il-1r1 mrna levels in mhf offspring compared to cont offspring ( figure 6(c ) ) .
there were no differences in il-6r , cardiotropin , il-1b , glut2 , gp130 , ctnf , lif , or tnf - r1 mrna expression between any of the offspring groups ( data not shown ) .
there is clear clinical and experimental evidence that maternal obesity predisposes to obesity and metabolic disorders in offspring [ 14 , 30 ] . however , despite the common phenotype of offspring obesity and hyperinsulinemia , the present study shows that at least in an animal model , that maternal nutritional history determines the pattern of mrna expression of key regulatory genes in the pancreas of adult offspring .
these data suggest that different regulatory pathways may lead to a similar metabolic phenotype and are in agreement with previous experimental studies suggesting that there may be separate influences of maternal obesity during the periconceptional period and late gestation on adiposity in offspring .
the present study further reinforces the importance of the timing of the dietary insult on the metabolic derangements that ensue in offspring during adulthood .
socs3 inhibition of obrb signaling has been proposed as a mechanism for leptin resistance in a range of tissues [ 3335 ] including pancreatic cells .
it is clear that leptin resistance in plhf pancreata was mediated by mechanisms that did not involve socs3 and this has been reported previously in rat models of hf feeding [ 37 , 38 ] .
socs3 is normally increased at sites of both acute and chronic inflammation and has been shown to specifically suppress stat3 activation .
this agrees with the present study ; a marked increase in socs3 mrna levels in mhf offspring is paralleled by stat3 levels similar to control offspring .
leptin is known to increase socs3 expression in isolated human pancreatic islets and in islets from ob / ob mice treated in vivo ; our data imply that leptin - induced socs3 stimulation appears intact in mhf offspring .
conversely , in plhf offspring , socs3 expression levels were similar to controls in the presence of markedly elevated stat3 expression .
of note , despite similar expression levels of socs3 between cont and plhf offspring , the positive correlations that were observed between socs3 and other key signaling genes were restricted mainly to cont and mhf offspring only .
this may point to a maladaptation in plhf offspring due to the acute hf dietary exposure and a change in homeostatic set - points in mhf offspring due to mothers entering pregnancy in an already obese state . despite the link between socs3 and inflammation
, there were no observed changes in tnf- mrna levels in the mhf group whilst there was a significant increase in tnf- expression in plhf offspring .
in addition , mrna levels of the macrophage marker cd68 were increased in both hf groups and taken together may indicate differing macrophage phenotypes / polarization states resulting in divergent offspring inflammatory profiles dependent upon prior maternal nutritional background . of note , despite marked changes in inflammatory markers at the tissue level , we observed no changes in circulating plasma tnf- concentration .
however , despite enhanced plasma il-1 concentrations pancreatic il-1 expression was unaltered between groups implicating other tissues as possible systemic sources of this proinflammatory cytokine .
induction of the obrb / pi3k signaling pathway prolongs potassium channel opening , leading to decreased insulin exocytosis , a direct action of leptin which inhibits insulin secretion .
moreover , the lowered pi3k mrna levels in plhf offspring were concomitant with a reduction in irs1 and irs2 mrna , whose phosphorylation is coupled to pi3k activation . of note ,
downstream of pi3k , pde3b gene expression levels remained similar to controls , suggesting that altered leptin signaling was achieved in earlier stages of this pathway .
thus , attenuation of the obrb / pi3k signaling pathway may mediate leptin resistance in these hyperleptinemic plhf offspring .
such a mechanism has been observed before , in hypothalamic neurons of rats with diet - induced obesity ( dio ) .
both are found downstream of the insulin receptor , in the ir / irs / pi3k pathway which mediates the autocrine actions of the insulin - ir complex [ 43 , 44 ] .
our observed attenuation of pi3k and irs mrna did not , however , affect insulin synthesis plhf animals were hyperinsulinemic , and preproinsulin mrna concentrations were elevated .
furthermore mrna concentrations of ir were unaffected potentially due to lack of socs3 induction in plhf offspring , similar to controls .
thus these mrna changes of pi3k coupled to irs likely reflect a mechanism of leptin resistance in this group .
mean concentrations of obrb mrna in plhf offspring were similar to controls , but a significant inverse relationship between plasma leptin and obrb mrna levels in the plhf group was suggestive of obrb downregulation .
a downregulation of receptor populations in the face of elevated ligand concentrations is not uncommon in resistant states .
it is seen , for example , in the hypothalamus in the presence of hyperleptinemia [ 4547 ] and during pregnancy , which is a natural state of leptin resistance .
a rise in irs1 and irs2 mrna associated with elevated obrb and socs3 mrna in mhf offspring may seem unexpected since socs3 is known to target insulin receptor signaling by interference with irs action .
however , several studies have demonstrated that socs3 mediates insulin resistance in both adipocytes and hepatocytes through post - translational modifications such as inhibition of irs tyrosine phosphorylation and targeted ubiquitin - mediated degradation of irs1 and irs2 without effecting transcription [ 49 , 51 ] .
irs proteins are involved in both leptin signaling and insulin signaling , so altered gene expression levels may represent changes in either ( or both ) pathways , although irs response to leptin is reportedly much lower than that seen with insulin .
this upregulation of irs1 expression may be related to the increased insulin production of the mhf offspring given the known role of irs1 in the regulation of insulin secretion .
socs3 is also reported to repress preproinsulin gene transcription , although other mechanisms clearly countered this action in mhf offspring , since ins1 and ins2 mrna expression levels remained elevated .
this observation was confirmed by the significantly raised levels of circulating plasma insulin and the lack of any correlation in levels of mrna between socs3 and ins1 or ins2 .
some of the observed differences may relate to the initial hyperphagia observed in the plhf maternal group as reported previously .
since rodents primarily eat on a calorific basis , they will passively over - consume calories when initially placed on an hf diet and self - regulate back to a normal total caloric intake over a period of a few days .
it has been shown in other rodent models that alterations in nutrition around the time of implantation can lead to lasting metabolic effects in the offspring .
maternal obesity has been shown to exert adverse effects as early as the oocyte and preimplantation embryo stage and that these effects may contribute to the lasting morbidity observed in offspring .
similarly , even a very short period of altered nutrition ( restricted to one ovulatory cycle ) prior to natural mating can elicit lasting effects on the offspring and this represents a further point of difference between the mhf and plhf groups .
the present study was not designed to incorporate the effect of a reversal paradigm whereby mothers fed the pre - conceptional hf diet were placed on a standard diet throughout pregnancy and lactation . however , while there is evidence that a period of dietary restriction in obese mothers may ablate the programming of obesity in offspring , it may be associated with an activation of the stress axis in the offspring .
further , the data are inconsistent ; it has also been shown that dams with diet - induced obesity , fed a standard diet throughout pregnancy and lactation , still transmit the propensity for obesity and metabolic disease to their offspring .
we recognize that gene expression analysis on whole pancreatic tissue is a limitation of the present study . due to the size of the cohort and logistical considerations ,
only whole pancreatic tissue was a possibility , whereas ideally only pure islet populations would have been examined .
however , for the key genes presented such as obrb , socs3 , and irs , expression has been shown to be primarily confined to the endocrine islets and not been detected in the exocrine pancreas [ 39 , 41 , 60 ] ; therefore we are confident that the observed changes in the genes studied represent changes in the islets .
further , although we have shown that differences in pancreatic gene expression in offspring are dependent upon the timing of maternal high fat diet exposure , this does not itself imply causation in the development of the obese phenotype other relevant pathways including liver , skeletal muscle , and fat metabolism will also be integrated into the programmed phenotype development .
this study has demonstrated enduring deleterious effects of a maternal hf diet during early windows of development .
maternal hf feeding both before conception and/or through pregnancy and lactation induced obesity , hyperinsulinemia , and hyperleptinemia in adult offspring , even though offspring were fed a standard diet postnatally .
timing of exposure to the maternal hf diet differentially altered gene expression in signaling pathways downstream of the pancreatic leptin receptor that mediated leptin resistance .
this also raises the possibility that despite common offspring obesity , treatment modalities may only be efficacious in a subpopulation of treated offspring as the current data suggest that the metabolic pathways disrupted will be dependent upon the timing of the early life dietary insult . | objective . offspring born to mothers either fed an obesogenic diet throughout their life or restricted to pregnancy and lactation demonstrate obesity , hyperinsulinemia , and hyperleptinemia , irrespective of their postweaning diet .
we examined whether timing of a maternal obesogenic diet results in differential regulation of pancreatic adipoinsular and inflammatory signaling pathways in offspring .
methods .
female wistar rats were randomized into 3 groups : ( 1 ) control ( cont ) : fed a control diet preconceptionally and during pregnancy and lactation ; ( 2 ) maternal high fat ( mhf ) : fed an hf diet throughout their life and during pregnancy and lactation ; ( 3 ) pregnancy and lactation hf ( plhf ) : fed a control diet throughout life until mating , then hf diet during pregnancy and lactation .
male offspring were fed the control diet postweaning .
plasma and pancreatic tissue were collected , and mrna concentrations of key factors regulating adipoinsular axis signaling were determined
. results .
mhf and plhf offspring exhibited increased adiposity and were hyperinsulinemic and hyperleptinemic compared to cont . despite a similar anthropometric phenotype , mhf and plhf offspring exhibited distinctly different expression for key pancreatic genes , dependent upon maternal preconceptional nutritional background .
conclusions .
these data suggest that despite using differential signaling pathways , obesity in offspring may be an adaptive outcome of early life exposure to hf during critical developmental windows . |
diabetes is a major threat to public health worldwide , and its prevalence has rapidly increased and even has influenced the younger population .
the world health organization estimates that about 366 million people worldwide will develop diabetes by 2030 , the majority of who develop type 2 diabetes ( t2d ) .
lifestyle negative changes such as obesity , weight gain , reduced physical activity , high - calorie diet and smoking contribute to the increased prevalence of the disease .
metformin is the most widely used antidiabetic medication among different medications administered to treat the disease , and it is considered as the first - line oral treatment for t2d . in general , the mechanisms of metformin action include reduction of hepatic glucose production , increased glucose uptake in skeletal tissue , suppression of the intestinal absorption of glucose , and improvement of insulin sensitivity .
metformin reduces triglyceride ( tg ) levels , free fatty acids , and low - density lipoprotein ( ldl ) in plasma and increases high - density lipoprotein ( hdl ) . due to slight hydrophobic property
a family of transporters called organic cation transporters ( octs ) is among transporters with an important role in bioavailability , clearance , and pharmacodynamics of metformin .
oct3 has a high - capacity for binding to the substrate and an extensive tissue distribution in the body , particularly in muscles and adipose tissue .
like two other members of oct family , oct3 is an excellent transporter for metformin .
various factors such as nature of the disease and organ function affect drug response , but estimates show that genetics can dictate 2095% of variability in different responses to the same medication .
oct3 - 564g > a is a noncoding variant in 3-untranslated region ( 3-utr ) of oct3 .
in other words , this variant contributes to the gene expression of oct3 and affects the final production of mrna .
so far , few studies have been conducted on the relationship between oct3 and metformin response .
therefore , similar studies on various variants of oct3 should be conducted in different ethnic populations .
hence , in this study , we evaluated whether the variant oct3 - 564g > a influences metformin response and also the distribution of genotypes of this variant in iranian patients with t2d .
this observational study conducted on 150 newly diagnosed patients with t2d ( according to the who criteria ) treated with metformin from march 2014 to august 2015 .
the patients were followed for 3 months and during this period , they received 1000 mg of metformin ( aria pharmaceutical company , iran ) twice a day .
information about medical history , personal habits , demographic parameters , and medication use was obtained through a questionnaire .
patients were divided into two groups based on the response to metformin : ( 1 ) responder group with a reduction in hba1c values by more than 1% compared to before taking metformin and ( 2 ) nonresponder group with a reduction in hba1c values < 1% compared to before taking metformin .
subjects with type 1 diabetes , and previous history of renal failure , chronic diseases , autoimmune , and liver diseases was not included in the study .
the study protocol was approved by the ethical committee at mazandaran university of medical sciences ( ethnic number : 93 - 1303 ) and informed consent was obtained from all participants .
blood samples were taken from subjects after an overnight fast ( for 10 - 12 h ) .
sera were isolated by low - speed centrifugation , and the serum aliquots were stored at 70c until use .
the hba1c levels were determined by boronate affinity technique ( axis - shield poc as , oslo , norway ; accuracy , failure < 5% ) .
hdl - cholesterol ( hdl - c ) , total cholesterol ( tc ) , triglycerids ( tgs ) , fasting blood glucose ( fbg ) , alanine aminotransferase ( alt ) levels were measured using an auto - analyzer ( prestige , japan )
. levels of ldl - cholesterol ( ldl - c ) were determined by the friedewald formula .
the oct3 - 564g > a variant was genotyped using the restricted fragment length polymorphism analysis after polymerase chain reaction amplification .
the designed primers used for this variant were : f 5'-agattgcatggaggatgaac-3 ' and r 5'-tgttacagaggaggtggacg-3 ' .
dna was amplified with an initial melting temperature of 93c for 3 min , followed by 35 cycles of 50 s at 93c , 40 s at 55c , and 40 s at 72c , with a final extension step of 5 min at 72c .
amplification products from each sample ( 171 bp ) were digested with the restriction enzyme acii ( thermoscientific ) at 37c for 16 h , and resulted in 67 and 104 bp fragments , which were subjected to electrophoresis on a 2.5% agarose gel .
wild - type ( gg ) patients were identified by the presence of 67 and 104 bp fragments .
heterozygous ( ga ) patients were identified by the presence of 171 , 104 , and 67 bp fragments , while the presence of an undigested fragment ( 171 bp ) was the basis for the identification of mutants ( aa ) .
smirnov test . to analyze the differences between the parametric variables , we used independent t - test or paired t - test .
statistical analyses were performed by spss ( version 16.0 ) software ( spss inc , chicago , il ) .
this observational study conducted on 150 newly diagnosed patients with t2d ( according to the who criteria ) treated with metformin from march 2014 to august 2015 .
the patients were followed for 3 months and during this period , they received 1000 mg of metformin ( aria pharmaceutical company , iran ) twice a day .
information about medical history , personal habits , demographic parameters , and medication use was obtained through a questionnaire .
patients were divided into two groups based on the response to metformin : ( 1 ) responder group with a reduction in hba1c values by more than 1% compared to before taking metformin and ( 2 ) nonresponder group with a reduction in hba1c values < 1% compared to before taking metformin .
subjects with type 1 diabetes , and previous history of renal failure , chronic diseases , autoimmune , and liver diseases was not included in the study .
the study protocol was approved by the ethical committee at mazandaran university of medical sciences ( ethnic number : 93 - 1303 ) and informed consent was obtained from all participants .
blood samples were taken from subjects after an overnight fast ( for 10 - 12 h ) .
sera were isolated by low - speed centrifugation , and the serum aliquots were stored at 70c until use .
the hba1c levels were determined by boronate affinity technique ( axis - shield poc as , oslo , norway ; accuracy , failure < 5% ) .
hdl - cholesterol ( hdl - c ) , total cholesterol ( tc ) , triglycerids ( tgs ) , fasting blood glucose ( fbg ) , alanine aminotransferase ( alt ) levels were measured using an auto - analyzer ( prestige , japan )
. levels of ldl - cholesterol ( ldl - c ) were determined by the friedewald formula .
the oct3 - 564g > a variant was genotyped using the restricted fragment length polymorphism analysis after polymerase chain reaction amplification .
the designed primers used for this variant were : f 5'-agattgcatggaggatgaac-3 ' and r 5'-tgttacagaggaggtggacg-3 ' .
dna was amplified with an initial melting temperature of 93c for 3 min , followed by 35 cycles of 50 s at 93c , 40 s at 55c , and 40 s at 72c , with a final extension step of 5 min at 72c .
amplification products from each sample ( 171 bp ) were digested with the restriction enzyme acii ( thermoscientific ) at 37c for 16 h , and resulted in 67 and 104 bp fragments , which were subjected to electrophoresis on a 2.5% agarose gel .
wild - type ( gg ) patients were identified by the presence of 67 and 104 bp fragments .
heterozygous ( ga ) patients were identified by the presence of 171 , 104 , and 67 bp fragments , while the presence of an undigested fragment ( 171 bp ) was the basis for the identification of mutants ( aa ) .
smirnov test . to analyze the differences between the parametric variables , we used independent t - test or paired t - test . mann
statistical analyses were performed by spss ( version 16.0 ) software ( spss inc , chicago , il ) .
as can be seen in the table , most of these parameters including systolic and diastolic blood pressure ( p < 0.001 ) , body mass index ( bmi ) ( p < 0.001 ) , fasting glucose ( p < 0.001 ) , and hba1c ( p < 0.001 ) were significantly reduced after 3 months of treatment .
compared with baseline levels before treatment , tg ( p < 0.001 ) , tc ( p = 0.001 ) , and ldl - c ( p
hdl - c levels were increased ( p = 0.101 ) after 3 months of treatment compared with baseline levels .
change in the study variables from baseline to 3 months of metformin treatment ( n=150 ) oct3 - 564g > a genotypes were in hardy weinberg equilibrium in the study patients .
the frequency of gg , ag , and aa genotypes were 51.3% , 36% , and 12.7% , respectively .
the allele frequency of g and a was found to be 0.69 and 0.31 , respectively . as shown in table 2 , the majority of the studied parameters such as hba1c , fasting glucose , bmi , and lipid profile in both gg genotypes and ga + aa group decreased significantly after 3 months of metformin therapy compared with baseline .
although there was an increase in hdl - c levels in both patients with the ga + aa genotype and gg genotype after 3 months of treatment with metformin , statistically significant differences were not found .
change in the study parameters from baseline to 3 months of metformin therapy according to the genotypes of organic cation transporter 3 - 564g > a in the current study , subjects were divided into two groups : metformin responders ( n = 69 ) and metformin nonresponders ( n = 81 ) . there were no statistically significant differences between responders and nonresponders with respect to the study parameters , except hba1c levels before treatment ( results not shown ) . when we analyzed the study parameters with respect to metformin response according to oct3 - 564g > a genotypes , our findings showed no statistically significant differences between responders and nonresponders with respect to the majority of the parameters of this study [ tables 3 and 4 ] . in both nonresponders [ table 3 ] and responders [ table 4 ] ,
fasting glucose levels were lower in patients with the ga + aa genotype than in those with the gg genotype ; however , the differences were not statistically significant .
furthermore , our results showed that in ga + aa group compared with gg group , reduction in average hba1c values was higher in responders than in nonresponders [ figure 1 ] , although were not found the statistically significant differences among the groups .
change in the study variables after 3 months of metformin therapy in metformin nonresponders according to the genotypes of organic cation transporter 3 - 564g > a change in the study variables after 3 months of metformin therapy in metformin responders according to the genotypes of organic cation transporter 3 - 564g > a change in glycated hemoglobin levels after 3 months of metformin therapy in metformin responders and nonresponders based on the genotypes of oct3 - 564g > a
to the best of our knowledge , there are few studies on the allele frequency of oct3 - 564g > a in different populations . in our study ,
the major allele frequency of g and minor allele frequency of a were 0.69 and 0.31 , respectively , in patients with t2d .
reported that g and a allele frequencies were 0.53 and 0.47 , respectively , in healthy caucasians in the united states .
moreover , in the study of aoyama et al . , g and a allele frequencies were 0.51 and 0.49 , respectively , in a healthy japanese population .
it should be noted that according to another study we are doing , the frequency of a allele in a healthy iranian population ( n = 152 ) was 0.38 .
it seems that minor allele frequency of a in iranian patients with t2d and also in healthy iranian population is lower than that of healthy caucasian and japanese populations . according to an investigation recently published by li et al .
, aa genotype of oct3 - 564g > a variant compared to gg genotype significantly reduces the risk of coronary heart disease ( chd ) in a chinese population . in other words
, they reported that this variant can contribute to reducing the risk of chd through its supporting role against inflammatory responses .
thus , lower frequency of a allele in iranian patients with t2d may contribute to the risk of chd in these patients .
it should be noted that mortality in diabetic patients is more due to cardiovascular complications .
for example , a sufficient response is not observed in certain metformin users that is one of the most important predicaments about efficacy of the important antidiabetic drug .
there are few studies on the effect of genetic variants of oct3 on the pharmacokinetics of metformin .
chen et al . showed that some different variants in oct3 such as rs68187715 and rs8187717 affect metformin uptake in vitro .
however , tzvetkov et al . investigated the effect of some variants of oct3 such as rs3123634 and rs2292334 on pharmacokinetics in healthy volunteers , but found no statistically significant relationship between these variants and clearance of metformin .
oct3 - 564g > a is one of the variants in 3-utr region of oct3 which affect the expression of this transporter .
li et al . showed that g > a displacement in the variant leads to binding mir-147 to mrna resulting in the decreased expression of oct3 .
moreover , nies et al . indicated that three variants in the noncoding region including oct3 - 564g > a are associated with decreased expression of mrna in oct3 in the liver cells . in the present study ,
given the importance of oct3 - 564g > a in oct3 expression , we investigated whether this variant can affect the response to metformin in patients with t2d .
as expected , the results showed that fasting glucose and hba1c levels were significantly decreased after 3 months of treatment with metformin .
when we analyzed these factors according to oct3 - 564g > a genotypes , it was found that fasting glucose and hba1c levels were significantly decreased in both gg and ga + aa groups after 3 months of metformin therapy .
in addition , our study are consistent with results obtained by other authors showed that metformin is effective in improving the lipid profile .
the results analysis according to the genotypes of oct3 - 564g > a showed that the lipid profile is improved after 3 months of treatment with metformin in both patients with gg and ga + aa genotypes .
further analyses according to responders and nonresponders to metformin revealed that fasting glucose and hba1c do not change significantly in responders and nonresponders with respect to the genotypes of study variant . in other words
, it seems that oct3 - 564g > a is not effective in the glycemic response to metformin .
however , it should be noted that metformin can be transported by two other members of the oct family , i.e. , oct1 and oct2 .
this issue is more important in the major organ of metformin activity , i.e. , the liver in which oct1 is the main transporter .
hence , for a more comprehensive review of the role of oct3 variants in response to metformin , these variants should be studied with functional variants of two other members particularly oct1 .
the relatively small sample size should be considered as a limitation of the study , and thus further research in different populations with a larger sample size is needed to clarify the role of the oct3 - 564g > a variant in metformin response . in general , considering the different consequences of the variations in drug transporters , the effect of oct3 variations as a high - capacity transporter widely expressed in various tissues can not be ignored .
the frequency of a allele ( which may be a protective allele against chd ) in the iranian diabetic patients was lower compared with caucasian , japanese , and iranian healthy populations .
metformin is useful in improving the lipid profile , in addition to its effects in glycemic control , and these impacts are regardless of the variant oct3 - 564g > a .
am contributed in the conception of the work , conducting the study , writing the article , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work mg contributed in the conception of the work , study design , samples and data collection , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work mbh contributed in the conception of the work , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work srh contributed in the conception of the work , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work aa contributed in the conception of the work , data analysis , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work smn contributed in the conception of the work , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . | background : organic cation transporter 3 ( oct3 ) is an excellent transporter for metformin , which is used as first - line therapy for type 2 diabetes ( t2d ) .
oct3 genetic variants may influence the clinical response to metformin .
this study aimed to determine the genotype and allele frequency of oct3 - 564g > a ( rs3088442 ) variant and its role in the glycemic response to metformin in patients with newly diagnosed t2d.materials and methods : based on the response to metformin , 150 patients were classified into two groups : sixty - nine responders ( decrease in glycated hemoglobin [ hba1c ] values by more than 1% from the baseline ) and 81 nonresponders ( decrease in hba1c values < 1% from the baseline ) .
hba1c levels were determined by chromatography .
the variant oct3 - 564g > a was genotyped using polymerase chain reaction - based restriction fragment length polymorphism.results:the genotypes frequencies were 51.3% gg , 36% ag , and 12.7% aa .
allele frequency of major allele ( g ) and minor allele ( a ) in oct3 - 564g > a variant was found to be 0.69 and 0.31 , respectively .
fasting glucose , hba1c , body mass index , and lipid profile in both gg genotypes and ga + aa group decreased significantly after 3 months of metformin therapy compared with baseline ( p < 0.05 ) . in both responders and nonresponders , hba1c and fasting glucose levels were lower in patients with the ga + aa genotype than in those with the gg genotype ; however , the differences were not statistically significant ( p > 0.05).conclusion : the a allele frequency ( which may be a protective allele against coronary heart disease ) in the iranian diabetic patients was lower than iranian , caucasian and japanese healthy populations .
metformin is useful in improving the lipid profile , in addition to its impacts in glycemic control , and these effects are regardless of oct3 - 564g > a variant . |
halogenated inhalational anesthetics are the most commonly used agents for induction and maintenance of general anesthesia .
the choice of anesthetic depends on many factors including ease of administration , cost , availability and permissibility of their effects . for more than 50 years
chloroform the original halogenated anesthetic abandoned for a long period is indicated to be hepatotoxic by different studies ( 1 , 2 ) .
halothane , introduced in 1950s as a safer alternative for chloroform , causes a well - documented syndrome of acute hepatotoxicity as well .
these agents are also shown to cause similar injury , but less than halothane ( 3 ) .
fortunately , fewer cases of hepatotoxicity have been reported for the newest agents , desflurane and sevoflurane ( 4 ) .
most hypotheses on the mechanism of halogenated anesthetics - induced hepatic injury are based on the production of metabolites causing immunoallergic reaction in patients .
all of these anesthetics undergo biotransformation , primarily involving cytochrome p-450 2e1 ( cyp 2e1 ) ( 5 ) .
the likelihood that each halogenated inhalational anesthetic causes liver injury correlates with the extent to which it is metabolized by this cytochrome ( 20% to 30% for halothane , 2% for enflurane , 1% for sevoflurane , and 0.2% or less for isoflurane and desflurane ) and the metabolic products ( 5 ) . in this study , we aimed to review available literature documenting the mechanism and extent by which these agents cause liver injuries .
electronic search was performed by searching several databases ( pubmed , google scholar , scopus , index copernicus , ebsco and the cochrane database ) using the keywords including inhalational anesthetics and liver injury ; inhalational anesthetics and hepatotoxicity ; volatile anesthetics and liver injury ; volatile anesthetics and hepatotoxicity ; for the period of 1966 to 2013 .
all randomized clinical trials , case series , case report and meta - analysis studies with the above mentioned contents were included in review process .
totally , 247 articles were found in primary search , from which 52 articles were eligible and enrolled in this study .
halothane is a classic form of halogenated anesthetics , which was introduced in 1956 and rapidly became the most commonly used agent for general anesthesia . within a few years of its introduction , cases of halothane - induced liver injuries were reported . in 1969 ,
the national institutes of health ( nih ) organized an epidemiological study on 250000 cases of halothane administration , which revealed an incidence of fatal hepatic necrosis of about 1 in 35000 individuals ( 6 - 8 ) .
concerns about its hepatotoxicity eliminated the use of this anesthetic for adults in the united states and many other countries .
two types of postoperative liver injuries have been reported to be associated with halothane administration .
twenty percent of patients receiving halothane anesthesia show a mild injury characterized by nausea , lethargy and fever .
the first is a transient elevation of liver enzymes , alanine aminotransferase ( alt ) and aspartate aminotransferase ( ast ) , which remain elevated for one to two weeks following halothane exposure and resolve without treatment ( 9 ) .
halothane associated hypoxia is indicated to be due to alteration in the hepatic oxygen demand and supply relationship ( 10 , 11 ) .
mild hepatic injury appears to result from direct toxic effect of halothane on hepatic cells ; however , evidence of immune activation is lacking in this type .
the other form of liver damage after halothane exposure is severe acute hepatitis with histological findings of massive hepatic necrosis .
this form is dose - independent and rare , occurring in approximately 1/15000 cases after initial exposure , but in about 1/1000 cases after repeated exposures ( 12 - 15 ) .
the severe type of injury is associated with acute elevations in alt and ast levels ( 5 to 50 fold ) and onset of jaundice within 2 to 14 days of halothane administration .
there is strong evidence that the severe type of liver injury is mediated by the patient 's own immune system .
approximately 60% to 80% of inhaled halothane is eliminated unchanged through the lungs , but about 20% of it undergoes metabolism either in an oxidative ( by cyp2e1 and cyp2a6 ) or reductive ( by cytochrome cyp2a6 and cyp3a4 ) pathways .
halothane oxidation leads to production of trifluoroacetic acid ( tfa ) , which acts on hepatocyte proteins to produce trifluoroacetylated components .
it is indicated that serum of patients with a clinical diagnosis of halothane - induced hepatitis contains circulating immunoglobulin g ( igg ) , reacting with liver microsomal trifluoroacetylated proteins ( 16 ) .
repeated halothane exposure stimulates the immune response , increasing the incidence of hepatitis , fever , rash , arthralgia , and peripheral eosinophilia ( 17 , 18 ) .
hepatic injury due to immune response is severe and even sometimes fatal ( 19 ) .
on the other hand , reductive pathways of halothane metabolism produce free radicals that can act as reactive metabolites causing minor injury .
different risk factors have been suggested to increase the incidence of halothane - induced hepatotoxicity .
this injury tends to be more severe in persons older than 40 years as they account for 80% of cases .
repeated exposure to halothane ( especially within a few weeks or months ) is documented in as many as 90% of cases .
obesity is another risk factor , possibly because of accumulation of halothane in adipose tissue .
induction of cyp2e1 by phenobarbital , alcohol , and isoniazid are all risk factors for halothane hepatitis ( 20 - 22 ) .
however , halothane is no longer used in many developed countries but continues to be used in some developing countries .
after halothane , other halogenated anesthetics , including enflurane ( 1972 ) , isoflurane ( 1979 ) , desflurane ( 1993 ) and sevoflurane ( 1995 ) have been introduced .
compared to halothane , these agents have the advantage of fewer metabolisms by the cyp2e1 system , which suggests less adverse effects on liver , following their administration ( 23 , 24 ) .
as it has a slow onset of action , it is used largely to maintain anesthesia after induction with other agents .
enflurane became available in 1972 and is still in use , although less widely than other more modern halogenated agents .
clinically apparent , very rare cases of severe hepatic injury from enflurane have been reported .
like halothane , enflurane is metabolized by the microsomal drug - metabolizing enzyme cyp2e1 to a trifluoroacetylated reactive intermediate , which is capable of binding to multiple intracytoplasmic proteins , forming potentially immunogenic adducts . as the tfa adducts induced antibodies
can be detected in patients with enflurane , as well as halothane , it is suggested that the mechanism of enflurane - induced liver injury resembles the mechanism of halothane hepatotoxicity .
however , the incidence of enflurane - induced hepatotoxicity appears to be less compared to halothane . as with halothane hepatotoxicity
, this injury is marked by acute elevations in serum alt ( 5- to 50-fold ) and appearance of jaundice 2 to 21 days after the operation and anesthesia ( 25 - 27 ) .
isoflurane , an isomer of enflurane , is a widely used anesthetic agent with rapid onset of action and rapid dispersal . it was approved in 1979 and widely accepted as a safer agent with less risk of hepatotoxicity . since its release by the food and drug administration
, controversy has existed about the extent to which isoflurane is capable of producing hepatotoxic effects ( 3 ) .
this agent is suggested to be less hepatotoxic compared to halothane and enflurane ; however , there are few case reports , describing fetal hepatotoxicity following single exposure to isoflurane ( 3 , 28 , 29 ) .
the mechanism of isoflurane hepatotoxicity is suspected to be similar to that of halothane and associated with production of reactive intermediates .
similar to previously described agents , desflurane is metabolized to a small but variable extent by the microsomal drug - metabolizing enzyme cyp2e1 to a tfa reactive intermediate , which is capable of binding to multiple intracytoplasmic proteins , forming potentially immunogenic adducts .
occurrence of hepatic injury following desflurane anesthesia was once thought to be rare . however , case reports reporting complications related to the use of this anesthetic continue to be accumulated ( 30 - 35 ) .
interestingly , in addition to circulating anti - tfa antibodies ( 4 , 5 ) , such as those associated with halothane or isoflurane , recently autoantibodies to cyp2e1 and 58 kda endoplasmic reticulum protein ( erp58 ) , have been reported after desflurane anesthesia ( 30 ) .
the presence of these antibodies suggests the induction of heterogeneous immune complexes following desflurane exposure .
sevoflurane has many desirable clinical properties , including a nonpungent odor and low solubility in blood , which provides rapid induction and recovery .
studies have indicated that its administration is not associated with elevated alt and ast levels ( 29 ) .
in contrast with older halogenated anesthetics , sevoflurane metabolism has not resulted in the formation of tfa ; hence , hepatotoxic potential of sevoflurane is considered to be low ( 36 , 37 ) .
a unique compound of sevoflurane metabolism , hexafluoroisopropanol ( hfip ) has significantly less protein binding capability than tfa .
in addition , hfip does not accumulate and rapidly undergoes phase ii biotransformation , specifically glucuronidation , to form hfip - glucuronide .
this compound is mostly excreted in the urine within 12 hours after anesthesia and is not detectable beyond 2 days ( 38 - 40 ) .
this is in contrast to previous anesthetics , where tfa is detectable in urine for up to 12 days after 75 minutes of anesthesia ( 41 - 43 ) .
however , sevoflurane has a low hepatotoxic potential as described in the current available literature ( 8 , 44 , 45 ) .
as apparently , sevoflurane is unlikely to cause severe postoperative hepatic injury and is an ideal anesthetic for patients with previous exposure to other halogenated anesthetics or hepatic disease ( 46 - 48 ) .
sevoflurane seems to be the best anesthetic choice in large size surgeries and liver transplants , intervention in which postoperative liver dysfunction could have harmful effects on patients .
interestingly , new studies demonstrated that sevoflurane pretreatment even exerts a protective effect on hepatic ischemia / reperfusion injury , which is a common problem in hepatic surgery ( 47 , 49 , 50 ) .
anesthesia - induce hepatotoxicity is a diagnosis of exclusion , so that ruling out other causes is essential .
clinical features similar to anesthesia - induced liver injury can be caused by acute viral hepatitis , exacerbation of previous chronic liver disease , hemolysis after blood transfusion , liver damage due to hypoxia ( caused by vasoconstriction , shock , hypoxia , cardiac arrest ischemia ) , hypotension or liver damage due to other hepatotoxic drugs ( antibiotics , antidepressants , phenothiazines , phenytoin , anticoagulants ) .
for instance , from a histopathological point of view , centrolobular necrosis with mild cholestasis and fatty infiltration are associated with both hepatic necrosis due to halothane and hepatic necrosis caused by viruses .
furthermore , an operation per se may cause changes in liver function regardless of the type of anesthesia .
using anamnestic or serological tests together with clinical and epidemiological criteria would be helpful to distinguish anesthesia - induced hepatitis from other causes of hepatitis .
features of anesthesia - induced hepatitis include high fever onset day 3 to 14 , incidence of jaundice 1 - 2 days after fever , presence of no viral antibodies , latent period less than 15 days , recent exposure in 80% of cases , leukocytosis with eosinophilia ( 20% following halothane but rare in viral hepatitis ) , fulminant course ( 40% mortality vs. 1% for viral ) and two times higher incidence in females .
halothane is a classic form of halogenated anesthetics , which was introduced in 1956 and rapidly became the most commonly used agent for general anesthesia . within a few years of its introduction , cases of halothane - induced liver injuries were reported . in 1969 ,
the national institutes of health ( nih ) organized an epidemiological study on 250000 cases of halothane administration , which revealed an incidence of fatal hepatic necrosis of about 1 in 35000 individuals ( 6 - 8 ) .
concerns about its hepatotoxicity eliminated the use of this anesthetic for adults in the united states and many other countries .
two types of postoperative liver injuries have been reported to be associated with halothane administration .
twenty percent of patients receiving halothane anesthesia show a mild injury characterized by nausea , lethargy and fever .
the first is a transient elevation of liver enzymes , alanine aminotransferase ( alt ) and aspartate aminotransferase ( ast ) , which remain elevated for one to two weeks following halothane exposure and resolve without treatment ( 9 ) .
halothane associated hypoxia is indicated to be due to alteration in the hepatic oxygen demand and supply relationship ( 10 , 11 ) .
mild hepatic injury appears to result from direct toxic effect of halothane on hepatic cells ; however , evidence of immune activation is lacking in this type .
the other form of liver damage after halothane exposure is severe acute hepatitis with histological findings of massive hepatic necrosis .
this form is dose - independent and rare , occurring in approximately 1/15000 cases after initial exposure , but in about 1/1000 cases after repeated exposures ( 12 - 15 ) .
the severe type of injury is associated with acute elevations in alt and ast levels ( 5 to 50 fold ) and onset of jaundice within 2 to 14 days of halothane administration .
there is strong evidence that the severe type of liver injury is mediated by the patient 's own immune system .
approximately 60% to 80% of inhaled halothane is eliminated unchanged through the lungs , but about 20% of it undergoes metabolism either in an oxidative ( by cyp2e1 and cyp2a6 ) or reductive ( by cytochrome cyp2a6 and cyp3a4 ) pathways .
halothane oxidation leads to production of trifluoroacetic acid ( tfa ) , which acts on hepatocyte proteins to produce trifluoroacetylated components .
it is indicated that serum of patients with a clinical diagnosis of halothane - induced hepatitis contains circulating immunoglobulin g ( igg ) , reacting with liver microsomal trifluoroacetylated proteins ( 16 ) .
repeated halothane exposure stimulates the immune response , increasing the incidence of hepatitis , fever , rash , arthralgia , and peripheral eosinophilia ( 17 , 18 ) .
hepatic injury due to immune response is severe and even sometimes fatal ( 19 ) .
on the other hand , reductive pathways of halothane metabolism produce free radicals that can act as reactive metabolites causing minor injury .
different risk factors have been suggested to increase the incidence of halothane - induced hepatotoxicity .
this injury tends to be more severe in persons older than 40 years as they account for 80% of cases .
repeated exposure to halothane ( especially within a few weeks or months ) is documented in as many as 90% of cases .
obesity is another risk factor , possibly because of accumulation of halothane in adipose tissue .
induction of cyp2e1 by phenobarbital , alcohol , and isoniazid are all risk factors for halothane hepatitis ( 20 - 22 ) .
however , halothane is no longer used in many developed countries but continues to be used in some developing countries .
after halothane , other halogenated anesthetics , including enflurane ( 1972 ) , isoflurane ( 1979 ) , desflurane ( 1993 ) and sevoflurane ( 1995 ) have been introduced .
compared to halothane , these agents have the advantage of fewer metabolisms by the cyp2e1 system , which suggests less adverse effects on liver , following their administration ( 23 , 24 ) .
as it has a slow onset of action , it is used largely to maintain anesthesia after induction with other agents .
enflurane became available in 1972 and is still in use , although less widely than other more modern halogenated agents .
. clinically apparent , very rare cases of severe hepatic injury from enflurane have been reported .
like halothane , enflurane is metabolized by the microsomal drug - metabolizing enzyme cyp2e1 to a trifluoroacetylated reactive intermediate , which is capable of binding to multiple intracytoplasmic proteins , forming potentially immunogenic adducts . as the tfa adducts induced antibodies
can be detected in patients with enflurane , as well as halothane , it is suggested that the mechanism of enflurane - induced liver injury resembles the mechanism of halothane hepatotoxicity .
however , the incidence of enflurane - induced hepatotoxicity appears to be less compared to halothane . as with halothane hepatotoxicity , this injury is marked by acute elevations in serum alt ( 5- to 50-fold ) and appearance of jaundice 2 to 21 days after the operation and anesthesia ( 25 - 27 ) .
isoflurane , an isomer of enflurane , is a widely used anesthetic agent with rapid onset of action and rapid dispersal . it was approved in 1979 and widely accepted as a safer agent with less risk of hepatotoxicity . since its release by the food and drug administration , controversy has existed about the extent to which isoflurane is capable of producing hepatotoxic effects ( 3 ) .
this agent is suggested to be less hepatotoxic compared to halothane and enflurane ; however , there are few case reports , describing fetal hepatotoxicity following single exposure to isoflurane ( 3 , 28 , 29 ) .
the mechanism of isoflurane hepatotoxicity is suspected to be similar to that of halothane and associated with production of reactive intermediates .
similar to previously described agents , desflurane is metabolized to a small but variable extent by the microsomal drug - metabolizing enzyme cyp2e1 to a tfa reactive intermediate , which is capable of binding to multiple intracytoplasmic proteins , forming potentially immunogenic adducts .
however , case reports reporting complications related to the use of this anesthetic continue to be accumulated ( 30 - 35 ) .
interestingly , in addition to circulating anti - tfa antibodies ( 4 , 5 ) , such as those associated with halothane or isoflurane , recently autoantibodies to cyp2e1 and 58 kda endoplasmic reticulum protein ( erp58 ) , have been reported after desflurane anesthesia ( 30 ) .
the presence of these antibodies suggests the induction of heterogeneous immune complexes following desflurane exposure .
sevoflurane has many desirable clinical properties , including a nonpungent odor and low solubility in blood , which provides rapid induction and recovery .
studies have indicated that its administration is not associated with elevated alt and ast levels ( 29 ) .
in contrast with older halogenated anesthetics , sevoflurane metabolism has not resulted in the formation of tfa ; hence , hepatotoxic potential of sevoflurane is considered to be low ( 36 , 37 ) .
a unique compound of sevoflurane metabolism , hexafluoroisopropanol ( hfip ) has significantly less protein binding capability than tfa .
in addition , hfip does not accumulate and rapidly undergoes phase ii biotransformation , specifically glucuronidation , to form hfip - glucuronide .
this compound is mostly excreted in the urine within 12 hours after anesthesia and is not detectable beyond 2 days ( 38 - 40 ) .
this is in contrast to previous anesthetics , where tfa is detectable in urine for up to 12 days after 75 minutes of anesthesia ( 41 - 43 ) .
however , sevoflurane has a low hepatotoxic potential as described in the current available literature ( 8 , 44 , 45 ) .
as apparently , sevoflurane is unlikely to cause severe postoperative hepatic injury and is an ideal anesthetic for patients with previous exposure to other halogenated anesthetics or hepatic disease ( 46 - 48 ) .
sevoflurane seems to be the best anesthetic choice in large size surgeries and liver transplants , intervention in which postoperative liver dysfunction could have harmful effects on patients .
interestingly , new studies demonstrated that sevoflurane pretreatment even exerts a protective effect on hepatic ischemia / reperfusion injury , which is a common problem in hepatic surgery ( 47 , 49 , 50 ) .
enflurane is a widely used halogenated inhalational anesthetic . as it has a slow onset of action , it is used largely to maintain anesthesia after induction with other agents .
enflurane became available in 1972 and is still in use , although less widely than other more modern halogenated agents .
approximately 2% of enflurane undergoes hepatic metabolism compared to 20 - 30% of halothane . clinically apparent
like halothane , enflurane is metabolized by the microsomal drug - metabolizing enzyme cyp2e1 to a trifluoroacetylated reactive intermediate , which is capable of binding to multiple intracytoplasmic proteins , forming potentially immunogenic adducts . as the tfa adducts induced antibodies
can be detected in patients with enflurane , as well as halothane , it is suggested that the mechanism of enflurane - induced liver injury resembles the mechanism of halothane hepatotoxicity .
however , the incidence of enflurane - induced hepatotoxicity appears to be less compared to halothane . as with halothane hepatotoxicity ,
this injury is marked by acute elevations in serum alt ( 5- to 50-fold ) and appearance of jaundice 2 to 21 days after the operation and anesthesia ( 25 - 27 ) .
isoflurane , an isomer of enflurane , is a widely used anesthetic agent with rapid onset of action and rapid dispersal . it was approved in 1979 and widely accepted as a safer agent with less risk of hepatotoxicity . since its release by the food and drug administration
, controversy has existed about the extent to which isoflurane is capable of producing hepatotoxic effects ( 3 ) .
this agent is suggested to be less hepatotoxic compared to halothane and enflurane ; however , there are few case reports , describing fetal hepatotoxicity following single exposure to isoflurane ( 3 , 28 , 29 ) .
the mechanism of isoflurane hepatotoxicity is suspected to be similar to that of halothane and associated with production of reactive intermediates .
similar to previously described agents , desflurane is metabolized to a small but variable extent by the microsomal drug - metabolizing enzyme cyp2e1 to a tfa reactive intermediate , which is capable of binding to multiple intracytoplasmic proteins , forming potentially immunogenic adducts .
occurrence of hepatic injury following desflurane anesthesia was once thought to be rare . however , case reports reporting complications related to the use of this anesthetic continue to be accumulated ( 30 - 35 ) .
interestingly , in addition to circulating anti - tfa antibodies ( 4 , 5 ) , such as those associated with halothane or isoflurane , recently autoantibodies to cyp2e1 and 58 kda endoplasmic reticulum protein ( erp58 ) , have been reported after desflurane anesthesia ( 30 ) .
the presence of these antibodies suggests the induction of heterogeneous immune complexes following desflurane exposure .
sevoflurane has many desirable clinical properties , including a nonpungent odor and low solubility in blood , which provides rapid induction and recovery .
studies have indicated that its administration is not associated with elevated alt and ast levels ( 29 ) .
in contrast with older halogenated anesthetics , sevoflurane metabolism has not resulted in the formation of tfa ; hence , hepatotoxic potential of sevoflurane is considered to be low ( 36 , 37 ) .
a unique compound of sevoflurane metabolism , hexafluoroisopropanol ( hfip ) has significantly less protein binding capability than tfa .
in addition , hfip does not accumulate and rapidly undergoes phase ii biotransformation , specifically glucuronidation , to form hfip - glucuronide .
this compound is mostly excreted in the urine within 12 hours after anesthesia and is not detectable beyond 2 days ( 38 - 40 ) .
this is in contrast to previous anesthetics , where tfa is detectable in urine for up to 12 days after 75 minutes of anesthesia ( 41 - 43 ) .
however , sevoflurane has a low hepatotoxic potential as described in the current available literature ( 8 , 44 , 45 ) .
as apparently , sevoflurane is unlikely to cause severe postoperative hepatic injury and is an ideal anesthetic for patients with previous exposure to other halogenated anesthetics or hepatic disease ( 46 - 48 ) .
sevoflurane seems to be the best anesthetic choice in large size surgeries and liver transplants , intervention in which postoperative liver dysfunction could have harmful effects on patients .
interestingly , new studies demonstrated that sevoflurane pretreatment even exerts a protective effect on hepatic ischemia / reperfusion injury , which is a common problem in hepatic surgery ( 47 , 49 , 50 ) .
anesthesia - induce hepatotoxicity is a diagnosis of exclusion , so that ruling out other causes is essential .
clinical features similar to anesthesia - induced liver injury can be caused by acute viral hepatitis , exacerbation of previous chronic liver disease , hemolysis after blood transfusion , liver damage due to hypoxia ( caused by vasoconstriction , shock , hypoxia , cardiac arrest ischemia ) , hypotension or liver damage due to other hepatotoxic drugs ( antibiotics , antidepressants , phenothiazines , phenytoin , anticoagulants ) .
for instance , from a histopathological point of view , centrolobular necrosis with mild cholestasis and fatty infiltration are associated with both hepatic necrosis due to halothane and hepatic necrosis caused by viruses .
furthermore , an operation per se may cause changes in liver function regardless of the type of anesthesia . using anamnestic or serological tests together with clinical and epidemiological criteria would be helpful to distinguish anesthesia - induced hepatitis from other causes of hepatitis
. features of anesthesia - induced hepatitis include high fever onset day 3 to 14 , incidence of jaundice 1 - 2 days after fever , presence of no viral antibodies , latent period less than 15 days , recent exposure in 80% of cases , leukocytosis with eosinophilia ( 20% following halothane but rare in viral hepatitis ) , fulminant course ( 40% mortality vs. 1% for viral ) and two times higher incidence in females .
halogenated anesthesia - induced liver injury has been well documented in available literature . beginning with halothane in the 1950s ,
however , postoperative liver injury was soon recognized , especially in patients re - exposed to halothane .
hepatitis followed by halothane anesthesia has high morbidity and mortality rates and survived patients may require a liver transplantation .
next generation of halogenated anesthetics including enflurane , isoflurane , desflurane , and sevoflurane have different molecular structures and are associated with less hepatotoxicity ; however , rare instances of acute liver injury have been reported with all of these agents .
the pattern of liver injury described with the newer halogenated anesthetics ( enflurane , isoflurane and desflurane ) has resembled that of halothane mechanism and evidence of autoimmune response to trifluoroacetylated liver proteins has been identified in patients serum .
a history of anesthesia - induced hepatitis is a reason to avoid subsequent exposure to halothane or other halogenated anesthetics , since crossed immunity can rarely occur . unlike other halogenated anesthetics
, sevoflurane is not metabolized to hepatotoxic trifluoroacetylated proteins ; however , very few reports have described liver injury after sevoflurane exposure . in spite of the fact that anesthesia - induced liver injury is not a common occurrence , we should be aware of the association between the disease and the use of halogenated anesthetics . | context : halogenated inhalational anesthetics are currently the most common drugs used for the induction and maintenance of general anesthesia . postoperative hepatic injury has been reported after exposure to these agents .
based on much evidence , mechanism of liver toxicity is more likely to be immunoallergic .
the objective of this review study was to assess available studies on hepatotoxicity of these anesthetics.evidence acquisition : we searched pubmed , google scholar , scopus , index copernicus , ebsco and the cochrane database using the following keywords : inhalational anesthetics and liver injury ; inhalational anesthetics and hepatotoxicity ; volatile anesthetics and liver injury ; volatile anesthetics and hepatotoxicity for the period of 1966 to 2013 .
fifty two studies were included in this work.results:all halogenated inhalational anesthetics are associated with liver injury .
halothane , enflurane , isoflurane and desflurane are metabolized through the metabolic pathway involving cytochrome p-450 2e1 ( cyp2e1 ) and produce trifluoroacetylated components ; some of which may be immunogenic .
the severity of hepatotoxicity is associated with the degree by which they undergo hepatic metabolism by this cytochrome . however , liver toxicity is highly unlikely from sevoflurane as is not metabolized to trifluoroacetyl compounds.conclusions:hepatotoxicity of halogenated inhalational anesthetics has been well documented in available literature .
halothane - induced liver injury was extensively acknowledged ; however , the next generation halogenated anesthetics have different molecular structures and associated with less hepatotoxicity .
although anesthesia - induced hepatitis is not a common occurrence , we must consider the association between this disorder and the use of halogenated anesthetics . |
the most prevalent cause of pelvic floor muscle dysfunction in females is the attenuation
of the pelvic floor muscle , resulting in a state in which the pelvic floor muscle is unable
to be independently recognized and contracted .
this results in pelvic girdle relaxation ,
pelvic organ prolapse , or stress urinary incontinence .
the pelvic floor muscle , in the form
of the pelvic floor and abdominal cavity , has the dual function of controlling cystic
incontinence and stabilizing the radial region1 .
dysfunction of the pelvic floor muscle is generally associated with
dysuria and radial pain2 .
a recent study has shown that the pelvic floor muscles are fundamentally important for
trunk core muscle strength and stabilization3 .
perri4 reported
that the transversus abdominis muscle , pelvic floor muscle , and deep spine intrinsic muscle
function in harmony with each other , and that dysfunction in one of them can produce related
effects , influencing spinal stability , and causing pain .
sapsford et al.5 found that during maximum contraction of the
pelvic floor muscles , all the abdominal muscles , including the transversus abdominis ,
internus obliquus , externus obliquus , and rectus abdominis , were activated .
recently , ultrasonography has become one of the instruments used widely in research and
clinical settings , since it allows muscle shape and reaction to be quantified , especially
the contraction of the abdominal muscles , in a non - invasive manner .
ultrasonography employs
a novel method to evaluate the provided morphological information of deep muscles6 .
it acquires objective and highly reliable
information , since the measurement is performed while visually checking the location and
shape of the deep muscles .
kiesel et al.7 )
reported that an ultrasonogram is useful for evaluating muscle thickness as the
changes in a muscle are measured related to contraction .
hence , this study employed
unltrasonography in order to objectively evaluate abdominal muscle contractions related to
changes in vaginal pressure . in cases of dysfunction ( e.g. ,
stress urinary incontinence ) , it is generally advised that
the rehabilitation of pelvic floor muscles be done in isolation8 .
however , it is now known that abdominal muscle activity
occurs in conjunction with pelvic floor muscle contraction , and there is preliminary
evidence that the reverse may also occur ( i.e. , pelvic floor muscle activity in response to
specific abdominal maneuvers ) .
many clinical exercise methods have been developed to use abdominal muscles in pelvic floor
muscle exercises .
however , studies have rarely been conducted to measure abdominal muscle
thickness in response to vaginal pressure generated by pelvic floor muscles . in this study
,
normal adult females were divided into different age groups , and changes in abdominal muscle
thickness in response to changing vaginal pressure were comparatively analyzed .
the subjects of this study were classified by age into two groups comprised of 11 subjects
in their 20s and 30s ( group i ) , and 13 subjects in their 40s and 50s ( group ii ) .
the average
ages of group i and group ii were 33.55 and 49.69 years , respectively , with a significant
difference .
however , there were no significant differences between the two groups in vaginal
pressure , height , weight , and body mass index ( bmi ) .
this study was approved by hospital ,
and all the participants provided their written informed consent .
vaginal pressure was
measured with a perineum vaginal pressure meter ( perineometer , peritron 9300 , cardio design
australia ) .
subjects were placed in a supine position with their hips flexed at approximately 60. each
subject was instructed to strongly tighten her vagina as if pulling it into her body . while
avoiding lumbar and pelvic motions , subjects received visual feedback about their
contraction force through a pressure gauge connected to a pressure biofeedback device .
the
pelvic floor muscle contraction was performed at five levels : resting ( 0 cmh2o ) ,
10 cmh2o , 20 cmh2o , 30 cmh2o , and maximum contraction .
the
subjects maintained the target pressure for five seconds and took a 10-second rest before
the next measurement as the muscles may have tired during each contraction .
three
measurements were taken for each subject , and the average values were calculated . an ultrasonography instrument ( hdi 5000 , philips atl ) was used with a linear array
transducer , which could be operated at 7 mhz .
the static muscle cross - sectional area was
represented by a b ( brightness)-mode scan . to minimize inter - observer variations , a physical
therapist who was skilled in ultrasonography performed the measurements on the basis of
basic anatomical knowledge of the abdomen .
gel for ultrasonography was applied between the
linear array transducer and the skin , and the right abdominal muscle was checked by
inspection through the ultrasonogram , and palpation by the hands of the tester .
the
thicknesses of the transversus abdominis muscle , internal oblique abdominal muscle , and
external oblique abdominal muscle were measured at the same position , which was 1 cm from
the boundary of the fascia to the center of the muscle9 .
the measurement position was in the anterolateral position of the
axillary line between the iliac crest and costal inferior horn10 .
the subjects were in the supine position with a
triangular support under the knee joints so that the position would remain fixed for the
duration of the measurement .
the mean and standard deviation of the general characteristics of the subjects were
calculated using descriptive analysis .
one - way anova was performed to compare the muscle
thickness changes between the isometric contractions at the five levels of vaginal pressure .
a least - square difference ( lsd )
the
independent t - test was performed to compare muscle thicknesses between the age groups .
the
reliability of the abdominal muscle thickness measurement by the tester according to vaginal
pressure was analyzed using the intraclass correlation coefficient ( icc ) .
all the
statistical processing was performed with spss version 12.0 software , and a significance
level of 0.05 .
the iccs of the tester s three times measurements of muscle thickness at each vaginal
pressure ranged from 0.900.98 for the transversus abdominis muscle , from 0.870.99 for the
internal oblique abdominal muscle , and from 0.880.97 for the external oblique abdominal
muscle .
i , the change in abdominal thickness dependent on vaginal pressure was significant
for the transversus abdominis muscle ( p<0.05 ) but not significant for the external
oblique abdominal muscle and the internal oblique abdominal muscle ( p>0.05 ) .
the change
in abdominal muscle thickness dependent on vaginal pressure was significant from resting to
a vaginal pressure of 30 cmh2o , from resting to maximum contraction , and from 10
cmh2o to maximum contraction for the transversus abdominis muscle ( p<0.05 ) .
however , no significant differences were found for the external oblique abdominal muscle and
internal oblique abdominal muscle ( p>0.05 ) ( table
1table 1 .
abdominal muscle thicknesses of each age group ( unit = cm)gi musclevpthicknessresting10 cmh2o20 cmh2o30 cmh2omaximumtraresting0.240.01**10 cmh2o0.260.0220 cmh2o0.300.0230 cmh2o0.320.03maximum0.350.03eoresting0.590.0210 cmh2o0.610.0320 cmh2o0.610.0330 cmh2o0.600.03maximum0.590.02ioresting0.430.0410 cmh2o0.440.0420 cmh2o0.460.0430 cmh2o0.470.04maximum0.500.04gii musclevpthicknessresting10 cmh2o20 cmh2o30 cmh2omaximumtraresting0.230.01***10 cmh2o0.300.02**20 cmh2o0.340.0330 cmh2o0.380.04maximum0.420.04eoresting0.550.0410 cmh2o0.530.0420 cmh2o0.510.0430 cmh2o0.510.04maximum0.510.04ioresting0.440.02 * 10 cmh2o0.440.03 * 20 cmh2o0.470.0230 cmh2o0.590.03maximum0.530.02vp : vaginal pressure , tra : transversus abdominis , eo : external oblique muscle , io :
internal oblique muscle . *
vp : vaginal pressure , tra : transversus abdominis , eo : external oblique muscle , io :
internal oblique muscle .
* p<0.05 in group ii , the change in thickness dependent on vaginal pressure was significant for the
transversus abdominis muscle and internal oblique abdominal muscle ( p<0.05 ) but not
significant for the external oblique abdominal muscle ( p>0.05 ) .
the change in abdominal
muscle thickness dependent on vaginal pressure was significant from resting to vaginal
pressures of 20 cmh2o , 30 cmh2o , and maximum contraction , and from
vaginal pressures of 10 cmh2o to 30 cmh2o and maximum contraction for
the transversus abdominis muscle ( p<0.05 ) .
additionally , a significant change was found
from resting to maximum contraction , and from 10 cmh2o to maximum contraction for
the internal oblique abdominal muscle ( p<0.05 ) .
however , no significant difference was
found for the external oblique abdominal muscle ( p>0.05 ) .
a comparison of the muscle thickness changes in the transversus abdominis muscle and
internal oblique abdominal muscle dependent on vaginal pressure between the two groups
showed that the thickness increase was greater in group ii , but the increase was not
significant ( p>0.05 ) .
for the external oblique abdominal muscle , while no thickness
change was found in group i , muscle thickness gradually decreased as vaginal pressure
increased in group ii , but the decrease was not significant ( p>0.05 ) .
the analysis of transversus abdominis muscle thickness depending on vaginal pressure showed
that the resting thickness was 0.24 cm in group i , and 0.23 cm in group ii , indicating a
similar starting line .
a significant difference was found in group i from resting to a
vaginal pressure of 30 cmh2o , and from resting to maximum contraction .
a
significant difference was found in group ii from resting to 20 cmh2o , 30
cmh2o , and maximum contraction , and from 10 cmh2o to 30
cmh2o and maximum contraction .
this was consistent with the finding of
sapsford5 that the pelvic floor muscle
and transversus abdominis muscle activate together when the pelvic floor muscle is
contracted .
this result is also in agreement with the result that the muscular activity of
the pelvic floor muscle can be increased to a greater degree when the pelvic floor muscle is
contracted using the abdominal muscle rather than when only the pelvic floor muscle is
contracted11 , 12 . in our study ,
the thickness of the external oblique abdominal muscle increased slightly or
did not change in group i , while it gradually decreased from resting to maximum contraction
in group ii .
the thickness of the internal oblique abdominal muscle significantly increased
from resting to maximum contraction , and from 10 cmh2o to maximum contraction in
group ii .
the result of group ii is consistent with previous results that the thickness of
the internal oblique abdominal muscle increases by 1020% in the performance of a hollowing
exercise , but , that the thickness of the external oblique abdominal muscle increases only
slightly or decreases13 . in group
ii , the
result of the pelvic floor muscle exercise was similar to that of a hollowing exercise ,
indicating that vaginal pressure was increased when the internal abdominal muscle was used
during the pelvic floor muscle contraction
. a morphometric study on the rhabdo - urethral muscle reported that the volume of the levator
anti - muscle decreases with age14 .
a
histological study found that as age increases , the transverse muscles decrease along the
back of the urethra and the bladder neck15 .
thus , we assume that the middle - aged women in this study increased
vaginal contraction pressure by using their abdominal muscles rather than their attenuated
pelvic floor muscles .
a comparison of the two age groups showed that the pelvic floor muscles were contracted
without using the transversus abdominis muscle from resting to 30 cmh2o in group
i , and from resting to 20 cmh2o in group ii .
the transversus abdominis muscle was
used at a vaginal pressure that was 10 cmh2o lower in group ii than in group i ,
but the difference was not significant .
the childbirth delivery method was not considered in the categorization of subjects . in
future studies , abdominal contraction during pelvic floor muscle contraction
should be
compared by considering how the childbirth delivery method affects pelvic floor muscle
contraction , and studies should also be conducted with patients with pelvic floor muscle
dysfunction . hung et al.16 suggested the retraining
of the combined functions of the diaphragm , deep muscle , and pelvic floor muscles as an
alternative treatment for urinary incontinence .
bo et al.17 also recommended the synergistic effects of the co - contraction of
the pelvic floor muscles and transversus abdominis in the treatment of urinary incontinence .
on the basis of our result
, it may be necessary to develop an exercise program that
maximizes the effect of the contraction of the abdominal muscle and pelvic floor muscle by
appropriately controlling the strength of the pelvic floor muscle contraction . | [ purpose ] the purpose of this study was to verify the efficacy of a pelvic floor muscle
exercise program by comparing subjects muscle thickness with changes in vaginal pressure .
[ subjects ] two groups of female participants without a medical history of pelvic floor
muscle dysfunction were evaluated .
the mean age of group i was 33.5 years and that of
group ii was 49.69 years .
[ methods ] the participants were instructed to perform a pelvic
floor muscle contraction . while measuring the vaginal pressure of the pelvic floor muscle ,
biofeedback was given on five levels , and the thicknesses of the transversus abdominis ,
external oblique , and internal oblique muscles were measured with ultrasound .
[ results ]
the thickness of the transversus abdominis muscle was significantly increased at 30
cmh2o in group i , and at 20 cmh2o in group ii .
the thickness of
the internal oblique abdominal muscle significantly increased at maximum contraction in
group ii .
[ conclusion ] different abdominal muscles contracted depending on vaginal
pressure .
the result may be used to create and implement an exercise program that
effectively strengthens the pelvic floor muscles . |
helminths employ a range of immunomodulatory strategies to modulate the host immune response and utilize it to extend their longevity in the host and facilitate transmission .
characteristic of infection with helminths such as schistosoma mansoni is the induction of a th2 response .
two products released from eggs have recently been shown to play important roles in the immune deviation induced by s. mansoni infection [ 1 , 2 ] .
the nod mouse provides a good animal model of the human autoimmune disease , type 1 diabetes .
we have previously shown that s. mansoni infection or exposure either to the helminth eggs or soluble extracts of worms ( swa ) or eggs ( sea ) can prevent diabetes onset in this mouse [ 46 ] .
as type 1 diabetes is a th1-mediated autoimmune disease , any skewing of the response towards th2 would result in diabetes prevention .
other responses that have the potential to impact on diabetes would be the induction of immunoregulatory cytokines such as il-10 and tgf and regulatory t cells ( tregs ) [ 8 , 9 ] .
we and others have shown that both sea and swa have profound effects on cells of the innate immune system including dendritic cells ( dcs ) and nkt cells [ 5 , 10 , 11 ] . in the case of bone marrow
derived dcs , it has been shown that exposure to s. mansoni antigens results in dc retention of a more immature phenotype while inducing them to mediate a th2 response in vivo [ 5 , 10 ] .
immature dcs have been associated with ability to induce a th2 response as well as immune tolerance through induction of tregs .
although there was little evidence of phenotypic change in sea exposed bone marrow derived murine dcs , recent studies showed increased expression of mannose receptor ( mr ) , dec-205 , and dc - sign as well as induction of il-10 .
sea has , furthermore , been shown to induce foxp3 expressing tregs in a tgf dependent manner .
most studies in mice have been carried out using bone marrow derived apcs and it could be argued that it would be more relevant to examine the effect of s. mansoni antigens on isolated splenic dcs as well as in vivo on macrophage ( m ) populations . in this manuscript
we have addressed in detail the effect of sea on functional and phenotypic changes in dcs and ms and the impact that this might have on the development of different t cell subpopulations including tregs .
female nod / tac mice were housed and barrier bred in the pathology department , university of cambridge animal facilities ( cambridge , uk ) and used between 412 weeks of age .
all work was conducted under uk home office project license regulations after approval by the ethical review committee of the university of cambridge .
briefly , for sea , eggs were harvested from the livers of outbred infected mice , which were treated to prevent granuloma formation .
livers were homogenized through sieves and the eggs were collected , washed , and sonicated in pbs on ice , prior to centrifugation to separate the saline soluble fraction .
both antigens were sterile filtered and endotoxin removed to below < 1 eu / mg using polymixin b agarose beads ( sigma ) .
cells were cultured in iscove 's modified dulbecco 's medium supplemented with 2 mm l - glutamine , 100 u / ml penicillin - streptomycin , 50 m 2-mercaptoethanol ( all sigma ) , and 5%10% fetal calf serum ( fcs ) ( gibco ) .
anti - cd3 ( cd3 ) ( 2c11 ) and cd28 ( 37.51 ) were obtained from bd pharmingen .
lps from salmonella enterica ( sigma ) and the tlr1/2 agonist pam3cysk4 ( invivogen ) were used to stimulate dcs .
anti - tgf ( 1d11.16 ) was precipitated from hybridoma supernatants and dialyzed against pbs before use .
the mle / pai cell line is derived from mink lung epithelial cells and contains firefly luciferase under pai-1 promoter control .
mle cells were cultured with 50 l of tissue culture supernatants or a double - dilution standard curve derived from recombinant human tgf1 ( r&d systems ) for 18 hours .
cells were then lysed and luciferase activity was measured using promega 's luciferase reporter system and a betalux scintillation counter ( wallac ) .
cells were stained with appropriate combinations of the following antibodies : cd4 ( rm4 - 5)-pcp - cy5.5 , cd25 ( pc61)-pe , cd44 ( im7)-fitc , cd11b ( m1/70)-pcp - cy5.5 , cd11c ( hl3)-fitc or apc , galectin-3 ( m3/38)-alexa647 , foxp3 ( fjk-16s)-alexa647 , cd205/dec-205 ( nldc-145)-fitc , cd206/mr ( mr5d3)-fitc , pd - l1 ( m1h5)-pe , cd1d ( 1b1)-fitc , sign - r1 ( 22d1)-alexa647 , il-4 ( 11b11)-pe , il-17 ( tc11 - 18h10)-pe , il-10 ( jes5 - 16a3)-apc , il-13 ( ebio13a)-alexa647 , and ifn ( xmg1.2)-pe - cy7 .
biotinylated polyclonal rat anti - mouse galectin-1 ( r&d systems ) was used in conjunction with streptavidin - pe ( bd biosciences ) .
intracellular foxp3 staining was according to the manufacturer 's instructions ( anti - mouse / rat foxp3 staining set ; ebioscience ) .
for intracellular cytokine staining , cells were stimulated for 5 hours with 0.5 g / ml pdbu , and ionomycin and cytokine secretion was blocked using 1 g / ml brefeldin a ( all sigma ) .
cells were fixed promptly in 1% paraformaldehyde pbs and permeabilized with 0.5% saponin ( sigma ) in staining buffer .
for all staining , fc receptor ligation was blocked using anti - fcr 2.4g2 supernatant grown in house .
cells were acquired using a bd facscalibur , a bd facscan ( bd biosciences ) , or a cyan - adp ( beckman coulter ) and analyzed using flowjo ( treestar ) software .
nave t cells ( > 98% pure ) were sorted from splenic cell suspensions on the basis of cd4cd44cd25 using a moflo ( beckman coulter ) .
splenic dendritic cells ( > 90% pure ) were purified by immunomagnetic selection on an automacs pro ( miltenyi biotec ) . briefly , spleens were digested using liberase ci ( roche ) and passed through a 70 m strainer to obtain a single - cell suspension .
nonspecific binding was blocked using 2.4g2 anti - fcr supernatant , and dcs were positively selected using pan - dc microbeads ( miltenyi biotec ) .
for t cell polarization , immature dcs were differentiated from bone marrow precursors using gm - csf as previously described .
peritoneal exudates cells , pecs ( 50% cd11b ms ) , were flushed from the peritoneal cavity by lavage with cold pbs .
pancreatic and hepatic leukocytes populations were isolated from collagenase digested samples by centrifugation through a 33% percoll ( ge healthcare ) gradient as previously described ( eji ) .
total rna was extracted using an rneasy mini kit , converted to cdna using a reverse transcription kit , and quantified in real time using sybr green fast pcr ( all qiagen ) .
cdna was analyzed in duplicate reactions with amplification of target gene and housekeeping gene ( hypoxanthine phosphoribosyl transferase 1 , hprt1 ) transcripts performed on the same reaction plate on a 7500 fast real - time pcr system ( applied biosystems ) .
data are presented after normalization to the threshold cycle ( ct ) for hprt1 , either as ct ct or as hprt re = 1000 2ct ) statistical analyses were performed using graphpad prism 4 software .
tests performed and calculated two - tailed p values are indicated in the individual figure legends . for nonparametric datasets , the mann whitney unpaired t test was employed .
sea triggers important changes in dendritic cell ( dc ) function , which are key in initiating modulation of the host immune system [ 1419 ] . in the absence of additional tlr stimuli
, we have shown that small consistent phenotypic changes occur when dcs are stimulated in vitro with sea .
these receptors such as dec-205 , sign - r1 , mr , and galectins 1 and 3 , expressed on the surface of apcs , have been shown to recognize glycans present in schistosome antigens and are important for adaptive responses induced by the parasite [ 2025 ] .
here we confirm that primary splenic dcs stimulated in vitro with sea also upregulate the surface expression of galectins 1 and 3 , and sign - r1 , thus possibly increasing their ability to bind schistosome self - glycans ( figure 1(a ) ) . using bone marrow generated dcs ,
we have shown that the il-10/il-12 axis can be tilted by sea [ 5 , 6 ] . in figures 1(b ) and 1(c ) we show that purified spleen cd11c dcs stimulated in vitro with sea in the presence of tlr ligands upregulate the secretion of bioactive tgf whereas il-12 ( p40 and p35 ) mrna expression is downmodulated .
interestingly , sea in association of pam3cysk4 ( a tlr1/2 ligand ) induces the secretion of bioactive tgf , but this effect is not seen in combination with lps ( tlr4 ) or fsl-1 ( tlr2/6 , figure 1(b ) ) .
no significant changes were found in il-10 mrna expression from purified splenic dcs ( figure 1(c ) ) .
we examined the effect of i.p . injected sea on cellular phenotypes and function in the peritoneal cavity of nod female mice .
examination of cytokine expression at the rna level revealed that , while il-4 mrna was not detected in the control peritoneal cells ( pecs ) , there was a substantial expression in pecs from sea - treated mice , together with increased expression of il-2 , il-6 , il-10 , and tgf ( figure 2(a ) ) .
interestingly , il-12p35 expression was not affected by sea while il-12p40 was upregulated ( figure 2(a ) ) .
expression of other subunits of the il-12 family ( ebi3 , p28 for il-27 and p19 for il-23 ) was not affected by sea treatment ( data not shown ) .
the significant upregulation of tgf suggests the presence of alternatively activated macrophages ( aams ) in pecs from sea - treated mice and the increased expression of integrin 8 ( figure 2(a ) ) indicates an increased capacity to activate tgf at the point of the immunological synapse .
further support for the presence of aams is the upregulated expression of arginase-1 and 2 as well as fizz-1 ( figure 2(b ) ) . by comparison , treatment of nod mice with lps increased inos expression but , unlike with sea ,
the full range of markers of alternative activation was not seen ( figure 2(c ) ) .
analysis of surface marker expression on pecs identified potentially relevant costimulatory molecules that might indicate the presence of aams as well as clrs involved in the binding of sea . after sea injection there is an increased percentage of cells expressing mr on peritoneal ms ( pms ) with no significant change in mean fluorescent intensity ( mfi ) ( figure 3(a ) ) .
this increased surface expression was mirrored by an augmented receptor mrna expression in whole pecs .
the expression of sign - r1 and dectin-1 was not altered by sea when measured by facs or by rt - pcr ( data not shown ) .
galectin-3 expression has been shown to be a feature of aams and its upregulation is il-4 dependent . following sea injection we find an increased percentage of galectin-3-expressing pms and upregulation of both galectin 1 and 3 mrna ( figure 3(b ) ) .
sea increased pd - l1 expression on pms , with an accompanying increase in message for pd - l1 and 2 in whole pecs ( figure 3(c ) ) . at this time point sea also increased the expression of mhc class ii ( data not shown ) and the nonclassical class i molecule cd1d ( figure 3(d ) ) .
the negative costimulatory molecule programmed death ligand 1 ( pd - l1 ) has been shown to be upregulated on aams . altogether
our findings indicate that sea alternatively activates ms in the peritoneal cavity and suggests that this anti - inflammatory signature might be tied to expansion of tregs and th2 cells .
schistosome antigens are powerful inducers of th2 responses , and this has been extensively explored using splenic and peripheral lymph node cells [ 29 , 30 ] . in this study , we have extended our analysis to the pancreas in order to assess the potential impact of exposure to helminth antigens on diabetes and on the liver , the major site of immunopathology in schistosome infection .
kupffer cells , specialized liver ms , have also been shown to initiate th2 responses to egg antigens , and we have previously observed changes in classical nkt cell populations in the liver following schistosome antigen treatment of nod mice .
after a short course of treatment with sea , the intracellular cytokine expression in cd4 t cells is profoundly changed in the spleen , pancreas , and liver of prediabetic nod mice ( figures 4(a ) , 4(b ) , and 4(c ) ) . the systemic th2/treg response induced by sea
is accompanied by the signature of m alternative activation in the pancreatic infiltrate of prediabetic nod mice and is presumably beneficial for diabetes prevention ( figures 4(b ) and 4(d ) ) .
together with the expected th2 and il-10 tr1 ( cd4 il-10 ) response , we have identified a population of cd4 t cells expressing both il-10 and ifn in response to sea ( figures 4(b ) and 4(c ) ) . as il-10-producing th1 cells
have been proposed as a self - regulating class of th1 cells , the presence of this cd4 t cell population in the pancreas might also mitigate th1 driven diabetes pathology .
we have previously demonstrated that tgf is important for the generation and expansion of foxp3 tregs in nod mice .
our preliminary experiments suggest that the th2 response induced by sea can also be regulated by tgf. figure 5(a ) shows that the increased percentage of il-4/il-13 double - producing cd4 t cells that we observe in sea - treated nod mice can be decreased by in vivo coadministration of sea together with a tgf neutralizing antibody .
this effect is particularly evident in the pancreas and not the spleen or pln ( figure 5(a ) and data not shown ) .
we have also confirmed our findings with an in vitro polarization assay . using bone marrow generated dcs and nave t cells from nod mice
, we have previously shown that sea increases foxp3 tregs in vitro and that tgf neutralization reverts this effect .figure 5(b ) shows that blocking tgf in vitro reduces the generation of il-10 and il-4 single- and double - producing cd4 t cells .
also in vitro we observe that sea can induce the generation of il-10/ifn double - expressing cd4 t cells and that tgf regulates this population .
these preliminary data suggest that the role of tgf in adaptive responses to schistosome antigens is quite complex and is not restricted to the generation / expansion of foxp3 tregs .
we have recently suggested that foxp3 tregs have an important role to play in sea immunomodulation and prevention of autoimmunity in the nod mouse .
we found that in vitro only sea , and not swa , was able to induce foxp3 in nave t cells from nod mice . to examine the effects of these helminth antigens on tregs in vivo , we injected sea and swa into nod mice .
figure 6(a ) shows that following a short in vivo treatment with s. mansoni antigens foxp3 tregs increase only in sea , but not swa - treated mice .
further analysis of the liver of sea - treated nod mice revealed a highly significant increase in foxp3 tregs , which is consistent with the fact that tregs are found in the egg - induced granulomas in the liver and regulate their formation ( figure 6(b ) ) [ 31 , 32 ] .
these in vivo studies , therefore , support our in vitro studies and emphasize differences between the modes of activity of sea and swa .
there has been considerable interest in defining the ways in which s. mansoni infection perturbs the immune system and induces a th2 response . as we have previously shown that s. mansoni antigens are able to inhibit the th1-mediated autoimmune disease type 1 diabetes , we were particularly interested in clarifying this in nod mice .
we focused our attention on dcs and ms as these cell types play a role in orchestrating the adaptive immune response .
much work has been carried out examining the effects of sea on in vitro generated dcs , and while this has produced valuable insights , we felt that it is important to analyze the effects on populations of cells which would be exposed to s. mansoni antigens in vivo . in our studies of splenic
dcs we showed that following exposure to sea in vitro these cells upregulated galectins 1 and 3 , sign - r1 , and dec-205 following exposure to sea in vitro .
this is interesting because these c - type lectin receptors ( clrs ) have been shown to recognize glycans present in schistosome antigens and are important for adaptive responses induced by the parasite .
galectins are of particular interest as they have the potential to alter sensitivity to cytokines and apoptosis [ 20 , 33 ] .
exposure to sea upregulates , therefore , the capacity of the dc to respond to further exposure to helminth glycans .
, we found that splenic dcs downmodulated il-12p35 and p40 in response to sea whereas the in vivo response of pms showed an upregulation of the il-12p40 response with no effect on il-12p35 ( figure 2(a ) ) .
this presents another interesting way in which the th1 response could be modulated as it has been suggested that excess il-12p40 in both a monomeric ( p40 ) and dimeric ( p80 ) forms might antagonize the il-12 receptor , providing a feedback mechanism to downmodulate th1 responses . with regard to effects of sea administration in vivo on apc phenotype and function ,
several were noted that again had the potential to modify a diabetogenic th1 response .
sea increased pd - l1 expression on pms , with an accompanying increase in message for pd - l1 and 2 in whole pecs ( figure 3(c ) ) .
the negative costimulatory molecule programmed death ligand 1 ( pd - l1 ) has been shown to be upregulated on aams , as well as playing a role in diabetes regulation in nod mice .
effects of i.p . injected sea were noted in several sites , some of which were particularly relevant to diabetes regulation . after a short course treatment of nod mice with sea , the intracellular cytokine expression in cd4 t cells
was profoundly changed in the spleen , pancreas , and liver of prediabetic nod mice ( figures 4(a ) , 4(b ) , and 4(c ) ) . the systemic th2/treg response induced by sea
was accompanied by the signature of m alternative activation in the pancreatic infiltrate of prediabetic nod mice and would be beneficial for diabetes prevention ( figures 4(b ) and 4(d ) ) .
together with the expected th2 and il-10 tr1 ( cd4 il-10 ) response , we have identified a population of cd4 t cells expressing both il-10 and ifn in response to sea ( figures 4(b ) and 4(c ) ) . il-10/ifn
double - producing cd4 t cells were first identified in murine model of leishmania infection and shown to mediate chronic pathology in the skin lesions with delayed / reduced clearance of infection [ 36 , 37 ] . il-10-producing th1 cells
have been proposed as a self - regulating class of th1 cells , and therefore , this cd4 t cell population in the pancreas might also mitigate th1 driven diabetes pathology .
we have previously demonstrated that sea has direct effects on dcs and t cells and suggest that the simultaneous interaction with both cell types is important for determining the host immune response .
these in vitro studies had highlighted a role for tgf in the sea - mediated induction of regulatory t cells ; here we provide data suggesting that this immunomodulatory cytokine may also play a role in modulating the th2 response in vivo as well as in vitro .
s. mansoni and its soluble antigens can prevent autoimmune diabetes in nod mice .
this paper summarizes and confirms our previous work demonstrating that sea modulates the immune response through alteration of clr expression , cytokine production , and treg expansion in nod mice .
in particular we show how functional and phenotypic changes on dcs and ms , the expansion of different t helper cell subsets ( th2 ) , and treg cell types ( il-10/ifn- double producers and foxp3 tregs ) might contribute to diabetes prevention in nod mice ( figure 7 ) . | we have shown that schistosoma mansoni egg soluble antigen ( sea ) prevents diabetes in the nonobese diabetic ( nod ) mouse inducing functional changes in antigen presenting cells ( apcs ) and expanding t helper ( th ) 2 and regulatory t cell ( treg ) responses . a th2 response to s. mansoni infection or its antigens is key to both the establishment of tolerance and successfully reproduction in the host .
more recently we demonstrated that sea treatment upregulates bioactive tgf on t cells with consequent expansion of foxp3 + tregs , and these cells might be important in sea - mediated diabetes prevention together with th2 cells . in this study
we profile further the phenotypic changes that sea induces on apcs , with particular attention to cytokine expression and markers of macrophage alternative activation .
our studies suggest that tgf from t cells is important not just for treg expansion but also for the successful th2 response to sea , and therefore , for diabetes prevention in the nod mouse . |
diabetic high blood glucose or hyperglycemia causes mortality and morbidity through dn and dr via disrupting the vascular function in the kidney and retina .
these pathologies are the major cause of death associated with renal failure and blindness among the type 1 and type 2 diabetes patients [ 13 ] .
similar molecular pathways appear to govern the development of diabetic renal and retinal microvascular injury .
this speculation arises from higher coincidence rates of dn and dr ; that is , patients with dn have already developed dr and patients with dr are vulnerable to develop dn [ 4 , 5 ] . according to the similarities in pathologic background affecting the retina and kidney , it was hypothesized that dn and dr could arise by injuries to perivascular supporting cells in glomeruli and retina .
the vulnerable vascular cell types affected by diabetic hyperglycemia are retinal pericytes ( rpc ) and their analogs within glomeruli are the mesangial cells . in dr ,
the rpc undergo cell death and disengage from the retinal vasculature , predisposing the retina to neoangiogenesis , vascular leakage , and tractional retinal detachment culminating in reduced vision or terminal blindness [ 68 ] .
dn is characterized by mesangial matrix expansion and the obstruction of the glomerular capillaries within the renal filtration units , the glomeruli .
the glomerulopathy in dn is associated with reduced efficiency of renal filtration , and in chronically established cases it triggers end - stage renal failure and mortality .
several biochemical mechanisms and pathways have been described in speculation of the pathogenesis caused by either dn or dr . these mechanisms include enhanced oxidative stress , enhanced polyol pathway , pkc activation , inflammation , and advanced glycation end product formation [ 3 , 1113 ] .
the most extensively investigated mechanisms associated with the development of both dn and dr are enhanced oxidative stress and inflammation caused by metabolic alterations .
however , a plethora of evidence indicates that immunological and inflammatory mechanisms are important factors in development and progression of dr and dn [ 1417 ] .
the recruitment of the activated macrophages and increased generation of inflammatory and proinflammatory mediators ( tnf- , il-1 , il-6 , mcp-1 , and opn ) in retinal and renal milieu is linked to the progression and exacerbation of the dn and dr [ 1722 ] . however , the exact molecular and cellular mechanisms involved have remained elusive .
oxidative stress is a phenomenon in which the balance between the generation of free oxidizing radicals and the system responsible for their removal in the cell is disturbed leading to enhanced formation of free reactive ions including reactive oxygen species ( ros ) .
the increased ros levels in cells subjected to diabetic hyperglycemia drive the antioxidant gene expression to protect the cells from oxidative injuries .
one of the target genes to be induced following oxidative stress is the nf - e2-related transcription factor 2 ( nrf2 ) .
two of the recently characterized genes that are regulated by the nrf-2 and provide protection against oxidative stress and stress - adaptation are proteasome activator genes , pa28 and pa28 .
it is speculated that pa28 proteins increase the proteasomal degradation activity to clear the oxidized or misfolded proteins [ 2426 ] .
the pa28/ genes were initially identified as components of immunoproteasomes which are induced in response to interferon-. pa28 and pa28 proteins form a heptameric complex ( 4/3 ) acting as the gate opener for the 20s proteasomes , hence stimulating the degradation of the nonubiquitinated short peptides . the well - characterized function for the pa28 proteins is the generation of the antigenic peptides to be presented by the mhc class i molecules [ 27 , 28 ] .
the pa28/ are upregulated in the cultured rpc under high glucose conditions and also in the intraglomerular capillaries of older type 1 diabetic akita mice . to understand the role of pa28/ genes in development of dn and dr ,
pa28dko mice were tested and their physiological and biometric indices were compared with stz - induced diabetic wild type mice .
the diabetic pa28dko mice provided higher survival rate , higher body weight , more efficient renal filtration function , and reduced microvascular damage in their retinae compared to diabetic wild type mice .
the glomeruli , mesangial cells , and the rpc isolated from pa28dko mice had lower levels of opn and mcp-1 under high glucose conditions .
opn is known as a proinflammatory protein associated with progression of diabetic microvascular injury [ 19 , 22 ] .
the pa28-dependent regulation of opn expression was stimulated by high glucose and abrogated by synthetic peptides that block the binding of pa28 to 20s proteasomes . therefore ,
the findings of this study provide novel insights into the role of the ubiquitin proteasome system ( ups ) and specially the pa28 proteins , in regulating the microvascular injury in diabetes .
animal maintenance , genotyping , treatments , and analytical procedures followed the guidelines accredited by institutional animal care and use committee of the university of wisconsin , school of medicine and public health .
all the mice used for the experiments were in c57bl/6 background . for cell isolation ,
6-week old male immorto mice ( stock number 006553 ) and to study diabetes , pa28dko male mice ( stock number 021202 ) were used .
diabetes was induced in animals with a single injection of streptozotocin ( stz ; 180 mg / kg in citrate buffer , ph 4.2 by ip injection ) .
three days following the treatment , the blood glucose of all stz - injected animals was above 400 mg / dl .
for acr analysis the urine samples were collected by housing each individual mouse in a metabolic cage ( tecniplast , italy ) for 24 hours .
urinary albumin and creatinine levels were measured by elisa ( albuwell m , exocell ) and the acr measurements were conducted according to the guidelines provided by the manufacturer .
mouse kidneys were sliced and immersion - fixed in a solution of 2% paraformaldehyde ( pfa ) and 2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer , ph 7.4 , overnight at 4c .
the tissue was postfixed at room temperature for 2 hours in 1% osmium tetroxide in the same buffer .
subsequently the samples were dehydrated in a graded ethanol series , then dehydrated in propylene oxide , and embedded in epon epoxy resin .
ultrathin sections were prepared using leica uc6 ultramicrotome and mounted on 200-mesh carbon - coated copper grids .
tissue sections were observed with a philips cm120 electron microscope , and images were captured with a megaview iii side - mounted digital camera . for pas and jms - h&e staining the formalin fixed ,
paraffin embedded , 5 - 6 m tissue sections were deparaffinized in xylene and rehydrated in descending graded percentage of ethanol ( 100% , 95% , 80% , and 70% ) and routinely stained with recommended reagents .
digital images were taken with na pl apo objectives ( 10x/0.25 na , 40x/0.95 na , and 63x/1.4 na oil ) on a scanscope xt system using imagescope version 10 software ( aperio technologies inc . ) .
briefly , the retinae from one litter ( 6 - 7 pups , 6-week old ) immorto mice were collected under a dissecting microscope .
the collected retinae were rinsed with serum - free dulbecco 's modified eagle 's medium ( dmem ) , pooled , minced , and digested for 45 min with collagenase type ii ( 1 mg / ml , worthington ) with 0.1% bsa in serum - free dmem at 37c .
cells were resuspended in equal amount of dmem containing 10% fetal bovine serum ( fbs ) and spun for 5 min at 400 g .
the pelleted cells were resuspended in 4 ml dmem containing 10% fbs , 2 mm l - glutamine , 100 g / ml streptomycin , 100 u / ml penicillin , and recombinant murine ifn- ( r&d systems ) at 44 u / ml .
cells were evenly divided into 4 wells of a 24-well tissue culture plate and maintained at 33c with 5% co2 .
cells were progressively passed to larger plates and maintained and propagated in 60 mm dishes .
the retinae from one litter ( 6 to 7 pups , 6-week old ) of immorto mice were dissected out aseptically under a dissecting microscope and kept in hbss buffer containing penicillin / streptomycin .
retinae were pooled together , rinsed with hbss buffer ( life technologies ) , minced into small pieces in a 60 mm tissue culture dish using sterile razor blades , and digested in 5 ml of collagenase type i ( 1 mg / ml in serum - free dmem , worthington ) for 45 min at 37c . following digestion , dmem with 10%
the cells were passed through a sterile 40 m nylon strainer ( bd falcon ) and spun at 400 g for 10 min to pellet cells , and cells were washed twice with dmem containing 10% fbs .
the cells were resuspended in 1.5 ml medium ( dmem with 10% fbs ) and incubated with anti - pecam-1 antibody - conjugated dynabeads ( life technologies ) . after affinity binding ,
magnetic beads were rinsed six times with dmem with 10% fbs and bound cells in endothelial cell growth medium were plated into a single well of a 24-well plate precoated with 2
endothelial cells were grown in dmem containing 20% fbs , 2 mm l - glutamine , 2 mm sodium pyruvate , 20 mm hepes , 1% nonessential amino acids , 100 g / ml streptomycin , 100 u / ml penicillin , freshly added heparin at 55 u / ml ( sigma ) , endothelial growth supplement 100 g / ml ( sigma ) , and recombinant murine ifn- ( r&d systems ) at 44 u / ml .
cells were progressively passed to larger plates , maintained , and propagated in 1% gelatin - coated 60 mm dishes .
briefly , 8 10 dynabeads ( life technologies ) were diluted in 40 ml of phosphate - buffered saline and perfused through the heart of the adult mice .
the kidneys were extracted , mechanically minced , digested with type 1 collagenase , and filtered through 100 m strainer .
the glomeruli were collected with a magnet and washed three times to eliminate the nonglomerular cells and debris .
the glomeruli were grown in either pericytes growth medium to establish the mesangial cell culture .
the kgec were isolated from the glomeruli similar to mesangial cell isolation following a procedure described before .
the purified glomeruli were seeded and allowed to grow in rec medium on coated 35 mm plates . upon 8085% confluency , the cells were incubated with anti - pecam-1 antibody - conjugated dynabeads , washed , and enriched similar to rec cultures .
the bpc , bec , kpc , hpc , and lpc were isolated by following a procedure similar to rpc isolation .
the peptides ( cpc scientific ) and the chariot transfection reagent ( active motif ) were reconstituted and used according to the manufacturer guidelines .
the enucleated eyes were fixed in 4% paraformaldehyde for at least 24 h. the dissected retinae were washed overnight in deionized water and incubated in 3% trypsin ( difco trypsin 250 , becton dickinson and company ) prepared in 0.1 m tris , 0.1 m maleic acid , and ph 7.8 containing 0.2 m naf for 2 h at 37c .
the neuroretinal tissue was gently brushed away , and the resultant isolated vascular tree was air dried onto a glass microscope slide .
total rna from cells was extracted by total rna isolation kit ( norgenbiotek ) according to the manufacturer 's instructions .
cells were allowed to reach 8590% confluence , rinsed twice with pbs , scraped off the plates , and transferred to rnase - free microfuge tubes .
one microliter of 1/10-diluted cdna was used as real - time pcr template in triplicate groups and reaction was completed using sybr green master mix in mastercycler realplex ( eppendorf ) with the specified primers in supplemental experimental procedures .
thermal cycles were programmed as 95c for 2 min ; 40 cycles of amplification ( 95c for 15 s and 60c for 40 s ) ; and a dissociation curve step ( 95c for 15 s , 60c for 15 s , and 95c for 15 s ) .
standard curves were generated from known quantities for each target gene of linearized plasmid dna .
tenfold diluted series were used for each known target to be amplified by sybr green qpcr premix .
the linear regression line for ng of dna was calculated from relative fluorescent units at a threshold fluorescence value ( ct ) to quantify amplification targets from cell extracts by comparing the relative fluorescent units at the ct to the standard curve , normalized by the simultaneous amplification of rpl13a ( a housekeeping gene ) for all samples . for protein isolation ,
the cells in 80% confluent dishes were harvested following adding the lysis buffer ( 150 mm nacl , 1% triton x-100 , 20 mm tris - hcl ( ph 7.4 ) , protease inhibitor cocktail ( 1 mm pmsf , 10 mm leupeptin , 10 mm pepstatin ) ) . for western blot analysis ,
50 g cell lysate or conditioned medium proteins were mixed with laemmli sample buffer and the reduced or nonreduced samples were boiled for 10 min .
the boiled samples were loaded into 420% tris glycine acrylamide gels ( life technologies ) and electrophoresis was performed using sds running buffer ( 25 mm tris , 190 mm glycine , 0.1% sds , ph 8.3 ) .
mouse cdna encoding the pa28 and pa28 were cloned into plvx - ires - hyg and plvx - ires - neo plasmids ( clontech ) , respectively .
the lentiviral particles were prepared by transfecting the lenti - x 293 t cells ( clontech ) and collecting the conditioned media . the pbabe - opn / puro plasmid and the packaging cells are described elsewhere .
cultured cells were transfected using the fugene6 reagent ( promega ) according to recommended protocol . after 48 h , hygromycin b ( 500 g / ml ; sigma - aldrich ) ,
neomycin ( 750 g / ml ; life technologies ) , and puromycin ( 5 g / ml ; sigma - aldrich ) were added to cell cultures . the data are represented as the mean sem .
statistical significance was determined by analysis of variance ( anova ) and tukey - kramer post hoc analysis for multiple comparisons using p value of 0.05 in graphpad prism software ( san diego , ca , usa ) .
the pa28 proteasome regulators were previously shown to be dramatically upregulated in the glomerular capillaries of akita mouse model with chronic hyperglycemia for 8 months but not in the same age wild type nondiabetic mice . in this study
the role of pa28/ in modulating diabetic microvascular disorders was investigated via streptozotocin ( stz ) injection to both wild type and pa28dko mice .
stz has cytotoxic effects on insulin - secreting pancreatic -cells and induces type i diabetes in experimental animals .
all the pa28dko / stz and wild type / stz mice had blood glucose levels above 450 mg / dl three days after stz injection and on the day of sacrifice ( table 1 ) . in comparison with pa28dko / stz mice ,
the wild type / stz mice exhibited significantly reduced survival rates and weight loss by four months of stz injection .
the pa28dko / stz mice were protected and had no obvious weight loss or reduced survival after six months of stz injection ( figures 1(a ) and 1(b ) ) .
it was assumed that improved renal function was responsible for the observed improvement in the health of the pa28dko / stz mice . to test this ,
acr is a cornerstone assay for the diagnosis of renal filtration disorders [ 36 , 37 ] and reflects the efficiency of the kidney filtration function .
the measured albuminuria was significantly higher in wild type / stz ( 291 8.54 g / mg ) compared to that of non - stz wild type ( 25.61 2.18 g / mg ) mice and 28dko / stz mice ( 119.6 5.61 g / mg ) ( figure 1(c ) ) .
next the histological and ultrastructural properties of the glomeruli in the non - stz and stz - injected mice were assessed .
renal histology in kidney sections was evaluated using the combined jones methenamine silver- ( jms- ) hematoxylin and eosin ( h&e ) staining and also periodic acid schiff ( pas ) staining .
the jms and pas staining is routinely used to detect the mesangial matrix increase and changes in the glomerular basement membrane ( gbm ) .
there were no discernible alterations in the appearance of gbms , the adjacent tubules , and also the mesangial or endothelial cells in different animal groups ( figure 2(a ) ) .
similar to light microscopic analysis , tem analysis did not show any significant difference in the thickness of gbm in wild type / stz and pa28dko / stz mice .
the lack of visible diabetic glomerular pathology in tem test despite the reduced renal filtration efficiency ( determined by acr test ) in mice studied here is likely due to the resistance of the c57bl/6 mouse line against morphological changes in gbm ( figures 2(b ) and 2(c ) ) .
however , there was a significant reduction in the relative number of intraglomerular endothelial fenestrae in four - month stz - injected wild type mice compared to those of pa28dko / stz mice ( 47.5 1.74 versus 60.3 0.67 ) ( figure 2(d ) ) . similarly , the six - month stz - injected pa28dko mice provided higher endothelial fenestrae compared with four - month stz - injected wild type mice ( 56 0.93 versus 47.5 1.74 ) ( figure 2(d ) ) .
since the inflammatory mediators opn and mcp-1 have been shown to be closely associated with the development and progression of the dn and dr , we investigated their levels using real - time pcr in the glomerular mrna isolated from stz - injected and nondiabetic wild type and pa28dko mice .
the qpcr analyses did not provide any differences in the levels of il-1 and tnf- mrna among wild type and pa28dko mice under both normal and stz- injected conditions ( not shown ) . however , the mrna for opn and mcp-1 showed significant increase in wild type / stz versus nondiabetic wild type glomeruli ( figures 3(a ) and 3(b ) ) . interestingly , unlike the wild type / stz glomeruli , in pa28dko / stz glomeruli the expression of opn and mcp-1 was not altered significantly .
these data demonstrate that pa28 and pa28 regulate the diabetic glomerular vascular injuries most likely through regulating the expression of proinflammatory agents . to understand how pa28 and pa28 genes promote glomerular injuries during diabetes , as an in vitro model , the mesangial cells from wild type and pa28dko mice
were isolated for further characterizations [ 29 , 31 , 39 ] . since the mrna levels for opn and mcp-1 in pa28dko / stz glomeruli were lower than those of wild type / stz mice and the expression of these proteins
has been linked to the development of dn and dr , we investigated whether higher glucose concentrations in vitro could induce the expression of opn and mcp-1 genes in isolated mesangial cells .
western blot and qpcr analysis of these genes were performed following three days of treatment with either normal ( 5 mm d - glucose ) , high glucose ( 25 mm or 40 mm d - glucose ) , or osmolarity control ( 5 mm d - glucose plus 20 mm l - glucose ) .
western blot analysis of the conditioned medium provided robust induction of opn expression in wild type but not in pa28dko mesangial cells following treatment with high glucose ( figure 4(a ) ) .
the qpcr analysis of wild type and pa28dko cells also confirmed the upregulation of opn expression in wild type but not in pa28dko mesangial cells treated with high glucose ( figure 4(b ) ) .
because mcp-1 protein could not be detected by western blot ( not shown ) , we used qpcr to evaluate changes in mcp-1 transcript levels in cultured mesangial cells under normal and high glucose conditions .
consistent with the qpcr data for opn , the mcp-1 transcripts were significantly increased in the wild type mesangial cells grown under high glucose conditions , whereas the pa28dko cells had markedly lower levels of mcp-1 transcripts at the same conditions ( figure 4(c ) ) .
it was next tested whether lentiviral - mediated reexpression of the pa28 and pa28 individually or simultaneously restored the opn expression in pa28dko mesangial cells .
the reexpression of pa28 alone or pa28 and pa28 together restored the opn release from pa28dko cells under both normal and high glucose conditions ( figure 4(d ) ) .
the overexpression of pa28 resulted in less vigorous expression of opn compared to simultaneous overexpression of both pa28 and pa28. this effect could be attributed to the ability of the pa28 subunits to form an active homoheptameric proteasome regulatory complex that has not been reported for pa28 monomers .
dr is another microvascular complication of diabetes attributed to the diminution of pericytes in retinal microvessels and subsequent death of endothelial cells . in retina
therefore , we investigated to test in respect of protection against glomerular microvascular injury in pa28dko / stz mice whether these mice were similarly protected against microvascular injury in the retina .
compared with four - month stz - injected wild type mice , the prepared retinal flat mounted microvessels of pa28dko mice showed remarkable reduction in the number of acellular capillaries after four months ( 8.71 0.43 versus 4.43 0.45 ) and six months of stz injection ( 8.71 0.43 versus 5.07 0.28 ) , respectively ( figures 5(a ) and 5(b ) ) .
reduction in the number of the acellular capillaries in pa28dko / stz retinae demonstrated that loss of pa28/ genes protected the rpc against cell death triggered by hyperglycemia .
to assay the effect of high glucose treatment on cultured rpc and investigate whether they respond to high glucose the same as to mesangial cells , rpc were isolated and cultured from wild type and pa28dko mice .
high glucose treatment of wild type rpc robustly induced the expression of opn and mcp-1 while the pa28dko rpc treated with high glucose did not manifest such increase in opn and mcp-1 expression ( figures 5(c)5(e ) ) . next it was tested whether increased expression of opn in response to high glucose was uniquely restricted to rpc and mesangial cells or other vascular cell types also responded similarly .
for this purpose , different mouse endothelial and perivascular cell types were isolated from various organs and exposed to high glucose similar to rpc and mesangial cells .
these cells included retinal endothelial cells ( rec ) , kidney glomerular endothelial cells ( kgec ) , total kidney nonglomerular pericytes ( kpc ) , brain pericytes ( bpc ) , brain endothelial cells ( bec ) , heart pericytes ( hpc ) , and lung pericytes ( lpc ) .
western blot analyses of opn demonstrated that any of these vascular cell types did not contain elevated opn production under high glucose conditions ( figure 5(f ) ) .
hence , increased production of opn under high glucose conditions is a unique characteristic of rpc and mesangial cells . to investigate if the opn release from mesangial cells exposed to high glucose was caused by the binding of pa28/ proteins to the 20s proteasomes , we examined peptides capable of disrupting the association of pa28/ with the 20s proteasome .
the pa28-inhibitory peptides fused to tat domain of the hiv virus were used for this assay .
tat sequence was added to the xapc7 and hbx peptides to promote the transduction of the cultured cells .
the xapc7-tat and hbx - tat peptides as well as scrambled control peptides fused to tat were used to transfect the cultured mesangial cells for three days under high glucose condition to promote opn release .
western blot analyses of conditioned media provided strong inhibition of opn release after treatment with xapc7-tat and hbx - tat at 5 m and 10 m concentrations , respectively ( figures 6(a ) and 6(b ) ) .
treatment with the liver x receptor ( lxr ) agonists , t0901317 and gw3965 , was shown to inhibit opn release and ameliorate the severity of dn . hence , as positive control for our peptide treatment experiments and suppression of opn release , we used lxr agonists in cotreatment of the mesangial cells with high glucose . as a result ,
almost both t0901317 and gw3965 completely inhibited the opn release at 10 m concentrations ( figures 6(a ) and 6(b ) ) .
interestingly , the tat - fused scrambled peptides also suppressed opn release in response to high glucose .
tat peptide had been previously demonstrated to inhibit the pa28 binding to the 20s proteasomes [ 43 , 44 ] .
therefore , the inhibitory effect of the tat - scrambled peptides could be attributed to the tat domain of the peptides .
however , treatment of the mesangial cells with tat - only peptide even at 200 m concentration did not inhibit the opn release ( figure 6(c ) ) .
the failure of the tat - only peptide in inhibiting the opn release under high glucose conditions suggests that tat requires fusion to a juxtaposing peptide to inhibit pa28 binding to the 20s proteasome . in order to exclude the inhibitory effect of the tat domain , xapc7 and hbx peptides without tat domain
treatment with xapc7 and its scrambled control resulted in complete dose - dependent inhibition of the opn release ( figure 6(d ) ) .
this effect of control peptide is possibly caused by the shorter size and positive charge of these peptides compared with hbx peptide .
the hbx showed marked reduction in opn release at various concentrations but this inhibition was less potent than xapc7 .
the scrambled hbx peptide did not markedly affect the opn release from the mesangial cells ( figure 6(d ) ) .
collectively , these findings demonstrate that high glucose - induced binding of pa28 and pa28 to 20s proteasomes in rpc and mesangial cells regulates opn production .
taking advantage of the pa28dko mice , the mesangial cells , and rpc isolated from pa28dko mice it was demonstrated in this study that pa28 and pa28 genes are key determinants of diabetic microvascular injuries .
evidence is provided for the involvement of pa28 proteasome regulators in exacerbating the pathogenesis of dn by demonstrating that , compared with wild type / stz mice , the pa28dko / stz mice do not develop severe albuminuria .
further testing provided that , compared to wild type , the opn and mcp-1 inflammatory mediators are markedly suppressed in the pa28dko / stz glomeruli , mesangial cells , and rpc under high glucose conditions .
opn secretion was regulated by pa28/ and , among different vascular cell types , was only detectable in cultured rpc and mesangial cells exposed to high glucose .
this is consistent with the higher sensitivity of rpc and mesangial cells to the cytotoxic effects of high glucose compared to other vascular cell types including the rec , kgec , bpc , and bec .
these findings have strong implications in addressing why the trigger for the development of diabetic microvascular complications lies in the rpc and mesangial cells .
the cell injury and death of these perivascular supporting cells in the retina and kidney caused by hyperglycemia precede the development of dr and dn .
the increased opn expression is reportedly associated with the development of dn and dr and pharmacological reagents capable of inhibiting opn release provide protection against dn and dr [ 21 , 42 , 45 ] .
opn has macrophage chemoattractive function and the progression of dn and dr is facilitated by the recruitment of the macrophages and monocytes into retina and kidney .
opn release by perivascular cells in diabetic kidney and retina might regulate the local inflammatory cues to increase macrophage recruitment and activation .
the novel function of pa28 and pa28 proteins in regulating opn expression and diabetic microvascular injury demonstrates that , apart from their antigen processing properties , these proteins possess unexplored functions in sensing and responding to metabolic changes in a certain subset of vascular cells .
importantly , the increased expression of opn following exposure to high glucose exclusively in rpc and mesangial cells that are main targets of diabetic hyperglycemia injury demonstrates a cell - specific function for pa28 and pa28 in sensing and responding to altered glucose levels .
similar to lxr agonists , t0901317 and gw3965 , that inhibit opn expression and the severity of dn in stz diabetic mice , the delivery of peptides that inhibit pa28 binding to 20s proteasomes , the xapc7 , hbx , and tat - conjugated peptides inhibited opn release from mesangial cells under high glucose conditions .
this suggests that opn expression under high glucose conditions from mesangial cells is regulated by pa28 and pa28 binding to the 20s proteasome . in agreement with the role of pa28 and pa28 in regulating the opn expression , the reexpression of pa28 alone or pa28 and
interestingly the expression of pa28 alone also stimulated the opn release from the pa28dko mesangial cells .
this observation can be explained by the exceptional ability of the pa28 monomers to form an active regulatory complex capable of binding and activating the 20s proteasomes .
presently , it is unclear whether the reduced expression of opn or mcp-1 alone is accountable for the ameliorated diabetic microvascular injury in pa28dko mice or other pathways are also involved .
nevertheless , since other groups showed that proteasome inhibition in mice attenuates the development of dn [ 4648 ] , the relative inhibition of the proteasomes in pa28dko rpc and mesangial cells would also provide a suitable explanation for their protection against diabetic renal and retinal injury .
this can be supported by previous study in which the pa28 proteins were shown to be upregulated in rpc and also in diabetic glomeruli where expression level of pa28 proteins correlated with the severity of diabetic renal injury [ 1 , 49 ] .
it therefore appears that excessive activation or deregulation of the proteasomes by upregulated pa28 proteins could possibly result in microvascular damage in the retinal and glomerular vasculature .
other investigators have shown that high glucose treatment of rpc and mesangial cells induces oxidative stress [ 50 , 51 ] .
the pa28 and pa28 were demonstrated to play key role in the clearance of misfolded and oxidized proteins and adaptation to oxidative stress in cultured cardiomyocytes .
similarly , in rpc and mesangial cells , the pa28 and pa28 proteins could assist the rpc and mesangial cells in their adaptation to oxidative stress caused by high glucose .
for instance , it was shown that rpc have lower proteasomal degradational capacity compared to other vascular cell types such as rec .
therefore , pa28-/- proteins could assist the degradation of proteins damaged by exposure of rpc to high glucose but overt activation of the proteasomes in rpc and mesangial cells provokes tragic consequences .
this study provides major insights into the role of pa28 and pa28 genes in promoting diabetic renal and retinal microvascular injury .
first of all , deletion of pa28 and pa28 genes protected diabetic animals against dn . secondly , the expression of mcp-1 and opn as important mediators of dn or dr was shown to be regulated by pa28 and pa28 in rpc and mesangial cells grown under high glucose .
thirdly , pa28dko mice showed reduced severity of dr reflected by reduced number of acellular capillaries in their retinae .
lastly , the suppressive effect of pa28-inhibitory peptides on opn release by mesangial cells grown in high glucose was demonstrated .
therefore , modulating the degradational activity of ubiquitin proteasome system mediated by pa28 and pa28 proteins offers a suitable candidate for the development of future treatments for microvascular complications of diabetes particularly in the retina and kidney . | diabetic nephropathy ( dn ) and diabetic retinopathy ( dr ) are major complications of type 1 and type 2 diabetes .
dn and dr are mainly caused by injury to the perivascular supporting cells , the mesangial cells within the glomerulus , and the pericytes in the retina .
the genes and molecular mechanisms predisposing retinal and glomerular pericytes to diabetic injury are poorly characterized . in this study ,
the genetic deletion of proteasome activator genes , pa28 and pa28 genes , protected the diabetic mice in the experimental stz - induced diabetes model against renal injury and retinal microvascular injury and prolonged their survival compared with wild type stz diabetic mice .
the improved wellbeing and reduced renal damage was associated with diminished expression of osteopontin ( opn ) and monocyte chemoattractant protein-1 ( mcp-1 ) in the glomeruli of stz - injected pa28/pa28 double knockout ( pa28dko ) mice and also in cultured mesangial cells and retinal pericytes isolated from pa28dko mice that were grown in high glucose .
the mesangial pa28-mediated expression of opn under high glucose conditions was suppressed by peptides capable of inhibiting the binding of pa28 to the 20s proteasome .
collectively , our findings demonstrate that diabetic hyperglycemia promotes pa28-mediated alteration of proteasome activity in vulnerable perivascular cells resulting in microvascular injury and development of dn and dr . |
adenocarcinoma of the vermiform appendix is a rare neoplasm of the gastrointestinal tract with an incidence of about 0.1% to 0.2% .
age - adjusted incidence of cancer of the appendix is 0.12 cases per 1 000 000 per year .
these rare tumors are seldom suspected before surgery , and < 1/2 are diagnosed intraoperatively .
the most common presentation of appendiceal malignancy is right lower abdominal pain that often mimics acute appendicitis .
right iliac fossa mass and intestinal obstruction have also been reported ; these presentations reflect various stages of a locally expanding tumor causing luminal obstruction of the appendix .
there are other clinical presentations , and we report herein a case of appendicular adenocarcinoma found unexpectedly in a patient who presented to the gastroenterology unit with per - rectal bleeding . the patient is a 55-year - old lady admitted with rectal bleeding due to the penetration of mucinous adenocarcinoma of the appendix into the sigmoid colon .
based on a review of the literature , this is the first reported case of an appendiceal malignancy presenting and being treated in this manner .
we diagnosed a case of appendicular mucinous cystadenocarcinoma found unexpectedly in 55-year - old lady admitted to the gastroenterology unit because of per rectal bleeding and a rectal polyp suspected on an outpatient colonoscopy .
colonoscopy revealed evidence of external compression in the rectosigmoid junction with tumor infiltration of the sigmoid mucosa . in the rest of the colon
, there was a small polyp in the ascending colon that was removed by snaring .
gastrografin enhanced ct - scan after per rectal filling and maximal distension of the rectum and sigmoid colon revealed a tightly stenosed 6.5-cm lumen in the distal part of the sigmoid colon .
specifically , this segment of the sigmoid colon was infiltrated by a 6 cm 6 cm large hypodense irregular tumor mass that also infiltrated part of the adjacent ileum .
this tumor mass was connected to the end of the appendix , which had a diameter of 1.2 cm ( figure 1 ) .
computed tomographic scan showing sigmoid colon infiltrated by 6 cm 6 cm large hypodense irregular tumor mass connected to the end of the appendix .
the clinical investigations , ct and colonoscopy , indicated the possibility of appendicular carcinoma with an infiltrated sigmoid colon .
a laparoscopic sigmoid colectomy was performed first . with the patient in the supine position ,
pneumoperitoneum was established with the so - called open technique , and 4 trocars were placed in the upper , lower , left , and right abdomen .
the chief surgeon operated from the right side via the first monitor placed at the left side of the patient , and the assistant was on the left side .
exploration of the abdomen showed a conglomerate tumor in the pelvis with involvement of the cecum and sigmoid colon .
for sigmoid and right colon resection , we mobilized , devascularized , divided , and resected the colon intracorporeally .
the retroperitoneum was incised medial to the left ureter , and the left hypogastric nerve was cautiously identified .
after the sigmoid colon had been mobilized , 2 mesenteric windows were created at the sigmoid mesentery , one at the level of the rectosigmoid junction , and the second at the midsigmoid level .
the inferior mesenteric vessel was divided with a vascular stapler , and the mesosigmoid was dissected up to the proximal part of the sigmoid colon .
after that , the distal part of the sigmoid colon and the proximal rectum were mobilized under the tumor mass attachment . following this
, we mobilized the right colon as described previously . the chief surgeon operated from the left side of the patient , and the endoscope
was placed into the left paraumbilical trocar . to carry out the lymphadenectomy simultaneously with the resection of the vascular stem , primarily the laterally running ileocolic / right
colic arteries and the branches of the superior mesenteric artery were sought out , exposed , and ligated using a window technique . the laterocaudal mobilization extended to the cecal pole and the terminal ileum , cranially to the ascending and meso - ascending colon until the mesenteric root in the outflow area of the lateral colonic vessels . after the terminal ileum was mobilized , the ileum was divided with a stapler . at this stage ,
the tumor mass including the sigmoid colon and fully mobilized cecum and distal part of the sigmoid colon under the tumor were well visualized and easily divided . with this action
as the preparation was advanced cranially , the lateral abdominal wall ties were released , at first , using the ultrasonic dissector or scissors step - by - step until the flexure .
then , the ascending colon was released of its retroperitoneal ties . with a properly layered dissection towards the aorta between the posterior side of the meso ascending colon and the renal fascia ,
the preparation was continued until the horizontal part of the duodenum came into view and the stump was detached from its mesenteric ties .
a dissection rod was indispensable for the traumatic dissection , because the embryonically preformed tissue strands were best detached from one another by blunt dissection . with detachment of the right colonic flexure , the status of the already mobilized colomesenteric segment changed in such a way that a caudal dissection of the flexure could become complicated , eg , at the vascular stems , due to visual obstruction by the falling intestinal convolution .
after complete mobilization of the right - sided colon intended for resection , the pneumoperitoneum was released , the trocar removed , and the incision extended for minilaparotomy .
externalization of the prepared sigmoid and right colon was carried out through a foil ring introduced into the minilaparotomy over the symphysis pubis for wound protection .
after the right transverse colon was divided extracorporeally and the specimen removed , the distal ileum was exteriorized ( figure 2 ) .
after the anvil was placed in the descending colon , then retained into the abdomen , the incision was closed .
we placed the stapler through the rectum and mated it with the anvil laparoscopically to complete the colorectal anastomosis .
first flatus was recognized on the second postoperative day , a solid diet was started on the third postoperative day , and the patient was discharged and went directly home on the eighth postoperative day ( figure 4 ) .
histopathological examination showed mucinous adenocarcinoma of the appendix with infiltration of the sigmoid colon of about 6 cm ( figure 3 ) .
the patient seen 2 months after surgery was in good general condition with no complications .
a laparoscopic sigmoid colectomy was performed first . with the patient in the supine position ,
pneumoperitoneum was established with the so - called open technique , and 4 trocars were placed in the upper , lower , left , and right abdomen .
the chief surgeon operated from the right side via the first monitor placed at the left side of the patient , and the assistant was on the left side .
exploration of the abdomen showed a conglomerate tumor in the pelvis with involvement of the cecum and sigmoid colon .
for sigmoid and right colon resection , we mobilized , devascularized , divided , and resected the colon intracorporeally .
the retroperitoneum was incised medial to the left ureter , and the left hypogastric nerve was cautiously identified .
after the sigmoid colon had been mobilized , 2 mesenteric windows were created at the sigmoid mesentery , one at the level of the rectosigmoid junction , and the second at the midsigmoid level .
the inferior mesenteric vessel was divided with a vascular stapler , and the mesosigmoid was dissected up to the proximal part of the sigmoid colon .
after that , the distal part of the sigmoid colon and the proximal rectum were mobilized under the tumor mass attachment . following this
, we mobilized the right colon as described previously . the chief surgeon operated from the left side of the patient , and the endoscope
was placed into the left paraumbilical trocar . to carry out the lymphadenectomy simultaneously with the resection of the vascular stem , primarily the laterally running ileocolic / right
colic arteries and the branches of the superior mesenteric artery were sought out , exposed , and ligated using a window technique . the laterocaudal mobilization extended to the cecal pole and the terminal ileum , cranially to the ascending and meso - ascending colon until the mesenteric root in the outflow area of the lateral colonic vessels .
after the terminal ileum was mobilized , the ileum was divided with a stapler . at this stage ,
the tumor mass including the sigmoid colon and fully mobilized cecum and distal part of the sigmoid colon under the tumor were well visualized and easily divided . with this action
as the preparation was advanced cranially , the lateral abdominal wall ties were released , at first , using the ultrasonic dissector or scissors step - by - step until the flexure . then , the ascending colon was released of its retroperitoneal ties . with a properly layered dissection towards the aorta between the posterior side of the meso ascending colon and the renal fascia ,
the preparation was continued until the horizontal part of the duodenum came into view and the stump was detached from its mesenteric ties .
a dissection rod was indispensable for the traumatic dissection , because the embryonically preformed tissue strands were best detached from one another by blunt dissection . with detachment of the right colonic flexure , the status of the already mobilized colomesenteric segment changed in such a way that a caudal dissection of the flexure could become complicated , eg , at the vascular stems , due to visual obstruction by the falling intestinal convolution .
after complete mobilization of the right - sided colon intended for resection , the pneumoperitoneum was released , the trocar removed , and the incision extended for minilaparotomy .
externalization of the prepared sigmoid and right colon was carried out through a foil ring introduced into the minilaparotomy over the symphysis pubis for wound protection .
after the right transverse colon was divided extracorporeally and the specimen removed , the distal ileum was exteriorized ( figure 2 ) .
side - to - side ileotransverse anastomosis was performed with a linear stapler . en bloc resection of the sigmoid and right colon .
after the anvil was placed in the descending colon , then retained into the abdomen , the incision was closed .
we placed the stapler through the rectum and mated it with the anvil laparoscopically to complete the colorectal anastomosis .
first flatus was recognized on the second postoperative day , a solid diet was started on the third postoperative day , and the patient was discharged and went directly home on the eighth postoperative day ( figure 4 ) .
histopathological examination showed mucinous adenocarcinoma of the appendix with infiltration of the sigmoid colon of about 6 cm ( figure 3 ) .
the patient seen 2 months after surgery was in good general condition with no complications .
primary adenocarcinoma of the appendix is a rare neoplasm of the gastrointestinal tract and constitutes about 0.5% of all gastrointestinal tract tumors .
these rare tumors are seldom suspected before surgery , and less than one - half are diagnosed intraoperatively .
several studies describing primary tumors of the appendix have appeared in the literature since 1903 , when elting published a review and case series .
there are 4 major histological subtypes : cystic , colonic , carcinoid , and adenocarcinoid .
carcinoids are most common , constituting nearly 90% of all the primary tumors of the appendix .
macroscopically , they produce mucin - filled cystic dilatation of the appendix indistinguishable from that associated with benign tumors .
diagnosis of malignancy is made using 2 features : invasion of the appendicular wall and identification of epithelial cells in peritoneal mucous collections .
spread of the neoplasm above the diaphragm or invasion of the abdominal viscera is exceptional , and lymphatic and hematogenous spread is also very rarely seen .
sometimes this tumor may present in an unusual manner like polyploidy infiltration of the abdominal wall , bladder infiltration mimicking bladder carcinoma , cecocecal intussusceptions , retroperitoneal abscess , vaginal hydronephrosis , or per - rectal bleeding .
sometimes they may present with distension of the abdomen due to pseudomyxoma peritoneum and obstruction of the gut due to involvement of the colon . even if an appendicectomy has been performed previously , cystadenocarcinoma of the appendicular stump may develop ; therefore
, the possibility of carcinoma in the appendicular stump that increases in size should be born in mind .
accurate and complete preoperative diagnosis has been rare in the past , but modern imaging techniques today allow us to recognize most of the complications and associated conditions .
also in such advanced cases with clear preoperative examination , it should not to be considered a contraindication to performing laparoscopic surgery .
due to intraperitoneal spread of the appendicular carcinoma , invasion of the abdominal viscera , as in our case , and the poor prognosis of this tumor , an aggressive approach should be taken when treating patients with this type of tumor .
further development of instruments and techniques has made it possible to apply laparoscopic surgery to malignant diseases in such complex cases as we have described .
compared with conventional surgery , laparoscopic surgery is beneficial with respect to short - term outcomes , including earlier recovery and less need for postoperative analgesia .
radiotherapy , chemotherapy , and new therapeutic modalities like radioimmunotherapy and matrix metalloproteinase inhibitors have still to be proven by prospective analysis . | adenocarcinoma of the vermiform appendix is a rare neoplasm of the gastrointestinal tract that most commonly presents as right lower abdominal pain , mimicking acute appendicitis .
presentation caused by loco - regional spread with involvement of adjacent structures is rare .
an accurate and complete preoperative diagnosis has been rare in the past ; however , modern imaging techniques allow recognition of most complications and associated conditions .
the diagnosis is confirmed postoperatively .
aggressive surgical management is the treatment of choice in appendicular adenocarcinoma .
we report the case of appendicular mucinous cystadenocarcinoma in a 55-year - old lady with penetration of the sigmoid colon treated with laparoscopic - assisted sigmoid and en block right hemicolectomy.it was possible to manage this complex case by using a laparoscopic procedure with all the known benefits of minimally invasive surgery . |
bronchogenic cysts are not uncommon in either children or adults . in children , they comprise approximately 6% of all mediastinal masses . their presentation can range from an asymptomatic incidental finding to sudden respiratory distress .
video - assisted thoracoscopy was utilized to remove a bronchogenic cyst that was densely adherent to the adjacent esophagus in a child .
the chest tube was removed on postoperative day 1 , and the patient was discharged on postoperative day 2 . an esophagogram obtained 2 weeks after surgery was normal , and the patient 's preoperative symptoms had not returned .
bronchogenic cysts should be considered in the differential diagnoses for mediastinal masses at any age . given their benign nature
. they may become symptomatic due to esophageal compression as they increase in size or from development of infection .
we report the case of a 9-year - old female with a symptomatic bronchogenic cyst , undiagnosed since the age of 2 years , who underwent successful thoracoscopic excision .
a 9-year - old girl was first seen at the age of 2 years for cough and fever in the emergency room .
a chest x - ray was obtained at that time , which was interpreted as normal .
nonetheless , the patient was diagnosed with pneumonia , given a prescription for antibiotics , and released .
subsequent to this , she presented several more times each year with similar complaints and with identical posteroanterior ( pa ) chest x - ray findings , resulting in several hospital admissions .
her examination revealed no abnormalities , and her laboratory work was within normal limits at the time of her presentation to the pediatric surgery service .
repeat chest x - rays were obtained ( figure 1 ) as well as a computed tomography ( ct ) scan of the chest ( figure 2 ) .
it revealed a well - circumscribed cyst in the right chest measuring approximately 3x4 cm directly adjacent to the esophagus in the midthoracic region .
it was unilocular and homogenous and did not appear to communicate with the esophagus or any neural structures .
given these findings , the diagnosis of either an esophageal duplication cyst or bronchogenic cyst was entertained .
a thoracoscopic approach was utilized to evaluate and remove the mass ( figure 3 ) .
the medial aspect of the cyst was intimately involved with the muscular wall of the esophagus for several centimeters .
a harmonic scalpel was used to separate the cyst , which was filled with a white mucoid material , from the esophagus .
a small portion of the cyst wall was left in place to avoid perforation of the esophagus .
the chest tube drainage was negligible , and it was removed on postoperative day one .
she was seen at follow - up 2 weeks after surgery where a normal barium esophagogram ( figure 4 ) was obtained , and her preoperative symptoms were completely resolved .
bronchogenic cysts are embryological remnants that are isolated from the normal development of the foregut . on rare occasions
they may be found incidentally on a radiographic study or once symptoms are produced as a result of compression or infection . if communication with the airways occurs , an air - fluid level is usually present .
histologically , they are lined with ciliated columnar epithelium and can contain cartilage and bronchial glands .
accepted treatment is excision because of concern about continued growth and subsequent compression of surrounding structures , the risk of infection , and the unlikely but reported possibility of malignant degeneration .
bronchogenic cysts comprise approximately 6% of all mediastinal masses in children , while foregut cysts as a group account for 15% .
traditionally , open thoracotomy has been the method by which these cysts have been removed .
even with increasingly sophisticated radiographic and percutaneous techniques , an exact diagnosis is often uncertain , and the unequivocal diagnosis must be made surgically .
thoracoscopy offers both a minimally invasive diagnostic modality and also a highly successful method of definitive therapy for patients with bronchogenic cysts .
although many case reports exist regarding bronchogenic cysts and their minimally invasive treatments , reports of precious few series have been published regarding their thoracoscopic excision . in 1993 , hazelrigg published a series of seven bronchogenic cysts removed thoracoscopically with favorable results .
martinod et al reported a series of 20 cases of bronchogenic cysts in adults , of which they successfully removed 13 thoracoscopically .
their reasons for conversion were bleeding in 2 cases and dense adhesions to surrounding vital structures in 5 . in 5 cases ,
thoracoscopy was performed on 22 children , 15 of which were found to have bronchogeni ccysts . of these
merry et al reported in their series of 8 foregut duplications 1 recurrence with incomplete excision 1 year after surgery .
two other reports of recurrence have been reported , , but so have several reporting successful obliteration of the mucosal lining with good results . reported complications in these articles include the following : bronchial / tracheal laceration ( both repaired endoscopically ) , , recurrence , esophageal injury , pneumothorax , , vascular injury .
bronchogenic cysts are not uncommon and , as this case illustrates , can be easily misdiagnosed .
subtle findings , as on this patient 's chest x - ray taken at 2 years of age , are overlooked if one fails to consider cystic mediastinal masses in the differential diagnosis .
we assert that thoracoscopy is a safe and effective method to deal with bronchogenic cysts and involves considerably less morbidity than thoracotomy does . as experience with thoracoscopy
improves , so too will its acceptance as a superior alternative to thoracotomy in most cases of bronchogenic cysts . | background : bronchogenic cysts are not uncommon in either children or adults . in children , they comprise approximately 6% of all mediastinal masses .
their presentation can range from an asymptomatic incidental finding to sudden respiratory distress.case report : video - assisted thoracoscopy was utilized to remove a bronchogenic cyst that was densely adherent to the adjacent esophagus in a child .
this was accomplished with a harmonic scalpel .
the chest tube was removed on postoperative day 1 , and the patient was discharged on postoperative day 2 . an esophagogram obtained 2 weeks after surgery was normal , and the patient 's preoperative symptoms had not returned.conclusions:bronchogenic cysts should be considered in the differential diagnoses for mediastinal masses at any age .
given their benign nature , thoracoscopy offers an excellent alternative to open thoracotomy for their removal . |
in recent years , medical anthropologists have been turning their ethnographic attention to issues of science and technology , including how science is produced , where it is produced , by whom , and when .
anthropologists have also highlighted how scientific and technological advancements spread around the world , gradually being incorporated into the lives of ordinary people in many global sites . in order to document the production and reproduction of new scientific and technological discoveries ,
medical anthropologists have begun to take their ethnographic research projects into previously unexplored terrains , including hospitals , scientific research laboratories , clinical consultation rooms , and other behind the scenes
furthermore , most of the medical anthropologists working in this field have documented the ways in which the scientific and the divine seem to have intermingled , not only among the patients who turn to religious theodicies to make sense of their suffering , but also among the scientists and clinicians who deliver medical services and attempt to offer patients hope . these recent medical anthropological efforts to document the intersections of science , technology , and religion
are clearly reflected in the theme of the 2004 annual meeting of the american anthropological association : magic , science , and religion . furthermore , this special issue of culture , medicine and psychiatry reflects the ongoing importance of religion to science , medicine , and biotechnology , including in the muslim world .
medical anthropology has much to offer to discussions of science , medicine , and religion , as demonstrated in a number of award - winning books on these subjects . to take but a few recent examples , in testing women , testing the fetus : the social impact of amniocentesis in america , medical anthropologist rayna rapp ( 1999 ) reveals the efforts of more than ten years worth of extensive ethnographic research conducted at new york city hospitals , genetic counseling and testing centers , and genetics laboratories themselves .
her book focuses on the difficult decision - making of pregnant women from multiple ethnic , religious , and economic backgrounds , who are asked by clinicians and advised by genetic counselors to undergo amniocentesis in order to detect genetic anomalies in their fetuses .
although the scientific field of genetics is burgeoning with excitement , rapp s intent in testing women , testing the fetus is to show how women who are expected to use new genetic tests during pregnancy are put in the difficult position of being moral pioneers : namely , they are forced to make often heart - wrenching moral decisions about what constitutes an acceptable human life . whereas rapp s book focuses on decisions about bringing life into the world
, a book by medical anthropologist margaret lock asks us to consider decisions about ending lives considered brain dead .
twice dead : organ transplants and the reinvention of death , lock s ( 2002 ) ethnography , examines the history of organ transplantation in north america over the past 20 years .
scientific advancements in transplant technology have made it possible for north american surgeons to perform thousands of transplants each year . in the majority of these cases ,
individuals diagnosed as brain dead are the source of the organs , without which transplants could not take place .
lock asks her readers to consider what is meant by brain death , and how it might be viewed differently in another culture , namely , japan . despite the availability of the necessary technology and expertise , japanese society has not accepted the notion of brain death as a sufficient criterion of death , following a well - informed but heated public debate on the topic .
although brain death was legally recognized in japan in 1997 , it is authorized only in limited circumstances , meaning that organ transplantation in japan is not widespread . drawing on extensive interviews conducted over ten years with intensive care unit ( icu ) physicians , transplant surgeons , organ recipients , donor families , japanese political activists opposed to the recognition of brain death , and members of the general public in both japan and north america , lock shows us that death itself is not a self - evident , biological event .
rather , it is surrounded by morally troubling cultural , medical , legal , and political dimensions .
lock s book clearly demonstrates the degree to which medical anthropology is concerned with comparative cultural perspectives , particularly in an era of rapid globalization .
indeed , medical anthropology as a discipline can be said to focus on the local in the global . in other words , local considerations , be they cultural , social , economic , or political , shape and sometimes curtail the way that western - generated scientific technologies are both offered to and received by non - western subjects .
new forms of science and technology are not transferred into cultural voids when they reach places like japan .
thus , the assumption on the part of global producer nations that new biotechnologies as value - free , inherently beneficial medical technologies are immune to culture and can thus be appropriately transferred and implemented anywhere and everywhere is subject to challenge once local formulations , perceptions , and actual consumption of these technologies are taken into consideration ( inhorn 2003a ) . indeed , the global spread of biotechnologies provides a particularly salient but little discussed example of what anthropologist arjun appadurai ( 1996 ) has termed a technoscape , or the global configuration , also ever fluid , of technology , and the fact that technology , both high and low , both mechanical and informational , now moves at high speeds across various kinds of previously impervious boundaries . appadurai reminds us that this movement of technologies around the globe is both a deeply historical and an inherently localizing process .
in other words , globalization is not enacted in a uniform manner around the world , nor is it simply culturally homogenizing necessarily westernizing or even americanizing in its effects . the global is always imbued with local meaning , such that local actors , living their everyday lives at particular historical moments in particular places , mold the very form that global processes take ( freeman 2000 ) .
this acknowledgment of the importance of locality in the global dispersion of modern biotechnologies has been a theme of much recent work in medical anthropology .
similarly , much of this anthropological concern with locality has to do with local moral systems , or what medical anthropologist / psychiatrist arthur kleinman ( 1995 ) has called local moral worlds of pain and suffering . according to kleinman ( 45 ) , local moral worlds are moral accounts , [ which ] are the commitments of social participants in a local world about what is at stake in everyday experience . through an
kleinman urges medical anthropologists to pay close attention to the moral issues that may accompany the arrival of new biotechnologies around the globe . in his recent powerful and path - breaking book ,
what really matters : living a moral life amidst uncertainty and danger , kleinman ( 2006 ) asks us to carefully consider what it really means to live a moral life , particularly in the midst of life - or - death decision - making .
this focus on the local moral is found in another award - winning book on the topic of ivf .
titled reproducing jews : a cultural account of assisted conception in israel , this book by medical anthropologist susan martha kahn ( 2000 ; see also her article in this special issue ) takes us into the often arcane world of jewish halakhic law , where male rabbis legislate on the appropriate uses of ivf for their followers .
kahn carefully describes how these rabbinical debates and decisions affect the actual practice of israeli ivf .
for example , third - party donation of gametes , including sperm donation , is allowed , since jewishness is seen to be conferred through the mother s side , particularly through the act of gestating and birthing the baby . however , most conservative rabbis prefer that non - jewish donor sperm be used , to prevent adultery between a jewish man and a jewish woman and to prevent future genetic incest among the offspring of anonymous donors in this small , intermarrying country .
furthermore , debates have revolved around whether surrogacy should be allowed for infertile couples , using single or married surrogates . generally speaking
, single jewish women are preferred as surrogates , both to avoid the implications of adultery for married surrogate women and to confer jewishness through a jewish woman s gestation of the fetus . finally , because the jewish state is pronatalist with the state subsidizing up to six cycles of ivf or up to the birth of two ivf children for any given jewish patient
rabbis have generally been permissive when it comes to single career women , as well as lesbian jewish mothers , conceiving children through assisted conceptive means .
kahn s fascinating and frankly funny book details the sometimes dizzying rabbinical arguments regarding morally appropriate and inappropriate reproduction . in so doing ,
the book bespeaks the importance of local religious moralities in the contemporary world of israeli assisted conception .
there , doctors in many clinics serving orthodox jewish patient populations attempt to practice ivf according to the moral dictates set forth by religiously conservative rabbis .
the ivf laboratories in these clinics are full of orthodox women called maschigots , who literally peer over the shoulders of laboratory technicians to make sure that the correct sperm and correct eggs are being united so as not to produce a mamzer , or an illegitimate child . in her book , kahn is explicit in stating that the american consumer model of free - market reproductive medicine has yet to take hold in israel , with its concern over religious guidelines . nonetheless , israel s relative permissiveness over the use of donor gametes , surrogacy , and single and lesbian motherhood stands in stark contrast to the muslim middle east , including both neighboring egypt and lebanon , where i have conducted my own ethnographic research on ivf .
with this background in mind , it is my goal in this article to take readers into the heart of the muslim middle east , thereby showing how the practice of ivf in israel , the country located between egypt and lebanon , differs significantly from that in both of its muslim neighbors . during more than two years of medical anthropological fieldwork in egypt ( 198889 , 1996 ) and
lebanon ( 2003 ) , i conducted in - depth , ethnographic interviews with infertile muslim ivf patients , both husbands and wives , now totaling nearly 500 patient couples . furthermore , since the mid-1990s , i have focused my research on the rapid expansion of ivf technologies into this region of the muslim world .
indeed , it is fair to assert that since the birth in 1978 of louise brown , the world s first test - tube baby , ivf has spread around the globe , reaching countries far from the technology - producing nations of the west .
perhaps nowhere is this globalization process more evident than in the 22 nations of the muslim middle east , where a private ivf industry is flourishing . as of 2003 , egypt alone ( population 70 million ) boasted 50 ivf centers , and the tiny country of lebanon ( population 4 million ) boasted more than 15 ivf centers , one of the highest per capita concentrations in the world . these global metrics are impressive compared to the ivf technology - producing nation of israel , where 24 ivf centers cater to a population of 6 million ( kahn 2000 ) . in both egypt and lebanon , as in other muslim countries , nonbinding but authoritative islamic religious proclamations called fatwas have profoundly affected the practice of ivf in ways that are not commonly seen in the west .
indeed , in the muslim world , infertile couples are usually extremely concerned about making their test - tube babies in the religiously correct fashion . to that end , they seek out the official islamic opinion on the practice of ivf in the form of a fatwa . in recent years , many such fatwas on a wide variety of reproductive health issues have been issued in egypt and other muslim countries ( lane and rubinstein 1991 ; zuhur 1992 ) . but as we shall see in this paper , major divergences have occurred between sunni and shiite religious authorities regarding the permissibility of third - party gamete donation , with new fatwas emerging from the shiite world in recent years .
it is the differences between sunni and shia islam with regard to third - party gamete donation that constitute the focus of this article .
ivf was first practiced in the sunni muslim world , with clinics opening in the mid-1980s in egypt , saudi arabia , and jordan , all sunni - majority countries .
the grand shaikh of egypt s famed religious university , al azhar , issued the first widely authoritative fatwa on medically assisted reproduction on march 23 , 1980 .
this fatwa issued only two years after the birth of the first ivf baby in england but a full six years before the opening of egypt s first ivf center has proved to be truly enduring in all its main points ( inhorn 2006a ) . in fact , the basic tenets of the original al - azhar fatwa on ivf have been upheld by other fatwas issued since 1980 and have achieved wide acceptance throughout the sunni muslim world .
sunni islam , it must be emphasized , is the dominant form of islam found in the middle eastern region and throughout the muslim world .
between 80 and 90 percent of the world s muslims are sunni , and more than 90 percent of egypt s citizens are sunni muslims , the rest being predominantly coptic christian .
the degree to which these official sunni islamic fatwas on ivf have affected the actual practices of the middle eastern medical profession is also quite striking . for physicians ,
the dominant sunni religious opinion on ivf has been made known to the middle eastern medical community through the writings of gamal i. serour , one of three founding members of the first egyptian ivf center and the director of al - azhar s international islamic center for population studies and research . in article after article ( serour 1992 , 1994 , 1996 ; serour and omran 1992 ; serour , el ghar , and mansour 1990 , 1991 ; serour , aboulghar , and mansour 1995 ) , serour has spelled out the main points of the sunni islamic position on medically assisted conception , as follows : artificial insemination with the husbandssemen is allowed , and the resulting child is the legal offspring of the couple.in vitro fertilization of an egg from the wife with the sperm of her husband followed by the transfer of the fertilized embryo(s ) back to the uterus of the wife is allowed , provided that the procedure is indicated for a medical reason and is carried out by an expert physician.no third party should intrude into the marital functions of sex and procreation , because marriage is a contract between the wife and husband during the span of their marriage .
this means that a third party donor is not allowed , whether he or she is providing sperm , eggs , embryos , or a uterus .
the use of a third party is tantamount to zina , or adultery.adoption of a donor child from an illegitimate form of medically assisted conception is not allowed .
the child who results from a forbidden method belongs to the mother who delivered him / her .
he or she is considered to be a laqit , or an illegitimate child.if the marriage contract has come to an end because of divorce or death of the husband , medically assisted conception can not be performed on the ex - wife even if the sperm comes from the former husband.an excess number of fertilized embryos can be preserved by cryopreservation .
the frozen embryos are the property of the couple alone and may be transferred to the same wife in a successive cycle , but only during the duration of the marriage contract.multifetal pregnancy reduction ( or so - called selective abortion ) is only allowed if the prospect of carrying a high - order pregnancy ( i.e. , twins , triplets , or more ) to viability is very small .
it is also allowed if the health or life of the mother is in jeopardy.all forms of surrogacy are forbidden.establishment of sperm banks is strictly forbidden , for such a practice threatens the existence of the family and the race and should be prevented.the physician is the only qualified person to practice medically assisted conception in all its permitted varieties . if he performs any of the forbidden techniques , he is guilty , his earnings are forbidden , and he must be stopped from his morally illicit practice . artificial insemination with the husbandssemen
is allowed , and the resulting child is the legal offspring of the couple . in vitro fertilization of an egg from the wife with the sperm of her husband followed by the transfer of the fertilized embryo(s ) back to the uterus of the wife is allowed , provided that the procedure is indicated for a medical reason and is carried out by an expert physician .
no third party should intrude into the marital functions of sex and procreation , because marriage is a contract between the wife and husband during the span of their marriage .
this means that a third party donor is not allowed , whether he or she is providing sperm , eggs , embryos , or a uterus .
adoption of a donor child from an illegitimate form of medically assisted conception is not allowed .
the child who results from a forbidden method belongs to the mother who delivered him / her .
if the marriage contract has come to an end because of divorce or death of the husband , medically assisted conception can not be performed on the ex - wife even if the sperm comes from the former husband .
the frozen embryos are the property of the couple alone and may be transferred to the same wife in a successive cycle , but only during the duration of the marriage contract .
multifetal pregnancy reduction ( or so - called selective abortion ) is only allowed if the prospect of carrying a high - order pregnancy ( i.e. , twins , triplets , or more ) to viability is very small .
it is also allowed if the health or life of the mother is in jeopardy .
establishment of sperm banks is strictly forbidden , for such a practice threatens the existence of the family and the race and should be prevented .
the physician is the only qualified person to practice medically assisted conception in all its permitted varieties .
if he performs any of the forbidden techniques , he is guilty , his earnings are forbidden , and he must be stopped from his morally illicit practice .
but to what degree are these fatwa declarations particularly the explicit prohibition on any form of third - party donation of reproductive materials actually followed by physicians in the muslim world ? a global survey of sperm donation among assisted reproductive technology centers in 62 countries provides some indication of the degree of convergence between official discourse and actual practice ( meirow and schenker 1997 ) . in all of the muslim countries surveyed in the mid-1990s
including the middle eastern countries of egypt , iran , kuwait , jordan , lebanon , morocco , qatar , and turkey , as well as a number of non - middle eastern muslim countries , including indonesia , malaysia , and pakistan sperm donation in ivf and all other forms of gamete donation were strictly prohibited . as the authors of this global survey , meirow and schenker ( 1997 ) , state , in many islamic countries , where the laws of islam are the laws of the state , donation of sperm was not practiced .
aid [ artificial insemination , donor ] is considered adultery and leads to confusion regarding the lines of genealogy , whose purity is of prime importance in islam ( 134 ) . in summary , in the sunni - majority countries of the middle east and the rest of the sunni muslim world , prohibitions against gamete donation have been enacted either in law or in professional medical codes of ethics . as a result , gamete donation is not legally practiced in the sunni muslim world , with clinics turning away ( or referring to other european countries ) couples who require these services . in sunni muslim countries such as egypt ,
the thought of using donor gametes is reprehensible to most infertile muslim ( and even christian ) couples , who agree completely with the bans on third - party donation dictated by the religion and upheld through medical codes of ethics ( inhorn 2003b ) . as shown in the next section , however ,
the situation is changing for shiite muslims , whose leading cleric has taken a bold step in a new direction .
for shiite muslims , attitudes toward gamete donation have changed considerably since the late 1990s .
shia is the minority branch of islam found in iran , parts of iraq , lebanon , bahrain , syria , and saudi arabia , as well as afghanistan , pakistan , and india .
it has been much in the news lately because of the united states led war in iraq , the conflict between lebanon and israel , and the current tensions between the united states and iran . until recently , most shiite religious authorities have supported the majority sunni view : namely , they have agreed with sunni clerics who say that third - party donation should be strictly prohibited . in the late 1990s , however , the supreme jurisprudent of the shia branch of islam , ayatollah ali hussein khamanei , the handpicked successor to iran s ayatollah khomeini , issued a fatwa effectively permitting donor technologies to be used .
this fatwa has proved to be very significant for those shia who follow the lead of ayatollah khamanei in iran .
this would include lebanon s hizbullah leaders , who consider ayatollah khamanei to be their marja taqlid , or spiritual reference ( literally , source of emulation ) .
with regard to egg donation , ayatollah khamanei stated in his initial fatwa that egg donation is not in and of itself legally forbidden . but he stated that both the egg donor and the infertile mother must abide by the religious codes regarding parenting .
thus , the child of the egg donor has the right to inherit from her , as the infertile woman who received the eggs is considered to be like an adoptive mother . with regard to sperm donation , ayatollah khamanei said in his original fatwa that the baby born of sperm donation will follow the name of the infertile father rather than the sperm donor .
however , as with egg donation , the donor child can only inherit from his biological father , the sperm donor , since the infertile father is considered to be like an adoptive father .
the situation for shiite muslims is actually much more complicated than this , however , given two shiite religious practices called ijtihad and muta . unlike sunni muslim scholars who are scripturally based in their thinking , shiite religious authorities give precedence to a form of individual religious reasoning known as ijtihad . through the use of aql , or intellectual reasoning ,
various shiite ulama have come to their own conclusions regarding the rightness or wrongness of gamete donation .
some shiite clerics continue to prohibit gamete donation for their followers , while others have allowed it under certain conditions .
as many scholars of shia have noted ( cole 2002 ; tober 2004 ) , the practice of ijtihad has allowed a certain flexibility and pragmatism toward new technological developments , including ivf and a number of other new medical technologies ( e.g. , contraception , organ transplants , transgender surgery ) .
furthermore , ijtihad has ultimately led to great heterogeneity of opinion and practice within the shiite community .
additionally , shia islam allows a form of temporary marriage called muta ( also called sigheh in iran ) , which is not recognized by sunni religious authorities ( zuhur 1992 ) . in shia islam
, muta is a union between an unmarried muslim woman and a married or unmarried muslim man , which is contracted for a fixed time period in return for a set amount of money .
it is practiced in iran ( haeri 1989 ) , as well as in other parts of the shiite world .
in the past , middle - aged and older women who were divorced or widowed often engaged in muta marriages for financial support . in iran , following the loss of men during the devastating , eight - year iran
iraq war , former iranian president rafsanjani recommended muta as a means of protecting the large numbers of single or widowed women who had no other source of income . for shiite men , muta marriages could be contracted while traveling , or as a way of achieving marital variety and sexual pleasure ( haeri 1989 ) . since the arrival of donor technologies , however , muta has also been invoked to make egg donation legal within the parameters of marriage . within this context of ijtihad and muta , shia religious authorities who now accept the idea of donation , but are strict in their interpretation of how donation should be practiced ,
argue that when a couple needs a donor , they should go to a shiite religious court , where a decision can be made on a case - by - case basis;there should be a determination about which religious reference
( i.e. , source of spiritual emulation ) the infertile couple follows;the decision should be made in the presence of witnesses , the ivf doctor , and with the agreement of both parties ( the infertile couple and the donor);the husband should do a muta marriage with the egg donor for the period of time in which the whole procedure ( egg retrieval to embryo transfer ) is taking place , because polygyny is legal in islam and avoids the implications of zina , or adultery;but because a married shiite muslim woman can not marry another man other than her husband ( since polyandry is illegal in islam ) , she can not do a muta marriage with a sperm donor .
technically , the child born of a sperm donor would be a laqit , or out - of - wedlock child , without a family name and without a father .
thus , in theory , only widowed or otherwise single women should be able to accept donor sperm , in order to avoid the implications of zina .
however , in the muslim countries , single motherhood of a donor child is unlikely to be socially acceptable ( inhorn 1996 ; zuhur 1992 ) .
when a couple needs a donor , they should go to a shiite religious court , where a decision can be made on a case - by - case basis ; there should be a determination about which religious reference
( i.e. , source of spiritual emulation ) the infertile couple follows ; the decision should be made in the presence of witnesses , the ivf doctor , and with the agreement of both parties ( the infertile couple and the donor ) ; the husband should do a muta marriage with the egg donor for the period of time in which the whole procedure ( egg retrieval to embryo transfer ) is taking place , because polygyny is legal in islam and avoids the implications of zina , or adultery ; but because a married shiite muslim woman can not marry another man other than her husband ( since polyandry is illegal in islam ) , she can not do a muta marriage with a sperm donor . technically , the child born of a sperm donor would be a laqit , or out - of - wedlock child , without a family name and without a father .
thus , in theory , only widowed or otherwise single women should be able to accept donor sperm , in order to avoid the implications of zina .
however , in the muslim countries , single motherhood of a donor child is unlikely to be socially acceptable ( inhorn 1996 ; zuhur 1992 ) .
be that as it may , divergent gamete donation practices are beginning to emerge in the shiite muslim world , as religious authorities come to their own conclusions about third - party donation . among shiite religious scholars , the major disagreements , or religious sticking points , revolve around the following set of issues : whether sperm donation should be allowed at all;whether the child should follow the name of the infertile father or the sperm donor;whether the child should inherit from the infertile father or the sperm donor;whether donor children and their social parents are related at all , and , if not , whether they could potentially marry each other , which has implications for proper comportment in domestic life ( e.g. , bathing , veiling , etc.);whether donation is permissible at all if the donors are anonymous;whether a financial transaction should be allowed between gamete donors and recipients;whether the husband of an infertile woman needs to do a temporary muta marriage with the egg donor , then divorce her after the embryo transfer ( 48 to 72 hours later ) , in order to avoid zina . for his part
, khamanei clearly stipulates that muta marriage is not required , for he believes that zina requires the physical act of intercourse ( clarke 2006 ) ; andwhether the wife of an infertile husband can temporarily divorce her infertile husband , remarrying him after accepting sperm from a donor ( tremayne , personal communication , 5/5/06 ) . whether sperm donation should be allowed at all ; whether the child should follow the name of the infertile father or the sperm donor ; whether the child should inherit from the infertile father or the sperm donor ; whether donor children and their social parents are related at all , and , if not , whether they could potentially marry each other , which has implications for proper comportment in domestic life ( e.g. , bathing , veiling , etc . ) ; whether donation is permissible at all if the donors are anonymous ; whether a financial transaction should be allowed between gamete donors and recipients ; whether the husband of an infertile woman needs to do a temporary muta marriage with the egg donor , then divorce her after the embryo transfer ( 48 to 72 hours later ) , in order to avoid zina . for his part , khamanei clearly stipulates that muta marriage is not required , for he believes that zina requires the physical act of intercourse ( clarke 2006 ) ; and whether the wife of an infertile husband can temporarily divorce her infertile husband , remarrying him after accepting sperm from a donor ( tremayne , personal communication , 5/5/06 ) .
as shown in anthropologist morgan clarke s ( 2006 ) recent research on the shiite religious discourses surrounding gamete donation , many shiite ulama do not agree with khamanei s permissive fatwa on donor technologies , because they do not regard him as a brilliant legal thinker .
for example , ayatollah muhammad husayn fadlallah , lebanon s most prominent shiite religious figure , does not agree with ayatollah khamanei s permission of sperm donation , although he agrees with the permission of egg donation .
neither of them requires the use of muta marriages to solve the zina issue ( clarke 2006 ) .
ayatollah fadlallah s positions opposing sperm donation but supporting egg donation square with the dominant religious discourse in iran . there ,
the religious rulings regarding gamete donation have evolved quickly , with sperm donation now effectively banned ( soraya tremayne , personal communication , 23 july 2004 , 31 july 2004 ) .
namely , a law on gamete donation passed in 2003 in the iranian parliament ( majlis ) and approved by the guardian council ( i.e. , a religious watchdog body that endorses every bill before it becomes law ) has restricted gamete donation to married persons . even though the law is brief ( less than one page ) , it states clearly and succinctly who can and can not donate and receive gametes .
egg donation is allowed , as long as the husband marries the egg donor temporarily thereby ensuring that all three parties are married .
sperm donation , on the other hand , is legally forbidden , because a sperm donor can not temporarily marry an already married woman whose husband is infertile . quite interestingly ,
however , embryo donation which involves both sperm and egg from another couple is allowed in order to overcome both male and female infertility . because an embryo comes from a married couple and
is given to another married couple , it is considered hallal , or religiously permissible .
the social and biological implications of embryo donation are quite interesting . for iranian couples unable to produce a child because of male infertility ,
embryo donation allows them to bypass the problem of the husband s weak ( or absent ) sperm .
however , embryo donation does not allow a presumably fertile wife of an infertile husband to contribute her own ova , in effect severing her biological ties to the donor child .
furthermore , and most strikingly , embryos donated from another married couple involve both egg and sperm donation .
even though direct sperm donation is bypassed via the injection of another man s sperm into the wife s womb / ova , embryo donation still disrupts male paternity and involves the acceptance by an already married woman of another man s ( and woman s ) gametes .
moreover , a woman s acceptance of another woman s egg is effectively like gestational surrogacy , which is strictly prohibited in sunni islam . indeed , in recent months
, cases of surrogate motherhood have occurred in iran , despite the lack of firm legislation regarding this practice . whether these problematic complications of embryo donation have been carefully thought through by the religious and legal authorities in iran
is unclear . based on her path - breaking research carried out at iranian ivf clinics , anthropologist soraya tremayne ( 2005 , 2006 ) notes ,
my first and possibly superficial interpretation at this stage can only be that the moral , ethical and legal aspects of the whole matter have not yet been thought through and the consequences dawned on either the doctors and legislators , or on people themselves .
these operations have been in place only for the past ten years ... and the cases are too new to cause problems as yet
it is interesting to note that many shiite religious leaders both inside and outside of iran do not agree with the relative iranian permissiveness
vis - - vis donor technologies ; instead , they abide by the dominant sunni muslim ban on all forms of third - party donation .
for example , i recently attended a two - day conference in tehran , iran , on gamete and embryo donation , sponsored by the avesina research institute in association with the law and political science faculty of the university of tehran .
the conference provided a fascinating example of ijtihad in action , with some ulama , dressed in their stately robes and turbans ( black for the sayyids , or descendants of the prophet muhammad ) , arguing against the moral permissibility of embryo and gamete donation .
the disagreements generated in public between pro and con ulama were also debated in the more private recesses of the conference .
for example , a shiite sharia judge from bahrain , who was staying at our guest residence , took great pains to describe to me his opposition to all forms of gamete donation . to prove this point
, he provided me with a copy of his book on islamic personal status law , which had been translated into english and which supported his anti gamete donation position based on evidence from the traditional islamic scriptures . according to him , iranian clergy , who speak farsi rather than arabic ,
are not as familiar with the original islamic scriptures ( in arabic ) that demonstrate the immorality of third - party donation .
thus , in his view , some iranian clergy are innovating in ways that are religiously unacceptable , and that are at odds with the rest of the muslim world . a case in point : ayatollahs ali al - sistani and muhammad said al - tabatabai al - hakim ,
both shiite religious authorities in iraq , advise caution against third - party donation practices , viewing them as largely unacceptable ( clarke 2006 ) .
indeed , ayatollah al - sistani s son , muhammad rida al - sistani , has devoted an entire volume of richly documented legal analysis to this debate , providing an invaluable resource for other scholars ( 26 ) .
sistani s work , while perhaps posing more questions than clear answers , opens up for other scholars a fascinating window into this area of shiite jurisprudential debate , at a time when the western media are just waking up to the vibrant engagement shiite scholars have had with other such new technologies ( 26 ) .
indeed , the degree to which some shiite clergy are pushing the envelope in the realm of reproductive science and technology is quite remarkable . at the recent gamete donation conference in iran , some iranian clergy and physicians present advocated for future laws permitting all forms of donation as well as surrogacy .
once passed into law , gamete donation of all kinds will be difficult to stop .
meanwhile , in the absence of formal legislation , some ivf physicians in tehran as well as in shiite - dominant lebanon , which is closely following the iranian lead are using the legal vacuum and the original permissive fatwa of ayatollah khamanei to practice all forms of gamete donation among their desperate infertile patients . as noted by clarke ( 2006 ) for lebanon ,
doctors keep khamanei s fatwa collection on the shelves of their surgeries to demonstrate the permissibility of such procedures to skeptical muslim patients ; and many such patients have profited from it to undertake donor sperm and egg procedures , even surrogacy arrangements , with a clear conscience ( 26 ) .
ivf was first practiced in the sunni muslim world , with clinics opening in the mid-1980s in egypt , saudi arabia , and jordan , all sunni - majority countries .
the grand shaikh of egypt s famed religious university , al azhar , issued the first widely authoritative fatwa on medically assisted reproduction on march 23 , 1980 .
this fatwa issued only two years after the birth of the first ivf baby in england but a full six years before the opening of egypt s first ivf center has proved to be truly enduring in all its main points ( inhorn 2006a ) . in fact , the basic tenets of the original al - azhar fatwa on ivf have been upheld by other fatwas issued since 1980 and have achieved wide acceptance throughout the sunni muslim world .
sunni islam , it must be emphasized , is the dominant form of islam found in the middle eastern region and throughout the muslim world .
between 80 and 90 percent of the world s muslims are sunni , and more than 90 percent of egypt s citizens are sunni muslims , the rest being predominantly coptic christian .
the degree to which these official sunni islamic fatwas on ivf have affected the actual practices of the middle eastern medical profession is also quite striking . for physicians ,
the dominant sunni religious opinion on ivf has been made known to the middle eastern medical community through the writings of gamal i. serour , one of three founding members of the first egyptian ivf center and the director of al - azhar s international islamic center for population studies and research . in article after article ( serour 1992 , 1994 , 1996 ; serour and omran 1992 ; serour , el ghar , and mansour 1990 , 1991 ; serour , aboulghar , and mansour 1995 ) , serour has spelled out the main points of the sunni islamic position on medically assisted conception , as follows : artificial insemination with the husbandssemen is allowed , and the resulting child is the legal offspring of the couple.in vitro fertilization of an egg from the wife with the sperm of her husband followed by the transfer of the fertilized embryo(s ) back to the uterus of the wife is allowed , provided that the procedure is indicated for a medical reason and is carried out by an expert physician.no third party should intrude into the marital functions of sex and procreation , because marriage is a contract between the wife and husband during the span of their marriage .
this means that a third party donor is not allowed , whether he or she is providing sperm , eggs , embryos , or a uterus .
the use of a third party is tantamount to zina , or adultery.adoption of a donor child from an illegitimate form of medically assisted conception is not allowed .
the child who results from a forbidden method belongs to the mother who delivered him / her .
he or she is considered to be a laqit , or an illegitimate child.if the marriage contract has come to an end because of divorce or death of the husband , medically assisted conception can not be performed on the ex - wife even if the sperm comes from the former husband.an excess number of fertilized embryos can be preserved by cryopreservation .
the frozen embryos are the property of the couple alone and may be transferred to the same wife in a successive cycle , but only during the duration of the marriage contract.multifetal pregnancy reduction ( or so - called selective abortion ) is only allowed if the prospect of carrying a high - order pregnancy ( i.e. , twins , triplets , or more ) to viability is very small .
it is also allowed if the health or life of the mother is in jeopardy.all forms of surrogacy are forbidden.establishment of sperm banks is strictly forbidden , for such a practice threatens the existence of the family and the race and should be prevented.the physician is the only qualified person to practice medically assisted conception in all its permitted varieties .
if he performs any of the forbidden techniques , he is guilty , his earnings are forbidden , and he must be stopped from his morally illicit practice .
artificial insemination with the husbandssemen is allowed , and the resulting child is the legal offspring of the couple . in vitro fertilization of an egg from the wife with the sperm of her husband followed by the transfer of the fertilized embryo(s ) back to the uterus of the wife
is allowed , provided that the procedure is indicated for a medical reason and is carried out by an expert physician .
no third party should intrude into the marital functions of sex and procreation , because marriage is a contract between the wife and husband during the span of their marriage .
this means that a third party donor is not allowed , whether he or she is providing sperm , eggs , embryos , or a uterus .
adoption of a donor child from an illegitimate form of medically assisted conception is not allowed .
the child who results from a forbidden method belongs to the mother who delivered him / her .
if the marriage contract has come to an end because of divorce or death of the husband , medically assisted conception can not be performed on the ex - wife even if the sperm comes from the former husband .
the frozen embryos are the property of the couple alone and may be transferred to the same wife in a successive cycle , but only during the duration of the marriage contract .
multifetal pregnancy reduction ( or so - called selective abortion ) is only allowed if the prospect of carrying a high - order pregnancy ( i.e. , twins , triplets , or more ) to viability is very small .
it is also allowed if the health or life of the mother is in jeopardy .
establishment of sperm banks is strictly forbidden , for such a practice threatens the existence of the family and the race and should be prevented .
the physician is the only qualified person to practice medically assisted conception in all its permitted varieties .
if he performs any of the forbidden techniques , he is guilty , his earnings are forbidden , and he must be stopped from his morally illicit practice . but to what degree are these fatwa declarations particularly the explicit prohibition on any form of third - party donation of reproductive materials actually followed by physicians in the muslim world ? a global survey of sperm donation among assisted reproductive technology centers in 62 countries provides some indication of the degree of convergence between official discourse and actual practice ( meirow and schenker 1997 ) . in all of the muslim countries surveyed in the mid-1990s including the middle eastern countries of egypt , iran , kuwait , jordan , lebanon , morocco , qatar , and turkey , as well as a number of non - middle eastern muslim countries , including indonesia , malaysia , and pakistan sperm donation in ivf and all other forms of gamete donation were strictly prohibited . as the authors of this global survey , meirow and schenker ( 1997 ) , state , in many islamic countries , where the laws of islam are the laws of the state , donation of sperm was not practiced .
aid [ artificial insemination , donor ] is considered adultery and leads to confusion regarding the lines of genealogy , whose purity is of prime importance in islam ( 134 ) . in summary , in the sunni - majority countries of the middle east and the rest of the sunni muslim world , prohibitions against gamete donation have been enacted either in law or in professional medical codes of ethics . as a result , gamete donation is not legally practiced in the sunni muslim world , with clinics turning away ( or referring to other european countries ) couples who require these services . in sunni muslim countries such as egypt ,
the thought of using donor gametes is reprehensible to most infertile muslim ( and even christian ) couples , who agree completely with the bans on third - party donation dictated by the religion and upheld through medical codes of ethics ( inhorn 2003b ) . as shown in the next section , however ,
the situation is changing for shiite muslims , whose leading cleric has taken a bold step in a new direction .
for shiite muslims , attitudes toward gamete donation have changed considerably since the late 1990s .
shia is the minority branch of islam found in iran , parts of iraq , lebanon , bahrain , syria , and saudi arabia , as well as afghanistan , pakistan , and india .
it has been much in the news lately because of the united states led war in iraq , the conflict between lebanon and israel , and the current tensions between the united states and iran . until recently ,
most shiite religious authorities have supported the majority sunni view : namely , they have agreed with sunni clerics who say that third - party donation should be strictly prohibited . in the late 1990s , however , the supreme jurisprudent of the shia branch of islam , ayatollah ali hussein khamanei , the handpicked successor to iran s ayatollah khomeini , issued a fatwa effectively permitting donor technologies to be used .
this fatwa has proved to be very significant for those shia who follow the lead of ayatollah khamanei in iran .
this would include lebanon s hizbullah leaders , who consider ayatollah khamanei to be their marja taqlid , or spiritual reference ( literally , source of emulation ) .
with regard to egg donation , ayatollah khamanei stated in his initial fatwa that egg donation is not in and of itself legally forbidden . but he stated that both the egg donor and the infertile mother must abide by the religious codes regarding parenting .
thus , the child of the egg donor has the right to inherit from her , as the infertile woman who received the eggs is considered to be like an adoptive mother . with regard to sperm donation ,
ayatollah khamanei said in his original fatwa that the baby born of sperm donation will follow the name of the infertile father rather than the sperm donor .
however , as with egg donation , the donor child can only inherit from his biological father , the sperm donor , since the infertile father is considered to be like an adoptive father .
the situation for shiite muslims is actually much more complicated than this , however , given two shiite religious practices called ijtihad and muta .
unlike sunni muslim scholars who are scripturally based in their thinking , shiite religious authorities give precedence to a form of individual religious reasoning known as ijtihad . through the use of aql , or intellectual reasoning ,
various shiite ulama have come to their own conclusions regarding the rightness or wrongness of gamete donation .
some shiite clerics continue to prohibit gamete donation for their followers , while others have allowed it under certain conditions .
as many scholars of shia have noted ( cole 2002 ; tober 2004 ) , the practice of ijtihad has allowed a certain flexibility and pragmatism toward new technological developments , including ivf and a number of other new medical technologies ( e.g. , contraception , organ transplants , transgender surgery ) .
furthermore , ijtihad has ultimately led to great heterogeneity of opinion and practice within the shiite community .
additionally , shia islam allows a form of temporary marriage called muta ( also called sigheh in iran ) , which is not recognized by sunni religious authorities ( zuhur 1992 ) . in shia islam
, muta is a union between an unmarried muslim woman and a married or unmarried muslim man , which is contracted for a fixed time period in return for a set amount of money .
it is practiced in iran ( haeri 1989 ) , as well as in other parts of the shiite world . in the past ,
middle - aged and older women who were divorced or widowed often engaged in muta marriages for financial support . in iran , following the loss of men during the devastating , eight - year iran
iraq war , former iranian president rafsanjani recommended muta as a means of protecting the large numbers of single or widowed women who had no other source of income . for shiite men , muta marriages could be contracted while traveling , or as a way of achieving marital variety and sexual pleasure ( haeri 1989 ) . since the arrival of donor technologies ,
however , muta has also been invoked to make egg donation legal within the parameters of marriage . within this context of ijtihad and muta , shia religious authorities who now accept the idea of donation , but are strict in their interpretation of how donation should be practiced ,
argue that when a couple needs a donor , they should go to a shiite religious court , where a decision can be made on a case - by - case basis;there should be a determination about which religious reference
( i.e. , source of spiritual emulation ) the infertile couple follows;the decision should be made in the presence of witnesses , the ivf doctor , and with the agreement of both parties ( the infertile couple and the donor);the husband should do a muta marriage with the egg donor for the period of time in which the whole procedure ( egg retrieval to embryo transfer ) is taking place , because polygyny is legal in islam and avoids the implications of zina , or adultery;but because a married shiite muslim woman can not marry another man other than her husband ( since polyandry is illegal in islam ) , she can not do a muta marriage with a sperm donor .
technically , the child born of a sperm donor would be a laqit , or out - of - wedlock child , without a family name and without a father .
thus , in theory , only widowed or otherwise single women should be able to accept donor sperm , in order to avoid the implications of zina .
however , in the muslim countries , single motherhood of a donor child is unlikely to be socially acceptable ( inhorn 1996 ; zuhur 1992 ) .
when a couple needs a donor , they should go to a shiite religious court , where a decision can be made on a case - by - case basis ; there should be a determination about which religious reference
( i.e. , source of spiritual emulation ) the infertile couple follows ; the decision should be made in the presence of witnesses , the ivf doctor , and with the agreement of both parties ( the infertile couple and the donor ) ; the husband should do a muta marriage with the egg donor for the period of time in which the whole procedure ( egg retrieval to embryo transfer ) is taking place , because polygyny is legal in islam and avoids the implications of zina , or adultery ; but because a married shiite muslim woman can not marry another man other than her husband ( since polyandry is illegal in islam ) , she can not do a muta marriage with a sperm donor . technically , the child born of a sperm donor would be a laqit , or out - of - wedlock child , without a family name and without a father .
thus , in theory , only widowed or otherwise single women should be able to accept donor sperm , in order to avoid the implications of zina .
however , in the muslim countries , single motherhood of a donor child is unlikely to be socially acceptable ( inhorn 1996 ; zuhur 1992 ) .
be that as it may , divergent gamete donation practices are beginning to emerge in the shiite muslim world , as religious authorities come to their own conclusions about third - party donation . among shiite religious scholars , the major disagreements , or religious sticking points , revolve around the following set of issues : whether sperm donation should be allowed at all;whether the child should follow the name of the infertile father or the sperm donor;whether the child should inherit from the infertile father or the sperm donor;whether donor children and their social parents are related at all , and , if not , whether they could potentially marry each other , which has implications for proper comportment in domestic life ( e.g. , bathing , veiling , etc.);whether donation is permissible at all if the donors are anonymous;whether a financial transaction should be allowed between gamete donors and recipients;whether the husband of an infertile woman needs to do a temporary muta marriage with the egg donor , then divorce her after the embryo transfer ( 48 to 72 hours later ) , in order to avoid zina . for his part
, khamanei clearly stipulates that muta marriage is not required , for he believes that zina requires the physical act of intercourse ( clarke 2006 ) ; andwhether the wife of an infertile husband can temporarily divorce her infertile husband , remarrying him after accepting sperm from a donor ( tremayne , personal communication , 5/5/06 ) .
whether sperm donation should be allowed at all ; whether the child should follow the name of the infertile father or the sperm donor ; whether the child should inherit from the infertile father or the sperm donor ; whether donor children and their social
parents are related at all , and , if not , whether they could potentially marry each other , which has implications for proper comportment in domestic life ( e.g. , bathing , veiling , etc . ) ; whether donation is permissible at all if the donors are anonymous ; whether a financial transaction should be allowed between gamete donors and recipients ; whether the husband of an infertile woman needs to do a temporary muta marriage with the egg donor , then divorce her after the embryo transfer ( 48 to 72 hours later ) , in order to avoid zina . for his part , khamanei clearly stipulates that muta marriage is not required , for he believes that zina requires the physical act of intercourse ( clarke 2006 ) ; and whether the wife of an infertile husband can temporarily divorce her infertile husband , remarrying him after accepting sperm from a donor ( tremayne , personal communication , 5/5/06 ) .
as shown in anthropologist morgan clarke s ( 2006 ) recent research on the shiite religious discourses surrounding gamete donation , many shiite ulama do not agree with khamanei s permissive fatwa on donor technologies , because they do not regard him as a brilliant legal thinker .
for example , ayatollah muhammad husayn fadlallah , lebanon s most prominent shiite religious figure , does not agree with ayatollah khamanei s permission of sperm donation , although he agrees with the permission of egg donation .
neither of them requires the use of muta marriages to solve the zina issue ( clarke 2006 ) .
ayatollah fadlallah s positions opposing sperm donation but supporting egg donation square with the dominant religious discourse in iran .
there , the religious rulings regarding gamete donation have evolved quickly , with sperm donation now effectively banned ( soraya tremayne , personal communication , 23 july 2004 , 31 july 2004 ) .
namely , a law on gamete donation passed in 2003 in the iranian parliament ( majlis ) and approved by the guardian council ( i.e. , a religious watchdog body that endorses every bill before it becomes law ) has restricted gamete donation to married persons . even though the law is brief ( less than one page ) , it states clearly and succinctly who can and can not donate and receive gametes .
egg donation is allowed , as long as the husband marries the egg donor temporarily thereby ensuring that all three parties are married .
sperm donation , on the other hand , is legally forbidden , because a sperm donor can not temporarily marry an already married woman whose husband is infertile . quite interestingly ,
however , embryo donation which involves both sperm and egg from another couple is allowed in order to overcome both male and female infertility .
because an embryo comes from a married couple and is given to another married couple , it is considered hallal , or religiously permissible .
the social and biological implications of embryo donation are quite interesting . for iranian couples unable to produce a child because of male infertility ,
embryo donation allows them to bypass the problem of the husband s weak ( or absent ) sperm .
however , embryo donation does not allow a presumably fertile wife of an infertile husband to contribute her own ova , in effect severing her biological ties to the donor child .
furthermore , and most strikingly , embryos donated from another married couple involve both egg and sperm donation .
even though direct sperm donation is bypassed via the injection of another man s sperm into the wife s womb / ova , embryo donation still disrupts male paternity and involves the acceptance by an already married woman of another man s ( and woman s ) gametes .
moreover , a woman s acceptance of another woman s egg is effectively like gestational surrogacy , which is strictly prohibited in sunni islam . indeed , in recent months
, cases of surrogate motherhood have occurred in iran , despite the lack of firm legislation regarding this practice . whether these problematic complications of embryo donation have been carefully thought through by the religious and legal authorities in iran
is unclear . based on her path - breaking research carried out at iranian ivf clinics , anthropologist soraya tremayne ( 2005 , 2006 ) notes ,
my first and possibly superficial interpretation at this stage can only be that the moral , ethical and legal aspects of the whole matter have not yet been thought through and the consequences dawned on either the doctors and legislators , or on people themselves .
these operations have been in place only for the past ten years ... and the cases are too new to cause problems as yet ( personal communication , 23 july 2004 ) .
it is interesting to note that many shiite religious leaders both inside and outside of iran do not agree with the relative iranian permissiveness
vis - - vis donor technologies ; instead , they abide by the dominant sunni muslim ban on all forms of third - party donation .
for example , i recently attended a two - day conference in tehran , iran , on gamete and embryo donation , sponsored by the avesina research institute in association with the law and political science faculty of the university of tehran .
the conference provided a fascinating example of ijtihad in action , with some ulama , dressed in their stately robes and turbans ( black for the sayyids , or descendants of the prophet muhammad ) , arguing against the moral permissibility of embryo and gamete donation .
the disagreements generated in public between pro and con ulama were also debated in the more private recesses of the conference .
for example , a shiite sharia judge from bahrain , who was staying at our guest residence , took great pains to describe to me his opposition to all forms of gamete donation . to prove this point
, he provided me with a copy of his book on islamic personal status law , which had been translated into english and which supported his anti gamete donation position based on evidence from the traditional islamic scriptures . according to him , iranian clergy , who speak farsi rather than arabic , are not as familiar with the original islamic scriptures ( in arabic ) that demonstrate the immorality of third - party donation .
thus , in his view , some iranian clergy are innovating in ways that are religiously unacceptable , and that are at odds with the rest of the muslim world .
a case in point : ayatollahs ali al - sistani and muhammad said al - tabatabai al - hakim , both shiite religious authorities in iraq , advise caution against third - party donation practices , viewing them as largely unacceptable ( clarke 2006 ) .
indeed , ayatollah al - sistani s son , muhammad rida al - sistani , has devoted an entire volume of richly documented legal analysis to this debate , providing an invaluable resource for other scholars ( 26 ) .
sistani s work , while perhaps posing more questions than clear answers , opens up for other scholars a fascinating window into this area of shiite jurisprudential debate , at a time when the western media are just waking up to the vibrant engagement shiite scholars have had with other such new technologies ( 26 ) .
indeed , the degree to which some shiite clergy are pushing the envelope in the realm of reproductive science and technology is quite remarkable . at the recent gamete donation conference in iran , some iranian clergy and physicians present advocated for future laws permitting all forms of donation as well as surrogacy .
once passed into law , gamete donation of all kinds will be difficult to stop .
meanwhile , in the absence of formal legislation , some ivf physicians in tehran as well as in shiite - dominant lebanon , which is closely following the iranian lead are using the legal vacuum and the original permissive fatwa of ayatollah khamanei to practice all forms of gamete donation among their desperate infertile patients . as noted by clarke ( 2006 ) for lebanon ,
doctors keep khamanei s fatwa collection on the shelves of their surgeries to demonstrate the permissibility of such procedures to skeptical muslim patients ; and many such patients have profited from it to undertake donor sperm and egg procedures , even surrogacy arrangements , with a clear conscience ( 26 ) .
although donor law and practice are headed in interesting new directions in both iran and lebanon , it must be reiterated that the vast majority of muslims , both shiite and sunni , do not accept the idea of third - party gamete donation .
why are they opposed to donation ? in the hundreds of interviews that i have conducted since 1996 in egypt ( with sunni muslims ) and lebanon ( with both sunni and shiite muslims ) , the majority of infertile couples were clear that donation is haram , or forbidden by the religion ( inhorn 2006b ) .
patient concerns revolve around three sets of related issues : ( 1 ) the moral implications of third - party donation for marriage , ( 2 ) the potential for incest , and ( 3 ) the moral implications of donation for kinship and family life . with regard to marriage ,
islam is a religion that can be said to privilege even mandate heterosexual marital relations . as is made clear in the original al - azhar fatwa , reproduction outside of marriage is considered zina , or adultery , which is strictly forbidden in islam .
although third - party donation does not involve the sexual body contact of adulterous relations , or presumably the desire to engage in an extramarital affair , it is nonetheless considered by most islamic religious scholars to be a form of adultery , by virtue of introducing a third party into the sacred dyad of husband and wife .
it is the very fact that another man s sperm or another woman s eggs enter a place where they do not belong that makes donation of any kind inherently wrong and threatening to the marital bond .
the other aspect of third - party donation that troubles marriage is the potential for incest among the offspring of unknown donors .
moral concerns have been raised about the potential for a single anonymous donor s offspring to meet and marry each other , thereby undertaking an incestuous union of half - siblings .
the final moral concern voiced by muslim ivf patients is that third - party donation confuses issues of kinship , descent , and inheritance . as with marriage , islam is a religion that can be said to privilege even mandate biological inheritance . preserving the origins of each child meaning its relationship to a known biological mother and father
is considered not only an ideal in islam , but a moral imperative . the problem with third - party donation , therefore , is that it destroys a child s lineage , which is immoral in addition to being psychologically devastating .
muslim ivf patients use the term mixture of relations to describe this untoward outcome .
such a mixture of relations , or the literal confusion of lines of descent introduced by third - party donation , is described as being very dangerous , forbidden , against nature , against godin a word , haram , or morally unacceptable .
it is argued that donation , by allowing a stranger to enter the family , confuses lines of descent in patrilineal islamic societies . for men in particular , ensuring paternity and the purity of lineage through known fathers is of paramount concern ( inhorn 2006b ) .
as one sunni muslim man , a high school biology teacher , summarized the problem :
the most important thing is that we are muslims .
if there is faith in carrying out this operation using sperm from the husband and ova from the wife , then this is okay .
we heard some women hire the womb of another woman , or take sperm . according to our religion ,
we consider it some kind of zina , prostitution . because there are many hadiths from the prophet muhammad that confirm this .
if you put your sperm in another woman besides your wife , you go to hell .
if you put your sperm in another woman other than your wife , you are going to commit a sin .
people asked the prophet , how ? he said , if you put it in your wife , you are going to be rewarded from allah .
they said , yes . he told them , but this is also the case if you put it in the wrong womb .
if there is faith in carrying out this operation using sperm from the husband and ova from the wife , then this is okay .
we heard some women hire the womb of another woman , or take sperm . according to our religion ,
we consider it some kind of zina , prostitution . because there are many hadiths from the prophet muhammad that confirm this .
if you put your sperm in another woman besides your wife , you go to hell .
if you put your sperm in another woman other than your wife , you are going to commit a sin .
people asked the prophet , how ? he said , if you put it in your wife , you are going to be rewarded from allah .
they said , yes . he told them , but this is also the case if you put it in the wrong womb .
you are going to have punishment . in addition to the consequences of mixed bloodlines and adultery , bringing such donor children into the world is considered unfair to the children themselves , who would never be treated with the love and concern parents feel for their real children .
thus , a child of third - party donation starts life off as an illegal
the child is deemed illegitimate and stigmatized even in the eyes of his or her own parents , who will therefore lack the appropriate parental sentiments ( inhorn 2006b ) .
as one sunni muslim ivf patient stated :
my baby must be mine , and from my husband .
a mother will never feel this is her child if it is from another [ man s ] donated sperm or ova .
if the child is from the father and mother , they will feel this is actually our baby . if not , we ll not be a family .
. you will feel like you re acting , making a movie , living a life that s not true .
of course , people take babies [ through adoption ] , but this is not so common here in egypt .
we do nt even want to think about this point ! we are making our trial [ of ivf ] and hoping god will help us .
a mother will never feel this is her child if it is from another [ man s ] donated sperm or ova .
if the child is from the father and mother , they will feel this is actually our baby . if not , we ll not be a family .
you will feel like you re acting , making a movie , living a life that s not true .
of course , people take babies [ through adoption ] , but this is not so common here in egypt .
we do nt even want to think about this point ! we are making our trial [ of ivf ] and hoping god will help us .
indeed , the firm conviction that parenthood of a donor child is an impossibility is clearly linked to the legal and cultural prohibitions against adoption throughout the muslim world .
the islamic scriptures , including the quran , encourage the kind fostering of orphans but do not allow legal adoption as it is known in the west , whereby a child takes its adoptive parents surname and is treated as one s own child ( inhorn 1996 ; sonbol 1995 ; zuhur 1992 ) . in the muslim middle east ,
few ivf patients , either sunni or shiite , will contemplate adoption , stating with conviction that it is against the religion and that the adopted child wo nt be my own son
even though legal adoption is practiced in iran with the child receiving a birth certificate in the adoptive couple s name after a six - month period of adjustment the social and cultural resistances to adoption remain strong in that country , making this a
last resort for infertile iranian couples ( janet heindl , personal communication , 8 july 2004 ; tober 2004 ) . nonetheless , iran s acceptance of adoption
has clearly paved the way for gamete donation , with ayatollah khamanei s initial fatwa deeming the infertile couple to be like adoptive parents . indeed , iran s clearly adventurous path regarding both adoption and donation is leading to social transformations in other parts of the shiite middle east , in ways described in the following section .
in considering infertility and ivf in the muslim world , it is important to ask : what happens to infertile middle eastern muslim couples who are not allowed to adopt and who do not accept the use of donor gametes ? in the absence of adoption and gamete donation , infertile muslim couples have no choice but to turn to ivf and other assisted reproductive technologies to solve their infertility problems using their own gametes . in the middle eastern muslim world , marriage is highly valued , and nearly all adults marry if possible in most middle eastern countries ( population reference bureau 2004 ; zuhur 1992 ) .
middle eastern societies are also pronatalist they highly value children for numerous reasons and expect all marriages to produce them ( inhorn 1996 ) .
children are desired from the beginning of marriage in most cases , and are usually loved and cherished once they are born . as a result ,
, infertile women often live in fear that their marriages will collapse , for islamic personal status laws consider a wife s barrenness to be a major ground for divorce .
although islam also allows women to divorce if male infertility can be proven , a woman s initiation of divorce continues to be so stigmatizing that women rarely choose this option unless their marriages are truly unbearable ( inhorn 1996 ) .
instead , they often cover for their infertile husbands , accepting the social responsibility for the infertility and diffusing the embarrassment of their husbands reproductive emasculation ( inhorn 2003b , 2004 ) .
the emergence of the revolutionary new ivf technology called intracytoplasmic sperm injection ( icsi ) , however , has ironically increased the potential for divorce in the muslim middle east .
namely , with icsi , infertile men with very poor sperm profiles even azoospermia , or lack of sperm in the ejaculate are now able to produce biological children of their own .
as long as a single viable spermatozoon can be retrieved from a man s body , including through painful testicular aspirations and biopsies , this spermatozoon can be injected directly into the ovum under a high - powered microscope . what icsi requires , then , is high - quality ova , despite low - quality sperm .
however , the wives of many of these men , who have stood by their infertile husbands for years , even decades in some cases , may have grown too old to produce viable ova for the icsi procedure . in the absence of adoption or of any kind of egg donation ,
infertile muslim couples with a reproductively elderly wife face four difficult options : ( 1 ) to remain together permanently without children ; ( 2 ) to legally foster an orphan , which is rarely viewed as an acceptable option ; ( 3 ) to remain together in a polygynous marriage , which is rarely viewed as an acceptable option by women themselves ; or ( 4 ) to divorce so that the husband can have children with a younger wife . in my research in egypt and lebanon ,
the first option has proven to be the most common namely , infertile husbands and their 40-something wives often love each other deeply , and remain together in long - term marriages without producing any children .
thus , divorce is not the immediate consequence of infertility that it is stereotypically portrayed to be . because of the sunni islamic restrictions on the use of donor eggs , however , as well as lack of acceptance of this option among some segments of the shiite population , at least some muslim men are choosing to divorce or take a second wife , believing that their own reproductive destinies lie with younger , more fertile women .
that being said , in the shiite muslim world , including in iran and lebanon , at least some shiite couples are beginning to receive both donor gametes and donor embryos , as well as donating their gametes to other infertile couples . for infertile shiite couples who accept the idea of third - party donation as well as for ayatollah khamanei , who originally introduced the idea of donation to the muslim world
the introduction of donor technologies has been described as a marriage savior , helping to avoid the marital and psychological disputes that may arise if the couple s case is otherwise untreatable .
such disputes are clearly dramatized in the popular iranian film laila , which documents the painful separation of an otherwise happily married but infertile couple , and which was released in the mid-1990s before the khamanei fatwa permitted such marriages to be saved through the use of donor technologies . in iran today , donor egg and donor embryo programs have been set up at most ivf clinics .
donor eggs come from three sources : other ivf patients , relatives , and unmarried women who agree to participate as egg donors in one - day muta marriages for a fee . such marriages only require a witness and are not officially registered ; thus , they take place in confidence in the back rooms of ivf clinics .
indeed , donors who wish to remain anonymous enter these muta marriages only by written agreement , without ever meeting the recipients of their eggs or their temporary husbands .
$ 550 ) , provide no personal information about themselves to the recipient couple , receive no information about the recipient couple , and go about their business ( soraya tremayne , personal communication , 31 july 2004 ) . in short , egg donation
as well as embryo donation from other couples is largely a financial transaction in iran , with very little regulation or control over who donates or how donation is enacted .
according to tremayne , the recent law in iran specifies clearly that couples desiring an embryo as a result of infertility must apply in writing to a court in order to receive permission for embryo transfer .
the law specifies that the couple must be morally sound and suitable as parents and must be iranian citizens , much like the law governing adoption in iran ( janet heindl , personal communication , 8 july 2004 ) .
still , the donor embryo law is so new that most ivf clinics in iran do not yet own a copy of the legislation and are not necessarily abiding by the legal requirements for donor embryo transfer at their clinics .
if the husband is infertile , the couple simply receives another couple s embryos , with most donor couples choosing to remain anonymous . as tremayne ( 31 july 2004 )
states ,
i did not get the impression that people desperate to have a child thought very far about the issues of kinship and family relations . as far as i could see
, the donation is considered more a financial transaction than a donation / gift , and once you have paid the couple to buy their embryo , or paid the temporary wife for her egg , they have no further claims on you and this is the end of the story .
the forms filled by the donors leave it to them to decide whether they want to give their name or not .
i did not get the impression that people desperate to have a child thought very far about the issues of kinship and family relations .
as far as i could see , the donation is considered more a financial transaction than a donation / gift , and once you have paid the couple to buy their embryo , or paid the temporary wife for her egg , they have no further claims on you and this is the end of the story .
the forms filled by the donors leave it to them to decide whether they want to give their name or not . in lebanon where i have conducted my own research ,
the situation is very similar , despite the lack of a national law governing any aspect of ivf or third - party donation . at lebanese ivf clinics providing donor technologies , some of the donors are other ivf patients ( mostly shiite muslims who accept the idea of donation ) , some are friends or relatives ( including egg - donor sisters ) , and some are anonymous donors , who provide their ova for a fee . in at least one clinic catering to a largely conservative shiite clientele , some of these donors are young non - muslim , american women who travel to lebanon for extra payment to donate their eggs anonymously to infertile lebanese couples .
ironically , those most likely to receive these american eggs are conservative shiite couples , who accept the idea of donation because they follow the teachings of ayatollah khamanei in iran .
thus , in lebanon , those most likely to follow the spiritual guidance of ayatollah khamanei and , hence , to receive american donor eggs are generally members of or sympathizers with lebanon s hizbullah political party , which is officially described by the u.s .
administration as a terrorist organization ! furthermore , quite interestingly , in multisectarian lebanon , the recipients of these donor eggs are not necessarily only shiite muslim couples .
some sunni muslim patients from lebanon and from other middle eastern muslim countries such as egypt and syria are quietly slipping across transnational borders to save their marriages through the use of donor gametes , thereby secretly going against the dictates of sunni muslim orthodoxy .
such border crossing has also been noted by clarke ( 2006 ) for lebanon ; he writes , indeed patients come from other countries to benefit from this relatively relaxed regime ( 26 ) .
the same is true in iran , where , according to ivf clinic staff , scores of persian gulf arabs from countries such as saudi arabia and kuwait are traveling to tehran in pursuit of donor gametes .
in conclusion , it is fair to state that global reproductive technoscapes ( appadurai 1996 ) are becoming increasingly expansive as we enter this new millennium . as this article has tried to show , the muslim world generally positioned on the receiving end of global reproductive technology transfers has nonetheless embraced assisted reproductive technologies with considerable enthusiasm while , at the same time , reconfiguring them in accordance with the local religious moralities so important in this region .
although generally portrayed as monolithic , islam itself takes several different forms , as evident in this essay . in the sunni muslim world , which includes most middle eastern countries ,
the use of ivf and related assisted reproductive technologies has clearly led to an entrenchment of deeply held religious beliefs about the importance of biologically based kinship , family life , and parenthood .
yet the globalization of these technologies to the shiite muslim world has fundamentally altered understandings of the ways in which families can be made and the ways in which marriages can be saved through the uses of assisted reproductive technologies . for shiite muslims , in particular , the frankly adventurous attitude on the part of some shiite religious leaders toward third - party donation has led to a potential transformation in gender relations among infertile muslim couples . for example , in lebanon , the recent shiite fatwas allowing egg donation have been a great boon to marital relations . there , both fertile and infertile men with reproductively elderly wives are lining up at ivf clinics to accept the eggs of donor women .
furthermore , in multisectarian lebanon , the recipients of donor gametes are not necessarily only shiite muslim couples , but include some sunni muslim and christian couples as well . in short ,
the arrival of donor technologies in the muslim middle east has led to a brave new world of reproductive possibility never imagined when these technologies were first introduced there nearly 20 years ago .
these technologies have engendered ( 1 ) significant medical transnationalism and reproductive tourism ; ( 2 ) mixing of gametes across ethnic , racial , and religious lines ; and ( 3 ) the birth of thousands of icsi and , now , donor babies to devout infertile muslim couples .
infertile couples have begun to reconsider traditional notions of biological kinship , even if social parenthood of a donor child is still not widely embraced ( inhorn 2006b ) . and because donor technologies are now widely available in both iran and lebanon , the power of the sunni muslim ban on third - party donation is being weakened across the region , with some infertile sunni muslim couples reconsidering their own antidonation moral stances . as a result , shiite gametes are finding their ways into sunni bodies , an interesting variation on the making of muslim babies . in my view , these multiple transformations are powerful indicators of the profound social effects that reproductive technologies may engender in the new world order .
as the assisted reproductive technologies become further entrenched in the muslim world , and additional forms of global reproductive technology become available , it is important to examine the new local moral worlds that are likely to arise in response to this variant of globalization . the pace of change evident in the production of assisted reproductive technologies themselves as highlighted on the recent nova special called 18 ways to make a babyas well as the rapid spread of these technologies into far reaches of the non - western world is , indeed , striking .
thus , as one science and technology studies scholar , david hess ( 1994 ) , rightly observes , anthropology brings to these discussions a reminder that the cultural construction of science is a global phenomenon , and that the ongoing dialogue of technoculture often takes its most interesting turns in areas of the world outside the developed west ( 16 ) .
to overcome the difficulties of raising a biologically unrelated donor child , some shiite physicians in lebanon are arguing for a variety of novel solutions , including inheritance through gifts and bequests ; institutions of rida ( milk kinship ) , whereby the mother of a donor child becomes related to it through breastfeeding ; and the notion of legal guardianship of foster children ( clarke 2006).i am deeply grateful to soraya tremayne , who , fresh from fieldwork in iran , has engaged in a lively e - mail discussion with me and has provided invaluable information on the practices of ivf and gamete donation in iran since 2004 .
i have tried to represent her findings as accurately as possible in this paper.in iran , i want to thank mohammad jalal abassi - shavazi , mohamad mehdi akhondi , and pegah ebadi for so generously inviting and hosting me at the avesina research center and university of tehran conference on embryo and gamete donation.in lebanon , anonymous sperm donation using frozen sperm from overseas sperm banks or fresh sperm samples from mostly medical and graduate students is quietly practiced at ivf clinics .
one of my azoospermic lebanese male informants produced a donor child in this way , and several others , both muslim and christian , had also made the decision to use donor sperm.in iran , women commonly bring their sisters as potential egg donors . but this is not allowed , as islam is explicitly against the marriage of one man to two living sisters .
apparently , men also bring their brothers as potential sperm donors in iran . according to tremayne
, she observed one case where the husband did so without his wife s knowledge ; the wife believed that she was receiving her husband s sperm instead of that of her brother-in-law.some leading members of the lebanese medical community are pushing for a law that bans all forms of third - party donation in the country .
however , this law has yet to be debated in the lebanese parliament and is unlikely to pass , according to some sources . to overcome the difficulties of raising a biologically unrelated donor child
, some shiite physicians in lebanon are arguing for a variety of novel solutions , including inheritance through gifts and bequests ; institutions of rida ( milk kinship ) , whereby the mother of a donor child becomes related to it through breastfeeding ; and the notion of legal guardianship of foster children ( clarke 2006 ) .
i am deeply grateful to soraya tremayne , who , fresh from fieldwork in iran , has engaged in a lively e - mail discussion with me and has provided invaluable information on the practices of ivf and gamete donation in iran since 2004 .
i have tried to represent her findings as accurately as possible in this paper . in iran ,
i want to thank mohammad jalal abassi - shavazi , mohamad mehdi akhondi , and pegah ebadi for so generously inviting and hosting me at the avesina research center and university of tehran conference on embryo and gamete donation . in lebanon ,
anonymous sperm donation using frozen sperm from overseas sperm banks or fresh sperm samples from mostly medical and graduate students is quietly practiced at ivf clinics .
one of my azoospermic lebanese male informants produced a donor child in this way , and several others , both muslim and christian , had also made the decision to use donor sperm . in iran , women commonly bring their sisters as potential egg donors . but this is not allowed , as islam is explicitly against the marriage of one man to two living sisters .
apparently , men also bring their brothers as potential sperm donors in iran . according to tremayne
, she observed one case where the husband did so without his wife s knowledge ; the wife believed that she was receiving her husband s sperm instead of that of her brother - in - law .
some leading members of the lebanese medical community are pushing for a law that bans all forms of third - party donation in the country .
however , this law has yet to be debated in the lebanese parliament and is unlikely to pass , according to some sources . | medical anthropological research on science , biotechnology , and religion has focused on the local moral worlds of men and women as they make difficult decisions regarding their health and the beginnings and endings of human life .
this paper focuses on the local moral worlds of infertile muslims as they attempt to make , in the religiously correct fashion , muslim babies at in vitro fertilization ( ivf ) clinics in egypt and lebanon .
as early as 1980 , authoritative fatwas issued from egypt s famed al - azhar university suggested that ivf and similar technologies are permissible as long as they do not involve any form of third - party donation ( of sperm , eggs , embryos , or uteruses ) . since the late 1990s , however
, divergences in opinion over third - party gamete donation have occurred between sunni and shiite muslims , with iran s leading ayatollah permitting gamete donation under certain conditions .
this iranian fatwa has had profound implications for the country of lebanon , where a shiite majority also seeks ivf services .
based on three periods of ethnographic research in egyptian and lebanese ivf clinics , this paper explores official and unofficial religious discourses surrounding the practice of ivf and third - party donation in the muslim world , as well as the gender implications of gamete donation for muslim marriages . |
rna interference ( rnai ) is a conserved cellular mechanism by which a small double stranded rna ( dsrna ) directs the degradation of complementary mrna and therefore inhibits the expression of a specific gene.(1 ) since its discovery , rnai has become a powerful tool to study gene functions in biological processes .
the ability to induce rnai in mammalian cells using synthetic small interfering rna ( sirna ) has stimulated great interest in therapeutic applications of rnai . in numerous studies ,
sirnas have shown promise for treating a variety of diseases , including influenza and hiv infection , cancer and genetic defects .
a key challenge of rnai - based therapeutic application is the efficient delivery of sirna into target cells .
sirna is usually 21 nucleotides in length and highly charged and therefore can not cross the cytoplasmic membrane by free diffusion . in the circulation and interstitial space , sirna is vulnerable to degradation by rnase.(11 ) although sirna can be delivered directly and locally to the target sites in limited applications , a carrier system is required in most applications to protect sirna from degradation and to facilitate its uptake by target cells .
the existing carrier systems usually contain a key cationic component , such as a cationic lipid , a cationic polymer or a cationic peptide , in order to bind sirna effectively .
other supplementary components help to improve the stability , solubility or pharmacological profiles of sirnacarrier complexes.(16 ) the use of carriers can dramatically increase the uptake of sirna . however , the efficacy of the carriers in mediating functional sirna delivery , i.e. , leading to the knockdown of the target transcript , remains low.(17 ) many of the existing carriers were originally designed for dna delivery , and hence have not been optimized to account for the differences between sirna and dna .
sirna mediates its effect in the cytosol,(18 ) while dna requires entry into the nucleus in order to gain access to the transcriptional machinery .
such differences between sirna and dna could have great influences on the functionality and efficacy of a carrier . to rationally develop efficient sirna carriers , it is necessary to elucidate the mechanisms by which the carriers mediate functional sirna delivery .
however , the current knowledge on the mechanisms of the carriers comes predominantly from studies with dna delivery .
although different carriers have vastly different chemical structures and physical properties , it is believed that a majority of the dnacarrier complexes is taken up into the cells by endocytosis . a primary focus in the research and development of carriers for dna delivery has been to facilitate the endosomal escape of the dnacarrier complexes prior to their degradation in the lysosomes . however , even with the most efficient dna carriers developed so far , it remains controversial to what extent the escape from endosome contributes to transfection efficiency .
considering the physical differences between sirna and dna and their sites of action , whether endosomal escape plays any significant role in functional sirna delivery by the various carrier systems has yet to be determined . in this report , we have determined the mechanisms by which a widely used cationic lipid carrier mediates functional delivery of sirna by a combination of imaging , pharmacological and genetic approaches .
we show that although most of the cellular uptake of sirna lipoplexes is via endocytic pathways , these pathways do not appear to play a significant role in the delivery of sirnas that mediate rnai . instead , a novel pathway , probably mediated by direct fusion between sirna lipoplexes and the plasma membrane , is responsible for the functional delivery of sirna into the cell .
african green monkey kidney epithelial cells bsc-40 , human embryonic kidney cells 293 ft , and human cervical carcinoma cells hela were purchased from the american type culture collection ( atcc ) .
the cells were maintained in dulbecco s modified eagle s medium ( dmem ) , supplemented with 100 iu / ml of penicillin , 100 iu / ml of streptomycin , 2 mm l - glutamine , and 10% heat - inactivated fetal bovine serum ( fbs ) ( biowest , miami , fl ) .
the cells were cultured in 6-well falcon tissue culture plates in a 5% co2 humidified incubator at 37 c .
sirnas specific for cyclophilin b ( cyb ) and green fluorescent protein ( gfp ) and cy5 conjugated gfp sirna were purchased from dharmacon ( lafayette , co ) .
the sirna sequences are as follows : gfp sirna : ( sense ) 5-ggcuacguccaggagcgcadtdt-3 and ( antisense ) 5-ugcgcuccuggacguagccdtdt-3. cyb sirna : ( sense ) 5-ggaaagacuguuccaaaaadtdt-3 and ( antisense ) 5-uuuuuggaacagucuuuccdtdt-3. the wild - type and mutant dynamin expression plasmids were generously provided by dr .
mark mcniven of the mayo cancer center(25 ) and were cloned into pegfp - n1 expression vector from clontech .
the vectors expressing the wild - type and mutant cav - fed and cav - dn caveolins were generously provided by dr .
jan eggermont(26 ) and were cloned into the pcineo / ires - gfp expression vector .
mutant cell lines were established by electroporation of bcs cells followed by selection in 2.5 mg / ml geneticin ( invitrogen , carlsbad , ca ) .
the day prior to experiments , the cells were sorted with a facsaria ( becton - dickinson and co. ) to enrich for the gfp positive cells .
uptake of fluorescence - labeled transferrin ( sigma - aldrich ) was used to test if the k44a mutant cells lacked the clathrin - mediated endocytosis .
uptake of fluorescence - labeled cholera toxin ( invitrogen ) was used to test if the caveolin mutant cells lacked the caveolin - mediated endocytosis .
sirna lipoplexes were prepared following the manufacturer s protocol . in brief , 4 l of dharmafect1
was diluted into 200 l of opti - mem i ( invitrogen ) and then mixed with an equal volume of opti - mem i with appropriate amount of sirna .
the sirna lipoplex solution was then diluted 5-fold in dmem , 10% fbs for transfection .
twelve hours prior to transfection , bsc cells were seeded in 6-well plates at a density of 2 10 cells / well so that , by the time of transfection , the cells were in the early log phase and reached 70% confluence . during transfection , cell culture media were aspirated and cells were washed with pbs . unless otherwise specified , 2 ml of 100 nm sirna lipoplexes was incubated with cells for 4 h at 37 c .
the cells were subsequently washed using cellscrub buffer ( genlantis , san diego , ca ) and pbs .
after washing , fresh media were added and cells were cultured at 37 c for the indicated length of time .
pharmacological inhibitors were purchased from sigma - aldrich ( st . louis , mo ) and were used at the following concentrations : cytochalasin d , 100 m ; chlorpromazine , 10 g / ml ; filipin , 10 g / ml ; nystatin , 100 g / ml ; amiloride , 3 mm ; d , l - threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( pdmp ) , 10 m ; sodium azide , 40 mm ; and 2-deoxy glucose , 40 mm .
the cells were pretreated with the inhibitors for 1 h at 37 c and then transfected with sirna lipoplexes for 4 h at 37 c in the presence of the inhibitors .
the cells were washed and cultured in fresh media in the absence of the inhibitors for another 20 h prior to rna isolation . in experiments where sirna transfection was carried out at 4 , 10 , 20 , and 37 c ,
the cells were incubated at specified temperatures for 1 h and then transfected with sirna lipoplex at the same temperatures for 4 h. the cells were washed , replenished with fresh media , and incubated for 20 h at 37 c prior to rna isolation . at indicated hours post - transfection , single cell suspension was prepared and total rna was extracted using an rneasy mini kit ( qiagen , valencia , ca ) according to the manufacturer s protocol .
rna concentration was determined by uv absorbance at 260 nm , and all samples had an a260/a280 ratio greater than 1.95 .
total rna was first converted into cdna using random hexamers and taqman reverse transcription reagents ( applied biosystems , foster city , ca ) .
the levels of cyb and actin mrnas were then measured using taqman gene expression assays ( applied biosystems ) .
the real time rt - pcr reactions were performed in applied biosystems microamp optical 96-well reaction plate , on a prism 7000 real - time pcr system .
unless specifically indicated , 2 ml of 100 nm sirna lipoplexes were incubated with cells for 4 h at 37 c .
the cells were washed with cellscrub buffer , trypsinized and suspended in pbs plus 2% bovine serum albumin and 0.05% nan3 .
the cells were analyzed with a facscaliber and cellquest software ( becton - dickinson ) .
cellular uptake of cy5 labeled sirna was quantified by measuring the relative fluorescence signal in cy5 channel .
cells incubated with nonlabeled sirna lipoplexes were used as a control , and their mean fluorescence values were subtracted from the uptake measurements of cy5-sirna lipoplexes .
cells were grown in 35 mm diameter mattek ( mattek , ashland , ma ) glass bottom culture dishes , transfected with cy5-sirna lipoplexes , and viewed on an inverted epifluorescence microscope ( olympus ix71 , olympus , center valley , pa ) , integrated in a deltavision deconvolution system ( applied precision , issaquah , wa ) .
cells were housed in an environmental chamber ( solent scientific , segensworth , u.k . ) to maintain constant temperature , humidity and co2 levels during the observation . unless specified ,
all experiments were conducted at 37 c , using an atmosphere of 95% air and 5% co2 . for fluorescence imaging , cell nuclei and endolysosomes
cells were viewed in opti - mem i medium with an olympus 63 planapo oil immersion objective with a numerical aperture of 1.40 , fitted with a coolsnap hq monochrome 12-bit cooled ccd camera ( photometrics , tucson , az ) .
images of 512 512 pixels were captured by applied precision s softworx software and analyzed using imaris 4.2 ( bitplane , saint paul , mn ) .
to elucidate the mechanisms underlying the functional sirna delivery , we focused on dharmafect1 , a widely used sirna transfection reagent based on cationic lipids . for easy imaging analysis , we used african green monkey kidney epithelial cells bsc-40 .
human embryonic kidney cells 293 ft and human cervical carcinoma cells hela were also used in certain experiments . to measure the functional sirna delivery , we assayed the knockdown of an endogenous gene cyclophilin b ( cyb ) because it is ubiquitously expressed and its knockdown has no apparent effect on cell survival and growth .
cyb is also highly conserved between human and african green monkey ; therefore , the same sirnas are effective in cells derived from both species .
we used quantitative pcr to measure the level of cyb transcript so as to detect the effect of functional sirna delivery as directly and quickly as possible .
in addition , we established that the uptake of sirna lipoplexes is proportional to sirna concentration in a range of 10 pm to 200 nm , and the target transcript knockdown is linear to sirna concentration in a range of 100 pm to 100 nm ( supplementary figure s1 in the supporting information ) . because 100 nm sirna produces the most dramatic knockdown of the target transcript but is still within the linear range , we used 100 nm sirna in most experiments . to determine the dynamics of sirna lipoplex transfection
, we analyzed the localization of sirna lipoplexes inside the cells at different times post - transfection .
bsc cells were incubated with cy5 labeled sirna lipoplexes for 1 , 2 , or 4 h , washed and then fixed for imaging analysis .
some of the 4 h transfected cells were washed and cultured for an additional 4 , 12 , and 46 h before fixation and imaging analysis .
sirna lipoplexes were detected in cells following 1 h transfection ; the number of sirna lipoplexes increased significantly following 4 h transfection ( figure 1a ) .
a significant number of sirna lipoplexes still persisted inside the cells 46 h post - transfection .
cy5-sirna lipoplexes were incubated with bsc cells for the indicated length of time , washed , and cultured for the indicated length of time before fixation and imaging analysis .
bsc cells were transfected with cy5-sirna lipoplexes for the indicated length of time in the presence of lysosensor .
the number of intracellular sirna complexes and the number of sirna complexes that overlapped with lysosensor staining were enumerated .
percentages of intracellular sirna complexes that colocalize with lysosensor are shown at the indicated time points .
bsc cells were transfected with cyb - specific sirna lipoplexes for 2 or 4 h , washed , were used for rna isolation immediately .
some 4 h transfected cells were cultured for another 2 or 20 h before rna isolation .
the levels of cyb transcript were quantified by real - time pcr and normalized to that in nontransfected cells .
thus , cells were stained with lysosensor specific for endolysosomes and then transfected with sirna lipoplexes for 1 , 2 , 3 , and 4 h. quantification of colocalization of lysosensor and cy5-labeled sirna lipoplexes revealed that > 90% sirna lipoplexes were localized in the endolysosomes ( figure 1b ) . to determine the relationship between the observed uptake of sirna lipoplexes and the target transcript knockdown , bsc cells were transfected for either 2 or 4 h and assayed for cyb transcript level immediately , or transfected for 4 h , washed and cultured for additional 2 or 20 h before measuring the level of cyb transcript . a decrease in cyb mrna level
was detected following 2 h transfection , but a significant decrease was clearly evident following 4 h transfection ( figure 1c ) .
additional culture of 4 h transfected cells resulted in further decrease of cyb transcript level . by comparing figures 1a , 1b and 1c ,
it is clear that significant target mrna knockdown occurred in the absence of significant change in the level of sirna lipoplexes in the endolysomes . because a 4 h sirna lipoplex transfection followed by a 20 h culture results in a consistent cyb mrna knockdown by 8090% , we used this assay format in the rest of the study .
one of the best characterized and most important endocytosis pathways is the clathrin - mediated endocytosis , which is responsible for the uptake of a wide variety of macromolecules from extracellular environment.(27 ) to quantify the uptake of sirna lipoplexes through this pathway , bsc cells were treated with chlorpromazine or cytochalasin d , which inhibit clathrin - mediated endocytosis,(28 ) followed by sirna lipoplex transfection .
both inhibitors reduced the uptake of cy5 labeled sirna lipoplexes by 50% ( figure 2a and supplementary figure s2a in the supporting information ) .
however , sirna mediated knockdown of cyb transcript was not diminished in the presence of these inhibitors ( figure 2b ) .
inhibition of clathrin - mediated endocytosis significantly blocks uptake of sirna lipoplexes but not functional sirna delivery .
bsc cells were treated with chlorpromazine ( chlorp , 10 g / ml ) or cytochalasin d ( cytod , 100 m ) for 1 h followed by sirna lipoplex transfection for 4 h in the presence of the inhibitors .
after washing , sirna lipoplex uptake was measured by flow cytometry ( see for details ) .
transfected cells were cultured for another 20 h before cyb transcript level was measured by real - time pcr .
( b ) relative levels of cyb transcript in the presence or absence of inhibitors as normalized to the level of untreated cells . ( c )
relative levels of cyb transcript in bsc cells that expressed the wild - type ( wt ) or k44a dynamin .
bsc cells that stably express either the wt or k44a dynamin were transfected with sirna lipoplexes for 4 h , washed , cultured for 20 h , and then assayed for cyb transcript level .
dynamin is required for the clathrin - mediated endocytosis . to confirm the results with pharmacological inhibitors
, we constructed bsc cells that constitutively expressed the wild - type or a dominant - negative form of dynamin with mutation of lysine at the position 44 to alanine ( termed k44a).(29 ) expression of the mutant but not the wild - type dynamin inhibited the uptake of transferrin ( supplementary figure s2b in the supporting information ) , a marker for the clathrin - mediated endocytosis .
however , sirna - mediated knockdown of the target transcript was not affected by expression of either the wild - type or k44a dynamin ( figure 2c ) .
similarly , transient expression of k44a dynamin in human embryonic kidney cells 293 ft did not inhibit the knockdown of target transcript following sirna lipoplex transfection ( supplementary figure s2c in the supporting information ) .
inducible expression of k44a dynamin in human cervical carcinoma cells hela also did not affect target transcript knockdown ( supplementary figure s2d in the supporting information ) .
together , these results show that although the clathrin - mediated endocytosis is a major pathway for cellular uptake of sirna lipoplexes , it does not appear to contribute significantly to the functional sirna delivery .
next , we determined if functional sirna delivery is dependent on cholesterol because cholesterol is a critical component of the cytoplasmic membrane and plays a critical role in many membrane - associated events , including membrane fusion , macropinocytosis , and caveolin- and lipid - raft - mediated endocytosis .
bsc cells were treated with nystatin or filipin for 1 h to deplete cholesterol from the plasma membrane and then transfected with sirna lipoplexes for 4 h in the presence of the inhibitors .
after washing , cells were assayed for sirna lipoplex uptake by flow cytometery or cultured for 20 h and then assayed for cyb transcript level . under these conditions , cytotoxicity was minimal because > 90% of the cells did not take up propidium iodide .
as shown in figure 3 , although treating cells with nystatin or filipin had minimal effect on the uptake of sirna lipoplexes , target mrna knockdown was completely abolished in the presence of filipin and significantly reduced in the presence of nystatin . to exclude the possibility that the inhibitors may have interfered with the functional sirna delivery by destabilizing the sirna lipoplexes , we measured the particle size of sirna lipoplexes in the presence of the inhibitors by dynamic light scattering .
when the inhibitors were added to sirna lipoplexes , the size of the particles remained the same , with > 95% of the particles were 100200 nm in diameter ( data not shown ) .
in addition , after the inhibitors were removed from the sirna lipoplexes suspension by dialysis , the sirna lipoplexes remained active and mediated efficient rnai ( supplementary figure s3 in the supporting information ) .
hence , depletion of cholesterol from the plasma membrane abolishes functional delivery of sirna mediated by cationic lipids .
( a ) uptake of sirna lipoplexes in the absence or presence of either nystatin ( 100 g / ml ) or filipin ( 10 g / ml ) as measured by flow cytometry .
( b ) relative cyb transcript level in the presence or absence of the inhibitors .
representative data from one of three experiments are shown . among the various endocytic pathways that are sensitive to cholesterol depletion ,
the caveolin - mediated endocytosis is perhaps the best characterized . to determine whether this pathway contributes to the functional delivery of sirna by cationic lipids , we constructed bsc cells that stably expressed one of the two dominant - negative caveolin constructs : one mutant , termed cav - dn , harbored a deletion of amino acid residues 181 at the n - terminal ; and the other mutant , termed cav - fed , had amino acid residues 6873 replaced with aaaaag.(26 ) expression of the mutant but not the wild - type caveolin in bsc cells resulted in the inhibition of the uptake of cholera toxin ( supplementary figure s4a in the supporting information ) , suggesting that caveolin - mediated endocytosis is inhibited significantly .
however , transfection of sirna lipoplexes into bsc cells expressing either the wild - type or the mutant caveolin resulted in cyb knockdown to the same extent ( figure 4a ) .
similarly , transient expression of the dominant negative caveolins in 293 ft cells did not diminish cyb transcript knockdown ( supplementary figure s4b in the supporting information ) .
functional delivery of sirna lipoplexes is not dependent on caveolin- or lipid - raft - mediated endocytosis or macropinocytosis .
( a ) relative levels of cyb transcript in bsc cells that express the wild - type ( wt ) or the mutant caveolins .
bsc cells that stably expressed either the wild - type or one of the mutant caveolins were transfected with sirna lipoplexes for 4 h , washed and cultured for 20 h. the level of cyb transcript was quantified .
( b , c ) effect of pdmp ( b ) or amiloride ( c ) on functional sirna delivery .
bsc cells were treated with pdmp ( 10 m ) or amiloride ( 3 mm ) for 1 h , followed by sirna lipoplex transfection for 4 h , washed , and cultured for 20 h. the level of cyb transcript was quantified .
( d ) uptake of cy5 labeled sirna lipoplexes in the absence or presence of inhibitors as quantified by flow cytometry .
d , l - threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( pdmp ) inhibits synthesis of sphingolipid,(37 ) a component enriched in domains of lipid raft.(38 ) treatment of bsc cells with pdmp had no effect on knockdown of target transcript following transfection with sirna lipoplexes ( figure 4b ) although it inhibited the uptake of sirna lipoplexes by approximately 20% ( figure 4d ) .
similarly , amiloride , a strong inhibitor of macropinocytosis,(39 ) showed no significant effect on either the knockdown of cyb transcript or sirna lipoplex uptake ( figure 4c , d ) .
together , these results suggest that functional delivery of sirna lipoplexes does not appear to depend on caveolin- or lipid - raft - mediated endocytosis or macropinocytosis . to determine
if the functional delivery of sirna lipoplexes is an energy - dependent process , we treated the cells with deoxy - glucose and sodium azide to inhibit atp biosynthesis .
bsc cells were treated with both inhibitors for 1 h and then transfected with sirna lipoplexes for 4 h in the presence of the inhibitors .
after washing , cells were assayed immediately for sirna uptake or cultured for 20 h in the absence of the inhibitors ( to avoid inhibition of rnai reaction ) and then assayed for cyb transcript level .
however , inhibition of atp biosynthesis during transfection did not diminish the target transcript knockdown ( figure 5b ) , suggesting that the functional delivery of sirna lipoplexes does not appear to depend on new atp biosynthesis .
( a ) uptake of sirna lipoplexes in the absence or presence of deoxy - glucose ( 40 mm ) and sodium azide ( 40 mm ) .
( b ) relative level of cyb transcript in the presence or absence of atp biosynthesis inhibitors . ( c )
representative data are shown as mean sd ( n = 3 or 4 ) .
bsc cells were precooled to 4 , 10 , or 20 c for 1 h and then transfected with sirna lipoplexes for 4 h at the same temperatures .
after washing , the cells were incubated at 37 c for 20 h and assayed for cyb transcript level .
knockdown of the target transcript was significantly reduced at low temperatures ( figure 5c ) .
curve fitting suggests that the reduction of target transcript level was exponential ( no sharp transition ) as temperature decreases , suggesting that the effect of temperature on functional delivery of sirna lipoplexes is gradual .
in this study , we examined pathways by which sirna lipoplexes are taken up by cells to determine whether specific uptake pathways contribute to functional sirna delivery .
our results show that 95% of sirna lipoplexes that enter cells end up in the endolysosomes , most of which are contributed by clathrin - mediated endocytosis ( 50% ) and lipid - raft - mediated endocytosis ( 20% ) .
these findings are consistent with observations that endocytosis is the major pathway for the uptake of dna lipoplexes . despite the contribution to the uptake of the majority of sirna lipoplexes
, the endocytotic pathways do not appear to contribute significantly to functional sirna delivery as measured by target transcript knockdown .
consistent with this observation , while endocytosis is an energy - dependent process , functional sirna delivery mediated by cationic lipids is independent of new atp biosynthesis during the transfection period .
furthermore , inhibition of clathrin - mediated endocytosis by either pharmacological inhibitors or expression of a dominant negative dynamin did not inhibit the knockdown of the target transcript ( figure 2 ) .
similarly , inhibition of caveolin - mediated endocytosis , lipid - raft - mediated endocytosis or macropinocytosis did not diminish the knockdown of the target transcript ( figure 4 ) .
while these results do not exclude the possibility that a small amount of sirna lipoplexes leaked out of endolysosomes following endocytosis and contributed to the target gene silencing , taking together , they suggest alternative mechanisms of cationic lipid - mediated functional sirna delivery . the functional delivery of sirna mediated by cationic lipids is shown here to be a rapid process as inferred from the rapid knockdown of target transcript ( figure 1c ) .
furthermore , the process is temperature sensitive but does not require new atp biosynthesis during the transfection period ( figure 5 ) .
based on these characteristics , the mechanism underlying the cationic lipid - mediated functional sirna delivery is probably through direct fusion between sirna lipoplexes and the plasma membrane .
membrane fusion requires cholesterol,(31 ) and it is notable that depletion of cholesterol from the plasma membrane by treating cells with nystain or filipin abolished or significantly inhibited target transcript knockdown ( figure 3 ) . because treating cells with nystain or filipin did not significantly inhibit the bulk uptake of sirna lipoplexes , the functional sirna delivery likely occurs through a minor uptake pathway , consistent with the mechanism of a direct fusion .
the mechanisms by which cationic lipids mediate dna delivery have been investigated extensively . during cationic lipid - mediated dna delivery , the lipids function by compacting plasmid dna into dna - containing liposomes or lipiddna complexes ( lipoplexes ) .
the lipoplexes facilitate cellular uptake of dna primarily via endocytosis following binding of positively charged lipiddna lipoplexes to the negatively charged cellular surfaces .
although fusion of dna lipoplexes with the plasma membrane was initially suggested as a way to deliver dna directly into the cytoplasm,(19 ) lipid mixing assays did not reveal a correlation between fusion of dna lipoplexes with plasma membranes and their transfection efficiency .
rather , it seems that when dna lipoplexes fuse with the cellular membrane , dna tends to be excluded at the cell surface and does not enter the cell.(48 ) because the majority of dna lipoplexes end up in the endolysosomes , most of the effort to enhance dna transfection has focused on stimulating the escape of dna lipoplexes from the endolysosomes . what could account for the differences in the mechanisms of cationic lipid mediated sirna and dna delivery ?
this similarity could account for the uptake of sirna or dna lipoplexes by the same endocytosis pathways .
however , sirna is much smaller than dna and functions in the cytosol whereas dna has to be transported into the nucleus to be transcribed . upon fusion of sirna lipoplexes with the plasma membrane ,
some sirna is likely delivered directly into the cytosol , where sirna can be readily recruited into rnai mechinary to exert its effect .
in contrast , dna delivered into the cytosol via fusion has to be transported into nucleus before it can be transcribed . even if some dna is delivered directly into the cytosol , it may not contribute significantly to the functional dna delivery .
this difference could potentially account for the different mechanisms by which cationic lipids mediate functional sirna versus dna delivery .
thus , one practical significance of our observations is that optimizing conditions for fusion between sirna lipoplexes and the plasma membrane should improve functional sirna delivery by cationic lipids . in summary ,
our studies show that although most sirna lipoplexes enter cells via endocytosis , this mode of entry does not appear to contribute significantly to functional sirna delivery . instead , a minor but rapid pathway , probably mediated by fusion of sirna lipoplexes with the plasma membrane , accounts for the finding that only a small fraction of the sirna lipoplexes that enter cells are responsible for most of the observed sirna - mediated target gene knockdown .
rnai , rna interference ; sirna , small interfering rna ; cytod , cytochalasin d ; chlorp , chlorpromazine ; cyb , cyclophilin b ; gfp , green fluorescent protein ; pdmp , d , l - threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol . | a key challenge for therapeutic application of rna interference is to efficiently deliver synthetic small interfering rnas ( sirnas ) into target cells that will lead to the knockdown of the target transcript ( functional sirna delivery ) . to facilitate rational development of nonviral carriers ,
we have investigated by imaging , pharmacological and genetic approaches the mechanisms by which a cationic lipid carrier mediates sirna delivery into mammalian cells .
we show that 95% of sirna lipoplexes enter the cells through endocytosis and persist in endolysosomes for a prolonged period of time
. however , inhibition of clathrin- , caveolin- , or lipid - raft - mediated endocytosis or macropinocytosis fails to inhibit the knockdown of the target transcript .
in contrast , depletion of cholesterol from the plasma membrane has little effect on the cellular uptake of sirna lipoplexes , but it abolishes the target transcript knockdown .
furthermore , functional sirna delivery occurs within a few hours and is gradually inhibited by lowering temperatures .
these results demonstrate that although endocytosis is responsible for the majority of cellular uptake of sirna lipoplexes , a minor pathway , probably mediated by fusion between sirna lipoplexes and the plasma membrane , is responsible for the functional sirna delivery .
our findings suggest possible directions for improving functional sirna delivery by cationic lipids . |
to study mitochondrial nuclear genetic interactions in the nematode caenorhabditis briggsae , our three laboratories independently created 38 intra - species cytoplasmic nuclear hybrid ( cybrid ) lines .
although the cross design combines maternal mitotypes with paternal nuclear genotypes , eight lines ( 21% ) unexpectedly contained paternal mitotypes .
all eight share in common ancestry of one of two genetically related strains .
this unexpected parallel observation of paternal mitochondrial transmission , undesirable given our intent of creating cybrids , provides a serendipitous experimental model and framework to study the molecular and evolutionary basis of uniparental mitochondrial inheritance . | |
simple anthropometric measures of body composition and fat distribution are widely accepted as being the most practical and valuable approach to diagnose excess body fat , usually classified as overweight and obesity .
excess body fat can have an adverse effect on health , leading to development of various chronic diseases , cardiovascular ( cv ) disease being the most typical [ 14 ] .
the oldest and the most commonly used anthropometric measure for this purpose is the body mass index ( bmi ) , an estimate of body fat calculated from height and weight .
it distinguishes 3 categories normal weight ( < 24.9 kg / m ) , overweight ( 2529.9 kg / m ) and obese ( 30 kg / m ) without precise distinction between body fat and other tissues , such as musculoskeletal . also , bmi is insensitive to the relative distribution of body fat .
the assessment of body fat percentage ( % bf ) is another measure often used , but has similar limitation as bmi .
because health risk seems to be primarily associated with abdominal fat [ 68 ] , measures for assessment of excess abdominal fat , such as waist circumference ( wc ) , waist - hip ratio ( whpr ) and waist - height ratio ( whtr ) were introduced as more specific alternatives .
all these measures were shown to be associated with metabolic disorders and cv risk factors in different patterns and extent [ 914 ] .
some of these measures were also reported to be related to arterial stiffness , an important , independent and proven cv risk factor [ 1517 ] .
several studies showed association of the anthropometric measures with arterial stiffness in obese population , with clear sex differences .
however , little is known about these associations in healthy and generally non - obese populations .
only a few studies have examined the association of several anthropometric measures with arterial stiffness in healthy populations close to normal weight , but their results were inconsistent [ 2022 ] .
for instance , in the study examining relation of bmi and bf content with stiffness index , augmentation index ( aix ) and pulse pressure ( pp ) , wykretowicz et al . , showed that bmi was not a good predictor of arterial stiffness . on the contrary , wildman et al
. found bmi together with wc and whpr was strongly associated with pulse wave velocity ( pwv ) , the direct marker of arterial stiffness .
further , maher et al . showed that none of the anthropometric measures was associated with aix .
arterial stiffness can be assessed by several approaches . a direct arterial stiffness index , pwv , considered as a gold standard method ,
aix is an indirect marker of arterial stiffness and a direct measure of wave reflection , also confirmed to be associated with cv risk .
central systolic blood pressure ( csbp ) , an indicator of central hemodynamics , is a complementary stiffness index , also used as a predictor of cv events .
the aim of our study was assessment of sex - specific association of different anthropometric measures of body composition ( bmi,%bf , wc , whtr and whpr ) with a spectrum of arterial stiffness indices ( pwv , aix and csbp ) in healthy and generally non - obese subjects . in order to clearly distinguish the effect of each anthropometric measure ,
the data were complemented with a wide set of known confounders involved in the complex pathophysiology of the increased arterial stiffness , such as age , heart rate , blood pressures , blood lipids and smoking .
the study was conducted at the university of split school of medicine , as a part of larger genetic epidemiology research program [ 2729 ] during 20092010 .
participants were unselected , healthy adults , > 18 years of age , originating from the city of split ( croatia ) and surrounding areas . after providing a signed informed consent for participation in the study ,
subjects with history of cardiovascular diseases ( hypertension , coronary artery disease , myocardial infarction , heart failure or arrhythmia ) , cerebrovascular diseases , cancer , thyroid disturbances , diabetes mellitus , elevated serum glucose , triglycerides and cholesterol were excluded from the study . also , those taking lipid - lowering , blood sugar - lowering , antihypertensive or other cardiovascular medications were excluded from the study .
blood sampling and anthropometric and arterial stiffness measurements were performed in the morning , after overnight fasting .
blood glucose , triglycerides , and total ldl and hdl cholesterol were measured by standard laboratory procedures .
smoking status was obtained from a questionnaire and was coded into categories : current smokers , former smokers and non - smokers .
height and weight were measured using a digital scale ( seca 789 bionetics , canada ) with the precision of 0.1 cm and 0.1 kg , respectively .
bmi was calculated by dividing the participant s weight ( kg ) by square of height ( m ) .
wc was measured using a tape measurer placed halfway between the lower border of the ribs and the iliac crest in a horizontal plane , and was measured to the nearest 0.1 cm .
percent bf was determined from measurements of skinfold thickness taken at 4 standard sites biceps , triceps , subscapular and suprailiac using harpenden skinfold caliper ( cms instruments , london , uk )
body density was calculated from the sum of the 4 skinfold measurements according to durnin and womersley , and final percentage of body fat was calculated by siri s equation .
all hemodynamic measurements were preformed in a temperature - controlled room , unaffected by external environmental influences , in a supine position after resting for 15 min .
arterial stiffness was measured non - invasively using arteriograph ( tensiomed ltd . , budapest , hungary ) , a recently introduced and validated device for arterial stiffness assessment [ 3436 ] .
arteriograph uses an oscillometric principle for measurement of the aortic pwv and pulse wave analysis . essentially , the device measures periodic pressure changes in the inflated cuff ( 35 mmhg above systolic pressure ) induced by fluctuations in pulsatile pressure in the brachial artery beneath .
the cuff was applied to the upper right arm . simultaneously with the parameters of arterial stiffness ( pwv ( m / s ) , caix ( % ) , csbp ( mmhg ) ) , the device also recorded systolic and diastolic blood pressures ( sbp and dbp , mmhg ) and heart rate ( min ) .
mean arterial pressure ( map ) was subsequently calculated by formula : map = dbp+(sbp - dbp)/3 .
all measurements were performed by 2 well - trained investigators ( db and gg ) .
the average values of 2 measurements that satisfied quality control , taken 5 minutes apart , were used .
the pearson correlations and multiple regression analysis ( mlr ) were used to assess the associations of each anthropometric measure of body composition with a particular arterial stiffness index within each sex group of subjects .
potential confounding effects adjusted for were age , heart rate , height , sbp , dbp , smoking , total ldl and hdl cholesterol and triglycerides . in order to avoid biased results ,
outliers were identified and removed from the initial dataset ( n=352 ) . in total , 7 univariate ( z>3.3 ) and 2 overlapping extreme multivariate outliers ( mahalanobis d2 , p0.001 ) were removed ( n=7 ) . since no a priori hypothesis was made to determine the order of entry of the potential predictor factors ( see above ) , a direct method for the regression analyses was used .
the entering of the predictor variable was followed by removing the one with the lowest non - significant regression coefficient and by subsequent regression analysis .
the procedure was repeated until all non - significant predictors were removed or until further exclusions caused a deterioration of the model according to f - test . in each case , the direct regression model was followed by backward method to check for inconsistencies .
finally , the optimal model of arterial stiffness index prediction with a particular anthropometric measure as independent variable was determined .
normal distributions were evaluated with determination of skewness and histograms as well as with one - sample kolmogorov - smirnov test .
independent sample t - tests as well as tests were used for quantitative and qualitative variables , respectively , when evaluating the differences between sexes .
all other statistical calculations were performed using spss software , version 13 ( spss inc . ,
the study was conducted at the university of split school of medicine , as a part of larger genetic epidemiology research program [ 2729 ] during 20092010 .
participants were unselected , healthy adults , > 18 years of age , originating from the city of split ( croatia ) and surrounding areas . after providing a signed informed consent for participation in the study ,
subjects with history of cardiovascular diseases ( hypertension , coronary artery disease , myocardial infarction , heart failure or arrhythmia ) , cerebrovascular diseases , cancer , thyroid disturbances , diabetes mellitus , elevated serum glucose , triglycerides and cholesterol were excluded from the study . also , those taking lipid - lowering , blood sugar - lowering , antihypertensive or other cardiovascular medications were excluded from the study .
blood sampling and anthropometric and arterial stiffness measurements were performed in the morning , after overnight fasting .
blood glucose , triglycerides , and total ldl and hdl cholesterol were measured by standard laboratory procedures .
smoking status was obtained from a questionnaire and was coded into categories : current smokers , former smokers and non - smokers .
height and weight were measured using a digital scale ( seca 789 bionetics , canada ) with the precision of 0.1 cm and 0.1 kg , respectively .
bmi was calculated by dividing the participant s weight ( kg ) by square of height ( m ) .
wc was measured using a tape measurer placed halfway between the lower border of the ribs and the iliac crest in a horizontal plane , and was measured to the nearest 0.1 cm .
biceps , triceps , subscapular and suprailiac using harpenden skinfold caliper ( cms instruments , london , uk ) .
body density was calculated from the sum of the 4 skinfold measurements according to durnin and womersley , and final percentage of body fat was calculated by siri s equation .
all hemodynamic measurements were preformed in a temperature - controlled room , unaffected by external environmental influences , in a supine position after resting for 15 min .
arterial stiffness was measured non - invasively using arteriograph ( tensiomed ltd . , budapest , hungary ) , a recently introduced and validated device for arterial stiffness assessment [ 3436 ] .
arteriograph uses an oscillometric principle for measurement of the aortic pwv and pulse wave analysis . essentially , the device measures periodic pressure changes in the inflated cuff ( 35 mmhg above systolic pressure ) induced by fluctuations in pulsatile pressure in the brachial artery beneath .
the cuff was applied to the upper right arm . simultaneously with the parameters of arterial stiffness ( pwv ( m / s ) , caix ( % ) , csbp ( mmhg ) ) , the device also recorded systolic and diastolic blood pressures ( sbp and dbp , mmhg ) and heart rate ( min ) .
mean arterial pressure ( map ) was subsequently calculated by formula : map = dbp+(sbp - dbp)/3 .
all measurements were performed by 2 well - trained investigators ( db and gg ) .
the average values of 2 measurements that satisfied quality control , taken 5 minutes apart , were used .
the pearson correlations and multiple regression analysis ( mlr ) were used to assess the associations of each anthropometric measure of body composition with a particular arterial stiffness index within each sex group of subjects .
potential confounding effects adjusted for were age , heart rate , height , sbp , dbp , smoking , total ldl and hdl cholesterol and triglycerides . in order to avoid biased results ,
outliers were identified and removed from the initial dataset ( n=352 ) . in total , 7 univariate ( z>3.3 ) and 2 overlapping extreme multivariate outliers ( mahalanobis d2 , p0.001 ) were removed ( n=7 ) . since no a priori hypothesis was made to determine the order of entry of the potential predictor factors ( see above ) , a direct method for the regression analyses was used .
the entering of the predictor variable was followed by removing the one with the lowest non - significant regression coefficient and by subsequent regression analysis .
the procedure was repeated until all non - significant predictors were removed or until further exclusions caused a deterioration of the model according to f - test . in each case , the direct regression model was followed by backward method to check for inconsistencies .
finally , the optimal model of arterial stiffness index prediction with a particular anthropometric measure as independent variable was determined .
normal distributions were evaluated with determination of skewness and histograms as well as with one - sample kolmogorov - smirnov test .
independent sample t - tests as well as tests were used for quantitative and qualitative variables , respectively , when evaluating the differences between sexes .
all other statistical calculations were performed using spss software , version 13 ( spss inc . ,
there were 152 ( 43.2% ) men and 200 ( 56.8% ) premenopausal women . in both sexes ,
body composition , hemodynamic and biochemical parameters were within the range expected for normally distributed population ( table 1 ) .
women had significantly lower csbp and greater aix values , while there was no difference in pwv between sexes .
our population sample was predominantly non - obese according to bmi , 52% of subjects had normal body weight , 38% were overweight and 10% were obese .
mean ( sd ) bmi was 23.953.60 in the female and 25.843.77 in the male subjects .
the analysis revealed several differences between the females and the males . in the female group
univariate analysis showed that most correlations between measures of body composition and arterial stiffness indices were positive and mainly significant ( table 2 , bold column ) .
however , application of a mlr analysis revealed quite different results . in general , after adjustment for known risk factors , multivariate models detected 3 measures of body composition ( bmi , wc and whtr ) as weak and inverse predictors of pwv and aix ( table 2 ) . in the csbp models ,
as independent predictors , the 3 measures of body composition ( bmi , wc and whtr ) , explained only several percentages of arterial stiffness variance ( sr=0.0030.02 ) .
other measures , such as % bf and whpr , were not significant predictors of arterial stiffness in the female group . in the male group ,
similar to the females , correlations between measures of body composition and arterial stiffness indices , determined by univariate analysis , were positive and mainly significant ( table 3 , bold column ) .
again , after application of the mlr and adjustment for known confounders , most of these univariate associations disappeared ( table 3 ) .
in contrast to females , bmi was the only common and inverse predictor of arterial stiffness indices , pwv and csbp .
similar to the female group , measures of body composition as independent predictors explained only several percent of arterial stiffness variance ( sr=0.0050.02 ) .
measures such as % bf and whpr were not significant predictors in the male group . finally ,
relative to the male models , female models in general explained 10% to 15% less variance , and differed in some predictors ( tables 2 and 3 ) .
the results of this study show that in the sample of healthy and generally non - obese subjects the positive univariate associations of anthropometric measures of body composition with arterial stiffness indices were not consistent , as their significance and strength depended on sex but also on the measure of body composition and arterial stiffness index .
in addition , mlr analysis revealed that these associations were strongly confounded by the arterial stiffness risk factors such as age , blood pressures , height , heart rate , blood lipids and/or smoking .
after removal of their confounding effects , the body composition measures turned out to be weak and inverse predictors of arterial stiffness , and their influence was sex - dependent .
namely , bmi , wc and whtr were key predictors of arterial stiffness in the females , while bmi was the principal predictor in the males .
these key findings of our study seemingly contradict the results of several other studies that showed stronger and positive association of anthropometric measures with the arterial stiffness indices .
in fact , our results indicate a possible explanation for discrepancies among these studies , as they did not completely distinguish the influence of other risk factors on arterial stiffness .
namely , these studies used univariate correlations or did not include all confounding factors within the best - fitted models of prediction , thus their data interpretation might be insufficient because confounding effects were not removed .
actually , the initial steps in our analysis also showed similar results until we applied mlr .
we therefore strongly suggest all further studies in this area to adopt a more critical approach when distinguishing body composition measures effects on arterial stiffness .
generally , the inverse association of anthropometric measures and arterial stiffness might appear to be surprising , but similar results were obtained by several other authors as well .
it was suggested that a decrease in peripheral resistance due to hyperinsulinemia or low - grade inflammation , that is actually accompanied with excess body fat , could lead to decreased wave reflection and consequently decreased arterial stiffness values .
this implies that the observed association was probably due to changes in peripheral resistance instead of direct changes in arterial stiffness . despite the bmi theoretical limitation
( it does not show relative fat distribution ) , it was the most strongly associated with arterial stiffness in both the female and male populations .
it had similar predictive value in the females as 2 central anthropometric measures , wc and whtr , while it was the only measure predicting stiffness in the males .
bmi has been in use for decades and its predictive value as a health risk factor has been well - investigated and confirmed .
although it has been also increasingly criticized , our results indicate that it is still a valuable predictor .
another parameter , wc , measures both abdominal subcutaneous and intra - abdominal amount of fat , and it has also been confirmed as a predictors of obesity - related health risk .
the weakness of the measure is that it does not take into account a person s stature .
two people with the same wc can have different amounts of fat depending on their height .
we found that the corrected measure , whtr , was as good a parameter as wc in the females , indicating their equal usefulness in practice .
this is in line with the study of nordstrand at al , who also showed whtr and wc to be predictors of arterial stiffness in obese females .
lack of association of the skinfold assessed % bf with arterial stiffness could be explained by the fact that it depends on the amount of peripheral subcutaneous fat , regardless of the amount of visceral fat .
difficulties in estimation of the fat amount and the use of standardized estimation formula , which were not validated in the investigated population , are likely reasons that % bf turned out to be an insignificant predictor .
the possible reason why another measure , whpr , was not predicting arterial stiffness may be that hip circumference reflects different aspects of body composition , including muscle mass , fat mass and skeletal frame .
thus , a reduction in weight usually results in a reduction of both waist and hip circumferences , and this will not necessarily result in changes in waist - hip ratio .
in addition , several studies showed that hip circumference is even negatively associated with health risk .
this may partially explain why whpr varies from weak to strong depending on population sample .
wc and whtr were the only measures predicting arterial stiffness in the female , but not in the male group .
this sex difference in association of anthropometric measures with arterial stiffness was also found by other authors , who showed that greater number of anthropometric measures were associated with arterial stiffness in women ( both non - obese and obese ) compared to men . in addition , our study also showed that different arterial stiffness indices were influenced by various confounding factors . underlining mechanisms explaining the described differences are yet to be examined
. the limitations of this study include the cross - sectional nature of the study design , without a possibility for longitudinal follow - up .
further , the estimates of percentage of body fat mass could have been a possible source of bias , since they were based on the anthropometric measurements of skinfold thickness and not other methods such as bioelectrical impedance or dual energy x - ray absorptiometry .
the association of anthropometric measures of body composition and arterial stiffness in healthy , generally non - obese subjects suggests that bmi , wc and whtr are predictors of arterial stiffness in the females and bmi in the males .
the associations of anthropometric measures with arterial stiffness confounded by different factors ( age , blood pressures , height , heart rate , blood lipids , smoking ) indicate the necessity for thorough regression analysis in similar studies . | summarybackgroundanthropometric measures of body composition and arterial stiffness are commonly used as indicators of cardiovascular risk .
little is known , however , about the association of the anthropometric measures with arterial stiffness , especially in a healthy , generally non - obese population.material/methodsin a sample of 352 healthy subjects ( 200 premenopausal women ) , 3 arterial stiffness indices were analyzed ( pulse wave velocity , augmentation index and central systolic blood pressure ) in relation to 5 anthropometric measures of body composition ( body mass index
bmi , body fat percentage by skinfold measurements %bf , waist circumference
wc , waist - hip ratio
whpr , and waist - height ratio
whtr ) .
data were analyzed using correlation and regression analyses , with adjustment for the following confounders : age , blood pressures , height , heart rate , blood lipids and smoking.resultsmost correlations between anthropometric measures and arterial stiffness indices were significant and positive in both sex groups ( r=0.140.40 , p<0.05 ) .
after adjustment for confounding effects , bmi , wc and whtr remained significant ( but inverse ) predictors of arterial stiffness ( from 0.06 to 0.16 ; p<0.05 ) in the females , while in the males bmi was the only measure inversely predicting arterial stiffness ( from 0.09 to 0.13 ; p<0.05).conclusionsmeasures of body composition are weak and inverse predictors of arterial stiffness and their influence is sex - dependent .
bmi , wc and whtr were key predictors of arterial stiffness in the females , while bmi was the principal predictor in the males .
the associations of anthropometric measures with arterial stiffness are strongly and differently confounded by various factors that have to be taken into account when explaining results of similar studies . |
subjects : this research was performed within the framework of the ( dpp ) diabetes prevention project , in isfahan , iran .
our aim in dpp was to prevent diabetes by some lifestyle measure or even medication interventions among at risk individuals ( ifg / igt patients ) . in the dpp ,
the first group was the lifestyle intervention that received intensive training in diet and physical activity .
our aim was , losing 7 percent of their body weight and maintaining that loss .
males and females 30 to 55 year of age who were first relative history of type 2 diabetes were invited to study via community announcement .
this population - based cross - sectional study was performed among 1450 people with first relative history of diabetes ( f.h.d ) . after excluding peoples using medications that would influence serum lipids ,
current analysis was done among 1323 individuals with f.h.d ( 332 males and 991 females ) .
the criterion for participants was having1 first - degree relative with a diagnosis of diabetes after 30 years old .
this study was approved by the research council of endocrine and metabolism research center ( emrc ) of isfahan university of medical science and each participant filled in consent .
anthropometric assessment : weight was measured by seca scale while subjects were lightly clothed and without shoes and recorded to the nearest .1 kg .
height was measured by seca stadiometer while subjects were in a standing position without shoes and their shoulders were in a normal position .
body mass index was calculated as weight ( in kg ) divided by height square ( in m2 ) .
wc was measured at minimal waist after a normal exhale and hip circumference was measured at the widest point over the buttocks with an un - scratched tape measure and without any pressure to body surface ; measurements were recorded to the nearest 1 cm .
data on physical activity were gathered by using participants responses to one query : how much time do you exercise in a week ? we categorized the replies of this query as never , < 3 h / wk , and 3 h / wk .
definition of terms : participants were categorized in 4 phenotype groups on the basis of the mentioned cutoff points : nwnt ( normal waist normal triglyceride ) : normal wc < 102 for man and for women < 88 and normal serum triglyceride concentrations ( < 150 mg / dl ) . ewnt ( enlarged waist normal triglyceride ) : enlarged wc for men 102 and for women 88 and normal serum triglyceride concentration ( < 150 mg / dl ) .
nwet ( normal waist elevated triglyceride ) : normal wc for men < 102 and for women < 88 and elevated triglyceride 150 mg / dl .
ewet ( enlarged waist elevated triglyceride ) : enlarged wc for men 102 and for women 88 and hyper serum triglyceride concentration ( 150mg / dl ) .
biochemical assessment : blood samples were taken after>10h overnight fasting while the participants were in a sitting and relax position and were centrifuged within 5 min .
the samples was analyzed by using autoanalyzer bt 3000 ( rome , italy ) by enzymatic glucose method using commercial kits ( chem enzyme , tehran , iran ) .
serum cholesterol and triglyceride levels were measured by enzymatic reagents ( chem enzyme , tehran , iran ) adapted to the selecta auto analyzer .
high - density lipoprotein cholesterol levels were measured using kits ( pars az - moon , tehran , iran ) .
inter - assay coefficients of variations ( cvs ) were 1.25 for tg , 1.2 for cholesterol and 1.2% for glucose .
glycated hemoglobin levels ( hba1c ) were assessed with ds5 and ds5 analyzer uses low pressure cation exchange chromatography in conjunction with gradient elution to separate human hemoglobin subtypes and variants from haemolysed whole blood.1920 statistical package for social sciences ( spss ) was used for statistical analyses .
general characteristics of individuals across different phenotypes of ewet were compared by oneway analysis of variance ( anova ) with tukey post - hoc correction .
multivariate - adjusted means for metabolic variables across various phenotypes of ewet was obtained by the use of analysis of covariance ( ancova ) in four models . in first model , we adjusted age ( y ) and physical activity ( never , < 3 hour / week , and 3 hour / week ) . in the second one
chi - square test was used for comparing prevalence of different phenotypes of ewet across different categories of ogtt test .
general characteristics of individuals across different phenotypes of ewet were compared by oneway analysis of variance ( anova ) with tukey post - hoc correction .
multivariate - adjusted means for metabolic variables across various phenotypes of ewet was obtained by the use of analysis of covariance ( ancova ) in four models . in first model , we adjusted age ( y ) and physical activity ( never , < 3 hour / week , and 3 hour / week ) . in the second one
chi - square test was used for comparing prevalence of different phenotypes of ewet across different categories of ogtt test .
general characteristics of individuals across different phenotypes of ewet were compared by oneway analysis of variance ( anova ) with tukey post - hoc correction .
multivariate - adjusted means for metabolic variables across various phenotypes of ewet was obtained by the use of analysis of covariance ( ancova ) in four models . in first model , we adjusted age ( y ) and physical activity ( never , < 3 hour / week , and 3 hour / week ) . in the second one
chi - square test was used for comparing prevalence of different phenotypes of ewet across different categories of ogtt test .
mean age of men and women was 43.5 5.5 and 43.0 5.6 y , respectively .
mean bmi , wc , whr and tg among men were 27.6 3.3 kg / m2 , 94.4 8.6 cm , 0.9 0.06 and 192.7 110 respectively and among women 29.6 4.5 kg / m2 , 87.9 9.1 cm , 0.8 0.05 and 158 83 respectively .
of the 1450 adult 332 ( 9.6% ) of men and 234 ( 23.6% ) of women had ewet phenotype .
general characteristics of male subjects separately in different phenotypes of wc and serum triglyceride levels have been presented in table 1 .
those with ewet phenotype had higher bmi , wc and whr as compared to other phenotypes ( p < 0.01 for all ) .
general characteristics of male participants by different phenotype of serum triacylglycerol concentration and waist circumference women 's general characteristics have been shown in table 2 .
those with the ewet phenotype were older and higher bmi , wc and whr as compared to other phenotypes .
characteristics female with phenotypes of serum triacylglyceral concentration and waist circumference men with the ewet phenotype had higher levels of serum triglyceride , cholesterol , and lower levels of hdl - cholesterol as compared to other groups ( table 3 ) .
such adjustments revealed significant differences in 2h - pg and hba1c between the ewet phenotype and other groups .
even after further adjustment for bmi , all the mentioned differences were significant except for serum hdl - cholesterol that was marginally non significant ( p = 0.057 ) .
crude means of all metabolic variables were of higher levels among women with the ewet phenotype as compared to other phenotypes ( table 4 ) .
controlling for age and physical activity and additional control for bmi had little impacts on these differences and they were still significant .
adjusted means for metabolic risk factors across four phenotypes of serum trigcylglycerol concentration and waist circumference in men multivariate adjusted means for metabolic risk factors across four phenotypes of serum trigcylglycerol concentration and waist circumference in women the prevalence of impaired glucose tolerance ( igt ) and diabetes across different phenotypes of wc and serum triglyceride levels have been provided in table 5 .
both men and women with the ewet phenotype had higher prevalence of type 2 diabetes compared to other phenotypes .
the prevalence of igt was not significantly different in four phenotypes of wc and serum tg concentration .
prevalence of diabetes and impaired glucose toleronce in male and female paticipants across different phenotypes of triacylglycerol conrentration ad waist circumference
findings of the current study suggest a positive association between hypertriglyceridemic waist phenotype and diabetes prevalence .
we observed that males and females with the ewet phenotype had higher metabolic abnormalities as compared to those without this phenotype .
this study is among the first study relating hypertriglyceridemic waist phenotype to igt and diabetesin a sample of individuals with first relative history of diabetes .
hypertriglyceridemic waist phenotype has been suggested as a screening tool for metabolic risk factors.21 considering the increasing trend in the prevalence of heart diseases and diabetes all over the world , developing simple and cheap screening tools for early identification of these chronic diseases is of great importance.17 ewet phenotype has the ability to recognize more subjects characterized by the metabolic traid than the presence of the metabolic syn - drome.21 some investigators have used this simple screening tool for the identification of metabolic triad and cardiovascular risk.15 among tehrani people , this phenotype have been reported as a valid predictor of cardiovascular disease , not only among adult,1617 but also among adolescents,22 such that those adolescents with the ewet phenotype had higher prevalence of other metabolic risks as well .
this phenotype has also been demonstrated as a simple method for early identification of those at higher risk of diabetes.9 lemieux et al have reported that two simple measures , waist an tg , might be an cheap clinical method identifying males with elevated insulin , apob and sldl concentration , and hence , increased chd and diabetes risk.15 however , this is the first among individuals with first relative history of diabetes relating this phenotype to igt and diabetes , among individual with fhd .
therefore , such a simple index can be used by health professionals in health care system to early identify those at higher risk of diabetes .
although waist circumference has been used as a screening measure for this purpose , it has been shown that the addition of a biomarker like serum triglyceride to this measure would increase its accuracy , sensitivity and specificity in this regard.22 in the current study , participants with the ewet phenotype had higher prevalence of metabolic risks .
although such findings have previously been reported by other investigators , the population studied in previous publications was not individuals with fhd .
the prevalence of ewet phenotype was respectively 9.6% and 23.6% among men and women in this study .
lemieux et al23 have reported the phenotype among 19% of 907 participants in the quebec health survey . among 3430 french men ,
the phenotype has been reported to be 12.1%.24 sumner et al25 found 21% of postmenopausal black women , 28.3% of white women and 42.3% of mexican women with this phenotype . in tehran
lipid and glucose study , the phenotype has been reported among 19% of adult men17 and 31.9% of women.16 this figure in isfahani women was 24%.26 as it comes from these figures , the prevalence is considerably different in different populations
it seems that the pattern of obesity among middle eastern population is something different from those in other places of the world . in other words
the so - called middle eastern pattern of obesity is highly prevalent in these countries .
this pattern of obesity is characterized by larger fat accumulation in the abdomen , particularly among women , such that women in these countries have higher prevalence of both general and central obesity .
different prevalence of this phenotype could also be explained by the use of different cut - off points for wc and serum triglyceride in the studies .
the age range of the studied population must also be taken into account . in this study ,
prevalence of diabetes among those with the ewet phenotype was 30.3% for men and 44.9% for women , respectively .
just 18.2% and 15.4% of those with nwnt phenotype were diabetic men and women , respectively .
st - piere at al,9 assessing 1190 individuals , introduced ewet phenotype as a predictor of cardiovascular risks among type 2 diabetic and igt patients .
they found that nearly 53% of males and 80% of females with the phenotype were diabetics or igt . in a study in us population ,
25.4% of those with the phenotype were diabetics and just 8% of diabetics were free of this phenotype.27 however all these studies have been performed among individuals without family history diabetes.all mentioned studies are in line with our findings in the current study suggesting the higher prevalence of diabetes among those with the phenotype .
it seems that enlarged waist circumference is associated with increased intra - abdominal fats which in turn could result in higher production of lipid products in the liver and therefore increasing levels of ldl and vldl . on the other hand
, high intra - abdominal fat would cause free fatty acid levels to increase in circulation which in turn could result in developing insulin resistance and hyperinsulinemia.22 the gene expression of fat / cd36 as a carrier of long chain fatty acids is regulated by insulin .
previous researches have shown that destruction of this protein is closely related to insulin resistance.28 others have shown that the activity of lipoprotein lipase ( lpl ) as a key enzyme for con - trolling serum tg levels is decreased among individuals with insulin resistance.29 increase levels of free fatty acids in beta cells could re - sult in decreased insulin secretions and there - fore increased risks of diabetes.30 some points needed to be considered in the interpretation of our findings .
the important limitation of this study is its cross - sectional na - ture which does not allow inferring causal rela - tions .
other point that must be kept in mind is the definition of ewet phenotype which might be different from other studies .
while the who expert committee31 on physical sta - tus recommends measurement midway be - tween the lower rib and the iliac crest , the nhanes iii guidelines32 prescribe use of a point just above the right ileum and the rec - ommendation of the north american associa - tion for the study of obesity ( naaso ) and the national heart , lung and blood institute ( nhlbi)33 is to use the right iliac crest .
the shortage of standard measurement for wc is problem , and makes comparison with other reasearches difficult .
it is believed that the use of narrowest waist measurement offers greater ease of acceptance and interpretation by the public and may facilitate self - measurement in addition to clinical use .
we measured wc at the point of noticeable waist because no stan - dard location has been reported for waist cir - cumference measuring .
we did not measure serum insulin levels and insulin sensitivity of tissues in the current study . therefore further studies in this field are warranted . in conclusion , there was a significant posi - tive association between hypertriglyceridemic waist phenotype and prevalence of diabetes .
therefore , using this phenotype for early iden - tification of individuals at high - risk of diabetes has important public health implications for prevention .
ma , ms , nm and ma participated in the collection of data , conception and design .
ae and mz contributed to conception and design , statistical analysis and data interpretation and manuscript drafting . | background : antropometric measures with biochemical indicators have been used as screening tools for metabolic abnormalities in adolescents and adults .
a few studies have assessed the relation of ewet ( enlarge waist elevated triglyceride ) phenotype with diabetes , especially among individuals with first relative history of diabetes .
this study aimed to evaluate the association of ewet phenotype with diabetes among individuals with family history of diabetes.methods:antropometric and biochemical measurments were evaluated in a population based cross sectional study of 332 male and 991 female isfahani adults aged 35 - 55 year .
the ewet phenotype was defined as serum trigcylglycerol concentrations 150 mg / dl and concurrent waist circumference ( wc ) 88 cm in females and 102 cm in males.results:the prevalence of ewet phenotype was respectively 9.6% and 23.6% among male and female .
individuals with the phenotype had significantly higher bmi and whr ( waist to hip ratio ) as compared to other groups .
after control for age and physical activity , male with ewet phenotype were significantly more likely to have high serum triglyceride levels ( p < 0.001 ) , cholesterol ( p < 0.001 ) . even after additional control for bmi , the significant associations remained except for low hdl cholestrol .
female with ewet phenotype had significantly adverse metabolic risks as compared to other groups , either before or after control for bmi ( p < 0.001 ) .
individuals with the phenotype were more likely to have diabetes ( both gender ) and ( igt ) impaired glucose tolerance ( female only).conclusions : our results showed that ewet phenotype has significantly associated with diabetes .
this phenotype could be used for early identification of diabetes and igt . |
the evolution of sexually dimorphic traits , such as armaments , bright coloration , or exaggerated traits has long been of interest to evolutionary biologists , as such traits are often thought to reduce fitness through natural selection ( e.g. , ) .
however , in spite of the potential negative consequences of these traits , many taxa exhibit not just one , but often multiple , sexually dimorphic traits . in nearly all systems
examined , there is evidence that these multiple , sexually dimorphic signals are used in both intrasexual ( competition , typically male - male competition ) and intersexual ( mate choice , typically female mate choice ) interactions .
traits traditionally thought to have evolved via competition include those that directly affect the physical competitive ability of males , such as larger body size , antlers , or other armaments , while traits typically associated with mate choice include signals such as coloration or specialized ornaments such as modified feathers that are unlikely to have arisen via natural selection .
there is a long - standing expectation that taxa may have multiple sexually dimorphic traits as a result of the separate action of competition and mate choice ( e.g. , ) .
if intrasexual and intersexual processes are acting independently , we would expect the evolution of dimorphism in a trait classically associated with competition to be unlinked ( over evolutionary time and in a given taxa ) with the evolution of dimorphism in a signaling trait that might be associated with mate choice .
although the evolution of a sexually dimorphic trait via competition and a second trait via mate choice may be expected within the same taxon , how can we explain the presence of multiple sexually dimorphic traits that are likely to have evolved through the same selective pressure ?
in the case of competition , where each individual trait may confer a net competitive advantage ( e.g. , each type of armament increases male competitive ability in an additive fashion ) , it would be predicted that multiple traits should accumulate over evolutionary time .
in contrast , assessing multiple signals used for intersexual interactions is likely to be costly for female receivers , making it difficult to understand why females would bear the cost of assessing multiple signaling traits in mate choice .
thus , the majority of theories to explain multiple sexual signals has developed in the context of mate choice ( reviewed in [ 2 , 3 ] ) . because signals can be costly to assess , produce , and maintain , the development of theory to explain the gain and maintenance of multiple traits used in intersexual signaling has focused on the identification of plausible mechanisms to explain why multiple signals , rather than a single signal , are used by a receiver .
candolin reviewed seven classes of explanations including ( 1 ) different messages are conveyed by the different signals , ( 2 ) that there are back - up or redundant signals , ( 3 ) some signals are used primarily in species recognition , ( 4 ) some signals are unreliable or fisherian cues and thus not informative , ( 5 ) two signals are received more efficiently than one ( receiver psychology ) , ( 6 ) different sensory environments favor different signals , and ( 7 ) intersexual conflict / antagonistic coevolution results in a dynamic in which older signals become uninformative antes and newer signals arise . in his more recent review , bro - jrgensen also summarizes this theory , albeit slightly differently .
in addition to the explanations discussed by candolin , bro - jrgensen expands the list to include the rare male effect ( i.e. , the hypothesis that an unusual signal is always advantageous ) and proposes a novel explanation for the advantage of multiple sexual signals : the idea that fluctuating signaling environments favor different signals at different times .
bro - jrgenson argues that since dynamic social and physical signaling environments are the rule , rather than the exception , this dynamic hypothesis may be a more general explanation for multiple signaling systems than what has been proposed previously .
as can be seen , there is a wealth of plausible hypotheses available to explain the use of multiple sexual signals ( table 1 ) .
a critical task at present is to evaluate whether or not there is any dominant mechanism at work across taxa , or whether many different hypotheses will be necessary to understand the ubiquity of multiple sexual signals .
the existing hypotheses are framed in a strongly microevolutionary context . as a result , we have detailed predictions about the expression of multiple traits and their use and costs within species ( or populations ) , but much less clarity about the expectations at the macroevolutionary ( above species ) scale regarding patterns of trait diversification , loss , or the association of traits in lineages where multiple sexual signals occur .
however , looking across lineages over evolutionary time should clarify whether there might be one or a few mechanisms , or whether multiple hypotheses are needed to explain the broad patterns seen across taxa . in an effort to relate this existing ( mostly microevolutionary ) theory to patterns quantified at the macroevolutionary scale
, we have reviewed the major classes of hypotheses available to explain the existence of multiple sexual signals ( table 1 ) and extended these ( when necessary ) to make predictions at the macroevolutionary scale .
although competition is expected to lead to the accumulation of traits associated with physical combat over evolutionary time ( as described above ) , we also recognize that intrasexual competition can lead to the evolution of signals that indicate competitive advantage ( e.g. , [ 14 , 15 ] ) . just as the assessment of multiple signals is likely to be costly for females choosing mates , the assessment of multiple signals indicating competitive advantages in intrasexual interactions are also likely to be costly .
thus , we argue that many of the hypotheses put forward to explain multiple cues in intersexual communication ( mate choice ) should also apply to the signals used in intrasexual interactions as well ( e.g. , ) . in extending the predictions of the existing theory to the macroevolutionary scale , we made the simplifying assumptions that signals of the same mode ( e.g. , coloration ) are more likely to be redundant in the information they convey , whereas the presence of multiple signals of different modes ( e.g. , color patterns versus specialized structures ) are more likely to communicate multiple different messages or to act in concert .
these assumptions can be logically argued and are supported by data ( e.g. , [ 17 , 18 ] but see ) .
the upper portion of table 1 focuses on the models that deal with signaling in general ( applicable to both mate choice and competition ) , summarizes the major classes of hypotheses to explain multiple sexual signals , and provides our macroevolutionary expectations for sexual dimorphism and the pattern of trait accumulation and loss over time from each of models . in the lower portion of table 1 ,
we discuss hypotheses that are specific just to mate choice . specifically , this includes rare male advantages in mate choice , antagonistic coevolution in sexual signaling [ 10 , 11 ] , and fisher runaway processes [ 4 , 12 , 13 ] .
these models do not make specific predictions about the types of traits that should evolve but have been framed in a dynamic context and thus make more explicit macroevolutionary predictions than the majority of hypotheses . to evaluate support for the macroevolutionary predictions of the models , we used a comparative framework to examine the distribution of sexual dimorphism in multiple traits in the family phasianidae ( chickens , pheasants , and allies ) .
although this is a very broad - brush approach , it has the potential to highlight general patterns and trends .
thus , this can be viewed as the first step for more detailed studies in specific clades that further refine our understanding of the evolution of multiple sexual traits .
the avian family phasianidae ( within the order galliformes ) contains some of the best - studied avian species , including the domestic chicken ( gallus gallus ) and turkey ( meleagris gallopavo ) , both of which now have complete genomes available [ 21 , 22 ] .
species within the family exhibit a wide range of variation in size and external morphology .
species range greatly in size , varying from relatively small ( ~35 g in coturnix chinensis ) to quite large ( up to 5 kg in the turkey ) .
while many species have little to no sexual size dimorphism , in others males may be more than 30% larger than females .
some species also exhibit one to several spurs on the tarsi , though these are absent in many species . in some cases
, spurs are found in both sexes , while in others they are restricted to males or males have more spurs than females ( e.g. , females typically have one , while males may have up to three ) .
plumage is also quite variable , ranging from species that are cryptically colored with no specialized feathers or extravagant coloration in either sex , to others in which males are strikingly colored , exhibit high levels of iridescence , or have specially modified feathers .
specialized feather types are found in many species , such as greatly elongated or modified tails as well as head modifications such as crests or ear tufts ; in some cases these can be found in both sexes , while in others they are either restricted to males or are more exaggerated in males .
additionally , some taxa have very uniquely modified feathers , typically found only in males , such as the highly modified tail coverts of peafowl ( pavo spp . ) , the elongated hackle and saddle feathers of most junglefowl species ( gallus spp . ) , or the elongated secondary wing feathers in the argus pheasant ( argusianus argus ) .
some species in the phasianidae ( and in galliformes overall ) also have regions on the head and neck that lack plumage , instead showing the flesh underneath .
these fleshy traits can vary from simply bare regions around the eye to the complete absence of feathers on the head and neck of the turkey and specialized structures such as the comb and wattle of junglefowl . in some phasianid species ,
these fleshy traits have the ability to be erected in which the trait can greatly change its size and appearance in a short time , and then be retracted ( often being barely visible in the nonerect state ) .
taxa that are able to rapidly erect these fleshy structures form a clade [ 25 , 26 ] , suggesting that the physiological basis for trait erection may have evolved a single time .
fleshy traits are typically found in both sexes when they are present in a species . however , the traits can still be dimorphic , since males often exhibit larger or more brightly colored fleshy regions than females .
moreover , the ability to erect these traits is found only in males . in part due to the large number of ornamental or exaggerated traits , the role of both competition and
results of these studies have varied and highlight the role of sexual selection in this group .
for example , in red junglefowl ( the ancestor to domestic chickens , also gallus gallus ) , the size of the male comb appears to be most consistently used by females in mating decisions ( e.g. , ) , and along with body size , correlates with dominance .
manipulation of social structure indicates that females prefer males with large combs , even when these are not the dominant male . in the indian peafowl ( p. cristatus ) , females prefer males with more ocelli ( eye - like spots ) in the modified tail coverts that form the train ( e.g. , [ 30 , 31 ] ) , though this may not be consistent across populations .
both train and tarsus length appeared to be involved in competition in the indian peafowl . in wild turkey ,
both competition and mate choice favor males with long snoods [ 33 , 34 ] , the fleshy protrusion above the beak .
studies in the ring - necked pheasant ( phasianus colchicus ) and various species of grouse have been less clear , but implicate various aspects of morphology and display behaviors in mate choice ( e.g. , [ 3538 ] ) .
early studies on relationships within the phasianidae showed little consensus ( reviewed by [ 20 , 39 ] ) .
the family exhibits limited osteological variation ( e.g. , [ 40 , 41 ] ) , and the group appears to have undergone a relatively rapid radiation , both of which probably contribute to the difficulties associated with resolving phylogenetic relationships .
more recent studies using both whole mitochondrial sequences , sequences from multiple nuclear loci , as well as insertions of transposable elements , have resulted in a better - resolved and more stable phylogeny [ 25 , 4349 ] . specifically , these studies agree on several key points .
first , the grouse and turkeys , traditionally separated into separate subfamilies or families , nest within the phasianidae .
second , the traditional grouping of pheasants ( dimorphic , highly ornamented , large bodied ) and the partridges and old world quail ( cryptic , little ornamentation , small bodied ) into separate clades within the family is erroneous . instead
, the characteristics that have traditionally been used to define pheasants and to differentiate them from partridges have been suggested to have arisen independently in multiple lineages , though this hypothesis has not been tested explicitly .
the suggestion that highly ornamented and dimorphic pheasants repeatedly evolved from more monomorphic partridge ancestors suggests that studying the phasianidae may provide insight into the forces that have led to complex patterns of sexual dimorphism and the evolution of multiple male secondary sexual traits .
recently , it has been shown that the evolution of sexual size dimorphism within the phasianidae may be related to mating system ( particularly lekking ; ) , though other types of traits that are potentially involved in sexual selection ( e.g. , plumage , fleshy traits , and spurs ) were not considered .
herein we use a comparative approach to examine the evolution of dimorphism in morphological traits that have been implicated in sexual selection , mapping the gain and loss of dimorphism across the family to generalize about the evolution of multiple sexual signals . to obtain a taxon - rich phylogeny
, we used a supermatrix analysis ( a large - scale data matrix ) rather than a supertree analysis ( meta - analysis of previous work ) [ 51 , 52 ] . to ensure all gene partitions
were represented by a good range of taxa in the supermatrix , we restricted our analyses to the six data partitions analyzed by kimball and braun , which included two mitochondrial coding regions ( cytochrome b [ cyb ] and nadh - ubiquinone oxidoreductase chain 2 [ nd2 ] ) and four nuclear introns ( ovomucoid intron g [ ovmintg ] , -fibrinogen intron 7 [ fgbint7 ] , pterin-4--carbinolamine dehydratase intron 3 [ pcbd1int3 ] , and rhodopsin intron 1 of [ rhoint1 ] ) .
kimball and braun obtained a complete data matrix comprising all of these gene regions for 44 galliformes , representing all galliform families but placing an emphasis on the phasianidae .
for the present study , we extended the taxonomic coverage by retrieving all sequences homologous to the six focal loci in the ncbi database using blastn and filtering the sequences to retain a single representative per species .
although the present study focused on phasianidae , all nonphasianid galliform species that were available for these partitions were included as outgroups .
cyb and nd2 sequences were equal in length ( with the exception of modest variation in the cyb stop codon ( see ) , making them straightforward to align .
the nuclear intron sequences were added to the kimball and braun alignment and then optimized by eye .
we were able to add data to all partitions in the kimball and braun matrix .
this resulted in a data matrix in which 51.8% of the cells were filled ; all taxa ( 170 species ) were represented by some mitochondrial data and 99 taxa ( 70 of which were phasianids ) also included nuclear intron data .
maximum likelihood ( ml ) phylogenetic analyses were performed using the gtrmix method implemented in raxml 7.0.4 , which performs searches using the gtrcat method to accommodate among - sites rate heterogeneity followed by a final optimization using the gtr+ model .
ml searches were conducted using 25 distinct randomized maximum parsimony ( mp ) starting trees .
partitioned analyses were conducted using 10 data partitions , one for each nuclear intron and three for each of the mitochondrial coding regions ( one partition for each of the codon positions ) .
bootstrap analyses used the gtrcat method and 500 replicates . to accommodate uncertainty in phylogenetic reconstruction when examining patterns of character change , we used mrbayes 3.1 [ 57 , 58 ] to generate a set of trees that accommodates the uncertainty in both topology and branch lengths .
for these analyses , the data were partitioned as described above for raxml . for each partition
, we used the aic to select the best fitting model that is implemented in mrbayes .
we ran four chains ( three of which were heated ) for 7.5 10 generations and discarded the first 1 10 generations .
we sampled every 1 10 generations , resulting in a set of 6500 trees that were used for trait reconstruction ( see below ) .
the focus of this study was to examine gains and losses in sexual dimorphism , so all traits were coded as binary characters , where 0 = monomorphic and 1 = dimorphic .
thus , a monomorphic score ( 0 ) could mean a trait was absent in both sexes or present and similar in both sexes ; a dimorphic score ( 1 ) could mean either that the trait was present in both sexes but larger or modified in one sex or present in one sex and absent in the other .
most information used to code traits was obtained from madge and mcgowan , though this source was supplemented with information in johnsgard [ 5961 ] .
although the phasianidae were the focus of this study , we also coded data for the five species of guineafowl ( numididae ) and six available new world quail ( odontophoridae ) represented in the sequence dataset to polarize traits .
five of these corresponded to plumage differences : ( 1 ) plumage color , ( 2 ) tail length , ( 3 ) head ornaments ( e.g. , crests and ear tufts ) , ( 4 ) other plumage differences ( e.g. , iridescence , unusual plumage features not covered in other traits ) , and ( 5 ) whether the male plumage was highly elaborated compared to females .
the remaining four traits included ( 6 ) fleshy traits , ( 7 ) spurs , ( 8) wing size , and ( 9 ) overall body size . body size is difficult to measure , and we examined several possibilities .
first , we looked at a qualitative difference based on descriptions in madge and mcgowan .
second , we took weight measurements for males and females from madge and mcgowan , although some of these were based on single individuals and/or captives whose weights may not be typical .
third , we considered the weights from lislevand et al . , which included values that were based on three or more individuals , primarily measured during the breeding season .
for the two weight measures , we considered species dimorphic if there was at least 10% difference between the sexes [ 20 , 50 ] . since the patterns using weight data were similar to those using qualitative measurements ( including in the relative differences between loss and gains ) , but were for many fewer species , we only report those results using the first approach . several additional characters were generated from this initial set , as defined in table 2 .
we also scored whether species were dimorphic in any plumage trait ( 10 ) and dimorphic in any competitive trait ( 11 ) . to highlight species that exhibited strong dimorphism in either plumage or competitive traits ,
species were scored for two additional binary characters : ( 12 ) high plumage dimorphism and ( 13 ) high competitive dimorphism .
to look at overall patterns , we also made several composite traits , including total dimorphism ( character 14 ; sum of the state of characters 19 ) , total signal dimorphism ( character 15 ; sum of characters 16 ) , total competitive dimorphism ( character 16 ; sum of characters 79 ) , and total plumage dimorphism ( character 17 ; sum of characters 15 ) . to assess overall rates of change and relative rates of gains and losses , we used maximum likelihood reconstruction in bayestraits using the 6500 phylogenetic trees from our bayesian analysis ( above ) .
from this , we estimated the transition rate and the bias ( the ratio of the gain rate to the loss rate ) for each binary character .
although the ml estimates obtained using this procedure do not represent posterior distributions in the bayesian sense , this approach captures the impact of phylogenetic uncertainty on our estimates and it has the advantage of being independent of priors .
we report the median estimate as well as the top 2.5 percentile and the bottom 2.5 percentile of the distribution ( 95% range ) for both the rate and bias . to determine whether there was a significant difference between gains and losses in the binary characters ( 113 ) , we used mesquite and the ml tree from raxml to test whether a two - rate model ( gains losses ; asymmmk ) was significantly better than a one - rate model ( gains = losses ) with a likelihood ratio test , assuming that twice the difference between models in their log likelihood is distributed with 1 degree of freedom . to understand the overall patterns in the ancestor
, we used the output from bayestraits to assess the probability of being dimorphic for characters 1 to 9 .
we did this at three possible ancestral states : ( 1 ) the ancestor of the outgroup taxa ( numididae and odontophoridae ) and the phasianidae , ( 2 ) the ancestor to the phasianidae , and ( 3 ) the ancestor to the core phasianidae ( excluding the arborophilinae from the remaining phasianids , figure 1 ) .
for each trait , we summed the probability of being dimorphic for all nine characters ( character 14 ) , total signal dimorphism ( character 15 ) , total competitive ( character 16 ) , and total plumage ( character 17 ) across all trees .
we then calculated the median value and the 95% range . to determine whether changes in one type of trait ( e.g. , plumage ) were associated with transitions in another ( e.g. , fleshy traits ) , we looked for correlations using correlated changes as implemented in mesquite 2.72 .
for each test , we used 10 likelihood iterations and ran 100 simulations to establish the distribution .
first , we examined whether there was a relationship between having dimorphism in any plumage trait ( 10 ) with any competitive trait ( 11 ) .
we also tested all pairwise combinations of high plumage dimorphism ( 12 ) , high competitive dimorphism ( 13 ) , and fleshy ( 6 ) characters to examine whether there was an association between being highly dimorphic in two different types of traits .
the combined alignment of 170 taxa ( 127 phasianids ) was 6296 bp in length , of which 2184 bp were mitochondrial and 4112 bp were nuclear .
the nuclear intron data included some regions that were difficult to align or present only in a small number of taxa ( e.g. , the mountain quail ( oreortyx pictus ) had an autapomorphic insertion that corresponded to a 579-bp segment of an endogenous retrovirus ( erv ) ) .
none of the rare genomic changes ( the erv insertion and microinversions ) united controversial groups : two were autapomorphic and one united the genus gallus . after the difficult - to - align regions and rare genomic changes were excluded from analyses , there were 2828 bp of nuclear intron data available for the phylogenetic analyses . for the data that were analyzed ,
there were 1298 variable mitochondrial sites ( 1159 were parsimony - informative ) and there were 1877 variable nuclear intron sites ( 1451 were parsimony - informative ) .
our analyses of the supermatrix yielded trees ( figure 1 ) that were more similar to other recent analyses of galliform phylogeny based upon sequence data ( e.g. , [ 25 , 26 , 43 , 44 , 46 , 49 , 66 , 67 ] ) than to the available galliform supertrees [ 68 , 69 ]
. points of agreement between the supermatrix trees and other studies include the strong support for dividing galliformes into five major clades : megapodidae ( megapodes ) , cracidae ( chachalacas , guans , and curassows ) , numididae ( guineafowl ) , odontophoridae ( new world quail ) , and phasianidae ( pheasants and partridges ) .
relationships among these groups ( especially the position of the new world quail ) are variable in supertrees [ 68 , 69 ] but relationships among these clades in our tree are consistent with studies based upon independent gene regions [ 25 , 44 , 46 , 67 ] , and the combination of both morphological and molecular data .
the deepest divergence within the phasianidae was between two well - supported clades , the arborophilinae and a large core phasianid clade .
arborophilinae includes hill partridges ( arborphilia spp . ) , crested wood - partridge ( rollulus roulroul ) , and recently discovered african forest partridges ( xenoperdix spp . ) , consistent with results from total evidence studies .
erectile clade , which includes a number of taxa that are able to rapidly erect fleshy traits [ 25 , 26 ] .
the erectile clade has also been found in other recent studies [ 43 , 67 ] .
the majority of the remaining core phasianids form a clade in both analyses presented here , with the exception of the argus pheasants ( argusianus argus and rheinardia ocellata ) which form the sister group of the remaining core phasianids in the partitioned analysis .
this agreement with prior estimates of galliform phylogeny ( e.g. , [ 25 , 43 , 49 , 67 ] ) indicates the trees are sufficiently accurate to allow rigorous examination of the evolution of sexual dimorphism in this order . despite the congruence between our supermatrix trees and prior estimates of galliform phylogeny
, the differences between trees obtained with and without partitioning ( figure 1 ) raise the question of whether the core phasianids can be divided into two large clades ( i.e. , the erectile clade and a second clade that comprises the remaining core phasianids ) or three clades ( the erectile clade , the argus pheasants , and the remaining core phasianids ) .
recent phylogenetic studies of galliformes ( e.g. , [ 25 , 26 , 43 , 4749 , 67 ] ) have reached contrasting conclusions regarding this question , but there is no consistency among studies . moreover , the relevant branches are short , and support for the conflicting relationships is limited both here and in previous studies .
given this region of conflict , we mapped traits on both trees ( figure 1 ) , though only the results from the unpartitioned tree are shown ( using the partitioned tree yielded similar results ) .
as can be seen when looking at total dimorphism ( character 14 ) , species that are highly dimorphic ( e.g. , dimorphic in seven or more characters ) can be found throughout the phasianidae ( figure 2 ) .
while some clades are comprised of species that are generally highly dimorphic , and others of species that exhibit little dimorphism , there are several cases in which taxa with relatively high levels of dimorphism are sister to taxa that have very low levels of dimorphism . as expected given this level of variation throughout the family , all nine dimorphism characters exhibit both gains and losses , and many are evolving at relatively high rates ( table 3 ) .
the lowest rate of change occurs in spur dimorphism , with the other traits typically exhibiting rates of change that are between 2- to 10-fold greater than for spur dimorphism ( although wing length shows a much greater relative rate of change , topological uncertainty had a relatively large impact upon the estimates on this character , and the 95% range is so broad , it is difficult to compare this to the other characters ) . although all characters exhibited gains and losses , the relative rate of gains and losses differed among characters ( table 3 ) .
signal traits ( plumage and fleshy traits ) exhibited a greater rate of loss than gain ( gain / loss values < 1 ) , while most competitive characters showed a greater rate of gains than losses ( the exception is spurs , where the median value is near one , indicating the rate of gains is similar to that of losses ) . however , in most cases , the difference between the rate of gains and losses was not substantial , and the estimated 95% range of gains to losses across the bayesian trees frequently included one .
although the 95% ranges that we report reflect the impact of topological uncertainty upon the ml estimate ( not the confidence interval on the ml estimate given a fixed topology ) , the results based upon the 95% range were typically consistent with the results of the likelihood ratio test when it was used to select the best fitting model .
all traits with a 95% range that included one did not show a significantly better fit to a two - rate model ( gains and losses occur at different rates ) relative to a one - rate model ( equal gain and loss rates ) .
in contrast , many traits in which the 95% range did not include one showed a significantly better fit to a two - rate model ( table 3 ; note that the likelihood ratio test for character 2 approached a significant improvement with a two - rate model ; 2 log likelihood = 3.70 , 2crit = 3.84 , df = 1 , p = .054 ) . directional selection for continued elaboration of sexually dimorphic traits
is assumed in many models of sexual selection ( e.g. , ) , suggesting that loss of dimorphism should be rare .
however , more recent studies have recognized that loss of dimorphism can occur [ 66 , 70 ] .
our study further emphasizes this point , as the rate of losses exceeded gains in most of the nine dimorphism characters ( table 3 ) .
moreover , even for characters where the rate of gains exceeded that of losses ( i.e. , characters 8 and 9 ) , the rate for gains was less than twice that of losses .
in contrast , the rate of losses was three to four times greater than that of gains for the several of the other characters ( i.e. , characters 2 through 5 ) .
this pattern of loss suggests that gains in dimorphism likely occurred early in the evolution of the phasianidae ( or earlier ) , making it more likely that subsequent transitions were losses rather than gains .
the summed probability of being dimorphic for all nine characters was low in the common ancestor of numididae , odontophoridae , and phasianidae ( table 4 ) , but increases in the common ancestor of the phasianidae and is even larger in the ancestor to the core phasianids ( table 4 , figure 2 ) .
similar patterns are seen when summing the signal and competitive traits separately ( table 4 , figure 2 ) , suggesting this early gain of dimorphism involved both types of traits .
these results suggest that the characteristics that have traditionally been used to define pheasants and differentiate them from partridges may not have arisen independently in multiple lineages , as suggested by kimball et al . . instead
, the common ancestor of the core phasianids may have been a dimorphic and pheasant - like , with the partridge lineages arising multiple times due to the loss of dimorphism . the only specific traits for which there is a low ( < 0.05 ) probability that the ancestor to all phasianids was dimorphic were fleshy traits ( character 6 ) and spurs ( character 7 ) . at the base of the core phasianids , only dimorphism in fleshy traits appears very unlikely , while the state for spurs is more equivocal ( probability of dimorphism is 0.39 ) .
the absence of dimorphism in fleshy traits is consistent with hypotheses that fleshy traits evolved through natural selection as a mechanism of thermoregulation in both sexes of galliformes ( and thus are present but monomorphic in many basal lineages ) , and only in some of the phasianidae were these co - opted for sexual selection [ 25 , 71 ] leading to the evolution of dimorphism for this trait . to assess
whether the pattern of transitions to and from a monomorphic state are similar to the patterns inferred using individual characters , we scored the presence or absence of any plumage dimorphism ( character 10 ) .
patterns of gains and losses of dimorphism in any plumage character exhibit roughly equal rates of gains and losses , in contrast to the pattern seen with the individual plumage characters that generally show a higher rate of losses ( table 3 ) .
the common ancestor to the core phasianids has a relatively high summed probability of dimorphism in the five individual plumage characters ( table 4 ) , making it likely the ancestor that had already gained multiple plumage characters . under this scenario
, any plumage character can only transition to monomorphism if all of the individual dimorphic characters are loss , whereas gain only requires a change in one trait .
thus , loss of dimorphism in any plumage is expected to be much less common for this character than in the individual characters , as we observe .
a very different pattern is seen when birds are scored for the presence of dimorphism in any competitive trait ( character 11 ) , where gains appear much more common than losses ( table 3 ) .
although the rate of gain is greater than that of loss , a one - rate model ( equal gains and losses ) could not be rejected for this character ( 2 log likelihood = 0.2 ; 2crit = 3.84 , df = 1 , p = .66 ) .
this suggests that the likelihood surface for this character is very flat , such that very different gain to loss ratios should result in only minor differences in likelihood values . since an equal rate of gains to losses
could not be rejected it remains possible that the gain to loss ratio is actually much lower than estimates obtained using the trees sampled by the bayesian analysis ( table 3 ) , making it difficult to interpret patterns of changes in this character at this time .
we examined transitions to and from high degrees of dimorphism in same type of trait .
species that exhibited high plumage dimorphism relative to other species ( character 12 ) showed a strong bias toward loss of dimorphism ( table 3 ) .
this pattern would be expected given the greater rate of loss for the individual plumage characters and the relatively high probability of plumage dimorphism in multiple traits in the ancestor to the phasianids ( table 4 ) .
similarly , as expected from the patterns observed in individual competitive traits , species that exhibited high competitive dimorphism ( character 13 ) showed the opposite pattern , with gains exceeding losses in extreme dimorphism ( table 3 ) .
strong sexual selection could select for increased dimorphism in two types of traits ( i.e. , one signal and one competitive trait ) .
an examination of the overall level of dimorphism in signal and competitive characters indicates that there are clades that are highly dimorphic for both types of traits , clades that exhibit little dimorphism in both types of traits , or clades that are highly dimorphic in one , but not both , types of traits ( figure 2 ) .
thus , there is not a strong , consistent pattern that emerges at this broad level .
however , there do appear to be some correlation among transitions in the characters ( table 5 ) suggesting that the transitions among character states may not be independent of other characters .
for example , gains in the any plumage ( character 10 ) and any competitive ( character 11 ) are correlated with fleshy traits ( character 6 ) , while being highly dimorphic for plumage ( character 12 ) appears to lead to more gains in both fleshy traits ( character 6 ) and being highly dimorphic for competitive traits ( character 13 ) . in summary ,
the patterns we observed are complex and suggest that no single dominant explanation will be sufficient to explain our observations .
both monomorphism and dimorphism evolved multiple times for all characters ( figure 2 and table 3 ) .
when all traits were considered together there was no clear trend towards gain or loss , although there were some weaker patterns .
consistent with the hypothesis that gains in dimorphism in traits that are directly involved in competition are expected , competitive characters exhibited a larger number of gains than signaling characters .
surprisingly , much of the gain in signal character dimorphism appears in the ancestor to modern phasianids ( figure 2 ) and losses predominate for these characters within the group ( table 3 ) .
this is in contrast both to expectation ( e.g. , ) and our previous hypothesis for the phasianidae . in table 1 , we summarize the patterns we identified that are either inconsistent with our predictions given specific models ( and would therefore falsify the model ) or consistent with our predictions .
it is immediately obvious by reference to table 1 that there was no single model that was consistently supported that also could not be refuted by our results .
the overall pattern of trait loss from an early and highly dimorphic ancestor or set of ancestors is one that has not been strongly considered in the field of sexual selection ( despite other studies that have highlighted the importance of loss of sexually dimorphic traits [ 66 , 70 ] ) .
since the sexual selection literature has driven the development of hypotheses to explain the presence of multiple sexual signals , loss of signaling traits has not been explicitly considered in these models as well .
thus , these results highlight the need for a more balanced treatment of trait loss and gain in this area .
furthermore , the diversity of observed patterns of gain and loss of dimorphism among lineages within the phasianidae suggests that the processes affecting the evolution of multiple signaling systems in any single lineage are either temporally variable or diverse .
thus , there appears to be no single , predominant mechanism to explain the evolution of multiple sexual signals .
that said , our test of the multiple / dynamic sensory environments hypothesis , essentially the idea that fluctuating signaling environments may favor different signals at different times , is particularly weak .
this hypothesis has the potential to be more general than the other hypotheses tested , due to its flexibility and the wide array of patterns it can explain as a result . within the hypothesis are nested the possibilities that different aspects of a mate or competitor are important in different contexts ( i.e. , the multiple messages hypothesis ) and that different environments may demand different signals to communicate the same message ( i.e. , receiver psychology ) .
thus , it is also more comprehensive than other proposed models , as well as being more dynamic .
however , adequate tests of this model demand a phylogenetic approach that considers environmental variables alongside signaling traits . without those comparisons , we can not seriously evaluate whether this more general model could explain the type of patterns we observed , and such analyses should be conducted in the future . in contrast , those models for which the phylogenetic predictions are best specified , that is , the models that specifically incorporate interspecific dynamics ( bottom portion of table 1 ) , have little support across the phasianidae . thus , none of these models appears to be sufficiently general so as to explain the distribution of multiple sexual signals within this family .
this is true , whether we focus exclusively on those characters that are most likely to be the result of mate choice or also include characters that are associated with competitive ability ( which females often consider in mate choice ; reviewed in ) .
thus , even within this one family , the diversity of factors contributing to the evolution and maintenance of multiple sexual signals across taxa appears to be greater than postulated by any single model .
however , it is also not possible to exclude any of these models as potential explanations for the patterns in at least some lineages within the phasianidae .
first , it may be that all of these processes contribute to the evolution of multiple signaling traits in this family to at least some degree , though the relative importance of each may vary across taxa .
alternatively , this may also reflect that the macroevolutionary predictions of each hypothesis are currently too imprecise to exclude some models .
additional refinement of the predictions each model makes over evolutionary time might allow the exclusion of some of these in the future .
taken together , this comparison of data with models to explain the evolution of multiple sexual signals suggests we need to focus on ( 1 ) a more explicit consideration of trait loss in the context of sexual selection and the evolution of multiple sexual signals and ( 2 ) formally extending the existing models on the evolution of multiple sexual signals to the macroevolutionary scale and thus , strengthening our predictions .
our observed pattern of diversification and losses of multitrait dimorphism in the phasianidae highlights the complexity of testing among the existing models , particularly as the models are currently described .
additionally , it is clear we need to incorporate environmental characters into these types of analysis to fully test existing models . finally , to better relate theoretical predictions about the functionality of signaling and competitive traits with their resulting phylogenetic distribution we also need to better understand the costs of different signal types to both signalers and receivers ( e.g. , ) , the effectiveness of different types of signals across environments , and the nature of interactions amongst signals .
if this is true , there are several expectations that should be apparent in a study such as this .
first , there may be gains of multiple sexual signals along a single branch in a phylogenetic tree , rather than a slow stepwise gain of such traits at the time that the strength of sexual selection increased .
second , a shift to increasing sexual selection in a lineage may drive rapid radiations and lead to short internodes shortly after that period . both of these patterns are consistent with our observations in the phasianidae .
the base of the core phasianids contains may short branches ( e.g. , figure 1 ) , and it is at this time that it appears that there was a gain in multiple sexually dimorphic traits . understanding whether this is a general phenomenon or a pattern specific to the phasianidae will require looking for similar associations between rapid evolutionary radiation and the gain of multiple sexually dimorphic traits in other groups . additionally , the potential to evolve sexual dimorphism in suites of traits in a short time period may depend on the genetic basis of such traits , which is likely to be complex . to understand the specific genetic changes responsible for sexually dimorphic characters is likely to require combining analyses like these with experimentation in specific species , but should lead to a greater knowledge of the processes that may lead to multiple sexual signals .
many models for the evolution of sexual signals have been phrased , either explicitly or implicitly , in a microevolutionary framework ( see table 1 ) .
this has led to tests using behavioral assays within species focused on assessing the strength and nature of sexual selection .
these types of studies are clearly important , but another area that will be important to explore in the future is the development of explicit mathematical models that can be used to examine patterns of sexual signal evolution in a macroevolutionary framework .
these can then be used to gain a greater understanding of the evolution of sexual dimorphism in the phasianidae and other groups of organisms , potentially revealing any unifying themes in the evolution of sexual signals over relatively large time scales . | traits involved in sexual signaling are ubiquitous among animals .
although a single trait appears sufficient to convey information , many sexually dimorphic species exhibit multiple sexual signals , which may be costly to signalers and receivers .
given that one signal may be enough , there are many microevolutionary hypotheses to explain the evolution of multiple signals . here
we extend these hypotheses to a macroevolutionary scale and compare those predictions to the patterns of gains and losses of sexual dimorphism in pheasants and partridges . among nine dimorphic characters ,
including six intersexual signals and three indicators of competitive ability , all exhibited both gains and losses of dimorphism within the group .
although theories of intersexual selection emphasize gain and elaboration , those six characters exhibited greater rates of loss than gain ; in contrast , the competitive traits showed a slight bias towards gains .
the available models , when examined in a macroevolutionary framework , did not yield unique predictions , making it difficult to distinguish among them . even with this limitation , when the predictions of these alternative models were compared with the heterogeneous patterns of evolution of dimorphism in phasianids , it is clear that many different selective processes have been involved in the evolution of sexual signals in this group . |
seasonal affective disorder ( sad ) was first time described as syndrome of recurrent episodes of depression that occur annually ( usually in winter season at same time each year ) by rosenthal et al . in 1984 .
diagnostic and statistical manual of mental disorders , 4 edition , text revision ( dsm - iv - tr ) describes sad not as a separate mood disorder but as
specifier referring to the seasonal pattern of major depressive episodes that can occur within major depression or bipolar disorders .
the specifier within seasonal pattern can be applied to pattern of major depressive episodes in bipolar affective disorders i , bipolar affective disorders ii and major depressive disorder ( recurrent ) .
the international classification of diseases , 10th revision ( icd-10 ) gives only provisional diagnostic criteria for sad on the grounds that its status is best regarded as uncertain , and its diagnostic criteria available in research domain of icd-10 manual are three or more episodes of mood ( affective ) disorder must occur with onset within same 90-day period of year , for three or more consecutive years .
remission also occurs within a particular 90-day period of the year and seasonal episodes substantially outnumber any non - seasonal episodes that may occur. although original description of sad included most people with diagnosis of bipolar affective disorder ( bpad ; most common bpad ii ) rather than recurrent major depressive disorder , who used to switch into hypomania ( or mania ) in summer season .
however , subsequent research into treatment strategies for sad almost exclusively focused on it as a subtype of recurrent depressive disorder as most of clinical trials involving sad focused on either bright light therapy ( blt ) , anti - depressant drug treatments , beta - blockers , vitamins and psychotherapy .
there is negligible data on role of mood stabilizers in management of sad ( winter depression ) . here ,
we describe response of 30 patients of recurrent major depressive disorder ( seasonal pattern winter ) ( winter sad ) to lamotrigine , who failed to respond to different anti - depressant treatment previously .
patients of sad ( winter depression ) fulfilling dsm - iv - tr criteria for major depressive disorder , recurrent , with seasonal pattern ( winter season ) , who failed to respond or responded partially to different anti - depressants were assessed from hospital records . the case records and assessment
was done by different members of the resident staff , but most of them were recorded by two of the authors .
they had already been assessed for mania / hypomania symptoms in current episode or in past ( particularly mania / hypomania in summer season ) using routine clinical interview and mini - international neuropsychiatric interview ( mini ) .
those having history of mania or hypomania and other axis i disorders were not taken up for this study .
selection criteria for this study :
patients having the onset of depression in autumn / winter lasting for whole of winter season and early spring.patients in whom progress was routinely monitored using hamilton depression rating scale ( ham - d).patients whose treatment was augmented with lamotrigine in addition to their routine anti - depressant medications.patients having minimum of three consecutive seasonal mood episodes.patient having a stable dose of other medications for minimum of 8 weeks and these doses were not changed during lamotrigine augmentation
.
patients having the onset of depression in autumn / winter lasting for whole of winter season and early spring .
patients in whom progress was routinely monitored using hamilton depression rating scale ( ham - d ) .
patients whose treatment was augmented with lamotrigine in addition to their routine anti - depressant medications .
patient having a stable dose of other medications for minimum of 8 weeks and these doses were not changed during lamotrigine augmentation .
these patients had been treated with lamotrigine augmentation for their partially responding or non - responding depressive episode .
they were assessed routinely at baseline , at 2 weeks , at 4 weeks and at 8 weeks of lamotrigine augmentation .
more than 50% decrease in ham - d score from baseline was taken as response and a final score of less than 8 was taken as remission .
lamotrigine was started at dose of 25 mg per day and gradually increased to 200 mg by 8 weeks with weekly increment of 25 mg .
lamotrigine was stopped in some patients owing to development of rash . in total , 30 patients who had completed follow - up for minimum of 8 weeks were assessed for this study .
mean / average and the deviations were calculated using statistical package for social sciences version 16 ( spss-16 ) .
single tailed t - test was used to study the difference of means to assess the therapeutic response and pre / post analysis of change .
thirty patients met the selection criteria , having a complete follow - up with fully maintained case sheets .
the sample included 9 males and 21 females with a mean age of 37.14 11.31 years ( 22 to 56 years ) . before the initiation of lamotrigine , the mean ham - d score was 20.93 4.93 , suggesting an ill state of most of these patients .
other variables , treatment history and treatment response are noted in table 1 . sociodemogarphic and clinical
variables the mean ham - d scores with standard deviation at start of treatment at 2 weeks , at 4 weeks and , at 8 weeks are 20.93 4.93 ( range = 14 - 35 ) , 19.63 5.82 ( range = 9 - 36 ) , 11.27 5.32 ( range = 3 - 23 ) and 9.57 6.16 ( range = 2 - 23 ) , respectively .
the difference in ham - d scores at 2 weeks of treatment was not significant compared to scores at baseline ( t = 0.93 , df = 58 , p = 0.177 ) .
however , there was significant difference in treatment outcome as per ham - d scores at 4 weeks of treatment compared to baseline scores ( t = 7.29 , df = 58 , p < 0.01 ) and this significant difference was maintained at 8 weeks of treatment ( t = 7.88 , df = 58 , p < 0.01 ) compared to start of treatment ( zero weeks ) .
an average decrease of 46.25% in ham - d score was achieved around 4 week and it was sustained to a further decrease in score by 54.27% by 8 week .
the mean time for responders to achieve an approximate 50% reduction on ham - d score was 4.2 1.2weeks at a mean dose of 150 mg / day of lamotrigine .
the mean dose of lamotrigine at 8 weeks was 200 mg / day with a 54.27% decline in ham - d score and 15 ( out 30 ) showed remission ( less than 8 score ) .
the difference in treatment response in different age - groups , gender groups and in groups of patients with different primary anti - depressant medication was not assessed because of small study group .
a few adverse effects were documented which included sedation ( seven patients ) , headache eight patients ) , fatigue ( one patient ) and benign skin rash ( one patient ) .
although first descriptions of sad presented it as more prevalent condition associated with bpad , however subsequent research into its therapeutics failed to focus it as such .
till now , most of the therapeutic research has failed to highlight it as bpad and there only few studies focusing on use of mood stabilizers in this condition .
this retrospective , non - blinded , open - label , non - comparative study in natural settings is an attempt to highlight role of mood stabilizers in sad .
this study proved significant improvement in depressive symptoms of sad patients ( winter depression ) with lamotrigine augmentation .
these were the patients who had previously shown poor response to anti - depressant ( medication ) alone treatment .
the role of early morning blt in winter depression may be actually an intervention based on social rhythm therapy which has been proven as effective strategy to deal with bipolar disorder patients because early morning blt in winter depression invariably promotes concept of chronotherapeutics in management of sad .
lamotrigine approved for the maintenance treatment of bpad i has also shown some efficacy for the treatment of bipolar depression .
limited data is available suggesting efficacy of lamotrigine in major depressive disorder ( mdd ) ( unipolar depression ) and these studies do not support lamotrigine use in unipolar depression robustly . in view of above findings and results of our study , the nosology and therapeutics of winter depression ( and other forms of depression not responding adequately to anti - depressant treatment ) needs to be reviewed as sad being a type of bipolar spectrum disorder even in absence of hypomania or mania symptoms .
the limitations of this study include the small sample size , lack of controls and randomization . in future
, this study needs to be replicated with better scientific design and these limitations need to be addressed .
this study brings to light the fact that sad needs to be looked as type of bipolar disorder for therapeutic purposes . | background : many therapeutic options have been evaluated and tried for seasonal affective disorder ( sad ) including bright light therapy ( blt ) , anti - depressants , beta - blockers and psychotherapy , but the data supporting use of mood - stabilizing agents is just handful in spite of this condition being understood most frequently to be associated with bipolar affective disorder ii ( bpad ii ) .
so we planned to study role of lamotrigine ( mood stabilizing agent ) in sad.materials and methods:30 patients of sad who were prescribed lamotrigine in addition to antidepressant medications for a minimum of 8 weeks and were assessed for severity using ham - d were selected retrospectively from the hospital records for this study .
ham - d scores at 2 , 4 and 8 weeks were compared to baseline scores.statistics analysis : single tailed t - test was used to study the difference of means to assess the therapeutic response and pre / post analysis of change .
statistical significance was set at p < 0.05.results:though no significant difference was seen in ham - d scores at 2 weeks of treatment compared to baseline , but results were statistically significant at 4 and 8 weeks of treatment with lamotrigine augmentation of antidepressant medications.conclusion:we conclude that lamotrigine augmentation was found to be effective treatment strategy for managing winter depression phase of seasonal affective disorder . |
in patients presenting with their first seizure , identifiable structural brain abnormalities are a risk factor for recurrent seizure and imaging helps guide clinicians in determining whether or not to recommend the initiation of chronic treatment with antiepileptic drugs ( aeds ) after an initial seizure .
the epidemiologic data that informs this decision - making process was obtained in studies of hiv negative individuals .
whether structural brain lesions in people with hiv , a significant proportion of which may represent transient , infectious phenomena , are predictive of recurrent seizures and indicate the need for chronic therapy is unknown . in resource - limited settings , where enzyme - inducing aeds may be the only available medications , initiating long - term epilepsy treatment carries the additional concern of adverse aed - antiretroviral ( arv ) interactions and warrants especially careful consideration . in the us , neuroimaging is indicated in hiv+ patients with new onset seizure for diagnostic purposes and to assure that lumbar puncture is not contraindicated . in sub - saharan african ( ssa ) countries , hiv remains the number one cause of disease and disability , despite the availability of arvs .
although neuroimaging was not routinely available in most african settings in 2004 , there is some evidence that , at least in tertiary care centers , access to imaging technology is improving .
there are few studies of hiv - related neuroimaging findings in africa , and viral clade differences may make extrapolations from the united states and european studies inappropriate . in african children with hiv , brain
atrophy with dilatation of the lateral ventricles , calcification of the basal ganglia , and periventricular white matter involvement are the most common neuroimaging findings .
we report the neuroimaging findings in a cohort study of hiv - associated seizures and epilepsy ( chase study ) which was conducted in zambia .
acute clinical and demographic risk factors for imaging abnormalities and the prognostic value of imaging for seizure recurrence and death are also described .
hiv+ adults who presented to the university teaching hospital in lusaka , zambia with new onset seizure between august 1 , 2011 and june 19 , 2013 were enrolled in chase .
additional inclusion criteria included age 18 years and no prior history of seizures except for childhood febrile seizures . written consent from the patient or their proxy was required . at enrollment ,
clinical and demographic characteristics were obtained as well as an electroencephalograph and , for patients who consented to lumbar puncture , cerebrospinal fluid ( csf ) analysis which included extensive studies using pcr technology to identify opportunistic infections ( ois ) .
after discharge , neuropsychiatric assessments with instruments previously used in zambia to detect hiv - associated neurocognitive disorders ( hand ) and psychiatric morbidity were conducted by a neuropsychologist ( lk ) including the shona symptom questionnaire ( ssq ) , the international hiv dementia scale ( ihds ) and the zambian mini - mental state exam ( zmmse).12 - 14 chase participants were followed prospectively with explicit assessments made during their hiv clinic visits to identify recurrent seizures .
death outcomes were obtained through clinic and hospital records and contact with family members through december 21 , 2013 .
when family members were interviewed after the death of a participant , specific questions were asked regarding recurrent seizures including seizures around the time of death .
a head computed tomography ( ct ) with contrast was performed as part of the study for chase participants in whom no seizure etiology was established based upon other diagnostic assessments , including csf analyses .
ct imaging was acquired on a siemens ct2007ys ct scanner and magnetic resonance ( mr ) imaging on a siemen s magnetom essenza 1.5 t mri scanner siemens , munich , germany ) .
ct protocols included : 1.5 mm contiguous axial imaging from the foramen magnum through the vertex for 3d reconstruction with 4 mm oblique axial imaging pre- and post - contrast .
mri protocols included sagittal t1 , axial t2 , flair , diffusion weighted imaging with apparent diffusion coefficients , and t1 pre and post contrast , as well as coronal t2 images .
several participants had neuroimaging studies , including 2 mris , obtained as part of their routine clinical care . in these patients
, there were no predetermined criteria for contrast administration and not all of them received contrast , possibly due to medical contraindications or cost incurred directly by the patient .
both ct and mri findings were interpreted and coded using an early version of neurointerp which codes dichotomous or ordinal values for specific anatomical findings .
these included the presence of cortical abnormalities , the presence of white matter abnormalities , the extent of white matter involvement , deep structural abnormalities , and posterior fossa abnormalities .
other radiographic findings captured included the presence of : abnormal masses or mass effect , abnormal fluid collections , intracranial bleeds , and calcifications . the extent of ventricular size ( decreased , normal or increased ) , and brain volume ( on a scale of 1 - 5 with 3 being normal ) were determined as well as contrast enhancement , if contrast was administered . the study radiologist ( mjp ) interpreted both the clinical and research images .
he was provided with age and gender and was aware of the patients hiv status and presenting symptoms , but was blinded to other clinical findings and outcome .
we used chi - square , t - test analysis or the kruskal - wallis test where population variances were non - homogenous to i ) identify clinical and demographic risk factors for imaging findings , and ii ) determine if imaging findings were predictive of death or recurrent seizure .
a p - value < 0.05 was considered statistically significant . prior to study initiation , the michigan state university biomedical institutional review board ( msu birb ) and the university of zambia , school of medicine biomedical research ethics committee ( unza brec ) provided ethical approval of the study .
written consent for participation was obtained from the participant or their proxy , if the participant was incapacitated .
a total of 43 chase participants were imaged : 41 with ct and 2 using mri . the mean age of participants was 37 years ; 24 were male .
approximately 80% had advanced hiv infection ( who stage iii or iv ; mean cd4 count 186 cells / mm ) and 44% had an oi identified .
the neuropsychiatric symptom burden was high , with over half of participants endorsing symptoms of anxiety and depression severe enough to warrant further clinical evaluation based upon who recommendations using the ssq
. over half also scored below normal values on the ihds and on the less specific zmmse suggesting a substantial burden of cognitive impairment in this cohort ( table 1 ) .
imaging abnormalities were identified in 70% of participants , with white matter abnormalities being the most common finding ( 56% ) , primarily from vasogenic edema .
deep gray ( 19% ) , posterior fossa ( 21% ) , and cortical abnormalities ( 28% ) were also common .
brain volumes were increased in 17% of subjects usually associated with cns infections , while other participants showed evidence of generalized atrophy .
isolated enlarged ventricles , indicative of subcortical atrophy , were seen in 16% ( table 2 ) . given the prevalence of imaging abnormalities in this cohort , our analyses had ~90% power to detect an effect size difference of 22% or more in the primary outcomes of interest ( death and recurrent seizure ) . during follow - up ,
patients with entirely normal brain images had a lower burden of depression and anxiety symptoms based upon the ssq ( 3.0 vs. 5.3 , p=0.04 ) and patients with subcortical atrophy had a lower mean ihds score ( 5.2 vs. 9.3 , p=0.002 ) .
although edema was associated with ois ( 36 vs. 6% , or 8.79 ( 95%ci : 1.03 - 236 , p=0.04 ) , none of the imaging findings , including cortical lesions , were predictive of seizure recurrence or death ( table 3 ) .
in this cohort of hiv - positive patients with their first seizure , most had advanced hiv disease and status epilepticus was common .
the imaging findings were diverse and included evidence of acute ois as well chronic atrophic changes .
structural brain lesions , evident primarily using ct , were not predictive of recurrent seizure or death in this cohort . this patient population was evaluated at zambia s only tertiary care referral center and as such they may not be representative of all adults with hiv in zambia . attempts to recruit from a community - based hiv clinic for more than 6 months failed to identify anyone with hiv presenting in the outpatient settings within 2 weeks of a new onset seizure .
mri imaging may have identified lesions not evident on ct . however , ct is more readily available and less expensive in most african tertiary care centers and therefore this data may be particularly useful in such resource limited settings .
imaging was preferentially obtained on chase patients who had no evident etiology for their seizure after a thorough work up including extensive csf studies .
recurrent seizures in participants who died may not have been adequately identified , but there was also no association between imaging abnormalities and death .
participants with any abnormality on their brain image had a higher burden of psychiatric symptoms .
this is particularly interesting since psychiatric morbidity , as measured by the ssq , has previously been shown to predict early mortality in rural zambians with hiv , even after controlling for hiv stage and socioeconomic status .
subcortical atrophy was associated with clinical evidence of cognitive impairment based upon lower ihds scores .
previous research in south africa using mri technology identified brain atrophy in several brain regions ( white matter , thalamus , gray matter , subcortical regions ) in hiv+ vs. hiv individuals .
using ct technology , we found that subcortical atrophy was specifically associated with the evidence of lower mean scores on the ihds but not zmmse .
neuropsychiatric morbidity , including cognitive impairment , depression and anxiety , are likely underdiagnosed and undertreated in most resource limited settings .
the association between psychiatric symptoms and structural brain lesions provides further support for the need to institute basic neuropsychiatric screening in hiv clinics .
epilepsy is a common non - communicable disease in ssa , where most people with hiv reside and the inevitable co - occurrence of two common conditions is further compounded by the high risk of provoked seizure in people with hiv due to ois , metabolic derangements , arvs which reduce the seizure threshold , and other challenges .
further studies are needed to determine neurologic vulnerabilities and outcomes in people with hiv , particularly in rural and pediatric populations . as hiv continues to evolve from a fatal to a chronic condition and neuroimaging
becomes increasingly available , clinicians and researchers need additional epidemiologic and natural history data relevant to this population to direct care and set research priorities . | in hiv - positive individuals with first seizure , we describe neuroimaging findings , detail clinical and demographic risk factors for imaging abnormalities , and evaluate the relationship between imaging abnormalities and seizure recurrence to determine if imaging abnormalities predict recurrent seizures . among 43 participants ( mean 37.4 years
, 56% were male ) , 16 ( 37% ) were on antiretroviral drugs , 32 ( 79% ) had advanced hiv disease , and ( 28 ) 66% had multiple seizures and/or status epilepticus at enrollment . among those with cerebrospinal fluid studies , 14/31 ( 44% ) had opportunistic infections ( ois ) . during follow - up ,
9 ( 21% ) died and 15 ( 35% ) experienced recurrent seizures .
edema was associated with ois ( odds ratio : 8.79 ; confidence interval : 1.03 - 236 ) and subcortical atrophy with poorer scores on the international hiv dementia scale ) ( 5.2 vs. 9.3 ; p=0.002 ) .
imaging abnormalities were not associated with seizure recurrence or death ( p>0.05 ) .
seizure recurrence occurred in at least a third and over 20% died during follow - up .
imaging was not predictive of recurrent seizure or death , but imaging abnormalities may offer additional diagnostic insights in terms of oi risk and cognitive impairment . |
renal cell carcinoma ( rcc ) is among the most frequent malignant tumors with significant morbidity and mortality .
more than 58,000 estimated new cases and more than 13,000 deaths occurred in the united states in 2010 . during the last decades , an increase in the incidence of all clinical stages of renal tumors was observed , with the greatest increase for localized tumors . owing to the wide use of cross - sectional abdominal studies such as ultrasound , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) ,
the detection rate of small solid lesions has increased , with up to 66% of tumors found incidentally .
the majority of incidentally diagnosed rcc tends to be of smaller size and thus is more likely to be asymptomatic , show a lower histological grade , and have a decreased incidence of metastasis .
radiofrequency ablation ( rfa ) is a novel minimally invasive therapeutic approach that should be offered to patients with small renal tumors with a size less than 4 cm in diameter or significant comorbidities precluding surgical resection . in the need for a therapeutic approach for such selected cases ,
rfa was established at our institution in 2006 . in the present study we sought to assess the efficacy , complications , and changes in renal function in our initial cases after an intermediate follow - up period .
percutaneous rfa was offered to highly selected patients whose renal tumors did not exceed 40 mm in diameter .
patient selection was limited to subjects with advanced age and severe comorbidities that would cause a high surgical risk , impaired renal function prior to treatment , a functional or anatomical solitary kidney , or bilateral renal tumors or patients who refused tumor resection .
after an initial implementation and learning process during which no renal biopsies were done , biopsies were routinely performed a few days before rfa under ct guidance .
all rfas were performed under general anesthesia with a rita device ( model 1500 rf generator , 25 cm starburst xl semi - flex rfa device , angiodynamics , queensbury , ny , usa ) by an interventional radiologist . according to the kidney protocol of the rita device , the maximum power to achieve a target temperature of 105 was 150 w. depending on the target size , the time of each cycle varied . for a desired ablation defect of 20 mm
, we used 5 minutes at the target temperature with a reset time of 5 minutes with a second identical cycle . for a 30-mm defect
, we analogously used 7 minutes , and for a 40-mm defect , 8 minutes .
if necessary , overlapping ablations were performed by repositioning the probe and restarting the procedure . at the end of the ablation ,
after the probe had been removed , a control ct scan verified the ablation and excluded complications .
patients were examined the day after the procedure through physical examination , ultrasound , and blood samples .
the patients ' renal function was assessed immediately before rfa , the day after rfa , and at follow - up by estimating the glomerular filtration rate ( egfr ) with the mdrd equation , as modified in 2005 : egfr=175(creatinine ) (age)(0.742 if female ) .
four definitions of treatment outcomes were used : complete and incomplete treatment and suspected and verified recurrence .
complete treatment was defined as a lack of contrast enhancement in combination with shrinkage or a stable size of the ablated tumor at the first follow - up mri / ct .
conversely , incomplete treatment was defined as contrast enhancement or progression in tumor size at the first follow - up imaging study after treatment
. suspected tumor recurrence was defined as contrast enhancement or extension in size in any subsequent imaging in initially completely treated patients
. suspected tumor recurrence turned into a verified recurrence either when a renal biopsy of the lesion was positive for vital tumor tissue or when the site of the formerly ablated lesion further increased .
patients with rcc or an unknown histology were followed with contrast mri or ct every 3 months after treatment in the first year and then every 6 months , whereas oncocytoma patients were followed every 6 months .
statistical analyses , including the related - samples wilcoxon signed - rank tests and the chi - square test , were performed with ibm spss ver .
after an initial implementation and learning process during which no renal biopsies were done , biopsies were routinely performed a few days before rfa under ct guidance .
all rfas were performed under general anesthesia with a rita device ( model 1500 rf generator , 25 cm starburst xl semi - flex rfa device , angiodynamics , queensbury , ny , usa ) by an interventional radiologist . according to the kidney protocol of the rita device , the maximum power to achieve a target temperature of 105 was 150 w. depending on the target size , the time of each cycle varied . for a desired ablation defect of 20 mm
, we used 5 minutes at the target temperature with a reset time of 5 minutes with a second identical cycle . for a 30-mm defect ,
we analogously used 7 minutes , and for a 40-mm defect , 8 minutes . if necessary
, overlapping ablations were performed by repositioning the probe and restarting the procedure . at the end of the ablation , after the probe had been removed , a control ct scan verified the ablation and excluded complications .
patients were examined the day after the procedure through physical examination , ultrasound , and blood samples .
the patients ' renal function was assessed immediately before rfa , the day after rfa , and at follow - up by estimating the glomerular filtration rate ( egfr ) with the mdrd equation , as modified in 2005 : egfr=175(creatinine ) (age)(0.742 if female ) .
four definitions of treatment outcomes were used : complete and incomplete treatment and suspected and verified recurrence .
complete treatment was defined as a lack of contrast enhancement in combination with shrinkage or a stable size of the ablated tumor at the first follow - up mri / ct .
conversely , incomplete treatment was defined as contrast enhancement or progression in tumor size at the first follow - up imaging study after treatment
. suspected tumor recurrence was defined as contrast enhancement or extension in size in any subsequent imaging in initially completely treated patients .
suspected tumor recurrence turned into a verified recurrence either when a renal biopsy of the lesion was positive for vital tumor tissue or when the site of the formerly ablated lesion further increased .
patients with rcc or an unknown histology were followed with contrast mri or ct every 3 months after treatment in the first year and then every 6 months , whereas oncocytoma patients were followed every 6 months .
statistical analyses , including the related - samples wilcoxon signed - rank tests and the chi - square test , were performed with ibm spss ver . 18.0 ( ibm co. , armonk , ny , usa ) .
mean tumor size was 26.2 mm ( range , 15 to 42 mm ) , mean length of hospital stay was 1.4 days ( range , 1 to 4 days ) , and mean follow - up was 23.8 months ( range , 3 to 59 months ) .
thirty - five of the 44 tumors were biopsied before rfa ( 79.6% ) . in biopsied patients ,
rcc was the prevalent histology in 68.6% and oncocytoma was the prevalent histology in 14.3% ; 17.1% of cases were benign or inconclusive .
grade i complications occurred in 11 patients ( 25% ) and grade ii complications in 1 case ( 2.3% ) .
all grade i complications ( pain or elevated temperature ) were treated conservatively with anti - inflammatory drugs , whereas the one patient with the grade ii complication ( perirenal bleeding ) received two units of blood without the need for any further surgical interventions ( table 2 ) .
overall changes in renal function and a precise breakdown of every patient are shown in table 3 . at more than 2 years after the treatment
, the mean serum creatinine increased by 0.14 mg / dl on average ( p<0.004 ) .
stratified by length of follow - up , the increase was not significant in the first 24 months but was after 24 months ( difference , 0.24 mg / dl ; p<0.001 ) .
mean egfr decreased significantly by 7.5 ml / min from 65.9 ml / min before treatment to 58.4 ml / min at maximum follow - up ( range , -55.2 to 17.3 ml / min ; standard deviation [ sd ] , 13.04 ; p=0.004 ) .
the distribution of patients in the corresponding egfr subgroups ( 60 , 59 - 30 , 29 - 15 , and 15 ml / min ) did not change significantly from before therapy to maximal follow - up ( p=0.08 ) .
forty - three of 44 ablated tumors ( 97.5% ) were classified as completely treated on the first imaging study at follow - up , which normally took place 1 to 3 months after rfa ( table 4 ) .
this patient is currently under active surveillance with no sign of tumor progression or recurrence . in 8 of 43 cases ( 18.6% )
the suspected recurrences were observed after an average of 23.9 months ( range , 11 to 43 months ) and had an average size of 22.6 mm ( range , 15 to 30 mm ; sd , 7.0 ) .
of those eight suspected recurrences , five ( 62.5% ) turned out to be false - positives whereas three ( 37.5% ) were confirmed ( table 5 ) .
the false - positive recurrences showed a negative ( repeat ) biopsy or , if the patient refused biopsy , showed no further contrast enhancement on subsequent imaging ( 10 or 12 months after suspected recurrence ) .
average follow - up until occurrence of the false - positive recurrences was 25.2 months ( range , 11 to 43 months ) .
the three proven recurrences ( 7.7% ) were noted at 12 , 23 , and 30 months after rfa .
one patient developed metastatic disease and is currently being treated with tyrosine kinase inhibitors . in our cohort ,
the overall survival was 87.5% ; five patients died from comorbidities , and renal tumors were not responsible for their deaths . in patients with
verified rcc , the tumor - free survival rate was 90% and the metastasis - free survival rate was 95.8% at a mean follow - up of 23.3 months .
grade i complications occurred in 11 patients ( 25% ) and grade ii complications in 1 case ( 2.3% ) .
all grade i complications ( pain or elevated temperature ) were treated conservatively with anti - inflammatory drugs , whereas the one patient with the grade ii complication ( perirenal bleeding ) received two units of blood without the need for any further surgical interventions ( table 2 ) .
overall changes in renal function and a precise breakdown of every patient are shown in table 3 . at more than 2 years after the treatment ,
the mean serum creatinine increased by 0.14 mg / dl on average ( p<0.004 ) .
stratified by length of follow - up , the increase was not significant in the first 24 months but was after 24 months ( difference , 0.24 mg / dl ; p<0.001 ) .
mean egfr decreased significantly by 7.5 ml / min from 65.9 ml / min before treatment to 58.4 ml / min at maximum follow - up ( range , -55.2 to 17.3 ml / min ; standard deviation [ sd ] , 13.04 ; p=0.004 ) .
the distribution of patients in the corresponding egfr subgroups ( 60 , 59 - 30 , 29 - 15 , and 15 ml / min ) did not change significantly from before therapy to maximal follow - up ( p=0.08 ) .
forty - three of 44 ablated tumors ( 97.5% ) were classified as completely treated on the first imaging study at follow - up , which normally took place 1 to 3 months after rfa ( table 4 ) .
this patient is currently under active surveillance with no sign of tumor progression or recurrence .
in 8 of 43 cases ( 18.6% ) initially classified as complete ablations , radiologists suspected tumor recurrences during follow - up .
the suspected recurrences were observed after an average of 23.9 months ( range , 11 to 43 months ) and had an average size of 22.6 mm ( range , 15 to 30 mm ; sd , 7.0 ) .
of those eight suspected recurrences , five ( 62.5% ) turned out to be false - positives whereas three ( 37.5% ) were confirmed ( table 5 ) .
the false - positive recurrences showed a negative ( repeat ) biopsy or , if the patient refused biopsy , showed no further contrast enhancement on subsequent imaging ( 10 or 12 months after suspected recurrence ) .
average follow - up until occurrence of the false - positive recurrences was 25.2 months ( range , 11 to 43 months ) .
the three proven recurrences ( 7.7% ) were noted at 12 , 23 , and 30 months after rfa .
one patient developed metastatic disease and is currently being treated with tyrosine kinase inhibitors . in our cohort ,
the overall survival was 87.5% ; five patients died from comorbidities , and renal tumors were not responsible for their deaths . in patients with verified rcc ,
the tumor - free survival rate was 90% and the metastasis - free survival rate was 95.8% at a mean follow - up of 23.3 months .
ablative techniques like percutaneous rfa are emerging because they offer an alternative to surgical excision , especially for elderly patients with impaired health . with the better understanding of the growth kinetics and behavior of small renal tumors [ 7 - 9 ]
tumor size seems to be a predictor of the tumor growth rate , whereas size alone is an insufficient parameter to distinguish between rcc with a so - called benign biological behavior from one with an aggressive behavior .
others have reported that the aggressive potential of rcc increases beyond a diameter of 30 mm .
imaging is not presently able to assess the malignancy of renal tumors : only angiomyolipoma can be diagnosed with sufficient accuracy .
renal biopsy is a safe procedure with an incidence of grade i complications of about 10% .
biopsy success depends on tumor size and is diagnostic in 81% in tumors with an average size of 25 mm ( showing rcc in about 75% and benign histology in about 20% ) . a repeat biopsy , after an initial nondiagnostic one , provides similar diagnostic rates so that diagnosis for most patients can be achieved . in terms of avoiding unnecessary and unjustified procedures owing to benign , unknown , or inconclusive histology , we recommend our strategy of taking a renal biopsy under local anesthesia a few days before rfa . with a biopsy rate of nearly 80% ,
our study differs from most of the previously published studies [ 12 - 19 ] . unlike most previously published series [ 12 - 22 ] ( table 6 )
, we used a generally accepted grading system of complications , thus guaranteeing transparency and comparability .
the minimally invasive nature of this procedure was confirmed by the low rate of complications in our setting : only minor complications occurred in 27% ( grade i , 25% ; grade ii , 2.3% ) .
rfa is associated with a significantly lower incidence of complications , morbidity , and mortality compared with tumor resection , especially laparoscopic nephron - sparing surgery .
complications after partial or radical nephrectomy are more likely to occur in older patients with preexisting comorbidities , and postoperative complications after nephrectomy are associated with a significantly higher risk of death .
this should be taken into consideration when patients with comorbidities present with small asymptomatic renal tumors .
the increase of serum - creatinine by 0.14 mg / dl at a 2-year follow - up and a slight decrease in egfr conforms to the literature .
ablative techniques have little impact on renal function in subjects with regular renal function as well as in those with a solitary kidney or renal insufficiency .
this is of particular interest because the association between impaired renal function and cardiovascular morbidity and mortality is well established . of note , we experienced a considerable discrepancy in suspected and proven recurrences during follow - up .
interestingly , 62.5% of all suspected recurrences ( mean follow - up , 25.2 months ) showed no evidence of malignancy in the ( repeat ) biopsy or no further contrast enhancement in subsequent imaging under active surveillance .
in one case , renal biopsy identified a focal inflammation that caused the contrast enhancement .
the considerable number of false - positive suspected recurrences must be emphasized , because immediate retreatment , either with a second ablation or even with tumor resection , might be an unnecessary overtreatment . in cases of suspected recurrences , a repeat biopsy or even temporarily active surveillance in selected patients might be justified , especially in small , asymptomatic tumors .
. stated that active surveillance up to 1 year after post - therapeutic enhancement could be justified , because most of these enhancements are not a sign of recurrence but a result of postoperative inflammation immediately after rfa .
however , their results relate to enhancements immediately after rfa and not to events occurring 11 to 43 months after rfa .
second , not every patient underwent renal biopsy before rfa or if a recurrence was suspected .
this was related to multiple factors such as the learning curve , the implementation process of rfa in our department , and patient 's choice .
however , this was mitigated because we separately analyzed patients undergoing rfa with biopsy - proven rcc .
third , renal function was assessed with serum creatinine and egfr and not with more accurate measures such as diethylene triamine pentaacetic acid - scans , thus diminishing the validity of our results in this field .
the primary intention of the study was to report the efficacy of rfa in highly selected patients who were offered a treatment option and not to assess the influence of rfa on renal function .
finally , follow - up was limited to a mean of 2 years ; changes in tumor - free survival or renal function might occur later on . owing to demographic changes , the increased incidence of renal tumors in the elderly , a better understanding of the growth kinetics and behavior of small renal tumors [ 7 - 9 ] , the low rate of complications [ 14,16 - 18,20 - 22 ] , and the only slight impairment of renal function , the role and importance of rfa might further increase in the future .
| purpose to evaluate the oncological outcomes , complications , and changes in renal function in patients treated with computed tomography - guided percutaneous radiofrequency ablation ( rfa ) for small renal tumors.materials and methodsthe charts of patients who underwent rfa from 2006 to 2011 at a single institution were reviewed .
oncological and functional outcomes were assessed .
statistical analyses were performed with ibm spss ver . 18.0
( ibm co. , armonk , ny , usa).resultsa total of 44 rfas were done in 40 patients .
biopsy prior to rfa was performed in 79.6% of procedures . of those , 68.6% had renal cell carcinoma ( rcc ) .
mean tumor diameter was 26.2 mm .
grade i complications occurred in 25% of cases ( n=11 , pain or elevated temperature ) and grade ii complications in 2.3% ( n=1 , perirenal bleeding needing two units of blood transfusion ) .
serum creatinine slightly increased by 0.14 mg / dl at 2 years after rfa ( p<0.004 ) .
tumor recurrences were suspected in 8 of 43 cases during follow - up . in five patients ,
the suspected recurrence was a false - positive as shown by a negative biopsy result or lack of contrast enhancement on subsequent imaging .
the verified recurrence rate was 7.7% in all tumors and 2.5% in rcc at a mean follow - up of 2 years .
tumor - free survival was 90% in all patients and 87.5% in those with rcc .
metastasis - free survival was 97.5% and cancer - specific survival was 100%.conclusionspercutaneous computed tomography - guided rfa shows promising results at intermediate follow - up .
suspected tumor recurrences are frequently false - positives findings . a longer follow - up
is required to verify the durability of these results . |
anti - n - methyl - d - aspartate receptor ( anti - nmdar ) encephalitis is an increasingly recognized etiology of previously unexplained encephalopathy and encephalitis , since its original description in 2007 , .
the disease has initially been described in women with an ovarian teratoma but can also be seen in seen in women without an ovarian teratoma , men .
the syndrome usually develops with a sequential presentation of symptoms including headache and fever followed by behavioral changes , psychosis , catatonia , decreased level of consciousness , dyskinesias , and autonomic instability .
extreme delta brush ( edb ) is a novel electroencephalogram ( eeg ) finding seen in 30.4% of patients with anti - nmdar encephalitis .
the presence of this pattern is associated with a more prolonged illness , although the specificity of this pattern is unclear .
additionally , the frequency and sensitivity of edb in anti - nmdar encephalitis , its implications for outcome , and whether there is a relationship between this pattern and antibody titers in serum and csf have yet to be determined .
we report a 27-year - old woman with no significant past medical history who was brought to the emergency department after becoming odder at home one week after the funeral of her boyfriend .
her family reported that she had not been sleeping or eating for a week , and she had been rambling wildly to herself ever since her boyfriend 's funeral .
she required chemical sedation in the emergency department as she became violent and hostile towards staff members .
she was admitted to the inpatient psychiatry ward for a presumed acute stress reaction with psychosis .
she remained uncooperative and was exhibiting delusions that she had to leave because her boyfriend had survived the shooting and was actually waiting for her at home .
two days later , she was noted to have sialorrhea and was picking at her clothes and bed sheets .
however , she was unresponsive after this seizure and would no longer verbalize or answer questions .
the neurology consultant observed that she had minimal responsiveness to voice , had intermittent picking movements at her bed sheets or clothing and was reaching for any instruments used to examine her [ i.e. , stethoscope or reflex hammer ] .
she also had a dysconjugate gaze when her eyelids were held open and was hyperreflexic throughout .
the initial electroencephalogram ( eeg ) , performed 3 days after presentation , demonstrated frequent electrographic seizures arising independently from the right and left hemispheres .
she was given intravenous ( iv ) lorazepam , started on intravenous fosphenytoin , and transferred to the neurological intensive care unit ( icu ) .
a lumbar puncture was performed , showing 76 white blood cells / mm with a lymphocytic predominance ( 11 red blood cells / mm , a protein level of 43 mg / dl , and a glucose level of 112 mg / dl ) .
the csf was sent for numerous studies including a full paraneoplastic panel , herpes simplex virus pcr , varicella zoster virus pcr , epstein barr virus pcr , cytomegalovirus pcr , arbovirus panel , anti - nmda receptor antibody titer , cytology , and flow cytometry .
brain mri with fluid - attenuated inversion recovery ( flair ) images on 7/6/2013 , 4 days after presentation , revealed abnormal high signal in the right medial temporal lobe . on repeat brain mri imaging done two days later on 7/8/2013 , she had hippocampal hyperintensity bilaterally on flair images and enhancement after gadolinium contrast administration ( fig
additionally , her continuous eeg monitoring within the first few days began to demonstrate frontally maximal high - voltage beta activity superimposed on frontally maximal delta waves ( fig . 2 ) .
this pattern of extreme delta brush had been described as highly suggestive of anti - nmdar encephalitis .
given suspicion for an autoimmune - mediated process causing her symptoms , 1000 mg of methylprednisolone was started on 7/9/2013 , 7 days after presentation .
in addition , she was then started on plasmapheresis once the steroid course was completed .
her csf result was positive for anti - nmda receptor antibodies , and her diagnosis was therefore confirmed as anti - nmda receptor encephalitis .
numerous studies including ct torso , pelvic and vaginal ultrasound , pelvis mri , and a pet scan either showed no evidence of malignancy or were limited by body habitus .
a pelvic ultrasound was repeated given persistent elevated concern for an occult malignancy and revealed a right ovarian mass that was concerning for an underlying tumor .
on 8/14/2013 , 43 days after presentation , she underwent laparoscopic right salpingo - oophorectomy .
surgical pathology report was consistent with a mature cystic teratoma , which was ultimately believed to be the etiology of her immune - mediated encephalitis .
unfortunately , there was no evident response to treatment . despite completing 15 rounds of plasmapheresis , high - dose steroids , cyclophosphamide , rituximab , and ketamine ,
her oral facial and limb dyskinesias continued to be pronounced , and she continued to have large fluctuations in temperature , blood pressure , and heart rate . her anti - nmdar antibody remains positive ( last tested on 11/11/2013 , 132 days after presentation ) .
additionally , follow - up eeg performed on 10/31/2013 , 121 days after presentation , continued to show pronounced extreme delta brush ( fig . 3 ) .
we report a 17.5-week follow - up of a young woman with anti - n - methyl - d - aspartate receptor ( anti - nmdar ) encephalitis associated with an ovarian teratoma .
the diagnosis was confirmed by persistence of a positive serum anti - nmdar antibody and the continued presence of extreme delta brush ( edb ) on continuous eeg .
extreme delta brush is a novel eeg finding seen in many patients with anti - nmdar encephalitis . in this particular patient ,
although specificity is uncertain , early diagnostic evaluation for malignancy should be pursued on the basis of clinical findings and edb before the titers return . because edb is associated with a more prolonged illness , whether the persistence of this pattern has further implications for outcome is unclear . whether there is a relationship between this edb pattern and antibody titers in serum and csf has yet to be determined .
additionally , whether there is a relationship between persistence of the anti - nmdar antibody and continued edb is also unclear .
however , the persistence of the finding has not been adequately correlated with outcomes or prognosis and requires further investigation .
| since its original description in 2007 , anti - n - methyl - d - aspartate receptor ( anti - nmdar ) encephalitis associated with an ovarian teratoma is an increasingly recognized etiology of previously unexplained encephalopathy and encephalitis .
extreme delta brush ( edb ) is a novel electroencephalogram ( eeg ) finding seen in many patients with anti - nmdar encephalitis .
the presence of this pattern is associated with a more prolonged illness , although the specificity of this pattern is unclear .
additionally , the frequency and sensitivity of edb in anti - nmdar encephalitis and its implications for outcome have yet to be determined .
we report a patient with early evidence of extreme delta brush and persistence of this pattern 17.5 weeks later with little clinical improvement . |
the european committee for standardization defines bitumen as a virtually nonvolatile , adhesive , and waterproofing dark brown viscous organic material derived from crude oil or present in natural asphalt . due to its binding and hydrophobic properties
, bitumen is commonly used for the construction of pavements and waterproof systems [ 2 , 3 ] .
bitumen properties are related to its composition , which in turn depends strongly on the type and origin of the crude oil .
however , the precise identification of the molecular composition and structure of bitumen is almost impossible ; therefore , they are often characterized by fractionation techniques [ 3 , 4 ] .
for instance , the sara method is based on sequential chromatographic separation of bitumen components into four generic groups according to their polarity and solubility , namely , saturates ( s ) , aromatics ( a ) , resins ( r ) , and asphaltenes ( a ) [ 3 , 5 , 6 ] .
commonly , the saturates fraction contains linear and branched alkanes eluted with a paraffinic solvent ( e.g. , n - hexane ) , the aromatics fraction is constituted mainly by alkylated c5 and c6 cyclic structures eluted with a moderately polar solvent ( e.g. , toluene ) , the resins are alkylated and cycloalkylated structures with 2 - 3 aromatic rings ( e.g. , eluted with tetrahydrofuran ) , and asphaltenes are heavier aromatic polycyclic structures
. moreover , bitumen contains heteroatoms ( such as sulfur , oxygen , and nitrogen ) normally embedded in the molecular structure of the aromatic rings [ 4 , 7 ] .
the structure of bitumen is controversial but the colloidal model , proposed by rosinger though generally attributed to nellensteyn , still prevails , especially for explaining the interactions between modifiers and bitumen fractions [ 6 , 10 ] . according to this model , bitumen is considered as a suspension of asphaltene micelles peptized by resins , dispersed in an oily medium constituted by saturates and aromatics [ 3 , 1012 ] .
depending on the relative contents of these fractions , bitumen exhibits different rheological properties used for its classification into three distinct types , specifically : sol ( viscous ) bitumen which has a colloidal structure constituted by noninteracting micelles and behaves as newtonian fluid ; gel ( elastic ) bitumen which is characterized by a three - dimensional colloidal structure and high resilience ; and sol - gel
( viscoelastic ) bitumen which exhibits an intermediate behavior with elastic effects in the initial stages of deformation and a colloidal structure characterized by the presence of supermicelles [ 13 , 14 ] . over the recent years
, bitumen has become a highly technical material with modifiers ( e.g. , polymers , acids , and mineral fillers ) gaining relevance in the control of the performance properties of modified bitumen [ 3 , 15 ] .
polyphosphoric acid ( ppa ) is an example of a reactive modifier that can be used by itself or in conjugation with a suitable polymer , in this case , with considerable economic benefits . in general , the concentrations of ppa in bitumen are in the range from 0.2 to 1.2 wt.% with an optimum at 1.0 wt.% . typically , commercial ppa is a mixture of phosphoric acid ( h3po4 ) , pyrophosphoric acid ( h4p2o7 ) , triphosphoric acid ( h5p3o10 ) , and higher oligomers .
bitumen refined from a specific crude oil has characteristic properties related to its consistency , namely , softening point and penetration value at 25c ( expressed in dmm ) which is the base for the european grading system .
these properties can be shifted up or down in the refining process or by posterior blending with different grade bitumen or an additive ( e.g. , a polymer , ppa , or a combination of both ) .
bitumen modification with a polymer or with ppa stiffens the product at higher temperatures , improving the resistance to permanent deformation , without negative effects at low temperatures ( i.e. , between 30 and 5c ) .
it is noteworthy that ppa has been reported to improve the behavior of modified bitumen also at low temperature ( 5c ) and to impart antioxidant properties . in this work ,
a detailed review of issued patents and scientific literature on the performance of straight - run bitumen ( i.e. , obtained directly from crude oil distillation ) modified with ppa has been accomplished and is presented in the appendix . on the other hand , since the influence of ppa on formulated bitumen is not reported in the scientific literature , this is the main focus of this paper .
it has been studied using phosphorous ( p ) and proton ( h ) nuclear magnetic resonance spectroscopy to evaluate structural modifications , sara and elemental analyses to evaluate chemical transformations , and standard grading tests ( softening point , penetration value , and penetration index ) for performance evaluation .
the interaction of ppa with bitumen model compounds was studied by several authors , as summarized in the appendix . from these studies ,
it became clear that ppa modification follows different mechanisms depending on the chemical composition of bitumen which is strongly related to the crude oil geographical source .
overall , the reactivity of ppa increases with the polarity of the asphaltene fraction because it enhances ppa dissociation ( into ppa and h ) disrupting the hydrogen bond network formed within the agglomerates of asphaltene micelles .
consequently , the molecular weight of the asphaltene fraction is lowered and the distribution of asphaltenes in the remaining fractions ( i.e. , saturates , aromatics , and resins , usually called maltene phase ) is improved , shifting the bitumen towards a more elastic gel - type structure , as represented schematically in figure 1 [ 13 , 17 ] .
there is a direct relationship between the chemical composition and colloidal structure of bitumen and its performance .
therefore , bitumen is often characterized by related parameters , such as the index of colloidal instability ( defined as the ratio of the sum of asphaltenes and saturates to the sum of aromatics and resins ) , the colloidal indices ip ( i.e. , resins - to - asphaltenes ratio ) and is ( i.e. , ratio of asphaltenes to maltenes ) , the penetration index ( pi ) , and other rheological measurements . several rheological studies of ppa - modified bitumen focused on its performance and stability .
for instance , giavarini et al . reported that ppa - modified bitumen exhibits higher penetration index ( pi ) and thus improved thermal susceptibility in comparison to unmodified bitumen .
and bonemazzi and giavarini performed rheological studies with polymer - modified bitumen and proved that ppa changes the bitumen structure towards gel type , thus improving the stabilization interactions between the polymer and the bitumen components .
edwards et al . performed rheological tests at low temperature ( 25c ) and established that bitumen modification is influenced by the amount and type ( grade ) of ppa used and also by the composition of bitumen .
validated the capacity of ppa to decrease the glass transition temperature ( tg ) and to increase the stiffness of modified bitumen .
another approach is to use spectroscopic techniques to detect specific functional groups in highly complex hydrocarbon mixtures such as bitumen [ 2326 ] . in particular , solution - state h and c nuclear magnetic resonance ( nmr ) spectroscopy can be used to identify and quantify the aromatic and aliphatic moieties .
this is of utmost importance to understand the molecular interactions between the sara fractions of bitumen and to explain the influence of modifiers on bitumen performance .
for instance , michon et al . [ 28 , 29 ] used quantitative c nmr data coupled with molecular weight distribution data to estimate structural parameters of bitumen such as , for example , aromaticity and the average number of naphthenic and aromatic rings per molecule .
their final purpose was to define a bitumen fingerprint , particularly for the aromatic part , in order to propose a mechanism for bitumen oxidation .
the chemical transformations associated with bitumen ageing were investigated by siddiqui and ali and siddiqui using h and c nmr .
curiously , molina et al . developed a very fast and reliable method that correlates the area of h nmr signals of petroleum residues to their sara fractions and to some physicochemical properties ( e.g. , density and s , n , and wax content ) .
related different h nmr spin - spin relaxation times ( evaluated by inverse laplace transform ) to different macroaggregates in ppa - modified bitumen .
moreover , bitumen modification with ppa can be analyzed by p nmr , a powerful tool to evaluate the structure of phosphorus - containing compounds .
the bitumen blends ( penetration grade 70/100 ) used in this work contained asphaltic residue ( ar ) , vacuum residue ( vr ) , and aromatic extracts sn1 , sn2 , or sn3 , in the proportions presented in table 1 .
bitumen modification with ppa was performed in a glass reactor , equipped with a mechanical stirrer and a temperature controller .
bitumen blends ( 1000 0.01 g ) were heated to approximately 135c and then ppa grade 105% ( supplied by innophos , cranbury , usa ) ( 8.00 0.01 g ) was added , which corresponds to 0.8 wt.% .
the mixture was allowed to react for 30 minutes at 135c with constant stirring ( ca .
the sara fractions of the individual components and the seven bitumen blends ( bitj , j = 17 ) were analyzed according to the international standard method ip-469 using iatroscan mk-6 tlc - fid instrument ( iatron laboratories inc . ) .
each sample was analyzed in triplicate and the results ( peak areas ) were averaged and considered as the weight percentage of the corresponding sara fractions .
elemental analysis of carbon , hydrogen , and nitrogen in bitumen samples was performed in a leco truspec instrument ( 628 series ) following the standard procedure astm d5291 .
sulfur content was determined by energy - dispersive x - ray fluorescence ( edxrf ) in oxford instruments lab - x3500 , following the international standard method ip 336 .
the bitumen samples ( 0.20 0.01 g ) were dissolved in deuteriochloroform ( cdcl3 , sigma - aldrich , 99.8 atom% d ) ( 1 ml ) and analyzed in triplicate .
solution - state h nmr measurements were obtained in a bruker avance 300 spectrometer ( operating at 300.13 mhz ) under the following conditions : spectral window of 6887 hz , with a 30 pulse width ( 3.63 s ) , acquisition time 2.4 s , relaxation delay 1 s , 32768 data points , and 128 scans .
solution - state p nmr spectra were acquired at room temperature in a bruker avance 300 spectrometer at a nominal frequency of 121.5 mhz , over a period of 7 minutes , using a pulse repetition rate of 1 s. chemical shifts were referenced to orthophosphoric acid at 0 ppm . bitumen penetration ( pen , expressed in dmm ) at 25c and the ring and ball softening point ( sp , in c ) were measured by standard methods . the penetration index ( pi )
was calculated by ( 1)pi = 12.04 sptp3002.903logpen0.602 sptp + 302.903logpen , where tp ( c ) is the temperature used to measure pen .
the sara fractions of the individual bitumen components and of the seven bitumen blends before and after ppa modification are presented in tables 2 and 3 , respectively .
the binary blends ( bit1 , bit2 , and bit3 ) prepared with asphaltic residue ( ar ) and one of the extracts ( sn1 , sn2 , or sn3 ) present similar contents of asphaltenes and resins ( within experimental error ) .
in contrast , the concentration of saturates and aromatics differs for the blends prepared with extracts sn1 and sn2 ( i.e. , bit1 and bit2 , resp . ) and with sn3 extract ( i.e. , bit3 ) . for the former ,
71 wt.% versus 65.5 wt.% for bit3 ) due to the nature of the extracts ( see table 2 ) . however , the differences in elemental composition of the three bitumen blends are negligible ( see table 3 ) .
it is noteworthy that the sara fractions for binary blend bit3 ( 78.39 wt.% ar + 21.61 wt.% sn3 extract ) are similar to those for bit7 , a binary blend prepared with asphaltic and vacuum residues ( 30 and 70 wt.% , resp . ; see table 1 ) .
the most significant difference is the high content of saturates ( 3.0 wt.% , table 3 ) in bit7 which can be related to its higher content of vacuum residue ( vr ) .
in fact , vr contains 5.5 wt.% of saturates whereas ar , the main component of the other blends , contains no saturates ( see table 2 ) .
again , there are no significant differences for the elemental chemical composition of bit7 in comparison to the other bitumen samples . the results for the ternary blends ( bit4bit6 ) prepared with ar , vr , and one of the extracts are quite similar ( within experimental error ) in what concerns the sara fractions and elemental composition ( table 3 ) .
overall , these results are consistent with the origin of the blends as they were all prepared with components derived from arabian light crude oil .
it is worth noting that the elemental analysis of formulated bitumen after ppa addition is not reported in table 3 , as the results were statistically equivalent .
in fact , from the literature review , it became clear that ppa modification follows different mechanisms , like redox reactions and disruption of hydrogen bond networks , and none of these mechanisms are expected to change the overall elemental composition of ppa - modified bitumen .
a final comment concerning the absence of saturates in bit1 and bit2 is in order .
in fact , these blends were prepared with extracts sn1 and sn2 which contain ca .
4 wt.% of saturates ( table 2 ) and thus the absence of saturates is puzzling .
a possible explanation is that the content of saturates is below the tlc - fid apparatus detection limit ( detection limit 0.8% ) .
the influence of ppa on bitumen composition ( sara fractions ) can be visualized in table 3 . in general ,
ppa modification decreases the content of saturates and resins and increases the fraction of asphaltenes in agreement with the literature [ 13 , 37 ] .
the higher content of asphaltenes in ppa - modified bitumen explains the experimental results ( table 4 ) , namely , higher softening points ( ca . 13c increment ) , lower penetration values ( ca
. 10 dmm ) , and , consequently , an improvement of the penetration index ( pi ) in accordance with the results reported by oyekunle . the higher values of pi indicate a change in the colloidal structure of ppa - modified bitumen blends which is expected to improve their thermal susceptibility .
the effect of ppa on bitumen modification has been associated with redox reactions involving polycondensed aromatic structures that act as reducing agents for phosphoric acid oligomers ( hpo3)n , which are therefore oxidized . as a result
, one would expect the conversion of part of the resins fraction into asphaltenes and the conversion of the saturates and aromatics fractions into resins .
however , the increase of the aromatics fraction observed in this work , after ppa addition , suggests that part of the resins were converted to asphaltenes and part were converted to aromatics .
the p nmr spectrum of the ppa used in this work is presented in figure 2 . according to the literature [ 37 , 39 ] ,
the resonance peak at 0 ppm is attributed to phosphorus in h3po4 , a smaller peak at 13 ppm is assigned to phosphorus in end groups of ppa chains , and a much smaller peak around 26 ppm is attributed to phosphorus in the middle groups of ppa chains .
the ppa - modified bitumen blends were also analyzed by p nmr spectroscopy , at room temperature .
the spectra for the seven blends ( see figure 3 ) are very similar revealing a broad signal approximately at 1 ppm .
this resonance peak , located in the vicinity of the phosphorous resonance peak in h3po4 , was assigned to hydrophobic interactions between phosphorous and polycondensed aromatic structures typically present in asphaltenes .
the absence of resonance peaks assigned to phosphorus in phosphate chains ( end and middle groups ) suggests that phosphoric esters were not formed during bitumen modification with ppa , in agreement with the results of miknis and thomas and in opposition to the earlier hypothesis of orange et al . .
furthermore , these results indicate that ppa is hydrolyzed completely back to h3po4 , probably due to the presence of residual water in bitumen . the broader spectral peaks for ppa - modified bitumen blends ( figure 3 ) , in comparison with ppa ( figure 2 ) , are probably due to higher viscosity of the ppa bitumen blends or to the formation of charge transfer complexes between phosphorous and the asphaltene polycondensed aromatic structures [ 39 , 41 ] .
another hypothesis is that small amounts of paramagnetic impurities typically present in bitumen ( e.g. , vanadium and nickel ) may decrease the relaxation time and , consequently , increase peak broadening due to the heisenberg uncertainty principle .
the h nmr results for unmodified and ppa - modified bitumen blends are presented in table 5 .
the spectra were divided into 12 unequal segments corresponding to well - defined chemical shifts associated with specific groups ( i.e. , hydrogen types ) , though minor overlapping may occur .
peak integration in the selected segments generated , after normalization , the relative content of the various proton types ( table 5 ) .
the mean values were obtained by averaging the results from the spectra of three aliquots of the same bitumen formulation .
some conclusions may be drawn from the h nmr results ( table 5 ) more explicitly presented in figure 4 .
in general , the content of aliphatic hydrogen ( segments 1 and 2 , table 5 ) in ppa - modified bitumen is different from that in the parent unmodified bitumen .
the signals in the segments associated with the branching ( 0.51.0 ppm ) and the length ( 1.01.7 ppm ) of paraffinic chains are systematically higher , though rather similar . the signals in segment 3 ( 1.71.9 ppm ) , that is , in the region of chemical shifts associated with sulfur adjacent hydrogen atoms ( 1.83.0 ppm ) , decrease after ppa modification , thus suggesting more substituted aromatic structures .
the slightly lower signals in segment 6 ( 2.43.5 ppm ) , figure 4 , which are associated with the chemical shifts of -hydrogen ( -ch , -ch2 ) indicate the occurrence of oxidation reactions , thus explaining the improved stability of ppa - modified bitumen .
the signals associated with bridging hydrogen atoms ( segment 7 , 3.54.5 ppm ) and hydrogen atoms in monoaromatic structures ( segment 9 , 6.07.2 ppm ) have both a tendency to decrease after ppa modification ( see figure 4 ) .
this might be related to a slight increase in the content of hydrogen atoms associated with polyaromatic structures ( segments 10 , 11 , and 12 , in table 5 ) , suggesting an increase in content of condensed structures as reported in the literature . a final note about the residual presence of olefins , detected in the region 4.56.0 ppm ( segment 8 , table 5 ) . in fact ,
since bitumen is allegedly free of olefins , due to the process conditions in the refinery , its presence can be attributed to cracking reactions eventually occurring during nmr sample preparation ( recall that bitumen reheating was necessary to facilitate sample handling ) .
binary and ternary bitumen blends formulated with asphaltic residue , vacuum residue , and aromatic- extracts derived from refined arabian crude oil were analyzed before and after modification with polyphosphoric acid ( ppa ) .
sara analysis revealed that , in general , ppa modification increased the fraction of asphaltenes in bitumen and decreased the saturates and resins fractions abundance .
these changes improved the performance properties , namely , the penetration index ( pi ) , which indicates higher thermal stability of ppa - modified bitumen blends . with respect to the mechanism of ppa actuation , the p nmr proved that ppa is hydrolyzed back to phosphoric acid and reacts with bitumen , probably in the form of charge transfer complexes with polycondensed aromatic structures .
this fact was disclosed from the presence of the single resonance peak assigned to phosphoric acid and the absence of peaks assigned to phosphorus in the end and middle groups of phosphate chains .
the ppa disrupts the agglomerates of asphaltene micelles , which promotes the distribution of asphaltenes in the maltene phase , thus increasing the content of the aromatic fraction .
furthermore , part of the resins is converted into the aromatic fraction . concerning the h nmr studies
, results suggest that ppa improves the oxidation stability of the bitumen blends and increases the concentration of condensed structures .
the former was evidenced by the content drop of -hydrogen ( range 2.43.5 ppm ) after ppa modification , as these hydrogen atoms are associated with oxidation susceptibility . | over the recent years , bitumen modification with polymers , acids , or mineral fillers has gained relevance to adjust its performance properties .
this work reports the use of polyphosphoric acid ( ppa ) for the modification of formulated bitumen . with this objective ,
an in - depth literature review on ppa modification was firstly performed .
subsequently , five individual refinery components were selected for the preparation of bitumen blends , namely , asphaltic residue , vacuum residue , and three lube oils extracts .
seven binary / ternary bitumen blends were prepared and then treated with ppa . afterwards
, the five components and the unmodified and ppa - modified bitumen were characterized by standard methods ( penetration , softening point , and penetration index ) , sara analysis , elemental analysis , and 31p and 1h nuclear magnetic resonance ( nmr ) spectroscopy .
the results evidenced higher asphaltenes and lower saturates / resins contents in ppa - modified bitumen .
the nmr data suggest that the paraffinic chains became longer , the content of condensed aromatics increased , more substituted aromatic structures appeared , and -hydrogen in aromatic structures diminished .
these findings disclosed the improved consistency and oxidation stability of ppa - modified bitumen blends . |
when a crime is committed and a biological trace such as blood stain or semen is found at the crime scene or the body of the victim , forensic scientists can link the case to the arrested suspect through the matching of the trace evidence to the suspect , using dna profiling . for cases in which no suspect can be identified based on non - dna evidences such as fingerprints or witness reports , the police force may search a database of dna profiles from previously convicted criminals or unsolved crime cases and open an investigation on individuals with perfectly matched profiles . since 1995 when the first national dna database came to operation , dna database search has become an important crime - solving tool for suspect identification , benefit from the amassing of large offender dna databases in many countries .
for example , as of june 2011 , the us federal dna database codis has assisted in over 141,300 investigations in the usa by more than 147,200 hits produced .
the evaluation of the evidentiary value of perfect matches from dna database search has been thoroughly discussed by balding and donnelly , stockmarr , and meester and sjerps , among many others .
if no offender profile in the database perfectly matches the crime trace , an additional search can be performed , hoping that an individual in the database is a close relative of the perpetrator and can be identified through the search .
the use of familial search on dna database starts to become popular in recent years .
as of may 2011 , about 40 serious crimes has been solved with the aid of about 200 familial searches in the uk , showing that the familial database search has potential to be an effective forensic tool that can increase the number of suspects identified based on dna evidences . since many and even all individuals in the database
may be qualified as the relative of the perpetrator , a scoring scheme is needed to rank the offender profiles , so that the police force can focus their investigation upon the top - listed candidates .
a widely adopted familial search score is the likelihood ratio ( lr ) of the following hypotheses :
hp : a relative of the individual is a contributor to the crime trace.hd : a relative of the individual is not a contributor to the crime trace.the lr for various two - person relationships can be easily calculated using the formulae provided by evett and weir . the performance of using lr scores for familial database searching has been evaluated in various articles [ 510 ] .
based on simulated and real dna databases , the familial searches were demonstrated to be able to identify the first - order relative such as parent / child or full sibling of the perpetrator in the 100 top - listed candidates in over 70% of the cases , provided that the database really contains the profile of the relative .
hp : a relative of the individual is a contributor to the crime trace .
hd : a relative of the individual is not a contributor to the crime trace . in some typical crime cases such as murder cases or rape cases , the biological trace is often observed as a dna mixture contributed by the victim and the perpetrator . over the years
, the evaluation of the dna mixtures in probable cause cases has been studied extensively [ 1114 ] , and various formulae have been developed to handle situations when relatives are involved [ 1517 ] . in two previous articles
[ 18 , 19 ] , we have reported our progress on the attempt to apply database search on cases with dna mixtures as part of the evidences and derived several formulae for evaluating the evidentiary values of cold hits . to go a further step , chung et al .
has extended the familial database search method so as to handle mixture cases , and the performance is shown to be as good as in single source cases .
based on the results of a familial search , the number of individuals to be further investigated may depend on the regional policy .
for example , according to the california familial search policy , after the list of top - ranked candidates is produced , lineage dna testing will be conducted on up to 168 candidates by using y - str typing .
in addition to familial search policy , the scale of the investigation will also vary based on practical considerations including the limitation of manpower , the resources of the police offices , and the scale of the crime severity .
the main cost factor is in the need to investigate the background of each candidate so that irrelevant candidates can be eliminated as being the possible relative of the perpetrator .
investigating on thousands of candidates for every case is impractical as the law enforcement resources will be overwhelmed by the heavy case load
. it would be more effective if a large number of candidates are investigated only for serious and high - profile criminal cases .
it is therefore necessary to determine the scale of the investigation so that the effectiveness of the search can be guaranteed under reasonable cost .
ge et al . had given some suggestions on the thresholds to the lr as well as the identity - by - state scores , with the aim of balancing the false - positive and false - negative rates .
these thresholds , however , are specific to a particular database as they are determined based on a simulation study using caucasian population data on the 13 codis str loci .
the primary aim of this work is to establish a general strategy on deciding the number of top - listed candidates to be investigated after the familial search , according to the statistical criteria on the true and false hit rates required by the police force .
this article is organized as follows : first , we describe the use of lr scores for producing a ranked list of candidates from familial search on dna mixtures , on the basis of our previous work in chung et al . .
we then establish an estimate of the true hit rate of the investigation on a specific number of top - listed candidates , from which a novel strategy is developed for determining an appropriate scale of the investigation .
the performance of the familial search on dna mixtures and the proposed strategy are demonstrated through an example using swedish data .
given a crime trace observed as a dna mixture m contributed by the perpetrator and the victim , the familial search score for a particular individual j in the database d is defined as a lr of the following hypotheses :
hj : the victim and a relative of individual j are contributors.hd : the victim and one unknown person are contributors.note that the prosecution hypothesis hj here states that a relative of individual j , rather than the individual , is the contributor and therefore is not the same as the hypothesis formulated in chung et al . and chung and fung . in general , a lr can be calculated for each of the possible genetic relationships for every member of the database .
however , the familial search is practically used only for parent / child and sibling relationships because it would become less effective for other degrees of relatedness below sibling that share less genetic similarity .
hd : the victim and one unknown person are contributors . at a particular locus l , denote ml as the set of alleles present in the mixture and vl and xjl as the genotype of the victim and individual j , respectively , for j d and l = 1, ... ,l . under linkage equilibrium assumption ,
presented the following formula for calculating the lr of hj versus hd :
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\begin{document}$$ \label{eqn : lr } { \rm lr}_j=\prod\limits_{l=1}^{l}{\frac{p(m_l|v_l , x_{j\,l},h_j)}{p(m_l|v_l , h_{\rm r } ) } } $ $ \end{document}where hr is the hypothesis that the victim and a random person contribute to the mixture .
the computation of p(ml|vl , xjl , hj ) and p(ml|vl , hr ) is based on the q - function presented in hu and fung and fung and hu :
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\begin{document}$$\begin{array}{lll } & & { \kern-6pt } p(m_l|v_l , x_{j\,l},h_j)\\ & & { \kern6pt}=k_0q(2,u_l)+k_1\big(i_m(t_1)q(1,u_l\setminus\{t_1\ } ) \\ & & { \kern138pt}+i_m(t_2)q(1,u_l\setminus\{t_2\})\big ) \\ & & { \kern18pt}+k_2i_m(t_1)i_m(t_2)q(0,u_l\setminus\{t_1,t_2\ } ) \end{array}$$\end{document}\documentclass[12pt]{minimal }
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\begin{document}$$ p(m_l|v_l , h_{\rm r})=q(2,u_l ) $ $ \end{document}where t1t2 is the genotype present in xjl , ul = ml vl is the set of alleles present in ml but absent in vl , and im(t ) is the indicator function defined by im(t ) = 1 if t m and 0 otherwise .
the quantities ( k0,2k1,k2 ) are the kinship coefficients for the relationship considered in hj .
in particular , ( k0,2k1,k2 ) take the values of ( 0.25,0.5,0.25 ) for full siblings and ( 0,1,0 ) for parent / child relationship .
table 1 shows the computational formulae of q ( . , . ) for a two - person mixture at a particular autosomal locus l with k alleles a1,a2, ... ,ak and corresponding allele frequencies p1,p2, ... ,pk\documentclass[12pt]{minimal }
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\begin{document}$(\sum_{i=1}^{k}p_i=1)$\end{document } , under the assumption of hardy weinberg equilibrium .
table 1the calculating formulae of q(j , b ) for different combinations of mixture ml and arbitrary set of alleles b at a particular autosomal locus l with alleles a1,a2, ... ,ak and corresponding allele frequencies p1,p2, ... ,pk , under the assumption of hardy
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\begin{document}$(p_i+p_j+p_k+p_t)^2$\end{document}ai0pipi(pi + 2pj + 2pk + 2pt)aj0pjpj(pj + 2pi + 2pk + 2pt)ak0pkpk(pk + 2pi + 2pj + 2pt)al0ptpt(pt + 2pi + 2pj + 2pk)ai , aj002pipjai , ak002pipkai ,
at002piptaj , ak002pjpkaj , at002pjptak , at002pkptthe indices i , j , k , and t are pairwise distinct the calculating formulae of q(j , b ) for different combinations of mixture ml and arbitrary set of alleles b at a particular autosomal locus l with alleles a1,a2, ... ,ak and corresponding allele frequencies p1,p2, ... ,pk , under the assumption of hardy weinberg equilibrium the indices i , j , k , and t are pairwise distinct the individuals in the database are ranked in descending order by the lr scores , and the individuals with highest scores will be preliminarily identified as the suspects that need further investigations by the police force . in case
when rare alleles are present in the unexplained profiles ml vl , the random match probability p(ml|vl , hr ) will become extremely small , and the unknown contributor s relative will be assigned a large lr score so that the perpetrator will be more likely to be identified .
therefore , the performance of the familial search depends much on the observed mixture m = { ml , l = 1, ... ,l } and the victim profile v = { vl , l = 1, ... ,l}. to predict the performance of the search , cowen and thomson suggested fitting the logistic regression model
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\begin{document}$$ \label{eqn : logistic_model } { \rm logit}(p(\theta_k=1))=\alpha-\beta \log_{10}{p(m|v , h_{\rm r } ) } $ $ \end{document}where k is an binary response variable taking the value of 1 if the relative of the unknown contributor is located within the top k profiles and 0 otherwise .
the model links the outcome of a particular search that limits the investigations to the k top - listed individuals to the random match probability p(m|v , hr ) and therefore can be used to predict the performance of the search given the observed dna profiles ( m , v ) . in particular ,
if the estimated probability p(k = 1 ) is too small even for large k , the familial search may fail to provide effective assistant for identifying the suspect .
this information can practically guide the police force to make decisions on whether it is worthwhile to open an investigation just based on the familial search results . the use of logistic regression for evaluating the general performance of a familial database search will be demonstrated through an example .
the logistic regression approach , however , uses only the random match probabilities but not the detail information provided in the dna evidence for a particular case . for practical crime cases , it may be also necessary to determine the most appropriate scale of the crime investigation by fully utilizing the available dna evidences . in the next section
, we develop a novel strategy on deciding the number of individuals to be investigated after the familial search , based on an estimate of the hit rate of the crime investigation on a specific number of top - listed candidates .
denote hd as the hypothesis that the sibling of the contributor is in the database , i.e. , hd = i dhi . under the assumption that the relative of someone in the database contributes to the mixture , the posterior probability that the contributor is the relative of individual j , given the mixture profile m , victim profile v and database profiles xd = { xjl , j d , l = 1, ... ,l } can be expressed as
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\begin{document}$$\begin{array}{lll } \label{eqn : posterior } & & { \kern-6pt } p(h_j|m , v,\mathbf{x_{\rm d}},h_{\rm d})\\ & & { \kern6pt } = \frac{p(m|v , x_j , h_j)p(h_j|h_{\rm d})}{\sum_{i\in d}p(m|v , x_i , h_i)p(h_i|h_{\rm d})}. \end{array } $ $ \end{document}whereexj = { xjl , l = 1, ...
,l } for j d. the proof of eq . 3 is given in the appendix . under most common scenarios ,
3 becomes
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\begin{document}$$ \label{eqn : posterior2 } p(h_j|m , v,\mathbf{x_{\rm d}},h_{\rm d})=\frac{p(m|v , x_j , h_j)}{\sum_{i\in d}p(m|v , x_i , h_i)}. $ $ \end{document}alternatively , other priors can also be used if the police authority has acquired more information about the family status details of each individual in the database . without loss of generality ,
suppose that the database is sorted in descending order according to the posterior probabilities such that
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\begin{document}$$\begin{array}{rll } p(h_1|m , v,\mathbf{x}_{\rm d},h_{\rm d})&\ge & p(h_2|m , v,\mathbf{x}_{\rm d},h_{\rm d})\\ & \ge & \cdots \ge p(h_n|m , v,\mathbf{x}_{\rm d},h_{\rm d } ) \end{array}$$\end{document}where n is the size of the database .
for any integer 1 k n , the function defined by
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\begin{document}$$ \label{eqn : hit_rate } q(k)=\sum\limits_{i=1}^{k}p(h_i|m , v,\mathbf{x_{\rm d}},h_{\rm d } ) $ $ \end{document}evaluates the probability that the relative of the contributor can be identified by investigating the top - k individuals with respect to their posterior probabilities , given that the relative is in the database .
in other words , q(k ) represents the hit rate of the crime investigation on k top - listed candidates , thereby providing a means for developing a practical crime investigation strategy under different criteria . for instance , a simple strategy is to investigate the top - k individuals in the database sorted with respect to their lrs or posterior probabilities , with k determined by
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\begin{document}$$ \label{eqn : kp0 } k(p_0)= \min\{k>0:q(k)\ge p_0\ } $ $ \end{document}where p0 is the hit rate required by the police force .
given a crime trace observed as a dna mixture m contributed by the perpetrator and the victim , the familial search score for a particular individual j in the database d is defined as a lr of the following hypotheses :
hj : the victim and a relative of individual j are contributors.hd : the victim and one unknown person are contributors.note that the prosecution hypothesis hj here states that a relative of individual j , rather than the individual , is the contributor and therefore is not the same as the hypothesis formulated in chung et al . and chung and fung . in general , a lr can be calculated for each of the possible genetic relationships for every member of the database .
however , the familial search is practically used only for parent / child and sibling relationships because it would become less effective for other degrees of relatedness below sibling that share less genetic similarity .
hd : the victim and one unknown person are contributors . at a particular locus l , denote ml as the set of alleles present in the mixture and vl and xjl as the genotype of the victim and individual j , respectively , for j d and l = 1, ... ,l . under linkage equilibrium assumption ,
presented the following formula for calculating the lr of hj versus hd :
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\begin{document}$$ \label{eqn : lr } { \rm lr}_j=\prod\limits_{l=1}^{l}{\frac{p(m_l|v_l , x_{j\,l},h_j)}{p(m_l|v_l , h_{\rm r } ) } } $ $ \end{document}where hr is the hypothesis that the victim and a random person contribute to the mixture .
the computation of p(ml|vl , xjl , hj ) and p(ml|vl , hr ) is based on the q - function presented in hu and fung and fung and hu :
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\begin{document}$$\begin{array}{lll } & & { \kern-6pt } p(m_l|v_l , x_{j\,l},h_j)\\ & & { \kern6pt}=k_0q(2,u_l)+k_1\big(i_m(t_1)q(1,u_l\setminus\{t_1\ } ) \\ & & { \kern138pt}+i_m(t_2)q(1,u_l\setminus\{t_2\})\big ) \\ & & { \kern18pt}+k_2i_m(t_1)i_m(t_2)q(0,u_l\setminus\{t_1,t_2\ } ) \end{array}$$\end{document}\documentclass[12pt]{minimal }
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\begin{document}$$ p(m_l|v_l , h_{\rm r})=q(2,u_l ) $ $ \end{document}where t1t2 is the genotype present in xjl , ul = ml vl is the set of alleles present in ml but absent in vl , and im(t ) is the indicator function defined by im(t ) = 1 if t m and 0 otherwise .
the quantities ( k0,2k1,k2 ) are the kinship coefficients for the relationship considered in hj .
in particular , ( k0,2k1,k2 ) take the values of ( 0.25,0.5,0.25 ) for full siblings and ( 0,1,0 ) for parent / child relationship .
table 1 shows the computational formulae of q ( . , . ) for a two - person mixture at a particular autosomal locus l with k alleles a1,a2, ... ,ak and corresponding allele frequencies p1,p2, ... ,pk\documentclass[12pt]{minimal }
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\begin{document}$(\sum_{i=1}^{k}p_i=1)$\end{document } , under the assumption of hardy weinberg equilibrium .
table 1the calculating formulae of q(j , b ) for different combinations of mixture ml and arbitrary set of alleles b at a particular autosomal locus l with alleles a1,a2, ... ,ak and corresponding allele frequencies p1,p2, ... ,pk , under the assumption of hardy
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at002piptaj , ak002pjpkaj , at002pjptak , at002pkptthe indices i , j , k , and t are pairwise distinct the calculating formulae of q(j , b ) for different combinations of mixture ml and arbitrary set of alleles b at a particular autosomal locus l with alleles a1,a2, ... ,ak and corresponding allele frequencies p1,p2, ... ,pk , under the assumption of hardy weinberg equilibrium the indices i , j , k , and t are pairwise distinct the individuals in the database are ranked in descending order by the lr scores , and the individuals with highest scores will be preliminarily identified as the suspects that need further investigations by the police force . in case
when rare alleles are present in the unexplained profiles ml vl , the random match probability p(ml|vl , hr ) will become extremely small , and the unknown contributor s relative will be assigned a large lr score so that the perpetrator will be more likely to be identified .
therefore , the performance of the familial search depends much on the observed mixture m = { ml , l = 1, ... ,l } and the victim profile v = { vl , l = 1, ... ,l}. to predict the performance of the search , cowen and thomson suggested fitting the logistic regression model
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\begin{document}$$ \label{eqn : logistic_model } { \rm logit}(p(\theta_k=1))=\alpha-\beta \log_{10}{p(m|v , h_{\rm r } ) } $ $ \end{document}where k is an binary response variable taking the value of 1 if the relative of the unknown contributor is located within the top k profiles and 0 otherwise .
the model links the outcome of a particular search that limits the investigations to the k top - listed individuals to the random match probability p(m|v , hr ) and therefore can be used to predict the performance of the search given the observed dna profiles ( m , v ) . in particular ,
if the estimated probability p(k = 1 ) is too small even for large k , the familial search may fail to provide effective assistant for identifying the suspect .
this information can practically guide the police force to make decisions on whether it is worthwhile to open an investigation just based on the familial search results . the use of logistic regression for evaluating the general performance of a familial database search will be demonstrated through an example .
the logistic regression approach , however , uses only the random match probabilities but not the detail information provided in the dna evidence for a particular case . for practical crime cases , it may be also necessary to determine the most appropriate scale of the crime investigation by fully utilizing the available dna evidences . in the next section
, we develop a novel strategy on deciding the number of individuals to be investigated after the familial search , based on an estimate of the hit rate of the crime investigation on a specific number of top - listed candidates .
denote hd as the hypothesis that the sibling of the contributor is in the database , i.e. , hd = i dhi . under the assumption that the relative of someone in the database contributes to the mixture , the posterior probability that the contributor is the relative of individual j , given the mixture profile m , victim profile v and database profiles xd = { xjl
, j d , l = 1, ... ,l } can be expressed as
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\begin{document}$$\begin{array}{lll } \label{eqn : posterior } & & { \kern-6pt } p(h_j|m , v,\mathbf{x_{\rm d}},h_{\rm d})\\ & & { \kern6pt } = \frac{p(m|v , x_j , h_j)p(h_j|h_{\rm d})}{\sum_{i\in d}p(m|v , x_i , h_i)p(h_i|h_{\rm d})}. \end{array } $ $ \end{document}whereexj = { xjl , l = 1, ... ,l } for j d. the proof of eq . 3 is given in the appendix . under most common scenarios , it is sensible to assume a uniform prior for hi so that eq . 3 becomes
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\begin{document}$$ \label{eqn : posterior2 } p(h_j|m , v,\mathbf{x_{\rm d}},h_{\rm d})=\frac{p(m|v , x_j , h_j)}{\sum_{i\in d}p(m|v , x_i , h_i)}. $ $ \end{document}alternatively , other priors can also be used if the police authority has acquired more information about the family status details of each individual in the database . without loss of generality ,
suppose that the database is sorted in descending order according to the posterior probabilities such that
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\begin{document}$$\begin{array}{rll } p(h_1|m , v,\mathbf{x}_{\rm d},h_{\rm d})&\ge & p(h_2|m , v,\mathbf{x}_{\rm d},h_{\rm d})\\ & \ge & \cdots \ge p(h_n|m , v,\mathbf{x}_{\rm d},h_{\rm d } ) \end{array}$$\end{document}where n is the size of the database . for any integer 1 k n , the function defined by
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\begin{document}$$ \label{eqn : hit_rate } q(k)=\sum\limits_{i=1}^{k}p(h_i|m , v,\mathbf{x_{\rm d}},h_{\rm d } ) $ $ \end{document}evaluates the probability that the relative of the contributor can be identified by investigating the top - k individuals with respect to their posterior probabilities , given that the relative is in the database .
in other words , q(k ) represents the hit rate of the crime investigation on k top - listed candidates , thereby providing a means for developing a practical crime investigation strategy under different criteria .
for instance , a simple strategy is to investigate the top - k individuals in the database sorted with respect to their lrs or posterior probabilities , with k determined by
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\begin{document}$$ \label{eqn : kp0 } k(p_0)= \min\{k>0:q(k)\ge p_0\ } $ $ \end{document}where p0 is the hit rate required by the police force .
we illustrate here the performance of the familial search and the use of eqs . 5 and 6 .
an offender database consisted of 50,000 unrelated dna profiles is generated according to the swedish allele frequencies given in montelius et al .
, at the 10 sgm plus str loci which are commonly used in european national dna databases .
the first 1,000 profiles in the database are used to represent the profiles of the relatives of the perpetrators in 1,000 different crime cases . for each of the first 1,000 profiles ,
a related profile ( full sibling or parent / child ) is generated to represent the unobserved profile of the unknown perpetrator in that particular case .
the observed dna mixtures are produced by mixing these relative profiles with independently generated victim profiles .
for each case , a familial search is performed , and a list of 50,000 lr scores is calculated by using eq . 1
. the rank of the lr score of the true relative of the unknown contributor indicates at least how many top - listed individuals must be investigated in order to successfully identify the relative of the perpetrator .
in general , such ranks recorded from the 1,000 cases can provide reasonable assessment of the effectiveness of the familial search .
for example , there are 704 cases in which the true sibling of the unknown contributor is within the top 100 individuals in the database ranked by the lr scores , showing a chance of 70.4% that the sibling of the unknown contributor can be successfully identified if the 100 top - listed individuals are investigated , provided that the unknown contributor is a sibling of one of the individuals in the database .
the chance of successful identification of the parent / child of the unknown contributor from the top 100 profiles is 88.0% .
1 shows the empirical probabilities of identifying the relative of the contributor using lr scores .
table 2empirical probabilities of identifying the parent / child and full sibling of the contributor in the top k profiles ranked by likelihood ratio scoresk% of identification in top k profilesparent
1empirical probability of identifying the parent / child and full sibling of the contributor in the topk profiles ranked by likelihood ratio scores in a one - unknown mixture case empirical probabilities of identifying the parent / child and full sibling of the contributor in the top k profiles ranked by likelihood ratio scores empirical probability of identifying the parent / child and full sibling of the contributor in the topk profiles ranked by likelihood ratio scores in a one - unknown mixture case clearly as shown in fig . 1 ,
the empirical probabilities indicate a more effective search for the parent / child of the unknown contributor than the sibling .
it is interesting to note that the empirical probabilities of successful identification are almost linearly related with logarithm of k , the number of top - listed individuals to be investigated .
this suggests that enlarging the scale of the investigation will have little improvement to the performance of familial search if the original scale is already substantially large . in roughly 46% of the cases ,
the sibling of the unknown contributor can be found within the top 20 profiles , while in 56% of the cases , investigating the top 10 profiles is already enough for identifying the parent / child of the unknown contributor .
, the performance of the search here is slightly inferior as we use a larger database and less loci for profiling in this work .
however , it is comparable to the result of curran and buckleton in a single - source case which also use a system of 10 str loci but a smaller database of about 24,000 profiles . therefore , in cases with dna mixture as part of the available clue , the familial dna database search can be still applied for identifying the suspect , with the false hit rate as low as in single - source cases . following cowen and thomson , logistic regression models in the form of eq .
2 are fitted to the data to estimate the dependence of the outcome with respect to the observed profiles ( m , v ) .
table 3 listed the coefficients of the fitted models , with p values all smaller than 0.001 .
the performance of the search is strongly associated with the value of the random match probability . as shown in fig . 2 , the smaller the random match probability , the more likely the relative of the unknown contributor can be identified .
in particular , if p(m|v , hr ) is less than 10 , which is about the median of the random match probabilities from the simulated profiles , there will be a 41% chance to identify the sibling and a 63% chance to identify the parent / child within the top 10 profiles of the database
. the performance would be worse if p(m|v , hr ) is as low as 10 .
the estimated probabilities of successfully identifying the parent / child and sibling are 24% and 17% , respectively . in such cases ,
an investigation on just 10 top - listed individuals from the familial search is definitely not enough .
table 3estimated parameters of the logistic regression models for the probability of identifying the relative of the contributor in the top k profilesrelationshipkparameter estimate ( se)parent / child5 6.0635 ( 0.5733)0.5114 ( 0.0492)10 6.1751 ( 0.5970)0.5593 ( 0.0522)20 6.1630 ( 0.6355)0.5969
( 0.0565)full sibling10 5.1906 ( 0.5513)0.4014 ( 0.0468)50 4.7499 ( 0.5610)0.4477 ( 0.0492)100 3.8619 ( 0.5926)0.4173 ( 0.0528)fig .
2predicted probability of identifying the a parent / child and b full sibling of the unknown contributor in the top k profiles estimated parameters of the logistic regression models for the probability of identifying the relative of the contributor in the top k profiles predicted probability of identifying the a parent / child and b full sibling of the unknown contributor in the top k profiles to determine an appropriate scale of the investigation , we can apply the proposed strategy .
for illustrative purpose , we consider a search that aims at identifying the parent / child or the full sibling of the contributor from the database , though the basic principle can be applied to any kind of relationship .
figure 3 shows the average of the estimated hit rates evaluated by using the q(k ) function in eq . 5 and the empirical hit rates obtained as in fig . 1 .
as can be seen , the average of the estimated hit rates are close to the empirical hit rates , suggesting reliable estimates of the hit rates by using q(k ) .
the number of top - listed individuals that needs investigation can be determined by k(p0 ) defined in eq . 6 for a particular required hit rate p0 . the empirical distributions of k(p0 ) for various values of p0
the distributions are all skewed toward the right as the averages are all greater than the medians .
for the search that aims at identifying the parent / child of the contributor , large proportions of k(p0 ) are found to be less than 100 with respect to p0 0.8 . when the required hit rate p0 is as high as 0.9 , there are still about 75% of the cases in which the values of k(p0 ) are less than 200 , i.e. , further investigations on less than 200 top - listed candidates will be suggested .
therefore , the investigation can be controlled under a reasonable scale even though a high hit rate is usually required .
3probabilities of identifying the a parent / child and b full sibling of the unknown contributor in the top k profiles estimated using empirical hit rates ( solid line ) and the average of the q(k ) function ( dashed line)table 4summary statistics of k(p0 ) from simulation resultsp0empirical probabilityaverage of k(p0)percentiles of k(p0 ) distribution10th25thmedian75th90thparent / child0.50.55317.1141123440.60.62525.8261838690.70.72445.131031621100.80.81675.2519531041790.90.925137114197192314full sibling0.50.51241.42112962970.60.61287.0527661301970.70.74419018701522834130.80.851458572083946699120.90.9561,3933137871,3131,9602,516the empirical hit rates are the probabilities of identifying the parent / child and full sibling of the contributor within the top - k(p0 ) individuals probabilities of identifying the a parent / child and b full sibling of the unknown contributor in the top k profiles estimated using empirical hit rates ( solid line ) and the average of the q(k ) function ( dashed line ) summary statistics of k(p0 ) from simulation results the empirical hit rates are the probabilities of identifying the parent / child and full sibling of the contributor within the top - k(p0 ) individuals on the other hand , the values of k(p0 ) determined in searching for the full sibling of the contributor are relatively larger . if p0
= 0.9 is required , the values of k(p0 ) are greater than 1,000 in the majority of the cases , indicating that it may not be feasible to achieve such a high hit rate with limited law enforcement resources .
the result is expected as the performance in searching for the full sibling should be inferior to the cases in searching for the parent / child .
nevertheless , lowering the requirement on the hit rate to p0 0.7 can still reduce the values of k(p0 ) to less than 150 in about 50% of the cases , which are more feasible scales .
based on this information , the decision to open an investigation can be taken by the police force after thorough consideration on the crime severity and the law enforcement resources .
it is remarkable to note that the empirical hit rates based on investigating the top - k(p0 ) individuals are all greater than the required hit rate p0 , further justifying our approach in providing information for the police force to determine an appropriate scale of crime investigation based on the results from a familial database search .
this article illustrates how the traditional familial database search methods that were used only for single - source samples can be extended so as to handle mixture cases .
it is demonstrated that the familial search applied to two - person mixture cases can perform as good as in single - source cases by using the lr scoring scheme .
the results presented in previous section are based on the most common scenario when the mixed stain is originated from the victim whose dna profile is available . for two - unknown mixture cases , the set of possible profiles for the unknown contributors will be less restrictive . as a result , the discriminatory power of the likelihood ratio score for database search in two - unknown mixture cases will become relatively lower , comparing to one - unknown mixture cases and single - source cases . to demonstrate this ,
consider the following hypotheses for a two - unknown mixture case :
hj : a relative of individual j and an unrelated unknown person are contributors.hd : two unrelated unknown persons are contributors.and the corresponding lr can be calculated as
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\begin{document}$$ { \rm lr}_j=\prod\limits_{l=1}^{l}{\frac{p(m_l|x_{j\,l},h_j)}{p(m_l|h_{\rm r } ) } } $ $ \end{document}where
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\begin{document}$$\begin{array}{lll } p(m_l|x_{j\,l},h_j)&=&k_0q(4,m_l)\\&&+k_1\big(i_m(t_1)q(3,m_l\setminus\{t_1\})\\ & & \qquad\;+\ ; i_m(t_2)q(3,m_l\setminus\{t_2\})\big ) \\ & & + k_2i_m(t_1)i_m(t_2)q(2,m_l\setminus\{t_1,t_2\ } ) , \end{array}$$\end{document}\documentclass[12pt]{minimal }
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\begin{document}$$ p(m_l|h_{\rm r})=q(4,m_l ) .
$ $ \end{document}figure 4 shows the empirical search results on a two - unknown case using the same generated database .
as expected , the performance is substantially inferior to the case in which the victim profile is available .
there is only a 36% chance to identify the sibling and a 54% chance to identify the parent / child within the top 100 profiles of the database .
4empirical probability of identifying the parent / child and full sibling of the contributor in the topk profiles ranked by likelihood ratio scores in a two - unknown mixture case hj : a relative of individual j and an unrelated unknown person are contributors .
hd : two unrelated unknown persons are contributors . empirical probability of identifying the parent / child and full sibling of the contributor in the topk profiles ranked by likelihood ratio scores in a two - unknown mixture case in addition to the logistic regression approach that models the predicted hit rate as a function of the random match probability , we have derived a formula that can accurately estimate the hit rate for a specific case . using this formula , a simple strategy is developed to determine the least number of individuals who should be included in the crime investigation , according to the desired hit rate required by the police force .
it should be noted that the formula for estimating the hit rate , as well as the proposed strategy , is also applicable in single - source cases .
as shown by the numerical example , the proposed strategy can provide useful information aiding the police force for deciding on the scale of investigation , thereby achieving desirable crime - solving rate with reasonable cost . as commented by chakraborty and ge
, it is necessary to point out that the familial dna database search is an auxiliary tool for crime solving when there is no clue on the source of the crime trace , rather than playing a decisive role to bring the suspects identified through database search to trials .
the evidentiary value of the cold hits from familial search should serve as supporting reference to aid the jury to make their decision , provided that a court case is raised after the investigation on the cold - hit suspects .
to clearly present our idea for the application of familial search to mixture cases , several key assumptions have been made in the works presented in this article .
these include the linkage equilibrium that assumes independence of alleles across all loci and the hardy weinberg equilibrium that assumes the independence between the two alleles of a genotype at a particular locus .
the former is usually guaranteed by the proper choice of str loci while the latter may fail to apply in case when the contributors of the mixture come from a small population or from different ethnic groups .
although it is not difficult to modify the formulae presented in earlier sections and table 1 to handle allele dependence , the impact of the deviation from hardy weinberg equilibrium to the performance of the familial search is yet to be studied . besides , the presented method is based on the common scenario that there is only one perpetrator committing the crime and a two - person mixture is included as part of the dna evidence .
more general approach that considers multiple perpetrators as in group rape cases can make familial search a more useful crime - solving tool .
therefore , another possible direction for future work is to develop general methodologies that can handle the complication arises due to the presence of multiple perpetrators .
under the assumption that the sibling of the contributor is someone in the database , the posterior probability of hj given the dna evidences m , v , xd can be evaluated by using the bayes rule :
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\begin{document}$$\begin{array}{rll } & & { \kern-6pt } p(h_j|m , v,\mathbf{x_{\rm d}},h_{\rm d})\\ & & { \kern6pt}=\frac{p(m|v,\mathbf{x_{\rm d}},h_j , h_{\rm d})p(h_j|h_{\rm d})}{\sum_{i=1}^n p(m|v,\mathbf{x_{\rm d}},h_i , h_{\rm d})p(h_i|h_{\rm d } ) } \\ & & { \kern6pt}=\frac{p(m|v,\mathbf{x_{\rm d}},h_j)p(h_j|h_{\rm d})}{\sum_{i\in d } p(m|v,\mathbf{x_{\rm d}},h_i)p(h_i|h_{\rm d } ) } \end{array}$$\end{document}where the equality results from the fact that hi hd = hi for i d and p(hi|hd ) = 0 for i d. when hi holds true , the mixture m will depend only on v and xi and is unrelated to the profiles of other individuals in the database .
substituting p(m|v , xd , hi ) = p(m|v , xi , hi ) into the above equation leads to eq . | the role of familial database search as a crime - solving tool has been increasingly recognized by forensic scientists . as an enhancement to the existing familial search approach on single source cases ,
this article presents our current progress in exploring the potential use of familial search to mixture cases .
a novel method was established to predict the outcome of the search , from which a simple strategy for determining an appropriate scale of investigation by the police force is developed . illustrated by an example using swedish data , our approach is shown to have the potential for assisting the police force to decide on the scale of investigation , thereby achieving desirable crime - solving rate with reasonable cost . |
human diseases can be treated by the transfer of therapeutic genes ( transgene ) into specific cells or tissues of patients to correct or supplement defective , causative genes .
the success of gene therapy depends on the ability to deliver therapeutic dna or sirna or other genetic materials to target site .
viral vectors ( e.g. , adenovirus ) are very effective in term of transfection efficiency , but they have limitations in vivo , particularly by their safety concern and nontissue - specific transfection .
nonviral gene transfer systems are limited by their lower gene transfer efficiency , low tissue specificity , and transient gene expression [ 1 , 2 ] .
cationic polymers , such as chitosan ( ch ) , are promising candidates for dna transport in nonviral delivery systems [ 3 , 4 ] . the enabling characteristics of ch - dna nanoparticles include biocompatibility , multiple ligand affinity , and a capacity of taking up large dna fragments , while remaining small in size .
genes can be delivered systemically ( intramuscularly , intravenously , subcutaneously , or , in animals , intraperitoneally ) .
otherwise , hydrodynamic - based gene delivery offers a convenient , efficient , and powerful means for high - level gene expression in animals [ 68 ] .
the highly effective delivery of naked plasmid into body or tissue with hydrodynamic limb vein injection technique has already been demonstrated for the treatment of muscular dystrophy .
however , the mechanism of hydrodynamic injection aiding the delivery of chitosan - based nanoparticles into targeted tissue and the use of this approach in other disease , treatment such as arthritis is unclear .
rheumatoid arthritis ( ra ) is a chronic , systemic autoimmune disorder that causes the immune system to attack the joints , where it causes inflammation ( arthritis ) and destruction .
other problems may also develop , including inflammation of blood vessels ( vasculitis ) , the development of bumps ( called rheumatoid nodules ) in various parts of the body , lung disease and blood disorders .
the recent report demonstrated that the muscle strength loss is strongly associated with the presence and duration of an inflammatory state like rheumatoid arthritis .
interleukin-1 ( il-1 ) , a key proinflammatory factor which consists of 2 subtypes , including il-1 and il-1 , is produced by monocytes macrophages .
il-1 remains mostly intracellular ( or expressed on the cell surface ) , while il-1 is secreted into the extracellular space .
prostaglandin e2 ( pge2 ) is considered to be of great importance in pathology of ra .
il-1 has been implicated in the pathological mechanisms of synovial tissue proliferation and joint destruction in ra [ 2 , 11 , 13 ] .
its physiologically competitive agent , il-1 receptor antagonist ( il-1ra ) , has been proven to act as a powerful inhibitor .
several therapeutic studies on the use of recombinant il-1ra , as an external source agent , have demonstrated its positive effect in controlling inflammation and symptoms of ra in animal models and in clinical practice [ 15 , 16 ] .
folate receptors ( fr ) are overexpressed on many human cancer cell surfaces , and the nonepithelial isoform fr( ) is expressed on activated synovial macrophages present in large numbers in ra .
folate - mediated transfection has been shown to facilitate dna and nanoparticle internalization into cells through membrane receptors for higher transfection yields both in vitro and in vivo . in this study
, 50 kda chitosan was conjugated with folic acid ( fa ) , a specific tissue - targeting ligand .
nanoparticles such as chitosan - dna ( ch - dna ) and chitosan - folate - dna ( ch - fa - dna ) were prepared by coacervation process .
the hydrodynamic limb intravenous injection of nanoparticles was performed in the right posterior paw in rats .
the first objective was to determine the distribution of these nanoparticles in normal rats following hydrodynamic injection .
the second objective of the study was to examine the in vivo anti - inflammaory effects of nanoparticles of plasmid il-1ra dna complexed to the ch or ch - fa in an adjuvant - induced arthritis ( aia ) rat model .
all animal experiments were approved by the animal ethics committee of the hpital du sacr - coeur de montral .
chitosan ( ch ) ( wako-10 , degree of deacetylation ( dd ) 85% , mw = 57 kda ) was purchased from wako chemicals usa ( richmond , va ) .
folic acid ( fa ) was purchased from sigma - aldrich chemical ( st - louis , mo , usa ) .
-amino , -carboxyl poly(ethylene glycol ) ( nh2-peg - cooh , mw = 3,400 dalton ) was purchased from shearwater inc . ( dallas , tx , usa ) .
ch was further deacetylated by treating with concentrated naoh solution ( 50% ) to obtain ~96% deacetylation degree according to the reported procedure . to prepare the ch - fa conjugate ,
folic acid was first attached to nh2-peg - cooh via the well - known carbodiimide chemistry to obtain folate - peg - cooh .
then , the folate - peg - cooh was again activated by n , n-dicyclohexylcarbodiimide ( dcc ) and n - hydroxysuccinimide ( nhs ) to convert to the reactive intermediate folate - peg - co - nhs and subsequently grafted onto chitosan to achieve the folate - peg - chitosan conjugate .
the level of folate - peg incorporation was determined to be 1.1 mol% with respect to the glucosamine unit of chitosan by uv - vis spectroscopy using folic acid as standard ( the extinction coefficient ( 363 nm ) of folic acid is determined to be 6165 m cm in ph 7.4 phosphate buffer ( 0.1 m ) ) .
we used human interleukin 1 receptor antagonist as a curing agent , with its secretive variant ( sil-1ra , 177aa , nm_173842 x52015 , genbank ) .
we subcloned this gene by pcr with oligos carrying hindiii and xbai sites ( il-1rah3 ccaagcttgaatggaaatctgcagaggcc , il-1rah4 gctctagactgggca gtactactcgtcctcc ) , using genomic dna of macrophage differentiated from monocyte cell line thp1 stimulated with pma as pcr dna template .
then , pcr product was inserted in the mammalian expression vector pcdna3 ( invitrogen , burlington , on , canada ) that drives overexpression of gene with cmv promoter . to reduce the size of pdna ,
the region of pvuii - pvuii was deleted . final plasmid ( 3.8 kb ) including targeted gene was verified by dna sequencing .
the plasmid dna vr1412 , encoding the -galactosidase ( -gal ) reporter gene with a cmv promoter and a backbone size of 8,100 kb , was obtained from vical inc .
the plasmid pdsred encoding the red fluorescent protein was obtained from clonetech ( mountain view , ca , usa ) .
the plasmid dnas were multiplied with escherichia coli bacteria then isolated and purified using the qiagen plasmid mega kit according to the manufacturer 's instruction ( qiagen , mississauga , on , canada ) .
the dna concentration was measured by uv absorption at 260 nm with a uv - visible 1601 spectrophotometer from shimadzu ( columbia , md , usa ) .
all bacteria were all grown in gibco invitrogen corporation lennox l broth base ( invitrogen canada inc . ,
ch or ch - fa was dissolved with low heating ( below 45c ) in a 20-mm acetic solution at ph 5.5 .
the solution was then adjusted to a final concentration of 0.02% chitosan in 5 mm acetic acetate and passed through a 0.22-m filter . the plasmid dna ( -gal , dsred , and il-1ra plasmid dnas ) employed for synthesis was a solution of 200 g dna / ml of 4.3 mm sodium sulfate solution .
after heating to 55c , 100 l of the dna solution was added to 100 l of ch or ch - fa solution and vortexed immediately at maximum speed for 1 min .
the nanoparticle solutions produced were deployed for stirring 30 minutes and allowing standing one hour before transfection without further modification .
two kinds of nanoparticles were synthesized : ch - dna and ch - fa - dna .
kb cells obtained from american type culture collection ( atcc ) ( rockville , md , usa ) were used for transfection in the media with or without folic acid supplement .
on 24-well plates at a density of 50,000 cells / well in 1 ml of roswell park memorial institute ( rpmi ) , 1640 medium supplemented with 10% fetal bovine serum ( fbs ) and 1% penicillin - streptomycin ( ps ) .
next day , medium was replaced with fresh rpmi medium 1640 or rpmi medium 1640 without folic acid , without fbs in two type media . after 2 hours
, kb cells received 500 l solution of ch- or ch - fa- plasmid il-1ra dna nanoparticles ( containing 5 g human il-1ra plasmid dna ) in the presence or absence of folic acid supplement .
after another two hours , 0.5 ml complete rpmi 1640 medium was added in each well .
after 24 hours , cell culture media and cells were collected separately for later analysis .
the hydrodynamic intravenous injection was performed in the right posterior paw , as previously published [ 2224 ] .
the injection point was located in the great saphenous vein inside the leg , in direction of the knee .
a tourniquet was located above the knee forcing the solution back into the smaller leg veins and ending up in muscle tissue cells .
the 4 ml transfection solution ( containing 200 g dna ) was injected into about 200 g rats , at a speed of 10 ml / minute , with infusion / withdrawal pump model 940 ( harvard apparatus , millis , ma , usa ) .
the eyes were humidified with liquifilm tears eyes drops from allergan ( markham , on , canada ) .
the female lewis rats were injected with the hydrodynamic technique via the femoral vein with desred plasmids dna , coding for fluorescent emitting proteins , measurable by detecting the emitted dsred fluorescence wave , with a maximum excitation at 555 nm and maximal emission at 583 nm .
we used 9 female lewis rats to administer dsred plasmid ( containing 200 g dna / rat ) alone via the hydrodynamic technique by the femoral vein , towards the direction of the knee .
3 experimental groups formed 3 rats for control , 3 rats for hydrodynamic injection , and 3 rats for normal femoral intravenous injection .
the rats were sacrificed 6 to 8 days following the injection ; the tissue sections of liver , kidney , gluteus superficialis muscle , and different skeletal muscle groups of two hind limbs ( femoris bicep , soleus , and gastro ) were removed , cleaned by dipping it in clean phosphate buffered saline ( pbs ) solution , homogenised with polytron , and centrifuged at 3000 rpm .
the samples suspensions were then stored in 80c until the day the measurements are done .
the fluorescence density was detected by photon technology international ( pti ) spectrofluorometer ( birmingham , nj , usa ) .
the female lewis rats were injected with the hydrodynamic technique via the femoral vein with 200 g naked -gal plasmids dna ( 3 rats ) , ch--gal plasmids dna ( 3 rats ) and ch - fa--gal plasmids dna ( 3 rats ) , respectively .
the sampled tissues ( soleus muscles ) were to be dissected into an ice cold pbs solution containing 2 mm mgcl2 .
the fixation process is done by dipping them , for 30 minutes and more , in a solution composed of 0.5% glutaraldehyde , reputed for its superior staining and endogenous enzyme preservation activity , up to the confounding point of the signal , compared to the other way by using paraformaldehyde .
afterwards , it is very important to carefully rinse of residual fixatives , which can inhibit the enzyme , by rinsing them many times with pbs . at some point
the x - gal stock was diluted into the x - gal reaction buffer ( 40 mg / ml in die methyl ) .
the tissues were then incubated in it for 24 hours at 37c before getting rinse in pbs .
the viewing step was done under the bright fluorescent white light for optimal visualisation and analysis .
one of section of specimens were embedded in paraffin , sectioned , and stained for -galactosidase protein expression stained with its corresponding polyclonal antibody ( sigma - aldrich , st - louis , mo , usa ) after hydrodynamic ch - fa- plasmid -gal dna injection .
the staining was undertaken with dako liquid dab+ substrate - chromogen system kit ( dako , carpinteria , ca , usa ) as directed by the manufacturer .
each section was examined under a light microscope ( zeiss axio - imager z1 , carl zeiss microimaging gmbh , gttingen , germany ) at x20 magnification and photographed with a zeiss axiocam camera and axio - vision software ( version 4.0 ) .
arthritis was induced in 150 to 200 g female lewis rats ( charles rivers , montreal , qc , canada ) .
briefly , on day 1 , rats were anaesthetized with 0.2 ml 0.5% ketamine and 0.5% xylazine hydrochloride , and 0.5 mg of heat - killed m butyrium suspended in mineral oil ( 5 mgml , difco , mi , usa ) was injected intradermically into the right hind foot .
arthritis usually develops in all treated animals , as evidenced by dramatic swelling in the injected paws and progressive swelling in all uninjected paws .
a control group ( naked dna , n = 5 ) , a ch - plasmid il-1ra dna nanoparticles group ( n = 5 ) , and a ch - fa - plasmid il-1ra dna nanoparticles group ( n = 7 ) were injected on day 18 post aia induction . a nontreated group ( ra ) ( n = 5 ) was used as an untreated control .
all animals were sacrificed on day 35 and clinical results were compared among the groups .
the evaluation of reduced adjuvant - induced arthritis was done by evaluating ankle inflammation , decreased articular index scores , ankle circumferences , and sequential calliper measurements of the ankle joints . while under anesthesia , 79 ml of whole rat blood was obtained by heart puncture .
blood was gelled at room temperature then sera were separated by centrifugation at 6000 rpm for 10 minutes .
il-1ra detection in culture medium and rat sera was carried out with elisa kit ( r&d systems , minneapolis , mn , usa ) .
blood serum samples were diluted 510 times in assay diluents ( biofx , owings mills , md , usa ) .
rat pge2 and il-1 were detected with elisa kit ( r&d systems ) , by diluting blood serum samples 100 times with pbs buffer containing 1% bovine serum albumin ( bsa ) .
all values are expressed as means sd and were subjected to t - test and one - way anova analysis .
zeta potential remained stable for all nanoparticles between + 4 and + 6 mv .
rats were injected with the hydrodynamic technique ( infusion / withdrawal pump model 940 from harvard apparatus co. , inc . , millis , mass .
, usa ) via the right femoral vein , toward the direction of the knee ( figure 1 ) . before performing the injection ,
this lowers the difficulty level when it comes to inserting the needle into the vein .
in addition , the leg is bound tightly , above the knee level with a tourniquet to force the solution back into the smaller leg veins and end up in tissue cells .
this is very crucial since that is what causes the hydrodynamic pressure , hence the whole point of this type of injection .
once the needle is properly inserted , the injection machine is turned on and its speed is set to three , around 0.3 - 0.4 ml / sec . after the injection , only the needle is taken out ; the elastic binder stays for an extra minute to let the plasmid settle in . for each rat , around 4 ml of the injection solution
is needed ; as a precaution , 5 ml is set per rat . to determine the tissue uptake of plasmid dna following hydrodynamic injection , we used 9 female lewis rats to administer dsred plasmid alone via the hydrodynamic technique by the femoral vein , towards the direction of the knee .
3 experimental groups formed 3 rats for control , 3 rats for hydrodynamic injection , and 3 rats for normal femoral intravenous injection .
the rats were sacrificed 6 to 8 days following the injection ; specific dsred plasmid expressed red fluorescent protein expression was measured by spectrofluorometer in various tissue extracts ( figure 2 ) .
the highest level of red fluorescent protein was observed in the soleus muscle of the right legs of hydrodynamic injected rat group .
the different sampled organs also show much lower red fluorescence intensities , like the liver that emits 12 times less .
lastly , by comparing the hydrodynamic injected rats with the control and normal intravenous injected rats , the majority of the red fluorescence values are higher for hydrodynamic injected rats . in order to study ch or ch - fa as gene delivery vector , three groups ( each consisted of 3 rats ) of the normal lewis rats treated with the hydrodynamic intravenous injection of 200 g naked -gal plasmid , ch--gal plasmid dna , and ch - fa--gal plasmid dna , respectively .
soleus muscles were isolated and visualized with x - gal expression 3 days post injection .
as shown in figure 3 , the naked dna can be expressed ( figure 3(a)2 ) .
the ch - fa - dna expression ( figure 3(a)4 ) is darkest in the soleus muscle compared to ch - dna expression ( figure 3(a)3 ) .
figure 3(b ) showed soleus muscle histological cross - sectional patterns of -gal protein expression stained with its corresponding antibody after hydrodynamic injection of nanoparticles of ch - fa - plasmid -gal dna with positive dark - brown staining within the muscle .
in vitro , to test the modification by adding folic acid on chitosan backbone , the nanoparticles made of two types of chitosan were transfected in kb cells with medium without folic acid supplement .
data showed the significantly higher expressive level in 1640 without folic acid medium ( n = 3 , p < .01 ) comparing to that in normal medium ( figure 4(a ) ) .
evidently folic acid in medium interferes the binding between folate - modified nanoparticle and cells .
in vivo , as shown in this figure 4(b ) , the naked plasmid is more rapidly expressed in greater quantity in the serum than the ch - nano and ch - fa - nanoparticles . despite the longer time taken by ch - dna and ch - fa - dna nanoparticles has taken the longer time to express themselves in the serum , but they presented more secreted il-1ra proteins at the day of 34 with compared to naked dna treated rats .
however , there were no significant differences in the il-1ra protein concentration across the three treatment groups ( naked dna , ch - dna , and ch - fa - dna ) .
decrease in macroscopic inflammation in various degrees in aia rat 's paws after treatment with ch - plasmid il-1ra dna , or ch - fa - plasmid il-1ra dna , as shown in figure 5(a ) .
there is a decrease in the percentage of ankle size in all treated groups compared to the untreated ra positive control group .
only the ch - fa - pil-1ra group showed significant decrease in ankle size ( p < .05 , n = 5 ) .
however , there was no significant difference between all other treatment groups ( naked dna , and ch - dna ) .
levels of serum proinflammatory factor il-1
and pge2 . to measure the inflammation changes after treatment , cytokine il-1 , a potent immunomodulator which mediates a wide range of immune and inflammatory responses , was detected in the serum of the experimental animal rat model .
a gradual increase of cytokine level was seen in the positive control group ( ra ) , while there was an abrupt decrease of il-1 levels following treatment with ch - plasmid il-1ra dna and ch - fa - plasmid il-1ra dna nanoparticles .
this bring to the fore the inflammation reduction , since il-1ra competes for the il-1 receptor and indirectly decrease il-1 production therein ( figure 5(b ) ) . as shown in figure 5(c ) , a gradual increase of pge2 levels is seen in the positive control group ( ra ) whereas there is a decrease of pge2 levels following treatment with ch - pil-1ra .
lower levels of pge2 synthesis were observed in all treated groups compared to positive control .
the hydrodynamic limb vein injection involves the rapid injection of a large volume of naked dna through a peripheral vein a limb that is temporarily isolate from normal blood flow by a tourniquet [ 68 ] . in this study
, we found that the expression of the targeted protein by a ch - nanoparticle is possible ( as reflected by dered protein expression and staining by -gal in the soleus muscle ) .
although hydrodynamic injection causes edema at the site of puncture ( data not show ) , injection skills may affect the effect of genetic material delivered .
however , in our study , this manipulation was performed by the same person , used to the technique , thereby reducing the effect of the learning curve on the quality of delivery .
in addition , the hydrodynamic injection allows the introduction of high volume solution into the muscle tissue .
this situation allows for a greater concentration of nanoparticles around cells suggesting a greater potential of capture through endocytosis .
the transfection efficiency of nonviral vectors may depend on several factors such as chemical structure of polycations , size and composition of complexes , interaction between cells and complexes and the cell type .
the small size complexes have the advantage of entering the cells through endocytosis or pinocytosis and crossing the nuclear pore complex , thereby increasing the transfection rate .
our results demonstrated that our nanoparticles ( average 108 nm ) obtained favored nanosize .
many researchers [ 27 , 28 ] , including us , are actually working on the synthesis of smaller size nanoparticles .
the hydrodynamic injection showed 5 to 12 times more red protein fluorescence in the leg muscles in proximity of the injected femoral vein than the other sampled parts ( ex : kidney ) .
it even goes up to 20 times more when comparing with the control ( ex : left gastro muscle ) .
this is what we hoped to obtain with the hydrodynamic vein tourniquet injection . a normal femoral vein injection done using these same manipulations
, the results would not appear much in the legs , but instead it will mostly be in the rat 's liver and spread across other body parts . in our case , the tourniquet made so that the solution travels back into smaller veins and end up in muscle cells . in the case where the tourniquet is well installed , directing the process towards good conditions by blocking the escape ,
the local injection load is administered gently enough to trap and spread the injected solution in and not beyond the tourniquet .
if some of the solution gets to escape , it would get filtered , stopped , in the lungs , liver , kidneys , and aorta , and in most cases , travel in the blood circulation system .
that is why in rat with normal intravenous injection , we were not able to observe the leg muscles swelling ( data not shown ) . instead , the whole leg got bigger in size , acting like a balloon being blown up . probably , the solution was at first trapped in the leg due to the tourniquet applied in the lymphatic and vein drainage system which stop the solution from spreading beyond the knee with high pressure .
the pressure does so that the injected solution in the femoral vein stops going forward and is forced back into the smaller veins in the opposite direction .
it has been demonstrated that naked dna express easily when injected into a muscle mass . according to the results obtained through -gal staining with x - gal
, we can see that without any treatment , the naked dna can be expressed . when surrounded by a layer of 50 kda chitosan as a nonviral approach , only fine blue lines can be seen on part of the targeted soleus leg muscle .
this means that the particles entered difficultly into the cells beyond the veins surrounding the leg muscle .
the answer to where they went is found by observing the final destination of the body fluid circulation , acting like body filter , the liver [ 3133 ] . once upgraded with folic acid ( fa )
when observing the hydrodynamic injection technique efficiency rate for this experiment , the injections technical skill play a very important role .
we observed that il-1ra protein expression was significantly higher ( p < .05 ) when transfection was prepared with chitosan - folate - il-1ra plasmid dna complexes in the culture medium without folic acid supplement ( figure 4(a ) ) .
these findings suggested that folic acid molecules supplemented in culture medium would prevent cellular uptake of the complexes by competitive binding to frs on the cell surface .
the in vivo study revealed that the il-1ra gene can be transduced in vivo exploiting nonviral chitosan and folate target technology via hydrodynamic limb vein injection .
the injected muscle was the predominant site of gene transfer and expression after hydrodynamic limb vein injection .
it is interesting to note that il-1ra protein can be detected in the serum of rats as long as 617 days following the injections of both chitosan - dna and chitosan - folate - dna nanoparticles , possibly offering a lasting , sustain release of il-1ra in the serum .
folate conjugation improves the transfection efficiencies of fa - chitosan for fr - positive cell gene silencing in conditions without folic acid supplement . because of fr overexpression in cancer cells and activated macrophages , a variety of therapeutic agents have been linked with folic acid for selective delivery to pathological cells , by avoiding normal tissues . among these therapies ,
folate - targeted nonviral gene therapy vectors are considered as promising treatments of cancer and inflammatory diseases [ 3335 ] .
the hydrodynamic limb vein injection technology has been shown to be effective for the delivery naked dna into the skeletal muscle of the limb .
this approach has a good profile for the treatment of muscular disease such as muscular dystrophy . in this study , the usefulness of hydrodynamic delivery to decrease inflammation in experimental arthritis treatment was also demonstrated . the first model of ra to be described was adjuvant arthritis , and the arthritis develops 1045 days after injection and generally subsides after a month . based on our experiences with
adjuvant induced arthritis in rats , disease is allowed to obtain maximum inflammation around 18 days , as evidenced by dramatic swelling in the injected paw measurements of proinflammatory biomarkers such as tnf- and il-1. so we inject chitosan / folate - chitosan - dna nanoparticles at day 18 to treat inflammation caused by adjuvant injection.our results showed that it is possible to express the il-1ra protein with a variety of cationic polymeric - based nanoparticles .
macroscopically , hydrodynamic injection of these nanoparticles in an aia rat model allow a significant decrease of the inflammation in the rats ' ankle compared to untreated rats , proving indirectly the efficacy of the il-1ra protein treatment .
various inflammation markers ( il-1 and pge2 ) showed a significant decrease in the muscle and serum after the injection of the il-1ra protein demonstrating by direct evidence the efficacy of the administration technique to deliver efficient nanoparticles .
therefore , it is possible to do gene therapy via hydrodynamic limb vein injection with il-1ra to decrease arthritis and have a positive effect on inflammation . in conclusion ,
the efficacy of chitosan - folate - based nanovectors was confirmed in vitro in kb cells and in vivo using hydrodynamic delivery of -gal dna nanocarriers in lewis rats .
this study demonstrated the efficacy of a therapeutic approach ( after maximum disease progression ) using hydrodynamic delivery of plasmid il-1ra dna and chitosan - dna - based nanocarriers in controlling the progression of inflammation in an aia rat model .
more research are needed toward the search for smaller and more performing nanoparticles in the treatment of arthritis using gene therapy . | 50 kda chitosan was conjugated with folate , a specific tissue - targeting ligand .
nanoparticles such as chitosan - dna and folate - chitosan - dna were prepared by coacervation process .
the hydrodynamic intravenous injection of nanoparticles was performed in the right posterior paw in normal and arthritic rats .
our results demonstrated that the fluorescence intensity of dsred detected was 5 to 12 times more in the right soleus muscle and in the right gastro muscle than other tissue sections .
-galactosidase gene expression with x - gal substrate and folate - chitosan - plasmid nanoparticles showed best coloration in the soleus muscle . treated arthritic animals also showed a significant decrease in paw swelling and il-1 and pge2 concentration in serum compared to untreated rats .
this study demonstrated that a nonviral gene therapeutic approach using hydrodynamic delivery could help transfect more efficiently folate - chitosan - dna nanoparticles in vitro / in vivo and could decrease inflammation in arthritic rats . |
contrast media can lead to acute deterioration in renal function particularly in patients with pre - existing chronic kidney disease .
about 1214% of patients who develop acute renal sufficiency during hospitalization do so after procedures involving radiographic contrast
. the incidence of progressive deterioration can be as high as 42% in patients with abnormal baseline renal function .
several protocols have been tested for the prevention of radiocontrast - induced acute kidney injury including periprocedural hydration with isotonic or hypotonic saline , antioxidant compounds such as n - acetyl cysteine ( nac ) , ascorbic acid or allopurinol and the use of low or iso osmolar contrast agents , hemofiltration , or dialysis .
few studies have shown that prophylactic oral administration of allopurinol , along with hydration , may protect against contrast - induced nephropathy ( cin ) in high - risk patients undergoing coronary procedures .
the results were disappointing or inconclusive and intravenous volume expansion remains to date the only measure of undisputed efficacy .
this study compared the efficacy of nac versus allopurinol in addition to intravenous hydration with normal saline ( ns ) as prophylaxis for cin in patients with impaired renal function undergoing planned coronary angiography or intervention .
this prospective , randomized controlled open - label parallel group study was undertaken at nizam 's institute of medical sciences , hyderabad , from june 2015 to december 2015 .
patients undergoing clinically driven nonemergent coronary angiography and percutaneous coronary interventions for both stable and unstable patients with angina , non - st - segment elevation myocardial infarction ( nstemi ) and acute myocardial infarction / stemi were included in this study .
they are as follows :
age more than 30 yearspatients should have their serum creatinine 1.2 mg / dl on their most recent sample drawn within 3 months of planned procedure .
age more than 30 years patients should have their serum creatinine 1.2 mg / dl on their most recent sample drawn within 3 months of planned procedure .
patients with acute renal failure , endstage renal disease requiring dialysis , intravascular administration of contrast material within previous 6 days , pregnancy , lactation , emergent coronary angiography , history of hypersensitivity reaction to contrast media , cardiogenic shock , pulmonary edema , mechanical ventilator , parenteral use of diuretics , recent use of nac , recent use of ascorbic acid , and use of metformin or nsaids within 48 h of procedure were excluded from the study .
they are as follows :
nac + ns : group of patients who received ns and nacall + ns : group of patients who received ns and allopurinolplacebo + ns : group of patients who received ns only
nac + ns : group of patients who received ns and nac all + ns : group of patients who received ns and allopurinol placebo + ns : group of patients who received ns only a total of 95 adult patients of age > 30 years with serum creatinine 1.2 undergoing iv contrast study for coronary angiography or interventions were enrolled in this study .
quantity of iv contrast given to all patients was recorded along with baseline and post procedure serum creatinine .
all the details of patients such as age , gender , and comorbidities ( presence of hypertension [ htn ] , diabetes , and coronary artery disease ) were noted .
uric acid levels were measured in all patients prior , and allopurinol was given irrespective of uric acid level .
nac : n - acetyl cysteine , ns : normal saline , all : allopurinol , cin : contrast - induced nephropathy serum creatinine , blood urea , electrolytes were done before and every 24 h for 2 days after the study .
the effect of modality was evaluated by observing the change in serum creatinine from baseline .
isotonic normal saline was infused at a rate of 0.5 ml / kg / h 12 h prior to the procedure and was continued for 12 h after contrast administration ( total 24 h ) .
those patients who had low ejection fraction ( < 40% ) received ns at rate of 0.3 ml / kg / h .
nac was given 600 mg orally twice daily , the day before and the day of the procedure .
single oral dose of 300 mg of allopurinol was given the day before the procedure .
cin , defined as either a relative increase in serum creatinine from baseline of 25% or an absolute increase of 0.3 mg / dl ( 44.2 mol / l ) during days 1 and 2 .
a total of 95 patients were enrolled into the study with confidence level of 95% and power of 80% to determine the significant change between treatment groups .
this study was planned as randomized pilot study in which the sample size calculation was based on standard measures of confidence levels and power , based on previous studies involving the study drugs .
odds ratio was calculated using medcalc for windows , version 16.4.3 ( medcalc software , ostend , belgium ) .
odds ratio was calculated using medcalc for windows , version 16.4.3 ( medcalc software , ostend , belgium ) .
as shown in table 2 , there was no significant difference between nac and allopurinol in the prevention of contrast nephropathy .
however , when compared with placebo alone only allopurinol was proved to be superior . as shown in tables 3 and 4 ,
odds ratio for age - cin was 1.07 , indicating an association between age > 60 years and the development of cin .
however , this was not determined to be statistically significant ( p > 0.05 ) , and a similar association between age > 60 years and development of cin was observed in all + ns group , without statistical significance .
the odds ratio for gender - cin was 1.2 , indicating an association between male sex and the development of cin .
however , this was not determined to be statistically significant ( p > 0.05 ) ; on analysis of the treatment groups , a similar association between male sex and development of cin was observed in all + ns group and nac + ns group , however without statistical significance .
the odds ratio for dm - cin was 0.62 , indicating no association between dm and the development of cin ; on analysis of the treatment groups , an association between dm and development of cin was observed in all + ns group , however without statistical significance .
the odds ratio for htn - cin was 1.14 , indicating an association between htn and the development of cin ; however , this was not determined to be statistically significant ( p > 0.05 ) ; on analysis of the treatment groups , a similar association between htn and development of cin was observed in all + ns group without statistical significance . the distribution of baseline characteristics in all three groups which were similar incidence of contrast - induced nephropathy in treatment groups risk factor analysis in general subgroup analysis of risk factors the odds ratio for preprocedural sbp - cin was 0.77 , indicating no association between preprocedural sbp and the development of cin ; on analysis of the treatment groups , an association between preprocedural sbp and development of cin was observed in all + ns group and nac + ns without statistical significance .
the odds ratio for preprocedural dbp - cin was 0.88 , indicating no association between preprocedural dbp and the development of cin ; on analysis of the treatment groups , no association was observed between preprocedural dbp and development of cin .
the odds ratio for contrast volume - cin was 0.8 , indicating no association between contrast volume and the development of cin ; on analysis of the treatment groups , no association between contrast volume and development of cin was observed in all three groups .
the odds ratio for anemia - cin was 1.8 , indicating an association between anemia and the development of cin .
however , this was not determined to be statistically significant ( p > 0.05 ) . on analysis of the treatment groups ,
an association between anemia and development of cin was observed in placebo group , however without statistical significance .
the odds ratio for uric acid - cin was 2.3 , indicating an association between uric acid and the development of cin and it was not statistically significant ( p > 0.05 ) .
incidence of cin in hyperuricemia group was 35% and 19% in normouricemic group . upon odds ratio analysis of the treatment groups ,
an association between uric acid and development of cin was observed in all + ns group and placebo , however without statistical significance in all + ns group and it was statistically significant in the ns group .
the odds ratio for albumin - cin was 0.91 , indicating no association between albumin and the development of cin ; on analysis of the treatment groups , no association between albumin and development of cin was observed in all three groups .
analysis shows that an association was found between baseline serum creatinine and cin , which was statistically significant ; upon odds ratio analysis of the treatment groups , an association between baseline serum creatinine and development of cin was observed in all three groups without statistical significance .
overall six patients needed dialysis ; one patient in nac group , one patient in allopurinol group , and four in placebo group .
four patients improved after few sessions of hemodialysis , two patients became dialysis dependent in placebo group .
the main objective of this study was to compare nac and allopurinol in the prevention of contrast nephropathy in patients with impaired renal function .
the major finding of this study was absence of difference between nac and allopurinol in the prevention of contrast nephropathy .
incidence of cin in nac + ns , all + ns , and placebo group were 20% , 16% , and 36% , respectively .
to the best of our knowledge , this is the first study comparing nac and allopurinol in the prevention of cin in patients with pre - existing ckd .
a similar study was done by kumar et al . in 2014 comparing nac and allopurinol in the prevention of contrast nephropathy in patients undergoing cardiac catheterization with normal baseline serum creatinine and showed that prophylactic administration of allopurinol along with hydration was better than nac and hydration for protection against cin .
they showed that in patients receiving iodixanol as contrast agent , the incidence of cin in allopurinol and hydration were 0% and 30% , respectively .
erol et al . analyzed efficacy of allopurinol pretreatment for the prevention of cin . in this study , 159 patients were enrolled with baseline serum creatine > 1.1 mg / dl .
cin occurred in 7.5% of patients in hydration alone group and 0% in allopurinol group .
erol et al . concluded that allopurinol along hydration may protect against cin in high - risk patients undergoing coronary procedures .
our study also showed similar results such that allopurinol along with hydration was superior to hydration alone in the prevention of cin . in our study , incidence of cin in allopurinol group was 16% compared to 36% in hydration group alone , which was statistically significant .
as shown in table 5 incidence of cin was high both in allopurinol and hydration group in our study when compared to studies done by erol et al . and
kumar et al . probably due to high baseline creatinine and increase of > 0.3 mg / dl being taken as cin .
allopurinol in the prevention of contrast nephropathy in our study , the incidence of cin in nac + ns subgroup was 20% , and in hydration only group was 36% .
our study showed that nac + ns was superior to hydration alone in the prevention of cin , although statistically not significant .
several studies compared hydration with ns , with or without nac in the prevention of cin in patients with elevated renal parameters .
ramesh et al . in 2006 compared nac with hydration in the prevention of contrast nephropathy in patients with elevated serum creatine ( 1.5 mg / dl ) .
the incidence of cin in nac + ns and hydration alone groups was 4% and 29% , respectively .
this study showed that nac was superior to hydration alone which was statistically significant . comparing various studies regarding n - acetyl cysteine in the prevention of contrast - induced nephropathy tepel et al .
prospectively studied 83 patients with chronic renal insufficiency with mean serum creatinine 2.4 1.3 mg / dl .
patients were randomly assigned to receive either nac and 0.45% ns or placebo with saline .
incidence of cin in nac + ns group was 2% , 21% placebo and saline group , which were statistically significant .
showed that intravenous hydration with saline and nac is an effective and low - cost tool in preventing cin in patients undergoing coronary artery angiography .
showed that intravenous and oral nac may prevent contrast nephropathy with a dose - dependent effect .
durham et al . conducted a randomized controlled trial of nac to prevent contrast nephropathy in cardiac angiography .
cin developed in 26.3% of nac group and 22% of placebo group ( hydration alone ) .
this study shows that nac was not effective for the prevention of cin after cardiac angiography ( p = 0.09 ) .
showed that in patients with chronic renal insufficiency , nac or fenoldopam offered no additional benefit over hydration with saline in preventing cin . in our study ,
age , male sex , history of hypertension , hemoglobin < 12 g / dl , diabetes mellitus , preprocedural systolic and diastolic blood pressure ( sbp and dbp ) , volume of contrast and serum albumin were were not significantly associated with cin .
cin occurred in 15.1% of hyperuricemic group and 2.9% of the normouricemic group ( p < 0.001 ) ; hypoalbuminemia and age > 70 years were other risk indicators . in our study , the incidence of cin in hyperuricemia group was 35% and 19% in normouricemic group .
cin incidence was high in our study due to high baseline serum creatinine and > 0.3 mg / dl increase being taken as cin .
toprak et al . in 2006 showed that patients with diabetes are at high risk of developing cin .
cin occurred in 20% of the dm patients and 11.4% of the pre - dm , and 5.5% of normal group . in our study ,
but there was no statistical difference between diabetics and nondiabetics developing cin may be due to other risk factors influencing cin .
showed that the risk of cin is minimal in patients receiving < 100 ml contrast volume .
klein et al . showed that in the diabetic population , cin developed every fifth , fourth , and second patient who received 200400 , 400600 , > 600 ml contrast study , respectively . in our study
, no significant association was found between patients who received contrast volume > 100 ml and cin .
the probable reason could be the mean contrast volume used in our study was low , around 70 ml .
was observed only for 48 h. in some patients , there could have been late rise in serum creatinine which were probably missed .
the type of acute renal failure ( ischemic , nephrotoxic , or atheroembolic ) could not be determined precisely as renal biopsies were not performed .
because of the nature of the patients and the procedures being performed , acute rise in serum creatinine was assumed to be multifactorial .
we concluded that there was no significant difference between nac and allopurinol in the prevention of contrast nephropathy in patients with impaired renal function .
| contrast media administration can lead to acute deterioration in renal function particularly in patients with pre - existing chronic kidney disease .
this prospective , randomized controlled open - label parallel group study was undertaken at nizam 's institute of medical sciences , hyderabad , from june to december 2015 .
a total of 95 patients were included , of which 35 received n - acetylcysteine ( nac ) + normal saline ( ns ) , 30 patients received allopurinol ( all ) + ns , and 30 patients received placebo . in our study , the overall incidence of cin was 24% .
incidence of cin in nac + ns , all + ns , and placebo group were 20% , 16% , and 36% , respectively .
the major finding of this study was there was no significant difference between nac and allopurinol in the prevention of contrast nephropathy .
however , only allopurinol was superior to placebo . in our study ,
hyperuricemia and baseline serum creatinine were the only risk factors associated with cin . |
h2o2 , an endogenous oxidant present in all aerobic cells , was traditionally viewed as a toxic substance ( winterbourn , 2008 ) .
however , research during the last two decades built a compelling case supporting the role of h2o2 as a diffusible signaling messenger that controls many cellular processes by modulating the redox state of cysteine residues in proteins ( ying et al . , 2007 ) .
accordingly , disruption of signaling pathways mediated by h2o2 and other oxidants would be a mechanism underlying diseases ( jones , 2006 ) .
h2o2 occurs in aerobic cells typically at steady state concentration in the 10 nm range ; and nadph oxidases and/or complex iii of the mitochondrial respiratory chain are frequently considered as the major sources , whereas catalase and multiple cys - based peroxidases exert a fine control of its levels ( reviewed by marinho et al . , 2014 ; sies , 2014 )
. h2o2 are neutral molecules that can cross biological membranes , although its diffusivity can be modulated by the lipid composition of biological membranes ( branco et al . , 2004 ; pedroso et al . , 2009 ) and aquaporins
a challenge in the field is the elucidation of the molecular mechanisms by which h2o2 is sensed and how this signal is transduced in cells .
furthermore , several chemical and biological aspects appear to be contradictory ( winterbourn , 2008 ) .
elucidation of these aspects is very relevant since redox regulated proteins are implicated in diverse pathologies . for instance , redox medicine is emerging as an innovative field supported by molecular evidence showing deregulated redox signaling pathways dependent on h2o2 to be at the basis of several pathologies , including obesity ( kaszubska et al . , 2002 ) and cardiovascular diseases ( schrder et al . , 2012 ) .
furthermore , peroxiredoxins ( prxs ) and its partner proteins , thioredoxins ( trxs ) , have been associated to cancer ( irwin et al . , 2013 ) ,
cardiovascular ( ahsan et al . , 2009 ) , and metabolic diseases ( abbasi et al . ,
2014 ) . in principle , understanding the mechanisms of h2o2 sensing and signal transduction may provide the basis for therapeutic interventions in redox - related diseases ( harald et al . , 2015 ; jones , 2006 ) .
prxs are highly abundant cys - based peroxidases with crucial roles in maintaining genome stability , protecting cells against cancer and promoting longevity ( nystrm et al . , 2012 ; rhee and woo , 2011 ) .
prxs are subdivided into subfamilies that present very divergent amino acid sequences but uniformly possess at least one conserved cysteine residue at the active site , the so - called peroxidatic cysteine , cp ( nelson et al . , 2011 ) .
reaction of cp with h2o2 is very fast , with second - order rate constants ranging from 10 to 10 ms ( ogusucu et al .
, 2007 ; parsonage et al . , 2005 ; peskin et al . , 2007 ; toledo et al . , 2011 ; trujillo et al . ,
this represents an extraordinary catalytic power , since free cys reacts with hydroperoxides with a rate constant equivalent to 10 ms ( winterbourn and metodiewa , 1999 ) .
remarkably for a protein involved in signal transduction , prx rapidly reacts with hydroperoxides , but not with other electrophiles such as iodoacetamides or chloroamines ( peskin et al . , 2007 ) .
high reactivity and specificity are related to the fact that prxs stabilize the transition state of a sn2 type of nucleophilic substitution reaction through the activation of not only cp , but also h2o2 ( hall et al . , 2011 ) .
besides reactivity and specificity , prxs are abundant proteins so they present several features appropriate for a sensor protein .
aspects related to how the oxidation signal is transmitted to other proteins are less understood .
it is well known that most prxs transmit oxidizing equivalents to trxs , especially in the case of typical 2-cys prx , which are prxs with intermolecular disulfide bonds ( in the context of this proposal , we refer to typical 2-cys prx as the proteins that belong to the ahpc / prx1 subgroup , according to nelson et al . , 2011 ) .
for some typical 2-cys prx , dual function ( moonlight property ) was reported as thiol peroxidase and chaperone ( holdase ) ( jang et al .
, 2004 ; rhee and woo , 2011 ) . in principle , prxs may mediate h2o2 signaling through three mechanisms that are not mutually exclusive ( fig .
1a ) : ( 1 , blue ) h2o2 reacts directly with the target signaling protein ( sp ) , and prxs control this process by regulating h2o2 tone in cells ; ( 2 , green ) h2o2 reacts with a prx which is oxidized and then relays this oxidation to the target signaling protein ( sp , for example a transcription factor or a phosphatase ) ; and ( 3 , orange ) h2o2 reacts with a prx which is oxidized , then this prx is reduced back by its partner thioredoxin ( trx ) , and it is the oxidized trx that relays the oxidation to the target sp .
some variations occur when prx enzymes are hyperoxidized , as will be further described below .
for instance , mechanism ( 1 ) also encompasses the so - called floodgate hypothesis ( rhee and woo , 2011 ; wood et al . , 2003 ) .
in this case , when h2o2 accumulates at high levels , cp could be hyperoxidized to sulfinic or sulfonic states ( fig .
adding more complexity to these mechanisms , it was found recently that glutaredoxin 1 ( grx 1 ) can also sustain the peroxidase activity of prxs by reducing the glutathionylated form of prx formed in the presence of h2o2 and glutathione ( gsh ) ( peskin et al .
1 , there are evidences supporting them , and most probably all three mechanism operate for different signal transduction proteins and probably depend on several conditions such as second - order rate constants , ph , as well as concentration of oxidants , reductants , sp and prx , among other factors . in principle , these three mechanistic models are not mutually exclusive , but obviously they should agree with chemical and biological data .
reactivities of h2o2 towards sp , like the phosphatases cdc25b , shp-2 and protein tyrosine phosphatase 1b ( ptb1b ) , are five to seven orders of magnitude lower than the reactivity towards prxs or glutathione peroxidases ( gpxs ) ( winterbourn and hampton , 2008 ) .
in addition , prxs are much more abundant than sp like phosphatases or transcription factors .
noteworthy , other thiol proteins than prxs and gpxs can also react faster with h2o2 than ordinary cys residues present in most sp . two cases deserve special attention here : glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) and oxyr .
oxyr is a transcription factor with highly reactive cys residues capable to react with h2o2 with a second order rate constant of 10 m s ( aslund et al . , 1999 ;
, oxyr is considered the h2o2 sensor in bacteria ( reviewed by marinho et al . , 2014 ) .
gapdh can be reversible inhibited by h2o2 at more moderate rates ( 500 m s ) ( little and obrien , 1969 ; winterbourn and hampton , 2008 ; peralta et al . , 2015 ) , still more reactive than free cys ; and gapdh
furthermore , most cells contain high amounts of gsh that can also outcompete for h2o2 ( winterbourne and hampton , 2008 ) .
therefore , there are multiple competitive routes that can detour h2o2 from the direct reaction toward ordinary thiols present in most sp .
however , sp like ptp1b and pten , which display low reactivity towards h2o2 ( denu and tanner , 1998 ) , are found oxidized in biological tissues ( kwon et al . , 2004 ; lee et al . , 1998 ;
therefore , attempts were made to reconcile both biological data ( showing that cys - based proteins are oxidized in cellular systems ) and chemical information ( low reactivity and low abundance of these cys - based proteins ) .
the so- called floodgate hypothesis is one of the most popular models ( rhee and woo , 2011 ; wood et al . , 2003 )
that could be considered a variation to pathway 1 , and it is schematically represented in ( fig .
first observation that gave support to this hypothesis , was that typical 2-cys prx can be divided into sensitive and robust enzymes and that this property is related with a gain of function ( wood et al . , 2003 ) .
sensitive prx are enzymes that are more easily hyperoxidized to sulfinic acids ( cp - so2h ) , inactivating these peroxidases and allowing the accumulation of h2o2 in cells . at higher levels , h2o2 would be able to attack other targets that in basal conditions would be outcompeted by sensitive prx .
the build - up of h2o2 is possible because sulfinic acid is not reducible by standard reductants such as trx and grx .
the identification of an enzyme capable to specifically reduce typical 2-cys prx sulfinic acid at the cp also supported the signaling role of their oxidative inactivation ( biteau et al . , 2003 ) .
besides hyperoxidation of human prx2 , phosphorylation of prx1 also leads to its inactivation ( rawat et al . , 2013 ;
woo et al . , 2010 ) , proving an alternative pathway for h2o2 accumulation inside cells .
a problem with this hypothesis is that even in the absence of highly reactive peroxidases , cells still have high amounts of gsh ( mm levels ) and gapdh that on chemical grounds can outcompete ptp-1b and pten for h2o2 ( winterbourne and hampton , 2008 ) .
another attempt to reconcile the low reactivity of h2o2 with sp and redox signaling is based on an extended kinetic analysis that focused not only on the routes of h2o2 elimination but also on the transmission of the oxidation signal .
the key notion is whether the fraction of h2o2 that escapes the action of prxs , glutathione peroxidases and also gsh is sufficient to alter the oxidation state of low - reactive sp , thus transmitting the oxidation signal to these target proteins ( marinho et al . , 2014 ) .
the h2o2 concentration needed to oxidized 50% of these targets within 5 minutes and in competition with all cellular h2o2-removing systems was estimated in the range 14 to 120 m for cdc25b and ptp1b , respectively ( marinho et al .
these h2o2 concentrations are much higher than the sub - micromolar concentrations usually assumed to occur intracellularly , but they may be reached in the vicinity of sp during signaling dependent on the local activation of nadph oxidases ( chen , 2008 ; mishina , 2011 ; paulsen , 2012 ) .
this analysis was extended by fitting ptp1b and shp-2 oxidation profiles obtained experimentally to a mathematical model that included not only the oxidation component of redox signaling , but also the reductive part , in which target sps are reduced back , switching - off the oxidation signal , thus taken into account all possible cellular biochemical reactions .
the results obtained suggest that while for ptp1b the redox signaling is compatible with a direct reaction between h2o2 and ptp1b , for shp-2 an alternative mechanism is more likely ( brito and antunes , 2014 ) .
1 , blue pathway ) may operate in vivo under some conditions , namely under relative high local h2o2 concentrations , even if sp display low reactivity towards h2o2 .
most of the knowledge related to pathway 2 ( fig . 1a , green pathway )
was initially gained through studies on the activation mechanism of yap1 transcription factors from saccharomyces cerevisiae ( boronat et al . , 2014 ) . like oxyr ,
however , apparently cys residues of yap1 are not highly reactive and therefore yap1 does not react directly with h2o2 , but instead undergo oxidative activation through thiol - disulfide reshuffling mediated by a cys - based peroxidase : glutathione peroxidase 3 ( gpx3 ) , also called oxidant receptor protein ( orp1 ) ( delaunay et al . , 2002 ) . in this signaling pathway ,
h2o2 oxidizes gpx3/orp1 into a sulfenic acid ( cys - soh ) that then condenses with a cys residue of yap1 , giving rise to a transient , mixed disulfide between the two proteins . through thiol
this oxidized form of yap1 has an altered structure that can not leave the nucleus .
the nuclear accumulation of yap1 facilitates its ability to induce the transcription of target genes , such as prx and catalase genes ( delaunay et al . , 2002 ) .
ybp1 is a third protein involved in this pathway that physically interacts with yap1 and thereby stimulates nuclear accumulation of this transcription factor in response to h2o2 ( veal et al . ,
interestingly , in strains where ybp1 gene is mutated , tsa1 ( by far , the most abundant cys - based peroxidase in yeast ) and not gpx3/orp1 is the protein that senses h2o2 and activates yap1 through thiol - disulfide exchange reactions ( tachibana et al . , 2009 ) .
these studies represented a breakthrough in the field , since they provide a pathway to redox regulate a sp presenting low - reactive thiols through a protein oxidation relay by a cys - based peroxidase .
therefore , in contrast to bacteria , cys - based peroxidases and not transcription factors are the major sensors of h2o2 in yeast .
indeed , a yeast strain deleted for the five prx genes and three gpx genes is still viable , but lost its capacity to transcriptionally respond to h2o2 stimuli ( fomenko et al . , 2011 ) , suggesting that green pathway ( fig .
1a ) is preponderant in the adaptive response of saccharomyces cerevisiae to h2o2 . according to this mechanism ,
h2o2 signaling is mediated through sequential transfers of oxidizing equivalents . upon oxidation , all prx enzymes are oxidized to sulfenic acids ( cys - soh ) that in most cases can be converted to intra- or inter - molecular disulfides .
oxidized prxs ( cys - soh or cys - ss - cys ) probably selectively transfers oxidizing equivalents to downstream regulatory proteins through specific protein - protein interactions and thiol - disulfide exchange reactions ( gutscher et al . , 2009
so far , the number of cases in which a high - reactive peroxidase was found to selectively transfer oxidizing equivalents to a sp is relatively low , but the descriptions of new cases are increasing .
remarkably , prx1 from mammals was shown to physically interact in a redox dependent manner with sp such as : ( 1 ) pten , a protein with phosphatase activity ( cao et al . , 2009 ) ;
( 2 ) apoptosis signaling kinase1 ( ask1 ) ( jarvis et al . , 2012 ) ; and ( 3 ) mapk phosphatases ( turner - ivey et al . , 2013 ) .
recently , the activation of a transcription factor ( stat3 ) by mammalian peroxiredoxin2 ( prx2 ) was also reported and present several features similar to the gpx3/orp1 yap1 pathway ( sobotta et al . , 2015 ) .
in all cases , the transfer of oxidizing equivalents involves physical interaction and thiol - disulfide exchange between the two partners .
a lot of attention has been centered on h2o2 in the signaling pathways that prx take place .
in contrast , the roles play by prx enzymes in regulating the redox state of its other substrate ( trx ) has been somehow overlooked .
this is surprising , since it is well established for a long time that several signal transduction pathways are activated by the oxidized but not by the reduced form of trx ( reviewed by berndt et al . , 2007 ) .
for instance , only reduced trx1 and trx2 bind ask-1 , thereby inhibiting its kinase activity .
the oxidation of trx1 leads to the physical dissociation of the trx1-ask1 complex and , consequently , to the activation of ask-1 ( saitoh et al . , 1998 ) .
the binding of nf-b subunit p50 to its target dna sequence requires the reduction of a single cysteinyl residue by trx1 ( hayashi et al . , 1993 ; matthews et al . , 1992 )
indeed , trx enzymes recognize the oxidized form of their target proteins with higher selectivity , than their corresponding reduced forms ( palde and carroll , 2015 ) .
it should be referred that trx itself reacts slowly with h2o2 ( chae et al . ,
therefore , typical 2-cys prxs that catalyze the oxidation of trx by hydroperoxides ( netto et al . , 1996 ; tairum et al . ,
therefore , another model can be envisaged , in which the redox pathway between h2o2 and the sp would be mediated not only by prx , but also by trx ( fig .
according to this prx - trx signaling pathway , the oxidizing equivalent from h2o2 would be transferred to prx and then to trx and finally to the sp .
alternatively , prx could oxidize trx , and thereby avoid the interaction of this thiol - disulfide oxidoreductase with a sp . more examples of redox - regulation by prx - trx pathway came from studies using fission yeast . in these organisms , two transcription factors have been found to be redox regulated by h2o2 : pap1 ( brown et al . ,
2013 ; calvo et al . , 2013 ) and msn2 ( boisnard et al . , 2009 ) .
relevant for the adaptive response of fission yeast to h2o2 , the formation of 2-cys prx disulfide triggers a signaling cascade that activates the otherwise unresponsive pap1 protein , but only when trx enzymes become transiently depleted ( brown et al .
2013 ) . as described for the model presented in the section 2.2 ( green pathway , fig .
1 ) , a feature underlying these pathways is the specificity provided by protein - protein interactions and thiol - disulfide exchange reactions .
proteins and their corresponding partners require to share complementary physico - chemical properties ( i.e. hydrophobicity , polarity ) in order to interact . as a matter of fact
, yeast trx reductase can only reduce yeast but not bacterial or mammalian trx , due to complementary properties ( oliveira et al . ,
noteworthy , a protuberance ( containing residues glu50 , arg146 and cys170 of yeast tsa1 ) present only in the oxidized form of 2-cys prx is involved in its recognition by yeast trx1 ( tairum et al . ,
therefore , physical proximity between proteins is a requirement to allow the transfer of oxidizing equivalents through pathways 2 and 3 .
the construction of artificial quimeras between cys - based proteins and redox sensitive gfp , gutscher et al .
( 2009 ) elegantly demonstrated the proximity - based protein thiol oxidation by h2o2-scavenging peroxidases concept that also form the basis of several genetically encoded fluorescent probes .
this prx - trx model also applies in conditions where cells are exposed to high levels of h2o2 , provoking prx hyperoxidation ( fig .
because trxr ( thioredoxin reductase ) levels are limiting , several proteins ( including disulfide form of 2-cys prx ; transcription factors and methionine sulfoxide reductase ) compete for reduced trx1 .
for instance , the hyperoxidation of tpx1 ( 2-cys prx from schizosaccharomyces pombe ) allowed accumulation of reduced trx ( since this oxidoreductase is not consumed by prx in the disulfide form ) that could be then used to repair other damaged proteins ( day et al . , 2012 ) .
in these conditions , the activity of enzymes such as methionine sulfoxide reductase and ribonucleotide reductase would not be limited by shortage of reduced trx . remarkably , when tpx1 is hyperoxidized , pap1 ( a transcription factor ) is no longer activated possibly because there is enough trx to keep this transcription factor in the reduced state and consequently out of the nucleus ( reviewed by veal et al . , 2014 ) .
other observations that supported the notion that prx participates in redox signaling by regulating the trx status were gained by studies with saccharomyces cerevisiae .
for instance , mutations that destroy cytosolic trxr activity rescue the growth of tsa1 rad51 double mutant and also suppress the genomic instability phenotype of tsa1 mutants ( ragu et al . , 2014 ) .
furthermore , a mutant of s. cerevisiae for cytosolic trxr suppressed the growth defect of tsa1 mutants in zinc - deficient conditions ( macdiarmid et al . , 2013 ) .
these studies are consistent with the idea that deletion of prx genes decreases the rates of trx oxidation , whereas deletion of trxr genes decreases rates of trx reduction and thereby compensates the absence of cys - based peroxidase by keeping the trx redox state similar to the levels observed in the wild type strain .
these studies suggest that the main role of prx enzymes at least in some cases may be regulating trx redox levels , rather than reducing hydroperoxides ( veal et al . , 2014 ) .
probably the redox status of trx is probably not affected by 2-cys prx peroxidase activity in bacteria , because in these microorganism there is a reducing system ( ahpf ) that is specifically dedicated to reduce this cys - based peroxidase ( veal et al . , 2014 ) .
this phenomenon is probably related to the fact that most 2-cys prx in bacteria are robust , meaning that they resist to hyperoxidation , even when exposed to high h2o2 levels .
in contrast , in eukaryotes , a selection pressure towards the evolution of 2-cys prxs that are sensitive to inactivation by h2o2 arose and according to this model could be involved to maintain trx in the reduced form , available to mediate repair damaged proteins under acute stress conditions ( veal et al . ,
2014 ) . not many examples of the prx - trx model are available in mammals .
but noteworthy , hstrx1 ( thioredoxin 1 from humans ) contains five cys residues : cys32 and cys35 are the two residues that belong to the conserved cgpc motif , whereas cys62 , cys69 and cys73 are three non - conserved residues .
hsprx1 ( 2-cys prx from homo sapiens ) specifically oxidizes cys32 and cys35 into an intramolecular disulfide .
in contrast , cys62 and cys69 is an additional dithiol - disulfide redox center . when a disulfide is formed between cys62 and cys69 , the ability of hstrx1 to bind a target is impaired ( watson et al .
indeed , cys62-cys69 non - active site disulfide is not a substrate for hstrxr1 , but to the gsh / hsgrx1 system , whereas the cys62-cys69 dithiol can be oxidized by hsprx1 ( du et al . , 2013 ) .
these multiple oxidative states of hstrx1 involving its five cys residues play a role in the fine tune of the redox regulation of sps such as ask1 ( kinases ) , pten ( phosphatases ) and ref-1 ( transcription factor ) in redox pathways that 2-cys prx enzymes also take place ( reviewed by lee et al .
a long way has come since h2o2 was seen as a toxicant , and today thiol switches are established cellular partners in signaling networks .
although the identification of the thiol redox proteome is advancing quickly , there is a massive lack of quantification , namely kinetic parameters .
these parameters entail dynamic information that helps to identify in the complex network of thiol switches , those that operate during physiology and those whose disruption is responsible for a pathological state .
the reactivity with h2o2 is known for only a few thiol sp ( ferrer - sueta et al . , 2011 ; tanner et al . , 2011 ) ;
rate constants for the thiol - disulfide exchange reactions in which a peroxidase transfers oxidizing equivalents to a sp are virtually unknown ; the reduction of the oxidized form of thiol sps , switching - off the oxidation signal ( dagnell et al . , 2013 ) , is known only for a couple of interactions ( parsons and gates , 2013 ) . determining true rate constants for these processes
entails experiments with purified systems that are complex to preform and can not cope with the rapid identification of new thiol switches provided by large - scale thiol proteomic experiments . as an alternative , the determination of apparent kinetic parameters under the typical redox experiments with intact cells or tissues ( brito and antunes , 2014 ) may provide a more practical approach so that quantitative knowledge can follow suit the rapidly expanding redox proteome landscape , establishing the basis for redox kinectomics . | a challenge in the redox field is the elucidation of the molecular mechanisms , by which h2o2 mediates signal transduction in cells .
this is relevant since redox pathways are disturbed in some pathologies .
the transcription factor oxyr is the h2o2 sensor in bacteria , whereas cys - based peroxidases are involved in the perception of this oxidant in eukaryotic cells .
three possible mechanisms may be involved in h2o2 signaling that are not mutually exclusive . in the simplest pathway ,
h2o2 signals through direct oxidation of the signaling protein , such as a phosphatase or a transcription factor .
although signaling proteins are frequently observed in the oxidized state in biological systems , in most cases their direct oxidation by h2o2 is too slow ( 101 m1s1 range ) to outcompete cys - based peroxidases and glutathione . in some particular cellular compartments ( such as vicinity of nadph oxidases ) , it is possible that a signaling protein faces extremely high h2o2 concentrations , making the direct oxidation feasible .
alternatively , high h2o2 levels can hyperoxidize peroxiredoxins leading to local building up of h2o2 that then could oxidize a signaling protein ( floodgate hypothesis ) . in a second model ,
h2o2 oxidizes cys - based peroxidases that then through thiol - disulfide reshuffling would transmit the oxidized equivalents to the signaling protein .
the third model of signaling is centered on the reducing substrate of cys - based peroxidases that in most cases is thioredoxin .
is this model , peroxiredoxins would signal by modulating the thioredoxin redox status .
more kinetic data is required to allow the identification of the complex network of thiol switches . |
a 33-year - old woman was admitted to our hospital with a two - month history of abdominal distension and edema . the patient had no prior history of drug usage , including oral contraceptives , and no relevant family medical history . upon admission ,
the patient underwent a physical examination , which revealed diffuse hepatomegaly , pitting edema in both legs , however no abnormal cardiac murmur .
the laboratory findings indicated abnormalities including a hemoglobin level of 7.5 g / dl , a platelet count of 18,000/mm , as well as a slightly elevated serum alanine aminotransferase ( 50 u / i ) and aspartate aminotransferase ( 56 u / i ) .
moreover , the hepatitis b virus antigen was negative , and the titer of the hepatitis b virus antibody and alpha - fetoprotein were normal .
the clinical and radiological evaluation showed no evidence of rendu - osler - weber disease or hemangiomas in the extrahepatic region .
an ultrasonography revealed a diffuse heterogeneous echoic infiltrative mass containing numerous and various - sized hypoechoic nodules involving the entire liver ( fig . 1a ) .
a doppler ultrasonography revealed no remarkable tumor vascularities except for the underlying hepatic parenchymal vascular flow ( fig .
a hepatic dynamic ct was performed and demonstrated the multifocal enhancing hepatic nodules on both the arterial and portal phases , as well as an increased area of contrast enhancement with a centripetal filling pattern of nearly the entire hepatic tumor on a delayed phase ct ( fig .
however , there was no remarkable tumor calcification or intervening normal hepatic parenchyma in either of the hepatic lobes .
subsequently , a 99mtc - labeled red blood cell scan was performed and the blood pool images were taken at five hours after inhomogeneous diffuse activity in the entire hepatic mass ( fig . 1 g ) .
however , no extrahepatic mass with abnormally increased radioactivity was observed on this 99mtc - labeled red blood cell scan .
our differential diagnoses included a diffuse hemangiomatosis , epithelioid hemangioendothelioma , and angiosarcoma . to rule out a hepatic angiosarcoma ,
an ultrasound - guided automated gun biopsy , using an 18-gauge core needle was performed in the right hepatic lobe . before the biopsy , the patient 's platelet count was 170,000/mm after infusion of fresh frozen plasma .
the patient 's histology revealed prominent cavernous vascular proliferation and fibrosis without angiosarcomatous components ( fig .
further , the patient showed no hemangiomas in other regions of the body , including the skin .
the patient continued to complain about continuous abdominal distension , which rapidly progressed to the deterioration of hepatic function .
it occurs in all age groups , although hepatic hemangiomatosis occurs predominantly in infants and may cause life threatening conditions ( 1 ) . however , diffuse hepatic hemangiomatosis in adults is very rarely encountered and moreso when observed without any other organ involvement ( 2 - 4 ) .
although several cases of long - term adult survival of diffuse neonatal hemangiomatosis have been reported ( 5 , 6 ) , the etiology and natural history of diffuse hepatic hemangiomatosis remains unclear .
the histological characteristics of hemangiomatosis includes the presence of large vascular channels in both the normal - appearing hepatic parenchyma and the cavernous tumor region ( 2 ) .
the histological findings of our ultrasound - guided biopsy specimen revealed prominently increased vascular proliferation and fibrosis with no sarcomatous change on staining ( fig .
1h ) , as well as vascular endothelial cells showing positive staining on immunochemical stains , which included masson 's trichrome stain and the gomori reticulin stain .
as no reliable clinical or radiological findings have been reported for the differential diagnosis of hepatic hemangiomatosis from rare primary vascular hepatic tumors such as hepatic hemangioendothelioma or angiosarcoma , the diagnosis relies solely on the histological findings ( 7 ) . also , because it is so rare , only a few reports document the imaging findings of diffuse hepatic hemangiomatosis .
however , these imaging findings are similar to those of other common hepatic hemangiomas , including variable echoic lesions on the ultrasound , vascular pooling of contrast material within the tumor on angiography , a centripetal enhancement pattern on dynamic enhancement studies , and bright tumor signal intensities on t2-weighted mr images ( 2 , 3 , 8) . in our patient ,
the ultrasonography findings included a heterogeneous echoic infiltrative hepatic mass containing numerous hypoechoic nodules . compared to the histological findings
our case showed a delayed centripetal contrast - enhancement pattern of the tumor on a dynamic ct and diffuse uptake on vascular pooling images from a 99mtc - labeled red blood cell scan . because our case revealed imaging findings similar to hepatic hemangiomas , epithelioid hemangioendotheliomas , and angiosarcomas , diffuse hepatic hemangiomatosis
although previous reports have cited the role of steroid medications in the development of hepatic cavernous hemangiomas ( 10 ) and the administration of metoclopramide medications in a patient with diffuse hepatic hemangiomatosis ( 8) , no history of steroid or estrogen use was documented in the reported cases of diffuse hepatic hemangiomatosis , including our patient .
the prognosis of diffuse hepatic hemangiomatosis without extrahepatic involvement is still unclear because it occurs so rarely and has variable clinical courses in affected patients . in the literature , various prognoses have been reported for diffuse hepatic hemangiomatosis without extrahepatic lesion in adults ( 2 , 3 , 8 , 11 ) . in our patient ,
the possibility exists that a minimal amount of the remaining normal hepatic parenchyma with extensive hepatic hemangiomatosis involving the entire liver may have caused hepatic failure and subsequent patient death .
more cases of diffuse hepatic hemangiomatosis are needed to establish their natural course and prognosis . despite its rarity
, we concluded that the diagnosis of diffuse hepatic hemangiomatosis in adults can be suggested in patients with a diffusely involved hepatic tumor showing radiologic findings of hepatic hemangioma . | we report an extremely rare case of a diffuse hepatic hemangiomatosis without extrahepatic involvement in an adult .
the imaging findings of this tumor were similar to those of a hepatic hemangioma and included contrast enhancement with a centripetal filling pattern of the entire hepatic tumor on the delayed phase of a dynamic ct and inhomogeneous diffuse uptake of the entire tumor on blood - pool images obtained five hours later on a 99mtc - labeled red blood cell scan . despite its rarity
, diffuse hepatic hemangiomatosis can be suggested in adult patients with diffusely involved hepatic tumors showing the radiological findings of a hepatic hemangioma . |
ectopic neurohypophysis may be accompanied by midline brain defects and some degree of endocrine disorders .
the associated endocrine disease varies from isolated growth hormone ( gh ) deficiency to multiple anterior pituitary hormone deficiencies , but posterior pituitary function remains unchanged .
anterior pituitary hormone deficiencies may present with varied clinical manifestations like growth failure , central hypothyroidism , or underdeveloped secondary sexual characters . at the same time , the disease starting with gh deficiency can progress to pan hypopituitarism with time .
neurohypophysis is observed as a bright dot behind normal posterior lobe of adenohypophysis on t1-weighted magnetic resonance ( mri ) images .
ectopic neurohypophysis is specifically monitored at the level of the median eminence of the hypothalamus in imaging studies .
because of its rareness , we present radiological findings of a case that showed coexistence of ectopic neurohypophysis , corpus callosum dysgenesis , and periventricular neuronal heterotopia , with accompanying literature review .
a 21-year - old female patient was admitted to the neurology clinic of our hospital with complaints of forgetfulness , twitching of eyes , and short stature .
laboratory measurements for the pituitary hormones uncovered a very low level of gh ( 0.06 ng / ml ) , while the other hormones and their levels were normal [ table 1 ] . in mr images of the brain and pituitary ( 1.5-t system , ge medical systems signa , milwaukee , wi )
, the neurohypophysis was seen at the ectopic site as a bright dot at the level of the optic chiasm on coronal t1 images .
infundibular part was thinner than usual [ figure 1 ] . at the same time , partial dysgenesis of corpus callosum and periventricular neuronal heterotopia were observed on the mr images [ figures 2 and 3 ] .
ophthalmologic examinations for a possible involvement of the optic discs , including visual acuity test , visual field test , and dilated fundoscopy were completely normal . also , the systemic evaluation of the patient for other possible associations was normal .
serum levels of anterior pituitary hormones in the patient 21-year - old female patient with complaints of forgetfulness , twitching of eyes , and short stature later diagnosed with coexistence of ectopic neurohypophysis , corpus callosum dysgenesis , and periventricular neuronal heterotopia .
a coronal non - contrast t1-weighted mri image reveals a hyperintense ectopic posterior pituitary adjacent to the optic chiasm , at the level of the median eminence of the hypothalamus ( arrows ) .
21-year - old female patient with complaints of forgetfulness , twitching of eyes , and short stature later diagnosed with coexistence of ectopic neurohypophysis , corpus callosum dysgenesis , and periventricular neuronal heterotopia .
sagttal t2-weighted mri image reveals partial dysgenetic view of the corpus callosum and protrusion of cingulated gyrus ( arrow ) .
21-year - old female patient with complaints of forgetfulness , twitching of eyes , and short stature later diagnosed with coexistence of ectopic neurohypophysis , corpus callosum dysgenesis , and periventricular neuronal heterotopia .
axal t2-weighted mri image reveals an appearance that is compatible with periventricular - subependymal heterotopic neuronal tissue ( arrows ) .
the neurohypophysis comprises the posterior pituitary lobe , the infundibulum , and the median eminence of the hypothalamus .
the neurohypophysis is formed by the evagination of neural tissue from the floor of the third ventricle .
it consists of the distal axons of the hypothalamic magnocellular neurones that shape the neurohypophysis .
after its downward migration , it is encapsulated together with the ascending ectodermal cells of rathke 's pouch , which form the adenohypophysis .
the pituitary gland acts as an endocrine gland under control of the hypothalamus via the infundibular stalk .
regardless of the underlying cause , the ectopic neurohypophysis results from incomplete downward growth and fusion of the neurohypophysis with the adenohypophysis in the sella .
possible pathophysiologic mechanisms for ectopic neurohypophysis development are transection of the pituitary stalk followed by hypertrophy of the proximal axons with subsequent reorganization . also trauma of the pituitary stalk due to vascular accidents , anoxia , or compression injuries have been suggested as acting through similar mechanisms leading to reorganization of the proximal neurons of the neurohypophysis .
although early studies describe a traumatic cause in the etiology of the ectopic neurohypophysis , more recent studies favor a genetic basis , supported by rare familial cases of ectopic neurohypophysis .
the ectopic neurohypophysis cases have been reported in idiopathic gh deficiency or in subjects with hesx1 , lhx4 , and sox3 gene mutations .
again , a few earlier reports have suggested that ectopic neurohypophysis may share a similar pathogenesis as septo - optic dysplasia , which classically has features of optic nerve hypoplasia , hypothalamic - pituitary dysfunction , and agenesis of the septum pellucidum .
the other well - known malformations associated with ectopic neurohypophysis include chiari 1 malformation , agenesis of the corpus callosum , persistent cranio - pharyngeal canal , kallmann syndrome , basilar impression , medial deviation of the carotid arteries , microcephaly , cerebellar atrophy , and vermian dysplasia . associated ophthalmic and midline facial abnormalities , such as a single central incisor tooth , are typical , and non - midline somatic conditions , including cardiac and musculoskeletal abnormalities ,
breech presentation and neonatal hypoglycemia are also commonly reported . as a result of ectopic neurohypophysis and associated stalk abnormalities , factors released by the hypothalamus , which usually travel down the portal circulation to the anterior pituitary
the patients with an ectopic neurohypophysis located at the median eminence and no visible pituitary stalk present a more severe form of the disease , with multiple anterior hormone deficiencies . whereas visibility of the pituitary stalk and an ectopic neurohypophysis located at some point along the stalk is associated with isolated gh deficiency . in our patient , there were radiological findings of coexistence of ectopic neurohypophysis , corpus callosum partial dysgenesis , and periventricular neuronal heterotopia .
dysgenesis of the corpus callosum represents an in utero developmental anomaly , which may be complete ( agenesis ) or partial .
it may occur in isolation or in association with other central nervous system or systemic malformations .
periventricular heterotopia is the most common form of gray matter heterotopia and is characterized by nodules of gray matter located immediately beneath the ependyma of the lateral ventricles .
previous reports of cases with coexistent periventricular heterotopia and ectopic neurohypophysis suggest both conditions can share a common underlying causative mechanism , even though pituitary development occurs earlier in fetal life than does neuronal migration
. another study has found hesx1 mutations in only 5 of 93 patients with ectopic neurohypophysis , indicating that other unrecognized genes or local environmental factors are likely to be involved in the ectopic neurohypophysis development . in ectopic neurohypophysis cases ,
normal neurohypophysis bright spot is absent in the sella and there is high t1 signal at the median eminence ( floor of third ventricle ) or along infundibuler stalk , instead of normal sellar position , on unenhanced t1-weighted images .
infundibuler stalk may be tiny or absent . although controversy still exists on the source of this distinct signal , it has most recently been attributed to phospholipids within the walls of the secretory vesicles containing the arginine vasopressin neurophysin complex .
distinction of the ectopic neurohypophysis from dermoid tumor or lipoma can be confirmed by images obtained using fat saturation . in this article , because of its rareness , we present mri findings of concurrence of ectopic neurohypophysis , dysgenesis of the corpus callosum , and periventricular neuronal heterotopia .
radiologists should carefully evaluate mr images in patients with epilepsy or hormone deficiency for the possibility of ectopic neurohypophysis and neuronal heterotopia . in the presence of ectopic neurohypophysis | ectopic neurohypophysis is a pituitary gland abnormality , which can accompany growth hormone deficiency associated with dwarfism .
here we present magnetic resonance imaging ( mri ) findings of a rare case of ectopic neurohypophysis , corpus callosum dysgenesis , and periventricular neuronal heterotopia coexisting , with a review of the literature . |
there are an estimated 150,000 surgical amputations per year in the us , most of which are secondary in nature , arising from diabetes mellitus , vascular disease , trauma , and cancer.1 residual pain in amputation stumps can extend beyond the normal recovery time , and is seen in 13%71% of cases.2,3 after the nerve injury , spontaneous nerve activity develops at the site of injury and the corresponding dorsal root ganglia .
these changes are followed by sensitization , reduction of the pain threshold , exaggerated response to nociceptive stimuli ( hyperalgesia ) or non - nociceptive ( alodinea ) , and phantom sensations.4 the syndrome of a phantom limb with associated pain is found in 70%85% of amputees , and pain persists in 5%10% of these people.3,5 sensory abnormalities are also described in more than 50% of patients with amputation stumps.6 in a study of 96 upper limb amputees , schley et al7 found that 44.6% of patients suffered from phantom pain , 53.8% had phantom sensations , 61.5% had stump pain , and 78.5% had stump sensation .
the authors reported that stump pain gradually decreased in 19 ( 47.5% ) of 40 amputees in the first year following amputation , but was stable in 12 ( 30% ) patients , and some may present with severe stump pain that is resistant to treatment .
the residual pain may be due to several factors , such as an underlying disease , bone deformities , wound healing , or neuropathic pain.3 many treatments have been reported for pain in a stump following amputation , and these can be divided into three categories : pharmacological , psychological , and physical .
the first includes the use of anti - inflammatory drugs , opioids , anti - depressants , and anti - convulsion drugs , in addition to the use of injections of steroids , analgesic nerve blocks , and painkiller adhesives.7,8 the second is represented mainly by antidepressant therapy , and the third included transcutaneous electrical stimulation of nerves , spinal electrical stimulation , low - intensity laser therapy , rehabilitation exercises and massage , and surgery.911 the use of low - intensity laser therapy ( lilt ) started in europe and russia in the 1960s . lilt also known as photobiology or biostimulation makes use of low levels of radiation , with the use of a single wavelength.12,13 it is suggested that the biological effect of this therapy is secondary to the direct effects of the light radiation , and not the result of thermal processes.14 clinically , there have been many uses of lilt demonstrated , but most require further studies before conclusions can be drawn .
one study indicated that tensor strength of wounds increased considerably after 12 weeks of laser treatment , with this improvement associated with a significant increase in collagen after 2 weeks of treatment.15 the effects of lilt on the peripheral nervous system are also encouraging . when treated with transcutaneous lilt ,
rats with facial nerve injuries caused by crushing had an increased rate of nerve regeneration.16 in a double - blind controlled study , lilt and transcutaneous electrical stimulation were associated with a significant reduction of subjective pain and improved sensory latency in patients with carpal tunnel syndrome.17 patients with maxillofacial pain including trigeminal neuralgia also reported decreased pain in response to laser therapy.18 it is unclear how lilt promotes an analgesic effect . in the case of neuropathic pain , lilt may affect the release of neurotransmitters such as serotonin,19 increase production of mitochondrial atp,20 increase the release of endorphins,21 and have anti - inflammatory effects.22 in controlled laboratory studies it has been shown that lilt may reduce inflammation by lowering the levels of prostaglandin pge2 and inhibiting cyclo - oxygenase -2 ( cox -2 ) in cell cultures.2326
three patients with neuropathic pain in an amputation stump were selected in the pain clinic of our hospital .
all patients experienced significant neuropathic pain in the form of the sensation of shock or needles on the scar of the amputation stump in all cases , this pain was represented by high scores on the visual analog scale ( vas ) of pain , and the pain persisted despite analgesics or functional rehabilitation .
the patients also had functional limitations in the activities they could perform as a result of this pain .
we selected three patients with higher pain intensity ( vas > 8) , pain that did not respond to medical treatment , homogeneity of previous treatment ( the patients were treated with the same medications and rehabilitation ) , and all three patients had undergone their traumatic amputation over 1 year prior to this study .
the first of these was the pain vas , which consists of a horizontal line ten centimeters long with no pain and pain as bad as it could be marked on the left and right ends of the line , respectively .
the patients were required to make a mark on the line indicating how bad the pain was in their amputation stump , and the distance from the left end to the mark on the scale was measured and recorded as the vas score .
the second assessment was the barthel scale , which aims to quantify the patient s ability to carry out daily living activities such as eating or using a toilet , and ranges from the highest dependency ( 0 points ) to the highest independency ( 100 points).27 the third assessment , the lawton scale , quantifies impairment to the carrying out of daily tasks and ranges from the highest dependency ( 8 points ) to the highest independency ( 24 points).28 after assessment , patients received nine sessions ( three treatment sessions per week for three weeks , as reported by lam and cheing)29 of low - intensity laser ( laser diode , = 830 nm , dose ranging from 815 j / cm , p = 70 mw ) on the amputation stump scar .
a continuous wave diode laser was used ( dmc , so paulo , brazil ) . following treatment ,
the patients were re - evaluated using the three scales described above , and patients were monitored for 4 months .
no side effects were observed in response to the lilt . this project was analyzed and approved by the ethics committee of our institution ( research protocol # 01254/09 ) .
the main objective of the study was to assess whether this treatment would result in pain relief in the amputated stump , expressed by a decrease in vas scores . as a secondary objective of the study ,
the effect of lilt on the improvement of independency and functionality of the patient was also assessed ( this would be expressed by an increase in the barthel and lawton scales ) .
all three patients reported subjective pain relief after nine sessions of low - intensity laser .
the patients had allodynia and hyperpathia prior to treatment , and the lilt treatment produced a verified remission in hyperpathia and significant control of allodynia .
the patients post - treatment reporting of pain through the vas also showed a significant reduction in pain in all three cases , as shown in figure 1 , with pain dropping by five to seven points .
measured on the barthel scale , improvements of 45 to 55 points were seen , and in the lawton scale the improvements ranged from eight to ten points ( figure 2 ) .
the application of low - intensity laser treatment contributed to an improvement in our patients reported pain intensity , their ability to carry out daily living activities ( barthel scale ) , and their impairment in the carrying out of daily tasks ( lawton scale ) .
reported pain was reduced by the lilt treatment , with 50%70% less pain after nine sessions .
this improvement should be interpreted with caution since all of the patients studied had high scores of pain intensity on the vas before treatment , and the same outcome should not be expected in patients who begin treatment with lower intensities of pain .
also , the study group is small , and the results should be seen only as a descriptive study of a new tool . although the exact mechanism is unknown , lilt has been beneficial in many studies that have used a range of different pain models .
generally these studies do not show a good evidence level due to a lack of randomization , doubleblind design , or the use of a placebo group.30 a reduction in pain without the use of analgesic drugs can help reduce the excessive use of medication , reducing the potential side effects of high doses . since no side effects were observed in the use of lilt , its use can probably be started at a very early stage and on patients suffering from less pain .
there are no similar studies presented in the current literature , and thus the results of the current study may not be directly compared to other work . in a meta - analysis of
the use of lilt in patients with low back pain by cochrane,31 laser treatment was beneficial in reducing pain unless patients were subjected to physical exercise as an adjunct treatment .
it is known that pain causes loss of function and decreased independence of the individual , often leading to reduced mobility and increased rates of depression .
the present study shows that lilt can result in an improvement in patient independence , with an average gain of 50% in the barthel scale and 37.5% in the lawton scale , and all improved to the point where they gave themselves the highest scores possible for the scales used in the study .
this indicates that lilt can be used in the future to decrease pain in patients following amputations .
this study s sample is small and no control group was used , which prevents any extrapolation of the results .
the reported results should be seen as a stimulus for randomized studies with larger sample sizes to confirm the effectiveness of lilt in the treatment of chronic pain in patients with amputated limbs . | debilitating stump pain following amputation surgery is a major problem when it affects the patient s quality of life , often making the patient totally dependent on others for their day - to - day care .
attempts have been made to treat those patients through pharmacological , psychological , and physical therapies , but in many cases these fail to relieve the pain .
this article focuses on three patients with chronic , intense , and debilitating stump pain who were previously treated with pain medications , but with little success .
these patients underwent nine sessions of low - intensity laser therapy ( lilt ) to the stump this is a new treatment that has been used to treat other pain disorders .
all patients reported a decrease in the intensity of their pain and increased ability to perform daily living activities during a 4-month follow - up . |
in excess of one billion people are infected with helminths in developing countries , where these diseases cause substantial morbidity and hundreds of thousands of deaths annually ( may , 2007 ; albonico et al . , 2008 ; hotez , 2011 ; bardosh , 2014 )
. helminths also plague livestock in developing and developed countries , with the global anthelmintic market for livestock and companion animals valued at $ us 3.7 billion in 2002 ( evans and chapple , 2002 ) . whereas chemotherapy is available for infection with most parasitic helminths , widespread use of anthelmintics in agriculture
has resulted in the emergence of drug - resistant parasites ( james et al . , 2009 ; molento , 2009 ) .
concern remains about the emergence of drug resistance in human helminth parasites , and as mass drug administration campaigns increase this worrisome prospect becomes more likely ( abdul - ghani et al .
this is due in part to the high cost and limited financial return from drug development , particularly for pathogens of medical importance .
a decade ago , the major component of this cost limitation was the lack of objective high - throughput screening methods for assessing drug effectiveness ( kotze et al . , 2006 ; abdul - ghani et al . , 2009 ; keiser , 2009 ) .
the gold standard for measuring drug effectiveness for helminth parasites was the costly , laborious and subjective in vitro assessment of worm motility , as measured visually via microscopy and larval development assays ( kotze et al .
, 2004 ; abdulla et al . , 2009 ; keiser , 2009 ) .
indeed , there are multiple requests in the peer - reviewed literature for high - throughput screening methods to facilitate drug development and to detect emerging resistance ( morel , 2003 ; sommerfeld and oduola , 2007 ; keiser , 2009 ) . in response
, the tropical diseases research network ( tdr ) of the who ( http://apps.who.int/tdr/ ) developed an international resistance screening network , but due to the limitations of available techniques , screening deployed to date has utilized only low - to medium - throughput approaches ( hopkins et al . , 2007 ; abdulla et al . ,
recently , a range of screening techniques have emerged , often adapted from methods to monitor cultured cells , which allow monitoring of several helminth parasite species and developmental stages by video ( paveley et al . , 2012 ) , impedance ( smout et al . , 2010 ) , enzymatic ( mansour and bickle , 2010 ) , colorimetric ( tritten et al . , 2012 ) , fluorescence ( peak et al . , 2010 ) and other mechanisms ( howe et al . , 2015 ; lalli et al . , 2015 ) .
we have previously adapted the impedance - based real time cell assay the xcelligence system to monitor the viability of a range of human and livestock parasitic helminths ( smout et al . , 2010 ) .
the xcelligence unit conventionally measures cell growth relaying on gold electrodes embedded in the base of tissue culture microplates that monitor changes in conductivity due to contact of the cells with the electrodes ( vistejnova et al .
with many species of parasitic helminths , the wild type phenotype is motile in tissue culture , and the movement of the worm is detected by change in the conductivity on the electrodes .
the xworm approach is simple , objective , high - throughput , relatively inexpensive , and applicable to many species of parasitic worms ( smout et al . , 2010 ) .
the technique has been favorably received in the field for its sensitivity , broad applicability and adaptability ( peak and hoffmann , 2011 ; silbereisen et al . , 2011 ;
, 2012 ; you et al . , 2013 ; zeraik et al . , 2014 ) .
however , alternate viability monitoring methods , such as energy metabolism and membrane permeability , have been shown to be more suitable for specific life cycle stages , notably the intra - mammalian larval stages of the human schistosome blood flukes ( mansour and bickle , 2010 ; peak et al . , 2010 ; howe et al .
xworm has been adapted for a range of applications that rely on measurement of a phenotypic change , including responses of genetically manipulated parasites ( you et al . , 2013 ) . moreover , developmental stages that originally were not envisioned suitable for the assay , such as eggs of schistosomes that require fresh water to hatch , have been successfully employed ( zeraik et al . , 2014 ) .
these new applications were successful but required further optimization as a low concentration buffered salt solution of 0.1 phosphate buffered saline ( pbs ) was necessary for egg hatching . in addition the signal : noise ratio at the default 10 khz frequency was sub - optimal for sensitive detection .
discussions with the developers of the xcelligence system ( acea ) , revealed alternate frequency settings of 25 and 50 khz available for monitoring cells or in our case monitoring parasitic helminths . in order to improve the sensitivity of the xworm assay we employed strictly standardized mean difference prime ( ssmd ) statistical analysis to compare different monitoring frequencies , i.e. the default 10 khz and alternate 25 khz and 50 khz .
in addition , low concentration salt motility assay facilitated accurate measurement of motility of cercariae , the infective stage of the schistosome for humans and other mammals .
mice infected with schistosoma mansoni were obtained from the biomedical research institute ( bri ) , rockville , md and housed at the animal research facility of the george washington university medical school , which is accredited by the american association for accreditation of laboratory animal care ( aaalac no .
000347 ) and has an animal welfare assurance on file with the national institutes of health , office of laboratory animal welfare , olaw assurance number a3205 - 01 .
all procedures employed were consistent with the guide for the care and use of laboratory animals .
maintenance of the mice and recovery of schistosomes were approved by the institutional animal care and use committee of the george washington university .
biomphalaria glabrata snails and swiss - webster mice infected with the nmri ( puerto rican ) strain of s. mansoni were supplied by the biomedical research institute , rockville , maryland usa under nih - niaid contract hhsn272201000005i .
four developmental stages collected and maintained as described ( mann et al . , 2010 ) were investigated : adults , eggs / miracidia , cercariae and schistosomula .
the motility assay was performed using the xcelligence dp platform ( acea biosciences , san diego , ca ) at 10 khz as described ( zeraik et al . , 2014 ) , and with the addition of 25 and 50 khz frequencies .
all motility index readings were monitored with sweeps at intervals of 15 s by the rtca software .
prior to the addition of parasites to e - plates , baseline motility using media alone was determined overnight in e - plate wells containing 100 l of relevant media but without parasites ( see sections 2.42.7 below ) .
treatments of forchlorfenuron in dmso ( fcf , sigma aldrich ) or dmso ( untreated vehicle controls ) were included when specified for the different life cycle stages .
developmental stages of s. mansoni were heat killed , 80 c for 15 min , immediately before the assay .
, 2010 ) , and egg hatching / miracidia motility was assessed as described ( zeraik et al . , 2014 ) . briefly
5000 eggs were seeded into 180 l of 0.1 pbs , ph 7.2 ( 13.7 mm nacl , 0.27 mm kcl , 1 mm nah2po4/na2hpo4 , 0.2 mm kh2po4 ) per well and supplemented with 20 l of dmso vehicle control , 50 m or 200 m of fcf ( final concentration ) in duplicate .
the eggs were induced to hatch under bright light at 23 c for 16 h. a replicate of the experiment was run in parallel in a 96-well microtitre plate for direct visual observation of the egg hatching using an axio observer a.1 inverted microscope fitted with an axiocam icc3 camera ( zeiss ) .
cercariae shed from b. glabrata snails infected with s. mansoni in water under bright light for 2 h at room temperature were washed three times in 0.1 pbs and 2% antibiotic / antimycotic ( life technologies ) , transferred to 0.1 pbs and dispensed in duplicate to e - plate wells in two - fold decreasing serial dilution of cercariae from 9000 - 281 cercariae / well .
the motility of the larvae was monitored overnight for 20 h at 23 c .
schistosomula were obtained by mechanical transformation of cercariae ( mann et al . , 2010 ) and cultured in modified basch 's medium under 5% co2 at 37 c ( dalton et al . , 1997 ; mann et al . , 2010 ; zeraik et al . , 2014 ) .
serial two - fold dilutions of 5000 - 78 schistosomula in 100 l of modified basch 's medium were added to e - plate wells containing 100 l of the same medium .
two wells containing 200 l of modified basch 's medium alone were included as empty well controls .
the parasites were monitored every 15 s at 37 c under 5% co2 for 16 h. the experiment was performed in duplicate .
adult worms were recovered from the hepatic veins of infected mice by portal perfusion ( mann et al . , 2010 ; zeraik et al . , 2014 ) .
thereafter , adult worms were washed and cultured in dulbecco 's modified eagle 's medium ( dmem - life technologies ) , supplemented with 10% fetal bovine serum and 2% antibiotic / antimycotic ( invitrogen , carlsbad , ca , catalogue no .
individual adult schistosomes were dispensed into wells of e - plates and the pre - treatment motility of flukes was registered for a minimum of 6 h before addition of fcf to 1 mm or dmso vehicle control with 68 replicates per condition . the motility index for each e - plate
well , i.e. individual flukes , 5000 eggs , schistosomula or cercariae , was calculated in excel ( microsoft ) as the standard deviation over 800 data points of the cell index ( ci ) difference from the rolling average over 30 data points as described ( smout et al . , 2010 ) .
the absolute ssmd value is the ratio of the difference of the sample mean to the standard deviation of the sample data and was generated as described by zhang ( 2011 ) .
treatment and negative control samples ( untreated parasites ) for egg hatch and cercariae motility have mean values designated as xt and xn with standard deviations of st and sn respectively . for egg hatch and cercariae motility ,
the total motility index over 16 and 20 h respectively was used for graphing and ssmd calculations.absolutessmd=|xtxnst2+sn2| ssmd prime ( ssmd ) is based upon the positive / negative controls and replaces xt with the heat - killed parasite positive control .
outliers encountered with the adult fluke assay require the use of robust ssmd value ( ssmd * ) , which uses a similar ssmd formula as shown below , where the mean and standard deviations are replaced with median ( x ) and median absolute deviation ( mad ) values ( s2 ) .
adult worm motility used the average motility index over 5 h for generation of ssmd * and ssmd* scores.absoluterobustssmd=|xtxnst2+sn2| the heat - killed parasites used as a positive control were an extremely strong control and used the most stringent category , shown in table 1 , for determination of assay quality .
treatment magnitude in table 2 delineates ssmd ranges for effect quality as recommended by zhang ( 2011 ) .
the xcelligence motility assay was developed ( smout et al . , 2010 ) for measuring motility of hookworms and adult blood flukes ( schistosomes ) ( fig . 1 ) , and adapted later to several helminth species and developmental stages ( silbereisen et al .
you et al . , 2013 ; zeraik et al . , 2014 ) .
the schistosome egg hatch assay requires 0.1 pbs as an approximation for the salinity of fresh water ( zeraik et al . , 2014 )
; tests using buffer alone ( 0.1 pbs ) at 25 khz and 50 khz showed both a lower and more temporally stable background motility index than the default 10 khz xcelligence frequency output ( fig .
assessment of the three frequencies with egg hatching showed an improvement for both 25 khz and 50 khz frequencies compared to 10 khz ( 68.9% and 70.4% total motility index respectively ) , although 50 khz resulted in higher variability ( fig .
furthermore , inhibition of egg hatching with 200 m fcf , reported by zeraik et al .
( 2014 ) measured at 10 khz , resulted in a similar 60% reduction of signal across all frequencies ( fig . 2c ) .
although it is worth noting the readings obtained at the two higher frequencies , 25 and 50 khz , demonstrated marginally steeper slopes than that at 10 khz ( fig .
this may represent the frequency influencing hatching rates and miracidial motility , and/or the higher sensitivity of the assay reflects improved detection resolution of normal hatching .
these xworm experiments were designed to determine which frequency generates the highest quality control parameters that is the largest difference between positive and negative controls relative to the variation within each group .
comparing assay quality using classic null hypothesis statistical significance tests with p - values is problematic ( cohen , 1994 ; kirk , 1996 ) given that the magnitude of the difference between groups is not measured , and only confirms that the difference is not zero ( zhang , 2007 ; goktug et al . ,
a range of statistical assessments has been developed for high - throughput assays , many compatible with xworm analysis ( birmingham et al .
z factor was initially used for quality control , but over the past decade the more meaningful strictly standardized mean difference ( ssmd ) parameter has become more commonplace ( zhang , 2007 ; goktug et al . , 2012 ) .
the ssmd value is the difference between groups relative to the variance within each group .
hence a high ssmd value represents a low variance within each group relative to the difference between the groups .
this allows the output to measure the magnitude of impact more effectively that other metrics based on null hypothesis ( zhang , 2007 ; birmingham et al . , 2009 ; kozak , 2012 ) . when used to evaluate the assay quality , the ssmd prime ( ssmd ) value is generated to compare the positive and negative control groups , i.e. heat killed and untreated parasites , respectively .
zhang and colleagues determined ssmd ranges to asses assay quality based upon the predicted effect size of the positive control ( zhang , 2011 ) . as heat - killed control parasites were expected to be completely immobile , the strictest assay criteria were selected , and a summary is shown in table 1 ( zhang , 2011 ) .
an absolute ssmd of 57 is considered a good assay rating , and values
> 7 indicate an excellent assay meaning that the difference in motility between groups is > 7-fold the square root of sum of the variance of the groups .
although the 25 and 50 khz groups had similar total motility indices ( fig . 2c
) , the lower variation obtained with the 25 khz frequency resulted in an excellent assay
screening of schistosome eggs in the presence of different compounds to detect a significant reduction in hatching / motility relies on the comparison of untreated negative control and treatment groups , in this case the drug fcf .
a positive hit for a significant motility reduction from a treatment is any ssmd > 1.645 , as mathematically this is the equivalent of the conventional t - test p < 0.05 ( zhang et al . , 2010 ; zhang , 2011 ) . we observed that the ssmd values for parasites treated with 200 m fcf were significant ( > 1.645 ) at every frequency ; the 10 and 50 khz ssmd scores of 35 are considered strong hits ( table 2 ) and the 25 khz score ( > 5 ) was considered an extremely strong hit ( fig . 2d ) .
comparison of control flukes and those treated with 50 m fcf did not reach significance at any frequency ( fig .
2 ) , the adult motility index score showed greater variability within groups at all the tested frequencies ( fig .
3a ) , probably due to differences in size / activity levels among individual worms . as
each worm generally displays similar motility index scores over time , this variability was reduced when post - treatment motility index scores were converted to percentages of pre - treatment values as previously described ( fig .
2010 ) . because the occasional parasite was overly active or subdued in the untreated groups we utilized median scores to counter outliers .
the subsequent ssmd values generated with median values have been designated robust ssmd ( zhang , 2011 ) and distinguished with an asterisk ( ssmd * and ssmd* ) . while the 10 khz frequency assay reached the good assay ( ssmd* 35 ) category with a score of 4.0 , both the 25 and 50 khz frequency reached the excellent assay range ( ssmd* > 7 ) with 7.3 and 7.9 scores respectively ( fig .
the complete ablation of schistosome movement when flukes were cultured in 1 mm fcf was observed at all three frequencies in fig .
3b . both the 25 and 50 khz frequencies scored extremely strong hits ( ssmd * >
5 ) with ssmd * scores of 6.9 and 8.8 respectively , while the 10 khz frequency scored ssmd * = 4.1 and is categorized a very strong hit ( ssmd * = 35 ) . given the success of xworm for measuring egg hatch in low salt conditions , we also tested the assay with the highly motile cercariae of s. mansoni , the fork - tailed , free - swimming larval stage that is infective to humans .
normally , when maintained in fresh water this developmental stage has minimal contact with the gold electrodes at the base of the wells , since it actively swims in the water column , and hence does not generate a substantial signal .
however , when cultured in 0.1 pbs rather than water , the cercariae still twitched rapidly ( as seen during swimming ) , but sunk to the base of the wells making contact with the sensors of the e - plate .
optimal numbers of cercariae per well , 2819000/well , were assessed at several frequencies . as with hatching of schistosome eggs , the motility was initially high but slowly declined over time ( fig .
the area under the curve was employed to generate a total motility index ( fig .
4b ) , further emphasizing the higher scores generated at 25 or 50 khz compared to 10 khz . as with the egg hatching , the downward motility index slope tended to increase as frequency
4c ) we further demonstrated that 25 khz with 4500 cercariae / well scored the highest ssmd value of 42.5 substantially greater than the
indeed , all three frequencies tested with 22509000 cercariae / well all scored above the excellent assay threshold ( table 2 ) . whereas the highest ssmd score would be optimal for applications requiring high sensitivity levels , a high - throughput screen that seeks to test thousands of samples would ideally consume fewer parasites / well .
our data showed that a good assay score in the assay ( ssmd 57 ) was achievable at 562 cercariae / well with 25 and 50 khz frequencies .
schistosomula of s. mansoni move in the bottom of the well by an expansion and contraction process rather than the period of rapid twitching exhibited by the cercariae in the water column since the tails of schistosomula were shed during the mechanical transformation .
testing of 1505000 schistosomula at the three discrete frequencies was unsuccessful , with minimal signals detected ( not shown ) .
( ssmd < 3 ) and only 625 schistosomula at 25 khz stimulated a score of 3.03 , the poor assay category ( ssmd = 35 ) .
this score did allow for detection of motility , but not at levels for reliable testing ( data not shown ) .
we previously developed a motility assay for adult and larval parasitic helminths ( smout et al .
our assay has been adapted by a number of other laboratories for a range of anthelmintics screens and other explorations of drug helminth interactions ( mansour and bickle , 2010 ; silbereisen et al . , 2011 ; tritten et al . , 2012 ;
you et al . , 2013 ; zeraik et al . , 2014 ) .
more recently the assay was adapted to monitor hatching of eggs of the human schistosome , s. mansoni , measuring a combination of direct miracidial electrode contact , the erratic movement of eggs induced by miracidial hatching as well as agitation of eggs by the highly motile hatched miracidia in the microplate well ( zeraik et al . , 2014 ) .
a low salt buffer is essential for schistosome egg hatching , as it mimics the natural fresh water body where hatching occurs .
however , xworm requires buffered salts for conductivity measurements by the xcelligence system . the alternate frequency option of 25 khz and 0.1 pbs was verified by ssmd comparisons as the optimal combination to measure egg hatching using xworm . when assessing the utility of xworm to monitor motility of cercariae , the 25 khz frequency was determined to be optimal with 4500 cercariae / well ; with good assay motility scores with as few as 562 cercariae / well .
although studying cercariae out of the natural fresh water environment is artificial , the motility assay in 0.1 pbs with the xcelligence system represents a surrogate approach to monitor motility of this developmental stage . despite this
, caution should be taken as the artificial conditions may confound interpretation of results . despite the improvements to the xworm sensitivity ,
the range of xcelligence systems that incorporate the three frequency options ( 10 , 25 and 50 khz ) required for this optimized xworm assay span from small lab scale ( dual - plate unit 3 16 wells and single plate unit 96 wells ) to larger systems ( multi - plate unit 6 96 well and high - throughput unit 384 wells ) .
the xworm assay is a robust yet sensitive technique that can now be predicted to be widely applicable across a range of species and life cycle stages , but recent progress has greatly expanded the available options to researchers seeking reliable methods to monitor parasitic helminths ( peak and hoffmann , 2011 ) .
initially , employing the model free - living helminth caenorhabditis elegans , methods that allowed microfluidics - based detection of motility from an individual worm were developed ( rohde et al .
subsequent approaches were adapted from techniques originally designed to count mammalian cells either based on mitochondrial enzymes ( lai et al . , 2014 ) or computer algorithms that automated detection in bright field images ( white et al . , 2013 ) .
these algorithmic and enzymatic techniques have been shown to be efficacious for monitoring the viability of schistosomula , the ideal developmental stage for high - throughput anthelmintic drug screening .
a range of cell health indicators are now available for monitoring schistosomula viability , such as the colorimetric alamar blue ( mansour and bickle , 2010 ) and the dual fluorogenesis assay using propidium iodide and fluorescein diacetate ( peak et al . , 2010 ; marxer et al . , 2012 ; rinaldi et al . ,
progress has been accomplished in developing assays to detect and quantify metabolism , notably luminescent quantitation of atp ( lalli et al . , 2015 ) and lactate the end product of glycolysis ( howe et al . , 2015 ) .
these provide a low - cost approach to screening schistosomula with a standard microplate reader , and while effective at detecting reduced metabolism or impaired cellular membranes , these assays have difficulty in detecting phenotypic or motility distortion ( marxer et al . , 2012 ; paveley et al . ,
this impediment was overcome by the development of algorithms that detected schistosomula movement or phenotypic change by using automated image acquisition systems ( paveley et al .
progress towards the screening of other schistosome life cycle stages is less advanced , and the detection of lactate ( howe et al . , 2015 ) is the only non - observational method that is also applicable to the adult schistosome . in terms of other flatworm genera , there has been some progress , such as screening for secreted enzymes in tapeworms ( mahanty et al . ,
so while xworm is not suitable for measuring the motility of the schistosomula , our assay complements the current range of schistosomula viability assays with wide applicability for the remaining major aspects of the schistosome life cycle ( eggs , cercariae , adults ) .
the widespread use of high - throughput methods to screen for new therapeutics or emerging drug resistance will facilitate greater capacity to drug screens to parse small compound libraries for novel interventions .
monitoring the efficacy of anthelmintic medicines and screening for new drugs was previously hindered by the lack of objective , high - throughput approaches .
here we present a more sensitive xworm assay for s. mansoni adults and egg hatch monitoring and expanded capabilities to monitor cercariae .
we focused on s. mansoni , but the xworm assay is envisaged to be applicable for the majority of helminth species and developmental stages where egg hatch assays and/or motility are deployed presently to evaluate parasite viability .
additionally , this assay further improves high - throughput helminth viability screening and will be an asset in the fight against the wide range of biomedical and veterinary helminths .
mjs helped conceive , design and analyze experiments , provided project oversight and wrote the manuscript .
gr and mjs have received travel funds to present at an xcelligence workshop , and the 2014 the international congress of parasitology meeting through the competitive acea travel award program , respectively .
acea played no role in performing the experiments , analyzing the data , or developing the manuscript other than providing raw data for fig . | infection with helminth parasites causes morbidity and mortality in billions of people and livestock worldwide . where anthelmintic drugs are available , drug resistance is a major problem in livestock parasites , and a looming threat to public health . monitoring the efficacy of these medicines and screening for new drugs
has been hindered by the lack of objective , high - throughput approaches .
several cell monitoring technologies have been adapted for parasitic worms , including video- , fluorescence- , metabolism enzyme- and impedance - based tools that minimize the screening bottleneck . using the xcelligence impedance - based system we previously developed a motility - viability assay that is applicable for a range of helminth parasites .
here we have improved substantially the assay by using diverse frequency settings , and have named it the xcelligence worm real - time motility assay ( xworm ) . by utilizing strictly standardized mean difference analysis we compared the xworm output measured with 10 , 25 and 50 khz frequencies to quantify the motility of schistosome adults ( human blood flukes ) and hatching of schistosome eggs .
furthermore , we have described a novel application of xworm to monitor movement of schistosome cercariae , the developmental stage that is infectious to humans .
for all three stages , 25 khz was either optimal or near - optimal for monitoring and quantifying schistosome motility .
these improvements in methodology sensitivity should enhance the capacity to screen small compound libraries for new drugs both for schistosomes and other helminth pathogens at large . |
bacteria and archaea exist in an incessant arms race with various selfish genetic elements ( phages , transposons , and plasmids ) and have evolved a variety of defense systems .
the best known ones probably are the numerous restriction - modification enzyme systems that exploit different methylation patterns of host and infecting agent dna to eliminate the invader .
recently , a novel widespread defense system that functions on a completely different principle was discovered ; it became known as the crispr ( clustered regularly interspaced short palindromic repeats)-associated system , usually referred to as crispr - cas ( where cas stands for crispr - associated proteins ) or , alternatively , as cass [ 2 - 4 ] .
distinct arrays of short repeats interspersed with unique spacers ( crispr ) have been seen in bacterial and archaeal genomes for years , with no clues as to their possible functions . independently , cas protein sequences encoded by putative operons adjacent to crispr were analyzed in detail and found to contain domains characteristic of several nucleases , a helicase , a polymerase , and rna - binding proteins ; it has been suggested that these proteins might belong to a novel repair system .
a new light was shed on the probable function of the crispr when it was observed that some of the unique inserts were ( nearly ) identical to fragments of phage and plasmids genes , a pivotal observation that immediately led to the idea that crispr might be involved in defense against selfish elements [ 9 - 11 ] .
these findings were combined with the results of comprehensive computational re - analysis of the cas proteins to develop a detailed hypothesis on the mechanism of crispr - cas .
this hypothesis drew a close analogy between the putative novel prokaryotic defense system and the eukaryotic rna interference ( rnai ) mechanisms , with the important difference that cass mediates integration of a piece of alien dna into the host genome as the first step in the sequence of events which leads to immunity to the given agent .
specific roles for individual cas proteins were proposed as well on the basis of their domain composition and by analogy with rnai components although the proteins involved are not homologous .
this hypothesis prompted direct experiments that demonstrated that engineering a specific bacteriophage sequence into the crispr locus of the lactic bacterium streptococcuss thermophilus indeed conferred resistance to the cognate phage , an effect that was abrogated by even a single mismatch between the insert and the target gene .
these key experiments clinched the case for the defense role of crispr - cas and triggered an avalanche of further genetic and biochemical experiments .
bacteria and archaea exist in an incessant arms race with various selfish genetic elements ( phages , transposons , and plasmids ) and have evolved a variety of defense systems .
the best known ones probably are the numerous restriction - modification enzyme systems that exploit different methylation patterns of host and infecting agent dna to eliminate the invader .
recently , a novel widespread defense system that functions on a completely different principle was discovered ; it became known as the crispr ( clustered regularly interspaced short palindromic repeats)-associated system , usually referred to as crispr - cas ( where cas stands for crispr - associated proteins ) or , alternatively , as cass [ 2 - 4 ] .
distinct arrays of short repeats interspersed with unique spacers ( crispr ) have been seen in bacterial and archaeal genomes for years , with no clues as to their possible functions . independently , cas protein sequences encoded by putative operons adjacent to crispr were analyzed in detail and found to contain domains characteristic of several nucleases , a helicase , a polymerase , and rna - binding proteins ; it has been suggested that these proteins might belong to a novel repair system .
a new light was shed on the probable function of the crispr when it was observed that some of the unique inserts were ( nearly ) identical to fragments of phage and plasmids genes , a pivotal observation that immediately led to the idea that crispr might be involved in defense against selfish elements [ 9 - 11 ] .
these findings were combined with the results of comprehensive computational re - analysis of the cas proteins to develop a detailed hypothesis on the mechanism of crispr - cas .
this hypothesis drew a close analogy between the putative novel prokaryotic defense system and the eukaryotic rna interference ( rnai ) mechanisms , with the important difference that cass mediates integration of a piece of alien dna into the host genome as the first step in the sequence of events which leads to immunity to the given agent .
specific roles for individual cas proteins were proposed as well on the basis of their domain composition and by analogy with rnai components although the proteins involved are not homologous .
this hypothesis prompted direct experiments that demonstrated that engineering a specific bacteriophage sequence into the crispr locus of the lactic bacterium streptococcuss thermophilus indeed conferred resistance to the cognate phage , an effect that was abrogated by even a single mismatch between the insert and the target gene .
these key experiments clinched the case for the defense role of crispr - cas and triggered an avalanche of further genetic and biochemical experiments .
crispr - cas is a highly diverse constellation of genes , with the number of crispr - cas loci , cas gene repertoire , and ( predicted ) operon organization often changing even between closely related strains [ 3,12,15 - 18 ] .
comparative analysis of operon architectures revealed seven distinct types of crispr - cas , each of which is characterized by a distinct signature of genomic architecture .
only two genes , cas1 and cas2 , are invariably present in each crispr - cas system so far detected and accordingly can be used as genomic markers of crispr - cas . in addition to the two universal genes , three genes ( cas3 , cas4 , and cas5 ) are present in the majority of crispr - cas , and approximately 20 other genes are found in various subsets of these systems .
cas5 and several other less common crispr - cas components belong to the large and extremely diverged superfamily of repeat - associated mysterious proteins ( ramps ) . using the highly conserved cas1 protein sequence as a marker , we detected crispr - cas in 297 of the 774 analyzed prokaryotic genomes ( 37% ) ; among archaea , crispr - cas is much more common than among bacteria : up to 90% of the available archaeal genomes carry crispr - cas ( figure 1 ) .
the representation of crispr - cas in the genomes of diverse groups of archaea and bacteria differs within a broad range , from ubiquity to complete absence ( although it should be noted that all groups completely lacking crispr - cas are currently represented by a small number of genomes ) ( figure 1 ) .
the bars show the fractions of sequenced genomes in each group that possess crispr - cas ( red ) and lack crispr - cas ( blue ) .
the actual numbers of available genomes are also indicated for both parts of each bar .
the figure is based on the results of screening of the proteins encoded in 774 archaeal and bacterial genomes for statistically significant sequence similarity to cas1 sequences using blastp .
phylogenetic analysis of core cas genes , such as cas1 and cas3 , fails to recover major bacterial and archaeal lineages , an observation that appears to be indicative of extensive mobility of crispr - cas modules via horizontal gene transfer ( hgt ) .
the cas genes are not only horizontally mobile but also typically show high rates of sequence evolution , with the partial exception of core genes , in particular cas1 . in many cases ,
this fast evolution renders sequence conservation between homologous cas proteins barely detectable , most conspicuously among the ramps , which are propagated by both gene duplication and hgt and constitute a large fraction of cas protein sets in most crispr - cas - carrying prokaryotes .
the ramps are extremely diverged in sequence , so that the demonstration that different ramp families were related and possessed the same fold required the careful use of the most sensitive sequence analysis methods ( and even so , it is likely that additional ramps have been missed ) .
conceivably , ramps and perhaps some other cas proteins evolve under positive selection dictated by the arms race with selfish elements .
this possibility is congruent with the observations that , although virus or plasmid origin is apparent for a considerable number of crispr spacers , the majority of the spacers are not significantly similar to any sequences in current databases .
in addition , deletion of crispr units ( a repeat with a spacer ) has been demonstrated .
thus , it appears , first , that the repertoire of selfish genetic elements encountered by archaea and bacteria is vast , and second , that the crispr - cas - mediated immunity is short - lived ; that is , spacers rapidly deteriorate by mutation once the cognate element is no longer a threat . taken together , these observations identify the crispr - cas as a bona fide component of the prokaryotic mobilome [ that is , the totality of genetic elements that are characterized by extensive horizontal mobility and include selfish elements ( viruses , plasmids , transposons , and so on ) as well as defense and stress response systems ] .
defense islands ( i.e. , regions of bacterial and archaeal genomes that consist primarily of genes encoding defense and stress response systems , such as restriction - modification and toxin - antitoxin modules ) .
comparative - genomic analysis of the crispr - cas loci is facilitated by the use of specialized databases and accompanying custom software tools for crispr detection .
crispr - cas systems mediate immunity to invading genetic elements via three distinct stages : ( a ) adaptation , ( b ) expression and processing of crispr , and ( c ) interference .
the full molecular picture is far from being clear for each of these stages , but recently several fundamental results , particularly on the processing of crispr transcripts , have been reported . with regard to the adaptation stage , following the original work that demonstrated the insertion of a phage - specific spacer into the crispr locus of streptococcus thermophilus ,
this process was explored systematically , leading to the conclusion that a phage challenge typically triggers insertion of a single phage - specific resistance - conferring spacer with a characteristic length of 30 base pairs ; successive infection of a bacterial culture with multiple phages led to the accumulation of the cognate spacers in the crispr loci .
furthermore , it has been shown that insertion of new spacers depends on short pams ( proto - spacer adjacent motifs ) , which differ between variants of the crispr - cas system and might determine the identity of the inserted spacer .
prokaryotic rnai hypothesis maintained that crispr - cas systems would target mrnas of invading agents .
however , the first experiments that , in general , validated the hypothesis have also shown that both strands of the crispr spacer dna were effective in conferring immunity to the cognate phage , an observation best compatible with a dna target .
a more direct experiment showed that the insertion of a self - splicing intron into the target gene made the respective plasmid resistant to the crispr - mediated immunity , a clear indication that the invading dna itself is targeted .
whether this conclusion is general and applies to all crispr - cas remains to be determined , especially given the extreme diversity of the architectures of these systems .
as of september 2009 , biochemical activities and/or crystal structures of several widespread cas proteins have been determined ( table 1 ) . in agreement with the computational predictions and nuclease activities , either rnase or dnase or both were demonstrated for several cas proteins .
specifically , cas1 has been shown to be a metal - dependent dnase with no sequence specificity and has been implicated in the integration of the alien dna into the crispr cassettes .
cas2 has been characterized as a metal - dependent endoribonuclease whose role in the crispr - cas mechanism remains unclear .
a striking finding is that some of the ramp proteins that contain a double ferredoxin - fold domain and were originally proposed to be non - enzymatic rna - binding proteins ( considering their extreme sequence divergence ) , actually possess rnase activity that is apparently involved in the processing of crispr transcripts . in particular , a ramp protein seems to be the active moiety of the cascade ( crispr - associated complex for antiviral defense ) complex that consists of five cas proteins ( table 1 ) and is the crispr - processing machine of escherichia coli . in concert with the cas3 protein that consists of (
predicted ) helicase and nuclease domains , cascade seems to be involved in the interference stage .
cas proteins and complexes : predicted and experimentally determined biochemical activities and possible functions in crispr - mediated immunity these are only the most widespread and experimentally characterized cas proteins ; there is no unified nomenclature of cas proteins ; the cas protein names are accompanied by the numbers of clusters of orthologous genes ( cogs ) where available .
cas , crispr ( clustered regularly interspaced short palindromic repeats)-associated protein ; cascade , crispr ( clustered regularly interspaced short palindromic repeats)-associated complex for antiviral defense ; cass , crispr ( clustered regularly interspaced short palindromic repeats)-associated system ; crispr , clustered regularly interspaced short palindromic repeats ; hd , hd ( histidine - aspartate)-family nuclease ; ramp , repeat - associated mysterious protein .
crispr - cas is a highly diverse constellation of genes , with the number of crispr - cas loci , cas gene repertoire , and ( predicted ) operon organization often changing even between closely related strains [ 3,12,15 - 18 ] .
comparative analysis of operon architectures revealed seven distinct types of crispr - cas , each of which is characterized by a distinct signature of genomic architecture .
only two genes , cas1 and cas2 , are invariably present in each crispr - cas system so far detected and accordingly can be used as genomic markers of crispr - cas . in addition to the two universal genes , three genes ( cas3 , cas4 , and cas5 ) are present in the majority of crispr - cas , and approximately 20 other genes are found in various subsets of these systems .
cas5 and several other less common crispr - cas components belong to the large and extremely diverged superfamily of repeat - associated mysterious proteins ( ramps ) . using the highly conserved cas1 protein sequence as a marker , we detected crispr - cas in 297 of the 774 analyzed prokaryotic genomes ( 37% ) ; among archaea , crispr - cas is much more common than among bacteria : up to 90% of the available archaeal genomes carry crispr - cas ( figure 1 ) .
the representation of crispr - cas in the genomes of diverse groups of archaea and bacteria differs within a broad range , from ubiquity to complete absence ( although it should be noted that all groups completely lacking crispr - cas are currently represented by a small number of genomes ) ( figure 1 ) .
the bars show the fractions of sequenced genomes in each group that possess crispr - cas ( red ) and lack crispr - cas ( blue ) .
the actual numbers of available genomes are also indicated for both parts of each bar .
the figure is based on the results of screening of the proteins encoded in 774 archaeal and bacterial genomes for statistically significant sequence similarity to cas1 sequences using blastp .
phylogenetic analysis of core cas genes , such as cas1 and cas3 , fails to recover major bacterial and archaeal lineages , an observation that appears to be indicative of extensive mobility of crispr - cas modules via horizontal gene transfer ( hgt ) .
the cas genes are not only horizontally mobile but also typically show high rates of sequence evolution , with the partial exception of core genes , in particular cas1 . in many cases ,
this fast evolution renders sequence conservation between homologous cas proteins barely detectable , most conspicuously among the ramps , which are propagated by both gene duplication and hgt and constitute a large fraction of cas protein sets in most crispr - cas - carrying prokaryotes .
the ramps are extremely diverged in sequence , so that the demonstration that different ramp families were related and possessed the same fold required the careful use of the most sensitive sequence analysis methods ( and even so , it is likely that additional ramps have been missed ) .
conceivably , ramps and perhaps some other cas proteins evolve under positive selection dictated by the arms race with selfish elements .
this possibility is congruent with the observations that , although virus or plasmid origin is apparent for a considerable number of crispr spacers , the majority of the spacers are not significantly similar to any sequences in current databases .
in addition , deletion of crispr units ( a repeat with a spacer ) has been demonstrated .
thus , it appears , first , that the repertoire of selfish genetic elements encountered by archaea and bacteria is vast , and second , that the crispr - cas - mediated immunity is short - lived ; that is , spacers rapidly deteriorate by mutation once the cognate element is no longer a threat . taken together , these observations identify the crispr - cas as a bona fide component of the prokaryotic mobilome [ that is , the totality of genetic elements that are characterized by extensive horizontal mobility and include selfish elements ( viruses , plasmids , transposons , and so on ) as well as defense and stress response systems ] .
defense islands ( i.e. , regions of bacterial and archaeal genomes that consist primarily of genes encoding defense and stress response systems , such as restriction - modification and toxin - antitoxin modules ) .
comparative - genomic analysis of the crispr - cas loci is facilitated by the use of specialized databases and accompanying custom software tools for crispr detection .
crispr - cas systems mediate immunity to invading genetic elements via three distinct stages : ( a ) adaptation , ( b ) expression and processing of crispr , and ( c ) interference .
the full molecular picture is far from being clear for each of these stages , but recently several fundamental results , particularly on the processing of crispr transcripts , have been reported . with regard to the adaptation stage , following the original work that demonstrated the insertion of a phage - specific spacer into the crispr locus of streptococcus thermophilus , this process was explored systematically , leading to the conclusion that a phage challenge typically triggers insertion of a single phage - specific resistance - conferring spacer with a characteristic length of 30 base pairs ; successive infection of a bacterial culture with multiple phages led to the accumulation of the cognate spacers in the crispr loci .
furthermore , it has been shown that insertion of new spacers depends on short pams ( proto - spacer adjacent motifs ) , which differ between variants of the crispr - cas system and might determine the identity of the inserted spacer .
prokaryotic rnai hypothesis maintained that crispr - cas systems would target mrnas of invading agents .
however , the first experiments that , in general , validated the hypothesis have also shown that both strands of the crispr spacer dna were effective in conferring immunity to the cognate phage , an observation best compatible with a dna target .
a more direct experiment showed that the insertion of a self - splicing intron into the target gene made the respective plasmid resistant to the crispr - mediated immunity , a clear indication that the invading dna itself is targeted .
whether this conclusion is general and applies to all crispr - cas remains to be determined , especially given the extreme diversity of the architectures of these systems .
as of september 2009 , biochemical activities and/or crystal structures of several widespread cas proteins have been determined ( table 1 ) . in agreement with the computational predictions and nuclease activities , either rnase or dnase or both were demonstrated for several cas proteins .
specifically , cas1 has been shown to be a metal - dependent dnase with no sequence specificity and has been implicated in the integration of the alien dna into the crispr cassettes .
cas2 has been characterized as a metal - dependent endoribonuclease whose role in the crispr - cas mechanism remains unclear .
a striking finding is that some of the ramp proteins that contain a double ferredoxin - fold domain and were originally proposed to be non - enzymatic rna - binding proteins ( considering their extreme sequence divergence ) , actually possess rnase activity that is apparently involved in the processing of crispr transcripts .
in particular , a ramp protein seems to be the active moiety of the cascade ( crispr - associated complex for antiviral defense ) complex that consists of five cas proteins ( table 1 ) and is the crispr - processing machine of escherichia coli . in concert with the cas3 protein that consists of (
predicted ) helicase and nuclease domains , cascade seems to be involved in the interference stage .
cas proteins and complexes : predicted and experimentally determined biochemical activities and possible functions in crispr - mediated immunity these are only the most widespread and experimentally characterized cas proteins ; there is no unified nomenclature of cas proteins ; the cas protein names are accompanied by the numbers of clusters of orthologous genes ( cogs ) where available . data are from references and
cas , crispr ( clustered regularly interspaced short palindromic repeats)-associated protein ; cascade , crispr ( clustered regularly interspaced short palindromic repeats)-associated complex for antiviral defense ; cass , crispr ( clustered regularly interspaced short palindromic repeats)-associated system ; crispr , clustered regularly interspaced short palindromic repeats ; hd , hd ( histidine - aspartate)-family nuclease ; ramp , repeat - associated mysterious protein .
comparative - genomic predictions validated by a rapidly growing body of experimental results indicate that the crispr - cas is an adaptive immunity system that is widely employed by archaea and bacteria for defense against diverse invading elements , in particular , viruses .
the system functions by integrating fragments of alien element genes into crispr loci and employing the resulting spacers , after transcription and processing , as guide rnas to abrogate the replication of the cognate elements by cleaving nucleic acid molecules complementary to the guide . in some cases
, at least , the target of crispr - cas is the genomic dna of an invading genetic element .
experiments aimed at molecular dissection of cass proved the predicted principle of its action and are starting to reveal multiple activities of the protein components of cass and the molecular architecture of complexes formed by these proteins .
however , an enormous amount of experimental work remains to be done to elucidate the mechanisms of cass , in particular , the molecular details of spacer incorporation into the crispr loci and the specific pathways of rna - guided destruction of alien genomes .
these experiments can be expected to reveal the considerable mechanistic diversity that reflects the extreme diversity of cas gene repertoires and operonic organization .
another important direction of future work is the characterization of the arms race between crispr - cas and viruses of prokaryotes and elucidation of putative mechanisms of counterdefense employed by the viruses .
finally , it is worth noting that , by integrating fragments of invaders ' genomes into the genomes of the archaeal and bacterial hosts , the cass effectively operates via a lamarckian - type inheritance of acquired characters .
| most of the archaea and numerous bacteria possess an elaborate system of adaptive immunity to mobile genetic elements known as the crispr ( clustered regularly interspaced short palindromic repeats)-associated system ( crispr - cas ) , which consists of arrays of short repeats interspersed with unique dna spacers and adjacent operons encompassing crispr - associated ( cas ) genes with predicted and , in some cases , experimentally validated nuclease , helicase , and polymerase activities .
the system functions by integrating fragments of alien dna between the repeats and employing their transcripts to degrade the dna of the respective invading elements via an rna interference - like mechanism .
the crispr - cas system is a case of apparent lamarckian inheritance . |
ductal carcinoma in situ ( dcis ) increases the risk for developing contralateral breast cancer ( cbc).1 the risk for developing either invasive cancer or dcis in the contralateral breast has been estimated at 0.6% per year.1,2 increasing numbers of women with dcis are electing contralateral prophylactic mastectomy ( cpm),2,3 with studies indicating a four - to five - fold increase in cpm in recent years.4 in a study of 2,037 dcis patients , 37% ( < 40 years ) elected cpm , and 82% ( < 40 years ) had immediate reconstruction.3 additionally , ashfaq et al
. found that a significantly higher proportion of patients undergoing cpm had reconstruction performed ( 46% ) than those who did not elect cpm ( 15%).5 another significant predictor of cpm among patients with dcis is having a germline brca mutation.3 defining brca1 and brca2 mutation carrier status is crucial , because mutation carriers have a 43% to 84% risk of developing breast cancer ( bc ) and up to a 65% risk for cbc.6,7 the prevalence of brca mutations in patients with dcis has been reported.8,9 our previous study8 indicated a 27% prevalence of deleterious brca mutations among 118 patients with dcis who were referred for genetic counseling .
we also recently reported a 27% cpm rate among 165 patients with dcis who were evaluated for brca genetic testing ( positive , negative , not tested ) .
predictors of cpm among that group were age 45 years or younger , brca positivity , and having one or more relatives with ovarian cancer ( oc).3 although a positive brca result has previously been shown to be associated with an increased likelihood of cpm election , our data also incidentally found a relatively high cpm rate for the brca - negative group . among women with dcis and a negative brca result ,
25% elected cpm.3 this relatively high rate warranted further investigation to elucidate possible predictive factors of cpm in this specific group .
it has been reported that women testing negative for a brca mutation and with a family history of bc ( 2 bc under age 50 , or 3 bc at any age ) have almost a four - fold increased risk of bc.10 while there is significant literature examining factors predicting cpm , there is scant literature related to the rate of cpm among this specific cohort of brca - negative patients with dcis .
howard - mcnatt et al.11 examined cpm decisions among brca - negative patients with bc , but their study did not distinguish dcis from invasive bc . among the brca - negative women with bc in their study , 37% chose cpm.11 in the current study , we further evaluated factors associated with cpm in patients with dcis who tested negative for brca mutation .
we specifically sought to identify differences between brca - negative patients who underwent cpm and those who did not .
one hundred women who had a diagnosis of dcis between 2005 and 2013 underwent genetic counseling and testing and were offered an opportunity to enroll in a registry study approved by the institutional review board at the university of texas md anderson cancer center .
each patient underwent genetic testing and received pre - test genetic counseling per standard of care .
this retrospective review included only patients with dcis who had negative test results for brca1 and brca2 .
we excluded patients with bilateral dcis , oc , micro - invasion , positive or variant of uncertain significance genetic test results , and patients who did not undergo genetic testing .
dcis diagnoses were based on pathologic evaluation by breast pathologists at md anderson cancer center .
genetic testing included comprehensive brca1 and brca2 gene sequencing and , when possible , large rearrangement testing ( bart ) . using the electronic patient medical records , we collected demographic and clinical information . variables included in the analysis were age at diagnosis , race , ethnicity ( ashkenazi jewish ancestry or not ) , marital status , educational level , family history of bc and/or oc in at least one first - degree relative , total number of relatives with bc and/or oc , patient 's brca1 and brca2 test results , tumor nuclear grade ( as defined by the modified nuclear grade system ) estrogen receptor and progesterone receptor status ( as determined by immunohistochemical analysis ) , surgical treatment , mastectomy of the affected breast , cpm , tamoxifen use , and reconstruction .
patient demographic and clinical characteristics were compared between the two groups defined according to cpm status .
univariate analyses were performed to determine the significance of each variable in relation to whether or not a patient had cpm , using chi - square tests for categorical variables and t - tests / anova or the counterpart nonparametric approaches ( wilcoxon rank - sum or kruskal - wallis ) for continuous variables.12 logistic regression analysis was used to assess multivariate relationships between patient demographic and clinical characteristics and cpm.13 a logistic regression model was obtained by first including an initial set of candidate predictor variables with p - value 0.05 in the univariate analysis .
stepwise backward elimination was then performed using 0.05 for the significance level of the wald chi - square for an effect to stay in the model .
table 1 outlines demographic and clinical characteristics . of the 100 patients with dcis who had negative brca genetic test results ,
sixty seven patients underwent mastectomy for the affected breast and 31 underwent only breast conservation surgery , ( unknown for two patients ) .
univariate analysis ( table 2 ) showed that married patients were more likely to elect cpm than those who were not married ( p = 0.0235 ) .
patients with a first - degree relative with oc were more likely to choose cpm than those without such a relative ( p = 0.0278 ) .
race , ethnicity , age at diagnosis , education , stage , and biomarker status were not significantly associated with cpm election .
moreover , patients who had any relatives with oc were more likely to choose cpm than those without any relatives with oc ( p = 0.0425 ) ; however , when analyzed by family history of bc ( first - degree relative or total number ) , the differences were not statistically significant ( p = 0.2436 and 0.7198 , respectively ) .
patients receiving tamoxifen were less likely to choose cpm than those who were not ( p = 0.0054 ) .
patients who underwent mastectomy were more likely to choose cpm than those who did not undergo mastectomy of the affected breast ( p < .0001 ) .
finally , patients who underwent reconstruction were more likely to choose to have cpm than those who did not ( p < .0001 ) .
reconstruction was not included in the final multivariate regression since reconstruction ( immediate vs. delayed ) was only a variable for those patients who had mastectomy .
when family history of oc in first - degree relatives , marital status , mastectomy , and tamoxifen use were included in the logistic regression model building procedure only mastectomy remained in the model ; patients who underwent mastectomy were more likely to elect cpm than patients who did not undergo mastectomy ( odds ratio [ or ] 51.267 , 95% confidence interval [ 95%ci ] 2.879 - 912.989 , p = 0.0074 , table 3 ) .
our study found a 31% cpm election rate among 100 dcis patients who tested negative for a brca mutation .
factors associated with cpm in this cohort were marital status , family history of oc , reconstruction , mastectomy of the affected breast , and tamoxifen use . to our knowledge
, our study uniquely examines cpm rates among dcis - only patients who are brca negative .
our recent previous study examined cpm among patients with dcis who were either brca - positive , brca - negative , or untested.3 in that study , 165 patients ( 27% ) elected cpm .
more specifically , cpm was elected by 12 ( 71% ) of 17 brca - positive patients , 23 ( 25% ) of 91 brca - negative patients , and 9 ( 16% ) of the 57 patients who did not undergo genetic testing .
factors associated with cpm in that study were age 45 years , family history of oc , and brca positivity .
the current study included only brca - negative patients , and the results demonstrated some findings distinguishable from those of our previous studies .
specifically , age ( < 45 years ) was significantly associated with cpm election in the previous article ; however , in the current study , while the trend remained as before , age was not a significant predictor of cpm .
marital status was a significant predictor in the current study but not in the previous one .
it is possible that current results differed slightly from previous results due to differences in the analyzed patient cohort , such as brca testing status and results .
both studies , however , found that family history of oc predicts cpm among patients with dcis , regardless of brca results .
brca positivity is known to be associated with significantly increased risks for bc and cbc,14 which may contribute to cpm election among brca - positive patients .
however , our current results suggest that family history of oc is associated with cpm even when patients are brca - negative . more specifically , a family history of 1 relatives with oc was significantly associated with increased rates of cpm election . to our knowledge , this is the first study to report an association between family history of oc and cpm election among brca - negative patients .
this significant finding poses the question of why a family history of oc is associated with increased cpm rates , particularly in brca - negative women .
it is possible that the low survival rate of women with oc may have contributed to the perspective on cancer of women in the current study , and thus this group was more willing to undergo preventive surgery to reduce chances of future cancer .
katapodi et al.15 observed that most women at high risk for bc underestimated their actual risk .
individuals with a family history of bc may have a more skewed perspective if their family members with bc survived , and this may contribute to the lack of significance of a family history of bc related to cpm election .
future studies are needed to analyze the effect of cancer deaths in families to verify this hypothesis . according to musiello and
associates,16 surgeons reported fear of developing another bc and a desire for breast symmetry as among the top reasons patients opted for cpm .
abbott et al.17 found that women with bc , including dcis , substantially overestimated their risk for developing cbc . in the presence of a negative brca test result ,
the experience of a personal bc diagnosis may trigger fear and anxiety concerning future risk of cbc and thus may lead to cpm election .
an individual patient 's concerns regarding the risk of cbc may be particularly warranted when there is a strong family history of breast cancer , which may be due to an unidentifiable hereditary cause , other than the brca genes .
thus , even though the patient is brca - negative , the decision to proceed with cpm may be appropriate due to concern about an unexplained family history .
furthermore a diagnosis of dcis increases the risk for invasive cancer and cbc by 0.5% to 1% per year regardless of family history1,2,8 ; therefore the reported statistics of additional cancer risk may also contribute to the rate of cpm among women with dcis .
the experience of frequent screening , the potential need for subsequent biopsies , and the worry and fear of anticipating test results are emotionally exhausting and may lead many women to undergo preventive surgeries .
many patients may feel burnout due to frequent cancer surveillance , and this may contribute to their desire for acceptance of cpm .
hoskin and greene18 indicated that preventive mastectomies could be a result of screening fatigue and/or stressful and costly screening .
future prospective study is needed to determine the significance of these factors in this cohort .
the various studies discussed here , including the results of our analysis , suggest that a personal history of bc and/or family history of oc may be sufficient to prompt the decision to undergo cpm despite a negative brca result .
subjective factors such as anxiety and fear , along with objective factors such as a family history of oc , being married , and choosing mastectomy , may explain the relatively high rate of cpm reported in this study .
it has been previously reported that marriage affects the decision to undergo cpm among dcis patients .
howard - mcnatt et al.11 examined cpm among patients with invasive bc who tested negative for a brca mutation ; among 110 women who underwent genetic testing , 37% of the brca - negative women chose cpm .
married women in that study were significantly more likely to elect cpm than those who were not married .
this finding is consistent with our finding that marital status is a significant predictor of cpm .
married women may have emotional support from their partner , as well as an established relationship ; thus their concerns related to body image may be less impactful than for single women .
further studies are needed to define more specifically the factors related to cpm election among married women .
the decision to proceed with mastectomy of the affected breast increases the rate of cpm.2 , 19 in this cohort , patients who had mastectomy were significantly more likely to elect cpm than those who had only breast - conserving surgery .
tuttle and associates2 analyzed the initial treatment of patients with unilateral dcis between 1998 and 2005 to determine the cpm rate .
a 13.5% cpm rate was found for patients who had a mastectomy on the affected side .
in addition , yao et al.20 observed a universal increase in cpm among surgically treated patients with invasive and noninvasive bc . among 23,218 surgically treated patients who elected cpm , election increased from 0.4% in 1998 to 4.7% in 2007 .
recent research has shown a similar rapid increase.21 the past two decades have witnessed increasing advances in reconstruction techniques and minimizing mastectomy scars , which may appeal to patients considering options for managing their cancers .
there is a known association between reconstruction and an increased cpm rate among women who have unilateral mastectomy for dcis.22,23 previous work reported that the use of tamoxifen reduces the 5-year rate of ipsilateral and contralateral bc in patients with dcis ; factors predicting tamoxifen use in their study were breast - conserving surgery and younger age.24 consistent with their conclusion , our study showed that patients who took tamoxifen were less likely to elect cpm than those who did not .
limitations of this study include the fact that this cohort included only patients referred for genetic counseling and testing .
we recognize the findings of this study are derived from a small sample , and are specific to brca - negative patients ; therefore , may not be generalized to all dcis patients .
moreover , given the uneven distribution of our groups limits our findings and their generalizability to a larger sample with a more even distribution .
additionally , future prospective studies are needed to evaluate the complex decision - making processes leading to cpm despite negative brca results .
a questionnaire or interview - based study could further elucidate which specific factors consciously affect patients ' decision for cpm .
the present study indicated a relatively high cpm rate among dcis patients with negative brca results which has not been clearly established among this specific cohort .
our analyses highlight not only the increasing trend of cpm among patients with dcis , but also the different predictive factors among brca carriers vs. non - carriers .
the predictors shown in this brca - negative cohort may need to be considered during discussions regarding surgical decision - making .
our results highlight the need for a more individualized and focused recommendation for this group . | the authors retrospectively examined the contralateral prophylactic mastectomy ( cpm ) rate among 100 women with ductal carcinoma in situ who are brca negative .
of 100 women with ductal carcinoma in situ , 31 elected contralateral prophylactic mastectomy ( cpm ) .
factors associated with increased likelihood of undergoing contralateral prophylactic mastectomy ( cpm ) among this cohort were : family history of ovarian cancer , marital status , reconstruction , mastectomy of the affected breast , and tamoxifen use . |
according to popular studies , the attempted resuscitation of patients in traumatic cardiac arrest in the prehospital care environment should not be pursued due to undo risk to the provider and has been shown to be ineffective . however , after review of current literature , sherren and colleagues developed and published in a recent edition of critical care a detailed treatment algorithm for their helicopter emergency medical service ( hems ) that encourages aggressive resuscitation for patients in traumatic cardiac arrest ( tca ) .
what is known in a review of the quoted literature is that in the prehospital care environment , the survival rate of tca patients is 0 to 3.7% , but in newer published studies survivability has risen to 7.5% .
what we already know is that prehospital medical cardiac arrest ( mca ) survival is approximately 9.8% , with in - hospital cardiac arrest survival at 24.2% . in mca ,
chest compressions , defibrillation , medication , and oxygenation are the mainstay treatment tools . where the tca patient differs is that many do not have extensive co - morbidities or coronary artery disease ; their arrest is primarily due to one of or a combination of factors : hypovolemia , obstruction of blood flow and hypoxia . in the patient with thoracic trauma ,
these causes are addressed by well described techniques of thoracostomies , endotracheal intubation and blood products .
this is followed by a clamshell thoracotomy , allowing for a better chance for hemorrhage control and treatment of hemopericardium if found .
sherren and colleagues discuss a very well outlined pathophysiology and rationale for tca survivorship in their article .
although mca and tca are not the same diseases , their survival statistics are interesting and possibly some inference can be made when mca data are more closely observed .
it appears that the differences between mca survival in the prehospital versus a hospital setting , where admitted patients are older with increased morbidities , might be due to the personnel performing the resuscitation .
staffing models for emergency medical services ( ems ) , especially for hems , differ between the us system and the european model . in the us , pre - hospital ems systems ( including most hems ) are staffed by allied health professionals and not by physicians . in the european model ( including australia ) hems are staffed by well trained physician / paramedic / nurse teams and this is possibly where the difference occurs for survival for tca patients . in multiple european studies
it was noted that there was a decrease in mortality of trauma patients without a decrease in scene time when a physician was part of the flight crew [ 6 - 9 ] . in the us ,
less than 5% of hems are staffed by physicians , and most of those involved in such teams are in their first few years out of medical school .
thus , no us ems systems have fully trained physicians as part of their standard ambulance crew . a reasonable question to consider
is whether staffing is the reason why tca statistics differ between the us and european models ?
paramedic training teaches needle thoracostomy for suspected tension pneumothorax , which in many jurisdictions do not need online direct physician permission as it is deemed life saving and with limited risk .
however , it is well known that in patients with larger chest walls , almost 50% of the time the needle fails to reach the plural space to relieve the obstruction of blood flow caused by the tension pneumothorax . on the contrary ,
the placement of a tube or finger thoracostomy are very successful and fairly routine in ems systems staffed with experienced physicians with excellent results .
it can not be fully applicable to hems not staffed by a well trained physician , as the proficiency of thoracotomy is an advanced skill that takes practice and years of anatomical training to be able to perform well .
as the authors stated , more study is needed to see if it is the depth of training or the protocols that are the reason for the increases in survivorship from tca .
i do not believe that this algorithm will be promoted in hems systems staffed only by paramedics and nurses , as their level of training does not allow for advanced procedures such as these .
therefore , one should expect that prehospital attempts at the resuscitation of the tca in those systems without physicians on board will remain futile .
ems : emergency medical services ; hems : helicopter emergency medical service ; mca : medical cardiac arrest ; tca : traumatic cardiac arrest .
| traumatic cardiac arrest resuscitation is considered a heroic and futile endeavor . however , newer articles have more promising statistics and divide between prehospital ground and helicopter transport . here
we discuss why there might be a difference in the survivability of this subset of trauma patients . |
following advances in information - tailoring tools and telemedicine , medical information becomes increasingly available and individuals take a more active role in managing their personal health . at the same time , the environment in which patients consume medical and health information has changed dramatically .
the internet access rate has continued to increase in all industrialized countries over the past 15 years and several international studies suggest that between 50 and 80% of adults in developed countries with internet access , use internet as the main tool for health care management . because of importance of identifying the most effective delivery method for each target group for an intervention and due to the small number of studies and their variable methodological quality , it is important that all avenues of primary health information be explored .
after a decade of using internet in iran , little is known about the effects of this new technology on health information - seeking behavior .
although there has been much conjectures , it is unclear exactly how using internet can affects the ways in which individuals acquire health or medical information and what demographic characteristic are associated with use of different information channels .
we conducted this cross - sectional community - based study to provide representative estimates of health information sources and the changes that were occurring in the health information environment in tehran . here
we report data from the baseline administration of this study during two periods : at the beginning and the end of the first decade of using internet in iran to make precise population estimates for the public use of different sources of health information .
this community - based cross - sectional study was conducted in tehran during two different periods of time- in august 2002 and in august 2010 .
tehran , the capital of iran , as a metropolitan has a population of more than 13.5 million in the area of 730 square km .
the samples were selected by multistage cluster sampling from three different socioeconomic neighborhood of tehran that best represented the general population of tehran .
squares of each part were coded based on municipal data and one neighborhood randomly selected from each part using random numbers .
each randomly selected neighborhood was the primary units ( clusters ) and squares within the neighborhood were the secondary units .
then squares in each neighborhood listed and one of them selected randomly . by this way
, three squares were chosen from three different socioeconomic part of tehran including tajrish square in the north , vali - asr square in the center and shoush square in the south of the city . during these two periods of time participants
it was based on considering prevalence of internet users of 15% in 2002 ( based on pilot study ) and forecasting increase to 20% in 2010 , error type 1 as 5% , error type 2 as 20% and cluster coefficient of 20% .
totally 3000 participants of 18-years - old or more were interviewed . after providing detailed oral information to respondents and
if they were ready to be a participant , a questionnaire in close - ended format was filled as data collecting tool .
they were asked to choose the most likely tools , services and resources to access health or wellness - related information among the detailed list . if participants could not find their personal preferred source in the list , they could write it at the end of the questionnaire in an open - ended question .
participants also reported their gender , age , educational status and household income . in this study age
categorized as young adult ( 18 - 39 years ) , older adult ( 40 - 59 years ) and elders ( 60 years ) ; the respondents education was defined as the total number of years of education and categorized into three groups of low ( 0 - 12 years ) , moderate ( 12 - 16 years ) or high ( more than 16 years ) education . considering gdp per capita ( ppp ) for iran
estimated as $ 11300 , annual minimum household income level defined in three levels of < $ 11 000 , $ 11 000-$22 999 , and $22 000 .
the study was approved by the medical research ethics committee of shahid beheshti university of medical sciences and was carried out in accordance with the declaration of helsinki ( 1989 ) of the world medical association .
descriptive statistics were used to describe data summaries as percent of frequencies , mean and standard deviation .
different health information sources of participants with respect to demographic characteristics were examined using chi - square , t - tests and analysis of variance , when appropriate .
the result adjusted using population characteristics for sex , age and education from data publicly available through the current national population survey .
logistic regression analysis initially included patient age , sex , educational level and household income .
chi - square test was used for comparing the results of two periods of time .
all statistical calculations were performed using spss software , version 19.0 ( spss inc . ,
logistic regression analysis results were presented as the calculated odds ratio with 95% confidence intervals .
totally 3000 participants , which adequately completed questionnaires , were collected and tabulated , 1500 participant during each period of study , included in study . in 2002 , 878 participants ( 58.5% ) were male and 622 ( 41.5% ) were females ; they had a mean age of 33.4 years ( sd = 11.3 ) . in 2010 , 927 participants ( 61.9% ) were males and 573 ( 38.1% ) were females ; they had a mean age of 33.8 years ( sd = 11.2 ) .
moderate educational degree reported in near 58% of participants in both periods of time . in 2002 , the mean household income was 13580 $ per year and in 2010 it grows to14340$. two groups were homogenous in basic characteristics as presented in table 1 .
basic characteristic of participants and sources of health information based on sex and year of study in 2002 , radio and television used by 266 ( 17.7% ) , caregivers by 223 ( 14.9% ) and internet by 213 ( 14.2% ) and in 2010 were radio and television used by 290 ( 19.3% ) , internet by 289 ( 19.3% ) and caregivers by 237 ( 15.8% ) as the most common sources of health information .
sources of health information among study population based on educational level , sex and year of study in 2002 , among different sources of health information , the most common used source for seeking health information among low educated women was their family and friends ; whereas in low educated men it was their family and caregivers ( p < 0.001 ) .
women with moderate educational level got their health information from radio and television and caregivers .
men with moderate educational level use radio and television and internet as main source of health information ( p > 0.05 ) .
high educated women and men mainly got their health information from internet and radio and television ( p > 0.05 ) . in 2010 ,
the most commonly used source for seeking health information among low educated women was their friends and family , whereas in low educated men it was their family and radio and television ( p = 0.001 ) . in moderate educational level women got their health information from radio and television and caregivers ; while men used radio and television and internet as main source of health information ( p = 0.005 ) .
highly educated women and men mainly got their health information from internet and radio and television ( p > 0.05 ) . in 2002 , among low - incomes , family and friends were the most common source of health information for both men and women ( p = 0.009 ) . in moderate average of household income , women got their health information from radio and television and caregivers , while men used radio and television and friends as main source of health ( p > 0.05 ) .
high - income women and men got their health information from internet as the first source and men preferred caregivers and women used radio and television as the second choice of health information ( p > 0.05 ) . in 2010 , among low - incomes , family and friends were the most common source of health information for both men and women ( p < 0.001 ) .
in moderate average of household income , women got their health information from radio and television and caregivers , while men used internet and radio and television as main source of health ( p = 0.006 ) .
high - income women and men got their health information from internet and radio and television ( p > 0.05 ) [ table 3 ] .
source of health information in study population based on household income , sex and year of study table 4 shows the sources of health information in different age categories . in 2002 , young adults female used television and radio and male used internet as the main source of health information ( p = 0.07 ) .
elderly women used book and men used television as the pattern for seeking health information ( p = 0.055 ) .
source of health information in the study population based on age , sex and year of study in 2010 , young adults female used television and radio and male used internet as the main source of health information ( p = 0.003 ) .
elderly women used internet and men used television as the pattern for seeking health information ( p = 0.052 ) . using chi - square for comparing resources in 2002 and 2010 showed that there is a change in pattern of using resources generally ( p = 0.005 for men and p
= 0.006 for women ) and also based on educational level , household income and age categories as showed in tables 24 .
source of health information in each country depends on many factors such as age , gender , cultural and educational level of people and accessibility , intelligibility and reliability of sources . since , to the best of our knowledge
, there is little evidence about the effect of new communication technologies in seeking health information in iran , the purpose of this study was to get a better understanding of main patterns of seeking health information among iranian people and tracking changes occur during first decade after implementing internet in iran .
our study reveals prominent differences between the sociodemographic determinants associated with health information seeking environments with past researches in developed countries , especially in variables such as age , gender , household income and educational level .
this study showed that source of health information in tehran was changing steadily during last decade .
in addition , we observed a steady alteration in health information sources from traditional ones such as published media and books to internet . more than 16% of our study respondents reported that they had used the internet as first tool to find information about their medical problems or other health - related concerns .
conceivably , limited access to broadband connection as an important factor underlying health information seeking , low speed of internet in iran and lack of persian language health related website , can describe this lower rate .
one the other hand , our study depicted noteworthy increase in use of internet from 14% in 2002 to 19% in 2010 .
first , although during 8-year - period of study , radio and television as main source of health information did not change , a trend is observed is that , while few people have generally been informed through the internet in the past , if they had a specific question today , internet is the source they would use .
this findings claim , health agencies can justify making health information available online because many people use this channel , often before talking to their clinicians .
second , based on importance of cultural beliefs in health care process such as the position of iranian traditional medicine in public attitudes , persian language health - related websites could play a key role in general public health matters .
this study also examined the prevalence of using health information sources among different socio - demographic groups .
internet users were more commonly found among , men , younger age , higher - level educational degree and richer groups than other counterparts .
an examination of the basic characteristic of those surveyed shows that health information - seeking sources appear some resemblance between men and women .
both groups use radio and television as main source- in contrast , men use internet as second main source of health information whereas woman prefer to get their health information from their care givers . our pattern were similar to last decade of 20 century in developed countries .
although in early 1990s internet users were predominantly men , recent studies in other countries showed that women are more likely to use the internet for seeking health information.[1416 ] in addition , our findings indicate many differences in health information - seeking behavior between older and younger ages .
as might be expected , dispute young people prefer use internet , older people tend to seek health information from sources such as health care givers .
young men aged 20 - 39 are more likely to use internet , while young women in same ages are more likely to use radio and television as first choice to intake health information .
although radio and television still is the most common source of health information among men aged 40 - 59 , our study elaborate a significant reduction of tendency on radio and television ( from 42.5% in 2002 to 26.1% in 2010 ) , and rapid grows on using of internet as an information source ( from 11% in 2002 to 22.9% in 2010).[1820 ] among women of 40 to 59 , caregivers had a main role in provide health information .
meanwhile radio and television are more apt to be cited as a source among men aged 60 and above , lower rate of using internet as general , unfamiliarity with new technologies and lack of english language knowledge can be the underline reasons for this lower rate .
caregivers are more apt to be cited as main source among women in same ages .
overall our study showed , online information - seeking behaviors slightly more prevalent among individuals aged-45 years than other age groups .
based on these data policy makers can organize their delivery channels of health information to each target group more efficient and with ease .
for instance , it is clear that some health topics such as sexually transmitted disease , in our culture are not suitable for exposing to discussion in public media like radio and television .
based on our results that showed , younger people use internet more than older ages , it seems good opportunity for policy makers to use internet for their sexual health educational programs without concerning of cultural taboos .
we also wish to argue that governments would not need to be the sole method for highlighting reputable health information sources . instead , online health providers and private sector can play an active role in this process based on demographic data and trends of each group of society .
the data also show that internet users are more likely to have higher educational attainment , and have higher incomes than those who do not use the internet as main source , which is compatible with other studies.[10172224 ] an implication of this finding may be that people with limited education have not enough information or knowledge about usage of internet .
second , the relation between higher education and higher income in most cases can illustrate the higher rate of use of internet in wealthier people
. however , this finding could also be the result of statistical error based on the low rate of participation in this study overall as well as the few number who did not report their income correctly .
information technologies such as the internet are receiving great attention for their potential impact on public health and health care in health care reform policy discussions , our result indicates that iranian pattern for seeking health information became similar to that observed in resource - rich countries , where consumers are using technology to expand and enhance rather than replace their existing sources of health information .
prominent dissimilarity between our results and other studies is about the role of published media as source of health information in iran .
this change might be reflective of developmental changes during past decade in urban area of iran .
in conclusion , this study found that , in the context of health , through in a recent decade in iran the internet became most widely used as an information resource . obviously , this is not an exhaustive study or analysis , but there is a noticeable point to consider here .
the difference of 5% in tendency to using internet among different sources during 8 years is very important for policy makers .
as our results show , it may be premature , however , to embrace the internet unilaterally as an effective asset for efforts that target broad segments of the public .
the internet is a promising tool for public health and health care and a potentially effective platform for health communication and education .
accessible and reliable environment for entrance to health information cause empowerment , lower health care costs , patient - driven , healthcare self - management and more easily disease prevention .
there is growing preliminary evidence that shows iranian urban citizens are increasingly relying on the internet to find health information . like the rest of the world
the internet has become a highly efficient tool for enhancing of health information . in the first half of the decade , the rate of increase of internet use in iran was 100% , although the rate of penetration was only 8% ( as a regional mean ) . in 2008 - 2009 ,
however , we have much to learn about the social reality of how diverse people communicate and change in the modern world .
while the findings of this study are meant to stimulate discussion about the role of the internet in changing source of health information among urban citizen of iran , there are some limitations .
because the sample in this study was limited to adults from three major geographic part of tehran , consequently the results may not be generalizable to broad populations of other cities , with less access to educational resources or more rural settings especially those whit - lacking access to the internet , more detailed information of the searching experience of people with different personal characteristics is needed .
additionally , although the data were produced using a rigorous methodology , they are from cross - sectional , self - report assessments and do not include medication conditions or past medical experience of subjects , as a result , may reflect certain confounder . furthermore , the measurement of health information seeking is rather crude in the current investigation .
it is possible that intensity and frequency of seeking behavior would indicate additional differences across age and sex , educational degree .
finally , we did not evaluate the ability of participant to judge information quality , for example , this brief measure does not capture specifically what types of medical information respondents were seeking , or if they found the information that was desired .
additional information about how information seeking is linked to other key health behaviors was not included in the current survey .
such information is an important next step in this area of survey research . modifying and adding survey items would enable better measurement of internet participation , especially in online support and social media mechanisms that could not be determined in the current research .
much work also remains to be done to evaluate the opportunities and pitfalls of electronic communication between patients and health professionals , and to integrate these tools into clinical practice if they prove to be effective , without disadvantaging those who have different preferences or those who benefit from more traditional modes of communication .
this kind of research has the potential to distinguish the role of employment status from having access to the internet at work . | background : following the entrance of new technologies in health information era , this study aimed to assess changes in health information sources of iranian people during past decade.methods:totally 3000 people were asked about their main sources of health information .
they were selected as two community - based samples of 1500 people of more than 18-years - old in two different periods of time in august 2002 and august 2010 from the same locations in tehran , the capital of iran .
data analyzed based on age group , sex , educational level and household income in two different periods of time using chi - square .
odds ratios associated with each basic characteristic were calculated using logistic regression.results:most common sources of health information in 2002 were radio and television ( 17.7% ) , caregivers ( 14.9% ) and internet ( 14.2% ) and in 2010 were radio and television ( 19.3% ) , internet ( 19.3% ) and caregivers ( 15.8% ) ( p < 0.001 ) . in 2010 , young adults female used television and radio and male used internet as the main source of health information ( p = 0.003 ) . in moderate educational level women got their health information from radio and television and caregivers ; while men used radio and television and internet as main source of health information ( p = 0.005 ) .
highly educated women and men mainly got their health information from internet and radio and television ( p > 0.05).conclusion : although during 8 years of study radio and television remained as main source of health information but there is an increasing tendency to use internet especially in men .
policymakers should revise their broadcasting strategies based on people demand . |
like in other branches of medicine , in psychiatry too , most clinicians and researchers are concerned , if not really obsessed , with what is the most recent advance in a particular field or area .
enlightened readers scan the references list and are suitably impressed if there is a generous sprinkling of recent ones .
writers , similarly , want to refer to recent works as far as possible , and sometimes may go out of their way to accommodate them in an effort to appear abreast of contemporary literature . the more recent the work quoted , the greater the impact , and equally greater the research writer 's satisfaction.subtle psychological factors play their role here no doubt , as does the not so subtle need to impress and hold the interest of one 's readers , and one 's peers .
ofcourse , in this there is always the danger that something time - tested and well - proven may get sidelined for the new and promising , yet unproven .
this can no doubt also disturb , if not damage , so many personal and collective equations .
the concern with keeping abreast of recent advances makes us knowledgeable , or at least seem so , and does serve to impress our audiences favourably .
but somewhere down the line is also involved the legitimate fear whether we sideline something proven for extra - scientific reasons .
when lithium was first introduced as the treatment of choice in bipolar disorders , there was a great welcome for its wonderful effects , both its anti - manic as well as its prophylactic properties . and justifiably so . over a period of years
, however , the enthusiasm seems to have settled to cautious questioning , and has even given way to skepticism in a number of quarters ( silverstone et al 1998 ; gershon and soares , 1997 ; moncrieff , 1995 ) . even if the us fda approved only of lithium as prophylactic for bipolar disorders , a number of studies in the last decade find that 20 - 40% of classical bipolar patients ( i.e. ones with clear cut episodes and remissions ) show less than desirable response to lithium , or develop undesirable side effects . and amongst the nonclassical cases ( like rapid cyclers , dysphoric or mixed states , and the ones with co- morbid substance abuse ) , the situation is still less satisfactory .
valproate and olanzapinehave been approved by the us fda as antimanic drugs but not as mood stabilizers .
number of clinicians down the years have switched over to carbamezapine for its anti - manic , antidepressant as well as mood stabilizing properties .
but now the emphasis seems to be shifting from carbamezapine to the other anticonvulsants , especially in the treatment resistant cases .
valproate , lamotrigine , gabapentin , topiramate and tiagabine are the ones holding promise . as also the atypical antipsychotics like clozapine , olanzapine , quetiapine , ziprasidone , risperidone and aripiprazole .
( see grunze and moller , 2003 , for a review ofrecent work in the field . )
has lithium suddenly become no good ? has carbamezapine remained only a hero of yester years ?
the new blue - eyed boys , the new stars on the firmament ? is every medicine to experience its hey days , and sink into oblivion , like film stars and other celebrities ? do we , as clinicians and researchers , unduly favour the new by neglecting the old but time -tested ? more pertinent to the issue here
the pharmaceutical industry , the superspecialist researcher committed to only one circumscribed area of research , who must keep churning out something new to capture audiences , get research grants , generate funds and profits ?
research in science has followed the path of refutation ( popper , 1968 ; 1969 ) . when something is systematically refuted , something new can , and must , be accepted .
sticking to the old and discarding the new in spite of evidence to the contrary can be as reactionary as believing in the new and rejecting the old to appear modern . or being coerced into accepting it because of impressive portrayals by
. for , just as obsession with theold is stifling , that with the new can be equally anarchical ( singh and singh,1988 ) .
this requires an attitude in the scientist of being willing to be proved wrong ; though he may believe a theory , he should not be committed to it , or give it his unreserved loyalty ( slater,1973 ) .
human beings have a strong tendency to look for explanations and to seek final answers .
it is often perplexing to find several competing explanations being advanced to account for the same phenomena .
in fact the more complex the phenomena , the greater the number of diverse opinions that emerge .
ofcourse all these viewpoints can not be valid . some will stand the test of time and scientific scrutiny , some must indeed be discarded , or become history . but the applicability of a viewpoint is often determined by the extent to which it seems helpful in understanding a given case .
historically , theoretical orientations in science typically retain a strong hold over their adherents , even in the face of discomforting evidence and equally plausible alternative explanations of observable phenomena ( kuhn , 1970 ) . they continue to do so until some new or fundamentally different insight is achieved that appears to resolve the problems left unsolved by the conflicting interpretations of the empirical data .
these new insights constitute paradigm shifts ( kuhn , 1970 ) , which involve fundamental reorganization of how people think about an entire field of science .
they parallel , in certain ways , the momentous cognitive shifts a child undergoes in gaining an adult understanding of the nature of the world , a process well described in the work of jean piaget ( carson , butcher and coleman , 1988 ) . hence ,
unless we change our paradigms , alter our frameworks of enquiry , raise new sets of questions , there can not be real breakthrough in scientific knowledge .
moreover , we must also understand why there is in science this constant concern with the new .
in fact we must thank some researchers for pointing it out relatively recently that it has indeed stood the test of time ( baldessarini and tondo , 2000 ) .
when we say that 20 - 40% of classical bipolar patients do not respond to lithium , we must not forget that it also means 60 - 80% of classical patients do . and
but what we can not also wish away is the fact that 20 - 40% of classical patients , and the majority of nonclassical ones , do not respond .
moreover , a problem that is solved no longer bothers us , but that which is not keeps rankling the mind .
patients who respond do not trouble us as much as those who do not . both the clinician and the researcher are concerned , and feel challenged , ( and rightly so ) , to find ways and means of resolving their difficulties .
hence the search for newer molecules , and newer treatment strategies , must legitimately be pursued .
the pharmaceutical industry and the superspecialist researcher can no longer remain whipping boys when viewed in this perspective , can they ?
also , let us not forget , that the numberof non - responders swells over the years , and that further adds to the discomfort , not only of patients and care - givers , but also of clinicians , researchers and their ancillaries .
moreover , subtle nuances of an illness , its processes and outcome , are only laid bare over a length of time .
the same must happen with bipolar disorders , lithium and the newer medications . or , for that matter , with any disorder and any treatment strategy . not just in psychiatry , but in medicine as a whole . and ,
both these needs are liable to be exploited due to unethical practices by market forces , both in the pharmaceutical industry and the research field .
while man worksmainly at the empirical level , pharmaceuticals work essentially at the business level , and science works fundamentally at the process level .
so , then , there we are . while obsession with the most recent can lead us astray and
should not become a fad or a game of one - up - manship , a healthy evidence - based acceptance of the new is essential , nay integral , to scientific advance and research in our field . in this
there is an inevitable process of sifting involved , as of personal liking , one 's vision for the future , contemporary predilections of peers and superiors , and the pulls and pressures of market forces , both in the research field and the pharmaceutical industry . *
the need for a healthy evidence - based acceptance of the new we must recognize , and encourage . this need the pharmaceutical industry also recognizes , and may sometimes be prone to exploit .
well , the branch of psychiatry , as indeed every other branch of medicine , has to beware of this .
q.1.how can we avoid exploitation of the needs of man and science by unethical practices carried out by market - forces , both in the pharmaceutical industry and the research field?q.2.is scientific advance always provisional ?
does it have no finality?q.3.are we justified in getting excited about recent advances , when what is recent today will become outdated tomorrow?q.4.will scientific progress ever lead to the best , or only , treatment , or explanation , in medicine in general and psychiatry in particular?q.5.how do we balance new knowledge with old findings?q.6.what is the litmus test on which every scientific breakthrough , old or new , should get tested?q.7.should be give up getting excited with recent advances ? then what should we get excited about , if at all?q.8.if refutation is the truth of science , then where is science ultimately going to lead us?q.9.paradigm shifts alter perceptions , reorient thinking .
but do they lead to real progress in knowledge , or does it simply mean giving up on one fad to get preoccupied with another?q.10.are there newer problems that need newer strategies in medicine , or most of the so - called new problems are generated by society and nurtured by man , or just recycled , and a whole pharmaceutical industry and the medical profession run their livelihood on it?q.11.are we becoming an overmedicated society in the name of newer and more advanced treatment strategies?q.12.is there any other way of looking at this problem ?
how can we avoid exploitation of the needs of man and science by unethical practices carried out by market - forces , both in the pharmaceutical industry and the research field ? is scientific advance always provisional ?
are we justified in getting excited about recent advances , when what is recent today will become outdated tomorrow ?
will scientific progress ever lead to the best , or only , treatment , or explanation , in medicine in general and psychiatry in particular ?
what is the litmus test on which every scientific breakthrough , old or new , should get tested ? should be give up getting excited with recent advances ?
then what should we get excited about , if at all ? if refutation is the truth of science , then where is science ultimately going to lead us ?
do they lead to real progress in knowledge , or does it simply mean giving up on one fad to get preoccupied with another ? are there newer problems that need newer strategies in medicine , or most of the so - called new problems are generated by society and nurtured by man , or just recycled , and a whole pharmaceutical industry and the medical profession run their livelihood on it ?
are we becoming an overmedicated society in the name of newer and more advanced treatment strategies ? | most clinicians and researchers are concerned with recent advances in psychiatry .
this involves the danger whether something time - tested may get sidelined for extra - scientific reasons .
that the pharmaceutical industry and superspecialist researcher may keep churning out new findings to impress audiences is only a partial truth .
research progresses by refutation and self - correction .
acceptance in science is always provisional ; changing paradigms , frameworks of enquiry and raising new questions is integral to break through in scientific knowledge .
hence , there is in science a constant concern with the new .
moreover , the number of treatment non- responders to the time - tested swells with time , and researchers feel challenged to find ways and means of resolving their difficulties .
newer challenges need newer strategies .
obsession with the most recent can lead us astray , but a healthy evidence - based acceptance of the new is essential for advancement in psychiatric research . as indeed of research in all fields of medicine . and of science in general .
the role of lithium and newer mood stabilizers in bipolar disorders are taken as examples to highlight this point . |
a 29-year - old woman , being artificially inseminated with twins , was hospitalized to get a cesarean section .
a cesarean section was scheduled for her on the next day , but sudden pain and progression of the childbirth delivery forced her to the childbirth delivery room early . as she had undergone a vaginal delivery 3 years ago ,
after entering the operating room , the surgeons made the parturient woman take the lithotomy position to try a vaginal delivery again , but it was unsuccessful .
spinal anesthesia was administered to the patient since she had spent only 5 hours fasting .
the surgeons put the patient in the right lateral decubitus position and disinfected the puncture part between the l3-l4 spinous processes with betadine solution .
then , the surgeons inserted 26 g quinke , a spinal needle , into the puncture part by the midline approach method to verify whether the cerebrospinalfluid ( csf ) would smoothly release . after seeing a smooth flow ,
the surgeons injected 10 mg of hyperbaric 0.5% bupivacaine and 20 g of fentanyl . during the insertion of the needle and injection of the drug ,
the mother did n't complain of any pains or paresthesia on her back and lower limbs .
following the verification of the sensory blockade up to t6 thoracic vertebra 10 minutes after the drug injection , the surgeons began the operation for cesarean section .
after the delivery , a hartmann solution containing 20 units of oxytocin was injected ; however , the vaginal contraction was still insufficient . therefore , the surgeons injected 0.2 mg of methylergometrine into her triceps brachialis muscle and 500 ml of normal saline containing 1 mg of sulprostone into her body at the rate of 100 ml per hour .
thirty minutes after the delivery , when her blood pressure declined to 70/40 mmhg , 8 mg of ephedrine was injected , and her blood pressure returned to the normal level and stayed at 110/60 mmhg afterward . for the rest of the operation time , there was nothing unusual . following the operation ,
the mother was brought to the postanesthetic recovery unit to be observed for 1 hour .
after the doctors found that her sensory blockade was maintained up to t12 thoracic vertebra and there was no abnormality , they decided to move the patient to her ward .
two days after the operation , the anesthesiology department was asked for a joint treatment because the patient complained of numbness and paresthesia on her left leg .
although the mother said that she had paresthesia on her left femoral region , the doctors could n't find any dyskinesia , decline in strength , lumbago , pooling of urine and feces , or paresthesia on the perineal region .
the authors scanned her lumbar vertebrae with magnetic resonance imaging ( mri ) to verify epidural hematoma or abscess , but nothing unusual was discovered .
the authors also conducted a blood test to determine infection and inflammatory condition , and obtained the result that her white blood cell ( wbc ) count was 6,400/l and erythrocyte sedimentation rate ( esr ) was 13 mm / hr , both of which are normal .
subsequently , the authors decided to remain on the watch without additional treatment , considering that , according to neurological and laboratory tests , the patient had little possibility of nerve injury from spinal anesthesia , epidural hematoma , and abscess . however ,
after the patient continuously complained of the symptom for days , needle electromyography and nerve conduction studies were conducted on her truncus and lower limbs to identify any lesions in the nerve root and peripheral nerve . as a result
, the sensory nerve conduction velocity ( sncv ) test discovered a left lateral cutaneous nerve injury while the needle electromyography study did n't find anything unusual ( fig .
, the patient said that her symptom was reduced when her left pelvis was compressed .
considering her symptom , which was limited to left lateral cutaneous nerve , and the result of the pelvic compression test , the authors diagnosed her as meralgia paresthetica and prescribed nonsteroidal anti - inflammatory drugs for conservative treatment .
she was allowed discharge and was traced as an outpatient for a month . according to the result of the observance after discharge , the numbness on her left femoral region disappeared without special treatment , and complete blood cell count ( cbc ) and esr were also within normal levels .
during pregnancy , skeletal muscles and ligaments of parturient women become relaxed , and , with the progress of pregnancy , uteri expand and lordoses of lumbar vertebrae increase . for those reasons ,
this can be attributed to the following accidents in the process of delivery or operation : neurotmesis , damage in kidneys or blood vessels , or compression .
postpartum nerve injury often occurs to primiparas and is related to cephalopelvic disproportion and midforceps delivery .
especially , it happens more frequently if primiparas have prolonged pains during their second stage of labor .
the nerve injury mainly results from endogenous factors , whereas direct damage caused by regional anesthesia is rare .
generally , neuraxial anesthesia is greatly favored to reduce pain during labor or to conduct a cesarean section .
if parturient women show some neurotic symptoms after neuraxial anesthesia , doctors need to verify mass lesions , such as epidural hematoma or abscess .
if diagnosis is delayed for more than 8 hours , the nerve injury can become permanent , thus early diagnosis and treatment is required . in this research ,
2 days after the operation , when the patient complained of paresthesia on her left femoral region , unaccompanied by any decline in strength and pain , the authors administered an mri scan and blood test although epidural hematoma or abscess seemed unlikely .
also , the authors reckoned that the possibility of cauda equina syndrome was low since there was no pooling of urine and feces , sense deprivation on the perineal region , or declined strength of the lower limbs .
moreover , the author excluded the possibility of direct damage to spinal cords , cornus medullaris , and spinal nerve root because the patient did n't complain of any pain during the insertion of the spinal needle .
then , the authors conducted needle electromyography and nerve conduction studies on the patient 's truncus and lower limbs since they suspected an endogenous nerve injury . as a result , the authors were able to exclude the possibility of radiculopathy since the needle electromyography showed a positive result . however , the nerve conduction study discovered a left lateral cutaneous nerve injury , leading the authors to diagnose meralgia paresthetica . after the series of tests , the patient fully recovered , along with conservative treatment and follow - up observation .
the patient 's such experience was also in line with the development of meralgia paresthetica .
meralgia paresthetica , which affects 4.3 out of 10,000 people annually , is a kind of peripheral nerve disorder and is caused by mononeuropathy of the lateral femoral cutaneous nerve .
this disease can be characterized by numbness limited to the front femoral region , weakened response to a pinprick , pain , or the sensation of burning fire . generally , the symptom is temporary but hardly clear .
the lateral femoral cutaneous nerve stretches from under the anterior iliac spine to the under the inguinal ligament .
a lot of entrapments happen in this part of body and result in meralgia paresthetica .
the examples of the former are mechanical factors that increase the pressure inside the stomach , such as obesity , pregnancy , tight clothes and belts , and pelvic benign tumors .
meanwhile , diopathic reasons include pelvic osteotomy , orthopedic treatments including spinal surgeries , laparoscopic cholecystectomy , laparoscopic myomectomy , coronary artery bypass grafting surgery , aortic valve replacement surgery , and bariatric surgery for extreme obesity [ 8 - 10 ] . in this research
, the authors thought that the meralgia paresthetica of the patient was caused by the increased intra - abdominal pressure due to pregnancy and labor , as well as the lithotomy position , which compressed the lateral femoral cutaneous nerve .
meralgia paresthetica is diagnosed by physical examination such as a pelvic compression test , which determines whether specific clinical symptoms ( e.g. , numbness of front thigh and paresthesia ) and symptoms of the patient are improved when she is laid in the lateral decubitus position and her pelvis is pressed .
if the patient has symptoms in bilaterality or complains of dyskinesia , and abnormal senses lead to distinct pain , other diseases that may cause sense paresthesia , such as a herniated intervertebral disk of the lumbar vertebra or radiculopathy , should be detected . during a clinical examination
in which it is difficult to clearly diagnosis the patient , the diagnosis can be confirmed by a neurophysiological test .
it is known that almost all patients with meralgia paresthetica show abnormality in the sensory nerve conduction study of the lateral femoral cutaneous nerve . a neurophysiological test other than the sensory nerve conduction study , such as a somatosensory evoked potential , can be also helpful in diagnosis . in treatment
, most patients with meralgia paresthetica fully recover ( nearly about 85% ) within 4 - 6 months with conservative treatment .
as for conservative treatment , nonsteroidal anti - inflammatory drugs , tricyclic antidepressants , and anticonvulsants can help ease symptoms . in this casuistics
, only conservative treatment brought about an improvement in the symptoms in times of diagnosing meralgia paresthetica , but in the case of severe symptoms , 1 ) local nerve block therapy that uses 0.25% of bupivacaine and corticosteroid or 2 ) ultrasound guidance block therapy on the lateral femoral cutaneous nerve are known as effective .
a local steroid reduces excessive irritability of neurons and c - fibers . if those nerve block therapies do not work - though this would be a rare case - then operational decompression of the inguinal ligament , neurolysis of the lateral femoral cutaneous nerve , or transposition are recommended .
nerve injury that the mother suffers after part of the regional anesthesia has been administered is not necessarily caused by the regional anesthesia but can be triggered by pregnancy , delivery , or the patient 's position during operation .
however , as temporary nerve injury is not infrequent , anesthesiologists should differentiate post - delivery nerve injury from injury caused by neuraxial anesthesia , in order to prevent that patient from developing complications that can result in permanent disability . | meralgia paresthetica is commonly caused by a focal entrapment of lateral femoral cuteneous nerve while it passes the inguinal ligament .
common symptoms are paresthesias and numbness of the upper lateral thigh area .
pregnancy , tight cloths , obesity , position of surgery and the tumor in the retroperitoneal space could be causes of meralgia paresthetica .
a 29-year - old female patient underwent an emergency cesarean section under spinal anesthesia without any problems .
but two days after surgery , the patient complained numbness and paresthesia in anterolateral thigh area . various neurological examinations and l - spine mri images were all normal , but the symptoms persisted for a few days .
then , electromyogram and nerve conduction velocity test of the trunk and both legs were performed .
test results showed left lateral cutaneous nerve injury and meralgia paresthetica was diagnosed .
conservative treatment was implemented and the patient was free of symptoms after 1 month follow - up . |
a large number of patients suffer from sciatica , low back , and postoperative pain , treating which is one of the most important topics for clinicians in orthopedic surgery or anesthesiology .
nonsteroidal anti - inflammatory drugs ( nsaids ) have been commonly used but are effective only for treating inflammatory pain by inhibiting prostaglandin production .
understanding endogenous pain modulatory systems that alter nociceptive transmission in the nervous system may lead to the development of pain treatment .
the opioid receptor ( or ) and monoamine systems are the main inhibitory transmission mechanisms against pain sensation .
endogenous opioid receptor agonists are very powerful analgesic mechanisms . at the spinal cord level ,
clinically , or agonists , such as morphine , are commonly used for treating rather serious pain , such as postoperative pain .
monoamines , such as noradrenalin and serotonin , are also strong endogenous pain - relieving agents .
increasing monoamines with reuptake inhibitors has been used for pain relief in patients with several neuropathic pains .
we previously reported that serotonin reuptake inhibitors ameliorated neuropathic pain induced by spinal cord injury . in the study reported here , we focused on adenosine receptors , another endogenous nerve transmission system
. adenosine is an endogenous neuromodulator that inhibits synaptic transmission in both the peripheral and central nervous systems .
adenosine a1 receptor activation can produce postsynaptic inhibition by activating potassium ( k ) channels .
the working mechanism of adenosine a1 receptors on pain signals is believed to inhibit pain signals by hyperpolarization of the neuronal membrane .
these reports indicate that adenosine a1 receptor signal activation may become a new therapeutic method for treating several kinds of pain .
we previously reported that selective adenosine a1 receptor agonists [ r - pia : r(-)n6-(2phenylisopropyl ) adenosine ] inhibited thermal hyperalgesia after spinal cord injuries in rats . to establish a clinical use for adenosine , we examined which kinds of pain are the ideal targets for adenosine treatment and the best timing for adenosine application . in the study reported here , to clarify the effects of adenosine on pain signals , we tested intrathecal adenosine injections in several kinds of animal pain models .
in addition , change of adenosine receptors in the spinal cord was assessed by real - time polymerase chain reaction ( pcr ) and immunohistochemistry .
one hundred and one female wistar rats ( 250 g , purchased from japan clea co. , japan ) were used for this study .
experiments were performed according to the ethical recommendations of the committee for research and ethical issues of the international association for the study of pain .
the research protocol was accepted by the ethical committee for animal experiments of ehime university ( ehime , japan ) . a plantar incision ( pi )
a 1-cm longitudinal incision through skin , fascia , and muscle was made in the left hind paw .
the spinal cord compression model was produced according to our previous reports [ 6 , 11 ] . under general anesthesia with halothane ,
the rat spinal cord was carefully exposed by removing the vertebral lamina at the 11th vertebra .
direct compression was performed using a 20-g weight , of which the point of contact to the dura consisted of very soft and rounded silicone .
the weight was gently placed on the thoracic spinal cord extradurally for 20 min ( sci ) .
we observed no serious damage , such as hyperextension , paresis of the hind limbs , or histological hemorrhage with tissue destruction at the point of compression . in some experiments ,
our cci model was produced according to our previous report . under general anesthesia with halothane ,
a 27-gauge needle was placed along the sciatic nerve and the nerve and needle were ligated using polyglycolic acid strings ( 4 - 0 , nesco company , osaka , japan ) at four points .
too much constriction produces irreversible nerve damage ; therefore , careful constriction is required to produce the appropriate level of damage .
usually , stable hypersensitivity , such as allodynia and hyperalgesia , was observed 3 days after operation .
cl - adenosine ( 10 nmol in 10 l ; research biochemicals inc .
ma , usa ) , a nonselective adenosine receptor agonist , was intrathecally injected through the intervertebral foramen between l3 and l4 at 24 and 72 h after surgery . in each model , animals injected with 10 l of saline instead of cl - adenosine solution were defined as vehicles . to evaluate the withdrawal threshold of thermal paw stimulation , we used the hargreaves plantar test apparatus ( ugo basile , varese , italy ) .
rats were placed on a 2-mm - thick glass floor . a mobile infrared heat generator with an aperture of 10 mm diameter was aimed at the rat s hind paw from under the floor .
when the rats felt pain and withdrew their paw , the power was shut off and the reaction time ( paw withdrawal latency ) was recorded automatically .
the temperature of the glass floor was kept at 22.523.5 c . in the cci and the pi models , l4 and l5 lumbar spinal segments
1 cm of the cord at the center of the compressed part was taken .
spinal cord tissue was dissected under rnase - free conditions , and samples were stored at 80 c before use .
total rna was extracted by tissue homogenization in trizol reagent , quantified by absorbance at 260 nm , normalized , and reverse - transcribed into first - strand complementary dna ( cdna ) using an rneasy mini kit ( qiagen , md , usa ) . for the a1r gene ( takara .
shiga , japan ) , the forward primer was 5-atcgatacctccgagtcaagatcc-3 and the reverse primer was 5-tccagtcttgctctaccacactcag-3. for the gapdh gene ( takara ) , the forward primer was 5-ggcacagtcaaggctgagaatg-3 and the reverse primer was 5-atggtggtgaagacgccagta-3. two - step real - time pcr denaturing , annealing , and extension reactions were performed for 40 cycles of 15 s at 95 c and then 1 min at 60 c ( for a1r and gapdh ) . increasing curves of reporter dye fluorescence emission were recorded and analyzed with the sybr premix ex taq ( takara ) to determine the threshold cycle ( ct ) value .
each sample was run and analyzed in triplicate , and ct values for a1r were subtracted from ct values of gapdh to yield ct values .
the average ct was calculated for the control group , and this value was subtracted from the ct of all other samples ( including the control group ) .
this resulted in a ct value for all samples , which was then used to calculate the fold induction of the mrna levels of a1r using the formula 2 , as recommended by the manufacture ( bio - rad , hercules , ca , usa ) [ 15 , 16 ] .
the spinal cord at the 11th vertebral level ( sci model ) and l4l5 lumbar segments ( lumbar enlargement ; cci and pi models ) were immediately removed , and axial freezing microtome sections of 10-m thickness were prepared .
the sections were fixed on glass slides with 4 % paraformaldehyde in phosphate - buffered saline ( pbs ) for 5 min .
then , after washing twice with pbs , slices on the slides were exposed to an anti - adenosine a1r antibody ( abcam , inc .
cambridge , uk : 1 g / ml in pbs ) overnight at 4 c .
slices were then washed twice with pbs and exposed to a fluorescein isothiocyanate ( fitc)-conjugated anti - rabbit immunoglobulin g ( igg ) antibody ( sigma , mo , usa : 20 g / ml in pbs ) for 30 min .
analysis of variance ( anova ) , followed by fisher s protected least significant difference ( plsd ) , was used .
one hundred and one female wistar rats ( 250 g , purchased from japan clea co. , japan ) were used for this study .
experiments were performed according to the ethical recommendations of the committee for research and ethical issues of the international association for the study of pain .
the research protocol was accepted by the ethical committee for animal experiments of ehime university ( ehime , japan ) .
a plantar incision ( pi ) was made similar to that described by brennan et al . .
a 1-cm longitudinal incision through skin , fascia , and muscle was made in the left hind paw .
the spinal cord compression model was produced according to our previous reports [ 6 , 11 ] . under general anesthesia with halothane ,
the rat spinal cord was carefully exposed by removing the vertebral lamina at the 11th vertebra .
direct compression was performed using a 20-g weight , of which the point of contact to the dura consisted of very soft and rounded silicone .
the weight was gently placed on the thoracic spinal cord extradurally for 20 min ( sci ) .
we observed no serious damage , such as hyperextension , paresis of the hind limbs , or histological hemorrhage with tissue destruction at the point of compression . in some experiments ,
our cci model was produced according to our previous report . under general anesthesia with halothane
a 27-gauge needle was placed along the sciatic nerve and the nerve and needle were ligated using polyglycolic acid strings ( 4 - 0 , nesco company , osaka , japan ) at four points .
too much constriction produces irreversible nerve damage ; therefore , careful constriction is required to produce the appropriate level of damage . after the strings were inserted ,
usually , stable hypersensitivity , such as allodynia and hyperalgesia , was observed 3 days after operation .
cl - adenosine ( 10 nmol in 10 l ; research biochemicals inc .
ma , usa ) , a nonselective adenosine receptor agonist , was intrathecally injected through the intervertebral foramen between l3 and l4 at 24 and 72 h after surgery . in each model , animals injected with 10 l of saline instead of cl - adenosine solution were defined as vehicles .
to evaluate the withdrawal threshold of thermal paw stimulation , we used the hargreaves plantar test apparatus ( ugo basile , varese , italy ) .
rats were placed on a 2-mm - thick glass floor . a mobile infrared heat generator with an aperture of 10 mm diameter was aimed at the rat s hind paw from under the floor .
when the rats felt pain and withdrew their paw , the power was shut off and the reaction time ( paw withdrawal latency ) was recorded automatically . shortening withdrawal latency indicated thermal hyperalgesia .
in the cci and the pi models , l4 and l5 lumbar spinal segments were taken for messenger rna ( mrna ) measurement . in the sci model ,
1 cm of the cord at the center of the compressed part was taken .
spinal cord tissue was dissected under rnase - free conditions , and samples were stored at 80 c before use .
total rna was extracted by tissue homogenization in trizol reagent , quantified by absorbance at 260 nm , normalized , and reverse - transcribed into first - strand complementary dna ( cdna ) using an rneasy mini kit ( qiagen , md , usa ) . for the a1r gene ( takara .
shiga , japan ) , the forward primer was 5-atcgatacctccgagtcaagatcc-3 and the reverse primer was 5-tccagtcttgctctaccacactcag-3. for the gapdh gene ( takara ) , the forward primer was 5-ggcacagtcaaggctgagaatg-3 and the reverse primer was 5-atggtggtgaagacgccagta-3. two - step real - time pcr denaturing , annealing , and extension reactions were performed for 40 cycles of 15 s at 95 c and then 1 min at 60 c ( for a1r and gapdh ) .
increasing curves of reporter dye fluorescence emission were recorded and analyzed with the sybr premix ex taq ( takara ) to determine the threshold cycle ( ct ) value .
each sample was run and analyzed in triplicate , and ct values for a1r were subtracted from ct values of gapdh to yield ct values .
the average ct was calculated for the control group , and this value was subtracted from the ct of all other samples ( including the control group ) .
this resulted in a ct value for all samples , which was then used to calculate the fold induction of the mrna levels of a1r using the formula 2 , as recommended by the manufacture ( bio - rad , hercules , ca , usa ) [ 15 , 16 ] .
the spinal cord at the 11th vertebral level ( sci model ) and l4l5 lumbar segments ( lumbar enlargement ; cci and pi models ) were immediately removed , and axial freezing microtome sections of 10-m thickness were prepared .
the sections were fixed on glass slides with 4 % paraformaldehyde in phosphate - buffered saline ( pbs ) for 5 min .
then , after washing twice with pbs , slices on the slides were exposed to an anti - adenosine a1r antibody ( abcam , inc .
cambridge , uk : 1 g / ml in pbs ) overnight at 4 c .
slices were then washed twice with pbs and exposed to a fluorescein isothiocyanate ( fitc)-conjugated anti - rabbit immunoglobulin g ( igg ) antibody ( sigma , mo , usa : 20 g / ml in pbs ) for 30 min .
for statistical analysis , analysis of variance ( anova ) , followed by fisher s protected least significant difference ( plsd ) , was used .
in all three kinds of pain models , significant shortening of withdrawal latencies were detected from 24 h to 1 week after the surgery ( fig . 1 ) .
hyperalgesia was seen only ipsilaterally to the surgery side in the pi and cci models . in the foot contralateral to the operation ,
withdrawal latencies against thermal stimulation was identical to that in normal animals ( 11.1 0.24 s).fig .
plantar incision ( pi ; n = 6 ) , chronic constriction injury ( cci ; n = 6 ) , and spinal cord injury ( sci ; n = 15 ) were performed .
statistical significance compared with preoperation levels for each time point is represented with an asterisk ( * p < 0.05 ) time course of withdrawal latency by thermal stimulation following three kinds of pain models .
plantar incision ( pi ; n = 6 ) , chronic constriction injury ( cci ; n = 6 ) , and spinal cord injury ( sci ; n = 15 ) were performed . measurements were taken every 24 h until the 7th day following operations .
data are the mean standard error of mean ( sem ) . statistical significance compared with preoperation levels for each time point
is represented with an asterisk ( * p < 0.05 ) in the postsurgical model ( pi model ; fig .
2 ) , apparent thermal hyperalgesia was seen in the foot ipsilateral to the pi in the vehicle animal both at 24 and 72 h after the operation . when cl - adenosine ( 10 nmol in 10 l ) was applied 24 h after surgery
, pain threshold returned to normal ( p < 0.05 , 11.37 0.97 s ) .
however , the antihyperalgesic action of cl - adenosine was not seen when the injection was done 72 h after the operation . in the cci model ( fig .
3 ) , hyperalgesia was also seen in the foot ipsilateral to sciatic nerve constriction in the vehicle animal both at 24 and 72 h after the operation .
intrathecal injection of cl - adenosine normalized withdrawal threshold against thermal stimulation at 24 ( 11.36 0.69 s ) and 72 h ( 12.07 0.19 s ) after the operation .
the difference of the cl - adenosine effect in the pi and cci models was seen at 72 h after the operation . in the sci model ( fig . 4 )
, thermal threshold decreased both at 24 and 72 h after the operation compared with that in the sham - operated animals .
intrathecal injection of cl - adenosine significantly ( p < 0.05 ) increased withdrawal latency both at 24 ( 11.12 0.55 s ) and 72 h ( 10.47 0.67 s ) after the operation.fig .
2effects of cl - adenosine on thermal stimulation in the plantar incision model at 24 h ( a ) and 72 h ( b ) after surgery .
data are mean standard error of mean ( sem ) ( n = 6 in each time point ) . asterisk statistical significance ( * p < 0.05)fig .
3effects of cl - adenosine on thermal stimulation in the chronic constriction injury model at 24 h ( a ) and 72 h ( b ) after surgery .
data are mean standard error of mean ( sem ) ( n = 6 in each time point ) .
4effects of cl - adenosine on thermal stimulation in the spinal cord injury model at 24 h ( a ) and 72 h ( b ) after surgery .
data are mean standard error of mean ( sem ) ( n = 6 in each time points ) .
asterisk , statistical significance compared vehicle animal ( * p < 0.05 ) effects of cl - adenosine on thermal stimulation in the plantar incision model at 24 h ( a ) and 72 h ( b ) after surgery .
data are mean standard error of mean ( sem ) ( n = 6 in each time point ) . asterisk statistical significance ( * p < 0.05 ) effects of cl - adenosine on thermal stimulation in the chronic constriction injury model at 24 h ( a ) and 72 h ( b ) after surgery .
data are mean standard error of mean ( sem ) ( n = 6 in each time point )
. asterisk statistical significance ( * p < 0.05 ) effects of cl - adenosine on thermal stimulation in the spinal cord injury model at 24 h ( a ) and 72 h ( b ) after surgery .
data are mean standard error of mean ( sem ) ( n = 6 in each time points ) . asterisk , statistical significance compared
vehicle animal ( * p < 0.05 ) real - time pcr analysis was represented as folds to normal mrna content ( fig .
twenty - four hours after operation , adenosine a1r mrna expression significantly ( p < 0.05 ) decreased in the pi model ( 0.2 folds compared with the normal level ) . in the sci model ,
adenosine a1r mrna level decreased to 0.7 folds . on the other hand , in the cci model it remarkably increased ( 2.5 folds ) , at 72 h after operation in all three models it moved toward the normal level .
however , in the pi model , it was still lower than normal ( 0.7 folds).fig .
5comparison of adenosine a1r messenger rna ( mrna ) expression by real - time polymerase chain reaction ( pcr ) analysis in the normal group and the three pain models .
data are mean standard error of mean ( sem ) ( n = 3 in each column ) .
asterisk statistical significance compared with normal group ( * p < 0.05 ) comparison of adenosine a1r messenger rna ( mrna ) expression by real - time polymerase chain reaction ( pcr ) analysis in the normal group and the three pain models .
data are mean standard error of mean ( sem ) ( n = 3 in each column ) .
asterisk statistical significance compared with normal group ( * p < 0.05 ) adenosine a1r protein expression was evaluated immunohistochemically . in the normal animals ,
twenty - four hours after operation , a1 receptors were maintained in all three models ( data not shown ) .
three days after operation , a1 receptors were rather increased in the dorsal horn of the cci model .
a1 receptor levels in the sci model were similar to that in the normal animal . on the other hand ,
a1 receptor proteins remarkably decreased in pi models 3 days after operation compared to the other two models and normal control ( fig .
seventy - two hours after operation , spinal cord sections from the 11th vertebra of the spinal cord mild - compression ( sci ) model and l4l5 lumbar segment [ lumbar enlargement ; plantar incision ( pi ) and chronic constriction injury of sciatic nerve model ( cci ) models and normal rat ] were stained by anti - adenosine a1r antibody .
normal rat ( a ) , pi model ( b ) , cci model ( c ) , sci model ( d ) .
apparent decrease in adenosine a1r expression was observed in pi model adenosine a1r protein expression in spinal cord dorsal horn .
seventy - two hours after operation , spinal cord sections from the 11th vertebra of the spinal cord mild - compression ( sci ) model and l4l5 lumbar segment [ lumbar enlargement ; plantar incision ( pi ) and chronic constriction injury of sciatic nerve model ( cci ) models and normal rat ] were stained by anti - adenosine a1r antibody .
normal rat ( a ) , pi model ( b ) , cci model ( c ) , sci model ( d ) .
the dorsal horn of the spinal cord is believed to be one of the most important areas for pain signal transmission . in the spinal cord ,
adenosine a1 receptors are mainly distributed in postsynaptic neuronal - cells bodies and processes in dorsal superficial layers ( lamina ii ) .
we also found that dense adenosine a1 receptor proteins existed in lamina ii of the dorsal horn ( fig .
this area is not only for adenosine signal transmission but is also the main area of adenosine production .
ecto-5-nucleotidase is a membrane - anchored protein that hydrolyzes extracellular adenosine 5-monophosphate ( amp ) to adenosine .
ecto-5-nucleotidase is located on nociceptive neurons in dorsal root ganglia and on axon terminals in lamina ii ( substantia gelatinosa ) of the spinal cord .
thus , this area should be a main spot of endogenous pain relief by adenosine signaling .
we found the adenosine a1 receptor mrna remarkably decreased in the spinal cord after pi but increased after sciatic nerve constriction ( fig .
this suggests that differential working mechanisms of adenosine in pain modulation existed in postoperative and neuropathic pain conditions .
it is not clear why adenosine a1 receptor expression in the spinal cord dorsal horn was enhanced in our cci models .
a remarkable difference between the cci and pi models is microglia activation in the spinal cord .
microglia proliferation and activation occurs in the spinal cord in the cci model . on the other hand ,
the activated microglia in the spinal cord may enhance the pain signal by releasing nitric oxide or cytokines .
therefore , it is possible that adenosine a1 receptor mrna expression enhanced to counteract the effect of activated microglia in our cci model .
the change in adenosine a1 receptor mrna expression of the sci model was not remarkable compared with that in the cci model ( fig .
although activated microglia also enhanced adenosine a1 receptor production in sci model , direct ischemic damage might scale back the amount of a1 receptors .
the pathological pain mechanism of the sci model is different from that in the cci model .
sci - induced pain is reported to induce inhibition of tonic descending inhibitory mechanisms , such as serotonergic and noradrenergic signaling , in the spinal cord .
therefore , the working mechanisms of injected cl - adenosine were different between cci and sci models .
in the sci model , injected cl - adenosine may compensate damaged descending inhibitory signaling from brain to spinal cord . in the cci model , in contrast , sensitivity to adenosine was increased in the dorsal horn by augmented adenosine a1 receptor expression .
the reason for decreased adenosine a1 receptor expression in our pi model also remains unclear .
hippocampal a1 receptor immunoreactivity in rats with cortex - kindled seizures remarkably decreased compared with that in normal rats .
the number of a1 receptors in the hippocampus was also decreased in aged rats compared with young adult rats . in aged rats ,
in addition , the amounts of adenosine in the synaptic cleft further increased due to the decrease in adenosine transporter activity .
it is possible that repetitive overstimulation of increased extracellular adenosine may induce adenosine a1 receptor expression inhibition in these models . in the pi pain model ,
stimulation from the hind paw may accelerate adenosine triphosphate ( atp ) release from the afferent nerve terminals in the dorsal horn .
then , adenosine a1 receptor mrna expression may be depressed by overstimulation of adenosine in the same manner as in aged or kindled rat .
released atp , which is the source of extracellular adenosine , may be depleted with time ; however , adenosine a1 receptor expression inhibition continued for several days after pi .
as adenosine is a common substance existing in the entire nervous system , it inhibits nerve transduction at the baseline level .
when the adenosine a1 receptor was blocked at the spinal cord level , the threshold of thermal pain decreased ( heat hyperalgesia ) .
therefore , when adenosine a1 signal is reduced by decreasing adenosine a1 receptors , pain signals should be enhanced .
if this phenomenon is a rational control of the self - protection system , a1 receptor inhibition may induce hyperalgesia as cautionary notices to avoid further nociception .
the striking decrease of a1 receptor mrna occurred 24 h after pi ( fig .
5 ) , mrna level recovered within another 2 days , and change in a1 receptor protein occurred later than that in mrna level .
decreased a1 receptor protein was apparent 3 days after pi ( fig . 6 ) .
this indicates that desensitizing signals from adenosine are not effective for several days after incision .
on the other hand , the desensitizing mechanism of adenosine signals was maintained after nerve injury .
therefore , although adenosine is a potential pain - relief agent , application of adenosine may not be effective in postoperative pain compared with that in neuropathic pain . or agonists have been used to treat several types of neuropathic or postoperative pain . however , several studies suggest that or agonists , like morphine , show decreased analgesic potency against neuropathic pain . after peripheral nerve injuries ,
these reports suggest that or agonist application for treating neuropathic pain after a peripheral injury is not rational .
relatively large amounts of the agonist may be required to achieve a large enough analgesic effect , which may produce considerable side effects such as constipation or nausea / vomiting .
these results suggest that adenosine may be a reasonable choice for treating neuropathic pain that has not responded well to the opioids .
clinically , chronic neuropathic pain is one of the most serious pathological conditions in the field of orthopedic surgery . as a result of this study
, we propose a new possible therapeutic module for treating neuropathic pain after peripheral nerve injuries .
brain barrier permeability , intrathecal injection of adenosine a1 receptor agonists , such as cl - adenosine or r - pia using a continuous infusion pump may effectively inhibit chronic neuropathic pain .
alternative ideas to increase the adenosine effect are to increase extracellular adenosine concentrations by amplifying production by ecto-5-nucleotidase or to inhibit adenosine uptake or degradation .
propentofylline , an adenosine reuptake inhibitor with a blood brain barrier permeability , is reported to inhibit pain behavior after peripheral nerve injury in the rat .
adenosine reuptake , kinase , and deaminase inhibitors may provide an avenue for the development of novel therapeutic methods against neuropathic pain .
our study indicates that intrathecally administered adenosine may be an effective pain - relief therapy for neuropathic pain but is not effective on postoperation pain .
we also found a decrease in adenosine a1 receptors in the spinal cord dorsal horn of the pi model , which may explain the variation of adenosine effects among the three pain models .
this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited . | backgroundadenosine is an endogenous neuromodulator in both the peripheral and central nervous systems .
adenosine inhibits pain signals by hyperpolarizing neuronal membrane.methodsto clarify the effects of adenosine on pain signals , we tested intrathecal adenosine injection in two neuropathic pains ( spinal cord compression and chronic constriction of sciatic nerve ) and postoperative pain ( plantar incision).resultsin all three kinds of pain models , significant shortening of withdrawal latencies to thermal stimulation were detected from 24 h to 1 week after the surgery .
significant improvements of pain sensation were observed in all three models after intrathecal injection of cl - adenosine 24 h after surgery . at 72 h after surgery , intrathecal cl - adenosine injection inhibited hyperalgesia in the two neuropathic pain models but not in the postoperative pain model .
adenosine a1r messenger rna ( mrna ) expression significantly decreased in the plantar incision model .
adenosine a1r protein levels also decreased compared with the other two models and normal control.conclusionsthese results suggest that adenosine effectively inhibits pain signals in neuropathic pain but is less effective in postoperative pain because of the decrease in adenosine a1 receptors . |
the phenomenon of
immigration has an immense impact on the health care of population in the united states .
cases of tuberculosis ( tb ) among foreign - born nationals , currently living in
the united states , account for more than half of the total .
also the absence of systemic symptoms does not rule out tb .
radiological imaging , sputum smear , and ppd only aid in the diagnosis but a
high degree of suspicion is required to ascertain an accurate diagnosis .
establishing an early diagnosis of tb infection and disease in a pregnant woman
a 21-year - old hispanic female with a 32-week twin pregnancy presented in
preterm labor .
she had immigrated to the united states
two years prior , and
her pregnancy course was uncomplicated except for complaints of an occasional
nonproductive cough .
she was a nonsmoker , denied any history of tb exposure ,
and was hbsag and hiv negative . on general exam , she was tachypneic and anxious
but in no apparent respiratory distress .
she was found to have a low oxygen
saturation of 89% on room air with a respiratory rate of 30/min and her blood
pressure was 127/76 mmhg .
her uterine
fundal height corresponded to 32-week twin pregnancy and she had bilateral
pedal edema .
she was initially admitted to the labor and delivery unit in our hospital and
subsequently transferred to the medical icu .
abg showed ph of 7.41 , paco2 of
29 , pao2 of 64 , bicarbonate of 18 , and the base excess of 6 , saturating at 93%
on 21% fio2 .
cbc showed wbc 9.9 th / mm , hb , and hct 10.8 g / dl and 32.8% ,
respectively , platelets 432 th / mm . the chest x - ray demonstrated a right
paratracheal soft tissue opacity measuring approximately
4 4 cm with a slight
elevation of the right hemidiaphragm with a linear density in the right lower
lung consistent with atelectasis ( see figure 1 ) .
patient was put in respiratory isolation early on and the pulmonary team was
consulted .
ct showed a large extensive mediastinal soft tissue mass in the
right paratracheal region extending into the hilar and subcarinal region .
an
associated atelectasis on the right upper and lower lobe with questionable
alveolar infiltrate in the right lower lobe was also found .
an abdominal
ultrasound showed twin - twin transfusion with reversed end - diastolic flow on
umbilical artery doppler in twin b and the patient had an emergent low
transverse cesarean section the next day with delivery of two male infants .
infant a had apgars of 7 and 9 , and infant b had apgars of 0 , 4 , and 5 ,
respectively .
both infants went to the nicu , and the patient returned to the
micu .
successful
samples of sputum could not be obtained but a bronchoscopy with endobronchial
biopsy and bronchoalveolar lavage ( bal ) was done .
bronchoscopy showed a trachea
with diffuse mucosal
swelling and the mucosal abnormalities persisted throughout the right bronchial
tree .
she also had irregular tumor growth from the anterior wall of the
proximal bronchus intermedius and the base of the proximal right upper lobe
bronchus .
there was an abundant growth of staphylococcus aureus but no acid - fast bacilli ( afb ) , fungus ,
or hyphae from the bal .
the patient was started on vancomycin and clindamycin
to cover methicillin - resistant staphylococcus aureus ( mrsa ) which was later changed to moxifloxacin based on
sensitivities .
transbronchial biopsy of the right bronchus intermedius showed
inflammatory and squamous debris with ulceration , reactive squamous epithelium ,
and colonizing coccal bacteria .
the treatment of this young hispanic immigrant
female with an occasional cough , positive ppd , questionable alveolar
infiltrate , caseating granulomatous inflammation and no significant evidence of
tb infection posed a definite challenge .
given her high risk for tb , she was
empirically started on inh , rifampin , ethambutol , and pyrazinamide .
it was only
on day 26 that mycobacterium tuberculosis was isolated by dna probe from her right lung tissue which was subsequently
confirmed on culture .
the infants were started on inh prophylaxis after
placental pathology did not demonstrate evidence of tb .
patient was discharged
to home in a stable condition to be followed up at the local tb center for
direct observation therapy .
among the communicable diseases , tb is the second leading cause of death
worldwide , killing nearly 2 million people each year .
the world health
organization estimates that 2 billion people have latent tb , in addition to the
3 million people worldwide who die each year due to tb . after a resurgence of
tb in the united states
between 19851992 ,
the decline
noticed during 19932005 has slowed
down causing concerns that the progress achieved in eliminating tb is slowing
down .
immigration demographics demonstrate that asians , blacks , and
hispanics essentially bear the burden of tb in immigrants .
the number of tb
cases due to foreign - born individuals has increased each year since 1993 . in
1996 , 10% of the 20,973 us tb cases were among foreign - born hispanic persons ,
with the four states bordering mexico
accounting for 83% of foreign - born hispanic cases .
mexicans accounted for 22.9%
of foreign - born patients or 8.4% of all us cases and persons born in central america comprised an additional 4.7% of foreign - born
patients or 1.7% of all us cases .
guatemala and el salvador contributed to nearly 75% of these
cases and the remaining were from the rest of central
america .
in 2005 , when
compared between foreign - born persons in the united states
and us - born persons ,
the tb rates were 8.7 times higher in
immigrants ; with hispanics , blacks , and asians having tb rates 7.3 , 8.3 , and
19.6 times higher , respectively , than those of whites . m. tuberculosis is transmitted by airborne droplet nuclei , which may
contain fewer than 10 bacilli and humans are the only known reservoir for m.
tuberculosis .
individuals at high risk for m. tuberculosis infection in industrialized countries include ( i ) individuals likely to be
exposed to or become infected with m. tuberculosis : close contacts of a
patient with infectious tb , foreign - born individuals from high - incidence areas ,
the elderly , residents of long - term care facilities ( e.g. , correctional
facilities and nursing homes ) , iv drugs abusers , other groups identified
locally as having increased prevalence of tb ( e.g. , migrant farm workers or
homeless persons ) , and individuals who may have occupational exposure to tb ; and
( ii ) individuals at high risk of developing tb disease once infected : individuals
recently infected with m. tuberculosis ( within the past 2 years ) ,
hiv - infected individuals , individuals with immunosuppressing conditions or
medication use , individuals with a history of inadequately treated tb , and
infants .
chances that an individual acquires infection depend on the
infectiousness of the index case , duration of the exposure , environment
( crowding , poor ventilation ) , and virulence of the organism .
atypical presentations and slow confirmation by culture often delay the
diagnosis and treatment of patients with tb .
other reasons include an under use
of tuberculin skin tests , misinterpretation of unusual chest x - rays , and
waiting for culture results in patients with afb - negative smears .
generally ,
in tb , a segmental pneumonic process is seen but may be too small to visualize
radiographically or complicated by atelectasis from the raised diaphragm
compressing the lower lung fields in pregnancy [ 7 , 8 ] .
upper zone shadowing ,
often bilateral and likely associated with cavitation or miliary shadowing are
the classic findings .
new findings like soft
shadowing amongst old , fibrotic changes generally indicate a relapse .
paratracheal , mediastinal , and hilar lymphadenopathy , though not unusual in
african and indian patients with tb or in children , are generally unusual in
immunocompetent adults like ours . in patients infected with hiv , the
radiological appearances could often be nonspecific . in our patient
the infiltrate could have been misdiagnosed as atelectasis in a woman with a 32-week
twin pregnancy .
five
millimeters is considered to be positive in individuals with hiv , on steroid
therapy , or in individuals in close contact with a person with active tb .
larger reactions of greater than or equal
to 10 mm are considered positive in recent ( within the last five years )
immigrants from high - prevalence countries , in individuals with diabetes , renal
failure , and health - care workers , among others .
our patient falls into this
category . in individuals with no known risks for tb
many patients with suspected pulmonary tb do not produce sputum spontaneously
or are smear - negative for afb .
we could not obtain sputum sample in our patient
due to absence of a productive cough and sputum induction was also
unsuccessful .
though the bal sample did not show any afb , the biopsy showed
caseating granuloma which could be found both in tubercular and nontubercular
mycobacterial infections or fungal infections due to coccidioides immitis and histoplasma capsulatum .
it was ultimately a dna probe from her lung
tissue that grew m. tuberculosis after 26 days . with the use of pcr
, nucleic acid sequences unique to m. tuberculosis can be detected directly in clinical specimens with better accuracy and urgency
than afb smear and culture , respectively .
probes are used for rapid
identification and maximizing cost effectiveness . used alone or in combination
with other identification methods , they serve as a substitute for biochemical
testing and are also more accurate .
can enhance the diagnostic ability particularly in
pauci - bacillary infections and in patients with atypical presentations like
ours .
the risk of tb to health - care workers is real . in the early 1990s ,
many urban us
hospitals reported purified protein derivative ( ppd ) conversion rates in health - care
workers of 35% .
a survey of us
hospitals conducted by the centers for disease control and prevention ( cdc )
found a mean conversion rate of 1.6% [ 16 , 17 ] .
efficient control of nosocomial tb is
compromised by the same difficulties complicating community control hence a high degree of suspicion is required to recognise and isolate patients with
tb .
an improved clinical acumen , development of rapid diagnostic tests , and the
institution of early empiric therapy are desirable to control this disease .
we
combined the use of bronchoscopy and m. tuberculosis complex - pcr which
provided a good diagnostic yield in our patient .
an early diagnosis of tb infection in a pregnant woman is important as better
results are seen in women who are detected with tb and have been treated before
the onset of pregnancy or earlier in its course , when compared with untreated
patients with tb .
infant and
maternal mortality are between 30% and 40% in untreated active tb cases .
treatment should also be initiated when the probability is moderate - to - high .
although the drugs in the initial treatment regime cross the placenta , these
concentrations do not appear to have harmful effect on the fetus .
pregnant
women with tb should also be tested for hiv as there is a higher incidence of
extrapulmonary tb and multidrug - resistant tb ( mdr tb ) in this set of patients .
breast feeding should not be discouraged in women being treated with first - line
antituberculosis drugs because the concentrations in the breast milk are
subtherapeutic and too low to produce toxicity in the nursing new born .
the effect would likely be much
lower if the mother breast feeds before taking the medication .
similarly ,
breast milk is also inadequate as a treatment option for tb or latent tb
infection in newborns .
close follow up of patients is essential since
current therapy for tb infection is long and suboptimal adherence may result in
failure of therapy .
congenital tb is rare with symptoms typically developing during the second or
third week of life which include poor feeding , poor weight gain , cough ,
lethargy , and irritability .
two possible routes of m. tuberculosis infection in utero
are ( a ) hematogenous infection through the umbilical vein , with primary lesions
in the liver and occasional porta hepatis lymphadenopathy ; and ( b ) prenatal
aspiration of infected fluid , with predominant pulmonary and gastrointestinal
disease [ 21 , 22 ] .
the criteria for congenital tb requires the infant to have a
tuberculous lesion ( e.g. , infiltrates on the chest radiograph or granulomas )
and at least one of the following : ( a ) onset during the first week of life , ( b )
presence of a primary hepatic tb complex or caseating hepatic granulomas , ( c )
infection of the placenta or maternal genital tract , or ( d ) exclusion of
postnatal transmission .
the role of bacillus of calmette - gurin ( bcg ) vaccine in preventing tb in
adults is debatable due to its variable efficacy ( 080% ) .
its efficacy in
prevention of tuberculous meningitis and miliary tb in young children has been
easier to document than in adults or in the prevention of pulmonary tb in both
children and adults .
the centers for disease control has made the
following recommendations concerning bcg vaccination in the us
.
( i )
bcg vaccination should be considered in infants and children 5 years if
the child is exposed continually to an untreated or ineffectively treated
patient with infectious pulmonary tb , if the child can not be separated from the presence
of the infectious patient or be given long - term primary preventive therapy , or if the child is exposed
continually to a patient with infectious pulmonary tb caused by m.
tuberculosis strains resistant to isoniazid and rifampin and the child
can not be separated from the presence of the infectious patient .
( ii ) health - care
workers in high - risk settings should be considered on an individual basis
in settings in which a high percentage of tb patients are infected with m.
tuberculosis strains resistant to both isoniazid and rifampin , transmission
and subsequent infection are likely and where comprehensive tb
infection - control precautions have been implemented and have not been
successful .
our case emphasizes the significance of identifying infectious diseases like tb
in developing countries due to a change in demographics .
it also underscores
the importance of an early diagnosis and treatment even in the backdrop of
confounding evidence in pregnant women due to better maternal and perinatal
outcomes .
a heightened index of suspicion and awareness of the rapid advances
and innovations made in the diagnosis of tb is also essential for better
control of this disease . | background . with the world becoming a global village ,
tuberculosis is no longer limited to endemic areas .
our case emphasizes the impact of immigration on infectious disease epidemiology and challenges associated with diagnosis and treatment in pregnancy . case .
a 21-year - old hispanic female presented in preterm labor and was found to be hypoxic .
chest x - ray revealed a paratracheal mass which a ct scan confirmed .
ppd test was positive .
bronchoalveolar lavage did not reveal acid - fast bacilli and biopsy revealed caseating granulomas .
diagnosis and treatment were challenging due to constraints in radiological investigations , lack of initial evidence of acid - fast bacilli , and toxic profile of medications . due to her high risk , she was started on antituberculosis regimen . the diagnosis was confirmed on day 26 when mycobacterium tuberculosis was isolated by dna probe .
conclusion .
a high index of suspicion is required to recognize the changing face and disease spectrum of tuberculosis and initiate treatment for better outcomes . |
core stabilization exercises may prevent or improve
symptoms of low back pain because it provides the basis for force production by the
limbs2 .
broad benefits of core
stabilization have been reported , including improvement of athletic performance and
prevention of injuries3 .
the core muscles include the abdominal muscles in the front , paraspinal and gluteal muscles
in the back , the diaphragm as the roof , and the pelvic floor and hip girdle musculature at
the bottom .
this core is composed of 29 muscle pairs that help to stabilize the spine ,
pelvis , and kinetic chain during functional movements . without these muscles ,
traditional core
exercises , core stability exercises , ball and device exercises , free - weight exercises , and
noncore free - weight training have been studied as methods for core stability1 .
methods of increasing core stability also
involve the use of an unstable support surface ( uss ) , such as a ball or balance
platform4 , 5 .
the use of uss that increases trunk muscle activity may also be
effective during core stability training6,7,8,9 .
in addition , movements of the limbs and
body weight during core stabilization exercises can be used to provide resistance to trunk
muscles10 , 11 .
moreover , shoulder resistance exercises increase the endurance and
strength of core stability muscles12 .
several studies have been performed using uss during core stability training13,14,15,16 ,
but no study has addressed the effects of a uss on core muscle activities during a shoulder
exercise . therefore , the purpose of this study was to determine the effects of uss on trunk
and lower extremity muscle activities during pulley - based shoulder exercises .
subjects were volunteers recruited
from a university in seoul and were accepted for this study if they were in good health , had
no current musculoskeletal , neuromuscular , or cardiovascular problems , and had a normal
range of shoulder joint motion .
subjects were excluded if they had trauma or pain in the
trunk or a shoulder joint or a history of surgery .
in addition , those who had experienced
core stabilization training or therapy within the last 3 months were excluded .
subjects
signed an informed consent form prior to participation , and the study protocol was approved
by the institutional review board of the university of sahmyook .
forty - eight hours before starting the study , all subjects abstained from excessive
exercise .
a 5-min warm - up stretching exercise was conducted prior to the performing of the
exercises .
prior to electrode placement , sites were prepared by abrading the skin with fine
sandpaper and cleaning it with 70% isopropyl alcohol .
muscle
activations of the trunk were measured during pulley - based shoulder exercise ( pbse ) using
14-kg resistance on the uss .
pbse comprised shoulder abduction , adduction , flexion ,
extension , internal rotation , and external rotation .
the orders of the six pbse exercises
were randomized , and each exercise was repeated five times .
a metronome , set at 80 counts
per minute , was used during the exercises to control speed .
all exercises were conducted with the feet apart ( within shoulder
width ) and parallel to the shoulders .
abduction and flexion exercises were conducted from 0
to 90 of the shoulder range of motion with the elbow joint in extension .
adduction exercise
involved 90 to 0 of the shoulder range of motion with the elbow joint in extension .
extension exercise involved 180 to 90 of the shoulder range of motion with the elbow joint
in extension .
external rotation exercise involved internal rotation 45 to external rotation
45 with the elbow joint in 90 of flexion .
internal rotation exercise was conducted from
external rotation 45 to internal rotation 45 with the elbow joint in 90 of flexion17 . a pulley machine ( pulley ex , sanimed ,
ibbenbren , germany ) was used for all shoulder exercises .
the flat floor of the laboratory
was used as the stable support surface , and a pedalo - vestimed # 50
( diameter 50 cm , height 19 cm ) was used for the uss during shoulder exercises .
the top and
bottom of the pedalo device were connected by four fixed springs , and the top of the pedalo
device was able to move up and down in all directions . to measure trunk muscle activities ,
emg data were collected from the rectus abdominis ( ra ) ,
external oblique ( eo ) , and erector spinae ( es ) ; to measure lower extremity muscle
activities , emg data were collected from the rectus femoris ( rf ) , tibialis anterior ( ta ) ,
and gastrocnemius ( ga ) . for the ra , electrodes were placed 2 cm lateral , 1 cm superior , and
1 cm inferior to the umbilicus . for the eo
, the electrode was placed midway between the
anterior superior iliac spine and the most inferior aspect of the rib cage . for the es ,
the
electrodes were placed 2 cm lateral to the 3rd lumbar vertebra and parallel to the
lateral - most high iliac crest . for the rf , the electrode was placed midway between the knee
and iliac crest on the front of the thigh . for the ta
, the electrode was placed on the
lateral side of the tibia three - fourths of the distance from the knee to the ankle . for the
the locations of
all electrodes were determined using the method described by cram and kasman18 , and all electrodes were placed on the
dominant sides . a datalog p3x8 data acquisition unit ( biometrics ltd , gwent , uk )
surface emg signals were extracted with the biometrics analysis
software ( v7.50 ) in ascii and were processed using a root mean square ( rms ) algorithm in
myoresearch xp master edition 1.06 ( noraxon u.s.a .
the sampling
rate was set at 1,000 hz per channel , emg signals were band - pass filtered from 20 to 450 hz ,
and a 60-hz notch filter was used to reduce noise .
descriptive statistics were used to analyze general subject characteristics , and a one - way
repeated - measures analysis of variance was used for each muscle .
twenty healthy adult male subjects ( height , 176 5 cm ; weight , 70 9 kg ; age , 22
3 years ; values are presented as means sd ) participated in this study ( table 1table 1.subject characteristicscharacteristicsage ( years)22.4 2.7height ( cm)176.4 5.1weight ( kg)69.8 8.6dominant siterightvalues are expressed as mean sd ) .
values are expressed as mean sd the ra and es showed significantly less activation on the uss ( p < 0.05 ) during shoulder
external rotation , but they showed no significant difference on the uss during shoulder
abduction , adduction , extension , flexion , and internal rotation .
the eo and rf showed no
significant difference on the uss during any shoulder exercises ( table 2table 2.mean ( sd ) of the emg activity of trunk muscles on ussshoulder exercisestable support surfaceunstable support surfacerectus abdominis1 .
however , the ta showed significantly greater activation ( p < 0.05 ) on the
uss during shoulder abduction , adduction , external rotation , and internal rotation . on the
other hand ,
the ta showed no significant difference during shoulder extension and flexion on
the uss , whereas the ga showed significantly greater activation ( p < 0.05 ) on the uss
during shoulder abduction , extension , and internal rotation .
however , during shoulder
adduction , flexion , and external rotation , the ga showed no significant difference on either
a stable surface or the uss ( table 3table 3.mean ( sd ) of the emg activity of lower extremity muscles on ussshoulder exercisestable support surfaceunstable support surfacerectus femoris1 .
. internal rotation23.4 10.930.5 8.2**statistical significance ( p > 0.05 ) ) . * statistical significance ( p < 0.05 ) * statistical significance ( p > 0.05 )
this study indicates that the use of uss during pbse did not increase trunk muscle
activity . on the contrary ,
this is probably because of the compensation strategy of the ankle rather than
because of that the trunk muscles .
this ankle compensation is a postural control strategy
that first appears when there is a slight instability in the support surface .
balance in the
upright posture can be restored through muscle contraction near the ankle joint , and a close
relationship exists between this ankle strategy and the activities of the ta and ga . in the
case of postural sway under unstable conditions ,
balance is recovered by an ankle strategy ,
a hip strategy , or both simultaneously19 .
in the case of rapid shaking of the support surface ,
balance recovery is achieved via an
integrated ankle / hip joint strategy , and in the case of swaying of the support surface in an
upright posture , balance can be maintained by ankle movement without hip joint
extension20 .
in addition , to maintain
postural control , normal adults mainly use an ankle strategy , whereas elderly individuals
and children mainly use a hip strategy21 .
in this study , we compared muscle activities in young male subjects on stable and unstable
support surfaces .
based on the results , it would appear that compensation for the uss
involved an ankle strategy rather than trunk muscle activity .
shoulder abduction and
adduction exercises involve movements in the frontal plane17 , and postural sway from the uss occurs in the medial / lateral
direction .
the results showed significantly increased ta and ga activities ( p < 0.05 )
during shoulder abduction when uss was used but only showed significantly increased ta
activity during shoulder adduction .
although ga activity did not significantly increase
during shoulder adduction , muscle activity increased slightly . in this study , the use of uss
significantly increased ga muscle activity during shoulder extension ( p < 0.05 ) .
the
extension and flexion exercises involve movements in the sagittal plane19 ; thus , postural sway occurs as the center of gravity moves
forward and backward on the support base .
the maintenance of balance against such
forward / backward sway is dependent on the alternating activities of the ta and ga .
ga
activity begins before the body collapses in the forward direction , whereas ta activity
appears before collapse in the backward direction23 . in the present study ,
shoulder flexion resulted in no significant
differences in lower extremity or trunk muscle activity on the unstable surface .
however ,
the most activity was seen in the ta compared with the other muscles . in the normal upright
posture ,
the center of gravity is on the front side22 , and ankle dorsiflexor activity is low ; on the other hand , ankle
plantar flexor activity is high21 , 23 , 24 .
the ankle plantar flexor plays an important role in torque
adjustment at the ankle joint and in the maintenance of upright posture25 .
therefore , back sway occurs during shoulder flexion due
to use of uss , but because of the influence of the center of gravity at the ankle joint , it
appears to have no significant impact on ta activity due to the ankle dorsiflexors .
shoulder
external and internal rotation exercises change the center of gravity with a complex form in
the forward / backward direction as well as in the medial / lateral direction . during shoulder
external rotation on uss ,
the body sways in the lateral backward direction , and an ankle
strategy can be used to maintain balance . in this study ,
the ta activity increased
significantly on the uss ( p < 0.05 ) , showing that the ta compensated for lateral backward
sway via eccentric contraction .
shoulder internal rotation causes the body to sway in a
forward medial direction , and the ankle strategy is used to maintain balance in a manner
similar to that during shoulder external rotation ( p < 0.05 ) . in this study
, pbse may have been able to increase core stability , but using a uss to
enhance core stability may not be effective .
further confirmation of these results is necessary in larger , more diverse populations ,
including females and older individuals , and there is a need to measure the deep muscles
such as the transverse abdominis and internal oblique . | [ purpose ] the aim of the present study was to identify the effects of an unstable support
surface ( uss ) on the activities of trunk and lower extremity muscles during pulley - based
shoulder exercise ( pbse ) . [ subjects ] twenty healthy college students were included in this
study .
[ methods ] surface emg was carried out in twenty healthy adult men .
the activities
of trunk and lower extremity muscles performed during pbse using a resistance of 14 kg on
a stable or unstable support surface were compared .
the pbse included shoulder abduction ,
adduction , flexion , extension , internal rotation , and external rotation .
[ results ] on the
unstable surface , the rectus abdominis and erector spinae showed significantly less
activation during shoulder external rotation , but the extent of activation was not
significantly different during other shoulder exercises .
the external oblique and rectus
femoris showed no significant difference during any shoulder exercises .
the tibialis
anterior showed significantly greater activation during all shoulder exercises , except
flexion and extension .
the gastrocnemius showed significantly greater activation during
shoulder abduction , extension , and internal rotation .
however , during shoulder adduction ,
flexion , and external rotation , the gastrocnemius showed no significant difference .
[ conclusion ] the use of uss to increase core stability during pbse is probably not
effective owing to compensatory strategies of the ankle . |
data prospectively collected in the canadian alteplase for stroke effectiveness study ( cases ) were analyzed ( 16 ) .
this was a national prospective cohort study that led to the full approval of iv - tpa for acute ischemic stroke in canada .
sixty sites across the country participated over a period of 2.5 years and , where relevant , each center obtained institutional ethics approval for data collection .
patients were treated at the discretion of the site neurologist according to canadian guidelines for intravenous thrombolytic treatment in acute stroke ( 17 ) .
baseline demographic data were obtained as were pretreatment basic blood tests and data regarding timing of drug administration and stroke subtype as de - termined by the site investigator using the oxfordshire community stroke project classification .
baseline glucose was determined before iv - tpa administration , either by laboratory blood testing or from capillary blood .
the particular method used was determined by the site 's own usual practice and was not specifically recorded .
the severity of the baseline neurological deficit was assessed with the national institutes of health stroke scale ( nihss ) before treatment .
the nihss is a validated 11-domain systematic assessment tool that provides a quantitative measure of stroke - related neurologic deficit , such that a higher score corresponds to a more severe deficit ( mild 015 , severe > 15 , and maximum 42 ) .
the outcome was measured using the 7-point modified rankin scale ( mrs ) at 90 days by clinicians who were not necessarily blinded to patients ' baseline glucose values .
all patients underwent a follow - up head computed tomography ( ct ) scan at 2448 h. baseline and follow - up ct scans were centrally reviewed by a stroke neurologist and neuroradiologist blinded to clinical information , and the alberta stroke program early ct score ( aspects)was applied .
aspects is a validated 10-point score assessing the extent of infarction in strokes of the middle cerebral artery using noncontrast ct .
a lower score suggests a more extensive area of infarction ( minimum score 0 ) ( 18 ) .
sich was defined as any hemorrhage documented on a follow - up ct scan that was associated with a decline in neurological status within the first 24 h after thrombolytic treatment as judged by the site investigator , who was not blinded to clinical information .
asymptomatic ich involved hemorrhage on a follow - up ct scan without associated clinical deterioration .
mmol / l in accordance with other studies ( 10,13,19 ) . as a primary analysis
, patients were dichotomized into those with admission hyperglycemia and those without hyperglycemia and compared with regard to baseline characteristics , rate of sich , functional outcome at 90 days , and death .
a secondary analysis examined the association of these outcomes with admission glucose as a continuous measure .
fisher 's exact test and student 's t test were used to compare the two groups .
both binomial regression and logistic regression were used to develop models for the three outcomes of interest depending upon use .
the large sample size provided sufficient power to perform multivariable analysis . for graphical descriptions ,
multivariable logistic regression was used to plot adjusted curves . for tabular description of risk ratios ( rrs ) , multivariable binomial regression was used to identify predictors of outcome using a log - link function .
the final models were all parsimonious models , meaning that variables that were not significant at p < 0.05 or variables that showed no evidence of confounding were eliminated from the final models .
models were developed by backwards stepwise elimination beginning with the variables listed in table 1 .
baseline patient characteristics data are means sd , median , or n ( % ) .
not all percentages are calculated from the total number of patients because certain clinical variables were not available for all patients .
standard descriptive statistics were used to report the data . fisher 's exact test and student 's t test were used to compare the two groups . both binomial regression and logistic regression were used to develop models for the three outcomes of interest depending upon use .
the large sample size provided sufficient power to perform multivariable analysis . for graphical descriptions ,
multivariable logistic regression was used to plot adjusted curves . for tabular description of risk ratios ( rrs ) , multivariable binomial regression was used to identify predictors of outcome using a log - link function .
the final models were all parsimonious models , meaning that variables that were not significant at p < 0.05 or variables that showed no evidence of confounding were eliminated from the final models .
models were developed by backwards stepwise elimination beginning with the variables listed in table 1 .
baseline patient characteristics data are means sd , median , or n ( % ) .
not all percentages are calculated from the total number of patients because certain clinical variables were not available for all patients .
of 1,135 patients enrolled in cases , 1,098 had adequate admission glucose data for inclusion in the current study .
of these patients , median age was 73 years , 45% were women , 96% had an anterior circulation stroke , and 16% had a known prior history of diabetes . a glucose value of 8.0 mmol / l corresponded to the 75th percentile and , overall , 296 ( 27% ) patients had admission hyperglycemia .
hyperglycemia was present in 70% of those with diabetes and 18% of those without diabetes .
hyperglycemic patients were older ; were more likely to be non - caucasian ; were more likely to have atrial fibrillation , hypertension , congestive heart failure , coronary artery disease , and known diabetes ; and were less likely to be current smokers .
the greater prevalence of comorbid illness in the hyperglycemic group is probably explained by older age and the higher proportion of diabetes .
there were no differences in sex , stroke subtype , or baseline stroke severity between groups ( table 1 ) .
a total of 49 ( 4.5% ) patients had sich . among hyperglycemic patients , this proportion was 6.8% compared with 3.6% for nonhyperglycemic patients
( p = 0.03 ) , resulting in an unadjusted rr of 1.87 ( 95% ci 1.073.25 ) for sich in patients with baseline glucose > 8.0 mmol / l ( table 2 ) .
multivariable regression attenuated this relationship somewhat , yielding an adjusted rr of 1.69 ( 95% ci 0.953.00 ) .
outcome at 90 days and sich in hyperglycemic ( baseline glucose > 8 mmol / l ) and nonhyperglycemic patients with unadjusted and adjusted rrs ( after multivariate logistic regression ) data are n ( % ) .
not all percentages are calculated from the total number of patients because certain outcome variables were not available for all patients .
clinical outcomes at 90 days , unadjusted for confounders , are shown in fig . 1 .
only 80 ( 27.7% ) hyperglycemic patients experienced a favorable functional outcome ( mrs 01 ) compared with 316 ( 40% ) nonhyperglycemic patients ( p = 0.0002 ) .
the proportion of patients who were dead at the 3 month follow - up was also significantly higher among patients with admission glucose > 8.0 mmol / l ( 30.8 vs. 18.7% , p < 0.0001 ) .
unadjusted risk ratios for favorable outcome and death among hyperglycemic patients were 0.69 ( 95% ci 0.560.85 ) and 1.64 ( 1.312.06 ) , respectively .
patient outcome at the 90-day follow - up by baseline glucose ( unadjusted for other predictors of outcome ) .
mrs 01 , excellent outcome ; mrs 23 , moderate disability ; mrs 45 , severe disability ; and mrs 6 , dead .
after multivariable regression , adjusted risk ratios were 0.7 ( 95% ci 0.50.9 ) for favorable outcome and 1.5 ( 1.21.9 ) for death .
multivariable analysis showed that independent predictors of 90-day outcome and death were age , baseline nihss , baseline aspects , and admission glucose .
each of these had a comparable magnitude of association with outcome . when dichotomized as age > 80 years
, baseline nihss > 15 , and aspects > 7 , unadjusted rrs for favorable functional outcome were 0.64 ( 95% ci 0.520.80 ) , 0.40 ( 0.320.48 ) , and 1.53 ( 1.261.86 ) , respectively .
the negative association between admission glucose and functional outcome and death persisted even after exclusion of patients with sich , a known independent predictor of death and disability after thrombolysis ( 4,20 ) .
these relationships were not different for patients with and without diabetes ( no evidence of an interaction effect ) .
no heterogeneity was found with regard to the association between baseline hyperglycemia and a favorable clinical outcome when analyzed by oxfordshire community stroke project stroke subtype ( test , p = 0.80 ) or when mild strokes ( nihss 5 ) and moderate - to - severe strokes were compared ( test , p = 0.29 ) , suggesting a deleterious effect of hyperglycemia on outcome regardless of stroke subtype or severity . with glucose as a continuous variable , the predicted probability of sich adjusted for age , atrial fibrillation ,
onset - to - treatment time , and sex was calculated and represented as a fractional polynomial estimate of best fit ( fig .
similarly , the predicted probability of death at 90 days according to admission glucose , adjusted for age , baseline nihss , aspects , onset - to - treatment time , and sex was calculated and represented using a linear least - squares line of best fit ( fig .
finally , the predicted probability of a favorable outcome at 90 days , adjusted for age , baseline nihss , aspects , congestive heart failure , atrial fibrillation , and diabetes was determined , yielding an inverse relationship between admission glucose levels and the probability of achieving an mrs of 01 ( fig .
the predicted probability of a favorable outcome was also determined for only those patients with baseline glucose < 8 mmol / l . even among these nonhyperglycemic patients , a linear decline in the probability of a good outcome was seen with increasing glucose levels .
quadratic polynomial line of best fit with range and 95% cis adjusted for age , baseline nihss score , sex , onset - to - treatment time , and atrial fibrillation .
mmol / l of serum glucose , the relative risk of sich rises by 10% .
the line and 95% cis are based upon a linear regression of predicted probability of death adjusted for age , baseline nihss score , sex , onset - to - treatment time , and baseline aspects score .
for every 1 mmol / l rise in the baseline serum glucose , the probability of death at 90 days increases by an absolute risk of 2% .
the line and 95% cis are derived from a fractional polynomial regression of baseline serum glucose and the predicted probability of good outcome adjusted for age , baseline nihss score , baseline aspects , sex , and onset - to - treatment time .
for every increase of 1 mmol / l of baseline serum glucose , the relative risk of a good outcome falls by 12% .
of 1,135 patients enrolled in cases , 1,098 had adequate admission glucose data for inclusion in the current study .
of these patients , median age was 73 years , 45% were women , 96% had an anterior circulation stroke , and 16% had a known prior history of diabetes . a glucose value of 8.0 mmol / l corresponded to the 75th percentile and , overall , 296 ( 27% ) patients had admission hyperglycemia .
hyperglycemia was present in 70% of those with diabetes and 18% of those without diabetes .
hyperglycemic patients were older ; were more likely to be non - caucasian ; were more likely to have atrial fibrillation , hypertension , congestive heart failure , coronary artery disease , and known diabetes ; and were less likely to be current smokers .
the greater prevalence of comorbid illness in the hyperglycemic group is probably explained by older age and the higher proportion of diabetes .
there were no differences in sex , stroke subtype , or baseline stroke severity between groups ( table 1 ) .
rates of ich , 90-day outcomes , and mortality are shown in table 2 . a total of 49 ( 4.5% ) patients had sich . among hyperglycemic patients , this proportion was 6.8% compared with 3.6% for nonhyperglycemic patients
( p = 0.03 ) , resulting in an unadjusted rr of 1.87 ( 95% ci 1.073.25 ) for sich in patients with baseline glucose > 8.0 mmol / l ( table 2 ) .
multivariable regression attenuated this relationship somewhat , yielding an adjusted rr of 1.69 ( 95% ci 0.953.00 ) .
outcome at 90 days and sich in hyperglycemic ( baseline glucose > 8 mmol / l ) and nonhyperglycemic patients with unadjusted and adjusted rrs ( after multivariate logistic regression ) data are n ( % ) . not all percentages are calculated from the total number of patients because certain outcome variables were not available for all patients .
hyperglycemic patients experienced a favorable functional outcome ( mrs 01 ) compared with 316 ( 40% ) nonhyperglycemic patients ( p = 0.0002 ) .
the proportion of patients who were dead at the 3 month follow - up was also significantly higher among patients with admission glucose > 8.0 mmol / l ( 30.8 vs. 18.7% , p < 0.0001 ) .
unadjusted risk ratios for favorable outcome and death among hyperglycemic patients were 0.69 ( 95% ci 0.560.85 ) and 1.64 ( 1.312.06 ) , respectively .
patient outcome at the 90-day follow - up by baseline glucose ( unadjusted for other predictors of outcome ) .
mrs 01 , excellent outcome ; mrs 23 , moderate disability ; mrs 45 , severe disability ; and mrs 6 , dead .
after multivariable regression , adjusted risk ratios were 0.7 ( 95% ci 0.50.9 ) for favorable outcome and 1.5 ( 1.21.9 ) for death .
multivariable analysis showed that independent predictors of 90-day outcome and death were age , baseline nihss , baseline aspects , and admission glucose .
when dichotomized as age > 80 years , baseline nihss > 15 , and aspects > 7 , unadjusted rrs for favorable functional outcome were 0.64 ( 95% ci 0.520.80 ) , 0.40 ( 0.320.48 ) , and 1.53 ( 1.261.86 ) , respectively .
the negative association between admission glucose and functional outcome and death persisted even after exclusion of patients with sich , a known independent predictor of death and disability after thrombolysis ( 4,20 ) .
these relationships were not different for patients with and without diabetes ( no evidence of an interaction effect ) .
no heterogeneity was found with regard to the association between baseline hyperglycemia and a favorable clinical outcome when analyzed by oxfordshire community stroke project stroke subtype ( test , p = 0.80 ) or when mild strokes ( nihss 5 ) and moderate - to - severe strokes were compared ( test , p = 0.29 ) , suggesting a deleterious effect of hyperglycemia on outcome regardless of stroke subtype or severity .
with glucose as a continuous variable , the predicted probability of sich adjusted for age , atrial fibrillation , onset - to - treatment time , and sex was calculated and represented as a fractional polynomial estimate of best fit ( fig .
similarly , the predicted probability of death at 90 days according to admission glucose , adjusted for age , baseline nihss , aspects , onset - to - treatment time , and sex was calculated and represented using a linear least - squares line of best fit ( fig .
finally , the predicted probability of a favorable outcome at 90 days , adjusted for age , baseline nihss , aspects , congestive heart failure , atrial fibrillation , and diabetes was determined , yielding an inverse relationship between admission glucose levels and the probability of achieving an mrs of 01 ( fig . 2c ) .
the predicted probability of a favorable outcome was also determined for only those patients with baseline glucose < 8 mmol / l . even among these nonhyperglycemic patients , a linear decline in the probability of a good outcome was seen with increasing glucose levels . a : probability of sich by baseline glucose level .
quadratic polynomial line of best fit with range and 95% cis adjusted for age , baseline nihss score , sex , onset - to - treatment time , and atrial fibrillation . for each increase of 1 mmol / l of serum glucose ,
the line and 95% cis are based upon a linear regression of predicted probability of death adjusted for age , baseline nihss score , sex , onset - to - treatment time , and baseline aspects score .
for every 1 mmol / l rise in the baseline serum glucose , the probability of death at 90 days increases by an absolute risk of 2% .
the line and 95% cis are derived from a fractional polynomial regression of baseline serum glucose and the predicted probability of good outcome adjusted for age , baseline nihss score , baseline aspects , sex , and onset - to - treatment time . for every increase of 1 mmol / l of baseline serum glucose
this study further confirms the relationship between admission hyperglycemia and sich , death , and poor functional outcome in ischemic stroke patients treated with iv - tpa .
our findings bolster and expand on those of previous studies , the majority of which examined smaller numbers of nonthrombolyzed stroke patients and some of which analyzed hemorrhagic and ischemic strokes together ( 13,10 ) . published studies of baseline hyperglycemia in stroke patients treated with thrombolysis have been few , were generally small , and often did not report 3-month outcomes ( 4,6,1215 ) .
however , all have suggested an increased risk of poor outcome with elevated glucose , despite using different cutoff levels to define hyperglycemia ( from 7.7 to 10 mmol / l ) ( 4,6,1215 ) .
one study included 312 tpa - treated patients from the original national institute of neurological disorders and stroke iv - tpa trial and analyzed these together with 312 placebo - treated patients ( 4 ) .
this study showed that regardless of treatment assignment , as admission glucose level increased , the odds for a favorable outcome progressively decreased and the odds of sich increased ( 4 ) .
a recent post hoc analysis of 748 patients from the european cooperative acute stroke study ( ecass - ii ) , a trial of iv - tpa given within 6 h of stroke onset , examined the prognostic value of hyperglycemia at baseline and 24 h ( 6 ) .
patients were classified into four groups : isolated baseline hyperglycemia , isolated 24-h hyperglycemia , hyperglycemia persisting at both time points , and persistent normoglycemia .
thrombolyzed ( n = 384 ) and nonthrombolyzed ( n = 364 ) patients were analyzed together .
interestingly , isolated baseline hyperglycemia was not found to independently predict poor outcome or ich . in this study ,
the strongest predictor of poor outcome , death , and ich was persistent hyperglycemia at baseline and 24 h , although this observation was only true in patients without known diabetes .
the prognostic utility of hyperglycemia exclusively in those patients treated with iv - tpa can not be determined because this subgroup was not analyzed separately .
two other studies of tpa - treated patients , one with intravenous treatment and the other with intra - arterial treatment , also showed that elevated glucose was an independent predictor of ich ( 5,11 ) .
what remains unclear is whether hyperglycemia is merely an epiphenomenon of underlying stroke severity or if it is itself directly harmful to ischemic brain tissue .
after adjustment for clinical stroke severity , a dose - response relationship between baseline glucose and unfavorable outcome is still suggested by our results and those of others ( 1,4,6,10 ) . there is ample animal literature suggesting plausible mechanisms by which glucose may exert a deleterious effect on ischemic brain , including cellular acidosis caused by anaerobic glycolysis , enhanced free radical production , increased blood - brain barrier permeability , impaired mitochondrial function , influx of intracellular ca , and cellular edema ( 21 ) .
baseline hyperglycemia may represent an acute stress response from activation of the hypothalamic - pituitary - adrenal axis causing a rise in cortisol and catecholamines and , therefore , may simply be indicative of underlying stroke severity .
furthermore , it may also be a result of injury or irritation of brain areas involved in glucose regulation , a theory supported by the association of hyperglycemia with strokes involving the insula ( 22 ) .
the argument for causality is also further mitigated by the equivocal results of trials targeting aggressive glucose - lowering therapy in the acute phase after stroke ( 8,9 ) .
the uk glucose insulin in stroke trial ( gist - uk ) was both stopped early and underpowered but suggested no difference in clinical outcome between patients randomly assigned to glucose - potassium - insulin infusion to maintain glucose levels at 47
mmol / l over the first 24 h and patients given saline without glucose - lowering interventions ( 9 ) .
the lack of benefit may be related to the relatively late initiation of therapy after stroke ( median 14 h ) and the modest mean reduction in glucose achieved in the treatment arm ( 0.57 mmol / l ) .
our prospective cohort study is the largest yet to report on the effect of admission glucose exclusively in stroke patients treated uniformly with iv - tpa .
our results confirm the notion that poststroke hyperglycemia is a predictor of death , worse functional outcome , and sich in thrombolyzed patients .
this association remains true regardless of stroke subtype or severity and in patients with and without diabetes .
nonetheless , the data were prospectively collected from a large number of representative patients drawn from 60 hospitals across canada .
contrary to data for acute coronary syndromes arguing that dynamic changes in glucose are not prognostically significant ( 23 ) , it has recently been suggested that changes in glucose over the first 24 h among stroke patients may provide additional prognostic value ( 6 ) . however , use of a single baseline glucose value presumably underestimates potential harm , because patients with the highest admission glucose levels would most likely have been treated earlier and more aggressively with glucose - lowering therapies .
also , because thrombolytic treatment is necessarily rapid , a single admission glucose level has greater clinical utility for guiding acute treatment decisions .
third , we did not collect a1c values and so we do not have any measures of chronic dysglycemia .
however , in acute coronary syndromes , a1c levels convey little short - term prognostic value with respect to mortality ( 24 ) .
fourth , the method of blood glucose determination was not uniform , with either capillary blood or laboratory glucose measurements used at different sites .
both methods have been shown to provide comparable results in critically ill patients ( 25 ) .
last , baseline differences between both groups may be potential confounders that can not be entirely accounted for using statistical adjustments .
although it remains unclear whether correcting elevated glucose in the acute phase after ischemic stroke is beneficial , it is apparent that admission hyperglycemia rapidly identifies patients at higher risk for poor outcomes in whom glucose levels should be closely monitored . | objectiveadmission hyperglycemia has been associated with worse outcomes in ischemic stroke .
we hypothesized that hyperglycemia ( glucose > 8.0
mmol / l ) in the hyperacute phase would be independently associated with increased mortality , symptomatic intracerebral hemorrhage ( sich ) , and poor functional status at 90 days in stroke patients treated with intravenous tissue plasminogen activator ( iv - tpa).research design and methodsusing data from the prospective , multicenter canadian alteplase for stroke effectiveness study ( cases ) , the association between admission glucose > 8.0 mmol / l and mortality , sich , and poor functional status at 90 days ( modified rankin scale > 1 ) was examined .
similar analyses examining glucose as a continuous measure were conducted.resultsof 1,098 patients , 296 ( 27% ) had admission hyperglycemia , including 18% of those without diabetes and 70% of those with diabetes .
after multivariable logistic regression , admission hyperglycemia was found to be independently associated with increased risk of death ( adjusted risk ratio 1.5 [ 95% ci 1.21.9 ] ) , sich ( 1.69 [ 0.953.00 ] ) , and a decreased probability of a favorable outcome at 90 days ( 0.7 [ 0.50.9 ] ) .
an incremental risk of death and sich and unfavorable 90-day outcomes was observed with increasing admission glucose .
this observation held true for patients with and without diabetes.conclusionsin this cohort of iv - tpa treated stroke patients , admission hyperglycemia was independently associated with increased risk of death , sich , and poor functional status at 90 days .
treatment trials continue to be urgently needed to determine whether this is a modifiable risk factor for poor outcome . |
in a retrospective observational study recently published in british medical journal , lockey et al . note that trauma patients intubated without sedation before reaching hospital had a poor prognosis .
they looked at the records of 1623 patients registered in a helicopter emergency medical service database over six years ; of these , 486 had data on survival .
out of 486 patients , only one survived ( 0.2% ) . from this , the authors associate non - drug - assisted airway management with poor prognosis in trauma patients .
the study design in the paper by lockey et al . was lacking ; observational and retrospective , it provided no information on the trauma patients intubated with sedation , no incidence rate of cardiac arrest in the study group , and no objective measure of the severity of injury of the study patients .
it is also not clear from the authors ' comments whether they are calling into question the procedure itself or the capacities of those performing it .
nevertheless , it did highlight the quandaries surrounding airway management in trauma patients before reaching hospital , and raised two fundamental questions .
first , is endotracheal intubation necessary , efficient and effective in the prehospital treatment of the trauma patient ?
second , is sedation or neuromuscular blockade useful for the intubation of the trauma patient ?
airway management is widely considered the highest priority in the management of the critically injured patient .
the rationale for invasive airway management includes facilitating oxygenation and ventilation , and protecting the patient from aspirating the contents of the stomach , or blood from the upper airway .
consequently , one might expect intubation and mechanical ventilation to be associated with decreased neurological damage as a result of preventing asphyxia and hypoxia .
however , some experts consider intubation before reaching the hospital as deleterious , dangerous , and a waste of precious time in trauma patients without a compromised airway .
although there are no broad , prospective , controlled trials comparing the basic and advanced prehospital management of adult trauma patients , several studies have highlighted the benefits of endotracheal intubation before the hospital has been reached .
for example , winchell and hoyt showed that intubation in the prehospital setting improved survival in patients characterized by a glagow coma scale score of 8 or less with blunt injury from 64% to 74% .
found that there were between 8 and 19 extra survivors per 100 blunt - trauma patients treated in the advanced prehospital management group ( 51% of intubated patients ) compared with more basic prehospital management ( 10% of intubated patients ; p < 0.001 ) .
others , however , have not found any advantage of intubation on the scene in terms of outcome .
for instance , eckstein et al . reported that patients who had bag - mask ventilation had 5.3-fold higher adjusted survival rates than patients who had on - scene tracheal intubation . in a randomized study ,
gausche et al . compared the survival and neurological outcomes of paediatric patients treated with bag - valve - mask ventilation with patients treated by tracheal intubation in a prehospital setting .
they found no significant difference between the two groups in survival or in the rate of achieving a good neurological outcome .
possibly the greatest factors in the incidence of failure in intubating trauma patients are inadequate sedation , patient combativeness , lack of practice and the concomitant need for in - line cervical spine stabilization . all this can be compounded by airway reactivity ( gag and cough reflexes ) , which can be present even in trauma patients with a glasgow coma scale score of 3 .
these reflexes contribute to the increase in difficulty of intubation and the rate of complications ( vomiting or aspiration ) .
the ability to intubate trauma patients with success without pharmacological aids is a reflection of the initial severity of patient .
thus , the poor prognosis of patients described in the paper by lockey et al . probably has little to do with the intubation technique , but instead reflects the patients ' pre - existing high probability of a fatal outcome and is therefore not surprising .
rapid sequence intubation ( rsi ) with succinylcholine has been associated with a decrease in intubation failures and complications in several studies , and has become the ' gold standard ' for emergency intubation attempts . in rsi , the patient is given a potent induction agent ( such as thiopental or etomidate ) followed immediately by administration of a rapidly acting neuromuscular blocking agent ( such as succinylcholine ) to induce unconsciousness and motor paralysis ; the patient is then intubated .
all this is preceded by a preoxygenation phase to permit a period of apnea to occur safely until intubation has been achieved . as well as fewer failures with rsi ,
emergency systems using emergency physicians in the prehospital setting seem have better success with intubation attempts .
however , it remains unclear whether such improved outcomes are due to additional training or are simply due to additional experience .
several studies suggest that timely intubation in the severely injured patient at the scene improves outcome .
conversely , inadequate airway management in this patient population is the primary cause of preventable mortality .
endotracheal intubation should therefore be considered necessary , efficient and effective in the treatment of the trauma patient before reaching hospital .
the use of rsi with neuromuscular blockade remains the procedure of choice , even in the unconscious trauma patient ; sedation or neuromuscular blockade therefore seems to be useful for the intubation of trauma patients . whether the presence of an emergency physician at the scene ( as opposed to a paramedic experienced in rsi ) is associated with better outcome merits additional study .
| endotracheal intubation is widely used for airway management in a prehospital setting , despite a lack of controlled trials demonstrating a positive effect on survival or neurological outcome in adult patients .
the benefits , in term of outcomes of invasive airway management before reaching hospital , remain controversial .
however , inadequate airway management in this patient population is the primary cause of preventable mortality .
an increase in intubation failures and in the rate of complications in trauma patients should induce us to improve airway management skills at the scene of trauma .
if the addition of emergency physicians to a prehospital setting is to have any influence on outcome , further studies are merited .
however , it has been established that sedation with rapid sequence intubation is superior in terms of success , complications and rates of intubation difficulty .
orotracheal intubation with planned neuromuscular blockade and in - line cervical alignment remains the safest and most effective method for airway control in patients who are severely injured . |
a 62-year - old female presented with gradually increasing swelling of the right side face for 5-years duration .
extra orally she had a diffuse , bony hard , non - tender swelling involving right side mandible ( expansion of ramus , angle , and posterior body region ) [ figure 1a ] . color and texture of the overlying skin was normal .
intraorally mild buccal vestibule obliteration was evident in relation to the edentulous molar region extending posteriorly to the ascending ramus area .
cortical expansion of right ramus ( a ) and computed tomography scan axial view showing an expansile osteolytic lesion involving ramus , angle , and body of right mandible ( b ) panoramic radiograph showed multilocular radiolucency involving the ascending ramus , angle and posterior body of the right side mandible .
computed tomography scan showed an ill - defined expansile lytic lesion seen within the ramus and angle of right side mandible measuring approximately of size 4.2 cm 3.2 cm 4.5 cm , with the lesional cavity partially filled with fluid and irregular soft tissue density in the residual portion with thinned out cortex .
aspiration yielded a fluid mixed with dark venous blood , and the mild oozing was stopped on applying gentle pressure for few minutes .
incisional biopsy was done , and the sample on histopathologic evaluation did not reveal any specific features except for loosely arranged fibro cellular areas with focal hemorrhage [ figure 2a ] .
blood filled spaces without endothelial lining in the fibro cellular area ( a ) and gross specimen showing central cavitation ( star ) , cystic spaces ( arrow ) , and cortical expansion ( b ) based on the incisional biopsy findings , a working diagnosis of aneurysmal bone cyst like change secondary to the unknown primary intraosseous lesion was made .
considering the clinical and radiographic features and the age of the patient it was decided to perform surgical resection and the patient underwent resection of the right side mandible ( ramus to the molar area ) .
macroscopically the resected specimen showed buccal cortical expansion with the cavitation of ramus and angle of the mandible with soft tissue mass confined to the peripheral parts of the lesion cavity [ figure 2b ] .
microscopic examination of the hematoxylin and eosin sections of the soft tissue of the resected specimen showed ameloblastic follicular islands showing extensive granular transformation of the central stellate reticulum like cells in a fibrous stroma [ figure 3a and b ] .
ameloblastic follicles are exhibiting cuboidal to columnar cells at the periphery and extensive granular cell changes at the center . ( a ) h and e , 4 and ( b ) h and e , 40 immunohistochemical study using anti - cytokeratin antibody showed a uniform and intense positive staining with peripheral ameloblastic cell and central granular cells [ figure 4a ] and anti cd-68 antibody showed intense positivity with central granular cells and negative reaction for peripheral ameloblastic cells [ figure 4b ] .
peripheral ameloblastic cell and central granular cell showing cytokeratin positivity ( a ) and cd-68 positivity in central granular cells alone ( b ) ( 40 ) with the support of histopathology and immunohistochemical findings , a final diagnosis of granular cell ameloblastoma was made .
ameloblastoma is one of the most common odontogenic tumors that do not always grow as uniform solid mass due its inherent potential to undergo cystic degeneration .
microscopically cystic degeneration usually starts as a small area within the ameloblastic follicle and as they grow gradually , lack of nutrition leads to complete degeneration of central region of the follicle leading to clear space centrally within the follicle .
the extent of cystic degeneration logically seems to be more in long standing cases , and it may not be rare to encounter a lesion with gross feature showing complete cavitations of the involved jaw bone . this macroscopic picture can potentially compromise the adequate representative sample procurement during the routine incisional biopsy . although any histopathological variant of ameloblastoma is prone for cystic degeneration , it can be speculated that the process is more aggressive and faster in granular cell variant of ameloblastoma .
this is based on the fact that , phagocytosis of apoptotic remnants and adjacent neoplastic cells ( digestion of solid tumor components ) by the granular cells within the follicle as evidenced by the macrophage - specific lysosomes within the granular cells .
macroscopic presentation of complete cavitations in the current case may be explained with the above said facts and the incisional biopsy sample did not show any evidence of ameloblastomous follicle , except for the presence of loosely fibrous stroma with multiple blood - filled spaces without any specific endothelial lining , simulating aneurysmal bone cyst histopathological feature .
these stromal features that is , areas of hemorrhage are generally considered to be a secondary phenomenon in long - standing lesion as similar to the present case that facilitated us to give a more practical working diagnosis for the further management .
ameloblastoma with its varied macroscopic presentation may offer difficulty in its representative sample procurement during the incisional biopsy and its accurate interpretation at the histopathological level
. this may be more significant in granular cell ameloblastoma , hence more practical approach with proper clinical correlation will avoid unnecessary advanced imaging modalities such as magnetic resonance imaging and repeat of surgical , diagnostic procedure , thereby reduce the cost and stress of the patients in its management . | granular cell ameloblastoma is a rare variant of ameloblastoma , which is histopathologically characterized by the presence of large eosinophilic granular cells within the ameloblastic follicle .
its accurate preoperative diagnosis is based upon clinical , radiological , and incisional biopsy findings .
this article reports a case of granular cell ameloblastoma in a 65-year - old female , which on incisional biopsy showed the features suggestive of aneurysmal bone cyst .
furthermore , the influence of macroscopic presentation of the current lesion on its accurate preoperative incisional biopsy diagnosis is discussed in detail . |
cardiovascular disease ( cvd ) including sudden cardiac death ( scd ) is a major cause of mortality in chronic kidney disease ( ckd ) .
apart from left ventricular dysfunction , unique factors such as autonomic imbalance and inflammation contribute to the high incidence of scd in ckd .
traditional risk factors for atherosclerosis , such as hypertension and dyslipidaemia , can to some extent explain the increased risk of atherosclerosis in ckd . however , nontraditional risk factors like oxidative stress and inflammation may also be of importance .
heart rate variability ( hrv ) is a reliable , noninvasive measurement of autonomic tone of the heart .
a reduction in hrv reflects an autonomic imbalance towards decreased vagal tone and subsequent sympathetic overactivity .
chronic , low - grade inflammation is involved in the development of both scd and atherosclerosis .
the most extensively examined marker of inflammation is high - sensitive c - reactive protein ( hs - crp ) , which is a predictor of cardiovascular mortality in different populations [ 12 , 13 ] including patients with ckd .
hs - crp may even be a risk marker for scd [ 10 , 15 ] .
haptoglobin ( hp ) is an abundant acute - phase protein as well as an innate antioxidant involved in the scavenging of free haemoglobin .
thus , hp prevents iron - mediated formation of free reactive oxygen species known to initiate oxidative stress . in humans
, hp is characterised by molecular heterogeneity with three common genotypes : 1 - 1 , 2 - 2 , and 2 - 1 .
the antioxidant ability of hp is genotype dependent with hp 2 - 2 being the weakest antioxidant .
there is evidence establishing hp 2 - 2 as a predictor of cvd in diabetes mellitus [ 20 , 21 ] .
however , results are inconsistent regarding hp genotypes and cardiovascular mortality in patients with ckd [ 22 , 23 ] .
a study in patients with ischemic heart disease ( ihd ) revealed a positive association between low hrv and hp 2 - 2 .
the aim of the present study was to determine whether hp genotype was associated with the cardiovascular risk markers hrv and hs - crp in patients with ckd .
sixty - four patients from the outpatient clinic at the department of nephrology , aalborg hospital , denmark , were included .
exclusion criteria were the start of dialysis within three months , nephrotic syndrome , malignancy , uncontrolled hypertension , or a previous renal transplantation .
patients were also excluded if they had an ongoing infection , a myocardial infarction during the last six months , or supraventricular arrhythmia .
the causes of ckd were chronic glomerulonephritis ( 32% ) , hypertensive nephropathy ( 23% ) , polycystic kidney disease ( 13% ) , and other causes including interstitial nephritis and chronic pyelonephritis ( 32% ) .
the study was conducted in accordance with danish legislation and the declaration of helsinki and approved by research ethics committee of the north denmark region .
creatinine clearance was calculated using a 24 h urine collection and a blood sample for serum creatinine .
hs - crp was measured using an advia 1650 analyzer ( bayer corp . , pittsburgh , pa , usa ) and an immunoturbidimetric assay ( randox laboratories ltd . , antrim , uk ) .
the hp genotype was determined by high - performance liquid chromatography as previously described , using a superdex 200 hr 10/30 column for chromatographic separation ( amersham pharmacia biotech ab , birkerd , denmark ) .
phosphate - buffered saline ( 50 mmol / l sodium phosphate , 150 mmol / l sodium chloride , and 0.01% sodium azide ; ph 7.3 ) was used as the mobile phase .
flow rate was 1 ml / min ( 76.4 cm / hour ) .
hp genotype was determined from the curve shape and relative retention time of the chromatograms .
a 24 h holter recording was obtained on a flash card using a 3-channel digital monitor dl-700 ( diagnostic monitoring , santa ana , ca , usa ) , and time domain variables were analysed using commercially available software ( diagnostic monitoring , santa ana , ca , usa ) .
if more than 20% of qrs complexes were abnormal or of poor technical quality , the analysis was excluded in the overall hrv analysis .
the time domain variables were identified as follows : ( 1 ) mean of all normal - to - normal rr intervals , ( 2 ) standard deviation ( sd ) of all normal - to - normal rr intervals ( sdnn ) , ( 3 ) mean of the sds of all normal - to - normal rr intervals ( sdnnindex ) , ( 4 ) sd of the mean normal - to - normal rr intervals for each 5-minute period ( sdann ) , ( 5 ) percentage of successive rr interval differences > 50 ms ( pnn50 ) , and ( 6 ) square root of the mean of the sum of squares of differences between adjacent rr intervals ( rmssd ) .
blood pressure was obtained with a 24 h ambulatory bp device , tm 2412 ( a&d co. ltd . , tokyo , japan ) . for analysis ,
the statistical analysis was performed using stata software , version 10 ( statacorp lp , tx , usa ) .
the hp alleles were tested for hardy - weinberg equilibrium using an asymptotic hardy - weinberg equilibrium test . for the comparison of sdnn , sdann , and hs - crp , respectively , between the three common hp genotypes , crude one - way analysis of variance was performed .
data was divided into two groups according to the presence of the hp allele 1 : one group including hp 1 - 1 and hp 2 - 1 patients and the remaining group consisting of hp 2 - 2 patients . for comparisons of groups ,
student 's t - test was used for continuous variables and fisher 's exact test for categorical variables .
linear regression was used to analyse the effect of hp 2 - 2 compared to hp 1 - 1 and hp 2 - 1 on sdnn , sdann , and hs - crp , respectively .
the analyses were performed with the hp group as the independent variable and sdnn , sdann , and hs - crp , respectively , as the dependent variables .
adjustments for potential confounders described in previous studies ( age , gender , heart rate , beta - blocker treatment , and creatinine clearance for hrv indices and age , gender , body mass index , and smoking status for hs - crp ) [ 2628 ] were performed .
the frequencies of the three common genotypes were hp 1 - 1 21% , hp 2 - 1 50% , and hp 2 - 2 29% , and the hp allele frequencies ( hp allele 1 0.457 and hp allele 2 0.544 ) were in hardy - weinberg equilibrium ( = 2.07 and p = 0.1 ) .
the genotype frequencies in the cohort were similar to those reported previously in northwestern european countries ( = 3.42 and p = 0.2 ) and similar to those reported previously in a danish population ( = 3.40 and p = 0.2 ) .
figure 1 depicts mean sdnn and sdann , and figure 2 mean hs - crp in the three common hp genotypes .
no differences were demonstrated between hp 1 - 1 and hp 2 - 1 regarding sdnn , sdann , and hs - crp , respectively . however , significant differences were observed between hp 1 - 1 and hp 2 - 2 and between hp 2 - 1 and hp 2 - 2 for both sdnn and hs - crp .
however , for sdann , a significant difference was demonstrated only between hp 2 - 1 and hp 2 - 2 .
table 1 shows clinical characteristics in the hp genotypes divided into two groups according to the presence of hp allele 1 .
the two groups were generally comparable , although there were fewer women in the hp 2 - 2 group .
sdnn and sdann were significantly lower in the hp 2 - 2 patients compared to the hp 1 - 1 and hp 2 - 1 patients ( table 1 ) .
the other time domain indices rr , sdnnindex , pnn50 , and rmssd showed a similar trend although not statistically significant .
multivariate linear regression analyses of hp 2 - 2 and sdnn and sdann , respectively , were performed adjusting for previously described confounders .
hp 2 - 2 was negatively and independently associated with both sdnn and sdann ( table 2 ) .
apart from hp 2 - 2 , the strongest determinants of sdnn in the model were age ( p < 0.001 ) and heart rate ( p = 0.04 ) , and the strongest determinants of sdann were age ( p < 0.001 ) , heart rate ( p = 0.05 ) , and beta - blocker treatment ( p = 0.04 ) .
after adjusting for age , gender , heart rate , creatinine clearance , and beta - blocker treatment , hp 2 - 2 was still significantly associated with both sdnn and sdann .
levels of hs - crp were significantly higher in the hp 2 - 2 patients compared to the hp 1 - 1 and hp 2 - 1 patients ( table 1 ) . in the multiple linear regression analysis of hs - crp and hp 2 - 2 ( table 2 ) , adjustments were made for confounders known from previous studies [ 27 , 28 ] .
only hp 2 - 2 revealed a positive and significant association with hs - crp in the model .
the present study demonstrated a close association between hp genotype and the cardiovascular risk markers hrv and hs - crp in patients with ckd .
thus , patients with hp 2 - 2 had significantly lower hrv and higher levels of hs - crp .
establishes an scd as one of the most single important causes of death in patients with ckd .
well - described risk factors of scd in the general population also apply to patients with ckd . however , several unique factors such as autonomic imbalance and inflammation are recognized .
hrv is being increasingly used as a valid and easily obtainable prognostic marker of autonomic alterations . as reviewed by ranpuria et al . , hrv is a promising surrogate marker for scd in patients with ckd and a potential modifiable outcome measure . in haemodialysis patients ,
oikawa et al . found decreased sdnn to be a predictor of all - cause and cardiovascular mortality .
recently , chandra et al . studied hrv in patients with ckd stage 35 demonstrating that lower hrv was independently associated with higher risk of cvd and mortality .
christensen et al . has previously addressed the issue of a possible association between hrv and hp genotype . in patients with ihd ,
a significant association between time domain variable rmssd and hp 2 - 2 was shown .
the present study did not show an association between rmssd and hp 2 - 2 .
however , a highly significant association between hp 2 - 2 and sdnn and sdann , primarily reflecting circadian rhythm and long - term fluctuations , was demonstrated .
a relationship between the autonomic nervous system and inflammation has been established showing increase in vagal tone as a counter - response to cytokine synthesis .
tracey first described this cholinergic anti - inflammatory pathway as a plausible , molecular mechanism for the association between hrv and hs - crp . in a cohort of patients with ckd , psychari et al . demonstrated a negative association between inflammatory marker interleukin-6 and sdnn and sdann , respectively
furthermore , in a large cohort of end - stage renal disease patients , parekh et al . found a positive association between hs - crp and scd , independently of other known cardiovascular risk factors .
direct effects on the vagal conduction system and resulting aggravation of sympathetic tone may contribute to the increased risk of scd .
being the weaker antioxidant of the three common hp genotypes , hp 2 - 2 may be unable to hinder oxidative damage to the autonomic nervous system , especially in patients with ckd exposed to a prooxidative milieu due to uremic toxins .
studies have established hp 2 - 2 as a potential risk predictor of cvd in patients without ckd .
chapelle et al . showed that more extensive myocardial infarction occurred in hp 2 - 2 patients .
demonstrated that hp 2 - 2 patient undergoing coronary bypass surgery needed more grafts and had shorter graft survival time . on the contrary , in a nested control study de bacquer et al .
showed that hp 1 - 1 patients had a higher risk of ihd compared to hp 2 - 2 and hp 2 - 1 patients .
studies on cvd and hp genotype in diabetes show more consistent results , both in human studies and recently in a mouse model .
levy et al . demonstrated in the strong heart study that hp 2 - 2 is a major predictor of cvd in patients with diabetes . yet , in a cross - sectional study of the framingham offspring cohort , levy et al . found that hp 2 - 2 and hp 2 - 1 patients with diabetes had decreased cvd prevalence .
a possible relation between hs - crp and hp 2 - 2 has been examined in several studies . in patients with peripheral occlusive disease ,
delanghe et al . found a higher , yet statistically insignificant , hs - crp in hp 2 - 2 patients compared to hp 1 - 1 and hp 2 - 1 patient . likewise , braeckman et al
. found a higher , but also statistically insignificant , hs - crp in apparently healthy hp 2 - 2 individuals compared to hp 1 - 1 and hp 2 - 1 individuals .
however , delanghe et al . demonstrated in patients with essential hypertension higher hs - crp in hp 2 - 2 patients .
the present study establishes a potential role for hp 2 - 2 as a risk predictor of cvd in patients with ckd through an association with hs - crp .
numerous studies have documented hs - crp to be a valid and well - documented marker of inflammation and hence a predictor of atherosclerosis and cardiovascular mortality in patients with ihd as well as in patients with ckd , as recently demonstrated by bazeley et al . . in patients with ckd
pernod et al . found a prevalence of hp 2 - 2 twice as high in end - stage renal disease patients with high cvd risk , defined as prior cardiovascular event , diabetes , or dyslipidemia , compared to end - stage renal disease patients with low cvd risk . in a group of end - stage
renal disease patients with diabetes , burbea et al . found a survival advantage in hp 1 - 1 patients above 60 years , whereas patients below 60 years demonstrated a better survival when having hp 2 - 2 .
however , pernod et al . found no association between hp genotype and cardiovascular mortality in patients with end - stage renal disease .
lipid oxidation plays a crucial role in the initiation of atherosclerosis , and the first step in lipid oxidation is mediated by redox - active metals such as iron .
brouwers et al . demonstrated that hp 2 - 2 is associated with high circulating levels of oxidised low - density lipoprotein thus posing a potential proatherogenic pathway for hp 2 - 2 . in an animal model of knock - in hp 2 - 2 mice ,
levy et al . demonstrated that hp contributes to atherosclerotic plaque modulation by increasing the local inflammation .
hp 2 - 2 plaques accumulate more macrophages and have decreased levels of antiinflammatory cytokines .
furthermore , hp 2 - 2 has been associated with diminished nitric oxide bioavailability [ 50 , 51 ] , a crucial step in endothelial dysfunction and hence initiation of atherosclerosis .
furthermore , the underlying renal disease pattern is divers and comprises diseases with both high and low levels of inflammation .
the design of the study is cross - sectional potentiating a risk of bias , and the causal inference can not be established .
in conclusion , in patients with ckd , a negative association was observed between hp 2 - 2 and hrv .
thus , hp 2 - 2 may indicate a poor outcome in patients with ckd , and one may speculate that hp 2 - 2 can help predict cardiovascular risk in patients with ckd as previously demonstrated in patients with diabetes . | sudden cardiac death and atherosclerosis have a major impact on cardiovascular mortality in chronic kidney disease ( ckd ) .
inflammation with elevated high - sensitive c - reactive protein ( hs - crp ) is involved in both sudden cardiac death and atherosclerosis , and decreased heart rate variability ( hrv ) is a predictor of both sudden cardiac death and atherosclerosis .
haptoglobin ( hp ) is characterised by three genotypes ( 1 - 1 , 2 - 1 , and 2 - 2 ) with different antioxidant abilities . the aim was to examine whether hrv and hs - crp were associated with hp genotype in ckd patients .
fifty - six patients with ckd stage 25 were included .
hp genotype was determined by high - performance liquid chromatography .
hrv was analysed from the 24 h holter recordings .
hs - crp was measured using an immunoturbidimetric assay .
the results show that the hrv indices sdnn and sdann were significantly lower in the hp 2 - 2 patients ( p = 0.02 and 0.04 , resp . ) . in an adjusted linear regression model , hp 2 - 2
was associated with both sdnn ( p = 0.005 ) and sdann ( p = 0.01 ) .
hs - crp was higher in the hp 2 - 2 patients ( p = 0.002 ) .
in an adjusted linear regression model , the association between hp 2 - 2 and hs - crp remained significant ( p = 0.003 ) .
in conclusion , a negative association was observed between hp 2 - 2 and hrv , and hp 2 - 2 was positively associated with hs - crp in ckd patients . |
it is generally established that the harmful effects of communicable disease and malnutrition on public health are of the utmost importance notably in the poor countries .
consequently , the priority of the health institutions and care workers has been focusing on control and preventive measures of the infectious diseases.[13 ] however , in many cases , health care promotion and enhancement of medical insurance have tackled these issues . on the other hand , rapid changes in lifestyle , nutrition , and physical activity , especially among the youngsters , have been accompanied by long - term change in disease patterns which is no longer allocated to industrialized countries . as a direct result of this , chronic noncommunicable disease ( ncd ) is becoming epidemic and global issue as two out of three deaths in the world are related to chronic diseases .
these are affecting an abundance of people of all ages , nationalities , and socioeconomic classes[68 ] in addition to developed countries , the prevalence of ncds is now increasing in developing countries , which would lead to increased morbidity and mortality , as well as to a high financial burden . a growing body of literature documented changes in lifestyle habits and the global increase in childhood overweight . given the long - term consequences of childhood obesity on morbidity and mortality , health care in the pediatrics group deserves further consideration .
similar to the children and adolescents of other developing countries , iranian youths are experiencing lifestyle changes , making them prone to risk factors of chronic diseases.[1419 ] considering the tracking of ncd - related risk behaviors and risk factors from childhood to adult life , surveillance of such factors , e.g. , blood pressure , avoidance of smoking , and overweight as well as encouraging regular physical exercise in children and adolescents , can provide useful information for long - term national planning .
some international organizations have implemented surveillance systems for tracking such factors . the global school - based student health survey ( gshs )
has developed and supported by the world health organization ( who ) in cooperation with united nations unicef , unesco , unaids , and centers for disease control and prevention ( cdc ) .
the youth risk behavior surveillance system ( yrbss ) aims to monitor those health - risk behaviors contributing to the young and adults morbidity and mortality .
they include behaviors related to unintentional injuries and violence , tobacco use , alcohol and other drug use , sexual risk behaviors , unhealthy dietary behaviors , and physical inactivity , as well as obesity and asthma . in iran , a school - based surveillance system entitled the childhood and adolescence surveillance and prevention of adult noncommunicable disease ( caspian ) study is implemented from 20032004 .
the surveys are repeated every 2 years , with blood sampling for biochemical factors every 4 years .
this paper presents the methods and primary findings of the third survey of this school - based surveillance system .
this school - based nationwide health survey was conducted in iran as the national survey of school student high risk behaviors ( 20092010 ) .
it was conducted as the third study of the school - based surveillance system entitled caspian - iii study .
it was performed with corporation of the ministry of health and medical education ; ministry of education and training , child health promotion research center , isfahan university of medical sciences ; and endocrinology and metabolism research institute of tehran university of medical sciences .
the survey was performed among 5570 students aged 1018 years , who were selected by multistage random cluster sampling from urban and rural areas of 27 provinces of iran .
eligible schools for our study were stratified according to information bank of ministry of education , and then , they were selected randomly . in selected schools ,
study protocols were reviewed and approved by ethics committees and other relevant national regulatory organizations was obtained after complete explanation of the study objectives and protocols for students and their parents , written informed consent was obtained from parents and oral assent from students , sampling and examinations and were begun .
we prepared the questionnaires in farsi based on and the who global school health survey , and added some more questions to the questionnaires of parents .
questions were about family dietary habits , students past history , and familial history of chronic diseases .
the validity of their content was affirmed based on observations of an experts panel and item analysis .
reliability measures were assessed based on a pilot study . under the supervision of expert health care professionals ,
the students filled out the self - administered questionnaire at school . a team of trained health care professionals recorded information in a checklist and conducted the examinations under standard protocol by using calibrated instruments
. weight was measured to the nearest 200 g in barefoot and lightly dressed condition .
body mass index ( bmi ) was calculated as weight ( kg ) divided by height squared ( m ) .
waist circumference ( wc ) was measured by a nonelastic tape to the nearest 0.2 cm at the end of expiration at the midpoint between the top of iliac crest and the lowest rib in standing position .
we used the who growth curves to define bmi categories , i.e. , underweight as age and sex - specific bmi < -2 z - score , overweight as sex - specific bmi for age of > + 1 z - score , and obesity as sex - specific bmi for > + 2 z - score .
we used the spss for windows software ( version 16.0 , spss , chicago , il ) for statistical analyses , and p < 0.05 was considered as statistically significant .
this school - based nationwide health survey was conducted in iran as the national survey of school student high risk behaviors ( 20092010 ) .
it was conducted as the third study of the school - based surveillance system entitled caspian - iii study .
it was performed with corporation of the ministry of health and medical education ; ministry of education and training , child health promotion research center , isfahan university of medical sciences ; and endocrinology and metabolism research institute of tehran university of medical sciences .
the survey was performed among 5570 students aged 1018 years , who were selected by multistage random cluster sampling from urban and rural areas of 27 provinces of iran .
eligible schools for our study were stratified according to information bank of ministry of education , and then , they were selected randomly . in selected schools ,
study protocols were reviewed and approved by ethics committees and other relevant national regulatory organizations was obtained after complete explanation of the study objectives and protocols for students and their parents , written informed consent was obtained from parents and oral assent from students , sampling and examinations and were begun .
we prepared the questionnaires in farsi based on and the who global school health survey , and added some more questions to the questionnaires of parents .
questions were about family dietary habits , students past history , and familial history of chronic diseases .
the validity of their content was affirmed based on observations of an experts panel and item analysis .
reliability measures were assessed based on a pilot study . under the supervision of expert health care professionals ,
the students filled out the self - administered questionnaire at school . a team of trained health care professionals recorded information in a checklist and conducted the examinations under standard protocol by using calibrated instruments
. weight was measured to the nearest 200 g in barefoot and lightly dressed condition .
body mass index ( bmi ) was calculated as weight ( kg ) divided by height squared ( m ) .
waist circumference ( wc ) was measured by a nonelastic tape to the nearest 0.2 cm at the end of expiration at the midpoint between the top of iliac crest and the lowest rib in standing position .
we used the who growth curves to define bmi categories , i.e. , underweight as age and sex - specific bmi < -2 z - score , overweight as sex - specific bmi for age of > + 1 z - score , and obesity as sex - specific bmi for > + 2 z - score .
we used the spss for windows software ( version 16.0 , spss , chicago , il ) for statistical analyses , and p < 0.05 was considered as statistically significant .
this national survey was conducted among 5570 school students ( 2784 girls and 2786 boys ) and one of their parents .
data of 5528 students ( 2726 girls and 2802 boys ) were complete and are reported .
the mean age of participants was 14.7 2.4 years without significant difference in terms of gender .
more than 90% of students were from public schools and the rest from private schools .
most students parents were literate ; 97.8% of fathers and 98.2% of mothers had at least elementary literacy .
the large number of students father was worker or employee ( 45.35% ) , whereas 90.7% of mothers were housewives .
characteristics of study participants : caspian - iii study overall , 17.3% ( 17.3% of girls and 17.5% of boys ) were underweight , and 17.7% ( 15.5% of girls and 19.9% of boys ) were overweight or obese . abdominal obesity was documented in 16.3% of students ( 17.8% of girls and 15% of boys ; 18.6% in urban and 11.2% in rural areas ) .
bread is the staple food for iranians , 42.4% of families consumed whole wheat bread , and the rest used breads prepared with white flour .
most families , i.e. , 43.8% in urban areas and 58.6% in rural areas used solid fats for food preparation at home .
some dietary habits of study participants : caspian - iii study according to the students report , 14.2% of them had a regular daily physical activity for at least 30-min a day ; 19.7% reported not to have any physical activity
. of students studied , 45% used computer in week days and 56.5% in weekends ; these frequencies were higher in urban areas and among girls [ table 3 ] . physical activity pattern of study participants : caspian - iii study regarding passive smoking , 48.5% of students reported to have at least one cigarette smoker in their relatives , the corresponding figure was 33.9% for waterpipe .
overall , 10.4% of students ( 11.7% in urban areas and 7.3% in rural areas ) reported that they used tobacco products , often waterpipe .
overall , 32.8% of students asserted that in the 3 months before the study , they experienced at least three times of bullying at school .
bullying was more frequent among girls than boys and decreased from elementary to high - school levels .
regarding injuries , 14.46% of students ( 14.8% elementary , 15.2% middle , and 14% in high school ) reported that during the year prior to the survey , they had suffered an injury needing the interference by school health providers .
the prevalence of these injuries was higher in girls than in boys , and in rural than in urban areas . in the most cases , the injury type has been cuts and scratching , and 42.4% of injuries occurred when students were exercising , running , and/or jogging .
the findings of this national survey can serve for action - oriented policy making for health promotion of children and adolescents , as well as for primordial / primary prevention of chronic diseases .
most students were in a good health condition ; however , in most of the time , the lifestyle habits of families exposed them to chronic diseases in their future adulthood .
, 80% of children and adolescents mentioned that they often add salt to their table food . despite that youths taste
have changed recently , and the preference for salty food has been escalated , our findings were about double in comparison with australian ( 10%39% ) or greek children ( 39% ) .
unhealthy eating habits among children and their families are considered as a problem for both developed and developing countries , which necessitate taking heed of needs for improvements in family and school food environments.[2729 ] in addition to some unhealthy dietary habits , the frequency and intensity of physical activity were low among children and adolescents .
furthermore , physical activity level was lesser in students of higher grades than in lower grades .
overall , the screen time , watching tv or computer games , was about 4 h a day , i.e. , twice than the recommended time . our findings are consistent with some other studies in showing low time allocated to physical activity among children and adolescents .
similar to the populations of most developing countries , in the current survey , we documented a dual burden of nutritional disorders in terms of underweight and overweight among iranian children and adolescents .
the epidemiologic transition with rapid changes in the habitual diet along with physical inactivity makes young individuals more susceptible to weight disorders.[3234 ] our findings also demonstrated a considerable prevalence of abdominal obesity , which even needs more attention for future chronic diseases .
principally , one of the leading causes of hypertension in this age group is the frequency of consuming fast food and solid hydrogenated fat .
many developing countries have encountered with dyslipidemia among children and adolescences , which can be justified by their sedentary lifestyle and high consumption of hydrogenated fat.[4042 ] thus , according to the association of dyslipidemia in childhood and its relation to the future cardiovascular morbidity and mortality , encouraging healthy lifestyle from childhood is of crucial importance .
a large number of students were exposed to secondhand smoking ; families should be aware of the hazards of their smoking habits on the health and the tendency of their children to begin smoking in the near future . although the prevalence of self - reported smoking was lower than some other countries,[4547 ] but
these statistics deserve more consideration for preventive measures in this vulnerable age group , particularly because of the young age of begging to smoke at about 11 years .
needless to say that exposure to violence and bullying is extremely likely to impose detrimental effects to the overall well - being and development of all youths .
moreover , to establish successful socioeconomic life and subsequently prosperous society , having a healthy educational system is essential .
our findings were consistent with studies conducted in some other countries.[4850 ] barriers to healthy lifestyles , e.g. , physical activity , should be recognized in each community , and facilities should be provided accordingly .
the current study documented important and preventable unhealthy lifestyle behaviors , which need proper attention both within the family and at school . living a healthier life is every society 's right , but it will not become true without amending the young 's lifestyle and behavior . this survey is confirmatory evidence on the importance of establishing surveillance systems for risk behaviors to implement action - oriented interventions for primordial and primary prevention of chronic diseases . | background : a school - based surveillance system entitled the childhood and adolescence surveillance and prevention of adult noncommunicable disease ( caspian ) study is implemented at national level in iran .
this paper presents the methods and primary findings of the third survey of this surveillance system.methods:this national survey was performed in 20092010 in 27 provinces of iran among 5570 students and one of their parents .
in addition to physical examination , fasting serum was obtained .
body mass index was categorized based on the world health organization growth charts.findings:data of 5528 students ( 2726 girls , 69.37% urban , mean age 14.7 2.4 years ) were complete and are reported .
overall , 17.3% ( 17.3% of girls and 17.5% of boys ) were underweight , and 17.7% ( 15.5% of girls and 19.9% of boys ) were overweight or obese .
abdominal obesity was documented in 16.3% of students ( 17.8% of girls and 15% of boys ) .
57.6% of families consumed breads , the staple food for iranians , prepared with white flour .
most families ( 43.8% in urban areas and 58.6% in rural areas ) used solid hydrogenated fats .
22.7% of students did not add salt to the table food .
14.2% of students reported to have a regular daily physical activity for at least 30 min a day .
overall , 10.4% of students ( 11.7% in urban areas and 7.3% in rural areas ) reported that they used tobacco products , often waterpipe .
32.8% of students experienced at least three times of bullying in the previous 3 months . during the year
prior to the survey , 14.46% of students had an injury needing the interference by school health providers.conclusion:this survey is confirmatory evidence on the importance of establishing surveillance systems for risk behaviors to implement action - oriented interventions . |
the effectiveness of endodontic files , rotary instrumentation , irrigating solutions , and chelating agents to clean , shape , and disinfect root canals underpins the success , longevity , and reliability of modern endodontic treatments .
the role of microorganisms in the development and perpetuation of pulp and periapical diseases has clearly been demonstrated in animal models and human studies [ 14 ] .
the chances of a favourable outcome with root canal treatment are significantly higher if infection is eradicated effectively before the root canal system is obturated . however
, if microorganisms persist at the time of obturation , or if they penetrate into the canal after obturation , there is a high risk of treatment failure [ 5 , 6 ] .
numerous measures have been described to reduce the number of microorganisms in the root canal system , including the use of various instrumentation techniques , irrigation regimens , and intracanal medicaments .
the use of chemical agents during instrumentation to completely clean all aspects of the root canal system is central to successful endodontic treatment .
irrigation dynamics plays an important role [ 9 , 10 ] ; the effectiveness of irrigation depends on the working mechanism(s ) of the irrigant and the ability to bring the irrigant in contact with the microorganisms and tissue debris in the root canal [ 11 , 12 ] .
the root canal system is complex and accessory features , such as fins , cul de sacs , and intercanal communications , are colonized by microorganisms once the tooth becomes infected [ 13 , 14 ] .
self - aggregates of monobacterial morphotypes and coaggregates of different bacterial morphotypes are also found adhering to teeth .
the interbacterial spaces are occupied by an amorphous material , spirochetes , and hyphal - like structures that are suggestive of fungi [ 15 , 16 ] .
bacteria within a biofilm have increased resistance to a variety of external hostile influences , such as the host defense responses , antibiotics , antiseptics , and shear forces , compared with isolated bacterial cells .
this article reviews recent developments in the identification of new agents to sterilize infected root canal .
the most frequently isolated microorganisms before root canal treatment include gram - negative anaerobic rods , gram - positive anaerobic cocci , gram - positive anaerobic and facultative rods , lactobacillus species , and gram - positive facultative streptococcus species .
the obligate anaerobes are rather easily eradicated during root canal treatment . on the other hand , facultative bacteria such as nonmutans streptococci , enterococci , and lactobacilli , once established , are more likely to survive chemomechanical instrumentation and root canal medication
. in particular enterococcus faecalis has gained attention in the endodontic literature , as it can frequently be isolated from root canals in cases of failed root canal treatments [ 22 , 23 ] .
in addition , yeasts may also be found in root canals associated with therapy - resistant apical periodontitis .
it is generally believed that mechanical enlargement of canals must be accompanied by copious irrigation in order to facilitate maximum removal of microorganisms so that the prepared canal becomes as bacteria - free as possible [ 25 , 26 ] .
ideally an irrigant should provide a mechanical flushing action , be microbiocidal and dissolve remnants of organic tissues without damaging the periradicular tissues if extruded into the periodontium .
a large number of substances have been used as root canal irrigants , including acids ( citric and phosphoric ) , chelating agent ( ethylene diaminetetraacetic acid edta ) , proteolytic enzymes , alkaline solutions ( sodium hypochlorite , sodium hydroxide , urea , and potassium hydroxide ) , oxidative agents ( hydrogen peroxide and gly - oxide ) , local anesthetic solutions , and normal saline . the most widely used endodontic irrigant is 0.5% to 6.0% sodium hypochlorite ( naocl ) , because of its bactericidal activity and ability to dissolve vital and necrotic organic tissue [ 28 , 29 ] . however , naocl solutions exert no effects on inorganic components of smear layer .
chelant and acid solutions have been recommended for removing the smear layer from instrumented root canals , including ethylene diaminetetraacetic acid ( edta ) , citric acid , and phosphoric acid [ 30 , 31 ] .
ideal requirement of root canal irrigantsit appears evident that root canal irrigants ideally should have a broad antimicrobial spectrum and high efficacy against anaerobic and facultative microorganisms organized in biofilms , dissolve necrotic pulp tissue remnants , inactivate endotoxin , prevent the formation of a smear layer during instrumentation or dissolve the latter once it has formed , be systemically nontoxic , be non caustic to periodontal tissues , be little potential to cause an anaphylactic reaction .
it appears evident that root canal irrigants ideally should have a broad antimicrobial spectrum and high efficacy against anaerobic and facultative microorganisms organized in biofilms , dissolve necrotic pulp tissue remnants , inactivate endotoxin , prevent the formation of a smear layer during instrumentation or dissolve the latter once it has formed , be systemically nontoxic , be non caustic to periodontal tissues , be little potential to cause an anaphylactic reaction .
have a broad antimicrobial spectrum and high efficacy against anaerobic and facultative microorganisms organized in biofilms , dissolve necrotic pulp tissue remnants , inactivate endotoxin , prevent the formation of a smear layer during instrumentation or dissolve the latter once it has formed , be systemically nontoxic , be non caustic to periodontal tissues , be little potential to cause an anaphylactic reaction .
it is not found in a free state in nature , but it exists in combination with sodium , potassium , calcium , and magnesium . in the human body ,
they are generated by neutrophils via the myeloperoxidase - mediated chlorination of a nitrogenous compound or set of compounds .
hypochlorite preparations are sporicidal and virucidal and show far greater tissue dissolving effects on necrotic than on vital tissues .
these features prompted the use of aqueous sodium hypochlorite in endodontics as the main irrigant as early as 1920 .
there has been much controversy over the concentration of hypochlorite solutions to be used in endodontics .
the antibacterial effectiveness and tissue dissolution capacity of aqueous hypochlorite is a function of its concentration , and so is its toxicity .
5.25% sodium hypochlorite as it is sold in the form of household bleach leading to several adverse reactions like irritation and decrease in flexural strength of dentin .
it must be realized that during irrigation , fresh hypochlorite consistently reaches the canal system , and concentration of the solution may thus not play a decisive role .
unclean areas may be a result of the inability of solutions to physically reach these areas rather than their concentration .
hence , based on the currently available evidence , there is no rationale for using hypochlorite solutions at concentrations over 1% wt / vol .
one of the methods to improve the efficacy of sodium hypochlorite was to use heated solution .
furthermore , heated hypochlorite solutions remove organic debris from dentin shavings more efficiently than unheated counterparts . however , there are no clinical studies available at this point to support the use of heated sodium hypochlorite .
accelerate chemical reactions , create cavitational effects , and achieve a superior cleansing action .
however , results obtained with ultrasonically activated hypochlorite versus irrigation alone are contradictory , in terms of both root canal cleanliness and remaining microbiota in the infected root canal system after the cleaning and shaping procedure .
it should also be noted that time is a factor that has gained little attention in endodontic studies .
even fast - acting biocides such as sodium hypochlorite require an adequate working time to reach their potential .
this should especially be considered in view of the fact that rotary root canal preparation techniques have expedited the shaping process .
the optimal time that a hypochlorite irrigant at a given concentration needs to remain in the canal system is an issue yet to be resolved .
although sodium hypochlorite appears to be the most desirable single endodontic irrigant , it can not dissolve inorganic dentin particles and thus prevent the formation of a smear layer during instrumentation .
demineralizing agents such as ethylenediamine tetraacetic acid ( edta ) and citric acid have therefore been recommended as adjuvants in root canal therapy [ 30 , 31 ] .
these are highly biocompatible and are commonly used in personal care products . although citric acid appears to be slightly more potent at similar concentration than edta , both agents show high efficiency in removing the smear layer . in addition to their cleaning ability , chelators may detach biofilms adhering to root canal walls . an alternating irrigating regimen of naocl and edta
may be more efficient in reducing bacterial loads in root canal systems than naocl alone .
antiseptics such as quaternary ammonium compounds ( edtac ) or tetracycline antibiotics ( mtad ) have been added to edta and citric acid irrigants , respectively , to increase their antimicrobial capacity .
the clinical value of this , however , is questionable [ 32 , 33 ] . generally speaking , the use of antibiotics instead of biocides such as hypochlorite or chlorhexidine appears unwarranted , as the former were developed for systemic use rather than local wound debridement and have a far narrower spectrum than the latter .
both citric acid and edta immediately reduce the available chlorine in solution , rendering the sodium hypochlorite irrigant ineffective on bacteria and necrotic tissue .
calt and serper demonstrated that 10 ml irrigation with 17% edta for 1 minute was effective in removal of smear layer , but a 10-minute application caused excessive peritubular and intertubular dentinal erosion . increasing contact time and concentration of edta from 10% to 17% as well as a ph of 7.5 versus ph 9.0 has been shown to increase dentin demineralization .
chlorhexidine was developed in the late 1940s in the research laboratories of imperial chemical industries ltd .
the original salts were chlorhexidine acetate and hydrochloride , both of which are relatively poorly soluble in water .
chlorhexidine is a potent antiseptic , which is widely used for chemical plaque control in the oral cavity .
aqueous solutions of 0.1 to 0.2% are recommended for that purpose , while 2% is the concentration of root canal irrigating solutions usually found in the endodontic literature .
a 2% chlorhexidine solution is irritating to the skin . as with sodium hypochlorite , heating a chlorhexidine irrigant of lesser concentration could increase its local efficacy in the root canal system while keeping the systemic toxicity low . despite its usefulness as a final irrigant
, chlorhexidine can not be advocated as the main irrigant in standard endodontic cases , because ( a ) chlorhexidine is unable to dissolve necrotic tissue remnants , and ( b ) chlorhexidine is less effective on gram - negative than on gram - positive bacteria .
all the irrigation solutions at our disposable have their share of limitations and the search for an ideal root canal irrigant continues with the development of newer materials and methods .
newer root canal irrigants in the horizon are as follows : electrochemically activated solutions , photon - activated disinfection , the article reviews the advantages and shortcomings of these newer irrigating agents and their potential role in endodontic irrigation in near future .
bio pure mtad ( dentsply , tulsa , ok ) is a mixture of a tetracycline isomer , an acetic acid , and tween 80 detergent ( mtad)was designed to be used as a final root canal rinse before obturation .
tetracycline has many unique properties of low ph and thus can act as a calcium chelator and cause enamel and root surface demineralization .
in addition , it has been shown that it is a substantive medication ( becomes absorbed and gradually released from tooth structures such as dentin and cementum [ 33 , 35 ] .
manufacturer instructions for using this irrigant were flooding the root canal with 1 ml of the irrigant and soaking for 5 minutes , and the remaining 4 ml is then delivered with continuous irrigation and suction .
mtad is effective in removing the smear layer along the whole length of the root canal and in removing organic and inorganic debris and does produce any signs of erosion or physical changes in dentine , whereas a mixture of 5.25% sodium hypochlorite and 17% edta does [ 3638 ] . in particular , mtad mixture is effective against e. faecalis , and it is also less cytotoxic than a range of endodontic medicaments , including eugenol , hydrogen peroxide ( 3% ) , edta , and calcium hydroxide paste [ 3942 ] .
torabinejad et al . showed that the effectiveness of the mtad was enhanced when low concentration of naocl is used as an intracanal irrigant before the use of mtad as a final rinse .
newberry et al . determined the antimicrobial effect of mtad as a final irrigant on eight strains of enterococcus faecalis .
after irrigating with 1.3% naocl , the root canal and the external surfaces were exposed to mtad for 5 minutes .
the results showed that this treatment regimen was effective in completely eliminating growth in seven of eight strains of e. faecalis .
compared the efficacy of bio- pure mtad , 17% edta , and 42% citric acid in endodontic smear layer removal and degree of erosion in the apical third of endodontic canals .
none of the agents were found to be efficient in the apical one third of the root canal .
showing an effective cleaning action with biopure mtad in the apical third [ 32 , 36 ] .
the placement of mtad with a cotton - wrapped barbed broach allows intimate contact of the solution even in the apical region of the canals and improves debridement of the entire root canal wall according to torabinejad et al . .
the effectiveness of sodium hypochlorite , chlorhexidine gluconate , and chlorhexidine acetate on c. albicans is documented in the literature [ 45 , 46 ] .
found that edta was the most effective irrigant against c. albicans using the agar diffusion test .
doxycycline is primarily a bacteriostatic antibiotic and inhibits bacterial protein synthesis by binding to the 30 s bacterial ribosome .
doxycycline is active against a wide range of gram - positive and gram - negative organisms , but it is not active against fungi . as with other antibiotic preparations , use of this
drug results in changes in the balance of normal flora and overgrowth of nonsusceptible organisms , including fungi .
it follows that antifungal efficacy of mtad when used in combination with naocl may be clinically insignificant . to further improve antisepsis ,
ruff et al . showed that 6% naocl and 2% chlorohexidine were equally effective and statistically significantly superior to biopure mtad and 17% edta in antifungal activity .
clegg et al . questioned the ability of mtad to remove or disrupt bacterial biofilms in root canals [ 29 , 42 ] .
mtad is less cytotoxic than eugenol , 3% hydrogen peroxide , calcium hydroxide paste , 5.25% naocl , peridex , and edta and more cytotoxic than 2.63% , 1.31% , and 0.66% naocl .
mtad can be a useful irrigant due to its antimicrobial property , less cytotoxic , but its effectiveness against fungi and value in the apical one third need to be assessed further .
tetraclean ( ogna laboratori farmaceutici , muggi ( mi ) , italy ) , like mtad , is a mixture of an antibiotic , an acid , and a detergent . however , the concentration of the antibiotic , doxycycline ( 50 mg / ml ) , and the type of detergent ( polypropylene glycol ) differ from those of mtad .
compared the surface tension of 17% edta , cetrexidin , smear clear , 5.25% naocl , mtad and tetraclean .
the naocl and edta had the highest surface tension , whereas cetrexedin and tetraclean had the lowest values .
in another study , they compared the antimicrobial efficacy of 5.25% naocl , mtad , and tetraclean against an e. faecalis biofilm generated on cellulose nitrate membrane filters .
only the naocl could disaggregate and remove the biofilm at every time interval tested although treatment with tetraclean caused a high degree of biofilm disaggregation at each time interval when compared with mtad .
electrochemically activated ( eca ) solutions are produced from tap water and low - concentrated salt solutions [ 5456 ] .
the eca technology represents a new scientific paradigm developed by russian scientists at the all - russian institute for medical engineering ( moscow , russia , cis ) .
principle of eca is transferring liquids into a metastable state via an electrochemical unipolar ( anode or cathode ) action through the use of an element / reactor ( flow - through electrolytic module or fem ) .
the fem consists of an anode , a solid titanium cylinder with a special coating that fits coaxially inside the cathode , a hollow cylinder also made from titanium with another special coating .
the fem is capable of producing types of solutions that have bactericidal and sporicidal activity ; yet they are odourless , safe to human tissue , and essentially noncorrosive for most metal surfaces .
electrochemical treatment in the anode and cathode chambers results in the synthesis of two types of solutions : that produced in the anode chamber is termed an anolyte , and that produced in the cathode chamber is catholyte .
anolyte solutions containing a mixture of oxidizing substances demonstrate pronounced microbiocidal effectiveness against bacteria , viruses , fungi , and protozoa [ 54 , 57 ] .
anolyte solution has been termed superoxidized water or oxidative potential water [ 58 , 59 ] . depending on the type eca device that incorporated the fem elements the ph of anolyte varies ; it may be acidic ( anolyte ) , neutral ( anolyte neutral ) , or alkaline ( anolyte neutral cathodic ) ; acidic anolyte was used initially but in recent years the neutral and alkaline solutions have been recommended for clinical application . under clean conditions ,
freshly generated superoxidized solution was found to be highly active against all these microorganisms giving a 99.999% or greater reduction in two minutes or less . that allowed investigators to treat it as a potent microbiocidal agent [ 58 , 60 ] .
it is nontoxic when being in contact with vital biological tissues [ 61 , 62 ] .
eca solutions demonstrated more pronounced clinical effect and were associated with fewer incidences of allergic reactions compared to other antibacterial irrigants tested .
cleaning efficiency and safety for surfaces of dental instruments and equipment has been demonstrated in a number of studies .
the experience of oxidative potential water application for irrigation of root canals has been reported . however , haga and coworkers studied the effect of acidic anolyte solutions .
the anolyte neutral cathodic solution ( anc ) provides an increased antiseptic effect and an enhanced cleaning ability at lower concentrations of active chlorine compared to the acidic anolyte and anolyte neutral solutions because of its higher concentration of peroxides . both electrolyzed neutral water and oxidative potential water
are claimed to be harmless to humans and are probably similar to eca water [ 33 , 37 , 54 ] .
the quality of debridement was better in the coronal and middle parts of canal walls where only scattered debris was noted in contrast to the apical part that contained numerous debris .
this observation confirms the previously published results . according to solovyeva and dummer naocl and eca solutions left a thinner smear layer with a smoother and more even surface .
the texture of the canal surfaces treated with eca solutions was relatively uniform in the various regions of the root canal and did not seem to be influenced by the method of instrumentation , that is , manually or mechanical .
it is important to note that irrigation with naocl resulted in open tubules predominantly in the coronal and middle thirds of root canals with no signs of tubule orifices were revealed in the apical third of canals .
irrigating with anolyte neutral cathodic as well as with alternate anc and catholyte resulted in more numerous open dentine tubules in the apical as well as in the coronal regions .
solovyeva and dummer studied the cleaning effectiveness of root canal irrigation with eca solution and found that it was similar to naocl in debris removal but was more effective than naocl in smear layer removal .
eca is showing promising results due to ease of removal of debris and smear layer , nontoxic and efficient in apical one third of canal .
ozone is a chemical compound consisting of three oxygen atoms ( o3triatomic oxygen ) , a higher energetic form than normal atmospheric oxygen ( o2 ) .
ozone is created when an oxygen molecule receives an electrical discharge breaking it into two oxygen atoms .
the individual atoms combine with another oxygen molecule to form an o3 molecule . the second from ultraviolet rays emitted from the sun which plays the role of electrical discharge over oxygen present in the stratosphere , thus , creating the ozone layer which absorbs most of the ultraviolet radiation emitted by the sun .
it was reported that ozone at low concentration , 0.1 ppm , is sufficient to inactivate bacterial cells including their spores .
it is present naturally in air and can be easily produced by ozone generator . when introduced in water
the concentration of ozone in ozonated water can be measured using a dissolved ozone meter .
although ozonated water is a powerful antimicrobial agent against bacteria , fungi , protozoa , and viruses , less attention has been paid to the antibacterial activity of ozonated water in bacterial biofilm and hence in root canal infection [ 66 , 67 ] .
cavitation is the formation of vapor - containing bubbles inside a fluid causing formation of pressure waves / shockwaves characterized by rapid changes in pressure and high amplitude .
a forced collapse of bubbles causes implosions that impact on surfaces , causing shear forces , surface deformation , and removal of surface material . in the root canal environment , such shockwaves could potentially disrupt bacterial biofilms , rupture bacterial cell walls , and remove smear layer and debris .
shockwave generation can also enhance the breakdown of agents such as hydrogen peroxide and ozone dissolved in water and thereby enhance their disinfecting and debriding actions [ 70 , 71 ] .
nagayoshi et al . found that killing ability of ozonated water and 2.5% of sodium hypochlorite was almost comparable when the specimen was irrigated with sonication .
study by hems et al . however found that naoci was superior to ozonated water in killing e. faecalis in broth culture and in biofilm .
ibrahim and abdullah studied that 1.31% naoci might allow passage of oxidation of ozonated water , thus increasing their antibacterial effect compared to 1.31% naoci or ozonated water alone .
cardoso evaluated the efficiency of ozonated water as an irrigating agent during endodontic treatment in an attempt to eliminate candida albicans and enterococcus faecalis and toneutralize lipopolysacharides ( lpss ) inoculated in root canals .
it was possible to see effective antimicrobial action after ten minutes of water ozonization on the microbial suspension .
however , ozonated water was not able to neutralize e. coli and lps inside root canals and the remaining amount of lps may have biological consequences such as apical periodontitis .
estrela et al . assessed the antimicrobial efficiency of aqueous ozone , gaseous ozone , 2.5% sodium hypoclorite , and 2% chlorexidine in human root canals infected with enterococcus faecalis .
none of the solutions tested were found to be effective against the bacterial suspension [ 74 , 75 ] .
there is need for further studies and modifications in ozonated water before it could be used as a root canal irrigant .
the use of photodynamic therapy ( pdt ) for the inactivation of microorganisms was first shown by oscar raab who reported the lethal effect of acridine hydrochloride on paramecia caudatum .
pdt is based on the concept that nontoxic photosensitizers can be preferentially localized in certain tissues and subsequently activated by light of the appropriate wavelength to generate singlet oxygen and free radicals that are cytotoxic to cells of the target tissue .
methylene blue ( mb ) is a well - established photosensitizer that has been used in pdt for targeting various gram - positive and gram - negative oral bacteria and was previously used to study the effect of pdt on endodontic disinfection [ 7885 ] .
several studies have shown incomplete destruction of oral biofilms using mb - mediated pdt due to reduced penetration of the photosensitizer [ 8689 ] .
used the combined effect of mb and red light ( 665 nm ) exhibited up to 97% reduction of bacterial viability .
the results suggested the potential of pdt to be used as an adjunctive antimicrobial procedure after standard endodontic chemomechanical debridement , but they also demonstrated the importance of further optimization of light dosimetry for bacterial photodestruction in root canals .
along with methylene blue , tolonium chloride has been also used as a photosensitizing agent .
it is applied to the infected area and left in situ for a short period .
the agent binds to the cellular membrane of bacteria , which will then rupture when activated by a laser source emitting radiation at an appropriate wavelength ( e.g. , 635 nm radiation emitted by savedent ; denfotex light systems ltd . , inverkeithing , united kingdom ) .
the light is transmitted into the root canals at the tip of a small flexible optical fiber that is attached to a disposable handpiece .
the laser emits a maximum of only 100 mw and does not generate sufficient heat to harm adjacent tissues .
lethal photosensitization of streptococcus intermedius biofilms in root canals is unable to achieve a total kill rate when a combination of a helium - neon laser and tolonium chloride is used .
leticia et al . investigated the antibacterial effects of photodynamic therapy ( pdt ) with methylene blue ( mb ) or toluidine blue ( tb ) ( both at 15 mg / ml ) as a supplement to instrumentation / irrigation of root canals experimentally contaminated with enterococcus faecalis .
the study revealed that pdt with either mb or tb may not exert a significant supplemental effect to instrumentation / irrigation procedures with regard to intracanal disinfection , until further adjustments in the pdt protocol are modified before clinical use is recommended .
in contrast , irrigation with sodium hypochlorite ( 3% ) eliminated the entire bacterial population . the difference could be because the optical fiber was not properly introduced into the root canals , and so the light could not transmit through the tooth structure .
thus , pad might not be able to achieve a 100% kill rate in infected root canals that have complex anatomic features and colonized by polymicrobial biofilms of varying properties .
pagonis et al . studied the in vitro effects of poly(lacticco- glycolic acid ) ( plga ) nanoparticles loaded with the photosensitizer methylene blue ( mb ) and light against enterococcus faecalis ( atcc 29212 ) .
the study showed that utilization of plga nanoparticles encapsulated with photoactive drugs may be a promising adjunct in antimicrobial endodontic treatment .
triphala ( impcops ltd , chennai , india ) is an indian ayurvedic herbal formulation consisting of dried and powdered fruits of three medicinal plants , terminalia bellerica , terminalia chebula , and emblica officinalis , and green tea polyphenols ( gtps ; essence and flavours , mysore , india ) ; the traditional drink of japan and china is prepared from the young shoots of tea plant camellia sinensis [ 9497 ] .
dimethyl sulfoxide ( dmso ) is used as a solvent for triphala and gtp , although they are readily soluble in water .
dmso is a clean , safe , highly polar , aprotic solvent that helps in bringing out the pure properties of all the components of the herb being dissolved [ 98 , 99 ] .
herbal alternatives showed promising antibacterial efficacy on 3- and 6-week biofilm along with mtad and 5% sodium hypochlorite .
although triphala and green tea polyphenols ( gtps ) exhibited similar antibacterial sensitivity on e. faecalis planktonic cells , triphala showed more potency on e. faecalis biofilm .
this may be attributed to its formulation , which contains three different medicinal plants in equal proportions . in such formulations , different compounds
may be of help in enhancing the potency of the active compounds resulting in an additive or synergistic positive effect . according to prabhakar et al .
5% of sodium hypochlorite exhibited excellent antibacterial activity in both 3-week and 6-week biofilm , whereas triphala and mtad showed complete eradication only in 3-week biofilm .
triphala and gtps are proven to be safe , containing active constituents that have beneficial physiologic effect apart from its curative property such as antioxidant , anti - inflammatory , and radical scavenging activity and may have an added advantage over the traditional root canal irrigants [ 100103 ] .
available literature and studies demonstrate advantages and limitations of each irrigant under consideration and none of them satisfy the requirements of the ideal root canal irrigant completely .
presently these newer irrigants could be used as an adjunct to naocl , with the hunt for the elusive ideal root canal irrigant continues . | sodium hypochloride is the most commonly used endodontic irrigant , despite limitations .
none of the presently available root canal irrigants satisfy the requirements of ideal root canal irrigant .
newer root canal irrigants are studied for potential replacement of sodium hypochloride .
this article reviews the potential irrigants with their advantages and limitations with their future in endodontic irrigation . |
recently , helper - dependent adenoviral vectors ( hdads ) have been used to deliver donor dna into human embryonic stem cells ( escs ) , induced pluripotent stem cells ( ipscs ) , and adult stem cells to achieve high efficiency gene editing by homologous recombination to a wide variety of transcriptionally active and inactive loci , to achieve knockins , knockouts , and corrections .
collectively , these studies have all consistently demonstrated that hdad - mediated gene editing of ipscs and escs is not associated with ectopic random hdad integrations , does not affect the undifferentiated state and pluripotency , and maintains genetic and epigenetic integrity .
indeed a recent study found that targeted gene correction in ipscs by hdad minimally impacts whole - genome mutational load as determined by whole genome sequencing .
the major appeal of hdad - mediated gene editing is that induction of an artificial double stranded break at the chromosomal target locus by a designer endonuclease is not required to achieve high targeting efficiency , thereby eliminating the potential for off - target cleavage .
hdads possess many features that explain their effectiveness as a gene editing vector reviewed in ref .
12 . they can very efficiently deliver foreign dna into the nucleus of target cells .
they are deleted of all viral - coding sequences thereby reducing their toxicity and increasing their cloning capacity to 37 kb .
this tremendous cloning capacity permits inclusion of long homology arms capable of correcting multiple mutations , as well as inclusion of multiple selectable markers and promoters / enhancers / cis - acting elements , and other transgenes to enhance their gene editing efficiency .
hdads also rarely integrate randomly into the host genome and they offer the potential for in vivo gene editing . as mentioned above , the large cloning capacity of hdad permits inclusion of long homology arms to maximize targeting efficiency , a common feature in all of the aforementioned studies .
however , the relationship between gene editing efficiency and the length of homology is not known .
this is an important issue because inclusion of long homologies renders vector construction and manipulation difficult and also complicates southern blot and polymerase chain reaction ( pcr ) analyses that are required to verify the genomic structure of targeted recombinants and in some cases inclusion of long homologies may not be possible . on the other hand , the efficiency of gene editing may be severely compromised by insufficient homology . clearly , the need to determine the relationship between targeting efficiency and homology length is important for advancing this technology .
another common feature of all hdads used for gene editing is inclusion of a positive selectable marker flanked by either loxp or frt sites which permit selection for vector integration and subsequent removal of the positive selectable marker by transient expression of cre or flp recombinase , respectively .
however , this strategy leaves behind a residual loxp or frt site at the target locus which is undesirable .
clearly , the ability to achieve footprintless genome modification is highly desirable , especially for potential clinic applications where introduction of only the minimally required genomic alteration would be desirable .
indeed it has been reported that the residual loxp site can interfere with expression of surrounding genes or potentially disrupt splicing elements . in this study
, we address the important issues raised above by investigating the relationship between the efficiency of hdad - mediated gene editing and the length of homology .
we also demonstrate that hdad can mediate footprintless gene editing by using piggybac ( pb)-mediated excision of the vector 's positive selection marker .
the objectives of this study were to determine the effect of homology length on the efficiency of hdad - mediated gene editing and to achieve hdad - mediated footprintless gene editing .
as a model system , we targeted the cystic fibrosis transmembrane conductance regulator ( cftr ) gene in a human ips cell line ( called cf17 ( ref .
16 ) ) that is compound heterozygous at the cftr locus , with one allele f508 and the other allele i507 in exon 10 for correction with hdads bearing the wild - type sequence in exon 10 ( figure 1a ) .
as shown previously , targeted correction of either mutant allele restores expression of the mature cftr glycoprotein and chloride channel function in epithelial cells derived from these ipscs . to achieve the aforementioned objectives ,
a panel of hdads bearing different total homology lengths to the cftr gene was generated , all bearing the wild - type cftr sequence ( figure 2 ) . to minimize variability
, each vector differs from the vector one size smaller and one size larger in only one of the two homology arms ; hd-23.8-cftr - neo contains a total of 23.8 kb of homology with 11.9 kb in the each of the two homology arms .
hd-21.4-cftr - neo contains a total of 21.4 kb of homology , having the same 11.9 kb right homology arm as hd-23.8-cftr - neo but a shorter left homology arm of 9.5 kb .
hd-14.8-cftr - neo contains a total of 14.8 kb of homology , with the same 9.5 kb left homology arm as hd-21.4-cftr - neo but a shorter right homology arm of 5.3 kb .
hd-9.6-cftr - neo contains a total of 9.6 kb of homology with the same 5.3 kb right homology arm as hd-14.8-cftr - neo , but a shorter 4.3 kb left homology arm .
hd-5.6-cftr - neo contains a total of 5.6 kb of homology , with the same 4.3 kb left homology arm as hd-9.6-cftr - neo but a shorter right homology arm of 1.3 kb .
all hdads possess a neomycin resistance marker ( neo ) to permit positive selection for hdad integration which is flanked by pb inverted terminal repeats to permit its footprintless excision by pb transposase .
all hdads possess the herpes simplex virus thymidine kinase ( hsv - tk ) cassette outside the region of homology to permit negative selection against random vector integration . to determine the effect of homology length on the efficiency of gene targeting by hdad
, 2 10 cf17 cells were transduced with each of the hdads shown in figure 2 at a multiplicity of infection ( moi ) of 175 vp / cell ( table 1 , transduction 1 ) or 350 vp / cell ( table 1 , transduction 2 ) . following selection ,
the total numbers of g418 resistant and ganciclovir resistant ( g418gcv ) colonies were enumerated ( table 1 , column 3 ) .
dna was extract from all g418gcv colonies , digested with apai and subject to southern analysis to identify targeted recombinants . for all hdads shown in figure 2 , targeted vector integration by a single crossover in each of the two homology arms results in the insertion of neo into the cftr gene , converting the endogenous 30.1 kb apai fragment ( revealed by the 5probe and 3 probe ) into a 19.1 kb ( revealed by the 5 probe and neo probe ) and a 14.2 kb apai fragment ( revealed by the 3 probe ) ( figure 1a ) .
the results of the southern blot analyses for all g418gcv colonies are summarized in table 1 .
representative southern blots are presented in figure 1b , which show that all 16 g418gcv colonies obtained from transduction with hd-23.8-cftr - neo at an moi of 175 vp / cell were correctly targeted into one of the two cftr alleles as evident by the presence of the 19.1 kb band when analyzed with the 5 probe and the presence of the 14.2 kb band when analyzed with the 3probe ( the 31 kb band in both blots is the unmodified allele ) .
this was further confirmed with the neo probe which hybridized to only the 19.1 kb band as expected .
the results revealed that 23.8 kb of total homology yielded the highest number of targeted recombinants and reducing total homology to 21.4 kb resulted in a ~30% reduction in the number of targeted recombinants ( table 1 , transduction 1 and 2 , column 4 ) .
however , both 23.8 kb and 21.4 kb of homology yielded comparable frequencies of targeted recombinants per g418 cell of 5056.3% ( table 1 , transduction 1 and 2 , column 5 ) , and also comparable frequencies targeted recombinants per g418gcv of 97.4100% ( table 1 , transduction 1 and 2 , column 6 ) . when total homology was reduced to 14.8 kb
, the total number of target recombinant decreased to less than half that obtained with 21.4 kb ( table 1 , transduction 1 and 2 , column 4 ) .
however , the frequencies of targeted recombinants per g418 cell remained relatively high at 26.957.1% ( table 1 , transduction 1 and 2 , column 5 ) and the frequency of targeted recombinants per g418gcv cell also remained high at 87.5100% ( table 1 , transduction 1 and 2 , column 6 ) . reducing total homology to
9.6 kb resulted in a further reduction in the total number of targeted recombinants ( table 1 , transduction 2 , column 4 ) as well as a reduction in the frequency of targeted recombinants per g418 cells to 21.4% ( table 1 , transduction 2 , column 5 ) but the frequency of targeted recombinants per g418gcv cell remained relatively high at 75% ( table 1 , transduction 2 , column 6 ) . further reducing total homology to only 5.6
kb did not appear to further reduce the total number of targeted recombinants compared with 9.6 kb total homology ( table 1 , transduction 1 and 2 , column 4 ) , but the frequency of targeted recombinants per g418 cells was reduced to 16.7% ( table 1 , transduction 1 and 2 , column 5 ) , and the frequency of targeted recombinants per g418gcv was reduced to 50% ( table 1 , transduction 1 and 2 , column 6 ) .
nevertheless , even with total homology as short as 5.6 kb , the frequency of targeted vector integration was still high enough to make identifying and isolating the targeted clones relatively easy and practical . as shown in table 1 , transduction with an moi of 350 vp / cell yielded twofold to fourfold more targeted recombinants compared with an moi of 175 vp / cell .
interestingly , we observed one clone from transduction with 350 vp / cell of hd-5.6-cftr - neo , which appeared to have undergone aberrant targeting and this is addressed in more detail below .
for all 110 correctly targeted clones from transduction 1 and 2 , southern analyses with the 5 and 3 probe revealed the presence of the 31 kb and this indicated that the vector had integrated into only one of the two cftr alleles in all cases ( figure 1b ) . to identify the targeted allele ,
pcr was used to amplify a 964 bp product specifically from the nontargeted allele and the product was sequenced to determine its identity .
the results are summarized in table 1 , column 4 and suggest that there did not appear to be an obvious allelic preference for targeted vector integration when all hdads were considered cumulatively ; overall , 56.4% ( 62/110 ) of the targeted integration occurred at the i507 allele while the remaining 43.6% ( 48/110 ) occurred at the f508 allele . however , although the numbers are small , a preference for targeting into the i507 allele might exist for the two vectors with the shortest homology lengths ( hd-9.6-cftr - neo and hd-5.6-cftr - neo ) .
we selected clone 26 , which had undergone targeted vector integration into the f508 allele , for pb - mediated excision of the neo cassette . to accomplish this
, 2 10 26 cells were transduced with hdad - cag - hypb - vai , a hdad expressing the hyperactive pb transposase , at an moi of 350 vp / cell .
the transduced cells were plated at low density in the absence of g418 and 107 colonies were picked , and of these 34 were determined to be sensitive to g418 ( g418 ) .
pb - mediated excision of neo converts the 19.1 and 14.2 kb apai fragments back to a 31 kb apai fragment ( figure 1a ) .
unexpectedly , southern analyses revealed that none of the 34 g418 clones had undergone pb - mediated excision of neo as evident by the continued presence of the 19.1 kb apai fragment revealed by the 5 and neo probes and the 14.2 kb apai fragment revealed by the 3 probe ( figure 3 ) . in an independent repeated attempt , another 2 10 26 cells were transduced with hdad - cag - hypb - vai , and this time 112 colonies were picked , 26 of which were determined to be g418 .
however , southern analyses again revealed none had undergone pb - mediated neo excision ( not shown ) . in a third attempt , we selected a different targeted clone , clone 14 , that had undergone targeted vector integration into the i507 allele .
following transduction of 2 10 14 cells with hdad - cag - hypb - vai at an moi of 350 vp / cell , 240 colonies were picked and 59 were determined to be g418 .
however , again none of the 59 clones had undergone pb - mediated neo excision as determined by southern analyses ( not shown ) .
we do not understand why these clones were sensitive to g418 even though excision of the neo cassette had not occurred . that no pb - mediated neo excision could be identified after screening a total of 459 colonies suggested that this reaction occurs at a relatively low frequency and that negative selection would be needed to isolate these events .
therefore , to accomplish this , the neo cassette in hd-23.8-cftr - neo was replaced with the positive / negative selectable pactk cassette to generate hd-23.8-cftr - pactk ( figure 4a ) .
pactk is a fusion of the puromycin n - acetyltransferase gene and the hsv - tk gene and therefore provides positive selection for vector integration by conferring puromycin resistance ( puro ) and negative selection for loss of pactk as a consequence of pb - mediated excision by conferring gcv .
unfortunately , the use of pactk meant that the hsv - tk cassette could no longer be used for negative selection against random vector integrations and thus was not included in hd-23.8-cftr - pactk .
fortunately , the relatively high frequencies of targeted integration after only positive selection ( 55.255.9% , table 1 , transduction 1 and 2 , column 5 ) should make identifying them relatively easy and practical even without negative selection against random integrations .
a total of 144 puro colonies were obtained following transduction of 2 10 cf17 cells with hd-23.8-cftr - pactk at an moi of 350 vp / cell ( table 1 , transduction 3 ) and southern analyses were performed on their extracted dna . as shown in figure 4a , targeted vector integration into the cftr gene converts the endogenous 31 kb apai fragment ( revealed by the 5 and 3 probes ) into a 19 kb ( revealed by the 5 probe ) and a 14.7 kb ( revealed by the 3 probe and pactk probe ) apai fragment .
the results of the southern analyses are summarized in table 1 ( transduction 3 ) and a representative blot is shown in figure 4b .
the southern analyses revealed that of the 144 puro clones , 93 of these were correctly targeted into one of the two cftr alleles ( no biallelic targeting was observed ) , 47 had random vector integration , and four were aberrantly targeted ( table 1 , transduction 3 ) .
next , we sought to achieve pb - mediated excision of the pactk marker to accomplish footprintless correction of the cftr mutation .
two correctly targeted clones , clone 7 and 27 , were selected for pb - mediated excision .
clone 7 and 27 had targeted vector integration into the f508 and i507 allele , respectively , as determined by sequencing of the 964 bp pcr product from the unmodified allele ( not shown ) .
2 10 clone 7 or clone 27 cells were transduced with hdad - cag - hypb - vai at an moi of 350 vp / cell .
because the frequency of gcv was unknown , the transduced cells were plated at a wide variety of cell densities in the absence of puromycin ( table 2 ) , and 48 hours later , gcv was added to the culture media .
if pb - mediated pactk excision was responsible for gcv , dna was extracted from 107 and 113 gcv colonies originating from clone 7 and 27 , respectively , and subjected to southern analyses . as shown in figure 4a , pb - mediated pactk excision converts the 19 kb and the 14.7 kb apai fragments back to a 31 kb apai fragment .
the results of the southern analyses revealed that pb - mediated pactk excision had occurred in all 220 gcv colonies and a representative southern blot is presented in figure 5 .
table 2 shows that pb - mediated pactk excision occurs at a frequency of 1 10 .
this explains why we were unable to identify pb - mediated excision of neo by simply screening 459 colonies .
thus the ability to perform negative selection afforded by pactk is essential for efficient isolation of clones that had undergone pb - mediated pactk excision . to confirm footprintless gene correction after pb - mediated pactk excision
, pcr was used to amplify a 964 bp region that encompassed both the site of i507/f508 mutation and the location of downstream pactk insertion from four gcv clones , two derived from clone 7 , and two from clone 27 .
the pcr product was then cloned into a bacterial plasmid ( required because both alleles are amplified after pb excision of pactk and this mixture can not be directly sequenced ) and sequenced and a representative result of this analysis is presented in figure 6 .
the sequence analyses revealed that in all four cases , correction of the i507 or f508 mutation had occurred and that pb - mediated excision was indeed footprintless .
as mentioned , four puro clones from transduction 3 appeared to have undergone aberrant targeting ( clones 21 , 57 , 34 , and 51 ) , two of these , clone 21 and 57 , are present in the southern blot presented in figure 4b . for clone 21 ,
the expected 31 and 19 kb bands are revealed with the 5 probe ( figure 4b ) indicating that the left homology arm of the hdad had integrated into one of the cftr alleles by correct homologous recombination .
however , the 3 probe and the pactk probe revealed a band that was larger than the expected 14.7 kb band ( figure 4b ) indicating the right homology arm of the hdad did not undergo correct homologous recombination with the target locus .
likewise , for clone 57 , the expected 31 and 19 kb bands are revealed with the 5 probe ( figure 4b ) indicating that the left homology arm of the hdad had integrated into one of the cftr alleles by correct homologous recombination .
however , the 3 probe revealed only the 31 kb band and the pactk probe revealed a band of high molecular weight band ( figure 4b ) indicating the right homology arm of the hdad did not undergo correct homologous recombination with the target locus . for clone 34 ,
the 5 probe revealed the expected 31 kb but not the expected 19 kb band , but instead an unexpected band between 31 kb and 19 kb was present ( not shown ) , the 3 probe revealed only the expected 31 kb band but not the expected 14.7 kb band , and the pactk probe revealed a band of high molecular weight ( not shown ) . for clone 51
the 5 probe revealed the expected 31 and 19 kb bands , but the 3 probe revealed only the expected 31 kb band but not the expected 14.7 kb band , and the pactk probe did not reveal any band ( not shown ) .
as mentioned above , transduction 2 with hd-5.6-cftr - neo also generated one aberrantly targeted clone which possessed the expected 31 and 19 kb band as revealed by the 5 probe , the expected 19 kb band revealed by the neo probe , but a band greater than the expected 14.1 kb band was revealed with the 3 probe ( not shown ) . in summary , in four of the five cases ( expected for clone 34 ) , the vector appears to have integrated into one of the cftr alleles by correct homologous recombination with the left homology arm but not the right homology arm . in the case of clone 34
, the vector appears to have integrated into one of the cftr alleles ( due to the presence of the expected 31 kb and a band of unexpected size with the 5 probe ) , but this integration did not occur by correct homologous recombination in either the left or right homology arms .
we made no further attempts to elucidate the genomic structure of these rare aberrantly targeted clones .
the objectives of this study were to determine the effect of homology length on the efficiency of hdad - mediated gene editing and to achieve hdad - mediated footprintless gene editing .
as a model system , we targeted the cystic fibrosis transmembrane conductance regulator ( cftr ) gene in a human ips cell line ( called cf17 ( ref .
16 ) ) that is compound heterozygous at the cftr locus , with one allele f508 and the other allele i507 in exon 10 for correction with hdads bearing the wild - type sequence in exon 10 ( figure 1a ) .
as shown previously , targeted correction of either mutant allele restores expression of the mature cftr glycoprotein and chloride channel function in epithelial cells derived from these ipscs . to achieve the aforementioned objectives ,
a panel of hdads bearing different total homology lengths to the cftr gene was generated , all bearing the wild - type cftr sequence ( figure 2 ) . to minimize variability
, each vector differs from the vector one size smaller and one size larger in only one of the two homology arms ; hd-23.8-cftr - neo contains a total of 23.8 kb of homology with 11.9 kb in the each of the two homology arms .
hd-21.4-cftr - neo contains a total of 21.4 kb of homology , having the same 11.9 kb right homology arm as hd-23.8-cftr - neo but a shorter left homology arm of 9.5 kb .
hd-14.8-cftr - neo contains a total of 14.8 kb of homology , with the same 9.5 kb left homology arm as hd-21.4-cftr - neo but a shorter right homology arm of 5.3 kb .
hd-9.6-cftr - neo contains a total of 9.6 kb of homology with the same 5.3 kb right homology arm as hd-14.8-cftr - neo , but a shorter 4.3 kb left homology arm .
hd-5.6-cftr - neo contains a total of 5.6 kb of homology , with the same 4.3 kb left homology arm as hd-9.6-cftr - neo but a shorter right homology arm of 1.3 kb .
all hdads possess a neomycin resistance marker ( neo ) to permit positive selection for hdad integration which is flanked by pb inverted terminal repeats to permit its footprintless excision by pb transposase .
all hdads possess the herpes simplex virus thymidine kinase ( hsv - tk ) cassette outside the region of homology to permit negative selection against random vector integration . to determine the effect of homology length on the efficiency of gene targeting by hdad
, 2 10 cf17 cells were transduced with each of the hdads shown in figure 2 at a multiplicity of infection ( moi ) of 175 vp / cell ( table 1 , transduction 1 ) or 350 vp / cell ( table 1 , transduction 2 ) . following selection ,
the total numbers of g418 resistant and ganciclovir resistant ( g418gcv ) colonies were enumerated ( table 1 , column 3 ) .
dna was extract from all g418gcv colonies , digested with apai and subject to southern analysis to identify targeted recombinants . for all hdads shown in figure 2 , targeted vector integration by a single crossover in each of the two homology arms results in the insertion of neo into the cftr gene , converting the endogenous 30.1 kb apai fragment ( revealed by the 5probe and 3 probe ) into a 19.1 kb ( revealed by the 5 probe and neo probe ) and a 14.2 kb apai fragment ( revealed by the 3 probe ) ( figure 1a ) .
the results of the southern blot analyses for all g418gcv colonies are summarized in table 1 .
representative southern blots are presented in figure 1b , which show that all 16 g418gcv colonies obtained from transduction with hd-23.8-cftr - neo at an moi of 175 vp / cell were correctly targeted into one of the two cftr alleles as evident by the presence of the 19.1 kb band when analyzed with the 5 probe and the presence of the 14.2 kb band when analyzed with the 3probe ( the 31 kb band in both blots is the unmodified allele ) .
this was further confirmed with the neo probe which hybridized to only the 19.1 kb band as expected .
the results revealed that 23.8 kb of total homology yielded the highest number of targeted recombinants and reducing total homology to 21.4 kb resulted in a ~30% reduction in the number of targeted recombinants ( table 1 , transduction 1 and 2 , column 4 ) .
however , both 23.8 kb and 21.4 kb of homology yielded comparable frequencies of targeted recombinants per g418 cell of 5056.3% ( table 1 , transduction 1 and 2 , column 5 ) , and also comparable frequencies targeted recombinants per g418gcv of 97.4100% ( table 1 , transduction 1 and 2 , column 6 ) . when total homology was reduced to 14.8 kb
, the total number of target recombinant decreased to less than half that obtained with 21.4 kb ( table 1 , transduction 1 and 2 , column 4 ) .
however , the frequencies of targeted recombinants per g418 cell remained relatively high at 26.957.1% ( table 1 , transduction 1 and 2 , column 5 ) and the frequency of targeted recombinants per g418gcv cell also remained high at 87.5100% ( table 1 , transduction 1 and 2 , column 6 ) . reducing total homology to
9.6 kb resulted in a further reduction in the total number of targeted recombinants ( table 1 , transduction 2 , column 4 ) as well as a reduction in the frequency of targeted recombinants per g418 cells to 21.4% ( table 1 , transduction 2 , column 5 ) but the frequency of targeted recombinants per g418gcv cell remained relatively high at 75% ( table 1 , transduction 2 , column 6 ) . further reducing total homology to only 5.6
kb did not appear to further reduce the total number of targeted recombinants compared with 9.6 kb total homology ( table 1 , transduction 1 and 2 , column 4 ) , but the frequency of targeted recombinants per g418 cells was reduced to 16.7% ( table 1 , transduction 1 and 2 , column 5 ) , and the frequency of targeted recombinants per g418gcv was reduced to 50% ( table 1 , transduction 1 and 2 , column 6 ) .
nevertheless , even with total homology as short as 5.6 kb , the frequency of targeted vector integration was still high enough to make identifying and isolating the targeted clones relatively easy and practical . as shown in table 1 , transduction with an moi of 350 vp / cell yielded twofold to fourfold more targeted recombinants compared with an moi of 175 vp / cell .
interestingly , we observed one clone from transduction with 350 vp / cell of hd-5.6-cftr - neo , which appeared to have undergone aberrant targeting and this is addressed in more detail below .
for all 110 correctly targeted clones from transduction 1 and 2 , southern analyses with the 5 and 3 probe revealed the presence of the 31 kb and this indicated that the vector had integrated into only one of the two cftr alleles in all cases ( figure 1b ) . to identify the targeted allele ,
pcr was used to amplify a 964 bp product specifically from the nontargeted allele and the product was sequenced to determine its identity .
the results are summarized in table 1 , column 4 and suggest that there did not appear to be an obvious allelic preference for targeted vector integration when all hdads were considered cumulatively ; overall , 56.4% ( 62/110 ) of the targeted integration occurred at the i507 allele while the remaining 43.6% ( 48/110 ) occurred at the f508 allele . however , although the numbers are small , a preference for targeting into the i507 allele might exist for the two vectors with the shortest homology lengths ( hd-9.6-cftr - neo and hd-5.6-cftr - neo ) .
we selected clone 26 , which had undergone targeted vector integration into the f508 allele , for pb - mediated excision of the neo cassette . to accomplish this
, 2 10 26 cells were transduced with hdad - cag - hypb - vai , a hdad expressing the hyperactive pb transposase , at an moi of 350 vp / cell .
the transduced cells were plated at low density in the absence of g418 and 107 colonies were picked , and of these 34 were determined to be sensitive to g418 ( g418 ) .
pb - mediated excision of neo converts the 19.1 and 14.2 kb apai fragments back to a 31 kb apai fragment ( figure 1a ) .
unexpectedly , southern analyses revealed that none of the 34 g418 clones had undergone pb - mediated excision of neo as evident by the continued presence of the 19.1 kb apai fragment revealed by the 5 and neo probes and the 14.2 kb apai fragment revealed by the 3 probe ( figure 3 ) . in an independent repeated attempt , another 2 10 26 cells were transduced with hdad - cag - hypb - vai , and this time 112 colonies were picked , 26 of which were determined to be g418 .
however , southern analyses again revealed none had undergone pb - mediated neo excision ( not shown ) . in a third attempt
, we selected a different targeted clone , clone 14 , that had undergone targeted vector integration into the i507 allele .
following transduction of 2 10 14 cells with hdad - cag - hypb - vai at an moi of 350 vp / cell , 240 colonies were picked and 59 were determined to be g418 .
however , again none of the 59 clones had undergone pb - mediated neo excision as determined by southern analyses ( not shown ) .
we do not understand why these clones were sensitive to g418 even though excision of the neo cassette had not occurred .
that no pb - mediated neo excision could be identified after screening a total of 459 colonies suggested that this reaction occurs at a relatively low frequency and that negative selection would be needed to isolate these events .
therefore , to accomplish this , the neo cassette in hd-23.8-cftr - neo was replaced with the positive / negative selectable pactk cassette to generate hd-23.8-cftr - pactk ( figure 4a ) .
pactk is a fusion of the puromycin n - acetyltransferase gene and the hsv - tk gene and therefore provides positive selection for vector integration by conferring puromycin resistance ( puro ) and negative selection for loss of pactk as a consequence of pb - mediated excision by conferring gcv .
unfortunately , the use of pactk meant that the hsv - tk cassette could no longer be used for negative selection against random vector integrations and thus was not included in hd-23.8-cftr - pactk .
fortunately , the relatively high frequencies of targeted integration after only positive selection ( 55.255.9% , table 1 , transduction 1 and 2 , column 5 ) should make identifying them relatively easy and practical even without negative selection against random integrations .
a total of 144 puro colonies were obtained following transduction of 2 10 cf17 cells with hd-23.8-cftr - pactk at an moi of 350 vp / cell ( table 1 , transduction 3 ) and southern analyses were performed on their extracted dna . as shown in figure 4a , targeted vector integration into the cftr gene converts the endogenous 31 kb apai fragment ( revealed by the 5 and 3 probes ) into a 19 kb ( revealed by the 5 probe ) and a 14.7 kb ( revealed by the 3 probe and pactk probe ) apai fragment .
the results of the southern analyses are summarized in table 1 ( transduction 3 ) and a representative blot is shown in figure 4b .
the southern analyses revealed that of the 144 puro clones , 93 of these were correctly targeted into one of the two cftr alleles ( no biallelic targeting was observed ) , 47 had random vector integration , and four were aberrantly targeted ( table 1 , transduction 3 ) .
next , we sought to achieve pb - mediated excision of the pactk marker to accomplish footprintless correction of the cftr mutation .
two correctly targeted clones , clone 7 and 27 , were selected for pb - mediated excision .
clone 7 and 27 had targeted vector integration into the f508 and i507 allele , respectively , as determined by sequencing of the 964 bp pcr product from the unmodified allele ( not shown ) .
2 10 clone 7 or clone 27 cells were transduced with hdad - cag - hypb - vai at an moi of 350 vp / cell .
because the frequency of gcv was unknown , the transduced cells were plated at a wide variety of cell densities in the absence of puromycin ( table 2 ) , and 48 hours later , gcv was added to the culture media .
if pb - mediated pactk excision was responsible for gcv , dna was extracted from 107 and 113 gcv colonies originating from clone 7 and 27 , respectively , and subjected to southern analyses .
as shown in figure 4a , pb - mediated pactk excision converts the 19 kb and the 14.7 kb apai fragments back to a 31 kb apai fragment .
the results of the southern analyses revealed that pb - mediated pactk excision had occurred in all 220 gcv colonies and a representative southern blot is presented in figure 5 .
table 2 shows that pb - mediated pactk excision occurs at a frequency of 1 10 .
this explains why we were unable to identify pb - mediated excision of neo by simply screening 459 colonies .
thus the ability to perform negative selection afforded by pactk is essential for efficient isolation of clones that had undergone pb - mediated pactk excision . to confirm footprintless gene correction after pb - mediated pactk excision
, pcr was used to amplify a 964 bp region that encompassed both the site of i507/f508 mutation and the location of downstream pactk insertion from four gcv clones , two derived from clone 7 , and two from clone 27 .
the pcr product was then cloned into a bacterial plasmid ( required because both alleles are amplified after pb excision of pactk and this mixture can not be directly sequenced ) and sequenced and a representative result of this analysis is presented in figure 6 . the sequence
analyses revealed that in all four cases , correction of the i507 or f508 mutation had occurred and that pb - mediated excision was indeed footprintless .
as mentioned , four puro clones from transduction 3 appeared to have undergone aberrant targeting ( clones 21 , 57 , 34 , and 51 ) , two of these , clone 21 and 57 , are present in the southern blot presented in figure 4b . for clone 21 ,
the expected 31 and 19 kb bands are revealed with the 5 probe ( figure 4b ) indicating that the left homology arm of the hdad had integrated into one of the cftr alleles by correct homologous recombination .
however , the 3 probe and the pactk probe revealed a band that was larger than the expected 14.7 kb band ( figure 4b ) indicating the right homology arm of the hdad did not undergo correct homologous recombination with the target locus .
likewise , for clone 57 , the expected 31 and 19 kb bands are revealed with the 5 probe ( figure 4b ) indicating that the left homology arm of the hdad had integrated into one of the cftr alleles by correct homologous recombination .
however , the 3 probe revealed only the 31 kb band and the pactk probe revealed a band of high molecular weight band ( figure 4b ) indicating the right homology arm of the hdad did not undergo correct homologous recombination with the target locus . for clone 34 ,
the 5 probe revealed the expected 31 kb but not the expected 19 kb band , but instead an unexpected band between 31 kb and 19 kb was present ( not shown ) , the 3 probe revealed only the expected 31 kb band but not the expected 14.7 kb band , and the pactk probe revealed a band of high molecular weight ( not shown ) . for clone 51 the 5 probe revealed the expected 31 and 19 kb bands , but the 3 probe revealed only the expected 31 kb band but not the expected 14.7 kb band , and the pactk probe did not reveal any band ( not shown ) .
as mentioned above , transduction 2 with hd-5.6-cftr - neo also generated one aberrantly targeted clone which possessed the expected 31 and 19 kb band as revealed by the 5 probe , the expected 19 kb band revealed by the neo probe , but a band greater than the expected 14.1 kb band was revealed with the 3 probe ( not shown ) . in summary , in four of the five cases ( expected for clone 34 ) , the vector appears to have integrated into one of the cftr alleles by correct homologous recombination with the left homology arm but not the right homology arm . in the case of clone 34
, the vector appears to have integrated into one of the cftr alleles ( due to the presence of the expected 31 kb and a band of unexpected size with the 5 probe ) , but this integration did not occur by correct homologous recombination in either the left or right homology arms .
we made no further attempts to elucidate the genomic structure of these rare aberrantly targeted clones .
we have systematically investigated the effects of homology length on gene targeting by hdad and found that there is positive correlation between targeting efficiency and the total length of homology in hdads . however , even with as little as 5.6 kb of total homology , the efficiency remains high enough to permit easy identification and isolation of the targeted recombinants .
this high targeting frequency may be attributed to the relatively low frequency of random hdad integration , a property that appears to be conferred by the adenoviral terminal protein that is covalently attached to the ends of the linear vector genome .
suzuki et al . compared the efficiency of targeting the hprt locus in mouse es cells with hdads bearing different homology lengths . in that study ,
hprt was nonfunctional due to an insertion of a 1.6 kb pgkneo cassette into exon 3 and hdads bearing the wild - type exon 3 within 18.6 kb , 6.7 kb or 1.7 kb of homology were used for targeted correction .
found that targeted correction was achieved only with 18.6 kb and not with 6.7 kb or 1.7 kb of homology , indicating that 6.7 kb of homology was not sufficient for targeting .
this is in contrast to our study showing that 5.6 kb of homology was enough to target the cftr locus .
however , one major difference between our two studies is that the 3 bp deletion ( i507 or f508 ) we are trying to correct is significantly more subtle than the 1.6 kb insertion of pgkneo that they were trying to correct and this might influence the amount of homology required .
another major difference is that mouse es cells might have different homology requirements than human ipscs .
we also employed pb technology to achieve footprintless removal of the positive selectable marker , but our initial attempts to identify pb - mediated excision of the neo cassette by screening clones transduced with an hdad expressing hyperactive pb transposase proved unsuccessful due to low efficiencies . to overcome this
, we replaced the neo marker with the pactk marker which permitted both positive selection for vector integration and negative selection for pb - mediated excision .
using pactk , we were able to easily select for gcv clones that had undergone pb - mediated excision despite the fact that we found pb - mediated pactk excision to occur at a low frequency of 1 10 per transduced cell which explains why we were unable to identify these rare clones based on screening alone .
others have reported frequencies of 8 10 to 1.1 10for excision using hyperactive pb transposase in ipscs .
there also appeared to be an inverse correlation between the number of gcv colonies and the number of cells plated per well ( table 2 ) and this is consistent with bystander killing , a known killing mechanism of the hsv - tk / gcv system in which cells that do no express hsv - tk are nevertheless killed by gcv because they are in gap - junction contact with cells that do express hsv - tk .
while the use of pactk permitted efficient isolation of pb - mediate pactk excision , it precluded the use of the hsv - tk for negative selection against random vector integrations . fortunately , this did not pose a significant problem because random vector integration with ad - based vectors is rare due to the covalently attached ad terminal proteins as discussed above . in the future , a diphtheria toxin or cytosine deaminase expression cassette
could be added to the hdad outside of the region of homology to serve as a negative selectable marker against random vector integrations if desired .
of which four of these five clones appear to have undergone correct homologous recombination in the left homology arm but not in the right homology arm .
we do not understand why homologous recombination appeared to occur with less fidelity in right arm in our targeting hdads .
aberrantly targeted ipscs have also been reported by others using hdad , and these also appear to have undergone correct homologous recombination in the left arm but not in the right arm .
recently , crane et al . reported zinc finger nuclease -mediated targeted correction of cftr mutations in the same cf ipscs used in this study . using the same pactk selectable marker used in this study , crane et al .
obtained an average of 2932 puro colonies per 2 10 ipscs nucleofected with wild - type donor dna and zinc finger nuclease expression plasmids , and found that 6.3% of the puro colonies were correctly targeted into one cftr allele . by comparison ,
hd-23.8-cftr - pactk yielded 144 puro colonies per 2 10 cells transduced , 64.6% of which were correctly targeted into one cftr allele .
however , the two studies differ in so many variables that direct comparison of their targeting efficiencies is difficult . nevertheless , an important advantage of hdad is avoidance of off - target cleavage associated with using a designer nuclease ( although none were found with the specific zinc finger nuclease used by crane et al . ) . on the other hand ,
crane et al . also reported a strong allelic bias in targeting which was attributed to a single base polymorphism 76 bp upstream of the zinc finger nuclease cleavage site .
specifically , the i507 allele has an adenine at this position while the f508 allele has a guanine , and strong preferential allelic targeting was observed depending on whether the donor dna possessed an adenine or guanine at that position .
the donor cftr dna in our hdads possess an adenine at that position and this could explain the apparent bias of targeting into the i507 allele with the two hdads bearing the shortest homologies .
however , because the numbers are small , generating and analyzing more targeted recombinants with these two hdads would be required to draw a definitive conclusion .
for example , ganciclovir which we used at the typical 2 mol / l concentration for negative selection against pactk , can induce chromosomal instability .
this may be avoided by using 1-(2-deoxy-2-fluoro--d - arabinofuranosyl)-5-iodouracil which is effective for negative selection against pactk at a 10-times lower concentration of 0.2
mol / l and was not found to be associated with genomic instability . in summary , we have found that there is a direct relationship between the total length of homology and the efficiency of hdad - mediated gene targeting in human ipscs . however , even with as little as 5.6 kb total homology , targeting is achieved at practical efficiencies to permit easy isolation of targeted recombinants .
we have also demonstrated for the first time , that hdad can mediate footprintless correction of the cftr mutation using pb - mediated excision of the selectable marker .
however , pb - mediated excision is relatively inefficient requiring negative selection for efficient isolation of the excision products .
nevertheless , the benefits of footprintless excision outweigh the disadvantage of low efficiency for many applications .
hd-23.8-cftr - neo was constructed as follows : first , an hdad plasmid containing the hsv - tk expression cassette was constructed from p28e4 ( ref .
next , a 23,786 bp fragment ( positon 67,95391,738 according to ncbi reference sequence : nc_000007.14 ) from bac clone rp11 - 1152a23 ( sigma # rpcl11.c , st .
next , the neomycin resistance marker flanked by pb inverted terminal repeats was inserted at position 79,857 ( according to ncbi reference sequence : nc_000007.14 ) by recombineering .
the hdads with shorter homology arms were derived from hd-23.8-cftr - neo with the new borders of homology indicated by the restriction enzyme site shown in figure 2 .
hd-23.8-cftr - pactk was derived from hd-23.8-cftr - neo by replacing the neo expression cassette with the pactk expression cassette .
cf17 , the feeder free human cf ips cell line used in this study is described elsewhere and was maintained in mtesr 1 ( stemcell technologies , vancouver , canada ) on matrigel ( corning , tewksbury , ma ) coated plates .
briefly , 2 10 cells were resuspended in 1 ml mtesr 1 supplemented with y27632 ( reagents direct , encinitas , ca ) to 10
mol / l in a 1.5 ml microfuge tube and infected with hdad at an moi of 175 or 350 vp / cell for 1 hour at 37c with gentle rocking .
following infection , cells were washed twice with 1 ml mtesr 1 supplemented with y27632 to 10 mol / l and plated into 1012 matrigel coated wells of six - well plates in mtesr 1 supplemented with y27632 to 10 mol / l . for transductions 1 and 2 ( table 1 ) , g418 was added to the media to a final concentration of 400 g / ml 48 hours post - transduction , and 14 days post - transduction gcv was added to the media to a final concentration of 2 mol / l and all surviving colonies were picked 21 days post - transduction . for transduction 3 ( table 1 ) , puromycin was added to the media to a final concentration of 0.5 g / ml 48 hours post - transduction and all surviving colonies were picked 13 days after transduction .
ipscs were transduced with hdad - cag - hypb - vai at an moi of 350 vp / cell as described above . for targeted clones 26 and 14 , generated with hd-23.8-cftr - neo
, cells were plated at 100 , 250 , 500 , and 1,000 cells per well in nonselective media following transduction with hdad - cag - hypb - vai .
a total of 459 well isolated colonies were picked from three independent transductions and these were screened for sensitivity to g418 and dna was extracted from a total of 119 g418 clones for southern analysis . for targeted clones 7 and 27 generated with hd-23.8-cftr - pactk , cells were plated in nonselective media following hdad - cag - hypb - vai transduction at the densities shown in table 2 .
48 hours post - transduction , gcv was added to the media to a final concentration of 2 mol / l .
220 well isolated colonies were picked and dna was extracted for southern analysis . dna analysis .
genomic dna from ipscs was extracted from a single confluent well of a 24 or 12 well plate as follows : cells were washed twice with 0.5 or 1 ml phosphate buffered saline ( pbs ) , and 0.4 ml lysis buffer ( 10 mmol / l tris - hcl ph 8.0 , 10 mmol
/ l ethylenediaminetetraacetate ( edta ) ph 8.0 , 100 mmol / l nacl , 0.5% sodium dodecyl sulfate ( sds ) , 100 g / ml proteinase k ) was added to the well .
the lysate was then transferred into a microfuge tube and incubated with gentle rocking overnight at 50 c. the next day , the dna was precipitated by the addition of 1 ml 95% ethanol , washed once with 1 ml 70% ethanol and resuspended in 10 mmol / l tris - hcl ph 8.0 .
nonradioactive digoxigenin - based southern blot hybridization was performed as recommended by the manufacturer ( roche , indianapolis , in ) .
all southern probes were digoxigenin - labelled pcr products generated according to manufacturer 's recommendation ( roche ) .
the 5 probe was generated using the primers 5-atttcaagtgtcttcgtcgg and 5-gtaaggtaagtccaggtgc and the plasmid plpbl-1-cftr - apai - bamhi .
the 3 probe was generated using the primers 5-tcattgccctttgtatgtgc and 5-catcctccactgccatttc and the plasmid plpbl-1-cftr - bstbi - bstbi .
the neo probe was generated using the primers 5-ggatcggccattgaacaag and 5-tgcgatgtttcgcttggtg and the plasmid pl451 .
the pactk probe was generated using the primers 5-atagagcccaccgcatcc and 5-aacggcgacctgtataacg and the plasmid plpbl-13-pactk .
thermocycling conditions were as follows ; 2 minutes at 95 c , followed by 30 cycles of 95 c for 30 seconds , 58 c for 40 seconds and 72 c for 40 seconds , and a final extension of 7 minutes at 72 c. primers 5-attggaggcaagtgaatcctgagc and 5-agcaataactactgaacccaccatc were used to amplify the unmodified cftr allele(s ) encompassing the i507/f508 mutation and the site of the selectable marker insertion from ipsc genomic dna using hotstarplus(qiagen , valencia , ca ) according to the manufacturer 's recommendations .
thermocycling conditions were as follows ; 5 minutes at 95 c , followed by 35 cycles of 94 c for 1 minute ; 68 c for 1 minute and 72 c for 1 minute , and a final extension of 10 minutes at 72 c. for dna extracted from the correctly targeted recombinants , the forward primer was used to sequence the pcr product to determine which allele vector integration had occur . for dna extracted from clones that underwent pb - mediated excision of the pactk expression cassette , the pcr products were first cloned into a plasmid ( because both alleles are amplified ) and then sequenced with the forward primer to verify footprintless gene correction . | helper - dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off - target cleavage . because of their large cloning capacity of 37 kb , helper - dependent adenoviral vectors with
long homology arms are used for gene editing . however
, this makes vector construction and recombinant analysis difficult .
conversely , insufficient homology may compromise targeting efficiency .
thus , we investigated the effect of homology length on helper - dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation . with 23.8 and 21.4 kb of homology ,
the frequencies of targeted recombinants were 5064.6% after positive selection for vector integration , and 97.4100% after negative selection against random integrations . with 14.8 kb ,
the frequencies were 26.957.1% after positive selection and 87.5100% after negative selection . with 9.6 kb ,
the frequencies were 21.4 and 75% after positive and negative selection , respectively . with only 5.6 kb ,
the frequencies were 5.616.7% after positive selection and 50% after negative selection , but these were more than high enough for efficient identification and isolation of targeted clones .
furthermore , we demonstrate helper - dependent adenoviral vector - mediated footprintless correction of cystic fibrosis transmembrane conductance regulator mutations through piggybac excision of the selectable marker . however , low frequencies ( 1 103 ) necessitated negative selection for piggybac - excision product isolation . |
effective occupational safety and health ( osh ) management requires appropriate and reliable osh performance measurement .
an indicator can be defined as a concise definition of a concept , meant to provide maximal information on an area of interest,[1 , p.36 ] or a quantitative or qualitative factor or variable that provides a simple and reliable means to measure achievement , to reflect the changes connected to an intervention , or to help assess the performance of a development actor.[2 , p.32 ] the indicators designed to measure osh performance in companies are intended to provide information on the extent to which a desired outcome is achieved or the quality of processes leading to that outcome .
different types of indicators showing osh inputs , processes and outcomes are used for the measurement of osh performance .
the indicators reflecting osh outcomes and applied during the process of reactive monitoring , with a focus on the measurement of losses incurred as a consequence of inappropriate osh performance , are frequently called lagging indicators and this term is applied in this article . in the literature on the subject , the term outcome indicators is also used along with the term negative performance indicators or trailing indicators .
examples of this type of indicator include the number of accidents at work or their frequency rates , the cost of compensations for workers , the number of days of absence owing to accidents at work , the number of occupational diseases and so forth . for years
, numerous researchers have emphasized that timely and accurate analysis of these indicators is essential for successful prevention ( e.g. , ) .
these traditional indicators are usually easy to calculate on the basis of data collected in company 's registers according to legal requirements .
to receive other lagging indicators , such as the number of incidents , number of work - related illnesses or costs of occupational accidents , an additional system of data collection is necessary .
the lagging indicators can provide information on the effectiveness of actions performed in the past and do not enable their current monitoring and correction .
the osh inputs are defined as all of the preventive potential and working conditions in a given company , which can be presented using input indicators .
a commonly known input indicator is the number of workers exposed to harmful factors in the working environment .
this indicator can be based on objective data received from measurements or on subjective data from questionnaire surveys and can be used , among others , as a predictor of future outcomes .
the osh processes comprise strategies , policies and measures implemented with a view to improving employees osh and are monitored thanks to the use of activities indicators .
for example , activities indicators related to actions taken with the aim of increasing employee involvement in osh , which is thought to be one of the most important and still insufficient implemented elements of the osh management system , can be the number of employees participating in osh training courses , consultations or the number of osh improvements proposed by workers .
the activities indicators are related to osh objectives established in a company and are defined in planning processes .
the indicators showing the inputs and processes are included in a group collectively called leading indicators and this term will be used in this article . in the subject literature ,
some research seems to confirm this assumption , among other research related to modifying unsafe behaviours in which positive changes in indicators of safety culture were accompanied by positive changes in lagging indicators . a general model for measuring osh performance using lagging and leading indicators is presented in figure 1 .
general model for measuring occupational safety and health ( osh ) performance using lagging indicators and leading indicators .
general model for measuring occupational safety and health ( osh ) performance using lagging indicators and leading indicators . for years
, guidance has been developed to support companies in osh performance measurement , among others by the british health and safety executive and the australian national health and safety commission .
the oecd also published guidance on safety performance indicators to assist companies in implementing measures to prevent chemical accidents . in the ilo - osh 2001 guidance on osh management systems and other guidance or requirements for osh management systems provided in various standards developed at international or national levels , such as , e.g. , voluntary polish pn - n-18000 series standards , the necessity of appropriate osh performance measurement
is emphasized . to evaluate the impact of adopting osh management rules according to these standards on using leading and lagging indicators for osh performance measurement and to check whether a relationship exists between using these indicators and the achieved osh outcomes , a study was conducted in 60 companies with different levels of osh performance .
to collect information on company osh performance and indicators used for its assessment , a questionnaire was developed in electronic form and circulated amongst executives in the studied companies .
the questionnaire contained questions concerning the following :
the company 's size , sector and management systems implemented;the company performance in the following areas of osh management , identified taking into account the osh management rules defined in the ilo - osh guidance :
commitment of the company 's top management to osh matters , including ensuring resources for osh , participation in training activities , receiving and analysing the osh information , taking up initiatives and personal involvement in the osh activities;the osh policy , including establishment of documented osh policy , its adjustment in line with the adopted requirements and its understanding by the company employees;participation of workers in efforts to enhance the osh , including information , consultation and participation in the decision - making process , appointment of representatives for the osh matters and direct involvement of employees in the health and safety efforts;planning in the area of osh , including the setting of long - term and short - term goals and plans considering current analyses of the company functioning and the employees opinions along with the documents updates , participation of company executives and employees in the goal - setting process;assignment of responsibilities and rights related to the osh efforts and activities for employees as well as subcontractors and suppliers;training in the area of osh , including performance of training courses required by law , identification of training needs on a regular basis and assessment of the training activities performance and quality;communication process , including the flow of information in all directions within the organization , in particular from the company personnel to management;procedures , including the development of documented procedures for crucial activities that ensure proper functioning of the company;management of occupational risks , including the assessment of occupational risks while ensuring the participation of employees and taking their opinions and feedback into consideration along with the implementation of measures resulting from risk assessment;preparedness and response to work accidents and failures , including the established principles of responsiveness , providing first aid and cooperation with external entities;investigation of accidents at work , occupational diseases and incidents , including the use of appropriate methods to identify their primary causes resulting from management deficiencies;monitoring in the area of osh , including regular inspections , reviews , measurements , researching health complaints related to work , incidents and so forth;corrective action , including successful elimination of non - compliances detected in the monitoring process ; andaudits .
the company 's size , sector and management systems implemented ; the company performance in the following areas of osh management , identified taking into account the osh management rules defined in the ilo - osh guidance :
commitment of the company 's top management to osh matters , including ensuring resources for osh , participation in training activities , receiving and analysing the osh information , taking up initiatives and personal involvement in the osh activities;the osh policy , including establishment of documented osh policy , its adjustment in line with the adopted requirements and its understanding by the company employees;participation of workers in efforts to enhance the osh , including information , consultation and participation in the decision - making process , appointment of representatives for the osh matters and direct involvement of employees in the health and safety efforts;planning in the area of osh , including the setting of long - term and short - term goals and plans considering current analyses of the company functioning and the employees opinions along with the documents updates , participation of company executives and employees in the goal - setting process;assignment of responsibilities and rights related to the osh efforts and activities for employees as well as subcontractors and suppliers;training in the area of osh , including performance of training courses required by law , identification of training needs on a regular basis and assessment of the training activities performance and quality;communication process , including the flow of information in all directions within the organization , in particular from the company personnel to management;procedures , including the development of documented procedures for crucial activities that ensure proper functioning of the company;management of occupational risks , including the assessment of occupational risks while ensuring the participation of employees and taking their opinions and feedback into consideration along with the implementation of measures resulting from risk assessment;preparedness and response to work accidents and failures , including the established principles of responsiveness , providing first aid and cooperation with external entities;investigation of accidents at work , occupational diseases and incidents , including the use of appropriate methods to identify their primary causes resulting from management deficiencies;monitoring in the area of osh , including regular inspections , reviews , measurements , researching health complaints related to work , incidents and so forth;corrective action , including successful elimination of non - compliances detected in the monitoring process ; andaudits .
commitment of the company 's top management to osh matters , including ensuring resources for osh , participation in training activities , receiving and analysing the osh information , taking up initiatives and personal involvement in the osh activities ; the osh policy , including establishment of documented osh policy , its adjustment in line with the adopted requirements and its understanding by the company employees ; participation of workers in efforts to enhance the osh , including information , consultation and participation in the decision - making process , appointment of representatives for the osh matters and direct involvement of employees in the health and safety efforts ; planning in the area of osh , including the setting of long - term and short - term goals and plans considering current analyses of the company functioning and the employees opinions along with the documents updates , participation of company executives and employees in the goal - setting process ; assignment of responsibilities and rights related to the osh efforts and activities for employees as well as subcontractors and suppliers ; training in the area of osh , including performance of training courses required by law , identification of training needs on a regular basis and assessment of the training activities performance and quality ; communication process , including the flow of information in all directions within the organization , in particular from the company personnel to management ; procedures , including the development of documented procedures for crucial activities that ensure proper functioning of the company ; management of occupational risks , including the assessment of occupational risks while ensuring the participation of employees and taking their opinions and feedback into consideration along with the implementation of measures resulting from risk assessment ; preparedness and response to work accidents and failures , including the established principles of responsiveness , providing first aid and cooperation with external entities ; investigation of accidents at work , occupational diseases and incidents , including the use of appropriate methods to identify their primary causes resulting from management deficiencies ; monitoring in the area of osh , including regular inspections , reviews , measurements , researching health complaints related to work , incidents and so forth ; corrective action , including successful elimination of non - compliances detected in the monitoring process ; and each of these listed areas was evaluated on a scale from 1 to 5 ( where 5 denotes the highest level ) .
the questionnaire also contained questions concerning the following :
lagging indicators used for the assessment and monitoring of osh performance in the company , identified on the basis of reviews with osh consultants supporting companies in the development of osh management systems , including :
number of occupational accidents , incidence rate of occupational accidents , severity rate of occupational accidents , number of sickness absence days , number of nonconformities with the legal requirements , cost of accidents at work , number of incidents , number of nonconformities with the requirements for the osh management system , andnumber of workers reporting work - related diseases .
leading indicators used for evaluating and monitoring the osh performance , identified on the basis of reviews with osh consultants supporting companies in the development of osh management systems , including :
a number of employees working in hazardous conditions ( in which maximum admissible concentrations or intensities are exceeded ) , anda number of workers reporting stress at work :
a number of osh training courses for the company executives, a number of osh reports referred to the company 's top management, a number or percentage of employees provided with the osh training courses, a number osh - related issues reported by workers, a number of occupational risk assessments, a number of workplaces where protection measures were improved , and a number of osh inspections .
lagging indicators used for the assessment and monitoring of osh performance in the company , identified on the basis of reviews with osh consultants supporting companies in the development of osh management systems , including :
number of occupational accidents , incidence rate of occupational accidents , severity rate of occupational accidents , number of sickness absence days , number of nonconformities with the legal requirements , cost of accidents at work , number of incidents , number of nonconformities with the requirements for the osh management system , andnumber of workers reporting work - related diseases .
number of occupational accidents , incidence rate of occupational accidents , severity rate of occupational accidents , number of sickness absence days , number of nonconformities with the legal requirements , cost of accidents at work , number of nonconformities with the requirements for the osh management system , and number of workers reporting work - related diseases .
leading indicators used for evaluating and monitoring the osh performance , identified on the basis of reviews with osh consultants supporting companies in the development of osh management systems , including :
a number of employees working in hazardous conditions ( in which maximum admissible concentrations or intensities are exceeded ) , anda number of workers reporting stress at work :
a number of osh training courses for the company executives, a number of osh reports referred to the company 's top management, a number or percentage of employees provided with the osh training courses, a number osh - related issues reported by workers, a number of occupational risk assessments, a number of workplaces where protection measures were improved , and a number of osh inspections .
a number of employees working in hazardous conditions ( in which maximum admissible concentrations or intensities are exceeded ) , and a number of workers reporting stress at work :
a number of osh training courses for the company executives, a number of osh reports referred to the company 's top management, a number or percentage of employees provided with the osh training courses, a number osh - related issues reported by workers, a number of occupational risk assessments, a number of workplaces where protection measures were improved , and a number of osh inspections .
a number of osh training courses for the company executives , a number of osh reports referred to the company 's top management , a number or percentage of employees provided with the osh training courses , a number osh - related issues reported by workers , a number of occupational risk assessments , a number of workplaces where protection measures were improved , and a number of osh inspections .
the questions asked about each indicator were whether it had been adopted for the measurement of osh performance , and whether it was regularly monitored and recorded .
a question was also asked about some other output and leading indicators if they were not included in the questionnaire but were used in a given company .
fifty per cent of them employed more than 250 workers , 35% fewer than 250 but more than 50 workers and 15% of companies employed more than 50 workers .
thirty - two of the studied companies have implemented an osh management system according to the voluntary standards and have received certificates confirming the implementation , whereas the other companies in the sample have not implemented this system .
the level of compliance with the standard was assessed in line with the responses to the questionnaire on the basis of the self - assessment performed by a representative of the company management .
next , the self - assessments were verified and confirmed by osh management systems consultants that cooperate with the companies .
the safety level in the studied companies , measured by the occupational accidents rate ( calculated as the number of occupational accidents related to 1000 workers ) , was very different : in over 20% of the companies , no occupational accident was registered in 2011 ; however , in the majority of them the occupational accident rates were higher than average at a national level ( which amounted to 8.35 in 2011 ) .
to evaluate the impact of the implementation of the osh management rules according to the voluntary standards pn - n-18000 on using leading and lagging indicators for osh performance measurement , the surveyed companies were divided into two groups : one for which the level of adopting these rules was rated higher than the median calculated for all the companies subject to the survey ( hereafter high - performing companies ) ; and the second group , for which the rating was lower than the median ( hereafter low - performing companies ) .
all of the high - performing companies and less than 60% of low - performing companies used more than four lagging indicators to measure their osh performance .
the relationship between the level of adopting osh management rules according to the voluntary standards and the number of adopted lagging indicators is statistically significant ( kendall 's
b = 0.72 , p < 0.01 ) .
the most frequently applied lagging indicators are the number of accidents at work ( in almost 97% of all the surveyed companies ) and the number of the absence days ( in over 61% of all the companies ) .
there are no statistically significant differences between using these indicators in high - performing and low - performing companies . for the other lagging indicators
, a test of independence indicates that the differences in using them are statistically significant ; the strongest correlation was found for the indicator number of incidents ( = 0.68 ; p < 0.01 , goodman and kruskal 's = 0.48 ; p < 0.01 ) .
the percentages of high - performing and low - performing companies in which specific lagging indicators are employed are shown in figure 2 .
percentages of high - performing and low - performing companies in which specific lagging indicators are used.note : ohs = occupational health and safety .
percentages of high - performing and low - performing companies in which specific lagging indicators are used .
leading indicators are applied less frequently than lagging indicators in all of the surveyed companies .
the number of leading indicators used increases in line with the level of adopting osh management rules according to the voluntary standards ; this relationship is statistically significant ( based on a
test of independence : kendall 's
b = 0.68 , p < 0.01 ) . the most frequently applied leading indicator the number of employees working in hazardous conditions refers to osh inputs .
this indicator is employed in more than 70% of high - performing as well as low - performing companies . for the other leading indicators included in the survey which refer to osh activities ,
the
test of independence confirmed statistically significant differences regarding the frequency of using them in both groups of companies .
the strongest relationship was found for the following indicators :
the number of documented procedures and/or safety instructions developed or verified at a given time ( = 0.67 , p < 0.01 , goodman and kruskal 's = 0.45 , p < 0.01).the number of reports on the osh situation submitted to top management ( e.g. , per year) ( = 0.61 , p < 0.01 ,
goodman and kruskal 's = 0.37 , p < 0.01).the number or percentage of employees participating in training on health and safety ( = 0.60 , p < 0.01 , goodman and kruskal 's = 0.36 , p < 0.01).the number of work stations for which risk assessment was carried out ( = 0.59 , p < 0.01 , goodman and kruskal 's = 0.35 , p < 0.01).the number of non - compliances with the requirements of the osh management system ( = 0.58 , p < 0.01 , goodman and kruskal 's = 0.34 , p < 0.01).the number of work stations , where better measures of protection against risks were implemented ( = 0.56 , p < 0.01 , goodman and kruskal 's = 0.31 , p < 0.01 ) .
the number of documented procedures and/or safety instructions developed or verified at a given time ( = 0.67 , p < 0.01 , goodman and kruskal 's = 0.45 , p < 0.01 ) .
the number of reports on the osh situation submitted to top management ( e.g. , per year) ( = 0.61 , p < 0.01 , goodman and kruskal 's = 0.37 , p < 0.01 ) . the number or percentage of employees participating in training on health and safety ( = 0.60 , p < 0.01 , goodman and kruskal 's = 0.36 , p < 0.01 ) .
the number of work stations for which risk assessment was carried out ( = 0.59 , p < 0.01 , goodman and kruskal 's = 0.35 , p < 0.01 ) .
the number of non - compliances with the requirements of the osh management system ( = 0.58 , p < 0.01 , goodman and kruskal 's = 0.34 , p < 0.01 ) .
the number of work stations , where better measures of protection against risks were implemented ( = 0.56 , p < 0.01 , goodman and kruskal 's = 0.31 , p < 0.01 ) . the percentages of high - performing and low- performing companies in which specific leading indicators are employed are shown in figure 3 .
figure 3 .
percentages of high - performing and low - performing companies in which specific leading indicators are used.note : ohs = occupational health and safety ; osh = occupational safety and health .
percentages of high - performing and low - performing companies in which specific leading indicators are used .
note : ohs = occupational health and safety ; osh = occupational safety and health . even though the performance indicators are defined and used for osh performance evaluation , they are not regularly monitored in over 40% of the surveyed companies .
approximately 46% of the high - performing companies and only 5% of the low - performing companies regularly monitor at least three indicators .
the relationship between the number of indicators monitored regularly and the level of adopting osh management rules according to the voluntary standards is not only statistically significant , but also the strongest of all the presented relationships ( kendall 's
b = 0.86 , p < 0.01 ) .
the greatest differences between the high - performing and low - performing companies occur for the following indicators :
the number or percentage of employees participating in training on occupational health and safety ( = 0.75 , p < 0.01 , goodman and kruskal 's = 0.56 , p < 0.01).the number of work stations for which risk assessment was carried out ( = 0.75 , p < 0.01 , goodman and kruskal 's = 0.56 , p < 0.01).the number of reports on the osh situation submitted to the top company management in a given year ( = 0.73 , p < 0.01 , goodman and kruskal 's = 0.53 , p < 0.01 ) .
the number or percentage of employees participating in training on occupational health and safety ( = 0.75 , p < 0.01 , goodman and kruskal 's = 0.56 , p < 0.01 ) . the number of work stations for which risk assessment was carried out ( = 0.75 , p < 0.01 , goodman and kruskal 's = 0.56 , p < 0.01 ) . the number of reports on the osh situation submitted to the top company management in a given year ( = 0.73 , p < 0.01 , goodman and kruskal 's = 0.53 , p < 0.01 ) .
the number of leading indicators used for osh performance evaluation is growing together with the number of lagging indicators used , and the relationship is statistically significant ( pearson 's correlation coefficient r = 0.70 ; p < 0.001 ) .
looking for the relationship between occupational accident rates and the number of leading indicators applied , statistical analysis of differences between groups of companies with different accident rates was performed .
the strongest differences was identified between the groups of companies with occupational accident rates higher than 10 ( mean number of applied leading indicators 3.5 ; sd 2.9 ) and lower than 10 ( mean number of applied leading indicators 5.5 ; sd 3.3 ) ( table 1 ) .
the strength of this relationship measured using cohen 's d effect size ( 0.7 ) is moderate .
differences between the number of leading indicators applied by the groups of companies with occupational accident rates ( per 1000 workers ) over and under 10 .
number of leading indicators appliedoccupational accident rates ( per 1000 workers)number of companies%statisticsse103151.7m5.50.59 mdn6.0 sd3.3 interquartile range6.0 asymmetry0.30.42 kurtosis1.20.82>102948.3m3.50.53 mdn3.0 sd2.9 interquartile range4.0 asymmetry0.60.43 kurtosis0.80.84
the differences in occupational accident rates were also identified between companies grouped into clusters as follows :
the companies which use the majority of the leading indicators listed in the questionnaire , andthe companies which use only three of the indicators that are the most frequently used .
the companies which use the majority of the leading indicators listed in the questionnaire , and the companies which use only three of the indicators that are the most frequently used .
the quality of the clustering was sufficient ( silhouette measure = 0.4 ) .
in the first group of companies the mean occupational accident rate amounts to 11 , and in the second group the rate is 26 .
this result indicates that companies which use more indicators for evaluating their performance can achieve a higher level of safety .
when analysing the influence of a particular , single leading indicator on occupational accidents rates , only for one of them number of risk assessments have statistically significant differences been identified between companies which use this indicator and the companies not using it ( table 2 ) , confirmed by the mann
whitney u test ( u = 238 ; p < 0.05 ) . the cohen 's d effect size ( which amounts to 0.83 ) indicates that the relationship is strong .
differences in occupational accident rates ( per 1000 workers ) between the group of companies which use the leading indicator number of risk assessments and the group of companies which do not use this indicator .
occupational accident rates ( per 1000 workers ) for the groups of the companiesgroup of companiesnumber of companies%statisticssecompanies in which the indicator number of risk assessments is not used2238m22.94.06 mdn27.8 sd19.0 interquartile range30.4 asymmetry0.80.49 kurtosis1.30.95companies in which the indicator number of risk assessments is used3662m10.31.67 mdn7.8 sd10.1 interquartile range16.2 asymmetry0.990.39 kurtosis0.160.78
to evaluate the impact of the implementation of the osh management rules according to the voluntary standards pn - n-18000 on using leading and lagging indicators for osh performance measurement , the surveyed companies were divided into two groups : one for which the level of adopting these rules was rated higher than the median calculated for all the companies subject to the survey ( hereafter high - performing companies ) ; and the second group , for which the rating was lower than the median ( hereafter low - performing companies ) .
all of the high - performing companies and less than 60% of low - performing companies used more than four lagging indicators to measure their osh performance .
the relationship between the level of adopting osh management rules according to the voluntary standards and the number of adopted lagging indicators is statistically significant ( kendall 's
b = 0.72 , p < 0.01 ) .
the most frequently applied lagging indicators are the number of accidents at work ( in almost 97% of all the surveyed companies ) and the number of the absence days ( in over 61% of all the companies ) .
there are no statistically significant differences between using these indicators in high - performing and low - performing companies . for the other lagging indicators
, a test of independence indicates that the differences in using them are statistically significant ; the strongest correlation was found for the indicator number of incidents ( = 0.68 ; p < 0.01 , goodman and kruskal 's = 0.48 ; p < 0.01 ) .
the percentages of high - performing and low - performing companies in which specific lagging indicators are employed are shown in figure 2 .
percentages of high - performing and low - performing companies in which specific lagging indicators are used.note : ohs = occupational health and safety .
percentages of high - performing and low - performing companies in which specific lagging indicators are used .
leading indicators are applied less frequently than lagging indicators in all of the surveyed companies .
the number of leading indicators used increases in line with the level of adopting osh management rules according to the voluntary standards ; this relationship is statistically significant ( based on a
test of independence : kendall 's
b = 0.68 , p < 0.01 ) . the most frequently applied leading indicator the number of employees working in hazardous conditions refers to osh inputs .
this indicator is employed in more than 70% of high - performing as well as low - performing companies . for the other leading indicators included in the survey which refer to osh activities ,
the
test of independence confirmed statistically significant differences regarding the frequency of using them in both groups of companies .
the strongest relationship was found for the following indicators :
the number of documented procedures and/or safety instructions developed or verified at a given time ( = 0.67 , p < 0.01 , goodman and kruskal 's = 0.45 , p < 0.01).the number of reports on the osh situation submitted to top management ( e.g. , per year) ( = 0.61 , p < 0.01 ,
goodman and kruskal 's = 0.37 , p < 0.01).the number or percentage of employees participating in training on health and safety ( = 0.60 , p < 0.01 , goodman and kruskal 's = 0.36 , p < 0.01).the number of work stations for which risk assessment was carried out ( = 0.59 , p < 0.01 , goodman and kruskal 's = 0.35 , p < 0.01).the number of non - compliances with the requirements of the osh management system ( = 0.58 , p < 0.01 , goodman and kruskal 's = 0.34 , p < 0.01).the number of work stations , where better measures of protection against risks were implemented ( = 0.56 , p < 0.01 , goodman and kruskal 's = 0.31 , p < 0.01 ) .
the number of documented procedures and/or safety instructions developed or verified at a given time ( = 0.67 , p < 0.01 , goodman and kruskal 's = 0.45 , p < 0.01 ) .
the number of reports on the osh situation submitted to top management ( e.g. , per year) ( = 0.61 , p < 0.01 , goodman and kruskal 's = 0.37 , p < 0.01 ) . the number or percentage of employees participating in training on health and safety ( = 0.60 , p < 0.01 , goodman and kruskal 's = 0.36 , p < 0.01 ) .
the number of work stations for which risk assessment was carried out ( = 0.59 , p < 0.01 , goodman and kruskal 's = 0.35 , p < 0.01 ) .
the number of non - compliances with the requirements of the osh management system ( = 0.58 , p < 0.01 , goodman and kruskal 's = 0.34 , p < 0.01 ) .
the number of work stations , where better measures of protection against risks were implemented ( = 0.56 , p < 0.01 , goodman and kruskal 's = 0.31 , p < 0.01 ) . the percentages of high - performing and low- performing companies in which specific leading indicators are employed are shown in figure 3 .
figure 3 .
percentages of high - performing and low - performing companies in which specific leading indicators are used.note : ohs = occupational health and safety ; osh = occupational safety and health .
percentages of high - performing and low - performing companies in which specific leading indicators are used .
note : ohs = occupational health and safety ; osh = occupational safety and health . even though the performance indicators are defined and used for osh performance evaluation , they are not regularly monitored in over 40% of the surveyed companies .
approximately 46% of the high - performing companies and only 5% of the low - performing companies regularly monitor at least three indicators .
the relationship between the number of indicators monitored regularly and the level of adopting osh management rules according to the voluntary standards is not only statistically significant , but also the strongest of all the presented relationships ( kendall 's
b = 0.86 , p < 0.01 ) .
the greatest differences between the high - performing and low - performing companies occur for the following indicators :
the number or percentage of employees participating in training on occupational health and safety ( = 0.75 , p < 0.01 , goodman and kruskal 's = 0.56 , p < 0.01).the number of work stations for which risk assessment was carried out ( = 0.75 , p < 0.01 , goodman and kruskal 's = 0.56 , p < 0.01).the number of reports on the osh situation submitted to the top company management in a given year ( = 0.73 , p < 0.01 , goodman and kruskal 's = 0.53 , p < 0.01 ) .
the number or percentage of employees participating in training on occupational health and safety ( = 0.75 , p < 0.01 , goodman and kruskal 's = 0.56 , p < 0.01 ) . the number of work stations for which risk assessment was carried out ( = 0.75 , p < 0.01 , goodman and kruskal 's = 0.56 , p < 0.01 ) . the number of reports on the osh situation submitted to the top company management in a given year ( = 0.73 , p < 0.01 , goodman and kruskal 's = 0.53 , p < 0.01 ) .
the number of leading indicators used for osh performance evaluation is growing together with the number of lagging indicators used , and the relationship is statistically significant ( pearson 's correlation coefficient r = 0.70 ; p < 0.001 ) .
looking for the relationship between occupational accident rates and the number of leading indicators applied , statistical analysis of differences between groups of companies with different accident rates was performed .
the strongest differences was identified between the groups of companies with occupational accident rates higher than 10 ( mean number of applied leading indicators 3.5 ; sd 2.9 ) and lower than 10 ( mean number of applied leading indicators 5.5 ; sd 3.3 ) ( table 1 ) .
the strength of this relationship measured using cohen 's d effect size ( 0.7 ) is moderate .
differences between the number of leading indicators applied by the groups of companies with occupational accident rates ( per 1000 workers ) over and under 10 .
number of leading indicators appliedoccupational accident rates ( per 1000 workers)number of companies%statisticsse103151.7m5.50.59 mdn6.0 sd3.3 interquartile range6.0 asymmetry0.30.42 kurtosis1.20.82>102948.3m3.50.53 mdn3.0 sd2.9 interquartile range4.0 asymmetry0.60.43 kurtosis0.80.84
the differences in occupational accident rates were also identified between companies grouped into clusters as follows :
the companies which use the majority of the leading indicators listed in the questionnaire , andthe companies which use only three of the indicators that are the most frequently used .
the companies which use the majority of the leading indicators listed in the questionnaire , and the companies which use only three of the indicators that are the most frequently used .
the quality of the clustering was sufficient ( silhouette measure = 0.4 ) .
in the first group of companies the mean occupational accident rate amounts to 11 , and in the second group the rate is 26 .
this result indicates that companies which use more indicators for evaluating their performance can achieve a higher level of safety .
when analysing the influence of a particular , single leading indicator on occupational accidents rates , only for one of them number of risk assessments have statistically significant differences been identified between companies which use this indicator and the companies not using it ( table 2 ) , confirmed by the mann
whitney u test ( u = 238 ; p < 0.05 )
. the cohen 's d effect size ( which amounts to 0.83 ) indicates that the relationship is strong .
differences in occupational accident rates ( per 1000 workers ) between the group of companies which use the leading indicator number of risk assessments and the group of companies which do not use this indicator . occupational accident rates
( per 1000 workers ) for the groups of the companiesgroup of companiesnumber of companies%statisticssecompanies in which the indicator number of risk assessments is not used2238m22.94.06 mdn27.8 sd19.0 interquartile range30.4 asymmetry0.80.49 kurtosis1.30.95companies in which the indicator number of risk assessments is used3662m10.31.67 mdn7.8 sd10.1 interquartile range16.2 asymmetry0.990.39 kurtosis0.160.78
the performance indicators commonly used in the studied companies include the number of accidents at work ( this lagging indicator is adopted in almost all companies ) and the number of people working in hazardous conditions ( this input indicator is used in more than 70% of all companies ) .
both indicators are used equally often in the high - performing and the low - performing companies .
these indicators have to be determined in line with law provisions that require the company to present relevant data for national statistics and for the needs of insurance .
the leading indicator that is among the most frequently used is the number of employees participating in training courses on health and safety issues .
the indicator is also linked to ensuring compliance with the requirements set out in the law under which companies have to conduct periodical training courses on health and safety matters .
these results point to the important role statutory requirements play in determining the indicators used to assess the osh performance in all companies .
the indicators that are in use even though they are not directly linked to achievement of compliance with the law requirements , in particular leading indicators related to osh activities , are adopted significantly more often in the companies which are better adjusted to the osh management rules presented in the standards for voluntary use ( such as pn - n-18000 ) . in this group , the most frequently adopted indicators are those directly required by the standards or reflecting the activities aimed at achieving compliance with them , e.g. , the number of incidents .
regular monitoring of indicators adopted for performance evaluation is necessary to make sure that responses to changes follow early enough .
the study shows that the adoption of a particular indicator does not always mean it is regularly monitored , especially in the companies with a low level of osh performance . the basic indicator adopted by almost all companies , which is the number of accidents at work ,
is regularly monitored in all companies with a high level of performance , and only in less than 60% of other companies .
in addition , over 80% of the companies with a highly rated performance monitor regularly some other indicators related to the law requirements or voluntarily adopted standards .
they include a number of risk assessments , a number of incidents and a number of employees participating in training activities .
therefore , better adjustment to the requirements and guidelines related to the osh management system means that the ongoing development of planning and monitoring processes is in place , which supports continuous improvement of the osh management .
some results achieved can also indicate that companies with lower occupational accidents rates more frequently employ more indicators to measure their osh performance .
the osh performance indicators are valuable tools in managing safety and health in a company .
the differences are particularly visible in the case of leading indicators , which can provide valuable information for proactive osh management .
however , it is rather difficult to evaluate the relationship between the various osh leading indicators and osh outcomes measured using lagging indicators . in this survey
the significant differences in accident rates have been identified between companies using the leading indicator number of risk assessments and companies not using it .
further research is needed to identify the most effective osh performance indicators and to support their practical implementation in companies .
| improvement of occupational safety and health ( osh ) management is closely related to the development of osh performance measurement , which should include osh outcomes ( e.g. , occupational accidents ) , osh inputs ( including working conditions ) and osh - related activities .
the indicators used to measure the osh outcomes are often called lagging indicators , and the indicators of inputs and osh activities are leading indicators .
a study was conducted in 60 companies in order to determine what kinds of indicators were used for osh performance measurement by companies with different levels of osh performance .
the results reveal that the indicators most commonly used in all of the companies are those related to ensuring compliance with the statutory requirements . at the same time , the leading indicators are much more often adopted in companies with a higher performance level .
these companies also much more often monitor on a regular basis the indicators adopted for the evaluation of their osh performance . |
polymer
nanocomposites that integrate nanoparticles ( nps ) into
polymer melts often possess superior mechanical , thermal , optical ,
or electrical properties in comparison with pure polymeric materials .
fabrication and processing of polymer nanocomposites
require a good understanding of their viscoelastic behavior .
central to the viscoelasticity of polymer nanocomposites is the
coupling between the motion of nps and the relaxation dynamics of
matrix chains .
the dynamical coupling between nps and linear
polymers depends
on the particle diameter d compared to the polymer
entanglement mesh size a , as shown in experiments , simulations , and
theoretical studies . for d < a , the motion of nps
is coupled to the unentangled dynamical modes of local chain segments
up to sizes d .
in contrast , nps with d > a are confined by the entanglement
mesh for time scales greater than the relaxation time e of an entanglement strand , as illustrated in figure 1a .
brochard - wyart and de gennes argued that nps with d > a have to wait for the complete relaxation of surrounding
entangled polymers to diffuse .
however , cai et al . recently proposed a hopping diffusion mechanism for nps
moderately larger than a to overcome the confinement
by the entanglement network .
a qualitatively different activated hopping
mechanism for nps to overcome confinement is described in a nonlinear
langevin equation theory for np motion in cross - linked networks and
entangled melts of polymers .
microscopic
force - level theories without considering the mechanism
of np hopping also predict a partial coupling between nps and the
entanglement network for d moderately larger than a. schematic sketch of a np ( light orange sphere ) in ( a )
entangled
linear polymers ( red lines ) and ( b ) entangled nonconcatenated ring
polymers ( green lines ) .
the discrepancy between the dynamics of nps smaller and larger
than the entanglement mesh size in linear polymer melts reflects the
impact of entanglements on np motion .
entanglements also play an important
role in the viscoelasticity of high molecular weight polymers with
nonlinear architectures , such as ring , star , and branched polymers .
one would anticipate intriguing dynamical couplings between nps
and entangled polymers with nonlinear architectures .
we study the
motion of nps in entangled nonconcatenated ring polymers as illustrated
in figure 1b .
ring
polymers are particularly interesting as their unique topology gives
rise to distinctive conformational and dynamic properties . entangled nonconcatenated ring polymers
adopt compact conformations rather than the almost gaussian random - walk conformations of linear
polymers .
the entanglements in ring polymers do not create long - lived
tubes that confine polymer motion as in entangled linear polymers .
the entangled dynamics of ring polymers is self - similar , and there
is a power - law time dependence of the stress relaxation function without
the rubbery plateau characteristic of entangled linear polymers .
nonconcatenated entangled ring polymers are
good models of the chromatin packed in cell nucleus and of the strands of deswollen gel networks .
molecular dynamics
( md ) simulations of nps in melts of entangled
nonconcatenated ring polymers and entangled linear polymers are performed .
the models of polymers and nps are similar to those in previous simulations . the bead
all beads of size and mass m interact via
the lennard - jones ( lj ) potential with interaction strength ,
cutoff distance rc = 2.5 , and characteristic
time .
neighboring beads in a polymer are connected
additionally by the finitely extensible nonlinear elastic ( fene ) potential .
a bond bending potential v = k(1 + cos ) is applied with k = 1.5. nps are modeled as smooth
spheres that interact with each other through a purely repulsive potential .
the mass density of a np is = m/. interactions between nps and polymers are weakly attractive ,
so nps are well dispersed in polymers
for the linear polymers , the number of monomers per entanglement
strand ne 28 , the number
of monomers per chain n = 800 30ne , the average spacing between entanglements a 5 , and the
entanglement time e
the root - mean - square end - to - end distance
of the linear polymers is re
41.2. the root - mean - square spanning distance of ring polymers
between pairs of beads separated by n/2 beads is r 15.3 and 7.0 for n = 800 and 100 , respectively .
the samples of nps in linear
polymers were built using the same
method in previous simulations by kalathi et al .
the
samples of nps in ring polymers were made by inserting nps into well - equilibrated
melts of nonconcatenated ring polymers from previous simulations by
halverson et al .
the insertion of nps into ring
polymers started with creating well - separated small voids with volumes
in a ring polymer melt .
then the volumes
of the voids were increased until each void was large enough to accommodate
a np . during the insertion ,
one ring of each concatenated pair was removed from the simulation
box to ensure nonconcatenation .
it is sufficiently
low for np motion to be barely affected by the interactions between
nps , while the corresponding number of nps nnp is sufficiently large for good statistics of np motion .
nnp ranges from 650 for d =
3 to 5 for d = 15. we also simulated
monomers with d = 1 dispersed in polymer melts
at volume fraction 10% .
subsequent simulations were run at constant
volume v for up to 10 depending
on d.
the mean - square
displacements ( msds ) of nps in entangled ring and linear polymer melts
are compared in figure 2a .
for nps in linear chains , the smallest nps with d = 3 < a move subdiffusively with the
minimum log log slope of msd min
0.7 .
e
4000 ( see dashed black line in figure 2a ) . larger nps with d a 5
also move subdiffusively after the ballistic
regime , but the crossover to fickian diffusion occurs at t > e ( see dashed color lines
in figure 2a ) .
min decreases from 0.58 for d = 5
to
0.14 for d = 15 , as shown in the inset
of figure 2a .
( a ) mean - square
displacements r(t) of nps with sizes d = 315
in entangled melts of nonconcatenated
ring polymers ( solid lines ) and linear polymers ( dashed lines ) with n = 800 .
thick red line indicates the log log slope
= 1 for fickian diffusion .
thick blue line indicates the expected
0.48 for complete dynamical coupling between nps
and rings .
the inset shows min vs d for np - linear ( filled circles ) and np - ring ( filled squares ) systems
and for np - linear systems ( open circles ) in previous simulations with smaller n / ne 9 and larger entanglement mesh size a 7. ( b ) ratio of the msds of nps in nonconcatenated
rings r(t)np - ring to the corresponding msds in linear
chains r(t)np - linear .
4000. the insets illustrate the trajectories of a
np with d = 10 in entangled rings ( dark yellow
line ) and linear chains ( violet line ) for a time period of 10 25e . for nps in linear chains ,
the subdiffusion of nps with d < a and the subdiffusion of nps with d > a for t < e arise from the coupling between np motion and the unentangled
dynamics of chain segments .
the theoretical prediction is
= 1/2 based on the rouse model of unentangled polymer dynamics .
this predicted asymptotic scaling regime is not well developed in
the present simulations . for d < a
, in the subdiffusive regime is affected by the crossover
to the fickian regime . for d
> a , in the subdiffusive regime for t <
e is affected by the crossover to the regime for t > e . the motion of nps with d > a in
linear chains is further suppressed for t > e because of confinement by the entanglement network . while
the theory by brochard - wyart and de gennes predicts that min decreases to 0 sharply at d a , min in
the simulations progressively decreases with d over
a broad range up to d 3a ( see the inset of figure 2a ) . for d moderately larger than a
, nps can overcome the confinement of the entanglement
network through the hopping diffusion mechanism . in hopping diffusion theory ,
the entanglement network is
described as many overlapping yet independent elementary networks ,
the centers of which are separated by an average distance .
each hop of a np confined by elementary networks corresponds to the
displacement from one network cage to a neighboring cage over a distance
. a successful hop needs to overcome a free energy
barrier associated with the fluctuation of one network strand from
the average size a to d. the barrier
height is proportional to d / a , and
therefore as d increases the probability of hopping
decreases as exp(d / a ) .
a
qualitatively different picture of activated np hopping is presented
in a microscopic force - level theory .
it
predicts that hopping distance of a large np in an entangled polymer
melt increases with d , and the hopping barrier height
is asymptotically proportional to the volume of a np .
as hopping becomes less likely with increasing d , the confinement of the entanglement network is enhanced ,
and min is reduced . for d
a , hopping diffusion is highly unlikely , and nps have to
wait for
the relaxation of the entanglement network to diffuse . in this case
,
min would be independent of d.
however , min is expected to be nonvanishing for n / ne 30 because the
entanglement network can partially relax through longitudinal rouse
modes in a confining tube , tube length fluctuation , and constraint
release .
hopping events are not
directly observable in the trajectories
of nps for a < d < a/ , as they
are masked by the thermal fluctuations of nps confined by individual
cages .
the msd due to thermal fluctuation of a np confined in an entanglement
cage is rfluct
a/(d ) . for a < d < a/ , rfluct is larger than resulting from one hopping event . for d
a/ , rfluct < , but
the propability of hopping exp(d / a ) is lower .
the contribution of hopping diffusion
to the msd is manifested
in the dependence of min on d.
kalathi et al .
have simulated nps in
linear polymers with k = 0.75 , ne 45 , n = 400
9ne , and a 7.
the dependence of min on d for
their simulations is shown in the inset of figure 2a by the open circles . compared to the previous
simulations , the present simulations with n / ne 30 and a
5 enable the exploration of hopping diffusion over a broader
range of d / a , as the largest d / a is increased from 2 to 3 .
higher n / ne is favorable
because the reptation time (n / ne ) is longer , and thus the contribution
to np mobility from hopping diffusion is more dominant over the competing
contribution from chain reptation .
min at the largest d / a
0.14 in the present simulations is reduced with respect to 0.3
in the previous simulations . to our knowledge
, this is the first time
such a strongly suppressed motion of nps in entangled linear polymer
melts is observed in simulations .
nps in entangled
nonconcatenated ring polymers differ from their counterparts in linear
chains . for d = 3 ,
np motion in a melt of
rings is comparable to but slightly faster than in a melt of linear
chains .
the ratio of the msds in rings and linear chains is 1.4
in the fickian regime ( see figure 2b ) .
more remarkable differences between msds in entangled
ring and linear polymers are observed for d
a 5. min for nps in rings
decreases slowly with d and is 0.6 for d = 15 ( see the inset of figure 2a ) .
unlike in entangled linear chains , the
msds are not strongly suppressed in entangled rings even for the largest d. the ratio of the msds in rings and linear chains is already
2 at t e and increases
with t for t > e as shown in figure 2b .
the trajectories of a np with d = 10 > a during a time period 10 25e in entangled rings and linear chains are illustrated in the
inset of figure 2b .
the absence of strongly suppressed np motion in entangled rings demonstrates
that there are no long - lived confining tubes in entangled nonconcatenated
rings at least for n / ne up to 30 , consistent with experiments and simulations of entangled ring melts . to understand the subdiffusive motion of nps in ring polymers
,
we developed a scaling model for the dynamical coupling between nps
and entangled rings .
entangled ring polymers relax in a self - similar
manner for t > e through relaxations
of progressively larger sections until the entire rings of size r are relaxed . the motion of
nps with size a < d < r is coupled to the relaxations of ring sections with sizes
up to d. this coupling gives rise to the subdiffusive
motion of nps for e < t < s , where s is the relaxation
time of a ring section with size d . for e
< t <
s , a ring section that has just relaxed contains g monomers .
the scaling ansatz is that the effective viscosity
eff experienced by a np at t is
the same as the melt viscosity of ring polymers with n = g. annealed lattice tree model and fractal
loopy globule ( flg ) model have been proposed
to describe the dynamics of multiple entangled nonconcatenated ring
polymers .
we use the flg model that self - consistently accounts for
the complete tube dilation with an appropriate renormalization of
monomeric friction at different time scales . in the flg model , the conformation of a ring polymer
the loops at different length scales
overlap with each other in a self - similar manner such that the overlap
parameter is the same at all scales and equal to the kavassalis
the constant overlap
parameter at the entanglement threshold okn for length scales above the entanglement spacing has been confirmed
in the simulations by rosa and everaers . during the relaxation of ring polymers ,
topological constraints
associated with rearranged smaller loops affect the dynamics of larger
loops through increasing the effective friction coefficient .
nevertheless ,
these topological constraints do not contribute to the tubes confining
the larger loops and thus are dynamically released .
the tube diameter
as the average spacing between dynamically relevant topological constraints
increases with time and is comparable to the size of a characteristic
loop that has just been rearranged .
this picture of complete tube
dilation self - consistently describes the dynamics of entangled rings
without long - lived tubes .
the flg model predicts that the relaxation
time of a ring section containing g monomers scales
as relax(g ) e(g / ne ) .
note that the exponent 7/3 is between 2 for rouse dynamics of unentangled
polymers and 3 for reptation dynamics of entangled linear polymers . according to the flg model ,
the number of monomers in a relaxed
ring section increases with time as g t. the terminal relaxation time for a ring
with n monomers is relax e(n / ne ) .
the stress relaxation
modulus g(relax ) kbt / vn at t relax is on the order of thermal
energy kbt per volume vn of a ring polymer , where v is the volume
of a monomer . as a result ,
the melt viscosity of ring polymers
relaxg(relax ) n / n n. using g t and n , we obtain eff g t for e < t < s . the effective
diffusion coefficient deff kbt/(effd ) t , and thus r(t) defft t for e < t < s . the resulting scaling exponent for the power - law subdiffusion
of nps in entangled rings is = 3/7 0.43 . recently ,
grosberg et al . have theoretically studied
the scale - dependent viscosity in polymer fluids . for the flg model
of nonconcatenated ring polymer melts , they obtained ( )
for 1/relax < < 1/e . since
1/t , this agrees with our
result eff t. the msd subdiffusive exponent is identical to the
exponent
for the power - law time dependence of the stress relaxation
modulus associated with the self - similar dynamics of entangled rings .
because of the finite polymer size , the relaxation time of ring polymers
is not well separated from e even for n / ne 30 , and thus the asymptotic
entangled dynamics is not well developed .
previous analysis has shown that 0.48 for n / ne 30 , and the asymptotic
theoretical value 3/7 0.43 could only be observed for n / ne > 100 .
for nps in rings
with n / ne 30 ,
it is expected that 0.48 for sufficiently
large nps that can be completely coupled to all the relaxation modes
of rings .
simulation results of min for nps
in ring polymers
( see the inset of figure 2a ) are all above the expected value 0.48 ( as indicated by
the thick blue line in figure 2a ) for n / ne
30 . this is attributed to the finite sizes of nps , which are not large
enough to allow the complete dynamical coupling between nps and relaxation
modes of rings .
the effects of d on the dynamical
coupling between nps and rings can be quantified by the dependence
of min on d. min decreases as d increases , indicating that the degree
of the dynamical coupling increases with d. extrapolation
of the d dependence of min yields
that the expected value 0.48 for complete coupling would occur for d > dc 45. dc is about 2.6 times the root - mean - square
average spanning distance r 15.3
between pairs of monomers separated by n/2 monomers
( see figure s1 ) .
the exact value of dc / r may be related to the shape
of nps and the boundary conditions between nps and polymers .
future
work is needed to elucidate the factors affecting dc / r . as shown
in figure 2a ,
the nps
have reached the fickian diffusive regime except for the largest nps
in linear chains .
results for diffusion coefficient , computed as d = limtr/(6 t ) , as a function
of d are shown in figure 3 by the solid symbols . for nps with d
> a in linear polymers , we can estimate
the upper bounds of d ( indicated by the arrows in figure 3 ) . as in previous
simulations , for the sake of consistency ,
we do not correct d for the long - range hydrodynamic
effects associated with the finite sample sizes . in the limit of very large d
, it is expected that
np mobility is completely coupled to the melt viscosity of
polymers , and thus d = kbt/(2d ) assuming the
stokes
the slip lengths for nps in the simulations are anticipated
to be much larger than d , resulting in the slip np - polymer
boundaries .
previous simulation results of nps in linear polymer melts have also been analyzed using the
slip boundary condition . for d 40 ,
the results of d calculated using the stokes
results for in simulations are
presented in section 2 of the supporting information .
simulation data for the d dependence of d can be extrapolated to match the stokes
diffusion coefficients d as functions of diameter d for monomers
( d = 1 ) and nps ( d
3 ) in ring polymers with n = 800 ( blue squares )
and n = 100 ( green triangles )
and in linear polymers with n = 800 ( red circles ) .
< a , the decrease
of d is almost the same for np - ring and np - linear
systems and
exhibits d d scaling ( see figure 3 ) , which agrees with the theoretical prediction for the coupling between np motion and unentangled rouse
dynamics .
however , for d > a ,
the
decrease is much steeper for nps in linear chains as the nps are confined
by the entanglement network before they are able to freely diffuse .
for d = 15 , even the estimated upper bound
of d in linear chains is smaller than d in rings with the same n by more than 2 orders
of magnitude .
recently , nahali and rosa have compared the motion of nps in concentrated solutions of semiflexible
linear polymers and ring polymers .
they observed that d for d a is about 3 times
larger in ring polymers than in linear polymers . in our simulations ,
the ratio of msds in ring and linear polymers is also 3 at d a. however , our simulations
go further up to d 3 and show much stronger
effects of polymer architecture on d. there
is a crossover of the d dependence of d from d d to d d for nps with d > a in linear
polymers .
the crossover is broad and extends at least
up to d 3a . according to
cai et al .
, hopping diffusion of nps
with d moderately larger than a results
in d exp(d / a ) that broadens the crossover to the stokes
the results of d for np - linear systems are
fitted by a crossover function d = c1(/)(/d ) exp(c2d / a ) + kbt/(2d ) , in which c1 and c2 are fitting parameters , and the exponential
term is motivated by the theory of hopping diffusion . the best - fit
result with c1 = 0.081 0.018 and c2 = 2.2 0.1 is shown by the red line
in figure 3 ( see figure s2 for the details of fitting ) .
for nps with a < d < r in ring polymers , our scaling analysis predicts d d. this
prediction is based on the ansatz that the effective viscosity eff that a np with a < d < r experiences in the fickian regime is equal
to the melt viscosity of ring polymers with size r d. a ring polymer with r d contains n d monomers , and the melt viscosity n d. therefore , eff d and d kbt/(effd ) d. grosberg et al
. found that the wave
vector q - dependent effective viscosity (q ) q for 1/r < d
< 1/a in the
flg model . based on their result , the effective viscosity for fickian
diffusion of a np with a < d < r is (q
1/d ) d , in
agreement with our result eff d. the d dependence of d for np - ring
systems with a < d < r in our simulations is weaker than the theoretical prediction d d. the crossovers
between the scaling regime for a < d < r and two neighboring regimes contribute to
the weakening of the d dependence . the results of d for nps in rings with n = 800 and n = 100 are fitted simultaneously using a crossover function d = b1(/)(/d)/(1 + b2a / d ) + kbt/(2d ) , in which the fitting parameters b1 and b2 are independent of n. the fitting function describes the crossover from d d for small
nps coupled to the unentangled dynamics of local ring sections to d d for nps
coupled to the self - similar entangled dynamics of rings and then to d d in the
stokes
the best - fit results with b1 = 0.039 0.003 and b2 = 0.05 0.03 are shown in figure 3 ( see figure s3 for the details of fitting ) .
d d dominates over d d only for d > 25.
this crossover occurs in the regime where d d dominates the overall d dependence .
therefore , there is no apparent d d regime . for n = 100 ,
the crossover to d d occurs at smaller d , and even the d d regime
is not well developed .
in conclusion ,
our simulations show that the motion of nps can
be strongly suppressed prior to fickian diffusion in entangled linear
polymers .
such a strong suppression occurs progressively as the np
diameter d becomes increasingly larger than the entanglement
spacing a. the motion of nps with d > a in entangled nonconcatenated ring polymers
is not strongly suppressed as in entangled linear polymers .
the decrease
of diffusion coefficient d with increasing d in entangled rings is more gradual compared to the steep
reduction of d in entangled linear chains as d exceeds a. np motion for d > a in entangled linear chains is understood
through
a hopping diffusion mechanism . for nps
with d > a in entangled rings ,
we
develop a scaling model for the coupling between np motion and the
self - similar dynamics of entangled rings .
our results demonstrate
the role of polymer architecture in the dynamical coupling between
nps and polymers in nanocomposites . | the motion of nanoparticles
( nps ) in entangled melts of linear
polymers and nonconcatenated ring polymers are compared by large - scale
molecular dynamics simulations .
the comparison provides a paradigm
for the effects of polymer architecture on the dynamical coupling
between nps and polymers in nanocomposites .
strongly suppressed motion
of nps with diameter d larger than the entanglement
spacing a is observed in a melt of linear polymers
before the onset of fickian np diffusion .
this strong suppression
of np motion occurs progressively as d exceeds a and is related to the hopping diffusion of nps in the
entanglement network .
in contrast to the np motion in linear polymers ,
the motion of nps with d > a in
ring polymers is not as strongly suppressed prior to fickian diffusion .
the diffusion coefficient d decreases with increasing d much slower in entangled rings than in entangled linear
chains .
np motion in entangled nonconcatenated ring polymers is understood
through a scaling analysis of the coupling between np motion and the
self - similar entangled dynamics of ring polymers . |
micrornas ( mirnas ) are small endogenous post - transcriptional regulators of gene expression found in a wide range of eukaryotes .
these 20- to 24-nucleotide ( nt)-long rnas regulate numerous physiological processes , including cell proliferation , differentiation , apoptosis and development [ 25 ] .
deregulation of mirna expression has been linked to many disorders , including cancer [ 68 ] and neurodegeneration [ 9 , 10 ] .
most of the mirna genes , which are located predominantly in introns of protein coding genes and intergenic regions , are transcribed by rna polymerase ii ( pol ii ) [ 11 , 12 ] , and some are transcribed by rna polymerase iii ( pol iii ) ( fig . 1 ) .
the primary transcripts ( pri - mirnas ) , which are typically capped and polyadenylated , contain one or more long hairpin structures .
the structural features of these hairpins are unique to pri - mirnas , thus distinguishing them from the various rna stem - loop - like structures present in the nucleus .
the pri - mirna hairpin typically contains a long imperfect stem of approximately 30 bp with flanking single - stranded rna segments at its base ( termed the single - stranded
this structure is recognized and cleaved by the microprocessor complex containing the ribonuclease drosha ( rnase iii enzyme ) , the rna binding protein dgcr8 ( digeorge syndrome critical region gene 8) [ 1618 ] and other proteins [ 17 , 19 ] .
drosha cleaves the pri - mirna hairpin at a distance of approximately 11 bp from the single - stranded rna
the drosha cleavage of intronic mirnas occurs co - transcriptionally before splicing of the host rna [ 20 , 21 ] . as a result , a long truncated hairpin of approximately 60 nt ( pre - mirna ) is generated , which usually has a 2-nt 3 overhang , the hallmark of rnase iii products .
the 3 overhang and the pre - mirna double - stranded stem with a minimal length of 16 bp are essential structural elements that are recognized by exportin-5 ( exp-5 ) [ 24 , 25 ] . in cooperation with
the guanine triphosphatase ( gtpase ) ran , exp-5 transports the pre - mirna from the nucleus to the cytoplasm irrespective of its nucleotide sequence and the presence of various structural motifs .
the high - resolution x - ray structure of the pre - mirna nuclear export machinery has recently been reported and the crucial role of the structural features of the precursor for exp-5 recognition confirmed .
however , the exact mechanism by which the pre - mirna is transferred from the microprocessor to exp-5 in the nucleus and from exp-5 to the risc loading complex ( rlc ) in the cytoplasm remains to be elucidated .
it is thought that exp-5 passes pre - mirna to dicer directly or via additional components .
the pre - mirna cutting machine of the rlc is the ribonuclease dicer ( another rnase iii enzyme ) , which converts the pre - mirna to mature mirna .
dicer recognizes the 3-ends generated by drosha and cleaves the pre - mirna two helical turns ( approx .
22 nt ) away , near the terminal loop , to produce a mirna mirna * duplex having 2-nt 3 overhangs at both ends [ 28 , 29 ] .
the human dicer is an approximately 200-kda multidomain protein that contains an n - terminal dead - box helicase domain , a domain originally named the domain of unknown function ( duf283 ) , a paz domain , two conserved catalytic rnase iii domains ( riiia and riiib ) and a c - terminal dsrna - binding domain ( dsrbd ) .
the two rnase iii domains of dicer form a single processing center for pre - mirna cleavage , and the 5 and 3 arms of the precursor are cleaved by domains riiib and riiia , respectively [ 31 , 32 ] .
dicer does not function alone ; it cooperates within the rlc with other proteins [ 33 , 34 ] .
its protein partners are members of the argonaute ( ago ) family [ 35 , 36 ] , hiv-1 transactivation response ( tar ) rna - binding protein ( trbp ) [ 37 , 38 ] and possibly other proteins ( see the section dicer partners in pre - mirna cleavage ) .
once dicer has cleaved the pre - mirna , only one mirna strand ( guide strand ) of the duplex is loaded onto ago to form the programmed risc ( referred to as the mirisc ) ; the other strand ( passenger strand or mirna * ) is released and degraded [ 37 , 39 , 40 ] . the thermodynamic stability of the ends of the mirna duplexes is thought to play a crucial role in mirna strand selection [ 4143 ] .
other factors contributing to strand selection are structural features of the mirna / mirna * duplex ( e.g. positions of base mismatches ) [ 4446 ] and sequence composition [ 27 , 47 ] .
these features may be disregarded , since there are cases where both mirna and mirna * strands may be involved in risc - mediated gene silencing [ 46 , 48 ] .
the finding of the cognate target is thought to be a diffusion - driven process , and the recognition of mrna by mirisc is based on the partial complementarity of mirna to mrna .
the seed region , i.e. nt 28 of the mirnas , typically forms perfect matches with their target sequences located in the 3 untranslated region ( utr ) of mrnas , but these interactions fall into several categories [ 5052 ] .
mirnas downregulate gene expression in several ways ; in animal cells , the downregulation is usually achieved by translational arrest , mrna deadenylation and degradation , or less frequently by mrna cleavage ( reviewed in ) .
more recently , mrna destabilization and degradation have been proposed to constitute the predominant mechanism of gene expression regulation by mirnas .
moreover , the existence of non - canonical pathways of mirna biogenesis has also been reported .
pre - mirna - like introns ( mirtrons ) are spliced out of mrna transcripts [ 5557 ] .
mirtrons bypass the drosha requirements , but other steps of their biogenesis , such as nuclear export and cleavage by dicer , follow the canonical pathway .
instead , an erythropoetic mir-451 circumvents the dicer step ; mir-451 is cleaved by ago2 within the 3 arm , and the exact mirna 3-end is generated by trimming , which is mediated by other 3-5 exonucleases [ 5961 ] . like all rnas ,
their turnover has been evaluated in various experiments with pol ii inhibitors and/or by the rnai - mediated depletion of mirna processing enzymes .
most mirnas are highly stable , and their half - lives range from hours to even days , but several mirnas with an accelerated turnover have also been reported [ 6266 ] . the different aspects of mirna biogenesis , function and regulation at multiple levels have been extensively reviewed elsewhere [ 6771].fig . 1canonical pathway of microrna ( mirna ) biogenesis and activity .
sequential processing reactions of the primary transcript by drosha in the nucleus and of pre - mirna by dicer in the cytoplasm are presented schematically ( details are given in the text ) .
the structural requirements for drosha , exportin-5 and dicer are highlighted in the yellow boxes canonical pathway of microrna ( mirna ) biogenesis and activity .
sequential processing reactions of the primary transcript by drosha in the nucleus and of pre - mirna by dicer in the cytoplasm are presented schematically ( details are given in the text ) .
the structural requirements for drosha , exportin-5 and dicer are highlighted in the yellow boxes here we review recent progress in understanding the mechanism of drosha and dicer cleavages from the perspective of their end - products .
we describe the specificity of these cleavages and analyze mirna length variety in terms of both diversity and heterogeneity , searching for their sources and consequences .
finally , we discuss the composition of the core rlc complex , the role of each dicer protein partner and the structure of this complex as a whole interacting with pre - mirna .
it was predicted 6 years ago that as many as 1,000 mirnas may regulate the expression of most human protein coding genes .
the latest release of the mirna repository ( mirbase , release 16 ) indeed matches that prediction , and the phenomenon of mirna end and length heterogeneity substantially increases this number .
this kind of mirna heterogeneity , i.e. the existence of several length variants and shifted sequence variants of the same mirna , has been known since early mirna studies and has recently been termed mirna end polymorphism or iso - mir formation [ 7577 ] . since the routine application of deep sequencing technology in mirna discovery studies [ 75 , 7882 ] , the scale of mirna heterogeneity has been found to be greater than anticipated ( fig .
at the same time , the proper interpretation of deep sequencing results and the separation of real biological effects from various deep sequencing artifacts remain ambiguous .
different deep sequencing platforms generate platform - specific biases [ 83 , 84 ] that stem from the methods of mirna library construction . among the biological effects involved , drosha- and dicer - induced cuts with relaxed specificity are one of the most likely explanations for the phenomenon of mirna heterogeneity [ 75 , 78 , 81 , 86].fig .
the number of sequence reads and fraction of mirna variants are indicated on the right .
variants represented by only one sequence read are not shown . the major contributor ( mirna - main )
is shown in boldface , and the mirna weighted average length ( mirna - wal ) is also indicated .
the reference precursor and annotated mirnas ( red type ) , along with the dot - bracket pattern of structure of the precursor , are shown below the aligned sequences . note that only the mirna
the figure was prepared from deep sequencing data deposited in mirbase [ 73 , 149 ] .
b drosha and dicer cleavages are the primary sources of mirna heterogeneity , and the ends generated by drosha are less heterogeneous than those generated by dicer .
c no matter which arm the mirna is generated from , the 5-end is always less heterogeneous than the 3-end .
d shifted drosha cleavages result in shifted dicer cleavages ( leading to mirna with two heterogeneous ends ) .
e ago2 loading is the selection step for binding mirnas with u and a at their 5-ends .
f nontemplated heterogeneity results from modifications of the 3-end occurring after drosha / dicer cleavages sources of mirna end heterogeneity . a example of deep sequencing data for an mirna generated from both precursor arms .
the number of sequence reads and fraction of mirna variants are indicated on the right .
variants represented by only one sequence read are not shown . the major contributor ( mirna - main )
is shown in boldface , and the mirna weighted average length ( mirna - wal ) is also indicated .
the reference precursor and annotated mirnas ( red type ) , along with the dot - bracket pattern of structure of the precursor , are shown below the aligned sequences .
the figure was prepared from deep sequencing data deposited in mirbase [ 73 , 149 ] .
b drosha and dicer cleavages are the primary sources of mirna heterogeneity , and the ends generated by drosha are less heterogeneous than those generated by dicer .
c no matter which arm the mirna is generated from , the 5-end is always less heterogeneous than the 3-end .
d shifted drosha cleavages result in shifted dicer cleavages ( leading to mirna with two heterogeneous ends ) .
e ago2 loading is the selection step for binding mirnas with u and a at their 5-ends . f nontemplated heterogeneity results from modifications of the 3-end occurring after drosha / dicer cleavages based on deep sequencing results , it has been proposed that drosha - induced cleavages generate much less mirna end heterogeneity than dicer cleavages ( fig .
other authors have concluded that the 5-ends of mirnas are always less heterogeneous than the 3-ends , regardless of the precursor arm from which the functional mirnas were generated [ 27 , 75 , 78 , 81 , 88 , 89 ] ( fig .
this conclusion is in agreement with the notion that evolutionary pressure favors homogenous mirna 5-end formation , which is important for specific target recognition .
however , the above propositions are mutually exclusive as drosha defines the 5-ends of mirnas from the pre - mirna 5 arm and the 3-ends of mirnas from the 3 arm , whereas dicer defines the 3 ends of mirnas from the pre - mirna 5 arm and the 5-ends of mirnas from the pre - mirna 3 arm ( see fig . 2 ) .
drosha and dicer belong to the same rnase iii class ; both possess a catalytic center formed by two rnase iii domains .
the difference in cleavage specificity for drosha and dicer may arise from their different strategies of substrate recognition .
dicer combines precursor recognition and cleavage activities , as it contains the paz and rnase iii domains within one protein .
this enzyme design makes the structure flexible and adaptable to various structures of pre - mirna substrates .
drosha requires another protein ( dgcr8 ) to recognize and bind to pri - mirnas .
it may be these differences between drosha and dicer which influence their cleavage specificities ( fig .
it would appear that the discriminative power of the drosha / dgcr8 complex between substrates and the multitude of nonsubstrate hairpins need to be very high ; also , the use of two specialized proteins to perform this task offers more precision in the cleavage .
however , it seems unlikely that drosha and dicer can sense which precursor arm will generate the functional mirna and that arm will be cleaved more precisely ( fig . 2c ) .
therefore , the hypothesis suggesting that mirna end heterogeneity is the result of different drosha and dicer cleavage specificities seems more appealing .
another model of mirna heterogeneity that was proposed is a parallel shift of drosha and dicer cuts to generate several mirnas from the same precursor ( fig .
this model simply reflects the fact that nonspecific drosha cleavages will have consequences for nonspecific dicer cleavages .
apart from the imprecise cleavages by drosha and dicer being the primary source of mirna heterogeneity , the ago binding step may also introduce a bias toward the u and a residues at the 5 nucleotides of the mirna ( fig .
most mirnas are represented by numerous length / sequence variants ; in the majority of cases , the mirnas are generated from one precursor arm ( 5 or 3 ) .
there are , however , examples of substantial amounts of mirnas generated from both arms of one precursor ( fig . 2a ) . the frequency of individual mirna variants varies strongly , with the most frequent approaching 100% and the rarest being represented by very small fractions ( considerably less than 1% of all the reads ) .
only the most frequent variants ( mirna - main and those that contribute to more than 5% of total reads ) can probably be considered functional .
if many mirnas are derived from one precursor arm , the parameter mirna - wal ( weighted average length ) would be appropriate to describe the lengths of mirnas obtained from deep sequencing and derived from single genes .
in addition to deep sequencing , conventional techniques of mirna detection have also been used to demonstrate and characterize mirna heterogeneity .
northern blotting , a gold standard in molecular biology , has been frequently used to detect newly identified mirnas [ 74 , 9295 ] .
recent improvements in northern blotting protocols have allowed us to distinguish not only mirnas but also pre - mirnas differing in length by 1 nt ; the northern blot method has also been used for the quantitative analysis of mirna and pre - mirna heterogeneity [ 9698 ] .
high - resolution northern blotting used in conjunction with primer extension ( which detects 5-end heterogeneity ) has made it possible to distinguish shares held by drosha and dicer in generating mirna heterogeneity .
both drosha and dicer generate substantial mirna end heterogeneity , but the contribution of dicer is slightly greater .
different nucleotides at the mirna 5-end may change the relative thermodynamic stability of the mirna / mirna * duplex ends and cause preferential risc activation by a different strand .
furthermore , the mirna 5-end , particularly its seed sequence , is responsible for the recognition of a complementary sequence and the binding to mrna .
mirnas with shifted 5-ends have different seed sequences and may regulate different targets [ 77 , 78 , 96 , 99 , 100 ] .
it has recently been shown that different mirna length variants ( iso - mirs ) may be loaded to different ago proteins .
thus , the generation of 5-end heterogeneity may be another mechanism of mirna activity regulation that functions either by increasing the number of targets regulated by one mirna or by decreasing the fraction and functional impact of a dominant , canonical mirna .
in addition to the frequently detected templated heterogeneity ( mirna end nucleotides match the genomic sequence ) , the presence of nontemplated nucleotides ( fig .
the nucleotides that most often differ from genomic dna are typically 3-end a and u [ 75 , 79 , 89 , 102 ] .
it is thought that these nucleotides are added to either mirna or pre - mirna ends by specific enzymes [ 102105 ] following drosha or dicer cleavage . in addition to contributing to the mirna
mrna interaction ( compensatory site effect ) [ 50 , 51 ] , the 3-end of the mirna may influence its localization .
extra nucleotides added at the 3-ends of some mirnas may also influence their stabilities [ 103106 ] , regulate the dicer step of biogenesis by blocking cleavage or modulate mirna uptake by risc bound with different ago proteins .
the 3-end modification has also been linked to a reduction in the efficiency of mrna targeting [ 102 , 104 ] .
the 5- and 3-end heterogeneity of the drosha and dicer cleavage products , as well as the nontemplated effects , may also have implications for the rna interference ( rnai ) and mirna technologies .
the end heterogeneity of biologically processed short rnai triggers or exogenous mirnas may create problems in reproducing the silencing effects achieved with synthetic small interfering rnas ( sirnas ) or mirnas having defined ends .
the nonspecific dicer and drosha cleavages generate a population of products , only a fraction of which may have the desired sequence and exert the expected effects .
another phenomenon related to the variation in the length of mirnas is their length diversity , i.e. the formation of mirnas differing in length from different mirna genes .
this effect originates from the structural features of the pre - mirna hairpins , which differ from each other in the number and localization of various types of structural motifs .
the range of human pre - mirna structural diversity has been estimated by the analysis of predicted structures of 460 pre - mirnas whose sequences were reconstructed from mature mirna sequences [ 108 , 109 ] .
the lowest energy structures of pre - mirnas were analyzed for the presence of various secondary structure motifs ( mismatches , bulges , symmetrical and asymmetrical internal loops ) .
all motifs identified were cataloged according to their type , size , position and orientation . of the 1,243 motifs ,
631 were symmetrical ( 1- to 5-nt - long mismatches and internal loops ) and 612 were asymmetrical ( bulges and internal loops ) .
3a ) . the number of structural motifs in the pre - mirna structures analyzed ranged from zero to seven , with an average of 2.7 motifs per precursor . based on the distribution , localization and sequence content of the structural motifs ,
the following interesting observations were reported : ( 1 ) the frequency of symmetrical motifs tended to increase and that of asymmetrical motifs decreased when proceeding from the pre - mirna hairpin base to its terminal loop ; ( 2 ) bulges were significantly overrepresented in the 5 arm of the precursor ( 262 ) compared to the 3 arm ( 172 ) ; ( 3 ) there were no strongly overrepresented specific sequences within the structural motifs analyzed .
the predicted structures of many pre - mirnas showed great variation , possibly explaining the length diversity of mature mirnas . to determine the specific structural motifs that could predominantly account for mirna length diversity ,
a large number of synthetic pre - mirnas selected to contain various structural motifs at specific locations were subjected to a cleavage assay with recombinant dicer .
prior to dicer cleavage , the structures of nearly 20 pre - mirnas were determined experimentally by chemical and biochemical methods .
3b ) , namely pb , ca , mg and mn ions [ 111 , 112 ] , was previously used to demonstrate the structural diversity of extended pre - mirnas . the ions mapped the single - stranded fragments , i.e. the terminal and internal symmetrical and asymmetrical loops .
when the structures of the terminal loops were probed , the best results were obtained with s1 and t1 nucleases , but v1 nuclease was used for mapping well - paired stem portions of the secondary structure .
the small differences between the predicted and experimentally determined structures were localized mainly in the hairpin terminal loops .fig .
3structure and dicing of mirna precursors . a structural diversity of mirna precursors based on bioinformatics analysis of the predicted structures of 460 human pre - mirnas .
lanes : ci incubation control ( no probe ) , f formamide ladder , t guanine - specific ladder .
s1 , t1 , t2 nucleases . the positions of selected g residues are shown .
the cleavage sites and intensities for the selected probes are indicated by the symbols described in the inset .
the precursor was incubated with human recombinant dicer for 1 , 2 and 5 min as described by starega
the dicer cleavage sites ( black arrowheads ) , are shown in the secondary structure model ; the reported mirna sequences are marked in red .
the values of the weighted average length of diced rna ( waldi ) parameter for mirnas generated from the precursor 5 and 3 arms are indicated .
other designations are as in 3b structure and dicing of mirna precursors . a structural diversity of mirna precursors based on bioinformatics analysis of the predicted structures of 460 human pre - mirnas .
lanes : ci incubation control ( no probe ) , f formamide ladder , t guanine - specific ladder .
s1 , t1 , t2 nucleases . the positions of selected g residues are shown .
the cleavage sites and intensities for the selected probes are indicated by the symbols described in the inset .
the precursor was incubated with human recombinant dicer for 1 , 2 and 5 min as described by starega
the dicer cleavage sites ( black arrowheads ) , are shown in the secondary structure model ; the reported mirna sequences are marked in red .
the values of the weighted average length of diced rna ( waldi ) parameter for mirnas generated from the precursor 5 and 3 arms are indicated .
3c ) of 19 pre - mirnas and several pre - mirna mutants revealed the important role of the structure of the precursor in determining the cleavage position .
in particular , the presence of asymmetrical structural motifs was found to be a major determinant of mirna length diversity .
precursor arms harboring excessive unpaired nucleotides gave rise to longer mirnas . to make the analysis more straightforward and quantitative ,
a new parameter was introduced in that study , namely , the weighted average length of diced rna
waldi , which facilitated finding the correlation between the pre - mirna structure and the lengths of products excised by dicer .
the results from the dicer cleavage assay of pre - mirnas were confirmed by bioinformatic analyses of mirnas deposited in mirbase .
the results of both approaches confirmed that the presence of excessive nucleotides in any pre - mirna arm results in the generation of longer mirnas from this arm by dicer .
most of the other information currently available on dicer cleavage of dsrna comes from biochemical studies in which the requirements for dicer binding and cleavage were determined [ 31 , 113115 ] .
the preferences toward dsrna over single - stranded rna were shown , and the binding of the paz domain to the 3 terminal single - stranded overhang was demonstrated .
dicer binding was not only shown to the typical 2-nt 3 overhang , but also to 1-nt and 3- to 5-nt protruding ends [ 113 , 116 ] as well as less efficient binding to blunt ends of rna [ 114 , 117 ] . not only the overhang structure itself , but also the base composition of the overhanging nucleotides influence the efficiency of dicer binding and cleavage [ 113 , 118 ] .
the pre - mirna terminal loop structure has also recently been shown to influence dicer s cleavage efficiency , and the existence of some local sequence preferences at dicer cleavage sites have been postulated ( our unpublished data ) .
additionally , the rnase iiib dicer domain was shown to prefer cleaving phosphodiester bonds adjacent to the structural distortions that occur in the 5 arm of the rna substrate , therefore determining the dicer cleavage site .
nevertheless , the 3 terminus of pre - mirnas is the major determinant of dicer s cleavage position as dicer measures the distance ( two helical turns ) from the 3 end to its cleavage site .
as mentioned earlier in this review , in cells , dicer functions within the rlc , which comprises the two components trbp and ago2 , as well as some additional proteins .
the role of dicer s protein partners in pre - mirna ( dsrna ) dicing has been addressed in several studies , but to date it has not been satisfactorily resolved . in this section , the protein components of the human dicing complex are characterized , and the results of studies aimed at resolving their role in pre - mirna processing are summarized . there are four argonaute proteins ( ago14 ) in human cells , but only ago2 has rna slicing activity .
ago2 is a major protein involved in the rnai mechanism whose main function is to induce the guide strand - mediated cleavage of target mrna by the catalytically competent risc .
the paz domain is responsible for the binding of the 2-nt overhangs of mirna and sirna duplexes [ 120123 ] , the mid domain interacts with the 5-phosphate group of rna and the piwi domain , located at the c - terminus , possesses the rnase h activity required for the endonucleolytic cleavage of the mirna passenger strand and subsequently the target mrna [ 121 , 124 ] .
two of the dsrbds can homodimerize or bind to the interferon ( ifn)-induced protein kinase r ( pkr ) and the protein activator of pkr kinase ( pact ) .
the exact function of trbp in mirna biogenesis has not yet been determined and remains controversial [ 37 , 38 ] .
this protein is believed to somehow cooperate with dicer to facilitate mirna / sirna production , probably by enhancing the stability of dicer substrate complexes .
based on the biochemical results and data from electron microscopy imaging of the rlc complex , trbp is the sensor for proper strand loading to risc , which can proofread incorrect strand loading .
trbp mutations observed in human cancers have been shown to cause defects in mirna biogenesis .
the localization and function of other accessory proteins , such as pact , in the complex are still poorly understood ; however , it has been shown that the loss of pact expression impairs mirna biogenesis .
since trbp and pact are very similar in terms of their domain composition , it is likely that they compete for the same binding site in dicer and that their functions may be mutually compensated .
trbp and pact may interact with each other and also associate with dicer to facilitate the cleavage of dsrna ; therefore , both proteins play a stimulatory role .
the following proteins may also be involved in the regulation of processing selected mirnas by dicer : ars2 , fmrp , ksrp , lin 28 or adar .
these proteins act by stimulating pre - mirna processing by dicer or by repressing mirna maturation ( reviewed in ) .
several experimental systems have been used to date to gain insight into the role of dicer s partners in its cleavage efficiency and specificity .
initially , the silencing of endogenous dicer partners resulted in decreased pre - mirna processing efficiency rather than in changed cleavage specificity . even with regard to cleavage efficiency ,
synthetic precursors were also injected or transfected to cells in culture to follow their cleavage by the endogenous dicing complex ( koscianska et al . , submitted ) .
the processing products of synthetic pre - mirnas in cells were found to correspond rather well to the cleavage products generated by the recombinant dicer ; however , the cleavage efficiency differed between these systems .
the experimental systems also included pre - mirna cleaved by an immunoprecipitate containing dicer with its protein partners [ 33 , 35 ] and pre - mirnas incubated in cellular extracts [ 138 , 139 ] .
human rlc was reconstituted in a 1:1:1 stoichiometry from recombinant dicer , ago2 and trbp proteins , and this rlc was used in a cleavage assay with synthetic pre - let-7 .
dicer s partners showed little or no effect on cleavage specificity compared to dicer s activity alone ( koscianska et al . ,
the successful reconstitution of rlc activity from recombinant proteins has prompted researchers to aim at acquiring deeper insights into the molecular architecture of the human dicer in the complex generated with its protein partners .
the high - resolution crystal structures of giardia intestinalis dicer [ 142 , 143 ] , thermus thermophilus ago2 and the dead - box helicase domain have provided useful information for developing the human dicer , dicer trbp and rlc models based on single particle electron microscopy images [ 131 , 146 ] .
these analyses yielded low - resolution ( 20 ) information on the mutual arrangement and possible interactions between the proteins within the binary and ternary complex .
multiple images obtained from electron microscopy suggest that rlc forms an l - shaped structure with an active rnase iii center of dicer in the back portion of the l - structure and in n - terminal domains localized at the base of the l - structure . using its piwi and mid domains , ago2 interacts with the dicer platform formed by the c - terminal region .
the n - terminal domain of ago2 , together with trbp , interacts with dicer s dead - box domain localized at the base of the l - structure .
s position in the rlc complex is flexible ; it can move upon the binding of rna and may play the role in increasing pre - mirna access to dicer [ 131 , 147 ] .
the three - component complex forms a stable , triangle - like architecture , with an inside channel with a diameter of about 20 and a length of > 100 .
this channel , which runs along the long edge of the l - shaped portion , may be used to bind and position the pre - mirna for catalysis .
attempts have been made to fit a hairpin structure into the cleft of the reconstructed dicer
most human pre - mirnas range from 57 to 66 nt and are approximately 7890 long ; they can , therefore , be accommodated within the channel .
the catalytic valley formed by the two rnase iii domains in giardia dicer is about 20 wide ( which is similar to the diameter of the rna - a helix ) and 50 long and covers about two - thirds of the length of a typical pre - mirna ( fig . 4 ) .
trbp structures [ 131 , 146 ] and better insight into pre - mirna structure and dicing will provide answers to the intriguing question of whether the formation of the complex with pre - mirna requires the structural adaptation of both the rna and protein components or whether structural changes in only one of them would be sufficient to provide an induced fit [ 30 , 143 ] .
previous studies that addressed this question focused mainly on the adaptive features in dicer s structure [ 142 , 148 ] .
protein flexibility was proposed to be a critical factor , allowing dicer to adjust its shape to accommodate the structural diversity of its pre - mirna substrates [ 142 , 148 ] . to excise the 20-nt mirnas from the pre - mirna hairpin with a fully base - paired stem in the rna - a conformation
, the catalytic site of the rnase iii domain has to be located approximately 56 from the pre - mirna 3-base . to excise 24-nt mirnas
thus , the amplitude of motion of the dicer catalytic center has to be at least 10 , i.e. approximately one - tenth the entire length of the substrate channel .
however , the movement of the dicer structure does not need to be as great .
only a few known human pre - mirnas have perfectly paired hairpin stems , and their derived mirnas vary in length from 21 to 22 nt [ 73 , 108 ] .
the stems of other human pre - mirnas are mosaics of base pairs and internal loops of various types and sizes ( fig .
the unmatched bases of asymmetrical motifs probably bulge out of the helix when the pre - mirna is accommodated within the substrate channel ( fig .
4 ) ; these bases are therefore not counted by dicer when it sets the distance to its cleavage site .
the accumulation of structural imperfections in pre - mirna hairpins results in a higher plasticity of the structures of the precursor ; thus , the pre - mirna may also contribute to the induced fit required for active complex formation.fig .
4a hypothetical model highlighting the role of structural plasticity of the precursors in the dynamics of the pre - mirna dicing complex .
the pre - mirna hairpin is forced to enter a narrow substrate channel formed by the dicer portion of risc loading complex ( rlc ) .
excessive nucleotides present in any precursor arm bulge out and are not counted by dicer , which measures the double - stranded rna ( dsrna ) distance from its anchoring site ( paz domain ) to the cleavage site .
riiia and riiib domains : rnase iiia and rnase iiib dicer domains a hypothetical model highlighting the role of structural plasticity of the precursors in the dynamics of the pre - mirna dicing complex . the pre - mirna hairpin is forced to enter a narrow substrate channel formed by the dicer portion of risc loading complex ( rlc ) .
excessive nucleotides present in any precursor arm bulge out and are not counted by dicer , which measures the double - stranded rna ( dsrna ) distance from its anchoring site ( paz domain ) to the cleavage site .
over the past several years numerous important advances have been made in the mirna field , and research focusing on mirna biogenesis is one of the most rapidly progressing areas in this field .
the discovery that many mirna variants containing slightly shifted sequences and differing in lengths are generated from individual mirna genes has brought an extra interest to the mechanism of mirna biogenesis and to the sources of mirna variety .
results of mirna deep sequencing studies have provided new insights into the specificity of mirna processing steps triggered by drosha and dicer and into the nature of post - cleavage modifications . on the other hand ,
insightful information regarding the structures of mirna precursors and their processing enzymes has also been obtained by the conventional molecular and structural biology methods . in this review , we have discussed structural aspects of mammalian microrna biogenesis , placing special emphasis on the cytoplasmic step triggered by rnase dicer and the role of precursor structure in generating mirna length variety .
it may be concluded from this article that the mechanism of mirna precursor cleavages induced by dicer is now fairly well established and that structural sources of mirna length diversity are well recognized .
there remains , however , a number of unresolved or poorly understood issues . among these
are the questions of sequence preferences for dicer - induced cleavages and the exact role of dicer protein partners in the dicing process .
the low - resolution structures of the dicing complex provide first insights into its functioning , but further refinement of these structures will be needed to learn more about its dynamics .
the recent identification of numerous auxiliary proteins implicated in the nuclear step of mirna maturation sets the stage for more challenging studies on the structure and dynamics of the microprocessor complex . | the human genome contains more than 1,000 microrna ( mirna ) genes , which are transcribed mainly by rna polymerase ii .
the canonical pathway of mirna biogenesis includes the nuclear processing of primary transcripts ( pri - mirnas ) by the ribonuclease drosha and further cytoplasmic processing of pre - mirnas by the ribonuclease dicer .
this review discusses the issue of mirna end heterogeneity generated primarily by drosha and dicer cleavage and focuses on the structural aspects of the dicer step of mirna biogenesis .
we examine the structures of mirna precursors , both predicted and experimentally determined , as well as the influence of various motifs that disturb the regularity of pre - mirna structure on dicer cleavage specificity .
we evaluate the structural determinants of the length diversity of mirna generated by dicer from different precursors and highlight the importance of asymmetrical motifs .
finally , we discuss the impact of dicer protein partners on cleavage efficiency and specificity and propose the contribution of pre - mirna structural plasticity to the dynamics of the dicing complex . |
atonic seizures are characterized by a sudden loss or diminution of muscle tone without an apparent preceding myoclonic or tonic event .
this phenomenon was first described as a postepileptic paralytic phenomenon and is now well known as todd 's paralysis .
currently , such an episode of atonia during an epileptic seizure is increasingly recognized as an ictal event .
focal atonic seizures are partial seizures in which the ictal manifestation consists of paresis or paralysis of one or more parts of the body .
it is crucial to recognize this limb atonia as an ictal event as it may easily be misdiagnosed as a nonepileptic condition , such as a transient ischemic attack associated with severe arterial stenosis in old age .
herein , we report the case of a patient with recurrent atonic seizures that presented as recurrent transient right upper limb paresis that was misdiagnosed as transient ischemic attacks .
an 86-year - old right - handed woman visited our neurology department complaining of recurrent transient right upper extremity paresis that had started 3 days previously .
the patient reported that the initial symptoms occurred after an unusual sensation in her chest that was accompanied by palpitations , and she experienced weakness for 10 min without loss of consciousness .
there were no abnormal movements or accompanying neurologic deficits , and similar phenomena occurred 2 more times before the hospital visit .
she had been treated for hypertension for 15 years , and she had a history of cerebral infarction for more than 5 years that was treated with an antiplatelet drug , but she had no prior history of a seizure disorder . on the neurologic examination , there were no neurologic deficits at the time of admission and her electrolytes , renal function , and complete blood count were within normal ranges . her systolic blood pressure at the time ranged between 120 and 150 .
the initial tentative diagnosis was recurrent transient ischemic attacks as she had obviously experienced multiple episodes of transient right - side weakness that persisted for less than 1 h with no residual deficits .
the magnetic resonance imaging ( mri ) of the brain performed at her admission showed no acute diffusion restriction in the diffusion restriction image ( dwi ) , but there was a complete obstruction of the right proximal internal carotid artery and an encephalomalacia of the right temporooccipital lobe , which was probably due to an old infarction .
in addition , a high - signal lesion on the left superior parietal cortical area was observed on the fluid - attenuated inversion recovery ( flair ) mri ( fig .
the patient again complained of a sense of palpitations , agitation , and general weakness that was dominant in the right arm and very similar to the initial symptoms that she experienced before admission .
however , the symptoms persisted for more than 1 h , and the patient gradually showed confusion with complete disorientation and right - side weakness with an mrc grade of ii . following these mental changes , focal repetitive jerky movements of the right extremity with right facial twitching was noted , which then progressed to generalized tonic
clonic movements accompanied by drooling and tongue biting with a duration of 1 min .
the episode was controlled after an intravenous injection of 5 mg of midazolam was administered .
the postictal period was characterized by confusion and memory lapses for approximately 12 h. after the secondary generalized seizure , the decision was made to evaluate the patient for an epileptic seizure disorder .
her electroencephalogram ( eeg ) showed several interictal spikes in the left temporal area ( t3 maximum ) ( fig .
she was initially treated with 1000 mg of levetiracetam per day , but the episodes of right - side weakness still occurred 2 times on the following day without abnormal movements .
an ictal single - photon emission ct ( spect ) combined with an mri ( siscom ) analysis showed an increased signal in the contralateral superior parietal area , which correlated well with a high - signal lesion in the flair and mesial frontal area ( fig .
the dosage of levetiracetam was increased to 2000 mg per day , and no further seizure episode or vascular event occurred during the 6-month follow - up .
the clinical semiology of the patient 's seizure was characterized by the abrupt onset of monolimb paresis lasting for less than 10 min .
these clinical features were remarkably similar to those of stroke , especially considering the patient 's past history and the complete obstruction of the ica .
oestreich et al . suggested that epileptic discharge in the contralateral temporal lobe causes ictal unilateral paresis . in this case , it was unlikely that the contralateral temporal area was the symptomatic zone of the ictal paresis because the ictal paresis produced from the temporal lobe is always associated with impairments of consciousness and automatism , and our patient had not lost consciousness .
however , the highlighted areas in the contralateral temporal lobe on the ictal spect analyzed using siscom ( the red arrow in fig . 1c ) and the interictal spikes in the contralateral temporal area on the eeg ( the red arrow in fig .
1b ) suggest that the contralateral temporal area may have played an important role in the generation of the transient monolimb weakness .
the primary negative motor area ( pnma ) and the supplementary negative motor area ( snma ) have been proposed as the inhibitory cortical areas responsible for atonic seizures , .
the pnma lies anterior to the primary motor face area , and the snma is mapped anterior to the face region of the supplementary sensorimotor area of the mesial frontal lobe .
stimulation of these areas produces not only contralateral but also , although to a lesser extent , ipsilateral atonia , predominantly of the limb muscles .
however , previous cases have reported that the primary somatosensory cortex is the possible generator of atonic seizures by activating the selective activation of the inhibitory motor system represented by the negative motor areas .
our patient 's unusual sensation in her chest accompanied by palpitations may be explained by the aura that originates from the somatosensory cortex .
the presence of the pathology in the corresponding somatosensory area on the flair mri ( the white arrows in fig .
1a ) and the increased signal in the contralateral somatosensory area in the analysis of the ictal spect in combination with the siscom analysis ( the red arrowhead in fig .
1c ) suggest that the contralateral somatosensory area was responsible for the generation of the ictal monoparesis .
moreover , the ictal manifestation is consistent with a previous report that ictal monoparesis is associated with lesions in the primary somatosensory area .
the contralateral mesial frontal area , in which the snma lies , also showed increased activity in the siscom analysis of the patient 's ictal spect ( the white arrowhead in fig .
therefore , we can hypothesize that the spike in the left temporal area stimulated the left superior parietal cortex , which was followed by the activation of the left mesial frontal cortex , which in turn elicited the contralateral monolimb paresis ( fig . 2 ) .
because focal epilepsy has been reported as a possible manifestation of a transient ischemic attack , , we should consider in this case the possibilities that either the monolimb weakness was the manifestation of a negative seizure provoked by a transient ischemic attack or a newly developed transient ischemic attack provoked the excitation of the left temporal area and the cascade hypothesized above .
these events may have been precipitated by the lesion in the superior parietal lobe , which presented as transient monolimb weakness in our case .
this case suggests that clinicians should consider the possibility of a seizure in the differential diagnosis when patients present with transient monolimb weakness in old age , and diagnostic tests for epileptic seizures , such as an eeg and ictal spect , may help to diagnose this treatable condition .
| a focal atonic seizure is a partial seizure in which the ictal manifestation consists of paresis of the extremities or muscles on one side of the body , and this phenomenon can easily be misdiagnosed as a transient ischemic attack .
an 86-year - old woman visited our hospital complaining of transient right upper extremity weakness lasting for 10 min following an unusual sensation in her chest accompanied by palpitations . on the third hospital day
, she again complained of right arm weakness , which progressed to jerky movements of her right extremity accompanied by facial twitching and then generalized into a tonic
clonic seizure .
the eeg displayed several interictal spikes in the contralateral temporal area , and the ictal spect , analyzed using the siscom system , showed an increased signal in both the contralateral superior parietal area and the mesial frontal area . in this case , the patient was diagnosed with focal atonic seizures as the cause of the monolimb weakness , which had been initially misdiagnosed aas transient ischemic attacks . in cases in which a patient presents with monolimb paresis , physicians should consider the possibility of an atonic seizure as the cause . |
inflammatory activation with increased serum cytokine levels has been described by several authors as an important factor in the progression of the syndrome of chronic heart failure ( chf ) [ 13 ] . in multifactorial analyses ,
elevated levels of tumour necrosis factor-(tnf- ) alpha and interleukin-(il- ) 6 have been identified as prognostic heart failure markers [ 46 ] .
cytokines act as catabolic factors involved in the pathogenesis of muscle wasting and cardiac cachexia [ 3 , 7 ] , and increased levels of serum tnf - alpha have been identified in patients with reduced skeletal muscle cross - sectional area and peripheral muscle strength .
there also exists a statistical significant association between elevated serum cytokine levels ( especially tnf - alpha ) and new york heart association ( nyha ) functional class as well as exercise intolerance .
inflammatory cytokines may alter skeletal muscle histology and have a negative impact on left ventricular remodelling and cardiac contractility [ 2 , 3 , 8 ] . the inflammatory response
is also associated with progression of atherosclerosis , oxidative stress , no impairment , vasoconstriction , endothelial cell apoptosis , and adverse vascular remodelling .
exercise training has been documented to improve the inflammatory profile in chf by inhibition of cytokine - chemokine production , regulation of monocyte activation and adhesion , inhibition of inflammatory cell - growth signals and growth factor production , reduction of soluble apoptosis signalling molecules , and attenuation of monocyte - endothelial cell adhesive interaction .
a study of 277 patients with coronary artery disease reported a significant 41% reduction in high - sensitivity c - reactive protein following exercise training .
a recent study of four - month duration , utilizing combined endurance / resistance training demonstrated reduced tnf - alpha receptor levels ( tnfr1 and tnfr2 ) and a significant ( 7.5% ) increase in peak vo2 in patients with ischemic cardiomyopathy , although changes in il-6 and tnf - alpha were not apparent . this effect on circulating levels of tnf - alpha receptors
, there were no alterations in il-6 , c - reactive protein ( crp ) , or tnf - alpha .
in addition , electrical muscle stimulation provided no changes in any of the aforementioned cytokines .
larsen et al . reported an 11% increase in peak vo2 following 3 months of endurance training ; tnf - alpha was significantly reduced , and this decrease was significantly correlated to the increase in peak vo2 .
adamopoulos reported a 13% increase in functional capacity with a 12-week cycle ergometry training program , which correlated with lower levels of soluble adhesion molecules .
the authors later reported a strong and highly significant correlation between improvements in peak vo2 ( 15% ) and reduction in tnf - alpha , soluble tnfr-1 and -2 , and il-6 .
plasma tnf - alpha is also documented to decrease after twice daily 6-minute walk tests in nyha ii / iii heart failure patients .
a recently published study reported absent von willebrand factor ( vwf ) release upon exercise testing in heart failure patients ; this normalised following 6 months of exercise training ; other plasma endothelial markers were unaltered .
changes in skeletal muscle , but not systemic expression of tnf - alpha , il-1-beta , and il-6 have been reported in heart failure patients undertaking a regimen of 10 minutes cycling , 46 times daily for 6 months .
this exercise program resulted in large changes in functional capacity ( 29% ) , nearly twice the mean expected increment ( 17% ) shown from our review of 81 heart failure exercise training studies .
this study suggested the existence of a cytokine cascade where levels may be changed at altered rates in different tissues .
as heart failure exercise training studies are often small , we sought by pooling data from four studies to establish whether exercise training programs were able to modulate systemic cytokine levels .
a second aim was to establish if differences in ext regimens are related to the degree of change in cytokines and peak vo2 .
we searched pubmed and medline for exercise training studies in heart failure patients that had measured one or more of the proinflammatory cytokines .
the focus of this work was interleukin-6 and tnf - alpha as these cytokines were measured in 10 exercise training studies , the correspondence authors of which were contacted for their cooperation in collaboration .
authors were requested to provide individual patient data from their study ; four centres provided data ( table 2 ) .
sufficient data were not available to analyse changes in other cytokines . in 3 studies ,
plasma or serum samples were obtained by venipuncture ( arterial cannula used in larsen 's study ) and stored on ice . in all studies ,
venipuncture collections were taken between 0900 , and 1200 , at least 24 hours and not more than 5 days after the last exercise session , thus negating the effects of the intervention .
within one hour , samples were centrifuged at 4c , 15002000 rpm for 10 minutes , and then separated into aliquots and stored at between 75c and 80c .
concentrations of il-6 or tnf - alpha were measured by commercially available enzyme - linked immunosorbent assays ( elisas ) ( r&d systems minneapolis , minnesota ) in all 4 studies .
the intra- and interassay coefficients of variation were < 10% for all assays . in one study , 16 healthy
, male volunteers of approximately the same age ( 62 5 years ) served as controls although this data was not included in our analyses .
all four collaborating investigators completed baseline and posttraining metabolic exercise tests to establish functional capacity .
larsen and smart used cycle ergometers with a 15 w and 10 w per min stepped protocol , respectively ; lemaitre and ferraz used a modified bruce treadmill protocol .
two studies used supervised cycle ergometry as the primary mode of exercise training [ 21 , 22 ] , and one study used both home - based and neuromuscular stimulation of the legs .
baseline and posttraining cytokine levels , peak vo2 , left ventricular ejection fraction ( lvef ) , clinical , demographic , and pharmacological characteristics of patients are shown in table 2 .
paired student t - tests were used to analyse baseline and postintervention changes in cytokines and peak vo2 .
anova ( 2 4 ) was used to analyse differences between the four datasets .
univariate and multivariate regression analysis with change in tnf - alpha as the dependent variable were used to determine factors leading to cytokine change .
the four collaborating authors provided data on 106 patients ( 98 male , age 62 10 yrs , body weight 79 14 kg ) .
mls / kg / min ) , with moderate lv systolic dysfunction ( ef 30
seventy eight % ( 83 ) had an ischaemic cardiomyopathy ( table 2 ) . adherence data relating to training regimes were 87.2 1.9% and 85 12% and were unavailable for the other 2 studies .
regimes varied between 3 and 5 exercise sessions per week , at an intensity of 5880% of peak vo2 .
program durations were between 6 and 24 weeks , 90150 minutes per week , and total program hours varied between 15 and 54 hours ( table 3 ) . after training , peak vo2 increased by 1.4 3.4 ml / kg / min or 9% ( p < .001 ) from 16.9 4.4 to 18.4 4.5 , serum tnf - alpha decreased from a baseline value of 13 15.2 pg / ml by 1.9 8.6 pg / ml ( p = .02 ) , and il-6 increased slightly from a baseline value of 7.8 11.4 pg / ml by 0.5 5.4 pg / ml ( p = .32 ) .
none of the clinical , demographic , or pharmacologic variables were correlated with changes in circulating il-6 or tnf - alpha following training .
the correlations between change in posttraining peak vo2 and changes in tnf - alpha ( r = 0.023 , p = .82 ) and il-6 ( r = 0.12 , p = .21 ) were not significant . change in tnf - alpha was correlated with exercise session duration and anerobic threshold ( both r = 0.21 , p = .31 ) , univariate but not multivariate analysis identified that previous myocardial infarction , longer exercise session duration , and higher body mass index predicted change in tnf - alpha ( r = 0.18 , p = .001 ) .
a total exercise program duration of 54 hours appeared to be superior than 15 or 18 hours in effecting change in peak vo2 ( p < .001 ) ; however , no difference was seen for change in cytokine levels .
the longest program duration resulted in a greater increment in peak vo2 compared to 12 weeks ( p = .003 ) and 6 weeks ( p = .001 ) , while peak vo2 or 6-minute walk distance was unchanged in the ext programs of 6 and 12 weeks duration .
pooled data from four studies demonstrated that alterations in levels of the cytokines il-6 and tnf - alpha are not necessarily uniform .
increments in peak vo2 following exercise training are widely accepted ; however , they may be unrelated to changes in cytokine levels .
. one can not be sure about the variable effects of the different program parameters and exercise adherence rates ; nevertheless , the mean change in functional capacity from the four studies was 8% , suggesting that cumulatively the four exercise programs provided stimulus for a possible favourable change in cytokine expression .
interpretation of this pooled data is limited by the fact that several other centres did not supply data .
table 2 suggests that study participants showed heterogeneity for age , peak , vo2 and beta - blocker use .
moderate endurance activity in frail , elderly , but otherwise healthy persons has previously been reported to influence circulating cytokine levels . as our patients had mild to moderate heart failure ,
it is not surprising to observe that levels of systemic tnf - alpha were decreased after training , thereby initiating anti - inflammatory effects .
however , one study has suggested that il-6 produced by exercising muscle is thought to exert an anti - inflammatory effect .
these data suggest that production and removal of tnf - alpha and il-6 may be , at least partially , from independent mechanisms and may have opposing effects ( inflammatory versus anti - inflammatory ) .
recent clinical trials have not shown benefit from treatments that target tnf - alpha . a clinical trial of etanercept ( a tnf - alpha antagonist ) therapy has cast doubt on the role of cytokines in the pathogenesis of heart failure .
there are then implications for health professionals or researchers in the process of designing an exercise program for heart failure patients .
primary end points of chf exercise programs should perhaps not include lowering cytokine levels as they may represent surrogate markers of efficacy ; this may be particularly true in patients with milder degrees of chf . in this population ,
program design may be better focussed on the parameters such as program frequency ( sessions / week ) , duration ( number of weeks ) , and intensity that may have a greater effect on peak vo2 changes .
peak vo2 improvement from exercise training may be linked to attenuated levels of oxidative stress which in turn may attenuate cytokine expression .
previous work in healthy older adults and heart failure patients has shown intermittent exercise programs to be at least more effective in improving peak vo2 than a continuous regime that would produce greater cumulative oxidative stress . in our work ,
peak vo2 was not significantly changed in patients who exercised despite utilizing a reasonable volume of exercise to elicit functional capacity changes . in heart failure , the effect of inflammation , which may be due partly to inactivity , may manifest in the terminal disease phase .
the study by adamopoulos et al . may provide the best evidence to date linking change in peak vo2 and cytokines in heart failure patients .
the small cytokine change shown in our studies may be due to the fact that our patients exhibited mild to moderate heart failure symptoms .
the participants in the study of adamopoulos et al . exhibited moderate to severe symptoms .
in addition , our participants had higher left ventricular ejection fractions ( 30% versus 24% ) than those of adamopoulos et al . .
exercise training has been shown to significantly reduce the local muscle expression of tnf - alpha , il-1-beta , il-6 , and inos in the skeletal muscle of chf patients . in turn
, physical exercise has been shown to improve both basal endothelial nitric oxide ( no ) formation and agonist - mediated endothelium - dependent vasodilation of the skeletal muscle vasculature in patients with chf .
these local anti - inflammatory and systemic effects of exercise may attenuate the catabolic wasting process associated with chf progression .
in addition to an overall beneficial effect on exercise capacity , combined endurance / resistance exercise training has an anti - inflammatory effect in patients with heart disease .
these skeletal muscle and anti - inflammatory changes may explain why alterations in tnf - alpha levels are most likely to be observed in patients with moderate or severe heart failure .
however , across a heterogenic patient group , change in peak vo2 was not correlated with alterations in cytokine levels .
while greater exercise volume ( number of hours ) was superior in improving peak vo2 , no particular characteristic of ext regimes appeared superior in effecting change in serum cytokines . | background . we pooled data from four studies , to establish whether exercise training programs were able to modulate systemic cytokine levels of tumour necrosis factor - alpha ( tnf - alpha ) and interleukin-6 ( il-6 ) .
a second aim was to establish if differences in ext regimens are related to degree of change in cytokines and peak vo2 .
methods .
data from four centres relating to training protocol , exercise capacity , and cytokine measures ( tnf - alpha and il-6 ) were pooled for analysis .
results .
data for 106 chf patients were collated ( 98 men , age 62 10 yrs , wt 79 14 kg ) . patients
were moderately impaired ( peak vo2 16.9 4.4 mls / kg / min ) , with moderate lv systolic dysfunction ( ef 30
6.9% ) , 78% ( 83 ) had ischaemic cardiomyopathy .
after ext , peak vo2 increased 1.4 3.4 ml / kg / min ( p < .001 ) , serum tnf - alpha decreased 1.9 8.6 pg / ml ( p = .02 ) and il-6 was not significantly changed ( 0.5 5.4 pg / ml , p = .32 ) for the whole group .
baseline and post - training peak vo2 changes were not correlated with change in cytokine levels . conclusions .
exercise training reduces levels tnf - alpha but not il-6 in chf .
however , across a heterogenic patient group , change in peak vo2 was not correlated with alterations in cytokine levels .
while greater exercise volume ( hours ) was superior in improving peak vo2 , no particular characteristic of ext regimes appeared superior in effecting change in serum cytokines . |
understanding the cellular and molecular basis of nociception is a challenge that has important implications for pain management and the development of new analgesics .
nociception plays a vital role in the survival of the organism , serving to transmit warning signals of impending harm.13 however , beyond this function , the experience of prolonged nociceptive and inflammatory pain , such as following surgery or other trauma , requires effective management , both to provide comfort for the patient and to reduce the possible risk of development of chronic pain.4,5 as somatosensory neurons of the peripheral nervous system , nociceptors innervate the periphery and function to detect noxious mechanical , temperature , and chemical stimuli . via communication with the central nervous system ,
nociceptor activity functions to evoke pain sensation at the site of injury , and nociceptor sensitivity ( ie , firing threshold ) to stimuli at the site can be modulated by soluble factors released by local nonneuronal cells , eg , keratinocytes , fibroblasts , and infiltrating immune cells.13 certain factors , such as nerve growth factor alpha , prostaglandins , eg , prostaglandin e2 , and proinflammatory cytokines , eg , tumor necrosis factor - alpha ( tnf- ) and interleukin ( il)-1 , can promote nociceptor hypersensitivity,1,3,6 whereas other factors , such as anti - inflammatory cytokines , eg , il-10 and il-4 , and endogenous opioids , can have the opposite effect on nociceptor firing , thereby acting as analgesics.3,6 new analgesic pharmaceuticals are actively being pursued .
many of these drugs fall into the class of nonsteroidal anti - inflammatory drugs , which primarily target cyclooxygenases ( ie , cox-1 and cox-2),7 which are enzymes involved in prostaglandin synthesis .
opioids are still the most widely used drugs for treatment of severe and pathological pain . however , important side effects associated with these pharmacological options continue to be a major drawback.8,9 this has spurred an interest in multimodal therapy as a way of facilitating decreased intake of any one type of analgesic.10 to optimize such an approach , novel adjunctive therapy options with limited side effects are needed .
recently , biophysical treatment modalities have been shown to be useful as an adjunctive analgesic therapy option .
one such modality , pulsed radiofrequency energy ( prfe ) , is an adjunctive therapy which involves local noninvasive delivery of prfe ( 27.12 mhz carrier frequency ) to superficial soft tissue , a treatment which has been reported to provide analgesic benefit in patients following surgery or other soft tissue trauma , with few reports of side effects.11 three recent , randomized , double - blind clinical trials that used prfe field treatment for pain in post - surgical patients found significantly reduced levels of reported pain as well as analgesic consumption in patients treated with prfe relative to control patients treated with a sham device.1214 a meta - analysis of studies assessing the clinical efficacy of prfe also found that a significant proportion of studies that assessed prfe use for pain reported a beneficial outcome ( 11 positive studies).15 while clinical results are promising , little is known regarding the underlying mechanism of action of prfe - mediated analgesia . in previous studies using cultured human keratinocytes and fibroblasts , prfe treatment was found to mediate rapid and widespread upregulation of endogenous transcripts of multiple factors that , in vivo , are involved in cutaneous wound healing.16,17 in the current study , we used the same system to examine whether prfe treatment has an effect on the expression of factors known to modulate nociception , including endogenous targets of other analgesic therapeutics .
the results suggest that , unlike nonsteroidal anti - inflammatory drugs , prfe does not inhibit cyclooxygenase enzymes , but instead has a positive effect on the expression of endogenous opioids as well as their secretion , possibly via an endothelin pathway - based mechanism .
culture medium ( minimal essential medium ) was purchased from cell applications inc ( san diego , ca ) and from mediatech inc ( herndon , va ) for general culture of human dermal fibroblasts ( hdf ) .
fetal calf serum , penicillin - streptomycin , trypsin , 1 phosphate- buffered solution , sodium pyruvate , and nonessential amino acids were purchased from hyclone ( logan , ut ) .
reagents for reverse transcription polymerase chain reaction ( rt - pcr ) were from life technologies ( carlsbad , ca ) .
oligonucleotide primers for pcr were purchased/ synthesized by real time primers ( elkins park , pa ) .
general chemicals were purchased from sigma- aldrich ( st louis , mo ) or vwr international ( radnor , pa ) .
endothelin a and b receptor antagonists were purchased from american peptide ( sunnyvale , ca ) .
routine culture was performed as recommended by the manufacturer in a 5% co2 humidified incubator at 37c.18 for the experiments , cells were cultured on 10 cm plates at a density of 1.14 10 cells / cm in minimum essential medium supplemented with 1 mm sodium pyruvate , 1 mm nonessential amino acids , 100 units penicillin , 100 g streptomycin , and 5% fetal calf serum .
cells were used for experimentation after 16 hours in a humidified incubator at 37c with 5% co2 .
routine culture was performed as recommended by the manufacturer in a 5% co2 humidified incubator at 37c . for experiments
, keratinocytes were cultured on 10 cm plates at a density of 0.75 10 cells / cm in keratinocyte growth medium supplied by the manufacturer .
hek were used for experimentation after 16 hours in a humidified incubator at 37c with 5% co2 .
treatment was performed by exposing cells to the prfe field of a provant device from regenesis biomedical inc ( scottsdale , az ) .
this device emits a 27.12 mhz radiofrequency signal transmitted from a flat spiral antenna with a radius of 7.5 cm made up of six turns at a width of 0.70 cm spaced with a separation of 0.3 cm .
this corresponds to energy parameters of 591 v / m and 6.7 a / m in electrical and magnetic fields , respectively , when measured 5 cm from the surface of the radiating antenna .
treatment was performed at room temperature for 30 minutes after which the cells were returned to the incubator .
cells were harvested for protein or total rna at the times indicated in the figures .
cells were treated with prfe for 30 minutes , and total rna was isolated using an rneasy mini kit ( qiagen , valencia , ca ) isolation kit .
total rna 1 g was reverse - transcribed using a high - capacity rna to cdna master mix ( life technologies ) following the manufacturer s instructions .
pcr was performed using the following reaction times and temperatures : denaturation for 15 seconds at 94c , annealing for 30 seconds at 55c , and elongation for 30 seconds at 72c in 20 l reaction volumes , and run out to 45 cycles using a rotor - gene device ( qiagen ) .
quantitation and analysis of relative gene expression was performed using the 2 method described by livak and schmittgen.19 glyceraldehyde phosphate dehydrogenase was used to normalize product expression between different rna samples and treatments .
rna was isolated from at least four independent experiments and pcr was performed in triplicate .
products were electrophoresed on 2% agarose gel and stained with ethidium bromide to determine the fidelity of the pcr reaction .
the relationship between the concentration of input rna and amount of pcr product was linear for each pcr product .
we used an enzyme - linked immunosorbent assay to measure cytokine and opioid levels in cell supernatants after treatment with prfe . for cytokine and opioid measurement ,
cells were plated at 5.0 10 per well in 24-well dishes ( bd biosciences , franklin lakes , nj ) .
the cells were incubated for 24 hours , after which 400 l of fresh medium was added .
cells were then treated with prfe , and the media and cell lysate were harvested at the times indicated in the figures and then analyzed for opioid , substance p ( phoenix pharmaceuticals , burlingame , ca ) , or endothelin-1 expression .
b- endorphin , substance p , dynorphin , and enkephalin ( wuhan eiaab science co , ltd , wuhan , china ) were analyzed by enzyme - linked immunosorbent assay .
assays were based on competition with a known concentration of a specific opioid or substance p standard .
cells were inoculated at a density of 5 10 cells per plate and grown for 24 hours .
prfe treatment was performed using standard conditions and cells were harvested at the indicated times .
cell lysates were made up in 0.1 m tris - hcl ( ph 7.4 ) containing aprotinin 3.3 g / ml , leupeptin 10 g / ml , and pepstatin 4 g / ml ( sigma - aldrich ) .
cells were lysed by freeze thawing three times followed by passage through a 26-gauge needle .
the protein concentration was then determined , and cyclooxygenase activity was assayed using a fluorescent activity assay kit ( cayman chemicals , ann arbor , mi ) by determining the conversion of 10-acetyl-3 , 7-dihydroxyphenoxazine to resorufin by the peroxidase component of cyclooxygenase .
resorufin fluorescence is then analyzed with an excitation wavelength of 530540 nm and an emission wavelength of 585595 nm using a biotek fl 800 fluorescent plate reader .
assays were performed with at least three separate protein isolations , and enzyme activity was run in triplicate .
enzyme - linked immunosorbent assay and rt - pcr data were analyzed using one - way analysis of variance . if the equal variance test failed for one - way analysis of variance , dunn s method for multiple comparisons
for all the experiments , reporting p < 0.05 was considered to be statistically significant .
culture medium ( minimal essential medium ) was purchased from cell applications inc ( san diego , ca ) and from mediatech inc ( herndon , va ) for general culture of human dermal fibroblasts ( hdf ) .
fetal calf serum , penicillin - streptomycin , trypsin , 1 phosphate- buffered solution , sodium pyruvate , and nonessential amino acids were purchased from hyclone ( logan , ut ) .
reagents for reverse transcription polymerase chain reaction ( rt - pcr ) were from life technologies ( carlsbad , ca ) .
oligonucleotide primers for pcr were purchased/ synthesized by real time primers ( elkins park , pa ) .
general chemicals were purchased from sigma- aldrich ( st louis , mo ) or vwr international ( radnor , pa ) .
endothelin a and b receptor antagonists were purchased from american peptide ( sunnyvale , ca ) .
routine culture was performed as recommended by the manufacturer in a 5% co2 humidified incubator at 37c.18 for the experiments , cells were cultured on 10 cm plates at a density of 1.14 10 cells / cm in minimum essential medium supplemented with 1 mm sodium pyruvate , 1 mm nonessential amino acids , 100 units penicillin , 100 g streptomycin , and 5% fetal calf serum .
cells were used for experimentation after 16 hours in a humidified incubator at 37c with 5% co2 .
routine culture was performed as recommended by the manufacturer in a 5% co2 humidified incubator at 37c . for experiments
, keratinocytes were cultured on 10 cm plates at a density of 0.75 10 cells / cm in keratinocyte growth medium supplied by the manufacturer .
hek were used for experimentation after 16 hours in a humidified incubator at 37c with 5% co2 .
treatment was performed by exposing cells to the prfe field of a provant device from regenesis biomedical inc ( scottsdale , az ) .
this device emits a 27.12 mhz radiofrequency signal transmitted from a flat spiral antenna with a radius of 7.5 cm made up of six turns at a width of 0.70 cm spaced with a separation of 0.3 cm .
this corresponds to energy parameters of 591 v / m and 6.7 a / m in electrical and magnetic fields , respectively , when measured 5 cm from the surface of the radiating antenna .
treatment was performed at room temperature for 30 minutes after which the cells were returned to the incubator .
cells were harvested for protein or total rna at the times indicated in the figures .
cells were treated with prfe for 30 minutes , and total rna was isolated using an rneasy mini kit ( qiagen , valencia , ca ) isolation kit .
total rna 1 g was reverse - transcribed using a high - capacity rna to cdna master mix ( life technologies ) following the manufacturer s instructions .
pcr was performed using the following reaction times and temperatures : denaturation for 15 seconds at 94c , annealing for 30 seconds at 55c , and elongation for 30 seconds at 72c in 20 l reaction volumes , and run out to 45 cycles using a rotor - gene device ( qiagen ) .
quantitation and analysis of relative gene expression was performed using the 2 method described by livak and schmittgen.19 glyceraldehyde phosphate dehydrogenase was used to normalize product expression between different rna samples and treatments .
rna was isolated from at least four independent experiments and pcr was performed in triplicate .
products were electrophoresed on 2% agarose gel and stained with ethidium bromide to determine the fidelity of the pcr reaction .
the relationship between the concentration of input rna and amount of pcr product was linear for each pcr product .
we used an enzyme - linked immunosorbent assay to measure cytokine and opioid levels in cell supernatants after treatment with prfe . for cytokine and opioid measurement ,
cells were plated at 5.0 10 per well in 24-well dishes ( bd biosciences , franklin lakes , nj ) .
the cells were incubated for 24 hours , after which 400 l of fresh medium was added .
cells were then treated with prfe , and the media and cell lysate were harvested at the times indicated in the figures and then analyzed for opioid , substance p ( phoenix pharmaceuticals , burlingame , ca ) , or endothelin-1 expression .
b- endorphin , substance p , dynorphin , and enkephalin ( wuhan eiaab science co , ltd , wuhan , china ) were analyzed by enzyme - linked immunosorbent assay .
assays were based on competition with a known concentration of a specific opioid or substance p standard .
cells were inoculated at a density of 5 10 cells per plate and grown for 24 hours .
prfe treatment was performed using standard conditions and cells were harvested at the indicated times .
cell lysates were made up in 0.1 m tris - hcl ( ph 7.4 ) containing aprotinin 3.3 g / ml , leupeptin 10 g / ml , and pepstatin 4 g / ml ( sigma - aldrich ) .
cells were lysed by freeze thawing three times followed by passage through a 26-gauge needle .
the protein concentration was then determined , and cyclooxygenase activity was assayed using a fluorescent activity assay kit ( cayman chemicals , ann arbor , mi ) by determining the conversion of 10-acetyl-3 , 7-dihydroxyphenoxazine to resorufin by the peroxidase component of cyclooxygenase .
resorufin fluorescence is then analyzed with an excitation wavelength of 530540 nm and an emission wavelength of 585595 nm using a biotek fl 800 fluorescent plate reader .
assays were performed with at least three separate protein isolations , and enzyme activity was run in triplicate .
enzyme - linked immunosorbent assay and rt - pcr data were analyzed using one - way analysis of variance . if the equal variance test failed for one - way analysis of variance , dunn s method for multiple comparisons
for all the experiments , reporting p < 0.05 was considered to be statistically significant .
prostaglandins are an important group of compounds that are closely involved in pain via the inflammation process , and inhibition of their synthesis is the target for nonsteroidal anti - inflammatory drugs .
there are many prostaglandins synthesized , all of which proceed through the rate - limiting enzyme , cyclooxygenase .
we treated hdf and hek cells in culture with prfe fields to determine the effect on cox gene expression .
hdf cells treated with prfe showed a 1.512.3 increase in inducible cox-2 gene expression and no increases in mrna expression of cox-1 ( figure 1b and c ) . in hek cells ,
no significant changes were observed in levels of either cox-1 or cox-2 gene expression ( figure 1a or c ) . because prfe field treatment had an increasing or no effect on cells in culture (
hdf and hek , respectively ) , expression of enzymes subsequently involved in the synthesis of prostaglandins was assayed .
prostaglandin e2 has been shown to be proinflammatory and involved in eliciting pain in the peripheral and central nervous systems .
prostaglandin d plays an important role in the resolution of inflammation . using rt - pcr , we determined the effects of prfe on mrna expression levels of these enzymes .
we found increases in prostaglandin e synthase levels in hdf cells ( 2.0-fold ) with no changes in prostaglandin d synthase levels . in hek
, we found no significant changes in prostaglandin e synthase expression levels and low mrna levels of prostaglandin d synthase .
we next determined the effects of prfe on protein activity to address any post - translation modification that prfe may induce in cyclooxygenase activity .
as shown in figure 2a and b , total cyclooxygenase activity changes mimic those found in changes in mrna levels of hdf and hek . peripheral expression of endogenous opioids is becoming recognized as an increasingly important analgesic mechanism in pain pathways due to tissue damage and injury , especially in skin , muscles , and viscera .
we determined the effect of prfe treatment on pro - opiomelanocortin ( pomc ) , pro - enkephalin ( penk ) , and prodynorphin ( pdyn ) gene expression in hek and hdf .
these endogenous opioids have been shown to be involved in antinociceptive pathways in peripheral tissue .
in particular , immune cells and keratinocytes have been shown to express endogenous opioid peptides , which are thought to elicit an analgesic effect during trauma to skin .
cells treated with prfe showed an increase in endogenous opioid gene expression with a maximum at 2 hours following treatment . both hek and hdf showed increases of all three opioid mrna levels as shown in figure 3a
this suggests that , at least in part , the analgesic effects of prfe fields may be through increased expression of pro - opioid mrna .
we next looked at the levels of -endorphin , enkephalin , and dynorphin peptides after prfe treatment .
both enkephalin and dynorphin peptides are increased in hek and hdf ( figure 3b and c ) .
we could not detect b - endorphin peptide , probably due to the sensitivity of the assay .
these results suggest a biological mechanism whereby treatment of injured tissue with prfe fields decreases pain associated with the trauma in a localized manner .
we have previously shown that prfe field treatment of hek and hdf increases many cytokines and other proteins involved in the inflammation phase of wound healing .
proinflammatory cytokines such as il-1 have been shown to be particularly important in the release of endogenous opioids from leukocytes .
we determined the effects of prfe field treatment on several cytokines and neurokine factors , which have been shown to be involved in pain produced by neuroinflammation and resolution of the inflammation process ( figure 4 ) .
the proinflammatory cytokine il-1 has been implicated in stimulating release of endogenous opioids from immune cells . in our studies ,
compared with control cells , prfe - treated hek and hdf expressed higher levels of il-5 , il-6 , and il-10 ( figure 4 ) .
nerve growth factor alpha mrna levels were increased in prfe - treated fibroblasts , while a lower level of nerve growth factor alpha mrna relative to control was observed following treatment of keratinocytes .
in addition , prfe - treated hek also had lower levels of tnf- mrna compared with the control .
substance p expression was not found in either of the cell types ( data not shown ) .
previous results of a microarray analysis found upregulation of several cytokine mrnas following prfe treatment;16 however , as here , these cytokines did not include those commonly implicated in stimulating endogenous opioid release from immune cells , eg , il-1 and tnf-. combined , these results indicate that prfe fields do not act through traditional cytokine mechanisms , which stimulate endogenous opioid release from immune system cells via factors such as il-1. endothelin-1 is associated with the pathogenesis of ischemia and cancer , and is a potent vasoconstrictor often associated with peripheral pain .
its pain response is associated with its binding to the endothelin a receptor ( eta ) on nociceptors .
in contrast , binding of et-1 to endothelin b ( etb ) receptors has been shown to produce localized analgesia by inducing expression of endogenous opioids in hek .
prfe treatment of hek increased expression of the etb receptor as well as mrnas for penk , pdyn , and pomc .
eta and endothelin-1 showed no significant increases in mrna levels . in hdf , prfe treatment showed increases in all three of the pro - opioid mrnas , with decreasing expression of endothelin-1 mrna ( figure 5a and b , p < 0.05 ) .
eta and etb receptors showed no statistically significant differences in expression after prfe treatment over controls in hdf .
these results suggest that prfe treatment produces peripheral analgesia by increasing endogenous opioid expression via endothelin-1 binding to its etb receptor in hek , but via multiple pathways in hdf .
khodorova et al20,21 have shown that increases in endogenous opioid expression via the endothelin-1 pathway are mediated through binding to the etb receptor .
bq-123 is a selective antagonist for eta receptors whereas bq-788 is a selective antagonist for etb receptors .
levels of endogenous opioid mrna expression were measured at 2 hours after treatment ( figure 6 ) . both antagonists inhibited prfe - induced increases in opioid expression in hek . in hdf , both bq-788 and bq-123 inhibited endogenous penk and pomc expression , but pydn expression was unaffected after prfe field treatment .
these results show that increases in opioid expression in hek are induced at least in part by an endothelin-1-dependent mechanism binding to its receptors , ie , eta and etb . in fibroblasts ,
the role for induction by endothelin-1 is less clear , with endothelin receptor antagonist blockade inhibiting pomc and penk expression but having no effect on induction of pydn after treatment with prfe .
prostaglandins are an important group of compounds that are closely involved in pain via the inflammation process , and inhibition of their synthesis is the target for nonsteroidal anti - inflammatory drugs .
there are many prostaglandins synthesized , all of which proceed through the rate - limiting enzyme , cyclooxygenase .
we treated hdf and hek cells in culture with prfe fields to determine the effect on cox gene expression .
hdf cells treated with prfe showed a 1.512.3 increase in inducible cox-2 gene expression and no increases in mrna expression of cox-1 ( figure 1b and c ) . in hek cells ,
no significant changes were observed in levels of either cox-1 or cox-2 gene expression ( figure 1a or c ) . because prfe field treatment had an increasing or no effect on cells in culture (
hdf and hek , respectively ) , expression of enzymes subsequently involved in the synthesis of prostaglandins was assayed .
prostaglandin e2 has been shown to be proinflammatory and involved in eliciting pain in the peripheral and central nervous systems .
prostaglandin d plays an important role in the resolution of inflammation . using rt - pcr , we determined the effects of prfe on mrna expression levels of these enzymes .
we found increases in prostaglandin e synthase levels in hdf cells ( 2.0-fold ) with no changes in prostaglandin d synthase levels . in hek
, we found no significant changes in prostaglandin e synthase expression levels and low mrna levels of prostaglandin d synthase .
we next determined the effects of prfe on protein activity to address any post - translation modification that prfe may induce in cyclooxygenase activity .
as shown in figure 2a and b , total cyclooxygenase activity changes mimic those found in changes in mrna levels of hdf and hek .
peripheral expression of endogenous opioids is becoming recognized as an increasingly important analgesic mechanism in pain pathways due to tissue damage and injury , especially in skin , muscles , and viscera .
we determined the effect of prfe treatment on pro - opiomelanocortin ( pomc ) , pro - enkephalin ( penk ) , and prodynorphin ( pdyn ) gene expression in hek and hdf .
these endogenous opioids have been shown to be involved in antinociceptive pathways in peripheral tissue .
in particular , immune cells and keratinocytes have been shown to express endogenous opioid peptides , which are thought to elicit an analgesic effect during trauma to skin .
cells treated with prfe showed an increase in endogenous opioid gene expression with a maximum at 2 hours following treatment . both hek and
this suggests that , at least in part , the analgesic effects of prfe fields may be through increased expression of pro - opioid mrna .
we next looked at the levels of -endorphin , enkephalin , and dynorphin peptides after prfe treatment .
both enkephalin and dynorphin peptides are increased in hek and hdf ( figure 3b and c ) .
we could not detect b - endorphin peptide , probably due to the sensitivity of the assay .
these results suggest a biological mechanism whereby treatment of injured tissue with prfe fields decreases pain associated with the trauma in a localized manner .
we have previously shown that prfe field treatment of hek and hdf increases many cytokines and other proteins involved in the inflammation phase of wound healing .
proinflammatory cytokines such as il-1 have been shown to be particularly important in the release of endogenous opioids from leukocytes .
we determined the effects of prfe field treatment on several cytokines and neurokine factors , which have been shown to be involved in pain produced by neuroinflammation and resolution of the inflammation process ( figure 4 ) .
the proinflammatory cytokine il-1 has been implicated in stimulating release of endogenous opioids from immune cells . in our studies ,
compared with control cells , prfe - treated hek and hdf expressed higher levels of il-5 , il-6 , and il-10 ( figure 4 ) .
nerve growth factor alpha mrna levels were increased in prfe - treated fibroblasts , while a lower level of nerve growth factor alpha mrna relative to control was observed following treatment of keratinocytes .
in addition , prfe - treated hek also had lower levels of tnf- mrna compared with the control .
substance p expression was not found in either of the cell types ( data not shown ) .
previous results of a microarray analysis found upregulation of several cytokine mrnas following prfe treatment;16 however , as here , these cytokines did not include those commonly implicated in stimulating endogenous opioid release from immune cells , eg , il-1 and tnf-. combined , these results indicate that prfe fields do not act through traditional cytokine mechanisms , which stimulate endogenous opioid release from immune system cells via factors such as il-1.
endothelin-1 is associated with the pathogenesis of ischemia and cancer , and is a potent vasoconstrictor often associated with peripheral pain .
its pain response is associated with its binding to the endothelin a receptor ( eta ) on nociceptors .
in contrast , binding of et-1 to endothelin b ( etb ) receptors has been shown to produce localized analgesia by inducing expression of endogenous opioids in hek .
prfe treatment of hek increased expression of the etb receptor as well as mrnas for penk , pdyn , and pomc .
eta and endothelin-1 showed no significant increases in mrna levels . in hdf , prfe treatment showed increases in all three of the pro - opioid mrnas , with decreasing expression of endothelin-1 mrna ( figure 5a and b , p < 0.05 ) .
eta and etb receptors showed no statistically significant differences in expression after prfe treatment over controls in hdf .
these results suggest that prfe treatment produces peripheral analgesia by increasing endogenous opioid expression via endothelin-1 binding to its etb receptor in hek , but via multiple pathways in hdf .
khodorova et al20,21 have shown that increases in endogenous opioid expression via the endothelin-1 pathway are mediated through binding to the etb receptor .
bq-123 is a selective antagonist for eta receptors whereas bq-788 is a selective antagonist for etb receptors .
levels of endogenous opioid mrna expression were measured at 2 hours after treatment ( figure 6 ) . both antagonists inhibited prfe - induced increases in opioid expression in hek . in hdf ,
both bq-788 and bq-123 inhibited endogenous penk and pomc expression , but pydn expression was unaffected after prfe field treatment .
these results show that increases in opioid expression in hek are induced at least in part by an endothelin-1-dependent mechanism binding to its receptors , ie , eta and etb . in fibroblasts ,
the role for induction by endothelin-1 is less clear , with endothelin receptor antagonist blockade inhibiting pomc and penk expression but having no effect on induction of pydn after treatment with prfe .
many analgesic therapies have historical derivations that are still used today , such as opioid - based medications and nonsteroidal anti - inflammatory drugs , which are currently the mainstay of treatment for postoperative pain .
although effective for many types of pain , the data suggest that adverse effects associated with postoperative pain medication are an issue of patient concern.22 a survey by apfelbaum et al found that 23% of their postoperative patients experienced side effects related to postoperative pain medication , with 72% of patients surveyed stating that , if given a choice , they would choose a non - narcotic pain medication.22 this highlights the need for novel analgesic therapies that are effective with fewer side effects . in our study , we sought to gain insight into the underlying molecular mechanism of a relatively new modality which uses pulsed electromagnetic f ields to treat pain due to postoperative or other soft tissue trauma .
unlike exogenous opioid and nonsteroidal anti - inflammatory therapeutics , little is known about the biological mechanism of action of prfe in mediating analgesia . in injured skin , factors expressed and secreted by non - neuronal cells ( ie , resident dermal and epidermal cells , such as fibroblasts and keratinocytes , as well as infiltrating immune cells ) in the proximity of nociceptive nerve termini can modulate nociceptor sensitivity , and thus can have an effect on downstream pain sensation , with either a proalgesic or analgesic effect . in the current study , we sought to gain insight into the molecular mechanism of prfe - mediated analgesia by investigating the effect of prfe field treatment on the expression of such factors by cultured fibroblasts and keratinocytes following prfe treatment .
this hypothesis was based on the previous finding that prfe mediates widespread mrna level increases for diverse endogenous factors in the same in vitro system .
unlike nonsteroidal anti - inflammatory drugs , which inhibit cyclooxygenase activity , prfe had a modestly positive effect on cyclooxygenase activity in hdf .
it also had a positive effect on mrna levels of enzymes involved in prostaglandin synthesis ( cyclooxygenase-2 , prostaglandin e synthase , figure 1 ) .
in contrast , prfe treatment of these cells led to significant increases in endogenous opioid precursor mrna ( figure 3a ) and secreted opioid peptides ( figure 3b and c ) , an effect that , in most cases , was dependent on endothelin receptors ( figure 6 ) .
in addition , prfe also had an effect on mrna levels of several cytokines and neurokines ( figure 4 ) .
it should be noted that it is not unusual that the two cell types , which in vivo are resident cells from different parts of the skin ( keratinocytes derived from the epidermal layer and fibroblasts derived from the dermis ) , were found to have different gene expression profiles for some of the mrnas examined here , considering the complexity of individual cell types .
for example , in other models , these two cell types have been found to show quantitative differences in levels of mrnas encoding oxidative stress response proteins23 and cytokines.24 although the role of immune cells in antinociception is well accepted,25 the precise role of opioid expression by keratinocytes and fibroblasts in peripheral analgesia is not well understood .
khodorova et al have previously shown that etb receptor activation by endothelin-1 stimulates -endorphin secretion by keratinocytes,20 supporting their finding that etb receptor activation mediates analgesia through a mechanism involving opioid receptors.20 in our study , using selective antagonists for endothelin receptors ( eta and etb ) , we found that the prfe - mediated increases in pyn , penk , and pomc mrna in hek were blocked by both eta and etb receptor antagonists ( figure 6 ) , suggesting that both types of endothelin receptors play a role in regulating endogenous expression of opioids at either the transcriptional or post - transcriptional level . in hdf , while endothelin receptor antagonists reduced the positive effect of prfe on levels of penk and pomc mrna , both receptor antagonists appeared to enhance the prfe - mediated increase in pydn mrna levels , suggesting that prfe can induce endogenous opioid expression through more than one pathway .
in both cell types , the prfe - mediated increase in opioid mrna occurred concomitantly with an increase in etb receptor mrna ( figure 6 ) , an effect which was also diminished by endothelin receptor antagonists .
while the effect of prfe on enzymes that synthesize prostaglandins may seem counter to what one might expect for an analgesic , recent findings by serhan et al show that prostaglandins e2 and d2 , while important for acute inflammation , upon modification ( lipid mediator class - switching ) could play an important role in the resolution of inflammation and return to homeostasis.26,27 both prostaglandins e2 and d2 are thought to act as important transcriptional regulators of enzymes involved in the generation of lipoxins and resolvins,8,28 which have been shown to play an important role in the resolution of acute inflammation and pain .
future studies to examine what effect prfe may have on the resolvin and lipoxin pathways are of interest . several cytokines and neurokines
for example , cytokines play an important role in the recruitment of immune cells to the site of trauma , and can function in stimulating these cells to release opioid peptides,29,30 and nerve growth factor alpha can bind directly to receptors on nociceptors to elicit a direct effect.1,3 using our in vitro culture system , we found that prfe can impact the endogenous expression of some of these factors as well .
il-1 mrna levels were lower in both cell types ( figure 4 ) following prfe treatment relative to control cells , a finding in agreement with clinical findings in which prfe therapy used postoperatively was associated with lower levels of il-1 in wound exudates of prfe - treated patients relative to control - treated patients as early as 1 hour after treatment was initiated , an observation which coincided with lower reported pain.14 prfe treatment of hek resulted in a lower level of nerve growth factor alpha and tumor necrosis factor alpha factor alpha mrna relative to control cells ( figure 4 ) , while there were higher levels of nerve growth factor alpha in prfe - treated hdf relative to control cells .
prfe treatment was also followed by an increase in il-5 , il-6 , and il-10 mrna in both cell types .
we found no detectible expression of substance p in either cell type . in order to get a sense of the direct effects of prfe treatment
, we evaluated the effect of prfe in our culture system at relatively early time points ( ie , hours ) after treatment .
while changes in mrna levels detected could be due either to an upregulation of transcription or greater mrna stability , an increase in peptide levels measured at 2 hours post - treatment reflects an increase in intracellular opioid precursor protein measured at 2 hours post - treatment is more likely to reflect a positive effect on translation or protein stability , indicating that prfe can have an effect on multiple steps involved in regulating the opioid pathway . because the current study was done out of interest to understand the molecular mechanism behind how prfe mediates analgesia in the clinical setting , it is worthwhile to compare the timing of prfe - mediated effects observed here relative to findings reported in vivo . in randomized ,
controlled postoperative studies , a clinically observable benefit ( eg , reduced pain scores and/or reduced analgesic consumption ) has been reported as early as 1 hour after initiation of prfe treatment.14 in other postoperative pain studies , a statistically significant difference in pain scores reported for prfe - treated versus sham device - treated control subjects occurred as early as 13 days postoperatively , depending on the study,12,13 and persisted through to the end of each study ( both studies followed patients through postoperative day 7 ) .
a rapid response to treatment , as reported by rohde et al,14 implies a mechanism that can , in a short amount of time , act on local nociceptors to promote analgesia ( such as promoting increased opioid levels ) .
later onset of analgesia in other studies , as well as long - lasting analgesic effects , could also involve a mechanism that requires more time before an effect on nociception occurs , such as changes in transcription .
in addition to reduced pain scores , each of these studies also reported lower narcotic pill consumption in prfe - treated patients following surgery relative to sham device - treated control subjects .
we propose a working model in which prfe treatment leads to an increase in both opioid precursor expression and opioid peptide release by keratinocytes and fibroblasts , an effect , which , in vivo , could mediate local analgesia .
upregulation of opioid precursor mrna in keratinocytes appears to be dependent on activation of endothelin receptors ( eta and etb ) , whereas an additional pathway also appears to be necessary for prfe - mediated opioid precursor expression in fibroblasts .
in addition , prfe modulation of cytokine expression in these cells may lead to an altered cytokine environment at the local site of injury , which could also affect analgesia . based on the finding that mrna levels of enzymes involved in prostaglandin synthesis are upregulated in fibroblasts following prfe treatment
, it needs to be investigated further whether prfe - mediated analgesia might also involve lipoxins and/or resolvins .
treatment of hdf and hek in culture with prfe led to changes in the expression of several endogenous factors , which , in vivo , may participate in modulating peripheral nociception .
notably , prfe treatment led to an increase in endogenous opioid expression and secretion in both cell types tested .
modulation of the endogenous opioid system in different in vivo models has been reported previously following exposure to certain types of extremely low frequency magnetic fields,31,32 as well as following electrical stimulation , such as electroacupuncture33,34 and transcutaneous electrical nerve stimulation.35 to our knowledge , this is the first report showing a positive effect of prfe on an endogenous opioid pathway .
these findings may provide a new insight into the molecular mechanism underlying prfe - mediated analgesia reported in the clinical setting . | backgroundpulsed radiofrequency energy ( prfe ) fields are being used increasingly for the treatment of pain arising from dermal trauma . however , despite their increased use , little is known about the biological and molecular mechanism(s ) responsible for prfe - mediated analgesia . in general , current therapeutics used for analgesia target either endogenous factors involved in inflammation , or act on endogenous opioid pathways.methods and resultsusing cultured human dermal fibroblasts ( hdf ) and human epidermal keratinocytes ( hek ) , we investigated the effect of prfe treatment on factors , which are involved in modulating peripheral analgesia in vivo .
we found that prfe treatment did not inhibit cyclooxygenase enzyme activity , but instead had a positive effect on levels of endogenous opioid precursor mrna ( proenkephalin , pro - opiomelanocortin , prodynorphin ) and corresponding opioid peptide . in hek cells , increases in opioid mrna
were dependent , at least in part , on endothelin-1 . in hdf cells
, additional pathways also appear to be involved .
prfe treatment was also followed by changes in endogenous expression of several cytokines , including increased levels of interleukin-10 mrna and decreased levels of interleukin-1 mrna in both cell types.conclusionthese findings provide a new insight into the molecular mechanism underlying prfe - mediated analgesia reported in the clinical setting . |
kimura 's disease is a rare benign haematological disorder , involving subcutaneous tissue , predominantly in the head and neck region and frequently associated with painless lymphadenopathies .
the disease is endemic in asia , but rare and sporadic in the rest of the world .
it can be associated with eosinophilia and increased total serum immunoglobulin e ( ige ) concentration .
renal manifestations include membranous glomerulopathy , mesangioproliferative glomerulonephritis , minimal change disease and focal segmental glomerulosclerosis .
the aetiology is unknown , but several reports suggest a t - cell disorder or an immunoallergic reaction .
we report the case of a young caucasian man presenting with the nephrotic syndrome , kimura 's disease , hyper ige and immune thrombocytopenia with signs of humoral autoimmunity .
a 29-year - old caucasian man was referred for nephrotic syndrome . in the last 2 months , he had received treatment with amoxicilline clavulanate and prednisone ( 1 mg / kg / day during 5 days ) for common acquired pneumonia .
cmv , parvovirus b19 , htlv1 serologies were negative , and ebv tests showed past infection , without replication .
antinuclear antibodies were positive ( 1/500 ) . anti - dna , anti - ect ,
bone - marrow aspirate showed a slight hypereosinophilia ( 3% ) and the presence of megakaryocytic lineage , which confirmed peripheral immune thrombocytopenia ( itp ) .
total plasmatic ige concentration was dramatically increased ( 3900 ui / ml , n < 20 ) .
a 18-fluoro - deoxy - glucose positron emission tomography ( fdg - pet ) was negative .
histologic analysis and immunohistochemistry ( cd3 , cd5 , cd10 , cd15 , cd30 , cd20 , ema , ebv - lmp , bcl2 ) excluded tumoral proliferation .
we found a preserved nodal architecture with hyperplastic follicules ( figure not shown ) , many eosinophilic infiltrates and vascular hyperplasia consistent with kimura disease ( figure 1 ) .
no clonal t - cell rearrangement was found , neither in the peripheral blood lymphocytes nor in the lymph node .
lymph node : paracortical zone showing interfollicular infiltrates with many eosinophils ( thin arrow ) mixed with small lymphocytes .
the renal biopsy specimen contained 18 glomeruli , and revealed segmental glomerular sclerosis in two of them ( figure 2 ) .
there was no mesangial proliferation , nor evidence of capillary basement membrane thickening or membranous spikes .
a diagnosis of kimura 's disease associated with focal segmental glomerulosclerosis ( fsgs ) and immune thrombocytopenia was retained .
the patient was treated with oral corticosteroids ( prednisone , 1 mg / kg / day ) , associated with an ace inhibitor ( ramipril ) .
after 1 month of treatment , while the nephrotic syndrome was in remission , antinuclear antibodies increased ( 1/1600 ) , and anti - dna antibodies appeared ( igg : 175
complement component c4 decreased to 0.10 g / l , whereas c3 remained in the normal range ( 0.85
the nephrotic syndrome relapsed twice , 12 and 18 months after the onset of steroid therapy when he was still receiving 20 mg prednisone per day .
so far , the patient has no sign of malignant lymphoproliferative disorder , and no systemic autoimmune disease .
kimura 's disease is a rare haematological disorder of unknown origin , occurring in young men of asian origin .
pathological features include preserved lymph node architecture , proliferative germinal centres , eosinophilic infiltration and postcapillary venule proliferation , which all were present in our case .
although peripheral eosinophilia was absent , most likely because of recent steroid treatment , our patient still had a slight medullar eosinophilia ( 3% ) .
kimura 's disease should not be confused with angiolymphoid hyperplasia with eosinophilia ( alhe ) .
this currently presents as a head or neck soft tissue swelling , with eosinophilic infiltrates and vascular proliferation .
other differential diagnosis include angioimmunoblastic t - cell lymphoma , castleman 's disease , parasitic lymphadenitis and drug - related hypersensitivity lymphadenopathy .
a total of 1216% of the patients present with proteinuria , and 5978% of them have the nephrotic syndrome .
the skin or lymph node lesions may precede the renal manifestations , or occur simultaneously or months later , but they are usually close in time , suggesting a pathogenic link between kimura 's disease and the renal manifestations .
an immunohistochemical study performed on 21 mass lesions failed to disclose any infectious agent and clonal proliferation .
no clonal rearrangement of t - cell receptor and ig heavy chain genes was found .
eosinophilia and high ige levels point to a th2 cell disturbance with hypersensitivity - type reaction .
. found high il-5 levels and il-5/ifn- ratio , while katagiri et al .
showed increased il-4 , il-5 and il-13 mrna expression in mononuclear cells , suggesting a role of th2 cytokines in the pathogenesis of kimura 's disease . in minimal change disease ( mcd )
also , there seems to be a th2 polarization that suggests that in kimura 's disease - related mcd and fsgs , the th2 imbalance may play an important role .
although kimura 's disease is not primarily a b - cell disorder , it is conceivable that the th2 imbalance may be associated with the production of autoantibodies in some patients .
thus , efficacy of rituximab in some case of recurrent fsgs may point to a role of b - cell disturbance in the pathogenesis of the nephrotic syndrome .
treatment of kimura 's disease consists in surgical resection of the lymph node , but recurrence may occur over time .
additional medical therapies , such as radiation or corticosteroids , have been used with good clinical response , but relapses are common when steroids are withdrawn . in summary , we present here the first case of kimura 's disease with fsgs , immune thrombocytopenia and signs of b - cell autoreactivity in a non - asian adult man . | we report the case of a young caucasian man presenting with diffuse oedema and nephrotic syndrome .
clinical examination revealed multiple lymphadenopathies .
histological examination was consistent with the diagnosis of kimura 's disease .
a renal biopsy showed focal segmental glomerulosclerosis .
immune thrombocytopenia and signs of humoral autoimmunity were discovered .
corticosteroid treatment induced remission of nephrotic syndrome but relapses occurred 12 and 18 months after onset of treatment while the patient was receiving 20 mg prednisone once a day . to our knowledge , this is the first case of kimura 's disease and nephrotic syndrome associated with b - cell autoreactivity . |
an increasing number of proteins with unknown function have their 3d structure solved at high resolution ( 1 ) .
this situation , largely due to structural genomics initiatives ( 2 ) , has been stimulating the development of automated structure - based function prediction methods ( 3 ) .
knowledge of residues important for function and more particularly for binding , can help automated prediction of function in different ways .
the properties of a binding site such as its shape , atomic group or amino acid composition can provide clues on the ligand that may bind to it . also , having information on functionally important regions in similar proteins can refine the process of annotation transfer between homologues ( 4 ) .
several methods for predicting the location of binding sites in protein structures are available and many more are currently being developed ( 57 ) .
most of these methods need examples of known binding sites in order to derive features , which can subsequently be used to distinguish between true binding sites and sites not involved in binding .
the adequate use of such methods requires the availability of valid benchmark datasets of known binding sites that can be used both to derive the appropriate combination of features and to test the prediction performance .
but none combine enough of the necessary attributes , namely to ( i ) be representative of the known structural data , ( ii ) be non - redundant , ( iii ) combine information on the apo- and holo - protein structures , ( iv ) consist only of biologically relevant binding sites , ( v ) take the description of the biological unit of the protein into account , ( vi ) consider data from all available holo - structures for each protein and lastly ( vii ) be updated automatically . among the available datasets gold
is a redundant set of binding sites manually checked for structural errors in the pdb files ( 9 ) .
sitesbase is a database of small ligand - binding sites specifically designed to enable structural comparison of known ligand - binding sites ( 10 ) .
the sc - pdb is a collection of binding sites specifically selected for their pharmacological interest ( 11 ) .
the pdbsite database attempts to annotate functional sites derived from pdb site records and contact residues in hetero - complexes ( 12 ) .
more recently , firedb ( 13 ) has been set - up to integrate data on binding sites from the pdb and catalytic residues from the csa ( 14 ) , and organizes these data so as to allow users to assess binding site similarity in homologous proteins .
to date , the only dataset of ligand - binding sites explicitly restricted to proteins with known apo - structures is the lig dataset of 154 unique protein sequences that was used by najmanovich et al .
to analyse side - chain flexibility upon ligand - binding however , the lig dataset has to our knowledge not been maintained or updated , and biologically irrelevant molecules are not consistently excluded from it ( with the exception of sulphate and phosphate ions ) . here
, we present ligasite ( ligand attachment site ) , a dataset of ligand - binding sites in protein 3d structures , which combines all of the seven attributes listed above . in particular , it contains the description of biologically relevant binding sites in proteins with at least one apo- and one holo - structure .
information on the apo - structure is key , since due to ligand - induced conformational changes , the structure of a protein without and with the ligand ( apo- and holo - structure , respectively ) can differ ( 16,17 ) . to mimic cases , where binding sites are truly unknown , binding site prediction methods should be validated by performing the predictions on apo - structures , and comparing them with known binding sites defined from all corresponding holo - structures .
another common problem when automatically defining binding sites from pdb holo - structures , is that some small molecules , which appear in these entries may have been introduced as part of the purification and or crystallization procedure and hence bind to the protein in a non - specific manner .
although the corresponding binding sites are usually not biologically relevant , some of these compounds may act as inhibitors or substrate analogues and hence bind to biologically relevant sites in the protein . with the aim of identifying all biologically relevant binding sites , we define them as sites bound by the biologically active ligands identified in vivo or in vitro , as well as by any compound that may act as inhibitor or substrate analogue .
it is admittedly not straightforward to distinguish between such relevant ligands and non - specific binders .
we however show that protocols developed in ligasite enable to automatically filter out non - specific binders with a high degree of accuracy as determined by manual validation ( see content and methods section ) . in all cases
, these protocols take into account the quaternary structures suggested by the pqs server ( 18 ) . using the correct quaternary structures has an impact on the definition of binding sites , as these can be located at the interface between protein subunits .
optimal coverage and adequate representation of the structural data available in the pdb at any given time is enabled through fully automated procedures , and no post - processing by the user is required .
ligasite is freely accessible at http://www.scmbb.ulb.ac.be/users/benoit/ligasite , and the data therein can be downloaded as a set of namespace - qualified schema - validated xml files .
the automated procedure used to generate the ligasite dataset from the pdb is summarized in the flowchart of figure 1 .
binding sites in the ligasite dataset are exclusively defined on the basis of structural data from the pdb ( 8) . in constructing the dataset , the first step involves selecting all structures with at least one protein chain , using the pdb search engine . as a starting point , ligands are defined as all non - water molecules that appear in the hetatm records of pdb entries .
such ligands are commonly referred to as het - groups. binding sites for nucleic acids and other proteins are hence ignored at this point .
quaternary structures suggested by pqs ( 18 ) are used for all structures in the dataset , unless pqs can not be applied ( e.g. nmr structures ) .
there are 27 913 pdb entries with at least one protein chain and at least one het - group in the pdb release of july 30 , 2007 , on which the current version of ligasite is based .
the total number of het - groups in these pdb entries amounts to 197 860 .
biologically relevant binding sites are defined as those bound by the biologically active ligands identified in vivo or in vitro , as well as by any compound that may act as inhibitor or substrate analogue .
biologically irrelevant sites are defined as sites bound non - specifically by compounds introduced by the purification or crystallization procedure . in order to select biologically relevant sites ,
first , het - groups are clustered together into het - clusters when any of their respective heavy atoms are located within < 4.0 from one another .
this clustering is performed to account for the fact that small solvent molecules often cluster together in biologically relevant binding sites and mimic parts of the natural ligand ( e.g. phosphate or sulphate ions , which mimic the phosphate groups of nucleotides ) .
the total of 197 860 het - groups in the pdb sums up to 148 879 het - clusters .
second , het - clusters with less than 10 heavy atoms are rejected on the basis that biologically irrelevant molecules in pdb files are generally very small . since
het - clusters are considered rather than single het - groups , het - groups with less than 10 heavy atoms , such as phosphate ions , can remain in the selected subset provided they are part of a het - cluster with a total of at least 10 heavy atoms ( e.g. two phosphate ions close to one another are kept ) .
finally , biologically relevant het - clusters are selected based on the number of inter - atomic protein ligand contacts . the underlying assumption being , that in general , biologically relevant ligands interact specifically with the proteins and should therefore make more inter - atomic contacts with protein atoms than their irrelevant counterparts .
we use the program lpc ( 19 ) to compute the inter - atomic contacts for the following reasons : it is not restricted to a specific subset of bonds ( e.g. hydrogen bonds ) ; it provides a detailed description of contacts based on the physico - chemical properties of contacting atoms and it is freely accessible and easy to run on a large number of structures . to perform the selection based on the above assumption
, we define a threshold number of inter - atomic contacts above , which an interaction is guaranteed to be relevant . to that end
, the number of inter - atomic contacts between het - cluster and protein is calculated for all het - clusters of the dataset .
the dataset is then divided into subsets based on ranges of 50 inter - atomic contacts , as indicated in table 1 . for each range , 1% of the het - cluster protein binding sites
have been manually inspected to assess whether the binding site is biologically relevant ( this manual inspection was completed for an initial version of ligasite , which was based on the december 22 , 2006 pdb release ) .
the 4th column in table 1 shows the fractions of binding sites that we manually annotated as biologically relevant . as expected , this fraction increases as the number of inter - atomic contacts increases .
since the fraction of biologically relevant binding sites steeply increases between the ranges of 150 and 51100 contacts , the subset of binding sites having between 30 and 80 contacts was subdivided into smaller bins of 10 inter - atomic contacts each , and re - analysed .
results ( table 1 ) show that above 70 inter - atomic contacts between het - cluster and protein , the het - cluster is biologically relevant in at least 95% of cases .
the small number of cases where we manually annotated the binding site as biologically irrelevant even though it consists of at least 70 inter - atomic contacts between protein and het - cluster , all correspond to membrane proteins in complex with membrane lipids , which we were therefore able to readily exclude on the basis of the ligand and/or protein type .
table 1.fractions of biologically relevant binding sites as a function of the number of inter - atomic contacts between het - cluster and protein , for steps of 50 inter - atomic contacts and steps of 10 inter - atomic contactsno . of contactsno . of sitesno . of inspectedfrelevantsteps of 50 inter - atomic contacts1506825680.5451100153261530.931011509786980.941512006531650.98>2003932390.98steps of 10 inter - atomic contacts31402145210.1041502793280.3251603287330.6461703129310.7771803293330.97the manual analysis
summarised in this table was conducted using an initial version of ligasite , which was based on the pdb release of december 22 , 2006.range of number of inter - atomic contacts between protein and het-cluster.total number of binding sites where number of inter - atomic contacts between protein and het - cluster is within range given in column 1.number of binding sites inspected manually from the literature , used for assessing biological relevance of het - cluster .
this number equals 1% of the number of sites in column 2.fraction of manually inspected binding sites that we annotated as biologically relevant .
fractions of biologically relevant binding sites as a function of the number of inter - atomic contacts between het - cluster and protein , for steps of 50 inter - atomic contacts and steps of 10 inter - atomic contacts the manual analysis summarised in this table was conducted using an initial version of ligasite , which was based on the pdb release of december 22 , 2006 .
total number of binding sites where number of inter - atomic contacts between protein and het - cluster is within range given in column 1 .
number of binding sites inspected manually from the literature , used for assessing biological relevance of het - cluster .
the resulting dataset is thus limited to binding sites for het - clusters that consist of at least 10 heavy atoms , and that make at least 70 inter - atomic contacts with protein atoms .
there are 32 656 such het - clusters , consisting of 63 105 individual het - groups and distributed among 14 459 pdb entries .
a gold - standard dataset of binding sites should include information on both the apo- and holo - forms of the protein , to enable taking into account the structural changes that can occur in a protein upon binding of its ligand(s ) . in order to derive the list of
true apo - structures in the pdb , we applied the following filtering procedure . from the pdb release of july 30 , 2007 , we selected all pdb entries with at least one protein chain that is not in complex with any other molecule , the latter being a small molecule , another protein or a nucleic acid chain .
this procedure resulted in a set of 10 046 pdb entries , which was then filtered to remove c-only and non - x - ray entries and x - ray entries with resolution better or equal to 2.4 , and r - value better or equal to 0.25 .
these quality filters were imposed to ensure that the apo - structures , which should be used to apply functional site prediction methods , are of sufficient quality to serve as input for any type of methods including those that are based on energy calculations ( 20 ) .
these apo - structures are then paired with the corresponding holo - structures containing the biologically relevant binding sites when they share 100% sequence identity , thus resulting in a set of 550 entries with one apo - structure and at least one holo - structure with a biologically relevant binding site . removing redundancy at 25% sequence identity [ using pisces ( 21 ) ] results in a non - redundant set of 286 proteins .
when several holo - structures are available for a given protein , all are used to define its binding site .
many holo - structures are bound to only a portion of the natural ligands , and the picture of the binding site that is obtained when considering all available holo - structures is therefore more complete and accurate . out of the 550 proteins in the redundant dataset , 291 have more than one holo - structure .
a frequency score is assigned to all binding site residues in a protein , based on the fraction of corresponding holo - structures in which the residue is observed to be part of a biologically relevant binding site ( figure 2 ) .
( a ) mapping of binding site residues on the apo - structure ( pdb entry 135l ) .
these residues are identified as part of the binding site from the three holo - structures in which the lysozyme is in complex with different ligands : ( b ) with three molecules of n - acetyl - d - glucosamine ( nag ) and a sulphate ion in pdb entry 1jef ; ( c ) with di(n - acetyl - d - glucosamine ) in pdb entry 1ljn ; and ( d ) with two molecules of nag in pdb entry 1lzy .
binding site residues are coloured red if they are in contact with ligand atoms in all three holo - structures ( i.e. frequency score of 1.0 = 3/3 ) , they are coloured orange if they are only in contact with ligand atoms in two of the holo - structures ( i.e. frequency score of 0.67 2/3 ) , and they are coloured yellow if they are in contact with ligand atoms in only one holo - structure ( i.e. frequency score of 0.33 1/3 ) .
het - groups considered as biologically relevant in ligasite are displayed and coloured in cpk .
sulphate ions filtered out as biologically irrelevant in pdb entry 1ljn are transparent and displayed in balls - and - sticks .
the figure was drawn with molscript ( 27 ) and rendered with raster3d ( 28 ) .
( a ) mapping of binding site residues on the apo - structure ( pdb entry 135l ) .
these residues are identified as part of the binding site from the three holo - structures in which the lysozyme is in complex with different ligands : ( b ) with three molecules of n - acetyl - d - glucosamine ( nag ) and a sulphate ion in pdb entry 1jef ; ( c ) with di(n - acetyl - d - glucosamine ) in pdb entry 1ljn ; and ( d ) with two molecules of nag in pdb entry 1lzy .
binding site residues are coloured red if they are in contact with ligand atoms in all three holo - structures ( i.e. frequency score of 1.0 = 3/3 ) , they are coloured orange if they are only in contact with ligand atoms in two of the holo - structures ( i.e. frequency score of 0.67 2/3 ) , and they are coloured yellow if they are in contact with ligand atoms in only one holo - structure ( i.e. frequency score of 0.33 1/3 ) .
het - groups considered as biologically relevant in ligasite are displayed and coloured in cpk .
sulphate ions filtered out as biologically irrelevant in pdb entry 1ljn are transparent and displayed in balls - and - sticks .
the figure was drawn with molscript ( 27 ) and rendered with raster3d ( 28 ) .
the fact that the construction of the dataset only relies on simple numerical cut - offs and automatic filters allows it to be automatically updated as new data become available in the pdb . at the time of writing the manuscript , the current release of ligasite
both the redundant and the non - redundant versions of the dataset can be browsed . for each protein ,
a front - page describes the apo - structure , and lists all binding site residues defined from the ensemble of corresponding holo - structures . binding site residue positions ( i.e. pdb serial numbers ) are coloured on a yellow - to - red scale , depending on the fraction of holo - structures in which the residue is found in contact with a biologically relevant het - cluster ( red when the fraction equals 1 ) .
pdb identifiers of corresponding holo - structures are listed together with unique identifiers describing the biologically relevant het - clusters used to define the binding site residues , the number of heavy atoms in the het - clusters and the number of inter - atomic contacts ( identified with lpc ) between each het - cluster and protein atoms . a pdb file providing coordinates of the binding site residues in the apo - structure
clicking on the details links associated with holo - structures leads to a holo - structure specific page , on which more details can be found for each structure .
residues identified as part of binding sites in the given holo - structure are listed , together with the unique identifier of the het - group with which each residue interacts .
residues interacting with het - groups in other holo - structures of the same protein are not listed .
further data on the het - groups that bind in biologically relevant binding sites is also available from these pages , i.e. pdb het identifiers , molecule name , chemical formula and ( non - stereo ) smiles string .
a pdb file providing coordinates of the binding site residues and of the het - groups labelled as biologically relevant is available for download for each holo - structure in the dataset .
cross - links to relevant databases [ e.g. pdbsum ( 22 ) , csa ( 14 ) ] are available on all pages , as well as links to ligplot ( 23 ) drawings of the interactions between het - groups and proteins .
a search facility is available and allows users to search ligasite for pdb identifiers ( both apo- and holo- ) , het - group identifiers ( e.g. atp ) or protein names .
the ligasite dataset can also be searched by sequence similarity using blast ( 24 ) .
all matches to a query sequence are returned to the user and are sorted by sequence identity and e - value .
finally , ligasite can be searched for proteins that have a domain in the same cath superfamily ( i.e. h level ) as the domains in the pdb entry input by the user ( 25 ) .
xml schema allows to define semantic constraints for the data contained in the xml files .
an xml file is not only humanly - readable , but can also be easily parsed , and consequently validated and transformed , by a computer .
an xml schema ( ligasiteml ) has been designed for a complete description of binding site information on the proteins of the dataset .
an xml file describing the complete ligasite database is generated for each update , and validated against the above - mentioned xml schema .
both the schema and xml files are available for download from the website , allowing users to easily use ligasite data locally . in addition , protein - specific xml files , validated against the same xml schema , and describing only binding site residues in a particular protein of the dataset are available for download from the pages describing the apo- and holo - structures of the corresponding protein . a comma - separated file containing the core data of ligasite , and created automatically from the xml file , is also available for download .
the het - groups featured in the biologically relevant binding sites of ligasite proteins are very diverse . in total ,
551 different het - groups appear in the holo - structures of the non - redundant ligasite dataset ( redundancy removed at 25% sequence identity ) .
this number exceeds the number of binding sites in the dataset because ( i ) several different het - groups can appear together in a binding site in a single holo - structure , e.g. a magnesium ion ( het i d
adp ) in the holo - structure 1pfk of phosphofructokinase ; and ( ii ) different het - groups can appear in a given binding site in different holo - structures of a protein ( figure 2 ) .
the majority of het - groups appear in the binding site of only one protein , suggesting an important diversity of ligands in the dataset .
table 2 gives the names , het ids and numbers of occurrences of the het - groups that appear most frequently in binding sites of the non - redundant version of ligasite .
when a het - group appears in several holo - structures of a given protein , only one occurrence is counted .
therefore , the number of occurrences in this table corresponds to the number of different proteins to which a given het - group binds .
the most common het - group is the magnesium ion , which occurs in the binding sites of 41 different proteins , as part of het - clusters consisting of more than 10 atoms ( e.g. mg together with
atp constitute the het - cluster in the binding site of pdb entry 1e4 g ) .
several nucleotides and derivatives thereof are also among the most frequent het - groups in ligasite ( i.e. atp , adp , amp , gdp , coa , nad and nap ) .
interestingly , a number of small molecules commonly ignored in existing datasets of binding sites because of their potential irrelevance , appear in several binding sites in ligasite ( i.e. phosphate ions and glycerol molecules , and sulphate ions which appear in the binding sites of six different proteins ) .
table 2.most frequent het - groups in ligasite ( nr25)namehet idno . of occurrencesmagnesium ionmg41adenosine-5-diphosphateadp19manganese ( ii ) ionmn13adenosine-5-triphosphateatp13n - acetyl - d - glucosaminenag12nicotinamide - adenine - dinucleotidenad11glycerolgol11guanosine-5-diphosphategdp11phosphate ionpo49glucoseglc9nadpnap8d - galactosegal8coenzyme acoa8adenosine monophosphateamp8phosphoaminophosphonic acid - adenylate esteranp7het - group name as provided in the pdb chemical component dictionary ( see http://deposit.pdb.org/cc_dict_tut.html).het-group i d ( i.e. residue name in pdb files).number of occurrences of het - groups in ligasite ( redundancy removed at 25% seq .
when a given het - group appears in several different holo - structures of a given protein , only one occurrence was counted to compute values in this table .
most frequent het - groups in ligasite ( nr25 ) het - group name as provided in the pdb chemical component dictionary ( see http://deposit.pdb.org/cc_dict_tut.html ) .
number of occurrences of het - groups in ligasite ( redundancy removed at 25% seq .
when a given het - group appears in several different holo - structures of a given protein , only one occurrence was counted to compute values in this table .
we used the pdbsprotec mapping ( 26 ) , in order to obtain the ec numbers for all 286 proteins in the non - redundant version of ligasite , and for all the proteins of a non - redundant version of the pdb [ redundancy removed at 25% sequence identity using pisces ( 21 ) ] ( figure 3 ) . only 48 proteins out of 286 ( i.e. 17% ) in the non - redundant version of ligasite are non - enzymes ( 26 ) . in the non - redundant version of the pdb , 39% of proteins are non - enzymes . among enzymes , transferases and hydrolases ( ec classes 2 . and 3 .
figure 3.distribution of ec classes among proteins in ( a ) the non - redundant version of the ligasite dataset ( redundancy removed at 25% sequence identity ) , and in ( b ) a non - redundant subset of the pdb ( redundancy removed at 25% sequence identity ) , which consists of 5180 pdb entries .
ec numbers were obtained from pdbsprotec , a mapping of pdb entries to ec numbers via swissprot ( 26 ) .
distribution of ec classes among proteins in ( a ) the non - redundant version of the ligasite dataset ( redundancy removed at 25% sequence identity ) , and in ( b ) a non - redundant subset of the pdb ( redundancy removed at 25% sequence identity ) , which consists of 5180 pdb entries .
ec numbers were obtained from pdbsprotec , a mapping of pdb entries to ec numbers via swissprot ( 26 ) .
binding sites in the ligasite dataset are exclusively defined on the basis of structural data from the pdb ( 8) . in constructing the dataset , the first step involves selecting all structures with at least one protein chain , using the pdb search engine . as a starting point , ligands are defined as all non - water molecules that appear in the hetatm records of pdb entries .
such ligands are commonly referred to as het - groups. binding sites for nucleic acids and other proteins are hence ignored at this point .
quaternary structures suggested by pqs ( 18 ) are used for all structures in the dataset , unless pqs can not be applied ( e.g. nmr structures ) .
there are 27 913 pdb entries with at least one protein chain and at least one het - group in the pdb release of july 30 , 2007 , on which the current version of ligasite is based .
the total number of het - groups in these pdb entries amounts to 197 860 .
biologically relevant binding sites are defined as those bound by the biologically active ligands identified in vivo or in vitro , as well as by any compound that may act as inhibitor or substrate analogue .
biologically irrelevant sites are defined as sites bound non - specifically by compounds introduced by the purification or crystallization procedure . in order to select biologically relevant sites ,
first , het - groups are clustered together into het - clusters when any of their respective heavy atoms are located within < 4.0 from one another .
this clustering is performed to account for the fact that small solvent molecules often cluster together in biologically relevant binding sites and mimic parts of the natural ligand ( e.g. phosphate or sulphate ions , which mimic the phosphate groups of nucleotides ) .
the total of 197 860 het - groups in the pdb sums up to 148 879 het - clusters .
second , het - clusters with less than 10 heavy atoms are rejected on the basis that biologically irrelevant molecules in pdb files are generally very small . since
het - clusters are considered rather than single het - groups , het - groups with less than 10 heavy atoms , such as phosphate ions , can remain in the selected subset provided they are part of a het - cluster with a total of at least 10 heavy atoms ( e.g. two phosphate ions close to one another are kept ) .
finally , biologically relevant het - clusters are selected based on the number of inter - atomic protein ligand contacts . the underlying assumption being , that in general , biologically relevant ligands interact specifically with the proteins and should therefore make more inter - atomic contacts with protein atoms than their irrelevant counterparts .
we use the program lpc ( 19 ) to compute the inter - atomic contacts for the following reasons : it is not restricted to a specific subset of bonds ( e.g. hydrogen bonds ) ; it provides a detailed description of contacts based on the physico - chemical properties of contacting atoms and it is freely accessible and easy to run on a large number of structures . to perform the selection based on the above assumption
, we define a threshold number of inter - atomic contacts above , which an interaction is guaranteed to be relevant . to that end , the number of inter - atomic contacts between het - cluster and protein is calculated for all het - clusters of the dataset . the dataset is then divided into subsets based on ranges of 50 inter - atomic contacts , as indicated in table 1 . for each range , 1% of the het - cluster protein binding sites
have been manually inspected to assess whether the binding site is biologically relevant ( this manual inspection was completed for an initial version of ligasite , which was based on the december 22 , 2006 pdb release ) .
the 4th column in table 1 shows the fractions of binding sites that we manually annotated as biologically relevant . as expected , this fraction increases as the number of inter - atomic contacts increases . since the fraction of biologically relevant binding sites steeply increases between the ranges of 150 and 51100 contacts , the subset of binding sites having between 30 and 80 contacts
was subdivided into smaller bins of 10 inter - atomic contacts each , and re - analysed .
results ( table 1 ) show that above 70 inter - atomic contacts between het - cluster and protein , the het - cluster is biologically relevant in at least 95% of cases . the small number of cases where we manually annotated the binding site as biologically irrelevant even though it consists of at least 70 inter - atomic contacts between protein and het - cluster , all correspond to membrane proteins in complex with membrane lipids , which we were therefore able to readily exclude on the basis of the ligand and/or protein type .
table 1.fractions of biologically relevant binding sites as a function of the number of inter - atomic contacts between het - cluster and protein , for steps of 50 inter - atomic contacts and steps of 10 inter - atomic contactsno . of contactsno . of sitesno . of inspectedfrelevantsteps of 50 inter - atomic contacts1506825680.5451100153261530.931011509786980.941512006531650.98>2003932390.98steps of 10 inter - atomic contacts31402145210.1041502793280.3251603287330.6461703129310.7771803293330.97the manual analysis
summarised in this table was conducted using an initial version of ligasite , which was based on the pdb release of december 22 , 2006.range of number of inter - atomic contacts between protein and het-cluster.total number of binding sites where number of inter - atomic contacts between protein and het - cluster is within range given in column 1.number of binding sites inspected manually from the literature , used for assessing biological relevance of het - cluster .
this number equals 1% of the number of sites in column 2.fraction of manually inspected binding sites that we annotated as biologically relevant .
fractions of biologically relevant binding sites as a function of the number of inter - atomic contacts between het - cluster and protein , for steps of 50 inter - atomic contacts and steps of 10 inter - atomic contacts the manual analysis summarised in this table was conducted using an initial version of ligasite , which was based on the pdb release of december 22 , 2006 .
total number of binding sites where number of inter - atomic contacts between protein and het - cluster is within range given in column 1 .
number of binding sites inspected manually from the literature , used for assessing biological relevance of het - cluster .
the resulting dataset is thus limited to binding sites for het - clusters that consist of at least 10 heavy atoms , and that make at least 70 inter - atomic contacts with protein atoms .
there are 32 656 such het - clusters , consisting of 63 105 individual het - groups and distributed among 14 459 pdb entries .
a gold - standard dataset of binding sites should include information on both the apo- and holo - forms of the protein , to enable taking into account the structural changes that can occur in a protein upon binding of its ligand(s ) . in order to derive the list of true apo - structures in the pdb , we applied the following filtering procedure . from the pdb release of july 30 , 2007 , we selected all pdb entries with at least one protein chain that is not in complex with any other molecule , the latter being a small molecule , another protein or a nucleic acid chain .
this procedure resulted in a set of 10 046 pdb entries , which was then filtered to remove c-only and non - x - ray entries and x - ray entries with resolution better or equal to 2.4 , and r - value better or equal to 0.25 .
these quality filters were imposed to ensure that the apo - structures , which should be used to apply functional site prediction methods , are of sufficient quality to serve as input for any type of methods including those that are based on energy calculations ( 20 ) .
these apo - structures are then paired with the corresponding holo - structures containing the biologically relevant binding sites when they share 100% sequence identity , thus resulting in a set of 550 entries with one apo - structure and at least one holo - structure with a biologically relevant binding site . removing redundancy at 25% sequence identity [ using pisces ( 21 ) ] results in a non - redundant set of 286 proteins .
when several holo - structures are available for a given protein , all are used to define its binding site .
many holo - structures are bound to only a portion of the natural ligands , and the picture of the binding site that is obtained when considering all available holo - structures is therefore more complete and accurate . out of the 550 proteins in the redundant dataset , 291 have more than one holo - structure .
a frequency score is assigned to all binding site residues in a protein , based on the fraction of corresponding holo - structures in which the residue is observed to be part of a biologically relevant binding site ( figure 2 ) .
( a ) mapping of binding site residues on the apo - structure ( pdb entry 135l ) .
these residues are identified as part of the binding site from the three holo - structures in which the lysozyme is in complex with different ligands : ( b ) with three molecules of n - acetyl - d - glucosamine ( nag ) and a sulphate ion in pdb entry 1jef ; ( c ) with di(n - acetyl - d - glucosamine ) in pdb entry 1ljn ; and ( d ) with two molecules of nag in pdb entry 1lzy .
binding site residues are coloured red if they are in contact with ligand atoms in all three holo - structures ( i.e. frequency score of 1.0 = 3/3 ) , they are coloured orange if they are only in contact with ligand atoms in two of the holo - structures ( i.e. frequency score of 0.67 2/3 ) , and they are coloured yellow if they are in contact with ligand atoms in only one holo - structure ( i.e. frequency score of 0.33 1/3 ) .
het - groups considered as biologically relevant in ligasite are displayed and coloured in cpk .
sulphate ions filtered out as biologically irrelevant in pdb entry 1ljn are transparent and displayed in balls - and - sticks .
the figure was drawn with molscript ( 27 ) and rendered with raster3d ( 28 ) .
( a ) mapping of binding site residues on the apo - structure ( pdb entry 135l ) .
these residues are identified as part of the binding site from the three holo - structures in which the lysozyme is in complex with different ligands : ( b ) with three molecules of n - acetyl - d - glucosamine ( nag ) and a sulphate ion in pdb entry 1jef ; ( c ) with di(n - acetyl - d - glucosamine ) in pdb entry 1ljn ; and ( d ) with two molecules of nag in pdb entry 1lzy .
binding site residues are coloured red if they are in contact with ligand atoms in all three holo - structures ( i.e. frequency score of 1.0 = 3/3 ) , they are coloured orange if they are only in contact with ligand atoms in two of the holo - structures ( i.e. frequency score of 0.67 2/3 ) , and they are coloured yellow if they are in contact with ligand atoms in only one holo - structure ( i.e. frequency score of 0.33 1/3 ) .
het - groups considered as biologically relevant in ligasite are displayed and coloured in cpk .
sulphate ions filtered out as biologically irrelevant in pdb entry 1ljn are transparent and displayed in balls - and - sticks .
the figure was drawn with molscript ( 27 ) and rendered with raster3d ( 28 ) .
the fact that the construction of the dataset only relies on simple numerical cut - offs and automatic filters allows it to be automatically updated as new data become available in the pdb . at the time of writing the manuscript ,
the current release of ligasite is based on the july 30 , 2007 release of the pdb .
both the redundant and the non - redundant versions of the dataset can be browsed . for each protein ,
a front - page describes the apo - structure , and lists all binding site residues defined from the ensemble of corresponding holo - structures . binding site residue positions ( i.e. pdb serial numbers ) are coloured on a yellow - to - red scale , depending on the fraction of holo - structures in which the residue is found in contact with a biologically relevant het - cluster ( red when the fraction equals 1 ) .
pdb identifiers of corresponding holo - structures are listed together with unique identifiers describing the biologically relevant het - clusters used to define the binding site residues , the number of heavy atoms in the het - clusters and the number of inter - atomic contacts ( identified with lpc ) between each het - cluster and protein atoms . a pdb file providing coordinates of the binding site residues in the apo - structure
clicking on the details links associated with holo - structures leads to a holo - structure specific page , on which more details can be found for each structure .
residues identified as part of binding sites in the given holo - structure are listed , together with the unique identifier of the het - group with which each residue interacts .
residues interacting with het - groups in other holo - structures of the same protein are not listed .
further data on the het - groups that bind in biologically relevant binding sites is also available from these pages , i.e. pdb het identifiers , molecule name , chemical formula and ( non - stereo ) smiles string .
a pdb file providing coordinates of the binding site residues and of the het - groups labelled as biologically relevant is available for download for each holo - structure in the dataset .
cross - links to relevant databases [ e.g. pdbsum ( 22 ) , csa ( 14 ) ] are available on all pages , as well as links to ligplot ( 23 ) drawings of the interactions between het - groups and proteins .
a search facility is available and allows users to search ligasite for pdb identifiers ( both apo- and holo- ) , het - group identifiers ( e.g. atp ) or protein names .
the ligasite dataset can also be searched by sequence similarity using blast ( 24 ) .
all matches to a query sequence are returned to the user and are sorted by sequence identity and e - value .
finally , ligasite can be searched for proteins that have a domain in the same cath superfamily ( i.e. h level ) as the domains in the pdb entry input by the user ( 25 ) .
xml schema allows to define semantic constraints for the data contained in the xml files .
an xml file is not only humanly - readable , but can also be easily parsed , and consequently validated and transformed , by a computer .
an xml schema ( ligasiteml ) has been designed for a complete description of binding site information on the proteins of the dataset .
an xml file describing the complete ligasite database is generated for each update , and validated against the above - mentioned xml schema . both the schema and xml files are available for download from the website , allowing users to easily use ligasite data locally . in addition ,
protein - specific xml files , validated against the same xml schema , and describing only binding site residues in a particular protein of the dataset are available for download from the pages describing the apo- and holo - structures of the corresponding protein . a comma - separated file containing the core data of ligasite , and created automatically from the xml file , is also available for download .
the het - groups featured in the biologically relevant binding sites of ligasite proteins are very diverse . in total ,
551 different het - groups appear in the holo - structures of the non - redundant ligasite dataset ( redundancy removed at 25% sequence identity ) .
this number exceeds the number of binding sites in the dataset because ( i ) several different het - groups can appear together in a binding site in a single holo - structure , e.g. a magnesium ion ( het i d mg ) and a molecule of adenosine-5-diphosphate ( het i d
adp ) in the holo - structure 1pfk of phosphofructokinase ; and ( ii ) different het - groups can appear in a given binding site in different holo - structures of a protein ( figure 2 ) .
the majority of het - groups appear in the binding site of only one protein , suggesting an important diversity of ligands in the dataset .
table 2 gives the names , het ids and numbers of occurrences of the het - groups that appear most frequently in binding sites of the non - redundant version of ligasite .
when a het - group appears in several holo - structures of a given protein , only one occurrence is counted .
therefore , the number of occurrences in this table corresponds to the number of different proteins to which a given het - group binds .
the most common het - group is the magnesium ion , which occurs in the binding sites of 41 different proteins , as part of het - clusters consisting of more than 10 atoms ( e.g. mg together with
atp constitute the het - cluster in the binding site of pdb entry 1e4 g ) .
several nucleotides and derivatives thereof are also among the most frequent het - groups in ligasite ( i.e. atp , adp , amp , gdp , coa , nad and nap ) .
interestingly , a number of small molecules commonly ignored in existing datasets of binding sites because of their potential irrelevance , appear in several binding sites in ligasite ( i.e. phosphate ions and glycerol molecules , and sulphate ions which appear in the binding sites of six different proteins ) .
table 2.most frequent het - groups in ligasite ( nr25)namehet idno . of occurrencesmagnesium ionmg41adenosine-5-diphosphateadp19manganese ( ii ) ionmn13adenosine-5-triphosphateatp13n - acetyl - d - glucosaminenag12nicotinamide - adenine - dinucleotidenad11glycerolgol11guanosine-5-diphosphategdp11phosphate ionpo49glucoseglc9nadpnap8d - galactosegal8coenzyme acoa8adenosine monophosphateamp8phosphoaminophosphonic acid - adenylate esteranp7het - group name as provided in the pdb chemical component dictionary ( see http://deposit.pdb.org/cc_dict_tut.html).het-group i d ( i.e. residue name in pdb files).number of occurrences of het - groups in ligasite ( redundancy removed at 25% seq .
when a given het - group appears in several different holo - structures of a given protein , only one occurrence was counted to compute values in this table .
most frequent het - groups in ligasite ( nr25 ) het - group name as provided in the pdb chemical component dictionary ( see http://deposit.pdb.org/cc_dict_tut.html ) .
number of occurrences of het - groups in ligasite ( redundancy removed at 25% seq .
when a given het - group appears in several different holo - structures of a given protein , only one occurrence was counted to compute values in this table .
we used the pdbsprotec mapping ( 26 ) , in order to obtain the ec numbers for all 286 proteins in the non - redundant version of ligasite , and for all the proteins of a non - redundant version of the pdb [ redundancy removed at 25% sequence identity using pisces ( 21 ) ] ( figure 3 ) . only 48 proteins out of 286 ( i.e. 17% ) in the non - redundant version of ligasite are non - enzymes ( 26 ) . in the non - redundant version of the pdb
, 39% of proteins are non - enzymes . among enzymes , transferases and hydrolases ( ec classes 2 . and 3 . ,
figure 3.distribution of ec classes among proteins in ( a ) the non - redundant version of the ligasite dataset ( redundancy removed at 25% sequence identity ) , and in ( b ) a non - redundant subset of the pdb ( redundancy removed at 25% sequence identity ) , which consists of 5180 pdb entries .
ec numbers were obtained from pdbsprotec , a mapping of pdb entries to ec numbers via swissprot ( 26 ) .
distribution of ec classes among proteins in ( a ) the non - redundant version of the ligasite dataset ( redundancy removed at 25% sequence identity ) , and in ( b ) a non - redundant subset of the pdb ( redundancy removed at 25% sequence identity ) , which consists of 5180 pdb entries .
ec numbers were obtained from pdbsprotec , a mapping of pdb entries to ec numbers via swissprot ( 26 ) .
ligasite is a publicly available dataset of binding sites in proteins with at least one known apo - structure and one known holo - structure .
the dataset relies on simple numerical cut - offs and is automatically updated regularly . in its current version
, ligasite contains binding sites for all ligands stored as het - groups in the pdb ( e.g. small organic compounds , oligo - saccharides , lipids , nucleotides and derivatives thereof , etc . ) .
we are presently working on extending our protocol , in order to include binding sites for peptides , proteins and nucleic acids .
we furthermore plan to derive an improved score for excluding biologically irrelevant binding sites from the dataset .
this score , in which the number of inter - atomic contacts between protein and ligand ( i.e. the one currently used ) is normalized by the number of heavy atoms in the ligand , should allow us to increase our coverage of the small biologically relevant ligands , which make few contacts with protein atoms ( table 1 ) .
we are also working on the development of a structure - based search facility to allow users to look for local structural matches with binding sites in ligasite .
ligasite should prove a highly valuable resource for validating and developing binding site prediction methods , and for the study of binding site properties in general . | better characterization of binding sites in proteins and the ability to accurately predict their location and energetic properties are major challenges which , if addressed , would have many valuable practical applications .
unfortunately , reliable benchmark datasets of binding sites in proteins are still sorely lacking . here , we present ligasite ( ligand attachment site ) , a gold - standard dataset of binding sites in 550 proteins of known structures .
ligasite consists exclusively of biologically relevant binding sites in proteins for which at least one apo- and one holo - structure are available . in defining the binding sites for each protein , information from all holo - structures
is combined , considering in each case the quaternary structure defined by the pqs server .
ligasite is built using simple criteria and is automatically updated as new structures become available in the pdb , thereby guaranteeing optimal data coverage over time .
both a redundant and a culled non - redundant version of the dataset is available at http://www.scmbb.ulb.ac.be/users/benoit/ligasite .
the website interface allows users to search the dataset by pdb identifiers , ligand identifiers , protein names or sequence , and to look for structural matches as defined by the cath homologous superfamilies .
the datasets can be downloaded from the website as schema - validated xml files or comma - separated flat files . |
vertebral artery injury is a catastrophic complication that can occur during anterior decompression of the cervical foramen .
the distance between the uncinate tip and the vertebral artery was found to be as short as 1 mm on cadaveric specimens.1
2 there are controversies regarding the width of the uncinate process that needs to be resected for adequate decompression of the intervertebral foramen ; the recommended widths based upon cadaveric studies has been variously reported as 3.5 to 4.5 mm,2 4 mm,3 5 mm,4
5 or 6 mm.6 none of these studies , however , provide the most important information about the location of the vertebral artery and the height of the uncinate process that needs to be resected to achieve adequate decompression of the intervertebral foramen .
the aim of the present study is to determine the anatomic parameters between the various structures near the intervertebral foramen and the transverse foramen where the vertebral artery lies .
this study was approved by the institutional review board ( number : 2013-i141 ) at the institution of the corresponding author .
it was a retrospective radiologic study of 51 patients who were randomly selected from a larger group of 1,939 patients .
they had undergone three - dimensional ( 3d ) computed tomography ( cts ) of the cervical spine as part of their workup for neck pain , radiating arm pain , or difficulty with ambulation , from january 2003 to january 2012 at the first author 's academic institution .
scanning parameters of the cervical spine 3d ct consisted of 2-mm thickness and 2-mm interval slices .
we excluded patients with a previous history of spinal trauma or any operations for trauma , infection , or tumor .
we measured the distance between the midline and the uncinate process , as well as the transverse foramen , which we used as the location of the vertebral artery . on the axial view
, we drew a line bisecting the vertebral body to identify the midpoints of the anterior and posterior cortices ( fig .
, we drew lines to the medial and lateral cortices of the pedicle bilaterally from c3 to c7 .
we measured the distance along the posterior cortex of the vertebral body to the structures involved in the foraminal decompression ( fig .
the distance from the midline to the medial pedicle cortex ( red line ) represents the central canal ( fig .
the distance from the midline to the lateral cortex of the pedicle ( yellow line ) represents the central canal plus the intervertebral foramen to the lateral border of the uncinate process ( fig .
the axial view of a cervical vertebra demonstrating all the measured parameters , the distance from the midline to the medial pedicle cortex ( red line ) , and the distance from the midline to the lateral pedicle cortex ( yellow line ) .
we measured the height of the uncinate process bilaterally from c3 to c7 on two coronal 3d ct views of the cervical spine : at the level of the posterior cortex of the vertebral body ( fig .
3 ) . these two views represent , respectively , the minimal and maximal heights of the uncinate process from the cranial cortex of the pedicles .
the horizontal line on the axial image on the left depicts the location of the coronal cut on the right .
the minimum height of the uncinate process ( red arrow ) is measured from a horizontal line connecting the cranial cortex of the pedicles .
the horizontal line on the axial image on the left depicts the location of the coronal cut on the right . on the right side
the maximal height of the uncinate process ( red arrow ) is measured from a horizontal line connecting the cranial cortex of the pedicles .
the mean distances from the midline to the medial and lateral cortices of the pedicle were 10.1 1.3 mm and 13.9 1.5 mm , respectively ( table 1 ) .
the mean distance from the midline to the medial cortex of the pedicle increased from c3 to c5 or c6 with a minimum of 9.2 1.1 mm on the right and a minimum of 9.3 1.1 mm on the left at c3 ( table 1 ) .
therefore , a complete decompression of the spinal canal would have to extend at least 20 mm from side to side ( figs .
the mean distance from midline to the lateral cortex of the pedicle gradually increased from c3 to c6 with a minimum 13.1 1.4 mm on the right and minimum 13.2 1.7 mm on the left at c3 ( table 1 ) .
this data suggests that to thoroughly decompress the foramina bilaterally , one has to extend a minimum of 26 mm from side to side .
paper ruler of 20 mm necessary for thorough decompression of the spinal canal .
the computed tomographic views showing the osteophyte ( black arrows ) medial to the right pedicle compressing spinal cord at c6c7 disk level before the decompressive surgery under the authors ' practical guide .
the computed tomographic views showing the complete removal of the osteophyte ( black arrows ) medial to the right pedicle compressing spinal cord at c6c7 disk level after the decompressive surgery under the authors ' practical guide of up to 10 mm laterally from the midline to medial cortex of the pedicle . note : results are mean standard deviation . to determine the width of the pedicles
, we took the difference between the distances to the medial and lateral cortices of the pedicle .
the size decreased from c3 to c5 , increased from c5 to c7 , and was smallest at c5 ( table 1 ) .
the mean minimal height of the uncinate process from the cranial cortex of the pedicle was 4.6 1.6 mm and the mean maximal height was 6.1 1.7 mm ( table 2 ) .
the mean minimal height of the uncinate process was highest at c3 and lowest at c6 ( table 2 ) .
the mean maximal height of the uncinate process decreased from c3 to c7 ( table 2 ) . because these are the normal values , in uncinate hypertrophy that causes root compression , the decompression of the uncinate process would need to extend more than 4 mm and less than 6 mm above the level of the pedicle to thoroughly decompress the foramen .
injury to the vertebral artery , which is at risk during anterior foraminal decompression , can result in catastrophic consequences such as massive bleeding , cerebellar or brainstem infarction , and death.1
2
7 previous studies have determined that the uncinate process has a width from 3.5 to 6 mm.2
3
4
5
6 however , the studies did not provide information regarding the location of the vertebral artery and the height of the uncinate process .
the purpose of the present study is to determine the anatomic parameters of the uncinate process and vertebral artery , which can be used as a guide for thorough decompression of the foramen .
we found that the mean distances from the midline to the medial and lateral cortices of the pedicle were 10.1 1.3 mm and 13.9 1.5 mm , respectively .
the mean height of the uncinate process above the cranial border of the pedicle ( measured from coronally reconstructed cts ) was 4.6 1.6 mm at the level of the posterior cortex and 6.1 1.7 mm at the midvertebral body level .
the mean distances from the midline to the medial cortex of the pedicle were longer at c5 and c6 and shorter at c3 .
these results were not consistent with the previous studies,8
9 which found that the interpedicular distance was smallest at c3 and greatest at c7 ( table 3).8
9 the difference might be explained by the fact that the interpedicular distance was measured by the maximal distance between the medial aspects of the pedicles in previous studies,8
9 and we measured the distances from the midline to the medial cortex of the pedicle where the pedicle meets the vertebral body ( fig .
the maximal interpedicular distance in the previous studies between the medial aspect of the pedicles ( black line ) and the interpedicular distance in the current study between the medial cortices of the pedicle where the pedicle meets the vertebral body ( white line ) .
the mean distance from the midline to the lateral cortex of the pedicle was longer at c6 and shorter at c3 .
these results showing increasing distance from c3 to c6 are similar to four of the previous studies ( table 3).10
11
12
13 two of these were cadaveric,10
11 and two were ct studies ( table 3).12
13 however , two other cadaveric studies found no obvious pattern of increasing or decreasing distances as one went from c3 to c7 ( table 3).7
14 it might be explained by the fact that russo et al had measured the distance from the midline to the medial border of the vertebral artery infused with colored silicone , not the distance between the lateral cortices of cervical pedicles.7 curylo et al had measured the distance between the right and left transverse foramena.14
in the current study , the pedicle width was 3.8 1.1 mm .
the pedicle width was smallest at c5 , decreasing from c3 to c5 and increasing from c5 to c7 .
in contrast to previous studies , we did not measure the width of the uncinates .
the purpose of the previous studies was to elucidate the anatomic features of the uncinate process including the uncinate width.1
2
6 in contrast , our purpose is to show the safe margin for avoiding vertebral artery injury . for this purpose ,
the most important landmark is the lateral margin of the pedicle , which is also the medial wall of the foramen transversarium , where the artery lies .
the height of the uncinate process was reported as 4.8 mm in a study with 40 cadaveric specimens and 6 mm in a study with 30 cadaveric specimens.15
16 the maximum height of the uncinate process gradually decreased from c3 to c7 in the current study . however , some previous studies showed results contrary to our results ( table 4).2
7 the other previous studies did not show any tendency of the uncinate process height to decrease from c3 to c7 ( table 4).1
6
17 this discrepancy might be explained by the fact that they had a different definition of uncinate process height.1
2
6
7
17 the height of the uncinate process was measured as the distance from the tip of the process to the superior surface of the vertebral body,2
6
17 or to the rostral edge of the superior surface of the vertebral body.1 the advantage to our measuring method is that if a spine surgeon wants to thoroughly decompress the foramen , they must remove any spurs that lie cranial to the pedicle and not just those above the edge of the vertebral body , because spurs often extend caudally down to the cranial border of the pedicle .
first , some of the measurements were made on normal uncinates . in such cases ,
the numbers are likely to differ in cases where there is severe uncinate hypertrophy , which is the kind of situation where one has to do an uncinate resection .
however , the dimensions of the pedicle would not be affected by uncinate hypertrophy and should be valid .
second , the study was done in korean subjects , and the size may be different in other races .
therefore , our measurements can not determine the true distance between the artery and the bony structures .
therefore , using the transverse foramen as the location of the artery is not entirely accurate .
nevertheless , in the vast majority of cases , it is reasonable to state that one should keep the decompression of the foramen posterior and medial to the wall of the transverse foramen .
there may also be individual variations based on body size such that one can not blindly trust the numbers that we describe for any given patient .
it is recommended that the surgeon preoperatively measure it in all cases where uncinate resection is being planned . therefore , the main value of our study is in defining the methodology for making the measurements , which surgeons should consider doing prior to uncinate resection surgery . despite these shortcomings , to our knowledge , this study is the first report providing anatomic measurements that can be used as a guide for decompressing the neural foramen without injuring the artery . in conclusion , our results suggest that in most cases , one can remove osteophytes between the cervical pedicles within the spinal canal up to 10 mm laterally from the midline and safely remove uncinate process osteophytes up to 13 mm laterally from the midline and up to 4 mm of the height of the uncinate process from c3 to c7 without violating the transverse foramen . it should be noted that these numbers should only serve as a guide to where the artery is most likely to lie but that individual measurements may differ due to anatomic variations .
therefore , it is recommended that the surgeon examine preoperative ct or magnetic resonance images prior to performing such decompressions . | study design computed tomography based cohort study .
objective although there are publications concerning the relationship between the vertebral artery and uncinate process , there is no practical guide detailing the dimensions of this region to use during decompression of the intervertebral foramen .
the purpose of this study is to determine the anatomic parameters that can be used as a guide for thorough decompression of the intervertebral foramen .
methods fifty - one patients with three - dimensional computed tomography scans of the cervical spine from 2003 to 2012 were included .
on axial views , we measured the distance from the midline to the medial and lateral cortices of the pedicle bilaterally from c3 to c7 .
on coronal reconstructed views , we measured the minimum height of the uncinate process from the cranial cortex of the pedicle adjacent to the posterior cortex of vertebral body and the maximal height of the uncinate process from the cranial cortex of the pedicle at the midportion of the vertebral body bilaterally from c3 to c7 .
results the mean distances from midline to the medial and lateral cortices of the pedicle were 10.1 1.3 mm and 13.9 1.5 mm , respectively .
the mean minimum height of the uncinate process from the cranial cortex of the pedicle was 4.6 1.6 mm and the mean maximal height was 6.1 1.7 mm .
conclusions our results suggest that in most cases , one can thoroughly decompress the intervertebral foramen by removing the uncinate out to 13 mm laterally from the midline and 4 mm above the pedicle without violating the transverse foramen . |
urethral catheterization is one of the most commonly performed procedures in healthcare settings worldwide , with more than 5 million urinary catheterization in the united states in the year 2000 alone ( 1 ) . adding to this , it is usually safe and relatively easy if performed by well - trained healthcare personnel .
although it is considered as simple and safe procedure , however , clinicians including urologists can encounter significant difficulties and traumatic catheterization is sometimes possible .
catheter related complications , such as infection , bleeding , injury to the urethra or bladder , or catheter malfunction can ensue ( 2 ) .
moreover , insertion of the urethral catheter can be exceedingly difficult particularly in patients with underlying problems such as existing urethral strictures and elevated bladder neck due to severely enlarged prostate .
likewise , placement of the urethral catheter in neuropathic bladder patients can also be challenging due to periodic catheterization leading to more traumatized urethra .
herein , we report a first case of intraperitoneally placed urethral catheter via verumontanum , which was presented as intraperitoneal bladder perforation , in a chronically malnourished bed - ridden elderly man .
a 82-yr old male was admitted to our department for sudden onset acute abdominal pain .
he also has a significant history of urinary incontinence which became progressively worse and an insertion of foley catheter was recommended at a private healthcare institute . briefly following this ,
a 14 fr foley catheter was placed after a second attempt by a non - trained orderly at the private healthcare institute .
two days later , slightly blood tinged urine was observed on his urinary drainage bag .
he had a past medical history of hypertension for 10 yr , brain surgery for intracranial hemorrhage 5 yr ago and total left hip arthroplasty 10 yr ago .
his body temperature was 36.7. there was mildly left - shifted leukocytosis ( wbc : 9,300/l ) on complete blood count but it was within upper normal limit . although the bladder integrity was checked with bladder irrigation and aspiration , we observed a significant discrepancy between input and output fluid volumes .
the retrograde cystography showed extravasation of the contrast material into the peritoneal cavity and the foley catheter balloon which was placed in peritoneal cavity ( fig .
subsequently , emergency laparatomy was performed . however , there was no bladder perforation noted and the tip of the foley catheter was noticed at the rectovesical pouch .
given the location of the foley catheter , we considered the possibility of false passage of the urethral catheter and subsequently removed the entire catheter . to further examine the extent of urethral injury , on - table , intraoperative urethrocystoscopy was performed .
the urethrocystoscopical examination revealed a tunnel - like false passage extending from the verumontanum into the rectovesical pouch between the posterior wall of the bladder and the anterior wall of the rectum .
furthermore , a small hole into the peritoneum was also identified during cystoscopy ( fig .
1b ) . following the identification of the extent of the urethral injury , a 16 fr open ended nephrostomy tube
was indwelled and safely secured into the bladder under guidance of amplatz guide wire after full cystoscopic examination .
in addition , a 16 fr cystostomy tube was placed into the bladder suprapubically to ensure adequate urinary drainage .
then , the perforated rectovesical pouch was repaired transperitoneally with simple continuous suture and a closed suction drain was left into the peritoneum .
the patient was discharged to the private care institution with only suprapubic cystostomy tube 7 days after the surgery .
the two most common complications related to foley catheters , particularly in males , are anterior urethral injuries which can lead to long - term sequelae , such as urethral stricture , and retention of the catheter balloon in the urethra ( 3 ) .
serious complications , indeed such as bladder perforation and/or peritonitis and rectovesical fistula have been previously reported ( 4 - 6 ) . however
, to date , urethral foley catheter misplacement into the peritoneum without bladder injury has not been yet reported in the literature .
this present patient was a chronically debilitated , malnourished and bed - ridden state for a relatively long period of time of 5 yr following brain surgery .
therefore , we can suggest that his body connective tissues could be loosened and weakened as a result . at the time of surgery
, cystoscopic findings revealed highly elevated bladder neck and destructed verumontanum area with a tunneling to the peritoneal cavity .
however , there was no structural abnormality with exception of moderate trabeculation on the bladder .
although it is difficult to fully understand the mechanism of this injury , we can hypothesize that the sheer force of the foley catheter allowed penetration into ejaculatory duct and peritoneum .
structurally , the existence of the highly elevated bladder neck as well as the widely opened verumontanum can partly be contributing to such an injury . another challenging aspect in this patient 's presentation was the partial drainage of urine via the foley catheter , but we presumed that this was possible because the leaked urine was back flowed around the catheter .
this case illustrates that misplacement of foley catheter via verumontanum through peritoneal cavity without bladder perforation in a chronically debilitated man might be possible .
it also shows that foley catheterization , though it is simple and usually safe procedure , it can potentially cause serious problem like this present case , if performed by non- or less - trained healthcare providers .
therefore , we would like to emphasize that the best way to avoid traumatic catheterization would be adherence to the basic principles of catheterization .
firstly , the procedure has to be performed by physician or very well - trained healthcare provider .
secondly , one must abandon the procedure and an urgent urological consultation should be immediately sought whenever any urethral resistance or bleeding during urethral catheterization encountered .
finally , we should pay particular attention to confirm the location of the foley catheter and/or balloon before inflation by the aspiration of urine . adding to this , clinicians ,
particularly urologist should increase awareness of the potential catheter related complications among other healthcare providers and perhaps encourage to adopting a proper technique of catheterization as well as provide adequate training and supervision of those healthcare and/or non - healthcare personnel who intent to perform this procedure in the future . | since urethral foley catheterization is usually easy and safe , serious complications related to this procedure have been rarely reported .
herein , we describe a case of intraperitoneally placed urethral catheter via verumontanum presenting as intraperitoneal bladder perforation in a chronically debilitated elderly patient .
a 82-yr - old male patient was admitted with symptoms of hematuria , lower abdominal pain after traumatic foley catheterization .
the retrograde cystography showed findings of intraperitoneal bladder perforation , but emergency laparotomy with intraoperative urethrocystoscopy revealed a tunnel - like false passage extending from the verumontanum into the rectovesical pouch between the posterior wall of the bladder and the anterior wall of the rectum with no bladder injury .
the patient was treated with simple closure of the perforated rectovesical pouch and a placement of suprapubic cystostomy tube . |
traditional bone setters ( tbs ) have been with us for a very long time and they still enjoy patronage by all categories of people , ranging from the uneducated to the very highly educated group in the society . there is a general belief in most african communities that tbs are better at fracture treatment than orthodox practitioners and that there is a supernatural influence in their management of fractures .
most patients with fractures present first to the traditional bonesetters before coming to the hospital ; it is therefore apt that we do not ignore this level of care .
this practice is usually within the family circle from father to son and sometimes extended family members with other people being trained through apprenticeship .
the challenge of the orthopedic surgeon is the attendant complications that are presented to him after the patient has been mismanaged by the tbs .
some of these complications include limb gangrene following very tight local splints , malunion , nonunion , osteomyelitis , contractures , and limb length discrepancies . despite these complications ,
the demand for tbs services remains on the increase with some patients on admission in orthodox hospitals opting for treatment by a tbs .
we set out to ascertain the reasons for the patronage of tbs by patients with fractures and assess patients impressions of them after the treatment .
this was a prospective study done at the university of calabar teaching hospital , calabar .
seventy - nine patients who presented at orthopedic out - patient clinic after attending traditional bone setting centers were recruited for the study .
a predesigned questionnaire was filled containing details about the demographic data of the patient , presentation to hospital before resorting to the tbs , reasons for patronizing tbs , duration of treatment at bonesetters centers , sponsors of the treatment , introduction to the tbs , reasons for returning to hospital if patient had presented initially to the hospital , impressions about outcome of treatment by the bone setters and finally , patient 's advice to others who have similar conditions .
the age range was 1783 years with a mean age of 36.8 11.8 years .
forty - six ( 58.2% ) patients were married , 32 ( 40.5% ) were single , while only one ( 1.3% ) was widowed .
there were 12 ( 15.2% ) students , 9 ( 11.4% ) artisans , and 16 ( 20.3% ) business men / women .
the others were 17 ( 21.5% ) civil servants , 5 ( 6.3% ) farmers , and 2 ( 2.5% ) drivers .
the educational status of the patients was as follows : primary education ( number [ n ] = 14 ; 17.7% ) ; secondary education ( n = 19 ; 24.1% ) ; tertiary education ( n = 44 ; 55.7% ) ; and no education ( n = 2 ; 2.5% ) .
others were falls from height ( n = 15 ; 19.0% ) , domestic falls ( n = 2 ; 2.5% ) , gunshots ( n = 2 ; 2.5% ) , and assaults ( n = 2 ; 2.5% ) .
of the 79 patients that consented for the study , 37 ( 46.8% ) accessed the hospital before patronizing tbs while 42 ( 53.2% ) did not .
the factors that influenced the decision to patronize the tbs [ table 2 ] are as follows : delay of treatment at hospital 5 ( 6.3% ) , not satisfied with hospital treatment 5 ( 6.3% ) , poor attitude of hospital staff 2 ( 2.5% ) , tbs are more competent 12 ( 15.2% ) , tbs fee being cheap 11 ( 13.9% ) , patients having faith in tbs 16 ( 20.3% ) , sponsors of treatment took the decision 19 ( 24.1% ) , brought to tbs against patient wish 5 ( 6.3% ) , unconscious when brought 2 ( 2.5% ) , and fear of amputation 2 ( 2.5% ) .
only occupation was significantly associated with reasons for the patronage of tbs ( p = 0.047 ) [ table 3 ] .
the duration of tbs treatment ranged from 1 to 6 months with a mean of 2.5 months .
more than half 39 ( 49.4% ) of the patients were introduced to tbs by family members , 34 ( 43.0% ) by friends , neighbors 2 ( 2.5% ) , unknown persons 2 ( 2.5% ) , hospital staff 1 ( 1.3% ) , and self 1 ( 1.3% ) .
the opinion of patients about outcome of tbs treatment as shown in table 4 is as follows ; hospital treatment more reliable 7 ( 8.9% ) , tbs are not competent 8 ( 10.1% ) , no one should go to tbs 32 ( 40.5% ) , competency of tbs 2 ( 2.5% ) , regret seeking treatment from tbs 12 ( 15.2% ) , tbs operating on trial and error 6 ( 7.6% ) , hospitals are more competent 2 ( 2.5% ) , hospitals and tbs are good and no comment 5 ( 6.3% ) , 5 ( 6.3% ) , respectively . types of injuries among traditional bone setters attendants ( percentage ) sociodemographic characteristics of patients factors that influenced the patients decision to patronize traditional bone setters bivariate analysis of factors associated with traditional bone setters patronage and sociodemographics general opinions of patients about traditional bone setters on treatment outcome
tbs still command the attention of most patients with fracture injury in west africa sub - region .
treatment of a fractures by a tbs is natural to the average nigerian irrespective of his level of education because the practice appears to be ingrained in the minds of people being what most people were born to see practiced commonly in this part of the world and the world generally for over 3000 years .
cultural and spiritual beliefs play a central role in the orientation of the people and their patronage of the tbs , as they consider the treatment as having a supernatural touch from the gods bringing about the healing of the bone .
although several complications follow the treatment by these tbs , the strong belief in the spirituality associated with their activities encourages patient to continue patronizing them .
the orthopedic surgeon today is constantly faced with the challenge of managing the attendant complications caused by the treatment of these tbs . in this study ,
young adult males constitute the bulk of those who patronized the tbs ( 59.5% ) with the mean age of 36.8 17.8 . the duration of stay with the tbs was between 1 and 6 months with a mean of 2.5 months .
this age group constitutes the workforce in any society and spending such time with tbs with no solution at the end of the treatment but rather ending up with complications is gross loss of man hours with resultant reduction in productivity .
their introduction to the tbs was most times from family members ( 49.4% ) and friends ( 43% ) , the result is similar with that by ogunlusi et al .
, who had 85.2% from middlemen and also solagberu 's work stating that external persons formed 75% of sources influencing contact with tbs .
this is because , in nigeria , there is still strong family bonds which allow extended family members and friends to influence very important decisions of an individual .
the majority of these patients ( 55.7% ) were those who had attained tertiary education which affirms the fact that educational attainment has not really changed the cultural beliefs that the tbs are better in the management of any bone injury .
this was different from the study by other authors and that of udosen et al .
where there was up to 50% illiterate motorcycle riders forming the bulk of those who patronized the tbs .
forty - two patients ( 53.2% ) were seen first by the tbs before presenting to the hospital ; this could have probably included some of the patients with open fractures which end up mismanaged with resultant wound infections and some with chronic osteomyelitis .
where 79.3% of the patients visited tbs from the scene of the accident before presenting in the hospital .
of utmost concern are the 46.8% of them who presented first for orthodox medical attention and were later taken away to the tbs because of unsatisfactory attention given to them by health care providers .
health - care providers need to review the attitudes of staff to patients and also ensure that prompt attention is given to patients on arrival at the health center .
the study reveals that 24.1% of the patients actually visited the tbs because of their sponsors who had to pay for their treatment ; this buttresses the fact that apart from the cultural beliefs , poverty plays a central role in the continued patronage of tbs .
another group constituting 20.3% of the those who visited the tbs did so because of their beliefs that they are more competent ( 15.2% ) , this has been documented by many other authors .
tbs being cheaper constituted 13.9% which also underscores the issue of poverty and the dependence on sponsors for treatment no matter where it is obtained .
others visited the tbs because of the delay in orthodox care and poor attitude of orthodox caregivers .
the fear of amputation ( 2.5% ) was the least in their reasons for patronage of tbs , which is different from reasons documented by some authors who had fear of amputation as one of the significant reasons for visiting the tbs .
the tbs managed the different bone injuries that presented to them with the use of herbal concoctions , wooden splints , fresh palm leaves , and some scarifications and application of special herbal balms .
the different impressions of the respondents about the outcome of tbs treatment were as follows : 40.5% concluded that no one should patronize tbs while 15.2% regretted ever being managed by the tbs , others felt orthodox medicine is more reliable .
however , there was still a 6.3% group who believed that both the tbs and orthodox medical practitioner are the same , and hence they could still visit either of them .
with recent advancements in the practice of orthopedics and trauma , there is still a very high patronage of the tbs by most of our patients irrespective of their educational status and position in the society .
the reasons here are largely due to an external locus of decision making for the patient owing to their financial constraints with associated influenced of cultural and religious beliefs in the tbs .
the present state of our hospitals also leaves nothing to be desired , as the patients wait endlessly for simple surgical procedures which are not done due to poor attitude of staff and undue delays .
most patients impressions about the outcome of treatment by tbs were that they were not competent , not reliable and many regretted ever seeking help from them , though few were still indecisive .
it is , therefore , pertinent that the relevant institutions of government ensure that the national health insurance scheme becomes fully operational to make health care affordable to all , especially trauma and orthopedic conditions that are relatively expensive .
| aims and objectives : the objectives of this study were to analyze the different reasons why patients with fractures patronize traditional bone setters ( tbs ) and their impression of the outcome of the treatment by the tbs.materials and methods : a 24 month prospective observational study was conducted from february 2012 to january 2014 .
all the patients were recruited from the orthopedics outpatient clinic .
the demographic data of each patient , the type of injury , presentation to hospital or not , reasons for leaving the hospital , reasons for patronage of the tbs and their impression of the outcome of tbs treatment , effect of educational background on patronage of tbs and reason for presenting to hospital for orthodox treatment.data analysis : analysis was done with spss software version 20.results:a total 79 patients were recruited for the study and they had different reasons for patronizing tbs .
these reasons include an external locus of decision making in 19 ( 24.1% ) patients , and greater faith in tbs compared to orthodox medicine in 16 ( 20.3% ) .
twelve ( 15.2% ) believed that tbs are more competent than orthodox medical practitioners while another group 11 ( 13.9% ) considered the fees of tbs cheaper than those in the hospital .
the delay in treatment in the hospital , forceful removal of patients from hospital against their will and nonsatisfaction with hospital treatment accounted for 5 ( 6.3% ) .
poor attitude of hospital staff , fear of amputation , and patients being unconscious during the injury accounted for 2 ( 2.5% ) .
their ages ranged from 17 to 83 years , with mean age of 36.8 11.8 years .
the male : female ratio was 1.5:1.conclusions and recommendations : with recent advancements in the practice of orthopedics and trauma , there is still a very high patronage of the tbs by most of our patients .
this is largely due to the dependence of the patients on their sponsors for treatment , while the influence of cultural and religious beliefs continues to play a major role in these decisions . |
spontaneous intracerebral hemorrhage ( s - ich ) accounts for 10% to 15% of all strokes and 78% to 88% of intracerebral hemorrhages24 ) .
the 30-day mortality rate of s - ich has been reported to range from 35% to 52%24 ) , and around half of all deaths occur during the first 2 days3 ) .
in addition , s - ich produces serious neurological sequelae that require long - term medical and social care2,5 ) .
several factors are known to predispose s - ich , such as , hypertension , diabetes mellitus , dyslipidemia , and overweightedness4,17,21,23,24,29 ) .
recently , several reports have been issued on the prognostic value of hemodialysis in s - ich patients with end - stage renal disease ( esrd ) .
cerebrovascular disease is the third most common cause of death in patients with esrd11,13 ) . in these patients ,
the rate of s - ich has been reported to range from 6.2 to 10.2 per 1000 individuals requiring chronic hemodialysis14,16,19 ) .
furthermore , the number of patients with esrd requiring maintenance hemodialysis has been increasing19 ) , as is the number of s - ich patients on chronic hemodialysis .
mortality among s - ich patients with esrd remains high and reportedly ranges from 50% to 90%14,16 ) .
the outcome of the s - ich patients on chronic hemodialysis treatment has been reported to be independently associated with high alcohol consumption , a short interval between onset and admission , impaired consciousness on admission , and a low fibrinogen level6,7 ) .
although some reports have described the risk factors and predictors of s - ich13,16,28 ) , relatively few have addressed management strategies for improving patient prognosis in such cases11,19 ) .
management of s - ich in hemodialysis patients is frequently complicated by several factors , such as hypotension during hemodialysis , active bleeding and coagulopathy that may be exacerbated by the systemic anticoagulant therapy used in hemodialysis , and dialysis disequilibrium syndrome attributing to edema of the central nervous system caused by a rapid osmolar shift , often resulting in increased peripheral edema and neurological deterioration20 ) .
although neurosurgeons occasionally encounter s - ich attacks in patients with esrd during hemodialysis in the clinical field , there have not been comprehensive studies show that the characteristics of this type of s - ich compared with those of general s - ich , nor been specific management guideline for these patients . additionally , the factors associated with mortality and functional recoveries in s - ich patients on hemodialysis for esrd have not been widely studied .
we retrospectively analyzed and compare the characteristics of s - ich patients with or without hemodialysis for esrd based on the data from a single institute .
the primary study end - points were 30-day mortality and the 6-month functional recovery rate in both patients .
in addition , we sought to identify predictors of 30-day mortality and 6-month functional recovery in patients with or without esrd .
we retrospectively studied consecutive patients with a diagnosis of s - ich admitted to the stroke unit at our single hospital ( a university hospital serving a population of 1500000 ) between january 2000 and december 2011 .
patients aged greater than 40 years were selected because of the likelihood of a secondary etiology in younger patients .
in addition , we excluded patients with an infratentorial hemorrhage , because small changes in hemorrhage size or location are believed to have greater impacts on survival than supratentorial hemorrhages in these patients .
patient related data were independently extracted from a computerized database ( pacs ; marosis m - view , marotech co. , seoul , korea ) by three clinical research coordinators using a structured form . to preserve patient confidentiality ,
the inclusion criteria applied were a diagnosis of supratentorial s - ich , confirmed by computerized tomography ( ct ) or magnetic resonance imaging , and admission to a stroke unit within 24 hours of symptom onset .
as recommended by the stroke council of the american heart association1,2 ) , selected patients underwent conventional angiography to identify secondary causes .
patients with a hemorrhage secondary to head trauma , a ruptured cerebral aneurysm , an arteriovenous malformation , a tumor , bleeding diathesis , or a hemorrhagic infarction were excluded .
this study was approved by the institutional review boards of our hospital ( 2012-scmc-065 - 00 ) .
hemodialysis was performed using gambro ak 95 units ( gambro , stockholm , sweden ) equipped with a polyamix dialysis membrane ( gambro ) using bicart ( gambro ) dialysis solution .
hemodialysis was performed three times a week at 4 hours / day at a mean blood flow rate of 150 - 200 ml / min .
the anticoagulant ( heparin ) loading dose was 1000 iu and was intravenously infused , and the maximum maintenance dose was 200 iu per hour .
in addition , nafamostat mesylate was intravenously infused at an hourly dosage of 35 mg without a loading dose .
demographic variables included ; age , gender , cigarette smoking and alcohol intake histories , body mass index ( bmi ) , level of consciousness , pupillary abnormalities , limb weakness , and underlying disease .
smoking status was classified as never smoker , ex - smoker ( a lifelong smoking history of 6 months ) , or current smoker ( 10 cigarettes / day for 6 months ) , and alcohol intake was dichotomized as < or 46 g / day 3 days / week12 ) .
pupillary abnormalities at admission were classified as 0 , 1 , or 2 based on pupil reactivity scores17 ) .
levels of consciousness at admission were assessed using the glasgow coma scale ( gcs)27 ) ; gcs scores of 3 - 8 , 9 - 12 , and 13 - 15 were defined as ' severely altered ' , ' moderately altered ' , or ' mildly altered or normal ' conscious states , respectively .
blood pressure at admission was not viewed as a potential prognostic variable because it is often markedly elevated during the first 1 to 2 days after a severe stroke .
ct variables , that is , side of hemorrhage , location of hemorrhage , volume of hematoma , lateral shift of cerebral midline structures , and ventricular extension of hemorrhage , were also included in the analysis .
hematoma volume was estimated from axial ct scans using the formula abc/2 , where a is greatest diameter on the largest hemorrhage slice , b is maximal diameter perpendicular to this , and c is vertical hematoma depth18 ) .
hematoma locations were defined based on sites of origin , such as , basal ganglia , thalamus , lobal , or ventricle alone .
ventricular extension of hemorrhage included a pure intraventricular hemorrhage and secondary spread of cerebral parenchymal hemorrhage into ventricular space .
a ct scan was performed whenever neurological status changed and 48 hours after the initial ct scan if neurological status was stable .
hematoma enlargement was defined as an increase in hematoma volume of 25% versus initial ct volume .
laboratory parameters obtained at admission were dichotomized during the analysis ; these were hemoglobin , leukocyte count , platelet count , c - reactive protein ( crp ) level , blood urea nitrogen ( bun ) , creatine , prothrombin time and activated partial prothrombin time . international normalized ratio ( inr ) , plasma total cholesterol level , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , blood plasma glucose , and hemoglobin a1c .
all patients ( medical and surgical ) were cared for in a neurosurgical intensive care unit until considered stable enough to move to a general unit .
treatment was administered according to current practices , but was not rigidly regimented as primary attending neurosurgeons were allowed to exercise medical judgment .
surgical intervention was undertaken in selected patients exhibiting clinical deterioration and a moderate or larger amount of lobar hemorrhage , and in patients with basal ganglionic or thalamic hemorrhage with a hematoma volume of > 30 ml , in patients with an expanding hematoma , and in patients with evidence of progressive neurological deterioration .
the techniques used were craniotomy , craniectomy , ct - guided stereotactic hematoma evacuation , and/or extraventricular drainage .
patients with a deep - seated s - ich usually underwent stereotactic evacuation when hematoma volume was from 30 to 60 ml by preoperative ct .
immediately after initial hematoma aspiration or evacuation , patients underwent repeat ct to check catheter placement .
when repeat ct showed remnant hematoma , a urokinase ( 6000 u ) injection was considered through the catheter to facilitate aspiration , and a ct scan was repeated 12 hours later .
patients with a hemorrhage of 10 to 30 ml and a severe neurological deficit ( limb weakness ) were also treated by stereotactic evacuation . on the other hand ,
when hematoma volume exceeded 60 ml , evacuation was performed via craniotomy or decompressive craniectomy .
the attending neurosurgeon decided whether to implant a bone - flap ( craniotomy or craniectomy ) depending on the intraoperative presence of cerebral swelling after s - ich removal .
mean arterial pressure was maintained in the range 100 to 140 mm hg using an antihypertensive ( e.g. , labetalol , amolodipine , or nicardipin ) .
in addition , a hypertonic agent ( e.g. , glycerol or mannitol ) was administered when ct indicated a mass effect or when clinical symptoms indicated an elevated intracranial pressure .
functional outcomes were assessed using the glasgow outcome scale ( gos ) in 6 months after ictus .
gos scores were defined as follows15 ) : 1=dead , 2=vegetative state , 3=severe disability ( able to follow commands , but unable to live independently ) , 4=moderate disability ( able to live independently , but unable to return to work or school ) , 5=good recovery ( able to return to work or school ) . for statistical purposes ,
patients were assigned to two outcome category groups , namely , a " good functional recovery " group ( gos score 4 to 5 ) , members of which were functionally independent , or a " poor functional recovery " group ( gos score 1 to 3 ) .
information on mortality and functional outcome was obtained from medical records , information obtained from family members was obtained by conducting telephone interviews , and information from patients was obtained by conducting direct examinations at our outpatient department after discharge .
our primary aim was to estimate 30-day mortality and functional recovery at 6-month post - s - ich in s - ich patients on chronic hemodialysis for esrd patients .
we defined 30-day mortality as death within 30 days of a radiological diagnosis of s - ich , and functional recovery was defined as described by gos at 6 months after a radiological diagnosis of s - ich .
chicago , il , usa ) was used for the analysis , and the student 's t test and a nonparametric test were used to analyze continuous and ordinal variables ( e.g. , mean levels of bun and creatine ) , respectively .
discrete variables , such as , gender , smoking , chronic alcohol intake , ventricular extension of hemorrhage , and hydrocephalus and categorical variables , such as , age subgroup , bmi , gcs subgroup , pupillary abnormality , underlying conditions , and location of hemorrhage , were analyzed using pearson 's test .
univariate analysis was used initially to identify relationships between potential prognostic factors and 30-day mortality and functional recovery at 6 months post - s - ich using the test . subsequently
, multivariate logistic regression analysis was used to identify variables independently associated with the above - mentioned outcomes .
statistical significance was accepted for p values of < 0.05 , and all tests were 2-tailed .
we retrospectively studied consecutive patients with a diagnosis of s - ich admitted to the stroke unit at our single hospital ( a university hospital serving a population of 1500000 ) between january 2000 and december 2011 .
patients aged greater than 40 years were selected because of the likelihood of a secondary etiology in younger patients .
in addition , we excluded patients with an infratentorial hemorrhage , because small changes in hemorrhage size or location are believed to have greater impacts on survival than supratentorial hemorrhages in these patients .
patient related data were independently extracted from a computerized database ( pacs ; marosis m - view , marotech co. , seoul , korea ) by three clinical research coordinators using a structured form . to preserve patient confidentiality ,
the inclusion criteria applied were a diagnosis of supratentorial s - ich , confirmed by computerized tomography ( ct ) or magnetic resonance imaging , and admission to a stroke unit within 24 hours of symptom onset .
as recommended by the stroke council of the american heart association1,2 ) , selected patients underwent conventional angiography to identify secondary causes .
patients with a hemorrhage secondary to head trauma , a ruptured cerebral aneurysm , an arteriovenous malformation , a tumor , bleeding diathesis , or a hemorrhagic infarction were excluded .
this study was approved by the institutional review boards of our hospital ( 2012-scmc-065 - 00 ) .
hemodialysis was performed using gambro ak 95 units ( gambro , stockholm , sweden ) equipped with a polyamix dialysis membrane ( gambro ) using bicart ( gambro ) dialysis solution .
hemodialysis was performed three times a week at 4 hours / day at a mean blood flow rate of 150 - 200 ml / min .
the anticoagulant ( heparin ) loading dose was 1000 iu and was intravenously infused , and the maximum maintenance dose was 200 iu per hour .
in addition , nafamostat mesylate was intravenously infused at an hourly dosage of 35 mg without a loading dose .
demographic variables included ; age , gender , cigarette smoking and alcohol intake histories , body mass index ( bmi ) , level of consciousness , pupillary abnormalities , limb weakness , and underlying disease .
smoking status was classified as never smoker , ex - smoker ( a lifelong smoking history of 6 months ) , or current smoker ( 10 cigarettes / day for 6 months ) , and alcohol intake was dichotomized as < or 46 g / day 3 days / week12 ) .
pupillary abnormalities at admission were classified as 0 , 1 , or 2 based on pupil reactivity scores17 ) .
levels of consciousness at admission were assessed using the glasgow coma scale ( gcs)27 ) ; gcs scores of 3 - 8 , 9 - 12 , and 13 - 15 were defined as ' severely altered ' , ' moderately altered ' , or ' mildly altered or normal ' conscious states , respectively .
blood pressure at admission was not viewed as a potential prognostic variable because it is often markedly elevated during the first 1 to 2 days after a severe stroke .
ct variables , that is , side of hemorrhage , location of hemorrhage , volume of hematoma , lateral shift of cerebral midline structures , and ventricular extension of hemorrhage , were also included in the analysis . hematoma volume was estimated from axial ct scans using the formula abc/2 , where a is greatest diameter on the largest hemorrhage slice , b is maximal diameter perpendicular to this , and c is vertical hematoma depth18 ) .
hematoma locations were defined based on sites of origin , such as , basal ganglia , thalamus , lobal , or ventricle alone .
ventricular extension of hemorrhage included a pure intraventricular hemorrhage and secondary spread of cerebral parenchymal hemorrhage into ventricular space .
a ct scan was performed whenever neurological status changed and 48 hours after the initial ct scan if neurological status was stable .
hematoma enlargement was defined as an increase in hematoma volume of 25% versus initial ct volume .
laboratory parameters obtained at admission were dichotomized during the analysis ; these were hemoglobin , leukocyte count , platelet count , c - reactive protein ( crp ) level , blood urea nitrogen ( bun ) , creatine , prothrombin time and activated partial prothrombin time .
international normalized ratio ( inr ) , plasma total cholesterol level , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , blood plasma glucose , and hemoglobin a1c .
all patients ( medical and surgical ) were cared for in a neurosurgical intensive care unit until considered stable enough to move to a general unit .
treatment was administered according to current practices , but was not rigidly regimented as primary attending neurosurgeons were allowed to exercise medical judgment .
surgical intervention was undertaken in selected patients exhibiting clinical deterioration and a moderate or larger amount of lobar hemorrhage , and in patients with basal ganglionic or thalamic hemorrhage with a hematoma volume of > 30 ml , in patients with an expanding hematoma , and in patients with evidence of progressive neurological deterioration .
the techniques used were craniotomy , craniectomy , ct - guided stereotactic hematoma evacuation , and/or extraventricular drainage .
patients with a deep - seated s - ich usually underwent stereotactic evacuation when hematoma volume was from 30 to 60 ml by preoperative ct . immediately after initial hematoma aspiration or evacuation , patients underwent repeat ct to check catheter placement .
when repeat ct showed remnant hematoma , a urokinase ( 6000 u ) injection was considered through the catheter to facilitate aspiration , and a ct scan was repeated 12 hours later .
patients with a hemorrhage of 10 to 30 ml and a severe neurological deficit ( limb weakness ) were also treated by stereotactic evacuation . on the other hand ,
when hematoma volume exceeded 60 ml , evacuation was performed via craniotomy or decompressive craniectomy .
the attending neurosurgeon decided whether to implant a bone - flap ( craniotomy or craniectomy ) depending on the intraoperative presence of cerebral swelling after s - ich removal .
mean arterial pressure was maintained in the range 100 to 140 mm hg using an antihypertensive ( e.g. , labetalol , amolodipine , or nicardipin ) .
in addition , a hypertonic agent ( e.g. , glycerol or mannitol ) was administered when ct indicated a mass effect or when clinical symptoms indicated an elevated intracranial pressure .
functional outcomes were assessed using the glasgow outcome scale ( gos ) in 6 months after ictus .
gos scores were defined as follows15 ) : 1=dead , 2=vegetative state , 3=severe disability ( able to follow commands , but unable to live independently ) , 4=moderate disability ( able to live independently , but unable to return to work or school ) , 5=good recovery ( able to return to work or school ) . for statistical purposes ,
patients were assigned to two outcome category groups , namely , a " good functional recovery " group ( gos score 4 to 5 ) , members of which were functionally independent , or a " poor functional recovery " group ( gos score 1 to 3 ) .
information on mortality and functional outcome was obtained from medical records , information obtained from family members was obtained by conducting telephone interviews , and information from patients was obtained by conducting direct examinations at our outpatient department after discharge .
our primary aim was to estimate 30-day mortality and functional recovery at 6-month post - s - ich in s - ich patients on chronic hemodialysis for esrd patients .
we defined 30-day mortality as death within 30 days of a radiological diagnosis of s - ich , and functional recovery was defined as described by gos at 6 months after a radiological diagnosis of s - ich .
chicago , il , usa ) was used for the analysis , and the student 's t test and a nonparametric test were used to analyze continuous and ordinal variables ( e.g. , mean levels of bun and creatine ) , respectively .
discrete variables , such as , gender , smoking , chronic alcohol intake , ventricular extension of hemorrhage , and hydrocephalus and categorical variables , such as , age subgroup , bmi , gcs subgroup , pupillary abnormality , underlying conditions , and location of hemorrhage , were analyzed using pearson 's test .
univariate analysis was used initially to identify relationships between potential prognostic factors and 30-day mortality and functional recovery at 6 months post - s - ich using the test .
subsequently , multivariate logistic regression analysis was used to identify variables independently associated with the above - mentioned outcomes .
statistical significance was accepted for p values of < 0.05 , and all tests were 2-tailed .
from january 2000 to december 2011 , 1943 cases of s - ich were diagnosed at the stroke units in our institute . of these , 1558 supratentorial s - ich patients were included in the present study ; 1456 ( 93.5% ) did not have esrd , and 102 ( 6.5% ) were esrd patients on hemodialysis .
mean patient age was 66.416.3 years ( range 40 to 91 years ) , and 771 ( 49.5% ) of the study subjects were male . among of the 102 s - ich patients with esrd , mean age was 62.318.6 years ( ranged 37 to 86 years ) and 55 ( 54.0% ) were male .
mean duration of hemodialysis in these patients was 65.2 months ( range from 1.0 month to 241.2 months ) .
causes of esrd were hypertension in 34 ( 33.3% ) , diabetes mellitus in 49 ( 48.0% ) , polycystic kidney disease in 10 ( 9.8% ) , and chronic glomerulonephritis in 9 ( 8.8% ) .
no significant intergroup differences were observed between demographic characteristics , that is , age , gender , chronic alcohol intake , body mass index , or new limb weakness .
however , esrd patients had a lower mean gcs score ( p=0.028 ) , and more esrd patients had nonreactive pupils ( p=0.040 ) and underlying diseases ( p=0.013 ) ( table 1 ) . in terms of radiologic characteristics , no significant intergroup difference was found for sidedness or hemorrhagic volume , midline shift , location of hemorrhage , or hydrocephalus .
however , ventricular extension of hemorrhages ( p=0.010 ) , and hematoma enlargement within 48 hours were more prevalent in the esrd patients ( table 2 ) . in terms of laboratory findings
, no significant intergroup difference was found for leukocytosis ( 1010/l ) , hepatopathy ( ast or alt > 40 iu / l ) , or elevated hemoglobin a1c ( 6.0% ) .
however , anemia ( < 11.7 g / dl in male , < 10.7 g / dl in female ) ( p<0.001 ) , thrombocytopenia ( < 15010/l ) ( p=0.032 ) , elevated inr ( > 1.2 ) ( p=0.004 ) , elevated crp ( > 5.0 mg / l ) ( p=0.002 ) , elevated blood plasma glucose ( > 126 mg / dl ) ( p<0.001 ) , elevated bun ( > 24 mg / dl ) ( p<0.001 ) , elevated serum creatine ( > 1.4 mg / dl ) ( p<0.001 ) , and abnormal findings of urine analysis ( p<0.001 ) were more prevalent in s - ich patients with esrd ( table 3 ) . in these patients ,
mean bun was 54.2 mg / dl ( range from 10.7 mg / dl to 145.9 mg / dl ) and mean serum creatine was 7.7 mg / dl ( range from 2.9 mg / dl to 27.7 mg / dl ) .
urine output was nil in 43 s - ich patients with esrd ( 42.2% ) ; proteinuria was present in 49 ( 48.0% ) , glucosuria in 24 ( 23.5% ) , and hematuria in 17 ( 16.7% ) ( table 3 ) .
regarding treatment modalities , 1005 ( 64.5% ) patients were treated conservatively and 553 ( 35.5% ) were treated surgically .
no significant intergroup difference was found in terms of treatment modality type ( p=0.667 ) .
furthermore , of the 553 s - ich patients that underwent surgical treatment , no significant difference was found between s - ich patients with or without esrd in terms of surgical techniques ( p=0.605 ) or time to surgery ( p=0.311 ) ( table 4 ) .
for all study subjects , 30-day , 3-month , and 6-month mortalities were 16.4% , 25.7% , and 27.3% , respectively .
overall , 256 patients died within 30 days , 401 patients within 3 months , and 425 patients within 6 months of s - ich .
a significant and meaningful intergroup difference was found between 30-day ( p<0.001 ) , 3-month ( p<0.001 ) , and 6-month ( p<0.001 ) mortalities ( table 5 ) . in terms of functional recovery , at 30 days after s - ich , gos scores were similar ( p=0.545 ) , but at 3 months ( p=0.046 ) and 6 months after s - ich gos scores were significantly different ( p=0.013 ) ( table 5 ) . in patients without esrd
, univariate analysis showed that the following variables were significantly associated with death within 30 days of s - ich ; gcs score , pupillary abnormality , underlying disease , ventricular extension of hemorrhage , hemorrhagic volume , thrombocytopenia , and treatment modality . in s - ich patients with esrd in addition to these factors , age , anemia , and c - reactive protein
were also found to be associated with death within 30 days of s - ich .
multivariate logistic regression analysis was performed on variables found to be significant by univariate analysis ( table 6 ) . in the s - ich patients without esrd
, multivariate analysis showed that gcs score ( 3 to 8 vs. 9 to 12 ; p=0.038 , and 3 to 8 vs. 13 to 15 ; p<0.001 ) , pupillary abnormality ( 0 reactive vs. 2 reactive ; p<0.001 ) , treatment modality ( surgery vs. conservative treatment ; p=0.006 , and craniotomy vs. other surgical modalities ; p<0.001 ) , underlying disease ( ischemic heart disease vs. other disease ; p=0.034 , and atrial fibrillation vs. other disease ; p=0.018 ) were independently associated with 30-day mortality . in the s - ich patients with esrd
, multivariate analysis showed that age ( 70 years vs. 40 to 54 years ; p=0.019 ) , gcs score ( 3 to 8 vs. 13 to 15 ; p<0.001 , and 9 to 12 vs. 13 to 15 ; p=0.014 ) , pupillary abnormality ( 0 reactive vs. 1 reactive ; p=0.032 , and 0 reactive vs. 2 reactive ; p<0.001 ) , ventricular extension of hemorrhage ( present vs. absent ; p=0.017 ) , hemorrhagic volume ( 60 ml vs. < 30 ml ; p=0.002 , and 30 - 59 ml vs. < 30 ml ; p=0.029 ) , anemia ( present vs. absent ; p=0.008 ) , and treatment modality ( surgery vs. conservative treatment ; p=0.034 ) , were independently associated with high 30-day mortality . associations between other variables and 30-day mortality by univariate analysis were not confirmed by multivariate analysis . in s - ich patients with esrd ,
actually , underlying disease , which was independently associated with high 30-day mortality in s - ich patients without esrd , was not associated with high 30-day mortality in s - ich patients with esrd .
conversely , several other variables , such as , ventricular extension of hemorrhage , hemorrhagic volume , and anemia , not associated with 30-day mortality in s - ich patients without esrd , were found to be significantly and independently associated with 30-day mortality in s - ich with esrd . of the variables
examined , the following were found to be significantly associated with functional recovery in s - ich patients with and without esrd by univariate analysis ; age , gcs score , location of hemorrhage , hemorrhagic volume , and treatment modality .
however , pupillary response , underlying disease , ventricular extension of hemorrhage , anemia , thrombocytopenia , and crp , which were all found to be associated with 30-day mortality , were not found to be associated with functional recovery in either group by univariate analysis . in patients without esrd , multivariate logistic regression analysis showed that the following five variables were independently associated with functional recovery at 6 months after s - ich : age ( 40 to 54 years vs. 70 years ; p<0.001 , and 40 to 54 years vs. 55 to 69 years ; p=0.038 ) , gcs score ( 9 to 12 vs. 3 to 8 ; p=0.009 , 13 to 15 vs. 3 to 8 ; p<0.001 , and 13 to 15 vs. 9 to 12 ; p=0.041 ) , location of hemorrhage ( thalamus vs. other regions ; p=0.022 , lobal vs. other regions ; p=0.042 , and ventricle only vs. other regions ; p=0.008 ) , volume of hemorrhage ( < 30 ml vs. 60 ml ; p=0.001 ) , and treatment modality ( conservative treatment vs. surgery ; p=0.039 , other surgical modalities vs. craniotomy ; p<0.001 , and stereotactic aspiration vs. other surgical modalities ; p=0.005 ) . in s - ich patients with esrd
, multivariate logistic regression analysis showed that the following four variables were independently associated with functional recovery at 6 months after s - ich ; gcs score ( 9 to 12 vs. 3 to 8 ; p<0.001 , and 13 to 15 vs. 3 to 8 ; p<0.001 ) , hemorrhagic volume ( < 30 ml vs. 60 ml ) , treatment modality ( conservative treatment vs. surgery ; p<0.001 ) , and duration of hemodialysis treatment ( < 50 months vs. 50 months ; p=0.027 ) .
although age and location of hemorrhage were associated with good functional recovery by univariate analysis in s - ich patients with esrd , multivariate analysis showed no such association .
thus , age and location of hemorrhage were associated with functional recovery in s - ich patients without esrd , but not in s - ich patients with esrd ( table 7 ) .
no significant intergroup differences were observed between demographic characteristics , that is , age , gender , chronic alcohol intake , body mass index , or new limb weakness .
however , esrd patients had a lower mean gcs score ( p=0.028 ) , and more esrd patients had nonreactive pupils ( p=0.040 ) and underlying diseases ( p=0.013 ) ( table 1 ) . in terms of radiologic characteristics ,
no significant intergroup difference was found for sidedness or hemorrhagic volume , midline shift , location of hemorrhage , or hydrocephalus .
however , ventricular extension of hemorrhages ( p=0.010 ) , and hematoma enlargement within 48 hours were more prevalent in the esrd patients ( table 2 ) . in terms of laboratory findings , no significant intergroup difference was found for leukocytosis ( 1010/l ) , hepatopathy ( ast or alt > 40 iu / l ) , or elevated hemoglobin a1c ( 6.0% ) . however , anemia ( < 11.7 g / dl in male , < 10.7 g / dl in female ) ( p<0.001 ) , thrombocytopenia ( < 15010/l ) ( p=0.032 ) , elevated inr ( > 1.2 ) ( p=0.004 ) , elevated crp ( > 5.0 mg / l ) ( p=0.002 ) , elevated blood plasma glucose ( > 126 mg / dl ) ( p<0.001 ) , elevated bun ( > 24 mg / dl ) ( p<0.001 ) , elevated serum creatine ( > 1.4 mg / dl ) ( p<0.001 ) , and abnormal findings of urine analysis ( p<0.001 ) were more prevalent in s - ich patients with esrd ( table 3 ) . in these patients ,
mean bun was 54.2 mg / dl ( range from 10.7 mg / dl to 145.9 mg / dl ) and mean serum creatine was 7.7 mg / dl ( range from 2.9 mg / dl to 27.7 mg / dl ) .
urine output was nil in 43 s - ich patients with esrd ( 42.2% ) ; proteinuria was present in 49 ( 48.0% ) , glucosuria in 24 ( 23.5% ) , and hematuria in 17 ( 16.7% ) ( table 3 ) .
regarding treatment modalities , 1005 ( 64.5% ) patients were treated conservatively and 553 ( 35.5% ) were treated surgically .
no significant intergroup difference was found in terms of treatment modality type ( p=0.667 ) .
furthermore , of the 553 s - ich patients that underwent surgical treatment , no significant difference was found between s - ich patients with or without esrd in terms of surgical techniques ( p=0.605 ) or time to surgery ( p=0.311 ) ( table 4 ) .
for all study subjects , 30-day , 3-month , and 6-month mortalities were 16.4% , 25.7% , and 27.3% , respectively .
overall , 256 patients died within 30 days , 401 patients within 3 months , and 425 patients within 6 months of s - ich .
a significant and meaningful intergroup difference was found between 30-day ( p<0.001 ) , 3-month ( p<0.001 ) , and 6-month ( p<0.001 ) mortalities ( table 5 ) . in terms of functional recovery , at 30 days after s - ich , gos scores were similar ( p=0.545 ) , but at 3 months ( p=0.046 ) and 6 months after s - ich gos scores were significantly different ( p=0.013 ) ( table 5 ) .
in patients without esrd , univariate analysis showed that the following variables were significantly associated with death within 30 days of s - ich ; gcs score , pupillary abnormality , underlying disease , ventricular extension of hemorrhage , hemorrhagic volume , thrombocytopenia , and treatment modality . in s - ich patients with esrd in addition to these factors , age , anemia , and c - reactive protein
were also found to be associated with death within 30 days of s - ich .
multivariate logistic regression analysis was performed on variables found to be significant by univariate analysis ( table 6 ) . in the s - ich patients without esrd
, multivariate analysis showed that gcs score ( 3 to 8 vs. 9 to 12 ; p=0.038 , and 3 to 8 vs. 13 to 15 ; p<0.001 ) , pupillary abnormality ( 0 reactive vs. 2 reactive ; p<0.001 ) , treatment modality ( surgery vs. conservative treatment ; p=0.006 , and craniotomy vs. other surgical modalities ; p<0.001 ) , underlying disease ( ischemic heart disease vs. other disease ; p=0.034 , and atrial fibrillation vs. other disease ; p=0.018 ) were independently associated with 30-day mortality . in the s - ich patients with esrd
, multivariate analysis showed that age ( 70 years vs. 40 to 54 years ; p=0.019 ) , gcs score ( 3 to 8 vs. 13 to 15 ; p<0.001 , and 9 to 12 vs. 13 to 15 ; p=0.014 ) , pupillary abnormality ( 0 reactive vs. 1 reactive ; p=0.032 , and 0 reactive vs. 2 reactive ; p<0.001 ) , ventricular extension of hemorrhage ( present vs. absent ; p=0.017 ) , hemorrhagic volume ( 60 ml vs. < 30 ml ; p=0.002 , and 30 - 59 ml vs. < 30 ml ; p=0.029 ) , anemia ( present vs. absent ; p=0.008 ) , and treatment modality ( surgery vs. conservative treatment ; p=0.034 ) , were independently associated with high 30-day mortality . associations between other variables and 30-day mortality by univariate analysis were not confirmed by multivariate analysis . in s - ich patients with esrd , duration of hemodialysis was not associated with 30-day mortality ( p=0.199 ) .
actually , underlying disease , which was independently associated with high 30-day mortality in s - ich patients without esrd , was not associated with high 30-day mortality in s - ich patients with esrd .
conversely , several other variables , such as , ventricular extension of hemorrhage , hemorrhagic volume , and anemia , not associated with 30-day mortality in s - ich patients without esrd , were found to be significantly and independently associated with 30-day mortality in s - ich with esrd .
of the variables examined , the following were found to be significantly associated with functional recovery in s - ich patients with and without esrd by univariate analysis ; age , gcs score , location of hemorrhage , hemorrhagic volume , and treatment modality .
however , pupillary response , underlying disease , ventricular extension of hemorrhage , anemia , thrombocytopenia , and crp , which were all found to be associated with 30-day mortality , were not found to be associated with functional recovery in either group by univariate analysis . in patients without esrd
, multivariate logistic regression analysis showed that the following five variables were independently associated with functional recovery at 6 months after s - ich : age ( 40 to 54 years vs. 70 years ; p<0.001 , and 40 to 54 years vs. 55 to 69 years ; p=0.038 ) , gcs score ( 9 to 12 vs. 3 to 8 ; p=0.009 , 13 to 15 vs. 3 to 8 ; p<0.001 , and 13 to 15 vs. 9 to 12 ; p=0.041 ) , location of hemorrhage ( thalamus vs. other regions ; p=0.022 , lobal vs. other regions ; p=0.042 , and ventricle only vs. other regions ; p=0.008 ) , volume of hemorrhage ( < 30 ml vs. 60 ml ; p=0.001 ) , and treatment modality ( conservative treatment vs. surgery ; p=0.039 , other surgical modalities vs. craniotomy ; p<0.001 , and stereotactic aspiration vs. other surgical modalities ; p=0.005 ) . in s - ich patients with esrd , multivariate logistic regression analysis showed that the following four variables were independently associated with functional recovery at 6 months after s - ich ; gcs score ( 9 to 12 vs. 3 to 8 ; p<0.001 , and 13 to 15 vs. 3 to 8 ; p<0.001 ) , hemorrhagic volume ( < 30 ml vs. 60 ml ) , treatment modality ( conservative treatment vs. surgery ; p<0.001 ) , and duration of hemodialysis treatment ( < 50 months vs. 50 months ; p=0.027 ) .
although age and location of hemorrhage were associated with good functional recovery by univariate analysis in s - ich patients with esrd , multivariate analysis showed no such association .
thus , age and location of hemorrhage were associated with functional recovery in s - ich patients without esrd , but not in s - ich patients with esrd ( table 7 ) .
of the estimated 67000 individuals who experienced s - ich in the united states during 2002 , only 20% were expected to be functionally independent 6 months later3 ) .
furthermore , unlike the declining mortality rates of subarachnoid hemorrhage and arteriovenous malformation , which have been attributed to improvements in surgical and critical care techniques , morbidity and mortality after s - ich have remained substantially unchanged for decades17 ) .
s - ich is relatively common in the far east4,23 ) , and in south korea , cerebrovascular disease is the second most common cause of death , for example , in 2009 , 90.1 deaths were recorded per 100000 population deaths26 ) .
several studies have reported the prognostic values of hemodialysis in s - ich patients with esrd . a 22-year ( 1980 - 2002 ) study performed toyoda et al.28 ) in 1740 esrd patients , it was found that stroke occurred in 61 hemodialysis patients during the first 17 years and in 90 patients during the just in the last 5 years .
furthermore , it has been reported that the incidence of hemorrhagic stroke among hemodialysis patients is approximately twice that of ischemic stroke16 ) .
although technical improvements in hemodialysis continue to prolong the life - spans of these patients , the rate of treated esrd continues to increase globally , and as a result , the numbers of esrd patients that experience spontaneous ich is set to increase .
this retrospective study , which is the largest of its type undertaken in korea , was performed to determine 30-day mortality and 6-month functional recovery after s - ich in esrd patients on chronic hemodialysis , and compare these results with those of the general s - ich population .
several studies have examined mortalities in s - ich patients on hemodialysis . in a prospective study , huang et al.10 ) found that overall 30-day mortality was 52.6% ( 20 deaths among 38 patients ) .
in addition , several retrospective studies have reported overall mortalities ranging from 36.5% to 78.9%8,9,16,20 ) . similarly , in the present study , 30-day mortality was 53.9% .
interestingly , molshatzki et al.20 ) reported that mortality is dependent on degree of renal function impairment . in this previous study ,
chronic kidney disease was categorized by estimated baseline glomerular filtration rate into moderate / severe impairment ( < 45 ) , mild impairment ( 45 - 60 ) , and no impairment ( > 60 ml / min/1.73 m ) .
the authors reported 30-day mortalities of 41% for patients with no impairment , 30% for mild impairment , and 83% for moderate / severe impairment20 ) . in the present study , gcs score , pupillary response , and treatment modality
were found to be independently associated with 30-day mortality in both of the study groups .
however , several factors not related to 30-day mortality in non - esrd patients ( age 70 , ventricular extension of hemorrhage , hemorrhagic volume 30 ml , anemia , and enlargement of hematoma within 48 hours ) were associated with 30-day mortality in esrd patients by multivariate analysis using a logistic regression model .
conversely , underlying diseases , such as , ischemic heart disease and atrial fibrillation , which were independently associated with 30-day mortality in non - esrd patients , were associated with 30-day mortality in esrd patients . additionally ,
although hemorrhagic locations were associated with functional recovery in s - ich patients without esrd , they were not associated with functional recovery in s - ich patients with esrd .
huang et al.9 ) suggested that initial gcs score , age > 80 years , an infratentorial location , hemorrhage volume , and ventricular extension of hemorrhage are associated with mortality rate in general s - ich population , but that these associations do not hold in s - ich patients with esrd . as a result
, these authors devised a scoring system based on a combination of initial gcs score , age , and initial systolic blood pressure ( sbp ) , and stratified esrd patients to determine their roles as predictors of mortality in s - ich patients with esrd .
it was concluded that the three above - mentioned factors are optimal predictors of 30-day mortality for s - ich patients with esrd .
similarly , we found that initial gcs score and age were independent predictors of 30-day mortality in these patients .
however , this previously study was limited by the exclusion of patients that underwent surgery or whose families requested dnr .
the gcs is a standard neurological assessment tool is often used to predict cerebrovascular disease outcome8 ) . like the majority of previous studies on predictive models
, we classified patients using gcs scores of 3 - 8 , 9 - 12 , and 13 - 15 .
the gcs scores of s - ich patients with esrd were found to be lower than those of non - esrd patients .
furthermore , we found that a lower gcs score was independently associated with high 30-day mortality rate and a low 6-month functional recovery rate by logistic regression analysis after adjustment .
the 30-day mortality rates of s - ich patients with esrd were 71.4% for a gcs score of 3 - 8 , 56.4% for a score of 9 - 12 , and 14.3% for a score of 13 - 15 .
hypertension may play an important role in the pathogenesis of cardiovascular and cerebrovascular morbidity and mortality ( which is approximately 24% among patients with cerebrovascular diseases and 60 - 90% among hemodialysis patients in us)22 ) .
furthermore , a ' u ' curve relationship between sbp and cerebrovascular mortality in hemodialysis patients has been shown30 ) .
huang et al.9 ) compared two subgroups in their hemodialysis cohort of s - ich patients in taiwan during 1994 - 2004 with a sbp of 130 - 199 mm hg vs. those with a sbp of < 130 mm hg or > 200 mm hg and found the latter was strongly related to 30-day mortality .
in fact , we tried to reduce elevated blood pressure after admission using anti - hypertensives , such as , labetalol and nicardipine .
the important point here is that concise information is required about the optimal control of blood pressure before a s - ich attack , because the continuous control of blood pressure may play a critical role in the development of cerebrovascular disease9 ) .
actually , hematomas in patients undergoing hemodialysis are larger and more likely to increase in size after symptom onset due to an increased bleeding tendency and increase the risk of death .
moreover , outcomes are especially poor in patients that demonstrate hematoma enlargement after hospitalization7 ) . in terms of hematoma enlargement , miyahara et al.19 )
reported that all s - ich patients exhibiting this condition died within 30 days of s - ich , and suggested that an elevated inr predicts hematoma enlargement .
although we also found that hematoma enlargement was associated with high mortality in s - ich patients with esrd , elevated inr was not found to be an independent predictor of mortality in these patients . in the study by miyahara et
al . , all patients received conservative treatment , but in the present study , some patients underwent surgical treatment , which might explain this discrepancy .
although the present study is the largest korean study conducted to date on the clinical outcomes of s - ich patients being treated by hemodialysis for esrd , it has several limitations .
we attempted to reduce this bias by collecting patient data from complete medical and radiological records and by recruiting patients treated using the same protocol .
however , the evaluation was not complete to compare the clinical outcome ( e.g. , 30-day mortality and 6-month functional recovery ) in both groups directly , because of basic differences in clinical characteristics between two groups . to overcome this limitation , prospective and randomized clinical trial must be essential .
second , although patients with esrd have markedly advanced atherosclerosis of the cerebral and coronary arteries and frequently succumb to cardiovascular disease , the conditions of their hearts and cardiovascular systems are not routinely monitored because echocardiography is not routinely carried out in emergency rooms .
third , although it is known that chronic hypertension is a critical contributor to the development of cerebrovascular disease , we were unable to investigate the relation between hypertension and clinical outcome in s - ich patients with esrd due to lack of long - term data before s - ich . forth , detailed analysis of the relation between renal function and clinical outcome was not performed , and as was reported by molshatzki et al.20 ) severity of renal impairment may influence outcomes .
this retrospective study shows that 30-day mortality was four times higher , and that the functional recovery rate was significantly lower in s - ich patients with esrd than in s - ich patients without esrd .
demographically , s - ich patients with esrd had poorer gcs scores , poorer pupillary response , and more underlying disease than s - ich patients without esrd , and in terms of radiologic and laboratory findings , intraventricular hemorrhage , hematoma enlargement within 48 hours , anemia , and thrombocytopenia were more common in s - ich patients with esrd than in those without . based on our comparison of s - ich patients with or without esrd , this study shows age , ventricular extension of hemorrhage , hemorrhagic volume , anemia , and hematoma enlargement within 48 hours increases the risk of mortality , and that duration of hemodialysis treatment affects functional recovery adversely in s - ich patients with esrd .
accordingly , although we think that the effective management of these risk factors may improve the outcomes of esrd patients on hemodialysis that experience s - ich , a prospective randomized clinical trial is needed to confirm our results . | objectivethe aim of this study was to determine 30-day mortality and 6-month functional recovery rates in spontaneous intracerebral hemorrhage ( s - ich ) patients undergoing hemodialysis treatment for end - stage renal disease ( esrd ) , and to compare the outcomes of these patients and s - ich patients without esrd.methodsthe medical records of 1943 s - ich patients from january 2000 to december 2011 were retrospectively analyzed with focus on demographic , radiological , and laboratory characteristics.resultsa total of 1558 supratentorial s - ich patients were included in the present study and 102 ( 6.5% ) were esrd patients .
the 30-day mortality of the s - ich patients with esrd was 53.9% , and 29.4% achieved good functional recovery at 6 months post - s - ich .
multivariate analysis showed that age , glasgow coma scale ( gcs ) score , pupillary abnormality , ventricular extension of hemorrhage , hemorrhagic volume , hematoma enlargement , anemia , and treatment modality were independently associated with 30-day mortality in s - ich patients with esrd ( p<0.05 ) , and that gcs score , volume of hemorrhage , conservative treatment , and shorter hemodialysis duration was independently associated with good functional recovery at 6 months post - s - ich in patients with esrd ( p<0.05).conclusionthis retrospective study showed worse outcome after s - ich in patients with esrd than those without esrd ; 30-day mortality was four times higher and the functional recovery rate was significantly lower in s - ich patients with esrd than in s - ich patients without esrd . |
g - protein
coupled receptors ( gpcrs ) are located in the cellular
membrane and act as mediators for cell signaling , making them promising
therapeutic targets . however , drug design for gpcrs has been challenging
due to the paucity of validated 3d structures .
although these integral
membrane proteins have long been difficult targets for structure determination
efforts , recent methodological developments now provide structures
for 33 of the 819 human gpcrs .
six of these , bovine rhodopsin , human 2 adrenergic receptor , human
adenosine a2a receptor , human muscarinic m2 receptor , human neurotensin nts1 receptor , and human p2y12 receptor , have been crystallized in an active or partially active
conformation , but only one , human 2 adrenergic receptor ,
has been cocrystallized with the full heterotrimeric gs protein . the human cannabinoid
1 ( cb1 ) is a gpcr with high therapeutic potential
as a drug target .
this receptor is activated by -tetrahydrocannabinol ( thc ) in marijuana and increases appetite for
aids and cancer patients .
the cb1-selective inverse agonist / antagonist
rimonabant ( also known as sr141716a ) is
an antiobesity drug that was available in europe and was in phase
iii of us fda clinical trials but had to be withdrawn due to severe
depressive effects .
there is some indication that these side effects
are caused by rimonabant antagonizing the cb1 receptor activity in
brain , underscoring the need for biased
inverse agonists for cb1 that are devoid of these unacceptable side
effects .
nonetheless , the intracellular pathways mediating the beneficial
and unwanted effects have not yet been elucidated .
cb1 has not
yet been crystallized ; however our previous studies ,
using the gensemble ( gpcr ensemble of structures in membrane bilayer
environment ) complete conformational sampling method , predicted the
ensemble of ten low energy cb1 conformations expected to play a role in binding various ligands and controlling
function .
briefly , gensemble carries out systematic sampling of trillions
of seven - helix bundles by rotating and tilting the transmembrane ( tm )
helices starting with templates from previous calculations or x - ray
experiments on other gpcrs .
we consider it important to examine this
full conformational space for cb1 , because gpcrs are dynamic and flexible ,
enabling different ligands to bind to and stabilize quite different
gpcr conformations .
this , in turn , facilitates coupling to multiple
types of intracellular proteins . for each of these
ten cb1 conformations we used the hierarchical
binding site prediction methods , darwindock and gendock , to predict the optimum structures for binding of rimonabant and
seven other ligands .
receptor complexes
are in good agreement with published site - directed mutagenesis data and structure activity relationship ( sar ) data .
we then used these predicted binding sites to design and identify
16 new ligands that we expected might act as inverse agonists upon
binding to the cb1 receptor .
subsequently we tested five of these
ligands experimentally and found that two compounds possess inverse
agonist properties including reduced g - protein coupling and changes
in cellular localization of the receptor .
this provides new design
strategies for developing more selective and potent cb1 inverse agonists
through computational optimization of chemical functionalities of
the compounds coupled with further pharmacological characterization .
we used our gensemble method to predict an ensemble of ten low energy cb1 receptor structures ,
described elsewhere .
these predictions started with four distinct templates [ bovine rhodopsin , turkey 1 adrenergic receptor , human 2 adrenergic receptor , and human adenosine a2a receptor ] , optimized the helix shapes within the turkey
1 adrenergic receptor template , sampled all ( 12 ) = 35 million rotations of each of the seven helices independently ,
followed by sampling of 13 trillion combinations of helix rotations
and tilts to select by energy the best ten conformations of the seven - helix
bundles .
we then docked various ligands , finding the best gpcr conformation
for each ligand .
previous papers have analyzed thoroughly this ensemble for the wild - type
and several mutant receptors , which were used successfully to predict
( in advance of experiment ) mutations that would bias the ensemble
toward the fully inactive , the constitutively active , or the essentially
fully active states . to obtain
a diverse set of ligand conformations for docking
the molecular structure
of rimonabant was constructed with maestro software , and a conformational search was performed with macromodel
software , which generated 83 conformations
of the ligand .
sytematic and extended torsional sampling options were
used where the selected rotatable bonds are rotated 360 in 30
increments .
ligand conformations that fall within an energy window
of 10 kcal / mol and an rmsd diversity of 0.5 were saved for
subsequent steps .
the conformational search was conducted with the
opls 2005 force field and in a dielectric
of 80.37 to match water .
subsequently , we preformed two rounds of
clustering for each ligand ; in the first round we clustered ligands
with a 2.0 diversity followed by another round of clustering
with a 1.0 diversity .
the mulliken populations of each atom
were calculated with jaguar software using
density functional theory ( dft ) with the b3lyp functional and the
6 - 31 g * * basis set .
an additional ligand conformation was constructed from existing coordinates
from the crystallized rimonabant in methanol solvate deposited in the cambridge structural database .
each ligand was minimized using the surface
generalize born ( sgb ) solvation model for 100 steps or to a convergence threshold of 0.2 kcal / mol /
rms force with the mpsim program . for
each of the ten conformations of rimonabant , we used the darwindock
and gendock methods to predict the optimum binding site for each of the ten
lowest energy protein structures predicted by gensemble for the cb1
receptor .
the darwindock method
aims at generating a complete set of poses for the binding pocket
while using rmsd clustering of the poses to dramatically reduce the
computational cost . to provide flexibility and space for the ligand
to identify favorable binding sites , we replaced the seven bulky hydrophobic
residues ( filmyvw ) with alanines .
the mutated residues are called
alanized residues , and the mutated protein is called alanized protein .
for each of the best 100 ligand poses in the alanized protein , we
then dealanized the mutated residues back to their original hydrophobic
identity and optimized their positions along with those of other residues
in the binding site using scream .
this
leads to a unique set of optimized residue side chains for each of
the 100 ligand poses . in this process
we did not replace the w5.43
residue with alanine because we consider this tryptophan to be critical
for ligand interaction based on site - directed mutagenesis data , leaving its side chain in the form predicted
by scream for each of the ten protein conformations . in the
pose generation step of darwindock
, a ligand pose is acceptable if
it clashed or bumped the receptor residues at six positions or less .
first , we used dock6 to generate 5,000
ligand poses ( without evaluating an energy ) and clustered them into
families , where every family member is within a 2.00 rmsd of
each other .
this procedure of adding 5,000
poses and reclustering is repeated until the number of new families
generated is less than 2% of the total number of families in the preceding
iteration .
at this point darwindock scores the energies
of one representative from each family , the family head , and selects
the 10% of family heads with the lowest dreiding energies .
all members of these respective families are
then scored energetically . from this list of approximately 5000 poses ,
we select the lowest 50 by each of three criteria : lowest hydrophobic
energy , lowest polar energy , and lowest total energy , giving at most
150 poses .
gendock
was used to refine
the 150 docked ligand receptor poses generated by darwindock .
in the scream step ,
residues were replaced with the original hydrophobic residues but
using the optimum side chain rotamers to avoid any clashes with the
ligand and other protein side chains .
then the entire complex was
minimized for 10 steps for each case to remove any bad contacts .
next ,
the receptor was neutralized , so that the acidic residues ( aspartic
acid and glutamic acid ) each gained a proton , and the basic residues
( lysine and arginine ) each lost a proton .
the resulting receptor ligand
complexes were minimized for 60 steps using the dreiding iii ff .
then for each of the ten receptor structures
and each of the ten ligand conformations ( 100 complexes total ) , we
selected the lowest binding energy structure , including strain and
ligand solvation , which we expected to best represent the binding
affinity . the binding energy with strain and ligand solvation is defined
as the energy difference between the complex and the sum of the receptor
and ligand energies with ligand strain and ligand solvation included .
the selected complex was minimized with the dreiding iii ff with the lj vdw term ( dreiding iii - lj ff ) in
vacuum for 50 steps or to an rms force threshold of 0.5 kcal / mol /
using the mpsim program .
we took the most stable predicted rimonabant - cb1
receptor complex shown in figure 1 and extracted the rimonabant .
then , we modified it
with maestro software to look like the new derivative , calculated
the mulliken charges at the b3lyp dft level ( with jaguar software ) ,
minimized this configuration with 100 steps of conjugate gradients
( or to a threshold of 0.2 kcal
/ mol / ) using the mpsim program ,
and predicted the binding site using darwindock as described above .
however , instead of docking the new ligands to all ten cb1 conformations ,
we docked only to the alanized cb1 receptor conformation wild - type
6 ( wt6 ) because it produced the lowest energy ( most stable ) complex
with rimonabant shown in figure 1 .
we repeated these steps for seven
derivatives and for the original rimonabant ligand as a control . for
rimonabant
, we did not alter the ligand structure but rather docked
the optimized ligand conformation .
the final complexes were chosen
according to the lowest binding energy including strain and ligand
solvation .
for comparison of the experimental binding affinities with
the computational energies , we used the binding energy , that is , the
difference in energy between the receptor ligand complex and
the receptor and the ligand structures calculated separately .
the
ligand strain and ligand solvation were ignored since we were comparing
energetics across different ligands based on the same rimonabant conformation . the experimental change in free energy upon ligand binding to the
receptor , gbindexp ,
is obtained
from the pki in eq 1:1 the pki is the experimental binding equilibrium constant of
the inhibitory
ligand . since the pki describes the strength
of the interaction between the receptor and ligand , we use it to derive
gbindexp .
the g , or the difference in the change in experimental
free energy upon ligand binding for a given inverse agonist ( gbindexp ) with respect to the corresponding
value of rimonabant , is determined by eq 2:2 this expression
was used to compare experimental changes in binding
energy with our predicted changes in binding energy for the series
of ligands .
our predicted rimonabant - cb1 binding site was used to identify
new potential cb1 inverse agonists .
the predicted pharmacophore ( shown
in figure 1b ) , was
used in a preliminary search over the 2 million compounds in pubchem for ligands that are similar to rimonabant according
to the tanimoto coefficient , an indicator of how
similar two 2d ligands structures are to one another .
the tanimoto
coefficient ranges from 0 to 1 , with 0 being no resemblance between
the molecules and 1 being identical molecules .
we wanted ligands that
are similar to rimonabant , but which could exploit nearby underused
polar and aromatic residues in the predicted binding site .
for example ,
k7.32 is located near rimonabant but does not form a hydrogen bond
or salt bridge with it ; thus we searched for ligands similar to rimonabant ,
with an appropriately placed functional group to create the new polar
interaction .
we also wanted a ligand that is commercially available
but has not been previously tested with cb1 , so that our prediction
can be tested easily .
once we had identified a ligand ( msc1 , described
later ) from pubchem that met the above criteria , we performed a search
in pubchem to identify ligands similar to msc1 ( scoring > 0.90 on
the
tanimoto coefficient ) or that have a 2d structure that is 90% similar
to the new pubchem ligand msc1 and satisfy specific constraints coming
from the cb1 binding site .
the 16 ligands found by using pubchem in
this protocol are denoted by the acronym
hek293 cells were maintained in
dulbecco s modified eagle s
medium supplemented with 10% fetal bovine serum and 3.5% mg / ml glucose
at 37 c in 5% co2 .
one day prior to transfection ,
cells were seeded at approximately 1 million cells/100 mm dishes .
the cells were transiently transfected by the calcium phosphate precipitation
method . at 24 h post - transfection
, the
cells were harvested in phosphate buffered saline ( pbs ) containing
mammalian protease inhibitor cocktail ( ( 42-aminoethyl)benzene - sulfonyl
fluoride , pepstatin a , e-64 , bestatin , leupeptin , and aprotinin ) ( sigma - aldrich ,
st .
louis , mo ) and lysed by nitrogen cavitation at 750 psi for 5 min .
the lysate was spun at 500 g for 10 min at 4 c , and the supernatant
was subsequently spun at 100,000 g for 45 min at 4 c .
the membrane - containing
pellet was resuspended in tme buffer ( 25 mm tris - hcl , 5 mm mgcl2 ,
and 1 mm edta , ph 7.4 ) containing 7% w / v sucrose .
competition
binding assays were performed as described previously to determine the binding affinity of the test compounds
to the receptor .
briefly , 6 g of membrane preparation was incubated
for 60 min in tme buffer containing 0.1% fatty acid - free bsa with
a fixed concentration of tracer [ h]sr141716a ( 43 ci / mmol ,
perkinelmer life sciences ( boston , ma ) ) typically at its kd which was determined from a saturation binding isotherm .
at least nine concentrations of the unlabeled test compound ( ranging
between 100 pm and 32 m ) were used for the binding assays .
nonspecific binding was determined in the presence of unlabeled sr141716a
( 1 m ) . the reaction was terminated by filtration with a brandell
cell harvester through whatman gf / c filter paper , and the radioactivity
was measured .
briefly , 6 g of membranes was incubated
for 60 min at 30 c in gtps binding assay buffer ( 50 mm
tris - hcl , ph 7.4 , 3 mm mgcl2 , 0.2 mm egta , and 100 mm nacl )
with the unlabeled 2-arachidonoylglycerol ( 2-ag ) ( at least nine different
concentrations were used ranging between 100 pm and 1 m ) , 0.1
nm [ s]gtps ( 1250 ci / mmol ; perkinelmer life sciences ,
boston , ma ) , 10 m gdp ( sigma , st .
louis , mo ) , and 0.1% ( w / v )
bsa in the absence and presence of 10 m test compounds .
the
effect of the compound on inhibiting the level of basal gtps
binding was evaluated in the absence of agonist .
nonspecific binding
was determined with 10 m unlabeled gtps ( sigma , st .
after rapid filtration through whatman gf / c filters the
radioactivity trapped in the filters was determined by liquid scintillation
counting .
data are presented as the mean se or
the mean with the corresponding 95% confidence limits from at least
three independent experiments .
the ki values
of the test compounds were calculated by nonlinear regression using
prism 6.0 ( graphpad software inc . , san diego , ca ) .
hek293 cells
expressing the cb1 receptor c - terminally fused to gfp were seeded
onto 35 mm glass - bottomed dishes ( mattek , ma ) precoated with poly - d - lysine .
cells were treated with 10 m msc compounds
or 1 m rimonabant for various lengths of time and then washed
three times with pbs , followed by fixation with 4% paraformaldehyde
for 10 min at room temperature .
cells were mounted in vectashield
mounting medium ( vector laboratories , ca ) and visualized using a leica
tcs sp2 confocal microscope ( leica microsystems , wetzler , germany ) .
images were collected from at least three independently transfected
cell dishes and processed for presentation in figures using adobe
photoshop 6.0 ( adobe systems , san jose , ca ) .
the predicted lowest energy pose
for the cb1-rimonabant complex is shown in figure 1a , and the corresponding pharmacophore is
shown in figure 1b .
we predict that rimonabant is anchored by hydrogen bonds to w5.43
and k3.28 [ residues are numbered according to the ballesteros
weinstein
scheme . ] , and indeed previous site - directed
mutagenesis data indicate that mutations of w5.43 and k3.28 to alanine
have the largest effect in decreasing experimental binding affinity
upon mutation to alanine with > 1000-fold and 17.2-fold , respectively .
the components of the predicted binding energies for this complex
are shown in table 1 , showing contributions from each residue in the binding site .
the
most important residue is w5.43 with a predicted binding contribution
to rimonabant of 9.36 kcal / mol , consistent with the decrease
by a factor of > 1000 upon mutation to alanine .
indeed our predicted pharmacophore for rimonabant ( figure 1b ) has the significant
polar and hydrophobic contacts with the cb1 receptor expected for
a strongly bonding ligand . according to the cavity analysis
in table 1
, k3.28 has
the third largest binding interaction energy ( 5.57 kcal / mol )
with rimonabant due to its hydrogen bond with the amide carbonyl of
the ligand .
the same experiments with the rimonabant derivative
vchsr , an analogue of rimonabant with hydrocarbons replacing the amide
and piperidine groups , showed that the binding affinity remained unaffected
upon mutation of k3.28 to alanine .
our study shows that k3.28 interacts
with the polar atoms of the amide group or piperidine ring , including
the carbonyl , which agrees with the mutagenesis study on this ligand
that lacks these polar atoms . in
our docked pose of rimonabant with the wt receptor
, the f3.36
residue has a significant interaction with the ligand , including a
van der waals component of 3.90 kcal / mol . in binding assays ,
the f3.36a mutation decreased the binding affinity of this inverse
agonist by 20-fold , or 1.78 kcal / mol ,
for the cb1 receptor .
interestingly , the f3.36l mutation decreased
the binding affinity by only 2-fold , indicating the importance of
the bulky hydrophobic residue for receptor ligand interaction .
( a ) the predicted
structures for inverse agonist rimonabant to
wt6 , the sixth best energetic conformation of the cb1 receptor .
( b ) our predicted
pharmacophore shows that in our gensemble - derived cb1 structure rimonabant
forms two hydrogen bonds , including one with w5.43 , and have strong
aromatic interactions with the receptor . only residues with energies with
contributions stronger than 1.0 kcal / mol are shown .
the nonbonding
( nonbond ) energy
in the far right column is the sum of the van der waals ( vdw ) energy ,
the electrostatic ( coulomb ) energy , and hydrogen bond ( h - bond ) energy .
we predicted that m6.55 has the second strongest interaction ( 6.8
kcal / mol ) with cb1 , which is almost entirely hydrophobic ( table 1 ) .
however , experiments
show that mutating m6.55 to alanine decreases the binding affinity
to cb1 by only 3-fold or by 0.65 kcal / mol
in energy .
one possible explanation is that the m6.55a mutation alters
the binding site and pose for rimonabant in a way that other residues
become available to compensate for the lost interaction with methionine .
a very important advantage of the gensemble
approach is that we have an ensemble of ten low energy structures
to which rimonabant is allowed to bind .
we showed for the adenosine
a3 receptor that neither the selective agonists nor the selective
antagonists prefer the lowest energy apoprotein structures .
also , for ccr5 we showed that the antagonists
all prefer to bind to structures that are not the lowest energy for
the apoprotein .
similarly for cb1 , the
most favorable binding is with wt6 , the sixth best conformation for
the apo - cb1 structure , not with the lowest energy apo - cb1 conformation
( wt1 ) ( table 2 ) .
since
the ligands do not preferentially bind to the lowest energy conformation ,
these docking results support the concept that gpcrs are highly dynamic
structures that can sample many conformations , providing an opportunity
for different ligands that can selectively bind to different conformations ,
and perhaps lead to different function .
the ten cb1 conformations
used in this study range in backbone rmsd from 0.42.1 .
of course the protein conformation may change when complexed with
the ligand . surprisingly , the wt6 cb1 structure , to which the
inverse agonist rimonabant preferentially binds , lacks the r3.50 and
d6.30 ionic lock , which is generally believed to be important for
preventing activation .
experimental data have shown that rimonabant
inactivates the cb1 receptor , while our calculations
find that the inverse agonist has the best binding energies with activated
cb1 conformations .
indeed of the top four rimonabant - cb1 complexes ,
we find that three conformations ( wt6 , wt1 , and wt5 ) do not form an
ionic lock to the conserved r3.50 and d6.30 .
the complexes are
ranked according
to best binding energy with strain and ligand solvation included .
receptor conformation numbers
that
do not contain the conserved r3.50 and d6.30 salt bridge are in bold . on the other hand , several
studies have suggested that a r3.50
to d6.30 salt bridge is not necessary for maintaining the inactive
conformation .
for example , audet and bouvier showed that the r3.50 and d6.30 salt bridge is broken in all but
three of the 36 crystallized gpcr structures bound to antagonists
and inverse agonists analyzed .
it should be noted that many crystallized
gpcr structures , such as the human adenosine a2a receptor ,
contain an inserted t4 lysozyme for stabilization , which may impact
the conformation of the cytocellular ends of the helices and thus
prevent the formation of the ionic lock . in our previous work with the human cb1 receptor ,
we showed
that
a different residue , r2.37 , plays an important role in preventing
receptor activation .
we found that this residue forms an ionic lock via
a salt bridge interaction with d6.30 to stabilize the inactive conformation .
indeed our calculations found that the rimonabant inverse agonist
did not prefer to bind to the conformations that contained this signature
contact as shown in table 2 . the rimonabant - bound cb1 conformation wt6 resembles the
predicted structure of a constitutively active mutant ,
the two structures have a c-rmsd of 1.2 , which is
smaller than most crystal structure resolutions suggesting that the two conformations are very similar .
for comparison ,
the difference between the wt receptor conformations
wt2 ( which contains the r3.50+d6.30 salt bridge ) and wt6 ( without
that salt bridge ) is 1.8 . to validate our proposed
binding site
, we docked seven known derivatives of rimonabant ( figure 2a ) to the cb1 conformation
and plotted the calculated binding energies against the experimental
binding affinity , pki , to find the correlation
between the two data sets ( figure 3 ) . for each ligand
, we found the same binding site
as for rimonabant ( figure 2b ) .
figure 3a shows a comparison between the pki of
the inverse agonists from hurst et al . and our calculated binding energies .
we observe a very high correlation
of 93.4% , indicating good agreement between the trends observed in
experimental binding affinities and our own calculated binding energies .
the portions of the ligand
that are different from rimonabant are colored in red .
the derivatives
in the first two rows were used in the hurst et al .
, 2006 study , and the derivatives in the third row were used
in the dantona et al .
comparison of experimental binding affinities and computationally
calculated energies between receptor and inverse agonists .
( a ) plot
showing the correlation of the experimental binding affinity , pki , for the six inverse agonists in hurst et
al .
this leads to r = 0.93 ,
indicating that the error bar for our calculations is 2 kcal / mol ,
while the range is 18 kcal / mol .
( b ) plot showing the correlation of
the g for seven previously
published inverse agonists with respect to that of rimonabant versus the calculated binding energy .
r = 0.89 , indicating that the error bar for our calculations is 2
kcal / mol , while the range is 14 kcal / mol . for the inverse agonists from dantona et al .
the gbindexp values are within 1 kcal / mol of each other since
the energies are 9.33 kcal / mol for am-281 , 10.0 kcal / mol
for am-251 , and 9.70 kcal / mol for rimonabant .
similarly , both
the calculated cavity and binding energies for the three ligands are
approximately within 1 kcal / mol .
the binding energies are 67.4
to 69.0 kcal / mol , which are very similar and agree with the
experimentally observed negligible changes in free energy upon binding .
this excellent correlation relies on the predicted energies that are
based purely on enthalpy and do not include entropy .
however , the
entropy change upon binding of all the ligands should be similar since
they all have the same number of rotatable bonds .
figure 3b combines
the results from figure 3a with those regarding the inverse agonists from dantona
et al . for a single comparison of our
calculated binding energies and the binding affinities from the two
experiments .
the respective g values from eq 2 are
plotted against the calculated binding energies for all eight ligands ,
leading to an 89.4% correlation .
the excellent agreement between our
predicted energies and the experimental binding affinity shown in figure 3b provides confidence
that our predicted binding site for rimonabant is reasonable . with
a reliable rimonabant binding pharmacophore for cb1 , which
is supported by site - directed mutagenesis and sar data , we aimed to
design a new , more selective cb1 inverse agonist .
after the informed
search in pubchem , described in the methods section , we selected zinc08587042 , which
we refer to as msc1 ( figure 4 ) , a ligand with a 68% similarity to rimonabant according
to the 2d tanimoto coefficient .
rather
than relying exclusively on pubchem s search algorithm , which
looks for molecules based on a 0.90 tanimoto similarity score with
respect to rimonabant , we used the predicted cb1 binding site and
searched for a ligand that maintained key structural aspects of rimonabant
and additionally took advantage of underused residues in the binding
site . for example , we wanted to find a ligand having the same amide
and pyrazole functional groups to maintain the hydrogen bonds with
k3.28 and w5.43 , respectively . yet , we wanted to replace the rimonabant
piperidine ring with a phenyl ring and a substituted polar group to
improve the interactions with the aromatic residues that we found
nearby and to gain an additional hydrogen bond with k7.32 , which is
within the rimonabant binding site .
furthermore , we wanted to replace
the methyl group of the pyrazole group with a polar substitute to
form a hydrogen bond with s7.39 .
msc1 is attractive because it contains
an acetylphenyl group , which we predicted would reach into the aromatic
pocket of the extracellular end , and a triazole ring , which would
replace a methyl group with a nitrogen atom .
we also wanted a small
molecule that was commercially available and that had not been previously
tested with cb1 in bioactivity assays .
portions of msc1 highlighted in red
are different from the corresponding groups in rimonabant .
pubchem also identified 15 other small molecule
ligands that have
a 2d structural similarity tanimoto score of 0.90 with msc1 or have
2d structures that are 90% similar to that of msc1 ( figure 5 ) .
we docked these 16 ligands
to the ensemble of predicted cb1 structures , found their corresponding
binding energies , and predicted their respective pki values based on figure 3 .
of the
16 msc small molecules , 14 compounds , msc1 msc6 , msc8msc11 ,
and msc13msc16 were commercially available .
pubchem identified
15 ligands that have 2d structures similar to msc1 . from the 16 compounds
identified from the computational analyses
, we chose five compounds
based on the 2d structural similarity , tanimoto score , and commercial
availability .
to evaluate
experimentally the binding affinities of the compounds , we performed
competition binding experiments using [ h ] rimonabant as
a tracer .
msc1 , msc3 , and msc9 bound the receptor with ki values of 502 nm , 495 nm , and 4619 nm , respectively
( table 3 ) .
these data
indicate that while removal of the chloro group from the chlorophenyl
ring of msc1 does not impact receptor binding ( msc1 versus msc3 ) ,
the position of the methyl group in one of the biphenyl rings and
the acetyl group in the amide - linked phenyl ring is critical for receptor
binding ( msc3 versus msc9 ) .
msc5 and msc8 failed to bind cb1 up to
32 m ( table 3 ) . unexpectedly , our experimental ki values
suggested a weaker affinity than rimonabant compared to the predictions .
this may be due to the limitations of force fields to reliably predict
relative binding affinities .
data are the median and corresponding 95%
confidence limits of three independent experiments performed in duplicate .
since the experimental binding affinities to the cb1 receptor were
higher for msc1 and msc3 than for msc5 , msc8 , and msc9 , we further
tested msc1 and msc3 using [ s]gtps binding assays
to evaluate changes in g - protein coupling .
we assessed the effect
of the compounds on the basal and the 2-ag - induced gtps binding .
we chose 2-ag , the endogenous eicosanoid cb1 agonist for the assay
since it showed binding affinity comparable to msc1 and msc3 .
the addition
of 10 m of msc1 and msc3 resulted in substantial reduction
in emax values with 90 fmol / mg and 81
fmol / mg , respectively , compared with that in the absence of these
msc compounds ( emax = 126 fmol / mg ) ( figure 6a ) .
these compounds
also exhibited small but not statistically significant shifts in ec50 values ( 785 nm for msc1 , 770 nm for msc3 , compared to 521
nm for the absence of any msc compound ) .
thus , the extent of g protein
coupling ( emax values ) changes , though
the amount of agonist needed to achieve this level of gtps
binding ( ec50 values ) does not . furthermore , figure 6b showed that both msc1 and
msc3 substantially decreased the basal [ s]gtps
binding ( 67 fmol / mg ) with specific binding of 50 fmol / mg and 47 fmol / mg ,
respectively . whereas prolonged agonist treatment can remove
receptors from the
cell surface by endocytosis , inverse agonists have been shown to enhance
the cell surface localization , consistent with receptor inactivation .
cb1 exhibits some constitutive activity , and the majority of cb1
receptors are localized on intracellular vesicles even in the absence
of agonist .
we tested if the msc compounds can affect cellular localization
of the receptor using confocal microscopy of cells expressing gfp - tagged
cb1 . similarly to rimonabant treatment , upon treatment with 10 m
msc1 or msc3 the level of cell surface localization of the receptor
was enhanced though the extent for msc3 is less than msc1 ( figure 6c ) .
collectively ,
although msc1 and msc3 exhibited somewhat lower potency and efficacy
compared to rimonabant , these data suggest that they possess inverse
agonist properties as evidenced by reduction in g - protein coupling
and enhancement of cb1 cell surface expression .
we took the msc1-bound
cb1 structure and relaxed it in an explicit lipid bilayer environment
using 50 ns of molecular dynamics ( md ) simulations .
isobaric conditions ( npt ) at
310 k and 1 atm , using periodic boundary conditions with particle - mesh
ewald summation method for calculating long - range coulomb interactions .
the temperature is controlled using langevin dynamics , and the pressure
is maintained using a langevin - hoover barostat .
these simulations
showed that binding of msc1 to the cb1 receptor constrains tm helix
6 more through additional intracellular couplings preventing the g - protein
from engaging the receptor , which is consistent with experimental
findings of msc1 ligand s inverse agonism .
these results offer
opportunities for developing new cb1 inverse agonists through the
optimization of chemical functionalities of the compounds and further
pharmacological characterization .
effect of msc1 and msc3 on the [ s]gtps binding
to hek293 cell membranes expressing the cb1 wt receptor .
2-ag - induced [ s]gtps binding in the
absence ( ) or presence of 10 m ( ) msc1 and
msc3 .
the levels of untransfected cells ( no cb1 ) and rimonabant
treated samples are shown for comparison .
all data are the mean se of at least three independent experiments performed in
duplicate .
( c ) cellular distribution of the wt receptor upon treatment
with msc compounds .
hek293 cells expressing the cb1 wt - gfp receptor
were incubated with vehicle alone ( 0.03% dmso ) , msc1 ( 10 m )
or msc3 ( 10 m ) for 6 h. rimonabant - treated ( 1 m ) cell
is shown for comparison .
our predicted rimonabant binding site
in cb1 is different from previous computational studies that used
the bovine rhodopsin template to create a cb1 homology structure for
docking rimonabant.supporting information figure s1 compares the rimonabant pharmacophore published by lange
and kruse ( figure s1a ) with the pharmacophore from our current study ( figure s1b ) .
both agree that rimonabant ( figure s1a ) spans the width of the binding site
with the chlorophenyl rings near tm5 and the piperidine ring near
tm3 .
however , we predict that rimonabant ( figure s1b ) lies parallel to the z - axis passing from
the extracellular to intracellular side of the membrane , which is
perpendicular to the previous models ( figure s1a ) .
although both agree that a hydrogen bond forms between k3.28 and
the amide carbonyl of rimonabant , the earlier models suggest that
k3.28 also participates in a salt bridge with d6.58 , which we do not observe .
in addition ,
the previous models have the w5.43 residue sandwiched by the two chlorophenyl
groups of rimonabant , leading to strong aromatic interactions .
furthermore ,
multiple other aromatic residues in the binding pocket ( f3.25 , f3.36 ,
w4.64 , y5.39 , w6.48 ) were found to participate in stacking with the
ligand , whereas in
our predicted binding site , it is w5.43 that has the biggest impact
on binding affinity , from the hydrogen bond with the pyrazole ring
of rimonabant . in our predicted structure , we find that the f3.36
residue is sandwiched by the chlorophenyl groups rather than w5.43 .
in addition , a paper from shim et al . based its cb1 model on the human
2 adrenergic receptor to identify a binding site similar to the one from lange and krause but with the ligand lying perpendicular to our
predicted structure , with the piperidine ring pointing to the tm127
binding pocket and the chlorophenyl rings pointing toward tm5 , where
they form aromatic stacks with w5.43 and w6.48
. one would expect
our predicted rimonabant binding site to be different from the previously
published ones since our predicted receptor structures are quite different .
specifically , we sample all rotations of all helices about their axes
and helix tilt and sweep angles , a total of nearly 13 trillion receptor
conformations . in contrast , the previous studies used homology models
based on the bovine rhodopsin or human 2 adrenergic
receptor structure templates , so that no rotations or tilting of the
helices were conducted .
these three gpcrs have very similar global
conformations , but we performed considerable sampling to obtain cb1
receptor conformations that were significantly different and more
stable than the starting template .
the method we used to identify our final receptor - inverse
agonist complex is also different .
the study from shim et al . used the selection criterion that one of the
two chlorophenyl rings should be near the tm356 hydrophobic
site , so their final pose was chosen based on the interactions of
rimonabant with residues for which there was site - directed mutagenesis
data .
however , the structures in our study were based on the binding
energy of the complexes , with no extra conditions imposed .
the previously
suggested rimonabant binding pose most similar to ours came from another
study in which the ligand was docked to a bovine rhodopsin structure - based
homology model .
this predicted binding
site is similar to the current model , with rimonabant sitting vertically
in the binding pocket
the piperidine ring points toward the
extracellular end and the chlorophenyl rings point toward the intracellular
end .
however , it does not lead to any hydrogen bonds with w5.43 and
finds a hydrogen bond with y5.39 , that we did not observe .
since many antiobesity drugs including
rimonabant have been suspended
from the market , there remains an enormous unmet need for compounds
that reduce food intake . in an effort to develop novel inverse agonists
for cb1 , we predicted the binding sites and energies for rimonabant
and structurally related molecules to our previously predicted and
validated cb1 receptor conformations .
our calculated binding energies
for the compounds are in good agreement with the previously reported
mutagenesis and sar data .
based on the predicted binding site of these inverse agonists , we
identified 16 new potential cb1 inverse agonists .
we tested five of
these experimentally and found that two of them ( msc1 and msc3 ) antagonized
g - protein coupling and enhanced surface localization of the receptor
suggesting they are inverse agonists .
the md simulations of msc1:cb1
complex in explicit lipid bilayer environment show that the relaxed
receptor exhibits additional intracellular couplings that will prevent
g - protein coupling , which is consistent with the characterization
of msc1 as an inverse agonist .
although msc1 and msc3 are less potent
than rimonabant , they provide new starting points for developing new
candidates for antiobesity drugs that target cb1 . | human
cannabinoid type 1 ( cb1 ) g - protein coupled receptor is a
potential therapeutic target for obesity .
the previously predicted
and experimentally validated ensemble of ligand - free conformations
of cb1 [ scott , c. e. et al .
protein sci.2013 , 22 , 10111323184890 ; ahn , k. h. et al .
proteins2013 , 81 , 1304131723408552 ] are used here to predict the binding sites
for known cb1-selective inverse agonists including rimonabant and
its seven known derivatives .
this binding pocket , which differs significantly
from previously published models , is used to identify 16 novel compounds
expected to be cb1 inverse agonists by exploiting potential new interactions .
we show experimentally that two of these compounds exhibit inverse
agonist properties including inhibition of basal and agonist - induced
g - protein coupling activity , as well as an enhanced level of cb1 cell
surface localization .
this demonstrates the utility of using the predicted
binding sites for an ensemble of cb1 receptor structures for designing
new cb1 inverse agonists . |
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